Acupuncture for poststroke hemiplegia focusing on bilateral cerebral connections: study protocol for a randomized controlled trial

Background: 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 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 bilateral cerebral connections, the underlying mechanism of acupuncture in stroke rehabilitation remains research space to date. Methods: This is a single-center, randomized controlled, paralleled neuroimaging trial, with patients, outcome assessors, and data statisticians blinded. Stroke patients with motor dysfunction meeting the inclusion criteria will be randomly assigned (2:1) to receive either true acupoints treatment or sham acupoints treatment (5 sessions per week for 2 weeks). All the patients will receive conventional treatment and rehabilitation. Participants will undergo the neuroimaging scanning before and after the entire acupuncture treatment. The voxel-mirrored homotopic connectivity (VHMC) will be the primary outcome and the primary effect indicator. The secondary outcomes comprise clinical part and neuroimaging part, which included Fugl-Meyer assessments (FMA), the National Institutes of Health stroke scales (NIHSS), fractional anisotropy (FA) and gray matter volume (GMV). The Needle Sensation Assessment Scale (NSAS) is an additional outcome. The correlation analysis will be explored between the neuroimage indicators and clinical motor assessments. Discussion: From the perspective of bilateral connections, this trial will investigate the cerebral influence of acupuncture treatment on poststroke patients with motor dysfunction, promoting application of acupuncture in stroke rehabilitation. Trial registration: Chinese Clinical Trials Registry, ChiCTR 1800016263. Registered on 22 May, 2018.


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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 scales evaluators, participants and data statisticians are blinded to the treatment group.

Interventions
All the participants will receive conventional therapy and true or sham acupuncture treatment.

Conventional Treatment
All the participants will receive conventional treatment 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. Besides, conventional standard medical care will comply with Chinese guidelines for diagnosis and treatment of acute ischemic stroke 2018 [15]. The frequency of use, the average daily dose, the time of administration and the condition of combination drug will be recorded during the entire study.

Acupuncture Interventions
We will take "Shou Zu Shi Er Zhen" as the intervention prescription. "Shou Zu Shi Er Zhen", a preferred acupuncture prescription for poststroke motor dysfunction, is set up by Wang Leting. Dr.
Wang is a famous acupuncturist owned more than 40-years practice experience from Beijing Hospital of Traditional Chinese Medicine. The prescription, named after the acupoints locations and its bilateral acupoints selection principles, consists of 6 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 neuroimage scanning, the participants will receive five-sessions acupuncture treatments per week for two weeks. 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 and consultation of two experienced acupuncturists [18], 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 acupoints location, other aspects of treatment procedures in SATG will be the same as TATG's.

MRI Examinations
MRI Examination will be performed with a 3.0 Tesla scanner (Siemens, Sonata Germany) before and after the entire acupuncture treatment at Dongzhimen Hospital affiliated to Beijing University of Chinese Medicine, Beijing, China. Before each scanning, participants will be informed of examination procedures and attentive matters. All participants will be asked to rest for 30 minutes before the scanning, wear earplugs to isolate noises, keep eyes closed, not fall asleep and stay still during the scanning process. 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.
Scanning examinations, including resting-state blood oxygenation level-dependent imaging (RS-BOLD fMRI), diffusion tensor imaging (DTI) and high-resolution anatomical T1-weighted imaging (T1W1), are designed for detecting cerebral functional and structural changes.

RS-BOLD fMRI
The functional images will be collected by using a T2×WI Gradient Echo-Planar Echo Imaging (GRE-EPI) sequence with parameters as follows: TR=2000ms, TE=30ms, Matrix=128×128, Fov=240×240mm 2 , Slice thickness=5.0mm, Gap: 0mm, Phase encode direction: A>>P, Flip angle= 90º, Fat suppr: Fat sat. The whole procedures will last for 8 min 10 s. The resting-state fMRI data are investigated to explore the change of functional connectivity between two brain hemispheres after treatment.

DTI and T1W1
The structural images will be collected by using a T1-weighted 3D anatomical sequence with parameters as follows: slice thickness=1.0mm, TR/TE:1900ms/3.93ms, Fov=240×240, Matrix=256×256. The T1-weighted and DTI scan are used to localize lesions and refer to the pathology. The DTI procedures will last for 5 min 10 s while the T1W1 procedures will last for 4 min 10 s.

Demographic characteristic and basic disease information
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.

Outcome measurements Primary Outcome
(VMHC) value is a functional parameter, expressed as the RSFCbetween homotopic cerebral regions, which will be the primary outcome and primary effect indicator. RSFC is defined as the temporal correlation between spatially remote neurophysiological events [19]. VMHC method analysis is a voxelwise computation method to qualtify the RSFC between any pair of symmetric interhemispheric voxels, providign an insight to explore not only where the asymmetry functional brain regions are but also exhibit how much intensity the fuction connectivity is [20,21].

Secondary Outcomes
All the motor scales assessments will be performed by a trained assessor who is blinded to the random sequence. FMA and NIHSS will be conducted at the baseline and after the entire acupuncture treatment.

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 [22]. The FMA scale is a 100-point rating system consisting of upper/lowerextremity 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. The higher the scores, the better the motor function [23].

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 [24]. Meaningfully, the NIHSS is recommended as a quick tool in the emergency department while regarded as a strong predictor of a stroke patient's recovery [25]. 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 15 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 stroke severity.
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.

Fractional Anisotropy (FA)
Fractional Anisotrophy (FA) is a diffusion tensor imaging parameter reflecting fiber density, axonal diameter, and myelination of white matter [26]. It is verified from the systematic reviews and metaanalysis that FA is a biomarker for the prognosis of stroke recovery [27].

