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Acupuncture for poststroke hemiplegia focusing on cerebral bilateral connections: study protocol for a randomised controlled neuroimaging trial.

Introduction Acupuncture is safe and effective for improving the motor function of poststroke hemiplegic patients, but there still exists a certain gap between clinical practice and understanding its neural mechanisms. The cerebral functional reconstruction after unilateral motor pathway injury exhibits a bilateral tendency, however current studies seldom pay attention to it. Hence, based on cerebral bilateral connections, the underlying mechanism of acupuncture in stroke rehabilitation remains an area for further research. The results of this study will increase our understanding of acupuncture-induced motor recovery in patients who had suffered a stroke and demonstrate the differences in brain response and clinical assessments. Methods and analysis This is a single-centre, randomised controlled, paralleled neuroimaging trial, with patients and outcome assessors blinded. Thirty patients who had a stroke with motor dysfunction meeting the inclusion criteria will be randomly assigned (2:1) to receive either 10 sessions true or sham acupoints treatments (five sessions per week for 2 weeks). All the participants will receive conventional standard medical care and rehabilitation. Motor function assessments and neuroimaging scanning will be conducted before and after the entire acupuncture treatment. The clinical and neuroimaging data will be analysed, respectively. The voxel-mirrored homotopic connectivity will be the primary outcome and the primary effect indicator. The secondary outcomes comprise clinical evaluations and neuroimaging assessments, which include Fugl-Meyer Assessment, the National Institutes of Health Stroke Scale, fractional anisotropy and gray matter volume. The Needle Sensation Assessment Scale is an additional outcome. The correlation analysis will be explored between the neuroimaging indicators, clinical motor assessments and needle sensation. Ethics and dissemination The protocol has been approved by the ethics committee of Dongzhimen Hospital affiliated to Beijing University of Chinese Medicine (DZMEC-KY-2018-04). The results of the neuroimaging trial will be disseminated through peer-reviewed publications and conferences. Trial registration number Chinese Clinical Trials Registry (ChiCTR 1800016263).

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The strength and limitations of this study
The application of randomized controlled neuroimaging trial will provide objective and valid evidence about how acupuncture treatment promotes the recovery of poststroke hemiplegia.
The assessment of motor function, needle sensation and multimode neuroimaging scans will be used to judge both cerebral response and motor-functional effects of course of acupuncture treatment on poststroke hemiplegia.
The research based on bilateral cerebral connections is closer to the actual state of coordinated regulation and integration of the acupuncture-induced brain recovery.

INTRODUCTION
In China, stroke often is the leading cause of death and disability, resulting in 70% of survivors living with motor function impairments. [1][2][3] Evidence-based medicine has shown that poststroke functional rehabilitation is the most primary method to F o r p e e r r e v i e w o n l y decrease the disability rate and get functional recovery. 4 5 Acupuncture plays an important role in hemiplegic recovery in long-term clinical practice, the history of which can date back thousands of years. Both World Health Organization (WHO) and National Institutes of Health (NIH) recommended the acupuncture treatment as an alternative or a part of a comprehensive program in stroke rehabilitation, 6 and its effect has been confirmed by systematic reviews and multi-center clinical study. 7 8 However, the underlying mechanisms of acupuncture for treating hemiplegia still remain research space, which partly restrains its widespread application in stroke recovery.
Brain functional reorganization and structural remodeling are closely related to the prognosis of stroke recovery. Cerebral bilateral modulation and cooperation have significant influence on motor recovery. Physiologically, every achievement of human activities is the process of resource allocation and execution controlled by right and left hemispheres. 9 Increasing neuroimaging fMRI researches reported that, along with poststroke motor recovery, there existed dynamic active or inactive status between ipsilesional primary motor cortex (M1) and contralesional M1. [10][11][12][13] In our previous studies, stroke patients with unilateral subcortical infarcts showed activations in multiple brain regions in both hemispheres. 14 All of the above proves that the bilateral issue matters in the brain functional reorganization. Besides, we found that instant acupuncture at Yanglingquan (GB34) will enhance decreased functional connectivity between bilateral M1 of right-hemispheric subcortical stroke patients. 15 16 However, whether the long-term acupuncture intervention will Based on the viewpoint of bilateral connections, this study aims to: 1) assess the differences of brain response, motor assessments and needle sensation between true and sham acupoints treatment group; 2) characterize the influence of acupuncture treatment on cerebral functional activities and structural changes; 3) analyse the possible relationship between the changes of brain activities and clinical assessments, so as to demonstrate the underlying mechanism of acupuncture treatment for hemiplegic patients and its effects on clinical motor performances.

Study design
This is a single-center, randomized controlled, paralleled neuroimaging study, with patients, assessors and data statisticians blinded to the group assignment. Stroke patients with motor dysfunction meeting the inclusion criteria will be randomly divided into true acupoints treatment group (TATG) and sham acupoints treatment group (SATG) by the ratio of 2:1. In addition to conventional standard medical care and rehabilitation, participants will receive 10 sessions of acupuncture treatment in F o r p e e r r e v i e w o n l y two weeks (5 sessions per week). Motor function scales and MRI scanning will be conducted before and after the entire acupuncture treatment. The needle sensation assessment scale will be assessed after each acupuncture treatment.
The study procedures is detailed in Figure 1.
Insert Figure 1 here.

Patients population and recruitment strategy
Participants will be screened and recruited among the department of Neurology, Rehabilitation and Acupuncture in Dongzhimen Hospital affiliated to Beijing University of Chinese Medicine via Electronic Medical Record from May 2018 to Dec 2020. Stroke patients with right or left hemiplegia who match the inclusion criteria will be regarded as the potential participants. If the patients are eligible and interested in taking part in the study, they will be fully informed of the project information including the study procedures, potential benefits and risks. Written informed consent will be obtained from the patient or their legally authorized representative before the allocation. Every participant has the right to withdraw from the study without any conditions.

Inclusion criteria
Patients will be included if they meet the following criteria: (1) patients with ischemic stroke; (2) single subcortical lesion restricted to the motor pathway involving the internal capsule (IC), basal ganglia (BG), corona radiate (CR),and its neighboring regions in the unilateral hemisphere; (3) right-handed before stroke; (4) age range from 40 to 75 years old; (5) within 6 weeks after the onset of stroke; (6) F o r p e e r r e v i e w o n l y being unconscious retardation and stable condition; (7) no psychiatric medications have been taken in one month; (8) informed consent is signed by the patient or his/ her immediate family.

Exclusion criteria
Patients will be excluded if they meet any of the following criteria: (1) a history of neurologic or psychiatric disorders; any other health problems or poor physical conditions that may influence participation; (2) being pregnant or lactation; (3) any other brain structure damage or abnormalities identified by MRI examinations; (4) any history of alcohol or drug dependency; (5) any MRI contraindications.

