A multimodal MRI study of XNKQ acupuncture for limb dysfunction after ischemic stroke: a randomized controlled study protocol
XNKQ针灸治疗缺血性中风后肢体功能障碍的多模态磁共振成像研究：随机对照研究方案

Introduction 导言

Data (1) from the Global Burden of Disease study show that the absolute number of stroke episodes increased by 70.0% from 1990 to 2019, while the age-standardized rates of stroke incidence decreased by 17.0%. Stroke (2) is the leading cause of disability and death in adults in China. By 2019, the number of stroke patients in China was about 28.76 million (3), of which cerebral infarction accounted for 24.18 million. If it is not treated and prevented in time, cerebral ischemic stroke (CIS) patients will be disabled or even have a recurrence of cerebral infarction in a short period, which will seriously affect their life and health and increase their economic burden.
全球疾病负担研究的数据（1）显示，从 1990 年到 2019 年，脑卒中发病的绝对数增加了 70.0%，而年龄标准化的脑卒中发病率却下降了 17.0%。脑卒中（2）是导致中国成年人残疾和死亡的首要原因。截至 2019 年，中国脑卒中患者人数约为 2876 万人（3），其中脑梗死患者人数为 2418 万人。脑缺血性卒中（CIS）患者如不及时治疗和预防，将在短期内致残甚至脑梗死复发，严重影响患者的生命健康，增加经济负担。

About 12.5% of stroke survivors left with a disability in 2020 (2), equivalent to file.2 million people. After 12 months of follow-up, the rate of disability among stroke survivors was 14.8% at 3 months and 14.0% at 12 months. Therefore, how to effectively improve limb dysfunction and help patients return to daily life still needs to be further explored. In this process, exploring the brain’s mechanistic changes to improve treatment options can provide better help to patients.
2020 年，约有 12.5% 的中风幸存者会留下残疾（2），相当于 120 万人。经过 12 个月的随访，中风幸存者的残疾率在 3 个月时为 14.8%，12 个月时为 14.0%。因此，如何有效改善肢体功能障碍，帮助患者重返日常生活仍需进一步探索。在这一过程中，探索大脑的机理变化，改进治疗方案，可以为患者提供更好的帮助。

Functional magnetic resonance imaging (fMRI) (4) is a non-invasive and radiation-free imaging technique, and multiple sequences can show different brain functional changes, which is widely used to study brain mechanisms (5). It can monitor the activation of brain regions, and brain functional connectivity, guide clinical rehabilitation and suggest patient prognosis.
功能磁共振成像（fMRI）( 4) 是一种无创、无辐射的成像技术，多种序列可显示不同的脑功能变化，被广泛应用于研究脑机制( 5)。它可以监测脑区激活情况和脑功能连接，指导临床康复和提示患者预后。

Acupuncture is a traditional Chinese medicine treatment technique that is applied to a variety of diseases. Xingnao Kaiqiao (XNKQ) acupuncture was founded by academician Shi Xuemin of Tianjin University of Traditional Chinese Medicine. Its clinical effects have been satisfactorily verified. It is now widely used in post-stroke rehabilitation. And clinical data show its safety and efficacy. Further research is needed to support the mechanism by which XNKQ acupuncture acts on the brain in CIS patients. We used multimodal MRI technology to further explore the central mechanism by which XNKQ acupuncture exerts action in patients with post-stroke limb dysfunction.
针灸是一种传统中医治疗技术，适用于多种疾病。针刺疗法由天津中医药大学石学敏院士创立。其临床疗效得到了令人满意的验证。目前，它已被广泛应用于中风后的康复治疗。临床数据显示其安全性和有效性。针刺 XNKQ 对 CIS 患者大脑的作用机制还需要进一步研究。我们采用多模态磁共振成像技术，进一步探讨了XNKQ针刺对中风后肢体功能障碍患者的中枢作用机制。

Methods and analyses 方法和分析

Design and setting 设计和环境

This is the version of the protocol, dated 1 April 2023. The study protocol was designed in accordance with standard protocol Items: Recommendations for Interventional Trials (6) (Supplementary File 1). The study protocol was approved by the Ethics Committee of IRB of The First Teaching Hospital of Tianjin University of TCM (TYLL2023[K]031). The study will be conducted in the First Teaching Hospital of Tianjin University of Traditional Chinese Medicine. In this study, 40 patients with post-stroke limb dysfunction and 20 healthy people will be included, and patients will be randomized into 2 groups in 1:1 ratio. The intervention group will receive XNKQ acupuncture therapy and basic therapy, the control group only will receive basic therapy. Acupuncture treatment will be given 6 times a week for 2 weeks. Outcome indicators include clinical scales and imaging indicators. Healthy people will be accessed at baseline, patients will be evaluated at baseline and after 2 weeks. The flow chart of this study is shown in Figure 1. The schedules of this study is shown in Table 1.
这是 2023 年 4 月 1 日的协议版本。研究方案是根据标准方案项目设计的：6）（补充文件 1）。研究方案经天津中医药大学第一教学医院 IRB 伦理委员会批准（TYLL2023[K]031）。研究将在天津中医药大学第一教学医院进行。本研究将纳入 40 名中风后肢体功能障碍患者和 20 名健康人，按 1:1 的比例将患者随机分为两组。干预组接受 XNKQ 针灸疗法和基础疗法，对照组仅接受基础疗法。针灸治疗每周 6 次，持续 2 周。结果指标包括临床量表和影像学指标。健康人将在基线时接受评估，患者将在基线时和 2 周后接受评估。本研究的流程图如图 1 所示。本研究的时间表见表 1。

