# NEURAL SUBSTRATES OF ACUPUNCTURE: FROM PERIPHERAL TO CENTRAL NERVOUS SYSTEM MECHANISMS

EDITED BY : Younbyoung Chae, Vitaly Napadow, Florian Beissner, Yi-Wen Lin and Richard E. Harris PUBLISHED IN : Frontiers in Neuroscience, Frontiers in Psychology, Frontiers in Cellular Neuroscience and Frontiers in Human Neuroscience

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ISSN 1664-8714 ISBN 978-2-88963-505-4 DOI 10.3389/978-2-88963-505-4

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# NEURAL SUBSTRATES OF ACUPUNCTURE: FROM PERIPHERAL TO CENTRAL NERVOUS SYSTEM MECHANISMS

Topic Editors:

Younbyoung Chae, Kyung Hee University, South Korea Vitaly Napadow, Harvard Medical School, United States Florian Beissner, Hannover Medical School, Germany Yi-Wen Lin, China Medical University, Taiwan Richard E. Harris, University of Michigan, United States

Citation: Chae, Y., Napadow, V., Beissner, F., Lin, Y.-W., Harris, R. E., eds. (2020). Neural Substrates of Acupuncture: From Peripheral to Central Nervous System Mechanisms. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88963-505-4

# Table of Contents

*05 Editorial: Neural Substrates of Acupuncture: From Peripheral to Central Nervous System Mechanisms*

Vitaly Napadow, Florian Beissner, Yiwen Lin, Younbyoung Chae and Richard E. Harris

*07 Neuropeptides SP and CGRP Underlie the Electrical Properties of Acupoints*

Yu Fan, Do-Hee Kim, Yeonhee Ryu, Suchan Chang, Bong Hyo Lee, Chae Ha Yang and Hee Young Kim

*19 Moxibustion Modulates Sympathoexcitatory Cardiovascular Reflex Responses Through Paraventricular Nucleus*

Ling Cheng, Peng Li, Yash Patel, Yiwei Gong, Zhi-Ling Guo, Huangan Wu, Shaista Malik and Stephanie C. Tjen-A-Looi

*29 Default Mode Network as a Neural Substrate of Acupuncture: Evidence, Challenges and Strategy*

Yuqi Zhang, Haolin Zhang, Till Nierhaus, Daniel Pach, Claudia M. Witt and Ming Yi


Yi Huang, Jui-Cheng Chen, Chun-Ming Chen, Chon-Haw Tsai and Ming-Kuei Lu

*51 Peripheral Sensory Nerve Tissue but Not Connective Tissue is Involved in the Action of Acupuncture*

Suchan Chang, O. Sang Kwon, Se Kyun Bang, Do-Hee Kim, Min Won Baek, Yeonhee Ryu, Jong Han Bae, Yu Fan, Soo Min Lee, Hyung Kyu Kim, Bong Hyo Lee, Chae Ha Yang and Hee Young Kim


Till Nierhaus, Yinghui Chang, Bin Liu, Xuemin Shi, Ming Yi, Claudia M. Witt and Daniel Pach


Juan Wan, Yi Ding, Sha Nan, Qiulin Zhang, Jinrui Sun, Chuanguang Suo and Mingxing Ding


So-Min Lee and Ji-Yeun Park


Suchan Chang, Yeonhee Ryu, Yu Fan, Se Kyun Bang, Nam Jun Kim, Jin Gyeom Lee, Jin Mook Kim, Bong Hyo Lee, Chae Ha Yang and Hee Young Kim

*171 Differential Influence of Acupuncture Somatosensory and Cognitive/ Affective Components on Functional Brain Connectivity and Pain Reduction During Low Back Pain State*

Jeungchan Lee, Seulgi Eun, Jieun Kim, Jun-Hwan Lee and Kyungmo Park


Zhi Ma, Zengli Zhang, Fuhai Bai, Tao Jiang, Chaoying Yan and Qiang Wang

# Editorial: Neural Substrates of Acupuncture: From Peripheral to Central Nervous System Mechanisms

Vitaly Napadow<sup>1</sup> \*, Florian Beissner <sup>2</sup> , Yiwen Lin<sup>3</sup> , Younbyoung Chae<sup>4</sup> and Richard E. Harris <sup>5</sup>

*<sup>1</sup> Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States, <sup>2</sup> Somatosensory and Autonomic Therapy Research, Hannover Medical School, Hanover, Germany, <sup>3</sup> Graduate Institute of Acupuncture Science, College of Chinese Medicine, China Medical University, Taichung, Taiwan, <sup>4</sup> Acupuncture and Meridian Science Research Center, Kyung Hee University, Seoul, South Korea, <sup>5</sup> Department of Anesthesiology, Chronic Pain and Fatigue Research Center, University of Michigan, Ann Arbor, MI, United States*

Keywords: acupuncture, neuroscience, somatosensation, endorphin, autonomic nervous system

#### **Editorial on the Research Topic**

#### **Neural Substrates of Acupuncture: From Peripheral to Central Nervous System Mechanisms**

"Acupuncture" is a therapeutic intervention involving percutaneous mechanical, thermal, or electrical stimulation via needling of specific locations on the body. Acupuncture has been used in China and other Asian countries for thousands of years, and is one component of traditional Chinese medicine. Usage of acupuncture in Western countries, such as the United States, has been growing slowly but steadily (Ma et al., 2016; Nahin et al., 2016), with most common applications for chronic pain and neurological conditions. Mechanistic research exploring how acupuncture may reduce pain and contribute to other clinically meaningful outcomes is ongoing. This special issue further explores the neural basis of acupuncture, focusing on both peripheral and central nervous system mechanisms.

Historically, in the West, acupuncture research began mainly in the 1970's. An early popular theory to explain acupuncture analgesia was the "gate control theory," which posited that stimulation from acupuncture needling produces pre-synaptic inhibition of nociceptive afference in the central nervous system (Melzack and Wall, 1965). However, the gate control theory would predict analgesia on the order of milliseconds, while acupuncture analgesia develops much more slowly, reaching a maximum analgesic effect 30 min after initial stimulation (Pomeranz, 1989). Such results seemingly refuted the previously popular gate control theory. However, other research studies in the 1970's clearly linked peripheral and central nervous system signaling in acupuncture mechanisms of action for pain relief. Naloxone, an opioid receptor antagonist, has been shown to block acupuncture analgesia by binding to selective opioid receptors (Mayer et al., 1977). In fact, acupuncture analgesia has been demonstrated to be mediated by afferent sensory nerves, as vascular occlusion has no effect on acupuncture analgesia (Chiang et al., 1973), while procaine (a local anesthetic) has indeed been shown to negate acupuncture analgesia (Ulett et al., 1998).

These early studies have been bolstered by more recent human and pre-clinical basic science studies, further defining the mechanistic role of endogenous opioid receptors in acupuncture analgesia (Harris et al., 2009), and suggesting other mechanisms of action including adenosine receptor modulation (Goldman et al., 2010), dopamine signaling for immune regulation (Torres-Rosas et al., 2014), and neuroplasticity in the primary somatosensory cortex of the brain (Maeda et al., 2017). Indeed, clinical trials of acupuncture for chronic pain conditions indicate that acupuncture analgesic effects persist for months following the end of treatment suggesting long term plastic changes in pain processing (MacPherson et al., 2017). This type of long term change

#### Edited and reviewed by:

*Rufin VanRullen, Centre National de la Recherche Scientifique (CNRS), France*

> \*Correspondence: *Vitaly Napadow vitaly@mgh.harvard.edu*

#### Specialty section:

*This article was submitted to Perception Science, a section of the journal Frontiers in Neuroscience*

Received: *02 December 2019* Accepted: *16 December 2019* Published: *17 January 2020*

#### Citation:

*Napadow V, Beissner F, Lin Y, Chae Y and Harris RE (2020) Editorial: Neural Substrates of Acupuncture: From Peripheral to Central Nervous System Mechanisms. Front. Neurosci. 13:1419. doi: 10.3389/fnins.2019.01419* in sensory processing is a hallmark of central neurobiology. These studies over the last 50 years of acupuncture research clearly highlight the nervous system as the main conduit for clinical efficacy underlying this therapeutic intervention.

This Issue furthers the research base on the neural basis of acupuncture mechanisms. Studies include both behavioral and neuroimaging research in humans, as well as basic molecular, cellular, and physiological research in animal models. Clinical applications included in this Issue extend beyond pain, to cover chronic itch, depression, stroke, drug addiction, and cardiovascular regulation. Molecular and cellular targets are identified for acupuncture analgesia and anti-depressive effects (Wan et al.; Zheng et al.), as well as inflammatory modulation (Ma et al.) and characterization of specific body locations (i.e., acupoints) for needle stimulation (Fan et al.). Other preclinical studies highlight specific brain regions such as paraventricular nucleus for cardiovascular modulation by acupuncture (Cheng et al.) and cuneate nucleus in drug dependence (Chang et al.). Human research studies explore the role of the brain's default mode network, previously implicated in acupunctureevoked brain response and analgesia (Hui et al., 2009; Napadow et al., 2012), in acupuncture clinical response more broadly (Zhang et al.). Human neuroimaging studies in this issue also evaluate potential brain-based mechanisms of acupuncture for motor outcomes (Nierhaus et al.) and itch (Min et al.). Human behavioral studies evaluate acupuncture modulation of temporal summation and its role in analgesia (Baeumler et al.). The role of cognitive effects and expectancy, vis-à-vis the placebo effect,

#### REFERENCES


are also investigated as they relate to acupuncture (Aichner et al.; Lee et al.; Musial; Song et al.). Other studies evaluate neural modulation by combined acupuncture with mental imagery (Takahashi et al.) or transcranial magnetic stimulation (Huang et al.), and the more general role of acupuncture in stress reduction (Lee et al.). Finally, structural parameters of the needle are evaluated for their role in analgesia (Bae et al.) and an interesting study explores how the connective tissue system and nervous system may be differentially involved in acupuncture efficacy (Chang et al.).

We hope the reader enjoys this compendium of novel acupuncture research studies, which both extends the substantive research base supporting acupuncture mechanisms of action, and bridges to future basic and translational research that more precisely defines the pathways and neural circuitry underpinning acupuncture clinical efficacy.

#### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

#### ACKNOWLEDGMENTS

We thank the following organizations for supporting the investigators on this editorial: Horst Görtz Stiftung to FB, NCCIH, NIH R01 AT007550 to RH and VN, and NCCIH NIH P01-AT009965 and R33-AT009306 to VN.


**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Napadow, Beissner, Lin, Chae and Harris. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Neuropeptides SP and CGRP Underlie the Electrical Properties of Acupoints

Yu Fan<sup>1</sup>† , Do-Hee Kim<sup>1</sup>† , Yeonhee Ryu<sup>2</sup>† , Suchan Chang<sup>1</sup> , Bong Hyo Lee<sup>1</sup> , Chae Ha Yang<sup>1</sup> and Hee Young Kim<sup>1</sup> \*

<sup>1</sup> Department of Physiology, College of Korean Medicine, Daegu Haany University, Daegu, South Korea, <sup>2</sup> Korean Medicine Fundamental Research Division, Korea Institute of Oriental Medicine, Daejeon, South Korea

Electrical skin measurements at acupuncture points (acupoints) have been utilized as a diagnostic and therapeutic aid for more than 50 years. Although acupoints are described as having distinct electrical properties, such as high conductance and low impedance, the underlying mechanisms are currently unknown. The present study investigated in a rat model of hypertension whether the high conductance at acupoints is a result of the release of the neuropeptides substance P (SP) and calcitonin generelated peptide (CGRP) during neurogenic inflammation in the referred pain area. When plasma extravasation from neurogenic inflammation was examined by exploring the leakage of intravenously injected Evans blue dye (EBD) to the skin, extravasated EBD was found most frequently in acupoints on the wrist. The increased conductance and temperature at these acupoints occurred during the development of hypertension. The increase in conductance and plasma extravasation at acupoints in hypertensive rats was ablated by cutting median and ulnar nerves, blocking small diameter afferent fibers with resiniferatoxin (RTX) injection into median and ulnar nerves, or antagonizing SP or CGRP receptors in acupoints. In turn, intradermal injection of SP or CGRP resulted in increased conductance and plasma extravasation in naïve rats. Elevated levels of SP and CGRP were found in the acupoints of hypertensive rats. These findings suggest that the high conductance at acupoints is due to vascular leakage following local release of SP and CGRP during neurogenic inflammation.

Keywords: substance P, CGRP, acupoints, electrical properties, skin conductance, Evans blue dye, plasma extravasation, neurogenic inflammation

#### SIGNIFICANCE STATEMENT

Electrical skin measurements at acupuncture points have been utilized as a diagnostic and therapeutic aid for more than 50 years. Although acupoints are described as having distinct electrical properties, such as higher conductance and lower impedance than that of surrounding skin, the underlying mechanisms are completely unknown. Using a newly constructed electrode, intravenous injection of Evans blue dye, and cutaneous thermal recordings and imaging, the present study suggests a novel mechanism underlying the electrical properties of acupoints: the neuropeptides SP and CGRP produce high conductance at acupoints by causing neurogenic inflammation, plasma extravasation and accumulation of subskin water contents.

#### Edited by:

Yi-Wen Lin, China Medical University, Taiwan

#### Reviewed by:

Yung-Hsiang Chen, China Medical University, Taiwan Dongwoon Han, Hanyang University, South Korea

> \*Correspondence: Hee Young Kim hykim@dhu.ac.kr

†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Perception Science, a section of the journal Frontiers in Neuroscience

Received: 19 September 2018 Accepted: 19 November 2018 Published: 12 December 2018

#### Citation:

Fan Y, Kim D-H, Ryu Y, Chang S, Lee BH, Yang CH and Kim HY (2018) Neuropeptides SP and CGRP Underlie the Electrical Properties of Acupoints. Front. Neurosci. 12:907. doi: 10.3389/fnins.2018.00907

#### INTRODUCTION

fnins-12-00907 December 12, 2018 Time: 12:37 # 2

Acupuncture, a therapeutic intervention of traditional medicine, has been used for centuries to relieve a variety of conditions. For acupuncture treatment, thin needles are inserted into specific but poorly defined sites on or under the skin called acupoints or acupuncture points. Based on acupuncture theory, there are about 360 acupuncture points, most of which lie along the Qi channels (called meridians) connecting the surface of the body to internal organs. Each acupoint communicates with a specific internal organ; an acupoint reflects the status of an internal organ, and the internal disorders can be treated by stimulating the acupoints (Stux and Pomeranz, 2012). In support of this, we and others have proven that acupoints become hypersensitive under abnormal visceral conditions and that stimulation of the acupoints can relieve the symptoms of the associated visceral organs (Chae et al., 2007; Kim et al., 2017).

As the acupoints themselves are grossly anatomically invisible, several scientific approaches, such as electrodermal measurements (Ahn et al., 2008) and infrared thermal imaging (Yang et al., 2007), have been attempted to identify the acupoints. Notably, numerous studies have reported the electrical properties of acupoints. Since the 1950s, when Nakatani (1956) reported that there were some points on the skin with special electrical properties, experimental and clinical studies have been carried out in many countries including China, Japan, France, Germany, and the United States and suggest that acupoints have distinct electrical properties, including a higher conductance, lower impedance and resistance and increased capacitance compared to the surrounding skin (Ahn et al., 2008). As this view gained traction, many instruments such as acupoint detectors and electrodiagnostic devices have been developed and are increasingly used in acupuncture clinics. However, the mechanisms by which acupoints have these distinct electrical properties are currently unknown.

Our previous studies showed that in rat models of hypertension or colitis, the skin over acupoints exhibits neurogenic inflammation due to viscerosomatic convergence in sensory pathways (Kim et al., 2006, 2017). Neurogenic inflammation is characterized by vasodilation and vascular leakage (plasma extravasation) in the skin arising from the release of neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP) from activated small diameter sensory afferents (Schmelz and Petersen, 2001). The insights that many acupoints show neurogenic inflammation under certain conditions (Kim et al., 2006, 2017) and electrically high conductance/low impedance (Ahn et al., 2008; Colbert et al., 2008, 2009) have led to the hypothesis that the neuropeptides CGRP and SP evoke vascular dilation and leakage, causing the increased subskin tissue water content in acupoints and thus producing electrically high conductance and low impedance, potentially underlying the electrical properties of acupoints. To prove this hypothesis, the present study used a rat model of immobilization-induced hypertension (IMH) to investigate (1) whether acupoints exhibit active neurogenic inflammatory responses by using intravenous injection of Evans blue dye (EBD) and cutaneous thermal recordings and imaging and (2) whether acupoints have high conductance by using a newly constructed electrode and plasma extravasation. Furthermore, we explored (3) whether the increased conductance at acupoints is mediated by activation of small diameter afferents and generated by localized release of CGRP and SP.

#### MATERIALS AND METHODS

#### Animals

Adult male Sprague-Dawley rats (Hyochang, Seoul, South Korea) weighing 250–350 g were used. Animals were housed at constant humidity (40∼60%) and temperature (22 ± 2 ◦C) on a 12-h light/dark cycle and allowed free access to food and water. All procedures were carried out in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee (IACUC) at Daegu Haany University.

#### Chemicals

Evans blue dye (50 mg/ml saline; Sigma-Aldrich, St. Louis, MO, United States), human calcitonin gene-related peptide (CGRP; 1 mg/ml saline; Bachem, Torrance, CA, United States; a 37 amino acid peptide), and α-CGRP 8-37 (1 mg/ml distilled water, Bachem, Torrance, CA, United States; a CGRP receptor antagonist) were used in this study (Shen et al., 2001). Substance P acetate salt hydrate (SP; 0.5 mg/ml saline; Sigma-Aldrich; a SP receptor agonist) and (+)-(2S,3S)-3-(2-methoxybenzylamino)-2 phenylpiperidine (CP-99994; 33 mmol/ml saline; Sigma-Aldrich; an SP receptor antagonist) were also used (McLean et al., 1993). Resiniferatoxin (RTX; 100 µg/ml vehicle; Sigma-Aldrich), an ultrapotent capsaicin analog known to block small diameter afferent fibers containing transient receptor potential vanilloid type 1 receptor (TRPV1) (Suter et al., 2009), was dissolved in a vehicle that contained 0.3% Tween 80, 10% DMSO and saline. Capsaicin (0.05 and 0.1%; Sigma-Aldrich; a TRPV1 agonist) was dissolved in a vehicle consisting of 10% alcohol and 10% Tween 80 in saline (Knotkova et al., 2008), and FITC-conjugated isolectin B4 (FITC IB4 tracer; 1%, 3 µl, Vector Laboratories, Burlingame, CA, United States) was used.

#### Immobilization Stress-Induced Hypertension (IMH) and Measurement of Systolic Blood Pressure

Hypertension was induced by immobilization with a coneshaped polyethylene bag, as described previously (Kvetnansky et al., 1979). Systolic blood pressure was measured non-invasively with a tail cuff blood pressure monitor (Model 47, IITC, Inc., Woodland Hills, CA, United States). Briefly, the rat was placed in a chamber, and an occluding cuff and a pneumatic pulse transducer were positioned on the base of the tail. A programmed electrosphygmomanometer (Narco Bio-Systems, Inc., Austin, TX, United States) was inflated and deflated automatically, and the tail cuff signals from the transducer were automatically collected every 10 min using an IITC apparatus (Model 47, IITC,

Inc.). The mean of two readings was taken at each blood pressure measurement.

# Detection of Neurogenic Inflammation in the Skin by EBD Injection

Neurogenic inflammatory sites were visualized by intravenously injecting EBD (50 mg/kg) in male Sprague-Dawley rats as described previously (Kim et al., 2017). While the rats were immobilized by the cone-shaped bags, the distal portion of the tail was dipped into 40◦C warm water for at least 30 s. EBD was then injected into the tail vein with a catheter (26 gauge), and skin color changes were observed up to 2 h after the injection. The blue-dyed areas on the skin were sketched using body charts, photographed and compared with a human acupoint chart based on the transpositional method, which locates acupoints on the surface of animal skin corresponding to the anatomic site of human acupoints (Yin et al., 2008).

## Measurement of Skin Surface Temperature

Skin temperature was measured using a K-type thermocouple microprobe (TC-11p, Minnesota Measurement Instruments, Minneapolis, MN, United States) coupled with an analog-digital interface converter (Physitemp BAT-12, American Laboratory Trading, San Diego, CA, United States) and digitized through a PowerLab 4/30 acquisition system (ADInstruments, Colorado Springs, CO, United States). While the rats were placed in a plastic chamber, a flexible thermoprobe was attached to the skin over wrist acupoints, mostly PC6 acupoints, the nearby site approximately 5 mm away from PC6 (**Figures 2A,B**) or capsaicin-injected sites (**Figure 2C**). Body temperature was also monitored with a regular thermocouple probe inserted into the rectum. For the experiment shown in **Figures 2A,B**, after the basal temperatures were recorded for at least 10 min, the rats were subjected to the IMH procedure, and the temperatures were measured up to 30 min after restraint. The skin-body temperature differentials were estimated by subtracting the body temperature from the skin temperature at each time point. At 30 min after restraint, infrared thermal images were obtained using a thermal camera (Ti55, Fluke IR Fusion Technology, Washington DC, United States) under slight isoflurane anesthesia. In another set of experiments (for **Figure 2C**), either capsaicin (0.05 or 0.1% capsaicin, 30 µl) or vehicle was intradermally injected into the bilateral PC6 acupoint near the wrist under slight isoflurane anesthesia, and temperature changes in the injected sites were monitored up to 30 min after injection.

### Measurement of Electrical Skin Conductance

Skin moisture and temperature in the rats may be influenced according to external environment. To control the factors, all experiments were carried out under constant humidity (40∼60%) and temperature (22 ± 2 ◦C). The animal hair was shaved prior to the placement of electrode. To simultaneously measure conductance and the applied pressure, a device was newly constructed by coupling a force transducer (FT-100, iWorx/CB Sciences, Inc., Dover, NH, United States) with an electrical conductance probe (3.7 mm diameter, stainless). The rats were placed in cone-shaped bags or plastic chambers. While the positive electrode was attached to the tail surface, the device (negative electrode) was placed on the skin over acupoints and was pressed at a force of 300 g. Signals from the conductance (current) probe and the force transducer were fed to an ETH-200 Bridge Amplifier (CB Science, Inc., Lemont, PA, United States) and a GSR AMP device (Model FE116, ADInstruments, Colorado Springs, CO, United States), respectively, and digitized through a PowerLab 4/30 acquisition system (ADInstruments). After basal electrical currents were recorded for at least 5 min, skin conductance (measured as a current) following treatments was recorded up to 30 min.

## Estimation of EBD Extravasation in Skin Tissues

To assess the extent of EBD extravasation in the skin, rats were sacrificed 120 min after intravenous injection of EBD. The skin near the wrist was photographed using a digital camera (SONY ILCE-5000, China). The original EBD images were used to obtain a three-dimensional (3D) surface plot with ImageJ software (National Institute of Mental Health, Rockville, MD, United States) using the command Plugins/3D/interactive 3D surface plot (parameters; Mesh, Spectrum LUT, Scale 1.75, and Z 0.19). To determine the concentration of EBD that infiltrated the skin tissues, the skin tissue was processed using the dyeextraction method described previously (Martin et al., 2010). In brief, the skin (approximately 3 mm in size) near the wrist was excised, dry-weighed, homogenized in 1: 3 volume of 50% trichloroacetic acid (TCA; dissolved in 0.9% saline) and centrifuged (10,000 rpm for 10 min). The supernatants were diluted with 1: 3 volume of 95% ethanol and measured using a spectrophotometric method (620 nm excitation/680 nm emission).

#### Surgical or Pharmacological Blockade of Median and Ulnar Nerves

Median and ulnar nerves were blocked surgically or pharmacologically as described previously (Kim et al., 2013). Briefly, under isoflurane anesthesia, a small skin incision was made longitudinally on the medial part of the elbow to expose the median and ulnar nerves. For surgical lesions of median and ulnar nerves, the nerves were bilaterally ligated with 4–0 silk and cut around the medial head of the triceps muscle of both forelimbs. The sham group underwent the same procedure but without nerve injury. For blocking small diameter afferent fibers, RTX (0.01%, 30 µl) was administered perineurally in the median and ulnar nerves. All incisions were closed aseptically and 2 or 3 days after surgery, experiments for skin conductance were performed.

Another set of experiments was performed to confirm the blockade of small diameter afferent fibers by RTX by using an IB4 tracing method (Pan et al., 2003). In brief, 48 h after perineural injection of either RTX (n = 3) or vehicle (n = 3) into the ulnar nerve, an FITC IB4 tracer (3 µl) was administered into ulnar

nerves under isoflurane anesthesia in rats (n = 3). Three to four days later, dorsal root ganglion (DRG) neurons of C8 and T1 were removed, post-fixed in 4% buffered paraformaldehyde (PFA) for 2 h, immersed in 30% sucrose overnight and cryosectioned at 30 µm. The cryosections were then mounted on gelatincoated glass slides. Skin images were taken from 3 to 4 sections per animal under an Olympus AX70 fluorescence microscope (Olympus, Japan) and quantified by using ImageJ software.

#### Immunohistochemistry for CGRP or SP in the Skin

One hour after restraint, skin samples were taken from the wrist, which most commonly showed EBD leakage in IMH rats (n = 6) and naïve rats (n = 6). The skin samples were paraffin-embedded, sectioned (5 µm), and incubated with either anti-CGRP mouse antibody (1:500; Chemicon, Temecula, CA, United States; RRID:AB\_1658411) or anti-SP mouse monoclonal antibody (1:500; GeneTex, Irvine, CA, United States; RRID:AB\_785913), followed by incubation with secondary antibody (1:500, Alexa Fluor 488-conjugated donkey anti-mouse IgG antibody, Thermo Scientific, Waltham, MA, United States; RRID:AB\_141607). The sections were mounted on gelatin-coated slides, air-dried, and coverslipped. Skin images were taken from three sections from each animal with a laser-scanning confocal microscope (LSM700, Carl Zeiss, Germany) and quantified by using ImageJ software (National Institute of Mental Health, Rockville, MD, United States). The number of pixels with green fluorescence intensity greater than the cut-off value (100) was counted to quantify positive staining. Data were expressed as the number of positive pixels over a field area of 1280 × 1024 pixels.

#### Statistical Analysis

Statistical analysis was carried out using SigmaStat 3.5 software (Systat Software, Inc., United States). All data are presented as the mean ± SEM (standard error of the mean) and analyzed by one or two-way repeated measures analysis of variance (ANOVA) with Tukey post hoc tests or unpaired t-tests where appropriate. Statistical significance was considered at p < 0.05.

# RESULTS

### Cutaneous Neurogenic Inflammation at Acupoints

First, we explored whether some of acupoints displayed neurogenic inflammation in IMH rats. When a rat was placed in an immobilization bag, systolic blood pressure gradually increased for the next several hours (**Figures 1A,B**), consistent with our previous study (Kim et al., 2017). Approximately 10 min after the initiation of restraint, cutaneous neurogenic inflammatory sites [neurogenic spots (Neuro-Sp)] were visualized by intravenous injection of EBD (50 mg/kg). The blue spots started to appear approximately 5 min after EBD injection, ranged in diameter from 0.5 to 3 mm and were maintained throughout the experiment. IMH rats exhibited approximately nine spots per animal (**Figure 1D**), while control rats only showed a few spots. When the Neuro-Sp in IMH rats (n = 21) were mapped and compared with the corresponding human anatomical acupoints, the majority appeared bilaterally or unilaterally on the wrist, and 69% (123 of 178 spots) were found in acupoints of the forelimbs, such as PC6 (33 spots), PC7 (25 spots), and HT7 (30 spots) (**Figures 1D–F**). These results indicate that acupoints, most frequently on the wrist, display cutaneous neurogenic inflammation and plasma extravasation in the rat model of IMH.

#### Increased Skin Temperature at Acupoints Following Neurogenic Inflammation

To determine whether the acupoints undergo active neurogenic inflammation processes, cutaneous temperature, as an outcome measure of inflammation (Birklein and Schmelz, 2008; Montalto et al., 2013), was compared between acupoints on the wrist and nearby sites approximately 5 mm away from the acupoints in IMH rats. Skin temperature rapidly elevated following restraint and then continued to increase slowly over 30 min after restraint. This increase was higher at acupoints than at nearby sites (two-way ANOVA; group F(1,4) = 26.231, p = 0.07; time F(15,60) = 28.589, p < 0.001; interaction F(15,60) = 1.061, p = 0.41; **Figure 2A**). Thermal infrared imaging found elevated temperature in the skin over wrist acupoints such as PC6 (**Figure 2B**). To verify whether neurogenic inflammation itself can increase skin temperature, the effect of intradermally injected capsaicin (0.05, 0.1%; 30 µl), which is known to trigger neurogenic inflammation (Lin et al., 1999), on skin temperature was investigated. Local injection of capsaicin into the skin of the wrist significantly increased temperature at the injection site in a dose-dependent manner compared to injection of vehicle (two-way ANOVA, group F(2,8) = 29.018, p < 0.001; time F(20,80) = 38.794, p < 0.001; interaction F(40,160) = 9.737, p < 0.001; **Figure 2C**), suggesting that acupoints on the wrist of IMH rats are undergoing active processes of neurogenic inflammation.

#### Increased Electrical Conductance and Plasma Extravasation at Acupoints in IMH Rats

Next, we investigated whether acupoints showed increased electrical conductance under pathological conditions. The pressure exerted by the probe on the skin is considered to be a potential confounder affecting the reproducibility and reliability of data on the measurement of electrical skin conductance/impedance (Ahn and Martinsen, 2007). To hold the electrode to the skin at a constant pressure at every measurement, we constructed a conductance probe that could measure electrical currents and touch pressure simultaneously (**Figure 3A**). Skin conductance was estimated as the plateau value of electrical current recorded while holding the electrode at a constant pressure of 300 g (**Figure 3B**). To assess the validity of the device, various amounts of distilled water (1∼10 µl) were applied to the skin near the wrist in isoflurane-anesthetized rats (n = 6), and the electrical conductance was measured at the wrist skin. Electrical currents proportionally increased with increasing water

unrestrained control rats (control). <sup>∗</sup>p < 0.001 vs. control. (C) A representative image of Neuro-Sp in an IMH rat. (D) Numbers of Neuro-Sp per animal (bar graph) and correlation of the anatomic location between Neuro-Sp and acupoints in hypertensive rats (n = 21 animals; pie graph). Numbers of Neuro-Sp corresponding to acupoints in the forelimb (E) or hind limb (F). Bar = 20 mm.

volumes, reaching a maximum level at approximately 5 µl water (**Figure 3C**), indicating an increase in conductance according to the extent of skin hydration.

To explore whether acupoints exhibit a high conductance under pathological conditions, electrical currents at an acupoint on the wrist were compared between IMH and control rats. When

FIGURE 2 | Increased temperature at acupoints in IMH rats. (A) Changes in temperature at acupoints on the wrist following restraint. Skin (acupoint)-body temperature differentials were calculated by subtracting the body temperatures from the temperatures over wrist acupoints. <sup>∗</sup>p < 0.05 vs. nearby site. (B) A representative thermal infrared image of the wrist 2 h after restraint. (C) Effect of intradermal capsaicin on skin–body temperature differentials. Capsaicin (0.05 or 0.1%) or vehicle was injected into the skin over the wrist of naïve rats. <sup>∗</sup>p < 0.05, 0.05% capsaicin vs. vehicle; #p < 0.05, 0.1% capsaicin vs. vehicle. Bar = 20 mm.

FIGURE 3 | Increased electrical conductance and plasma extravasation at acupoints in IMH rats. (A) A newly constructed electrode for the simultaneous measurement of conductance and applied pressure. (B) A representative trace of applied force and electrical currents. Conductance was estimated as the maximum current value (µA) reached when a pressure of 300 g on the electrode was held constant. (C) Changes in conductance induced by various amounts of water topically applied to rat skin. (D–F) Increased conductance at acupoints following IMH. A representative trace of applied pressure (upper panels) and electrical currents (low panels) recorded in wrist acupoints of control and IMH rats. Significantly increased conductance at the wrist acupoint was observed in IMH rats compared to that in control (F). p < 0.05 vs. control. (G) Extravasation of EBD over the wrist acupoint of hypertensive rats. Representative photographs of wrist areas in control and IMH rats 2 h after intravenous injection of EBD and the 3D images created from the circles in the photographs by using ImageJ. (H) Concentration of EBD in the wrist acupoints of naïve control and IMH rats. <sup>∗</sup>p = 0.027 vs. control.

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the conductance probe was applied over the PC6 acupoint on the wrist at a constant pressure of 300 g, the acupoint in the IMH rats showed a higher electrical conductance than that in the control rats (two-way ANOVA; group F(1,4) = 1147.149, p < 0.001; time F(19,76) = 228.471, p < 0.001; interaction F(19,76) = 232.043, p < 0.001; **Figures 3D–F**). In another set of experiments, we imaged and quantified neurogenic extravasation at acupoints on the wrist 2 h after EBD injection with or without restraint. **Figure 3G** shows that there was blue EBD staining over the wrist acupoints in IMH rats (hypertension) but not naïve control rats (control). The 3D plots derived from the photographs also show the predominant EBD staining in IMH rats (right panels in **Figure 3G**). When measured by spectrophotometry, the EBD concentration at the wrist acupoints was significantly higher in IMH rats (hypertension, n = 6) than in naïve control rats (control, n = 6; p < 0.001; **Figure 3H**). These results suggest that both conductance and plasma extravasation were enhanced at the acupoints of IMH rats.

#### C-Fiber Mediation of the High Electrical Conductance of Acupoints

To identify whether afferent nerves mediate the development of high conductance at acupoints, surgical lesions of the ulnar and median nerves were made 48∼72 h prior to restraint and skin conductance measurements (**Figure 4A**). While conductance at the wrist acupoint wrist gradually increased following restraint (sham; **Figure 4B**), such effects were not observed in the rats with nerve lesions (nerve lesion; two-way ANOVA; group F(1,4) = 939.849, p < 0.001; time F(13,52) = 64.358, p < 0.001; interaction F(13,52) = 32.538, p < 0.001; **Figure 4B**). To further examine the role of small diameter afferent fibers in producing high acupoint conductance, we injected a specific C/Aδ-fiber blocker RTX into ulnar and median nerves 48–72 h prior to the IMH procedure. Unlike pretreatment of the nerves with saline (vehicle), pretreatment with RTX abolished the development of the high conductance at the acupoint in IMH rats (two-way ANOVA; group F(1,4) = 398.84, p < 0.001; time F(13,52) = 82.402, p < 0.001; interaction F(13,52) = 25.643, p < 0.001; **Figure 4C**). Furthermore, Aδ/C-fiber blockade by RTX was confirmed by significantly less FITC IB4 tracer labeling in the DRG in RTX-treated rats (RTX/IB4) compared to in the vehicle group (vehicle/IB4, **Figures 4D,E**). Taken together, our results indicate that small diameter afferent fibers mediate the development of high conduction at acupoints.

### Production of High Conductance by SP and CGRP

To explore whether the increased conductance at acupoints is associated with levels of SP and CGRP, we injected either SP or CGRP intradermally into the skin on the wrist and

vs. vehicle. Bar = 100 µm.

FIGURE 5 | Increased conductance at acupoints by SP or CGRP. (A–C) Effect of intradermal substance P (SP) on electrical conductance in naïve rats. Injection of SP into the skin of the wrist acupoints significantly increased conductance compared to injection of vehicle (A). p < 0.05 vs. vehicle. The three-dimensional (3D) images of EBD extravasation in the wrist skin in vehicle- or SP–treated rats (B) and the concentration of EBD in the wrist acupoints of vehicle- or SP-treated rats (C). <sup>∗</sup>p < 0.001 vs. vehicle. (D–F) Effect of intradermal calcitonin gene-related peptide (CGRP) on electrical conductance in naïve rats. Injection of CGRP into the skin of the wrist acupoints significantly increased conductance compared to injection of vehicle (D). The 3D images of EBD extravasation in the wrist skin in vehicle- or CGRP-treated rats (E) and the concentration of EBD in the wrist acupoints of vehicle- or CGRP-treated rats (F). <sup>∗</sup>p < 0.001 vs. vehicle. (G–I) Effects of intradermal SP or CGRP antagonists on the development of high conductance in hypertensive rats. Injection of SP or CGRP antagonists into the skin of the wrist acupoints prevented the development of high conductance in IMH rats compared to injection of vehicle (G). The 3D images of EBD in the wrist skin in vehicle- or SP or CGRP antagonist-treated rats (H) and the concentration of EBD in the wrist acupoints of vehicle- or SP or CGRP antagonist-treated rats (I). <sup>∗</sup>p = 0.003 vs. vehicle.

measured the skin conductance. An artificial increase in SP in the skin significantly increased conductance (two-way ANOVA; group F(1,4) = 63.285, p = 0.001; time F(19,73) = 117.661, p < 0.001; interaction F(19,76) = 22.732, p < 0.001; **Figure 5A**). In another set of experiments, we determined the levels of plasma extravasation 2 h after the injection of vehicle (n = 6) or SP (n = 6) and found significantly higher levels of plasma extravasation in the wrist acupoints of SPinjected rats than in those of vehicle-injected rats, as shown in 3D skin images (**Figure 5B**) and the EBD concentration assessed by spectrophotometry (t-test, p < 0.01; **Figure 5C**). Such effects were replicated by injection of a CGRP agonist (two-way ANOVA; group F(1,4) = 20.372, p = 0.011; time F(19,76) = 122.989, p < 0.001; interaction F(19,76) = 18.115, p < 0.001; **Figures 5D–F**).

To see if inhibition of SP or CGRP prevents the increase in conductance at acupoints, we injected either vehicle (saline), a SP antagonist CP-99,994 or a CGRP antagonist α-CGRP 8- 37 into acupoints on the wrist prior to restraint and measured conductance up to 30 min after restraint. Saline-injected rats showed enhanced conductance at the wrist acupoints following restraint, which was significantly reduced by pretreatment with the SP or CGRP antagonist (two-way ANOVA; group F(2,8) = 2.307, p = 0.162; time F(19,76) = 277.166, p < 0.001; interaction F(38,152) = 2.361, p < 0.001; **Figure 5G**). This decrease was further confirmed by 3D skin images (t-test; <sup>∗</sup>p < 0.05,

**Figure 5H**) and the EBD concentration in the skin (one-way ANOVA; <sup>∗</sup>p < 0.05 vs. Vehicle, **Figure 5I**). Taken together, these results suggest that local release of SP and CGRP in acupoints leads to an increase in conductance by inducing vascular leakage.

#### Increased Levels of SP and CGRP at Acupoints in IMH Rats

Finally, to confirm the increased expression of SP and CGRP at acupoints in IMH rats, we compared the expression of CGRP and SP in the skin on the wrist between normal (n = 6) and IMH rats (n = 6). Significantly greater SP (**Figures. 6A,B**) and CGRP (**Figures 6C,D**) fluorescence was found in the dermis of IMH rats than in that of control naïve rats (t-test; p < 0.001 in **Figures 6B,D**). Notably, the dermal blood vessels in IMH rats but not control naïve rats were predominantly enlarged (**Figures 6A,C**).

#### DISCUSSION

Our findings demonstrated that SP and CGRP released from afferents fibers increased electrical conductance at acupoints in IMH rats. In the IMH rat model, cutaneous neurogenic inflammation was found most frequently in the acupoints on the wrist. These acupoints revealed higher temperature and more plasma extravasation than the acupoints in naïve rats. Electrical conductance at the acupoints gradually increased with the development of hypertension but was blocked by surgical transection of the median and ulnar nerves or blockade of small diameter afferent fibers with RTX. Skin conductance and plasma extravasation were increased by intradermal injection of SP or CGRP in normal rats. In turn, inhibition of SP or CGRP by antagonists prevented the increase in both the conductance and plasma extravasation at acupoints in IMH rats. Levels of SP and CGRP were elevated in the dermis of acupoints in hypertensive rats. Our findings suggest that the local release of SP and CGRP induces vasodilation and plasma extravasation, resulting in accumulation of subskin tissue water content, thereby leading to an increase in electrical conductance at acupoints (**Figure 6E**).

While traditional acupoints have long been thought to be anatomically invisible, our recent study suggested that acupoints can be identified as neurogenic inflammatory spots (Neuro-Sp) on the skin, which are produced by activation of somatic afferents in abnormal conditions of visceral organs and can be visualized by intravenous injection of EBD (Kim et al., 2017). Consistent with our previous study (Kim et al., 2017), hypertensive rats

revealed highly localized neurogenic inflammation (indicated by Neuro-Sp) in the dermatome of the forelimbs (**Figure 1**), which is innervated by the same spinal segments (C8-T2) that innervate the heart (Alles and Dom, 1985). Cutaneous neurogenic inflammation was observed most frequently in acupoints on the wrist, such as PC6, HT7, and PC7 (**Figures 1D,E**), which are commonly used in acupuncture clinics for cardiac disorders (Stux and Pomeranz, 2012). Furthermore, neurogenic inflammation tended to rapidly develop in acupoints after an IMH procedure and to be maintained during IMH, as shown by EBD staining and thermal recordings. Similar to our findings, a previous study reported that neurogenic inflammation appears in the skin of the abdomen, groin, lower back, and perineal areas several minutes after uterine inflammation with mustard oil, as assessed by EBD extravasation (Wesselmann and Lai, 1997). Cutaneous neurogenic inflammation is manifested as flare, vasodilation and increased local skin temperature (Wesselmann and Lai, 1997; Serra et al., 1998). In our thermal recordings and imaging, a rapid and long-lasting rise in temperature at acupoints was observed after the IMH procedure and was mimicked by intradermal injection of capsaicin, which produces cutaneous neurogenic inflammation (Lin et al., 1999). These results suggest that acupoints on the wrist displayed active neurogenic inflammation characterized by plasma extravasation in this rat model of hypertension.

Multiple studies have suggested that acupoints have higher conductance and lower impedance (resistance) than the surrounding skin (Reichmanis et al., 1975; Colbert et al., 2008, 2009), although this issue is currently controversial (Ahn et al., 2008; Wong, 2014). Reichmanis et al. (1975) reported that certain acupoints had significantly higher electrical conductance than nearby sites in healthy subjects. Colbert et al. (2008, 2009) recorded skin impedance at multiple acupoints simultaneously by using a fully automatic multichannel device and reported that several acupoints showed lower impedance than nearby sites in healthy subjects. The above studies are supported by our previous and current studies showing a high conductance of acupoints in IMH rats (Kim et al., 2017) (**Figures 3D–F**). However, others have also shown inconsistent results in the electrical conductance and impedance of acupoints. Pearson et al. (2007) observed that none of the three acupoints tested had lower skin impedance than the surrounding skin in healthy subjects. Kramer et al. (2009) reported that when skin impedance was measured in healthy subjects by using an array electrode of 64 channels, the majority of acupoints tested showed no changes in impedance, but some showed transient high or low impedance. A systematic review found that five out of nine studies showed a positive association between acupoints and low electrical impedance (Ahn et al., 2008). In these previous studies, a major problem is that almost all were conducted in healthy subjects and not in disease states, which may generate mixed results in electrodermal measurements at acupoints. As acupoints are generally accepted to reflect pathological states of the body and to become hypersensitive under pathological conditions (Stux and Pomeranz, 2012; Kim et al., 2017), pathological body conditions may cause considerable changes in skin conductance or impedance at acupoints. In support of this, our previous and present studies found that the conductance of acupoints significantly increased with the development of hypertension in rats, and such an increase was not seen in the same acupoints of control rats (Kim et al., 2017) (**Figures 3D–F**). In addition, the acupoints with high conductance in IMH rats but not naïve rats showed higher neurogenic inflammation and plasma extravasation than those of naïve rats (**Figures 3G,H**). Our hypothesis is further supported by another study showing that more significant changes in skin impedance at acupoint GB34 were observed in patients that had undergone surgery than in healthy subjects (Kramer et al., 2012). Importantly, previous studies point out that the precision of skin conductance measurements can be influenced by numerous factors such as skin dryness, skin thickness, size of the sensing electrode, pressure applied on the electrode, interelectrode distance, room temperature, and humidity (Ahn and Martinsen, 2007). In the present experiment, we developed a device that applied constant pressure on the electrode and performed the experiments under controlled environmental conditions, which might rule out the impact of the above factors. Therefore, the present study suggests that during diseases, the conductance at acupoints is abnormally high and that these electrical changes are associated with neurogenic inflammation and plasma extravasation.

In the somatic areas of referred pain from viscera, Neuro-Sp are generated by activation of small diameter sensory afferents (C/Aδ-fibers) in the dermatome convergent with visceral afferents (Wesselmann and Lai, 1997; Arendt-Nielsen et al., 2008). The sensory neurons are branched, with one projection leading to the internal organs and the other extending to the skin. The visceral inputs activate the viscerosomatic convergent neurons in the sensory pathway, and the neurons antidromically activate the branches, leading to the release of neuropeptides (e.g., SP and CGRP) from small diameter sensory fibers and subsequent neurogenic extravasation (Wesselmann and Lai, 1997; Arendt-Nielsen et al., 2008). Linkage of Neuro-Sp to internal organs was proven by our previous study showing convergent DRG neurons innervating both the heart and the Neuro-Sp (Kim et al., 2017). In the present study, the development of high conductance at acupoints in hypertensive rats was almost completely ablated by surgical lesions of the median and ulnar nerves (**Figures 4A,B**), suggesting that the afferent nerves mediate the high conductance at acupoints. Furthermore, pretreatment of the median and ulnar nerves with a specific C/Aδ-fiber blocker RTX prevented the development of high conductance at acupoints (**Figure 4C**), while RTX effectively blocked transmission of small sensory afferents in the median and ulnar nerves (**Figures 4D,E**), as reported previously (Suter et al., 2009). Taken together, these findings indicate that high conductance at acupoints is caused by antidromic activation of peripheral nerves, especially small diameter sensory afferents, in the dermatome associated with visceral disorders.

Activation of small diameter sensory afferents is known to induce the release of neuropeptides SP and CGRP into the periphery and lead to the development of neurogenic inflammation (Richardson and Vasko, 2002). SP as well as other tachykinins activates neurokinin receptors to increase microvascular permeability and edema formation, while CGRP

acts on CGRP1 receptors to dilate arterioles (Schmelz and Petersen, 2001). The neuropeptides released by activated afferent fibers evoke neurogenic inflammation in the skin by activating vasodilation, axon reflex flare, and microvascular plasma extravasation (Wesselmann and Lai, 1997; Schmelz and Petersen, 2001). In the present study, intradermal injection of SP or CGRP increased both conductance and plasma extravasation in naïve rats, similar to the pattern observed in hypertensive rats (**Figures 5A–F**). In contrast, intradermal injection of SP or CGRP antagonists into acupoints prevented the development of high conductance and plasma extravasation at acupoints (**Figures 5G–I**). Moreover, increased levels of SP and CGRP were found in the acupoints of hypertensive rats. Thus, our findings suggest that SP and CGRP induce vasodilation and plasma extravasation to increase skin hydration, resulting in the development of high conductance at acupoints. Paradoxically, the locally released SP and CGRP into acupoints may in turn play a role in acupuncture effect. It has been suggested that active acupoints are associated with tissues where the sensory nerve endings are sensitized by neurogenic inflammatory mediators (Rong et al., 2013; He et al., 2017). Given that the sensitized sensory nerve endings are more sensitive to external stimuli than intact sensory nerves, we suggest that sensory nerve endings in acupoints would be sensitized by SP or CGRP released during neurogenic inflammation. Accordingly, stimulation of these sensitive acupoints would evoke the therapeutic effects of acupuncture by reaching physiological thresholds quickly, compared with stimulation of normal surrounding tissues including as sham or inactive acupoints.

A limitation of this study is that neurogenic extravasation of EBD was examined in only the skin over acupoints. Acupuncture needles often penetrate multiple layers including skin, subcutaneous tissue and muscles. As these layers may

#### REFERENCES


contain structures that respond to needling and produce acupuncture effects, future study will be needed to identify whether the neurogenic inflammatory processes also occur in the tissues below skin (i.e., subcutaneous tissue and muscles) and in turn are associated with acupuncture effects.

#### CONCLUSION

The present study suggests a novel mechanism underlying the electrical properties of acupoints: the neuropeptides SP and CGRP produce high conductance at acupoints by causing neurogenic inflammation, plasma extravasation and accumulation of subskin water contents. This study would help solve some of controversial issues concerning electrical properties of acupoints.

#### AUTHOR CONTRIBUTIONS

HK designed the experiment, responsible for the overall direction of the project, and for edits to the manuscript. YF, D-HK, YR, SC, BL and CHY performed the experiments and analyzed the data. YF and HK drafted the manuscript.

#### FUNDING

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF; 2018R1A5A2025272, and 2018R1E1A2A02086499), the Korea Institute of Oriental Medicine (KIOM; K18181) and Korea Brain Research Institute (KBRI; 18-BR-03).


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer Y-HC and handling Editor declared their shared affiliation.

Copyright © 2018 Fan, Kim, Ryu, Chang, Lee, Yang and Kim. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fnins-12-00907 December 12, 2018 Time: 12:37 # 12

# Moxibustion Modulates Sympathoexcitatory Cardiovascular Reflex Responses Through Paraventricular Nucleus

Ling Cheng<sup>1</sup> \* † , Peng Li<sup>2</sup> , Yash Patel<sup>2</sup> , Yiwei Gong<sup>2</sup> , Zhi-Ling Guo<sup>2</sup> , Huangan Wu<sup>3</sup> , Shaista Malik<sup>2</sup> and Stephanie C. Tjen-A-Looi<sup>2</sup> \* †

<sup>1</sup> Eastern Hospital Affiliated to Tongji University, Shanghai, China, <sup>2</sup> Susan Samueli Integrative Health Institute, University of California, Irvine, Irvine, CA, United States, <sup>3</sup> Shanghai Research Institute of Acupuncture and Meridian, Shanghai University of Traditional Chinese Medicine, Shanghai, China

#### Edited by:

Younbyoung Chae, Kyung Hee University, South Korea

#### Reviewed by:

Hee Young Kim, Daegu Haany University, South Korea Min-Ho Nam, Korea Institute of Science and Technology (KIST), South Korea

#### \*Correspondence:

Ling Cheng chlrosy.east@sina.com Stephanie C. Tjen-A-Looi stjenalo@uci.edu

†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Perception Science, a section of the journal Frontiers in Neuroscience

Received: 27 June 2018 Accepted: 31 December 2018 Published: 21 January 2019

#### Citation:

Cheng L, Li P, Patel Y, Gong Y, Guo Z-L, Wu H, Malik S and Tjen-A-Looi SC (2019) Moxibustion Modulates Sympathoexcitatory Cardiovascular Reflex Responses Through Paraventricular Nucleus. Front. Neurosci. 12:1057. doi: 10.3389/fnins.2018.01057 Electroacupuncture (EA) point specific (ST36-37) stimulation decreases cardiovascular reflex responses through supraspinal regions such as the hypothalamic paraventricular nucleus (PVN) while mechanical stimulation of acupoints decreases pressor responses through peripheral thermal transient receptor potential vanilloid type-1 (TRPV1). Moxibustion generating heat applied at acupoint in combination with antihypertensive drugs decreases elevated blood pressure. We hypothesized that moxibustion modulates sympathoexcitatory cardiovascular responses through the hypothalamic PVN and peripheral heat sensitive TRPV1 in the absence of antihypertensive drugs. Rats were anesthetized, ventilated, and heart rate and mean blood pressure were monitored. Gastric distention induced consistent pressor reflex responses every 10 min. Thirty-minutes of bilateral moxibustion at the acupoint ST36, overlying the deep peroneal nerves, reduced the gastric distention evoked elevation in blood pressure. Blood pressure reflex responses were not reduced by both EA and moxibustion at G39. The moxibustion inhibition but not EA inhibition of the cardiovascular responses was reversed with blockade of local heat sensitive TRPV1 at ST36. Accordingly, activation of thermal TRPV1 by moxibustion at an average of 44.2◦C in contrast to 40◦C reduced the pressor responses. Naloxone, an opioid receptor antagonist, microinjected into PVN inhibited transiently the effect of moxibustion. Thus, activation of peripheral heat sensitive TRPV1 mediated the moxibustion-inhibition, but not EA-inhibition, of sympathoexcitatory cardiovascular reflex responses through hypothalamic PVN opioid system.

Keywords: gastric distention, deep peroneal nerve, acupoint specific, paraventricular nucleus, sympathoexcitatory cardiovascular reflex response

# INTRODUCTION

Moxibustion has been used to treat gastrointestinal problems such as irritable bowel syndrome (Liu et al., 2015; Shi et al., 2015) but not much is known about its effect on hypertension. Few clinical studies indicate that moxibustion in the presence of antihypertensive drugs may reduce hypertension, but the efficacy and mechanisms underlying moxibustion in the absence of drugs are

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unknown (Xiong et al., 2014; Lee et al., 2016). We have shown in a series of studies in men and animals that electroacupuncture (EA) at specific acupoints activating peripheral nerves modifies sustained hypertension and reflex elevated blood pressure through reduction of sympathetic activity (Chao et al., 1999; Tjen-A-Looi et al., 2004; Li et al., 2004, 2016; Crisostomo et al., 2005). In this respect, specific central regions and neurotransmitter systems in the hypothalamus and medulla participate in the central processing of the action of acupuncture (Chao et al., 1999; Crisostomo et al., 2005; Li et al., 2009, 2010, 2015; Tjen-A-Looi et al., 2009; Moazzami et al., 2010; Tjen-A-Looi et al., 2013). The current study investigates in the absence of antihypertensive drugs the effect as well as underlying mechanisms of moxibustion at specific acupoint on sympathoexcitatory cardiovascular reflex responses in rats.

Cardiovascular regions in the central nervous system (CNS) such as rostral ventrolateral medulla (rVLM), and paraventricular nucleus (PVN) are activated during stimulation of visceral spinal afferents that leads to increases in blood pressure (Tjen-A-Looi et al., 2003, 2016). A series of studies have shown that through actions in rVLM stimulation of both gallbladder and gastric afferent activates splanchnic nerve and increases blood pressure (Li et al., 2002, 2010; Tjen-A-Looi et al., 2004; Crisostomo et al., 2005; Zhou et al., 2005a; Tjen-A-Looi et al., 2006; Moazzami et al., 2010). The PVN projects directly to the rVLM and to a lesser extent to the intermediolateral column of the spinal cord (Pyner and Coote, 1999; Hardy, 2001; Pan, 2004; Chen and Toney, 2010) and contributes to the descending neuronal pathway regulating sympathetic outflow in the rVLM (Tjen-A-Looi et al., 2016). In particular, the parvocellular PVN is critical in regulation of sympathoexcitatory cardiovascular pressor reflex responses such as during activation of splanchnic and cardiac sensory fibers (Xu et al., 2012; Tjen-A-Looi et al., 2016).

Manual acupuncture (MA) and EA modulate pressor responses that are induced by chemical, mechanical, or electrical stimulation of visceral afferent nerves (Li et al., 1998, 2001, 2002; Chao et al., 1999; Tjen-A-Looi et al., 2003, 2004; Crisostomo et al., 2005; Zhou et al., 2005a,b). We observe that EA at P5-6 decreases ischemic myocardial dysfunction (Chao et al., 1999) through decrease of oxygen demand demonstrating that acupuncture reduces demand-induced myocardial ischemia and blunts the sympathoexcitatory reflex elevation of blood pressure (Li et al., 1998). EA stimulation at P5-6 or ST36-37 activating respectively median or deep peroneal nerves and reducing elevated sympathetic activity decreases cardiovascular reflex vasoconstriction and elevation in blood pressure (Li et al., 1998, 2002; Chao et al., 1999; Tjen-A-Looi et al., 2004). With this regard, sympathoexcitatory cardiovascular responses are decreased by EA stimulation at both P5-6 and ST36-376. In addition, sympathetic premotor cardiovascular rVLM cells important for sympathetic outflow participate in central processing of the actions of EA and MA (Tjen-A-Looi et al., 2003, 2004; Zhou et al., 2005b). Recently, we demonstrate that EA-modulation of visceral induced excitatory cardiovascular responses also involves the hypothalamic PVN (Tjen-A-Looi et al., 2016). We showed that the rVLM-projecting PVN neurons important in regulation of cardiovascular responses participate in central processing of the modulatory actions of EA (Tjen-A-Looi et al., 2016). Thus, parvocellular PVN appears to be an important cardiovascular region in the hypothalamus that processes cardiovascular reflex responses and contributes to the actions of acupuncture.

Moxibustion, like EA and MA, is a form of acupoint stimulation. The acupoints are stimulated with heat generated by moxibustion (Wang et al., 2013). Moxibustion at specific acupoints relieves pain and modulates gastric motility and irritable bowel syndrome (Su et al., 2014; Liu et al., 2016; Zhao et al., 2016). Moxibustion applied at acupoint ST36 modulated gastric motility in rats (Su et al., 2014) suggesting that activation of peripheral nerves underlying the acupoint modulates gastrointestinal responses (Tjen and Fu, 2017). Moreover, stimulation of acupoints with either EA or MA modulates elevated blood pressure (Tjen-A-Looi et al., 2004; Zhou et al., 2005b) but through different peripheral mechanisms (Guo et al., 2018). In this regard in contrast to EA, peripheral transient receptor potential vanilloid type-1 (TRPV1) at acupoint P6 contributes to the effect of MA (Guo et al., 2018) suggesting differential underlying mechanisms are involved during stimulation of acupoints. The TRPV1 polymodal receptor is activated by heat greater than 42◦C, mechanical stimuli, during EA in analgesia at ST36, and others (Tominaga et al., 2001; Birder et al., 2002; Pan and Chen, 2004; Nakagawa and Hiura, 2006; Caterina, 2007; Xin et al., 2016). Although activation of mechanosensitive TRPV1 during MA at P6 reduces elevated blood pressure, no information is available on the role of thermosensitive TRPV1 at acupoint ST36 during EA or moxibustion on sympathoexcitatory cardiovascular reflex responses. We hypothesize that moxibustion activating peripheral thermal-sensitive TRPV1 at acupoint ST36 decreases reflex hypertension through the opioid system in the PVN. A preliminary report of this work has been presented (Gong et al., 2018).

# MATERIALS AND METHODS

# Surgical Procedures

Experimental preparations and protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the University of California, Irvine, CA, United States. The study conformed to the American Physiological Society "Guiding Principles for Research Involving Animals and Human Beings." The minimal possible number of rats was used to obtain reproducible and statistically significant results. Studies were performed on adult Sprague-Dawley male rats (400–600 g). Rats were anesthetized with ketamine (100 mg/kg im) and anesthesia was maintained with αα-chloralose (5 mg/kg iv). Additional doses of α-chloralose were given as necessary to maintain an adequate level of anesthesia, as determined by the lack of response to noxious toe pinch and corneal reflex, and the ability to artificially maintain a consistent respiratory rate. The trachea was intubated for artificial respiration using a ventilator (Model 661, Harvard Apparatus). The respiration was maintained with room air enriched with oxygen. The right jugular vein was cannulated for administration of fluids. The

right or left carotid artery was cannulated and attached to a pressure transducer (P23XL, Ohmeda) to monitor arterial blood pressure. Heart rate was derived from the pulsatile blood pressure pulse using a biotech Gould Instrument (Cleveland, OH, United States). Blood pressures and heart rates were recorded and analyzed offline with a computer and CED Spike 2 Windows software. Throughout the experiment arterial blood gases and pH were measured periodically with a blood gas analyzer (ABL5, Radiometer America) and were kept within normal physiological limits (PCO<sup>2</sup> 35–40 mmHg, and PO<sup>2</sup> 100–150 mmHg) by adjusting ventilatory rate or volume and enriching the inspired O<sup>2</sup> supply. Arterial pH was maintained between 7.35–7.45 by infusion of 8% sodium bicarbonate. Body temperature was kept between 36◦ and 38◦C with a heating pad and an external heat lamp. Fur was removed in the region of the ST 36 or G39 acupoint located bilaterally on the hind limbs.

To stimulate gastric afferent by gastric distention, a 2-cm diameter unstressed dimension latex balloon attached to a polyurethane tube (3 mm diameter) was inserted into the stomach through the mouth and esophagus (Tjen-A-Looi et al., 2009). During insertion and passage through the esophagus, the balloon was palpated manually to confirm positioning of the balloon inside the stomach. A syringe was attached to the cannula to inflate and deflate the balloon with air, while a manometer through a T-connection was used to monitor balloon pressure. Transmural pressure was determined by measuring the pressure required to inflate the balloon with the various volumes of air before it was inserted into the stomach (Li et al., 2002). Distention pressures were selected to fall within the range that a rat normally experiences during ingestion of food and fluids in a single meal (Davison and Grundy, 1978; Baird et al., 2001). The balloon was deflated within 30 s after reaching the maximum increase in blood pressure. The positioning of the balloon was verified post mortem to be in the stomach.

To investigate the action of moxibustion in the hypothalamus, the animal was stabilized with a Kopf stereotaxic head frame. A craniotomy was performed to expose the dorsal surface of the cortex and gain access to the PVN. An injection cannula and microsyringe (Hamilton) fixed to a microinjection probe with inner diameter of 0.4 mm was inserted into the PVN to deliver antagonist, vehicle control or Chicago blue dye. Using visual approximation, the microinjection probe was positioned perpendicularly to the dorsal surface of the cortex 1.8 mm posterior to the Bregma, 0.5 mm lateral to the midline, and advanced ventrally 7.5–7.8 mm to reach the PVN. These coordinates provide access to a region that has been found by others to contain sympathetic related cells (Kenney et al., 2003; Chen and Toney, 2010; Cardoso et al., 2012). At the end of experiment, the microinjection site was marked with Chicago blue dye for later histological confirmation following delivery of drugs into the PVN.

# Stimulating Methods

Gastric distensions were repeated 10 times to establish consistent pressor responses. Recovery of at least 10 min between gastric distensions prevented tachyphylaxis of the cardiovascular responses. To examine effects of moxibustion, acupoint ST36 (the anterolateral side of the hind-limb near the anterior crest of the tibia below the knee, under the tibialis anterior muscle that overlies the deep peroneal nerves) was stimulated with moxibustion for 30 min during three gastric distensions following two initial consistent pressor responses. Additional five pressor responses were evaluated after cessation of thermal stimulation to determine long-lasting effect of thermal stimulation at ST36. Total of 10 gastric distention induced pressor responses were evaluated. To examine the point specific thermal-sensitive effect, acupoint G39 (above the ankle at the lower one third of the hind limb overlying superficial peroneal nerves) also was stimulated similarly with heat generated by moxibustion. Responsiveness to specific temperature was evaluated with moxibustion–induced heat at 1 or 2 cm distance from the surface of the hind limbs at acupoint ST36 or G39 (Su et al., 2014). Temperature was measured with probe and thermometer (Fluke 51 II Thermometer) placed on the skin without fur during thermal stimulation at 15 and 30 min. To achieve and maintain the temperatures, the adjustment of the distance from the moxibustion stick to the surface of the skin and the tapping off the ash at end of the stick were performed frequently. Once the temperatures were achieved, the distance was determined and held constant. To determine the similarity of EA and moxibustion on sympathoexcitatory reflex responses, EA at G37-39 and ST36-37 on increases in blood pressure were evaluated in 11 other rats. Needles (40-gauge made by Suzhou Medical Appliance, Suzhou, China) were inserted at ST36-37 at a depth of 3–5 mm and G37-39 at depth of 1–2 mm bilaterally and connected to an isolation unit and stimulator (model S88, Grass, West Warwick, RI) to deliver bipolar stimuli at frequency of 2 Hz, duration of 0.5 ms, and current of 1–4 mA for 30 min. To ensure that twitches by the stimulation of motor fibers (Li et al., 2016) do not contribute to the effects of EA, gallamine triethiodide (4 mg/kg) was administered into the vein after insertion of needles and before the application of EA (Li et al., 2006).

# Methods of Blockade

The role of TRPV1 at acupoint ST36 during action of bilateral moxibustion on gastric distention induced pressor responses was determined with bilateral administration of TRPV1 antagonist iodoresiniferatoxin (Iodo-RTX; 0.1 mM; 10 µl) (Guo et al., 2018). The vehicle control 5% dimethylsulfoxide (DMSO) was administered bilaterally at ST36. The antagonist or vehicle was administered with a hypodermic needle at acupoint ST36 following moxibustion or EA. The proper site and depth of hypodermic needle insertion was confirmed with brief EA stimulation at ST36-37 (placed at a depth of about 3–5 mm) inducing slight repetitive paw twitches in the rats treated with moxibustion. The twitches confirmed stimulation of motor fibers in the mixed deep peroneal nerve bundle (Li et al., 2016). In the hypothalamus, the roles of opioid receptors in the PVN during effect of moxibustion were evaluated by microinjection of the non-specific antagonist naloxone (100 nM, 50-75 nl, Sigma Aldrich, St. Louise, MO, United States) (Tjen-A-Looi et al., 2007) or 0.9% saline vehicle control. An injection cannula and microsyringe (Hamilton) fixed to a pipette were used to administer naloxone or saline into the PVN. Unilateral microinjection allows maintenance of optimal physiological state in studies of EA-modulation of cardiovascular responses (Li et al., 2001; Tjen-A-Looi et al., 2012). With this regard, we investigated the PVN during the effect of heat generating moxibustion on pressor responses with unilateral blockade of opioid receptors.

#### Experimental Protocols

fnins-12-01057 January 21, 2019 Time: 13:27 # 4

#### **1. Gastric distention evoked reflexes**

After the surgical procedures, experiment was conducted following a 30 min stabilization period. Repeated gastric distention was evaluated for consistent increase in blood pressure (Li et al., 2002). After obtaining the maximal cardiovascular pressor response, air was withdrawn from the balloon. Typically, the pressor response was observed within 10 sec following inflation of the balloon. The pressor reflex in mean arterial pressure (MAP) was calculated as the difference in MAP before gastric distention and MAP at the peak of the reflex response. After the completion of each experiment, rats were euthanized with intravenous KCl under deep anesthesia.

#### **2. Moxibustion-inhibition of gastric distention responses**

Consistency of increase in blood pressure to gastric distention was evaluated in a group of five animals. Moxibustion-inhibition (ST36) of the increases in MAP was examined in nine other rats. Five of the nine rats were treated with moxibustion 1-cm distance from acupoint ST36 while the other four rats were subjected to the thermal stimulation at 2-cm distance from the skin.

#### **3. Point specific moxibustion-inhibition of pressor responses**

Point specific moxibustion-modulation of gastric distention reflex responses was determined by stimulating two different acupoints, including ST36 or G39 at 1-cm distance from the skin. The moxibustion effect at ST36 was evaluated in five rats listed in above protocol (2). In four other animals, eight pressor responses were evaluated during and after 30 min moxibustion at acupoint G39 after obtaining two consistent responses to gastric distension.

#### **4. Electroacupuncture-inhibition of gastric distention responses**

Following consistent increases in blood pressure with at least two gastric distentions, cardiovascular reflex responses were measured during 30 min EA at G37-39 (n = 5) or EA at ST 36-37 (n = 6) in rats. The reflex responses to EA were determined during three gastric distentions. Thereafter, five more gastric distentions were performed every 10 min to evaluate the long lasting inhibitory responses.

#### **5. Thermal sensitive TRPV1 in moxibustion-inhibition and EA-inhibition of pressor responses**

Role of TRPV1 during moxibustion-inhibition of the increases in MAP was examined in 12 animals. At ST36 acupoint, TRPV1 was blocked with Iodo-RTX 5 min following termination of moxibustion to evaluate its role in mediating the actions of moxibustion on pressor responses in six of the 12 rats. As a control for the receptor blockade study, DMSO (5%) was injected at the acupoint in six of the 12 subjects tested for ST36 moxibustion-inhibition. In addition, TRPV1 was blocked with Iodo-RTX 5 min following termination of EA ST36-37 to evaluate its role in EA-inhibition of pressor responses in six other rats, listed in protocol (4).

#### **6. Influence of opioid receptor blockade in PVN on moxibustion-inhibition of pressor responses**

Role of PVN in moxibustion-inhibition of the increase in MAP was evaluated in eight animals. Two reproducible premoxibustion control values were obtained followed by 30 min of moxibustion at the ST36 acupoints. Saline was microinjected into PVN after 20 min of moxibustion. Five min after the end of moxibustion, naloxone was administered into the PVN of five rats. To confirm the role of opioids in moxibustion-inhibition, the effect of naloxone on the pressor responses in the absence of moxibustion was examined in four other animals.

#### **7. Histology**

At the end of each experiment, animals were euthanized under deep α-chloralose anesthesia followed by iv injection of saturated KCl. Microinjection sites were marked by microinjection of 2% Chicago blue dye. The brain was removed and fixed in 10% paraformaldehyde for at least 48 hr. Brains were sliced with a microtome cryostat into 40 µm sections. The sites of microinjection were identified and reconstructed with the aid of a microscope (Nikon) and software (Corel presentation) according to the atlas of Paxinos and Watson (2009).

#### Statistical Analysis

Means and standard errors of mean blood pressure and heart rate at rest were compared over time using a repeated-measures analysis of variance (ANOVA) followed by the Student Newman Keuls Test to examine for non-random variation. In the distension-response protocol, comparisons by ANOVA were made between blood pressure responses before and after EA at each increment in gastric volume. Student t-test was used to compare responses among groups. The 0.05 probability level was chosen to determine statistical significance.

# RESULTS

### Moxibustion-Inhibition of Gastric Distention Evoked Pressor Responses

Gastric distention increased blood pressure through activation of splanchnic afferent and induced sympathoexcitatory cardiovascular reflex responses (Li et al., 2002). Repeated stimulation of gastric afferent every 10 min displayed consistent increases in MAP during application of 30-min moxibustion 2-cm distance from the skin at acupoint ST36 showing temperatures lower than 40 ± 0.4◦C (MAP, **Figure 1A**). On the other hand, application of 30-min moxibustion 1-cm distance from the skin at acupoint ST36 yielding temperature of 44.2 ± 0.6◦C reduced the elevated blood pressure responses for 40 min (**Figure 1B**). Thus, moxibustion applied with different temperature at acupoint ST36 displayed differential inhibitory outcomes. Baseline blood pressures throughout the experiments were not significantly different. Heart rates also were not altered during the cardiovascular responses.

# Point Specific Moxibustion-Inhibition of Pressor Responses

Consistent gastric distention sympathoexcitatory cardiovascular reflex responses were examined for point specific effects. Moxibustion at average temperature of 44.2 ± 0.6◦C applied at ST36 or G39 modulated the reflex responses differentially. Heat stimulation with moxibustion at G39 acupoint did not reduce the pressor responses (**Figure 1C**) in contrast to ST36 (**Figure 1B**). Thirty minutes of EA (G37-39) stimulating superficial peroneal nerve also did not reduce the blood pressure reflex responses (**Figure 1D**) supporting the importance of acupoints ST36 in reducing pressor responses.

# Thermal Sensitive TRPV1 in Moxibustion-Inhibition of Pressor Responses

To determine the peripheral mechanism at acupoint ST36 on the inhibitory effect of moxibustion on sympathoexcitatory cardiovascular responses, the heat sensitive TRPV1 receptor underneath the acupoint was blocked with iodoresiniferatoxin. The TRPV1 blockade transiently reversed the inhibitory effect of moxibustion in contrast to DMSO vehicle (**Figures 2A,B**). The reversal of moxibustion-inhibition with the TRPV1 antagonist compared with vehicle DMSO on the pressor responses was significantly different (P = 0.019).

# Thermal Sensitive TRPV1 in EA-Inhibition of Gastric Distention Responses

To examine the peripheral mechanism at acupoint ST36 on the inhibitory effect of EA on sympathoexcitatory cardiovascular responses, the heat sensitive TRPV1 receptor underneath this acupoint was blocked with iodoresiniferatoxin. Our previous study shows that EA ST36-37 reduced the elevated blood pressure responses during repeated gastric distention (Zhou et al., 2005a). Present study showed that the TRPV1 blockade did not reverse the inhibitory effect of EA (**Figure 2C**). Since DMSO did not affect the inhibition on the cardiovascular reflex responses, DMSO was not re-examined in the presence of EA. In addition, the reversal of moxibustioninhibition compared with EA-inhibition on the pressor responses following TRPV1 antagonist administration was significantly different (P = 0.012) suggesting that the peripheral mechanisms at ST36 in reducing elevated blood pressure are dissimilar.

# Role of Opioids in PVN on Moxibustion-Inhibition of Pressor Responses

To establish participation of the hypothalamus during the moxibustion inhibition on elevated blood pressure responses, opioid receptors were examined in the PVN. Naloxone in

application of moxibustion. # indicates significant reversal of moxibustion-inhibition compared with pressor response immediately before the TRPV1 blockade.

contrast to saline in the PVN reversed the moxibustion inhibition on pressor responses (**Figure 3**).

# Histology

Sites of microinjection marked with Chicago blue dye were confirmed histologically. The sites were determined by the location of microinjection tracks and dye spots. The PVN microinjection sites were confirmed to be 0.4–0.6 mm lateral to the midline and 1.6–1.75 mm from the ventral surface. Data from two microinjections of naloxone that were outside the PVN and did not reverse the effects of moxibustion were not included. **Figure 4A** displays a coronal section of 60 microns showing the site of microinjection. **Figure 4B** represents a composite map of all sites closely matched with the rat brain atlas (Paxinos and Watson, 2009).

# DISCUSSION

The present study investigates the effect and underlying mechanisms in the reduction of elevated blood pressure by heat generating moxibustion. The study examined four aspects that are important in the actions of moxibustion inhibition of sympathoexcitatory cardiovascular reflex responses. First, we have observed that the effect of moxibustion is temperature dependent. A temperature greater or at 43◦C at the surface of the skin at acupoint ST36 decreases gastric distention induced reflex increases in blood pressure. Secondly, the temperature-dependent moxibustion inhibitory effect on gastric distention induced cardiovascular responses also is related to point specificity. Moxibustion similar to EA applied at ST36, in contrast to G39, significantly decreases the gastric distention induced pressor response (Tjen-A-Looi et al., 2004; Zhou et al., 2005a). As such, previous (Tjen-A-Looi et al., 2004; Zhou et al., 2005a) and current data show that EA and moxibustion share similar point specific modification of pressor responses. Third, blockade of TRPV1 receptor at acupoint ST36 reverses the inhibitory effect of moxibustion. On the other hand, blockade at ST36 is unable to reverse EA-inhibition of reflex elevation of blood pressure. In this respect, heat sensitive receptor TRPV1 activation by moxibustion appears to be important in the inhibition of the pressor response while this receptor activation is not involved during EA-inhibition. Importantly, we demonstrate that the inhibitory effect of moxibustion on cardiovascular reflex responses includes opioid receptor activation in hypothalamic PVN. We have observed that blockade of opioid receptors reverses the moxibustion blood pressure lowering effect suggesting that the action of moxibustion, in part, likely is processed in this brain region. Thus, temperature-dependent moxibustion at specific acupoint reduces elevated blood pressure through hypothalamic opioid receptors in the PVN and, unlike cardiovascular EA-inhibition, peripheral local TRPV1 receptors.

Point specific stimulation with moxibustion or EA differentially inhibits elevated blood pressure. Current study shows that stimulation at ST36 acupoint with moxibustion

is effective in reducing elevated blood pressure in contrast to moxibustion application at G39. In line with these observations, moxibustion at different acupoints induces facilitatory and inhibitory effects on gastric motility (Su et al., 2015) supporting the point specific effect of moxibustion. In addition, stimulation with EA at P5-6 or ST36-37 acupoints inhibits sympathoexcitatory blood pressure responses (Tjen-A-Looi et al., 2004; Zhou et al., 2005a) while current data show the inability of EA-inhibition at G37-39. EA modulates also reflex hypotension and bradycardia with point specific actions (Tjen-A-Looi et al., 2018) suggesting that EA influences both cardiovascular inhibitory and excitatory reflex responses. Collectively, these observations suggest that both moxibustion and EA modify excitatory as well as inhibitory reflex responses by stimulation at specific acupoints.

Transient receptor potential vanilloid 1, a known capsaicin receptor, is heat-sensitive and depolarizes neurons at temperatures greater than 42◦C. Interestingly, studies have suggested that ruthenium red, the non-competitive antagonist for the capsaicin receptor, abolish the capsaicin but not the heat induced reflex response (Hiura, 2009). The polymodal TRPV1 receptor, sensor of chemical, mechanical and heat stimuli, is expressed most abundantly in small diameter peripheral sensory neurons (Sanchez et al., 2001; Caterina, 2007). The TRPV1 located on sensory neuronal tissue is a ligand-gated, voltagegated and cationic channel (Voets et al., 2004; Matta and Ahern, 2007). TRPV1 located on sensory fibers is important during 50 Hz EA ST36 on systemic analgesia (Xin et al., 2016). In these respects, current study shows an important role for TRPV1, likely located in the small diameter fibers underneath acupoint ST36, during moxibustion inhibition of pressor responses, however,

this receptor does not participate during the inhibitory actions of 2 Hz EA. Notably, MA also modulates sympathoexcitatory cardiovascular reflex responses through peripheral TRPV1 underneath the blood pressure lowering acupoint P6 (Guo et al., 2018). Hence, TRPV1 activated by heat or mechanical stimuli during moxibustion or MA application respectively modulates sympathoexcitatory cardiovascular reflex responses in contrast to EA-inhibition on pressor responses. Further studies are warranted to examine the role of TRPV1 during actions of EA focusing on physiological conditions and stimulating parameters.

The importance of supraspinal regions during effects of moxibustion has been unclear while EA inhibits cardiovascular responses, in part, through hypothalamus, midbrain and medulla. Stimulation of ST36-37 with EA activates hypothalamic arcuate nucleus, modulates brainstem rVLM activity and decreases sympathoexcitatory cardiovascular reflex responses (Tjen-A-Looi et al., 2004; Li et al., 2006). We also have demonstrated that the PVN projecting to rVLM is activated during EA (P5-6) while the present study shows that moxibustion through local TRPV1 at ST36 reduces reflex increases in blood pressure through the hypothalamic PVN. In accordance with the previous and current studies, activation of peripheral TRPV1 at ST36 modifies hypothalamic PVN, PVN-rVLM projection and hence cardiovascular reflex responses. However, current observation indicates that activation of ST36 by EA does not include the activation of the heat sensitive TRPV1. Future studies are needed to determine the peripheral mechanisms during EA activation of ST36 involving central regions such as PVN in modulating the reflex elevation of blood pressure.

The PVN is rich in opioids. In particular, the opioids, enkephalin and β-endorphin, are present in the PVN and participate in regulation of cardiovascular function (Kiss et al., 1984; Lessard and Bachelard, 2002; Bowman et al., 2013). Furthermore, opioids reduce neuronal activity in PVN in rat hypothalamic brain slice preparation (Muehlethaler et al., 1980; Pittman et al., 1980). In intact animal, we show the relevance of PVN opioid receptors during effects of EA at P5-6 (Tjen-A-Looi et al., 2016). Although stimulation with EA at P5-6 decreases the sympathetic activity of cardiovascular neurons in PVN that, in turn, reduces pressor responses through activation of PVN opioid receptors (Tjen-A-Looi et al., 2016), the actions and effects during ST36 stimulation and activation of the local TRPV1 has been unclear. We currently demonstrate a reversal of the moxibustion-inhibition activating temperature specific TRPV1 on sympathetic cardiovascular responses following non-specific blockade of opioid receptors in the PVN. Thus, the present data show that moxibustion inhibition by stimulation of ST36 and activation of peripheral TRPV1 employs the opioid system in the PVN to reduce sympathoexcitatory reflex responses. The

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specific opioid receptor subtype(s) in the PVN relevant to the moxibustion-inhibition warrants further investigation.