Gray Matter Volume (GMV)
GMV (gray matter volume) is a morphological parameter indicating the volumetric change of gray matter. It has been verified that the increased GMV in motor-related and cognitive-related cerebral regions are in positive correlation with motor recovery [28,29].

Needle Sensation Assessment Scale (NSAS)
The needle sensation assessment scale will be conducted after every acupuncture treatment by the acupuncturists.
"Deqi" is a needle response describing how the patients feel when they receive the needle insertion, which is regarded as a uniquely key predictor and an essential role in therapeutic effectiveness of acupuncture for stroke recovery [30]. Until now, there isn't a standard needle sensation assessment scale but all the self-designed scales consist of two parts. One is the kind of sensation, and the other is the intensity of the sensation. The self-designed Needle Sensation Assessment Scale we adopt has been tested in our previous studies [31]. We list the sensation including sourness, numbness, tingling, aching and propagated feeling along the meridians for option and make patients evaluate the intensity of sensation by visual analogue scale (VAS) with Chinese description.

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 clinical study, ensuring members involved to get fully understand of 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 underwent the double entry verification in EpiData Entry software. If the inconformity 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.

Data processing and analysis
For clinical data including demographics and motor function scales variables, statistical analyses will be conducted with Statistical Package for the Social Science 20.0 (IBM Corp, Armonk, NY, USA).

All the reported neuroimage statistics will be colored and mapped in MNI (Montreal Neurological
Institute) space.

Resting-state fMRI data processing
The preprocessing procedures of functional data are as follows: Firstly, discard the first-10-time points image to eliminate the effect of inhomogenous magnetization. Then, the slice timing and head motion would be corrected and data with excessive head motion (> 3.0 mm) or rotation ( > 3º) will be excluded. After that, the images will be normalized into the Montreal Neurological Institute template and resampled to 3×3×3 mm 3 . Finally, the data will be smoothed with a Gaussian kernel of 4×4×4 mm 3 full width at half-maximum.
VMHC values will be computed by the DPARSF software. For each subject, firstly, we will calculate the Pearson's correlation between preprocessed time-series of each pair of symmetric interhemispheric voxels [20]. The results will be transformed by Fisher z to get VMHC values, which will be used for further group-level analysis [32].

Structural data processing
For DTI analysis, we will calculate the diffusion tensor in AFNI firstly. Then, FA images will be generated by applying Tract-Based Spatial Statistics (TBSS) method in FSL, of which undergoes nonlinear alignment into MNI152 space. Next, do the affine transformation and create the mean of all aligned FA images. Apply thinning and get the mean FA "skeleton" representing the centers of all the reacts common to the group. Finally, threshold the FA skeleton at FA 0.2 to suppress areas of low mean FA and high intersubject variability [33,34].
For GMV processing and analysis, we will run SPM12 software on Matlab 2014a (Mathworks, Inc., Natick, MA, USA). Firstly, all the raw data will be co-registered and segmented into gray matter, white matter and CSF before normalization into the standard MNI152 space with resample of 2 mm ×2 mm ×2 mm. Then, smoothing will be performed within a Gaussian kernel of 4×4×4 mm 3 full width at halfmaximum. Voxel-based morphometry (VBM) analysis will be performed at voxel-wise level using SPM12.

Statistical analysis and correlation analysis
We will apply two-sample T test to compare FMA, NIHSS and Fisher's transformed z value by VMHC between TATG and SATG. While, linear regression analysis between motor function scales, NSAS and neuroimaging properties will be conducted in each group.

Discussion
Brain plasticity takes priority in the neuroscience field in the 21 st century, being the most important component of application and cognition of the brain. The revelation of how two hemispheres cooperate after damage will contribute to the recovery of hemiplegic patients and better use of rehabilitation therapies. In the guidance of the National Institute of Neurological Disorders and Stroke (NINDS) [35], many countries, including China, USA, Korea, Japan and so on, have made great efforts on studies of mechanism on experience-based method following stroke [36][37][38]. Since acupuncture therapy owns a long history in treating poststroke hemiplegia in China, the exploration of its underlying mechanism becomes a spotlight and can help to maximize the treatment outcome. The neuroimage evidence will facilitate to illustrate the central principles of acupuncture.
The introduction of two special MRI techniques, including functional magnetic resonance imaging (fMRI) and diffusion tensor imaging (DTI), has opened up visible avenues for exploring how the human brain works in physiological or pathological conditions, improving the precision of clinical decision, as well as helping develop rehabilitation strategies [27,39,40]. 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 [41,42].
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. The integrated research of function and structure reorganization will give us a comprehensive presentation of acupuncture-induced brain recovery.
Deqi, or called qi arrival in Traditional Chinese Medicine (TCM) ancient books, is an indispensable part of acupuncture treatment, which is defined as a diverse sensation (e.g. sourness, numbness, tingling, aching and propagated feeling along the meridians) [43]. 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 [44,45]. Similarly, compared to the sham acupuncture treatment, hemiplegic patients treated with verum acupuncture exhibited a tendency to a larger activation in contralateral motor cortex and a better motor function outcome [46], indicating that the deqi sensation may become a stable predictor of therapeutic effects [31]. Hence, deqi is crucial not only in clinical practice but also in related scientific researches. In our study, we also record the needle sensation and its intensity to preliminarily probe the essential association between deqi and the neuroimage phenomenon. reports [18], we perform the sham acupoints locating 1 cun beside the true one in this study.
In summary, under the bilateral observations on brain functional activations and structural changes, and the assessment on motor function of limbs, 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. Written informed consent will be obtained from the patient or their legally authorized representative before the allocation. Every participant will be fully informed of the project information including the potential benefits and risks. Table 1 Due to technical limitations, Table 1 is only available as a download in the supplemental files section. Figure 1