Recruitment and informed consent
招募和知情同意

Recruitment information will be available to patients in the form of posters at various locations in the hospital, and interested patients can contact the program leader by phone and e-mail. Eligible patients must meet the inclusion and exclusion criteria. If an applicant meets the study criteria, they will be invited to participate and sign written informed consent before the start of the study (Supplementary File 2). The following basic personal information will be collected: sex, age, height, weight, educational background, occupation, marital status, health insurance and related past health history, etc. These details of the participants will be for the record only and will never be revealed to any individual or organization not connected to the study.
招募信息将以海报的形式张贴在医院的各个位置，感兴趣的患者可以通过电话和电子邮件联系项目负责人。符合条件的患者必须满足纳入和排除标准。如果申请人符合研究标准，他们将被邀请参与研究，并在研究开始前签署书面知情同意书（补充文件 2）。我们将收集以下个人基本信息：性别、年龄、身高、体重、教育背景、职业、婚姻状况、医疗保险和相关既往健康史等。参与者的这些详细信息仅供记录之用，绝不会透露给任何与本研究无关的个人或组织。

Diagnostic criteria 诊断标准

The diagnostic criteria are as follows: Refer to the diagnostic criteria for CIS in Chinese Guidelines for Diagnosis and Treatment of Acute Ischemic Stroke in 2018, all patients had acute onset and neurological deficits lasting more than 24 h. Imaging showed the presence of infarct lesions and ruled out non-vascular causes.
诊断标准如下：参照《中国急性缺血性脑卒中诊治指南（2018年版）》中CIS的诊断标准，所有患者均急性起病，神经功能缺损持续时间超过24 h，影像学检查显示存在梗死病灶，排除非血管性原因。

Inclusion criteria 纳入标准

The inclusion criteria are as follows:
纳入标准如下

1. Patients group: 1.患者组：

1. Meets the above diagnostic criteria;
1.符合上述诊断标准；

2. First-ever clinical CIS occurred ≤2 weeks, the number of times ≤ 1;
2.首次临床 CIS 发生时间≤2 周，次数≤1；

3. 40–70 years old, right-handed;
3.40-70 岁，右撇子；

4. Unilateral basal ganglia infarction and the area of infarction is 3.0 ~ 5.0 cm in diameter at the largest level, with motor dysfunction and manual muscle testing showing muscle strength grades 1 to 4;
4.单侧基底节梗死，梗死面积最大处直径为 3.0 至 5.0 厘米，伴有运动功能障碍，徒手肌力测试显示肌力为 1 至 4 级；

5. No severe cognitive impairment and also mini-mental state examination (MMSE) with a score of 17 or above.
5.没有严重的认知障碍，同时小型精神状态检查（MMSE）得分在 17 分或以上。

6. Provided written informed consent to participate.
6.提供了参与的书面知情同意书。

2. Healthy people group
2.健康人群

1. Age and sex were matched with the patient group, right-handed;
1.患者组的年龄和性别与右撇子相匹配；

2. Subjects are in good health and free of organic and significant functional diseases;
2.受试者身体健康，无器质性和重大功能性疾病；

3. No previous family history of the mental and nervous system;
3.无精神和神经系统家族病史；

4. No stimulant drugs have been taken in the past 2 months;
4.过去两个月内未服用过兴奋剂；

5. Provided written informed consent to participate.
5.提供了参与的书面知情同意书。

Exclusion criteria 排除标准

The exclusion criteria are as follows:
排除标准如下

1. Patients with a history of stroke, or diseases such as brain tumors or rheumatoid arthritis that result in an mRS Score of ≥2;
1.有中风病史，或患有脑肿瘤或类风湿性关节炎等疾病，导致 mRS 评分≥2 分的患者；

2. Patients with severe heart, liver, kidney, blood system, endocrine system, and mental illness, or with other diseases that prevent them from cooperating with treatment;
2.患有严重的心脏、肝脏、肾脏、血液系统、内分泌系统和精神疾病的患者，或患有其他无法配合治疗的疾病的患者；

3. Patients with a history of metal implants in the past or with claustrophobia who are not able to finish in the MRI scanning room;
3.既往有金属植入史或有幽闭恐惧症，无法在核磁共振扫描室完成扫描的患者；

4. Persons with hearing and vision disorders that obstruct communication;
4.有听力和视力障碍，妨碍交流的人；

5. Pregnant and lactating women;
5.孕妇和哺乳期妇女；

6. Currently participating or have participated in other clinical studies that have not yet been completed for more than 3 months.
6.目前正在参与或曾经参与其他尚未完成超过 3 个月的临床研究。

Removed criteria 删除标准

Patients will be removed if they have:
如果患者有以下情况，将被移除

1. Patients with poor compliance during clinical trials who voluntarily withdrew from the study;
1.在临床试验期间依从性较差并自愿退出研究的患者；

2. Patients are not appropriate to continue the research who are with serious adverse reactions, serious complications, or deterioration of condition that occurred in the study;
2.在研究中出现严重不良反应、严重并发症或病情恶化的患者不适合继续研究；