### PERSPECTIVE AND SUMMARY

Our results suggest that thermal dependent moxibustion stimulating ST36 activates TRPV1 in peripheral sensory nerves, reduces PVN neuronal activity through the opioid system, and decreases sympathoexcitatory cardiovascular reflex responses. Decrease in these cardiovascular reflex responses by stimulation of acupoint ST36 with EA or moxibustion appears to occur through different peripheral mechanisms. These observations imply that the activation of sensory neuron during acupoint stimulation by different modes such as EA, MA, or moxibustion eventually decreases sympathoexcitatory responses possibly through similar central processes (Tjen-A-Looi et al., 2016; Guo et al., 2018). Although previous reports show moxibustion blood pressure lowering effect in combination with antihypertensive drugs, present data show that moxibustion alone decreases sympathoexcitatory blood pressure responses. Studies are warranted to examine the long lasting effect of moxibustion on sustained hypertension.

# AUTHOR CONTRIBUTIONS

ST-A-L, LC, and PL were responsible for experimental design and data evaluation. The experiments were conducted by ST-A-L, YG, Z-LG, and YP. All authors reviewed the manuscript.

# FUNDING

National Heart, Lung, and Blood Institute and National Center for Complimentary and Integrative Health, Bethesda, MD, United States, grants HL-72125 and AT-009347. Shanghai Natural Science Foundation: Moxibustion at Zusanli on IML-rVLM neural regulation pathway in hypertensive rats (16ZR1428300). The National Basic Research Program of China, the "973 plan" project, the mechanism of the moxibustion effect and the mechanism of its endogenous regulation (2015CB554501).

### ACKNOWLEDGMENTS

The authors are grateful for technical input from Sixian Audrey Li.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Cheng, Li, Patel, Gong, Guo, Wu, Malik and Tjen-A-Looi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Default Mode Network as a Neural Substrate of Acupuncture: Evidence, Challenges and Strategy

Yuqi Zhang<sup>1</sup> , Haolin Zhang<sup>2</sup> \*, Till Nierhaus3,4, Daniel Pach5,6, Claudia M. Witt5,6,7 and Ming Yi1,8 \*

<sup>1</sup> Department of Neurobiology, School of Basic Medical Sciences, Neuroscience Research Institute, Peking University, Beijing, China, <sup>2</sup> Department of Traditional Chinese Medicine, Peking University Third Hospital, Beijing, China, <sup>3</sup> Neurocomputation and Neuroimaging Unit, Department of Education and Psychology, Freie Universitat Berlin, Berlin, ˝ Germany, <sup>4</sup> Department of Neurology, Max Planck Institute for Human Cognitive and Brain Sciences, Leipzig, Germany, 5 Institute for Social Medicine, Epidemiology, and Health Economics, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany, <sup>6</sup> Institute for Complementary and Integrative Medicine, University Hospital Zurich, University of Zurich, Zurich, Switzerland, <sup>7</sup> Center for Integrative Medicine, University of Maryland School of Medicine, Baltimore, MD, United States, <sup>8</sup> Key Laboratory for Neuroscience, Ministry of Education, National Health Commission, Peking University, Beijing, China

#### Edited by:

Florian Beissner, Hannover Medical School, Germany

#### Reviewed by:

Norman Waldemar Kettner, Logan College of Chiropractic, United States Jeungchan Lee, Harvard Medical School, United States

#### \*Correspondence:

Haolin Zhang zhanghaolin@bjmu.edu.cn Ming Yi mingyi@hsc.pku.edu.cn; mingyi@bjmu.edu.cn

#### Specialty section:

This article was submitted to Perception Science, a section of the journal Frontiers in Neuroscience

Received: 11 September 2018 Accepted: 28 January 2019 Published: 11 February 2019

#### Citation:

Zhang Y, Zhang H, Nierhaus T, Pach D, Witt CM and Yi M (2019) Default Mode Network as a Neural Substrate of Acupuncture: Evidence, Challenges and Strategy. Front. Neurosci. 13:100. doi: 10.3389/fnins.2019.00100 Acupuncture is widely applied all over the world. Although the neurobiological underpinnings of acupuncture still remain unclear, accumulating evidence indicates significant alteration of brain activities in response to acupuncture. In particular, activities of brain regions in the default mode network (DMN) are modulated by acupuncture. DMN is crucial for maintaining physiological homeostasis and its functional architecture becomes disrupted in various disorders. But how acupuncture modulates brain functions and whether such modulation constitutes core mechanisms of acupuncture treatment are far from clear. This Perspective integrates recent literature on interactions between acupuncture and functional networks including the DMN, and proposes a back-translational research strategy to elucidate brain mechanisms of acupuncture treatment.

Keywords: acupuncture, default mode network, neuroimaging, pain, fMRI

#### COMPLEX BRAIN MECHANISMS OF ACUPUNCTURE

Acupuncture, an important component of traditional Chinese medicine, has been practiced in China for more than 3000 years, and is now widely applied all over the world (Zhuang et al., 2013). Studies have shown that for disorders such as chronic pain, the effects of acupuncture cannot be fully attributed to placebo (Vickers et al., 2012, 2018). Neuroimaging studies have revealed significant brain activity changes in response to acupuncture, indicating possible brain contribution to its effects.

Intriguingly, brain responses to acupuncture stimuli encompass a broad network of regions involving not only somatosensory, but affective and cognitive processing. A meta-analysis of brain activities associated with acupuncture stimulation reveals activation in the sensorimotor cortical network, including the insula, thalamus, anterior cingulate cortex (ACC), and primary and secondary somatosensory cortices, and deactivation in the limbic-paralimbic neocortical network, including the medial prefrontal cortex (mPFC), caudate, amygdala, posterior cingulate cortex

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(PCC), and parahippocampus (Chae et al., 2013). These findings indicate multi-dimensional brain responses to acupuncture. However, contribution of each dimension to acupuncture effects is poorly defined.

Additional complexity stems from differences between various acupuncture paradigms (Huang et al., 2012). Such variations may stem from (but not restricted to) manual versus electro-acupuncture, electro-acupuncture of different stimulating frequencies and intensities, acupuncture in different points, responders versus non-responders of acupuncture, and acupuncture in healthy versus morbid participants (Han, 2003; Yi et al., 2011; Huang et al., 2012; Xie et al., 2013). Thus, common and specific brain responses need to be clarified between these conditions for delicate mechanistic understanding of acupuncture.

#### DEFAULT MODE NETWORK AS A NEURAL SUBSTRATE OF ACUPUNCTURE

Default mode network (DMN) is a recently appreciated brain system, which shows strong activity at rest but deactivates upon externally oriented attention (Buckner et al., 2008; Northoff et al., 2010). Resting state functional magnetic resonance imaging has identified key clusters of human DMN including mPFC, ACC, PCC, orbital frontal cortex, lateral temporal cortex, inferior parietal lobe, retrosplenial cortex and precuneus (Buckner et al., 2008). Simultaneous with signal attenuation in the DMN, a significant signal potentiation in the salience network can be observed (Napadow et al., 2009; Nierhaus et al., 2015), with anterior insula initiating dynamic switching between these intrinsic networks (Bai et al., 2009).

We could note that brain regions within the DMN overlap to a large extent with acupuncture-responsive regions (Chae et al., 2013), which leads to the hypothesis that acupuncture exerts effects through its modulation over the DMN (Otti and Noll-Hussong, 2012; Zhao et al., 2014). In addition to local activation/deactivation, the functional connectivity within and across DMN is also modulated by acupuncture (Dhond et al., 2008; Zyloney et al., 2010; Long et al., 2016; Shi et al., 2016). More importantly, acupuncture-induced deactivation of DMN is stronger than sham acupuncture or tactile stimulation, but attenuated or reversed in direction if sharp pain occurs during acupuncture practice (Hui et al., 2010). In addition, increasing the "dose" of acupuncture, by increasing the number of needles or the intensity of needle stimulation, might induce an enhanced modulation of DMN that persisted even after the termination of acupuncture stimulation (Lin et al., 2016).

Disrupted DMN activities have been observed in various diseases including pain (Dhond et al., 2008; Kucyi et al., 2014; Alshelh et al., 2018), autism (Kennedy and Courchesne, 2008), schizophrenia (Bluhm et al., 2009), Alzheimer's disease (Sorg et al., 2007), depression (Liston et al., 2014), attentiondeficit/hyperactivity disorder (Norman et al., 2017), insomnia (Yu et al., 2018), multiple sclerosis-related fatigue (Jaeger et al., 2018), and posttraumatic stress disorder (Sripada et al., 2012; Akiki et al., 2018). Chronic low back pain is associated with less connectivity within DMN, mainly in the dorsolateral prefrontal cortex, mPFC, ACC and precuneus (Baliki et al., 2008, 2014; Loggia et al., 2013; Ceko et al., 2015; Jiang et al., 2016; Alshelh et al., 2018). Acupuncture reverses these changes almost to the levels seen in healthy controls, and reductions in clinical pain are correlated with increases in DMN connectivity (Li et al., 2014). Similar results are also reported in chronic sciatica patients (Li et al., 2012). In another study on experimental acute low back pain (Shi et al., 2015), pain state induces higher regional homogeneity values in the limbic system and DMN, and acupuncture yields broad deactivation in DMN, consistent with other research as previously described. Apart from pain, acupuncture has also been evaluated in other disorders. In patients with depression, acupuncture induced wide posterior DMN activation (Quah-Smith et al., 2013) and increased functional connectivity between PCC and bilateral ACC (Deng et al., 2016). In stroke patients, enhanced interregional interaction between ACC and PCC, two key DMN hubs, was observed after acupuncture (Zhang et al., 2014). Finally, acupuncture attenuates impaired DMN connectivity seen in patients with Alzheimer disease (Liang et al., 2014).

If DMN is generally affected by acupuncture, we might observe both common and specific modulation of DMN by stimulation at different acupuncture points. Liu et al. performed electro-acupuncture stimulation at three acupuncture points (GB37, BL60, and KI8) and observed consistently interrupted correlation between PCC and ACC, two key nodes of the DMN (Liu et al., 2009b). However, stimulating these three points produced different correlation strength between other nodes in DMN. In addition, visual cortical regions and mPFC are specifically responsive to the stimulation of GB37, whereas KI8 is more associated with activity changes in insula and hippocampus (Liu et al., 2009a). This modulatory pattern is consistent with clinical practice that GB37 is one of the important acupuncture points for eye diseases whereas KI8 is related to gynecological disorders such as menstrual pain. Claunch et al. (2012) examined the specificity and commonality of the brain response to manual acupuncture at LI4, ST36, and LV3, and found clusters of deactivation in the mPFC, medial parietal and medial temporal lobes showing significant convergence of two or all three of the acupuncture points. For differences, LI4 predominated in the pregenual cingulate and hippocampal formation, ST36 predominated in the subgenual cingulate, and LV3 predominated in the posterior hippocampus and PCC. Similar commonality and specificity of brain responses to different acupuncture points, with DMN regions as crucial hubs, are also reported by a series of studies on PC6, PC7, and GB37 (Bai et al., 2010; Ren et al., 2010; Feng et al., 2011).

#### A BACK-TRANSLATIONAL STRATEGY FOR FUTURE RESEARCH

Despite these correlative observations, direct evidence to causally validate DMN as a neural substrate of acupuncture is lacking:

the modulation of DMN by acupuncture could only reflect indirect consequences of other more specific therapeutic effects, or even some insignificant by-products of the stimulation. Additional complexity stems from the fact that DMN changes upon acupuncture could be directly driven by somatosensory afference of acupuncture (i.e., stimulation intensity or deqi sensation), or indirectly caused by affective or cognitive processes related to the therapeutic effect. Caution should be taken to differentiate between these mechanisms using sham acupuncture methodology. Indeed, it remains a tremendous challenge to causally elucidate brain mechanisms of acupuncture. In the first instance, mechanisms of both physiological and pathological brain networks are still under investigation, before we superimpose acupuncture stimulation above them. For example, the molecular and cellular architecture of DMN is far from clear, despite the discovery of DMN-like networks in laboratory animals (Hayden et al., 2009; Popa et al., 2009; Northoff et al., 2010; Lu et al., 2012; Sforazzini et al., 2014) and some pilot mechanistic findings (Nair et al., 2018; Turchi et al., 2018; Yang et al., 2018). Indeed, mechanisms of acupuncture, pain and other neural processes could not be fully clarified without understanding these network substrates, since the same brain region could participate in distinct processes through different microcircuits (Zheng et al., 2017; Jiang et al., 2018). In addition, acupuncture may exert its effects at multiple levels ranging from local stimulation sites to higher centers in the brain. For example, adenosine locally released in acupuncture sites is sufficient to induce analgesia (Goldman et al., 2010; Takano et al., 2012), in which case brain activity changes may only reflect secondary responses of this peripheral mechanism. However, ACC and other brain regions have a crucial role in at least some forms of acupuncture-induced analgesia (Yi et al., 2011). It is challenging to differentiate between causal brain mechanisms of acupuncture stimulation and secondary responses of peripheral effects.

Despite these challenges, novel techniques, especially those targeting neural circuitries, are becoming available to solve the problem. We propose a back-translational strategy involving several key experimental steps toward scientific verification of brain mechanisms of acupuncture, including the possible role of DMN or other functional networks.

First, the architecture of functional neuronal networks requires elucidation at neuronal and molecular levels. Taking the DMN as an example, the concept of default mode stems from neuroimaging studies primarily based on blood oxygen metabolism, which only indirectly reflects neuronal activities. Recent years have witnessed several intriguing studies linking blood oxygen level-dependent signals with electrophysiological measures of neuronal ensembles, especially high frequency neuronal oscillations in the gamma band (Niessing et al.,

#### REFERENCES

Akiki, T. J., Averill, C. L., Wrocklage, K. M., Scott, J. C., Averill, L. A., Schweinsburg, B., et al. (2018). Default mode network abnormalities in posttraumatic stress disorder: a novel network-restricted topology approach. Neuroimage 176, 489–498. doi: 10.1016/j.neuroimage.2018.05.005

2005; Scholvinck et al., 2010). Key brain regions in the DMN revealed from neuroimaging studies in humans could first be confirmed with in vivo multi-channel electrophysiological recording in freely behaving animal models, taking advantages of accurate evaluation of cross-regional interactions and their behavioral correlates (Li et al., 2017). Neuronal and molecular substrates of these networks could be further examined with pharmacological and genetic techniques. Special attention may focus on activity- and metabolism-associated molecules such as adenosine triphosphate, adenosine and neurosteroids (Goldman et al., 2010; Zhang et al., 2016, 2017).

With the same techniques, the multi-dimensional brain responses of various acupuncture paradigms could be evaluated at both neuronal network and single cell levels. Such "mapping" studies in animals would complement neuroimaging studies in humans, and form the basis for following causal verification. Computational methods including pattern recognition and machine learning would show their strength in differentiating common and specific brain responses between various stimulating paradigms and to isolate key electrophysiological features.

Finally and most importantly, interventional techniques such as opto- and chemo-genetics are required to causally verify the molecular and neuronal mechanisms of functional networks, the overlying acupuncture effects, and the contribution of different dimensions of brain responses to acupuncture effects. Basal forebrain has been suggested to underlie DMN-like activities in rodents (Nair et al., 2018; Turchi et al., 2018), but causal evidence for this hypothesis is still missing. Similarly, causal contribution of brain activity changes in acupuncture is also lacking. These techniques would finally demonstrate causal contribution of DMN activity changes to acupuncture effects.

With this strategy, one might elucidate brain mechanisms of acupuncture in animal models. This knowledge could then be used to improve future acupuncture studies in humans.

#### AUTHOR CONTRIBUTIONS

MY, DP, and CW designed the perspective. YZ, HZ, TN, and DP reviewed the relevant literature. YZ, HZ, and MY drafted the manuscript. All authors read and confirmed the manuscript.

#### FUNDING

This work was supported by the National Natural Science Foundation of China (81603446 and 31872774) and Beijing Natural Science Foundation (7174363 and 5182013).

Alshelh, Z., Marciszewski, K. K., Akhter, R., Di Pietro, F., Mills, E. P., Vickers, E. R., et al. (2018). Disruption of default mode network dynamics in acute and chronic pain states. Neuroimage Clin. 17, 222–231. doi: 10.1016/j.nicl.2017.10.019

Bai, L., Qin, W., Tian, J., Dong, M., Pan, X., Chen, P., et al. (2009). Acupuncture modulates spontaneous activities in the anticorrelated resting brain networks. Brain Res. 1279, 37–49. doi: 10.1016/j.brainres.2009.04.056



**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Zhang, Zhang, Nierhaus, Pach, Witt and Yi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Expectations of the Physiological Responses Can Change the Somatosensory Experience for Acupuncture Stimulation

Hyun-Seo Song<sup>1</sup> , Won-Mo Jung<sup>1</sup> , Ye-Seul Lee1,2, Seung-Woo Yoo<sup>1</sup> and Younbyoung Chae<sup>1</sup> \*

<sup>1</sup> Acupuncture and Meridian Science Research Center, College of Korean Medicine, Kyung Hee University, Seoul, South Korea, <sup>2</sup> Department of Anatomy and Acupoint, College of Korean Medicine, Gachon University, Seongnam, South Korea

Objective: Humans interpret sensory inputs based on actual stimuli and expectations of the stimuli. We investigated whether manipulating information related to the physiological response could change the somatosensory experience of acupuncture.

#### Edited by:

Vitaly Napadow, Harvard Medical School, United States

#### Reviewed by:

Meryem A. Yucel, Harvard University, United States Shellie Ann Boudreau, Aalborg University, Denmark

> \*Correspondence: Younbyoung Chae ybchae@khu.ac.kr

#### Specialty section:

This article was submitted to Perception Science, a section of the journal Frontiers in Neuroscience

Received: 16 July 2018 Accepted: 23 January 2019 Published: 12 February 2019

#### Citation:

Song H-S, Jung W-M, Lee Y-S, Yoo S-W and Chae Y (2019) Expectations of the Physiological Responses Can Change the Somatosensory Experience for Acupuncture Stimulation. Front. Neurosci. 13:74. doi: 10.3389/fnins.2019.00074 Methods: Twenty-four participants received tactile stimulations with a von Frey filament on the left arm. Participants were informed that they would receive acupuncture stimulations at different angles while they were presented with changes in their peripheral blood flow (PBF) measured with Laser Doppler perfusion imaging. However, in reality, they were observing premade pseudo-biosignal images (six sessions: one circular, two rectangular elongated, two diagonally elongated, and one cross-fixation [control] shape). After each session, the participants reported the intensity and location of the de qi sensations perceived on their arm using a bodily sensation mapping tool. The spatial patterns of the somatic sensations were visualized using statistical parametric mapping. The F1 score was calculated to measure the similarity between the presented pseudo-biosignals and reported de qi response images.

Results: The spatial configurations of the presented pseudo-biosignal images and de qi response images were similar. The rectangular elongated pseudo-biosignal shape had a significantly higher F1 score compared to the control. All tactile stimulations produced similar levels of enhanced PBF regardless of the pseudo-biosignal shape.

Conclusion: The spatial configurations of somatic sensations changed according to the presented pseudo-biosignal shape, suggesting that expectations of the physiological response to acupuncture stimulation can influence the perceived somatic sensation.

Keywords: acupuncture, biosignal, de qi, expectation, psychophysics

# INTRODUCTION

A fundamental characteristic of acupuncture treatment is the elicitation of unique somatic sensations around points (i.e., de qi sensations) stimulated with the insertion of a needle (Chae et al., 2013; Chae and Olausson, 2017). Achieving an appropriate de qi sensation is believed to be a key component of acupuncture treatment (Kong et al., 2005; Mao et al., 2007). In a clinical research

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data-mining study, 82.1% of studies supported that de qi sensations were closely related to clinical efficacy (Pan et al., 2017). De qi sensations include a combination of various sensations, such as heaviness, numbness, soreness, distention, and even a spreading sensation far from the stimulus site (MacPherson and Asghar, 2006). Recently, spatial patterns of acupuncture-induced sensations, including sensations propagated along the acupuncture meridians, have been demonstrated based on a geographic information system using a bodily sensation map (BSM) (Beissner and Marzolff, 2012; Jung et al., 2016). The BSM, one of a digital pen-and-paper platform, is useful to measure location and intensity of the bodily sensation (Jung et al., 2017a,c).

Humans perceive somatic sensations by integrating afferent signals and higher cognitive processes. For example, placebo stimulations have been reported to cause considerable and widespread sensations despite a lack of peripheral stimulus (Beissner et al., 2015). Moreover, in a clinical trial using laser acupuncture, remarkable de qi sensations were elicited without any cutaneous sensory input (Salih et al., 2010). Furthermore, brain activation associated with acupuncture stimulation was influenced by enhanced bodily awareness and bodily attention around the acupoint (Chae et al., 2015; Jung et al., 2016). In a recent study, expectations of acupuncture stimulation elicited a distinct somatic sensation experience and activation of the salience network in the brain, even without an afferent somatosensory signal (Jung et al., 2018). Together, these results suggest that experiences and expectations prior to tactile stimulation can influence the perception of somatic sensations.

These properties of somatic sensory experience suggest that de qi sensation patterns can be influenced by expectations of psychophysiological responses to acupuncture stimulations. In this study, we hypothesized that biosignal information (i.e., peripheral blood flow change) presented as a psychophysiological response to acupuncture would alter the spatial configurations of somatic sensations in response to tactile stimulations. More specifically, since propagated sensations induced by acupuncture were prominently along perpendicular and horizontal lines on the body, it can be assumed that rectangular elongated pseudo-biosignal of acupuncture can efficiently influence on the perceived somatic sensations. When tactile stimulations were applied to the arm, the perceived sensation showed circularshaped spatial patterns. Thus, it is assumed that there will be no differences in the spatial patterns of the de qi sensations between the circular images session and the control session.

To test this hypothesis, we presented participants with different Laser Doppler perfusion images of pseudo-biosignal shapes mimicking changes in peripheral blood flow (PBF) and compared the spatial configurations of the somatic sensations induced by tactile stimulation.

#### MATERIALS AND METHODS

#### Subjects

We recruited 24 healthy volunteers via advertisements targeting students of Kyung Hee University and Korea University. None of the participants of this study had any history of cardiac, neurological, psychiatric, or visual disorders. Participants refrained from drinking alcohol or caffeine or from taking any drugs or medications for 12 h before the experiment. All participants received a detailed explanation of the experiment and provided written informed consent. This experiment was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Kyung Hee University. All procedures were conducted in a quiet and temperature-controlled room (24 ± 2 ◦C) throughout the experiment.

#### Experimental Design and Procedure

Each participant was asked to sit in a comfortable position in front of a computer monitor before the experiment. Participants were informed that they would receive acupuncture stimulation at various angles and that they would be presented with changes in their PBF measured using Laser Doppler perfusion imaging. However, in reality, the participants were presented with premade pseudo-biosignal images (one circular, two rectangular elongated, two diagonally elongated, and one cross-fixation [control] shape). A total of six sessions were conducted and all participants were unaware of the procedures. The pseudo-biosignals and control image were presented in a pseudorandom order. The experiment followed a within-subject crossover design, in which the independent variable was the pseudo-biosignal shape. All participants were unaware of the procedures. The pseudobiosignals were presented in a pseudorandom order.

Each experimental session lasted for 315 s, during which the participants looked at the cross fixation for the preparation period for 60 s and they received tactile stimulation for 15 s. Right after stimulation, participants were presented with the pseudobiosignal for 120 s. They were required to fix their gaze on the computer monitor to view the change of the pseudo-biosignal immediately following the tactile stimulation. The actual PBF around the stimulation site was measured before and after stimulation. Finally, participants were asked to mark the areas of induced sensation on a BSM for 120 s. A 2-min break was given between sessions to allow the participants to relax (**Figure 1A**).

Basic demographic data were collected from all participants before commencing the experiments. Participants also evaluated the expectancy and fear of the acupuncture treatment using the Acupuncture Expectancy Scale (AES) and Acupuncture Fear Scale (AFS) (Kim et al., 2013, 2014).

#### Tactile Stimulation

Each participant received a tactile stimulation at the same site of the left arm (TE5 acupoint: on the posterior aspect of the forearm, at the midpoint of the interosseous space between the radius and ulna, 2 B-cun proximal to the dorsal wrist crease). All tactile stimulations were delivered using a 60-g von Frey filament (North Coast Medical Inc., San Jose, CA, United States). Tactile stimulations designed to mimic acupuncture stimulation were given at a frequency of 1 Hz for 15 s. The stimulated sites on the arm were hidden from the participants' sight to make them believe that they were receiving real acupuncture stimulations. All participants were told that they would receive

area. The F1 score is calculated as (2 × precision × recall)/(precision + recall), where precision is the area of the correct positive results divided by the area of all relevant samples from the perceived de qi sensation area (c/a) and recall is the area of correct positive results divided by the area of all positive results from the

different acupuncture stimulations at various angles in each session. However, we did not inform the participants which angle of the needle was being inserted. In acupuncture practice, it is common to change the angle of needle insertion according to the site and the disease. Thus, participants might have supposed that various directions of the pseudo-biosignal image are derived from various angle of the needle stimulation.

region of interest of the presented pseudo-biosignal image (c/b).

# Measurement of Perceived Tactile Sensations Using the Bodily Sensation Map

Following stimulation, the participants were asked to report the locations and intensity of bodily sensations using the BSM<sup>1</sup> . This

<sup>1</sup>http://cmslab.khu.ac.kr/downloads/bsm

tool presents a template of the human body as two-dimensional images, including a lateral view of the left arm. The perceived sensations after each session were recorded in 1,024 × 512 matrices on an iPad (Apple Inc., Cupertino, CA, United States). Participants could express the intensity of the somatic sensation by changing the color of the points on a continuous color map via successive strokes with a touch pen (Adonit Inc., Austin, TX, United States).

#### Measurement of Peripheral Blood Flow Using Laser Doppler Perfusion Imaging

Measurements of peripheral blood perfusion around the stimulation site were performed using a Laser Doppler perfusion imager (PeriScan PIM 3 System; Perimed AB, Järfälla, Sweden). The left arm of the participants was stabilized with a kapok-filled

vacuum cushion to maintain its position, and measurements were conducted every 10 s for 180 s before and after stimulation. PBF (symbolized as Ri, [i = 1, 2, 3, . . ., 18]) was calculated as the average perfusion value during each 10-s scan in the region of interest (ROI) (a 2 cm × 2 cm square centered on the TE5 acupoint), and the change in PBF was defined as 1PBF = (Ri − R1)/R1. Each 10 s PBF was subtracted and divided by a baseline (collected over 30 s) in each session, which yielded the change in scores reflecting the increase in PBF from the baseline.

#### Data Analysis

To determine the spatial patterns of the sensations under each condition, we extracted parametric maps of bodily sensation using the BSM tool. Individual datasets for each subject were normalized within the range of 0–1. The normalized BSMs were subjected to group-level analysis of the statistical parametric maps. The group-level analysis consisted of a random effects analysis using the pixel-wise univariate t-test of individual BSMs for each session (3dttest++, AFNI<sup>2</sup> ). False discovery rate (FDR) corrections were carried out to account for false positives due to multiple comparisons (FDR-corrected p < 0.05). The resulting t-value maps were transformed into Z scores that

<sup>2</sup>http://afni.nimh.nih.gov/afni

reflected significant spatial information of the bodily sensation to tactile stimulation. The information was color-coded based on the Z score.

The regions of color changes for each pseudo-biosignal image were extracted as the ROI of each condition. A larger intersection area and smaller complementary area between the ROI and reported somatic sensation area indicated a greater similarity between them. We used the F1 score to evaluate the similarity between the presented pseudobiosignal images and reported areas of somatic sensation. The F1 score is generally used as a measure of a test's accuracy in binary classifications (Lipton et al., 2014), and is the harmonic mean of precision and recall, calculated as (2 × precision × recall)/(precision + recall). Here, precision is defined as the area of correct positive results divided by the area of all positive results (from the area of the reported sensation), and recall is the area of correct positive results divided by the area of all relevant samples (from the ROI of the presented pseudo-biosignal image). In this study, precision was the fraction of the intersection area of the reported sensation area and recall was the fraction of the intersection area of the ROI of PBF changes for a given pseudo-biosignal image. The F1 score was calculated for each participant and pseudobiosignal type (i.e., circular, rectangular elongated, and diagonally elongated shapes) (**Figure 1B**).

presented pseudo-biosignal images and de qi response images for the rectangular elongated shape. The group-level analysis consisted of a random effects analysis using a pixel-wise univariate t-test of individual sensation maps for each session (FDR-corrected p < 0.05). The color coding represents the Z score.

Statistical analyses of the F1 score and PBF data were performed using the R software package<sup>3</sup> . The F1 score for each pseudo-biosignal type was compared to the F1 score extracted from the control session with the same ROI. Paired t-test was used to identify significant differences. We conducted repeated measures analysis of variance (ANOVA) to compare the intensities of the change in PBF under each condition over time to determine the effect of time and biosignal type (i.e., the four conditions).

#### RESULTS

#### Baseline Characteristics

This study was carried out in 24 right-handed participants (aged 18–37 years; mean = 23.4, standard deviation = 4.0; 13 females). The AES score was 12.6 ± 0.6 and the AFS score was 29.0 ± 2.2.

# Effect of Expectation on Tactile Stimulation Sensory Experience

We demonstrated individual spatial patterns of somatic sensation following tactile stimulation in each condition (**Figure 2**). After group level analysis, we found that the spatial configurations of the presented pseudo-biosignal images and the de qi response images were similar in the rectangular elongated shape condition, suggesting that presentation with different pseudo-biosignal shapes could influence the spatial configuration of the somatic sensation to the same tactile stimulation (**Figure 3**).

F1 score was used to evaluate the similarity between the presented pseudo-biosignal images and reported areas of somatic sensation. Based on the F1 score, the association between the rectangular elongated pseudo-biosignal image and resulting de qi response image exhibited a significant similarity compared to control (0.445 ± 0.039 vs. 0.388 ± 0.043, t = 2.168, p < 0.05). By contrast, the circular and diagonally elongated pseudo-biosignal images and de qi response images did not exhibit any significant similarity (**Figure 4**). These findings suggest that the rectangular elongated pseudo-biosignal image, but not other conditions, can enhance similarity between the pseudo-biosignal images and perceived somatosensation.

### Peripheral Blood Flow Response to Tactile Stimulation

All tactile stimulations produced similar levels of enhanced PBF around the acupoint regardless of the pseudo-biosignal shape, and participants exhibited a 40% increase in PBF within 30 s under all conditions (**Figure 5**). Repeated measures ANOVA revealed a significant effect of time [F(3,13) = 14.877, p < 0.001]; however, the interactive effect of biosignal type × time [F(3,39) = 0.386, p > 1.000] and biosignal type [F(3,3) = 0.193, p > 0.901] showed no significant effects.

FIGURE 4 | Similarity between the presented biosignal images and de qi response images. The F1 score indicates the similarity between the presented pseudo-biosignal image and reported somatic sensation area. Presentation with the rectangular elongated-shaped pseudo-biosignal resulted in a greater similarity between the presented biosignal image and de qi response pattern compared to the control. The red bars represent the similarity with the pseudo-biosignal images and the blue bars represent the similarity without presentation of pseudo-biosignal images (control). A paired t-test was conducted to identify significant differences between the pseudo-biosignal image and control conditions. <sup>∗</sup> represents p < 0.05.

## DISCUSSION

The purpose of this study was to determine whether the spatial configurations of de qi sensations could be influenced by manipulating the shape of pseudo-biosignal images presented to participants. The results revealed similarities between the spatial configurations of the presented pseudo-biosignal images and somatic sensation response patterns. In particular, presentation with the rectangular elongated pseudo-biosignal resulted in a similar somatic sensation spatial configuration. These findings suggest that biosignal information of psychophysiological responses to acupuncture can change somatic sensation spatial configurations. Overall, this study provides evidence that information from prior experiences and expectations can influence the interpretation of sensory inputs.

Somatic sensations of acupuncture stimulations can be established not only by afferent signals, but also by higher cognitive process, such as expectations. We previously found that expectations of acupuncture stimulations could trigger considerable de qi sensations and brain activations in the salience network, even in the absence of actual afferent signals (Jung et al., 2015, 2018). The main finding of the present study is that the spatial configurations of sensory experiences can be altered by biosignal information. To test this, we presented participants with different pseudo-biosignal shapes mimicking Laser Doppler perfusion images to manipulate their expectations of the physiological changes to acupuncture stimulation. Even though we did not explicitly explain the associations between the pseudo-biosignal shapes and de qi sensation patterns,

different expectations of physiological changes to acupuncture stimulation successfully induced different somatic sensation spatial configurations. Recent findings in cognitive neuroscience suggest that the brain can actively make inferences based on prior experiences and expectations (Buchel et al., 2014). In other words, information from prior experiences can be used to generate expectations about future perception and interpret sensory inputs (Wiech, 2016). Therefore, we assume that the altered sensory experiences in this experiment might have been derived from the implicit expectation of the association between the physiological response to acupuncture stimulations and the experience of somatic sensations.

When somatic sensations are induced by an acupuncture needle, de qi sensations and brain activations to acupuncture stimulation can vary among sessions and participants (Kong et al., 2007; Napadow et al., 2009). Therefore, to control the intensity of the tactile stimulation and induce the same intensity of somatic sensations among tests, we applied a von Frey filament to the TE5 acupoint in the left arm. This method can control for both the tactile stimulation of the cutaneous somatosensory receptor at the acupuncture point and the cognitive processing of participants expecting an acupuncture stimulation (Napadow et al., 2013; Chae et al., 2015). The stimulated sites on the arm were hidden from sight and all participants believed that they received acupuncture stimulations. Moreover, this experiment showed that all tactile stimulations produced, similarly, enhanced PBF around the acupoint regardless of the presented pseudobiosignal shape, supporting that alterations of spatial patterns to tactile stimulation in rectangular elongated shape condition were not induced by differences in tactile stimulation evoked physiological responses.

The spatial configurations of the presented pseudo-biosignal images and de qi response images were similar, especially in the case of the rectangular elongated shape. Meanwhile, the circular pseudo-biosignal shape was created based on preliminary data of the spatial configurations of de qi sensations to tactile stimulations. When tactile stimulations were applied to the left arm, the de qi sensation elicited circular-shaped spatial configurations around the stimulation site. As expected, there were no differences in the spatial configurations of the de qi sensations between the session with the circular pseudo-biosignal image and the control session without pseudo-biosignal images. Meanwhile, the participant somatic sensation pattern responses to tactile stimulation were highly similar to the presented rectangular elongated (but not diagonally elongated) pseudobiosignal pattern. In this experiment, the participants expected that they would receive acupuncture treatment at various angles. Interestingly, propagated sensations induced by acupuncture have been reported along perpendicular and horizontal lines on the body (Beissner and Marzolff, 2012; Jung et al., 2016). Since diagonal spatial patterns of de qi sensations are unusual, the participants may have had weaker expectations of diagonally elongated-shaped response perception patterns.

Perceived sensation to acupuncture stimuli, like other pain perceptions, can be influenced by prior knowledge or expectations (Buchel et al., 2014; Jung et al., 2017b; Ongaro and Kaptchuk, 2019). In this study, we manipulated participants' expectation by using pseudo-biosignal images mimicking the changes in the blood flow during Laser Doppler perfusion imaging. We did not tell them any association between the biosignal from blood flow change and perceive sensations. However, the spatial patterns of de qi sensation in response to tactile stimulation were influenced by the information, especially in more plausible response condition. These findings suggest that the implicit expectations from the physiological changes from their own body can have influence on the perception of the tactile stimulation. From this study, the prospective studies include manipulating expectation by alterations of biosignals from the body can be applied to the modulations of other somatosensation areas such as pain control.

In summary, this study reveals that the spatial configurations of de qi sensations in response to tactile stimulation can be influenced with visual biosignal information. This suggests that information on physiological responses to acupuncture stimulations can change participants' expectations of the perception of somatic sensation and interpretation of the stimulations. In future studies, it will be necessary to further clarify the characteristics of the cognitive factors in the perception of acupuncture stimulations.

#### AUTHOR CONTRIBUTIONS

fnins-13-00074 February 9, 2019 Time: 17:9 # 8

H-SS, Y-SL, and YC conceived and designed the experiments. H-SS and S-WY performed the trial. H-SS, W-MJ, and YC

#### REFERENCES


analyzed the data. H-SS, W-MJ, and YC discussed the data. H-SS and YC wrote the first draft of the paper. H-SS, W-MJ, Y-SL, S-WY, and YC revised the paper and approved the final version.

#### FUNDING

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (No. 2018R1D1A1B07042313).

electro, and sham acupuncture treatment on experimentally induced thermal pain. J. Pain 6, 55–64. doi: 10.1016/j.jpain.2004.10.005


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling Editor is currently editing a Research Topic with one of the authors YC, and confirms the absence of any other collaboration.

Copyright © 2019 Song, Jung, Lee, Yoo and Chae. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Paired Associative Electroacupuncture and Transcranial Magnetic Stimulation in Humans

Yi Huang<sup>1</sup> , Jui-Cheng Chen2,3 , Chun-Ming Chen<sup>4</sup> , Chon-Haw Tsai 2,3 and Ming-Kuei Lu1,2 \*

<sup>1</sup>Graduate Institute of Biomedical Sciences, Medical College, China Medical University, Taichung, Taiwan, <sup>2</sup>Neuroscience Laboratory, Department of Neurology, China Medical University Hospital, Taichung, Taiwan, <sup>3</sup>School of Medicine, Medical College, China Medical University, Taichung, Taiwan, <sup>4</sup>Department of Radiology, China Medical University Hospital, Taichung, Taiwan

Pairing transcutaneous electric nerve stimulation (TENS) and transcranial magnetic stimulation (TMS) with specific stimulus-intervals induces associative motor plasticity at the primary motor cortex (M1). Electroacupuncture (EA) is an established medical technique in the eastern countries. This study investigates whether EA paired with TMS induces distinct M1 motor plasticity. Fifteen healthy, right-handed subjects (aged 23.6 ± 2.0 years, eight women) were studied. Two-hundred and twenty-five pairs of TMS of the left M1 preceded by right EA at acupoint "Neiguan" [Pericardium 6 (PC6), located 2 decimeters proximal from the wrist wrinkle] were respectively applied with the interstimulus interval (ISI) of individual somatosensory evoked potential (SSEP) N20 latency plus 2 ms (N20+2) and minus 5 ms (N20-5) with at least 1-week interval. The paired stimulation was delivered at a rate of 0.25 Hz. Sham TMS with a sham coil was adopted to examine the low-frequency EA influence on M1 in eleven subjects. M1 excitability was assessed by motor-evoked potential (MEP) recruitment curve with five TMS intensity levels, short-interval intracortical inhibition (SICI), intracortical facilitation (ICF) and cerebellar inhibition (CBI) at the abductor pollicis brevis (APB) muscle of the right hand before and after the EA-M1 paired associative stimulation (PAS). In addition, median nerve SSEPs and H-reflex were respectively measured to monitor somatosensory and spinal excitability. The MEP showed significantly facilitated after the sham EA-M1 PAS while tested with 80% of the TMS intensity producing on average 1 mV amplitude (i.e., MEP1 mV) in the resting APB muscle. It was also facilitated while tested with 90% MEP1 mV irrespective of the stimulation conditions. The SSEP showed a higher amplitude from the real EA-M1 PAS compared to that from the sham EA-M1 PAS. No significant change was found on SICI, ICF, CBI and H-reflex. Findings suggest that repetitive low frequency EA paired with real TMS did not induce spike-timing dependent motor plasticity but EA paired with sham TMS induced specific M1 excitability change. Complex sensory afferents with dispersed time locked to the sensorimotor cortical area could hamper instead of enhancing the induction of the spike-timing dependent plasticity (STDP) in M1.

Keywords: electroacupuncture, motor cortex, motor evoked potential, paired associative stimulation, transcranial magnetic stimulation

#### Edited by:

Florian Beissner, Hannover Medical School, Germany

#### Reviewed by:

Antonio Ivano Triggiani, University of Foggia, Italy Matt J. N. Brown, California State University, Sacramento, United States

#### \*Correspondence:

Ming-Kuei Lu d4297@mail.cmuh.org.tw

Received: 05 November 2018 Accepted: 29 January 2019 Published: 12 February 2019

#### Citation:

Huang Y, Chen J-C, Chen C-M, Tsai C-H and Lu M-K (2019) Paired Associative Electroacupuncture and Transcranial Magnetic Stimulation in Humans. Front. Hum. Neurosci. 13:49. doi: 10.3389/fnhum.2019.00049

#### INTRODUCTION

Primary motor cortex (M1) receives multi-direction gated sensory information and executes the final motor command in humans (Cheng et al., 2017; Lei et al., 2018). It plays a key role in motor learning and motor performance. Nowadays there are several non-invasive techniques capable of inducing M1 excitability change, such as fixed-frequency repetitive transcranial magnetic stimulation (rTMS), paired associative stimulation (PAS; Stefan et al., 2000), theta burst stimulation (Huang et al., 2005) and so on (for a review see Quartarone et al., 2006; Huang et al., 2017). The modification of abnormal M1 excitability by these non-invasive brain stimulation techniques has shown possible clinical benefits on neuropathic pain and Parkinson's disease (Lefaucheur et al., 2012; Brys et al., 2016; Goodwill et al., 2017).

PAS is one of the well-known non-invasive brain stimulation techniques with which to investigate Hebbian principles of neural plasticity in humans (Stefan et al., 2000). The most traditional form of PAS consists of repeated pairing of a single electric stimulus at the peripheral median nerve and a TMS pulse on the contralateral M1 with a specific interstimulus interval (ISI) between these two stimuli. It induces aftereffects representing the associative long-term potentiation (LTP)- and depression (LTD)-like phenomenon that bears resemblance to spike-timing dependent plasticity (STDP) as it has been elaborated in animal models (Carson and Kennedy, 2013). An ISI of 25 ms or individual N20 latency plus 2 ms (N20+2) results in arrival of the afferent sensory signal elicited by the peripheral median nerve stimulation before or almost at the same time in M1 when the TMS of the M1 generates actions potentials in excitatory interneurons and corticospinal neurons. The order of the events in M1 is reversed if an ISI of 10 ms or individual N20 latency minus 5 ms (N20-5) is applied (Müller-Dahlhaus et al., 2010). Nevertheless, the PAS effect can be affected by many factors and identifying these factors is a challenge in the current clinical practice (Murase et al., 2015; Huang et al., 2017). Among the factors relevant to the PAS effects, attention and the afferent somatosensory stimulation play an instrumental role in determining the magnitude of the PAS effects (Carson and Kennedy, 2013). In the original PAS protocol, the afferent somatosensory input is mediated through transcutaneous electric nerve stimulation (TENS) at wrist median nerve. The electric stimulation is prone to be adapted for most of the subjects so an additional device for maintaining the subject's attention during the experiment is usually required (Stefan et al., 2004; Lu et al., 2009).

Acupuncture is an ancient medical technique frequently applied for pain control in the eastern countries. Evidences from functional brain imaging have suggested that acupuncture needle stimulation actually modulates specific neural networks in the brain (Hui et al., 2000; Fang et al., 2009), and different acupuncture modalities recruit different brain networks (Jiang et al., 2013). The invention of electric power allows acupuncture delivering a repetitive and constant stimulation at the specific stimulation site which is called ''acupoint.'' Stimulation at different acupoints may have distinct associations with neurocircuits. For example, acupuncture at acupoint ''Shenmen; HT7,'' but not ''Neiguan; PC6,'' improves the ventral tegmental area (VTA)-nucleus accumbens dopaminergic function via inhibition of brain-derived neurotrophic factor (BDNF) expression in the VTA (Zhao et al., 2015). Intriguingly, the acupoint ''Neiguan; PC6'' is approximately located in the same site where median nerve TENS is adopted for PAS. PC6 has been documented with a capacity to change brain activation patterns relevant to attention and improve cognition for stroke patients (Chou et al., 2009; Jung et al., 2015). A study compared the median nerve somatosensory evoked potentials (SSEPs) between TENS, electroacupuncture (EA) and sham stimulation of the specific acupoints (i.e., ST36 and ST37) in the leg (Kang et al., 2015). The results showed that EA, but not TENS nor sham stimulation, alters the mean amplitude of N20 and N30 during and post the stimulation periods (Kang et al., 2015). The findings also suggest a possibility that EA may have different influences on the somatosensory cortex from TENS stimulation. In a recent study, recruitment of additional corticospinal pathway has been achieved by the state-dependent PAS protocol in which sensorimotor event-related desynchronization (ERD) of the βband was used to trigger peripheral stimulation (Kraus et al., 2018). Gathering evidences raise an issue: whether the peripheral EA stimulation carries a distinct impact on the PAS effect? If the answer is yes, in which level the influence may occur? Since the central mechanism of EA remains not clear, we wondered that EA paired with TMS has a consistent STDP-like effect similar to the traditional PAS. Complex sensory afferents might disrupt instead of enhancing STDP in M1. This study aims to clarify this issue.

#### MATERIALS AND METHODS

#### Subjects

In total 15 right-handed (Oldfield, 1971) healthy subjects (23.6 ± 2.0 years, eight women) were recruited in this study. They all received both real and sham stimulation conditions including two different stimulation protocols in each [see ''Paired Associative Electroacupuncture and TMS (PAET)'' section]. This study was carried out in accordance with the recommendations of the local ethics committee of the China Medical University Hospital with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the local ethics committee of the China Medical University Hospital (CMUH104-REC2-164). They all received a brain MRI examination to exclude structure lesion.

#### Procedures

#### Measurement of Motor Cortical Excitability

TMS was delivered through a focal figure-of-eight stimulating coil (inner diameter of each wing, 70 mm) connected via a BiStim module to two Magstim 200 magnetic stimulators (Magstim Co., Carmarthenshire, Wales, UK). The optimal coil position (''hot spot''; M1HAND) is determined as the site where TMS at a slightly supra-threshold intensity produced consistently the largest motor-evoked potentials (MEPs) in the right abductor pollicis brevis (APB). The intensity of TMS adjusted to produce MEPs of on average 1 mV in peak-to-peak amplitude in the resting APB is defined as the MEP1 mV. The individual resting motor threshold (RMT) and active motor threshold (AMT) was determined over the left M1HAND. AMT was additionally determined over the inion (inion AMT) prior to the baseline recording. The detailed procedure for determining RMT and AMT has been described elsewhere (Lu et al., 2012).

MEP IO curve is a quantitative measurement for corticospinal excitability. It was measured by stimulation at five intensity levels ranging from two levels below and two levels above MEP1 mv with a step of 20% MEP1 mv (i.e., five levels of stimulus intensity). Due to the constraint of the whole experiment time, eight stimuli were recorded at each intensity level. The ISI was determined as 7 s with a 25% variance to limit the anticipation effect.

#### Short-Interval Intracortical Inhibition (SICI)/Intracortical Facilitation (ICF)

Short-interval intracortical inhibition (SICI) and intracortical facilitation (ICF) were studied using an established paired-pulse TMS protocol (Kujirai et al., 1993; Ziemann et al., 1996). In brief, the two magnetic stimuli were given through the same figure-of-eight stimulating coil over the left M1HAND and the effect of the sub-threshold conditioning stimulus on the test MEP elicited by the subsequent supra-threshold test stimulus (TS) was investigated. SICI was assessed at an ISI of 2.0 ms because at this interval SICI is not contaminated by SICF (Peurala et al., 2008). At the baseline recording, the condition stimulation (CS) intensity was adjusted to produce approximately 50% inhibition in order to provide highest sensitivity for detection of changes in SICI after PAS. The CS intensities usually ranged from 70 to 90% AMT in different individuals (Lu et al., 2012). This CS intensity was kept constant throughout the experiment. ICF was assessed at an ISI of 10 ms (Ziemann et al., 1996). The CS intensities usually ranged from 75 to 95% AMT in different individuals to produce consistent test MEP facilitation (Ziemann et al., 1996; Lu et al., 2012).

#### Cerebello-Motor Cortical Inhibition (CBI)

Cerebellar inhibition (CBI) was measured with a double-cone coil positioned at the midpoint of the inion and the right incisura mastoidea for CS (Ugawa et al., 1995). 95% AMTinion was adopted for the CS intensity to avoid any corticospinal excitability (Ugawa et al., 1995; Lu et al., 2012). The TS was delivered at left M1 and kept an intensity to elicit an average MEP of ∼0.7 mV while delivered alone. The ISI between CS and TS was 6 ms.

#### Median Nerve Somatosensory-Evoked Potentials (SSEPs)

The early component of median nerve SSEP (N20-P25) is an index of the somatosensory cortex (S1) excitability. It was recorded while the subjects voluntarily relaxed with eyes closed (Krivanekova et al., 2011). The active electroencephalography electrode was placed at C3', 2 cm posterior to C3 according to the International 10–20 system, corresponding to the putative site of the left S1. The reference electrode was placed on the frontal midline (Fz). The right median nerve was stimulated through a bipolar electrode (cathode proximal) with a constant current square pulse of 0.2 ms duration at rate of 3 Hz (Digitimer DS7A, Digitimer Ltd., UK). Stimulus intensity is adjusted to 110% of twitching threshold in the right thenar muscle. Six-hundred trials were recorded and averaged for offline analysis.

#### H-Reflex

The H-reflex is an electrical analog of the spinal stretch reflex and an index of spinal excitability. Subjects were positioned in sitting with the elbow at 45◦ flexion. A stimulus location in the medial bicipital groove was determined where a maximal H-reflex without an M-wave was evoked on the flexor carpi radialis muscle. Stimulation intensity was sequentially increased with a step of 1 mA from a sub-H-reflex threshold intensity to the level when H-reflex amplitude began to decline. The intensity which provokes a maximal H-wave peak-to-peak amplitude (HMax) was determined and used for the following measurement.

#### Acupuncture

Acupoint ''Neiguan'' (Pericardium 6; PC6) is located 2 decimeters (i.e., 5.08 cm) proximal from the wrist wrinkle (Chou et al., 2009; **Figure 1**). This location is approximately

FIGURE 1 | Localization of the acupoint "Neiguan" (Pericardium 6, PC6; <sup>∗</sup> ).

the same site where median nerve SSEP is stimulated. Prior to the EA, an acupuncture needle (sterile stainless needle, 0.3 mm diameter, 50 mm length, Yuguang Corporation, Taiwan) was inserted into the right PC6 with a depth of 10 mm. The de-chi sensation of the acupuncture is somewhat subjective and difficult to be quantitatively defined. Therefore, it was not essential for subjects to report de-chi sensation in this study.

#### Paired Associative Electroacupuncture and TMS (PAET)

The conventional PAS protocol consists of 225 pairs of a cutaneous electric stimulation of the median nerve at the wrist followed by a single TMS at the contralateral M1HAND (Stefan et al., 2000). The cutaneous nerve stimulation was replaced by the EA at PC6 in the current PAET paradigm. The needle for EA was connected to the stimulator (Digitimer DS7A, Digitimer Ltd., UK) which was triggered by a computer-based interface (Spike2 for Windows, Version 3.05, CED, UK). The stimulus intensity was adjusted to evoke a same level of the right-hand muscle twitching observed during the SSEP recording. There were two intervals between the EA and the TMS, individual N20 latency plus 2 ms (N20+2) and minus 5 ms (N20–5). Based on the STDP principle and the previous evidences, the N20+<sup>2</sup> interval produces LTP-like and the N20–5 interval produces LTD-like plasticity (Ziemann et al., 2004; Müller-Dahlhaus et al., 2010; Lu et al., 2016). The intensity of TMS was adjusted to produce MEPs of on average 1 mV in peak-to-peak amplitude in the resting APB. To carefully examine the effect of the repetitive low-frequency EA stimulation, we designed sham TMS by using a sham TMS coil (Magstim Co., Carmarthenshire, Wales, UK). In total 225 pairs were delivered at a rate of 0.25 Hz (i.e., duration of PAET, 15 min; **Figure 2**).

#### Statistical Analysis

Repeated measures analyses of variance (rmANOVA) was applied to test the effects of PAET on the five intensity steps of the MEP, SICI, ICF, CBI, SSEP and H-reflex. The within-subject effects were TIME (Pre-PAET vs. Post-PAET), PROTOCOL (PAETN20+2 vs. PAETN20-5) and CONDITION (Real vs. Sham). Conditional on a significant F value, post hoc comparisons were performed using paired-sample t-tests with Bonferroni's correction. Violation of sphericity was checked with Mauchly's test and degrees of freedom were adjusted whenever Mauchly's W < 0.05 using the Greenhouse-Geisser correction (SPSS 22.0). Data are reported as means ± SD if not stated otherwise.

### RESULTS

All of the subjects were cooperative throughout the experimental procedures. None of them reported any noticeable adverse effects during or after the study.

**45**

The mean RMT, AMT and inion AMT of the 15 participants were 54 ± 7.9%, 45 ± 7.0% and 39 ± 4.8%, respectively. The mean MEP1 mv was 65 ± 12.0%. The intensities applied for measuring SICI, ICF and CBI were listed in **Table 1**. We analyzed the data of the 15 subjects for three main effects (i.e., CONDITION, PROTOCOL and TIME). RmANOVA of the MEP amplitude revealed a significant effect of TIME (F(1,14) = 8.8, P = 0.01) and a significant CONDITION × TIME interaction at TMS intensity of 80% MEP1 mV (F(1,14) = 5.63, P = 0.03), and a significant effect of TIME at TMS intensity of 90% MEP1 mV (F(1,14) = 12.73, P = 0.003; **Table 2**). RmANOVA of the SICI did not show any significant effect (all P > 0.08). A significant PROTOCOL × TIME interaction was found for the analysis of ICF (F(1,14) = 7.46, P = 0.02). A significant main effect of CONDITION was found on SSEP (F(1,14) = 8.97, P = 0.01). RmANOVA of the HMax did not show any significant effect (all P > 0.3). The statistical power reaches 0.93 with the effect size of 0.4 for the three-way rmANOVA.

Post hoc comparisons showed a significant MEP amplitude increase after Sham condition at 80% MEP1 mV (Pre/Post: 0.36 ± 0.16/0.47 ± 0.25 mV, P < 0.01 by paired t-test; **Figure 3**). At 90% MEP1 mV, the MEP amplitude showed a significant increase after the PAET intervention irrespective of CONDITION and PROTOCOL (Pre/Post: 0.58 ± 0.26/0.77 ± 0.49 mV, P < 0.01 by paired t-test; **Figure 3**). There was no significant difference from the post hoc comparisons on ICF (all P > 0.05 by paired t-test; **Figure 4**). The post hoc comparison of SSEP revealed a significant difference between the real condition and the sham condition (Real/Sham: 1.5 ± 0.8/1.1 ± 0.6 µV, P < 0.01 by paired t-test; **Figure 4**).

#### DISCUSSION

#### PAET Effect on M1 Excitability

The current data support the notion that the PAET intervention may have complex influences on the M1 excitability. While M1 excitability was investigated with a relatively low TMS intensity such as 80% or 90% MEP1 mV, the PAET, particularly the sham PAET, significantly facilitated the MEP amplitude. Following the increase of the TMS test intensity, the aftereffect of PAET was not detectable with the MEP recording.

It has been known that acupuncture has clinical effects on analgesia (Xiang et al., 2017; Pang et al., 2018). The rTMS protocols which facilitate M1 excitability also revealed a benefit response on neuropathic pain (Hosomi et al., 2013; Boyer et al., 2014). Whether the analgesic effect of acupuncture is mediated through a similar mechanism with rTMS remains not clear and

TABLE 1 | The transcranial magnetic stimulation (TMS) intensity<sup>∗</sup> adopted for measuring short-interval intracortical inhibition (SICI), intracortical facilitation (ICF) and cerebello-motor cortical inhibition (CBI).


<sup>∗</sup>Presented as percentage of maximal stimulator output.


ICF, intracortical facilitation; MEP, motor evoked potential; SICI, short-interval

 intracortical inhibition; SSEP, somatosensory

 evoked potential.

FIGURE 3 | Post hoc comparisons of the motor evoked potential (MEP) evoked with different TMS intensities for the 15 subjects. MEP1 mV means the TMS intensity inducing on average 1 mV MEP amplitude in the resting abductor pollicis brevis (APB) muscle. The MEP amplitude showed a significant facilitation after the sham PAET intervention with 80% MEP1 mV and the PAET intervention with 90% MEP1 mV. Data are shown by means ± standard errors. ∗∗P < 0.01 by paired t-test with Bonferroni's correction.

needs further investigation. Our findings on the sham PAET provided evidence showing that EA may facilitate M1 excitability and the change of the M1 excitability is only detectable with a low to moderate TMS intensity. In addition to EA, the trains of 1 Hz electrical stimulation and 25 Hz repetitive magnetic stimulation on the peripheral nerve also have shown ability to modulate sensorimotor excitability (Kaelin-Lang et al., 2002; Gallasch et al., 2015). Since the low intensity TMS activates interneurons instead of corticospinal neurons (Di Lazzaro and Rothwell, 2014), it is likely that the PAET-induced M1 plasticity is mediated through the interneurons. SICI and ICF are two common parameters representing the function of the interneurons (Chen et al., 1998).

The reason why the sham PAET induced a higher MEP amplitude compared to the real PAET can be explained by the frequency of the repetitive TMS. In this study the real PAET consists of 225 TMS pulses with 0.25 Hz. It has been known that the supra-threshold rTMS of low frequency (i.e., 1 Hz or less) at M1 suppresses corticospinal excitability and reduced MEPs (Chen et al., 1997). It might be achieved by suppressing the amplitude of later I-waves, which have been proposed based on the epidural observation (Di Lazzaro et al., 2008). The reduction of the corticospinal neuronal excitability was supposed to be robust enough to block or erase the EA-induced interneuron plasticity mentioned above.

The current findings suggest that there is no significant role on the protocol difference. That is, we did not find any M1 excitability change following the spike-timing dependent pattern. One of the possible explanations is that EA carries complex sensory modalities including somatosensory, pain and variable attention. These different sensory afferents are actually not homogeneously time-locked to the cortical sensorimotor area. Despite the EA location (i.e., PC6) is very close to that adopted for the traditional PAS, the subtle difference of the location between these two methods could result in different findings. Therefore, it would be difficult to detect typical M1 motor plasticity similar to that induced by the traditional PAS protocols. Another possibility for the absence of a significant spike-timing dependent pattern is probably due to the intersubjects variability, which has been recognized as a notable issue for the non-invasive brain stimulation protocols including PAS (López-Alonso et al., 2014; Guerra et al., 2017).

#### SICI/ICF

The current findings on SICI are agree with the previous report which revealed SICI was not consistently affected by the traditional PAS intervention (Russmann et al., 2009). SICI has been found mediated by the GABA<sup>A</sup> receptors (Di Lazzaro et al., 2006; Florian et al., 2008). In a recent animal study, EA may increase GABA<sup>A</sup> receptors but the influence is only restricted in the spinal cord (Jiang et al., 2018).

The PAS protocol facilitating M1 excitability increases ICF, which suggests that the modification of M1 excitability is contributed from the intracortical excitatory circuits (Pyndt and Ridding, 2004). In the post hoc analysis we could not find any significant change following the PAET (**Figure 4**). A further study with more conditioning and test TMS intensities would be helpful to clarify the role of ICF in the M1 excitability change induced by the PAET.

#### SSEP/H-Reflex

Low frequency (0.05 Hz) EA intervention for 4 months can induce fluctuating cortical plasticity in the somatosensory area, pain-related areas and limbic/paralimbic areas in rats (Wu et al., 2018). In the previous report the amplitude of the median SSEP was found significantly increased by 2 Hz EA at the leg acupoint for 15 min (Kang et al., 2015). However, the SSEP finding in this study did not support the notion that the EA per se induce a significant plasticity in the somatosensory area. The main effect of CONDITION instead of TIME from the current rmANOVA analysis suggests that EA paired with real or sham M1 TMS may induce different excitability in the somatosensory area (**Table 2**, **Figure 4**). The finding is consistent with the previous report showing that the traditional PAS consisting of paired median nerve stimulation and TMS at M1 changes SSEP (Murakami et al., 2008). Since the SSEP amplitude may depend on the adopted method (Macerollo et al., 2018), a more sensitive method such as using a non-cephalic reference would be better to measure the excitability change of the somatosensory area in this regard.

H-reflex represents the spinal excitability. Fifty hertz EA can modulate H-reflex response in the experimental rat (Escobar-Corona et al., 2017). The current low frequency (i.e., 0.25 Hz) EA did not significantly alter H-reflex, suggesting that the observed MEP changes are induced in the cortical level, probably the M1 instead of the spinal cord.

There are limitations in this study. As a proof-of-concept study, we merely recruited a limited number of subjects. This inevitably constrained our findings and interpretations. In addition, there was no control condition (e.g., sham or TENS) for

#### REFERENCES


the EA stimulation. This also diminishes the significance of the current findings. Nevertheless, the method adopted in this study could shed light on how to combine EA and TMS approaches in the future.

#### CONCLUSION

Repetitive EA paired with sham TMS at M1 induces specific motor cortical plasticity which could be only detectable with moderate TMS intensities. EA paired with real low-frequency (i.e., 0.25 Hz) rTMS may interrupt instead of enhancing this kind of plasticity. That is, there is no spike-timing dependent character for this plasticity. Complex sensory afferents with dispersed time locked to the sensorimotor cortical area may hamper the induction of the STDP-like plasticity in M1. GABAergic, glutamatergic, cerebellar afferent and spinal excitability respectively examined by SICI, ICF, CBI and H-reflex were not significantly affected.

#### DATA AVAILABILITY

All datasets generated for this study are included in the manuscript.

### AUTHOR CONTRIBUTIONS

YH: subject recruitment, acquisition of data, writing first draft of the manuscript. J-CC: experimental design, critical review of the manuscript, revision of the first draft. C-MC: experimental execution, acquisition of data, review of the manuscript. C-HT: experimental design, critical review of the manuscript, comments on the manuscript. M-KL: study concept and experimental design, data analysis and interpretation, critical revision of the manuscript.

#### FUNDING

This study is supported in part by grants from the Ministry of Science and Technology (MOST105-2314-B-039-002-MY2 and MOST105-2632-B-039-003) and the Brain Disease Research Center in China Medical University Hospital (DMR-107-190, CRS-106-060), Taichung, Taiwan.

#### ACKNOWLEDGMENTS

We thank Miss Nai-An Yang for her assistance on data collection.

disease: a randomized trial. Neurology 87, 1907–1915. doi: 10.1212/wnl. 0000000000003279


Evid. Based Complement. Alternat. Med. 2017:3837194. doi: 10.1155/2017/38 37194


**Conflict of Interest Statement**: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Huang, Chen, Chen, Tsai and Lu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Peripheral Sensory Nerve Tissue but Not Connective Tissue Is Involved in the Action of Acupuncture

Suchan Chang1†, O. Sang Kwon2†, Se Kyun Bang<sup>1</sup> , Do-Hee Kim<sup>1</sup> , Min Won Baek <sup>1</sup> , Yeonhee Ryu<sup>2</sup> , Jong Han Bae<sup>3</sup> , Yu Fan<sup>1</sup> , Soo Min Lee<sup>1</sup> , Hyung Kyu Kim<sup>1</sup> , Bong Hyo Lee<sup>1</sup> , Chae Ha Yang<sup>1</sup> and Hee Young Kim1,3 \*

*<sup>1</sup> College of Korean Medicine, Daegu Haany University, Daegu, South Korea, <sup>2</sup> Clinical Medicine Division, Korea Institute of Oriental Medicine, Daejeon, South Korea, <sup>3</sup> Department of Physics, Yeungnam University, Gyeongsan, South Korea*

#### Edited by:

*Florian Beissner, Hannover Medical School, Germany*

#### Reviewed by:

*Helene M. Langevin, Brigham and Women's Hospital, Harvard Medical School, United States Stephanie Tjen-A-Looi, University of California, Irvine, United States*

> \*Correspondence: *Hee Young Kim hykim@dhu.ac.kr*

*†These authors have contributed equally to this work*

#### Specialty section:

*This article was submitted to Perception Science, a section of the journal Frontiers in Neuroscience*

Received: *03 November 2018* Accepted: *29 January 2019* Published: *20 February 2019*

#### Citation:

*Chang S, Kwon OS, Bang SK, Kim D-H, Baek MW, Ryu Y, Bae JH, Fan Y, Lee SM, Kim HK, Lee BH, Yang CH and Kim HY (2019) Peripheral Sensory Nerve Tissue but Not Connective Tissue Is Involved in the Action of Acupuncture. Front. Neurosci. 13:110. doi: 10.3389/fnins.2019.00110* Acupuncture has been used to treat a variety of diseases and symptoms for more than 2,500 years. While a number of studies have shown that nerves are responsible for initiating the effects of acupuncture, several lines of study have emphasized the role of connective tissue in the initiation of acupuncture signals. To determine whether nerves or connective tissue mediate the action of acupuncture, we constructed a robotic acupuncture needle twister that mimicked the twisting of the needle by an acupuncturist, and we examined the role of nerves and connective tissues in the generation of acupuncture effects in rat cocaine-induced locomotion, stress-induced hypertension, and mustard oil-induced visceral pain models. Robotic or manual twisting of acupuncture needles effectively suppressed cocaine-induced hyperactivity, elevated systemic blood pressure or mustard oil-induced visceral pain in rats. These acupuncture effects were completely abolished by injecting bupivacaine, a local anesthetic, into acupoints. However, disruption of connective tissue by injecting type I collagenase into acupoints did not affect these acupuncture effects. Our findings suggest that nerve tissue, but not connective tissue, is responsible for generating the effects of acupuncture.

Keywords: acupuncture, peripheral sensory nerve, connective tissue, robotic acupuncture needle twister, collagenase

#### INTRODUCTION

Acupuncture has been used to treat various diseases in Asian countries over the last 2,500 years and has been widely practiced in many countries. In 1997, the United States' National Institutes of Health (NIH) reported acupuncture's safety and efficacy for treating a wide range of conditions, including drug abuse, stroke rehabilitation, and headache (Nih Consensus Conference, 1998). In a bibliometric review of the publications on acupuncture research in PubMed over the last 20 years, acupuncture research has increased markedly in the past two decades, with twice the growth rate of overall biomedical research. While pain (approximately 38% of publications) is consistently the most common topic of acupuncture research, the publications of randomized clinical trials have been increasing in numbers at a high rate (Ma et al., 2016). Although these studies have shown acupuncture's effectiveness, the mechanisms by which the acupuncture signals are initiated and conveyed remain contradictory and inconclusive.

Acupuncture is a therapeutic intervention for improving the efficiency of homeostatic mechanisms of the body by stimulating acupoints. There are two leading models of the initiation of acupuncture signals: the neurologic model and the connective tissue model. The neurologic model, which is by far the more widely accepted and studied, posits mediation by the nervous system, with acupuncture signals being initiated by the activation of sensory nerve endings and specific nerve fibers and transmitted to the brain through nervous pathways, resulting in a variety of physiological effects. In support of this view, it was reported that acupuncture produces an antinociceptive effect by releasing adenosine at peripheral nerve terminals in rodents (Goldman et al., 2010). Acupuncture stimulation at acupoints in the wrist, PC5-6, increased Aδand C-fiber activity to evoke cardiovascular effects (Zhou et al., 2005). Our previous studies showed that acupuncture at HT7 attenuates drug-induced or drug-seeking behaviors for substances such as cocaine, morphine and ethanol (Yoon et al., 2004, 2010, 2012; Yang et al., 2010). During acupuncture stimulation, peripheral sensory afferents, such as Pacinian and Meissner's corpuscles, are activated, and the afferent signals are transmitted via the A-fibers of the ulnar nerve (Kim et al., 2013) and the somatosensory pathway in the spinal dorsal column (Chang et al., 2017). While cumulative evidence has supported the neurological model, in the last two decades another hypothesis, known as the "connective tissue model," has emerged to address possible roles of connective tissue in the initiation of acupuncture's signals (Langevin and Yandow, 2002; Langevin, 2014). According to this model, twisting or rotation of the acupuncture needle creates winding of connective tissue around the needle and deformation of collagen fibers, which initiates mechanical signals transmitted via the whole-body fascia network, the layers of connective tissue surrounding muscle, organs, and blood vessels. Research groups have proposed that the connective tissue may mediate the effects of manual acupuncture through a number of pathways: transmission of a mechanical signal from the needle to nearby sensory afferent nerves, potential purinergic signaling within connective tissue fibroblasts with local effects on sensory nerves, and direct effects on connective tissue pathology, possibly involving reductions in inflammation and/or fibrosis (Langevin and Yandow, 2002; Langevin et al., 2007; Langevin, 2014; Wu et al., 2015). However, it remains to be established whether connective tissue mediates the therapeutic effects of acupuncture. While these models have been thought to provide plausible explanations for the initiation of acupuncture signals, evidence is currently lacking as to whether these two models work in separate or synergistic ways.

To explore whether acupuncture effects are mediated through nerve or connective tissue, we used our newly constructed robotic needle twister to test the effects of twisting acupuncture in the rat models of cocaine-induced locomotion, immobilization-induced hypertension and mustard oil-induced visceral pain, and explored whether the effects of acupuncture are altered by a blockade of nerves or disruption of connective tissue.

# MATERIALS AND METHODS

#### Animals

Male Sprague-Dawley rats (weight 270–320 g, Daehan Animal, Korea) were used, and each group consisted of 5–8 rats. Animals were housed at a constant humidity (40∼60%) and temperature (22 ± 2 ◦C), with a 12 h/12 h light/dark cycle and allowed free access to food and water until use. All procedures were carried out in accordance with the NIH Guide for Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee (IACUC) at Daegu Haany University.

# Chemicals

Cocaine (15 mg/ml saline, MacFarlan Smith Ltd., UK), mustard oil (30∼50 µl; allyl isothiocyanate, Sigma Aldrich, USA), and bupivacaine (20 µl/loci, 5 mg/ml, Huons Pharm, South Korea; a long-acting local anesthetic) were used. Type I collagenase (20 µl/loci, 5 mg/ml saline, Sigma Aldrich, USA) or together with toluidine blue (20 µl/loci, 1 mg/ml, Sigma Aldrich) was injected. Bupivacaine or type I collagenase was injected into acupuncture points to a depth of 3 mm, perpendicular to the skin surface, by using a syringe with a 32-gauge needle.

### Measurement of Rotational Duration and Angle During Manual Needle Twisting

A needle (0.20 mm in diameter, needle length of 30 mm and handle length of 20 mm; Dongbang Medical Co., Korea) with red tape on the shaft was inserted into a Styrofoam board to measure the rotational duration (speed) and angle during manual needle twisting (**Figure 1A**). Acupuncturists (n = 3) were asked to twist the handle of the acupuncture needle for at least 20 s and video recorded. This procedure was repeated two times per person. Rotation duration and angle were analyzed using a media player program (Gom Player, Gom and Company, Korea).

# A Robotic Acupuncture Needle Twister (RANT) and Acupuncture Treatment

A robotic twister was constructed to mimic a needle twisting technique that is commonly performed by acupuncturists. This device consisted of a handheld needling instrument coupled to a servo motor (ez-SERVO, Fastech, Korea), a personal computer connected with a servomotor controller (ez-SERVO, Fastech, Korea) and a custom-made control software program (LabVIEW, National Instruments, Austin, TX, USA). The rotation shaft of the servo motor was coupled to a needle holder (**Figure 1D**). A rubber grommet was fixed to the needle at a distance of 2 or 3 mm from the tip, as described previously (Kim et al., 2013), to control the depth of acupuncture needle insertion.

In the rat model of cocaine-induced locomotor activity, acupuncture needles (0.10 mm in diameter, needle length of 10 mm and handle length of 10 mm; Dongbang Medical Co., South Korea) were inserted perpendicularly into the HT7 acupoint at a depth of 3 mm, twisted for 20 s with the RANT device, maintained in place for up to 60 s after needle insertion and withdrawn. In the animal model of immobilization-induced hypertension, needles were inserted perpendicularly into the

PC6 acupoint at a depth of 3 mm, twisted for 10 min with the RANT device and withdrawn. In the rat model for mustard oil-induced visceral pain, acupuncture needles were inserted 2– 3 mm deep into BL62-64 and manually twisted for 30 s at 10 min intervals, which was repeated 4 times for a total of 30 min. Type I collagenase or bupivacaine was injected into acupoints 30 min before acupuncture treatments.

#### Cocaine-Induced Locomotor Activity

Locomotor activity was measured through a video tracking system (EthoVision, Noldus Information Technology BV, Netherlands). Briefly, in a dimly lit room, each animal was placed in a square open field box (40 × 40 × 45 cm) made of black acrylic. Video tracking software (EthoVision 3.1) measured the distance traveled (cm). On the testing day, animals were habituated for at least 60 min. After baseline activity was recorded for 30 min, the animal received an intraperitoneal injection of cocaine (15 mg/kg) and acupuncture treatment and was monitored for up to 60 min after injection. Data are reported as the total distance traveled (cm) in 1 h or the distance traveled (cm) during each 10 min.

# Immobilization-Induced Hypertension and Measurement of Blood Pressure

Hypertension was induced by restraining in animals a coneshaped plastic bag, as described previously (Kim et al., 2017). Systolic blood pressure was measured noninvasively with a tail cuff blood pressure monitor (Model 47, IITC). Briefly, the restrained rats were placed in a chamber kept at 27◦C, and an occluding cuff and a pneumatic pulse transducer were positioned on the base of the rat tail. A programmed electrosphygmomanometer (Narco Bio-Systems Inc., USA) was inflated and deflated automatically, and the tail cuff signals from the transducer were automatically collected every 10 min using an IITC apparatus (Model 47, IITC Inc., USA). The mean of the two readings was taken at each blood pressure measurement.

# Mustard Oil-Induced Visceral Pain and Measurement of Electromyographic Response to Colorectal Distension

A pair of silver wire electrodes (0.20 mm in diameter, Nilaco, Japan) were inserted into the external oblique abdominis muscle, buried for 10 mm of their length and separated by 10 mm for electromyography (EMG) recording. Colorectal distension (CRD) was applied using an inflatable balloon (3 cm in length, 1 cm in diameter) attached to an intravenous line via a T connector that was connected to a bulb with a valve and a sphygmomanometer. EMG recordings in response to CRD stimulation for 5 s at strengths of 20, 40, 60, and 80 mmHg were carried out. The EMG signal was amplified (×10,000, 300–1,000 Hz; ISO-80, World Precision Instruments, USA), digitized and analyzed with a data acquisition and analysis interface (Micro1401, CED, UK). After measurement of baseline, for induction of colonic pain, a mustard oil (30∼50 µl; Sigma Aldrich)-soaked Qtip was inserted into the colon, approximately 5 cm proximal to the anus, kept for 30 min and withdrawn. Acupuncture was then applied, and EMG was recorded up to 30 min after acupuncture treatment.

#### Measurement of Needle Rotational Force

The rotational force (torque) acting on the needle in the tissue was measured through a custom-made needle force measurement system. The torque sensor was simply constructed by fixing a strain gauge (KFH-C1 Series, OMEGA Engineering, USA) to a plunger of a 50 ml syringe connected to a needle holder (**Figure 4A**) and wired to a Wheatstone bridge. The signals generated from the torque sensor during needle twisting were digitalized using a data acquisition system (DAQ-NI USB-6200, National Instruments, USA) and analyzed with a customized LabVIEW program (National Instruments; **Figure 4B**).

#### Hematoxylin-Eosin (H&E) Staining and Immunohistochemical Staining of PGP9.5 Expression in the Skin of Type I Collagenase-Treated Rats

At the end of the experiments, the rats were sacrificed under sodium pentobarbital (90 mg/kg, IP) anesthesia. The skin (10 × 10 × 10 mm) over the PC6 acupoints injected with either type I collagenase or bupivacaine was removed and fixed in 4% buffered paraformaldehyde (PFA) for 2 h. The samples were processed, embedded in paraffin wax, and cut into 5 µm-thick serial sections. The skin samples were subjected to H&E staining or immunohistochemistry for PGP9.5. For immunohistochemistry of PGP9.5, the sections were incubated with anti-PGP9.5 mouse monoclonal antibodies (1:1000; Abcam, Cambridge, UK), followed by incubation with a biotinylated donkey anti-mouse Alexa Fluor 488 (1:200; Invitrogen, USA). Images were taken from 4–5 sections from each animal under an epifluorescence microscope (AX70, Olympus, USA) and analyzed with ImageJ software (NIH, USA). A mixture of type I collagenase and toluidine blue (20 µl/locus) was injected into the PC6, HT7, and BL62 acupoints to a depth of 3 mm in a normal rat to further evaluate the extent of the diffusion of type I collagenase after the injection. Thirty minutes after the injection, the tissues were removed, post-fixed, protected with sucrose, and cryosectioned at a thickness of 15µm. The blue-stained areas were examined under a microscope.

#### Data Analysis

All data are presented as the mean ± SEM (standard error of the mean) and analyzed by one- or two-way repeated measurement analysis of variance (ANOVA) followed by post hoc testing using Fisher's least significant difference (LSD) method or paired or unpaired t-test, where appropriate. Statistical significance was considered at P < 0.05.

#### RESULTS

#### Construction of a Robotic Acupuncture Needle Twister (RANT) That Mimicked Human Needle Twisting Manipulation

To evaluate the rotational duration (speed) and angle generated during needle twisting, 3 well-trained acupuncturists were asked to manually twist the acupuncture needle (**Figure 1A**), videorecorded and analyzed using media player software. Across 58 instances, the needles were rotated for an average of 0.972 ± 0.034 s (n = 30) in a clockwise direction (CW), left stationary for an average of 0.035 ± 0.001 s (n = 52) and then rotated for an average of 0.936 ± 0.028 s (n = 28) in an anticlockwise direction (ACW) (**Figure 1B**). The angles of rotation were 356◦ ± 4.37◦ CW and 322.32◦ ± 6.19◦ ACW (**Figure 1C**). However, there were no significant differences in rotation duration or angles between CW and ACW.

To control the rotation speed and angle objectively, a robotic twister was constructed with a servo motor and interfaced with a LabVIEW program written by our laboratory (**Figure 1D**). When the above parameters for the device were set, the robotic twister replicated the motion of human acupuncture needle manipulation (**Supplementary Movie 1**).

