Edited by: Xinglong Wang, Case Western Reserve University, United States
Reviewed by: Timothy J. Collier, Michigan State University, United States; Kaneyasu Nishimura, Kyoto Pharmaceutical University, Japan; Heather Boger, Medical University of South Carolina, United States
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.
Spinal cord stimulation (SCS) exerts neuroprotective effects in animal models of Parkinson’s disease (PD). Conventional stimulation techniques entail limited stimulation time and restricted movement of animals, warranting the need for optimizing the SCS regimen to address the progressive nature of the disease and to improve its clinical translation to PD patients.
Recognizing the limitations of conventional stimulation, we now investigated the effects of continuous SCS in freely moving parkinsonian rats.
We developed a small device that could deliver continuous SCS. At the start of the experiment, thirty female Sprague-Dawley rats received the dopamine (DA)-depleting neurotoxin, 6-hydroxydopamine, into the right striatum. The SCS device was fixed below the shoulder area of the back of the animal, and a line from this device was passed under the skin to an electrode that was then implanted epidurally over the dorsal column. The rats were divided into three groups: control, 8-h stimulation, and 24-h stimulation, and behaviorally tested then euthanized for immunohistochemical analysis.
The 8- and 24-h stimulation groups displayed significant behavioral improvement compared to the control group. Both SCS-stimulated groups exhibited significantly preserved tyrosine hydroxylase (TH)-positive fibers and neurons in the striatum and substantia nigra pars compacta (SNc), respectively, compared to the control group. Notably, the 24-h stimulation group showed significantly pronounced preservation of the striatal TH-positive fibers compared to the 8-h stimulation group. Moreover, the 24-h group demonstrated significantly reduced number of microglia in the striatum and SNc and increased laminin-positive area of the cerebral cortex compared to the control group.
This study demonstrated the behavioral and histological benefits of continuous SCS in a time-dependent manner in freely moving PD animals, possibly mediated by anti-inflammatory and angiogenic mechanisms.
Parkinson’s disease manifests as a progressive neurodegenerative disease resulting from the loss of dopaminergic neurons in the nigrostriatal system. Cardinal symptoms of PD include bradykinesia, rigidity, resting tremor, and postural instability. Levodopa treatment stands as the first-line therapy for PD. However, long-term medication often results in adverse events, including motor fluctuation and dyskinesia.
Deep brain stimulation (DBS) improves motor symptoms in advanced PD patients. In animal models of PD, DBS may increase BDNF (
Spinal cord stimulation in the management of intractable neuropathic pain demonstrates a solid track record of effectiveness and safety. Although neurological injuries account for the most serious complication in SCS procedure, they are rare with an incidence rate of only 0.6% (
Electrical stimulation shows efficacy in PD animal models. However, technical problems plague the SCS animal model, including the short duration of the stimulation (no more than 1 h per day) and the highly restricted movement of animals (i.e., due to anesthesia) (
All animal procedures in this study followed specifically the approved guidelines by the Institutional Animal Care and Use Committee of Okayama University Graduate School of Medicine (Protocol# OKU-2018807). Adult female Sprague-Dawley rats (Shimizu Laboratory Supplies Co., Ltd., Japan) weighing 200–250 g at the beginning of the study served as subjects for all experiments. Animal housing consisted of individual cages in a temperature and humidity-controlled room and maintained on a semidiurnal light-dark cycle.
We developed an electrical stimulation device called SAS-200 (Unique Medical Co., Ltd., Japan) that offered convenient adjustment of stimulation conditions via Bluetooth and allowed free movement of rats owing to its small size. The SAS-200SCS, which was attached to the back of the rats and connected to the SCS electrode, delivered the stimulation. This stimulation required no anesthesia, thereby allowing rats to freely move around, making continuous stimulation possible. Additionally, the stimulation conditions could be easily adjusted wirelessly.
The SAS-200 measured 20 mm × 40 mm × 20 mm, with a net weight of 26 g (including the battery) (
Wireless controllable electrical stimulation system (SAS-200).