3. The subjects did not complete the tasks as required (less than 50% percent of the tasks or receiving treatment of other trials) or had incomplete image observation data.
3.受试者未按要求完成任务（任务完成率低于 50%或接受其他试验的治疗）或图像观察数据不完整。

Randomization and blinding
随机化和盲法

An independent researcher will be responsible for generating random numbers and sealed in opaque envelope. After subjects agree to participate in this study, they will randomly obtain an assigned serial number. The identification cord and basic information about each enrolled patient will be recorded on the case report form. Indicator assessors will be not aware of specific subgroups. Information exchange between patients will be not allowed. An independent researcher will perform data management and statistical analysis tasks.
独立研究人员将负责生成随机数字，并将其密封在不透明的信封中。受试者同意参与本研究后，将随机获得分配的序列号。病例报告表将记录每位入组患者的识别码和基本信息。指标评估人员不知道特定的亚组。患者之间不允许交换信息。独立研究人员将负责数据管理和统计分析工作。

Interventions 干预措施

All patients will receive the conventional basic medications which refer to the Chinese guidelines for diagnosis and treatment of acute ischemic stroke2018 (7). All of these medications will be individualized to control blood pressure, blood sugar and lipid, and prevent platelet aggregation.
所有患者都将参照《中国急性缺血性脑卒中诊治指南2018》（7）接受常规基本药物治疗。所有这些药物都将进行个体化治疗，以控制血压、血糖和血脂，防止血小板聚集。

The difference is that the XNKQ group will receive 30 min of acupuncture treatment. We will use stainless steel disposable acupuncture needles (size 0.25 mm × 40 mm or 0.30 mm × 75 mm) (Changchun Aikang Medical Instrument Factory) for the treatment. All acupuncture therapists have more than 5 years of clinical experience and have uniform training to ensure that the treatments will be administered consistently.
不同之处在于，XNKQ 组将接受 30 分钟的针灸治疗。我们将使用不锈钢一次性针灸针（规格为 0.25 mm × 40 mm 或 0.30 mm × 75 mm）（长春爱康医疗器械厂）进行治疗。所有针灸治疗师都有 5 年以上的临床经验，并经过统一培训，以确保治疗效果的一致性。

Acupuncture group 针灸小组

Acupoints of XNKQ acupuncture included bilateral PC6, DU26, and affected side SP6. Patients will be in the supine position. Acupoints will be sterilized with 75% alcohol before acupuncture. The first acupuncture point to be needled will be PC6, then the needle body will be rotated to stimulate the acupoints with a speed of 40-60/min and a rotation angle of more than 180°. The second will be DU26, the needle body will be rotated 360° and fixed to the skin, and moved to stimulate the acupoint until the eye is moist or weeping. Finally, SP6 will be stimulated until the leg twitches 3 times.
针刺 XNKQ 的穴位包括双侧 PC6、DU26 和患侧 SP6。患者取仰卧位。针刺前用 75% 的酒精消毒穴位。首先针刺 PC6，然后旋转针体刺激穴位，速度为 40-60/分钟，旋转角度大于 180°。其次是 DU26，针体旋转 360°，固定在皮肤上，移动针体刺激穴位，直至眼睛湿润或流泪。最后刺激 SP6，直至腿部抽搐 3 次。

Depending on each patient’s tolerance level, the therapist adjusts the acupuncture strength until the patient achieves “deqi” without significant pain. Leave the needle in place for 30 min per treatment. Acupuncture 6 times per week for 2 weeks for a total of 12 times.
根据每位患者的耐受程度，治疗师会调整针灸力度，直到患者在无明显疼痛的情况下达到 "出气 "的效果。每次治疗留针 30 分钟。每周针灸 6 次，连续 2 周，共 12 次。

Non-acupuncture group 非针灸组

In the non-acupuncture group, patients will receive the same basic treatments as the acupuncture group except for acupuncture.
在非针灸组，除针灸外，患者将接受与针灸组相同的基本治疗。

Healthy group 健康组

The healthy group will not receive any interventions.
健康组不接受任何干预。

Sample size 样本量

This study focuses on MRI data. The sample size was calculated based on similar previous studies. Through reading the research literature on the mechanism of the functional effects of acupuncture on the brain, we found that the acute phase of stroke is generally characterized by an acute condition and poor compliance. Desmond et al. (8) showed that when analyzing functional MRI data with a free threshold of 0.05, it is probably necessary to include at least 12 subjects to achieve an activation efficacy of 80% or more at the single voxel level. Anecdotal evidence (9) suggests that fMRI studies typically involve 12 to 16 subjects per group. Therefore, we included 16 subjects in each group. Considering the high dropout rate, we set the 20% dropout rate and finally included 20 subjects per group. So, a total of 40 CIS patients and 20 healthy people will be enrolled.
本研究侧重于核磁共振成像数据。样本量是根据以往类似研究计算得出的。通过阅读有关针灸对大脑功能影响机制的研究文献，我们发现中风急性期一般具有病情急、依从性差的特点。Desmond 等人（8）的研究表明，在对自由阈值为 0.05 的功能磁共振成像数据进行分析时，可能需要纳入至少 12 个受试者，才能在单个体素水平上达到 80% 或以上的激活效果。轶事证据（9）表明，fMRI 研究通常每组包括 12 到 16 名受试者。因此，我们在每组中纳入了 16 名受试者。考虑到辍学率较高，我们设定了 20% 的辍学率，最终每组纳入 20 名受试者。因此，我们将总共纳入 40 名 CIS 患者和 20 名健康人。