#### Twisting Acupuncture Suppressed Cocaine-Induced Locomotor Activity

To test whether the effect of acupuncture effect is dependent on the needle rotation duration (and speed), we evaluated the effects of needle rotation at various durations on cocaine-enhanced locomotor activity. After baseline recording of locomotor activity for 30 min, rats were given an intraperitoneal (IP) injection of cocaine (15 mg/kg). One minute after injection, acupuncture needles were inserted into the bilateral HT7 and twisted for 20 s at different rotation durations (**Table 1**) using the robotic twister. Control rats received the cocaine injection without acupuncture treatment. An acute cocaine injection rapidly increased locomotor activity, which lasted up to approximately 60 min after injection, with a peak at 10 min (cocaine group in **Figures 2A,B**). Acupuncture applied with the Duration-D3 condition (fastest rotation speed) significantly decreased cocaine-induced locomotion compared to the control group [one-way ANOVA, F(3, 21) = 3.706; #p < 0.05 vs. Control; **Figures 2C,D**]. However, needle twisting at other durations (Duration-D1 and D2) or needle placement without twisting (**Supplementary Figure 1**) failed to inhibit the cocaine-induced



increase in locomotor activity, suggesting that the inhibitory effects were not stimulus-duration dependent. To determine whether the inhibitory effects of twisting acupuncture are dependent on the needle rotation angle, rats (n = 29) were randomly divided into the following groups: control and 180◦ -, 360◦ -, and 720◦ -rotation angle groups (**Table 2**). Although acupuncture stimulation tended to decrease the locomotor response to cocaine in an angle-dependent manner, significant inhibitory effects of acupuncture on locomotor activity were found in the 360◦ - and 720◦ -rotation angle groups compared to the control [one-way ANOVA, F(3, 21) = 4.998; #p < 0.05 vs. Control; **Figures 2E,F**]. Because a twisting condition of Duration-D3 produced consistent, reproducible acupuncture effects, the parameter was used in subsequent experiments.

# Acupuncture Effects Were Prevented by Blockade of the Nerve but Not Disruption of Connective Tissue

To investigate whether acupuncture effects are mediated via nerve or connective tissue, we injected either bupivacaine (a local anesthetic) or type I collagenase (a proteolytic enzyme) into acupoints 30 min before acupuncture treatment. The experiment was first performed in the rat model of cocaine locomotion. While acupuncture at HT7 attenuated cocaineinduced enhancement of locomotor activity, such effects were inhibited by pretreatment of bupivacaine, but not collagenase, indicating mediation of the afferent nerve [one-way ANOVA, F(3, 21) = 4.998; <sup>∗</sup>p < 0.05 vs. Normal; #p < 0.05 vs. Control; \$p < 0.05 vs. BUPIVA+Acup; **Figures 3A–C**]. To further confirm this,


\**Note that rotational speeds (time/turn) were the same in all groups.*

we repeated the experiments in the rat models of immobilizationinduced hypertension (IMH) or mustard oil-induced visceral pain. For the IMH model, when a rat was placed in an immobilization bag, systolic blood pressure (SBP) gradually increased for the next several hours (Control; **Figures 3D–F**), consistent with our previous study (Kim et al., 2017). Twisting acupuncture at PC6 with the robotic device prevented the development of hypertension compared to the control (∗p < 0.05 vs. Control; **Figure 3F**). When either bupivacaine or collagenase was injected into PC6 30 min before acupuncture treatment, the acupuncture effects were prevented in bupivacaine-treated rats but not in collagenase-treated rats (**Figures 3E,F**). Unexpectedly, significant differences were observed at the time points of 100– 120 min between the control and BUPIVA+Acup groups (\$p < 0.05 compared to the control group; **Figure 3F**). Compared to our previous study (Kim et al., 2017), 2 of 5 control rats developed borderline hypertension (140–160 mmHg) and did not reach an SBP of 160–180 mmHg at the end of the 2 h immobilization, which would yield a significant difference at the later time points between the control and Bupiva+Acup groups. In the rat model of mustard oil-induced visceral pain, acupuncture needles were inserted into BL62-64 and manually rotated, and visceral sensitivity was assessed in recording the electromyographic (EMG) responses to graded colorectal distensions (CRD) of 20, 40, 60, and 80 mmHg in conscious rats. A significant increase in the EMG discharges in response to CRDs was observed in mustard oil-treated rats (Control) compared to normal rats (Normal). The elevated EMG responses were attenuated in the rats given acupuncture treatment [one-way ANOVA, 20 mmHg; F(4, 30) = 4.755; #p = 0.025, 40 mmHg; F(4, 30) = 7.273; #p = 0.003, 60 mmHg; F(4, 30) = 8.047; #p < 0.001, 80 mmHg; F(4, 30) = 8.845; #p < 0.001 vs. Control], which was reversed by pretreatment of bupivacaine [one-way ANOVA, 20 mmHg; F(4, 30) = 4.755; p = 0.514, 40 mmHg; F(4, 30) = 7.273; p < 0.001, 60 mmHg; F(4, 30) = 8.047; p = 0.009, 80 mm Hg; F(4, 30) = 8.845; p = 0.002 vs. Acup]. However, the inhibitory effects of acupuncture on visceral pain were not altered by treatment with collagenase prior to acupuncture treatment (**Figures 3G–I**). This observation indicates the mediating role of afferent nerves in the effects of acupuncture.

### Type I Collagenase Disrupted Connective Tissue in Acupoints Without Morphological Changes in the Afferent Nerve

To assess the functional alteration of connective tissue in the acupoints injected with collagenase or bupivacaine, we constructed a torque sensor (**Figures 4A,B**) and measured rotational force (torque) acting on the needle in the tissue. The acupuncture needle inserted into the HT7 acupoint over the wrist was connected to the needle holder of the torque device and manually twisted. The bidirectional needle twisting generated sawtooth waves, with amplitudes in the range of −15 to 15 mN.mm in the untreated normal rats (Normal, **Figure 4C**). While needle rotational force was not changed in bupivacaine-injected acupoints compared to those of normal rats (BUPIVA; **Figures 4E,F**), a significant drop in rotational force was found in collagenase-treated acupoints (COLG; <sup>∗</sup>p < 0.05 vs. Normal; **Figures 4D,F**). This observation indicates that type I collagenase eliminated the pulling of collagen fibers created during needle manipulation.

To identify whether collagenase causes morphological damage of connective tissue or peripheral nerve in the acupoints, the skin over acupoints was evaluated through histological or immunohistochemical methods. In H&E staining, the skin treated with collagenase displayed faint staining, with sparse thin collagen bundles and a significantly decreased density of collagen fibers compared to that of normal, untreated rats (**Figures 5A,B**; <sup>∗</sup>p < 0.05). On the other hand, in a quantitative immunohistochemical analysis for PGP 9.5, a marker for nerve fibers (Beiswenger et al., 2008), the morphological changes of the peripheral nerve were not observed in collagenase-treated skin compared to the normal group (**Figures 5C,D**). In addition, the histological examination revealed that the type I collagenase that had been injected into the acupoints diffused into the skin, including the epidermis, dermis, and subcutaneous tissues (**Figure 5E**). This suggests that injection of type I collagenase into acupoints disrupted the connectivity of collagen fibers but did not induce morphological damage of peripheral nerves in skin.

#### DISCUSSION

Using our robotic device that mimicked needle twisting by a human acupuncturist, we demonstrated that twisting acupuncture effectively suppressed cocaine-induced locomotor activity, immobilization-induced hypertension and mustard oil-induced visceral pain, which was completely prevented by blocking afferent nerves with bupivacaine but not by degrading connective tissue with type I collagenase. Furthermore, collagenase destroyed collagen in the skin without damaging the peripheral nerve. Our findings suggest critical mediation of the nerve in producing acupuncture effects. Additionally, modulation of connective tissue does not seem to be sufficiently reliable to serve as the principal explanation for acupuncture effects, at least in the animal models tested.

Acupuncture needles are manually manipulated to obtain therapeutic effects after needle insertion into acupoints (Pomeranz and Berman, 2003). Various manipulation techniques, such as twisting (also known as bidirectional twisting-rotation), vibrating and lifting and thrusting, have been used in clinics. Among them, manual twisting has been popular because it is a simple and effective technique to provoke the needling sensation called deqi, which acupuncturists believe to be essential for obtaining effective acupuncture stimulation

FIGURE 3 | Effect of preinjection of collagenase or bupivacaine into acupoints in various animal models. (A–C) Effect of pretreatment of type I collagenase or bupivacaine in HT7 acupoint on cocaine-induced locomotor activity in rats. Inhibition of cocaine-induced locomotion by acupuncture at HT7 (Acup) was prevented by pretreatment of bupivacaine (BUPIVA) into HT7 acupoint (A), but not type I collagenase (COLG; B,C). \**p* < 0.05 vs. Normal; #*p* < 0.05 vs. Control; \$*p* < 0.05 vs. BUPIVA+Acup; *n* = 5 per group. Representative tracings (B). (D–F) Effect of pretreatment of type I collagenase or bupivacaine in the PC6 acupoint on immobilization-induced hypertension in rats. Bupivacaine, but not collagenase, inhibited the acupuncture effects of PC6 (D) on elevated systolic blood pressure in rats (E,F) Representative pulse signals measured from tail (E) \**p* < 0.05 vs. Control (immobilization only without acupuncture); #*p* < 0.05 vs. BUPIVA+Acup; *n* = 5 per group. (G–I) Effect of pretreatment of type I collagenase or bupivacaine in acupoints on mustard oil-induced visceral pain in rats. Electromyogram (EMG) in each group was recorded during colorectal distension of 20, 40, 60, and 80 mmHg for approximately 5 s. Normal, normal rats; Control, mustard only without acupuncture; Acup, acupuncture in mustard-treated rats; COLG+Acup, pretreatment of collagenase prior to acupuncture in mustard-treated rats; BUPIVA+Acup, pretreatment of bupivacaine prior to acupuncture in mustard-treated rats. *n* = 7 per group. Acupoints used. (G) Representative EMG signals recorded during colorectal distension of 80 mmHg (H). Bupivacaine, but not collagenase, inhibited the acupuncture effects on colorectal distension-induced visceral motor responses in rats (I). \**p* < 0.05 vs. Normal; #*p* < 0.05 vs. Control; \$*p* < 0.05 vs. BUPIVA+Acup; *n* = 7 per group. Acup, acupuncture; COLG, collagenase; BUPIVA, bupivacaine.

(Pomeranz and Berman, 2003; Stux and Pomeranz, 2012). (Kim G. H. et al., 2010) reported that manual twisting is more effective than the lifting-thrusting technique in producing analgesic effects on formalin-induced pain in rats. We and others have shown that manual twisting of needles applied to HT7 acupoints suppresses addictive behaviors induced by abused drugs, including cocaine, methamphetamine, and ethanol (Yang et al., 2010; Jin et al., 2018; Kim et al., 2018). Moreover,

acupuncture at HT7 attenuates drug-induced dopamine release and metabolic neuronal activity in the nucleus accumbens (Yoon et al., 2004; Jin et al., 2018) through group II metabotropic glutamate receptors (Kim et al., 2018) and modulation of GABA neuron activity in the ventral tegmental area (Yang et al., 2010). While these studies provide evidence that twisting acupuncture needle manipulation produces therapeutic effects, manual acupuncture stimulation could alter its effect by affecting many vagaries, including rotation durations and angles. Unfortunately, few studies have quantified the twisting technique performed by practitioners. Therefore, the present study video-analyzed the motion of needle twisting performed by acupuncturists and found that the duration of needle rotation ranged from 0.75 to 1.21 s (mean value of 0.975 ± 0.073 s) and rotation angles ranged from 325◦ to 350◦ (mean value of 340◦ ± 4.27◦ ). However, considerable variations in both durations and angles were observed within each acupuncturist or across acupuncturists (data not shown). The variations may lead to low reproducibility and high individual variations among practitioners (Napadow et al., 2005; Kim et al., 2013). As our robotic twister has an advantage in controlling rotation duration and angle, it would help solve the control issues of manual needle manipulation.

Researchers have suggested that needle twisting in different stimulation parameters [including durations (or frequency, Hz) and angles, etc.] could produce different physiological responses and therapeutic effects (Kim G. H. et al., 2010; Chang et al., 2017). Hong et al. (2015) showed that the responses of spinal dorsal horn neurons following gastric distension are enhanced by manual acupuncture at 0.5 and 1 Hz, while the dorsal horn neurons are inhibited by manual acupuncture at 2 and 3 Hz. In the present study, the effects of acupuncture were not dependent on the rotation duration or angle when various durations or angles of needle twisting were tested in three different animal models. Similarly, a previous study attempted to determine the analgesic effects of acupuncture in 4 different stimulation conditions (angle and frequency of rotation: 90◦ + 1 Hz, 90◦ + 1/4 Hz, 360◦ + 1 Hz, and 360◦ + 1/4 Hz)

by using a computer-controlled rotational device in rats. This study showed that only the 90◦ + 1/4 Hz condition generates a statistically significant effect compared to plain acupuncture (Kim et al., 2006). These findings indicate that there does not appear to be a correlation between rotation durations or angles and the degree of response alteration in the twisting needle manipulation. Rather, acupuncture effects may require specific stimulation parameters, in accordance with what has been found in animal models. Additionally, in accordance with a previous study (Kim et al., 2006), our data showed that simple needle placement did not generate acupuncture effects on cocaine-induced locomotion in rats. It supports

the observations that needle manipulations such as rotation and electrical stimulation are needed to achieve the desired effects by activating sensory nerve fibers and cells, in contrast to needle placement (sham acupuncture) (Kim et al., 2006; Guo et al., 2018).

In the present study, injection of a long-lasting local anesthetic bupivacaine into acupoints effectively blocked acupuncture effects in the rat models of cocaine-induced locomotion, hypertension, and mustard oil-induced visceral pain, suggesting that acupuncture signals are conveyed via afferent nerve fibers. These results agree with reports that application of a local anesthetic, 1% lidocaine, to acupoints prior to acupuncture treatment inhibits antiemetic action by acupuncture at PC6 in patients undergoing gynecological surgery (Dundee and Ghaly, 1991), and that injection of a local anesthetic procaine into ST36 blocks acupuncture analgesia in animal models (Ulett et al., 1998). Additionally, the role of afferent nerve fibers in acupuncture-initiated impulses was supported by several observations (given below). Acupuncture analgesia is reported to be ablated after sectioning nerve innervating acupoints (Noguchi and Hayashi, 1996; Kim H. Y. et al., 2010) and is reproduced by direct stimulation of exposed nerves (Li et al., 1998; Kim H. Y. et al., 2010). Manual and electroacupuncture at PC6-7 acupoints activate Aδ and C fibers to evoke a cardiovascular effect (Zhou et al., 2005), which is eliminated by neonatal capsaicin to block C-fiber afferents (Tjen-a-Looi et al., 2005). The acupoint stimulation activates opioid receptors in the rostral ventrolateral medulla, a central site regulating blood pressure (Sved et al., 2003), and suppresses stimulationinduced hypertension (Tjen-a-Looi et al., 2003; Kim et al., 2017). As shown in our previous study, mechanical stimulation of HT7 acupoints activates peripheral sensory afferents, such as Pacinian and Meissner's corpuscles, which are conveyed via large A-fibers of the ulnar nerve and spinal dorsal column somatosensory pathway. These signals enhance γaminobutyric acid (GABA) neuronal activity in the ventral tegmental area (VTA) and suppress extracellular dopamine release in the nucleus accumbens (Jin et al., 2018), resulting in the attenuation of cocaine-induced locomotor activity or cocaineseeking behaviors (Kim et al., 2013; Chang et al., 2017; Jin et al., 2018). Indeed, the above evidence indicates that acupuncture stimulates afferent nerve fibers which, in turn, send messages to the brain.

An emerging theory proposes a mediating role of connective tissue in the effects of acupuncture, wherein acupuncture transmits signals through the layers of connective tissue surrounding muscle groups, organs, and blood vessels. This theory posits that acupuncture needle manipulation creates winding of connective tissue around the needle, known as "needle grasp," and in turn activates signal pathways by deformation of connective tissue, resulting in therapeutic effects (Langevin and Yandow, 2002; Langevin et al., 2007). Previous studies have also found that needle twisting produces an active cytoskeletal response in a manner dependent on the number of rotations and the angle of the needle (Langevin et al., 2007) This procedure also induces degranulation of the mast cells in the tissues of acupoints through an interaction with the collagen fibers (Zhang et al., 2008; Wu et al., 2015). Although many studies, mostly led by the Langevin research group, have proposed biomechanical actions of connective tissue during acupuncture manipulation (Langevin and Yandow, 2002; Langevin et al., 2007; Langevin, 2014), to date, it has not been determined fully whether the connective tissue is associated with therapeutic effects of acupuncture. In the present study, a peak-to-peak torque value of about 30 mN.mm was generated by twisting a needle inserted into HT7, which was decreased to about 3 mN.mm after injection of type I collagenase, similar to those reported in a previous study (Yu et al., 2009). Thus, an injection of type I collagenase into acupoints disrupted the phenomenon of "needle grasp" by causing connective tissue breakdown. Type I collagenase did not affect the expression of the neuronal marker PGP9.5 in skin, indicating that the afferent nerve in the collagenase-treated skin was intact. Importantly, disruption of connective tissue by type I collagenase failed to block the acupuncture effects generated by needle twisting in the rat models tested. On the other hand, bupivacaine, a long-acting local anesthetic (Rosenberg and Heinonen, 1983), injected into acupoints effectively blocked the acupuncture effects without affecting needle grasp of connective tissue. Taken together, our data suggest that acupuncture may not recruit connective tissue to produce therapeutic effects. In contrast to our results, Yu et al. reported that the destruction of collagen fibers with type I collagenase at ST36 acupoints inhibited the effects of acupuncture on inflammatory pain in rats (Yu et al., 2009). The reason for this discrepancy is not completely understood at present, but a possible explanation is that the acupuncture points chosen in this experiment were located on distal limbs (HT7, PC6, and BL62-64), which contain relatively less subcutaneous tissue, while the experiments in the paper by Yu et al. used ST36, which is a more proximal acupoint enriched in loose connective tissue. Considering that the interaction of the needle with collagen fibers mainly occurs within the loose subcutaneous tissue, but not within the dermis (Langevin and Yandow, 2002), the interaction between the needle and loose connective tissue in proximal acupoints such as ST36 may be greater than the distal acupoints used in the current study. However, our ability to draw conclusions about the importance of afferent nerve fibers in acupuncture-mediated responses, rather than connective tissue, was hampered by the major limitation of the present study, in that 5 points of approximately 360 acupoints were investigated using three animal models. More extensive studies using various animal models and acupoints will be required.

In conclusion, twisting acupuncture produced therapeutic effects in rat cocaine-induced locomotion, hypertension and mustard oil-induced visceral pain models, and these effects were abolished by a blockade of the afferent nerve in the vicinity of acupoints but not by disruption of collagen fibers. Accordingly, it seems reasonable to propose that the effects of acupuncture require mediation by nerve tissue but not connective tissue. This study helps to understand the mechanisms involved in the initiation of acupunctur signals.

#### AUTHOR CONTRIBUTIONS

HYK and CY designed the experiment. SC, YR, SB, JB, YF, OK, MB, D-HK, SL, HKK, and BL conducted the experiments. HYK was responsible for the overall direction of the project and for edits to the manuscript.

#### FUNDING

This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2018R1A5A2025272 and 2018R1E1A2A02086499), the KBRI basic research program through the Korea Brain Research Institute funded by the Ministry of Science and ICT (18-BR-03), and the Korea Institute of Oriental Medicine (KIOM) (K18181).

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# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnins. 2019.00110/full#supplementary-material

Supplementary Movie 1 | Needle twisting by a robotic acupuncture needle twister or an acupuncturist.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Chang, Kwon, Bang, Kim, Baek, Ryu, Bae, Fan, Lee, Kim, Lee, Yang and Kim. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Effects of Leg Motor Imagery Combined With Electrical Stimulation on Plasticity of Corticospinal Excitability and Spinal Reciprocal Inhibition

Yoko Takahashi1,2, Michiyuki Kawakami<sup>1</sup> \*, Tomofumi Yamaguchi1,3, Yusuke Idogawa<sup>2</sup> , Shigeo Tanabe<sup>4</sup> , Kunitsugu Kondo<sup>2</sup> and Meigen Liu<sup>1</sup>

<sup>1</sup> Department of Rehabilitation Medicine, Keio University School of Medicine, Tokyo, Japan, <sup>2</sup> Tokyo Bay Rehabilitation Hospital, Chiba, Japan, <sup>3</sup> Department of Physical Therapy, Yamagata Prefectural University of Health Sciences, Yamagata, Japan, <sup>4</sup> Faculty of Rehabilitation, School of Health Sciences, Fujita Health University, Toyoake, Japan

#### Edited by:

Younbyoung Chae, Kyung Hee University, South Korea

#### Reviewed by:

Kyungmo Park, Kyung Hee University, South Korea Junsuk Kim, Max Planck Institute for Biological Cybernetics, Germany

\*Correspondence: Michiyuki Kawakami michiyukikawakami@hotmail.com

#### Specialty section:

This article was submitted to Perception Science, a section of the journal Frontiers in Neuroscience

Received: 12 October 2018 Accepted: 08 February 2019 Published: 21 February 2019

#### Citation:

Takahashi Y, Kawakami M, Yamaguchi T, Idogawa Y, Tanabe S, Kondo K and Liu M (2019) Effects of Leg Motor Imagery Combined With Electrical Stimulation on Plasticity of Corticospinal Excitability and Spinal Reciprocal Inhibition. Front. Neurosci. 13:149. doi: 10.3389/fnins.2019.00149 Motor imagery (MI) combined with electrical stimulation (ES) enhances upper-limb corticospinal excitability. However, its after-effects on both lower limb corticospinal excitability and spinal reciprocal inhibition remain unknown. We aimed to investigate the effects of MI combined with peripheral nerve ES (MI + ES) on the plasticity of lower limb corticospinal excitability and spinal reciprocal inhibition. Seventeen healthy individuals performed the following three tasks on different days, in a random order: (1) MI alone; (2) ES alone; and (3) MI + ES. The MI task consisted of repetitive right ankle dorsiflexion for 20 min. ES was percutaneously applied to the common peroneal nerve at a frequency of 100 Hz and intensity of 120% of the sensory threshold of the tibialis anterior (TA) muscle. We examined changes in motor-evoked potential (MEP) of the TA (task-related muscle) and soleus muscle (SOL; task-unrelated muscle). We also examined disynaptic reciprocal inhibition before, immediately after, and 10, 20, and 30 min after the task. MI + ES significantly increased TA MEPs immediately and 10 min after the task compared with baseline, but did not change the task-unrelated muscle (SOL) MEPs. MI + ES resulted in a significant increase in the magnitude of reciprocal inhibition immediately and 10 min after the task compared with baseline. MI and ES alone did not affect TA MEPs or reciprocal inhibition. MI combined with ES is effective in inducing plastic changes in lower limb corticospinal excitability and reciprocal Ia inhibition.

Keywords: motor imagery, motor-evoked potential, H-reflex, disynaptic reciprocal inhibition, peripheral nerve electrical stimulation

# INTRODUCTION

Motor imagery (MI) has been described as a dynamic state during which the representation of a given motor act is internally rehearsed within working memory without any overt motor output (Decety and Grezes, 1999). Brain activation during MI is similar to that observed during motor execution (Jackson et al., 2001; Chen et al., 2016; Sacheli et al., 2017). MI has been shown to increase

corticospinal excitability, the H-reflex, spinal stretch reflex, and reciprocal inhibition (Kiers et al., 1997; Hale et al., 2003; Bakker et al., 2008; Aoyama and Kaneko, 2011; Lebon et al., 2012; Kato and Kanosue, 2017; Ruffino et al., 2017; Kawakami et al., 2018). Mental practice using MI is widely used in sports and rehabilitation (Jackson et al., 2001; de Lange et al., 2008; Malouin and Richards, 2010; Mrachacz-Kersting et al., 2012, 2016; Grospretre et al., 2016; Guerra et al., 2017; Ruffino et al., 2017).

Introducing the plasticity of neural circuits involved in motor activation is important for motor recovery (Stefan et al., 2000; Wolpaw and Tennissen, 2001; Wolpaw, 2007; Di Pino et al., 2014). Previous studies using a paired associative stimulation (PAS) protocol (Stefan et al., 2000; Stinear and Hornby, 2005) have shown that spike-timing-dependent input to the motor cortex and peripheral nerves is important for the induction of plasticity in the motor cortex. Input to the motor cortex and spinal cord is also important for inducing plastic changes in spinal circuits (Wolpaw and Tennissen, 2001; Chen et al., 2016; Yamaguchi et al., 2018). One previous study used MI as the input to the motor cortex and ES as the input to the peripheral nerve (Saito et al., 2013). Indeed, a combination of finger movement MI and ES was shown to increase corticospinal excitability to a greater extent than MI or ES alone (Saito et al., 2013). Finger movement MI during action observation combined with ES for 20 min induced plastic change of corticospinal excitability at the time just after the end of the intervention, but each intervention alone was ineffective (Yasui et al., 2018). In the lower limb, Mrachacz-Kersting et al. (2012) showed that the corticospinal excitability during the application of MI using electroencephalography combined with peripheral nerve ES was greater than that during the period without MI. However, whether these effects remain after the task is currently unknown. Systematic reviews have reported the effects of MI on the upper or lower limb hemiparesis in patients with stroke (Braun et al., 2006; Guerra et al., 2017). The number of studies in the lower limb were less than those in upper limb; therefore, the therapeutic effect has not been established. Basic knowledge on the neurological effects of MI and MI combined with ES are needed to develop therapeutic strategies for lower limb MI.

The neural control of the upper and lower limb is differ depending on the task (Arya and Pandian, 2014). For motor control of the lower limb (such as that required for walking), both spinal and descending neural circuits from the motor cortex are important. Spinal reciprocal inhibition between agonist and antagonist muscles is responsible for the achievement of smooth movements (Crone and Nielsen, 1989; Morita et al., 2001). Reciprocal inhibition of calf muscles is often reduced or facilitated in patients with stroke and spinal cord injury (Crone and Nielsen, 1994; Okuma et al., 2002; Crone et al., 2003). Normalization of the reciprocal inhibition is as important as that of the neural circuits in the brain for motor recovery in the lower limb or for gait rehabilitation (Dietz and Sinkjaer, 2007).

In this study, we aimed to examine the effects of MI combined with ES on the plasticity of both corticospinal excitability and spinal reciprocal inhibition in the lower limb. We hypothesized that MI combined with peripheral nerve electrical stimulation (ES) may be superior in inducing plastic changes in cortical and spinal neural circuits than MI or ES alone.

#### MATERIALS AND METHODS

The Tokyo Bay Rehabilitation Hospital ethics committee approved the study protocol (approval number: 175-2). All tests were performed at the Tokyo Bay Rehabilitation Hospital. This study was registered in the University Hospital Medical Information Network (UMIN; registration number: 000028087). All participants provided written informed consent prior to enrolment. The procedures complied with the Declaration of Helsinki.

Seventeen healthy adults (mean age: 24.6 ± 2.1 years, seven females) participated in this study. None of the participants had history of neurological disease or was receiving any acute or chronic medications that could affect the central nervous system.

All participants performed the following three tasks, in a random order: (1) MI alone; (2) peripheral nerve ES alone (ES alone); and (3) MI combined with ES (MI + ES; **Figure 1A**). Tasks were performed on different days, and were separated by more than 7 days to minimize carry-over effects. Participants performed each task for 20 min. Motor-evoked potential (MEP) and disynaptic reciprocal inhibition were assessed before, immediately after (post0), and 10 (post10), 20 (post20), and 30 min (post30) after the task.

During the experimental sessions, participants remained seated on a chair with a backrest in a relaxed position with 80◦ hip flexion, 80◦ knee flexion, 10◦ ankle plantar flexion and their feet on the floor (**Figure 1B**).

#### Task

#### Motor Imagery

Participants were instructed to imagine dorsiflexion of their right ankle with the help of a video, for 20 min (kinesthetic motor imaging of ankle dorsiflexion). A trial consisted of imagination for 2 s and a rest period of 4 s. The full task consisted of 200 trials that were repeated over a 20 min period. To avoid muscle contraction during the MI, we monitored the electromyographic recording of the tibialis anterior (TA) and soleus (SOL) muscles and provided verbal feedback to avoid muscle contraction when signs of muscle contraction were identified.

#### Peripheral Nerve Electrical Stimulation

Electrical stimulation was applied to the common peroneal nerve at the fibular head. ES was delivered with a frequency of 100 Hz (pulse width 1 ms) for 2 s at an intensity of 120% of the sensory threshold of the TA at rest without muscle contraction. This stimulus intensity was determined so that MEP can be increased to a level greater than those at rest when combined with MI (Yamaguchi et al., 2012). A trial consisted of stimulation for 2 s and a rest period of 4 s. 200 trials were conducted over 20 min.

#### Motor Imagery Combined With Electrical Stimulation

MI was applied in the same manner as in the MI alone paradigm. Participants were asked to imagine dorsiflexion of their right

ankle at the same time that ES was applied. 200 trials were conducted over 20 min.

#### Assessment

#### Motor-Evoked Potential

The schema of the experimental measurements is presented in **Figure 1B**. MEPs were assessed using transcranial magnetic stimulation. Since the MI task consisted of ankle dorsiflexion, we selected the TA as a task-related muscle, and the SOL as a task-unrelated muscle. We stimulated the left primary motor cortex using a Magstim 200 magnetic stimulator (The Magstim Company, Whitland, Dyfed, United Kingdom) and a doublecone coil. The stimulation site was selected to coincide with the point where MEP amplitude of the right TA was the largest. The stimulus intensity was defined as 120% of the resting motor threshold of each muscle. The resting motor threshold was defined as the intensity that evoked responses of 100-µV (at 5 of 10) in the TA or SOL muscle at rest. A total of 15 MEPs were recorded for each muscle. Peak-to-peak amplitudes were averaged for each time point.

#### Reciprocal Inhibition

We measured spinal reciprocal inhibition of the calf muscles, i.e., reciprocal inhibition from the TA to the SOL. Reciprocal inhibition was assessed using a soleus H-reflex conditioningtest paradigm. The H-reflex in the soleus muscle was elicited by stimulating the tibial nerve at the popliteal fossa (1 ms rectangular pulse). A positive (anode) stimulation electrode was set above the patella. Throughout the experiment, the test H-reflex amplitude was maintained at 15–20% of the amplitude of the maximum motor response for the SOL, as previously described (Crone et al., 1990). The conditioning stimulus was delivered to the common peroneal nerve using surface electrodes positioned below the fibular head. The conditioning stimulus intensity was defined

as 1.0 × motor threshold. The motor threshold was defined as the intensity that evoked a response of 100 µV in the TA at rest. The common peroneal nerve-stimulating electrode was carefully positioned to avoid activation of the peroneus muscles, thereby ensuring selective stimulation of the deep branch of the peroneal nerve (Crone et al., 1990). The conditioning stimulus was repeatedly checked during the experiments by monitoring the M wave of the TA. The interval between the conditioning and test stimulus was set at 0, 1, 2, and 3 ms (Fujiwara et al., 2011). The optimal interval to produce disynaptic reciprocal inhibition by stimulating the common peroneal nerve was determined at the beginning of each session and used throughout. Ten conditioned and 10 test H-reflexes were recorded at each time point. Reciprocal inhibition was defined as the mean conditioned H-reflex amplitude/mean test H-reflex amplitude.

#### Motor Imagery Ability

To assess MI ability, all participants answered the Vividness of Movement Imagery Questionnaire-2 (VMIQ-2, Roberts et al., 2008) at the beginning of the experiment. The VMIQ-2 is one of the most commonly used questionnaires to assess the vividness of MI. This questionnaire is used to determine the level of vividness with which the 12 motor tasks can be imagined from the viewpoint of the following three factors: internal visual imagery, external visual imagery, and kinesthetic imagery. Subjects rate the vividness of their MI on a five-point scale (1 = perfectly vivid and as clear as normal vision, 5 = no image at all). The maximum score is 60 points per factor. A low score indicates greater imagery ability.

#### Statistical Analysis

We calculated the sample size of the study based on our previous study that examined the after-effects of reciprocal inhibition by ES combined with voluntary contraction (Takahashi et al., 2017). We performed the power analysis based on the following: effect size, 1.39; α error, 0.05; and statistical power, 0.95 using G∗Power 3.1.9.2 for Windows (Heinrich Heine University, Dusseldorf, Germany). The power analysis indicated that 12 samples were needed. We recruited 17 participants, which fully satisfied the power sample size requirements.

A two-factor repeated-measures analysis of variance (ANOVA) was used to analyze the main and interaction effects of task (MI alone, ES alone, and MI + ES) and time (before, post0, post10, post20, and post30) on TA MEP amplitudes, test H amplitudes, and reciprocal inhibition. Paired t-tests, with Bonferroni correction for multiple comparisons, were performed as post-hoc tests, when a significant result was obtained in the primary analyses. Prior to ANOVA, we confirmed that all TA MEPs and reciprocal inhibition variables were normally distributed using Shapiro–Wilk tests. SOL MEPs were not normally distributed; therefore, we used Wilcoxon signed rank tests to compare SOL MEPs before and at each time point after the task.

To assess the relationship between MI ability and the effects of the MI + ES task, a Spearman's rank correlation coefficient was performed. We compared the correlation between the VMIQ-2 mean score (internal visual imagery, external visual imagery, and kinesthetic imagery) and changes in TA MEPs and reciprocal inhibition after the intervention. Changes in TA MEPs and reciprocal inhibition after the intervention were calculated as the difference between the valuables before and post0.

Statistical analyses were performed using SPSS 24 (IBM Corp., Armonk, NY, United States) for Windows. Results with p < 0.05 were considered statistically significant.

#### RESULTS

#### MEP Amplitudes

**Figure 2** shows the representative change in TA MEPs in one participant. We found a significant main effect of task (F2,<sup>32</sup> = 7.733, p = 0.002) and a significant interaction between task (MI alone, ES alone, and MI + ES) and time (before, post0, post10, post20, and post30) (F8,<sup>128</sup> = 2.892, p = 0.005) for MEPs of the task-related TA muscle. No significant main effect of time (F4,<sup>64</sup> = 0.854, p = 0.497) was observed. Post hoc paired t-test comparisons revealed that TA MEPs were significantly higher at post0 and post10 after MI + ES than at baseline (post0: p = 0.009, post10: p = 0.009; **Table 1** and **Figure 3A**). MI alone and ES alone did not produce any significant changes in TA MEPs at any time point. TA MEPs of MI + ES were significantly higher than that of MI alone at post0 (p = 0.014). TA MEPs of MI + ES were also significantly higher than that of ES alone at post0 (p = 0.002), post10 (p = 0.004), post20 (p = 0.027), and post30 (p = 0.011).

We could only measure SOL MEPs in 12 of the 17 participants. A Wilcoxon signed rank test revealed no significant difference among SOL MEPs at any time point (**Figure 3B**).

#### Reciprocal Inhibition

No significant main effects of task (F2,<sup>32</sup> = 0.339, p = 0.715) or time (F4,<sup>64</sup> = 0.971, p = 0.43), and no significant interactions

TABLE 1 | Statistical results of the post hoc paired t-tests with Bonferroni correction in tibialis anterior motor-evoked potentials.



MI, motor imagery; ES, electrical stimulation; MI + ES, motor imagery combined with electrical stimulation; ∗∗p < 0.01, <sup>∗</sup>p < 0.05.

between task and time (F8,<sup>128</sup> = 0.909, p = 0.511) were identified for the test H amplitudes.

We found significant main effects of task (F2,<sup>32</sup> = 13.764, p < 0.001) and time (F4,<sup>64</sup> = 3.384, p = 0.014) for reciprocal inhibition. A significant interaction between task and time was also observed (F8,<sup>128</sup> = 13.764, p = 0.004). Post hoc paired t-test comparisons showed that reciprocal inhibition was significantly higher at post0 and post10 in the MI + ES group than at baseline (post0: p = 0.003, post10: p = 0.032; **Table 2** and **Figure 3C**). MI alone and ES alone showed no significant changes in reciprocal inhibition in comparison with baseline at any of the time point evaluated. Reciprocal inhibition in MI + ES was significantly higher than in MI alone at post0 (p = 0.007), post10 (p = 0.003), and post20 (p = 0.028). Reciprocal inhibition in MI + ES was also significantly higher than in ES alone at post0 (p = 0.027), post10 (p = 0.035), post20 (p = 0.026), and post30 (p = 0.04).

#### Motor Imagery Ability

The VMIQ-2 mean score was 26.9 ± 8.7 (mean ± SD) for the internal visual imagery subscale, 28.5 ± 11.0 for the external visual imagery subscale, and 26.1 ± 8.4 for the kinesthetic imagery subscale. There were no significant correlations between changes in TA MEPs or reciprocal inhibition after intervention and the VMIQ-2 scores.

#### DISCUSSION

We found that ankle dorsiflexion MI combined with ES increased task-related muscle (TA) MEPs and reciprocal inhibition from the TA to the SOL. Intervention had no effect on task-unrelated muscle (SOL) MEPs. MI and ES alone produced no effects on TA or SOL MEPs or reciprocal inhibition. Our study provides the first evidence that a combination of MI and ES is effective at inducing plasticity of both lower limb corticospinal excitability and spinal reciprocal inhibition circuits.

#### Corticospinal Excitability

We found that MI + ES resulted in significantly higher TA MEPs immediately after the task than at baseline. These effects remained for up to 10 min after the task. These results indicate that MI combined with ES can induce plastic changes such as long-term potentiation of the primary motor cortex and/or corticospinal circuits (Huang et al., 2011). Our results align with those previously presented by Khaslavskaia and Sinkjaer (2005), where the authors reported that voluntary dorsiflexion combined with ES of the common peroneal nerve maintained the enhanced corticospinal excitability of the TA for longer than voluntary dorsiflexion or ES alone. The combination of the activities of the somatosensory afferents and intrinsic motor cortical circuits may be important for inducing cortical plasticity (Stefan et al., 2000). It is known that MI alone and ES alone (Bakker et al., 2008; Yamaguchi et al., 2012), and MI using electroencephalography combined with ES (Mrachacz-Kersting et al., 2012) increase the excitability of a region of the primary motor cortex in real time. It is possible that the spike-timingdependent plasticity resulting from combined inputs may have occurred at a synapse somewhere in corticospinal pathway of the TA (Stefan et al., 2000; Stinear and Hornby, 2005). Few studies have investigated the effects of ES alone on MEPs after intervention. One study indicated that ES applied to the common peroneal nerve for 30 min induced plastic changes in TA MEPs (Khaslavskaia and Sinkjaer, 2005). In another study targeting the upper extremity, ES alone for 14 min did not induce any plastic changes (Taylor et al., 2012). Thus, it is possible that the timewindow of stimulation used in our study (20 min) was insufficient to induce plastic changes in the corticospinal excitability of the TA following ES. Kaneko et al. (2014) showed that MEP during ES alone was not higher than at rest. The short-term effect of ES alone on corticospinal excitability may be less than that of MI + ES. Yasui et al. (2018) demonstrated that MI during action observation combined with ES significantly increased MEP by 10 min, however, MI during action observation or ES alone for

TABLE 2 | Statistical results of the post hoc paired t-tests with Bonferroni correction in reciprocal inhibition.

#### Comparison with the baseline within task


#### Comparison of the value among tasks at the same time points


MI, motor imagery; ES, electrical stimulation; MI + ES, motor imagery combined with electrical stimulation; ∗∗p < 0.01, <sup>∗</sup>p < 0.05.

20 min did not increase MEP. The effect of MI alone for 20 min on corticospinal excitability might be insufficient, just as ES alone.

As expected, we could not find any changes in the taskunrelated muscle (SOL) MEPs following any of our tasks. One previous study examining corticospinal excitability during MI showed that MI increased corticospinal excitability in both task-related and task-unrelated muscles (Bakker et al., 2008). Although we cannot completely discard the possibility of an increase in corticospinal excitability for the task-unrelated muscle given the limited amount of data we were able to collect, we hypothesize that combining ES with MI may help to focalize the effects on excitability in the areas of the primary motor cortex or corticospinal tract related to the task-relevant muscle.

#### Reciprocal Inhibition

We found that MI + ES also significantly increased reciprocal inhibition immediately after the task when compared to the baseline. This effect remained up to 10 min after the task. Spinal Ia inhibitory interneurons projecting to the antagonist (SOL) motor neuron receive convergent inputs from the motor cortex and Ia afferents of the agonist muscle (TA) (Nielsen et al., 1993; Masakado et al., 2001). It has been shown that combining descending inputs coming from the corticospinal tracts during voluntary movements with those coming from Ia afferents during ES induces plastic changes of the Ia inhibitory circuit (Yamaguchi et al., 2013; Takahashi et al., 2017, 2018). This mechanism may underlie the effects on reciprocal inhibition we have reported herein. MI and ES alone did not affect reciprocal inhibition. Long-term interventions that target the upper extremities using MI have been known to induce plasticity of the reciprocal inhibition circuits in patients after stroke (Kawakami et al., 2018). However, the effect of short-time MI on reciprocal inhibition remains unknown. Motor cortex excitability is important for maintaining plastic changes in spinal reciprocal inhibition (Wolpaw and Tennissen, 2001; Chen et al., 2006; Fujiwara et al., 2011; Yamaguchi et al., 2013, 2016). Based on our findings, we suggest that short-term motor descending inputs during MI alone are not sufficient to induce plastic changes of the reciprocal inhibition spinal circuits – and that longer times may be needed for the effects to emerge.

In our study, ES alone also did not change reciprocal inhibition. Perez et al. (2003) showed that ES that mimicked aspects of sensory feedback from muscle spindle during walking was effective in inducing plastic changes in reciprocal inhibition, while ES with constant period parameters was ineffective. In our study, stimulus intensity was lower than that found by Perez et al. (2003). The parameters used for the stimulation may be important determinants of the ES effects on the plasticity of the reciprocal inhibition circuits.

#### Motor Imagery Ability

Although we first hypothesized that the increase in MEP and reciprocal inhibition after MI + ES would be correlated with an individual's MI ability, we did not find any significant relationship between the effects on TA MEPs or reciprocal inhibition after MI + ES and the VMIQ-2 scores. MI ability, as assessed by the VMIQ-2, and corticospinal excitability during MI have been known to correlate positively, at least when the upper extremities are the targets for the MI intervention (Lebon et al., 2012; Williams et al., 2012). Previous studies have focused on skillful finger movement imagery. To our knowledge, no previous reports have examined the relationship between MI ability and corticospinal excitability during MI, when the lower limb are the targets for the MI intervention. It is possible that the absence of a correlation between MI + ES effects on MEPs and reciprocal inhibition and MI ability in our study derives from the fact that the movement of the lower limb is coarse when compared with that of skillful finger movement. Future studies examining and comparing different movements will be needed to clarify whether this association is found only in some cases.

### Comparison With Results in the Upper Limb

The present study showed that MI alone and ES alone did not change MEPs or reciprocal inhibition, and that MI + ES effectively increased TA MEP and reciprocal inhibition. The combined effects found were similar to those from a previous study in the upper limb (Yasui et al., 2018). These results suggest that combining ES with MI rehabilitation in the lower limb might be effective for neuromodulation in patients with central nervous system lesions. For gait rehabilitation in patients with central nervous system lesions, some strategies such as robotic gait training (Cheung et al., 2017; Holanda et al., 2017; Bruni et al., 2018) or body-weight support treadmill gait training (Mehrholz et al., 2017) can be used; however, the therapy for lower limb paralysis itself is limited. Our results, which indicated that MI + ES might be an effective treatment of lower limb paralysis, are of clinical importance.

#### Clinical Implications

The induction of long-term potentiation in neural circuits is important for motor recovery in patients with central nervous system lesions. Reduced reciprocal inhibition correlates with the development of hyperactive reflexes and spasticity (Crone and Nielsen, 1994; Okuma et al., 2002; Crone et al., 2003). Furthermore, restoring reciprocal inhibition is important to improve the movement of paretic ankles. We suggest that MI + ES is a useful and safe rehabilitation method that can be performed even in patients who are severely paralyzed and have difficulty performing voluntary movements. As a next step, we would like to investigate the effects of MI + ES on reciprocal inhibition in patients with central nervous system lesions.

#### Limitations

In this study, we measured the effects of MI + ES in healthy subjects. It is necessary to investigate the effects of MI + ES in patients with central nervous system lesions, i.e., those who would be the target of neuromodulation in clinical practice. We could not measure SOL MEPs from all subjects, which limited our power to detect potential effects on the task-unrelated muscle. A similar problems was faced by Bakker et al. (2008), who was unable to reliably record MEPs of the medial gastrocnemius muscle during ankle dorsiflexion MI. Future studies should consider this limitation when selecting about the MI task and the muscle used to record MEPs.

#### CONCLUSION

fnins-13-00149 February 19, 2019 Time: 17:7 # 8

Ankle dorsiflexion MI combined with ES enhanced TA MEPs and reciprocal inhibition; these effects persisted after the task. In addition to other descending modulation methods such as brain stimulation or voluntary muscle contraction, the combination of MI with afferent input stimulation through ES may be effective in inducing plasticity of the corticospinal excitability and spinal reciprocal inhibition circuits.

#### AUTHOR CONTRIBUTIONS

YT, MK, and TY contributed to the concept, idea, research design, and project management. YT, MK, TY, and ML wrote the manuscript. YT, YI, and KK recruited participants and collected data. YT and YI performed data analysis. MK, TY, and

#### REFERENCES


KK provided facilitates and equipment. ST wrote the program for data collection. ML procured funding. YT, MK, TY, KK, and ML provided consultation (including review of manuscript before submission).

## FUNDING

This work was partially supported Japan Agency for Medical Research and Development (AMED): Research and Development of Advanced Medical Devices and Systems to Achieve the Future of Medicine-Development of advanced medical equipmentresearch and development into medical device systems to restore function in patients with motor and sensory paralysis under Grant Number JP18he0402255.

#### ACKNOWLEDGMENTS

The authors would like to thank Ms. Saeko Kawamoto for her descriptive illustration.



stimulation on the motor cortex. Exp. Brain Res. 227, 333–342. doi: 10.1007/ s00221-013-3513-5


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Takahashi, Kawakami, Yamaguchi, Idogawa, Tanabe, Kondo and Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Somatosensory Stimulation With XNKQ Acupuncture Modulates Functional Connectivity of Motor Areas

Till Nierhaus 1,2 \* † , Yinghui Chang3,4†, Bin Liu<sup>3</sup> , Xuemin Shi <sup>3</sup> , Ming Yi 5,6, Claudia M. Witt 4,7† and Daniel Pach4,7 \* †

<sup>1</sup> Neurocomputation and Neuroimaging Unit, Department of Education and Psychology, Freie Universität Berlin, Berlin, Germany, <sup>2</sup> Department of Neurology, Max Planck Institute for Human Cognitive and Brain Sciences, Leipzig, Germany, <sup>3</sup> First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, China, <sup>4</sup> Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health, Institute for Social Medicine, Epidemiology and Health Economics, Berlin, Germany, <sup>5</sup> Neuroscience Research Institute and Department of Neurobiology, School of Basic Medical Sciences, Peking University, Beijing, China, <sup>6</sup> Key Laboratory for Neuroscience, Ministry of Education/National Health Commission, Peking University, Beijing, China, <sup>7</sup> Institute for Complementary and Integrative Medicine, University of Zurich and University Hospital Zurich, Zurich, Switzerland

#### Edited by:

Florian Beissner, Hannover Medical School, Germany

#### Reviewed by:

Yihuai Zou, Beijing University of Chinese Medicine, China Ming-Kuei Lu, China Medical University, Taiwan

#### \*Correspondence:

Till Nierhaus till.nierhaus@fu-berlin.de Daniel Pach daniel.pach@charite.de

†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Perception Science, a section of the journal Frontiers in Neuroscience

Received: 19 November 2018 Accepted: 08 February 2019 Published: 11 March 2019

#### Citation:

Nierhaus T, Chang Y, Liu B, Shi X, Yi M, Witt CM and Pach D (2019) Somatosensory Stimulation With XNKQ Acupuncture Modulates Functional Connectivity of Motor Areas. Front. Neurosci. 13:147. doi: 10.3389/fnins.2019.00147 Xingnao Kaiqiao (XNKQ) acupuncture is an acupuncture technique used for stroke patients. In 24 healthy volunteers, we applied this complex acupuncture intervention, which consists of a manual needle-stimulation on five acupuncture points (DU26 unilaterally, PC6, and SP6 bilaterally). XNKQ was compared to three control conditions: (1) insertion of needles on the XNKQ acupuncture points without stimulation, (2) manual needle-stimulation on five nearby non-acupuncture points, and (3) insertion of needles on the non-acupuncture points without stimulation. In a within-subject design, we investigated functional connectivity changes in resting-state functional magnetic resonance imaging (fMRI) by means of the data-driven eigenvector centrality (EC) approach. With a 2 × 2 factorial within-subjects design with two-factor stimulation (stimulation vs. non-stimulation) and location (acupuncture points vs. non-acupuncture points), we found decreased EC in the precuneus after needle-stimulation (stimulation<non-stimulation), whereas the factor location showed no statistically significant EC differences. XNKQ acupuncture compared with needle-stimulation on non-acupuncture points showed decreased EC primarily in subcortical structures such as the caudate nucleus, subthalamic nucleus, and red nucleus. Post-hoc seed-based analysis revealed that the decrease in EC was mainly driven by reduced temporal correlation to primary sensorimotor cortices. The comparison of XNKQ acupuncture with the other two (non-stimulation) interventions showed no significant differences in EC. Our findings support the importance of the stimulation component of the acupuncture intervention and hint toward the modulation of functional connectivity by XNKQ acupuncture, especially in areas involved in motor function. As a next step, similar mechanisms should be validated in stroke patients suffering from motor deficits.

#### ClinicalTrials.gov ID: NCT02453906

Keywords: resting-state fMRI, acupuncture, functional connectivity, centrality, stroke, red nucleus, precuneus

# BACKGROUND

The acupuncture procedure consists of at least two components: stimulation and point location (Nierhaus et al., 2016; Langevin and Wayne, 2018). According to Chinese Medicine (TCM) theory, different types of stimulation will provide different clinical effects. The stimulation of the needle is mostly accompanied by a needle sensation that is called "deqi" in Chinese Medicine (Kong et al., 2007; Pach et al., 2011). Stimulation that elicited deqi was shown in a PET study to increase blood flow in the hypothalamus, insula, and subcortical structures compared with minimal or non-stimulation after needle insertion (Hsieh et al., 2001). Another study showed that acupuncture impacted selective attention networks, enhancing the efficiency of the alerting and executive control networks, and that acupuncture had a significantly greater effect on the alerting network compared to painful stimulation (Liu et al., 2013). Therefore, the subjective quality and the intensity of the stimulation seem to have an impact on the brain activity changes observed (Hui et al., 2005, 2009; Huang et al., 2012).

The role of location or point specificity in acupuncture is still controversial (Choi et al., 2012; Langevin and Wayne, 2018) and might depend on whether this role is evaluated (i) from the perspective of acupuncture with its concept of meridians and extra points or (ii) from the perspective of modern anatomy and physiology, which can take into account dermal, muscular, and neural components as well as connective tissue and chemical aspects (Nierhaus et al., 2016). In a previous trial of our group, point-specific cerebral responses were shown for one acupuncture point (ST36) in comparison to two control locations (Nierhaus et al., 2015b; Long et al., 2016).

Numerous neuroimaging studies that evaluated the impact of acupuncture on the brain hinted toward specific brain activity and functional connectivity changes due to acupuncture (Dhond et al., 2007; Huang et al., 2012; Chae et al., 2013). Most of these studies either investigate manual acupuncture only on one single point or investigate the effects of electroacupuncture (Huang et al., 2012). The latter applies an electrical current between acupuncture needles inserted into the skin and is therefore easier to evaluate due to its better potential for blinding, standardization, and easier simultaneous multipoint application. However, in most clinical settings (in China and the West), manual needle-stimulation on multiple acupuncture points is applied.

Xingnao Kaiqiao (XNKQ) acupuncture is a semi-standardized manual acupuncture technique developed in Tianjin, China, by Professor Shi Xuemin using a specific set of acupuncture points and strong needle-stimulation for different neuropathological conditions such as acute and chronic stroke symptoms (Shi, 2013) and multiple sclerosis. In a clinical setting, it was shown that stroke patients suffering from motor deficits react especially well to XNKQ acupuncture (杜蓉 et al., 2015). This suggests an impact of XNKQ on central mechanisms. However, modulation of brain activity following XNKQ acupuncture has not yet been fully investigated.

In the present study, we aimed to evaluate the impact of XNKQ acupuncture (and its components "stimulation" and "point location") on resting-state functional MRI connectivity. For this, we developed a 2 × 2 design that varied the stimulation component (stimulation vs. non-stimulation) and the location component of acupuncture (acupuncture points vs. non-acupuncture points). We applied data-driven eigenvector centrality mapping (ECM) to evaluate functional connectivity differences for the stimulation component (comparison of stimulated and non-stimulated acupuncture conditions) as well as the point location component (comparison of conditions with acupuncture points and non-acupuncture points).

We had assumed that it would be possible to detect a difference between stimulated and non-stimulated acupuncture conditions as well as between acupuncture conditions which used the established acupuncture point location according to Chinese medicine and conditions which used non-acupuncture point locations. Moreover, we hypothesized that XNKQ acupuncture differed from the three control conditions.

# MATERIALS AND METHODS

#### Subjects

We studied 24 healthy volunteers between 18 and 36 years of age (26.1 ± 4.3 years (SD); 12 females). They gave written informed consent to participate in the experiment according to the declaration of Helsinki. The ethics committee of Charité - Universitätsmedizin Berlin approved the study (Ethics No EA1/338/14) and the study was registered (ClinicalTrials.gov NCT02453906).

Prior to participation, all volunteers underwent a clinical neurological examination and they confirmed they were not taking any medications for acute or chronic diseases.

According to the Edinburgh inventory for the assessment of handedness (Oldfield, 1971), all subjects were right-handed [laterality score: 90.1 ± 17.3 (SD) over a range of −100 (fully left-handed) and +100 (fully right-handed)].

### Design

In a 2 × 2 factorial within-subject design we evaluated four different interventions in four different sessions (four different days with at least 24 h in between each) in a random order: (a) XNKQ acupuncture as manual needle-stimulation on five acupuncture points (DU26 unilaterally, PC6 and SP6 bilaterally); (b) insertion of needles on the five XNKQ acupuncture points without stimulation; (c) manual needle-stimulation on five nearby non-acupuncture points; and (d) insertion of needles on five non-acupuncture points without stimulation. The intervention had a duration of 5 min and was applied in the MRI scanner room. Resting-state fMRI was acquired before and after each intervention to compare changes (post minus pre) in functional connectivity between interventions. The break between pre- and post-resting state scans was about 10 min. Before informed consent, subjects were informed that they would receive acupuncture with five needles at the lower leg, the forearm, and above the upper lip on four separate days, on 2 days with stimulation of the needle and on the other two without. Subjects were blinded regarding the point specificity (acupuncture points vs. non-acupuncture points).

#### Point Locations

The following acupuncture points have been used: PC6 (nei guan, bilateral), DU26 (ren zhong, unilateral), and SP6 (san yin jiao, bilateral) (Shi, 2002, 2013), see **Figure 1**.

DU26 is located at the junction of the upper 1/3 and middle 1/3 of the philtrum. PC6 is located 2 cun above the transverse crease of the wrist, between the tendons of radial wrist flexor and palmaris longus. SP6 is located 3 cun above the tip of the medial malleolus, behind the posterior border of the medial aspect of the tibia (Shi, 2002).

The control points have been used in an earlier study on XNKQ acupuncture (李筱媛 and 李军, 2009) and were chosen after a discussion process with acupuncture experts. They are not located on a meridian or above a main nerve and are within a radius of 2.5 cm of the respective acupuncture point (**Figure 1**). Control point 1 (control of PC 6) is located laterally to PC 6, between the lung meridian of hand-taiyin and the pericardium meridian of hand-jueyin (李筱媛 and 李军, 2009). Control point 2 (control of DU 26) is located on the vertical line of the mouth, left-horizontal to DU26 (李筱媛 and 李军, 2009). Control point 3 (control of SP 6) is located six cun above the tip of the medial malleolus, between the spleen meridian of foot-taiyin and the liver meridian of foot-jueyin. It is three cun above the tip of the medial malleolus, in front of the inner of the tibia, 1.25 cun in front of SP 6 (李筱媛 and 李军, 2009).

## Acupuncture Procedures

The acupuncture was performed while the subjects lay in a supine position on the scanner bed by an acupuncturist from Tianjin University of Traditional Chinese Medicine (YC) trained for 12 years and with 8 years of clinical experience. The acupuncturist was trained directly by the developer of the XNKQ acupuncture and had long-time experience in the application of XNKQ acupuncture in patients in China and was also familiar with German study settings. For the acupuncture, sterile, single use, individually wrapped acupuncture needles (0.20 × 30 mm; titanium, DongBang, Acupuncture, Inc., Boryeong, Korea) were used.

For the XNKQ acupuncture PC6 was punctured bilaterally to a depth of 0.5–1.0 cun and stimulated with the reducing method by lifting and thrusting with simultaneous twirling manipulation for 1 min (twirling anticlockwise with the left hand and clockwise with the right hand). After this, DU26 was punctured obliquely toward the nasal septum to a depth of ∼0.3–0.5 cun with birdpecking needling until the eyes became wet or developed tears. Subsequently, SP6 was punctured on both sides obliquely along with the medial border of the tibia to a depth of ∼0.5–1.0 cun, with lifting and thrusting reinforcing manipulation, thrusts with heavy strength and lifting with gentle strength for 1 min. The needles were removed directly after stimulation [called "quick needles" technique (Shi, 2013)].

Control condition 1 consisted of the insertion of needles on the same five acupuncture points in the same order as used for XNKQ acupuncture (PC6 bilaterally, DU26 unilaterally, SP6 bilaterally), but without needle-stimulation.

Control condition 2 consisted of manual needle-stimulation on the five nearby non-acupuncture points (control point 1 bilaterally, control point 2 unilaterally, control point 3 bilaterally), identically to the XNKQ needle-stimulation.

Control condition 3 consisted of the insertion of needles on the five non-acupuncture points (control point 1 bilaterally, control point 2 unilaterally, control point 3 bilaterally) without manual needle-stimulation.

Needle sensation as a proxy for deqi and pain sensation was measured after each session by the Massachusetts General Hospital Acupuncture Sensation Scale [MASS, (Kong et al., 2007)].

# MRI Data Acquisition

Before all measurements, participants were instructed to keep the eyes open and to stay relaxed. Data was acquired using a 3T Tim Trio Siemens MRI System (Siemens Medical, Erlangen, Germany) equipped with a 12-channel head coil. For restingstate fMRI images, we used a T2<sup>∗</sup> -weighted echo planar imaging (EPI) sequence (37 axial slices, in-plane resolution is 3 × 3 mm, slice thickness = 3 mm, flip angle = 70◦ , gap = 0.3 mm, repetition time = 2,000 ms, echo time = 30 ms). A structural image was acquired for each participant using a T1-weighted MPRAGE sequence (repetition time = 1,900 ms, inversion time = 900 ms, echo time = 2.52 ms, and flip angle = 9, voxel size 1 × 1 × 1 mm). Subjects' heads were immobilized by cushioned supports, and they wore earplugs to protect against MRI gradient noise throughout the experiment.

#### Resting-State fMRI Data Analysis

We removed the first ten volumes of each resting-state scan (RS\_pre and RS\_post for each subject and all four interventions) to account for adaptation of the participant to scanner noise and environment. We performed slice time correction, head motion correction, and spatial normalization to MNI152 space with SPM12 (www.fil.ion.ucl.ac.uk/spm/). The toolbox REST (www.restfmri.net) was used for temporal band-pass filtering (0.01–0.08 Hz). We did not regress out the global mean signal since this step might affect the correlation between time courses (Buckner et al., 2009; Lohmann et al., 2010; Fransson et al., 2011; Taubert et al., 2011). The anatomical T1-images were normalized to MNI152 space and then segmented into gray matter, whiter matter and cerebral spinal fluid (CSF). Average masks were generated for gray matter, white matter, and CSF derived from the segmented T1 images of all subjects. Principal component analysis (CompCor) was done by the DPABI toolbox (toolbox for Data Processing & Analysis of Brain Imaging, http://becs.aalto. fi/~eglerean/bramila.html) within the CSF/white matter mask on the resting-state data (Behzadi et al., 2007). The first five principal components and six head motion parameters were used as nuisance signals to regress out associated variance. We did not apply spatial smoothing before the centrality analysis, as this could generate artificially high correlation coefficients (Zuo et al., 2012).

To compare differences in head motion across restingstate scans, we calculated the frame-wise displacement (FD) using BRAMILA tools (Power et al., 2012) (http://becs.aalto. fi/~eglerean/bramila.html). The average FD for all scans was examined with two two-factorial ANOVAs, including (a) the factors "session" (1–4) and "time" (pre-post) and (b) the factors "condition" and "time."

We used the data-driven ECM approach to characterize whole-brain functional connectivity without prior assumptions (Nierhaus et al., 2015a; Long et al., 2016; Antonenko et al., 2018). This graph theoretical network approach quantifies the correlation of each voxel with all other voxels in the brain, aiming to identify how "central" (or prominent) this region is within the whole-brain network (Lohmann et al., 2010). For each individual resting-state scan, the EC map has been generated within the gray matter mask by using fastECM, which provides a more efficient way to perform the centrality analysis without calculating the voxel-wise correlation matrix (Wink et al., 2012). Z-standard transformation (i.e., for each voxel, subtract the mean value of the whole brain then divide by the standard deviation of the whole brain) and 6 mm FWHM smoothing was performed on the individual ECM maps (Zuo et al., 2012; Yan et al., 2013). To evaluate the impact of the four different acupuncture conditions on EC, we analyzed the difference EC-maps (post minus pre) in a 2 × 2 flexible factorial design [correlated repeated measures (Gläscher and Gitelman, 2008)] with the factors "stimulation" (stimulation vs. non-stimulation of the needles) and "location" (acupuncture points vs. non-acupuncture points) within SPM12 with age, gender, and MASS index as covariate. For statistical analysis, we determined cluster-extent thresholds with Monte Carlo simulation (AlphaSim procedure) as implemented in Neuroelf Version 1.1 (http://neuroelf.net) using a family-wise error (FWE) cluster level correction of pFWE <0.05.

The ECM analysis identifies brain regions with altered overall (whole-brain) connectivity, however it does not show to which specific brain areas the connectivity has changed. A complementary seed-based functional connectivity analysis can be applied to characterize the "origin" of observed EC changes.

The comparison of XNKQ with stimulation on nonacupuncture points revealed EC changes in subthalamic brain regions which are known to be involved in motor control, such as the subthalamic nucleus and red nucleus. In order to show that this result might be connected to cortical motor areas, we have performed a complementary seed-based analysis. As two examples, we chose the red nucleus and subthalamic nucleus as seed regions: For all resting-state scans, we calculated the temporal correlation between the time series of the seed region and all other voxels within the gray matter mask. The difference in the resulting correlation maps were analyzed in a 2 × 2 flexible factorial design with the factors "stimulation" and "location" with age, gender, and MASS index as covariate (similar to the ECM analysis). It should be noted that no formal statistics was applied in this step (double dipping), rather it was used to identify the most affected connections, which contributed most to the statistically significant effect of the ECM analysis. For visualization of the most prominent clusters, we used voxel-wise whole brain FWE correction with peak-level p < 0.05.

# RESULTS

#### Head Motion

There was no significant difference in head motion (mean FD) across all resting-state scans (4 pre- and 4 post-acupuncture scans) for either the comparison between the four session or the comparison between the four acupuncture conditions (all p > 0.46). Over all 8 scans, the mean FD was 0.14 ± 0.06 mm [mean ± std] and the average percentage of volumes exceeding an FD-threshold of 0.5 mm was 1.9 ± 2.8% [mean ± std].

## Needle Stimulation Decreases Eigenvector Centrality in Precuneus

For the factor "stimulation," we found a significant cluster of decreased eigenvector centrality (stimulation < nonstimulation, **Figure 2**) in the parietal lobe mainly including the precuneus (maximum T value −4.01, left hemisphere (LH), MNI-coordinates X = −3, y = −78, Z = 54) and the cuneus (Tmax= −4.68, LH, X = −6, y = −81, Z = 42).

Further evaluation of the four different conditions showed that the decreased eigenvector centrality in the precuneus was driven by the stimulation of non-acupuncture points (non-points with stimulation, npws). It was significant for the stimulation of

non-acupuncture points compared to the two non-stimulation conditions (npws vs. points non-stimulation, pns and npws vs. non-points non-stimulation, npns), and it was not significant when XNKQ was compared to the two non-stimulation conditions (XNKQ vs. pns and XNKQ vs. npns). However, the comparison of the two stimulation conditions (XNKQ vs. npws) also showed no significant difference in precuneus.

The factor location showed no statistically significant different eigenvector centrality. Additionally, no effect was found for the comparison of the non-stimulation conditions (pns vs. npns).

### XNKQ Modulates Functional Connectivity of Motor Areas

The EC analysis for XNKQ compared with the stimulation of non-acupuncture points (XNKQ vs. npws) showed one significant cluster (mainly subcortical) of decreased centrality (**Figure 3A**). This cluster included subcortical structures such as the caudate (right hemisphere (RH), Tmax = −3.71, MNIcoordinates X = 12, y = 15, Z = −3), the subthalamic nucleus (RH: Tmax = −3.57, X = 9, y = −12, Z = −18), the thalamus (LH: Tmax = −3.48, X = −6, y = −27, Z = −3; RH: Tmax = −3.42, X = 15, y = −24, Z = −6), the lentiform nucleus (RH, Tmax = −3.80, X = 27, y = 3, Z = −12), and the red nucleus (RH, Tmax = −3.13, X = 9, y = −30, Z = −18), as well as the claustrum (RH, Tmax = −5.06, X = 36, y = 0, Z = −15), the anterior cingulate cortex (RH, Tmax = −2.94, X = 12, y = 36, Z = 9), and the cingulate cortex (RH, Tmax = −2.74, X = 9, y = −3, Z = 30). No significant effect was found for XNKQ compared to the two non-stimulation conditions (XNKQ vs. pns and XNKQ vs. npns).

The complementary seed-based functional connectivity analysis revealed that the decreased EC in subthalamic areas was mainly driven by decreased temporal correlation of the respective seed region to primary sensorimotor areas (S1/M1); the red nucleus showed significantly decreased correlation mainly to bilateral S1/M1 (RH: Tmax = −6.00, X = 42, y = −19, Z = 55; LH: Tmax = −6.05, X = −51, y = −22, Z = 46; **Figure 3B**) and the subthalamic nucleus mainly to left S1/M1 (LH: Tmax = −7.04, X = −39, y = −22, Z = 49; **Figure 3C**).

# Needle Sensation

Needle sensation was measured for all four conditions using the MASS index: XNKQ mean 4.17 95%CI[3.34;5.01]; npws 3.44[2.54;4.34]; pns 3.52[2.65;4.39]; and npns 2.29[1.58;3.00]. The statistical analysis showed a significant main effect for both factors (stimulation and location) in our 2 × 2 factorial design (stimulation vs. non-stimulation conditions p=0.030; acupuncture points vs. non-acupuncture points p = 0.018). The comparison of the four conditions showed significant differences only for npns comparisons (XNKQ vs. NPNS, p = 0.001; NPWS vs. NPNS p = 0.043; PNS vs. NPNS p = 0.028).

# DISCUSSION

To elucidate cerebral effects of manual acupuncture using more than one acupuncture point, we applied XNKQ acupuncture and three control conditions in a neuroimaging study in healthy subjects. In a 2 × 2 factorial within-subject design, we investigated the impact of the factors stimulation and location, and of XNKQ acupuncture specifically, on resting-state functional connectivity.

While the factor location appears to have no significant effect on centrality, we found decreased eigenvector centrality in the precuneus for the factor stimulation. This result was driven by the stimulation of non-acupuncture points, as the comparison of XNKQ acupuncture with the two non-stimulation interventions showed no significant differences. However, when comparing XNKQ acupuncture with manual needle-stimulation on nonacupuncture points, we found significantly decreased functional connectivity for areas involved in motor function.

Our results support the assumption that (1) needlestimulation drives the cerebral effects, (2) point location only impacts connectivity when the acupuncture points are stimulated, and (3) XNKQ acupuncture, as a complex form of acupuncture, modulates functional connectivity in motor areas minutes after the acupuncture.

Our study design shows strengths and weaknesses that should be considered when interpreting the results. With our factorial within-subject design including a relatively large number of healthy subjects measured on four separate days, we were able to separate the factors stimulation and location, as well as to reduce variance and carry over effects. The design we chose aimed at a study question relevant for the understanding of clinical acupuncture which primarily uses manual needle-stimulation on more than one point. So far, numerous imaging studies evaluate only one-point acupuncture and/or apply electro-acupuncture (Huang et al., 2012; Chae et al., 2013). Such a setting might be better suited for standardization and blinding options but does not represent clinical acupuncture in usual care settings.

Although in our study design only five locations were acupunctured for a relatively short time interval (hence still does not fully represent the clinical setting), we were able to observe cerebral changes that illustrate the impact of a complex acupuncture, such as XNKQ acupuncture, on the brain. Because the duration of the sustained effect of acupuncture is not known, we do not know whether pausing the intervention for at least 24 h is sufficient to avoid carry-over effects. However, the order of interventions was randomized to minimize the risk of a systematic impact.

We chose a design that can evaluate rapid effects on restingstate functional connectivity, which are observable after the intervention, but not the instant evoked responses of the different acupuncture conditions. This design decision may decrease the sensitivity to identify differences between the conditions. For the evaluation of instant effects, an event-related design with needling during the scanning phase would have been necessary. However, this would be much more difficult to achieve, especially when evaluating manually stimulated acupuncture on multiple acupuncture points.

Although we included a relatively large number of subjects, the sample size might be too low to show robust effects. The level of statistical significance we chose was liberal. For future studies with a similar design, an even larger sample size might be recommended, especially for the evaluation of effects in patients. Based on our findings, it is now possible to evaluate a hypothesis-driven approach, which might create more robust results in contrast to the data-driven approach we chose as the primary analysis.

In our study, we included only healthy subjects for an easyto-standardize setting to understand the neurophysiology of the different acupuncture conditions. However, usually XNKQ acupuncture is only applied in a clinical setting for patients with neurological deficits such as multiple sclerosis or stroke as part of a multi-component intervention that also includes physiotherapy. Therefore, it is possible that effects in healthy subjects differ from effects expected in patients. However, a study on patients would have created more variance and is more prone to bias, which is not ideal as a first step.

However, only the subjects were blinded for the applied acupuncture conditions as well as the researchers analyzing the data during the first stages of analyses. The acupuncturist applying the manual acupuncture could not be blinded for the different conditions and this might have had an effect on needlestimulation. However, we measured needle sensation as a proxy for stimulation strength and included it into our statistical model.

The choice of control points for an acupuncture study is very challenging because it is still not clear what constitutes an acupuncture point, and it is difficult to combine the traditional concept of acupuncture with modern anatomy (Nierhaus et al., 2016; Langevin and Wayne, 2018). Therefore, it is possible that the control points chosen for our study were not inert, either from the perspective of acupuncture or from the perspective of anatomy.

To our surprise, we found no significant differences between XNKQ and the two non-stimulated acupuncture conditions. This means that the main effect that we found in precuneus for the factor "stimulation" is driven by the needle-stimulation on non-acupuncture points. However, the comparison within the two stimulated acupuncture conditions (XNKQ vs. stimulation on non-acupuncture points) revealed a significant difference mainly in subcortical regions—that is not observed in the other comparisons. It seems that the stimulation of acupuncture points (XNKQ) induces subcortical connectivity changes (decreased centrality) that are opposite to the connectivity changes induced by needle-stimulation of "neutral" non-acupuncture points. This result supports the view that both "stimulation" and "point location" contribute to the acupuncture effect.

Other studies have also shown that acupuncture can affect functional connectivity of brain networks such as the default mode network (DMN) or sensorimotor network in pain, stroke, or mental conditions (Dhond et al., 2008; Bai et al., 2009; Hui et al., 2009; Chae et al., 2013; Napadow et al., 2013; Liang et al., 2014; Li et al., 2014; Zhao et al., 2014; Deng et al., 2016). Numerous studies could show that the precuneus (as part of the DMN) is frequently affected by acupuncture (Chae et al., 2013; Nierhaus et al., 2015b). So far, the specific role of the precuneus is not fully understood, however for pain it might be involved in the assessment and integration of pain (Goffaux et al., 2014). The reduced centrality that we found for the precuneus in resting-state after needle-stimulation might hint toward such cerebral processing induced by the strong and (sometimes) painful stimulation.

Functional connectivity is regularly affected by stroke (Grefkes and Fink, 2011; Rehme and Grefkes, 2013; Baldassarre et al., 2016; Almeida et al., 2017), and brain imaging studies have revealed functional brain reorganization in relation to recovery (Schaechter, 2004; Almeida et al., 2017). In a stroke mouse model, it could be shown that multisensory input can improve functional recovery and resting-state functional connectivity after stroke (Hakon et al., 2018). Acupuncture can be regarded as a complex somatosensory input with needle-stimulation. According to a study by Li et al. both acupuncture and somatosensory stimuli to the contralesional side produce hyperactivation in the ipsilesional primary sensorimotor cortex and SII (Dhond et al., 2007). A study by Schaechter et al. (2007) revealed that after acupuncture intervention (verum or sham), patients exhibited changes in motor cortex activity associated with the stroke-affected hand that were positively correlated with changes in somatosensorymotor function of the affected upper limb. There was a trend toward greater increases in motor cortex activity in patients treated with verum acupuncture than sham acupuncture (Dhond et al., 2007). XNKQ is an acupuncture technique specially designed for different neuropathological conditions such as acute and chronic stroke symptoms (Shi, 2013), and moreover seems to impact on patients suffering from motor deficits. Therefore, our results are well in line with the existing literature and support the assumption that XNKQ affects the motor system.

Our data-driven analysis (ECM) showed that XNKQ acupuncture affects functional connectivity of subcortical areas (e.g., red nucleus or subthalamic nucleus) that are known to be involved in motor function (Milardi et al., 2016). This is supported by our complementary seed-based analysis, which showed reduced functional connectivity between the seed regions and primary sensori-motor areas after XNKQ acupuncture. Maybe this reduced functional connectivity in the motor systems allows for a better

# REFERENCES


reorganization during recovery from motor deficits in stroke. Of course, it needs to be proven if this can be translated to stroke patients.

# CONCLUSION

Our findings support the importance of the stimulation component of the acupuncture intervention and hint toward the modulation of functional connectivity by XNKQ acupuncture, especially in areas involved in motor function. As a next step, similar mechanisms should be validated in stroke patients suffering from motor deficits.

### DATA AVAILABILITY

The datasets generated for this study are available on request to the corresponding author.

# AUTHOR CONTRIBUTIONS

TN, YC, CW, and DP conceived and designed the experiments. TN, YC, BL, and DP performed the trial. TN and DP analyzed the data. TN, DP, YC, and CW wrote the first draft of the paper. TN, YC, BL, XS, MY, CW, and DP discussed the data, revised the paper, and approved the final version.

# FUNDING

YC and BL had a scholarship from First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, China.

### ACKNOWLEDGMENTS

We thank Christian Kainz for his support during the experiments.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Nierhaus, Chang, Liu, Shi, Yi, Witt and Pach. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Acupuncture for Histamine-Induced Itch: Association With Increased Parasympathetic Tone and Connectivity of Putamen-Midcingulate Cortex

Seorim Min1,2† , Koh-Woon Kim3,4† , Won-Mo Jung1,2, Min-Jung Lee<sup>2</sup> , Yu-Kang Kim1,2 , Younbyoung Chae1,2, Hyangsook Lee1,2 and Hi-Joon Park1,2 \* †

 Department of Science in Korean Medicine, Graduate School, College of Korean Medicine, Kyung Hee University, Seoul, South Korea, <sup>2</sup> Acupuncture and Meridian Science Research Center, Kyung Hee University, Seoul, South Korea, Department of Korean Rehabilitation Medicine, College of Korean Medicine, Kyung Hee University, Seoul, South Korea, East-West Medical Research Institute, Kyung Hee University, Seoul, South Korea

#### Edited by:

Florian Beissner, Hannover Medical School, Germany

#### Reviewed by:

Hee Young Kim, Daegu Haany University, South Korea Yi-Hung Chen, China Medical University, Taiwan

> \*Correspondence: Hi-Joon Park acufind@khu.ac.kr †These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Perception Science, a section of the journal Frontiers in Neuroscience

Received: 27 July 2018 Accepted: 25 February 2019 Published: 12 March 2019

#### Citation:

Min S, Kim K-W, Jung W-M, Lee M-J, Kim Y-K, Chae Y, Lee H and Park H-J (2019) Acupuncture for Histamine-Induced Itch: Association With Increased Parasympathetic Tone and Connectivity of Putamen-Midcingulate Cortex. Front. Neurosci. 13:215. doi: 10.3389/fnins.2019.00215 Previous studies have suggested that acupuncture is effective for ameliorating itch intensity. However, factors associated with the antipruritic effects of acupuncture have yet to be clarified. In a randomized, sham-controlled, crossover trial, we investigated the antipruritic effects of acupuncture against histamine-induced itch in healthy volunteers. Autonomic changes using heart rate variability (HRV) and brain connectivity using functional magnetic resonance imaging (fMRI) were also assessed to identify physiological factors associated with the acupuncture response. Acupuncture significantly reduced itch intensity and skin blood perfusion as assessed by laser Doppler perfusion imaging compared to sham control, indicating the antipruritic effects of acupuncture. In responder and non-responder analysis, the power of normalized high frequency (HF norm) was significantly higher, while the power of normalized low frequency (LF norm) and LF/HF ratio were significantly lower in responders compared to non-responders, suggesting the acupuncture response involved parasympathetic activation. In fMRI analysis, the putamen and the posterior part of the midcingulate cortex (pMCC) were positively connected to itch and negatively correlated with itch intensity in responders. These results suggest that parasympathetic activity and functional connectivity of the putamen and pMCC could be associated with antipruritic response to acupuncture.

Keywords: acupuncture, histamine, itch, heart rate variability, functional magnetic resonance imaging, putamen, midcingulate cortex

# INTRODUCTION

The sensation of itch, defined as "an unpleasant sensation associated with the desire to scratch," is the most prevalent subjective symptom of inflammatory skin diseases and cause of suffering in many dermatologic and some allergic conditions (Charlesworth and Beltrani, 2002). Itching leads to scratching, consequently interfering with skin barrier function. With the loss of cutaneous integrity

and softening of the skin surface, a vicious cycle of skin damage and inflammation occur. This cycle induces a state of complex dermatitis, creates more itching, resulting in a scratch-itch cycle, and leads to serious quality-of-life problem. Patients with atopic dermatitis (AD) suffer from severe itch, most notably hampering their quality of sleep (Arck and Paus, 2006; Bender et al., 2008).

The autonomic nervous system (ANS) is the primary mechanism for the response to external stimuli. The response results in a fight-or-flight system from the sympathetic nervous system (SNS) or freeze-or-dissociate response from parasympathetic nervous system (PNS). Some studies suggest that the ANS functions in the itch mechanism (Stander and Steinhoff, 2002; Chida et al., 2007; Cicek et al., 2008). A previous study (Kirchner et al., 2002) found that the vagus nerve, which is the main parasympathetic outflow, controls histamine-induced itch, probably by a central mechanism. The brain circuitry involved in the itch sensation is an active topic of study of the peripheral and central sensitization for itch (Desbordes et al., 2015). Itch is usually perceived when inflammatory mediators activate a specific subset of peripheral sensory nerve endings, an activation that is transmitted to the brain via the spinothalamic tract (Andrew and Craig, 2001; Davidson et al., 2007; Napadow et al., 2014).