Sample size considerations
When calculating the sample size in acupuncture-neuroimaging studies, the number in researches of public peer review and previous studies is often taken into consideration, for two main reasons. On the one hand, in fMRI studies, millions of voxels are used to estimate the blood oxygen level dependent (BOLD) signal indirectly, and conventional power calculations often make no sense. On the other hand, the number of participants is often constrained by scanning time and costs. Therefore, considering the published acupuncture-neuroimaging literature focusing on stroke patients, 16-32 subjects show enough statistical power for brain functional analysis; 17 18 besides, according to our previous studies, 20 subjects represent a reasonable sample size for stable cerebral response. 14 15 Under the condition of high homogeneity of lesion location in this study, we finally assumed 30 patients could ensure enough statistical power and provide repeatable results.

Randomization and blinding
An external professional statistician will generate a random sequence using Excel's rand function. Thirty patients met the inclusion criteria will be randomly assigned into either true acupoints treatment group (TATG) or sham acupoints treatment group (SATG) (2:1 ratio). The TATG and SATG will be coded A and B respectively. The code will be kept in an opaque envelope and subsequently sealed. These envelopes will be sent to the chief principal who won't take part in the recruitment and will take direct charge of treatment allocation. The acupuncturists are aware of the treatment group. While, rehabilitation therapists, motor function evaluators, participants and data statisticians are blinded to the treatment group.

Patient and public involvement
Patients in this trial will not be involved in the design, recruitment or conduction of the study. After the MRI scanning, the patients will be informed of the results of neuroimaging data in the form of pictures. The doctor in charge of the patient will be also informed of the results to optimize treatment and rehabilitation scheme.

INTERVENTIONS
In addition to conventional standard medical care and rehabilitation, all the participants will receive true or sham acupoints treatment.

Acupuncture Interventions
"Shou Zu Shi Er Zhen", a preferred acupuncture prescription for poststroke motor dysfunction established by Leting Wang, will be taken as the acupuncture intervention prescription. Dr. Wang is a famous acupuncturist owned more than The prescription, named after the acupoints locations and its bilateral acupoints selection principles, consists of 12 acupoints bilaterally from five transport acupoints and original acupoints of twelve meridians. Focused on the pathogenesis of stroke, the prescription is designed to regulate qi and blood, balance yin and yang and attach importance to spleen and stomach which has the function of nourishing muscles.
Two doctors held a master's degree with more than 3 years of clinical experience in acupuncture will be trained together and use the same techniques. After the first MRI scanning, the participants will receive five-sessions acupuncture treatments per week for two weeks. 2) Acupuncture procedures ： All participants will be in supine position. After skin disinfection routinely, 12 sterile acupuncture needles for single use (size 0.25 mm×40 mm, Ande brand, manufactured by Ande Medical Appliance in Guiyang, Guizhou Province, China) will be inserted vertically for 1.0-1.5 cun with 6-8 times reinforcing-reducing manual stimulation at the frequency of 1 Hz until the patients experience "Deqi" (a sensation including sourness, numbness, tingling, aching and propagated feeling along the meridians). The needles will be left in the acupoints for 3) Treatment course: Every participant will receive five sessions of treatments per week for two weeks. A fixed acupuncturist will conduct the whole treatment course for one participant. After each acupuncture treatment, participants will be asked to express the kind and intensity of "Deqi" so as to finish the needle sensation assessment scale. The acupuncturist will give a treatment compliance judgment (completion rates) at the end of acupuncture treatment.

Sham acupoints treatment group (SATG)
Referring to Liu's method of sham acupoints 19 and consultation of two experienced acupuncturists, we take the location where is one inch beside the true point as the sham acupoint. Hence, we don't overemphasize the deqi experience of the participants in SATG. Except for the locations of acupoints, other aspects of treatment procedures and course in SATG will be the same as TATG's.

Conventional Treatment
All the participants will receive conventional treatment including the conventional standard medical care and rehabilitation.
Conventional rehabilitation treatment, comprised of physical therapy (PT) and occupational therapy (OT), will be applied by qualified rehabilitation therapists. The rehabilitation programs will be carried out five times per week for 2 weeks and the rehabilitation treatment (PT and OT) will last for approximately 1 hour every time.
Conventional standard medical care will comply with Chinese guidelines for diagnosis and treatment of acute ischemic stroke 2018. 20 The frequency of use, the average

MEASUREMENTS
The measurements consist of three parts including neuroimaging scanning assessments, motor function assessments, and needle sensation assessment. The study schedule is exhibited in Table 2 Table 1 Study schedule of enrollment, intervention, and assessments. At the baseline, and after the whole acupuncture treatment, Fugl-Meyer assessments (FMA) and NIH stroke scales (NIHSS) will be used to evaluate the motor function. MRI scanning will be performed to detect the brain functional and structural change before and after the treatment period. Needle Sensation Assessment Scale (NSAS) will be conducted after every treatment session.

Demographic and basic clinical information collection
The demographic information (name, sex, age, height, weight and working status), the history of stroke (time since the attack, lesions location and symptoms), and the history of other concomitant diseases will be obtained at the baseline. Vital signs (blood pressure, pulse, respiration rate and temperature) will be measured before each scanning.

Neuroimaging outcomes
The neuroimaging outcomes will be acquired after the MRI scanning and analysis, including voxel-mirrored homotopic connectivity (VMHC), Fractional anisotropy (FA) and Gray matter volume (GMV). VMHC will be taken as the primary outcome and the primary effect indicator. GMV and FA will be regarded as secondary outcomes.
As a functional parameter, VMHC is expressed as functional connectivity between any pair of symmetry interhemispheric voxels, which is an optimal parameter to reflect the bilateral functional connections. 21

Motor function assessments and outcomes
The following motor assessment scales, including FMA and NIHSS, will be conducted at the baseline and after the entire acupuncture treatment by a trained assessor who is blinded to the random sequence. The results of FMA and NIHSS will be taken as the secondary outcome in this trial.

Fugl-Meyer Assessment (FMA)
The Fugl-Meyer Assessment is highly recommended as a clinical and research tool for measuring sensorimotor stroke recovery, which has been demonstrated worldwide for more than 40 years in stroke population. 27 The FMA scale is a 100-point rating system consisting of upper/ lower-extremity motor function, extensor and flexor muscle synergy, stability, balance, reflex and joint function in supine, sitting and standing position. The motor function is ranged from 0 to 2 points according to the level of completion. The total scores of upper extremity range from 0 to 66, and total scores of lower extremity range from 0 to 34

NIH Stroke Scale (NIHSS)
The National Institutes of Health Stroke Scale (NIHSS) is a reliable and valid scale for measuring different levels of stroke severity. 29 The NIHSS comprises 15 items including consciousness, eye movements, visual field, facial movements, muscular strength of extremities, ataxia, sensation, language, speech and neglect. Depending on the level of completion, every item scores range from 0 to 1, 0 to 2, 0 to 3, 0 to 4, or 0 to 9. The total fifteen items scores sum up to a range from 0 to 58. The level of scores indicates the level of neurological impairments. The higher the scores, the worse the illness.
The assessment of NIHSS for every patient with cerebral infarction has become routine in our clinical work. Although NIHSS is an extra non-fund supported outcome in this study, we still regard it as one of the secondary outcome indicators.