Outcome measures 成果措施

The study period is 2 weeks. Patients in both groups will be evaluated at baseline and week 2, the healthy individuals only at enrollment. All outcomes will be measured by evaluators who will be blinded to group allocation. All evaluators will receive the same training on these evaluations.
研究时间为两周。两组患者都将在基线和第 2 周接受评估，健康人只在注册时接受评估。所有结果都将由评估人员进行测量，他们对组别分配不设盲区。所有评估人员都将接受相同的评估培训。

Primary outcomes 主要成果

Blood oxygen level-dependent functional magnetic resonance imaging (BOLD-fMRI) indirectly evaluates neuronal activity by monitoring changes in blood oxygenation (10). Amplitude of low frequency fluctuation (ALFF) and regional homogeneity (ReHo) are common evaluation indicators (11). ALFF represents the spontaneous activity of neurons, and its derivative, fraction amplitude of low frequency fluctuation (fALFF), can better remove noise signals such as heartbeat and respiration, and is more sensitive and precise. ReHo responds to intervoxel activity synchronization by calculating the consistency of voxels over time series. The two indicators have better repeatability and clear physiological significance. Functional connectivity (FC) reflects functional integration between regions.
血氧水平依赖性功能磁共振成像（BOLD-fMRI）通过监测血氧变化间接评估神经元活动（10）。低频波动幅度（ALFF）和区域均匀性（ReHo）是常用的评估指标（11）。ALFF 代表神经元的自发活动，其衍生物低频波动幅度分数（fraction amplitude of low frequency fluctuation，fALFF）能更好地去除心跳和呼吸等噪声信号，灵敏度和精确度更高。ReHo 通过计算体素在时间序列上的一致性来反应体素间活动的同步性。这两个指标具有更好的可重复性和明确的生理意义。功能连通性（FC）反映了区域之间的功能整合。

Voxel-based morphometry (VBM) is a structural MRI quantitative analysis technique in which gray matter volume or gray matter density can be obtained by calculating the gray matter.
基于体素的形态计量学（VBM）是一种结构磁共振成像定量分析技术，通过计算灰质，可获得灰质体积或灰质密度。

Diffusion tensor imaging (DTI) technique traces fiber bundle alignment and identifies structural changes in white matter fibers (12), and fractional anisotropy (FA) is the most commonly used measure reflecting the integrity of white matter fiber bundles (13).
弥散张量成像（DTI）技术可追踪纤维束的排列并识别白质纤维的结构变化（12），而分数各向异性（FA）是反映白质纤维束完整性最常用的测量方法（13）。

Arterial spin labeling (ASL) is a magnetic resonance perfusion imaging technique that can be used to detect cerebral blood perfusion (14). As an important indicator, cerebral blood floot (CBF) can reflect cerebral blood flow perfusion (15).
动脉自旋标记（ASL）是一种磁共振灌注成像技术，可用于检测脑血流灌注（14）。作为一项重要指标，脑浮游血流（CBF）可以反映脑血流灌注情况（15）。

Secondary outcomes 次要结果

National Institutes of Health Stroke Scale (NIHSS), modified Rankin Scale (mRS), Fugl–Meyer Assessment (FMA), Modified Barthel Index (MBI) and EuroQol Five Dimensions Questionnaire (EQ-5D) will be assessed before and after 2 weeks of treatment as secondary outcomes.
美国国立卫生研究院卒中量表（NIHSS）、改良兰金量表（mRS）、福格尔-迈耶评估（FMA）、改良巴特尔指数（MBI）和欧洲五维问卷（EQ-5D）将作为次要结果在治疗前和治疗 2 周后进行评估。

FMA (16) is widely used in motor function assessment which can reflect the level of limb function recovery in patients with post-stroke disability. The scale focuses on limb joint flexibility and muscle strength, including 66 points for the upper limbs and 34 points for the lower limbs, with a total score of 100 as normal. Currently, the Spanish, Russian, and German versions of the FMA are used for clinical assessment with high sensitivity and validity. Consistency of motor scores in patients with post-stroke dysfunction due to harmonization of kinematic measurement tools (17).
FMA（16）广泛应用于运动功能评估，可反映脑卒中后残疾患者的肢体功能恢复水平。该量表侧重于肢体关节灵活性和肌肉力量，其中上肢 66 分，下肢 34 分，总分 100 分为正常。目前，西班牙文、俄文和德文版的 FMA 被用于临床评估，具有较高的灵敏度和有效性。由于运动学测量工具的统一，卒中后功能障碍患者的运动评分具有一致性（17）。

NIHSS (18) can quickly measure the severity of stroke and is recommended as a valid tool for stroke in the emergency department. It consists of 15 items with a total score of 42, the higher the score, the more severe it is. Numerous data demonstrate its reliability, validity, ability to predict stroke outcomes, and strong correlation with infarct foci on imaging. The NIHSS only provides a cursory assessment of the patient’s impairment and does not provide detailed information.
NIHSS ( 18) 可快速测量中风的严重程度，被推荐为急诊科治疗中风的有效工具。它由 15 个项目组成，总分为 42 分，分数越高，病情越严重。大量数据证明了其可靠性、有效性、预测卒中预后的能力以及与影像学梗死灶的强相关性。NIHSS 仅能粗略评估患者的损伤程度，不能提供详细信息。