Many studies have focused on itch itself, but appropriate treatments are far from satisfactory. A systematic review (Yu et al., 2015) suggested that acupuncture is effective for ameliorating itch intensity in itch-related diseases. Topical antihistamines have limited use for acute itch only and have relatively high costs and risks for allergic contact dermatitis sensitization (Callahan and Lio, 2012). A previous study showed that acupuncture has better antipruritic effects than third generation antihistamines (Napadow et al., 2014). Another study comparing acupuncture and second generation antihistamine found that acupuncture demonstrated a greater effect during peak itch intensity, while antihistamine had a stronger effect during lower itch intensity suggesting different mechanisms of action (Pfab et al., 2012a). Acupuncture for experimentally induced itch in healthy participants (Belgrade et al., 1984; Lundeberg et al., 1987; Pfab et al., 2005) and patients with AD (Pfab et al., 2010, 2011, 2012a; Napadow et al., 2014) has a significant antipruritic effect compared to placebo. Possible mechanisms for itch reduction by acupuncture in patients with AD include reduction of in vitro allergen-induced basophil activation and modulation of neurotransmitters, peripheral hormone levels, and brain areas that are involved in itch processing (Pfab et al., 2013). Furthermore, acupuncture may reduce itch by creating an inhibitory input called gate-control theory through the response of the sensory nerve with a one-toone correspondence between dermatomes and spinal segments (Bear et al., 2016). However, factors related to the antipruritic effects of acupuncture remain unclear.

Acupuncture stimulation influences organs and functions including ANS activity (Kawakita et al., 2006) and brain processing (Dhond et al., 2007a,b). Several studies suggest that acupuncture influences ANS functions such as blood pressure, skin conductance, skin temperature, heart rate, and heart rate variability (HRV) (Malliani et al., 1991; Lee et al., 2010; Chung et al., 2014). Studies found that press needle stimulation induces alterations in vagal function (Noda et al., 2015) and acupuncture stimulation at HT7 affects cardiac autonomic neural regulation in healthy individuals, mainly via the PNS (Huang et al., 2015). Acupuncture activates the PNS and default mode network connectivity (Dhond et al., 2008). It induces changes in heart rate and brain stem structures (Beissner et al., 2012).

Using functional magnetic resonance imaging (fMRI), we investigated changes in cerebral perfusion to identify the critical brain areas mediating the antipruritic effect of acupuncture. The fMRI method for imaging brain functions is increasingly used to investigate dynamic brain patterns with acupuncture stimulation (Malliani et al., 1991; Park et al., 2005; Dhond et al., 2007b; Colombo et al., 2015). In clinics, acupuncture treatment typically involves a phase of needle retention following acupuncture insertion and manipulation. Thus, functional connectivity indicating characteristics of acupuncture is better suited for a block design of fMRI analysis. Functional connectivity analysis explores the relationship between neuronal activation patterns that functionally linked, but anatomically separated brain areas (Colombo et al., 2015). The striatum, which consists of the putamen and the caudate, is critical for striato-thalamo-cortical circuits for itch. An fMRI study (Napadow et al., 2014) found itch reduction following acupuncture is associated with reduced activation of the putamen response. Thus, our study chose the putamen as a seed ROI.

Since individually differing responses to acupuncture have been reported and itch is a subjective symptom, an individualistic approach is needed (Park et al., 2005; Chae et al., 2006). Our individualistic approach classified participants into responders and non-responders (Scholz and Woolf, 2002; Woodcock et al., 2007; Schork, 2015). Analysis of the differences between responders and non-responders strengthens therapeutic effects by managing response-related factors (Kim et al., 2007).

The aim of this study was to test the antipruritic effects of acupuncture on experimentally induced itch. Subsequently, we investigated the factors that influenced these effects via ANS response and functional connectivity of the brain by analyzing responder and non-responder groups.

#### MATERIALS AND METHODS

The study was conducted in two separate steps. Study 1 was followed by Study 2.

#### Participants

For Study 1, 20 healthy participants between 18 and 50 years of age were recruited using an advertisement. Participants with no present history of asthma and AD were screened by telephone. Study 2 had 15 participants who met the schedule for fMRI scans and retested for MRI compatibility. All participants refrained from alcohol or caffeine for 12 h before experiments. Participants with skin problems on the stimulated site were excluded. Participants had to stop antihistamine medications at least 3 days before the study to avoid potential influence on normal itch perception or flare and wheal response to SPT, nevertheless, in case of no itch response, the applicable data would be excluded for analysis. Detailed explanations of experimental procedures such as the two conditions and four visits were given. This study was carried out in accordance with the recommendations of the guideline for clinical researches, Kyung Hee University Ethics Committee with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Kyung Hee University Ethics Committee (KHSIRB-15-056).

#### Experimental Design

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The study design was a randomized, crossover trial in which each participant served as his or her own control. Participants received acupuncture and placebo stimulation in random order. Session orders were determined with a random number table generated by Excel (Microsoft, Redmond, WA, United States). Each experiment was separated by 7 days and occurred at the same time of the day. Tests were carried out in winter, in January to March 2016, to minimize the influence of environmental allergens.

Study 1 was in a light-conditioned and quiet room. Room temperature was controlled at 23 ± 1 ◦C (mean ± SD). Demographic data including gender, age, height, and weight and psychological factors such as credibility, expectation, and fear of acupuncture and perception of bodily sensation were collected from all participants. After 15 min of resting, participants were seated on a chair throughout and remained comfortable in that position. Baseline skin blood perfusion and HRV measurements were obtained for 5 min and interventions were subsequently performed on the non-dominant side. Skin blood perfusion and HRV were obtained (A1 and A2 phase) for 10 min. After itch induction, HRV, skin blood perfusion and itch intensity were measured for 10 min (H1 and H2 phase). The intervention was removed and skin blood perfusion, HRV, and itch intensity were obtained (H3 and H4 phase) (**Figure 1A**).

For Study 2, room temperature was controlled at 11 ± 1 ◦C (mean ± SD). As participants lay in the magnet, they viewed a screen using prism glasses to respond to situations by pressing a button. After 15 min of resting, participants underwent three fMRI scanning sessions. Each session included a 4-min baseline resting state scan, intervention (5 min), and itch resting state scan (0–4, 4–8 min) (**Figure 1B**). The order of acupoints and rest periods within each session were counterbalanced and randomized across participants. Itch was induced as in Study 1. The study flow is depicted in **Figure 2**.

# Acupuncture and Placebo Control

For Study 1, manual acupuncture stimulation was used on the arm and leg of the participant's non-dominant side at LI11 (on the elbow at the midpoint of the end of the transverse cubital crease and lateral epicondyle of the humerus when the elbow is flexed); SP10 (on the medial aspect of the thigh, 6.6 cm above the medio-superior border of the patella, on the bulge of the medial portion of muscle quadriceps femoris when the knee is flexed); PC6 (on the palmar aspect of the forearm, 6.6 cm proximal to the middle point of the carpal fold, on the line connecting PC3 and PC7, between the tendons of muscle palmaris longus and muscle flexor carpi radialis); HT7 (on the wrist, at the ulnar end of the transverse crease, in the depression on the radial side of the tendon of the ulnar flexor muscle); and ST36 (on the anterior aspect of the lower leg, 9.9 cm below ST35, middle finger from the anterior crest of the tibia). Acupoints based on a standard acupuncture textbook, previous study, and clinical experience, are important for treating cutaneous itch. LI11 and SP10 are used frequently to treat itch in clinical and experimental studies (Pfab et al., 2005, 2010, 2011, 2012a). To induce activation of the PNS, HT7 (Huang et al., 2015) and PC6 (Huang et al., 2005; Li et al., 2005) were chosen. ST36 is the most frequently used acupoint for immune regulation and anti-inflammatory effects, and reducing SNS (Michikami et al., 2006). For Study 2, due to fMRI system structural problems, LI11 was excluded.

For Study 1, sterile stainless steel needles (0.20 mm in diameter, 30 mm in length; Haeng Lim Seo Won, South Korea) were inserted to depth (LI11, 20 mm; SP10, 15 mm; PC6, 15 mm; HT7, 6 mm; ST36, 20 mm) controlled by an empty guide tube and manipulated for 9 s repeated every 5 min over 20 min (**Figure 3A**). For Study 2, non-magnetic titanium sterile acupuncture needles (0.20 mm diameter, 40 mm length; DongBang Acupuncture Inc., Boryeong, South Korea) were used. Manipulation techniques were performed as tonifying and reducing, which involved bidirectional rotation (1 Hz). A previous study (Takakura et al., 1995; Min et al., 2015) showed repeated manipulation improves acupuncture effects and traditionally, manual acupuncture uses manipulation to reinforce effects (Kim et al., 2000). The same doctor of Korean medicine with a certified license performed all acupuncture treatments. Prior to baseline, skin was cleaned with alcohol at the acupoint and histamine stimulus locations.

As a placebo control, tactile stimulation was performed by gentle tapping with a size 5.46 von Frey filament (Touch-Test Sensory Evaluator Instructions, North Coast Medical, Inc., CA, United States) every 5 min over 20 min for Study 1 and over 5 min for Study 2. Placebo acupoints were on the arm and leg using the same meridian system, but not acknowledged as acupoints by textbooks. The non-acupoint for LI11 was on the lateral side of the arm at the midpoint of the line connecting LI11 and LI15. The non-acupoint for SP10 was on the medial aspect of the thigh at the midpoint of the line connecting SP10 and SP12. The non-acupoint for PC6 was on the palmar aspect of the forearm at the midpoint of the line connecting PC3 and PC7. The non-acupoint for HT7 was on the palmar aspect of the forearm at the midpoint of the line connecting HT3 and HT7. The non-acupoint for ST36 was on the anterior aspect of the lower leg in the center of the belly of the muscle tibialis anterior. Placebo procedures were carried out as acupuncture procedures. The von Frey filament and acupuncture materials were prepared in advance and placed on a tray and covered so materials could not be seen by participants. Locations of acupoints and nonacupoints were blinded by self-produced blinding box. More detailed procedures according to the Standard for Reporting Interventions in Clinical Trials of Acupuncture (STRICTA) are attached in **Supplementary Appendix 1**.

was subsequently performed on the non-dominant side. Skin blood perfusion and HRV were obtained (A1 and A2 phase) for 10 min. After itch induction, HRV, skin blood perfusion, and itch intensity were measured for 10 min (H1 and H2 phase). The intervention was removed and skin blood perfusion, HRV, and itch intensity were obtained (H3 and H4 phase). (B) In Study 2, after 15 min resting, participants underwent three fMRI scanning sessions. Scans for each session were for 4-min baseline resting state, intervention (A, 5 min), and itch resting state (H1, 0–4; H2, 4–8 min). HRV, heart rate variability; s, seconds; min, minutes; fMRI, functional magnetic resonance imaging.

# Itch Induction

After acupuncture procedures, 1% histamine (10 mg/ml histamine dihydrochloride, Lofarma, Milan, Italy) was applied to induce itch on the volar aspect of the forearm and medial aspect of the foot of the participant's non-dominant side using SPT. A single drop of histamine in aqueous solution was applied to the skin, followed by puncture with a lancet for SPT as in routine allergy diagnosis. The technique was performed by the same investigator.

### Itch Intensity

For Study 1, itch intensity was rated at 20-s intervals for 20 min after SPT. Participants were seated with the left arm resting on the cushion of a chair arm and the other hand selecting a number from 0 (no itch) to 10 (worst imaginable itch). One-third of the scale (3.3/10) was the intervention point "scratch threshold," above which the individual felt a strong desire to scratch, which was not allowed. Area under the rating curve above the scratch threshold was calculated as itch intensity multiplied by time (in min). For Study 2, participants could view a screen in the scanner through prism glasses in the supine position. Itch intensity was quantitatively expressed in percent of a visual analog scale (VAS) at 4 and 8 min after SPT. Participants reported perceived itch intensity by pressing one of four buttons in an MRI-compatible button box (Current DesignTM). Participants were familiarized with the rating procedure, which was a different reporting method than a previous study, with exercises before the fMRI session. This study did not record continuous ratings to minimize other effects.

# Skin Blood Perfusion Using Laser Doppler Perfusion Imaging

A PIM3 system (Perimed AB, Järfälla, Sweden) was used to measure skin blood perfusion in this study. The area of interest was 4.5 cm × 4.5 cm at the stimulus site on the non-dominant foot. The distance between the detector and the tissue was fixed to 45 cm with normal resolution. To minimize movement, the foot was fixed with a kapok-filled vacuum cushion. Skin blood perfusion was measured at baseline for 10 min after intervention (A1 and A2 phase), and 20 min post prick testing (H1, H2, H3, and H4 phase) in Study 1.

# Heart Rate Variability Using MP150

For electrocardiographic recordings, three electrodes were attached to participants' chests. The sampling rate of the electrocardiogram (ECG) was 1000 samples/s and signals were amplified with a BIOPAC (Biopac Systems, Inc., Goleta, CA, United States) ECG100C amplifier. HRV data were calculated from a series of 5-min epochs of ECG signals. Spectral HRV components were evaluated and obtained as absolute values of power (ms<sup>2</sup> ) based on frequency to one of three bands: very low frequency component (0.00–0.04 Hz), low frequency (LF) component (0.04–0.15 Hz), and high frequency (HF) component (0.15–0.40 Hz). HF and LF components of HRV were conventionally observed in normalized units. HF norm is an indicator of parasympathetic tone and LF norm is a measure of sympathetic regulation (Malliani et al., 1991; Camm et al., 1996). LF/HF ratio, an estimate of the balance between sympathetic and parasympathetic activities, was calculated from the absolute power of both frequency components.

### Blinding

At the end of the study, participants were asked if they believed they received "acupuncture treatment" or "placebo treatment." Bang's blinding index, ranging from −1 to 1, was calculated: 0 was random guessing (50% correct and 50% incorrect); 1 was complete unblinding (all responses correct); and −1 was all responses incorrect (Bang et al., 2010).

#### Statistical Analysis

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Statistical analyses were carried out using SPSS version 22.0 software (IBM SPSS Statistics, IBM Corporation, Armonk, NY, United States). Unless otherwise stated, data are mean values (95% confidence intervals). P-values <0.05 were considered statistically significant.

Data on itch intensity, skin blood perfusion, and HRV parameters were divided into 5-min intervals for ease of data management and analysis and changes were calculated using the formula: (change from baseline)/baseline X 100. Measurement times were divided into seven phases: (1) baseline, (2) A1 (0– 5 min after acupuncture), (3) A2 (5–10 min after acupuncture), (4) H1 (0–5 min after histamine stimulus), (5) H2 (5–10 min after histamine stimulus), (6) H3 (10–15 min after histamine stimulus), (7) H4 (15–20 min after histamine stimulus). For responder and non-responder analyses, entire samples were separated into responders (N = 10) and non-responders (N = 10) based on the mean of sum values from arm and foot itch intensity at H1 using peak value.

Analyses were performed after checking all parameters were normally distributed using the Shapiro–Wilk one-sample test. Itch intensities for entire measurements were analyzed by two-way analysis of variance (ANOVA). Effects of time and time × treatment interaction using data divided into 5 min intervals were analyzed by repeated measure ANOVA. If Mauchly's test of sphericity was significant, Greenhouse– Geisser correction for degrees of freedom was used. Differences between acupuncture and placebo conditions were evaluated using a paired sample t-test or Wilcoxon signed rank test. Blinding integrity was analyzed using a chi-square test. Differences between responder and non-responder groups were evaluated using independent sample t-test or Mann– Whitney test. Correlation analysis used Pearson's correlation for parametric analysis.

# fMRI Data Acquisition

Scanning used a 3-axis gradient head coil in a 3 Tesla Siemens MRI scanner with echo planar imaging. Structural scans were acquired using a magnetization prepared rapid gradient echo sequence with TR = 2000 ms, TE = 2.37 ms, flip angle 9 ◦ , field of view 240 mm, and slice thickness 1.0 mm. For resting state analysis, 37 slices were acquired with parameters: TR = 2000 ms, TE = 30 ms, flip angle 90◦ , field of view 240 mm, and slice thickness 4.0 mm, with in-plane spatial resolution at 3 mm × 3 mm × 3 mm.

#### fMRI Data Preprocessing and Analysis

This study focused only on resting state scans (baseline) before acupuncture and induced 8-min resting state scans (itch).

Preprocessing of resting state images used SPM12 software<sup>1</sup> implemented in a MATLAB suite (Mathworks, Inc., Natick, MA, United States). Preprocessing included slice time correction, head motion correction, coregistration to patients' structural images, segmentation, normalization, linear detrending, and smoothing (FWHM = 8 mm).

Functional connectivity analysis used the CONN toolbox<sup>2</sup> (Whitfield-Gabrieli and Nieto-Castanon, 2012). Time courses from components associated with white matter and cerebrospinal fluid were regressed from whole-brain gray matter activity and 12 motion regressors (6 realignment parameters and first derivatives) were used to control for correlations during movement. Data were filtered between 0.008 and 0.09 Hz and global brain signal was not subtracted.

Functional connectivity analysis used a seed-to-voxel approach. The left putamen was used as a seed. The segmentation mask of the left putamen in the Harvard-Oxford atlas is implemented in the CONN toolbox by default, so connectivity of the putamen with the anatomically defined brain areas was examined. The putamen mask used in our study (center MNI coordinates of X = 26, Y = 3, Z = −1) includes the MNI coordinates (X = −32, Y = −14, Z = 6) where is reported to be significantly correlated with itch rating in a previous study (Napadow et al., 2014). In first-level analysis, we produced a correlation map for each subject by extracting blood-oxygen-level dependent time courses from the putamen seed. Connectivity values between the putamen seed and other brain areas for the baseline and itch sessions (baseline-acupuncture and itchacupuncture) were extracted for each participant. Furthermore, we performed Pearson correlation to explore the relationship between connectivity values of the putamen to VAS at 8 min, to determine if behavioral measures correlated with connectivity in the acupuncture conditions. Second-level connectivity analysis was performed with a between group factor (responder minus non-responder) and a within-subject factor treatment (itch minus baseline) to identify effects compared to baseline by two-way ANOVA. For all analyses, a threshold of p < 0.05, FDR corrected, was used based on the intensity threshold p < 0.001.

## RESULTS

This study was conducted in two separate steps: Study 1 was followed by Study 2. Study 1 was 20 healthy participants (10 women, 10 men), mean age 22.5 (95% CI, 21.8–23.2) years, mean height 167.4 cm (95% CI, 163.5–171.3), mean weight 60.8 kg (55.8–65.9), and mean BMI 21.6 (95% CI, 20.3–22.9). From the 20 participants in Study 1, 15 (6 women, 9 men) who met the schedule for fMRI scans and retested for MRI compatibility, were mean age 22.8 (95% CI, 22.0–23.5) years, mean height 170.1 cm (95% CI, 166.0–174.1), mean weight 64.1 kg (59.0–69.2), and

<sup>1</sup>http://www.fil.ion.ucl.ac.uk/spm

<sup>2</sup>www.nitrc.org/projects/conn

mean BMI 22.1 (95% CI, 20.7–23.5) and included in Study 2. Study 1 and Study 2 courses are in **Figure 2**. Participants had more than one experience with acupuncture and no present history of asthma or AD.

# Acupuncture vs. Placebo: Antipruritic Effects of Acupuncture

#### Changes in Itch Intensity

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All of the 20 participants reported itch without pain 40 s after SPT. Itch intensities for the entire measurement were significantly lower in the acupuncture condition than the placebo condition for the arm (p < 0.001) and foot (p < 0.001) by two-way ANOVA (**Figures 3B,C**). Repeated-measure ANOVA showed significant effects on mean itch intensity for time (Greenhouse–Geisser corrected: F = 70.536, p < 0.001) and time × treatment interaction (Greenhouse–Geisser corrected: F = 7.486, p = 0.002) on the arm. Repeated-measure ANOVA showed significant effects on mean itch intensity for time (Greenhouse–Geisser corrected: F = 69.654, p < 0.001) and no significant effects on mean itch time × treatment interaction (Greenhouse–Geisser corrected: F = 2.150, p = 0.120) on the foot. At H1 and H2 phases, itch intensities were lower in the acupuncture condition (2.1 [95% CI, 1.4–2.7]; 1.3 [95% CI, 0.8– 1.8]) compared to the placebo condition (3.8 [95% CI, 2.9–4.6]; 2.5 [95% CI, 1.5–3.5]) (p < 0.001 and p = 0.015, respectively) on the arm (**Figure 3D**). At H1, H2, and H3 phase, itch intensities were lower in the acupuncture condition (2.5 [95% CI, 1.7–3.4]; 1.6 [95% CI, 1.0–2.3]; 0.8 [95% CI, 0.4–1.2]) compared to the placebo condition (3.4 [95% CI, 2.6–4.1]; 2.6 [95% CI, 1.9–3.4]; 1.7 [95% CI, 1.0–2.3]) (p = 0.001, p = 0.001, and p = 0.002, respectively) on the foot (**Figure 3E**).

Time above scratch threshold was significantly lower in the acupuncture condition (21.7 [95% CI, 5.1–38.3]) than

the placebo condition (71.4 [95% CI, 31.5–111.2], p = 0.002) on the arm. Time above scratch threshold was significantly lower in the acupuncture condition (29.3 [95% CI, 4.6–53.9]) than in the placebo condition (74.3 [95% CI, 33.3–115.3], p = 0.008) on the foot. The area under the rating curve above the scratch threshold for itch intensity was significantly lower in the acupuncture condition (94.2 [95% CI, −0.3 to 188.6]) than the placebo condition (568.3 [95% CI, 155.7–980.9], p = 0.001) on the arm (**Figure 3F**). The area under the rating curve above the scratch threshold for itch intensity was significantly lower in the acupuncture condition (178.1 [95% CI, −64.3 to 420.5]) than the placebo condition (473.7 [95% CI, 193.2–754.2], p = 0.008) on the foot (**Figure 3G**).

#### Changes in Skin Blood Perfusion

Mean perfusion units in the acupuncture condition were 323.3 (95% CI, 221.1–425.5), 391.5 (95% CI, 223.8–559.2), and 310.2 (95% CI, 166.9–453.6), and in the placebo condition 427.9 (95% CI, 283.6–572.1), 587.1 (95% CI, 419.7–754.4), 455.2 (95% CI, 318.6–591.8) (p = 0.049 at H1; p = 0.021 at H2; p = 0.011 at H3; p = 0.022 at H4) (**Figure 4A**). Maximum perfusion units were 642.8 (95% CI, 436.0–849.7) in the acupuncture condition and 840.6 (95% CI, 631.4–1050.0) in the placebo condition, with a significant difference (p = 0.036) (**Figure 4B**). Itch intensity was significantly correlated with skin blood perfusion (r = 0.36, p = 0.02) (**Figure 4C**). Representative images of skin blood perfusion from a participant are in **Figure 4D**.

#### Changes in Heart Rate Variability

Acupuncture compared to placebo showed no significant change on normalized high frequency (HF norm), normalized low frequency (LF norm), or LF/HF ratio. Participants in the acupuncture condition experienced increased HF norm and decreased LF norm and LF/HF ratio from baseline at H1, while participants in the placebo condition did not.

#### Assessment of Blinding

Comparisons of blinding rates showed no significant difference between conditions, as assessed by chi-square test with p = 0.068.

5–10 min, H3 is 10–15 min, and H4 is 15–20 20 min. PU, perfusion unit.

Thus, participants could not predict if they received acupuncture or placebo. In the acupuncture condition, 75% of participants correctly guessed the treatment (Bang's blinding index = 0.75 [95% CI, 0.52–0.98]). Placebo treatment had successful blinding (Bang's blinding index = 0 [95% CI, −0.39 to 0.39]).

#### Seed-Based Functional Connectivity Analysis Using fMRI

Acupuncture stimulation ("itch" minus "baseline") compared to placebo stimulation ("itch" minus "baseline") revealed no significant change in functional connectivity of the putamen.

#### Responder vs. Non-responder Analysis: Factors Influencing Acupuncture Response

For responder and non-responder analysis, the entire sample was separated into 10 responders and 10 non-responders in Study 1. They were based on a mean of the sum values of arm itch intensity and foot itch intensity at H1 in Study 1 involving peak value in the acupuncture condition. Mean itch intensities at H1 phase in Study 1 were 2.5 (95% CI, 1.9–3.1) in responders and 6.7 (95% CI, 4.9–8.5) in non-responders for sum values from arm and foot itch intensity in the acupuncture condition.

Baseline data for psychological factors influencing response to acupuncture are in **Figure 5**. Questionnaire results for acupuncture credibility were not significantly different between responder and non-responder groups: values were 17.5 (95% CI, 15.9–19.1) in the responder group and 17.9 (95% CI, 15.9– 19.9) in the non-responder group (**Figure 5A**). Acupuncture expectancy for the treatment indicated no significant difference between the responder (13.6 [95% CI, 11.9–15.2]) and nonresponder groups (12.6 [95% CI, 9.9–15.3]) (**Figure 5B**). Perception of bodily sensation showed no significant difference between responder and non-responder groups: the value was 39.9 (95% CI, 31.9–47.9) in the responder group and 37.0 (95%

CI, 31.0–43.0) in the non-responder group (**Figure 5C**). Fear of acupuncture was not significantly different between responder and non-responder groups. The scale was 25.0 (95% CI, 19.3– 30.7) in the responder group and 32.3 (95% CI, 22.6–42.0) in the non-responder group (**Figure 5D**).

#### Changes in Itch Intensity

In Study 1, mean itch intensities at H1 phase were lower in responders (2.5 [95% CI, 1.9–3.1]) compared to non-responders (6.7 [95% CI, 4.9–8.5]) for sum values from arm and foot itch intensity (p < 0.001) in the acupuncture condition. In Study 2, mean itch intensities at 8 min were lower in responders (2.7 [95% CI, 0.7–4.6]) compared to non-responders (6.0 [95% CI, 4.0–8.1]) for sum values for arm and foot itch intensity (p = 0.021) in the acupuncture condition.

#### Changes in Heart Rate Variability

HF norm in the responder group was 32.9 (95% CI, 3.0–62.9) and 49.5 (95% CI, 8.7–90.3). In the non-responder group, HF norm was −1.0 (95% CI, −11.7 to 9.8) and 0.8 (95% CI, −10.9 to 12.5) at A1 and A2 phase with a significant difference (p = 0.009 for A1 and p = 0.035 for A2). HF norm in the responder group was 81.2 (95% CI, −22.6 to 184.9) and 34.9 (95% CI, −24.8 to 94.7). HF norm in the non-responder group was −9.9 (95% CI, −17.2 to 2.6) and −22.1 (95% CI, −34.7 to 9.5) after histamine stimulus (H1 and H3 phase) with a significant difference (p = 0.007 for H1 and p = 0.029 for H3) between responder and non-responders to acupuncture (**Figure 6A**). HF norm values were not significantly different between responders and non-responders for placebo (**Figure 6B**).

LF norm for responders to acupuncture was −16.18 (95% CI, −25.3 to 7.1) for A1 phase, −12.4 (95% CI, −27.7 to 2.9) for H1 and 5.8 (95% CI, −16.4 to 28.0) for H3. LF norm for nonresponders was 7.1 (95% CI, −12.1 to 26.3) for A1, 13.8 (95% CI, −2.7 to 30.2) for H1 and 42.9 (95% CI, 10.1–75.7) for H3. Differences were significant (p = 0.028, p = 0.015, and p = 0.023, respectively) (**Figure 6C**). LF norm values were not significantly different between responders and non-responders in the placebo condition (**Figure 6D**).

The LF/HF ratios in responders to acupuncture were −31.8 (95% CI, −46.9 to 16.7) for A1 phase, −29.0 (95% CI, −58.1 to 0.0) for H1 and 11.2 (95% CI, −49.9 to 72.4) for H3. LF/HF ratios for non-responders to acupuncture were 22.8 (95% CI, −19.9 to 65.5) for A1, 29.3 (95% CI, 2.9–55.8) for H1, and 1127 (95% CI, 26.2–199.3) for H3. Differences were significant (p = 0.019, p = 0.003, and p = 0.019, respectively) (**Figure 6E**). HF norm values were not significantly different between responders and non-responders for placebo given (**Figure 6F**).

#### Seed-Based Functional Connectivity Analysis Using fMRI

Responder ("itch" minus "baseline") compared to nonresponder showed significant changes in putamen-posterior part of the midcingulate cortex (pMCC) connectivity (MNI coordinates of the peak voxel: X = −6, Y = 0, Z = 40, cluster p-FDR = 0.043). More positive putamenpMCC connectivity was observed in the responder group following acupuncture stimulation compared to the nonresponder group. Connectivity correlated negatively with itch intensity (r = −0.573 p = 0.032) (**Figure 7**). Functional connectivity changed significantly from baseline to itch for several of seed regions including basal ganglia (peak p-unc <0.001) (**Table 1**).

#### DISCUSSION

This study showed that experimentally induced itch and skin blood perfusion that were increased by histamine injection were significantly reduced after acupuncture compared to placebo.

Because itch is not a local symptom but a systemic symptom in clinical situations, we induced itch on both the arm and the foot simultaneously. Acupuncture reduced itch in both areas, indicating the systemic antipruritic effects of acupuncture. This study also showed via responder and non-responder analysis that itch intensity reduction was associated with PNS activation and positive functional connectivity of the putamen-pMCC following acupuncture. Psychological factors such as credibility, expectation, and fear of acupuncture and perception of bodily sensation were not related to itch response to acupuncture. To our knowledge, this is the first study to investigate factors related to acupuncture by distinguishing differences between responders and non-responders to itch.

In previous studies, the effects of acupuncture stimulation on histamine-induced itch were observed in healthy participants: Belgrade et al. (1984) and Lundeberg et al. (1987) reported reduced itch intensity using electroacupuncture stimulation during intradermal histamine injection. Pfab et al. (2005) showed significant reduced histamine-induced itch and wheal formation after acupuncture-point treatment compared to placebo-point treatment and no treatment; Kesting et al. (2006) showed ipsilateral acupuncture treatment significantly reduces areas

(bottom), (D) Graph shows differences in connectivity from baseline to itch as a function of differences in perceived itch intensity from baseline to itch, as reported by participants for each scan session.

of alloknesis (a sensory phenomenon that appears in area surrounding an itching source) after histamine application compared with contralateral and no-acupuncture treatment. Pfab et al. (Pfab et al., 2010, 2012a) showed that patients had significantly reduced type I hypersensitivity itch, which is commonly seen in AD. Acupuncture could therefore be useful for managing itch, urticarial or eczema in patients with AD. Pfab et al. (2011) also showed that itch intensity and basophil activation decreased with acupuncture in patients with AD from IgE-mediated allergy. And as did in the previous study, our study chose a protocol of pretreatment acupuncture and skin prick test (SPT) for histamine, to investigate the preventive antipruritic effects of acupuncture. Because the itch VAS is a subjective outcome, we used the laser Doppler perfusion imaging detecting skin blood perfusion to measure itch objectively.

The ANS innervates almost every part of the body and interacts with the immune system. In particular, the parasympathetic division of the ANS facilitates the immune response (Bear et al., 2016). Recent studies elucidated the role of the ANS in the itch mechanism (Chida et al., 2007; Cicek et al., 2008). In healthy participants, histamine stimulus induces an increase in the SNS from baseline (Tran et al., 2010). Stander and Steinhoff (2002) suggested that ANS dysfunction may contribute to itch in patients with AD. Kirchner et al. (2002) showed that the vagus nerve controls histamine-induced itch. Several studies (Backer et al., 2008) explored the action of acupuncture on the ANS by analyzing changes in HRV. These studies suggest that acupuncture stimulates parasympathetic activity (Kitagawa et al., 2014). In our study, participants in the responder group had an activated PNS and deactivated SNS, but non-responder groups did not. These results imply that an activated PNS might be associated with the antipruritic effect of acupuncture.

The PNS was more highly activated during the first 5 min after acupuncture insertion (A1 phase) in the placebo condition


TABLE 1 | Brain regions showing functional connectivity of basal ganglia.

FDR, false discovery rate; unc, uncorrection; l, left; r, right; pMCC, posterior part of the midcingulate cortex; aPaHC, anterior parahippocampal gyrus; SubCalC, subcallosal cortex; PaCiG, paracingulate gyrus; FP, frontal pole.

than in the acupuncture condition. A possible explanation for this phenomenon of slightly higher activation after acupuncture stimulation could be a belief in an acupuncture effect, which was shown in another placebo study (Darragh et al., 2015). A study demonstrated that the needling-specific component (somatosensory needling stimulation) induces sympathetic activation, whereas needling non-specific components (needling credibility by ritual/contextual influence) result in increased parasympathetic activation after acupuncture needling (Lee et al., 2014). This result could also explain the more strongly activated PNS in the placebo condition. In this study, placebo treatment was successfully blinded. A Bang's blinding index of 0 indicated that it might be a credible placebo (Bang et al., 2010). After histamine stimulus, acupuncture resulted in more activation of the PNS than placebo (H1 phase), showing that acupuncture increased parasympathetic activity; however, no significant change was observed in HF norm, LF norm, or LF/HF ratio between the acupuncture and placebo conditions. This result was partially because of high deviations in the HRV of participants, as examined by Kim and Woo (2011) in a normal Korean population. The impact of age, gender, and mean heart rate on short-term HRV measurement might have affected the results (Tsuji et al., 1996; Jensen-Urstad et al., 1997; Agelink et al., 2001; Kuch et al., 2001; Parati and Di Rienzo, 2003).

This study indicated that acupuncture had antipruritic effects via the positive connectivity of putamen and the pMCC. Itch is perceived by activation of inflammatory mediators via the spinothalamic tract to the brain (Davidson et al., 2007), influencing cortical and subcortical structures (Dhand and Aminoff, 2013). The striatum has a prominent function in striato-thalamo-cortical circuits (Koob and Volkow, 2010). The basal ganglia consists of the caudate nucleus, the putamen, the globus pallidus (consisting of an internal segment and an external segment), and the subthalamic nucleus (Bear et al., 2016). The putamen and caudate together are called the striatum, which is the target of cortical input to the basal ganglia. The globus pallidus is the source of output to the thalamus. Previous studies indicate that experimentally induced itch activates a brain network that includes a premotor and supplementary motor area, the thalamus, and the cingulate, insular, inferior parietal, and prefrontal cortices in healthy individuals (Pfab et al., 2012b). Electroacupuncture activates the putamen (Napadow et al., 2005), which is implicated in salience detection, particularly for stimuli such as pain (Downar et al., 2003) and itch. Recent fMRI study of patients with AD (Napadow et al., 2014) showed that acupuncture reduced itch and itch-evoked activation of parts of the putamen, insula, prefrontal, and premotor cortex (Napadow et al., 2014). Among these brain regions, greater reduction in putamen response was associated with greater decrease of itch intensity following acupuncture. The putamen is the region implicating the motivation and habitual behavior underlying the urge to scratch, and thus they suggested that the changes of putamen response are closely correlated with the antipruritic effects of acupuncture (Napadow et al., 2014). Based on this previous finding, we selected the putamen as seed for functional connectivity analysis using fMRI. The cingulate cortex consists of anterior cingulate cortex (ACC), midcingulate cortex (MCC), and posterior cingulate cortex (PCC). Among them, MCC or posterior part of the ACC was activated during itch induction or itch related behaviors (i.e., scratch), and the activation in this regions might be linked with cognition or evaluation of itch (Hsieh et al., 1994; Drzezga et al., 2001; Mochizuki et al., 2003, 2017; Herde et al., 2007; Papoiu et al., 2012). In a previous study, seed-based functional connectivity from the right anterior MCC showed a significant cluster in the right putamen in AD (Desbordes et al., 2015). In contrast, in this study, the significant connectivity of putamen and pMCC was found especially in responder to acupuncture during itch. The primary role of pMCC is reflexive orientation of the body in space to sensory stimuli including noxious ones (Vogt, 2016). Numerous neuroimaging studies have reported the activation of the pMCC during pain processing including the heat pain (Erpelding et al., 2012), short-duration and early noxious stimuli (Peyron et al., 2000; Bentley et al., 2003; Vogt et al., 2003; Frot et al., 2008). The MCC areas are thought to be numerous submodalities of sensory cortical processing (Vogt, 2014) and be involved in premotor planning and in affective-motivational processing in itch (Schneider et al., 2008). The motor-related regions such as the MCC along with the supplementary motor area and reward system including the putamen are associated with the reflexive process of scratching an itch (Mochizuki et al., 2015; Vogt, 2016). Thus, the antipruritic effect of acupuncture may be associated with the pMCC. Identifying negative correlations with clinical outcomes would allow us to understand how the positive connectivity of the putamen and pMCC lead to reduced itch.

Still, our results cannot exclude the possibility of the influence of individual difference of itch perception and responsiveness to histamine apart from the factors related with response to acupuncture. In this protocol of pretreatment acupuncture and SPT for histamine to investigate the preventive antipruritic

effects of acupuncture, as the baseline itch sensation data before acupuncture are not exist, responders and non-responders were determined using absolute values of the itch sensation obtained in the acupuncture condition rather than the optimal way of using subtracted value between baseline and endpoint. And we decided not to use the subtracted value between placebo and acupuncture condition as we thought it was inappropriate for responder analysis of this study based on the clinical perspective. Though we investigated the antipruritic effects of acupuncture against experimentally induced itch in healthy volunteers, we purposed not to distinguish acupuncture-specific factors, but to explore factors contributing the clinical antipruritic effects of acupuncture and we tried to focus on the total effect of acupuncture not excluding placebo factors in responder analysis. However, this might have led to another possible conclusion that responders in this study may be simply less responsive to itch. There are several studies on the individual difference of itch sensitivity in patients with AD or psoriasis which conclude that patients with chronic itch reacted with a higher itch response to histamine, suggesting somatosensory stimuli are processed in line with that patients' main symptom through generic sensitization processes (van Laarhoven et al., 2007, 2013). A seed-based analysis of a previous fMRI study (Desbordes et al., 2015) on evoked itch perception in chronic itch patients revealed decreased functional connectivity from baseline resting state to the evoked-itch state between several itch-related brain regions, particularly the insular and cingulate cortices and basal ganglia, where decreased connectivity was significantly correlated with increased levels of perceived itch. In contrast, evoked itch increased connectivity between key nodes of the frontoparietal control network (superior parietal lobule and dorsolateral prefrontal cortex), where higher increase in connectivity was correlated with a lesser increase in perceived itch, suggesting that greater interaction between nodes of this executive attention network serves to limit itch sensation via enhanced top-down regulation. Although these result cannot be directly applied to our study with healthy participants, individual difference of itch perception can naturally exist among healthy individuals. Further studies on brain regions or network in healthy participants that explain why some subjects perceive less itch are needed. Another fMRI study (Napadow et al., 2015) with AD patients demonstrated that expectations and other psychological factors play a role in modulating itch perception in chronic itch patients. In our study, psychological factors such as credibility, expectation, and fear of acupuncture and perception of bodily sensation were not related to itch response to acupuncture. The level of perceived bodily sensation varies from person to person and might lead to different responses of brain activation. In this study, perception of bodily sensation showed no significant difference between responder and non-responder groups: the value was 39.9 (95% CI, 31.9–47.9) in the responder group and 37.0 (95% CI, 31.0–43.0) in the non-responder group (**Figure 5C**).

In this study, acupuncture response to anti-pruritic effects might be associated with an anti-inflammatory effect mediated by an activated PNS and increased functional connectivity of putamen-pMCC. Acupuncture is thought to multidimensionally modulate anticipatory, somatosensory, and cognitive re-appraisal circuitries. Considering that the putamen and the MCC are related with somatosensory processing (Downar et al., 2003; Almeida et al., 2004; Vogt, 2014), the somatosensory aspect of acupuncture might be important for its antipruritic effects. However, while acupuncture may also have peripheral mechanisms as a counter stimulus (Ward, 1996), in this study, we induced itch on both the arm and the foot simultaneously and acupuncture reduced itch in both areas, suggesting that the systemic antipruritic effects of acupuncture are mediated by multiple central nervous pathways (Davidson and Giesler, 2010). Other studies indicated that antipruritic effects of acupuncture may modulate TRPV1 activation (McDonald et al., 2015, 2016) or endogenous opioid peptides in the central nervous system (Han, 2004; Wang et al., 2005; Dougherty et al., 2008; Guo et al., 2008; Zheng et al., 2008; Pfab et al., 2012a). Han et al. (2008) suggested that the antipruritic effects of acupuncture treatment are mediated by kappaopioid receptor activation in a rat AD model. In addition to anti-inflammatory effects, the response of the sensory nerve could be a possible explanation for the antipruritic effects of acupuncture.

This study had several limitations that warrant consideration. First, we performed responder and non-responder analysis of itch intensity. Because we did not have a clinical standard due to the experimentally induced itch, entire participants were separated into responders and non-responders based on a mean of the sum values of arm and foot itch intensities. As mentioned above, responders and non-responders were determined using absolute values of the itch sensation obtained in the acupuncture condition, the possibility of the influence of individual difference of itch perception and responsiveness to histamine apart from the factors related with response to acupuncture still remains. Second, our model was not clinical itch symptoms, but experimentally induced itch. Third, this study also had a small sample size. By classifying into responder and non-responder participants, the sample numbers became even smaller considering that recommended sample size for fMRI studies is usually 16 or more. Thus, this study should be regarded as a pilot study requiring further studies with larger sample size and additional normal control group without any interventions to compensate the defect appeared in this pilot study and to induce definite conclusion. Next, according to the Bang's blinding indexes for this study, the experimental condition was unblinded with 75% of correct guessing, while the control condition was completely blinded. Although this result can be interpreted as indicating that tactile stimulation acted as a credible placebo, the greater treatment effect in the experimental condition compared to control condition might have affected the study results (Park et al., 2008). Finally, no differences were seen in the analysis of the acupuncture and placebo conditions using HRV and fMRI data. As an explanation for the HRV results, acupuncture can be divided into needling-specific and non-specific components and it is equally applied to the placebo tactile stimulation (Langevin et al., 2011). Considering that the putamen and the pMCC are somatosensory processing regions related to afferent pain stimulation (Downar et al., 2003; Almeida et al., 2004; Vogt, 2014), the acupuncture and placebo condition might have had common brain responses.

#### CONCLUSION

fnins-13-00215 March 8, 2019 Time: 17:22 # 14

In conclusion, the results suggested that acupuncture treatment was useful against histamine-induced itch. Additionally, an activated PNS and the functional connectivity of putamen-pMCC could be considered factors related to the antipruritic response of acupuncture. Explaining clear mediators of the observed effects is still difficult. Thus, further work on the mechanisms involved in specific regulatory factors is needed. Further study to identify the most important brain region for the antipruritic effects of acupuncture with more participants is warranted. These factors might allow better prediction of the therapeutic effects of acupuncture.

# DATA AVAILABILITY

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

# REFERENCES


# AUTHOR CONTRIBUTIONS

SM and K-WK involved in the manuscript preparation. SM and H-JP conceived and designed the experiments. W-MJ, M-JL, and Y-KK made substantial contributions to the study conception and performed the overall experiments. K-WK, YC, and HL provided the critiques and revised the article for important intellectual content. H-JP participated in the critical revision of the manuscript and had the final responsibility for the decision to submit for publication. All authors read and approved the final manuscript.

## FUNDING

This research was supported by the National Research Foundation of Korea funded by the Korean Ministry of Science, ICT and Future Planning (NRF-2015M3A9E052338).

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnins. 2019.00215/full#supplementary-material

evoked potential source localisation study using realistic head models. Pain 102, 265–271. doi: 10.1016/S0304-3959(02)00405-0



**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling Editor is currently organizing a Research Topic with one of the authors YC and confirms the absence of any other collaboration.

Copyright © 2019 Min, Kim, Jung, Lee, Kim, Chae, Lee and Park. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fnins-13-00215 March 8, 2019 Time: 17:22 # 16

# Thymosin Beta 4 Is Involved in the Development of Electroacupuncture Tolerance

Juan Wan, Yi Ding, Sha Nan, Qiulin Zhang, Jinrui Sun, Chuanguang Suo and Mingxing Ding\*

*College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China*

Background: Electroacupuncture (EA) tolerance, a negative therapeutic effect, is a gradual decline in antinociception because of its repeated or prolonged use. This study aims to explore the role of thymosin beta 4 (Tβ4), having neuro-protection properties, in EA tolerance (EAT).

Methods: Rats were treated with EA once daily for eight consecutive days to establish EAT, effect of Tβ4 on the development of EAT was determined through microinjection of Tβ4 antibody and siRNA into the cerebroventricle. The mRNA and protein expression profiles of Tβ4, opioid peptides (enkephalin, dynorphin and endorphin), and anti-opioid peptides (cholecystokinin octapeptide, CCK-8 and orphanin FQ, OFQ), and mu opioid receptor (MOR) and CCK B receptor (CCKBR) in the brain areas (hypothalamus, thalamus, cortex, midbrain and medulla) were characterized after Tβ4 siRNA was administered.

Edited by: *Yi-Wen Lin, China Medical University, Taiwan*

#### Reviewed by:

*Senthilkumar Rajagopal, Rayalaseema University, India Hongwei Dong, Vanderbilt University Medical Center, United States*

> \*Correspondence: *Mingxing Ding dmx@mail.hzau.edu.cn*

Received: *10 December 2018* Accepted: *14 February 2019* Published: *26 March 2019*

#### Citation:

*Wan J, Ding Y, Nan S, Zhang Q, Sun J, Suo C and Ding M (2019) Thymosin Beta 4 Is Involved in the Development of Electroacupuncture Tolerance. Front. Cell. Neurosci. 13:75. doi: 10.3389/fncel.2019.00075* Results: Tβ4 levels were increased at day 1, 4, and 8 and negatively correlated with the changes of tail flick latency in all areas. Tβ4 antibody and siRNA postponed EAT. Tβ4 siRNA caused decreased Tβ4 levels in all areas, which resulted in increased enkephalin, dynorphin, endorphin and MOR levels in most measured areas during repeated EA, but unchanged OFQ, CCK-8, and CCKBR levels in most measured areas. Tβ4 levels were negatively correlated with enkephalin, dynorphin, endorphin, or MOR levels in all areas except medulla, while positively correlated with OFQ and CCK-8 levels in some areas.

Conclusion: These results confirmed Tβ4 facilitates EAT probably through negatively changing endogenous opioid peptides and their receptors and positively influencing anti-opioid peptides in the central nervous system.

Keywords: electroacupuncture tolerance (EAT), opioid peptides, anti-opioid peptides, mu opioid receptor (MOR), CCK B type receptor (CCKBR)

#### INTRODUCTION

Electroacupuncture (EA), derived from traditional hand acupuncture, has been widely used for treating various pains with few side effects (Xum et al., 2009; Zeng et al., 2016; Zhao et al., 2017). EA stimulation for a short time (10–40 min) can induce analgesic effect (Ulett et al., 1998; Qiu et al., 2015; Hu et al., 2016). However, prolonged or repeated EA stimulations would attenuate and finally nullify analgesic effect, which is termed "EA tolerance" (EAT) (Han et al., 1979, 1981b).

Because the tolerance to EA results in the decrease or even loss of its treatment effects, it has attracted more attention from practitioners and researchers. To explore EAT mechanism, some studies focus on the roles of analgesic and anti-analgesic neuromodulators in the central nerve system (CNS). It has been verified that EA produces analgesic effect through the release of endogenous opioid peptides (EOP) (Chen and Han, 1992b; Han, 2003). Tang et al. (1979) found repeated EA induced the higher levels of EOP in EAT rats. Further studies demonstrated that EA induced the release of anti-opioid substances including cholecystokinin octapeptide (CCK-8) and orphanin FQ (OFQ) (Bian et al., 1993; Tian et al., 1998) while it provoked the release of opioid peptides. The increase in anti-opioid peptides is believed to be due to the feedback regulation of opioid peptides. Tang et al. (1997) microinjected CCK antisense RNA into the lateral cerebral ventricle of rats to block the CCK gene expression, and found the development of tolerance elicited by prolonged EA stimulation was delayed. Tian and Han (2000) reported that intracerebroventricularly injecting OFQ antibody partially reversed tolerance to chronic EA. These studies indicate that the tipping of the balance to anti-opioid peptides may attenuate EA analgesia and contribute to EAT.

Opioid peptides or anti-opioid peptides exert analgesic or anti-analgesic effect through binding to their corresponding receptors. Therefore, some researchers have paid attention to the roles of these receptors in EAT. Ni et al. (1987) developed EAT and found that the opioid receptors decreased in the brain of rats. Dong et al. (1998) also found the decreased level of opioid receptors in the midbrain and striatum of rats after repeated application of EA. Huang et al. (2007) reported that CCK B type receptor (CCKBR) antagonist (L365, 260) can potentiate EA-induced analgesia and reverse chronic EAT. These findings showed that EAT was related to a change in opioid and anti-opioid receptors levels. However, the specific mechanism deserves further investigation.

Recently, Hu et al. (2018) found thymosin beta 4 (Tβ4) gene differentially expressed in the periaqueductal gray at 4 h in EAtreated goats. Tβ4, a 44-amino acid pleiotropic polypeptide, has important roles in neurobiological processes, including neurogenesis, neuronal developing, metabolism plasticity, and apoptosis (Sun and Kim, 2007; Chopp and Zheng, 2015). Recent studies reported that Tβ4 had a neuro-protective effect (Xiong et al., 2012a,b). EAT induced by prolonged or repeated EA stimulation can be considered as a neuro-protection response. Therefore, there may be a potential association between Tβ4 and EAT, which is worthy being studied.

In the present study, rats were stimulated with EA for 30 min once daily for eight consecutive days to establish EAT. Effect of Tβ4 on the development of EAT was determined through microinjection of Tβ4 antibody and siRNA into the cerebroventricle. The expression profiles of Tβ4, opioid peptides (endorphin, encephalin, and dynorphin), anti-opioid peptides (CCK-8 and OFQ) and related receptors (mu receptor and CCKBR) in the brain were characterized at mRNA and protein levels after Tβ4 siRNA was intracerebroventricularly administered to determine effects of Tβ4 on these proalgesic or analgesic substances and to further explore the role of Tβ4 in EAT.

#### MATERIALS AND METHODS

#### Animals

The study was conducted under the guidelines approved by Institutional Animal Care and Use Committee of Huazhong Agricultural University, Wuhan, China and adhered to the guidelines of the Committee for Research and Ethical Issues of the International Association for the Study of Pain.

Female Sprague–Dawley rats (No. 42000600024665) weighing 200–220 g were purchased from the laboratory animal center of Huazhong Agricultural University. Rats were housed six per cage with food pellets and water ad libitum. One week was allowed for adaptation to the surroundings.

#### Experiment Design

To explore the dynamic expression of Tβ4 in the brain of rats induced by repeated EA, thirty rats were randomly divided into sham group (n = 6) and EA group (n = 24). The rats in EA group were treated with EA once per day for 8 days consecutively. The rats in sham group were treated in the same manner as the rats in EA group, but without electricity. The tail-flick latency (TFL) was detected everyday immediately before and after EA, respectively, and the change rates of TFL were calculated. Six rats from EA group at day 0 (before EA), 1, 4, and 8, respectively, were euthanized.

To investigate the effect of Tβ4 neutralizing antibody on the development of EAT. Thirty-six rats were randomly classified into five groups: Sham + PBS (Sh- PBS, n = 6), EA + PBS (EA-PBS, n = 6), EA + IgG (EA-IgG, n = 6), EA + 0.1 µg Tβ4 antibody (EA-0.1 µg Ab, n = 6), EA + 1 µg Tβ4 antibody (EA-1 µg Ab, n = 6), and EA + 10 µg Tβ4 antibody(EA-10 µg Ab, n = 6). The rats in EA-0.1 µg Ab, EA-1 µg Ab, EA-10 µg Ab, or EA-IgG group were intracerebroventricularly injected with 15 µL 0.1 µg, 1 µg, 10 µg Tβ4 neutralizing antibody or isotype IgG, respectively. The rats in Sh-PBS and EA-PBS groups were treated with 15 µL PBS. The rats except those in Sh-PBS group were treated with EA 30 min after intracerebroventricular (icv) injection, for total 8 times. The rats in Sh-PBS group were treated as the same as the rats in EA-PBS group, but without electricity. TFL was examined every day immediately before and after EA, respectively.

To further detect the effect of Tβ4 silencing on the formation of EAT and expression pattern of Tβ4, endorphin (END), encephalin (ENK), dynorphin (DYN), CCK-8, OFQ, MOR, and CCKBR in EA-treated rats, 93 rats were randomly classified into five groups: sham EA (Sh-EA, n = 18), EA treatment (EA-tr, n = 18), EA treated with lipofection (EA-L, n = 18), and EA treated with lipofection mixture with control siRNA (EA-C-si, n = 18),

**Abbreviations:** CCK-8, Cholecystokinin octapeptide; CCKBR, CCK B type receptor; CNS, Central nerve system; DYN, Dynorphin; EA, Electroacupuncture; EAT, EA tolerance; EOP, Endogenous opioid peptides; ENK, Enkephalin, END, Endorphin; icv, Intracerebroventricular; MOR, Mu opioid receptor; OFQ, Orphanin FQ; PONC, Prepronociceptin; PDYN, Prodynorphin; PENK, Proenkephalin; POMC, Proopiomelanocortin; TFL, Tail flick latency; Tβ4, Thymosin beta 4.

FIGURE 1 | The scheme of experiment. (I) The rats in EA group were treated with EA (2/15 Hz, 30 min) once per day for 8 days consecutively. The rats in sham group were treated in the same manner as the rats in EA group, but without electricity. (II) The rats in EA-0.1 µg Ab, EA-1 µg Ab, EA-10 µg Ab, or EA-IgG group were intracerebroventricularly injected with 15 µL 0.1 µg, 1 µg, 10 µg Tβ4 neutralizing antibody or isotype IgG, respectively. The rats in Sh-PBS and EA-PBS groups were treated with 15 µL PBS. The rats except those in Sh-PBS group were treated with EA (2/15 Hz, 30 min) 30 min after intracerebroventricular (icv) injection, for total 8 times. The rats in Sh-PBS group were treated as the same as the rats in EA-PBS group, but without electricity. (III) The rats in EA-L, EA-C-si, or EA-Tβ4-si group were intracerebroventricularly injected with lipofection, lipofection mixture with control siRNA or Tβ4 siRNA, respectively. The rats except those in Sh-EA group were treated with EA (2/15 Hz, 30 min) at the day after icv injection and thereafter every day, for total 8 times. The rats in Sh-EA group were treated as the same as the rats in EA-tr group, but without electricity. The tail flick latency (TFL) was examined every day immediately before and after EA, respectively. The symbols have the same meanings in Figures 2–5.

or Tβ4 siRNA (EA-Tβ4-si, n = 18). The rats in EA-L, EA-C-si, or EA-Tβ4-si group were intracerebroventricularly injected with lipofection (15 µL), lipofection (10 µL) mixture with control siRNA (5 µL) or Tβ4 siRNA (5 µL), respectively. The rats except those in Sh-EA group were treated with EA at the day after icv injection and thereafter every day, for total 8 times. The rats in Sh-EA group were treated as the same as the rats in EA-tr group, but without electricity. TFL was examined every day immediately before and after EA, respectively. Six rats from each group at day 1, 4, and 8, respectively, were euthanized. Additional three rats were used to verify a fluorescence-conjugated siRNA transfection into the brain at 24 h after icv injection (**Figure 1**).

#### Intracerebroventricular Injection

The surgery for icv injection was conducted based on a previous method (Cui et al., 2017a). Briefly, rats were anesthetized with sodium pentobarbital (40 mg/kg, Sigma, USA) and then mounted on a stereotaxic apparatus (RWD, Shenzhen, China). Skin over the skull was incised, and a small hole was micro-drilled on the skull 1.5 mm lateral to and 0.8 mm posterior to the bregma. Then, a small cannula was inserted 4.0 mm below the skull surface and placed in the position. All rats were allowed for recovery for 7 days and for adaption to the cylinder for 3 days before the formal experiment. For icv administration, an injection needle was inserted into the cannula to reach the target site. PBS, Tβ4 antibody (Santa Cruz, CA, USA), lipofection (Santa Cruz, CA, USA) or lipofection mixture with control siRNA (Santa Cruz, CA, USA) / Tβ4 siRNA (Santa Cruz, CA, USA) was administered at a rate of 1 µL/15 min. After injection, the needle was kept in place for 5 min to reduce the backflow of the solution. To verify if siRNA could be transfected into brain areas after icv injection, a fluorescence-conjugated siRNA (Santa Cruz, CA, USA) was used in rats (n =3).

#### Electroacupuncture Application

EA stimulation was conducted at a fixed time of a day (9:00 a.m.), according to the method reported by Cui et al. (2017a). Briefly, each rat was gently placed into a specially designed polyethylene holder, with the hind legs and tail exposed. Before EA, rats were adapted to the holder for 3 days, and the room temperature was controlled at 22 ± 1 ◦C. The skin of the hind legs was sterilized with 75% alcohol. Stainless-steel needles (0.30 mm in diameter, 13 mm in length) were inserted into bilateral Zusanli points (ST36, 4 mm lateral to the anterior tuber point of the tibia, which is marked by a notch, 6–7 mm depth) and Sanyinjiao points (SP6, 3 mm proximal to the medial malleolus at the posterior border of the tibia, 4–5 mm depth). Rats were administered electrical impulses for 30 min with WQ-6F Electronic Acupunctoscope (Beijing Xindonghua Electronic Instrument Co., Ltd., Beijing, China). The stimuli were set as square waves with 2/15 Hz in frequency (dense-and dispersemode) and 3 mA in amplitude. Throughout the EA, rats were kept in the holder without anesthesia.

−0.40, −1.30, −2.40, −4.80, −8.60, −10.30, and −14.2 mm rostral to the transverse plane of bregma, respectively. (B) Change rates of tail flick latency after repeated EA (Mean ± SD, %, *n* = 6). The significance of differences was calculated by a *t*-test between sham and EA groups or a one-way ANOVA among different time points in EA group followed by Bonferroni's post-test.\**P* < 0.05 compared with sham group. The values with different letters (a–d) within the EA treatment differ. (C) The Tβ4 expressions in the brain areas after repeated EA (Mean ± SD, *n* = 6). The values with different letters differ significantly among different time points within the same brain area (*P* < 0.05). One-way ANOVA followed by Bonferroni's post-test.

#### Measurement of Tail Flick Latency

The nociceptive threshold was assessed using the TFL response elicited by radiant heat with the YLS-12A Tail Flick Analgesia Instrument (ZS Dichuang Science and Technology Development Co., Ltd., Beijing, China). Focused light from a projection bulb was applied to the proximal third of the tail and the TFL was measured. The intensity of the thermal stimulus was adjusted to obtain a basal TFL within the range of 4–6 s. A cutoff limit of 15 s was set to avoid tissue damage. Before EA, the basal TFL was determined by averaging three consecutive measures at 5 min intervals. TFL after EA was measured every 10 min during the 30 min of EA application. The change rate of TFL was taken as EA-induced antinociception and was calculated as the formula: TFL (%) = (TFL after EA–basal TFL)/basal TFL × 100%.

#### Sample Collection

At day 1, 4, and 8, immediately after TFL measurement, six rats from each group were anesthetized and euthanized by an overdose of sodium pentobarbital and their brains were quickly removed on a DEPC water-treated icebox. According to the atlas of Paxinos and Watson (1986), the hypothalamus, thalamus, cortex (sensory region), midbrain, medulla were taken immediately and stored in liquid nitrogen for western blotting and qPCR detection (**Figure 2A**).

Three rats were euthanized by an overdose of sodium pentobarbital at 24 h after fluorescence-conjugated siRNA injection, and intracardiac perfused ice-cold 4% paraformaldehyde in PBS. The brain was removed, cryoprotected in 30% sucrose in PBS and embedded in TissuTek. Cryostat sections (10µm) were collected on slides and observed with an OLYMPUS BX51 microscope with fluorescence light.

#### Western Blotting

Brain sample was weighed, grinded in liquid nitrogen, then protein extracted from the grinded brain tissue using the RIPA buffer according to the manufacturer's instruction (Beyotime Biotech, Nantong, China). The protein concentration was measured with Nano Drop Spectrophotometer (Thermo Fisher Scientific, Inc., USA). Equal amounts of protein sample (40 µg) was loaded in 15% SDS polyacrylamide gel and transferred to a PVDF membrane with the Mini-PROTEIN Tetra Cell (Bio-Rad, CA, USA). The membrane was blocked for 2 h at the room temperature in 5% skimmed milk, and was subsequently immunolabeled overnight at 4◦C with rabbit anti-endorphin IgG (1:500, ABclonal, Wuhan, China), rabbit anti-enkephalin IgG (1:500, ABclonal, Wuhan, China), rabbit anti-dynorphin IgG (1:500, ABclonal, Wuhan, China), rabbit anti-CCK-8 IgG (1:500, ABclonal, Wuhan, China), rabbit anti-OFQ IgG (1:500, ABclonal, Wuhan, China), rabbit anti-CCKBR IgG (1:500, ABclonal, Wuhan, China), mouse anti-MOR IgG (1:1,000, Novus, CO, USA), mouse anti-Tβ4 IgG (1:300, Santa Cruze, CA, USA), or rabbit anti-beta-actin (1:2,000, Boster biotech, Wuhan, China), respectively. The membrane was washed and treated with horseradish-peroxidase-conjugated anti-rabbit secondary

Wan et al. Tβ4 Involved in Electroacupuncture Tolerance



*T*β*4, Thymosin beta 4; POMC, proopiomelanocortin; PENK, proenkephalin; PDYN, prodynorphin; PONC, prepronociceptin; CCK, cholecystokinin ; MOR, mu opioid receptor; and CCKBR, CCK B receptor.*

antibody (1:5,000, Boster biotech, Wuhan, China) for 1 h at the room temperature. Visualization of the antigen-antibody complex was conducted with a horseradish peroxidase substrate (Millipore, MA, USA) with the Image Quant LAS 4,000 min CCD camera (GE Healthcare, CHI, USA). The bands were analyzed with Quantity One software (Bio-Rad, CA, USA). Beta-actin was used as the internal control. Values of these substances were represented as the ratio of the optical density of the bands to the density of the related beta-actin band.

#### RT-PCR

Total RNA was extracted from the each brain area of each group using Trizol reagent (Invitrogen, CA, USA). Subsequently, cDNA was synthesized from 900 ng of total RNA using a First Strand cDNA Synthesis Kit (TOYOBO, Osaka, Japan). The primer sequences of proopiomelanocortin (POMC), proenkephalin (PENK), prodynorphin (PDYN), CCK, prepronociceptin (PONC), Tβ4, MOR, CCKBR, and GAPDH were shown in **Table 1**. RT-PCR was performed with Step One PlusTM Real-Time PCR System (Applied Biosystems, CA, USA) using SYBR Green RT-PCR kit (Takara, Dalian, China). The mRNA of POMC, PDYN, PENK, CCK-8, PONC, Tβ4, MOR, and CCKBR relative to GAPDH mRNA were quantified with the 2 <sup>−</sup>1Ct method, where 1Ct= Ct target gene–Ct GAPDH.

#### Statistical Analysis

All data were expressed as mean ± SD. Statistical analyses were performed with SPSS version 18.0 (SPSS Inc., CHI, USA). Independent T-test was used to analyze variables, including Tβ4 and its mRNA levels between EA treatment and sham treatment. One-way ANOVA was used to analyze protein and mRNA levels of opioid and anti-opioid peptides and related receptors among the groups with different treatments. Data including TFL changes after repeated EA were analyzed with repeated ANOVA. Bonferroni post-test was used when significant differences were found. The correlation coefficient (Pearson's) was used to examine the correlations. A difference was considered significant if P was < 0.05.

## RESULTS

### Repeated EA-Induced Tβ4 Expression and Change Rate of Tail Flick Latency

The TFL was measured every day throughout the experiment (**Figure 2B**). No change (P > 0.05) was found in TFL in sham treatment during this trial. EA-induced TFL change rate was 49.6 ± 7.6% at day 1, then declined (P < 0.001) to 30.7 ± 8.5% at day 4 and fell (P < 0.001) to 1.8 ± 1.2% at day 8, implying EAT formation. TFL change rates in EA treatment were higher (P < 0.05) than those in sham treatment at day 1 to 6.

The dynamic expression of Tβ4 induced by repeated EA was determined in cortex, thalamus, hypothalamus, midbrain and medulla at day 0, 1, 4, and 8. EA increased (P < 0.05) the expression of Tβ4 in the measured areas at day 1–8 (**Figure 2C**). Statistical analysis showed that TFL change rates had a negative correlation with Tβ4 levels in cortex (r = −0.774, P < 0.001), in thalamus (r = −0.689, P = 0.002), in hypothalamus (r = −0.705, P = 0.001), in midbrain (r = −0.709, P = 0.001) and in medulla (r = −0.612, P = 0.007).

### The Effect of Intracerebroventricularly Injection of Tβ4 Antibody on Repeated EA-Induced Tail Flick Latency

TFL change rates in Sh-EA and EA-PBS groups showed the same change pattern as those in **Figure 2B**. TFL change rate of EAtreated rats was decreased in a Tβ4-antibody-dose- and timedependent manner. EA-treated rats with icv injection of Tβ4 antibody showed higher (P < 0.05) TFL change rates than rats with PBS or isotype IgG at day 3–7. TFL change rates in 10 µg antibody group were higher (P < 0.05) than that in 1 µg or 0.1 µg antibody group at day 4–7 (**Figure 3A**).

### The Effect of Tβ4 siRNA on Repeated EA-Induced Tail Flick Latency

To verify siRNA was able to transfect, the fluorescenceconjugated control siRNA was intracerebroventricularly injected, the fluorescence in hypothalamus, thalamus, cortex, midbrain, and medulla was observed at 24 h after injection (**Figure 3B**).

TFL change rates in Sh-EA and EA-tr groups showed the same change pattern as those in **Figure 2B**, and the development of EAT at day 8. TFL change rates in EA-tr, EA-L, and EA-C-si groups were higher (P < 0.05) than those in Sh-EA group at day 1–5, but lower (P < 0.05) than those in EA-Tβ4-si group at day 3–8. No change was observed in TFLs among EA-tr, EA-L, and EA-C-si groups (**Figure 3C**).

### The Effect of Tβ4 siRNA on Protein Expressions of Opioid- and Anti-opioid Peptides and Related Receptors in the Brain Areas of Rats With Repeated EA

The protein expressions of END, ENK, DYN, CCK-8, OFQ, Tβ4, MOR, and CCKBR were observed in hypothalamus, thalamus, cortex, midbrain and medulla at day 1–8. No difference (P > 0.05) was observed in the expressions of these eight substances among EA-tr, EA-L, and EA-C-si groups in the measured areas during the experiment.

The protein levels of those substances in cortex were shown in **Figure 4A**. Compared with sham treatment, EA treatment induced an increase in OFQ, CCK-8, and CCKBR levels (day 4 and 8), and Tβ4, ENK, DYN, and END levels (day 1–8), and MOR level (day 1 and 4). EA-Tβ4 siRNA-treated rats exhibited lower Tβ4 level (day 1–8), but higher levels of CCKBR (day 4), DYN (day 1), ENK, END, and MOR (day 1 and 4) than EA-treated rats. TFL change rates were negatively (P < 0.05) correlated with Tβ4, OFQ, CCK-8, or CCKBR levels, but positively (P < 0.05) correlated with ENK, DYN, END, or MOR levels. Tβ4 levels were negatively correlated with ENK, DYN, END, or MOR levels (**Figure 4B**).

In thalamus, compared with Sh-EA treated rats, EA-treated rats showed an increase in Tβ4 and OFQ (day 1–8), and CCK-8 and CCKBR (day 4 and 8), and ENK, DYN, END, and MOR (day 1 and 4). Compared with EA-treated rats, **EA**-Tβ4 siRNA-treated rats showed a decrease in Tβ4 (day 1–8), but an increase in OFQ and ENK (day 4 and 8), DYN and MOR (day 1–8), and END (day 4). TFL change rates were negatively (P < 0.05) correlated with Tβ4, OFQ, CCK-8, or CCKBR levels, but positively (P < 0.05) correlated with ENK, DYN, END, or MOR levels. Tβ4 levels were positively correlated with OFQ levels, but negatively correlated with ENK, DYN, END, END, or MOR levels (**Table 2**).

In hypothalamus, rats in EA-tr group showed an increase in CCK-8 (day 4 and 8), and Tβ4, OFQ, CCKBR, and MOR (day 1– 8), and ENK, DYN, and END (day 1 and 4) compared with rats in Sh-EA group. **EA**-Tβ4 siRNA treatment caused a decrease (P < 0.05) in Tβ4 (day 1 and 4), but an increase in OFQ, CCK-8, and MOR (day 8), in ENK and DYN (day 4 and 8), and END (day 4) and CCKBR (day 1) Compared with EA treatment. TFL change rates were negatively (P < 0.05) correlated with Tβ4, OFQ, or CCK-8 levels, but positively (P < 0.05) correlated with ENK, DYN, END, or MOR levels. Tβ4 levels were positively correlated with OFQ or CCK-8 levels, but negatively correlated with ENK, DYN, END, or MOR levels (**Table 2**).

In midbrain, EA treatment caused increased Tβ4, OFQ, CCK-8, CCKBR, ENK, DYN, and END (day 1–8) and MOR levels (day 1 and 4) compared with sham treatment. EA-Tβ4 siRNA treatment induced a decrease in Tβ4 (day 1–8), but an increase in OFQ and CCK-8 (day 1 and 4), ENK, END, and MOR (day 4 and 8), and DYN (day 4) compared with EA treatment.

encephalin (ENK), dynorphin (DYN), and mu opioid receptor (MOR) were observed. The values with different letters (a-c) differ significantly in different groups at the same day (*P* < 0.05). One-way ANOVA followed by Bonferroni's post-test. (B) The correlation coefficients between the levels of opioid or anti-opioid peptides or their receptors and the change rates of tail flick latency (TFL) or Tβ4 levels. \**P* < 0.05, \*\**P* < 0.01. The correlation coefficient was analyzed with Pearson's correlation coefficient.

TFL change rates were negatively (P < 0.05) correlated with Tβ4 levels, but positively (P < 0.05) correlated with ENK, DYN, END, or MOR levels. Tβ4 levels were positively correlated with CCK-8 levels, but negatively correlated with ENK, DYN, END, or MOR levels (**Table 2**).

In medulla, Tβ4, OFQ, CCK-8, CCKBR, ENK, and DYN levels (day 1–8) and END and MOR levels (day 1 and 4) in EA-tr group were enhanced compared with those in sham group. Rats in EA-Tβ4-si group exhibited a decrease in Tβ4 (day 1–8), but an increase in CCK-8, CCKBR, and ENK (day 4), DYN (day 8), END (day 1 and 4), and MOR (day 4 and 8) compared with rats in EA-tr group. TFL change rates were negatively (P < 0.05) correlated with Tβ4, OFQ or CCKBR levels, but positively (P < 0.05) correlated with ENK, DYN, END, or MOR levels. Tβ4 levels were positively correlated with OFQ levels, but negatively correlated with ENK, DYN, END, or MOR levels (**Table 2**).

## The Effect of Tβ4 siRNA on Gene Expressions of Opioid- and Anti-opioid Peptides and Related Receptors in the Brain Areas of Rats With Repeated EA

PNOC, PENK, POMC, and PDYN are the precursors of OFQ, ENK, END, and DYN, respectively. The mRNA levels of POMC, PENK, PDYN, CCK, Tβ4, MOR, and CCKBR are measured in hypothalamus, thalamus, cortex, midbrain, and medulla at day 1–8 (**Table 3**). No difference (P > 0.05) was observed in the expression of these eight genes among EA-tr, EA-L, and EA-C-si groups in the measured areas during the experiment.

In cortex, EA treatment induced an increase in Tβ4, CCK, and MOR mRNAs (day 1–8), PNOC and POMC mRNAs (day 4 and 8), PENK and PDYN mRNAs (day 4), and CCKBR mRNA (day 8) compared with sham treatment. EA-Tβ4 siRNA treatment caused a decrease in Tβ4 mRNA (day 1–8), but an increase PDYN and MOR mRNAs (day 1 and 4) and PENK mRNA (day 4) compared with EA treatment.

In thalamus, compared with rats in Sh-EA group, rats in EA-tr group showed increased Tβ4 and PNOC (day 1–8), CCK (day 4 and 8), and CCKBR, PENK, POMC, and MOR (day 4) mRNAs. Compared with rats in EA-tr group, rats in EA-Tβ4-si group showed decreased Tβ4 mRNA (day 1 and 4), but increased CCK (day 4), POMC (day 4 and 8), and PDYN and MOR (day 1) mRNAs.

In hypothalamus, mRNA levels of Tβ4 (day 1–8), PNOC and CCK (day 4 and 8), CCKBR (day 1 and 8), and PENK, PDYN, POMC, and MOR (day 4) mRNAs in EA-tr group were higher than those in Sh-EA group. EA-Tβ4 siRNA treatment induced a decrease in Tβ4 (day 1), but an increase in PENK (day 1), and PDYN and MOR (day 1 and 4) mRNAs compared with EA treatment.

In midbrain, EA-treated rats exhibited higher Tβ4, PNOC, CCK, CCKBR, PENK, and MOR (day 4 and 8), PDYN (day 1 and



Areas Sh-EA

 EA-tr

 EA-L-si

 EA-C-si

 EA-Tβ4-si

 Sh-EA

 EA-tr

 EA-L-si

 EA-C-si

 EA-Tβ4-si

 Sh-EA

 EA-tr

 EA-L-si

 EA-C-si

 EA-Tβ4-si

 r(TFL)

r(Tβ4)

Day 1

Day 8

Correlation coefficients

Day 4

THALAMUS

Tβ4

OFQ

CCK-8

CCKBR

ENK

DYN

END

MOR

 1.03 ± 0.12

 1.04 ± 0.11

HYPOTHALAMUS

Tβ4

OFQ

CCK-8

CCKBR

ENK

DYN

END

MOR

 0.98 ± 0.06

 1.03 ± 0.09

 1.01 ± 0.08

 1.03 ± 0.09

 1.04 ± 0.09

MIDBRAIN

Tβ4

OFQ

CCK-8

CCKBR

ENK

DYN

END

MOR

 1.04 ± 0.07

 1.02 ± 0.13

 1.05 ± 0.15

 1.05 ± 0.14

 1.02 ± 0.11

MEDULLA

Tβ4

OFQ

CCK-8

CCKBR

ENK

DYN

END

MOR

*The measured brain areas included* 

*encephalin;*

*respectively.*

*group, but without electricity. The symbols*

\**means the levels of opioid or anti-opioid*

*Pearson's correlation coefficient.*

 *The rats except those in* 

 *DYN, dynorphin; MOR, mu*

 0.99 ± 0.13

b 2.08 ± 0.2

b 2.18 ± 0.28

c 2.02 ± 0.26

b 2.24 ± 0.38

 1.01 ± 0.1

 1.02 ± 0.07

 1.04 ± 0.12

 1.03 ± 0.09

 1.01 ± 0.09

 1.01 ± 0.12

 0.99 ± 0.09

b 1.3 ± 0.17

b 1.49 ± 0.22

b 1.45 ± 0.2

b 1.52 ± 0.15

a 1.3 a 2.24

b 2.09

a 2.14

a 2.12

a 1.56

a 1.42

a 1.48

 1.04 ± 0.07

 1.01 ± 0.11

 1.02 ± 0.07

b 1.48 ± 0.25

c 1.41 ± 0.18

c 1.52 ± 0.24

b 1.47 ± 0.29

b 2.05 ± 0.28

b 2.21 ± 0.2

b 2.13 ± 0.29

b 2.65 ± 0.19

a 1.51 a 2.69

a 2.11

a 2.22

a 2.1

a 1.42

b 1.53

b 1.46

 1.03 ± 0.08

 1.04 ± 0.11

 1 ± 0.13

b 1.36 ± 0.11ab 1.33

c 1.42 ± 0.23

b 2.64 ± 0.33

b 2.31 ± 0.26

b 2.74 ± 0.28

b 2.73 ± 0.3

b 1.47 ± 0.21

b 1.3 ± 0.08

a 1.48 a 2.74

a 2.74

a 2.34

a 2.76

b 1.44

a 1.33

 0.99 ± 0.09 1.1 ± 0.18 1.12

b 2.01 ± 0.13

c 2.28 ± 0.23

 1.01 ± 0.11

 1 ± 0.11

b 2.55 ± 0.28

c 2.34 ± 0.22

 1.05 ± 0.11 1.04 ± 0.17 1.06

 1.01 ± 0.06

b 1.58 ± 0.11

> 0.99 ± 0.1

b 1.5 ± 0.39

a 1.52 b 2.31

a 2.58

b 2.11

a 1.97

a 1.52


TABLE

3


 peptides and related receptors.

9 March 2019 | Volume 13 | Article 75

*ANOVA followed by* 

4), and POMC (day 4) mRNAs than sham-treated rats. EA-Tβ4 siRNA-treated rats showed decreased Tβ4 (day 4), PNOC (day 1), CCK, and PDYN (day 1 and 4), CCKBR and PENK (day 8) and MOR (day 1–8) mRNAs compared with those in EA-treated rats.

In medulla, EA treatment caused an increase in Tβ4, PNOC, CCK and CCKBR mRNAs (day 4 and 8), in POMC mRNA (day 4) and MOR mRNA (day 1 to 4) compared with sham treatment. EA-Tβ4 siRNA treatment induced decreased Tβ4 (day 8), CCK (day 1), PDYN (day 1–8), POMC (day 4), and MOR (day 4 and 8) mRNAs compared with EA treatment.

# DISCUSSION

EA has been extensively used for treating various diseases (Liu et al., 2009; Shah et al., 2016; Cui et al., 2017b; Wan et al., 2018), especially pain disorders (Zeng et al., 2016; Hu et al., 2017; Wan et al., 2017). However, it also can provoke EAT, which is a negative effect for its application. Some researchers use different EA modalities to establish EAT. Tian et al. (1998) used 100 Hz EA to stimulate rats for consecutive 6 h, and found that the pain threshold decreased at 1 h (105%) and approximated to the level before EA (25%) at 6 h, suggesting that continuous EA can provoke a rapid EAT. Repeated EA is more commonly used than continuous EA for pain therapy in clinical practice. Cui et al. (2016, 2017a) stimulated rats with 2/15 Hz and 2 Hz EA (30 min/day, total 8 days), and found that the pain thresholds decreased from 59.6 ± 4.6% and 61.4 ± 5.5% at day 1–2.1 ± 4.1% and 1.9 ± 7.4% at day 8, respectively, showing that repeated EA can elicit chronic EAT. Wang et al. (2002) used 2 Hz EA for 30 min every time for total 6 times with different intervals (0, 1, 2, and 3 days, respectively) in rats, and found that EA with the interval of 1 day induced EAT whereas EA with the interval of 3 days produced potent analgesia, showing that EA interval is an important factor influencing the development of EAT. In the present study, 2/15 Hz was used to stimulate rats for 30 min once daily for consecutive 8 day. The TFL change rate was reduced from 47.0 ± 3.4% at day 1 to 1.5 ± 1.5% at day 8, indicating the formation of EAT. The result in our experiment is similar to the report by Cui et al. (2016, 2017a).