Needle Sensation Assessment
The needle sensation assessment scale (NSAS) will be conducted after every acupuncture treatment by the acupuncturists. The results of NSAS is a possible prognostic marker associated with the effectiveness of acupuncture and recovery of motor function, which will be taken as the additional outcomes.
"Deqi" is a needle response describing how the patients feel when they receive the needle insertion, which is regarded as a unique key predictor and an essential role in the therapeutic effectiveness of acupuncture for stroke recovery. 30 The self-designed Needle Sensation Assessment Scale we adopt has been tested in our  F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y previous studies, 31 which includes "Deqi" features ( sourness, numbness, tingling, aching and propagated feeling along the meridians) and rating of "Deqi". Patients will describe the intensity of needle sensation by visual analogue scale (VAS) (0 stands for none, 10 stands for unbearable).

Incidence of adverse events
All adverse events happened during the acupuncture treatment, including acupuncture syncope reaction (e.g. sweating, fainting, dizziness, etc.), broken needle, infection, and local hematoma, will be recorded with details such as the date of occurrence, time, degree, measurement related to the acupuncture treatment and causality with the acupuncture treatment.
All adverse events happened during the MRI scanning, mainly the occurrence of claustrophobia will also be recorded with details as above after the scanning.
Serious adverse events will be reported to the principal investigator immediately.

Quality control, data collection and management
All researchers will receive a series of training sessions before the start of this study, ensuring members involved to get fully understand of the study protocol and standard procedures. All the rehabilitation treatment will be conducted by qualified physical therapists who will receive standard operating procedures of physical rehabilitation techniques before the start. The data are required to be recorded on printed CRFs simultaneously at the visit points. The completed CRFs will be undergone the double-entry verification in EpiData Entry software. If the unconformity is found, the third person will search for printed CRFs to ensure. The medical imaging technicians will monitor the qualities of neuroimage data after every scanning. If the data are of quality dissatisfaction (such as head motion), we will call for the patients to receive an additional scanning. All the neuroimage data will be stored in dedicated hard drives after every scanning finished.

Clinical data analysis
Clinical data including demographic information, motor function scales variables and needle sensation rating, will be analysed by a statistician blinded to the group allocation using Statistical Package for the Social Science 20.0 (IBM Corp, Armonk, NY, USA). All the clinical data will be presented as mean±SD. Clinical data among the two groups will be compared by the two-sample t-test. A paired t-test will be carried out to make comparisons between the baseline and at the end of acupuncture treatment. A p value < 0.05 will be considered statistically significant.

Neuroimaging data preprocessing and analysis
Resting-state fMRI data processing For fMRI data, preprocessing and statistical analysis will be performed with Data  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y   resampled to 3×3×3 mm 3 . Finally, the data will be smoothed with a Gaussian kernel   of 6×6×6 mm 3 full width at half-maximum. For VMHC computation, briefly, after normalizing to the symmetric template and applying the refined transformation to the functional data, we will calculate the Pearson's correlation between preprocessed time-series of each pair of symmetric interhemispheric voxels. 21 Correlation values will be then Fisher Z transformed. The resultant values are referred to as the VMHC, which will be used for further group-level analysis. 32

DISCUSSION
Brain plasticity, strongly linked to the prognosis of poststroke hemiplegia, is becoming a research highlight worldwide. [35][36][37] Since acupuncture therapy owns a long history in treating poststroke hemiplegia in China, great efforts have been made to explore its underlying mechanism. However, there still remains some uncertain points. Luckily, the rapid development of MRI techniques provide a direct tool for investigating how the brain response towards acupuncture intervention. The neuroimage evidence will facilitate to illustrate the central principles of acupuncture.
Previous fMRI studies in stroke patients have shown that functional reorganization coupled with structural remodeling during the motor recovery, indicating the necessity of combined researches about brain function and structure. 38 39 In this study, the application of VMHC method will add to the understanding of functional changes of bilateral homotopic regions following the subcortical infarction, and the DTI technic will display the integrity of bilateral corticospinal tracts and their interactions for motor output after stroke, both of which will help to reflect bilateral function and structure after stroke objectively.
Based on bilateral connections, the integrated research of function and structure reorganization will give us a comprehensive presentation of acupuncture-induced brain recovery.
Deqi (needle sensation), or called qi arrival in Traditional Chinese Medicine  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y   (TCM) ancient books, is an indispensable part of acupuncture treatment, which is defined as a diverse sensation. 40 In theory of TCM, deqi is a sign that qi and blood of meridians and collaterals are activated to regulate the function of internal organs and to balance yin and yang, which is confirmed by the clinical trials and literature reviews that deqi is strongly linked to the efficacy. 41 42 Compared to the sham acupuncture treatment, hemiplegic patients treated with verum acupuncture exhibited a tendency to a larger activation in the contralateral motor cortex and a better motor function outcome, 43 indicating that the deqi sensation may become a stable predictor of therapeutic effects. 31 In our study, we also record the needle sensation and its intensity to preliminarily probe the essential association between deqi and the neuroimage phenomenon. In summary, under the bilateral observations on brain functional activations  Time schedule of enrolment, interventions (including any run-ins and washouts), assessments, and visits for participants. A schematic diagram is highly recommended (see Figure) _ Table1, Figure 1 Page 32 of 34

Methods: Assignment of interventions (for controlled trials)
Allocation: Sequence generation 16a Method of generating the allocation sequence (eg, computer-generated random numbers), and list of any factors for stratification. To reduce predictability of a random sequence, details of any planned restriction (eg, blocking) should be provided in a separate document that is unavailable to those who enrol participants or assign interventions _____ 9 ____ Allocation concealment mechanism 16b Mechanism of implementing the allocation sequence (eg, central telephone; sequentially numbered, opaque, sealed envelopes), describing any steps to conceal the sequence until interventions are assigned ____ 9 ____ Implementation 16c Who will generate the allocation sequence, who will enrol participants, and who will assign participants to interventions ____ 9,22 ____ Blinding (masking) 17a Who will be blinded after assignment to interventions (eg, trial participants, care providers, outcome assessors, data analysts), and how If blinded, circumstances under which unblinding is permissible, and procedure for revealing a participant's allocated intervention during the trial ____ N/A_____

Data collection methods 18a
Plans for assessment and collection of outcome, baseline, and other trial data, including any related processes to promote data quality (eg, duplicate measurements, training of assessors) and a description of study instruments (eg, questionnaires, laboratory tests) along with their reliability and validity, if known. Reference to where data collection forms can be found, if not in the protocol

11-16, Table1__
18b Plans to promote participant retention and complete follow-up, including list of any outcome data to be collected for participants who discontinue or deviate from intervention protocols ____ 17 _ Definition of analysis population relating to protocol non-adherence (eg, as randomised analysis), and any statistical methods to handle missing data (eg, multiple imputation) ____ N/A _

Methods: Monitoring
Data monitoring 21a Composition of data monitoring committee (DMC); summary of its role and reporting structure; statement of whether it is independent from the sponsor and competing interests; and reference to where further details about its charter can be found, if not in the protocol. Alternatively, an explanation of why a DMC is not needed Description of any interim analyses and stopping guidelines, including who will have access to these interim results and make the final decision to terminate the trial  patients and demonstrate the differences in brain response and clinical assessments.