The mRS (19) is highly reproducible and has convergence with other disability scales. Compared to the Barthel Index, mRS is reliable in that it predicts outcomes more accurately. The World Health Organization (WHO) (20) recommends the mRS as an indicator of stroke outcome. The mRS is categorized into 7 grades covering physical function and ability to live, allowing for assessment of patient prognosis.
mRS ( 19) 具有高度的可重复性，并与其他残疾量表趋同。与 Barthel 指数相比，mRS 的可靠性在于它能更准确地预测预后。世界卫生组织（WHO）( 20) 推荐使用 mRS 作为卒中预后的指标。mRS 分为 7 级，涵盖身体功能和生活能力，可用于评估患者的预后。

The MBI is a 100-point scale that assesses 10 essential abilities of patients in their daily lives, and is one of the more widely used and researched methods of assessing activities of daily living. EQ-5D has five dimensions to measure health, including mobility, self-care, daily activities, pain/discomfort, and anxiety/depression. EQ-5D-5L (21) scale which each dimension is divided into five levels, increasing the reliability, validity, and sensitivity of the measurement tool.
MBI 是一个 100 分的量表，用于评估患者在日常生活中的 10 种基本能力，是一种应用较广、研究较多的日常生活活动评估方法。EQ-5D 有五个维度来衡量健康状况，包括行动能力、自理能力、日常活动能力、疼痛/不适以及焦虑/抑郁。EQ-5D-5L ( 21) 量表将每个维度分为五个等级，提高了测量工具的可靠性、有效性和敏感性。

Data acquisition 数据采集

Data will be acquired by a Siemens Skyra 3.0 T MRI scanner (Siemens Healthineers) equipped with a 32-channel head coil. For BOLD-fMRI images, we used an echo-planar imaging (EPI) sequence (repetition time (TR) = 2,000 ms, echo time (TE) = 30 ms, flip angle = 90°, matrix = 64 × 64, field of view (FOV) = 220 mm × 220 mm, resolution = 3.4 × 3.4 × 3.0 mm³, gap = 0.99 mm, 240 time points, 37 interleaved axial slices). The structural image of the whole brain will be acquired for each participant using the T1 sequence (TR = 2,000 ms, TE = 1.97 ms, FOV = 256 mm × 256 mm, matrix = 192 × 192, voxel size = 1 × 1 × 1 mm³). White matter fiber bundle structure will be acquired from the DTI sequence (voxel size = 2 × 2 × 2 mm³, TE = 75 ms, TR = 8,200 ms, 70 slices, matrix = 110 × 110, FOV = 220 × 220 mm², b = 0,1,000 s/mm², and 64 diffusion directions). The cerebral perfusion imaging will be acquired by ASL sequence (TR = 4,000 ms, TE = 16.18 ms, matrix = 64 × 64, FOV = 220 × 220 mm², voxel size = 3.4 × 3.4 × 3.0 mm³ TI1/TI = 700/1990 ms). Subjects’ heads will be immobilized by cushioned supports, and they will wear earplugs to protect against MRI gradient noise throughout the experiment.
数据将由配备 32 通道头部线圈的西门子 Skyra 3.0 T MRI 扫描仪（Siemens Healthineers）采集。对于BOLD-fMRI图像，我们使用回声平面成像（EPI）序列（重复时间（TR）= 2,000 ms，回波时间（TE）= 30 ms，翻转角= 90°，矩阵= 64 × 64，视场（FOV）= 220 mm × 220 mm，分辨率= 3.4 × 3.4 × 3.0 mm ³ ，间隙= 0.99 mm，240个时间点，37个交错轴向切片）。将使用 T1 序列（TR = 2,000 ms，TE = 1.97 ms，FOV = 256 mm × 256 mm，矩阵 = 192 × 192，体素大小 = 1 × 1 × 1 mm ³ ）为每位参与者采集全脑结构图像。白质纤维束结构将通过 DTI 序列获取（体素大小 = 2 × 2 × 2 mm ³ ，TE = 75 ms，TR = 8,200 ms，70 个切片，矩阵 = 110 × 110，FOV = 220 × 220 mm ² ，b = 0,1,000 s/mm ² ，64 个扩散方向）。脑灌注成像将采用 ASL 序列（TR = 4,000 ms，TE = 16.18 ms，矩阵 = 64 × 64，FOV = 220 × 220 mm ² ，体素大小 = 3.4 × 3.4 × 3.0 mm ³ TI1/TI = 700/1990 ms）。受试者的头部将由软垫支撑固定，并在整个实验过程中佩戴耳塞以防止核磁共振成像梯度噪声。

For the observation steps, we will communicate with the patient in advance about the length of the scanning, so that the patient will not feel anxious if the time is too long. The subject’s ears will be plugged with cotton balls. When the patient is ready, the patient’s entire brain will be scanned. During resting-state scanning, patients will be instructed to relax, close their eyes, remain still, and avoid thinking activities as much as possible.
在观察步骤中，我们会事先与患者沟通扫描时间的长短，以免患者因时间过长而感到焦虑。我们会用棉球塞住受试者的耳朵。当患者准备就绪后，将对患者的整个大脑进行扫描。在静息状态扫描过程中，患者会被要求放松、闭眼、保持静止，并尽量避免思考活动。