Intracerebroventricular injection has been frequently used to study neural substrates in acupuncture analgesia (Tang et al., 1997; Cui et al., 2017a). Icv injection of antibody is a classical method for verifying the function of neural substrates. SiRNA has been shown to be a powerful technology allowing the silencing of mammalian genes with great specificity and potency (Alisky and Davidson, 2004; Aigner, 2006). A number of experiments have demonstrated the potential of appropriately designed siRNAs (Karagiannis and Elosta, 2005). Several formulations, including liposomal and viral vectors, have been shown to be efficacious for delivering siRNAs to local sites of the CNS (Bumcrot et al., 2006). Intracereboventricularly injected siRNA can maintain a longer effect because the cerebrospinal fluid lacks a significant nuclease (Guo et al., 1996). In the present study, strong fluorescence at 24 h after icv injection of the mixture of fluorescenceconjugated siRNA and lipofection was observed in several brain areas, including hypothalamus, thalamus, cortex, midbrain and medulla, which implied that siRNA was successfully transfected into the cells in targeted brain tissues via the cerebrospinal fluid. The dosage (5 µL Tβ4 siRNA mixture with 10 µL lipofection) and a single application of the mixture solution were determined according to fluorescence intensity in brain areas and the duration of Tβ4 siRNA effect (a lower level of Tβ4 lasted for 8 days), respectively, in our pretest.

Numerous studies confirmed that EOPs in the CNS were main mediators to participate in EA analgesia regulation (Han et al., 1984; Han, 2003). Hence, opioid tolerance may be an important mechanism for EAT. In the present study, EOPs levels in the measured areas were positively correlated with the change rates in TFLs of rats treated with repeated EA.Tβ4 siRNA injection evoked an increase in ENK, DYN and END levels in most measured areas. ENK, DYN, or END level was negatively correlated with Tβ4 levels in the measured areas except in medulla where no correlation was observed between ENK and Tβ4 level. These results obviously displayed that the inhibition of Tβ4 partly reversed the expression of EOPs, hereby delayed the formation of EAT.

It has been found that EA induces the release of anti-opioid peptides (CCK-8 and OFQ) as it increases levels of opioid peptides in the CNS (Bian et al., 1993; Tian et al., 1998). Therefore, the roles of these anti-opioid peptides in EAT have attracted more attention. Han et al. (1985) injected CCK-8 into the cerebroventricle or spinal subarachnoid space in rats, and found that CCK-8 dose-dependently antagonized EA-induced analgesic effect. They also confirmed that icv or intrathecal injection of the antiserum against CCK-8 postponed or reversed the EAT (Han et al., 1986). Microinjection of OFQ into rats' periaqueductal gray remarkably antagonized EA analgesia in a dose-related manner (Zhi-Qi, 2008). Tian et al. (1998) reported that icv injection of OFQ antibody reversed EAT. These studies indicate that endogenous CCK-8 and OFQ participate in the formation of EAT. In this trial, Tβ4 siRNA treatment caused unchanged OFQ and CCK-8 levels in most brain areas except midbrain and hypothalamus. Studies found that the increased CCK-8 and OFQ were along with the increment of EOPs, which is believed to be a negative feedback of the former (Huang et al., 2003; Wang et al., 2006). Therefore, the increase of CCK-8 and OFQ in midbrain and hypothalamus may be also due to negative feedback on increased opioid peptides induced by Tβ4 siRNA.

Since opioid and anti-opioid peptides exert biological functions through their receptors, the roles of opioid and anti-opioid receptors in the development of EAT appeal to researchers. MOR, one of classic opioid receptors with a high affinity for ENK and END, is involved in EA-induced analgesia (Han et al., 1981a; Chen and Han, 1992a; Huang et al., 2000). CCK-8 could interact with CCK receptors (especially CCKBR) in the specific sites of the CNS to interfere with the functions of MOR, resulting in anti-analgesic effect (Wang et al., 1990; Shen et al., 1995; Huang et al., 2007). MOR and CCKBR can be selected as representatives of EOP- and anti-EOP- receptors, respectively. Ni et al. (1987) found that repeated EA resulted in EAT and decreased opioid receptors in rats' brain. Huang et al. (2007) demonstrated that CCKBR antagonist (L365, 260) potentiated 100 Hz EA-induced analgesia and reversed chronic tolerance to 100 Hz EA in mice. These findings demonstrated that MOR and CCKBR were involved in EAT. In the current study, the level of MOR decreased as EA application frequencies increased, which was consistent with the report of Ni et al. (1987). However, Tβ4 siRNA treatments caused increased MOR level; MOR levels were negative correlation with Tβ4 levels in the measured brain areas. Tβ4 siRNA caused CCKBR levels to be unchanged in thalamus and midbrain, and to be increased in cortex, hypothalamus and medulla at some specific time points. However, CCKBR levels were not correlated with Tβ4 levels. These results indicated that Tβ4 facilitated EAT probably via influencing opioid receptors. However, the underlying mechanisms need to be further studied.

Tβ4 is a small molecule polypeptide that widely exists in the CNS. Tβ4 can be released from neurons, but the targets it works on are unclear. Xiong et al. (2012a,b) reported that Tβ4 provided neuro-protection and neuro-restoration in experimental traumatic brain injury. Santra et al. (2012) reported that Tβ4 mediated oligodendrocyte differentiation via the inhibition of p38MAPK and the reduction of phosphorylated JNK accumulation. Jeon et al. (2013) found that Tβ4 facilitated mesenchymal stem cells proliferation through activation of ERK. These studies suggested that Tβ4 exerted neuro-protection via MAPK (ERK1/2, P38, and JNK) signal pathways. (Chen and Sommer, 2009) reported that MAPK signaling pathways were activated in the CNS and the periphery in the chronic morphine tolerance which was reduced by the inhibitors of these pathways. The similarity between EA and morphine tolerance has been demonstrated in a cross-tolerance study (Han et al., 1981b). Cui et al. (2017a) reported that the MAPK signaling pathway was targeted by miRNAs involved in chronic EAT. The previous studies suggest that there is a potential relationship between MAPK signal pathways and chronic EAT (Han et al., 1981b; Cui et al., 2017a). However, whether Tβ4 activates these pathways in chronic EAT needs to be confirmed.

In summary, repeated EA can result in decreased TFL, which is a negative effect of its treatment. Studies have showed that Tβ4 has neuroprotective and neuro-nutritional effects. Repeated EA, as a stimulus, caused increased Tβ4 in brain areas. Tβ4 siRNA icv injection postponed the formation of EAT, reversed decreased opioid- and their receptors induced by EAT in most brain areas. These results confirmed Tβ4 facilitated EAT probably through negatively changing endogenous opioid peptides and their receptors and positively influencing antiopioid peptides in the CNS. Neuromodulators (opioid peptides and non-opioid peptides) in the CNS are distributed in different regions in which the neurons communicate with each other through neural fibers, and constitute a regulating network. EA induces the release of the neuromodulators in the specific regions, thereby exerts the analgesic modification. In the present study, the expression levels of opioid- and anti-opioid-peptides and their receptors induced by Tβ4 siRNA were fluctuated in some brain areas, indicating the complex regulations of these neuromodulators and brain areas in the formation of EAT. Although the present study exhibited that Tβ4 siRNA delayed the formation of EAT through affecting the expression of opioid peptides, anti-opioid peptides and opioid receptors in a wide range of the brain regions. Since substances are not evenly distributed in the specific pain-regulated nuclei, the brain area sampling may result in their homogenization; it may not accurately reflect their levels in the specific nuclei. It will be more important to investigate the effect of Tβ4 on neuromodulators in the specific nuclei in the formation of EAT. In addition, the role of Tβ4 in anti-analgesic neural circuits or cellular signaling pathways needs to be further studied. Anyhow, our results provided an important foundation for investigating the molecular mechanisms by which Tβ4 modifies EAT in the future.

#### AUTHOR CONTRIBUTIONS

MD contributed to the conception and design of the study. JW, YD, QZ, SN, JS, and CS performed animal experiments, collected

#### REFERENCES


samples and accomplished the laboratory investigations. YD, JW, and SN performed the acquisition of data. YD, JW, and QZ conducted the data analysis and interpretation of data. WJ drafted the manuscript. MD, YD, and JW revised the manuscript. All authors read and approved the final manuscript.

#### FUNDING

This study was supported by the National Natural Science Foundation of China (Nos. 31672615 and 31272619).


system are involved in electroacupuncture tolerance. Front. Neurosci. 12:902. doi: 10.3389/fnins.2018.00902


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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# Transcriptome Analysis on Maternal Separation Rats With Depression-Related Manifestations Ameliorated by Electroacupuncture

Yuanjia Zheng<sup>1</sup>† , Jiang He<sup>1</sup>† , Lili Guo<sup>1</sup> , Lin Yao1,2, Xiaorong Zheng<sup>1</sup> , Zhihua Yang<sup>1</sup> , Yucen Xia<sup>1</sup> , Xiaoli Wu<sup>1</sup> , Yang Su<sup>1</sup> , Nenggui Xu<sup>1</sup> \* and Yongjun Chen1,3 \*

<sup>1</sup> South China Research Center for Acupuncture and Moxibustion, Medical College of Acu-Moxi and Rehabilitation, Guangzhou University of Chinese Medicine, Guangzhou, China, <sup>2</sup> School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, China, <sup>3</sup> Center for Brain Science and Brain-Inspired Intelligence, Guangdong-Hong Kong-Macao Greater Bay Area, Guangzhou, China

#### Edited by:

Yi-Wen Lin, China Medical University, Taiwan

#### Reviewed by:

Sin-Jhong Cheng, Academia Sinica, Taiwan Dongwoon Han, Hanyang University, South Korea

#### \*Correspondence:

Nenggui Xu ngxu8018@163.com Yongjun Chen chyj417@126.com

†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Perception Science, a section of the journal Frontiers in Neuroscience

Received: 15 November 2018 Accepted: 19 March 2019 Published: 05 April 2019

Citation:

Zheng Y, He J, Guo L, Yao L, Zheng X, Yang Z, Xia Y, Wu X, Su Y, Xu N and Chen Y (2019) Transcriptome Analysis on Maternal Separation Rats With Depression-Related Manifestations Ameliorated by Electroacupuncture. Front. Neurosci. 13:314. doi: 10.3389/fnins.2019.00314 Maternal separation (MS), a stressful event in early life, has been linked to neuropsychiatric disorders later in life, especially depression. In this study we investigated whether treatment with electroacupuncture (EA) could ameliorate depression-related manifestations in adult animals that had adverse early life experiences. We demonstrated depression-like behavior deficiencies in a sucrose preference test and a forced swimming test in a rat model with neonatal MS. Repeated EA treatment at the acupoints Baihui (GV20) and Yintang (GV29) during adulthood was shown to be remarkably attenuated above behavioral deficits. Using unbiased genome-wide RNA sequencing to investigate alterations in the transcriptome of the prefrontal cortex (PFC), we explored the altered gene sets involved in circadian rhythm and neurotransmitter transporter activity in MS rats, and their expression tended to be reversed after EA treatment. In addition, we analyzed the interaction network of differentiated lncRNA– or circRNA–miRNA–mRNA by using the principle of competitive endogenous RNA (ceRNA). These results suggest that EA at GV20 and GV29 ameliorates depression-related manifestations by regulating the expression of multiple genes.

Keywords: electroacupuncture, transcriptome, prefrontal cortex, maternal separation, depression

# INTRODUCTION

The mother–child relationship has been reshaped by rapid societal changes. Mother-newborn separation shortly after birth, for example, has become routine following hospital births (Csaszar-Nagy and Bokkon, 2018). In the past 30 years, the phenomenon of rural children living apart from their parents, who work in cities, has become so widespread that as many as 37.7% of children in China are affected (Wang, 2015). A growing body of literature indicates that adverse early life experience is significantly associated with susceptibility to stress-related psychopathologies such as depression (Gibb et al., 2007) and anxiety (Coplan et al., 2014; Ishikawa et al., 2015)

disorders. Similarly, parental loss due to sudden death increases the vulnerability of children to depression (Brent et al., 2009). Although the molecular basis has not been fully elucidated, neonatal maternal separation (MS), an early-life adverse event, can have long-lasting effects on neural development and increases risk of adult psychopathology in human adults (Hanson et al., 2012).

Depression is a common mental disorder worldwide, with over 322 million people being diagnosed with it every year. As the leading cause of disability worldwide (Cai et al., 2015) participants with depression had greatly higher total healthcare costs than those without (\$20,046 vs. \$11,956; p < 0.01) in previous study (Unutzer et al., 2009). The selective serotonin reuptake inhibitors (SSRIs) and serotonin and noradrenaline reuptake inhibitors (SNRIs) that are currently the first-line treatment options for major depressive disorder (MDD) (Dale et al., 2015) usually require 4–6 weeks, and sometimes longer, to be effective (Liu et al., 2015). The side effects of their longterm use include sleep disturbance (Roohi-Azizi et al., 2018) and non-response to other classes of antidepressants (Li Q. et al., 2013). To induce a depression-like phenotype for investigating the antidepressant effects of the drugs, various rodent models were used, such as being exposed to chronic unpredictable mild stress, learned helplessness, chronic social defeat stress and so on (Cao et al., 2013; Alboni et al., 2017; Post and Warden, 2018). Adverse experience in early life is associated with stressrelated psychopathologies, and previous studies have shown neonatal rats or mice exposed to MS were displayed depressionlike behavior in adulthood (Vetulani, 2013; Sadeghi et al., 2016; Tchenio et al., 2017).

Acupuncture as a well-known useful treatment for depression has been proven. For example, acupuncture (electro- and manual) may moderately reduce the severity of depression by the end of treatment (SMD −0.66, 95% CI −1.06 to −0.25, five trials) in total 488 participants (Smith et al., 2010). According to a report from the Department of Veterans' Affairs from Washington, DC, United States, depression is one of the nine clinical indications relevant to mental health for acupuncture (Hempel et al., 2014). However, the molecular mechanisms through which electroacupuncture (EA) modulates depressive behaviors are largely uncharacterized. Rat is an organism that provides a model with clinically relevant phenotypes for exploring new therapeutics (Jacob and Kwitek, 2002) and for studying the mechanism of acupuncture (Kou et al., 2017; Zhang Z.Y. et al., 2018). Furthermore, as acupuncture or EA can ameliorate depressive-like behaviors, a rat model of depression is a tool that has been widely used to investigate the antidepressant effects of acupuncture (Lu et al., 2016, 2017; Jiang et al., 2017). "Baihui" (GV 20) – "Yintang" (GV 29) are considered to be the optimized acupoint modules for mental illness (Duan et al., 2009a, 2014; Jiang et al., 2017), and our previous study indicates that EA at GV 20–GV 29 acupoints ameliorates cognitive deficits and improves hippocampal synaptic plasticity in adult rats with neonatal MS (Guo et al., 2018).

To investigate whether acupuncture can alleviate psychopathology in adults with stress-related adverse early life experiences, as well as to detail the mechanism by which acupuncture might regulate gene expression, we generated rat neonatal MS models and applied EA treatment at the GV20 and GV29 acupoints. Additionally, to explore experimentally the mechanisms involved with MS-induced depression, unbiased RNA sequencing (RNA-Seq) was used to identify rat genomewide alterations in the prefrontal cortex (PFC) after MS and EA treatments. Furthermore, analysis of the interaction network of differentiated long non-coding RNA (lncRNA)- or circular RNA (circRNA)-miRNA-mRNA was performed using the principle of competitive endogenous RNA (ceRNA). Our study is the first to provide new information on the mechanism underlying anti-depressive effects of EA in adult rats with neonatal MS experience.

# MATERIALS AND METHODS

# Experimental Animals

Male and nulliparous female Wistar rats of 180∼220 g were obtained from the Guangdong Medical Laboratory Animal Center. Animals were housed in the standard cages in controlled temperatures (20–22◦C) and a 12-h light–dark cycle room. Food and water were available ad libitum. Animals entered the study at 8 weeks of age following a week-long acclimatization period and were mated at a 2:1 ratio of male to female rats. The female rats were raised alone once found to be pregnant. Wistar dams were assigned partly to control groups, with most to the molding groups. For each litter, the day of birth was named as postnatal day 0 (PND0) and the day after PND0 was the first day of molding, which was set as PND1. The experimental procedure was approved by the Animals Care and Use Committee of Guangzhou University of Traditional Chinese Medicine.

# Maternal Separation Model

From PND1 to PND21, MS that kept the mothers from their filial generation of molding groups into another cage for 4 h (9:00–13:00) was conducted every day. The mothers and litters in control groups were under no disturbance until weaning. All pups were weaned at PND21 and the males were housed four or five per cage until adult age, and the females were eliminated. The experimental procedure was approved by the Animals Care and Use Committee of Guangzhou University of Traditional Chinese Medicine. All efforts were made to minimize the animals' suffering and to reduce the number of animals used for experiments.

#### Animal Groups

#### Vehi, MS+Vehi, and MS+Flu

To determine whether the MS model induced depression in adulthood, at PND60, the litters belonging to healthy reservation groups were assigned to Vehicle group (Vehi), and the molding rats were assigned randomly into two groups: vehicle group (MS+Vehi) and Fluoxetine group (MS+Flu).

#### Cont, MS, EA, and Sham-EA

To examine whether the EA stimulation had effect on MS rats, in another trial, at the PND60, the litters belonging to

healthy reservation groups were assigned to control group (Cont), and the molding rats were assigned randomly into three groups: maternal separation group (MS), MS with electroacupuncture treatment group (EA) and MS with sham EA treatment group (Sham-EA).

#### Treatment

All the treatments were performed from PND61 to PND81 every other day.

#### EA Stimulation

Using isoflurane (RWD, Shenzhen, China), the EA rats were positioned in the induction case with an anesthetized concentration of 5% for the initial 5 min for deep anesthetized condition, and then they were moved to the EA operating platform with a 2% concentration for the middle 10 min, decreasing to 1.5% for the last 5 min. Disposable acupuncture needles as previous reported (Guo et al., 2018) were inserted to the acupoints of GV20 and GV29. The Master-8 Stimulator (Master-8, AMPI, Israel) was connected to deliver electrical current to the needles. We set the output parameters as follows: holding the frequency constant at 2 Hz and intensities at 2 mA, for 15 min. EA stimulation was administered every other day for 15 min starting at 8:30 a.m. Rats in the Sham-EA group were anesthetized with isoflurane as the EA group. The difference was rats in the Sham-EA group received no electrical stimulation; a disposable acupuncture needle was attached to the surface near GV20 and GV29 but apart from any other known acupoints. The Cont rats and MS rats only received anesthetization conduction.

#### Fluoxetine Administration

The MS+Flu group were given injections of fluoxetine (10 mg kg−<sup>1</sup> , i.p.) while rats in the Vehi group and the MS+Vehi group received injections of an equal volume of saline (i.p.) from PND61 to PND81.

## Behavioral Tests

#### Body Weight Measurement

The body weights of rats in each group were measured every week during this period at 9 a.m. by balances (MS3002ts/00, Mettler Toledo).

#### Sucrose Preference Test (SPT)

In training days, each rat was exposure to two bottles of 1% sucrose solution for 24 h in the first day, and two bottles of tap water in the second day. In the third day, the bottles and food were withdrawn to make the rats hungry and thirsty. On the test day, bottle A contained 1% sucrose solution, and bottle B contained water. The fluid that was consumed from each bottle was measured after 24 h. The sucrose preference of each rat was calculated as 100 × [VolA/(VolA + VolB)], and the total fluid intake was calculated as VolA + VolB.

#### Forced Swimming Test (FST)

The FST was performed in a clear glass cylinder (height 45 cm, diameter 20 cm), which was filled to 30 cm with water (22–25◦C). The test lasted for 5 min. The duration of immobility was recorded by JLBehv-FSR-4 (Shanghai Jiliang Software Technology Co., Ltd.).

#### Elevated-Plus-Maze Test (EPMT)

The elevated-plus-maze test consisted of two opposing open arms (50 cm × 15 cm) and two opposing enclosed arms (30 cm × 50 cm × 15 cm) that were connected by a central platform (15 cm × 15 cm), forming the shape of a plus sign. All of the measurements were taken in a dimly lit experimental room, in which the rats where acclimatized for at least 30 min before testing. The times that were spent in the open arms and the enclosed arms were recorded over a 10 min test period. The maze was cleaned with a solution of 20% ethanol in water between the sessions.

#### Open Field Test (OFT)

The open field presented an open box structure (80 cm × 80 cm × 40 cm) with a black square at the bottom. A camera device was installed directly above the central area of the open field. Before the experiment, the rats were conditioned for 60 min in advance in the test room. Uniform light and a quiet environment throughout the test were ensured. The rats were gently lowered into the central part of the square and allowed to move freely in the open field for 10 min. Its total distance and the time in the central area were recorded. The area was cleaned with 20% alcohol between the sessions. Only after the alcohol smell dissipated and the bottom of the box was without obvious signs of moisture were the rats tested.

#### Light-Dark Box Test

The test was carried out in a soundproof box with a light box (25 cm × 25 cm × 40 cm) and a dark box (25 cm × 25 cm × 40 cm). The dividing wall was inserted with an opening hole (8 cm × 7.5 cm) to allow the animal's free movement from one compartment to another. The illumination was above the light box. The animal was released into the center of the light compartment (facing away from the opening) and was allowed to explore the area for 10 min. Distance traveled and time spent in different compartments were recorded by JLBehv-FSR-4 (Shanghai Jiliang Software Technology Co., Ltd.). The box was cleaned with a solution of 20% ethanol in water between the sessions.

#### Radioimmunoassay

The reagent kits of corticosteroid (CORT) and adrenocorticotropic hormone (ACTH) for measurement of amounts were purchased from IZOTOP Institute of Isotopes Ltd. and Beijing North Biotechnology Research Institute, respectively. All determination procedures were according to the manufacturers' instructions.

#### Tissue Extraction and RNA Sequencing

Three samples per group (Cont, MS, and EA group) were sent for RNA sequencing. Rats were anesthetized with isoflurane (RWD, Shenzhen, China). The induced anesthesia concentration was 5% to perform tissue extraction. PFC was removed quickly and then put in liquid nitrogen for quick freezing.

Then the PFC was stored at −80◦C until tissue processing. Total RNA was extracted by Trizol reagent (Invitrogen) from tissue. RNA samples were prepared by using rRNA Depletion Kit (NEBNext <sup>R</sup> ). The DNA libraries were applied to cluster generation and sequencing using cBot Operation for HD V2.5 Reagent and HiSeq X Operation for HD v2.5 reagent\_v1.3 (Illumina). Sequencing was performed using Hiseq X ten (Illumina, United States). The raw data were deposited onto NCBI's Read Gene Expression Omnibus (GEO) database and the accession number is GSE124387. RPKM (Reads Per kb per Million reads) was used to calculate gene expression from RNA-Seq data, which can eliminate the influence of gene length and sequencing amount for calculating gene expression. If multiple transcripts exist in a gene, we select the longest one to calculate sequencing depth and expression.

# Data Analysis

#### Statistical Analyses

All of the results are expressed as the means ± SEM. The statistical analyses were performed using SPSS 17.0 software. Potential differences between the mean values were evaluated using two-way or one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test for post hoc comparisons when equal variances were assumed. Non-parametric Kruskal–Wallis test was used to compare differences through groups when there was heterogeneity of variance. The significance level for all of the tests was set at p < 0.05.

#### Computational Analysis for RNA-Seq Data

Reads were filtered for quality by Fast-QC program and mapped to the rat genome (NCBI assembly Rnor\_6.0) by the Hisat2 program. EB-Seq algorithm (Leng et al., 2013) was applied to filter the differentially expressed genes after the significant analysis and false discovery rate (FDR) analysis under the following criteria: fold change >1.5 or <0.667; FDR <0.05. According to the NCBI Gene Ontology database, GO analysis was performed by using Fisher's exact test. It classifies the GO terms, and the FDR was calculated to correct the p-value. We utilized Miranda (Picao-Osorio et al., 2015) and RNAhybrid (Rehmsmeier et al., 2004) as the tools for predicting differentially expressed miRNA targets on circRNA, lncRNA, and mRNA.

For Series Cluster analysis, the raw expression values were converted into log2 ratio. Using a strategy for clustering short time-series gene expression data, we defined some unique profiles. Significant profiles have higher probability than expected by Fisher's exact test and multiple comparison test (Ramoni et al., 2002). The following expression tendencies are what we had interest in: the genes decreased in MS rats compared to control rats but increase in EA rats contrasted with MS rats (decreaseincrease type); the genes increased in MS rats compared to control rats but decreased in EA rats in contrast to MS rats (increase-decrease type). Based on that, the expression of mRNA and circRNA as well as mRNA and lncRNA satisfying this relationship are positively correlated; series cluster analysis is performed to identify a set of unique expression tendencies.

# RESULTS

## Neonatal MS Induced Depression-Like Behavioral Deficits in Adults

To confirm whether neonatal MS affects the onset of affective disorders in adults, we replicated the MS model to monitor depression- and anxiety-related behaviors in Wistar rats (**Figure 1A**). Before the beginning of the behavior test, body weight was measured every week. We found that the weight of rats with MS (MS+Vehi) increased slightly in adulthood compared with control (Vehi) and MS rats with Fluoxetine treatment (MS+Flu) (**Figure 1B**). In tests for depression-like behaviors, we applied SPT and FST, two well-known tests for detecting anhedonia and despair symptoms of depression disorder (Tye et al., 2013; Liu et al., 2018; Ji et al., 2019). Rats in the MS+Vehi group showed a significantly reduced sucrose consumption rate in SPT and increased duration of immobility in FST compared to Vehi. The administration of Fluoxetine restored sucrose consumption and immobility behavior deficits in both instances (**Figures 1C,D**). We next performed the EPM task, a well-established test of anxiety-like behavior (Walf and Frye, 2007). We found that rats from the three groups spent similar amounts of time in open arms (**Figure 1E**). To further detect whether MS rats have anxiety-like behaviors, the OFT and light/dark box test were performed (Bourin and Hascoet, 2003). There was no difference between the control and MS rats on the total distance, time in center in OFT, and time in light in light/dark box test (**Supplementary Figure S1**). Together, these results suggested that the MS model rat in the current study was successfully replicated for depression, but not anxiety.

#### EA Induced Antidepressant-Like Effects

To assess the effect of EA treatment on depression-like behavioral deficits in adult rats that suffered from MS, rats were tested for sucrose consumption in SPT and the total duration of immobility in FST (**Figure 2A**). Rats in the MS and the Sham-EA groups exhibited a decreased rate of sucrose consumption compared to controls (**Figure 2B**). However, the level of sucrose consumption in the MS+EA group was similar to the control group (p > 0.05), suggesting the anhedonia was attenuated after the EA treatment. Similarly, rats in the MS and Sham-EA groups showed increased immobility time compared to controls in FST. However, EA treatment dramatically decreased the total duration of immobility in rats with neonatal MS (**Figure 2C**). These results suggested that MS-induced depression-like behavior deficits were ameliorated after EA treatment.

To further demonstrate that EA could attenuate depressionlike impairment in rats with neonatal MS, we measured the plasma ACTH and CORT levels, which is a biological indicator of depression and in part explains the relationship between hypothalamic-pituitary-adrenal (HPA) axis regulation and MSinduced depression (Mirescu et al., 2004; van der Doelen et al., 2014; Gururajan et al., 2016; Salvat-Pujol et al., 2017). Rats in the MS and Sham-EA groups showed dramatically increased plasma ACTH and CORT levels, which were significantly decreased by EA treatment (**Figures 2D,E**). These results indicated that

repeated EA intervention induced antidepressant-like effects in rats with neonatal MS.

# RNA-Seq of PFC Transcriptome

We further investigated the possible mechanisms after confirming the anti-depressive effects of EA on rats with neonatal MS. RNA was extracted from the PFC, a brain area which is highly associated with the onset of depression (Kumar et al., 2015; Seo et al., 2017), in control, MS and EA therapy rats. After depletion of rRNA, unbiased deep sequencing was performed to an average depth of ∼89 million reads per sample in the three groups. Approximately 68 million reads mapped to unique locations on the NCBI assembly Rnor\_6.0 reference genome (**Supplementary Table S1**).

# Identification of Differentially Expressed Genes Under EA Treatment in PFC of Rats With Neonatal MS

To determine the regulation of mRNA expression, we performed an unsupervised clustering analysis of the significantly regulated genes in the PFC (**Figure 3A**). The EBseq algorithm was applied to filter the differentially expressed genes after the significant and FDR analyses (fold change >1.5 or <0.667; FDR <0.05). Forty-eight genes were significantly and differentially expressed in MS rats relative to control rats, with 39 upregulated genes and 9 downregulated genes. Thirty-nine genes in the EA rats relative to the MS rats show differential expression, with 21 genes upregulated and 18 genes downregulated (**Supplementary Table S2**). Venn analysis was additionally applied to learn

the possible marker that participates in the exertion of acupuncture's antidepressant effect. We found three of them (Ucp3, Cplx3, Dbp) can be reversed by EA treatment in the nine downregulated genes following MS. Similarly, EA treatment reversed 2 (LOC102555167, Syt6) of the 39 upregulated genes following MS (**Figures 3B,C**).

To show the possible cellular functions linked to differentially expressed genes, we used Gene Ontology (GO) enrichment analysis for the differentially expressed genes across three domains, including molecular functions (MF), cellular components (CC), and biological processes (BP) (**Table 1**). The genes that differentially decreased in the MS group but tended to be reversed after EA treatment were involved in synaptic vesicle exocytosis, circadian rhythm, syntaxin binding and synapse, and so on. Upregulated gene sets in the MS rats and reversed in the EA group were involved in mitochondrial transport, oxidative phosphorylation uncoupler activity, etc.

Long non-coding RNA is a type of RNA molecule that is more than 200 bp in length and has no protein coding ability. Using the same method as above, we found 11 genes increased significantly in the MS rats relative and 4 genes decreased relative to controls. Thirty-one genes increased in the EA rats relative to the MS rats while one gene decreased. Venn analysis shows that 1 gene co-occurred in two comparison groups: Rps2-ps2 (**Figure 4** and **Supplementary Table S2**).

Circular RNA is a new class of RNA that differs from traditional linear RNA. It has a closed loop structure and is abundantly present in the eukaryotic transcriptome. CircRNAs were predicted by finding reads that cover both exons, but the direction is opposite to linear RNA. Twenty genes increased

TABLE 1 | The significant GO terms downregulated in MS but upregulated in EA or upregulated in MS but downregulated in EA (p < 0.05).


significantly in the MS rats relative to control rats and 17 genes decreased. Eighteen genes increased in the EA rats relative to the MS rats while 28 genes decreased. Venn Analysis shows that expression of two genes increased in the MS group but decreased in the EA group, while one gene decreased in the MS group but increased in the EA group. According to the position of the gene

loop, these three genes were derived from LOC102555866, Npepo, Cdh12 (**Figure 5** and **Supplementary Table S2**).

MicroRNA (miRNAs) is a short-chain RNA about 22 nt in length that can reverse the expression of the target gene by inhibiting its translation or degrading it. Competitively binding miRNAs to regulate the expression of target genes is called competitive endogenous RNA (ceRNA). To explore the relationship between mRNA expression and its regulation, coexpression analysis of lncRNA/circRNA, miRNA and mRNA was carried out using the principle of ceRNA. First, the Miranda and the RNAhybrid algorithms were used to predict the target miRNAs for the circRNA, lncRNA, and mRNA, respectively. The combination of the two algorithms was used as the final result (**Supplementary Table S3**). Then,

the genes with the same expression trend (both belong to decrease/increase – increase/decrease type) of lncRNA/circRNA and mRNA were extracted, and the lncRNA/circRNA, miRNA, and mRNA were combined to form an interaction network after Series Cluster analysis (**Supplementary Table S3**). In the lncRNA-miRNA-mRNA predicted interaction network, seven lncRNAs and eight mRNAs had the same expression trend (belonging to the increase-decrease type/decrease-increase type). Thirty miRNAs were predicted to be possible target genes for regulation (**Figure 6A**). In the circRNA-miRNA-mRNA predicted interaction network, 28 circRNAs and 17 mRNAs had the same trend. One hundred sixteen miRNAs were predicted to be potential target genes to regulate these circRNA and mRNA changes (**Figure 6B**).

# DISCUSSION

Maternal separation rats exhibited a significantly lower preference for sucrose and higher immobility time (**Figures 1C,D**), but these behaviors were reversed in the fluoxetine injection group, suggesting MS is a stable and reliable means to model depression-like behaviors. These results are consistent with previous studies that showed chronic stress in early life could induce depressive-like behaviors in adults rats or mice (Arborelius et al., 2004; Vetulani, 2013; Masrour et al., 2018; Ruiz et al., 2018). However, some animal studies have shown that neonatal MS induced anxiety-like behaviors (Banqueri et al., 2017; Auth et al., 2018). Contrastingly, our result in the EPM task, OFT and Light-dark box test showed that rats with neonatal MS spent similar amounts of time in the open arms, center area, and light box, respectively, compared to controls (**Figure 1E** and **Supplementary Figure S1**). This difference may be due to a variation of species of animals (rats vs. mice), the MS protocol (longer vs. shorter duration of separation), and the different postnatal periods (PDN1-21 vs. PDN2-15) of MS (Auth et al., 2018). Meanwhile, the difference of housing conditions during the separation period (staying in home cage under 20–22◦C vs. being kept in a new cage under 30◦C) may also contribute to the variation of behavior phenotypes in adult MS (Banqueri et al., 2017). In addition, we found that the weight of rats with MS increased slightly in adulthood compared to control mice, which is consistent with previous studies (Macri et al., 2008; Yoo et al., 2013; Gonzalez-Pardo et al., 2019). However, other studies show normal body weight or reduced weight of MS rats in adulthood (Ryu et al., 2009; Paternain et al., 2016). Therefore, the relationship between body weight, located environment and MS-induced behavior deficits of animals still needs to be further studied.

Acupuncture (electro- and manual) is a treatment generally recommended for mental illnesses, including depression (Smith et al., 2010; Hempel et al., 2014). Recently, EA at the GV20 and GV29 acupoints has been reported to alleviate depression-related symptoms in model rats induced by chronic unpredictable mild stress (Duan et al., 2009b). Our results are in line with this finding, showing that EA significantly enhanced the sucrose uptake rate in the SPT and decreased the immobility duration in the FST, enabling the most characteristic presentations of depression to be reversed (Han et al., 2015). In addition, early-life stress influenced mature adults with sustained hyper-activation of the HPA axis (Maniam et al., 2014). Consistent with this conclusion, we found rats with MS induced a higher level of ACTH and

CORT in their plasma compared to healthy rats (**Figures 2D,E**), which may in part reveal the potential relationship between early life adverse stress and depression in adulthood (Choi et al., 2018; Frost et al., 2018). Notably, the EA treatment can reverse the increased concentration of ACTH and CORT in rats with MS (**Figures 2D,E**), which is compatible with other studies showing that EA regulates the function of the HPA axis to treat depression (Tanahashi et al., 2016). Together, these results further confirmed that EA is an effective therapy for depression induced by neonatal MS.

Little is known of the mechanism of depression caused by MS, and the mechanism of anti-depression after EA. To the best of our knowledge, this was the first study to show EA has anti-depressive effects in a depression rat model induced by early MS. We detected the PFC genome-wide transcriptome of male rats suffering from MS to reveal the antidepressant effects of EA at the molecular level. Previous studies found EA can alter gene expression in a chronic unpredictable mild stress-induced rat model for mRNA or miRNA by using microarray or RNA-sequencing (Duan et al., 2014, 2016). However, instead of constructing the cDNA libraries by extracting poly-A (Wang et al., 2017), we constructed them after rRNA depletion to obtain data at a larger scale. Sequencing depth involves mRNA (**Figure 3**), lncRNA (**Figure 4**), and circRNA (**Figure 5**), which allowed us to detect differences in gene expression at the transcriptome level and predict the regulatory mechanisms that may mediate changes to their expression by ceRNA (**Figure 6**). These results suggest more comprehensive perspectives to explore the antidepressant mechanisms of acupuncture.

By GO enrichment analysis, we found altered gene sets are involved in circadian rhythm (**Table 1**), which is in accordance with other studies. From the dataset of transcripts from postmortem brains, it has been observed that >100 transcripts exhibited rhythmicity across six brain regions, including the dorsolateral PFC, while rhythmicity was much weaker in these brain areas of patients with MDD compared to those with no history of psychiatric disorders. Correspondingly, the canonical clock genes, including Arntl, Per2-3, and Dbp found in subject transcripts were observed in our analysis (Li J.Z. et al., 2013; Bunney et al., 2015). Furthermore, the clock genes (Arntl, Per2-3) of animals suffering from chronic mild stress (CMS) were strongly expressed in the PFC, but did not follow a circadian rhythm. The other study also found a reduction of the BMAL1 protein (Arntl) in the PFC of CMS rats (Calabrese et al., 2016; Christiansen et al., 2016). Consistent with our conclusions, one previous study reported EA can ameliorate the bowel dysfunction in spinal cord injury rats, and increase Per2 expression (Cheng et al., 2016). Another reported that EA has regulatory effects on the circadian rhythm of temperature in CMS rats (Yao et al., 2014).

Additionally, we found the altered genes enrich in the neurotransmitter transporter activity, syntaxin binding, synaptic vesicle membrane. The genes corresponding to the GO terms are Cplx3 and Syt6. Cplx3 (complexin 3) is a subunit of the presynaptic protein that affects synaptic transmission. Previous studies indicated that Cplx3 can affect the neurotransmitter release process by modulating exocytosis (Vaithianathan et al., 2015; Mortensen et al., 2016). Also, activity-dependent BDNF release via endocytic pathways can be regulated by syt6 and complexin (Wong et al., 2015), which is closely linked to depression (Gururajan et al., 2016). Thus, the above altered expression of the syntaxin binding may provide a means to investigate EA anti-depression effects.

Some previous studies suggested lncRNAs with mouse depression models may relate to depression pathologies (Bond et al., 2009; Huang et al., 2017). Furthermore, a study on peripheral blood profiling also found the expression of certain lncRNAs was changed in patients with MDD (Liu et al., 2014). In CMS mice, total saponins from the leaves of Panax notoginseng inhibited depression by regulating circRNA expression (Zhang H. et al., 2018). In the peripheral blood mononuclear cells of MDD patients, hsa\_circRNA\_103636 was significantly altered, suggesting that circRNA may be a potential novel biomarker (Cui et al., 2016). This suggests that lncRNA and circRNA play a non-negligible role in the mechanisms of pathological depression.

There are many examples of using transcriptome data analysis to investigate the mechanism of acupuncture without coexpression analysis, which could lead to the limitation of unclear and false positives targets (Fu et al., 2014; Hansen et al., 2014; Wang et al., 2015). Using ceRNA, we narrowed the targets of mRNA from 31 to 17 genes with a circRNA\_miRNA\_mRNA network, and narrowed the targets of mRNA from 31 to 8 genes with a lncRNA\_miRNA\_mRNA network (**Figure 6**), pointing the way for further studies to verify the genes and describe their function.

# CONCLUSION

In summary, our investigation indicates that attenuated neonatal MS induced depression-related manifestation by repeated EA treatment at the acupoints GV20 and GV29 during adulthood. Furthermore, we explored the altered gene sets involved in circadian rhythm and neurotransmitter transporter activity in MS rats through unbiased genomewide RNA sequencing to investigate alterations in the transcriptome of the PFC.

# ETHICS STATEMENT

The experimental procedure was approved by the Animals Care and Use Committee of Guangzhou University of Traditional Chinese Medicine.

# AUTHOR CONTRIBUTIONS

YC and NX designed the experiments. YZ and JH conducted the EA treatment and data analysis. ZY, XZ,

and YS conducted the behavioral tests. YC and LG performed the radioimmunoassay analysis. YZ, JH, and YC wrote the manuscript. LY, XW, and YX helped revise the manuscript. All authors read and approved the final manuscript.

#### FUNDING

This work was supported by the National Natural Science Foundation of China (31571041 and 3160060271); Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2016); Scientific Research and Innovation Team Program of Guangzhou University of Chinese Medicine (2017KYTD03); the Innovation Team Program of Guangdong Provincial Department of Education (2017KCXTD006); and Science and Technology Program of Guangdong (2018B030334001).

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TABLE S1 | Statistical results by sequence quality control and the mapping rate by comparing with reference genes.

TABLE S2 | Differentially expressed mRNA/lncRNA/circRNA in rats.

TABLE S3 | Detail of the lncRNA/circRNA-miRNA-mRNA predicted interaction network.


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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Zheng, He, Guo, Yao, Zheng, Yang, Xia, Wu, Su, Xu and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Augmented Mechanical Forces of the Surface-Modified Nanoporous Acupuncture Needles Elicit Enhanced Analgesic Effects

Sun-Jeong Bae1,2, Junsik Lim1,3, Sangmin Lee1,4, Hansaem Choi<sup>5</sup> , Jae-Hwan Jang1,6 , Yu-Kang Kim1,2,6, Ju-Young Oh1,2,6, Jeong Hun Park<sup>6</sup> , Hyuk-Sang Jung<sup>2</sup> , Younbyung Chae1,2,6, Su-Il In<sup>5</sup> \* and Hi-Joon Park1,2,6 \*

<sup>1</sup> Acupuncture and Meridian Science Research Center, Kyung Hee University, Seoul, South Korea, <sup>2</sup> College of Korean Medicine, Kyung Hee University, Seoul, South Korea, <sup>3</sup> College of Korean Medicine, Semyung University, Jecheon, South Korea, <sup>4</sup> College of Korean Medicine, Dongguk University, Goyang, South Korea, <sup>5</sup> Department of Energy Science and Engineering, DGIST, Daegu, South Korea, <sup>6</sup> Graduate School of Korean Medicine, Kyung Hee University, Seoul, South Korea

#### Edited by:

Florian Beissner, Hannover Medical School, Germany

#### Reviewed by:

Jui-Hung Hung, China Medical University Hospital, Taiwan Karen M. von Deneen, Xidian University, China

#### \*Correspondence:

Su-Il In insuil@dgist.ac.kr Hi-Joon Park acufind@khu.ac.kr

#### Specialty section:

This article was submitted to Perception Science, a section of the journal Frontiers in Neuroscience

Received: 11 December 2018 Accepted: 06 June 2019 Published: 20 June 2019

#### Citation:

Bae S-J, Lim J, Lee S, Choi H, Jang J-H, Kim Y-K, Oh J-Y, Park JH, Jung H-S, Chae Y, In S-I and Park H-J (2019) Augmented Mechanical Forces of the Surface-Modified Nanoporous Acupuncture Needles Elicit Enhanced Analgesic Effects. Front. Neurosci. 13:652. doi: 10.3389/fnins.2019.00652 Over the past several decades, clinical studies have shown significant analgesic effects of acupuncture. The efficacy of acupuncture treatment has improved with the recent development of nanoporous needles (PN), which are produced by modifying the needle surface using nanotechnology. Herein, we showed that PN at acupoint ST36 produces prolonged analgesic effects in an inflammatory pain model; the analgesic effects of PN acupuncture were sustained over 2 h, while those using a conventional needle (CN) lasted only 30 min. In addition, the PN showed greater therapeutic effects than CN after 10 acupuncture treatments once per day for 10 days. We explored how the porous surface of the PN contributes to changes in local tissue, which may in turn result in enhanced analgesic effects. We showed that the PN has greater rotational torque and pulling force than the CN, particularly at acupoints ST36 and LI11, situated on thick muscle layers. Additionally, in ex vivo experiments, the PN showed greater winding of subcutaneous connective tissues and muscle layers. Our results suggest that local mechanical forces are augmented by the PN and its nanoporous surface, contributing to the enhanced and prolonged analgesic effects of PN acupuncture.

Keywords: acupuncture analgesia, nanoporous needle, muscle, connective tissue, torque, complete Freund's adjuvant

# INTRODUCTION

Acupuncture treatment has been used as a therapeutic modality in East Asia since 6000 BC, starting with a sharpened stone and progressing to stainless steel needles (White and Ernst, 2004; Wang et al., 2008). Acupuncture is of continuing global interest; the World Health Organization stated that acupuncture can be used to treat 63 diseases. Controlled clinical studies have shown its effectiveness in treating 28 diseases, including depression, nausea, vomiting, insomnia, and

pain (Lao, 1996; Leake and Broderick, 1999). Among these conditions, treating pain is one of the most common uses of acupuncture therapy (Wang et al., 2008; Han, 2011; Vickers et al., 2012; Lee et al., 2013), particularly for treating lower back pain (Gunn et al., 1980; Macdonald et al., 1983; Manheimer et al., 2005; Lee et al., 2013) and osteoarthritis (Casimiro et al., 2005; Kwon et al., 2006).

Acupuncture is the practice of inserting needles into specific areas of the body surface, called acupoints, by penetrating the epidermis, dermis, subcutaneous layer, and/or muscle (Kimura et al., 1992; Park et al., 2011; Zhang et al., 2015). To enhance its therapeutic effect, acupuncture is often accompanied by manipulation of the needle involving rotation, vibration, and in-and-out movements (Stux et al., 2003; Meridians and Acupoints Compilation Committee of Korean Oriental Medical Colleges, 2015). Although details of the underlying mechanisms remain unclear, the impact of acupuncture is believed to involve various molecules [e.g., adenosine (Fredholm, 2007; Goldman et al., 2010), pERK (Park et al., 2014), and/or TRPV1 (Abraham et al., 2011; Wu et al., 2014; Liao et al., 2017)] and structures [e.g., subcutaneous connective tissue (Shen et al., 1973; Langevin et al., 2001b, 2002; Zhao, 2008), muscle (Shen et al., 1973), nerve (Kim et al., 2013), vessels (Huang T. et al., 2013), fibroblasts (Langevin et al., 2007, 2011), mast cells (Zhang et al., 2007; Fung, 2009), and/or keratinocytes (Geoffrey, 2009; Park et al., 2014)] near the acupoints, resulting in reconstitution and activation of various signals (Lin and Chen, 2008; Zhao, 2008; Park et al., 2014; Shu et al., 2016).

Because local changes induced by needle insertion and/or manipulation initiate the therapeutic effects of acupuncture, we hypothesized that potentiating the intensity of acupuncture stimulation could enhance its therapeutic efficacy. Thus, we developed a novel acupuncture needle, the micro/nanoporous acupuncture needle (PN). Unlike the smooth surface of a conventional acupuncture needle (CN), the PN surface has micro/nano-sized holes that provide a 20-fold increase in surface area. Previous studies have explored the properties of the PN surface produced using electrochemical techniques (Lee et al., 2017). Acupuncture using the PN showed a greater therapeutic effect than did the CN in an addiction model (In et al., 2016) and a colorectal cancer model (Lee et al., 2017). Other than the electrical conductivity (Not understood; how is porosity related to electrical conductivity?), the micro/nanoporous surface of PN has different physical characteristics that affect the therapeutic efficacy of acupuncture treatment. However, no studies have explored the physical impact of PN acupuncture, including the pullout force, mechanical load, and structural deformation induced by surface modification of local tissues during PN acupuncture.

In this report, we (i) analyzed various aspects of PNs and CNs on four different acupoints by measuring the pullout force and rotational torque using an Acusensor; (ii) compared the morphological deformation of local tissues induced by PN and CN acupuncture; and (iii) investigated changes in therapeutic effects induced by PN acupuncture by assessing the analgesic effects in an inflammatory pain model.

# MATERIALS AND METHODS

# Experimental Animals

All experiments were performed using male C57BL/6 mice aged 7–10 weeks (Samtaco, Seoul, Korea). The mice were housed under 12-h light–dark cycle conditions with continuous access to chow and water. The mice were acclimated to the housing facility for 1 week before the experiments. All experiments were approved by the Dongguk University Animal Care Committee for Animal Welfare, and were performed according to the guidelines of the National Institutes of Health and the Korean Academy of Medical Sciences (IACUC-2017-022-1).

### Preparation of the PNs

To manufacture the PNs, conventional stainless steel acupuncture needles (8-mm length and 0.30-mm diameter; Dong Bang Acupuncture Inc., Boryeoung, South Korea) were anodized. First, the needles were washed sequentially with acetone, ethanol, and deionized (DI) water. Then, the stainless steel needles were anodized using a two-electrode cell. The cell was composed of a needle for the working electrode, carbon paper (2 cm × 0.5 cm × 0.042 cm carbon and fuel cell; CNL Technology, Suwon, South Korea) for the counter electrode, electrolytes with 0.2 wt.% NH4F (98%; American Chemical Society reagent, Alfa Aesar; Thermo Fisher Scientific, Waltham, MA, United States), and 2.0 vol% DI water in ethylene glycol. The needles were anodized for 30 min with 20 V (Paulose et al., 2006). Then, the anodized needles were rinsed with acetone, ethanol, and DI water, and dried in a nitrogen gas stream.

# Characterization of the PNs

The morphology of the needle surface was evaluated using a high-resolution scanning electron microscope (S-4800; Hitachi, Tokyo, Japan) operated at 3 kV. EIS measurements were obtained using a Biologics SAS (Model VSP-1158; Biologics, Cary, NC, United States) three-electrode workstation with a platinum wire as the counter electrode, Ag/AgCl electrode as the reference electrode and the needles as the working electrodes. The system was operated using EC Lab software in the frequency range of 100 kHz to 200 MHz. The electrolyte consisted of a saline solution (0.9 g NaCl in 100 mL DI water) purchased from JW-Pharma (Seoul, South Korea) and was used without further modifications.

### Measurements of the Rotational Torque and Pulling Force

Mice were anesthetized by intraperitoneal (i.p.) injection of Zoletil/Rompun and acupuncture needles (CN and PN, 8 mm in length, 0.18 mm in diameter; Dongbang Co., South Korea) were inserted to a depth of 3 mm at the ST36, LI11, ST25 and BL23 acupoints; **Figure 2b** shows the acupoints used in this study. Acupuncture using CNs and PNs was performed as follows: (1) without manipulation, or (2) with manipulation (consisting of clockwise rotations of 360◦ /s for nine spins). Pulling and rotational forces of the acupuncture needling were measured using the Acusensor (Stromatec, Inc., Burlington, VT, United States) motion sensor system (**Figure 2a**). The vertical

force acting on the needle to resist its insertion and pulling, and the rotational force acting on the needle to resist its rotation, were measured using a motion sensor. The data of each acupoint were analyzed respectively. And overall values were also calculated using average data.

# Histochemical Staining of Abdominal Tissues

#### Sampling

We performed histochemical analyses of the abdominal tissues around the needle. The large surface area and lack of bones in the abdominal tissues allowed for rapid histological analyses of acupuncture treatment (Langevin et al., 2001a). Mice were anesthetized by Zoletil/Rompun (i.p.). The CNs and PNs (or NN) were inserted on both sides of the abdomen in a random order. Then, the needles were manipulated by nine unilateral spins, with one spin consisting of a clockwise rotation of 360◦ . For all insertions, the needle depth was held constant at 3 mm, with the needle penetrating the entire abdominal wall, and the track was labeled with tissue-marking dye (Davidson Marking System, Bradley Products, Inc., Bloomington, MN, United States). After the manipulation, the mice were sacrificed without removing the needle, and the whole body was immersion-fixed in 10% formalin for 48 h. Then, the abdominal tissues were excised, including the epidermis to the abdominal muscle layer and the needle, and fixed again in 10% formalin overnight.

#### Histological Analysis

The needles were removed after paraffinization of the samples and before paraffin embedding. Tissue blocks were cut parallel to the needle axis at 8-µm thickness using a manual rotary microtome (Shandon Finesse 325; Thermo Fisher Scientific Inc.). Every fifth slide with a needle track label was stained with hematoxylin (Merck Co., Darmstadt, Germany) and eosin (Sigma–Aldrich Co., St. Louis, MO, United States), or Masson trichrome-counterstained with aniline blue (Masson Trichrome Stain Kit, Nova UltraTM; IHC World, Ellicott City, MD, United States), which stains collagen as blue and muscle as red. Then, the thicknesses of the epidermis, dermis, subcutaneous, and abdominal muscle layers along the needle track were measured using a light microscopy (BX51, Olympus, Japan), with the subcutaneous layer divided into adipose tissue, cutaneous muscle, and subcutaneous connective tissue.

#### Behavioral Experiments

#### Experimental Groups and Acupuncture Treatment

Acupuncture was performed at the ST36 (Joksamli or Zusanli) acupoint, located 3–4 mm below and 1–2 mm lateral to the midline of the knee (Lim, 2010) (**Figure 2b**). ST36 is one of the most effective points in East Asian medicine and its anti-nociceptive effects in rodent CFA models of chronic pain are well-established (Goldman et al., 2010; Wu et al., 2014; Lu et al., 2016).

The mice were divided into five treatment groups: treatment with CFA only (CFA), treatment with CFA and non-invasive acupuncture (NN) needling at the ST36 acupoint (CFA+NN), treatment with CFA and CN at the ST36 acupoint (CFA+CN), treatment with CFA and PN at the ST36 acupoint (CFA+PN), and control (CON) (n = 7–8 in each group).

The mice in the CFA+CN and CFA+PN groups were immobilized by hand, and the needles were inserted to a depth of 3 mm at the bilateral acupoints. Then, the acupuncture needles were turned at a rate of two spins/s bidirectionally (one spin consisted of clockwise rotation of 180◦ and a counterclockwise rotation of 180◦ ) for 30 s. The needles were removed immediately after manipulation. To create a blunt non-invasive acupuncture needle, the sharp end of the acupuncture needle was snipped. The mice in the CFA+NN group were pressed with 50 mN of force non-invasively, and no manipulation was done for 30 s. The mice in the CON and CFA groups were also immobilized by hand for 1 min to ensure an equal amount of stress among the acupuncture groups. The treatments began at 4 days after CFA injection once per day for 10 days. **Figures 7A,B** shows the schedule of the acupuncture treatments and behavioral experiments.

#### CFA-Induced Mouse Pain Model

CFA oil suspension was diluted 1:1 with saline and used to induce chronic inflammation. The mice were anesthetized with ether before the injection. The mice in the CFA, CFA+NN, CFA+CN, and CFA+PN groups received a subcutaneous injection of 100 µL CFA emulsion solution into the plantar of both hind paws, and the mice in the CON group received identical injections with equal amounts of saline. CFA-treated mice developed signs of inflammation that were prominent at 4 days after the injection.

#### Mechanical Threshold Assessment

Mechanical allodynia was assessed by testing the withdrawal response to von Frey filaments. Before the baseline test, the mice were habituated in 8 × 10 × 10 cm acrylic boxes with gridded floors, daily for 1 h for 2 days. Mechanical thresholds were assessed before CFA injection, as well as and after acupuncture treatment (**Figure 7A**). Prior to each test, the mice were habituated again for 1 h. The mechanical allodynia of the hind paws was assessed using an electronic von Frey filament (**Figure 7B**). The filament was applied to the plantar surface of the hind paw with 0.6 g of force for 1 s. All mice underwent 10 applications on both hind paws, with a 5 s interval between each application. Frequency was defined according to positive responses from 10 applications. The assessor was blinded to the group assignment, and an experienced investigator who was completely blinded counted the withdrawal frequencies.

To observe the short-term changes of allodynia induced by acupuncture, tests were performed before an acupuncture treatment and every 30 min for 2 h after the treatment (0, 30, 60, 90 and 120 min after acupuncture treatment). We tested these short-term effects only after the 1st and the 8th acupuncture treatment, to minimize the stress. And to test the long-term changes, we tested mechanical allodynia on the 1st, 3rd, 7th, and 10th day of acupuncture treatment (**Figure 7A**).

#### Statistical Analyses

GraphPad Prism 7 software (GraphPad Software Inc., La Jolla, CA, United States) was used for the statistical analyses. All data

are expressed as means and standard error of the mean (SEM). Statistical analyses were performed using one-way analysis of variance (ANOVA), two-way ANOVA or repeated measures of ANOVA, where appropriate. The Bonferroni post hoc test was followed. In all analyses, p < 0.05 was considered to indicate significant differences.

#### RESULTS

#### Physical Properties of PNs

**Figures 1a–d** shows field emission scanning electron microscopy (FE-SEM) images of stainless steel CNs compared with PNs, fabricated by anodizing CNs using an electrolyte of 0.2 wt.% NH4F and 2 vol.% DI water in ethylene glycol at room temperature for 30 min at 20 V (Paulose et al., 2006). Initially, the CN had a smooth surface (**Figure 1a**); after anodization, the needle showed a micro-nanoscale porous surface topology with a high surface area (**Figure 1b**). High-resolution images of the PN showed uniform formation of conical-shaped pores (**Figure 1c**); a cross-sectional image of the porous topology is shown in **Figure 1d**.

Electrochemical impedance spectroscopy (EIS), a powerful technique for characterizing surfaces (Ehsani et al., 2014), was used to investigate CNs and PNs. **Figure 1e** shows the fitted Nyquist plots corresponding to the EIS results for CNs and PNs immersed in saline solution. The Nyquist plots for both needles show similar shapes, indicating that saline had no effect on the needle surface. Depressed semicircles are formed due to the combination of the charge transfer resistance (RCT) and the constant phase element of the working electrode (acupuncture needles) and the electrolyte interface (Ehsani et al., 2013). A reasonable decrease was observed in the semicircle diameter in the PNs, indicative of a significant decrease in RCT.

### Measurements of Rotational Torque and Pullout Force at Four Representative Acupoints

The rotational torque and pullout force of PNs and CNs were measured using the Acusensor motion sensor system (**Figure 2a**). Four representative acupoints at the mouse hindlimb (ST36), forelimb (LI11), abdomen (ST25), and back (BL23) (**Figure 2b**) were used to identify and compare mechanical loads between PN and CN at each acupoint.

The rotational torque was measured during nine rotations at each acupoint (**Figures 3A–D**). The torque induced by PN and CN was enhanced as the number of rotations was increased in both PN and CN groups, and the rate of increase was much higher in the PN than in the CN group. Especially, the PN group showed significantly higher torque than the CN group when acupuncture was administered to ST36 (P < 0.05 at both the 6th and 8th rotation) and to ST25 (P < 0.05 at the 8th rotation).

The pullout force was measured after nine rotations of the needles (**Figures 3E–H**). These rotations induced trends of increased pullout force compared to no rotations for both the PN and CN groups; the increase was significant at acupoint ST36 (only PN: P < 0.001), LI11 (CN: p < 0.05, PN: P < 0.001), ST25 (only PN: P < 0.05), and BL23 (only PN: P < 0.01). As shown in **Figures 3E–H**, compared with the CN group, the PN group showed significantly higher pullout force at ST36 (P < 0.001) and LI11 (P < 0.01). No increase in pullout force was found for either needle group when the needle was inserted without rotation.

To explore differences in mechanical load between PNs and CNs on local tissues, we calculated the overall pulling force and rotational torque by averaging data of each location (**Figure 4**). The results showed that the overall rotational torque of the PN group increased to a more significant degree than did that of the CN group (P < 0.01, P < 0.001, P < 0.01, and P < 0.001 at the 5th, 7th, 8th, and 9th rotations, respectively; **Figure 4A**). The overall

pulling force of the PN group was significantly higher than that of the CN group (P < 0.01, **Figure 4A**).

## Histological Analysis in Local Tissues After Acupuncture

Histological analysis using hematoxylin and eosin staining (**Figures 5a,c,e**) or Masson's trichrome staining (**Figures 5b,d,f**) was performed to investigate acupuncture-induced morphological changes in local tissues, including the epidermis, dermis, subcutaneous tissue (adipose, cutaneous muscle, and subcutaneous connective tissue), and muscle layers. Masson's trichrome staining of the samples revealed that collagencontaining layers such as dermis, adipose, and subcutaneous connective tissue were stained blue; the epidermis, cutaneous muscle, and abdominal muscle were stained red. The measured thicknesses of the layers are shown in **Figure 6**.

Histological analysis showed that the thickness of subcutaneous layers increased after rotating CNs and PNs. In the adipose layer, both CNs and PNs increased the thickness, but only PNs showed a significant change compared to the CON group (CON: 69.4 ± 10.3 µm; CN: 173.6 ± 42.1 µm; PN: 218.5 ± 28.1 µm, P < 0.05 CON vs. PN). In the cutaneous muscle layer, both CNs and PNs increased the thickness, but only PNs induced a significant difference compared to the CON group (CON: 42.9 ± 4.3 µm; CN: 64.2 ± 7.0 µm; PN: 78.1 ± 4.1 µm, P < 0.01 vs. CON). In subcutaneous connective tissue, both CNs and PNs induced significantly greater thickness compared to the CON group (CON: 58.0 ± 11.7 µm; CN: 259.2 ± 77.0 µm; PN: 339.2 ± 53.3 µm, all P < 0.05 vs. CON); there was no difference between CNs and PNs (**Figures 5**, **6**). Masson's trichrome staining showed a whorl pattern in collagen fibers in subcutaneous connective tissues in the CN and PN groups (**Figures 5b,d,f**).

Based on muscle layer analysis, morphological changes in muscle thickness were only significant with PNs: the thickness using PNs (942.7 ± 44.8 µm) was significantly greater than that using CNs (665.4 ± 122.0 µm, PN vs. CN: P < 0.01) and that for the CON group (524.7 ± 38.3 µm, PN vs. CON: P < 0.001, **Figures 5**, **6**). Masson's trichrome staining analyses showed significant whorl patterns in muscles layers in the PN group (**Figures 5b,d,f**).

No morphological changes in the dermis and epidermis were observed in either group.

## Effects of PN Acupuncture on the Mechanical Pain Threshold in Complete Freund's Adjuvant (CFA)-Induced Inflammatory Pain Model

We investigated the short-term and long-term analgesic effects of CN and PN acupuncture in a CFA-induced inflammatory pain model. Acupuncture was performed at acupoint ST36 (**Figure 2b**), and mechanical allodynia was measured using Von Frey filaments (**Figure 7B**). The baseline mechanical threshold before CFA injection was similar across all groups. Acupuncture treatments with CN and PN started 4 days after CFA injection, when the pain reached a stable level.

At the 1st acupuncture treatment, both CN and PN attenuated mechanical allodynia at 30–60 min post-acupuncture; however, only the PN group (5.9 ± 0.4) showed a significant difference compared to the CFA group (7.8 ± 0.4, P < 0.001) at 30 min post-acupuncture. Moreover, allodynia scores of the PN group were significantly lower than those of the CN group (7.0 ± 0.3, P < 0.05) at the same time point. At 120 min after treatment, mechanical allodynia in both the CN (7.69 ± 0.2) and PN (7.57 ± 0.3) groups returned to the pre-treatment values (**Figure 7A**).

FIGURE 4 | Overall value of rotational torque (A) and pulling force (B). PNs had significantly stronger pulling force and rotational torque than CNs (##P < 0.01, ###P < 0.001, CN vs. PN; two-way ANOVA followed by the Bonferroni test; each n = 16). Error bars indicate the SEM.

After the 7th acupuncture treatment, the short-term effects of PN and CN acupuncture were tested again using the same protocol. As previously shown (Zhao, 2008; Goldman et al., 2010),

muscle (M). The dotted blue line indicates the inserted trace of acupuncture needle. Scale bar, 500 µm.

the most effective time for CN acupuncture was 30 min after acupuncture (4.4 ± 0.6, P < 0.001 vs. CFA group), with the effect returning to baseline after 90 min. In contrast, in the PN group,

FIGURE 6 | Thickness of each layer in the abdominal wall. The thickness of each tissue layer was measured at the epidermis (E), dermis (D), adipose (A), cutaneous muscle (CM), subcutaneous connective tissue (SC), and abdominal muscle (M). SCL represents the subcutaneous layer. <sup>∗</sup>P < 0.05, ∗∗∗P < 0.001, compared to the CON group; one-way ANOVA followed by the Bonferroni test; each n = 4–5. Error bars indicate the SEM.

the level of mechanical allodynia decreased gradually until 90 min after acupuncture (4.0 ± 0.3, P < 0.001 vs. CFA group), and the decrease continued until 120 min after acupuncture.

A comparison of the pain levels of those in the PN group with that of those in the CN group showed that the PN group exhibited significantly less pain than did the CN group at 90 and 120 min after acupuncture (both P < 0.001, **Figure 7B**).

In addition to the short-term analgesic effect, we observed long-term therapeutic effects measured 24 h after each acupuncture treatment. Throughout 10 acupuncture treatments (once per day for 10 days), both the PN and CN groups showed gradual alleviation of mechanical allodynia (PN vs. CFA, P < 0.01 after the 1st acupuncture session; CN vs. CFA, P < 0.05 after the 2nd acupuncture session). The difference between the PN and CN groups gradually increased as the number of treatments increased; the anti-allodynic effect of PN (4.5 ± 0.4) was significantly greater than that of CN (5.8 ± 0.4) after 10 acupuncture sessions (measured 1 day after the 10th acupuncture treatment, P < 0.05, **Figure 8**).

FIGURE 7 | The short-term effects of porous and conventional acupuncture needles in the CFA-induced inflammatory pain model. Timeline of the mechanical threshold assessment in the complete Freund's adjuvant (CFA)-induced pain model (A) Starting 4 days after CFA treatment, acupuncture treatment was administered once per day for 10 days (A1–A10, total 10 times). The Von Frey test was conducted as follows: at 2 days before CFA injection (•, baseline); at 30, 60, 90, and 120 min after acupuncture treatment (}); and at 1 day after acupuncture (◦). Schematic diagram of the von Frey test (B). The analgesic effect of acupuncture under the CFA-induced inflammatory pain model was assessed after the 1st (C) and 7th (D) acupuncture treatments (∗∗P < 0.01, ∗∗∗P < 0.001, compared to the CFA; #P < 0.05, ###P < 0.001, CN vs. PN; two-way repeated measures ANOVA followed by the Bonferroni test; each n = 7–8) (C,D). Error bars indicate the SEM.

needles in the CFA-induced inflammatory pain model. The paw withdrawal frequency of each mouse was measured 24 h after acupuncture treatments. The PN group showed a greater therapeutic effect than the CN group ( <sup>∗</sup>P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, compared to the CFA group; #P < 0.05, CN vs. PN; two-way repeated measures ANOVA followed by the Bonferroni test; each n = 7–8). Error bars indicate the SEM.

#### DISCUSSION

Although PNs have been developed and applied for improving acupuncture treatment efficacy (In et al., 2016; Lee et al., 2017; Sorcar et al., 2018), no study has explored their analgesic effect, mechanical load, or influence on tissues. Therefore, we investigated the local impact and morphological changes caused by PN acupuncture and measured the difference in analgesic effects between PN and CN acupuncture. The results are summarized as follows: (1) PN induced larger mechanical load than CN; (2) likewise, the PN treatment tended to produce greater morphological changes in muscle layer thickness around the needle track than the CN treatment; and (3) in the CFA inflammatory pain mouse model, PN acupuncture at ST36 resulted in a longer duration of analgesic effect and longer-term treatment efficacy compared with CN acupuncture.

The different impact of PN on local tissues compared with that of CN is likely due to the high surface area of the needle. In et al. reported that the surface area of a PN is 20× greater than that of a CN of the same diameter (In et al., 2016). FE-SEM images confirm the presence of micro-nano porosity on the PN surface. This porosity significantly increases the PNs contact with the surrounding environment; the smaller RCT of EIS implies a greater surface area (Ehsani et al., 2013, 2014). The larger surface area enhances the manipulation-induced frictional force between the needle and adjacent tissues, in turn enhancing the needle-grasp force (Kwon et al., 2017). PNs showed greater deformation of tissues (subcutaneous and muscle layers) around the needle and higher needle-grasp compared to CNs, as measured by the pulling force and torque on four different acupoints.

Histological examination of mouse abdominal tissues indicated that PN acupuncture significantly thickened the subcutaneous connective tissues and muscle layer, resulting in thicker layers than those induced by CN acupuncture, especially in the muscle. Subcutaneous connective tissue and muscle are the two main components of needle grasp, facilitating the therapeutic effect (Shen et al., 1973; Langevin et al., 2002; Zhao, 2008). Rotational manipulation of the acupuncture needle facilitates the gathering of collagen fibers of connective tissues around the needle (Langevin et al., 2006), transmitting mechanical stimulation to the surrounding cells and activating a cellular response in the fibroblasts of connective tissues (Burridge et al., 1988; Banes et al., 1995). Muscle contraction is also believed to induce needle grasp, and its polymodal-type receptor in deep tissue is associated with needle grasp and therapeutic effects (Shen et al., 1973; Lin et al., 1979). Furthermore, other tissue layers (e.g., the epidermis and dermis) may play a role in the effects of acupuncture (Langevin et al., 2007, 1; Abraham et al., 2011; Park et al., 2014); however, morphological changes were minimal in this study.

The exact mechanism through which PNs exert a greater effect cannot be determined based on morphological observation. However, our results are consistent with previous reports suggesting that a greater dose of acupuncture stimulation leads to greater changes at the molecular level, increasing signal transduction and thus exerting an enhanced effect (Goldman et al., 2010; Zylka, 2010; Abraham et al., 2011; Park et al., 2014; Wu et al., 2014). This newly developed PN acupuncture method results in greater stimulation of tissues around the needle track and enhances spinal dorsal horn neuronal activity by acupuncture stimulation in rats (In et al., 2016). Several studies have shown that PNs enhance the therapeutic effect of acupuncture (In et al., 2016; Lee et al., 2017; Sorcar et al., 2018); however, this study is the first to describe the analgesic effect of PNs, the most representative effect of acupuncture.

This study showed that the short-term analgesic effect of PN acupuncture at ST36 was greater than that of CN acupuncture in the CFA-induced pain mouse model. PN acupuncture showed greater immediate (30 min after treatment) and prolonged (90–120 min after treatment) analgesic effects. Our results are in agreement with studies showing higher efficacy and greater therapeutic effects of PN acupuncture in an addiction model (In et al., 2016) and a colorectal cancer model (Lee et al., 2017) compared with CN acupuncture.

Morphologically, CN acupuncture needle rotations induce collagen winding in subcutaneous connective tissues with no structural changes in the muscle layers (Langevin et al., 2001a). In contrast, PN acupuncture resulted in winding of both muscle and subcutaneous connective tissues. The difference in needle grasp between the PN and CN was noticeable at ST36 and LI11. Anatomically, ST36 and LI11 are situated on thicker muscle layers (i.e., the tibialis anterior muscle for ST36 and extensor carpi radialis longus muscle for LI11) compared to ST25 and BL23. In the upper skin layer, the collagen fibers are relatively small, and only needle tracks can be observed rather than tissue twirling; thus, morphological changes appear to be relatively minimal. However, the lower degree of morphological change

does not indicate that there are no biological changes associated with the therapeutic effects of acupuncture. Park et al. reported that acupuncture manipulation increased the expression level of pERK in keratinocytes of the epidermis, which may be associated with the analgesic effects of acupuncture. Further studies should be performed simultaneously considering both morphological and molecular changes. Although our results do not preclude the participation of other layers in the acupuncture effects, this study supports that muscle layers may play an important role in the improvements induced by PNs. Taken together, these findings suggest that both subcutaneous connective tissue and muscle contribute to the effects of acupuncture; however, the enhanced therapeutic effects by PNs may be associated with the effects on muscle layers.

Changes in subcutaneous connective tissue in the CN group were less marked than those reported previously (Li et al., 2005; Langevin et al., 2011; Huang C.-P. et al., 2013; Lu et al., 2016). This difference is likely due to the less marked manipulation in the present versus previous studies (Langevin et al., 2002, 2007) because a smaller manipulation using PNs was sufficient to produce an effective change similar to that reported previously (Kwon et al., 2017).

Our study has several limitations. First, to perform the experiments more precisely, sample size calculation is recommended based on a previous study (Festing and Wilkinson, 2007; Kar and Ramalingam, 2013). Since no previous studies have evaluated the analgesic effects of PN and CN, we could not calculate the sample size for this experiment. For subsequent large-scale studies of CN and PN groups, the sample size was calculated using G∗Power (G∗Power 3.1.9.2 for Windows 10, Faul et al., 2007) 1 . The measurement of mechanical pain in the CN and PN groups was used as ground data. The sample size was calculated using the independent t-test with an α-level of 0.05 and power of 80%. The number calculated was 10 per group, with an effect size d:1.219424, critical t: 1.7340636, Df: 18, noncentrality parameter δ:2.7267150, and actual power: 0.8357533. Next, our results did not reveal whether the PN had better effects than the CN at the other acupoints (excluding ST36). Therefore, further studies are required to compare the effects of the two types of needles using clinically relevant disease models with each acupoint. For example, the effects of ST25 can be tested in a visceral pain or colitis model (Shi et al., 2011; Zhu L. et al., 2018; Zhu X. et al., 2018; Ji et al., 2019). Differences in structural

1 http://www.gpower.hhu.de

#### REFERENCES


changes according to the modified needle surfaces may be associated with greater analgesic efficacy, but the detailed mechanisms should be explored. In addition, further experiments are required to characterize the enhanced analgesic effect of PN and its detailed mechanism in humans.

Overall, this study is the first to report that the PN, with its large surface area, shows a greater needle grasping power than the CN, thus producing greater morphological changes in subcutaneous and muscle tissues. More importantly, we showed that this treatment increases the analgesic effects with prolonged time. This study suggests that the PN is an effective tool for improving acupuncture efficacy in clinical medicine.

#### ETHICS STATEMENT

All experiments were approved by the Dongguk University Animal Care Committee for Animal Welfare, and were performed according to the guidelines of the National Institutes of Health and the Korean Academy of Medical Sciences (IACUC-2017-022-1).

#### AUTHOR CONTRIBUTIONS

JP, S-JB, JL, and SL conceived and designed the study. S-II, J-HJ, S-JB, JL, SL, and YC developed the methodology. SB, JL, SL, Y-KK, and HC acquired the data. S-JB, JL, SL, H-SJ, and J-YO analyzed and interpreted the data. SB, JL, SL, H-JP, and S-II wrote, reviewed, and revised the manuscript. H-JP, S-II, and JP administrated the technical or material support. H-JP supervised the study. S-JB, JL, and SL drew the **Figures 2**, **7**. All authors had input into the manuscript and have approved the manuscript for publication.

# FUNDING

This work was supported by grants from the National Research Foundation of Korea funded by the Korean Government (2017R1A2B4009963) and the Korea Institute of Oriental Medicine (Grant K18182) to H-JP, and by grants of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare (HI19C0506) to S-II.

cytoskeleton. Annu. Rev. Cell Biol. 4, 487–525. doi: 10.1146/annurev.cb.04. 110188.002415


electroactive film: comparative study. J. Electroanal. Chem. 710, 29–35. doi: 10.1016/j.jelechem.2013.01.008


fnins-13-00652 June 19, 2019 Time: 15:19 # 11


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling Editor is currently organizing a Research Topic with one of the authors, YC, and confirms the absence of any other collaboration.

Copyright © 2019 Bae, Lim, Lee, Choi, Jang, Kim, Oh, Park, Jung, Chae, In and Park. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fnins-13-00652 June 19, 2019 Time: 15:19 # 12

# Effects of Acupuncture on Chronic Stress-Induced Depression-Like Behavior and Its Central Neural Mechanism

*Min-Ju Lee1† , Jae-Sang Ryu1† , Seul-Ki Won1 , Uk Namgung1 , Jeeyoun Jung2 , So-Min Lee2 and Ji-Yeun Park1 \**

*1 Department of Korean Medicine, College of Korean Medicine, Daejeon University, Daejeon, South Korea, 2 Clinical Medicine Division, Korea Institute of Oriental Medicine, Daejeon, South Korea*

#### *Edited by:*

*Yi-Wen Lin, China Medical University, Taiwan*

#### *Reviewed by:*

*Dae Hahm, Kyung Hee University, South Korea Xinjing Yang, The University of Hong Kong, Hong Kong*

*\*Correspondence: Ji-Yeun Park jypark@dju.kr These authors have contributed equally to this work*

*†*

#### *Specialty section:*

*This article was submitted to Health Psychology, a section of the journal Frontiers in Psychology*

*Received: 14 December 2018 Accepted: 24 May 2019 Published: 05 July 2019*

#### *Citation:*

*Lee M-J, Ryu J-S, Won S-K, Namgung U, Jung J, Lee S-M and Park J-Y (2019) Effects of Acupuncture on Chronic Stress-Induced Depression-Like Behavior and Its Central Neural Mechanism. Front. Psychol. 10:1353. doi: 10.3389/fpsyg.2019.01353*

Depression is a serious psychiatric disorder with an enormous socioeconomic burden, and it is commonly comorbid with pain, chronic fatigue, or other inflammatory diseases. Recent studies have shown that acupuncture is an effective therapeutic method for reducing depressive symptoms; however, the underlying mechanism remains unknown. In this study, we investigated the effects of acupuncture on chronic stress-induced depression-like behavior and its central neural mechanisms in the brain. We induced chronic restraint stress (CRS) in male C57BL/6 mice for 14 or 28 consecutive days. Acupuncture treatment was performed at KI10·LR8·LU8·LR4 or control points for 7 or 14 days. Depression-like behavior was assessed with the open field test. Then, brain neural activity involving c-Fos and serotonin-related mechanisms *via* the 5-HT1A and 5-HT1B receptors were investigated. Acupuncture treatment at KI10·LR8·LU8·LR4 points rescued the depressive-like behavior, while control points (LU8·LR4·HT8·LR2) and non-acupoints on the hips did not. Brain neural activity was changed in the hippocampus, cingulate cortex, motor cortex, insular cortex, thalamus, and the hypothalamus after acupuncture treatment. Acupuncture treatment increased expression of 5-HT1A receptor in the cortex, hippocampus, thalamus, and the hypothalamus, and of 5-HT1B in the cortex and thalamus. In conclusion, acupuncture treatment at KI10·LR8·LU8·LR4 was effective in alleviating the depressive-like behavior in mice, and this therapeutic effect was produced through central brain neural activity and serotonin receptor modulation.

Keywords: acupuncture, brain neural activity, chronic restraint stress, depressive-like behavior, serotonin receptor modulation

#### INTRODUCTION

Depression is a common mood disorder, which has a high mortality and recurrence rate (Reddy, 2010). According to a previous study, about 10% of the world's population has major depressive disorder (Riolo et al., 2005), and continued depression can lead to suicide, sleep disorders, anorexia, anxiety, and gangrene (Hammen, 2005). Depression is not only a serious disease *per se* but also has a major impact on the occurrence of other conditions, such as pain (Bair et al., 2003; Goldenberg, 2010), chronic fatigue syndrome (Bram et al., 2018), neurodegenerative disease (Modrego and Ferrández, 2004), and inflammatory diseases (Baerwald et al., 2019). The etiology of depression involves genetic, environmental, socioeconomic, and stress-related factors (Dunn et al., 2015). As stress is emerging as a major cause of depression (Yang et al., 2015), there is an interest in developing treatment methods for depression and elucidating the pathological and neurobiological mechanism of stress-induced depression. However, much of the fundamental mechanisms of stressinduced depression have yet to be elucidated.