Methods and analysis
This is a single-center, randomized controlled, paralleled neuroimaging trial, with patients, and outcome assessors blinded. Thirty stroke patients with motor dysfunction meeting the inclusion criteria will be randomly assigned (2:1) to receive either 10 sessions true or sham acupoints treatments (5 sessions per week for 2 weeks). All the participants will receive conventional standard medical care and rehabilitation. Motor function assessments and neuroimaging scanning will be conducted before and after the entire acupuncture treatment. The clinical and neuroimaging data will be analyzed respectively. The voxel-mirrored homotopic connectivity (VHMC) will be the primary outcome and the primary effect indicator. The secondary outcomes comprise clinical evaluations and neuroimaging assessments, which include Fugl-Meyer assessments (FMA), the National Institutes of Health stroke scales (NIHSS), fractional anisotropy (FA) and

The strength and limitations of this study
The application of randomized controlled neuroimaging trial will provide objective and valid evidence about how acupuncture treatment promotes the recovery of post-stroke hemiplegia.
The assessment of motor function, needle sensation and multimode neuroimaging scans will be used to judge both cerebral response and motor-functional effects of acupuncture treatment on post-stroke hemiplegia.
The research based on bilateral cerebral connections is closer to the actual state of coordinated regulation and integration of the acupuncture-induced brain recovery.
The settings of 2-weeks acupuncture treatment intervention mainly focused on the background of post-stroke hemiplegia, while it was a little slightly insufficient for one individual's whole recovery procedure. shown that post-stroke functional rehabilitation is the most primary method to decrease the disability rate and get functional recovery. 4  and National Institutes of Health (NIH) recommend acupuncture treatment as an alternative or as part of a comprehensive program in stroke rehabilitation, 6 and its positive effect has been confirmed by systematic reviews and multi-center clinical studies. 7 8 However, the underlying mechanisms of acupuncture for hemiplegia remain unclear, which partly restricts its widespread application in stroke recovery.

INTRODUCTION
Brain functional reorganization and structural remodeling are closely related to the prognosis of stroke recovery. Cerebral bilateral modulation and cooperation have significant influences on motor recovery. Physiologically, every achievement of human activities is the process of resource allocation and execution controlled by right and left hemispheres. 9 Increasing fMRI researches reported that, along with post-stroke motor recovery, there existed a pathologically enhanced or decreased activation pattern between ipsilesional primary motor cortex (M1) and contralesional M1. [10][11][12][13] In our previous studies, stroke patients with unilateral subcortical infarcts showed activations in multiple brain regions in both hemispheres. 14 All of the above proves that the bilateral issue is really important in the brain functional reorganization. To observe the changes of strength and location of bilateral connections, voxel-mirrored homotopic connection (VMHC) is an optimal neuroimaging indicator.
It reflects functional connectivity between any pair of symmetry interhemispheric voxels, which is a fundamental characteristic of the intrinsic functional architecture of the brain. 15 16 During the stroke hemiplegia recovery, local infarct always affects the remote cerebral function, not only involving the sensorimotor networks but also the cognitive and executive networks. 17 VMHC, the whole-brain based functional connectivity method, will provide more global information than other data processing methods (ReHo, ALFF, et al).
In our previous studies, we found that instant acupuncture at Yanglingquan (GB34) will improve the decreased functional connectivity between bilateral M1 of right-hemispheric subcortical stroke patients. 18 19 Whether long-term acupuncture intervention will facilitate the bilateral functional reorganization and structural remodeling, is worthy to be explored.
Consequently, from the perspective of bilateral cerebral connections, combined with changes in function and structure, figuring out what happens after unilateral cerebral infarction, is much closer to the distribution and integration of motor function in the actual recovery, which can better illustrate the central mechanism of acupuncture treatment.
Based on the viewpoint of bilateral connections, this study aims to: 1) assess the differences of brain response, motor assessments and needle sensation between true and sham acupoints treatment group; 2) characterize the influence of analyze the relationship between changes in brain activities and clinical assessments, in order to demonstrate the underlying mechanism of acupuncture treatment and its effects on clinical motor performances in hemiplegic patients.

Study design
This is a single-center, randomized controlled, paralleled neuroimaging study, with patients and assessors blinded to the group assignment. Stroke patients with motor dysfunction meeting the inclusion criteria will be randomly divided into true acupoints treatment group (TATG) and sham acupoints treatment group (SATG) by the ratio of 2:1. In addition to conventional standard medical care and rehabilitation, participants will receive 10 sessions of acupuncture treatment in two weeks (5 sessions per week). Motor function scales and MRI scanning will be conducted before and after the entire acupuncture treatment. The needle sensation assessment scale will be assessed after each acupuncture treatment.
The study procedures is detailed in Figure 1.
Insert Figure 1 here.

Patients population and recruitment strategy
Participants will be screened and recruited among the department of Neurology, participants. If the patients are eligible and interested in taking part in the study, they will be fully informed of the project including the study procedures, potential benefits and risks. Written informed consent will be obtained from the patient or their legally authorized representative before the allocation. Every participant has the right to withdraw from the study without any conditions.

Inclusion criteria
Patients will be included if they meet the following criteria: (1)

Exclusion criteria
Patients will be excluded if they meet any of the following criteria: (1) a history of neurologic or psychiatric disorders; any other health problems or poor physical conditions that may influence participation; (2) being pregnant or lactating; (3) any other brain structure damage or abnormalities identified by MRI examinations; (4) any history of alcohol or drug dependency; (5) any MRI contraindications.

Sample size considerations
When calculating the sample size of acupuncture-neuroimaging studies, the number  20 21 in addition, according to our previous studies, 20 subjects represent a reasonable sample size for stable cerebral response. 14 18 Under the condition of high homogeneity of lesion location in this study, we conclude 30 patients would ensure enough statistical power and provide repeatable results.

Randomization and blinding
An external professional statistician will generate a random sequence using Excel's rand function. Thirty patients who meet the inclusion criteria will be randomly assigned into either true acupoints treatment group (TATG) or sham acupoints treatment group (SATG) (2:1 ratio). The TATG and SATG will be coded A and B respectively. The code will be kept in an opaque envelope and subsequently sealed.
These envelopes will be sent to the chief principal who is not involved in patient recruitment and will take direct charge of treatment allocation. The acupuncturists are aware of the treatment group. While, rehabilitation therapists, motor function evaluators and participants are blinded to the treatment group. Patients in this trial will not be involved in the design, recruitment or conduction of the study. After the MRI scanning, the patients will be informed of the results of neuroimaging data in the form of pictures. The doctor in charge of the patient will be also informed of the results to optimize treatment and rehabilitation scheme.

INTERVENTIONS
In addition to conventional standard medical care and rehabilitation, all the participants will receive true or sham acupoints treatment.

