Effects of Optimized Acupuncture and Moxibustion Treatment on Depressive Symptoms and Executive Functions in Patients With Post-Stroke Depression: Study Protocol for a Randomized Controlled Trial

Background Post-stroke depression (PSD), a common neuropsychiatric comorbidity after stroke, has a negative impact on the functional recovery and quality of life of survivors. It lacks effective therapeutic drugs with good curative effects and few adverse reactions. Preliminary experiments have shown that the optimized acupuncture and moxibustion treatment (OAMT), including acupuncture, moxibustion, and auricular intradermal acupuncture, improved depressive symptoms and neurological deficits in patients with PSD. However, the evidence for its effectiveness is still insufficient. Hence, we designed this study to evaluate the efficacy and safety of the OAMT in the treatment of PSD and to explore its possible mechanism from the perspective of executive functions. Methods/Design This is a randomized controlled trial, which comprises a total of 134 patients with PSD. Participants are randomized into intervention group and control group at a 1:1 ratio. All treatments are given five times per week for 4 weeks. The primary outcome is the severity of depression, which is evaluated by the Hamilton Depression Scale-17 (HAMD-17) and the Beck Depression Rating Scale (BDI). Secondary outcomes are executive abilities, which are measured by several neuropsychological tests, including the Stroop Color and Word Test (SCWT), the Trial Making Test (TMT), the Digit Symbol Substitution Test (DSST), and the Matrix Reasoning Test (MRT). All outcomes have been evaluated at baseline and weeks 4, 8, 12, and 20. At the same time, functional MRI (fMRI) is used to measure the functional connectivity in the cognitive control network (CCN) at baseline and 4 weeks after intervention. Discussion This study aims to provide high-quality evidence for the efficacy and safety of the OAMT for treating PSD. In addition, this trial is the first trial to explore if the improvement condition of depression in the OAMT group is related to the improvement of executive functions and the favorable changes in the structure. Clinical Trial Registration Chinese Clinical Trial Registry, identifier: ChiCTR2100048431.


INTRODUCTION
Depressive symptoms after stroke have been reported to be closely related to executive dysfunction (21). Pohjasvaara et al. (22) showed that executive dysfunction was detected in 40.6% (n = 104) of 256 patients 3-4 months after stroke, and this dysfunction was proven to be related to depressive symptoms. In the same vein, patients with PSD with executive dysfunction show more severe depressive symptoms and have a significantly higher incidence of cerebral infarction in the frontal lobe-subcortical circuit compared with patients with PSD without executive dysfunction (21). In neuroanatomy, links between the two have also been reported (23). Both are associated with alterations in intrinsic and extrinsic structural and functional connectivity in the convolutional neural network (CNN). Specifically, both may have structural disconnections in frontal, parietal, and subcortical areas (24)(25)(26), and these disconnections have contacts with lower intrinsic functional connectivity in the CNN (27). Cognitive control network (CCN) is the frontal parietal loop (28), which participates in topdown, attention-dependent executive functions such as decisionmaking and task switching (29)(30)(31). The dorsolateral prefrontal cortex (DLPFC) is an important node of this network (32), and it has been proven that the left DLPFC functional connectivity is negatively correlated with the severity of PSD (33). Several studies using DLPFC as a seed have reported the decreased functional connectivity within the CCN after depression (34)(35)(36). Alexopoulos et al. (35) pointed out that lower CCN connectivity can predict the lower recovery rate and symptom improvement of depressed individuals after taking escitalopram. Ye et al. (36) found that the node centrality of DLPFC in patients with depression is lower than that of normal people, which also reflects the weakening of the network function of CCN in patients with depression. Therefore, DLPFC is used as a seed to observe the functional connectivity in the CCN, which may be a key for exploring the neural mechanism of the OAMT in the treatment of patients with PSD. In addition, studies have shown that acupuncture is conducive to relieving executive dysfunction (37,38), which may be resulted from taking an effect on the central nervous system through local reflex, affecting neurotransmitter levels, etc., to regulate the executive control system (39,40). Therefore, this trial not only evaluates the degree of depression and executive functions in patients with PSD but also observes the CCN network's functional connectivity, providing stronger evidence for the efficacy and mechanism of the OAMT from multiple perspectives.