Adverse events and safety monitoring
不良事件和安全监测

Adverse events will be assessed and managed by designated personnel within 24 h. Patients may voluntarily inform their physician of the presence of an AE at the time of the visit and during follow-up, or they may be screened for the presence of an AE by physical examination, laboratory tests, or other methods of assessment. These data will be evaluated by two investigators and documented on the CRF, including those taken by the instructing practitioner for acupuncture treatments after an AE occurs, such as discontinuance of acupuncture, resumption of acupuncture after a pause, reduction in the frequency or adjustment of the treatment regimen, continuation of acupuncture, and other interventions taken. If the adverse event is accompanied by medication or other treatment, it should be documented at the same time. Subjects may withdraw from the trial at their own discretion, or the physician may decide whether the patient should continue or terminate the study by evaluation.
患者可在就诊时和随访期间主动告知医生是否存在不良反应，也可通过体格检查、实验室检测或其他评估方法筛查是否存在不良反应。这些数据将由两名研究人员进行评估，并记录在 CRF 上，包括发生 AE 后针灸治疗指导医师采取的措施，如停止针灸、暂停后恢复针灸、减少频率或调整治疗方案、继续针灸以及采取的其他干预措施。如果不良事件伴随药物或其他治疗，则应同时记录。受试者可自行决定退出试验，或由医生通过评估决定患者是否应继续或终止研究。

Data collection and statistical analysis
数据收集和统计分析

Data will be collected using the paper CRFs which will be kept in a locked cabinet at the end of the trial. Statistical analysis of data will be performed by an independent researcher. For dislocated subjects, we will record the number and reason for termination, especially adverse events. Data will be analyzed using the R software version R-4.2.2. Statistical significance is defined as a p value of <0.05.
数据收集将使用纸质 CRF，试验结束后，CRF 将保存在上锁的柜子里。数据统计分析将由独立研究人员进行。对于脱位的受试者，我们将记录终止试验的人数和原因，尤其是不良事件。数据将使用 R 软件 R-4.2.2 版进行分析。统计学意义的定义是 p 值小于 0.05。

For comparisons of baseline material, measurements that follow a normal distribution overall will be expressed as mean ± standard deviation ( $\overline{X}$ ± SD), and the whole data that do not follow a normal distribution will be expressed as median (interquartile range, IQR) [M (P25-P75)]. Count data will be expressed as the number of cases (n) and percentage (%) using the X² test. The t-test or Mann–Whitney U-test will be chosen to compare the two groups’ scores on each scale based on a normal distribution and homogeneous variance. For within-group comparisons, changes in scores will be assessed by paired t-test or Wilcoxon rank-sum test. For subjects who discontinued or deviated from the intervention, listwise deletion or multiple interpolation will be used.
对于基线材料的比较，总体符合正态分布的测量数据将以平均值±标准差（ $\overline{X}$ ±SD）表示，不符合正态分布的全部数据将以中位数（四分位间，IQR）[M（P25-P75）]表示。计数数据将用 X ² 检验法表示为病例数（n）和百分比（%）。根据正态分布和同质性方差，将选择 t 检验或 Mann-Whitney U 检验来比较两组在每个量表上的得分。对于组内比较，得分变化将通过配对 t 检验或 Wilcoxon 秩和检验进行评估。对于中断或偏离干预的受试者，将采用列表删除或多重内插法。

The multimodal MRI images will be analyzed by MATLAB 2023a and the statistical parameter graph software Statistical Parametric Mapping (SPM)12. Before data processing, all data will be removed the first 10 time points to retain the stabilized images; and adjust for differences arising from the time points, after which orientation alignment will be performed; and all aligned functional images will be aligned to the montreal neurological institute (MNI) space with a voxel standard size of 3 mm × 3 mm × 3 mm, and then the functional images will be smoothed with a Gaussian kernel of 6 mm × 6 mm × 6 mm. Afterward, the images will be datamined by extracting the indicators. Data will be analyzed with two independent samples t-tests to compare the differences between the patient group before treatment and the healthy group, as well as the differences between the acupuncture group and the no-acupuncture group after treatment. The longitudinal changes in MRI indexes before and after the respective treatments of the two groups of patients will be observed with paired t-tests. Age, gender, BMI, blood pressure, and history of diabetes will be used as covariates in data analysis.
多模态 MRI 图像将通过 MATLAB 2023a 和统计参数图软件统计参数图（SPM）12 进行分析。数据处理前，所有数据将去除前 10 个时间点，以保留稳定图像；并调整时间点产生的差异，然后进行方向对齐；所有对齐后的功能图像将对齐到蒙特利尔神经研究所（MNI）空间，体素标准尺寸为 3 mm × 3 mm × 3 mm，然后用 6 mm × 6 mm × 6 mm 的高斯核对功能图像进行平滑处理。然后，通过提取指标对图像进行数据化。数据将通过两个独立样本 t 检验进行分析，以比较治疗前患者组与健康组之间的差异，以及治疗后针刺组与未针刺组之间的差异。用配对 t 检验观察两组患者各自治疗前后 MRI 指标的纵向变化。年龄、性别、体重指数、血压和糖尿病史将作为协变量进行数据分析。

Finally, the statistically significant MRI index values of the acupuncture group will be extracted and analyzed by Pearson correlation with the clinical scores of this group before and after treatment.
最后，将提取针灸组具有统计学意义的磁共振成像指标值，并与该组患者治疗前后的临床评分进行皮尔逊相关分析。