Rats were randomly divided into three groups: the control, 8-h stimulation, and 24-h stimulation groups (30 rats total,
Time course of this study.
All rats received anesthesia with 0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol by intraperitoneal injection and placed in a stereotaxic instrument (Narishige, Japan). The animals underwent a midline head skin incision on and a small hole drilled in their skull. Twenty μg of 6-OHDA (4 μl of 5 mg/ml dissolved in saline containing 0.2 mg/ml of ascorbic acid; Sigma, United States) was injected into the right striatum (1.0 mm anterior and 3 mm lateral to the bregma and 5.0 mm ventral to the surface of the brain with the tooth-bar set at −1.0 mm) with a 28G Hamilton syringe that delivered an injection rate of the drug at 1 μl/min. Syringe withdrawal commenced after a 5-min absorption time following injection.
Following 6-OHDA injection, animals received a midline skin incision that extended to the back, and carefully dissecting the spinal muscles to expose and to eventually perform a C2 laminectomy. We implanted a silver bipolar ball electrode, with a diameter of 2 mm, epidurally on the dorsal surface of the spinal cord and fixed to the muscle using a 5-0 silk thread (
An electrode and images of surgery.
After recovery from anesthesia, the stimulation device commenced by wireless command from Windows PC via Bluetooth in the 8- and 24-h stimulation groups. In the 8-h stimulation group, the stimulator automatically delivered biphasic square pulses for 8 h then switched off for 16 h. Stimulation continued for 14 consecutive days, and with battery changed every 3 days. Stimulation consisted of 50 Hz pulses in 100 μs. Intensities corresponded to the 80% of motor threshold (
To assess the degree of forepaw asymmetry, we performed the cylinder test on days 7 and 14. This test involved placing individual animals in a transparent cylinder (diameter: 20 cm, height: 30 cm) for 3 min and recording the number of forepaw contacts on the cylinder wall (
Rats received an intraperitoneal injection of methamphetamine (3.0 mg/kg; Dainippon Sumitomo Pharma, Japan) on days 7 and 14. We assessed for 90 min with a video camera the full 360° turns in the direction ipsilateral to the lesion. Such drug-induced ipsilateral rotations also indicated successful 6-OHDA-induced unilateral nigrostriatal dopaminergic depletion.
Processing for immunohistochemistry started after completion of behavioral tests on day 14. Animals underwent euthanasia with an overdose of pentobarbital (100 mg/kg). The rats then received transcardial perfusion with 150 ml of cold phosphate-buffered saline (PBS) and 150 ml of 4% paraformaldehyde (PFA) in PBS. We then harvested the brains carefully, post fixed in 4% PFA in PBS overnight at 4°C, and subsequently stored in 30% sucrose in PBS until completely submerged. Thereafter, we sectioned the brains coronally at a thickness of 40 μm.
For assessing nigrostriatal dopaminergic pathways, we used TH staining. We initially exposed free-floating sections to a blocking solution using 3% hydrogen peroxide in 70% methanol for 7 min. After three washes in PBS, we incubated the sections overnight at 4°C, with rabbit anti-TH antibody (1:500; Chemicon, Temecula, CA, United States) with 10% normal horse serum. We then washed the sections three times for 5 min in PBS and incubated them for 1 h in biotinylated donkey anti-rabbit IgG (1:500; Jackson ImmunoResearch Lab, West Grove, PA, United States), followed by 30 min in avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA, United States). We next treated the sections with 3, 4-diaminobenzidine (DAB; Vector) and hydrogen peroxide, then mounted on albumin-coated slides, and embedded them with cover glass.
Next, we performed Iba-1 and laminin staining to evaluate activated microglial cells and blood vessels, respectively. We initially washed 40-μm-thick sections three times in PBS and incubating them in 10% normal horse serum and primary antibodies: rabbit anti-Iba1 antibody (1:250; Wako Pure Chemical Industries, Osaka, Japan) and rabbit anti-laminin antibody (1:500; AB11575, Abcam plc, Cambridge, United Kingdom) overnight at 4°C. Thereafter, we washed the sections three times in PBS, incubated them for 1 h in FITC-conjugated affinity-purified donkey anti-rabbit IgG (H + L) in a dark chamber, then washed them three more times in PBS and finally mounted and embedded them with cover glass as above.