Acupuncture Interventions
"Shou Zu Shi Er Zhen", a preferred acupuncture prescription for post-stroke motor dysfunction established by Leting Wang, will be taken as the acupuncture intervention prescription. Dr. Wang is a famous acupuncturist with more than 40 years of experience practicing in the Beijing Hospital of Traditional Chinese Medicine.
The prescription, named after the acupoints locations and its bilateral acupoints selection principles, consists of 12 acupoints bilaterally from five transport acupoints and original acupoints of twelve meridians. Focused on the pathogenesis of stroke, the prescription is designed to regulate qi and blood, balance yin and yang and attach importance to spleen and stomach which has the function of nourishing muscles.
In addition, two doctors with master's degrees and more than 3 years of clinical experience in acupuncture will be trained together and will use the same techniques.
After the first MRI scanning, the participants will receive 10-sessions acupuncture treatment (five times per week for two weeks).  3) Treatment course: Every participant will receive five sessions of treatment per week for two weeks. A fixed acupuncturist will conduct the whole treatment course for one participant. After each acupuncture treatment, participants will be asked to express the kind and intensity of "Deqi" so as to finish the needle sensation assessment scale. The acupuncturist will give a treatment compliance judgment (completion rates) at the end of acupuncture treatment.

Sham acupoints treatment group (SATG)
Based on Liu's method of sham acupoints 22 and after consultation with two experienced acupuncturists, we will take the location which is one inch beside the true point as the sham acupoint. Hence, we don't overemphasize the deqi aspects of treatment procedures and course in SATG will be the same as TATG's.

Conventional Treatment
All the participants will receive conventional treatment including the conventional standard medical care and rehabilitation.
Conventional rehabilitation treatment, comprised of physical therapy (PT) and occupational therapy (OT), will be applied by qualified rehabilitation therapists. The rehabilitation programs will be carried out five times per week for 2 weeks and the rehabilitation treatment (PT and OT) will last for approximately 1 hour each time.
Conventional standard medical care will comply with Chinese guidelines for diagnosis and treatment of acute ischemic stroke 2018. 23 The frequency of use, the average daily dose, the time of administration and the condition of drug combination will be recorded during the entire study.

MEASUREMENTS
The measurements consist of three parts including neuroimaging scanning assessments, motor function assessments, and needle sensation assessment. The study schedule is exhibited in Table 1.

SAFETY
Adverse events × × Table 1 Study schedule of enrollment, intervention, and assessments. At the baseline, and after the whole acupuncture treatment, Fugl-Meyer assessments (FMA) and NIH stroke scales (NIHSS) will be used to evaluate the motor function. MRI scanning will be performed to detect the brain functional and structural change before and after the treatment period. Needle Sensation Assessment Scale (NSAS) will be conducted after each treatment session.

Demographic and basic clinical information collection
The demographic information (name, sex, age, height, weight and working status), the history of stroke (time since the attack, lesions location and symptoms), and the history of other concomitant diseases will be obtained at the baseline. Vital signs (blood pressure, pulse, respiration rate and temperature) will be measured before each scanning. Germany) before and after the entire acupuncture treatment at Dongzhimen

MRI scanning and neuroimaging outcomes
Hospital, Beijing, China. Before each scanning, participants will be asked to rest for 20 minutes, and will be informed of MRI examination procedures and attentive matters. During the scanning process, all participants will be required to wear earplugs to isolate noises, keep eyes closed, not fall asleep and stay still. All participants will be in the supine position with the head fixed by foam pads to minimize the head movements as far as possible during the scanning.
The functional images will be collected by using a T2×WI Gradient Echo-Planar

Neuroimaging outcomes
The neuroimaging outcomes will be acquired after the MRI scanning and analysis, including voxel-mirrored homotopic connectivity (VMHC), Fractional anisotropy (FA) and Gray matter volume (GMV). VMHC will be taken as the primary outcome and the primary effect indicator. GMV and FA will be regarded as secondary outcomes.
As a functional parameter, VMHC is expressed as functional connectivity between any pair of symmetry interhemispheric voxels, which is an optimal parameter to reflect the bilateral functional connections. 15 16 FA is a diffusion tensor imaging parameter reflecting fiber density, axonal diameter, and myelination of white matter. 24 It has been verified from the systematic reviews and meta-analysis that FA is a biomarker for the prognosis of

Motor function assessments and outcomes
The following motor assessment scales, including FMA and NIHSS, will be conducted at the baseline and after the entire acupuncture treatment by a trained assessor  The assessment of NIHSS for every patient with cerebral infarction has become in this study, we still regard it as one of the secondary outcome indicators.

Needle Sensation Assessment
The needle sensation assessment scale (NSAS) will be conducted after every acupuncture treatment by the acupuncturists. The results of NSAS is a possible prognostic marker associated with the effectiveness of acupuncture and recovery of motor function, which will be taken as the additional outcomes.
"Deqi" is a needle response describing how the patients feel when they receive the needle insertion, which is regarded as a unique key predictor and an essential role in the therapeutic effectiveness of acupuncture for stroke recovery. 31 The self-designed Needle Sensation Assessment Scale we adopt has been tested in our previous studies, 32 which includes "Deqi" features ( sourness, numbness, tingling, aching and propagated feeling along the meridians) and rating of "Deqi". Patients will describe the intensity of needle sensation by visual analogue scale (VAS) (0 stands for none, 10 stands for unbearable).

Incidence of adverse events
Any adverse events which may happen during the acupuncture treatment, including acupuncture syncope reaction (e.g. sweating, fainting, dizziness, etc.), broken needle, infection, and local hematoma, will be recorded with details such as the date of occurrence, time, degree, measurement related to the acupuncture treatment and causality with the acupuncture treatment.
Any adverse events which may happen during the MRI scanning, mainly the occurrence of claustrophobia will also be recorded with details as above after the scanning.
Serious adverse events will be reported to the principal investigator immediately.

Quality control, data collection and management
All researchers will receive a series of training sessions before the start of this study, to ensure that they fully understand of the study protocol and standard procedures.
All the rehabilitation treatments will be conducted by qualified physical therapists who will receive standard operating procedures of the required physical rehabilitation techniques before the start of the study. The data are required to be recorded on printed CRFs simultaneously at the visit points. The completed CRFs will undergo the double-entry verification in EpiData Entry software. If the unconformity is found, the third person will search for printed CRFs to ensure consistency.
The medical imaging technicians will monitor the qualities of neuroimaging data after every scanning. If the data are of unsatisfactory quality (such as head motion), we will call for the patients to receive an additional scanning. All the neuroimaging data will be stored in dedicated hard drives after every scanning completed.

Clinical data analysis
Clinical data including demographic information, motor function scale variables and needle sensation rating, will be analyzed by a statistician blinded to the group allocation using Statistical Package for the Social Science 20.0 (IBM Corp, Armonk, NY, USA). All the clinical data will be presented as mean ± SD. Clinical data of the two A p value < 0.05 will be considered statistically significant.

Neuroimaging data preprocessing and analysis
Resting-state fMRI data processing For fMRI data, preprocessing and statistical analysis will be performed with Data For DTI data, preprocessing and statistical analyses will be processed with FSL (FMRIB Software Library) (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/) and SPM12 (https://www.fil.ion.ucl.ac.uk/spm). FA will be obtained using the tract-based spatial statistics (TBSS) analysis in FSL. 34 35 GMV will be generated by voxel-based morphometry (VBM) analysis using SPM12 software.