Trial status 试用情况

The trial will begin recruitment and treatment on 7 December 2023, and is expected to be completed by 30 December 2025.
试验将于 2023 年 12 月 7 日开始招募和治疗，预计将于 2025 年 12 月 30 日完成。

Quality control 质量控制

In order to implement the project smoothly, we will rigorously standardize the intervention and evaluation processes. Acupuncturists with more than five years of experience will be familiar with the conduct of intervention and control group interventions to ensure consistency in the study. The assessors will be uniformly trained on the scales to ensure the reliability of the data. Ethics committee members will also review implementation throughout the trial.
为了顺利实施项目，我们将严格规范干预和评估流程。拥有五年以上经验的针灸师将熟悉干预组和对照组干预的操作，以确保研究的一致性。评估人员将接受统一的量表培训，以确保数据的可靠性。伦理委员会成员还将在整个试验过程中对实施情况进行审查。

Discussion 讨论

Due to the specificity of the intervention, we were unable to achieve a double-blind design. Therefore, we design a single-blind, randomized controlled trial to evaluate the brain mechanism of the acupuncture group in the treatment of limb dysfunction in comparison to the non-acupuncture group. Patients who meet the inclusion criteria will be enrolled and randomly assigned to either the XNKQ group or the control group. Indicator assessors who are not aware of the subgroups will be objective in making indicator evaluations to avoid bias.
由于干预的特殊性，我们无法实现双盲设计。因此，我们设计了一项单盲随机对照试验，以评估针灸组与非针灸组治疗肢体功能障碍的脑机制。符合纳入标准的患者将被随机分配到 XNKQ 组或对照组。不了解亚组情况的指标评估员将客观地进行指标评估，以避免偏差。

The fMRI is widely used in the study of brain mechanisms of acupuncture therapy for stroke. Past studies of cerebral mechanisms in patients with cerebral infarction, were focused on single acupoint such as Zusanli (ST36) and Yanglingquan (GB34). Zou et al. (22–24) studied GB34 and found that this acupoint can activate multiple sensory and motor imagery areas in patients. The FC between bilateral M1 was decreased in patients with cerebral infarction and increased after acupuncture on GB34. The researchers also found that patients tended to have increased needling effects early in the stroke period (within 1 month) and decreased responses thereafter (1–3 months). Therefore, it is necessary to intervene acupuncture treatment as soon as possible. ST36 (25) has been shown to improve post-stroke motor deficits by activating the primary motor cortex and somatosensory-motor areas. In another randomized controlled trial, ST36 (26) also increased the compensatory function of the lateral circulation in the brains of stroke patients. In addition, researchers have studied the combination of multiple acupoints and found that the combination of Baihui-Yintang-Neiguan (27) improves cerebral blood perfusion in post-stroke patients.
fMRI 广泛应用于针灸治疗中风的脑机制研究。以往对脑梗死患者脑机制的研究主要集中在单个穴位上，如足三里（ST36）和阳陵泉（GB34）。Zou 等人 ( 22- 24) 对 GB34 进行了研究，发现该穴位能激活患者的多个感觉和运动意象区。在针刺 GB34 后，脑梗塞患者双侧 M1 之间的 FC 值降低，而双侧 M1 之间的 FC 值升高。研究人员还发现，患者往往在中风早期（1 个月内）针刺效果增强，之后（1-3 个月）反应减弱。因此，有必要尽快干预针刺治疗。ST36 ( 25) 通过激活初级运动皮层和躯体感觉运动区，改善了中风后的运动障碍。在另一项随机对照试验中，ST36 ( 26) 还增强了中风患者大脑侧支循环的代偿功能。此外，研究人员还对多个穴位的组合进行了研究，发现白会-印堂-内关（27）的组合能改善中风后患者的脑血流灌注。

In this trial, we will study the brain mechanisms produced by the main point of XNKQ acupuncture in cerebral infarction patients. The XNKQ acupuncture method produces therapeutic effects by stimulating the combination of three acupoints: Neiguan (PC6), Shuigou (DU26), and Sanyinjiao (SP6). It has been widely used in the rehabilitation of stroke patients and has shown remarkable effects. In a meta-analysis of 12 randomized controlled studies of acute stroke, Yang et al. (28) found that XNKQ acupuncture significantly reduced the disability rate and improved the clinical efficacy and quality of life. In another systematic review (29), they analyzed the long-term efficacy of XNKQ and found reduced recurrence, disability, and mortality rates. Scholars also have conducted a lot of basic research (30) on acupuncture to explain the mechanism of XNKQ acupuncture. However, its brain mechanisms remain unknown. Till Nierhaus et al. (31) studied healthy individuals using the combination of PC6-DU26-SP6 and found that this combination could have an effect on functional connectivity in motor areas minutes after acupuncture. They also found that the point location only has an impact when the acupuncture points are stimulated. Based on this, we investigate the sustained effect of this combination of acupoints in stroke patients with motor dysfunction.
在本试验中，我们将研究针刺 XNKQ 主穴对脑梗塞患者产生的脑机制。XNKQ 针刺法通过刺激三个穴位的组合产生疗效：内关（PC6）、水沟（DU26）和三阴交（SP6）三个穴位的组合产生治疗效果。该方法已被广泛应用于中风患者的康复治疗，并取得了显著效果。Yang 等人 ( 28) 对 12 项急性中风随机对照研究进行了荟萃分析，发现针刺 XNKQ 能显著降低致残率，提高临床疗效和生活质量。在另一篇系统综述（29）中，他们分析了 XNKQ 的长期疗效，发现其降低了复发率、致残率和死亡率。学者们还对针灸进行了大量基础研究（30），以解释 XNKQ 针灸的机制。然而，其大脑机制仍然未知。Till Nierhaus 等人（31）使用 PC6-DU26-SP6 组合对健康人进行了研究，发现这种组合可以在针刺后几分钟对运动区的功能连接产生影响。他们还发现，只有在刺激穴位时，穴位位置才会产生影响。在此基础上，我们研究了这种穴位组合对运动功能障碍的中风患者的持续影响。