METHODS AND ANALYSIS Design and Setting
This is a prospective, randomized controlled trial conducted by the First Affiliated Hospital of Henan University of Chinese Medicine. A total of 134 patients who meet the inclusion and exclusion criteria are randomly divided into two groups. One group receives the OAMT and routine medicine and rehabilitation treatment, and another group receives routine medicine and rehabilitation treatment only. All treatments are provided five times per week for 4 weeks. The primary outcome is the severity of depression, which is evaluated by the HAMD-17 and the Beck Depression Rating Scale (BDI). Secondary outcomes are executive abilities, which are measured by several neuropsychological tests, including the Stroop Color and Word Test (SCWT), Trial Making Test (TMT), Digit Symbol Substitution Test (DSST), and Matrix Reasoning Test (MRT). All outcomes are evaluated at baseline and weeks 4, 8, 12, and 20. At the same time, functional MRI (fMRI) is used to measure the functional connectivity in the CCN at baseline and 4 weeks after intervention. The study's flow chart is shown in Figure 1, and the process chart is shown in Table 1.

Recruitment of Participants
This trial is conducted at the First Affiliated Hospital of Henan University of Chinese Medicine, China. Patients who meet the criteria are recruited through the outpatient and inpatient systems and advertisements. The recruitment begins on January 1, 2022 and is expected to end in June 2023.

Inclusion Criteria
Participants who meet the following criteria are included

Exclusion Criteria
Participants with the following conditions are excluded: (1) The HAMD-17 score ≤7 points, or ≥24 points.
(2) Patients with obvious suicidal tendencies assessed by specialists.

Randomization
The randomization sequence is generated by an independent statistician from the Henan Evidence-based Medicine Center of Traditional Chinese Medicine, using the PROCPLAN process of the SAS statistical analysis system. Then, the randomization sequence is placed in opaque sealed envelopes and assigned to the eligible patients who can be included.

Blinding
Due to the characteristics of the OAMT and the technical limitations, we are unable to conduct a double-blind study design. However, evaluators and statisticians of the outcome are blinded to the assignments. Patients are treated separately to avoid communication, and the OAMT sessions are strictly performed by licensed and experienced acupuncturists.
Acupuncturists provide any information about the allocation to the patients, evaluators, or statisticians. In the process of data management and statistical analysis, a professional statistician who is not in this study is invited to undertake analysis tasks.

Interventions
Patients receive treatments in separate rooms five times a week for 4 weeks. All treatments are performed by licensed acupuncturists with more than 3 years of experience in practice. The location of the acupoints follows the WHO standards (42)(43)(44). At the same time, routine medicine and rehabilitation treatment of each patient is performed by physicians and therapists who do not know the allocation. The interventions of the two groups are as follows.

Intervention Group
Patients receive the OAMT and routine medicine and rehabilitation treatment once a day, five times a week, and for 4 weeks. The OAMT consists of acupuncture, moxibustion, and auricular intradermal acupuncture. The specific operations are as follows:

Control Group
Patients receive routine medicine and rehabilitation treatment for 4 weeks. For stroke and underlying diseases (such as hypertension and diabetes), we have provided symptomatic treatments, including antiplatelet aggregation, lowering lipids and stabilizing plaques, blood pressure, and blood sugar treatment, and have tried to avoid the drugs that affect mental factors, referring to the Guidelines for the Prevention and Treatment of Cerebrovascular Diseases in China (45). At the same time, routine rehabilitation treatment, such as exercise therapy and physical therapy, is provided equally.

Sample Size
The main outcome of this study is the improvement of depressive symptoms. Our team's preliminary pre-experiment (n = 30) showed that the OAMT for 4 weeks can reduce the HAMD-17 score by 14.53 ± 1.66. When α = 0.05, β = 0.1, 1β = 0.9, and the sample size of the intervention group and the control group are equal, the normal distribution quantile table shows 66 means the SD of the intervention group and δ = 1.28 means the mean difference of the intervention group. Bring the data in the following formula: After the calculation, the sample size is N = 60. Taking the 10% dropout rate into account, the sample size is 67 patients per group (134 in total). All 134 participants receive fMRI scans, and this sample size is much larger than the 12 cases per group required by the technical requirements of quality control and network analysis of acupuncture brain functional imaging (46), which can make the test results more reliable.