Although the cause of depression remains unclear, the brain is known to play a fundamental role in the mechanism of depression (Leuchter et al., 1997). Changes in brain neural circuits or a chemical imbalance in the brain are involved in the onset of depression (Palazidou, 2012). Recent brain imaging studies have identified that several brain regions (Pandya et al., 2012), such as the hippocampus, amygdala, and anterior cingulate cortex are involved in depression (Drevets et al., 2008; Eisch and Petrik, 2012). Hippocampal neurons have bilateral connections to the amygdala, and relay the signals to several brain areas (Fried et al., 1997; McEwen et al., 2016). It also receives synaptic inputs through the cingulate cortex. Hippocampal activity is affected by a stressed state and is involved in stress-induced depression (Krugers et al., 2010; Kim et al., 2015). Since hippocampal neurons can be altered by serotonergic and adrenergic inputs, as well as by the corticotropin-releasing hormone, many antidepressants are aimed at blocking or modulating serotonin or serotonin receptors (Kohler et al., 2016).

The serotonergic system is one of the major neurotransmitter systems involved in depression. Monoaminergic neurotransmission imbalance is causally related to the clinical features of depression, and depletion of monoamines such as serotonin (5-hydroxytryptamine, 5-HT) in the brain is a widely accepted hypothesis in the field of depression (Rot et al., 2009). Conventional antidepressants that improve 5-HT transmission or inhibit 5-HT reuptake, such as selective serotonin-reuptake inhibitors (SSRIs), are the most commonly used pharmacotherapy (Gijsman et al., 2004; Holtzheimer and Nemeroff, 2006). However, the efficacy of these drug in some patients remains controversial (Torrens et al., 2005; Papakostas et al., 2008), and they also induce side effects, such as a lack of concentration, gastrointestinal disorder, recurrence of depression, and involve the inconvenience of long-term administration (Penn and Tracy, 2012). Therefore, various alternative therapies for depression are sought.

Acupuncture has been used to treat various disorders, including pain (Vickers et al., 2012; Park et al., 2014), neurodegenerative disease (Cho et al., 2012; Park et al., 2017a), and psychological disorders, such as depression and stressinduced symptoms (Mukaino et al., 2005; Leo and Ligot, 2007), with a very rare occurrence of adverse events. Previous studies have reported that acupuncture is an effective therapeutic approach for improving symptoms of depression (Pilkington, 2010; Zhang et al., 2010; Qu et al., 2013; Chan et al., 2015; Li et al., 2018) by regulating the mTOR signal (Oh et al., 2018), glial glutamate transporter (Luo et al., 2017), neuropeptide Y (Eshkevari et al., 2012), and ERK-CREB pathways in the brain (Lu et al., 2013), as well as the expression of hippocampal brain-derived neurotrophic factor (Yun et al., 2002). However, the biological basis of its efficacy remains largely unknown. Most *in vivo* acupuncture studies aimed at elucidating the effect and the fundamental mechanism underlying acupuncture effects have used a single acupoint for therapy. ST36 has been most commonly used, while some studies used PC6, GB20, and EX-HN3 (Yun et al., 2002; Eshkevari et al., 2012; Lu et al., 2013, 2016). In the clinic, however, practitioners generally use a combination of acupoints, selected on the basis of traditional Korean or Chinese medical theory. Therefore, it is necessary to select acupuncture combinations that are frequently used in clinics when conducting efficacy evaluation and mechanistic studies.

In this study, we investigated the effect of acupuncture treatment consisting of a combination of specific acupoints (KI10·LR8·LU8·LR4) in a mouse model of chronic stress-induced depressive-like behavior. We also analyzed neural activation and the changes in 5-HT receptor expression in various brain regions, to elucidate the central neural mechanism of this acupuncture treatment.

# MATERIALS AND METHODS

#### Animals

Male C57BL/6 mice (age: 6–7 weeks old; weight: 20–25 g) were used in this study (Daehan Biolink, Eum-seong, Korea). Mice were acclimated for at least 1 week before the experiments and maintained with a 12-h light/dark cycle with free access to water and food. The animals were randomly divided, and five mice were placed in each cage (room temperature 25°C and humidity of 55%) in a clean room. Their weight was monitored every 2 days (**Supplementary Figure S1**). This study was carried out in accordance with recommendations in the guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health. All experimental protocols used in this study were approved by the Institutional Animal Care and Use Committee (IACUC) at Daejeon University (approval no. DJUARB2016-40).

#### Chronic Restraint Stress

All mice were exposed to chronic restraint stress (CRS). CRS was induced by placing mice individually into a 50-ml Falcon tube and the tubes were slightly tilted for 6 h per day (10:00–16:00), for 14 or 28 consecutive days to induce depressivelike behavior. Mice subjected to CRS could not access water and food during the CRS but could freely access water and food at the end of it.

#### Behavioral Test

Mice were challenged in the open-field test (OFT) before and after the repeated restraint stress procedure to measure depressivelike behavior (**Figure 1A**). Mice were stabilized in the test room for more than 1 h prior to behavioral testing. Then, they were placed in a box (30 cm × 30 cm × 30 cm) which

was made of white plastic and the total distance and number of zone transitions measured for a period of 10 min using a video camera system to track movement (SMART 3.0; Panlab S. L., Barcelona, Spain). The tracking images showed that the mice had locomotor activity and explorational ability. All the tests were performed between 10:00 am and 12:00 pm.

#### Acupuncture Treatments

After CRS inducing, mice were randomly assigned to each group (*n* = 5–8 per group) according to the acupuncture treatment performed: CRS without acupuncture (CRS group), at the acupuncture points (KI10·LR8·LU8·LR4; AP group), at modified-acupuncture points (KI10·LR8; mAP group), at control points (LU8·LR4·HT8·LR2; CP group), or at non-acupoints (on the hips, located midway between the coccyx and hip joints; NP group) for 7 or 14 consecutive days. For acupuncture treatment, mice were slightly immobilized by fixing their neck. Then, acupuncture needles (0.18 mm in diameter and 8 mm in length; Dong-Bang Acupuncture Inc., Boryung, Korea) were inserted bilaterally at the appropriate positions according to group assignment. Needles were turned at a rate of two spins per second for 30 s, and then immediately removed (Yin et al., 2008). The CRS group was immobilized for the same amount of time to induce an equal amount of stress.

The location of each acupoint is as follows:

KI10 is located on the posteromedial aspect of the knee, just lateral to the semitendinosus tendon, in the popliteal crease. LR8 is located on the medial aspect of the knee, in the depression medial to the tendons of the semitendinosus and the semimembranosus muscles, at the medial end of the popliteal crease. LU8 is located on the anterolateral aspect of the forearm, between the radial styloid process and the radial artery, 1 cun superior to the palmar wrist crease. LR4 is located on the anteromedial aspect of the ankle, in the depression medial to the tibialis anterior tendon, anterior to the medial malleolus. HT8 is located on the palm of the hand, in the depression between the fourth and fifth metacarpal bones, proximal to the fifth metacarpophalangeal joint. LR2 is located on the dorsum of the foot, between the first and second toes, proximal to the web margin, at the border between the red and white flesh (**Figure 1B**; WHO, 2008; Yin et al., 2008).

# Brain Tissue Section Preparation

On the last day of the experiment, mice were anesthetized with an intraperitoneal injection of zoletil (30 mg/kg) and xylazine (10 mg/kg). Mice were transcardially perfused with 0.05 M phosphate-buffered saline (PBS) buffer followed by 4% paraformaldehyde (PFA). Their brains were removed, postfixed overnight in cold 4% PFA, and then cryo-protected in a gradient of 10–30% sucrose solution until the brain sank. Brain tissues were subsequently cut into 40-μm-thick sections using a cryostat at −20°C (Leica Microsystems, Wetzlar, Germany), on the coronal plane, and sections were prepared in free-floating sections.

#### Immunohistochemistry

Tissue sections were incubated in 1% H2O2 to reduce endogenous peroxidase activity, followed by incubation in 1% bovine serum albumin for 1 h at room temperature. Tissue sections were incubated with primary antibodies at 4°C overnight. The details of the primary antibodies were as follows: c-Fos monoclonal antibody (1:150, Santa Cruz, Dallas, TX, USA), 5-HT1A (1:100, Novus Biologicals, Centennial, CO, USA), and 5-HT1B (1:100, Abcam, Cambridge, UK). Thereafter, tissues were incubated with secondary antibodies at room temperature for 2 h; these included biotinylated goat anti-rabbit IgG (H + L) or antimouse IgG (H + L) (Vector Laboratories, Burlingame, CA, USA). After washing in PBS, tissue sections were incubated with ABC reagent (Vector Laboratories), with 0.02% diaminobenzidine and 0.003% hydrogen peroxide in 1 M Trisbuffered saline (pH 7.5). The tissues were then dehydrated by immersion in a gradient of 70–100% ethyl alcohol, followed by 100% xylene, before being mounted with Permount solution. Photographs of the stained brain sections were obtained under a microscope (Nikon, Minato, Japan).

The number of c-Fos positive cells on 30 brain regions of the cortex, cerebral nuclei, hippocampus, thalamus, hypothalamus, and midbrain, and the 5-HT1A- and 5-HT1B receptor-positive cells in 25 brain regions of the cortex, hippocampus, thalamus, and hypothalamus were manually counted, within a square of 32 μm × 32 μm. The mean values for the left and right regions were calculated and used for analysis. When the area was wider than the 32 μm × 32 μm square, three regions were randomly selected, and the average value was obtained. All measurements were randomly confirmed. All the brain regions assessed, and their abbreviations are shown in **Table 1**.

#### Immunoblotting Assay

Proteins were extracted from the brain tissues using RIPA buffer containing a protease and phosphatase inhibitor. The protein amount was measured by a BCA protein assay kit (Thermo Fisher Scientific). Protein lysates (10 μg) were loaded into a 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred on polyvinylidene difluoride (PVDF) membranes. Membranes were incubated overnight at 4°C with primary antibodies of c-Fos (1:1,000, Santa Cruz). The secondary antibodies of anti-mouse IgG HRP (1:5,000, Sigma-Aldrich) and anti-rabbit IgG HRP

TABLE 1 | Brain region and its abbreviation used in this study.


(1:5,000, Sigma-Aldrich) were used. The bands were detected with an ECL western blotting substrate kit (1:1 solution, GE Health Care). All the results were normalized to the respective GAPDH.

#### Statistical Analyses

All data are expressed as mean ± standard error (SEM). The statistical data were analyzed by two-way ANOVA followed by Bonferroni post-tests or one-way ANOVA, followed by Newman-Keul's post-tests using GraphPad Prism 5.0 (GraphPad Software Inc., CA, USA). The significance was set at *p* < 0.05 for all experiments.

#### RESULTS

#### AP Acupuncture Treatment Improves CRS-Induced Depressive-Like Behavior

To investigate the anti-depressive effect of acupuncture treatment, we established a murine model of depressive-like behavior by 2 or 4 weeks of CRS. Then, acupuncture treatment, consisting of a specific combination of acupoints (KI10·LR8·LU8·LR4) was performed for 1 week (on the 2-week CRS model) or 2 weeks (on the 4-week CRS model). In the 2-week CRS model, CRS significantly decreased the total distance traveled and the zone transition number, as compared to the NOR mice (*p* < 0.001). Acupuncture treatment at the KI10·LR8·LU8·LR4 points significantly restored the total distance and the zone transition number as compared to the CRS group (*p* < 0.001). However, acupuncture treatment at the non-acupoints (NP group) did not have this rescue effect (**Figures 2A**–**C**). In the 4-week CRS model, acupuncture treatment at KI10·LR8·LU8·LR4 significantly rescued the decreased total distance as compared to the CRS and NP group (*p* < 0.05) (**Figures 2D**–**F**).

#### Effects of Acupuncture Treatment at AP and mAP Points on 2 Weeks CRS-Induced Depressive-Like Behavior

Since AP treatment involved a combination of four acupoints and NP involved a single point, there was a possibility that the effect of AP treatment was more pronounced than NP treatment due to the intensity of the stimulation. Thus, we validated the therapeutic effect of AP in comparison to other combinations of four acupoints (CP; LU8·LR4·HT8·LR2). We found that AP treatment significantly rescued the decreased total distance (*p* < 0.001); the total distance traversed was significantly higher in the AP group than in the CP and NP groups (both *p* < 0.001). There was a similar trend in the zone transition number, but it did not reach statistical significance (**Figures 3A,B**).

Since acupuncture treatment at the AP (KI10·LR8·LU8·LR4) improved depression, while that at the CP (LU8·LR4·HT8·LR2) did not, we hypothesized that among the four acupoints in AP, KI10 and LR8 (mAP) were the major points involved in the therapeutic effect. As expected, the mAP group showed a significant improvement in depressive-like behavior (*p* < 0.01), similar to the AP group, and the AP while the mAP groups were significantly higher in both total travel

FIGURE 2 | Acupuncture treatment at the AP (KI10·LR8·LU8·LR4) can reverse chronic restraint stress (CRS)-induced depressive-like behavior, as compared to non-acupoint treatment. Acupuncture treatment at KI10·LR8·LU8·LR4 significantly reversed the decrease in the total distance and the number of zone transitions caused by CRS (AP vs. CRS and non-acupoint treatment group) in both the 2-week (A–C) and 4-week CRS models (D–F). NOR, normal; AP, CRS and acupuncture treatment at KI10·LR8·LU8·LR4; NP, CRS and acupuncture treatment at non-acupoints on the hips. \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001 vs. NOR. # *p* < 0.05, ###*p* < 0.001 vs. CRS. &*p* < 0.05, &&*p* < 0.01 vs. AP. Two-way ANOVA followed by the Bonferroni test. Error bars indicate SEM.

FIGURE 3 | Acupuncture treatment at AP (KI10·LR8·LU8·LR4) and mAP (KI10·LR8) can reverse chronic restraint stress (CRS)-induced depressive-like behavior. (A,B) Acupuncture treatment at AP (KI10·LR8·LU8·LR4) significantly reversed the decrease in the total distance and the number of zone transitions caused by CRS (AP vs. CRS, CP, and NP treatment groups). Acupuncture treatment at AP and mAP significantly reversed the decrease in the total distance and the number of zone transitions compared to the CRS and CP treatment groups (C,D). NOR, normal; AP, CRS and acupuncture treatment at KI10·LR8·LU8·LR4; mAP, CRS and acupuncture treatment at KI10·LR8; CP, CRS and acupuncture treatment at LU8·LR4·HT8·LR2; NP, CRS and acupuncture treatment at non-acupoints on the hips. \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001 vs. NOR. ##*p* < 0.01, ###*p* < 0.001 vs. CRS. &&&*p* < 0.001 vs. AP. +++*p* < 0.001 vs. mAP. Two-way ANOVA followed by the Bonferroni test. Error bars indicate SEM.

hips. \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001 vs. NOR. # by the Newman-Keuls test. Error bars indicate SEM.

distance and zone transition number than that of the CP group (both *p* < 0.001) (**Figures 3C, D**). This indicated that KI10·LR8·LU8·LR4, but particularly at KI10 and LR8, are the acupuncture points involved in the improvement of symptoms of depression.

# Acupuncture Treatment at AP Induces Brain Neural Activity in the CRS Model

To investigate the therapeutic mechanism of acupuncture, we analyzed how brain neural activity changed after acupuncture treatment. We used activation of the immediate early gene

Newman-Keuls test. Error bars indicate SEM.

compared to the CRS group. NOR, normal; AP, CRS and acupuncture treatment at KI10·LR8·LU8·LR4; NP, CRS and acupuncture treatment at non-acupoints on the hips. \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001 vs. NOR. # *p* < 0.05, ##*p* < 0.01, ###*p* < 0.001 vs. CRS. One-way ANOVA followed by the Newman-Keuls test. Error bars indicate SEM.

c-Fos as a marker of neuronal activity, according to previous studies (Chen et al., 2013; Wheeler et al., 2013). c-Fos activation was slightly lower in the hippocampus and hypothalamus of the 2-week CRS group, and AP increased the c-Fos activation in the hippocampus, thalamus, hypothalamus, and anterior cingulate cortex, as compared to the CRS group, but the difference was not statistically significant (**Supplementary Figures S2A–D**).

For more detailed observations, we observed c-Fos activity by subdividing the cortex, cerebral nuclei, hippocampus,

thalamus, hypothalamus, and the midbrain regions into a total of 30 regions. We observed significant changes in 15 of the 30 regions in the 2-week CRS model. c-Fos activation in the CA1 and CA3 of the hippocampus (HIP-CA1, HIP-CA3), cingulate cortex area 1 (CC-1), secondary motor cortex (MC-2), and paraventricular thalamic nucleus (TH-PV) was significantly increased after AP, but no other treatment. c-Fos activation in the dentate gyrus (HIP-DG), dorsal part of the insular cortex (IC-AID), ventral part of the insular cortex (IC-AIV), primary somatosensory cortex (SC-1), primary motor cortex (MC-1), arcuate nucleus of the hypothalamus (HyTH-ARC), lateral hypothalamic area (HyTH-LH), and dorsomedial periaqueductal gray (PAG-DM) was increased by both of AP and NP treatment. c-Fos activation in HIP-CA2 and the dorsomedial part of the striatum (ST-DM) was increased only after NP treatment (**Figures 4A**–**C**). Among the brain regions changed by AP, CA1 demonstrated the greatest change in comparison to the CRS group (**Figure 4D**). In the CRS, AP, and NP groups, c-Fos expression in CA1 showed a positive correlation with the results of the total distance travelled in the open field test (*p* < 0.05) (**Figure 4E**).

In the 4-week CRS model, c-Fos activation in the HIP-CA1, HIP-CA2, CC-1, cingulate cortex area 2 (CC-2), MC-1, MC-2, granular insular cortex (IC-Gi), piriform cortex (Pir), PAG-DM, mediodorsal thalamic nucleus (TH-MD), central medial thalamic nucleus (TH-CM), dorsomedial hypothalamic nucleus (HyTH-DM), ventromedial hypothalamic nucleus (HyTH-VM), HyTH-LH, and posterior hypothalamic area (HyTH-PH) was significantly increased only after AP treatment. c-Fos activation in the HIP-CA3 and IC-AIV was increased by both AP and NP treatment. c-Fos activation in the IC-AID was increased only after NP treatment (**Figures 5A**–**C**). Among the brain regions changed by AP treatment, HyTH-VM demonstrated the most change as compared to the CRS group (**Figure 5D**). In the CRS, AP, and NP groups, c-Fos expression in the HyTH-VM showed a positive correlation with the total distance travelled in the open field test (*p* < 0.01) (**Figure 5E**).

These results indicated that the cortex, hippocampus, thalamus, and the hypothalamus, which were the only regions changed by AP treatment, were the main regions mediating the therapeutic effect of acupuncture treatment.

#### Regulation of 5-HT1A and 5-HT1B Receptors by AP Treatment

We further investigated whether acupuncture treatment at KI10·LR8·LU8·LR4 affected the expression of serotonin receptors. We analyzed the activation of 5-HT1A and 5-HT1B receptors in 25 brain regions of the cortex, hippocampus, thalamus, and the hypothalamus, which were identified as the main regions involved in mediating the effect of acupuncture on c-Fos expression. CRS significantly decreased 5-HT1A receptor activation in the CC-1, CC-2, MC-2, HIP-DG, HIP-CA2, TH-MD, TH-CM, and paraventricular hypothalamic nucleus (HyTH-PVN) as compared to the NOR group. Acupuncture treatment at KI10·LR8·LU8·LR4 significantly increased 5-HT1A receptor activation in the CC-1, CC-2, MC-2, HIP-DG, HIP-CA2, HIP-CA3, TH-MD, TH-CM, TH-PV, HyTH-PVN, HyTH-DM, and HyTH-ARC as compared to the CRS group (**Figures 6A,B**).

CRS significantly decreased 5-HT1B receptor activation in the CC-1 and MC-1 compared to the NOR group. AP treatment significantly increased 5-HT1B receptor activation in the CC-1, CC-2, MC-1, MC-2, and TH-PV as compared to the CRS group (**Figures 7A,B**).

These results indicate that the effect of acupuncture treatment was mediated *via* central brain neural activity and modulation of serotonin receptors.

# DISCUSSION

To the best of our knowledge, this study is the first to identify the effect of acupoint combination of KI10·LR8·LU8·LR4 in chronic stress-induced depressive-like behavior and its neural mechanism. In this study, we found that acupuncture treatment at KI10·LR8·LU8·LR4 was effective in improving CRS-induced depressive-like behavior, and that among these four acupoints, KI10 and LR8 were the major acupoints effective in the treatment of depressive-like symptoms. Moreover, we also found that this behavioral improvement was mediated by brain neuronal activity involving the modulation of serotonin receptor expression in specific brain regions.

Sa-am acupuncture is a unique Korean traditional acupuncture method developed by Sa-am in the seventeenth century A.D. and most widely adopted by Korean medical doctors in the clinic (Han et al., 2005). Sa-am acupuncture uses *five-transporting points* according to the principle of tonification and sedation. The combination of KI10·LR8·LU8·LR4, called Liver-tonification, has been reported as the most commonly used acupoint combination in Sa-am acupuncture (Yoon et al., 2018), and is used to treat a variety of pain and psychological conditions, such as depression (Kim, 2006; Yoon et al., 2018). However, despite being widely used clinically, few studies have reported the effect of the KI10·LR8·LU8·LR4 acupoint combination, and no animal studies to date have identified its therapeutic effects and underlying biological mechanisms.

In our study, we found that the acupuncture treatment at KI10·LR8·LU8·LR4 significantly improved depressive-like behavior in mice, as compared to non-acupoint treatment on the hips and at control points (LU8·LR4·HT8·LR2). The LU8·LR4·HT8·LR2 combination has been reported to be effective in reducing anxiety (Kim, 1998, 2006; Yoon et al., 2018), which shows a different pathological phenotype to that of depression. This combination also involves four acupoints and can thus exert the same amount of stimulation as the AP combination. Therefore, the difference in effectiveness between the two groups is not due to the amount of stimulation, but due to specific acupoints. We demonstrated that KI10·LR8·LU8·LR4, and particularly KI10 and LR8, play a pivotal role in improving depressive-like behavior. In order to establish a more precise basis in future, it will be necessary to study the therapeutic efficacy of each acupuncture point specifically and define the related mechanism.

To elucidate the therapeutic mechanisms of depression, researchers have focused on the brain, which controls all sensory and behavioral modulations (MacPherson et al., 2016). Since previous studies have reported that hippocampal atrophy occurs with depression (Bremner et al., 2000), and that the hippocampus plays an important role in the pathophysiology of major depressive disorder (Campbell and MacQueen, 2004), the hippocampus has been studied as a major component in the mechanism involved in the treatment of depression. Recently, research has shown that not only the hippocampus but also the prefrontal cortex, thalamus, and hypothalamus are closely related to depression (Pang et al., 2015; Treadway et al., 2015;

Kasahara et al., 2016). Stress response is mainly mediated by the hypothalamic-pituitary-adrenal (HPA) axis, and the hypothalamus is a key brain region that has various functions in modulating the endocrine and autonomic nervous systems. The anterior cingulate cortex, amygdala, and hippocampus are anatomically linked to the hypothalamus and midbrain, and interconnected with the prefrontal-limbic network that is strongly related to depressive disorders (Bao et al., 2008; Bennett, 2011).

Previous studies indicated that acupuncture modulates activity within specific brain areas, including the somatosensorymotor cortices, limbic system, basal ganglia and brain stem (Chae et al., 2009; Huang et al., 2011) in healthy subjects. These brain responses reflect acupuncture stimuli-related brain regions. In particular, acupuncture treatment has been reported to cause therapeutic effects by activation of neurons in brain regions such as the somatosensory and motor cortex, prefrontal cortex, cingulate cortex, insular cortex, limbic area, and the hypothalamus (Park et al., 2017a,b). Based on this preliminary report, we found that the therapeutic effect of acupuncture is exerted by a combination of acupuncture stimulated brain regions and disease-related brain regions. Therefore, we broadly selected brain regions that are related to acupuncture stimuli and depression, and then analyzed neural activation in each brain region to identify the acupuncture mechanism by assessing central neural activity. We noted changes in neuronal activity in 15 (2-week CRS model) and 18 (4-week CRS model) of the 30 brain regions. The altered brain area could be divided into the area changed only by AP treatment (at KI10·LR8·LU8·LR4), the area changed only by NP treatment (non-acupoints on the hip), and the area changed by both AP and NP stimulation. The effects of acupuncture therapy included the specific effects of acupuncture as well as nonspecific effects (placebo effect) of acupuncture. Therefore, the brain areas changed by non-acupoint stimulation is thought to be related to the nonspecific effect of acupuncture treatment, and the brain areas changed only by AP treatment (HIP-CA1, HIP-CA3, CC-1, MC-2, and TH-PV in the 2-week CRS model; HIP-CA1, HIP-CA2, CC-1, CC-2, MC-1, MC-2, IC-Gi, IC-Pir, TH-MD, TH-CM, HyTH-DM, HyTH-VM, HyTH-LH, and HyTH-PH in the 4-week CRS model), are considered to be the main brain areas representing the specific effects of acupuncture.

From these results, we could identify that the hippocampus, cingulate cortex, motor cortex, and the thalamus function as important brain regions that exert acupuncture effects. The thalamus acts as a hub that relays information between different subcortical brain areas and the cerebral cortex. It receives sensory signals and sends them to the cortical area (Hwang et al., 2017). The cingulate cortex receives inputs from the thalamus and the neocortex, and projects them to the other brain regions. It integrates the limbic system, which is involved in emotional changes, learning, and memory (Hayden and Platt, 2010). These brain regions are related to both acupuncture signaling transduction and modulating depression. Changes in the motor cortex may be particularly associated with the behavioral improvement of mice.

The neuronal activity in the brain regions associated with the specific effects of acupuncture was prominently increased in the 4-week model as compared to the 2-week model, suggesting that, the longer the treatment period of acupuncture, the more prominent the specific rather than the nonspecific effect was. Moreover, the insular cortex, PAG, and hypothalamic brain areas with increased c-Fos in the 4-week model may be associated with long-term therapeutic effects of acupuncture.

For further mechanistic studies, we observed the expression of serotonin receptors in the core brain regions associated with acupuncture treatment effects. Among the serotonin receptors, 5-HT1A and 5-HT1B receptors have been extensively studied (Savitz et al., 2009). Increased 5-HT1A receptor expression is known to be associated with an improvement in both depressive and anxiety behaviors, while the 5-HT1B receptor plays a role in regulating impulsive behavior, reward, and depression (Heisler et al., 1998; Parks et al., 1998; Knobelman et al., 2001; Parsons et al., 2001). We found that AP treatment increased 5-HT1A receptor expression in the hippocampus, cingulate cortex, motor cortex, thalamus, and the hypothalamus, and 5-HT1B receptor expression in the cingulate cortex, motor cortex, and the thalamus.

Based on our results, we hypothesize that a central neural mechanism underlies the effect of KI10·LR8·LU8·LR4 acupuncture treatment (**Figure 8**). Acupuncture stimulation at KI10·LR8·LU8·LR4 is likely received by peripheral nerve endings, which is then transmitted to the afferent sensory nervous tract. These mechanical signals from the spinal cord might be transmitted to the thalamus, then to the cingulate cortex, somatosensory/ motor cortex, insular cortex, and the hippocampus. The thalamus transmits signals to the hypothalamic area, which are then relayed back to the pons and medulla, activating the descending neuronal pathway that provokes the acupuncture effect.

Thus, we found that signal transmission in the brain of a depressive mouse model differed, depending on the acupuncture points used, and the effect of acupuncture correlated with the expression of serotonergic receptors. Although depression studies have mainly focused on the hippocampus, further studies of the prefrontal cortex, thalamus, and hypothalamus are needed. In addition, an analysis of correlations of proteins such as CREB, ERK, and BDNF, in addition to serotonin, is necessary to elucidate the mechanism underlying acupuncture therapeutic effects.

Acupuncture treatment at KI10·LR8·LU8·LR4 was effective in alleviating depressive-like behavior in mice. This therapeutic effect was produced by the modulation of central neural activity and 5-HT1A/B receptor expression in various brain regions of the hippocampus, cortex, thalamus, and the hypothalamus. In future, the effect of the acupoint combination KI10·LR8·LU8·LR4 needs to be clarified in various diseases, and its neurobiological mechanism of acupuncture requires further elucidation to improve our knowledge of the fundamental mechanism.

#### ETHICS STATEMENT

This study was carried out in accordance with recommendations in the guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health. All experimental protocols used in this study were approved by the Institutional Animal Care and Use Committee (IACUC) at Daejeon University (approval no. DJUARB2016-40).

#### AUTHOR CONTRIBUTIONS

M-JL performed immunostaining, data analysis and drafted the manuscript. J-SR performed animal behavioral tests, immunostaining and data analysis. S-KW performed animal behavioral tests and western blotting. JJ, S-ML, and UN contributed to the conception of the work and assisted with protocol development. J-YP designed the study, coordinated

#### REFERENCES


the acquisition of all study data, interpretation of results and writing of the article. All co-authors were involved in critical revision of initial drafts.

#### FUNDING

This research was supported by the Korea Institute of Oriental Medicine (K17860) and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2018R1C1B3004424 and NRF-2018R1A 6A1A03025221).

#### ACKNOWLEDGMENTS

The authors would like to thank Mr. Se-gun Kim and Ms. Geun-Hyang Eom for their help with the animal experiment and all the present and past members of the labs for support and insightful discussions.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyg.2019.01353/ full#supplementary-material

evidence of the neuronal connection between groin A-shi point and uterus. *Evid. Based Complement. Alternat. Med.* 2013:429186. doi: 10.1155/2013/821813


a rat post-traumatic stress disorder model. *Sci. Rep.* 8:11864. doi: 10.1038/ s41598-018-30337-5


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2019 Lee, Ryu, Won, Namgung, Jung, Lee and Park. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# High Temporal Summation of Pain Predicts Immediate Analgesic Effect of Acupuncture in Chronic Pain Patients—A Prospective Cohort Study

#### Petra Iris Baeumler <sup>1</sup> \*, Peter Conzen<sup>2</sup> and Dominik Irnich<sup>1</sup>

*<sup>1</sup> Multidisciplinary Pain Center, Department of Anaesthesiology, University Hospital Ludwig-Maximilians-University, Munich, Germany, <sup>2</sup> Department of Anaesthesiology, University Hospital Ludwig-Maximilians-University, Munich, Germany*

#### Edited by:

*Younbyoung Chae, Kyung Hee University, South Korea*

#### Reviewed by:

*Kyungmo Park, Kyung Hee University, South Korea In-Seon Lee, National Center for Complementary and Integrative Health (NCCIH), United States*

> \*Correspondence: *Petra Iris Baeumler petra.baeumler@ med.uni-muenchen.de*

#### Specialty section:

*This article was submitted to Perception Science, a section of the journal Frontiers in Neuroscience*

Received: *14 December 2018* Accepted: *30 April 2019* Published: *11 July 2019*

#### Citation:

*Baeumler PI, Conzen P and Irnich D (2019) High Temporal Summation of Pain Predicts Immediate Analgesic Effect of Acupuncture in Chronic Pain Patients—A Prospective Cohort Study. Front. Neurosci. 13:498. doi: 10.3389/fnins.2019.00498* Objectives: This prospective cohort study explored whether two distinguished sensory parameters predicted acupuncture effects in chronic pain patients; namely high temporal summation of pain (TS) indicating spinal synaptic facilitation as well as a low vibration detection threshold (VDT) indicating a loss of Aβ-fiber function.

Methods: Pinprick induced TS and VDT were assessed by standardized, validated methods at the most painful body site and a pain free control site in 100 chronic pain patients receiving six acupuncture sessions as part of an interdisciplinary multimodal pain treatment (IMPT). Immediate change in pain intensity after the first acupuncture session (first treatment on the first day of IMPT) was assessed by the verbal rating scale (VRS, 0–100). After 4 weeks of treatment, patients indicated in a questionnaire whether acupuncture had relieved pain immediately and whether it had contributed to overall pain reduction and well-being after IMPT.

Results: Logistic regression analysis revealed an association between high TS at the control site and a reduction in pain intensity of at least 30% (VRS) after the first acupuncture (OR [95%-CI] 4.3 [1.6–11.8]). Questionnaire ratings of immediate pain relief after acupuncture were associated with high TS at the control site (OR [95%-CI] 3.8 [1.4–10.2] any pain relief, OR [95%-CI] 5.5 [1.7–17.1] over 50% pain reduction) and at the pain site (OR [95%-CI] 3.2 [1.2–8.9] any pain relief). Appraisals of the contribution of acupuncture to overall pain reduction and well-being after IMPT were not associated with TS. The VDT was not associated with any outcome.

Conclusion: This explorative study provides first-time evidence that high TS, especially at a pain free control site, but not VDT, might predict immediate analgesic response to acupuncture in chronic pain patients. Thus, highly centrally sensitized chronic pain patients might respond particularly well to acupuncture.

Keywords: quantitative sensory testing, wind-up ratio, vibration detection threshold, responder, sensitization

# INTRODUCTION

In the management of chronic pain it is crucial to identify effective treatments in order to minimize the patient's risk of further chronification and to reduce health care costs (Main et al., 2008; Institute of Medicine, 2011). Acupuncture has been shown to be effective in treating chronic pain conditions (MacPherson et al., 2017; Vickers et al., 2018) with responder rates of 50% (Vickers and Linde, 2014), but little is known about predictors of treatment response. The effectiveness of acupuncture is thought to be based among other mechanisms on its potential to reduce central sensitization processes that sustain chronic pain states. This involves a reduction of spinal synaptic facilitation and reestablishment of endogenous pain control (Zhao, 2008; Zhang et al., 2014) up to demodulation of maladaptive functional brain neuroplasticity (Maeda et al., 2017). Thus, it stands to reason that chronic pain patients exhibiting strong central sensitization might respond particularly well to acupuncture.

Quantitative sensory testing has proven to permit conclusions about sensitization mechanisms contributing to chronic pain (Arendt-Nielsen, 2015) and standardized methods have been developed. Those suggested by the German Research Network for Neuropathic Pain (DFNS) are easily applied in clinical practice (Rolke et al., 2006), show good test-retest and interobserver reliability (Geber et al., 2011) and come with the advantage of established reference values (Rolke et al., 2006).

Increased temporal summation of pain (TS) is a sensory sign observed in various chronic pain conditions (Eide et al., 1994; Nikolajsen et al., 1996; Pud et al., 1998; Graven-Nielsen et al., 2000; Staud et al., 2001; List et al., 2008; Edwards et al., 2011; Gerhardt et al., 2015) and considered to reflect advanced spinal synaptic facilitation. TS describes the rise in pain intensity during repetitive nociceptive stimulation and is regarded as the physiological correlate of wind-up (Eide, 2000). This phenomenon is caused by a rapid facilitation of synaptic transmission between nociceptive afferents and spinal projection neurons during repetitive C- and Aδ-fiber activation (Mendell, 1966; Clarke et al., 2002). If spinal synaptic strength is permanently augmented through long-term potentiation (LTP) in chronic pain states, TS was observed to be elevated (Herrero et al., 2000). Furthermore, high TS was found to be associated with further signs of central sensitization such as hyperalgesia (Rabey et al., 2015), number of pain sites (Gerhardt et al., 2015; Vaegter and Graven-Nielsen, 2016) and impaired endogenous pain control (Staud et al., 2001; Lev et al., 2010; Daenen et al., 2014). Thus, the first hypothesis addressed in this study is that high TS is associated with the response to acupuncture in chronic pain patients.

Additionally, considering that acupuncture effects rely on sensory input elicited by the needle stimulus, it can be proposed that patients exhibiting a loss of function of sensory afferents might respond less to acupuncture. In this regard we obtained hints from a previous study in which acupuncture effects on sensory thresholds were assessed by QST in healthy volunteers. This study provided first-time conclusive support for the importance of segmental inhibition for the analgesic effect of electroacupuncture (Baeumler et al., 2015). In contrast, effects of manual acupuncture varied largely. A sub-analysis revealed a particularly poor vibration detection threshold (VDT), indicating a loss of Aβ-fiber function, in volunteers who did not experience a change in heat or pressure pain thresholds in the segment of needle placement after manual acupuncture (Baeumler et al., 2014). Segmental inhibition describes the reduction of nociceptive transmission in the spinal dorsal root through homosegmental A-fiber activation via inhibitory interneurons (Sandkuhler, 1996; Melin et al., 2013). While Aβfiber activation seems mainly responsible for an immediate but transient analgesic effect, low intensity Aδ-fiber activation can induce long-term depression of synapses between primary nociceptors and spinal projection neurons (Sandkühler, 2000). In line with these mechanisms as crucial components of the analgesic effects of acupuncture and other naturopathic stimulation therapies (White et al., 2011; Musial et al., 2013) segmental needling represents an integral part of acupuncture practice (Wancura-Kampik, 2012). Thus, the second hypothesis tested in this study is that a loss in vibration detection might be associated with a poor response to acupuncture in chronic pain patients.

In this study, we explored the association of TS and the VDT, as evaluated by DFNS standards, with the immediate analgesic response to acupuncture and with the subjective evaluation of acupuncture effects after a whole treatment in chronic pain patients undergoing multimodal pain therapy.

### MATERIALS AND METHODS

#### Study Design

In this prospective cohort study TS and VDT were assessed as predictors for the analgesic effect of acupuncture. The study population consisted of chronic pain patients who received six to eight acupuncture treatments in the course of a 4 week interdisciplinary multimodal pain treatment (IMPT). Acupuncture was applied as the first treatment modality on the first day; meaning that effects of other therapies were excluded. The percentage change in pain intensity after this first acupuncture treatment was evaluated by the verbal rating scale (VRS). In addition, the subjectively perceived immediate pain relief experienced through acupuncture and its contribution to the overall pain reduction as well as to the general well-being during the whole 4-week treatment period was assessed by a patient questionnaire.

TS and VDT were evaluated at the most painful site (PS) and a pain free control site (CS) before the first acupuncture treatment and were analyzed as predictors for the acupuncture effect. TS and VDT were also assessed at the last day of IMPT and are not subject of this publication.

#### Study Setting

Recruitment of study participants was performed among chronic pain patients undergoing a multimodal pain treatment program, the Munich Outpatient Program in Complementary and Alternative Medicine (MOCAM) at the Multidisciplinary Pain Center at the Department of Anesthesiology, University Hospital of the Ludwig-Maximilians University of Munich, Germany. The MOCAM follows the national criteria for IMPT, combining education, physical exercises, relaxation and cognitive behavioral therapy. MOCAM also integrates complementary methods of Traditional Chinese Medicine (acupuncture, qigong) and classical naturopathy (poultices and packs). It is carried out in an outpatient setting. Recruitment period of this study was from 2013, 3rd June to 2015, 23rd February. Data acquisition was completed on 2015, 20th March.

### Patients

Inclusion criteria were age between 18 and 75 years, chronic pain condition (>3 months), command of the German language and written informed consent. General exclusion criteria were malignant, neurodegenerative or chronic inflammatory diseases. Patients suffering from pain in the hands were also excluded, because the dorsum of the dominant hand served as a pain free control site. Patients who had received less than six acupuncture treatments in the course of the MOCAM were retrospectively excluded. Patients were informed about conduct and purpose of the study in oral and written form after the introductory seminar of 75 min on the first day of the multimodal pain treatment program. Inclusion was performed after the subsequent coffee break in case that written informed consent was obtained.

#### Acupuncture

The schedule of the MOCAM stipulates that participating chronic pain patients receive eight acupuncture treatments during the 4-week multimodal pain program. Study documentation included the number of treatments that were actually received. For the course of this study acupuncture was scheduled on the first treatment day as the first intervention. Acupuncture in the MOCAM is exclusively performed by experienced medical acupuncturists with at least 200 h of acupuncture training (A-Diploma of the German Medical Acupuncture Association, DÄGfA). Treatments are usually performed in a group setting and point localization, needling depth as well as type and strength of needle stimulation are chosen individually. The individual treatment regimens integrate elements of TCM based acupuncture and western acupuncture techniques. Treatments rely on a differentiation of symptoms according to ba gang as well as on differential point selection according to zang-fu in case of internal diseases or according the theory of qi, blood and fluids. In addition, treatment included meridian orientated choice of points according to the site of pain as well as microsystem and trigger points. Five to ten needles are inserted per treatment. Needling depth is on average 0.5–1 cm and needle stimulation is performed by up and down movement as well as twisting of the needle. Acupuncturists attempt to elicit deqi, but this is not mandatory. Electrical or laser stimulation devices are available, but were not used in any of the patients included in this study. With the needles in place patients relax for 20–40 min.

#### Outcome Parameters

#### Acupuncture Responder Based on the Reduction in Pain Intensity After the First Acupuncture Treatment

The pain intensity directly before and after the first acupuncture treatment on the first treatment day of the multimodal pain program was evaluated by the verbal rating scale (VRS, 0 = "no pain," 100 = "worst pain imaginable"). According to recommendations of the US Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials (IMMPACT) (Dworkin et al., 2005) two definitions for clinically relevant pain relief were applied; reduction in pain scores >30 and >50%. A reduction of pain scores of more than 30% has been shown to represent a clinically meaningful pain relief independent from baseline pain (Farrar et al., 2001). Accordingly, percent change in pain intensity was calculated as (VRSpost – VRSpre)/VRSpre <sup>∗</sup> 100 to classify patients as acupuncture responders and non-responders. Patients with a reduction in pain intensity of over 30% after the first acupuncture treatment are referred to as Resp30 and those with a reduction in pain intensity of over 50% are referred to as Resp50.

#### Patient Questionnaire on Acupuncture Effects

After the closing seminar on the last day of the multimodal pain treatment program patients were asked to fill in a questionnaire in order to assess the subjective perception of their response to acupuncture. Patients who were not able to attend this posttreatment visit were offered a separate study visit within the next days, at most 2 weeks later. The questionnaire was designed for the purpose of this study in order to assess the patients' subjective appraisal of immediate pain relief achieved through acupuncture, its contribution to the overall pain reduction during the 4 weeks of treatments as well as its contribution to general well-being. Questionnaire items translated into English read as follows:


#### Predictor Assessment

Assessments of TS and VDT were performed by two examiners (PB and a study nurse) during the waiting time before the first acupuncture treatment on the first day of the multimodal pain treatment program. Both sensory signs were determined at the most painful body site (PS) and at the dorsum of the dominant hand, which served as a pain free control site (CS). In headache cases, the temple was classified as the pain site. Measurements were performed according to the protocol for quantitative sensory testing (QST) designed by the German Research Network on Neuropathic Pain (DFNS) (Rolke et al., 2006). According to DFNS reference data, a certain value range at the bottom of the TS scale and at the top of the VDT scale can be regarded as not aberrant. Just exceedingly high TS indicates advanced spinal synaptic facilitation and low VDT a loss of Aβ-fiber function. Thus from a physiological point of view, the postulated relationship between the probability to respond to acupuncture and these sensory parameters is most likely not linear. This was accounted for by categorizing TS and VDT values in the statistical analysis (see section Data Analysis).

## Temporal Summation of Pain

As a measure for TS, the pin-prick evoked wind-up ratio (WUR) was applied. The WUR represents the quotient of the pain intensity evoked by 10 pin-prick stimuli and the pain intensity evoked by one single pin-prick stimulus. For all measurements a pin-prick of 256 mN (MRC Systems GmbH-Medizintechnische Systeme, Heidelberg, Germany) was used. Patients were asked to rate the pin-prick evoked pain intensity on the VRS (0–100). First, the researcher applied a single pin-prick stimulus followed by a series of 10 pin-prick stimuli applied with a frequency of 1 Hz within an area of 1 cm<sup>2</sup> . WUR assessments were repeated three times per measurement site at intervals of at least 15 s. The WUR was calculated by division of the arithmetic mean of the three VRS ratings of the 10 stimuli by the arithmetic mean of the three VRS ratings of the single stimuli. This is indicated by the denotation [VRS-ratio], since the wind-up ratio is a dimensionless quantity.

#### Vibration Detection Threshold

The VDT was evaluated by the Rydel-Seiffer tuning fork (Aesculap AG, B.Braun Melsungen AG, Melsungen, Germany) which allows the application of vibration stimuli of 64 Hz. The continuously decreasing vibration amplitude can be read from a gauged scale ranging from 0 to 8 with an accuracy of 0.5 points. Patients were asked to notify the examiner as soon as their sensation of vibration stopped, and the corresponding amplitude was recorded. The final VDT value also represents the arithmetic mean of three measurements.

### Assessment of Patient Characteristics

Sociodemographic and pain diagnostic data were extracted from the patients' medical records. These data included age in years, sex, right-handedness, pain relevant, and psychological diagnoses according to ICD-10 coding, stage of chronicity according to the Mainz-Pain Staging System (MPSS, 1–3), previous surgery (yes/no) and use of analgesics (yes /no; if yes, class of drugs was recorded). Pain duration in months, the most painful body site as well as the average and maximum pain intensity (VRS, 0-100) were assessed during the first study visit on the first treatment day.

# Sample Size Estimation

Sample size estimation was performed on the basis of reference data for the WUR provided by the DFNS which suppose a lognormal data-distribution (Rolke et al., 2006). Given a target difference of 0.4 of WUR raw-values (0.15 in log-values), a standard deviation (SD) of 0.234 in log-values, an α-error of 5% and a power of 80%, the total sample size was estimated as 78 patients. In addition, a correction for an unequal responder/nonresponder ratio of 33 to 66% and a drop-out rate of 15% resulted in a final sample size of 101 patients (calculations according to Altman, 1991).

#### Data Analysis

Data analysis was exploratory and performed in IBM SPSS Statistics for Windows (Version 22.0. Armonk, NY: IBM Corp) as well as in R version 3.5.2 (R Development Core Team, 2008). Categorical study variables are described as absolute and relative frequencies. Assumption of normal distribution of metric study variables was estimated by Shapiro-Wilk test. Besides age all metric study variables (pain intensity measures, pain duration, WUR and VDT) were not normally nor log-normally distributed. Descriptive statistics are therefore given as median with interquartile range (IQR) for the remaining metric variables.

A responder analysis was carried out to evaluate whether the predictors were associated with clinically relevant treatment response. As described above, percent changes in VRS sores were categorized (≥30 and ≥50%) to represent responders and nonresponders. As recommended for analyses of pain data (Farrar et al., 2006), cumulative proportion of responder analysis graphs are used to illustrate that cut-off selection did not bias results. That this constitutes the best strategy to present our data is based on two main considerations. First, neither raw post-treatment scores nor raw change scores nor percent change scores provide an unbiased estimate of treatment effects (Vickers, 2001). Second, regression analyses with adjustment for baseline scores is not recommended in observational studies, in particular if covariates are likely to be related to baseline scores (Cribbie and Jamieson, 2000; Cole et al., 2010).

Logistic regression was used to assess confounder adjusted associations between the dichotomous outcome parameters representing acupuncture effects (dependent variables) and the VDT as well as the WUR at the control site and at the pain site (independent variables). Based on the neurophysiological considerations introduced above, VDT and WUR data were categorized before application as independent variables in the logistic regression models. We pragmatically chose the 33% percentile for the VDT and the 66%-percentile for the WUR as cut-off values in order to identify patients with the highest WUR and lowest VDT. These cut-off values were verified by visual inspection of scatter plots of predictors against percent change in pain intensity (**Figure 2**) and ROC-analyses.

Assessed confounders included age, gender, pain duration, pain related surgery before participation in the multimodal pain treatment and intake of analgesics. First, the crude associations of acupuncture effects and patient characteristics were assessed in an exploratory analysis. Subgroups formed according to the different dichotomous outcome variables were compared for metric variables by Mann-Whitney-U test and for categorical variables by Fisher test. Subsequently, final logistic regression models were identified by automated covariate selection using the R-function bestglm (McLeod and Changjiang, 2018). Model fit was evaluated by Omnibus test and goodness of fit by Hosmer-Lemeshow test. Odds-ratios (OR) with 95% confidence intervals (95% CI), the Nagelkerke's pseudo R 2 (R 2 ) as well as correctly specified cases (sensitivity & specificity) were used to describe the predictive value of the VDT and the WUR at the control site and at the pain site. Collinearity was assessed by the variation inflation factor (VIF). Effect modification was assessed by introduction of interaction terms in the final models. For additional graphical

ratio; CS, control site; PS, pain site.

illustration (e.g., cumulative response curves), identified metric confounders were categorized with cut-off values identified by the receiver operating characteristic (ROC-) analysis.

#### RESULTS

#### Patient Characteristics

Of 171 screened patients 116 met the inclusion criteria and gave written informed consent. Of these 16 were excluded from the analysis. A late detection of exclusion criteria accounted for exclusion in 12 cases. Three patients had received less than six acupuncture treatments during the multimodal treatment program for organizational reasons, and one patient discontinued the acupuncture treatment after experiencing a strong vegetative reaction during the first session. Four patients did not attend the follow-up visits for personal reasons (**Figure 1**).

Patient characteristics of the 100 patients available for analysis are displayed in **Table 1**. Overall, patients were middle-aged, mainly females, right-handed, with a long history of chronic pain and relevant pain intensity. Most patients (n = 77) were diagnosed with persistent pain with somatic and psychological factors (F45.41). The second most frequent diagnosis (n = 64) was myofascial pain (M79.1-) followed by dorsopathies (M40– 54, n = 42). Migraine or other headache syndromes (G43.- or G44.-) were present in 21, arthropathies (M00–25) in 18 and disorder of the trigeminal nerve (G50.-) in 12 patients. The most frequent mental and behavioral diagnoses were mood or affective

reference line indicates a 30% reduction in pain intensity as the cut-off for clinically relevant pain relief. WUR, wind-up ratio [ratio of the pain intensities evoked by one and by 10 pin-prick stimuli of 256 mN as evaluated by the verbal rating scale (0–100)]; VDT, vibration detection threshold; CS, control site; PS, pain site; filled points, (A,B) subjects with high TS (WUR > 2.5 VRS-ratio) as identified by the 66%-percentile (C) low VDT at the CS (≤7.7/8) (D) low VDT at the PS (≤4.3/8) as identified by the 33%- percentile; empty points, subjects with low TS or high VDT, respectively.

disorder (F30–39) present in 34 patients. The most frequently reported area of the main pain problem was the lower back (25%) followed by the neck and cervical-spine area (22%), the shoulderarm region (10%), the head (9%) and the area of the thoracic spine (8%).

#### Descriptive Analysis of Acupuncture Effects, TS and the VDT

Pain intensity as evaluated by the VRS (0–100) was significantly reduced after the first acupuncture treatment when compared to baseline (median (IQR) 30 (10–50) vs 45 (30–60), p < 0.001).

Six patients had no pain on the first day of MOCAM and were therefore excluded from responder rate calculations. Of those suffering from pain on the first day of the multimodal pain treatment, 38 (40.4%) experienced a pain reduction of at least 30% (Resp30) and 25 (26.6%) of at least 50% (Resp50) after the first acupuncture treatment as evaluated by the VRS (0–100).

Thirty seven patients (38.5%) reported that they had experienced an immediate pain relief through acupuncture in the questionnaire filled in after the end of the 4 weeks of treatment. Twenty-four patients (25%) affirmed that acupuncture had reduced their pain immediately by over 50%. Subjective rating of the contribution of acupuncture to the overall pain relief over the whole 4-week treatment period was positive in 52 cases (54.7%). Seventeen patients (17.9%) perceived acupuncture to be responsible for an overall pain relief of over 50%. Seventy-five patients (80.6%) experienced a beneficial effect of acupuncture on general well-being.

TS was similar at the control site and at the pain site with a median WUR (IQR) of 1.9 (1.3–3.0) and 2.0 (1.5–3.1), respectively. Insensitivity to pin-prick stimuli (VRS rating of 1 stimulus with 0) leading to missing values of the WUR were observed in one patient at the control site and in two patients at the pain site. In two patients TS assessments at the pain site could not be performed due to strong hyperalgesia.

Median VDT at the control site was 8.0 with an IQR of 7.0– 8.0 indicating a ceiling effect (accumulation of scores at the upper limit of the 8-point scale). At the pain site median VDT was 5.4 (IQR 4.0–6.6).

#### TABLE 1 | Patient characteristics (*n* = 100).


*n, absolute frequency corresponding to relative frequencies since the total number of patients was 100; a, years; m, months; mdn, median; IQR, interquartile range; VRS, verbal rating scale; MPSS, Mainz Pain Staging System; ICD-10, 10th revision of the International Statistical Classification of Diseases and Related Health Problems.*

# Crude Association of TS and the VDT With Acupuncture Effects

Comparisons of TS and the VDT at the control site and at the pain site were performed between patient subgroups formed according to the different dichotomous outcome variables: reduction of the pain intensity as evaluated by the VRS after the first acupuncture treatment over 30% (Resp30) or 50% (Resp50), subjective perception of acupuncture effects as assessed by the questionnaire—immediate pain relief, immediate pain relief of over 50%, contribution to overall pain relief after 4 weeks of treatment, overall pain relief over 50% and contribution to overall well-being (**Table 2**). TS at the control site was significantly higher in patients who reported an immediate pain relief through acupuncture in the questionnaire than in those who did not experience an immediate analgesic acupuncture effect (median WUR (IQR) 2.4 (1.5–3–6) vs. 1.6 (1.3–2.8); p = 0.050). Patients who had reported an immediate pain relief of over 50% were even more demarked form the remaining patients in terms of a higher TS at the control site (median WUR (IQR) 2.5 (1.6– 4.4) vs. 1.6 (1.3–2.9); p = 0.029). In addition, there was a trend toward higher TS at the pain site in patients with an immediate reduction in their pain intensity after the first acupuncture treatment of more than 50% (Resp50) as evaluated by the VRS (p = 0.085). Conversely, patients with and without acupuncture effects differed neither with regard to the VDT at the control site nor with regard to the VDT at the pain site (p > 0.05).

Plotting percent change in pain intensity against the predictor variables showed a non-linear relationship between the WUR

and the immediate reduction in pain intensity after acupuncture. The density of data points representing no or only poor acupuncture response decreases in the value range between 2 and 3 of the WUR at the control site (**Figure 2A**). A similar but less pronounced association was seen for TS at the pain site (**Figure 2B**). In contrast, visual inspection does not suggest an association between percent change in pain intensity and the VDT at the pain site or at the control site (**Figures 2C,D**).

Comparisons of frequencies of low VDT and high TS between patient subgroups with and without acupuncture effects (**Table 3**) support observations from **Figure 2**. High TS (WUR > 2.5) at the control site was more frequent in patients with a reduction in pain intensity of at least 30% (Resp30) as evaluated by the VRS than in patients not responding to the first treatment (OR 2.6 [1.1–6.4]; p = 0 0.045). The proportion of patients with high TS at the control site was also larger among those who subjectively rated the immediate pain relief through acupuncture as positive (OR 2.6 [1.1–6.3]; p = 0.043) or even over 50% (OR 2.8 [1.1–7.5]; p = 0.043). High TS at the pain site was merely associated with the report of an immediate pain relief through acupuncture of over 50% in the questionnaire (OR 3.4 [1.3–9.1]; p = 0.021). In addition, a ROC-analysis confirmed the WUR cut-off of 2.5 for all outcomes. Both a low VDT at the control site (≤7.7) and a low VDT at the pain site (≤4.3) were equally frequent in patients with and without acupuncture effects.

### Associations Between Acupuncture Effects and Patient Characteristics

Acupuncture effects were similarly distributed between men and women. There was only a trend toward a more frequent perception of an immediate pain relief of over 50% through acupuncture in females (OR 6.6 [0.8–52.5]; p = 0.062). All 24 patients who had reported an immediate pain relief through acupuncture of over 50% in the questionnaire took analgesics, while 20.8% (15 out of 72) of patients who experienced less or no immediate pain relief after one of the acupuncture sessions were not on pain medication (p = 0.019). Patients of chronicity stage 2 and 3 as defined by the MPSS rated the contribution of acupuncture to their general wellbeing more frequently as positive than patients of chronicity stage 1 (OR 7.7 [1.3–45.4]; p = 0.031 and OR 6.4 [1.2–33.5]; p = 0.036, respectively). Furthermore, older age was associated with immediate analgesic response to acupuncture. Patients with immediate, clinically relevant reductions in VRS pain scores were older than patients without such response (Resp30: mean (SD) 51.7 (9.4) vs. 46.4 (10.8) years, p = 0.005; Resp50: (52.8 (9.6) vs. 47.0 (10.6) years, p = 0.006). The subjective experience of an immediate pain relief through acupuncture of over 50% was also associated with older age (53.3 (8.4) vs. 46.3 (11.3) years; p = 0.010). A similar age trend was also observed between patient subgroups formed according to whether an immediate pain relief was experienced at all (50.8 (11.2). vs 46.3 (10.7) years; p = 0.063) (**Table 4**).


*Resp30*/*50, Acupuncture responders defined according to a reduction in pain intensity after the first acupuncture treatment of at least 30 and 50%, respectively; VDT, vibration detection threshold; x/8, score out of eight on the Rydel-Seiffer tuning fork; WUR, wind-up ratio; VRS-ratio, ratio of the pain intensities evoked by one and by ten pin-prick stimuli of 256 mN as evaluated by the verbal rating scale (0–100); CS, control site; PS, pain site; n, case number; mdn, median; IQR, interquartile range;* \**statistically significant at an* α*-error of 5% according to Mann-Whitney-U test.*

### Adjusted Analysis of the Association Between TS and the VDT With Acupuncture Effects

Logistic regression confirmed the positive association of high TS at the control site as well as at the pain site with an immediate reduction in pain after acupuncture and identified age as a relevant confounder (Model 1–4; **Table 5**). Age-adjusted ORs were larger than crude ORs (**Table 4**). All models showed a good model fit (Omnibus test p < 0.01 and H-L test > 0.07). There was no collinearity between explanatory variables (VIF < 1.1). Explained variance according to Nagelkerkes R 2 was between 18 and 25%. As identified by automated covariate

#### TABLE 3 | Crude association between acupuncture effects and high TS and low VDT.


*Resp30*/*50, Acupuncture responders defined according to a reduction in pain intensity after the first acupuncture treatment of at least 30 or 50%, respectively; VDT, vibration detection threshold; x/8, score out of eight on the Rydel-Seiffer tuning fork; WUR, wind-up ratio; VRS-ratio, ratio of the pain intensities evoked by one and by ten pin-prick stimuli of 256 mN as evaluated by the verbal rating scale (0–100); CS, control site; PS, pain site; OR, odds ratio; 95% CI, 95% confidence interval;* \**statistically significant at an* α*-level of 5% according to Fisher test.*

selection, age was the only additional significant explanatory variable. Effect modification by any patient characteristic was not observed. Specificity of prediction (80–94%) was usually larger than sensitivity (13–55%).

Model 1 describes the association between a high TS at the control site and the outcome Resp30. It indicates that patients with a high TS (WUR > 2.5) at the control site had a 4.0 (95% CI [1.6–11.8]; p = 0.005) times higher chance that their pain intensity was reduced by at least 30% after the first acupuncture treatment. With each year of age the chance to belong to the Resp30 subgroup increased by 8% (95% CI [2–13%]; p = 0.004). The model correctly classified 55% of responders and 80% of non-responders.

#### TABLE 4 | Crude associations between acupuncture effects and patient characteristics.




*(Continued)*

#### TABLE 4 | Continued


*Resp30*/*50, Acupuncture responders defined according to a reduction in pain intensity after the first acupuncture treatment of at least 30 or 50%, respectively; MPSS, Mainz Pain Staging System; m, months; a, years; ref, reference category; mdn, median; IQR, interquartile range; OR, odds ratio; 95% CI, 95% confidence interval;* \**statistically significant at an* α*-level of 5% according to Fisher test for dichotomous variables and according to Mann-Whitney-U test for continuous variables.*

TABLE 5 | Logistic regression models of age-adjusted associations between immediate analgesic acupuncture effects and high TS.


*Resp30, Acupuncture responders defined according to a reduction in pain intensity after the first acupuncture treatment of at least 30%; WUR, wind-up ratio; VRS-ratio, ratio of the pain intensities evoked by one and by ten pin-prick stimuli of 256 mN as evaluated by the verbal rating scale (0–100); CS, control site; PS, pain site; a, years; OR, odds ratio; 95% CI, 95% confidence interval; H-L test, Hosmer-Lemeshow test; VIF, variation inflation factor;* \**statistically significant at an* α*-level of 5%.*

There was no significant association between a reduction in pain intensity after the first acupuncture treatment of over 50% (Resp50) and a high TS (WUR > 2.5). However, when subgrouping patients according to an age cut-off identified by a ROC-analysis (53 years), the cumulative response function (**Figure 3**) shows a trend toward higher frequencies of responders among those with a WUR over 2.5 irrespective of the responder definition. Responder rates in the subgroup of patients over 53 years of age displaying a high WUR are only based on eight cases.

Model 2 indicates that the subjective experience of an immediate pain relief through acupuncture was reported with a 3.8 (95% CI [1.4–10.2%]; p = 0.007) times higher odds in patients with a high TS (WUR > 2.5) at the control site. With every year of age the likelihood of such positive appraisal of the immediate analgesic effect of acupuncture increased by 6% (95% CI [1–11%]; p = 0.014). Specificity was slightly higher (86%) and sensitivity lower (36%) than in model 2.

The association between a subjective immediate pain relief of over 50% after one of the acupuncture sessions with a high TS at the control site is described by model 3. Patients with a WUR of over 2.5 at the control site were 5.5 (95% CI [1.7–17.1]; p = 0.003) times more likely to report such strong immediate analgesic response. The influence of age was larger when compared to the other final models (OR 1.11 [1.04–1.189]; p < 0.001). With 78% the percentage of correctly classified cases was also high when compared to the other models. Specificity was 94%, while sensitivity was lower than in the other models (26%).

According to model 4, a high TS (WUR > 2.5) at the pain site increased the chance to experience an immediate pain relief of over 50% after one of the acupuncture treatments by 3.2 (95% CI [1.2–8.9]; p = 0.024) times. The odds was further increased by 7% (95% CI [1–14%]; p = 0.026) with every year of age. As in model 3, specificity was as high as 94% while the sensitivity of 13% was the lowest among all models.

## DISCUSSION

To our knowledge, this is the first study evaluating temporal summation of pain (TS) and the vibration detection threshold (VDT) as predictors for acupuncture effects. The results support the hypothesis that high TS might predict response to acupuncture in chronic pain patients with regard to immediate effects. The presumed association of vibration detection with acupuncture effects was not ascertained.

#### Association of High Temporal Summation of Pain With Acupuncture Effects

A WUR over 2.5 indicating elevated TS at a pain-free control site and at the most painful body site increased the likelihood to experience immediate analgesic effects through acupuncture by three to five times. This suggests that in chronic pain patients in whom spinal synaptic transmission is facilitated acupuncture may be particularly effective for immediate pain relief. This appears plausible considering that immediate analgesic acupuncture effects are mainly mediated by bottomup and top-down mechanisms which reduce spinal transmission (Zhao, 2008; Zhang et al., 2014). These include the activation of the descending inhibitory pain control, segmental inhibition, a reduction of spinal pronociceptive peptides and local modulation of primary afferents mediated by purinergic signaling.

Increased TS has been shown to be associated with the efficacy of drugs i.e., Ketamin (Lavand'homme and Roelant, 2009), Pregabalin (Olesen et al., 2013) and Oxycodon (Eisenberg et al., 2010). It is argued that this fact relates to the ability of these substances to reduce spinal synaptic fascilitation (Yarnitsky, 2015). According to numerous animal experiments (mostly conducted in rats), the same can be assumed for acupuncture (Zhang et al., 2014). In particular, electroacupuncture was found to mediate its analgesic effect in different animal pain models (cancer pain, muscle pain and inflammatory pain) through reducing spinal substance P concentration (SP) (Lee et al., 2009; Hsieh et al., 2014) and counteracting the upregulation of the neurokinin receptor 1 (NK1) (Zhang et al., 2005) as well as SP-NK1 downstream targets such as phosphatidylinositol 3-kinase and Akt (Kim et al., 2012). Additionally, reduced hyperalgesia after electroacupuncture was associated with lower upregulation of NGF in a diabetes model (Manni et al., 2011) and decreased NMDA receptor phosphorylation (Tian et al., 2008) as well as lower density of the NMDA subunit (NR2B) in a model of irritable bowel syndrome (Liu et al., 2016). Further acupuncture mechanisms counteracting spinal synaptic facilitation seem to include inhibition of glia cell activation (Lin et al., 2016). In line with this, electroacupuncture reduced spinal concentrations of interleukin-1β in a bone-cancer model (Zhang et al., 2007) and of nitric oxide in a neuropathic pain model (Cha et al., 2010).

Investigations of acupuncture effects on TS in humans that would provide further evidence of its spinal anti-fascilitatory effects are sparse. According to one trial electroacupuncture, but not manual acupuncture decreased TS in healthy volunteers (Zheng et al., 2010), while four studies, two also in healthy volunteers (Lang et al., 2010; Baeumler et al., 2015), one in whip-lash patients (Tobbackx et al., 2013) and one recent trial in patients with chronic low back pain (Leite et al., 2018) found neither electroacupuncture nor manual acupuncture to affect TS. Two main legitimate objections have been raised regarding these conflicting results (Kong et al., 2013). First, except for one trial, effects of a single acupuncture treatment were assessed. However, a clinically relevant reduction of spinal synaptic facilitation would rather be expected after a series of acupuncture sessions. Second, wind-up in healthy volunteers is already low and, thus, may hardly be affected by regulatory interventions such as acupuncture. In addition, methodological differences between studies such as the nociceptive stimulus used to elicit TS and the use of absolute pain scores elicited by repetitive stimuli instead of the ratio between single and repetitive stimuli accounting for overall pain sensitivity may give rise to further source of bias.

High TS at the control site was positively associated with reduced pain intensity after the first acupuncture treatment as indicated by both logistic regression and by the cumulative response function. Additionally, high TS at the control site was associated with subjective pain relief as evaluated by the questionnaire. In contrast, the association of high TS at the most painful body site was only associated with the subjective experience of an immediate pain relief of over 50%. This indicates that propagated synaptic facilitation throughout the spinal cord, e.g., through volume transmission (Zieglgänsberger, 2009), as a sign of advanced central sensitization is more important for the prediction of immediate analgesic acupuncture effects than primary synaptic facilitation. External validity of these results can be assumed high, since no specific selection criteria were applied. The cut-off definition for a high TS as a WUR of over 2.5 applied in our study may also be useful in future trials, as it was confirmed by an ROC-analysis and it matches the population mean established in healthy controls which appears constant over various body sites (Rolke et al., 2006; Puta et al., 2013; Pfau et al., 2014).

Few studies had previously assessed sensory signs as evaluated by QST as predictors for the analgesic effect of acupuncture. Two trials from the same research group identified a low pressure pain threshold at the thumbnail to predict a good response to sham-acupuncture (needling at non-acupuncture points or non-penetrating sham) in fibromyalgia (Harte et al., 2013; Zucker et al., 2017). In their second trial (Zucker et al., 2017), conversely, a low pressure pain threshold predicted a nonresponse verum acupuncture and strong needle sensations even caused an increase in clinical pain. In addition, a low electrical pain threshold in a pain-free body site has also been found to be lower in electroacupuncture non-responders than responders among patients suffering from painful burn scars (Cuignet et al., 2015). Authors discuss that the strength of needle stimulation needs to be adapted to the patients' sensitivity in order to maximize treatment effects and to avoid unfavorable outcomes. As treatments in our trial were individually tailored, it can be assumed that bias caused by needle stimulation exceeding the patients' comfort zone was minimal.

We found no association of TS with the patients' subjective appraisal of the contribution of acupuncture to overall pain relief during the 4-week multimodal pain treatment program. However, it needs to be emphasized that our study was not designed to confirm predictors for pain reduction after a series of acupuncture treatments. The appraisal of the contribution of acupuncture to overall pain reduction is likely to be affected by treatment success of the whole multimodal pain program involving a variety of therapy modalities. Therefore, the prediction of the long-term effects of acupuncture by TS and other somatosensory thresholds remain subject of further investigations.

#### Confounding by Age

Age confounded the association of high TS at the control site with the immediate analgesic acupuncture effect and ageadjusted ORs were larger than crude OR. Collinearity was ruled out according to the VIF of the respective logistic regression models. Thus, one can conclude that older age was positively associated with response to acupuncture and marginally more frequent in patients with high TS at the control site. The latter is in line with previous findings indicating that age does not substantially influence heat evoked TS in fibromyalgia patients (Staud et al., 2008) and pressure evoked TS in patients suffering from knee osteoarthritis (Petersen et al., 2017). In contrast, the relation of age with TS remains elusive in healthy adults in which heat evoked TS was found to be positively associated with age (Edwards and Fillingim, 2001) while pin-prick evoked TS seems age-independent (Magerl et al., 2010).

It can only be speculated why the chance to experience an immediate analgesic effect through acupuncture increased by 6–10% with every year of age. We are only aware of previous studies that assessed the influence of age on long-term pain relief achieved by acupuncture. A reanalysis of the large German acupuncture trials revealed that pain reduction after a series of verum- as well as after a series of sham-acupuncture was weakly associated with younger age (Witt et al., 2011). A more recent qualitative patient assessment of acupuncture effects revealed no association with age (Cramer et al., 2015). Our finding of a more pronounced immediate acupuncture effect in older patients might be explained by the fact that sensory stimulation, such as applied during needling, might have stronger impact on older patients. Sensory perception is known to decrease with age (Magerl et al., 2010), but was shown to be more efficiently modulated by conditioning stimuli in older than in younger subjects (Naugle et al., 2015).

#### No Association Between Vibration Detection Threshold and Acupuncture Effects

The hypothesis that poor Aβ-fiber function indicated by a loss in vibration detection would predict a poor acupuncture response was not confirmed. However, the large variance in VDT data particularly at the pain sites hampers interpretability of this finding. It is known that the VDT varies largely between body sites (Rolke et al., 2006). Our results once again suggest that a meaningful interpretation of the VDT in clinical practice would require reference values for a large variety of body sites. These are not yet established. To fully reject or approve our hypothesis, patient populations with consistent pain sites might be assessed. It should be considered that supra-spinal and local acupuncture mechanisms might compensate the lack of segmental inhibition related to poor Aβ-fiber function. Consequently, poor VDT might still be associated with immediate analgesic effects of a purely segmental needling regimen. An association between long-term acupuncture effects and the VDT in turn appears unlikely, as segmental effects inducing long-term depression of spinal synaptic pain transmission rely on the activation of Aδ- rather than Aβ-fibers. Furthermore, a loss of mechanical detection that has been described in several also non-neuropathic chronic pain conditions (Geber et al., 2008; Westermann et al., 2011; Puta et al., 2013) might not necessarily be caused by a loss in Aβ-fiber function but rather by a presynaptic inhibition linked to the constant nociceptive input (Janig and Zimmermann, 1971). If the latter hypothesis proves true, a reduced activation of the nociceptive system after a successful acupuncture treatment would also be reflected by an increased ability to detect mechanical stimuli. Future studies on the effectiveness of acupuncture in neuropathic pain conditions might explore this hypothesis.

# LIMITATIONS

A source of bias in this study is that recruitment and data collection were integrated into the course of a multimodal pain treatment program. Thus, overall treatment satisfaction might have influenced patients' rating of acupuncture effects. Social desirability bias (Furnham, 1986) might have been caused by the fact that baseline and follow-up examinations were performed by the same examiner. However, evaluations of the reduction in pain intensity after the first acupuncture treatment were free from such bias, as acupuncture was always applied as the first treatment modality on the first day of the multimodal pain program. Furthermore, patients took their usual pain medication. However, it seem unlikely that the intake of analgesics has affected the results. First, short-term drug level changes were avoided as stable pain medication intake is a precondition to take part in the multimodal pain treatment program performed at the study center. Second, the use of analgesics was neither a confounder nor an effect modifier for the associations under investigation, nor was it confirmed to be an independent predictor for pain relief after acupuncture in the multivariate analysis. Additionally, excluding patients on pain medication would have rendered our sample not representative for the average population of chronic pain patients that an acupuncturist encounters in daily clinical practice.

Additionally, it is known that multiple testing might lead to false inferences in observational studies, as they require adjustment for various covariates which automatically increases the number of hypotheses tested (Patel and Ioannidis, 2014; Gruber and Tchetgen, 2016). Consensus on strategies for a meaningful p-value adjustment has not been found. However, Bonferroni correction of each predictor hypothesis test for the four outcome parameters (VRS pain score, subjective immediate and overall pain relief and effect on wellbeing) would results a critical p-value of 0.0125. Under this assumption, associations between the WUR at the control site but not the pain site would remain statistically significant; supporting the notion that WUR in a pain free control site is a more promising predictor for response to acupuncture. It is argued that hypothesis driven analyses are best to omit bias arising from multiple testing. The design of this study is based on hypotheses driven by clinical and neurophysiological considerations and previous observations. Nevertheless, statistical analyses were performed in an exploratory manner to optimally explain variation in the data. Thus, these results require replication by future confirmatory research and validation of the proposed predictor models in an independent population.

#### Perspectives

Our results are an important step toward an individualized pain therapy in regard to non-pharmacological treatments based on sensory profiles. Expenditure of resources to explore TS in chronic pain patients as a predictor also for long-term acupuncture effects is now justified. In addition, further research is needed to explore whether effectiveness of acupuncture in certain pain conditions such as neuropathic pain, low back pain etc. can be predicted by strong signs of central sensitization. Another sensory sign of interest in this regard might be the conditioned pain modulation (CPM) indicating descending pain control. Acupuncture has been shown to activate descending pain control mechanisms, but it has not yet been investigated whether CPM predicts acupuncture effects, or whether a lack in CPM can be reestablished by continuous acupuncture treatment.

#### CONCLUSION

Strong central sensitization reflected by high TS, in particular at pain free control sites, seems to predict the immediate analgesic

#### REFERENCES


response to acupuncture. Clinically spoken, acupuncture could be especially effective in highly sensitized chronic pain patients.

#### ETHICS STATEMENT

Study conduct was in accordance with the Declaration of Helsinki formulated by the World Medical Association [updated version, Seoul (The World Medical Association, 2008)] and was approved by the ethics committee of the Medical Faculty of the Ludwig-Maximilians University of Munich. Written informed consent was obtained from all participants. Withdrawal from study procedures was open to participants at any time. In case of withdrawal all data recorded in the course of the study were deleted. Data handling was performed in pseudonymous form and followed the German data-protection act.

#### AUTHOR CONTRIBUTIONS

PB, DI, and PC conceptualized the study design, contributed to interpretation of the results and drafted the manuscript. PB was in charge of patient recruitment, data-acquisition, and data-analysis.

#### ACKNOWLEDGMENTS

We thank the acupuncturists of the MOCAM (Mrs. Gesa Harreus MD, Mrs. Barbara Jopen-Wolff MD, Mrs. Maria Durand MD, Mrs. Franziska Portzky MD and Mr. Christoph Colling MD) for making it possible to conduct the study during their daily clinical practice. We also thank Mrs. Adelheid Wanninger for her support in data-acquisition as well as Mrs. Erika Weber and Mrs. Christina Deisenrieder for their organizational support.

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and desensitizing trends during response dependent stimulation. Behav. Brain Res. 289:61–68. doi: 10.1016/j.bbr.2015.04.014


**Conflict of Interest Statement:** PB was presented with the Young Scientists Award (sponsored by 3B-Scientific) for an oral presentation of the results presented in this manuscript at the congress of the International Council for Acupuncture and Related Techniques (ICMART) in June 2017. PB and DI receive honorarium and travel costs from non-profit academic organizations, physician chambers and universities for teaching and lecturing.

The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Baeumler, Conzen and Irnich. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Involvement of the Cuneate Nucleus in the Acupuncture Inhibition of Drug-Seeking Behaviors

Suchan Chang<sup>1</sup>† , Yeonhee Ryu<sup>2</sup>† , Yu Fan<sup>1</sup> , Se Kyun Bang2,3, Nam Jun Kim<sup>1</sup> , Jin Gyeom Lee<sup>1</sup> , Jin Mook Kim<sup>1</sup> , Bong Hyo Lee<sup>1</sup> , Chae Ha Yang<sup>1</sup> and Hee Young Kim<sup>1</sup> \*

<sup>1</sup> College of Korean Medicine, Daegu Haany University, Daegu, South Korea, <sup>2</sup> Korean Medicine Fundamental Research Division, Korea Institute of Oriental Medicine, Daejeon, South Korea, <sup>3</sup> Korean Convergence Medicine, University of Science and Technology, Daejeon, South Korea

#### Edited by:

Yi-Wen Lin, China Medical University, Taiwan

#### Reviewed by:

Antonio Luchicchi, VU University Medical Center Amsterdam, Netherlands Eric Andrew Engleman, Indiana University Bloomington, United States Sheketha R. Hauser, Indiana University Bloomington, United States

#### \*Correspondence:

Hee Young Kim hykim@dhu.ac.kr; vet202001@gmail.com

†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Perception Science, a section of the journal Frontiers in Neuroscience

Received: 10 November 2018 Accepted: 19 August 2019 Published: 29 August 2019

#### Citation:

Chang S, Ryu Y, Fan Y, Bang SK, Kim NJ, Lee JG, Kim JM, Lee BH, Yang CH and Kim HY (2019) Involvement of the Cuneate Nucleus in the Acupuncture Inhibition of Drug-Seeking Behaviors. Front. Neurosci. 13:928. doi: 10.3389/fnins.2019.00928 Our previous studies have shown that acupuncture suppresses addictive behaviors induced by drugs of abuse, including cocaine, morphine and ethanol, by modulating GABA neurons in the ventral tegmental area (VTA) and dopamine (DA) release in the nucleus accumbens (NAc). The mechanisms by which the peripheral signals generated by acupoint stimulation are transmitted to brain reward systems are largely unexplored. The present study aims to investigate the role of spinal dorsal column (DC) somatosensory pathways in the acupuncture inhibition of drug addictive behaviors. Thus, we tested whether acupuncture at Shenmen (HT7) points reduces drug-seeking behaviors in rats self-administering morphine or ethanol and whether such effects are inhibited by the disruption of the cuneate nucleus (CN). The stimulation of HT7 suppressed morphine and ethanol self-administration, which were completely abolished by surgical lesioning of the CN. In in vivo extracellular recordings, singleunit activity of the CN was evoked during acupuncture stimulation. The results suggest that acupuncture suppresses morphine- and ethanol-seeking behaviors through the modulation of the CN, second-order neurons of the DC somatosensory pathway.

Keywords: acupuncture, dorsal column pathway, cuneate nucleus, morphine, ethanol, self-administration

# INTRODUCTION

Over the last three decades, there has been an increasing interest in the treatment of substance abuse by acupuncture (Cui et al., 2013). We and others have demonstrated that acupuncture attenuates drug-seeking behaviors and relapse through the modulation of the mesolimbic dopamine (DA) system in animals and humans (Yoon et al., 2004, 2010, 2012; Yang et al., 2010). While the fundamental relationship between somatic input signals induced by acupuncture and brain reward systems are not largely understood, our previous studies have suggested peripheral and spinal mechanisms underlying the inhibitory effects of acupuncture on acute cocaine-induced locomotor activity. Our previous studies have shown that acupuncture at Shenmen (HT7) points activates peripheral sensory afferents and that acupuncture-initiated impulses in turn activate large A-fibers within the ulnar nerve trunk, resulting in the inhibition of acute cocaine-induced locomotion (Kim et al., 2013). Furthermore, our recent study revealed that the modulation of the dorsal column (DC) somatosensory pathway by acupuncture signals that stimulate HT7 suppresses cocaine-induced locomotor activity, an effect that is abolished by lesioning of the DC pathway including cuneate nucleus (CN, second order neurons of DC), but not to the spinothalamic tract (Chang et al., 2017). Acupuncture can suppress acute cocaine-induced locomotion through the spinal DC pathway

(Kim et al., 2013; Chang et al., 2017). However, there is no direct evidence that acupuncture signals conveyed by the spinal DC pathway attenuate the reinforcing effects of drugs of abuse. Therefore, the present study extended prior work by testing the effects of acupuncture on drug-taking behaviors in rats selfadministering morphine or ethanol. A role for the CN in the effects of acupuncture is also explored.