Neuroimaging data analysis
A paired t-test will be used to detect the differences of neuroimaging variables between pre-and after-treatment within a group. The two-sample t-test will be carried out to explore the differences of neuroimaging variables between TATG and SATG. Z-statistic maps of clusters significant will be defined by a cluster-level threshold of P < 0.05 and a voxel-wise level threshold of P < 0.005 based on Gaussian Random Field (GRF) theory. Pearson correlation analyses were performed with a threshold cluster level of P < 0.05 (Gaussian Random Field, GRF corrected) to test the correlations between the clinical data and neuroimaging properties. All the reported neuroimaging statistics will be colored and mapped in MNI (Montreal Neurological Institute) space.

DISCUSSION
Brain plasticity, strongly linked to the prognosis of post-stroke hemiplegia, is becoming a research highlight worldwide. [36][37][38] Since acupuncture therapy has a long history in treating poststroke hemiplegia in China, great efforts have been made to explore its underlying mechanism. However, there still remains some uncertain indicating the necessity of researches about the unity of the brain's function and brain's structure. 39 40 In this study, the application of VMHC method will add to the understanding of functional changes of bilateral homotopic regions following the subcortical infarction. The DTI technique will display the integrity of bilateral corticospinal tracts and their interactions for motor output after stroke. These MRI techniques will help to reflect bilateral function and structure after stroke objectively.
Based on bilateral connections, the integrated research of function and structure reorganization will give us a comprehensive presentation of acupuncture-induced brain recovery.
Deqi (needle sensation), or called qi arrival in Traditional Chinese Medicine (TCM) ancient books, is defined as a diverse sensation, and is an indispensable part of acupuncture treatment. 41 In theory of TCM, deqi is a sign that qi and blood of meridians and collaterals are activated to regulate the function of internal organs and to balance yin and yang, which is confirmed by the clinical trials and literature reviews that deqi is strongly linked to the efficacy. 42 43 Compared to the sham acupuncture treatment, hemiplegic patients treated with verum acupuncture In summary, under the bilateral observations on brain functional activations and structural changes, and the assessment of motor function and deqi, this study aims to investigate the neural mechanism of acupuncture treatment and express its effect of acupuncture treatment in an objective and visual way. We expect that our findings can provide a new perspective to illustrate the mechanism of acupuncture treatment and promote the widespread application of it.  Competing interests None declared.       Time schedule of enrolment, interventions (including any run-ins and washouts), assessments, and visits for participants. A schematic diagram is highly recommended (see Figure) _ Table1, Figure 1 Page  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59

The strength and limitations of this study
The application of the randomized controlled neuroimaging trial will provide objective and valid evidence about how acupuncture treatment promotes the recovery of post-stroke hemiplegia.
A series of motor function scales, the needle sensation assessment scale and multi-mode neuroimaging scanning will be used to judge the effects of acupuncture treatment on post-stroke hemiplegia.
The research based on cerebral bilateral connections is better for understanding the mechanism of coordination and integration in the acupuncture-induced brain recovery.
The 2-week treatment design is set up for the purpose to study the mechanism of acupuncture on post-stroke motor impairment, which is not the recommended course for stroke hemiplegia recovery.  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  and National Institutes of Health (NIH) recommend acupuncture treatment as an alternative or as part of a comprehensive program in stroke rehabilitation, 6 and its positive effect has been confirmed by systematic reviews and multi-center clinical studies. 7 8 However, the underlying mechanisms of acupuncture for hemiplegia remain unclear, which partly restricts its widespread application in stroke recovery.
Brain functional reorganization and structural remodeling are closely related to the prognosis of stroke recovery. Cerebral bilateral modulation and cooperation have significant influences on motor recovery. Physiologically, every achievement of human activities is the process of resource allocation and execution controlled by the right and left hemispheres. 9 Increasing fMRI researches reported that, along with post-stroke motor recovery, there existed a pathologically enhanced or decreased activation pattern between ipsilesional primary motor cortex (M1) and contralesional M1. [10][11][12][13] In our previous studies, stroke patients with unilateral subcortical infarcts showed activations in multiple brain regions in both hemispheres. 14 All of the above proves that the bilateral issue is really important in  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n l y the brain functional reorganization.
To observe the changes in strength and location of cerebral bilateral connections, voxel-mirrored homotopic connection (VMHC) is an optimal neuroimaging indicator. It reflects functional connectivity between any pair of symmetry interhemispheric voxels, which is a fundamental characteristic of the intrinsic functional architecture of the brain. 15 16 During the stroke hemiplegia recovery, local infarct always affects the remote cerebral function, not only involving the sensorimotor networks but also the cognitive and executive networks. 17 VMHC, the whole-brain based functional connectivity method, will provide more global information than other data processing methods (ReHo, ALFF, et al).
In our previous studies, we found that instant acupuncture at Yanglingquan (GB34) will improve the decreased functional connectivity between bilateral M1 of right-hemispheric subcortical stroke patients. 18 19 Whether long-term acupuncture intervention will facilitate the bilateral functional reorganization and structural remodeling, is worthy to be explored.
Consequently, from the perspective of cerebral bilateral connections, figuring out the changes in brain function and structure after unilateral cerebral infarction via neuroimaging tools helps to illustrate the central mechanism of acupuncture treatment.
Based on the viewpoint of bilateral connections, this study aims to: 1) assess the differences of brain response, motor assessments and needle sensation between true and sham acupoints treatment group; 2) characterize the influence of  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y   acupuncture treatment on cerebral functional activities and structural changes; 3) analyze the relationship between changes in brain activities and clinical assessments, in order to demonstrate the underlying mechanism of acupuncture treatment and its effects on clinical motor performances in hemiplegic patients.

Study design
This is a single-center, randomized controlled, paralleled neuroimaging study, with patients and assessors blinded to the group assignment. Stroke patients with motor dysfunction meeting the inclusion criteria will be randomly divided into true acupoints treatment group (TATG) and sham acupoints treatment group (SATG) by the ratio of 2:1. In addition to conventional standard medical care and rehabilitation, participants will receive 10 sessions of acupuncture treatment in two weeks (5 sessions per week). Motor function scales and MRI scanning will be conducted before and after the entire acupuncture treatment. The needle sensation assessment scale will be assessed after each acupuncture treatment.
The study procedures is detailed in Figure 1.
Insert Figure 1 here.

Patients population and recruitment strategy
Participants will be screened and recruited among the department of Neurology,

Rehabilitation and Acupuncture in Dongzhimen Hospital (Beijing, China) via
Electronic Medical Record from May 2018 to Dec 2020. Stroke patients with right or left hemiplegia who match the inclusion criteria will be regarded as the potential  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y participants. If the patients are eligible and interested in taking part in the study, they will be fully informed of the project information including the study procedures, potential benefits and risks. Written informed consent will be obtained from the patient or their legally authorized representative before the allocation. Every participant has the right to withdraw from the study without any conditions.