Outcome Measures
Participants are evaluated at baseline and weeks 4, 8, 12, and 20. All evaluations are conducted by researchers who are blinded to the treatment allocation.

Primary Outcomes
Primary outcomes are the HAMD-17 and the BDI.

Beck Depression Rating Scale
The BDI is a commonly used self-evaluation scale to measure depression. It measures the intensity of depression by judging the main symptoms of depression syndrome (50). The advantages of the BDI lie in its international spread, high internal consistency of psychiatric and non-psychiatric samples, high content validity, high sensitivity to changes, and high convergence validity. The entire scale includes 21 groups of items, each group has four options, and each option corresponds to a certain score. A total score of <10 means no depression. The higher the score, the more severe the depression (51).

Secondary Outcomes
Secondary outcomes will be obtained using several neuropsychological tests and fMRI scanning. Neuropsychological tests include SCWT, TMT, DSST, and MRT.

Stroop Color and Word Test
The SCWT is a neuropsychological test widely used for experimental and clinical purposes. It can measure not only the ability to suppress cognitive interference but also a variety of cognitive functions (such as attention, processing speed, cognitive flexibility, and working memory) (52). The test is generally divided into three parts: (1) quickly name the color pictures; (2) quickly read the nouns that represent the name of the color; and (3) a set of cards have been presented with the nouns representing the color names written in colors different from the meaning of the words. A researcher checks the patient's ability to distinguish color names from actual colors and quickly read nouns representing color names. The degree to which the patient is affected by the color of the words is used as an index to measure his/her cognitive control ability.

Trial Making Test
The TMT is a commonly used neuropsychological test (53), and its reliability and effectiveness have been previously proven (54). TMT consists of two parts, involving visual search and scanning capabilities, processing speed, mental flexibility, and executive functions. TMT-A requires a patient to connect the circled numbers distributed on a piece of paper one by one. The requirements of TMT-B are similar to those of TMT-A, but the patient must alternate between numbers and graphics. The total score is calculated based on the task completion time and the accuracy rate.

Digit Symbol Substitution Test
The DSST was originated from the Wechsler Adult Intelligence Scale (55), which can evaluate participants' abilities related to digital decoding, memory, attention, and operating speed (56).
Patients are provided with a table showing various symbols and matching numbers, and they are asked to write down matching  symbols for each number in several rows of numbers. The score is the number of correctly coded numbers completed in 120 s (57).

Matrix Reasoning Test
The MRT is a sub-test of the Wechsler Intelligence Abbreviation Scale (58,59), which measures non-verbal reasoning ability. It is composed of a sequence or a set of graphic matrixes. Patients are required to select the patterns or symbols that can be filled in the vacant part according to the pattern or symbol change rules in the incomplete sequence or graphic matrix. The score is the number of items completed correctly.

Safety Evaluation and Adverse Events
Safety indicators are tested before and after the treatment, including general physical examination (blood pressure, pulse, and breathing), ECG, blood routine, urine routine, stool routine, and liver and kidney function tests. In the case report forms (CRFs), adverse events such as pain, fainting, local infection, and allergies are recorded in detail. Serious adverse events, such as death or life-threatening events, are reported to the researcher immediately and reported to the ethics committee within 24 h. In those circumstances, the research team gives the patient treatment and suggestions based on the situation, evaluates whether he/she continues to participate in the research, and compensates him/her accordingly.

Data Management
The trained evaluators use CRFs to record patients' information and data in detail and import them into an electronic database. After the study is completed, the paper CRFs are stored in a locked cabinet. Meanwhile, the electronic database has also been locked, so researchers do not able to modify the data. Participants' personal data are kept anonymously and strictly confidential. For patients who discontinue or leave the trial, we obtain their data by telephone with their consent. The Data Monitoring Committee of the Rehabilitation Center of the First Affiliated Hospital of Henan University of Chinese Medicine is established. They are independent of researchers to monitor the trial progress, regularly monitor the safety of the trial, and check the completeness and accuracy of the CRFs.