### MATERIALS AND METHODS

#### Subjects

Male Sprague-Dawley rats (n = 64; Daehan Animal, Korea) weighing 250–280 g at the beginning of the experiment were housed singly on a 12-hr light and dark cycle with free access to food and water. All procedures were approved by the Institutional Animal Care and Use Committee at Daegu Haany University and conducted in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals (National Research Council, 2010).

# Acupuncture Treatment

Acupuncture was performed as described elsewhere (Kim et al., 2013). Briefly, while an assistant lightly restrained the rat, a needle (0.10 mm thick, 10 mm long; Dongbang Medical, South Korea) was bilaterally (except during the CN recordings) inserted 3 mm deep into the HT7 or LI5 acupoint, located on the transverse crease of the wrist of the forepaw, and stimulated for 20 sec (10 s for in vivo extracellular recordings) in duration and 1.3 m/sec<sup>2</sup> in intensity by using a mechanical acupuncture instrument (MAI) that was developed by our laboratory (Kim et al., 2013; **Figure 1A**). The needle was maintained in place for up to 1 min after insertion and then withdrawn. The rats received acupuncture treatment only once.

#### Cocaine-Induced Locomotor Activity

Locomotor activity was measured as previously described (Kim et al., 2013; Chang et al., 2017). Briefly, each animal was placed in an open field box and monitored with an image analysis system. After recording baseline activity for 30 min, the animal was given an intraperitoneal injection of cocaine (15 mg/kg) alone or in combination with acupuncture at HT7 or LI5 and monitored for up to 60 min after injection. The distance traveled was analyzed. The data are expressed as a percentage of the baseline activity.

### In vivo Extracellular Recording of the Cuneate Nucleus

Extracellular single-unit recordings of the CN were performed in 11 rats as previously described (Qin et al., 2010). In brief, under isoflurane anesthesia, a carbon-filament glass microelectrode (0.4–1.2 M, Carbostar-1, Kation Scientific, United States) was stereotaxically inserted in the CN (0–0.8 mm deep from the dorsal surface of medulla, 1 mm caudal to the obex and 1– 2 mm lateral from the midline) (Paxinos and Watson, 1998). Single-unit activity from the discharges was isolated, recorded and analyzed via a CED 1401 Micro3 device and Spike2 software (Cambridge Electronic Design, Cambridge, United Kingdom). After the basal single-unit neuronal activity of the CN neurons was recorded for 20 s prior to stimulation, we monitored singleunit activity following mechanical stimulation (10-sec duration) induced by brushing the receptive area, pressure stimulation (10-sec duration) and pinch stimulation (10-sec duration). The phenotypes of the cuneate neurons were classified according to previous studies (Qin et al., 2010) as follows: high-threshold (HT), wide-dynamic-range (WDR), and low-threshold (LT). The rats were then given MAI acupuncture at unilateral HT7 for 10 s, and the mean values of the firing rates in the 20 s before, 10 s during and 10 s after acupuncture were compared.

# Surgical Transection of the Cuneate Nucleus

Bilateral lesions of the CN were made as described elsewhere (McKenna and Whishaw, 1999; Wang and Thompson, 2008) with slight modifications. The animal was placed in a stereotaxic frame with its head tilted forward, the obex was exposed surgically and the CN was macerated along its length with a fine forcep under a microscope. Sham lesions were made using the same procedure (incision of skin, muscle and occipital bone) except the lesion was not created in CN. For histological confirmation of the lesions, all animals were sacrificed at the end of experiment and the brain stems, including the CN, were removed, postfixed in 4% paraformaldehyde and cryoprotected in 30% sucrose. The tissue was then cryosectioned into 30 µmthick sections and stained with toluidine blue. Only the rats with confirmed CN lesions were included in the data analysis.

# Morphine Self-Administration Procedure

Morphine self-administration was carried out in operant chambers (MED Associates Inc., Georgia, VT, United States) equipped with two response levers as described previously (Yoon et al., 2010). Briefly, a total of five rats were allowed to press a lever for the self-administration of morphine (0.5 mg/kg in 0.1 ml over 5 s, time-out period of 10 s) on a fixed ratio 1 (FR1) schedule in daily 1-h sessions for 6 days a week. Responding to the active lever caused 5 s of illumination of the cue light and 15 s of extinction of the house light. After the establishment of the baseline (defined as a mean value in three consecutive lever responses that varied less than 20%), the rats underwent procedures as shown in **Figures 2A,E**. The groups were assigned as follows; Con (control manipulation without the insertion of needles), HT7 (HT7 acupuncture), CN X+HT7 (HT7 acupuncture in CN lesioned rats) and Sham+HT7 (sham lesion and HT7 acupuncture).

#### Ethanol Self-Administration Procedure

Another set of animals were trained to self-administer ethanol orally in operant chambers using a modified sucrose-fading procedure as previously described (Yang et al., 2010). In brief, rats were initially allowed to press a lever to receive a sucrose solution (20% w/v) on an FR1 schedule to facilitate the acquisition of ethanol self-administration. Following the establishment of a stable response, the sucrose concentration was gradually

decreased to 0%, and the ethanol concentration was raised to 10%. After acquiring stable self-administration baselines (with the total varying less than 20% from the average of three consecutive sessions), the rats were exposed to acupuncture and CN lesioning using the same experimental procedure as described above. The groups were assigned as follows; Con (control manipulation), HT7 (HT7 acupuncture), and CN X+HT7 (HT7 acupuncture in CN lesioned rats).

#### Data Analysis

Statistical analysis was carried out using SigmaStat 3.5 software (Systat Software Inc., United States). All data are presented as the mean ± SEM (standard error of the mean) and were analyzed by one or two-way repeated measures analysis of variance (ANOVA) followed by post hoc testing using the Tukey method. Statistical significance was considered at p < 0.05.

#### RESULTS

The systemic injection of cocaine increased locomotor activity, and the effect lasted for approximately 60 min from the peak magnitude at 10 min. Acupuncture at HT7, but not at LI5, attenuated the cocaine-induced enhancement of locomotor activity (**Figures 1A,B**). Thus, HT7 was used as the verum acupoint in the following experiments.

In total, 27 neurons in the CN were isolated and examined for responses to somatic stimuli. The activity of WDR neurons (n = 12 cells) in the CN increased during brushing (Br), light pressure (Pr) and noxious pinch (Pi) stimulation of the somatic field. LT neurons (n = 15 cells) were activated by hair movement or light pressure but not by noxious pinch (**Figures 1C,D**). However, we did not record any HT neurons. The WDR and LT neurons exhibited spontaneous basal firing rates of 4.16 ± 0.27 Hz and 2.44 ± 0.21 Hz, respectively. When an acupuncture needle was inserted into the HT7 acupoint and stimulated for 10 s with the MAI (**Figure 1A**), the firing rates of WDR and LT neurons increased to 19.21 ± 1.76 Hz and 18.42 ± 1.79 Hz, respectively, and they quickly returned to baseline levels after the termination of stimulation (p < 0.05, **Figures 1D,E**), indicating the excitation of the CN during acupuncture at HT7.

Next, to determine whether acupuncture suppresses morphine-seeking behaviors via the modulation of the CN, rats were divided into four groups: control manipulation (Con, n = 5), acupuncture treatment (HT7, n = 5), CN

#### FIGURE 2 | Continued

fnins-13-00928 August 28, 2019 Time: 17:5 # 5

(Sham+HT7; paired t-test, <sup>∗</sup>p = 0.017 vs. Baseline) (C4). There were no differences in the number of inactive lever presses among the groups or before and after HT7 (D1–D4). A representative toluidine blue-stained image of a CN lesion (B). (E–H) The effect of cuneate nucleus (CN) lesions on the inhibition of ethanol self-administration behaviors by acupuncture at HT7. A schematic of the procedure of the ethanol self-administration experiment (E). The sham operation without HT7 acupuncture did not affect the number of active lever presses (Con) (G1). The numbers of active lever presses in the HT7-treated group was significantly reduced after HT7 stimulation compared to baseline (HT7; paired t-test, <sup>∗</sup>p = 0.042 vs. Baseline) (G2). This effect was not seen in the rats with CN injury (CN X+HT7; G3). There were no differences in the number of inactive lever presses among the groups or before and after HT7 (H1–H3). A representative toluidine blue-stained images of a CN lesion (F).

sham lesion/acupuncture treatment (Sham, n = 5), and CN lesion/acupuncture treatment (CN X+HT7, n = 5). The effect of acupuncture at HT7 with or without surgical lesioning of the CN was compared in the rats self-administering morphine. While control manipulation without acupuncture did not affect morphine self-administration (Con group in **Figure 2C1**), acupuncture at HT7 reduced the number of active lever presses (9.00 ± 2.76), but not inactive lever presses (**Figures 2D1–D4**), compared to baseline (15.80 ± 2.39) (paired t-test, p = 0.008; HT7 group in **Figure 2C2**). On the other hand, in the animals given bilateral CN lesions (**Figure 2B**), the inhibitory effects of acupuncture on morphine self-administration behaviors were attenuated compared to the corresponding baseline levels (CN X+HT7 group in **Figure 2C3**), whileas the group with the sham surgery (same surgery except CN lesion) showed inhibitory effects of acupuncture on morphine intake (Sham+HT7; P = 0.017, paired t-test; **Figure 2C4**).

The experiment was repeated in the rats self-administering ethanol. The rats were divided into three groups: control manipulation (Con, n = 5), acupuncture treatment (HT7, n = 5) and CN lesion/acupuncture treatment (CN X+HT7, n = 5; **Figure 2F**). While control manipulation without acupuncture did not affect morphine self-administration (Con group in **Figure 2G1**), Acupuncture stimulation of HT7 significantly suppressed active lever responses (28.00 ± 11.96), but not inactive lever responses (**Figures 2H1–H3**), compared to baseline (52.80 ± 13.50) (paired t-test, p = 0.04; HT7 group in **Figure 2G2**). This effect was abolished by the surgical dissection of the bilateral CN before acupuncture at HT7 (CN X+HT7 group in **Figure 2G3**). These results suggest that the CN is modulated during the acupuncture inhibition of the reinforcing effects of drugs.

#### DISCUSSION

We have shown that acupuncture at HT7 can suppress selectively morphine and ethanol self-administration, but not general consummatory behaviors, through GABA receptors in the ventral tegmental area (VTA) and DA release in the nucleus accumbens (NAc) induced by drugs of abuse (Kim et al., 2005; Yang et al., 2010; Yoon et al., 2010). Consistent with our previous studies, the present study showed that acupuncture at HT7 attenuated morphine and ethanol self-administration behaviors. Most importantly, the inhibition of drug-taking behaviors by acupuncture at HT7 was inhibited by the surgical lesioning of the CN, suggesting the involvement of sensory inputs to the CN in the effects of acupuncture. Previously, we reported that the dorsal column-medial lemniscus (DC-ML) pathway is involved in the inhibitory effects of acupuncture at HT7 on cocaine-induced locomotor activity (Chang et al., 2017). Mechanical stimulation of an acupuncture needle inserted into the HT7 acupoint attenuates cocaine-induced locomotor activity via A-fiber modulation of the ulnar nerve (Kim et al., 2013), which is reversed by surgical lesioning of the CN (Chang et al., 2017) and second-order neurons of the DC-ML pathway. The CN has also been reported to be anatomically connected to certain acupuncture points on the forelimb. For example, a neuronal tracing study revealed that, when cholera toxin subunit B, a retrograde tracer, is injected into the PC8 acupoint, which is approximately 5 mm from HT7 in rats, transganglionically labeled axonal terminals are found mainly in the CN and the medial part of the deep laminae of the spinal dorsal horn (Cui et al., 2013), suggesting that the CN can be excited by the stimulation of acupoints on the forelimb in rats. This finding was further confirmed by our previous and present data showing that the mechanical stimulation of HT7 activates both LT (A-fibers) and HT afferent fibers (Aδ and C fibers) (Kim et al., 2013) and excites WDR (responsive to both lowand high-threshold stimuli) and LT (responsive to LT stimuli) neurons in the CN during in vivo extracellular recordings. Moreover, the present study showed that reduction of morphineor ethanol-seeking behaviors by acupuncture at HT7 was ablated by surgical CN lesioning. This finding suggests the involvement of CN neurons in the effects of acupuncture on the reinforcing effects of drugs of abuse. However, one of the limitations of this study is that the electrophysiological recordings of WDR and LT neurons were conducted in anesthetized rats, whereas the behavioral studies were carried out in freely moving rats. It is possible that the state of the rats during the electrophysiological recordings may not accurately reflect the state of the rats during behavioral testing.

While the CN may be particularly important for the acupuncture inhibition of drug-taking behaviors, the exact mechanism by which afferent inputs from the CN function in the mesolimbic DA system to influence addictive behaviors remains elusive, but some evidence suggests that the mechanism is dependent on the lateral habenula (LHb). Our recent study revealed that acupuncture at HT7 activates LHb neurons that project to the VTA and that the electrolytic lesioning of the LHb reduces acupuncture inhibition of cocaine-induced locomotion (Chang et al., 2017). The LHb is known to convey inhibitory reward signals to the VTA that inhibit DA release in the NAc (Velasquez et al., 2014). Thus, the results suggest that the effects of acupuncture on morphine- and ethanol-seeking behaviors might

result from the activation of CN inputs to the LHb-VTA/RMTg pathway via direct or indirect projections. Although the LHb is thought to be sensitive to somatosensory inputs (Benabid and Jeaugey, 1989; Gao et al., 1996), it is not known yet how these inputs enter the LHb. It has been shown that the LHb receives input from various structures, such as the lateral hypothalamus, entopeduncular nucleus and prefrontal cortex (Shelton et al., 2012). As none of these structures provide direct sensory inputs (projections) to the LHb, neural connections that conduct impulses from the CN to the LHb may require multisynaptic pathways. Determining the specific neural circuits between the CN and LHb that are involved in the acupuncture-mediated inhibition of drug-taking behaviors will require additional study.

In conclusion, the results suggest that the CN is involved in the effects of acupuncture on drug-seeking behaviors in rats.

#### ETHICS STATEMENT

Male Sprague-Dawley rats (Daehan Animal, Korea) weighing 250–280 g at the beginning of the experiment, were housed on a 12 h light and dark cycle and freely accessed to food and water. All

#### REFERENCES


procedures were approved by the Institutional Animal Care and Use Committee at the Daegu Haany University and conducted in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals.

# AUTHOR CONTRIBUTIONS

HK and CY designed the experiments. SC, YR, SB, YF, NK, JL, JK, and BL conducted the experiments. HK was responsible for the overall direction of the project and for edits to the manuscript.

#### FUNDING

This research was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean Government (MSIT) (Nos. 2018R1A5A2025272 and 2018R1E1A2A02086499), the KBRI basic research program through the Korea Brain Research Institute funded by the Ministry of Science and the ICT (19-BR-03-01), and the Korea Institute of Oriental Medicine (KIOM; KSN 1812181).


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Chang, Ryu, Fan, Bang, Kim, Lee, Kim, Lee, Yang and Kim. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Differential Influence of Acupuncture Somatosensory and Cognitive/ Affective Components on Functional Brain Connectivity and Pain Reduction During Low Back Pain State

#### Jeungchan Lee<sup>1</sup> , Seulgi Eun<sup>2</sup> , Jieun Kim<sup>3</sup> , Jun-Hwan Lee<sup>3</sup> and Kyungmo Park<sup>2</sup> \*

<sup>1</sup> Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Boston, MA, United States, <sup>2</sup> Department of Biomedical Engineering, Kyung Hee University, Yongin, South Korea, <sup>3</sup> Clinical Research Division, Korea Institute of Oriental Medicine, Daejeon, South Korea

#### Edited by:

Yi-Wen Lin, China Medical University, Taiwan

#### Reviewed by:

James Liao, China Medical University, Taiwan In-Seon Lee, National Center for Complementary and Integrative Health (NCCIH), United States

> \*Correspondence: Kyungmo Park saenim@khu.ac.kr

#### Specialty section:

This article was submitted to Perception Science, a section of the journal Frontiers in Neuroscience

Received: 14 November 2018 Accepted: 20 September 2019 Published: 04 October 2019

#### Citation:

Lee J, Eun S, Kim J, Lee J-H and Park K (2019) Differential Influence of Acupuncture Somatosensory and Cognitive/ Affective Components on Functional Brain Connectivity and Pain Reduction During Low Back Pain State. Front. Neurosci. 13:1062. doi: 10.3389/fnins.2019.01062 The underlying mechanism of pain reduction by acupuncture is still unclear, because acupuncture treatment involves multidimensional factors. In this study, we investigated the differential influence of acupuncture components on brain functional connectivity and on pain reduction. We used a specific form of sham acupuncture (phantom acupuncture; PHNT), which only has a needling-credibility (a belief that they were treated with real acupuncture needles), while real acupuncture (REAL) has a somatosensory needling stimulation, as well as a needling-credibility. Forty-three patients with low back pain were randomized into the REAL group (n = 25) and the PHNT group (n = 18). They underwent two pain steady-state fMRI runs implemented by a low back extension (LBE) pain model (lifting the low back using air-cuff inflation) before and after REAL or PHNT stimulation. Subjective pain ratings, perceived throughout the LBE runs due to the posture, were reported (LBEpain). The regions of interest (ROI) were (1) the main nodes of the default mode network (DMN) – the medial prefrontal cortex (mPFC), posterior cingulate cortex (PCC), (2) the main nodes of the salience network (SN) – the anterior/posterior insular cortices (a/pINS), and (3) the low back-specific region of sensorimotor network (SMN), S1back. Significant reductions in LBEpain were observed in both groups (REAL = −1.02 ± 1.53, PHNT = −1.26 ± 2.20). In REAL group, decreased LBEpain was positively correlated with decreased functional connectivity between the mPFC and pINS (r = 0.58, P < 0.05). Reduced LBEpain in PHNT was negatively correlated with increased PCC–aINS connectivity (r = −0.48, P < 0.05) and tended toward positive correlation with decreased S1back–pINS connectivity (r = 0.44, P = 0.07). Our findings might suggest different brain mechanisms of observed pain reduction; REAL seems to involve detachment of the self from the sensory aspect of pain, while PHNT does to shift attention to self and disengages physical pain processing hubs. This exploratory study proposes a sham methodology to dissociate the influence of different acupuncture components in acupuncture research. Further studies need to be followed with more elaborated hypothesis, study design, and analysis considering various cognitive/affective factors for better understanding of brain mechanisms of pain reduction regarding the different acupuncture aspects.

Keywords: functional connectivity, default mode network, sensorimotor network, salience network, somatosensory afference, needling credibility

#### INTRODUCTION

fnins-13-01062 October 3, 2019 Time: 18:1 # 2

Acupuncture treatment, sham as well as real, is known to modulate pain, but the underlying brain mechanism is not clear, probably because multidimensional factors are involved (Kaptchuk, 2011), and it is not easy to dissociated them from each other. To provide a standard for investigation into the acupuncture mechanism, acupuncture components have been defined as needling-specific (e.g., somatosensory needling, which is exclusive to acupuncture needling such as insertion and manipulation), specific non-needling (e.g., theory-based diagnosis and palpation, which has been considered to be related to treatment efficacy), and non-specific [e.g., needling credibility (patients' belief that they were treated with real acupuncture needles) and visual feedback (observation of treatment procedure), which is not exclusively driven by or related to needling itself] components (Langevin et al., 2011). However, it has not yet been possible to differentiate needling-specific from non-specific components, because tactile (touch) stimulation cannot be excluded as a factor in the proposed sham acupunctures (Lee et al., 2014; Makary et al., 2018). Thus, to differentiate acupuncture needling-specific (somatosensory needling) from non-specific components (needling credibility and visual feedback) (Langevin et al., 2011), phantom acupuncture (PHNT), which can produce non-specific effects without the acupuncture-specific components, has been devised to contrast with real acupuncture (REAL) (Lee et al., 2014; Makary et al., 2018). The comparison between real and PHNT stimulation has shown that somatosensory needling stimulation (acupuncture needling-specific components) produces sympathetic activation (e.g., greater skin conductance response) as well as greater acupuncture-related sensations (overall acupuncture sensation measured as Mass Index (Kong et al., 2007), soreness, tingling, and sharp pain) than other non-specific components (Lee et al., 2014). The posterior insular (pINS) and anterior cingulate cortices (ACC), which process ascending somatosensory and pain signals, showed acupuncture needling-specific brain responses in our previous study (Makary et al., 2018). Conversely, needling credibility has been shown to induce parasympathetic activation (decreased heart rate and pupil size responses), as well as vicarious acupuncture (deqi) sensations (deep pressure, heaviness, fullness, and numbness) (Lee et al., 2014). These vicarious sensations have been linked to increased brain response in the primary and secondary somatosensory cortices (S1 and S2) (Kerr et al., 2011; Beissner et al., 2015; Makary et al., 2018), and the evoked sensations may result from a top-down mechanism related to sensory imagery (Beissner et al., 2015) and attention toward stimulated body parts (Kerr et al., 2011). The previous study also showed that vicarious brain response (e.g., S1) and acupuncture sensations play an important role in creating and enhancing needling credibility in PHNT (Makary et al., 2018). Patients' needling credibility was developed by the instruction that they would receive REAL stimulation and by the visual feedback being stimulated. Vicarious acupuncture (deqi) sensations induced by the visual feedback of acupuncture stimulation, mediated by the involvement of expectation (Song et al., 2019), mirror neuron system, or mirror-touch synesthesia (Beissner et al., 2015), seemed to bolster the needling expectancy and credibility.

Functional connectivity analysis has been used to investigate interactions between brain regions to better understand chronic pain mechanisms. Various clinical outcomes have been reported to be correlated with the functional connectivity within or between subregions in the default mode network (DMN), sensorimotor network (SMN), and salience network (SN). The strength of functional connectivity (between the DMN and SMN, as well as within DMN subregions) has been shown to be significantly correlated with the reported pain intensity in patients with chronic back pain (Hemington et al., 2018). In patients with chronic pelvic pain, functional connectivity between the anterior insular cortex (aINS; a subregion of the SN) and medial prefrontal cortex (mPFC; a subregion of the DMN) was positively correlated with the levels of anxiety, depression, and pain (As-Sanie et al., 2016). In patients with fibromyalgia, decreased DMN– insula connectivity was significantly positively correlated with decreased pain after acupuncture stimulation (Napadow et al., 2012). Functional connectivity between the insula and the somatotopic region of the leg in the S1 has been correlated with pain sensitivity in healthy controls (Kim et al., 2013) and with pain intensity in fibromyalgia patients (Kim et al., 2015).

Our previous study mainly focused on the brain responses to REAL and PHNT, as well as their neural correlates with clinical LBP levels (Makary et al., 2018). In this functional MRI (fMRI) study, however, the low back extension (LBE) pain model was used to evoke normalized back pain levels across patients, and functional connectivity analysis was performed to investigate the brain mechanism of pain modulation after REAL and PHNT. This allowed us to dissociate acupuncture-needling specific and non-specific components experimentally, as well as to understand the functional brain mechanisms of the shortterm pain modulation in different brain hubs. In other words, the REAL was designed to encompass not only all of physical components of acupuncture stimulation (e.g., palpation, needle insertion, manipulation technique) as well as psychological

components (e.g., needling credibility), while the PHNT was designed as a control in that this only has psychological components without any physical components. Thus, the aim of this study was to investigate the association between the physical components of acupuncture (controlled by the influence of psychological components) and brain functional connectivity.

### MATERIALS AND METHODS

This study protocol was approved by the Institutional Review Board of the Kyung Hee University (KHNMC-OH-IRB 2010- 013), and all participants provided written informed consent in accordance with the Declaration of Helsinki. This study is registered at the clinical research information service (CRIS)<sup>1</sup> (registration number: KCT0002253).

#### Participants

Fifty-six patients with non-specific low back pain (LBP; 31 men, age = 38.4 ± 12.7 years old, mean ± SD) were enrolled in this study. All patients completed prescreening for MRI eligibility and were included if they reported LBP of greater than four on a visual analog scale (VAS; 0 = no pain, 10 = most pain imaginable) when their low back was lifted (4–7 cm) in the supine position (for more on the LBE pain model, see below). Patients were excluded if they met the following exclusion criteria: (1) LBP greater than four during baseline, (2) severe pain other than LBP (e.g., neck pain) at scan, (3) severe radicular pain extending into lower leg, (4) psychiatric or cardiovascular disorders, (5) accident- or surgery-related back pain, (6) back pain from metastatic cancer, vertebral fracture, spinal infection, inflammatory spondylitis etc., (7) taking medication for pain management (e.g., corticosteroid, narcotics, muscle relaxants, and any herbal medicine), (8) receiving acupuncture treatment for back or neck pain within a month, and (9) participating any previous acupuncture studies.

#### Experimental Paradigm

The 56 enrolled non-specific LBP patients were randomized into either the REAL (n = 33) or PHNT (n = 23) group. Patients in both groups completed four fMRI runs: a resting-state run (REST, 6 min), an acupuncture stimulation run (REAL or SHAM, 7 min), and two continuous pain runs using the LBE pain model (6 min before and after the acupuncture stimulation run; these were named the LBEpre and LBEpost runs; **Figure 1A**). The perceived pain ratings throughout the LBE runs (LBEpain) were collected and its change (i.e., LBEpost – LBEpre) was analyzed as the main outcome measure in this study (**Figure 1A**).

All the setup equipment needed for the REAL and PHNT runs (MR-compatible acupuncture needles, camera, beam projector, screen, visual barrier, and blanket) was prepared before the MRI session (Makary et al., 2018). An MR-compatible cuff bladder was also placed on the MRI scanner bed for the two LBE runs (LBEpre and LBEpost), under the most painful area of the patients' low back.

Before the LBEpre run, the cuff pressure for stimulation was calibrated in each individual patient. Air pressure was slowly applied to the cuff bladder until patients reported the target pain rating (4/10 on a 11-point VAS; 0 = no pain, 10 = most pain imaginable). In this way, we tried to standardize the intensity of induced LBP across all patients (**Figure 1B**). During this procedure, all patients confirmed that the exacerbated pain was due to their change in posture (extended back angle) rather than the force of the pressure. This information was recorded to ensure that the induced pain was not influenced by differing pain thresholds stimulating different layers of the body (e.g., skin, soft tissue, dura, disk, and deep muscle). During the LBEpre run, patients were asked to stay still with their eyes open, staring at crosshairs on a screen while the individually calibrated pressure was applied. They were also instructed to focus on the perceived LBP intensity and sensations. After the run, to prevent additional pain, the air pressure was cautiously removed so that the cuff bladder deflated slowly.

During the setup procedure for the REAL and PHNT runs, a video recording the patient body was projected onto the screen in real time to consolidate the video-body link between the patient's own body and the displayed video. This visual feedback process was designed to initiate/boost needling credibility. Patients randomized into the REAL group were told that they would be stimulated with acupuncture needles (i.e., instruction) and received four real acupuncture needles (diameter = 0.3 mm, length = 30 mm; DongBang Co., Seongnam, South Korea): bilateral SP13, left SP11, and left ST36. Before the REAL run started, the needles were inserted at acupoints (which were chosen by a licensed and experienced acupuncturist based on their clinical effectiveness and easy access during the fMRI scanning) for clinical relevance and manipulated using the traditional technique by an acupuncturist to induce acupuncture (deqi) sensations. During the REAL run, the acupuncturist, who were trained for the stimulation paradigm, stimulated each needle in a random order [i.e., somatosensory needling afference; depth = 2–3 cm, 2 s per stimulation at 1 Hz rotation (±180◦ ), five stimulations per acupoint, interstimulation interval = 7.9 ± 1.7 s], and the procedure was video-recorded and simultaneously played on the screen (i.e., visual feedback).

Before the PHNT run started, patients in PHNT group were also told that they would be stimulated with acupuncture needles (i.e., the same instruction given to REAL group) and after the video-body link was built by visual feedback procedure, the acupuncturist mimicked the needling ritual (i.e., insertion and manipulation) without actual needles. Patients in PHNT group were told that the needles had been inserted, as in the REAL run. The acupuncturist then pretended to stimulate the needles according to the stimulation paradigm while the previously recorded video from the other patient (in the REAL group, with real needles) was displayed on the screen during the PHNT run (i.e., visual feedback). Thus, the procedure was designed to build needling credibility based on the previous videobody link (during setup) and visual feedback (during the run). Importantly, this was carried out without any somatosensory needling afference.

<sup>1</sup>http://cris.nih.go.kr/

For the LBEpost run, patients were stimulated with the same air pressure intensity with that was used in the LBEpre run. Subjective pain ratings (LBEpain, 0-10 VAS), as perceived throughout the LBE runs, were reported at the end of each scan, and the changes in LBEpain between LBEpost and LBEpre were calculated as the primary clinical outcome for pain reduction in REAL and PHNT (**Figure 1A**).

At the end of the experiment, patients' needling credibility during REAL or PHNT runs was assessed retrospectively through an in-depth interview whether they believed that they had experienced REAL stimulation during the acupuncture stimulation run. If patients reported that they had any doubt or strong belief during the run that the stimulation was not happened to them actually (for example, due to no needling sensations), they were classified as non-credible patient group (i.e., without needling credibility) and removed from the analysis. Thus, our study simply hypothesized that REAL has somatosensory tactile stimulation, visual feedback, and needling credibility, whereas PHNT has only visual feedback and needling credibility.

#### MRI Data Acquisition

MRI data were collected using a 3T Philips Achieva MRI scanner (Philips Medical Systems, Netherlands) with an 8-channel head coil at the Kyung Hee University Hospital, Gangdong. For anatomical localization of the results, structural T1-weighted images were collected using an MP-RAGE pulse sequence [repetition time (TR) = 9886 ms, echo time (TE) = 4.59 ms, flip angle = 8◦ , field of view (FOV) = 256 × 256 mm, voxel size = 1 × 1 × 1 mm], and functional images were acquired for 6 min using a T2<sup>∗</sup> -weighted echo planar imaging pulse sequence (TR = 2000 ms, TE = 35 ms, flip angle = 90◦ , FOV = 230 × 230 mm, voxel size = 2.875 × 2.875 × 4 mm, 34 interleaved axial slices) during the REST and LBE runs (LBEpre and LBEpost) for functional connectivity analysis. During fMRI, physiological data (electrocardiogram and respiration signal) were collected using a data acquisition system (PowerLab ML800; ADInstruments, Inc., Australia) to calibrate cardiorespiratory artifacts (Glover et al., 2000).

#### Data Processing and Analysis

Collected MRI data were processed using conventional analysis software, such as the Functional Magnetic Resonance Imaging of the Brain (FMRIB), Software Library (FSL)<sup>2</sup> , Analysis of Functional NeuroImages (AFNI)<sup>3</sup> , and FreeSurfer<sup>4</sup> . The fMRI

<sup>2</sup>https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/

<sup>3</sup>https://afni.nimh.nih.gov/

<sup>4</sup>https://surfer.nmr.mgh.harvard.edu/fswiki

data were corrected for cardiorespiratory artifacts (3dretroicor, AFNI) (Glover et al., 2000), and for head motion (MCFLIRT, FSL; ICA-AROMA). They were also preprocessed for skull stripping (BET, FSL), spatial smoothing (Gaussian kernel, full width at half maximum = 6 mm; fslmaths, FSL) and temporal filtering (high-pass cut-off frequency = 0.006 Hz; 3dBandpass, AFNI). Individual structural and functional data were aligned first (bbregister, FreeSurfer). They were then co-registered to the standard Montreal Neurological Institute (MNI) space (FNIRT, FSL) to allow dual-regression independent component analysis (ICA) (Filippini et al., 2009) and seed-voxel connectivity analysis.

In the dual-regression ICA, all fMRI data from the REST, LBEpre, and LBEpost runs were temporally concatenated and then fed into the group ICA (MELODIC, FSL). The group ICs of the DMN, SMN, and SN were selected based on the spatial templates of the resting state networks (Beckmann et al., 2005). ROIs in the DMN (posterior cingulate cortex, PCC; mPFC) and SN (anterior and middle cingulate cortices, aINS and pINS) were decided based on the group map of REST run to ensure they were independent of influence from the pain model and acupuncture stimulation. ROIs in the aINS and pINS were combined bilaterally. The ROI in the SMN was located in the bilateral low back region of the primary somatosensory cortex (S1back; MNI X = ± 18 mm, Y = −38 mm, Z = 72 mm) (**Table 1**) based on a localization run in an independent LBP study (Lee et al., 2018a) that investigated low back-specific functional connectivity. The average time-series brain activities (sphere mask, radius = 4 mm) were extracted from each of the ROIs, and the correlation coefficients (Pearson's r) of crosscorrelation between the ROIs were converted into z scores using Fisher z-transformation. The changes in withinand between-network connectivity (LBEpost vs. LBEpre) after REAL and PHNT runs, as well as their association with changes in LBEpain (primary clinical outcome), were calculated (Hemington et al., 2018). Significance was defined at P-values <0.05.

### RESULTS

Of the 56 enrolled LBP patients (33 in the REAL group and 23 in the PHNT group), 43 were included in this analysis (25 in the REAL group, 38.4 ± 13.2 years old, mean ± SD, 13 men; 18 in the PHNT group, 38.3 ± 13.0 years old, 8 men; P for age [REAL vs. PHNT] = 0.98). Eight patients were excluded from the REAL group: four due to incomplete scanning, three due to uncorrectable distortions, and one due to excessive head motion (>2 mm). Five were excluded from the PHNT group: four due to the absence of needling credibility (patients reported that the acupuncture procedure did not happen to them, and no acupuncture stimulation was delivered because they did not feel any sensation during the PHNT run) and one due to excessive head motion (>2 mm) (**Figure 2**).

#### Decreased Pain Model-Induced Pain Intensity (LBEpain) After the REAL and PHNT Stimulation

While the applied air pressure for pain model was individually calibrated and applied with the same intensity in LBEpre and LBEpost [REAL: 118.4 ± 72.4 mmHg, PHNT: 94.4 ± 57.2 mmHg; P (REAL vs. PHNT) = 0.25], the PHNT group reported significantly greater LBEpain than the REAL group during the LBEpre run [REAL: 4.74 ± 1.00/10, PHNT: 5.54 ± 0.83; P (REAL vs. PHNT) = 0.01], and the reported pain was significantly greater than the target value (4/10) in both groups [P (REAL) < 0.005, P (PHNT) < 0.001]. None of the patients reported any pain during the REST run.

Importantly, however, the subjective pain (LBEpain) which patients felt during the continuous pain runs, as measured using the VAS, was significantly reduced after both REAL and PHNT runs [1 = LBEpain(LBEpost) – LBEpain(LBEpre); 1REAL: −1.02 ± 1.53, P < 0.005; 1PHNT: −1.26 ± 2.20, P < 0.01; P (1REAL vs. 1PHNT) = 0.67] (**Figure 3**).


ROI, region of interest; MNI, Montreal Neurological Institute; DMN, default mode network; SMN, sensorimotor network; SN, salience network; mPFC, medial prefrontal cortex; PCC, posterior cingulate cortex; S1back, low back region in primary somatosensory cortex; dACC, dorsal anterior cingulate cortex; aMCC, anterior middle cingulate cortex; aINS, anterior insular cortex; pINS, posterior insular cortex; R, right hemisphere; L, left hemisphere.

In spite of the randomization of the groups, the LBEpain in LBEpre was significantly different in the REAL and PHNT. Thus, for the analysis of brain correlates, we investigated how the LBEpain reduction was correlated with the change of functional connectivity (LBEpost vs. LBEpre).

## Changes in Functional Connectivity and Its Association With Clinical Outcome

Twenty-five ICs were derived from the dual-regression group ICA, and the ICs in the DMN, SN, and SMN were identified. The ROIs were then decided in the submodules of each network (**Table 1** and **Supplementary Figure S1**). The DMN mainly comprised the PCC, inferior parietal lobule, and the mPFC. The ROIs in the PCC and mPFC were localized in DMN. The SMN encompassed the pre- and post-central gyri, including the predefined S1back ROI. The SN consisted of the dorsal anterior and anterior middle cingulate cortices, as well as the supplementary motor area, ventrolateral prefrontal cortex, and the aINS and pINS on both sides. The pINS is the main subregion of the SMN, as reported in a previous study involving 1000 healthy controls (Yeo et al., 2011). However, in the present study involving patients with LBP, a substantial portion of the pINS was intrinsically connected with the SN (**Supplementary Figure S1**).

While no significant differences in functional connectivity were found between the LBEpre and LBEpost runs, we found that the REAL group showed significantly positive correlation (r = 0.58, P < 0.01) between the reduction in LBEpain and the change of functional connectivity between the nodes of the DMN (mPFC) and SN (pINS) (**Figure 4**).

In the PHNT group, the change in LBEpain was negatively correlated with the change of DMN–SN connectivity (PCC– aINS, r = −0.48, P < 0.05), and it showed a trending positive correlation with the change of SMN–SN functional connectivity (S1back-pINS, r = 0.44, P = 0.07) (**Figure 4**).

Interestingly, there was no significant difference between the REAL and PHNT groups in terms of changes in functional brain connectivity (LBEpost – LBEpre) as well as any other significant correlations between the reduction in LBEpain and the change of functional connectivity between the nodes of the DMN, SN, and SMN.

# DISCUSSION

In the present study, we investigated the differential influence of needling-specific and non-specific components in acupuncture treatment using REAL and PHNT, whereby REAL induced needling credibility by combining somatosensory needling afference and visual feedback from the video, while PHNT did so through visual feedback only. The LBE pain model was used to modulate and standardize the patients' back pain levels, while functional connectivity analysis was performed to investigate the brain mechanism of the short-term pain modulation effects of REAL and PHNT. We found that the LBE pain model significantly increased clinical pain levels (LBEpain) in patients with back pain, and that this elevated LBEpain was reduced after both REAL and PHNT stimuli, indicating that both methods shared a common, short-term, pain-relieving effect. However, the corresponding brain mechanisms for pain reduction differed from each other. Somatosensory afference seemed to play an important role in pain modulation in REAL, because the physical-pain processing area (pINS) was engaged during corresponding functional connectivity alteration. In PHNT, a cognitive/affective factor (needling credibility and anticipation) seemed to be involved in the relevant connectivity changes, namely the salience-processing area (aINS). These results allowed us to identify short-term brain connectivity changes related to acupuncture-specific or non-specific effects on back pain intensity (LBEpain).

# Clinical Pain Reduction in REAL: Detachment of Self From Pain

In REAL, greater LBEpain reduction was associated with decreased connectivity between the mPFC and pINS. Metaanalysis of acupuncture stimulation has shown significant deactivation in the mPFC and PCC, indicating that these areas modulate pain by shifting the focus between internal physical/mental states and external stimulation (Chae et al., 2013). Deactivation in the DMN subregion has also been observed both during real and sham acupuncture stimulation (Jung et al., 2015; Makary et al., 2018; Zhang et al., 2019). Previous studies have shown that the mPFC plays an important role in self-referential processing, which is critical for physical signal regulation, including pain (Stankewitz et al., 2018). Such processing includes both positive and negative selfappraisal (Dixon et al., 2017), as well as self-rumination and pain catastrophizing (Lee et al., 2018b). This consistent deactivation in the mPFC was interpreted as an attentional shift from self-referential to external-focused attention for a better understanding of given events and stimulations. The pINS shows stimulation-specific responses to the physical aspects of pain, representing bottom-up processing of sensory information. Thus, activity in the pINS has been considered

a proxy of somatosensory (Christopher et al., 2014), pain (Morel et al., 2013), pain ratings (Segerdahl et al., 2015), and cortical amplification (Lee et al., 2017) processing. It is likely for this reason that activation in the pINS has been reported in most acupuncture neuroimaging studies (Chae et al., 2013). Indeed, it has recently been closely linked to the needlingspecific component of acupuncture (somatosensory afference and corresponding arousal) (Jung et al., 2015; Makary et al., 2018). Interestingly, the pINS has been reported to be the main subregion of the SMN in healthy controls (Yeo et al., 2011), because it is significantly engaged in the physical aspects of pain and sensory processing. However, in the present study, a significant portion of the pINS was a part of the SN, rather than the SMN. The physical aspect of back pain, including painful sensations, seemed to capture the patients' attention, at least during the scan runs, in contrast to the healthy controls. This speculation should be further investigated.

Interestingly, the association between DMN and INS functional connectivity and clinical pain levels has been reported in other studies. For example, in patients with fibromyalgia, reduced clinical pain has been associated with reduced DMN–INS connectivity, and the same connectivity has been suggested as a possible marker for chronic pain (Napadow et al., 2012). In patients with low back pain, DMN– pINS connectivity, as well as changes thereof, has been shown to be significantly correlated with baseline (and increased) clinical pain (Loggia et al., 2013). The same relationship has also been reported in patients with LBP, complex regional pain syndrome, and knee osteoarthritis (Stankewitz et al., 2018). Thus, it has been speculated that a greater awareness of pain and the stronger incorporation of pain within the self (increased DMN–INS connectivity) can predict higher pain ratings (Napadow et al., 2012; Loggia et al., 2013). In other words, patients detach themselves from the pain, they report lower levels.

Our findings in REAL are in line with previous studies. Thus, we speculated that detachment of self from the physical aspects of pain contributes to the reduction in back pain (LBEpain) after REAL. As somatosensory tactile input is a predominant factor in creating sensory perception (e.g., reported strong acupuncture

sensations), we speculated that the influence of somatosensory afference may play a crucial role in the relationship between changes in functional connectivity and low back pain.

# Clinical Pain Reduction in PHNT: Endorsement of Self and Disengagement in Pain Processing

In PHNT, we found that the increase in functional connectivity between PCC and aINS was associated with reduced LBEpain. The PCC is the main node of the DMN (Zhang et al., 2019), which is associated with self-reflection (Vogt and Laureys, 2005), internally directed cognition (Leech and Sharp, 2014), bodily attention (Jung et al., 2015), and self-referential pain catastrophizing (Lee et al., 2018b). In many acupuncture studies, consistent deactivation in the PCC has been reported in both real and sham acupuncture stimulation (Huang et al., 2012; Chae et al., 2013; Makary et al., 2018). The aINS, as part of the SN, is a key region in directing cognitive process (Christopher et al., 2014), transient attention (Cauda et al., 2012), significance of stimulus (Christopher et al., 2014), and empathy with perceptivetaking (Nieuwenhuys, 2012; Christopher et al., 2014), as well as in the integration of anticipation and sensory inputs (Jung et al., 2015), cognitive and affective processing of pain (Morel et al., 2013; Kuehn et al., 2016; Stankewitz et al., 2018), and self-awareness of physical condition (Stankewitz et al., 2018). In our previous study, increased activity in the aINS and decreased activity in the PCC were observed during both REAL and PHNT, and aINS activity was positively correlated with changes in low back pain (Makary et al., 2018). Thus, neither region is specific to the physical aspects of stimulation, but rather to the cognitive/emotional aspects of it – the PCC to self-referential processing and endorsement of self, and the aINS to processing of the salient/emotional aspects of pain events.

We observed a different pattern during PHNT than during REAL. Specifically, the aINS, which is the center of cognitive/emotional pain processing, as well as salience, plays an important role in pain reduction during PHNT, whereas the pINS, which is the center for physical pain processing does so during REAL. In PHNT, needling credibility can be consolidated by visual information and feedback from acupuncture stimulation, without somatosensory tactile inputs. This needling credibility might be built as a result of an internal decision-making process against incongruent visual and tactile information (i.e., between visual information which shows stimulation ritual and no/less sensations felt in their body) (see section "Empirical Account for Needling Credibility," **Figure 5**). In addition, vicarious sensations might be a result of shift the focus (the aINS) from the physical aspects of pain to the self (the PCC) as they believed they were actually stimulated with needles. Thus, this attentional shift from external pain to the self (increased PCC–aINS connectivity) might be related to LBEpain reduction in PHNT. We speculated that engagement of emotional regulation and subsequent disengagement between pain processing regions may contribute to the improvement in symptoms when there is a cognitive/affective component induced by needling credibility, not by needling (placebo effect).

Interestingly, the decreased level of functional connectivity between the S1back and pINS, which was transited from SMN to SN, tended toward correlation with the reduction in LBEpain (P = 0.07) in PHNT. A functional connection between the pINS and S1 has been reported, and it may result in precise perception and awareness of pain (Christopher et al., 2014). In one study involving healthy controls, S1–pINS connectivity that was somatotopically specific to the leg was negatively correlated with pain sensitivity when cuff stimuli were applied to the leg (Kim et al., 2013), and the S1 has shown significantly stronger pain-specific functional connectivity to the pINS (Peltz et al., 2011). S1–aINS connectivity has been reported to be significantly correlated with clinical pain in fibromyalgia, suggesting that synchronization between pain-processing regions influences exacerbated pain states (Kim et al., 2015). Thus, our results might suggest that bottom-up nociceptive sensory information is delivered to the corresponding S1 subregion (S1back), as well as to the physical pain-processing area (pINS), and desynchronization between these two regions may ameliorate back pain (LBEpain) in PHNT. Further study, however, should be followed to investigate this speculation.

# Empirical Account for Needling Credibility

Our experimental paradigm was designed to build and enhance needling credibility by an interaction between somatosensory needling afference from needling, visual feedback from the video, and instruction from the acupuncturist. Different combinations of components have been defined and used to model REAL and PHNT conditions and to explain the influence of each component experimentally (**Figure 5A**). In the first PHNT acupuncture experiment (Lee et al., 2014), 20 healthy participants received both REAL and PHNT stimulation in a counterbalanced design. After an in-depth interview with all subjects, eleven subjects of 20 were found to have needling credibility during the experiment and nine were not. Interestingly, some of the subjects believed they were actually stimulated with REAL during PHNT stimulation; some reported retrospectively that they thought the acupuncture stimulation was applied with new and advanced techniques so that they didn't feel much sensations, and some actually reported vicarious sensations. This phenomenon– reporting sensations in observing other's stimulation–has been reported and discussed as results of expectation (Song et al., 2019), sensory mirror system or mirror-touch synesthesia (Beissner et al., 2015; Makary et al., 2018), or body-ownership (Botvinick and Cohen, 1998). This implies that instruction to the subjects and visual feedback are also important factors to create those sensations, where the contribution of needling credibility to vicarious sensation is not significant (i.e., not all subjects with needling credibility reported vicarious sensations). However, four subjects who didn't believe that they were stimulated emphasized synchronization problem between the visual feedback from the video and the actual movement of an acupuncturist. Other five answered that they did not feel any sensation at the stimulation site or surrounding body region during PHNT stimulation, thus they thought the stimulation was not actually applied to

them. In the second study (Makary et al., 2018), four LBP patients were excluded from the analysis as they reported the absence of needling credibility during the PHNT run. The major reason was no acupuncture sensations during the stimulation period, too. This shows that the existence of acupuncture sensations is an important factor for needling credibility. Rest of the patients, who believed that they were actually stimulated with needles, reported vicarious sensations or did believe even without any sensations from the stimulation. Thus, once the sensations (either actual or vicarious) were perceived, subjects tend to believe that they were actually stimulated with needles. If there were no sensations felt from the stimulation (while the instruction was given for needling), their needling credibility seemed to rely on the individually different weighting/priority of multisensory information in decision making (i.e., shift attention to self). This gives clue to estimate who will be likely to have needling credibility and who would not in the PHNT stimulation paradigm.

of treatment from the in-depth interview with subjects.

Taken together, (1) instructions from acupuncturist that they would be stimulated with real acupuncture needles and (2) visual feedback that is a simultaneous display of acupuncture stimulation (i.e., observation of treatment procedure with REAL) contributes to induce vicarious sensations and to initiate needling credibility (even without actual needling). (3) Actual or vicarious acupuncture sensations play an important role in enhancing needling credibility. (4) If tactile/acupuncture sensations are not congruent with visual feedback (for example, when they are observing the treatment procedure but do not feel any sensation from it), individual priority of multisensory information might affect maintaining the needling credibility or diminishing/eliminating of it (**Figures 5B,C**). Further studies should be done to confirm this hypothesis with several experimental conditions: (1) for better explanation and understanding of clinical condition, other components can be added to our model (**Figure 5A**), (2) conditions with only acupuncture needling (without any needling credibility/therapeutic expectancy and visual display of needling) and with only visual display of needling (without actual needling and needling credibility/therapeutic expectancy) (**Figure 5B**, i and j, respectively), (3) with controlling the influence of instructions (Cheon et al., 2018), (4) with different contents of provided visual information (i.e., treatment-relevant or not-relevant, or incongruent with actual stimulation), and (5) with different extents to which patients pay attention to the given visual or perceived sensations.

#### Limitations

Several limitations in the present study must be noted. Firstly, we had no untreated control group in which to investigate the effect of elapsed time (e.g., habituation or sensitization to our pain model), nor did we include a healthy control group to identify patient-specific brain responses (e.g., the involvement of the pINS in SN and connectivity changes to the pain model). Secondly, the number of subjects who experienced no needling credibility was too small (n = 4) to dissociate the effect of needing credibility from the effect of visual feedback in PHNT. As most of the patients who received PHNT believed that they had been treated with REAL and reported vicarious acupuncture

(deqi) sensations, a separate group with enough number of the subject must be included in future studies to explicitly exclude needling credibility during REAL/PHNT session to investigate the influence of needling credibility. Thus, several experimental conditions (with and without somatosensory needling, needling credibility, instruction, and visual feedback etc.) with a sufficient number of the subject are needed to investigate the analgesic effects of somatosensory and cognitive/affective components on back pain. For example, shallow (minimal acupuncture or tactile stimulation) acupuncture to investigate the influence of the amount of somatosensory afference, acupuncture methodology (manual- vs. electrical-acupuncture stimulation), acupoint specificity (by comparing sham points and acupoints), and no/irrelevant display to patients to inquire into the influence of the visual feedback can be used. Thirdly, along with the limitation of experimental conditions, several cognitive/affective factors were not considered in this analysis. Thus, for better understanding future study should also investigate factors which are clinically relevant and influential: doctor–patient relationship (rapport), previous acupuncture experience and its efficacy, expectancy of acupuncture efficacy, accuracy and consistency of sensation reporting, degree of sleepiness/awakeness/engagement during the experiment, and eagerness to be cured. Specific nonneedling factors (i.e., acupuncture theory-based factors such as diagnosis and palpation) were not considered in this study. Fourthly, the acupoints were decided for the general purpose of back pain reduction and were not individualized to maximize its efficacy. Further study needs to be done with a set of individualized acupoints to further understand the influence of different aspects of acupuncture. Lastly, randomization should have performed more accurately and systematically. Unbalanced number of patients were allocated for REAL (n = 33) and PHNT (n = 23). Also, the significantly different baseline (LBEpre) pain scores made it difficult to investigate its direct comparison between groups, and thus the analysis of this study was limited to the changes in pain scores.

#### Conclusion

In the present study, we found significant back pain reduction and corresponding changes in functional connectivity after REAL and PHNT. It suggests that the involvement of different brain regions is related to improved symptoms, for example by detaching self (DMN) from the sensory aspect of pain (pINS)

#### REFERENCES


in REAL, by shifting attention (aINS) to self (DMN), and by disengaging between physical pain processing hubs (e.g., pINS and S1back region) in PHNT. We also speculated the relationship between visual and tactile information from video and needling, respectively, contextual conditioning from treatment instruction, and belief of treatment. This experimental paradigm might provide an appropriate sham methodology to dissociate the influence of different acupuncture components in acupuncture study, and the findings might help to understand corresponding brain mechanisms of acupuncture analgesia.

# ETHICS STATEMENT

This study protocol was approved by the Institutional Review Board of the Kyung Hee University (KHNMC-OH-IRB 2010- 013), and all participants provided written informed consent in accordance with the Declaration of Helsinki. This study is registered at the clinical research information service (CRIS; http: //cris.nih.go.kr/) (registration number: KCT0002253).

# AUTHOR CONTRIBUTIONS

JL, JK, and KP contributed to the conception and design of the study. JL, SE, J-HL, and KP conducted the experiment and collected the data. JL and SE performed the statistical analysis. JL, SE, and KP interpreted the results and wrote the manuscript. All authors contributed to the manuscript version, read, and approved the submitted version.

# FUNDING

This study was supported by grant from the Korea Institute of Oriental Medicine (K18052).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnins. 2019.01062/full#supplementary-material


cingulate cortex in fibromyalgia. Arth. Rheumatol. 70, 1308–1318. doi: 10.1002/ art.40507


**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Lee, Eun, Kim, Lee and Park. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fnins-13-01062 October 3, 2019 Time: 18:1 # 11

# Acupuncture for the Treatment of Pain – A Mega-Placebo?

#### Frauke Musial\*

Department of Community Medicine, National Research Center in Complementary and Alternative Medicine, NAFKAM, Faculty of Health Science, UiT – The Arctic University of Norway, Tromsø, Norway

Several control conditions, such as penetrating sham acupuncture and non-penetrating placebo needles, have been used in clinical trials on acupuncture effects in chronic pain syndromes. All these control conditions are surprisingly effective with regard to their analgesic properties. These findings have fostered a discussion as to whether acupuncture is merely a placebo. Meta-analyses on the clinical effectiveness of placebo revealed that placebo interventions in general have minor, clinically important effects. Only in trials on pain and nausea, including acupuncture studies, did placebo effects vary from negligible to clinically important. At the same time, individual patient meta-analyses confirm that acupuncture is effective for the treatment of chronic pain, including small but statistically significant differences between acupuncture and sham acupuncture. All acupuncture control conditions induce de qi, a distinct stimulation associated with pain and needling which has been shown to be a nociceptive/pain stimulus. Acupuncture therefore probably activates the pain matrix in the brain in a bottom-up fashion via the spino-thalamic tract. Central nervous system effects of acupuncture can be modulated through expectations, which are believed to be a central component of the placebo response. However, further investigation is required to determine how strong the influence of placebo on the attenuation of activity in the pain matrix really is. A metaanalysis of individual participant functional magnetic imaging data reveals only weak effects of placebo on the activity of the pain network. The clinical acupuncture setting is comprised of a combination of a distinct neurophysiological stimulus, the needling stimulus/experience, and a complex treatment situation. A broader definition of placebo, such as that proposed by Howick (2017) acknowledges a role for expectation, treatment context, emotions, learning, and other contextual variables of a treatment situation. The inclusion of particular treatment feature as a definitional element permits a contextual definition of placebo, which in turn can be helpful in constructing future clinical trials on acupuncture.

Keywords: acupuncture, sham acupuncture, placebo needles, pain, placebo, expectation, pain matrix

# THE EFFECTS OF ACUPUNCTURE AND PLACEBO IN CLINICAL PAIN STUDIES

#### Clinical Acupuncture Studies on Chronic Pain Conditions

In clinical acupuncture studies on pain, acupuncture control conditions (often called sham acupuncture) show strong clinical effects. The three most frequently applied acupuncture control conditions are: (i) points that differ from the acupuncture points are needled; (ii) true acupuncture points are needled, but only very gently, and with very thin needles (minimal acupuncture) and

#### Edited by:

Florian Beissner, Hannover Medical School, Germany

#### Reviewed by:

Karin Meissner, Hochschule Coburg, Germany Paul Enck, University of Tübingen, Germany

> \*Correspondence: Frauke Musial frauke.musial@uit.no

#### Specialty section:

This article was submitted to Perception Science, a section of the journal Frontiers in Neuroscience

Received: 14 December 2018 Accepted: 01 October 2019 Published: 17 October 2019

#### Citation:

Musial F (2019) Acupuncture for the Treatment of Pain – A Mega-Placebo? Front. Neurosci. 13:1110. doi: 10.3389/fnins.2019.01110

**182**

(iii) so-called placebo needles that do not penetrate the skin are used (for further discussion see paragraph "Needling associated procedures as sham acupuncture conditions in clinical trials").

In many of these clinical pain studies on acupuncture, acupuncture control conditions are more effective than standard medical care. In the German Acupuncture Randomized Trials (ART) studies, for example, in which true acupuncture was compared with minimal acupuncture and waiting list control, both true and minimal acupuncture were superior to being on a waiting list for the diagnosis of migraine, tension headache, or chronic back pain (Linde et al., 2005; Melchart et al., 2005; Brinkhaus et al., 2006). The only exception was knee arthritis, where only true acupuncture brought significant relief (Witt et al., 2005).

The German Acupuncture trials (GERAC studies) followed a slightly different design by comparing true acupuncture with minimal-acupuncture and standard medical care (Molsberger et al., 2006a,b). The results were similar: The treatment effect of acupuncture or minimal acupuncture was significantly superior to standard medical care and the effect was strong with no significant differences between the acupuncture conditions (Haake et al., 2007). The close similarity between true acupuncture, sham- and minimal acupuncture has raised the question as to whether the actual procedure of acupuncture is a placebo. Nonetheless, one of the consequences of these trials, which were funded by German health insurance companies, was that acupuncture is now recognized for by many German health insurance companies as a viable treatment for several conditions.

One of the most central questions in clinical studies is, however, whether and how the treatment which is to be tested compares to the available gold standard. A systematic review (Linde et al., 2009) consisting of twenty-two trials with a total of 4,419 patients came to the conclusion that acupuncture is not only at least as effective for migraine prophylaxis as prophylactic drug treatment but that it also has less side effects. Once again, no difference was established between true acupuncture and sham acupuncture procedures. In a systematic review on acupuncture treatment for tension type headache (Linde et al., 2009), the trials included were more heterogeneous (11 trials with 2,317 patients) with regard to the control conditions while the results were less homogeneous. The authors nonetheless came to the conclusion that acupuncture constitutes a valuable non-pharmacological treatment alternative.

Unlike the results from the large clinical acupuncture studies on pain syndromes, several brain-imaging studies in defined patient populations revealed relatively clear therapeutic acupuncture effects. For example, acupuncture therapeutic brain imaging studies in neuropathic pain (carpal tunnel syndrome) (Maeda et al., 2013, 2017) showed distinct true acupuncture-related neuroplasticity in the somatosensory cortex. This neuroplasticity is specifically related to increased neural functionality in the periphery (median nerve function) as well as to symptom improvement. One particularity of these two studies may be partly due to the use of electro acupuncture which constitutes a somewhat powerful acupuncture treatment.

In conclusion, the fact that sham acupuncture was equally effective as true acupuncture in almost all of the clinical trials on acupuncture has fostered a discussion as to whether acupuncture constitutes a particularly effective placebo, or whether sham acupuncture is an inadequate control.

## How Powerful Is Placebo? Meta-Analyses on Clinical Placebo Effects

At the same time, the placebo effect, and in particular its potential clinical relevance was under discussion. While basic scientists claimed that a strong placebo effect existed (Benedetti et al., 2005), the authors of the first major Cochrane review on the topic (Hrobjartsson and Gotzsche, 2003), concluded that there was no evidence to suggest that placebo interventions have clinically important effects in general. Moreover, in the only clinical condition in which a significant placebo effect could be detected, i.e., pain, the result was restricted to subjective continuous outcomes, and could not be clearly distinguished from bias (Hrobjartsson and Gotzsche, 2003). The authors came to the same conclusion after incorporating new evidence and trials (Hrobjartsson and Gotzsche, 2004a,b). Despite extending the search to more clinical conditions, the conclusion that placebo interventions in general have no important clinical effects still prevailed (Hrobjartsson and Gotzsche, 2010).

However, on the basis of the new data material, the authors qualified their conclusion as follows: "in certain settings placebo interventions can influence patient-reported outcomes, especially pain and nausea, though it is difficult to distinguish patientreported effects of placebo from biased reporting. The effect on pain varied, even among trials with low risk of bias, from negligible to clinically important. Variations in the effect of placebo were partly explained by variations in how trials were conducted and how patients were informed" (Hrobjartsson and Gotzsche, 2010). It is important to bear in mind that, since these meta-analyses contained acupuncture trials, in particular with regard to pain, they included and calculated sham- acupuncture conditions as placebo interventions. As discussed later in more detail, non-pharmacological placebos such as sham acupuncture as well as sham surgery maybe clinically more effective than oral pharmacological placebos (Meissner et al., 2013). This has certainly been the case in trials on migraine prophylaxis using network meta-analysis. The interpretation that the effect of non-pharmacological placebos is similar to pharmacological placebos may, therefore, be misleading and may undermine the effectiveness of the verum condition, here true acupuncture.

Consequently, the discussion on the relevance of placebo in general – and its role for acupuncture in particular – was intense and at times heated (Hrobjartsson and Gotzsche, 2006). Nonetheless, even a meta-analysis on acupuncture treatment for pain which included a systematic review of randomized clinical trials with true acupuncture, sham acupuncture, and no acupuncture groups did not reach any conclusion (Madsen et al., 2009). The aim was to explore the analgesic effect of acupuncture and sham acupuncture and to ascertain whether the type of the placebo acupuncture applied is associated with the estimated effect of acupuncture. Only three-armed randomized clinical trials consisting of a true acupuncture,

a sham acupuncture (or other similar controls), and a nonacupuncture group were included. However, this did not alter the results and conclusion: "A small analgesic effect of acupuncture was found, which seems to lack clinical relevance and cannot be clearly distinguished from bias. Whether needling at acupuncture points, or at any site, reduces pain independently of the psychological impact of the treatment ritual is unclear" (Madsen et al., 2009). The general conclusion from meta-analytic approaches to placebo is therefore that placebo effects are small, usually not clinically significant, and that the acupuncture effects in pain trials are inconsistent, dependent on study design and outcome, and very probably the result of bias.

# Sham Acupuncture: A Particularly Powerful Placebo?

While several meta-analyses on acupuncture pain trials did not differ, a meta-analyses technique combining individual patient data (Vickers et al., 2010) came to a completely different conclusion. The first individual patient data meta-analyses, using data from 29 of 31 eligible RCTs, with a total of 17,922 patients, established that acupuncture is effective for the treatment of chronic pain (Vickers et al., 2012). Furthermore, they noted small, but statistically significant differences between true- and sham acupuncture. The authors state that "these differences are relatively modest, suggesting that factors in addition to the specific effects of needling are important contributors to the therapeutic effects of acupuncture." An update confirms the results and conclusion (Vickers et al., 2017) and a further detailed analysis of the data set suggests that the clinical benefit persists over time (MacPherson et al., 2017).

The interpretation that acupuncture in general is a particularly powerful placebo is predominantly due to the strong effects of sham acupuncture conditions as control conditions in clinical trials. Nonetheless, with regard to the question as to the clinical relevance of these effects, the comparison of true acupuncture/sham acupuncture conditions to other control conditions, in particular no-treatment controls or routine care controls, is relevant.

In their meta-analysis over 37 trials involving 5,754 patients Linde et al. (2010a) concluded that sham acupuncture conditions have much greater non-specific effects than placebo interventions. This meta-analysis also focused on three-armed trials, including non-acupuncture control groups. Unlike Madsen et al. (2009) the authors distinguished sham acupuncture conditions from inert placebo conditions in the discussion and raised the question as to whether sham acupuncture conditions differ from other placebos in that the former induce particularly large unspecific effects. Such an interpretation would have substantial methodological consequences for the design of clinical trials. The authors also raised the issue as to how such a finding should be evaluated in the light of the clinical relevance of sham acupuncture (Linde et al., 2010a,b). The same issue was raised by Moffet (2009) who, in his systematic review, also deemed sham acupuncture conditions to be as clinically relevant as true acupuncture conditions.

To answer the question as to whether sham acupuncture interventions are generally more clinically effective than other placebos (Linde et al., 2010b), reanalyzed the data from the Cochrane review of Hrobjartsson and Gotzsche (2010) on placebo effects in all clinical conditions. They concluded, with all due caution given the heterogeneity of the included trials, that sham acupuncture conditions are in fact associated with stronger non-specific effects than other physical and pharmacological placebos. As already mentioned above, the phenomenon that sham acupuncture conditions are more clinically effective than, for example, oral placebos, was confirmed by Meissner et al. (2013) in their elegant systematic review on the differential effectiveness of different placebo treatments in trials on migraine prophylaxis. This network meta-analysis revealed that both sham acupuncture and sham-surgery were clinically more effective than oral pharmacological placebos.

Penetrating sham acupuncture has been criticized on account of the fact that it may actually constitute a less effective form of acupuncture treatment. Therefore, non-penetrating devices which mimic the acupuncture procedure but which do not actually penetrate the skin have been developed (Streitberger and Kleinhenz, 1998; Park et al., 2002). Nonetheless, a systematic review by Zhang et al. (2015) reveals precisely the same effect for non-penetrating needles as for other sham acupuncture procedures. They conclude that neither the Streitberger nor the Park device is an inert control intervention. Moreover, sixteen studies recorded adverse events in true and sham acupuncture alike.

Nonetheless, while focusing on the issue of sham acupuncture conditions, it should be borne in mind that acupuncture has also been shown to be clinically more effective than for example non-steroidal anti-inflammatory drugs (NASIDs). In a study on chronic back pain patients, Toroski et al. (2018) ascertained that electro-acupuncture treatment was clinically effective and, moreover, more cost effective than treatment with non-steroidal anti-inflammatory drugs.

In summary, it is generally accepted that acupuncture is clinically effective in treating chronic pain states. There is a significant, but rather small difference between acupuncture and sham acupuncture. At the same time, sham acupuncture conditions appear to be more effective than other physical and pharmacological placebos.

#### NEEDLING-ASSOCIATED PROCEDURES SUCH AS SHAM ACUPUNCTURE CONDITIONS IN CLINICAL TRIALS

Three frequently employed control strategies are generally defined as sham acupuncture. Two of these methods penetrate the skin: (i) other points than acupuncture points, are needled. This type of control procedure is used to investigate the site (or point)-specific effects of acupuncture (Moore and McQuay, 2005); (ii) applying minimal acupuncture; true acupuncture points are needled, but only very gently and with extremely thin needles. This procedure, which is used for mode-specific control of acupuncture effects, should enable the physician to define the appropriate "dose" of peripheral sensory stimulation (Backer et al., 2002). A combination of these two strategies is sometimes used. A third procedure utilizes devices with non-penetrating devices similar to the so-called "Streitberger needle" or the "Park-Sham" device. These procedures are similar in that they have the appearance of an acupuncture needle but do not penetrate the skin (Streitberger and Kleinhenz, 1998; Park et al., 2002; Chae et al., 2018). These devices are often called "placebo needles."

On the basis of the findings that sham acupuncture conditions appear to be more effective than other physical and pharmacological placebos, the issue has been raised as to whether sham acupuncture is physiologically active and, as such, not acceptable as a placebo control (Birch, 2006; Langevin et al., 2006, 2011; Lundeberg et al., 2008, 2011; Lund et al., 2009), Moreover, the choice of appropriate control conditions in clinical acupuncture trials on chronic pain syndromes poses a particular challenge, given that it is not possible to blind the acupuncturist. Double blinding is therefore essentially impossible which, in turn, substantially increases the risk of bias. In their recent review Chae et al. (2018) not only discuss the problems associated with non-penetrating sham acupuncture devices but also focus on the methodological challenges associated with trial design while introducing possible solutions with their benefits and shortcomings.

Several of the physiological mechanisms identified by Chae et al. (2018) are liable to account for some of the effects of placebo needles and cover the physiological effects of penetrating sham acupuncture: tactile stimulation is believed to be specific to the intervention and divided into the sensory discriminative and affective social component of the procedure. Some of the particularities of all forms of acupuncture/sham acupuncture/minimal acupuncture is the impossibility of blinding, possibly resulting in greater expectation-related placebo effects than when using a placebo pill. Nonetheless, expectation is also a neurophysiological effect which originates in the brain and which thus exerts its effect top-down.

#### POSSIBLE NEUROBIOLOGICAL MECHANISMS OF ACUPUNCTURE AND SHAM ACUPUNCTURE

#### C-Fiber Touch as Potential Mediator for the Affective-Social Dimension of Acupuncture

C-fiber touch (CT's) are tactile low threshold mechanosensitive fibers which are sensitive to gentle touch and which form a second touch system, with pleasant touch as a modality. They are assumed to represent the positive hedonic aspects of gentle touch (McGlone et al., 2007, 2014; Abraira and Ginty, 2013; Musial and Weiss, 2014). Thanks to animal experiments, this system has been known for quite some time and its role for humans has already been acknowledged. In humans, this system has been proposed to represent the neurobiological substrate, inducing feelings of calm and well-being related to gentle touch (McGlone et al., 2007, 2014; Abraira and Ginty, 2013; Musial and Weiss, 2014).

The particular role of touch for humans is not new to us, even though the particular C-fiber touch system has not yet been fully described for humans. For example, holding hands reduces the neural response to threat in an fMRI paradigm, even if the person holding the hand is a complete stranger (Coan et al., 2006). As Chae et al. (2018) point out, touch is a central factor in many medical procedures and an integral factor in all acupuncture conditions, regardless of whether or not the device penetrates the skin.

The touch component per se therefore constitutes a distinct neurobiological pathway, uncommon in all non-touch placebos such as a placebo pill. Moreover, it is represented by a specific pathway where the origin of stimulation is generated in the periphery of the body and travels in a bottom-up fashion to the brain. From here, it exerts a complex affective-emotional reaction (McGlone et al., 2007, 2014; Abraira and Ginty, 2013; Musial and Weiss, 2014). At the same time, this pathway is common to all acupuncture control conditions, thereby threatening the validity of virtually all of these conditions (Campbell, 2006; Lund et al., 2009).

### De qi and the Sensory-Discriminative Aspects of Acupuncture

Inserting a needle and penetrating the skin is a stimulus of vital importance. Such a procedure will stimulate pain pathways and signal information of an injury, and is therefore recognized by the brain as a potential threat to the body's integrity. It therefore comes as no surprise that acupuncture procedures induce the strong and distinct activation of pain-related areas in the brain (Zhao, 2008; Chae et al., 2013). According to Zhao "Acupuncture analgesia is manifested only when the intricate feeling (soreness, numbness, heaviness, and distension) of acupuncture in patients occurs following acupuncture manipulation" (Zhao, 2008). This sensation, known as de qi, is believed to be fundamental to the therapeutic outcome of acupuncture (Choi et al., 2013; Chae et al., 2018).

To date, several studies have shown that even nonpenetrating placebo needle procedures induce de qi sensations that are indistinguishable from the effect of real acupuncture needles (Chae, 2017; Chae et al., 2018). Taken together, these findings indicate that even non-penetrating acupuncture placebos are capable of stimulating sensory-discriminative pathways, including pain sensation. The current evidence therefore suggests that acupuncture – or indeed any other kind of needling procedure – constitutes a nociceptive and/or pain signal: Acupuncture (e.g., ST 36) activates pain-related brain structures (Biella et al., 2001; Pariente et al., 2005; Beissner et al., 2012; Theysohn et al., 2014); for overview and discussion see Wang et al. (2008), Zhao (2008), and the activation is dependent on the needling sensation (Beissner et al., 2012). Moreover, a metaanalysis across 28 fMRI studies of acupuncture needling showed an activation of the pain matrix (sensorimotor cortical network, including the insula, thalamus, and anterior cingulate cortex (ACC), as well as both the primary and secondary somatosensory cortices) (Chae et al., 2013). In summary, there is ample evidence

that acupuncture per se constitutes a nociceptive/pain stimulus which influences brain networks in a bottom-up fashion.

Under the premise that the therapeutic acupuncture effects are at least partly mediated through the pain pathway of the sensorydiscriminative system, the processing of nociceptive signals in the spinal cord must be taken into account to explain the effect of needling and its related de qi sensation. A needling stimulus in a painful situation, be it clinically manifested such as in a pain syndrome or experimentally induced in the laboratory, is liable to excite two mechanisms on the level of the spinal cord: segmental (gate-control) (Mayer et al., 2000; Irnich and Beyer, 2002; Stux et al., 2003) and spino-medullary (diffuse noxious inhibitory control, DNIC) (Le Bars et al., 1989; Le Bars, 2002; Le Bars and Willer, 2002, 2007; Le Bars and Cadden, 2007) processes.

While gate control is, neurophysiologically speaking, somewhat short-lived (Fields et al., 2005), DNIC is a strong long-term effect, inasmuch as it persists for several minutes after its induction. Such a response is typical of neuroplastic changes (Le Bars and Willer, 2002, 2007). DNIC characterizes an intrinsic mechanism for inhibiting pain that becomes activated when an additional nociceptive stimulus is applied to a region far removed (heterotopic) from the initial region receiving nociceptive stimulation (Le Bars et al., 1989; Le Bars, 2002; Le Bars and Willer, 2002; Le Bars and Cadden, 2007; Le Bars and Willer, 2007). This phenomenon is often classified under the superordinate concept of "counter irritation" (Le Bars et al., 1989) and is mediated via a spino-medullary loop (Le Bars, 2002; Le Bars and Willer, 2007).

In humans, experimental paradigms which test the effect of additional pain stimuli on an already existing experimental pain stimulus, and which are thus homologous to DNIC are often called "heterotopic noxious conditioning stimulations (HNCS)" (Sprenger et al., 2011) or "conditioned pain modulation (CPM)" (Bjorkedal and Flaten, 2012). Sprenger et al. (2011) used a tonic cold pressor task and phasic additional pain stimulation in an fMRI paradigm and noticed a clear HNCS effect with marked endogenous analgesia. This outcome was accompanied by reduced activity in classical pain-related brain structures and the recruitment of an opiate-dependent, descending pain control system (naloxone blockade). In women Bjorkedal and Flaten (2012) were able to show that a CPM response can be reduced by expectations.

Acupuncture has been shown to exhibit rather strong effects in an HCNS/CPM design (Choi et al., 2011a,b; Musial et al., 2012). Moreover, acupuncture decreased somatosensoryevoked potential amplitude to noxious stimuli in anesthetized volunteers in an HCNS/CPM design (Meissner et al., 2004), thus showing clear acupuncture effects in unconscious humans. This is an interesting observation, given that all cortical effects such as expectations, suggestions, beliefs etc, can be excluded as confounding factors in subjects under deep anesthesia. Further evidence from fMRI studies shows less brain activity in somatosensory areas in response to pain following acupuncture stimulation (modulation of cold pain: (Zhang et al., 2003) and electrical pain: (Theysohn et al., 2014).

In conclusion, acupuncture therapeutic effects are seen to be related to the de qi sensation, which is characterized as a pain-related sensation. There is evidence that acupuncture and related conditions act via a pain signal elicited in the periphery and traveling bottomup to the brain. These peripheral nociceptive signals may undergo modulation via spinal and spino-medullary mechanisms. Since virtually all placebo/sham acupuncture procedures are known to induce de qi ensations that are indistinguishable from real acupuncture, it should be borne in mind that this pain-related, somatosensory-discriminative mechanism akin to acupuncture is common to all true acupuncture/sham acupuncture/minimal acupuncture, and placebo acupuncture conditions.

#### Enhanced Expectation as a Mediator for the Strong Effects of "Sham"- and Placebo Acupuncture Conditions?

Despite various creative approaches such as the above discussed non-penetrating placebo needles (Streitberger and Kleinhenz, 1998; Park et al., 2002; Chae et al., 2018), an adequate experimental control of acupuncture for the treatment of chronic pain is scarcely possible, particularly since the acupuncturist cannot be blinded. One of the characteristics of all acupuncture and related procedures is, therefore, the impossibility of blinding, which can lead to greater expectation-related placebo effects than with other placebos (for overview and discussion see Chae (2017), Chae et al. (2018).

Expectation is a key mediator of the placebo response (Kirsch, 1999; Enck et al., 2008; Colloca and Miller, 2011a,b) and its relevance for the clinical effectiveness has already been well documented [(Moffet, 2009; Linde et al., 2010a; Chae et al., 2018)]. Interestingly enough, while it is widely accepted within placebo research that enhancing the placebo effect during drug treatment is desirable (Enck et al., 2013; Evers et al., 2018), the possibility that expectation – and thus placebo – may play a role in clinical acupuncture effects has been held against acupuncture as a treatment, in particular in public discourse. The argument is that acupuncture may merely represent a "placebo" without so-called "specific effects." This is contradictory in view of the fact that, physiologically speaking, the expectancy-mediated placebo effect per se has meanwhile been reasonably well defined and represents a rather distinct neurophysiological mechanism (Enck et al., 2008; Wager and Atlas, 2015; Geuter et al., 2017; Schafer et al., 2018).

The individual placebo response has been under close scrutiny ever since the 1990s (Benedetti et al., 1995; Benedetti, 1996; Amanzio and Benedetti, 1999; Benedetti et al., 2005). In an experimental pain-tolerance setting, Benedetti et al., showed how conditioning and expectation processes both play a role in placebo analgesia. The expectationinduced placebo response can be completely blocked by the opioid antagonist naloxone (Benedetti, 1996), whereas the conditioning-dependent placebo response can be blocked by naloxone only when conditioning was carried out with an opioid (Amanzio and Benedetti, 1999). Thus, expectationdependent placebo responses are, in principle, mediated by the

endogenous opioid system, whereas conditioning-dependent placebo effects depend on the particular subsystem that had been conditioned. The opioid-dependent mechanism of expectation-induced placebo analgesia is also supported by neurophysiological imaging studies in man, showing that an opioid-dependent cortical network becomes activated during a placebo response (Petrovic et al., 2002; Zubieta et al., 2005). Acupuncture analgesia is similar to the expectationdependent placebo effect in that it too can be blocked by naloxone, i.e., it is also opioid-dependent [for an overview, see (Mayer et al., 2000)].

#### ACUPUNCTURE, PLACEBO, AND THE PAIN MATRIX

Data from imaging studies have confirmed that not only expectation but also learning and context factors play a role in placebo effects [for overview and discussion see Enck et al. (2008), Wager and Atlas (2015), Geuter et al. (2017), Schafer et al. (2018)]. Moreover, context and social factors are welcome modulators for expectation and associative learning. Most of these models are based on results from pain studies.

With regard to the placebo response, the ventromedial prefrontal cortex (vmPFC), the dorsolateral prefrontal cortex (dlPFC), the lateral orbitofrontal cortex (lOFC), nucleus accumbens, insula, amygdala, hypothalamus, and periaqueductal gray are generally regarded as crucial (Wager and Atlas, 2015; Geuter et al., 2017; Schafer et al., 2018). All these areas increase activity in response to placebo manipulations related to pain stimulation (Wager and Atlas, 2015), albeit the periaqueductal gray (PAG) and the dorsal anterior cingulate cortex (dACC) respond in a more differentiated fashion, and appear to have a modulatory function within the placebo network (Wager and Atlas, 2015). The vmPFC seemingly plays a particularly essential role in the mediation of expectation (Geuter et al., 2017). Thus, the vmPFC represents the part of the placebo brain network that connects situational factors to neurophysiological responses (Geuter et al., 2017).