Inclusion criteria
Patients will be included if they meet the following criteria: (1)

Exclusion criteria
Patients will be excluded if they meet any of the following criteria: (1) a history of neurologic or psychiatric disorders; any other health problems or poor physical conditions that may influence participation; (2) being pregnant or lactating; (3) any other brain structure damage or abnormalities identified by MRI examinations; (4) any history of alcohol or drug dependency; (5) any MRI contraindications.

Randomization and blinding
An external professional statistician will generate a random sequence using Excel's rand function. Thirty patients who meet the inclusion criteria will be randomly assigned into either true acupoints treatment group (TATG) or sham acupoints treatment group (SATG) (2:1 ratio). The TATG and SATG will be coded A and B respectively. The code will be kept in an opaque envelope and subsequently sealed.
These envelopes will be sent to the chief principal who is not involved in patient recruitment and will take direct charge of treatment allocation. The acupuncturists are aware of the treatment group. While, rehabilitation therapists, motor function evaluators and participants are blinded to the treatment group.

Patient and public involvement
Patients in this trial will not be involved in the design, recruitment or conduction of

INTERVENTIONS
In addition to conventional standard medical care and rehabilitation, all the participants will receive true or sham acupoints treatment.

Acupuncture Interventions
"Shou Zu Shi Er Zhen", a preferred acupuncture prescription for post-stroke motor dysfunction established by Leting Wang, will be taken as the acupuncture intervention prescription. Dr. Wang is a famous acupuncturist with more than 40 years of experience practicing in the Beijing Hospital of Traditional Chinese Medicine.
The prescription, named after the acupoints locations and its bilateral acupoints selection principles, consists of 12 acupoints bilaterally from five transport acupoints and original acupoints of twelve meridians. Focused on the pathogenesis of stroke, the prescription is designed to regulate qi and blood, balance yin and yang and attach importance to spleen and stomach which has the function of nourishing muscles.
In addition, two doctors with master's degrees and more than 3 years of clinical experience in acupuncture will be trained together and will use the same techniques.
After the first MRI scanning, the participants will receive 10-sessions acupuncture treatment (five times per week for two weeks).  3) Treatment course: Every participant will receive five sessions of treatment per

True acupoints treatment group (TATG)
week for two weeks. A fixed acupuncturist will conduct the whole treatment course for one participant. After each acupuncture treatment, participants will be asked to express the kind and intensity of "Deqi" so as to finish the needle sensation assessment scale. The acupuncturist will give a treatment compliance judgment (completion rates) at the end of acupuncture treatment.

Sham acupoints treatment group (SATG)
Based on Liu's method of sham acupoints 22 and after consultation with two experienced acupuncturists, we will take the location which is one inch beside the true point as the sham acupoint. Hence, we don't overemphasize the deqi experience of the participants in SATG. Except for the locations of acupoints, other aspects of treatment procedures and course in SATG will be the same as TATG's.

Conventional Treatment
All the participants will receive conventional treatment including the conventional standard medical care and rehabilitation.
Conventional rehabilitation treatment, comprised of physical therapy (PT) and occupational therapy (OT), will be applied by qualified rehabilitation therapists. The rehabilitation programs will be carried out five times per week for 2 weeks and the rehabilitation treatment (PT and OT) will last for approximately 1 hour each time.
Conventional standard medical care will comply with Chinese guidelines for diagnosis and treatment of acute ischemic stroke 2018. 23 The concomitant medications will be recorded in printed CRFs with details (the using-frequency, the dosage and the administration).

MEASUREMENTS
The measurements consist of three parts including neuroimaging scanning assessments, motor function assessments, and needle sensation assessment. The study schedule is exhibited in Table 1.

SAFETY
Adverse events × × Table 1 Study schedule of enrollment, intervention, and assessments. At the baseline, and after the whole acupuncture treatment, Fugl-Meyer assessments (FMA) and NIH stroke scales (NIHSS) will be used to evaluate the motor function. MRI scanning will be performed to detect the brain functional and structural change before and after the treatment period. Needle Sensation Assessment Scale (NSAS) will be conducted after each acupuncture treatment session.

Demographic and basic clinical information collection
The demographic information (name, sex, age, height, weight and working status), the history of stroke (time since the attack, lesions location and symptoms), and the history of other concomitant diseases will be obtained at the baseline. Vital signs (blood pressure, pulse, respiration rate and temperature) will be measured before each scanning. The MRI examination will be performed with a 3.0 Tesla scanner (Siemens, Sonata

MRI scanning protocol
Germany) before and after the entire acupuncture treatment at Dongzhimen Hospital, Beijing, China. Before each scanning, participants will be asked to rest for 20 minutes, and will be informed of MRI examination procedures and attentive matters. During the scanning process, all participants will be required to wear earplugs to isolate noises, keep eyes closed, not fall asleep and stay still. All participants will be in the supine position with the head fixed by foam pads to minimize the head movements as far as possible during the scanning.
The functional images will be collected by using a T2×WI Gradient Echo-Planar

Neuroimaging outcomes
The neuroimaging outcomes will be acquired after the MRI scanning and analysis, including voxel-mirrored homotopic connectivity (VMHC), fractional anisotropy (FA) and gray matter volume (GMV). VMHC will be taken as the primary outcome and the primary effect indicator. GMV and FA will be regarded as secondary outcomes.
As a functional parameter, VMHC is expressed as functional connectivity between any pair of symmetry interhemispheric voxels, which is an optimal parameter to reflect the bilateral functional connections. 15 16 FA is a diffusion tensor imaging parameter reflecting fiber density, axonal diameter, and myelination of white matter. 24 It has been verified from the systematic reviews and meta-analysis that FA is a biomarker for the prognosis of

Motor function assessments and outcomes
The following motor assessment scales, including FMA and NIHSS, will be conducted at the baseline and after the entire acupuncture treatment by a trained assessor who is blind to the random sequence. The results of FMA and NIHSS will be taken as

Fugl-Meyer Assessment (FMA)
The Fugl-Meyer Assessment is highly recommended as a clinical and research tool for measuring sensorimotor stroke recovery, which has been demonstrated worldwide for more than 40 years in stroke population. 28 The FMA scale is a 100-  in this study, we still regard it as one of the secondary outcome indicators.

Needle Sensation Assessment
The needle sensation assessment scale (NSAS) will be conducted after every acupuncture treatment by the acupuncturists. The result of NSAS is a possible prognostic marker associated with the effectiveness of acupuncture and recovery of motor function, which will be taken as the additional outcome.
"Deqi" is a needle response describing how the patients feel when they receive the needle insertion, which is regarded as a unique key predictor and an essential role in the therapeutic effectiveness of acupuncture for stroke recovery. 31 The self-designed Needle Sensation Assessment Scale we adopt has been tested in our previous studies, 32 which includes "Deqi" features ( sourness, numbness, tingling, aching and propagated feeling along the meridians) and rating of "Deqi". Patients will describe the intensity of needle sensation by visual analogue scale (VAS) (0 stands for none, 10 stands for unbearable).

Incidence of adverse events
Any adverse events which may happen during the acupuncture treatment, including acupuncture syncope reaction (e.g. sweating, fainting, dizziness, etc.), broken needle, infection, and local hematoma, will be recorded with details such as the occurrence date, lasting time, severity degree, and the causality with the acupuncture treatment.