Clinical Data Analysis
We invite third-party professional statisticians who do not know the trial protocol to conduct a statistical analysis and participate in the whole process comprising trial design, implementation, and data analysis. If the necessary data are available, subgroup analyses are performed by different clinicopathological features, such as gender, age, time of stroke onset, and stroke severity. A statistical analysis is performed using SPSS 22.0. The data are statistically described by mean ± SD. Continuous variables are compared using Student t-test or Wilcoxon rank sum test, and categorical variables are compared using Pearson χ 2 test or Wilcoxon rank-sum test. The comparison between the groups is carried out by an independent sample t-test or the Mann-Whitney U-rank-sum test based on whether the measurement data present a normal distribution and homogeneous variance. When doing a statistical analysis with two-tailed testing, the significance level is set at 5%. When p < 0.05, differences have been considered as statistically significant.

MRI Data Analysis
The SPM8 software platform (SPM8, Wellcome Department of Imaging Neuroscience, London, UK; http://www.fil.ion.ucl.ac. uk/spm/) is used to preprocess the MRI data. Referring to the study of DLPFC resting-state connectivity of patients with PSD (33), we select the seeds from bilateral DLPFC, draw the region of interest (ROI) in WFU Pickatlas (60) to tap into the CCN, and perform a whole-brain regression analysis. The DLPFC resting-state connectivity in the CCN is compared between the intervention group and the control group. A paired t-test is used to evaluate brain changes in each group through an intragroup analysis (before and after the treatment). We include factors such as age and gender as covariates in the data analysis. The Pearson correlation coefficient is used to analyze the relationship between the improvement value of the correlation scales and the change of fMRI image data.

Quality Control
Qualified acupuncturists, physicians, therapists, and statisticians in the First Affiliated Hospital of Henan University of Chinese Medicine monitored and revised this trial protocol. All relevant personnel are trained in accordance with the prepared standard operating procedure (SOP) to ensure consistency in the comprehension and implementation of interventions and evaluations. At the same time, a quality control team is established to conduct quality control, and the qualified clinical trial experts are invited to supervise the trial once a month.

DISCUSSION
There is a large volume of published studies showing that acupuncture and related therapies effectively improved depression, and the combination therapy appeared to have superior efficacy (14-16, 19, 20). However, these studies used diverse acupoint selection plans and intervention methods, and lacked a unified standard acupuncture program. Moreover, they remain narrow in focus dealing only with efficacy without exploring the mechanism. The OAMT is a combination therapy based on the acupuncture theory, our previous experiments, and practical experience. It was developed according to the theory of "ShuGanTiaoShen" (smoothing the liver and regulating mental activities). Chinese doctor believes that the cause of PSD is closely related to "Liver Failing to Maintain Normal Flow of Qi" and "Disorder of Cerebral Soul" (61,62). Referring to this, this study select acupoint plans with the effect of "ShuGanTiaoShen." Based on the clinical practice and previous experimental results of the First Affiliated Hospital of Henan University of Chinese Medicine, we completed the first draft of the protocol for this study. Subsequently, the ethics committee of the hospital discussed and revised the draft, and the final version of the protocol was unanimously approved by the ethics committee.
This trial is the first trial to explore the OAMT treatment of the PSD population from the executive control system. It has been proven that the reduction of functional connectivity in the CCN affects depression vulnerability and maintenance through an impact on the cognitive control of emotional information (63,64). In other words, neurological deficits are considered as a basis for the difficulty of cognitive control mechanisms (e.g., attention control, inhibition, and reassessment) that support emotion regulation. These deficits may hinder the function of CNN. Therefore, observing neurological defects of CCN through fMRI is of certain significance to explore the mechanism of the OAMT in the treatment of PSD.
However, this study has some limitations. First, it is a singlecenter study in which patients from different areas cannot be recruited. Second, although this study followed the principle of separation of researchers, operators, and statisticians due to the nature of acupuncture and moxibustion treatment, we did not use blind methods in the OAMT. This may cause partial deviations in the results. Despite all of these, we strive to standardize the process of this study to provide high-quality medical evidence for the OAMT, as well as an optimized plan as a complementary therapy for PSD.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Henan University of Chinese Medicine (reference number: 2021HL-184-01). The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
XF and JG designed this study. ML and ZD drafted the manuscript together. XS revised this manuscript and conducted the preliminary pre-experiment. CL, RL, KS, and XW recruited patients and provided treatment. WF and YB conducted a statistical analysis in the pre-experiment. All authors agreed to the final version of this manuscript.