Zhang et al. (5) concluded that in terms of experimental design, previous research commonly used fMRI, while in terms of analytical indicators, ALFF, ReHo, and FC were more commonly used In our experiments, we will use multimodal MRI techniques to analyze brain function, also focusing on gray matter, white matter fiber tracts, and cerebral blood flow perfusion. Finally, in order to explore the predictive power of the above indexes for patient prognosis, we will evaluate the correlation of brain indicators with the clinical symptom indexes.
在我们的实验中，我们将使用多模态 MRI 技术分析脑功能，同时关注灰质、白质纤维束和脑血流灌注。最后，为了探索上述指标对患者预后的预测能力，我们将评估脑部指标与临床症状指标的相关性。

In summary, the results of this study will be expected to provide an argument for the mechanism of the action of XNKQ. Exploring its central mechanisms with multimodal MRI can provide an evidence-based basis for future clinical promotion.
总之，本研究的结果将有望为 XNKQ 的作用机制提供论证。利用多模态磁共振成像探索其中心机制，可为今后的临床推广提供循证依据。

Ethics statement 道德规范声明

The study involving human subjects was reviewed and approved by the Ethics Committee of IRB of The First Teaching Hospital of Tianjin University of TCM (TYLL2023[K]031). This study complied with the Declaration of Helsinki. Written informed consent about this study was provided by the participants. The investigators will report any changes of protocol or informed consent to the Ethics Committee and Registry.
本研究涉及人类受试者，经天津中医药大学第一教学医院IRB伦理委员会审查批准（TYLL2023[K]031）。本研究符合《赫尔辛基宣言》。参与者对本研究提供了书面知情同意书。研究者将向伦理委员会和注册处报告方案或知情同意书的任何变更。

Author contributions 作者供稿

CT: Methodology, Writing – original draft. LX: Writing – original draft. RL: Methodology, Project administration, Writing – review & editing. YC: Methodology, Writing – review & editing. ZL: Project administration, Writing – review & editing. LL: Project administration, Writing – review & editing. SZ: Project administration, Writing – review & editing. XD: Resources, Funding acquisition, Methodology, Writing – review & editing.
CT：方法论，写作 - 原稿。LX：写作--原稿。RL：方法论、项目管理、写作--审阅和编辑。YC：方法论、写作--审阅和编辑。ZL：项目管理、写作--审阅和编辑。LL：项目管理、写作--审阅和编辑。SZ：项目管理、写作 - 审核与编辑。XD：资源、资金获取、方法、写作 - 审核与编辑。

Funding 资金筹措

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study was supported by the Tianjin Science and Technology Program (18PTLCSY00050) and National Key Research and Development Program of China (2022YFC3500500, 2022YFC3500501). The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.
作者声明本文的研究、撰写和/或发表得到了资助。本研究得到了天津市科技计划项目（18PTLCSY00050）和国家重点研发计划项目（2022YFC3500500、2022YFC3500501）的支持。资助方未参与本研究的设计、数据收集、分析和解释，也未参与本文的撰写或决定是否将其发表。

Acknowledgments 致谢

We thank Qinyi, Liu Jihua, and all the patients and medical workers who contributed to our study.
我们感谢秦怡、刘继华以及所有为我们的研究做出贡献的患者和医务工作者。

Conflict of interest 利益冲突

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
作者声明，本研究在进行过程中不存在任何可能被视为潜在利益冲突的商业或经济关系。

Publisher’s note 出版商说明

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
本文中表述的所有主张仅代表作者本人，并不一定代表其附属机构的主张，也不代表出版商、编辑和审稿人的主张。本文可能评估的任何产品，或其制造商可能提出的任何主张，均未得到出版商的保证或认可。

Supplementary material 补充材料

The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fneur.2024.1367654/full#supplementary-material
本文的补充材料可在线查阅： https://www.frontiersin.org/articles/10.3389/fneur.2024.1367654/full#supplementary-material

Abbreviations 缩略语

CIS, Cerebral ischemic stroke; XNKQ, Xingnao-Kaiqiao acupuncture; fMRI, Functional magnetic resonance imaging; FMA, Fugl–Meyer Assessment; mRS, Modified Rankin Scale; NIHSS, National Institutes of Health Stroke Scale; MBI, Modified Barthel Index; EQ-5D, EuroQol Five Dimensions Questionnaire.
CIS，缺血性脑卒中；XNKQ，星火-开窍针灸；fMRI，功能磁共振成像；FMA，Fugl-Meyer评估；mRS，修正Rankin量表；NIHSS，美国国立卫生研究院卒中量表；MBI，修正Barthel指数；EQ-5D，EuroQol五维问卷。

References 参考资料