Quality control, data collection and management
All researchers will receive a series of training sessions before the start of this study, to ensure that they fully understand the study protocol and standard procedures. All the rehabilitation treatments will be conducted by qualified physical therapists who will receive standard operating procedures of the required physical rehabilitation techniques before the start of the study. The clinical data are required to be recorded in printed CRFs simultaneously at the visit points. The completed CRFs will undergo the double-entry verification in EpiData Entry software. If the unconformity is found, the third person will search for printed CRFs to ensure consistency.
The medical imaging technicians will monitor the qualities of neuroimaging data after every scanning. If the data are of unsatisfactory quality (such as obvious head motion), we will call for the patients to receive an additional scanning. All the neuroimaging data will be stored in dedicated hard drives after every scanning completed.

Clinical data analysis
Clinical data including demographic information, motor function scale variables and needle sensation rating, will be analyzed by a statistician using Statistical Package for the Social Science 20.0 (IBM Corp, Armonk, NY, USA). All the clinical data will be presented as mean ± SD. Clinical data of the two groups will be compared by the two-sample t-test. The paired t-test will be carried out to make comparisons  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y between the baseline and the end of acupuncture treatment. A p value < 0.05 will be considered statistically significant.

Neuroimaging data processing and analysis
Resting-state fMRI data processing and analysis For fMRI data, processing and statistical analysis will be performed with Data  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y The two-sample t test will be used to compare VMHC values between TATG and SATG. The paired t test will be conducted when comparing VMHC values before and after acupuncture treatment within the groups. All brain maps of clusters significant will be defined by a cluster-level threshold of P < 0.05 and a voxel-wise level threshold of P < 0.005 based on Gaussian Random Field (GRF) theory. Pearson correlation analysis will be performed with a threshold cluster level of P < 0.05 (GRF corrected) to test the correlations between the clinical data and VMHC. All the reported neuroimaging statistics will be colored and mapped in MNI space.
For DTI data analysis, nonparametric, permutation-based tests were carried out in group comparison by Randomize tool in FSL with 5000 permutations and threshold-free cluster enhancement (TFCE). The threshold for statistical significance will be set at P < 0.01, corrected for multiple comparisons. For GMV analysis, two sample t-test will be conducted between TATG and SATG. A paired t-test will be applied to test GMV change before and after acupuncture intervention in both groups respectively. The threshold for statistical significance will be set at P < 0.05 using family-wise error correction. Pearson correlation analysis will be performed  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y with a threshold cluster level of P < 0.05 (GRF corrected) to test the correlations between the clinical data and structural data.

DISCUSSION
Brain plasticity, strongly linked to the prognosis of post-stroke hemiplegia, is becoming a research highlight worldwide. [36][37][38] Acupuncture is an effective therapeutic strategy for post-stroke motor impairment but its mechanism remains elusive. Luckily, the rapid development of MRI techniques has provided a direct tool for investigating how the brain responds towards acupuncture intervention. The neuroimaging evidence will help illustrate the central principles and the effects of acupuncture.
Previous fMRI studies on stroke patients have shown that functional reorganization is coupled with structural remodeling during the motor recovery; this indicates the necessity of a combined research of the brain function and structure. 39 40 In this study, the application of VMHC method will add to the understanding of functional changes of bilateral homotopic regions following the subcortical infarction.
The DTI technique will display the integrity of bilateral corticospinal tracts and their interactions for motor output after stroke. These MRI techniques will help to reflect bilateral function and structure after stroke objectively. Based on bilateral connections, the integrated research of function and structure reorganization will give us a comprehensive presentation of acupuncture-induced brain recovery.
Deqi (needle sensation), or called qi arrival in Traditional Chinese Medicine (TCM) ancient books, is defined as a diverse sensation, and is an indispensable part  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y of acupuncture treatment. 41 In theory of TCM, deqi is a sign that qi and blood in meridians and collaterals are activated to regulate the function of internal organs and to balance yin and yang. It has been confirmed by clinical studies and literature reviews that deqi is tightly linked with the effectiveness of treatment. 42 43 Compared to the sham acupuncture treatment, hemiplegic patients treated with true acupuncture exhibited a tendency to a larger activation in the contralateral motor cortex and a better motor function outcome, 44 indicating that the deqi sensation may become a stable predictor of therapeutic effects. 32 In our study, we will also record the needle sensation and its intensity to preliminarily probe the essential association between deqi and the neuroimaging phenomenon.  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   F  o  r  p  e  e  r  r  e  v  i  e  w  o  n  l  y neuroimaging trial aims to investigate the neural mechanism of acupuncture treatment for post-stroke hemiplegia from the viewpoint of cerebral connections.
We expect that our findings can provide a new perspective to illustrate the mechanism of acupuncture treatment and promote the widespread application of it.

Ethics and dissemination
The protocol has been approved by the ethics committee of Dongzhimen Hospital affiliated to Beijing University of Chinese Medicine (DZMEC-KY-2018-04). The results of the neuroimaging trial will be disseminated through peer-reviewed publications and conferences.

Methods: Assignment of interventions (for controlled trials)
Allocation: Sequence generation 16a Method of generating the allocation sequence (eg, computer-generated random numbers), and list of any factors for stratification. To reduce predictability of a random sequence, details of any planned restriction (eg, blocking) should be provided in a separate document that is unavailable to those who enrol participants or assign interventions _____ 9 ____ Allocation concealment mechanism 16b Mechanism of implementing the allocation sequence (eg, central telephone; sequentially numbered, opaque, sealed envelopes), describing any steps to conceal the sequence until interventions are assigned ____ 9 ____ Implementation 16c Who will generate the allocation sequence, who will enrol participants, and who will assign participants to interventions ____ 9,23 ____ Blinding (masking) 17a Who will be blinded after assignment to interventions (eg, trial participants, care providers, outcome assessors, data analysts), and how ____ 9 _ ____ 17b If blinded, circumstances under which unblinding is permissible, and procedure for revealing a participant's allocated intervention during the trial ____ N/A_____

Methods: Data collection, management, and analysis
Data collection methods 18a Plans for assessment and collection of outcome, baseline, and other trial data, including any related processes to promote data quality (eg, duplicate measurements, training of assessors) and a description of study instruments (eg, questionnaires, laboratory tests) along with their reliability and validity, if known. Reference to where data collection forms can be found, if not in the protocol

12-17, Table1__
18b Plans to promote participant retention and complete follow-up, including list of any outcome data to be collected for participants who discontinue or deviate from intervention protocols ____ 18 _ Definition of analysis population relating to protocol non-adherence (eg, as randomised analysis), and any statistical methods to handle missing data (eg, multiple imputation) ____ N/A _

Methods: Monitoring
Data monitoring 21a Composition of data monitoring committee (DMC); summary of its role and reporting structure; statement of whether it is independent from the sponsor and competing interests; and reference to where further details about its charter can be found, if not in the protocol. Alternatively, an explanation of why a DMC is not needed Description of any interim analyses and stopping guidelines, including who will have access to these interim results and make the final decision to terminate the trial ____ N/A ____  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  46