We assessed the density of TH-positive fibers in the striatum with a computerized analysis system as described previously (
We used the software package SPSS 20.0 (SPSS, Chicago, IL, United States) to perform one-way analysis of variance (ANOVA) with subsequent Tukey’s tests, with significance set at
Body weight decreased at day 7 and nearly recovered at day 14 in all groups (
Changes in body weight.
The 24-h stimulation group performed significantly better in the cylinder test than the control group on days 7 and 14. In the 8-h stimulation group, the treated animals displayed significant improvement in the contralateral bias on day 14 compared to the control group (contralateral bias: control group: 25.0 ± 10.1 and 47.6 ± 28.4%; 8-h stimulation group: 22.7 ± 14.7 and 23.3 ± 12.3%; 24-h stimulation group: 11.6 ± 9.56 and 9.80 ± 6.39% at 1 and 2 weeks, respectively;
Spinal cord stimulation and behavioral outcomes.
The number of methamphetamine-induced rotations on days 7 and 14 in the 8- and 24-h stimulation groups statistically decreased compared to that of the control group (control group: 1292 ± 239 and 1518 ± 172 turns/90 min; 8-h stimulation group: 893 ± 217 and 1,020 ± 146 turns/90 min; 24-h stimulation group: 670 ± 244 and 820 ± 289 turns/90 min at 1 and 2 weeks, respectively;
The stimulation groups exhibited significant preservation of TH-positive fibers in the striatum and TH-positive neurons in the SNc compared to the control group (control group: 21.9 ± 7.16%; 8-h stimulation group: 45.3 ± 12.6%; 24-h stimulation group: 57.2 ± 9.11% relative to the intact side of TH-positive fibers in the striatum,
Spinal cord stimulation and TH staining in the striatum.
Spinal cord stimulation and TH staining in the SNc.
The number of Iba1-positive cells in the striatum and the SNc of rats in the 24-h stimulation group decreased significantly compared to the control group. In the 8-h stimulation group, the number of Iba1-positive cells tended to decrease in the striatum, and was significantly decreased in the SNc (control group: 37.9 ± 7.55; 8-h stimulation group: 31 ± 8.73; 24-h stimulation group: 23.5 ± 6.13 cells/field of view in the striatum; control group: 40.6 ± 6.26; 8-h stimulation group 32.4 ± 6.30; 24-h stimulation group 25.1 ± 5.62 cells/field of view in the SNc;
Spinal cord stimulation and Iba1 staining in the striatum and SNc.
The laminin-positive area in the lesioned cortex significantly increased in the 8- and 24-h stimulation groups compared to the control group of the intact and lesion side (control group intact side: 4.59 ± 1.89%; control group lesion side: 6.23 ± 2.63%; 8-h stimulation group intact side: 7.90 ± 2.82%; 8-h stimulation group lesion side: 8.04 ± 3.19%; 24-h stimulation group intact side: 9.12 ± 2.58%; 24-h stimulation group lesion side: 10.8 ± 3.90%;
Spinal cord stimulation and laminin staining in the cerebral cortex.