At the same time, the placebo network appears to be independent from the pain network, even though some structures, primarily the PAG and the dACC, seem to have a modulatory function in both networks (Wager and Atlas, 2015; Geuter et al., 2017). This conceptualization fits well with a number of experimental findings, indicating that the perception of pain is also dependent on the context (Keltner et al., 2006). In one fMRI study, for instance (Koyama et al., 2005) were able to show that, to a great extent, the expectation of one particular stimulus intensity determined its cortical representation, regardless of whether or not it was anticipated to be painful. As the magnitude of the expected pain increased, activation increased simultaneously in the thalamus, insula, prefrontal cortex, and ACC. Pain-intensityrelated brain activation was identified in a widely distributed set of brain regions and overlapped partially with expectation-related activation in other regions, including the anterior insula and ACC (Koyama et al., 2005).

Thus, the evidence available with regard to placebo effects on pain in experimental human and animal studies suggests that placebo effects are mediated by a network of brain structures that differ from the pain network. However, the placebo network influences and modulates the pain network, relying on several factors such as context, learning and prior experiences, instructions, etc. These factors probably influence the placebo network by way of expectations. Expectations are fundamental inasmuch as they may signal reward (e.g., reduced pain). Anticipated reward will activate the reward system in the brain, a system that involves the nucleus accumbens, the amygdala, the ventral tegmental area, and the orbitofrontal cortex (Enck et al., 2008). The neurotransmitters of this system are dopamine and opioid peptides, and this network is usually investigated with regard to drug dependencies and reinforcement. In summary, instructions, suggestions, and other types of social information can have powerful effects on pain and these changes are mediated by top-down control via the involvement of prefrontal regions, particularly the dorsolateral and ventromedial prefrontal cortex (Koban et al., 2017).

As already elucidated, current evidence suggests that any kind of needling procedure constitutes a nociceptive and/or pain signal (Biella et al., 2001; Pariente et al., 2005; Beissner et al., 2012; Theysohn et al., 2014); for overview and discussion see Wang et al. (2008), Zhao (2008), Beissner et al. (2012), and that acupuncture needling (a meta-analysis across 28 fMRI studies) induces an activation of the pain matrix (sensorimotor cortical network, including the insula, thalamus, anterior cingulate cortex, and both primary and secondary somatosensory cortices) (Chae et al., 2013). Moreover, data from healthy humans subjects in response to needling of commonly used acupuncture points for the treatment of pain syndromes (manual acupuncture at LI4, ST36) revealed a partial overlap with the pain matrix as well as a deactivation of a limbic-paralimbic-neocortical network (Hui et al., 2009; Claunch et al., 2012). This limbic deactivation is correlated to the psychophysical response modulating the bottom-up nociceptive signal. It therefore modulates brain networks in response to acupuncture stimulation. Assuming that acupuncture is a pain stimulus that travels bottom-up and activates the pain networks, it must also be assumed that expectations, instructions, emotions, and other context factors modulate acupuncture effects.

In an elegant study Lee et al. (2015) modulated and manipulated an acupuncture stimulus by instruction. One group was told that the needling was part of an acupuncture treatment, whereas the other group was informed about the needle insertion only, and that this might be a painful intervention. In addition to their acupuncture stimulation, all participants received the same sensory stimuli which included a pain stimulus. The brain activation in response to the pain stimulus in parts of the pain network was significantly lower in those participants who had been informed that the needle insertion was part of an acupuncture treatment. At the same time, the needling within a therapeutic context activated reward circuits in the brain and modulated the pain matrix.

The results from brain imaging studies emanating from placebo research as well as the results of Chae et al. (2013) and Lee et al. (2015) suggest that: (i) the pain matrix and the placebo network are primarily independent brain networks; (ii) the pain matrix and the placebo network interact and the placebo-induced modulation of the pain-matrix is mediated by expectation; (iii) acupuncture activates the pain matrix and is a nociceptive/pain stimulus, and (iv) acupuncture as a nociceptive/pain stimulus is modulated by placebo effects, in particular expectation.

Nonetheless, how powerful the modulation of the painmatrix through placebo effects actually is remains to be elucidated. A meta-analysis of individual participant functional magnetic resonance imaging data combined the results of 603 healthy individuals from 20 studies and investigated the effects of placebo intervention on the pain matrix (Zunhammer et al., 2018). The analysis revealed rather moderate analgesic effects on subjective pain reports and only a very small effect of the placebo condition on the pain matrix. The authors concluded that placebo treatments affect pain through brain mechanisms (top down) and that these influences are largely independent from the bottom-up effects of nociceptive processing.

### IS ACUPUNCTURE A MEGA-PLACEBO? ON THE RELATIVITY OF PLACEBO

In particular the neurobiological developments in the understanding of the mechanisms of the placebo response have confirmed that the concept placebo encompasses a wide variety of modalities such as emotions, instructions, suggestions, and other types of social information mediated by alterations in prefrontal brain areas (Koban et al., 2017). These factors are often described as the "unspecific" effects of a particular treatment setting, indicating that the treatment effects are mainly psychological in nature and independent from the treatment provided.

To date, the concept of specificity at brain level, which is reminiscent of the historic theory of phrenology, may be questioned. Defined brain areas, such as the PAG or the dACC play a role in a number of brain functions. This fact tallies well with the concept that brain functions are performed by the activity of neural networks. The concept of neural networks also explains why the perception of pain is context-dependent (Koyama et al., 2005; Keltner et al., 2006); a finding that is relevant for the explanation of pain-related placebo effects.

Howick (2017) proposes a broader definition of placebo which goes beyond the claim that a placebo should consist of an inert "intervention simulation." His modified version of Grünbaum's definition of placebo emphasizes the role of expectations, and thus includes emotions, learning, and other contextual variables. Moreover, his definition assigns a special role to the peculiarities of a treatment situation, including special treatment features, and thus partly allows for a contextual definition of placebo. However, it is worth bearing in mind that Grünbaum's original article (Grunbaum, 1986), which relates to placebo effects in psychiatry and psychotherapy, remains somewhat controversial (Enck and Zipfel, 2019). To what extent this concept can be applied to other non-pharmacological treatments such as acupuncture – which is in many ways more "physical" than psychotherapy – may not be entirely consensual in the field of placebo research.

Nonetheless, Howick's interpretation of Grünbaum's definition (Grunbaum, 1986) provides an opening for the role of treatment theory and thus calls for a broadening of the definition of placebo beyond the role of expectation. In his paper, Howick (2017) uses the acupuncture/sham acupuncture/placebo needle discussion as a case study to illustrate that treatment theory is fundamental to the definition of a placebo condition and that it does not suffice to control exclusively for expectation. In his example, point specificity as treatment theory will lead to an acupuncture-related placebo concept which interprets needling at non-acupuncture points as "placebogenic" [page 1371, Howick (2017)]. In his case study on acupuncture, he argues that independent evidence supports the assumption that the placebo needle itself (Streitberger needle) cannot be a true placebo. He pinpoints that the core challenge in the interpretation of the clinically effective control procedures for the acupuncture treatment of pain states is the lack of an accepted therapeutic theory for acupuncture.

Howick's broadened concept of placebo not only allows for the defined use of placebo controls beyond the pharmacological context but also includes also nonpharmacological interventions. Nonetheless, as he demonstrates in his acupuncture case study, his definition demands and includes a defined therapeutic theory. With regard to many interventions out of the spectrum of complementary and alternative medicine, this may still constitute a challenge, such as in the case of acupuncture. Further clinical studies addressing treatment theory of acupuncture as well as the placebo/expectation issue are therefore required.

The field of placebo research has accepted that placebo might not be as distinctively defined as it is necessary for conducting a clinical trial in the non-pharmacological arena. Placebo effects are viewed as positive and useful factors of a treatment, particularly in clinical practice, and are considered to be a part of every regular treatment (Evers et al., 2018). With this definition, the debate as to what extent a treatment is dependent on placebo becomes less pungent and the focus is relocated to the most important question, namely to: what helps patients, what relieves their pain, and how can they be treated most effectively. This pragmatic approach aims to maximize all the positive factors of a treatment that can be recruited while minimizing risk and negative effects, including nocebo (Evers et al., 2018).

# AUTHOR CONTRIBUTIONS

The author confirms being the sole contributor of this work and has approved it for publication.

#### FUNDING

The publication charges for this article were funded by a grant from the publication fund of UiT – The Arctic University of Norway.

#### REFERENCES


antiinflammatory drugs in the treatment of chronic low back pain. J. Acupunct. Meridian Stud. 11, 62–66. doi: 10.1016/j.jams.2018.01.003


**Conflict of Interest:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Musial. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Role of Tactile Stimulation for Expectation, Perceived Treatment Assignment and the Placebo Effect in an Experimental Nausea Paradigm

Simone Aichner<sup>1</sup> , Anja Haile<sup>1</sup> , Verena Hoffmann<sup>1</sup> , Elisabeth Olliges1,2 , Matthias H. Tschöp3,4,5 and Karin Meissner1,2 \*

1 Institute of Medical Psychology, Faculty of Medicine, LMU Munich, Munich, Germany, <sup>2</sup> Division of Health Promotion, Coburg University of Applied Sciences, Coburg, Germany, <sup>3</sup> Institute for Diabetes and Obesity, Helmholtz Diabetes Center, Helmholtz Zentrum München, Munich, Germany, <sup>4</sup> German Center for Diabetes Research (DZD), Munich, Germany, <sup>5</sup> Division of Metabolic Diseases, Department of Medicine, Technische Universität München, Munich, Germany

#### Edited by:

Younbyoung Chae, Kyung Hee University, South Korea

#### Reviewed by:

Jose Pablo Ossandon, Universität Hamburg, Germany Yi-Hung Chen, China Medical University, Taiwan

> \*Correspondence: Karin Meissner karin.meissner@med.lmu.de

#### Specialty section:

This article was submitted to Perception Science, a section of the journal Frontiers in Neuroscience

Received: 12 February 2019 Accepted: 28 October 2019 Published: 13 November 2019

#### Citation:

Aichner S, Haile A, Hoffmann V, Olliges E, Tschöp MH and Meissner K (2019) The Role of Tactile Stimulation for Expectation, Perceived Treatment Assignment and the Placebo Effect in an Experimental Nausea Paradigm. Front. Neurosci. 13:1212. doi: 10.3389/fnins.2019.01212 Introduction: Tactile stimulation during a placebo treatment could enhance its credibility and thereby boost positive treatment expectations and the placebo effect. This experimental study aimed to investigate the interplay between tactile stimulation, expectation, and treatment credibility for the placebo effect in nausea.

Methods: Ninety healthy participants were exposed to a 20-min vection stimulus on two separate days and were randomly allocated to one of three groups on the second day after the baseline period: Placebo transcutaneous electrical nerve stimulation (TENS) with tactile stimulation (n = 30), placebo TENS without tactile stimulation (n = 30), or no intervention (n = 30). Placebo TENS was performed for 20 min at a dummy acupuncture point on both forearms. Expected and perceived nausea severity and further symptoms of motion sickness were assessed at baseline and during the evaluation period. At the end of the experiment, participants in the placebo groups guessed whether they had received active or placebo treatment.

Results: Expected nausea decreased significantly more in the placebo groups as compared to the no treatment control group (interaction day × group, F = 6.60, p = 0.003, partial η <sup>2</sup> = 0.20), with equal reductions in the two placebo groups (p = 1.0). Reduced expectation went along with a significant placebo effect on nausea (interaction day × group, F = 22.2, p < 0.001, partial η <sup>2</sup> = 0.35) with no difference between the two placebo groups (p = 1.0). Twenty-three out of 29 participants in the tactile placebo group (79%) but only 14 out of 30 participants (47%) in the non-tactile placebo group believed that they had received the active intervention (p = 0.015). Bang's blinding index (BI) indicated random guessing in the non-tactile placebo group (BI = 0; 95% CI, −0.35 to 0.35) and non-random guessing in the direction of an "opposite guess" in the tactile placebo group (BI = −0.52; 95% CI, −0.81 to −0.22).

Conclusion: Tactile stimulation during placebo TENS did not further enhance positive treatment expectations and the placebo effect in nausea but increased the credibility of the intervention. Further trials should investigate the interaction between perceived treatment assignment, expectation, and the placebo effect during the course of a trial.

Keywords: placebo effect, expectation, nausea, motion sickness, tactile stimulation, acupuncture

#### INTRODUCTION

fnins-13-01212 November 11, 2019 Time: 14:7 # 2

In randomized controlled trials (RCT), the specific effect of acupuncture compared with placebo acupuncture is usually small. An individual patient data meta-analysis with nearly 18.000 patients revealed standardized differences (SD) for the specific effect of acupuncture between 0.15 and 0.23 SD for various chronic pain conditions (Vickers et al., 2012). Compared to no treatment, however, the overall improvement after acupuncture is usually large (Linde et al., 2010a,b; Meissner et al., 2013). The small specific effect of acupuncture compared to the large overall effect indicates the involvement of "nonspecific" factors, such as expectation and tactile stimulation, which contribute to the success of acupuncture treatment.

There is increasing evidence that positive treatment expectations influence the non-specific response to acupuncture treatment. In four large RCT of acupuncture, for example, patients with chronic pain conditions and high outcome expectations at baseline showed larger pain reduction at follow-up regardless of whether they were allocated to active acupuncture or placebo acupuncture (Linde et al., 2007). Research from the last decades clearly indicates that the effects of expectation, referred to as "placebo effects," are deeply rooted in our brains. Placebo effects in pain, for example, are accompanied by the activation of a specific pain modulating network that extends from cortical areas to the spinal cord (Geuter et al., 2017). Expectation can be formed via various mechanisms, including verbal suggestions and conditioning (Colloca and Miller, 2011). Also the non-verbal cues of a treatment, such as prizing, labeling, and dosing, can increase outcome expectations and thus the placebo effect (Meissner and Linde, 2018). Placebo effects have been demonstrated for various clinical conditions including nausea (Quinn and Colagiuri, 2014, 2016; Müller et al., 2016), depression and anxiety (Meyer et al., 2015; Peciña et al., 2015; Rutherford et al., 2016), Parkinson's disease (De la Fuente-Fernández et al., 2001; Benedetti et al., 2004; Lidstone et al., 2010), as well as for gastrointestinal and cardiovascular systems (Meissner, 2009, 2011; Meissner and Ziep, 2011; Meissner et al., 2011; Ronel et al., 2011; Meissner, 2014; Rief et al., 2017) and immune responses (Hadamitzky et al., 2018).

In addition to positive expectations, skin penetration during needling appears to contribute to the non-specific effects of acupuncture. A re-analysis of the individual patient data metaanalysis by Vickers et al. (2012) revealed a considerably smaller specific effect of acupuncture in RCTs with control groups using skin penetrating needles (SD acupuncture vs. control 0.17, 95% CI 0.11–0.23; n = 9) compared to control groups using non-penetrating needles (SD acupuncture vs. control 0.43, 95% CI 0.01–0.85; n = 4) (MacPherson et al., 2014). An update of the individual patient data meta-analysis by Vickers et al. (2018) further confirmed this finding, with smaller effects sizes for sham controlled trials that used a penetrating needle in the sham groups compared to trials that used non-penetrating or non-needle sham (difference in SD −0.30, 95% CI −0.60, −0.00, p = 0.047). The authors argue that this difference could be either due to the unblinding of patients in the control groups with nonpenetrating needles, or to the physiologic effects of skinpenetrating placebo needles, which may have still some therapeutic activity against pain. They argue further that the possibility of unblinding is unlikely, since non-penetrating needles, such as the Streitberger needle, have been confirmed as credible placebos (MacPherson et al., 2014).

However, an imbalance due to differences in blinding could still occur: Patients receiving penetrating needles may be more prone to believe that they have received true acupuncture than patients receiving non-penetrating needles. An "opposite guess" – that is, a higher probability to guess "active treatment" than would be expected by chance (Bang et al., 2010) – could strengthen the outcome expectations of patients in the control groups of RCTs and thereby enhance the placebo effect. First support for this notion comes from experimental placebo studies, showing that "active" placebos that mimic the side effects of pharmacological drugs were more effective in reducing pain than placebos without such side effects (Bjorkedal and Flaten, 2011; Rief and Glombiewski, 2012).

To better understand the role of tactile stimulation, expectation, and perceived treatment assignment for the overall response to acupuncture point stimulation, we conducted a placebo study using an established experimental nausea design (Gianaros et al., 2001; Levine et al., 2006). We recently showed that a placebo transcutaneous electrical nerve stimulation (TENS) stimulation at a dummy point, which elicited slight tactile stimulation, induced a large placebo effect on experimentally induced nausea in comparison to a no-treatment control condition in female participants (Müller et al., 2016). In the present study, we focused on the role of tactile stimulation for the size of the placebo effect and included a second placebo group without tactile stimulation by the TENS device. We hypothesized that tactile stimulation during the placebo intervention would enhance the placebo effect in nausea due to the development of higher outcome expectations. With regard to blinding effectiveness, we expected that the tactile stimulation would lead more participants to believe that they had received the active treatment ("opposite guess") as compared to the placebo TENS intervention without tactile stimulation, for which we expected a "random guess" (Bang et al., 2010).

# MATERIALS AND METHODS

fnins-13-01212 November 11, 2019 Time: 14:7 # 3

#### Participants

Healthy adult participants between 18 and 50 years with a history of motion sickness [score ≥80 in Motion Sickness Susceptibility Questionnaire (MSSQ); Golding, 1998] were recruited. Further inclusion criteria were normal or corrected-to-normal vision and hearing, right-handedness, and normal weight (BMI 18– 25 kg/m<sup>2</sup> ). Exclusion criteria comprised implanted devices (e.g., pacemaker, insulin pump) or metal implants, a history of diseases of the inner ear (e.g., Morbus Menière, acute hearing loss), bloodclotting disorders or a tendency for thromboembolic diseases, and the presence of skin disease, diabetes, cardiovascular disease, epilepsy, or cancer. Further exclusion criteria comprised surgery during the past 4 weeks, current pregnancy or breast feeding, alcohol or drug abuse, inability to imply with the specific instructions, the regular intake of drugs except of hormonal contraceptives, thyroid hormones, and anti-allergic drugs, and anxiety and/or depression scores above the clinically relevant cut-off, as assessed with the Hospital Anxiety and Depression Scale (Zigmond and Snaith, 1983). Participants, who fulfilled all inclusion criteria and none of the exclusion criteria, were invited to participate in a 20-min screening session, during which their susceptibility for the visual vection stimulus was tested. Participants, who developed at least moderate nausea (≥"5" on a 11-point NRS, with "0" indicating "no nausea" and "10" indicating "maximal tolerable nausea"), were invited to participate in the core experiment.

The study protocol was approved by the ethical committee of the Medical Faculty at Ludwig Maximilian University of Munich and was registered retrospectively at the German Clinical Trials Register (no. DRKS00015192). All participants provided written informed consent.

#### Study Design

The experiment consisted of a baseline session (day 1) and a testing session (day 2) on two separate days at least 24 h apart. On day 2 after the resting period, participants were randomly allocated to one of four treatment arms: placebo intervention with somatosensory stimulation (n = 30), placebo intervention without somatosensory stimulation (n = 30), no treatment (n = 30), or active treatment (n = 10; results not reported). The active treatment arm was implemented to avoid deceptive administration of the placebo treatment. All groups were stratified by sex (50% women, 50% men).

#### Experimental Procedure

Participants were tested on two separate days at least 24 h apart (median, 7 days) at the same daytime between 2:00 to 7:00 pm and were instructed to fast at least 3 h before the experiment. Nausea was induced through a standardized visual presentation of alternating black and white stripes with circular motion at 60◦ , which induces a circular vection sensation (Napadow et al., 2012). The visual stimulus was projected onto a semicylindrical and semitransparent screen placed around the volunteer at a distance of 30 cm to the eyes. Participants were asked to keep their eyes open and look straight ahead without fixating the stripes.

On both testing days upon arrival in the laboratory, participants were seated in a recliner and asked to fill out several questionnaires. Electrodes for psychophysiological assessments were placed, and an indwelling catheter was fixed at the forearm to allow for repeated blood drawings (results of physiological parameters will be reported elsewhere). On day 1, the session started with a 10-min baseline period, followed by a 10 min resting period and a 20-min presentation of the visual vection stimulus. The session ended with a 15 min resting period. On day 2 after the 10-min baseline period, participants were randomized to one of the experimental groups. The experimenter opened the first randomization envelope and delivered standardized information according to group allocation ("treatment" or "no treatment"). Then a medical assistant opened the second randomization envelope, placed the TENS electrodes according to group allocation and started the TENS device for 20 min, if applicable. After 10 min the visual stimulus was presented for 20 min. The experiment ended with a 15-min resting period. For security reasons, the vection stimulus was stopped on both testing days, if nausea ratings indicated severe nausea (ratings of 9 or 10 on 11-point NRS). **Table 1** summarizes the time course of the experiment as well as the different times of symptom assessment.

#### Interventions

**Supplementary Table S1** shows the main characteristics of the interventions and the content of the verbal suggestions for each of the three study groups. In short, participants in the treatment groups were informed that the nausea treatment would consist of either an active treatment or a placebo treatment and that the active treatment would reduce nausea by electrical stimulation of an acupuncture point, while the placebo treatment would

TABLE 1 | Timeline of the experiment on day 1 and day 2 and time points of behavioral assessments.


NRS, 11-point numeric rating scale; SSMS, Subjective Symptoms of Motion Sickness. <sup>a</sup>Only day 2, according to group allocation. <sup>b</sup>Last minute. <sup>c</sup>Every minute (ratings were averaged prior to analysis). <sup>d</sup> Immediately after verbal suggestions and applying/testing the TENS device, if applicable.

consist of a placebo acupoint stimulation. Subjects were further informed that the best effects were to be expected when the treatment is begun before exposure to the nauseogenic stimulus (Ezzo et al., 2006). Participants in the no-treatment control group were informed about the rationale and value of a notreatment control group.

All interventions were conducted using a programmable TENS device (Digital EMS/TENS unit SEM 42, Sanitas, Uttenweiler, Germany). In the placebo groups, two electrodes were attached proximal and distal to a generally accepted dummy point in the context of acupuncture research located on the ulnar side of both forearms (Witt et al., 2012). In the placebo group with somatosensory stimulation ("tactile placebo"), the superficial massage program of the TENS device was turned on for 20 min in order to induce a slight tingling sensation at the electrode site. In the placebo group without somatosensory stimulation ("non-tactile placebo"), electrodes were connected to the TENS device but the device was only allegedly turned on. The active treatment group received real TENS at the acupoint "PC6" (Lee and Fan, 2009) on both forearms for 20 min.

#### Randomization and Blinding

Random allocation was accomplished using sealed and numbered envelopes. A person not directly involved in the experiments prepared the randomization envelopes based on a computerderived randomization list. The interventions were performed in a single-blind design. Participants in the no-treatment control group were necessarily unblinded.

#### Ratings and Questionnaires

Expected nausea intensity was rated on 11-point NRS, with "0" indicating "no expected nausea" and "10" indicating "maximal tolerable expected nausea." Perceived nausea and dizziness intensities were rated at baseline and every minute during the nausea period on 11-point NRSs, with "0" indicating "no nausea/dizziness" and "10" indicating "maximal tolerable nausea/dizziness." Symptoms of motion sickness were assessed using the "Subjective Symptoms of Motion Sickness" (SSMS) questionnaire (adapted from Graybiel et al., 1968), with scores of 0–3 assigned to responses of none, slight, moderate, and severe for symptoms of dizziness, headache, nausea/urge to vomit, tiredness, sweating, and stomach awareness, respectively. At the end of day 2, participants in the treatment groups were asked to guess whether they had received active or placebo treatment as well as to rate the perceived effectiveness of treatment on an 11-point NRS, with "0" indicating "not effective at all" and "10" indicating "highly effective." They were furthermore asked to rate on 11-point NRS, how sure they were about their treatment guesses, with "0" indicating "not sure at all" and "10" indicating "very sure." Bang's blinding index (BI) was used to estimate the proportion of participants who guess their treatment incorrectly beyond chance level. Random guess exists if the BI's confidence interval covers 0 (Bang et al., 2010).

#### Statistical Analyses

Sample size calculation was performed for baseline-adjusted nausea scores. Assuming a medium effect size partial eta-squared of 0.13 for the difference in baseline-adjusted nausea scores between the tactile and the non-tactile placebo group, 28 subjects per group would be needed to give 80% power to detect a significant difference (with a type 1 error of 5%) (calculated by G∗Power Version 3.1.7). We increased the sample size to 30 per group to compensate for possible attrition rates. Statistical analyses were performed with SPSS statistics software (version 24, IBM).

Prior to the analyses, nausea and dizziness ratings were averaged for the evaluation period, which comprised minutes 11 to 20 of visual nausea induction. A period without placebo-TENS intervention was chosen in order to avoid distraction effects by tactile stimulation. Expected nausea as well as nausea, dizziness, and the SSMS sum score were subjected to separate 2 × 3 × 2 mixed-design analyses of variance (ANOVA), with day (day 1: baseline, day 2: intervention) as the withinsubject factor and group (placebo TENS with tactile stimulation, placebo TENS without tactile stimulation, no treatment) and sex (male, female) as between-subject factors. Significant effects were followed-up by Bonferroni-corrected post hoc tests. For all statistical tests, a p-value of ≤0.05 (two-tailed) was considered statistically significant.

# RESULTS

#### Sample

The flow of study participants is shown in **Figure 1**. In total, 494 volunteers were assessed for eligibility and 245 were invited to participate in the screening session to assess their susceptibility for the visual vection stimulus. 104 volunteers were included in the study, four of whom were excluded before randomization on Day 2. Hundred participants were randomized and completed the experiment. Analyses were based on the data from 90 participants assigned to placebo treatment or no treatment [45 males, 45 females; age (M ± SD), 23.5 ± 3.2 years]. The experimental groups were comparable with regard to sociodemographic, physical and psychological characteristics (**Table 2**).

#### Nausea Expectation

The 2 × 3 × 2 mixed-design ANOVA indicated differential changes of expected nausea from day 1 to day 2 in the experimental groups (interaction day × group, F = 6.60, p = 0.003, partial η <sup>2</sup> = 0.20) without a difference between male and female participants (interaction day × group × sex, F = 1.8, p = 0.564, partial η <sup>2</sup> = 0.02). Post hoc tests indicated significantly lower levels of expected nausea in both placebo groups in comparison to the no treatment group (Bonferroni-corrected p-values 0.002 and 0.021 vs. no treatment for the tactile and the non-tactile placebo groups, respectively). The decrease in expected nausea did not differ between the tactile and the non-tactile placebo groups (p = 1.0) (**Table 3**).

#### Nausea and Dizziness

The 2 × 3 × 2 mixed-design ANOVA for baseline-adjusted nausea scores indicated a significant group-by-day interaction

#### TABLE 2 | Sample characteristics at baseline.


MSSQ, Motion Sickness Susceptibility Questionnaire; HADS, Hospital Anxiety and Depression Scale; STAI, State-Trait Anxiety Inventory; PSQ, Perceived Stress Questionnaire. <sup>∗</sup>Results of χ 2 tests, Kruskal–Wallis tests, or univariate ANOVA, as appropriate.

(F = 22.2, p < 0.001, partial η <sup>2</sup> = 0.35) with no evident sex difference (interaction day × group × sex, F = 1.0, p = 0.390, partial η <sup>2</sup> = 0.02). The decrease in baseline-adjusted nausea scores from day 1 to day 2 was significantly larger in the placebo groups as compared to the no treatment group, confirming the occurrence of a placebo effect in nausea (both Bonferronicorrected p's < 0.001). The placebo effect did not differ between the two placebo groups (p = 1.0) (**Table 3**; **Figure 2**).

The 2 × 3 × 2 mixed-model ANOVA for baseline-adjusted dizziness scores likewise revealed a significant group-by-day interaction (F = 14.2, p < 0.001, partial η <sup>2</sup> = 0.25), without a difference between male and female participants (interaction day × group × sex, F = 1.7, p = 0.190, partial η <sup>2</sup> = 0.04). Significantly larger decreases of baseline-adjusted dizziness scores were observed in both placebo groups in comparison to the no treatment group (both Bonferroni-corrected p's < 0.001), while no differences between the two placebo groups occurred (p = 1.0) (**Table 3**).

#### Subjective Symptoms of Motion Sickness

The 2 × 3 × 2 mixed-design ANOVA for sum scores in the baseline-adjusted SSMS scores revealed a significant

TABLE 3 | Expected and perceived symptoms (day 1, day 2, and changes) in each experimental group.


NRS, numeric rating scale (0–10), average of 11 individual NRS ratings assessed during the target period on the control and intervention days; SSMS, Subjective Symptoms of Motion Sickness Questionnaire. <sup>∗</sup>p < 0.05 (vs. no treatment, Bonferroni-corrected). ∗∗p < 0.01 (vs. no treatment, Bonferroni-corrected). ∗∗∗p < 0.001 (vs. no treatment, Bonferroni-corrected).

FIGURE 2 | Individual changes in baseline-adjusted nausea score from day 1 (control) to day 2 (intervention) in the control group, the non-tactile placebo group, and the tactile placebo group. Nausea was assessed on 11-point numeric rating scales.

group-by-day interaction (F = 7.9, p = 0.001, partial η <sup>2</sup> = 0.16), comparable in size for male and female participants (interaction day × group × sex, F = 1.1, p = 0.333, partial η <sup>2</sup> = 0.03). Bonferroni-corrected post hoc t-tests indicated significantly larger decreases of baseline-adjusted SSMS scores in both placebo groups in comparison to the untreated control group (p = 0.012 and 0.014 vs. untreated controls for the tactile and non-tactile placebo groups, respectively). Again, changes in baseline-adjusted SSMS scores did not differ between the two placebo groups (p = 1.0) (**Table 3**).

# Perceived Treatment Effectiveness and Treatment Guesses in the Placebo Groups

Twenty-three out of 29 participants in the tactile placebo group (79%) believed that they had received the active intervention as compared to 14 out of 30 participants (47%) in the nontactile placebo group, the difference was significant (χ <sup>2</sup> = 6.72, p = 0.015). Participants in the two placebo groups were equally sure about their treatment guesses (non-tactile placebo, 5.3 ± 2.6

(mean ± SD); tactile placebo: 5.9 ± 2.4; Z = −1.12, p = 0.363). Explorative analyses revealed no relationship between changes in baseline-adjusted nausea scores from day 1 to day 2 and treatment guess (guess "placebo," −3.0 ± 1.9; guess "verum," −3.5 ± 1.5; F = 1.0, p = 0.313).

At the end of the experiment, participants in the tactile placebo group rated the treatment as significantly more effective than did participants in the non-tactile placebo group (mean ± SD, 6.9 ± 2.2 vs. 5.7 ± 2.5; Z = −1.99, p = 0.046). At the same time, larger reductions in baseline-adjusted nausea scores from day 1 to day 2 correlated with higher effectiveness ratings in both placebo groups (non-tactile placebo, r<sup>s</sup> = −0.446, p = 0.013; tactile placebo, r<sup>s</sup> = −0.402, p = 0.027). An exploratory regression analysis on perceived treatment effectiveness with the factors "type of placebo" and "reduction in baseline-adjusted nausea" included as independent variables revealed that both factors contributed significantly to subjective effectiveness (F = 7.2, p = 0.001; type of placebo, β = 0.257, p = 0.034; reduction in baseline-adjusted nausea, β = −0.377, p = 0.002). These results suggest that ratings of perceived effectiveness are driven by both, perceived improvement and sensory characteristics of the placebo intervention.

## Bang's Blinding Index in the Two Placebo Groups

Bang's BI in the non-tactile placebo group was 0 (95% CI, −0.35 to 0.35), indicating random guessing (**Table 4**). In the tactile placebo group Bang's BI was −0.52 (95% CI, −0.81 to −0.22), indicating non-random guessing in the direction of an "opposite guess," that is, the probability to guess "active treatment" was significantly higher than would be expected by chance.

#### DISCUSSION

In this randomized controlled placebo study, we aimed to vary the credibility of two placebo interventions by combining, or not combining it with tactile stimulation elicited by a TENS device. We hypothesized that 20 min of tactile stimulation would increase positive outcome expectations and thus the placebo effect. Results confirmed a large effect of the tactile stimulation by TENS on the credibility of the placebo treatment: Significantly more participants in the tactile placebo group believed that they


<sup>∗</sup>Two participants in each placebo group indicated that they were "not sure at all" (NRS = "0") about their treatment guess and were re-assigned to category "don't know."

had received the active intervention as compared to the nontactile placebo group. In addition, the tactile placebo intervention was perceived as more effective. Neither expectations nor the placebo effect, however, differed between the two placebo groups.

The placebo effect as the difference between the placebo groups and the no-treatment control group was consistent for different outcome parameters and effect sizes were generally large (partial η 2 , 0.16–0.35; Richardson, 2011). Results thus confirm the findings of our pilot study that placebo TENS induces a large placebo effect in experimentally induced nausea (Müller et al., 2016) and further extend them to male volunteers and to a placebo TENS intervention without tactile stimulation. The medical environment, in which the experiment took place – with many factors present that are known to boost placebo effects, such as a room full of sophisticated electrical equipment as well as prolonged interaction with the experimenters (Burke et al., 2019) – may have contributed to this large placebo effect.

As hypothesized, somatosensory stimulation during the placebo intervention increased blinding effectiveness: Bang's BI indicated random guessing in the non-tactile placebo group but non-random guessing in the direction of an opposite guess in the tactile placebo group. Our results thus lend support to the view that somatosensory stimulation during acupuncture point stimulation challenges the goal of patient blinding by enhancing the chance for a non-random guess. Given that most placebo acupuncture procedures are associated with random-guesses (Zhang et al., 2015), this discrepancy could result in a problematic blinding scenario with enhanced expectations and placebo effects in the true acupuncture groups (Bang et al., 2010; Chae et al., 2018). Contrary to our expectations, however, tactile stimulation by the TENS device during the placebo intervention did neither enhance outcome expectations nor the placebo effect during the evaluation period. Possibly, participants with opposite treatment guesses after the first placebo application may develop higher treatment expectations only with respect to subsequent placebo interventions. In a recent RCT in depression, for example, perceived treatment assignment affected symptom improvement only in the second half of the trial (Laferton et al., 2018). Furthermore, a large RCT in patients with chronic arm pain found no evidence that sham acupuncture was associated with an enhanced placebo effect during the 2 week placebo run-in period; however, sham acupuncture was significantly more effective than placebo pills during the further 6 weeks of the trial (Kaptchuk et al., 2006). Future studies are warranted to disentangle the putative interaction between expectation, perceived treatment assignment, and the placebo effect during the course of a trial.

Several possible limitations have to be considered. The medical setting of our experiment may have resulted in a ceiling effect, thereby preventing further enhancement of the placebo effect by tactile stimulation. Most acupuncture trials, however, are performed in comparable medical settings, emphasizing the external validity of our results. Furthermore, the gentle touch when placing the electrodes of the TENS device at the participants' skin could have initiated physiological responses by activating unmyelinated C tactile fibers in the body, resulting in feelings of calm and well-being as well as lower heart rate and blood pressure (Campbell, 2006; Kang et al., 2011;

Chae et al., 2018). Such physiological effects may have contributed to the improvement in the placebo groups independently from expectation. However, also the participants in the untreated control group received a variety of skin electrodes to measure the EEG, the EKG and the electrogastrogram and were provided with an indwelling catheter for repeated blood drawings during the experiment. Therefore, the gentle touch when placing the TENS electrodes was not unique to the placebo groups and the only difference between placebo and no treatment groups was the therapeutic meaning of placing the TENS electrodes. Finally, placing the TENS electrodes in the non-tactile placebo group also involved some amount of tactile stimulation and may thereby have enhanced the placebo effect. Compared with 20 min of somatosensory stimulation stimulation in the tactile TENS placebo group, however, somatosensory stimulation in the non-tactile TENS placebo group was considered to be only minor. The differential pattern of treatment guesses in the two placebo groups further supports the conceptual difference between the two placebo interventions.

#### CONCLUSION

Electrical stimulation during a placebo TENS intervention did not enhance the placebo effect in nausea but increased the credibility of the treatment. Further experimental trials are needed to investigate the putative interaction between perceived treatment assignment, expectation, and the placebo effect during the course of a trial.

# DATA AVAILABILITY STATEMENT

The datasets generated for this study are available on request to the corresponding author.

#### REFERENCES


### ETHICS STATEMENT

All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Ethics Committee of the Medical Faculty (LMU Munich) and was registered at the German Clinical Trials Register (no. DRKS00015192).

#### AUTHOR CONTRIBUTIONS

KM and MT designed the study. SA, AH, and VH conducted the experiment. KM, SA, and EO analyzed and interpreted the data. KM and SA drafted the manuscript. All authors revised the manuscript for critical intellectual content and approved the final version.

## FUNDING

The study was supported by a research grant (ME-3675/1-1) from the German Research Foundation [Deutsche Forschungsgemeinschaft, DFG (FOR 1328)]. KM received additional financial support from the Theophrastus Foundation (Germany), and the Schweizer-Arau-Foundation (Germany). The funders had no role in study design, data collection or analysis, decision to publish, or in writing this manuscript.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnins. 2019.01212/full#supplementary-material



randomized controlled pilot study. J. Psychosom. Res. 91, 9–11. doi: 10.1016/j. jpsychores.2016.09.012


**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Aichner, Haile, Hoffmann, Olliges, Tschöp and Meissner. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Electroacupuncture Pretreatment Alleviates Cerebral Ischemic Injury Through α7 Nicotinic Acetylcholine Receptor-Mediated Phenotypic Conversion of Microglia

Zhi Ma<sup>1</sup>† , Zengli Zhang<sup>2</sup>† , Fuhai Bai<sup>2</sup> , Tao Jiang<sup>1</sup> , Chaoying Yan<sup>1</sup> and Qiang Wang<sup>1</sup> \*

<sup>1</sup> Department of Anesthesiology, Center for Brain Science, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China, <sup>2</sup> Department of Anesthesiology, Xijing Hospital, Fourth Military Medical University, Xi'an, China

#### Edited by:

Younbyoung Chae, Kyung Hee University, South Korea

#### Reviewed by:

Nadine Ahmed Kerr, University of Miami, United States Marta Olah, Columbia University Irving Medical Center, United States Donald Stein, Emory University, United States

\*Correspondence: Qiang Wang dr.wangqiang@139.com †These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Cellular Neuropathology, a section of the journal Frontiers in Cellular Neuroscience

Received: 14 March 2019 Accepted: 19 November 2019 Published: 06 December 2019

#### Citation:

Ma Z, Zhang Z, Bai F, Jiang T, Yan C and Wang Q (2019) Electroacupuncture Pretreatment Alleviates Cerebral Ischemic Injury Through α7 Nicotinic Acetylcholine Receptor-Mediated Phenotypic Conversion of Microglia. Front. Cell. Neurosci. 13:537. doi: 10.3389/fncel.2019.00537 Electroacupuncture (EA) pretreatment alleviates cerebral ischemic injury through α7 nicotinic acetylcholine receptor (α7nAChR). We attempted to investigate whether the phenotypic conversion of microglia was involved in the therapeutic effect of EA pretreatment in cerebral ischemia through α7nAChR. Adult male Sprague–Dawley (SD) rats were subjected to middle cerebral artery occlusion (MCAO) after EA or α7nAChR agonist N-(3R)-1-azabicyclo[2.2.2]oct-3-yl-furo[2,3 c]pyridine-5-carboxamide hydrochloride (PHA-543,613 hydrochloride) and antagonist α-bungarotoxin (α-BGT) pretreatment. Primary microglia were subjected to drug pretreatment and oxygen-glucose deprivation (OGD). The expressions of the classical activated phenotype (M1) microglia markers induced nitric oxide synthase (iNOS), interleukin-1β (IL-1β), and cluster of differentiation 86 (CD86); the alternative activated phenotype (M2) microglia markers arginase-1 (Arg-1), transforming growth factorβ1 (TGF-β1), and cluster of differentiation 206 (CD206); and the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and anti-inflammatory cytokines interleukin-4 (IL-4) and interleukin-10 (IL-10) in the ischemic penumbra or in the supernatant of primary microglia were analyzed. The infarction volume and neurological scores were assessed 72 h after reperfusion. The cell viability and lactate dehydrogenase (LDH) release of neurons co-cultured with microglia were analyzed using cell counting kit-8 (CCK-8) and LDH release assays. EA pretreatment decreased the expressions of M1 markers (iNOS, IL-1β, and CD86) and pro-inflammatory cytokines (TNF-α and IL-6), whereas it increased the expressions of M2 markers (Arg-1, TGF-β1, and CD206) and anti-inflammatory cytokines (IL-4 and IL-10) by activating α7nAChR. EA pretreatment also significantly reduced the infarction volume and improved the neurological deficit. The activation of α7nAChR in microglia relieved the inflammatory response of primary microglia subjected to OGD and attenuated the injury of neurons co-cultured with microglia. In conclusion, EA pretreatment alleviates cerebral ischemic injury through α7nAChR-mediated phenotypic conversion of microglia, which may be a new mechanism for the EA pretreatment-induced neuroprotection against cerebral ischemia.

Keywords: stroke, electroacupuncture, α7 nicotinic acetylcholine receptor, microglia, inflammation

# INTRODUCTION

fncel-13-00537 December 5, 2019 Time: 15:38 # 2

Stroke is one of the leading causes of death worldwide (Mozaffarian et al., 2016). High mortality and disability after stroke significantly increase the financial burden of the society and threaten public health. In response to ischemic stroke, microglia in the ischemic penumbra are excessively activated and show two different phenotypes in the early and late stages after ischemic stroke. The classical activated phenotype (M1) microglia are immediately activated after ischemic stroke and release robust inflammatory cytokines, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and induced nitric oxide synthase (iNOS), which contribute to neuronal apoptosis and aggravate brain injury (Denes et al., 2007; Hu et al., 2012; DeGracia, 2013; Garden, 2013; Wang et al., 2013). At later time points after ischemic stroke, M1 phenotype microglia converted to the alternative activated phenotype (M2) microglia, which release anti-inflammatory cytokines and neurotrophic substances and contribute to repair of injury and neuroprotection (Kawabori and Yenari, 2015; Zhai et al., 2017). It may be a potential therapeutic target for the treatment of ischemic stroke to facilitate the phenotypic conversion of microglia from M1 to M2.

Electroacupuncture (EA), as a combination of traditional acupuncture and electrotherapy, not only inherits the benefits of traditional acupuncture but also integrates with the physiological effects of electric stimulation (Napadow et al., 2005). Previous studies have reported that EA effectively ameliorates TNFα and interleukin-6 (IL-6) expressions in activated microglia (Liu et al., 2016; Tang et al., 2016) and increased antiinflammatory cytokines, such as interleukin-4 (IL-4) and interleukin-10 (IL-10) (Jiang et al., 2017). The α7 nicotinic acetylcholine receptor (α7nAChR) is highly expressed in the central nervous system (CNS) and plays an important role in cholinergic anti-inflammatory pathway. Studies have showed that activating α7nAChR in microglia and macrophage inhibits the release of NO and TNF-α (Lutz et al., 2014; Baez-Pagan et al., 2015), suggesting that α7nAChR might be a target to regulate microglial phenotypes. Our previous study found that activating α7nAChR reduced cerebral ischemic injury (Wang et al., 2012). Therefore, we investigated the roles of microglial α7nAChR in the phenotypic conversion of microglia and the neuroprotective effects of EA pretreatment against ischemic stroke.

# MATERIALS AND METHODS

#### Animals

Male Sprague–Dawley (SD) rats (weighing 280–300 g) were obtained from the Experimental Animal Center of the Xi'an Jiaotong University (Xi'an, Shaanxi Province, China) and were maintained under a 12-h light–dark cycle, a temperature of 21 ± 2 ◦C, and humidity of 60–70% for at least 1 week before any drug treatment or surgery was conducted. All animal experiments were approved by the Ethics Committee for Animal Experimental Center of the Xi'an Jiaotong University and were conducted according to the Chinese National Institutes of Health Guide for the Care and Use of Laboratory Animals<sup>1</sup> .

# Culture of Cells and Co-culture of Neuron–Microglia

Culture of cells and co-culture of neuron–microglia were performed as previously described (Zhang Z. et al., 2018). Briefly, mixed primary glial cultures were isolated from the cerebral cortex of 1-day-old SD pups. After the meninges and hippocampus were removed, the cortical tissues were subjected to enzymatic digestion and mechanical isolation. Then, the mixed cells were passed through a 70-µm nylon mesh cell strainer and were seeded into the poly-L-lysine-coated cell culture flask in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin). Cells were maintained in a humidified incubator at 37◦C enriched with 5% CO<sup>2</sup> and fed every 3– 5 days with fresh medium. After 14 days, microglia were harvested from the mixed glial cultures by mild shaking at 220 r/min for 1 h and were seeded into cell culture containers. The purity of the microglia was confirmed by Iba1 staining (**Supplementary Figure S1**).

Primary neurons were isolated from the cerebral cortices of E17–E19 rat embryos and were seeded into the poly-L-lysine-coated cell culture plates. Then, cells were cultured in Neurobasal medium (Gibco, Invitrogen Corp., Carlsbad, CA, United States) containing 2% B27, 1% glutamine, and 1% penicillin/streptomycin (Sigma–Aldrich, St. Louis, MO, United States). Cells were kept in a humidified incubator at 37◦C enriched with 5% CO2.

For indirect co-culture of neuron–microglia, the primary microglia (microglia:neurons = 1:2) were added to 0.4-µm pore sized Transwell inserts (Costar, United States) 2 days after the neurons were seeded into the 24-well plates. After the primary microglia and neurons were co-cultured for 2 days, the microglia and neurons were separated to receive drugs or oxygen-glucose deprivation (OGD) treatment. Then, the neurons and microglia were co-cultured again for 24 h.

#### Groups and Drug Treatment

In vivo, first, to observe the neuroprotection of EA pretreatment and whether EA pretreatment induced the phenotypic conversion of microglia from M1 to M2 after ischemia– reperfusion injury, rats were randomly divided into three groups: sham, middle cerebral artery occlusion (MCAO), and EA + MCAO. The rats in the EA + MCAO group received EA for five consecutive days and were then subjected to MCAO for 90 min 1 day after the last EA pretreatment. The infarction volume and neurological scores were assessed 72 h after reperfusion. The expressions of α7nAChR; M1 microglia markers iNOS, IL-1β, and cluster of differentiation 86 (CD86); and M2 microglia markers arginase-1 (Arg-1), transforming growth factor-β1 (TGF-β1), and cluster of differentiation 206 (CD206) in the ischemic penumbra

<sup>1</sup>https://grants.nih.gov/grants/olaw/Guide-for-the-Care-and-use-of-laboratoryanimals.pdf

were analyzed. The pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 in the ischemic penumbra were analyzed. Second, to determine whether α7nAChR was associated with the phenotypic conversion of microglia and the neuroprotection of EA pretreatment after ischemia– reperfusion injury, rats were randomly divided into the following groups: sham, MCAO, N-(3R)-1-azabicyclo[2.2.2]oct-3-ylfuro[2,3-c]pyridine-5-carboxamide (PHA-543,613) + MCAO, vehicle + MCAO (saline), EA + MCAO, and sham, MCAO, EA + MCAO, α-bungarotoxin (α-BGT) + EA + MCAO, and vehicle + EA + MCAO (saline). Rats in the PHA-543,613 + MCAO group received intraperitoneal injection of PHA-543,613 (1.0 mg/kg, Tocris Bioscience, Bristol, United Kingdom) once a day for five consecutive days (Wang et al., 2012) and were then subjected to MCAO 24 h after the last injection. Rats in the α-BGT + EA + MCAO group received intracerebroventricular injection of α-BGT (0.5 µg/kg, Tocris Bioscience, Bristol, United Kingdom) for 30 min before the onset of EA for five consecutive days (Wang et al., 2012) and were then subjected to MCAO 24 h after the last EA treatment. The expressions of M1 microglia markers (iNOS, IL-1β, and CD86), M2 microglia markers (Arg-1, TGF-β1, and CD206), pro-inflammatory cytokine TNF-α, and anti-inflammatory cytokine IL-10 in the ischemic penumbra were accordingly analyzed. The infarction volume and neurological scores were assessed 72 h after reperfusion.

In vitro, to further confirm whether the α7nAChR in microglia was associated with the conversion of microglial phenotypes, α7nAChR agonist PHA-543,613 (100 µM) and antagonist α-BGT (10 nM) were used. Primary cultured microglia were randomly divided into five groups: control, OGD, PHA-543,613 + OGD, α-BGT + PHA-543,613 + OGD, and α-BGT + OGD. The PHA-543,613 (100 µM) was added into the cultured medium of microglia 24 h before OGD treatment, and the α7nAChR antagonist α-BGT (10 nM) was added to cells 30 min prior to PHA-543,613 to block the α7nAChR (Macpherson et al., 2014; Kimura et al., 2019). The M1 microglia markers (IL-1β and CD86) and M2 microglia markers (Arg-1 and CD206) were detected by western blot analysis and immunofluorescence staining. In addition, a neuron–microglia co-culture system was established to investigate the neuroprotection of α7nAChR in microglia. After the primary microglia and neurons were co-cultured for 2 days, the microglia were subjected to PHA-543,613 or α-BGT separately, and then the primary microglia and neurons were exposed to OGD for 4 and 1 h, respectively (Zhang Z. et al., 2018). The pro-inflammatory cytokines (TNF-α and IL-6) and anti-inflammatory cytokines (IL-4 and IL-10) in the supernatants of cultured microglia were detected by enzyme-linked immunosorbent assay (ELISA). The cell viability and lactate dehydrogenase (LDH) release of neurons were detected 24 h after the reintroduction of oxygen and glucose.

All drugs were administered as previously described (Wang et al., 2012; Macpherson et al., 2014; Kimura et al., 2019). All experiments followed the principles of randomization and double blindness.

# Intracerebral-Ventricular Injection

The rats were anesthetized with isoflurane and fixed on a stereotaxic apparatus (Narishige, Tokyo, Japan). A scalp incision was made, and the bregma was exposed. The stereotaxic apparatus was located at bregma and punched in the position of 1.5 mm lateral and 1.0 mm posterior to bregma at the left hemisphere. A stainless steel 26-gauge cannula (C315G; Plastics One Inc., Roanoke, VA, United States) was slowly introduced through the hole into the left lateral ventricle (3.8 mm below the cerebral dura mater). The cannula was fixed by dental cement, and four stainless steel screws were secured to the skull and occluded. The rats were allowed to recover from the operation 3 days before the next treatment.

# Electroacupuncture Pretreatment

Electroacupuncture was conducted as previously described (Li et al., 2012). Briefly, the rats were anesthetized, fixed on the animal thermostatic operating table, and maintained at 37.0 ± 0.5◦C. The acupoint "Baihui (GV 20)," which is located at the intersection of the sagittal midline and the line linking the two ears, was stimulated using the Hwato Electronic Acupuncture Treatment Instrument (model no. SDZ-V, Suzhou Medical Appliances Co., Ltd., Suzhou, China) at an intensity of 1 mA and dense-disperse frequency of 2/15 Hz for 30 min per day for 5 days (Li et al., 2012).

# Middle Cerebral Artery Occlusion

The focal cerebral ischemia model of rats was induced by MCAO as described previously (Wang et al., 2009). Briefly, the rats were anesthetized with isoflurane. The right internal carotid artery was separated and occluded using an intraluminal filament technique. Regional cerebral blood flow (rCBF) was monitored using a transcranial Doppler ultrasound flowmeter (PeriFlux System 5000; PERIMED, Stockholm, Sweden). The body temperature of the rats was maintained at 37.0–37.5◦C. The rats were available if the rCBF showed a sharp drop to 20 and were recovered up to more than 80% of baseline (pre-ischemia) level. There were no significant differences in physiological parameters during EA pretreatment (at the onset of EA, 15 min after EA, and at the end of EA) or surgery (at the onset of ischemia, 60 min after the onset of ischemia, and 30 min after reperfusion) according to our previous study (Wang et al., 2012).

# Neurobehavioral Evaluation

The neurological score was assessed 72 h after reperfusion using an 18-point neurobehavioral scoring system, which contained spontaneous activity, limb symmetry, forepaw outstretching, climbing, body proprioception, and response to vibrissae touch by another experimenter who was blind to the experimental groups as previously described (Garcia et al., 1995). The details of the score criterion are provided in **Supplementary Table S1**.

#### Infarct Measurement

The infarct measurement was assessed by 2,3,5 triphenyltetrazolium chloride (TTC; Sigma–Aldrich, St. Louis, MO, United States) staining. The brain sections were stained for 15 min at 37◦C, followed by overnight immersion in 4% paraformaldehyde. The stained sections were photographed, and the unstained areas were measured as the infarcted volume using an image analysis software (Adobe Photoshop 8.0 CS, Adobe Systems, San Jose, CA, United States) by an experimenter who was blind to the experimental groups. Infarct volume was quantified as follows: relative infarct volume = (contralateral area − ipsilateral non-infarct area)/contralateral area.

#### Oxygen-Glucose Deprivation

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The cells were switched to a serum- and glucose-free medium and were then transferred into a humidified hypoxic chamber, which contained mixed gas (5% CO<sup>2</sup> and 95% N2) for 15 min at room temperature. The chamber was then sealed and placed in a container at 37◦C. The OGD was carried out for 4 h (microglia) or for 1 h (neurons). Following the OGD, cells were incubated with normal medium for an additional 24 h after the reintroduction of oxygen and glucose.

### Assessment of Neuron Cell Viability

The survival of neuron cells was evaluated by cell counting kit-8 (CCK-8) (Seven Sea Biotech, China) 24 h after OGD. The procedures were strictly conducted according to the manufacturer's instructions. Briefly, after the CCK-8 solution was added to each well of the 24-well plates, neurons were incubated for 4 h at 37◦C. Eventually, the absorbance was measured at 450 nm using a microplate reader (Infinite M200; TECAN, Männedorf, Switzerland).

#### Lactate Dehydrogenase Release Assay

The LDH-Cytotoxicity Colorimetric Assay Kit II (#K313-500; BioVision Inc., Milpitas, CA, United States) was used to detect cell injury 24 h after OGD. The LDH reaction mixture was mixed according to the manufacturer's instructions and added to a 96-well plate (100 µl per well). Then, 10 µl of cell-free supernatant of the samples was incubated with this reaction mixture for 30 min at 37◦C. Finally, the absorbance was measured at 450 nm using a microplate reader (Infinite M200; TECAN, Männedorf, Switzerland).

# Analysis of Inflammatory Factors

Enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN, United States) was used to measure the levels of TNF-α and IL-10 in the ischemic penumbra, and also the levels of TNF-α, IL-4, IL-6, and IL-10 in the supernatants of cultured microglia according to the manufacturer's instructions.

#### Immunofluorescence Staining

Immunofluorescence staining was performed on frozen coronal sections of rat brains or on primary cultured microglia plated on cover slips. The rat brains were fixed with 4% paraformaldehyde. After fixation and concentration gradient dehydration, the brains were cut into 12-µm-thick sections using a Leica CM1900 frozen slicer. The cells were fixed with 4% paraformaldehyde for 30 min. The brain sections and cell cover slips were washed three times with phosphatebuffered saline (PBS) and then incubated overnight at 4◦C in a humidified atmosphere with primary antibodies. The following primary antibodies were used: rabbit anti-iNOS antibody (1:100; Abcam, Cambridge, United Kingdom), rabbit anti-liver arginase antibody (1:100; Abcam, Cambridge, United Kingdom), rabbit anti-IL-1β antibody (1:50; Santa Cruz Biotechnology, Dallas, TX, United States), rabbit antiα7nAChR antibody (1:50; Santa Cruz Biotechnology, Dallas, TX, United States), and goat anti-Iba1 antibody (1:50; Santa Cruz Biotechnology, Dallas, TX, United States) were used. The Alexa Fluor 488-labeled donkey anti-goat secondary antibody (1:200; Santa Cruz Biotechnology, Dallas, TX, United States), Alexa Fluor 594-labeled goat anti-rabbit secondary antibody (1:200; Santa Cruz Biotechnology, Dallas, TX, United States), and Alexa Fluor 488-labeled goat antirabbit secondary antibody (1:200; Santa Cruz Biotechnology, Dallas, TX, United States) were incubated for 2 h at room temperature in the dark. 4<sup>0</sup> ,6-Diamidino-2-phenylindole (DAPI) (ZLI-9557, Zsbio) was used to stain nuclei. The sections were mounted with 50% glycerol. Finally, the sections were viewed and photographed using an Olympus BX51 (Japan) fluorescence microscope.

## Western Blot Analysis

To determine the expressions of α7nAChR, iNOS, IL-1β, CD86, Arg-1, TGF-β1, and CD206 in the ischemic penumbra of rats or in the microglia, proteins were extracted and western blot analysis was performed. Briefly, the extracted proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electrically transferred to polyvinylidene difluoride (PVDF) membranes. Then, the membranes were blocked in TBST containing 5% non-fat dry milk for 1 h at room temperature and were successively incubated with the dilute primary and secondary antibodies. The following primary antibodies were used: anti-α7nAChR rabbit antibody (1:50; Santa Cruz Biotechnology, Dallas, TX, United States), anti-iNOS rabbit antibody (1:500; Abcam, Cambridge, United Kingdom), anti-IL-1β rabbit antibody (1:200; Santa Cruz Biotechnology, Dallas, TX, United States), anti-CD86 rabbit antibody (1:500; Proteintech, United States), antiliver arginase rabbit antibody (1:1,000; Abcam, Cambridge, United Kingdom), an anti-TGF-β1 rabbit antibody (1:1,000; GeneTex, Irvine, CA, United States), anti-CD206 rabbit antibody (1:500; Proteintech, United States), and an anti-tubulin rabbit antibody (1:1,000; Beijing ComWin Biotech Co., Ltd, Beijing, China). In addition, horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:10,000; Beijing ComWin Biotech Co., Ltd, Beijing, China) were used. Protein bands were visualized using the LI-COR Odyssey System (LI-COR Biotechnology, Lincoln, NE, United States). The relative changes in protein expression were expressed as the ratio of the integrated optical density (OD) of the target protein band to that of β-tubulin.

#### Statistical Analysis

GraphPad Prism 7.0 software was used for performing statistical analysis. Data were collected by two independent and blind investigators. Comparisons among multiple groups were

undertaken using one-way ANOVA followed by Tukey's post hoc test. Neurological scores were presented as medians and were analyzed using two-tailed Mann–Whitney U-tests, and the other values were presented as the mean ± SD. p-Value < 0.05 was considered statistically significant.

# RESULTS

## Electroacupuncture Pretreatment Ameliorated Cerebral Ischemia Injury and Upregulated Microglial α7 Nicotinic Acetylcholine Receptor Expression in Ischemic Penumbra After Stroke

The influence of EA pretreatment on cerebral ischemia injury and α7nAChR expression in the ischemic penumbra was evaluated 72 h after reperfusion (**Figure 1B**). As shown in **Figure 1**, EA pretreatment, compared with the MCAO group, significantly ameliorated the infarction volume (**Figures 1A,C**, ∗∗p < 0.01) and increased the neurological scores after stroke, which mean that the neurological deficits had been improved (**Figure 1D**, ∗∗p < 0.01). Moreover, the protein expression of α7nAChR in the ischemic penumbra was significantly increased in the EA group compared with the MCAO group (**Figure 1E**, <sup>∗</sup>p < 0.05). In addition, we detected the expression of α7nAChR in microglia by immunofluorescent double labeling of α7nAChR and Iba1 (microglial marker). It showed that EA pretreatment, compared with the MCAO group, upregulated microglial α7nAChR expression in the ischemic penumbra (**Figure 1F**). These results indicated that EA pretreatment exerted neuroprotective effects and reversed the effects of MCAO on the expression of α7nAChR in the ischemic penumbra of rats subjected to ischemia injury.

### Electroacupuncture Pretreatment Induced the Phenotypic Conversion of Microglia From M1 to M2 and Relieved Inflammatory Response in the Ischemic Penumbra After Stroke

The time point of 72 h after ischemia–reperfusion was the key time point for microglial transformation from M1 to M2 (Zhai et al., 2017); thus, this specific time point was selected for subsequent experiments. At 72 h after ischemia– reperfusion, the expression of M1 microglia markers iNOS and IL-1β in the ischemic penumbra were significantly decreased in the EA + MCAO group compared with the MCAO group (**Figures 2A,B**, ∗∗p < 0.01), whereas the expressions of M2 microglia markers Arg-1 and TGF-β1 were remarkably increased (**Figures 2C,D**, ∗∗p < 0.01), which indicated that EA pretreatment induced the phenotypic conversion of microglia from M1 to M2. The pro-inflammatory cytokine TNF-α was significantly decreased and anti-inflammatory cytokine IL-10 was notably increased after EA pretreatment in the ischemic penumbra as detected by ELISA (**Figures 2E,F**, <sup>∗</sup>p < 0.05, ∗∗∗p < 0.001).

# The α7 Nicotinic Acetylcholine Receptor Was Involved in the Conversion of the Microglial Phenotype After Oxygen-Glucose Deprivation in vitro

We used primary microglia to further investigate whether the microglial phenotypic conversion was caused by the activation of α7nAChR in microglia. The purity of cultured primary microglia was confirmed by Iba1 staining (**Supplementary Figure S1**). The α7nAChR was expressed in the microglia (**Figure 3A**). Compared with the OGD group without drug treatment, pretreating microglia with 100 µM of α7nAChR agonist PHA-543,613 24 h before OGD treatment induced the phenotypic conversion of microglia from M1 to M2 as assessed by the expression of Arg-1 in microglia exposed to OGD injury (**Figure 3B**, ∗∗∗p < 0.001); thus, the concentration of PHA-543,613 was chosen for the following experiments. PHA-543,613 pretreatment significantly attenuated M1 microglia markers IL-1β and CD86 protein expressions (**Figures 3C,D** and **Supplementary Figure S2C**, ∗∗p < 0.01) and increased M2 microglia markers Arg-1 and CD206 expressions (**Figures 3E,F** and **Supplementary Figure S2C**, ∗∗∗p < 0.01) in the microglia exposed to OGD. When the function of α7nAChR was blocked by adding its antagonist α-BGT to cells 30 min prior to PHA-543,613, PHA-543,613 pretreatment lost its influence on the expression of M1 and M2 microglia markers (**Figures 3C– F** and **Supplementary Figure S2C**, PHA-543,613 + OGD vs. α-BGT + PHA-543,613 + OGD). No significant differences were found between the OGD group and the α-BGT + PHA-543,613 + OGD group. These results suggested that microglial α7nAChR contributed to the phenotypic conversion of microglia.

### Activating Microglial α7 Nicotinic Acetylcholine Receptor Relieved Inflammatory Response and Induced Neuroprotection Against Oxygen-Glucose Deprivation Injury in vitro

The levels of pro-inflammatory cytokines TNF-α and IL-6 and the levels of anti-inflammatory cytokines IL-10 and IL-4 in the supernatants of primary microglia were detected 24 h after the reintroduction of oxygen and glucose. The levels of TNF-α and IL-6 were significantly decreased whereas the levels of IL-4 and IL-10 were remarkably increased in the PHA-543,613 + OGD group when compared with the OGD group (**Figures 4A,B**, <sup>∗</sup>p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). After the function of α7nAChR was blocked by its antagonist α-BGT, the PHA-543,613 pretreatment lost its influence on the expressions of TNF-α, IL-6, IL-4, and IL-10 (**Figures 4A,B**; PHA-543,613 + OGD vs. α-BGT + PHA-543,613 + OGD). To further observe the neuroprotection of microglial α7nAChR, neurons and microglia were co-cultured. During the drug treatments and OGD, neurons and microglia were fully separated to avoid activating α7nAChR in neurons owing to the leakage of drugs through the 0.4-µm pore-sized membrane of the Transwell inserts. According to the CCK-8 cell viability assay, activation of α7nAChR in microglia

(D) Neurological deficit scores were evaluated 72 h after reperfusion. The data were expressed as the median and were analyzed by the Mann–Whitney U-test. n = 8. ∗∗p < 0.01 compared with the MCAO group. (E) Western blot analysis of the expression level of α7nAChR protein in the ischemic penumbra 72 h after reperfusion. The data were expressed as the mean ± SD and were analyzed by one-way ANOVA with Tukey's post hoc test. n = 5. <sup>∗</sup>p < 0.05 compared with the MCAO group. (F) Representative immunofluorescence images showing the expression of α7nAChR in microglia in the ischemic penumbra after stroke. Microglial cells were labeled by Iba1 (microglia marker, green). n = 5. Scale bars = 20 µm.

significantly improved the viability of neurons compared with that in the OGD group (**Figure 4C**, ∗∗p < 0.01). According to the LDH release assay, the levels of LDH release in the PHA-543,613 + OGD group were markedly lower than those in the OGD group (**Figure 4D**, ∗∗∗p < 0.001). After the function of α7nAChR was blocked by its antagonist α-BGT, the PHA-543,613 pretreatment lost its influence on the viability and injury of neurons exposed to OGD (**Figures 4C,D**; PHA-543,613 + OGD vs. α-BGT + PHA-543,613 + OGD). No significant differences were observed between the OGD group and the α-BGT + PHA-543,613 + OGD group. These results indicated that activation of α7nAChR relieved inflammatory response and protected neurons from OGD injury.

### Electroacupuncture Pretreatment Induced the Phenotypic Conversion of Microglia From M1 to M2 via α7 Nicotinic Acetylcholine Receptor After Stroke

The α7nAChR agonist PHA-543,613 and antagonist α-BGT were used to detect whether α7nAChR was associated with the phenotypic conversion of microglia in vivo. As illustrated

in **Figure 5**, 72 h after ischemia–reperfusion, the expressions of M1 microglia markers iNOS, IL-1β, and CD86 in the ischemic penumbra were significantly reduced in the PHA-543,613 + MCAO group compared with the MCAO group (**Figure 5A** and **Supplementary Figure S2A**, <sup>∗</sup>p < 0.05, ∗∗p < 0.01), whereas the expressions of M2 microglia markers Arg-1, TGF-β1, and CD206 were notably increased (**Figure 5B** and **Supplementary Figure S2A**, <sup>∗</sup>p < 0.05, ∗∗∗p < 0.001). The EA and PHA-543,613 pretreatment had the same influence on the expression of iNOS, IL-1β, CD86, Arg-1, TGF-β1, and CD206 (**Figures 5A,B** and **Supplementary Figure S2A**). The expressions of M1 microglia markers iNOS, IL-1β, and CD86 in the ischemic penumbra were markedly increased in the α-BGT + EA + MCAO group compared with the EA + MCAO group (**Figure 5C** and **Supplementary Figure S2B**, #p < 0.05, ##p < 0.01, ###p < 0.001), whereas the expression of M2 microglia markers Arg-1, TGF-β1, and CD206 were significantly decreased (**Figure 5D** and **Supplementary Figure S2B**, ##p < 0.01, ###p < 0.001). The immunofluorescence assay revealed that the percentage of iNOS+/Iba1<sup>+</sup> microglia in the ischemic penumbra of the EA + MCAO group was less than that in the MCAO group, and the percentage of Arg-1+/Iba1<sup>+</sup> microglia in the ischemic penumbra of the EA + MCAO group was more than that in the MCAO group (**Figures 5E,F**, ∗∗∗p < 0.001). After the function of α7nAChR was blocked by its antagonist α-BGT, the EA pretreatment lost its influence on the percentage of iNOS+/Iba1<sup>+</sup> microglia and Arg-1+/Iba1<sup>+</sup> microglia in the ischemic penumbra (**Figures 3E,F**, α-BGT + EA + MCAO vs. EA + MCAO). No significant differences were observed between the MCAO group and the α-BGT + EA + MCAO group. These findings suggested that EA pretreatment induced the phenotypic conversion of microglia from M1 to M2 via α7nAChR.

#### Electroacupuncture Pretreatment Relieved Inflammatory Response and Alleviated Cerebral Ischemia Injury via α7 Nicotinic Acetylcholine Receptor

The pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 in the ischemic penumbra 72 h after ischemia– reperfusion were measured by ELISA. The level of proinflammatory TNF-α was significantly decreased whereas the level of anti-inflammatory IL-10 was remarkably increased in the PHA-543,613 + MCAO group compared with the MCAO group (**Figures 6A,B**, ∗∗p < 0.01, ∗∗∗p < 0.001). The level of pro-inflammatory TNF-α was notably increased whereas the level of anti-inflammatory IL-10 was significantly decreased in the α-BGT + EA + MCAO group compared with the

Tukey's post hoc test. The data were pooled from five independent experiments. ∗∗∗p < 0.001 compared with the OGD group without drug treatment. (C) Immunofluorescence staining of M1 microglia marker IL-1β in primary microglia 24 h after the reintroduction of oxygen and glucose. The α7nAChR agonist PHA-543,613 (100 µM) was administrated 24 h before OGD. Scale bar = 5 µm. (D) Western blot analysis of the protein expression of IL-1β in primary microglia 24 h after the reintroduction of oxygen and glucose. The α7nAChR agonist PHA-543,613 (100 µM) was administrated 24 h before OGD. The α7nAChR antagonist α-BGT (10 nM) was administrated 30 min prior to PHA-543,613 to block the function of α7nAChR. The data were expressed as the mean ± SD and were analyzed

(Continued)

#### FIGURE 3 | Continued

fncel-13-00537 December 5, 2019 Time: 15:38 # 9

by one-way ANOVA with Tukey's post hoc test. The data were pooled from five independent experiments. ∗∗p < 0.01 compared with the OGD group, ##p < 0.01 compared with the PHA-543,613 + OGD group. (E) Immunofluorescence staining of M2 microglia marker Arg-1 in primary microglia 24 h after the reintroduction of oxygen and glucose. Scale bar = 5 µm. (F) Western blot analysis of the protein expression of Arg-1 in primary microglia 24 h after the reintroduction of oxygen and glucose. The data were expressed as the mean ± SD and were analyzed by one-way ANOVA with Tukey's post hoc test. The data were pooled from five independent experiments. ∗∗∗p < 0.001 compared with the OGD group, ##p < 0.01 compared with the PHA-543,613 + OGD group.

FIGURE 4 | Activating α7 nicotinic acetylcholine receptor (α7nAChR) in microglia relieved inflammatory response and induced neuroprotection against oxygen-glucose deprivation (OGD) injury in vitro. (A) The levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the supernatants of primary microglia were detected by ELISA 24 h after the reintroduction of oxygen and glucose. The data were expressed as the mean ± SD and were analyzed by one-way ANOVA with Tukey's post hoc test. The data were pooled from five independent experiments. <sup>∗</sup>p < 0.05, ∗∗p < 0.01 compared with the OGD group, #p < 0.05 compared with the PHA-543,613 + OGD group. (B) The levels of the anti-inflammatory cytokines IL-4 and IL-10 in the supernatants of primary microglia were detected by ELISA 24 h after the reintroduction of oxygen and glucose. The data were expressed as the mean ± SD and were analyzed by one-way ANOVA with Tukey's post hoc test. The data were pooled from five independent experiments. ∗∗p < 0.01, ∗∗∗p < 0.001 compared with the OGD group, ##p < 0.01, ###p < 0.001 compared with the PHA-543,613 + OGD group. (C) The cell viability of neurons co-cultured with microglia was detected by cell counting kit-8 (CCK-8) 24 h after the reintroduction of oxygen and glucose. The data were expressed as the mean ± SD and were analyzed by one-way ANOVA with Tukey's post hoc test. The data were pooled from five independent experiments. ∗∗p < 0.01 compared with the OGD group, #p < 0.05 compared with the PHA-543,613 + OGD group. (D) The injury of neurons co-cultured with microglia was detected by lactate dehydrogenase (LDH) release assay 24 h after the reintroduction of oxygen and glucose. The data were expressed as the mean ± SD and were analyzed by one-way ANOVA with Tukey's post hoc test. The data were pooled from five independent experiments. ∗∗∗p < 0.001 compared with the OGD group, #p < 0.05 compared with the PHA-543,613 + OGD group.

PHA-543,613 + MCAO group (**Figures 6A,B**, ##p < 0.01, ###p < 0.001).

The infarct volume was significantly decreased and the neurological deficit was dramatically relieved in the PHA-543,613 + MCAO group compared with the MCAO group, whereas the infarct volume was increased and the neurological deficit was aggravated in the α-BGT + EA + MCAO group compared with the PHA-543,613 + MCAO group (**Figures 6C– E**, ∗∗p < 0.01, #p < 0.05, ##p < 0.01). No significant differences were observed between the MCAO group and the α-BGT + EA + MCAO group. These results further demonstrated that EA pretreatment displayed neuroprotective effect by regulating inflammatory response via α7nAChR.

#### DISCUSSION

In the present study, we investigated the roles of microglial α7nAChR in the neuroprotection of EA pretreatment against ischemic injury by in vitro and in vivo experiments. We found that EA pretreatment decreased the expression of M1 microglia markers (iNOS, IL-1β, and CD86) and proinflammatory cytokines (TNF-α and IL-6), whereas it increased the expression of M2 microglia markers (Arg-1, TGF-β1, and CD206) and anti-inflammatory cytokines (IL-4 and IL-10) in the ischemic penumbra and microglia by activating α7nAChR. EA pretreatment also significantly reduced the infarct volume and improved the neurological deficit. Activation of α7nAChR in microglia relieved the inflammatory response of primary microglia subjected to OGD and attenuated the injury of neurons co-cultured with microglia. Thus, although failing to completely cure stroke, EA pretreatment exerted neuroprotective effects against cerebral ischemic injury by promoting α7nAChRmediated microglial phenotype conversion, suggesting a potential therapeutic target in the treatment of ischemic stroke.

Electroacupuncture confers strong neuroprotection against cerebral ischemic injury by inhibiting the inflammatory responses (Xiong et al., 2003; Wang et al., 2005, 2011;

Hong et al., 2013; Zhu et al., 2013). A previous study has found that EA pretreatment suppresses the NF-κB signaling pathway by upregulating cylindromatosis to alleviate inflammatory injury after cerebral ischemia/reperfusion (Jiang et al., 2017). In addition, EA treatment inhibits microglia-mediated neuroinflammation through inactivation of p38 MAPK and MyD88, accompanied by the decrease of IL-1β, IL-6, and TNF-α after ischemic stroke (Liu et al., 2016). A randomized clinical trial provides evidences for the effectiveness of EA pretreatment on urinary leakage among women with stress urinary incontinence (Liu et al., 2017). In the current study, we demonstrated that EA pretreatment significantly decreased the expression of M1 microglia markers iNOS, IL-1β, and CD86, whereas it increased the expression of M2 microglia markers Arg-1, TGF-β1, and CD206 in the ischemic penumbra. Microglial cells are the pivotal mediators of neuroinflammation, performing functions by adopting different activation states, M1 phenotype with cytotoxic properties, and M2 phenotype with regeneration and repair (Colonna and Butovsky, 2017). The iNOS is transiently expressed during immune activation. Overproduction of NO by iNOS acts as a cytotoxic agent in pathological processes (Mukherjee et al., 2014). IL-1β is mainly produced by M1 microglia, expanding the microglia population in an autocrine manner and amplifying the production of inflammatory cytokines (Zhang C. J. et al., 2018). Arg-1 is expressed in the immune system and participates in a variety of inflammatory diseases by downregulation of nitric oxide synthesis, induction of fibrosis, and tissue regeneration (Munder, 2009). TGF-β1 has extensive immunomodulatory and anti-inflammatory properties and is abundantly produced in the ischemic penumbra by microglia (Islam et al., 2018). EA pretreatment transformed microglial phenotype from M1 to M2; thus, the expression of M1-related proteins decreased and M2-related proteins increased. Whether the inhibition of p38 MAPK-, MyD88-, and NF-κB-mediated signaling pathways is involved in the EA pretreatment-induced expression changes of iNOS, IL-1β, Arg-1, and TGF-β1 needs further investigation.

The α7nAChR is the pivotal receptor in the cholinergic anti-inflammatory pathway and is widely expressed in the CNS (Sharma and Vijayaraghavan, 2001; Gotti and Clementi, 2004; Shytle et al., 2004; Huston et al., 2006; De Jaco et al., 2017). Pharmacological stimulation of α7nAChR and stimulation of vagus nerve exert neuroprotective effects against cerebral ischemic injury (Kalappa et al., 2013; Parada et al., 2013; Jiang et al., 2014). Moreover, the α7nAChR signaling modulates the inflammatory phenotype of microglia in fetal brain, thereby providing a therapeutic approach for neuroinflammation in utero (Cortes et al., 2017). Consistent with these studies, we found that microglial α7nAChR played a key role in the neuroprotection of EA pretreatment against ischemic injury. In our study, activating α7nAChR in microglia promoted conversion of microglial phenotypes from M1 to M2 as assessed by the expression of M1 and M2 microglia markers, the pro-inflammatory cytokines (TNF-α and IL-6), and anti-inflammatory cytokines (IL-4 and IL-10) in the ischemic penumbra or in the supernatant of primary microglia. Furthermore, by establishing the microglia-neurons co-culture system, we found that activating α7nAChR in microglia increased neuronal cell viability and attenuated neuronal injury, which indicated that microglial α7nAChR exerted neuroprotection effects against OGD injury.

Microglial phenotypic transformation plays an important role in stroke, but the specific molecular mechanisms are not fully understood. A study has shown that cannabinoid receptors drive the acquisition of M2 polarization in microglia (Mecha et al., 2015). Inhibition of MyD88 signaling attenuates neuronal death in the hippocampus after status epilepticus in mice by skewing microglia/macrophage polarization (Liu et al., 2018). Our previous study found that activation or upregulation of triggering receptor expressed on myeloid cells 2 (TREM2) promoted the phenotypic conversion of microglia and thus decreased the number of apoptotic neurons in stroke mice (Zhai et al., 2017). Studies have also found that α7nAChR activation altered microglial phenotype by modulating intracellular iron load through α7nAChR-ferroportin signaling pathway, thus playing a neuroprotective role (Cortes et al., 2017). In addition, activation of the α7nAChR strongly inhibits the phosphorylation of p38 MAPK and upregulates phosphorylation of JAK2 and STAT3, thus transforming LPS-activated M1 microglia to the M2 phenotype (Zhang et al., 2017). Whether these pathways and signaling molecules are associated with the α7nAChR-mediated conversion of microglial phenotypes requires further clarification.

Our study includes some limitations. First, utilizing a rat model with a conditional knockout of the α7nAChR in microglia could better verify the effect of microglial α7nAChR. Second, as estrogen is an independent factor affecting the outcomes of stroke and the inflammatory signaling pathways, we failed to account for females. And long-term sensory or motor impairments detecting would be helpful to assess potential future therapies of EA pretreatment.

# CONCLUSION

We found that EA pretreatment alleviated cerebral ischemic injury through the α7nAChR-mediated phenotypic conversion of microglia, which might be a new mechanism for EA pretreatment-induced neuroprotection against cerebral ischemia.

# DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

# AUTHOR CONTRIBUTIONS

Study supervision and study design was performed by QW. The study was conducted by ZM and ZZ. Data collection

and analysis were performed by FB, TJ, and CY. Writing and critical revision of the manuscript were performed by ZM, ZZ, and QW.

#### FUNDING

This work was supported by the National Natural Science Foundation of China (Grant Nos. 81473488 and 81774113) and

#### REFERENCES


the Overseas, Hong Kong and Macao Scholars Collaborative Researching Fund (Grant No. 81228022).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fncel. 2019.00537/full#supplementary-material

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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Ma, Zhang, Bai, Jiang, Yan and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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