The present study demonstrated that a small mobile device efficiently delivered continuous SCS and exerted neuroprotective effects behaviorally and immunohistochemically on PD rats in a time-dependent manner. While both SCS-treated groups generally improved their performance in both contralateral bias and methamphetamine rotations, and displayed an increase in laminin-labeled cerebral blood vessels, The 24-h stimulation group conferred better therapeutic effects than the 8-h stimulation group, in that the longer continuous SCS regimen significantly reduced microglial cells both in the lesioned striatum and SNc compared to rats in the control group (
Until now, conventional SCS machines allow limited control of stimulation parameter and highly restrict the movements of animals. Current SCS machines consist of a large electrical stimulator and an electrode implanted in the animals with wire connections (
A small mobile electrical stimulator may circumvent the technical limitations of current SCS machines. Indeed, such mobile device shows efficacy as a DBS apparatus for PD animals (
Although neuroprotective effects of SCS have been documented in PD animals, the optimal electrical stimulation conditions remain unclear. Effective electrical stimulation parameters in PD rats vary in pulse width (400–1,000 μs), frequency (300–333 Hz), stimulation duration (30 min at 2 times/week for 4.5 weeks – 30 min at once a week for 5 weeks) (
Parkinson’s disease neurodegeneration manifests in part as a chronic neuroinflammation characterized by activated microglial cells in the striatum and SNc (
Low-frequency cervical SCS increases cerebral blood flow (
Neuroinflammation in PD pathogenesis may involve multi-pronged neurodegenerative processes, such as inflammation and downregulation of neurotrophic factors (
Deep brain stimulation stands as an effective treatment for motor symptoms in advanced PD patients. SCS offers a less invasive approach compared to DBS in that the procedure spares the brain from surgical manipulations. Such minimally invasive SCS may be equally effective as DBS in reducing the hallmark PD motor deficits. Indeed, SCS alleviates motor deficits in PD marmosets (
In this study, we used PD model of rats induced by 6-OHDA. The main advantages of this model include the ease of creating the lesion that produces loss of dopaminergic fibers in the striatum and of dopaminergic neurons in the substantia nigra. One of the disadvantages of this model is that it does not resemble the natural pathology of PD, which is slow progression of the degeneration of nigrostriatal dopaminergic neurons with degradation of α-synuclein. Therapeutic potentials of the SCS should be explored with other PD models of neurodegeneration and α-synucleinopathy reminiscent of the clinical scenario.
The aim of this study was to explore the neuroprotective effects of the SCS with duration of treatment as a factor. Here, treatment was started immediately after 6-OHDA lesion induction, which may not be applicable in the clinical setting since PD symptoms do no manifest when at least 80% of the dopaminergic neurons have already been depleted. Testing SCS in a late-stage PD model is warranted. Another limitation is that elucidating the therapeutic mechanism of SCS will require additional studies. In our study, the neuroprotective effects with angiogenic potentials were shown, but whether the neuroprotective effects of SCS during the pre-symptomatic phase is sustained during the symptomatic stage warrants further examination. In the future, behavioral changes over time after discontinuation of the SCS may reveal long-lasting effects of SCS, as well as its mechanism of actions, on PD symptoms.
We demonstrated that a small mobile stimulator afforded continuous SCS and exerted neuroprotective effects in PD rats in a time-dependent manner. SCS attenuated behavioral and histological deficits associated with 6-OHDA-induced PD symptoms, possibly by mitigating microglial activation while enhancing angiogenesis. The newly developed device for continuous SCS serves as a useful tool for basic research in our understanding of interplay across electrical stimulation, neurodegeneration, and neural repair, but also advances its utility as a therapeutic modality for PD.
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.
The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Okayama University Graduate School of Medicine (Protocol# OKU-2018807).
KeK and TS contributed conception and design of the study. KeK, TY, YO, KH, IK, MO, SY, SK, YT, and MU performed the experiments. KeK and JM collected the data. KyK and NT performed the statistical analysis. KeK wrote the first draft of the manuscript. KyK, TS, TY, and J-YL wrote sections of the manuscript. CB performed the critical editing. ID supervised the study. All authors contributed to manuscript revision, read and approved the submitted version.
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 Supplementary Material for this article can be found online at:
The graphic abstract showing therapeutic effects of SCS against 6-OHDA-induced PD model of rats through angiogenesis and anti-inflammation.
The video showing twitching rats with SCS. The intensities corresponded to the 80% of motor threshold were used for each rat.
brain-derived neurotrophic factor
dopamine
deep brain stimulation
ionized calcium-binding adaptor molecule 1
Parkinson’s disease
spinal cord stimulation
substantia nigra pars compacta
tyrosine hydroxylase
vascular endothelial growth factor
6-hydroxydopamine.