Effects of repetitive transcranial magnetic stimulation on electroencephalographical measures of post-stroke upper limb dysfunction: study protocol for a randomized controlled trial

Effects of repetitive transcranial magnetic stimulation on electroencephalographical measures of post-stroke upper limb dysfunction: study protocol for a randomized controlled trial | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of repetitive transcranial magnetic stimulation on electroencephalographical measures of post-stroke upper limb dysfunction: study protocol for a randomized controlled trial Sam Chi Chung Chan, Bo Chen, Heng Ming Wu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5345711/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Mar, 2026 Read the published version in Trials → Version 1 posted 6 You are reading this latest preprint version Abstract Background Patients with stroke would experience upper limb dysfunction, leading to functional impairments in daily living and placing a burden on society and families. Repetitive transcranial magnetism stimulation (rTMS), a non-invasive neuromodulation tool, has been shown to upper limb dysfunction. However, the clinical efficacy and the underlying neurophysiological mechanisms of optimal intensity of rTMS to promote motor recovery of stroke patients with upper limb function need further investigations. This study aims to investigate the changes in the brain neurophysiological mechanisms ranging from cortical oscillatory activity to the effectiveness of the complex network after low frequency (LF)- and high frequency (HF)-rTMS on motor areas of patient post-stroke. Methods A total of 42 stroke patients with upper limb dysfunction will be randomized into high frequency (HF) rTMS group, low frequency (LF) rTMS group (1:1 ratio). The HF-rTMS group will use 90% rest motor threshold (RMT), 10 Hz acting with the ipsilesional M1 for a total of 1500 pulses for 2 weeks, and the LF-rTMS group acting on the contralesional M1 with the same parameters, except that 1 Hz was used. The National Institutes of Health Stroke Scale (NIHSS), The motor deficit (the Fugl–Meyer Assessment upper Extremity (FMA-UE)), Modified Barthel Index (MBI), and resting-state electroencephalogram (EEG) signals will be obtained at the baseline and within one week after the rTMS. Discussion This study will contribute to the understanding of the neurophysiological changes in the brain corresponding to the clinical effects of HF- and LF-rTMS in patients with stroke. Trial registration This study was approved by the Ethics Committee of the Hong Kong Polytechnic University and the Ethics Committee of the Affiliated Hospital of Southwest Medical University (HSEARS20220303002). This study was registered in November 2022 (https://www.chictr.org.cn). Clinical trial registration number is ChiCTR2200065639. Stroke Resting-state electroencephalography Brain network Non-invasive brain stimulation Figures Figure 1 Administrative information Title {1} Effects of repetitive transcranial magnetic stimulation on electroencephalographical measures of post-stroke upper limb dysfunction: study protocol for a randomized controlled trial Trial registration {2a and 2b}. This study was registered in November 2022 (https://www.chictr.org.cn). Clinical trial registration number is ChiCTR2200065639 Protocol version {3} Date:11/01/2022, version 1 Funding {4} Sichuan Provincial Innovative Scientific Research Program for Medical Youth (Q21100) Sichuan Science and Technology Program (2023NSFSC1496) Other: Program of Doctor of Health Science of the Hong Kong Polytechnic University Author details {5a} Bo Chen 1,2,3 , Heng Ming Wu Heng 4 ,Sam C.C. Chan 1 *. 1 Department of Rehabilitation Sciences, The Hong Kong Polytechnic University 2 Department of Rehabilitation, The Affiliated Hospital of Southwest Medical University 3 Department of Rehabilitation, The Affiliated Hospital of Southwest Medical University Laboratory of Neurological Diseases and Brain Function 4 Department of Rehabilitation, The First Affiliated Hospital of Xinjiang Medical University Name and contact information for the trial sponsor {5b} Department of Rehabilitation Sciences, The Hong Kong Polytechnic University Department of Rehabilitation, The Affiliated Hospital of Southwest Medical University Role of sponsor {5c} The study sponsor and funder have approved the study design but do not participate in data collection, management, analysis, or interpretation of results, writing the report, or deciding whether to submit the report for publication. Background and rationale {6a} Stroke is one of the common non-communicable diseases that causes long-term disability in people[ 1 ]. The prevalence of stroke in adults is expected to increase significantly by 2030, compared to data reported in 2012[ 2 ]. Despite various approaches of rehabilitation interventions, a high proportion of patients with stroke still have residual functional impairment[ 2 ]. Those patients with stroke manifest limited potential for functional recovery and present as a challenge for rehabilitation interventions. With the advent of an aging society, stroke, especially with persistent physical dysfunction, is bound to place a heavier burden on society in the future. Thus, there is a need to explore better treatment modalities and to understand the underlying neurological mechanisms[ 3 ]. Since the 80s in the last century, transcranial magnetic stimulation (TMS) machines were introduced in rehabilitation field to serve a non-pharmaceutical treatment[ 4 ]. Through continuous developments, repetitive TMS (rTMS) has gradually been applied in the rehabilitation of stroke. Accumulative evidence suggested that high-frequency-TMS (HF-rTMS) (≥ 3 Hz) would induce an excitatory effect of the synaptic transmission at the cortical level while low-frequency-rTMS (LF-rTMS) (≤ 3 Hz) would lead to an inhibitory effect[ 5 – 8 ]. Stroke patients with different lesions in the brain respond differently to rTMS. Previous studies have shown that stroke patients with subcortical lesions have better clinical outcomes than those with cortical lesions alone after rTMS[ 8 – 10 ]. This may be explained that better clinical outcomes may be attributed to more preserved and intact cortical network [ 8 ] and more hyperactive contralateral motor cortex in stroke patients with subcortical lesions[ 11 ]. Besides, some observational studies have found that stroke patients with subcortical and cortical lesions show different activated brain areas in fMRI[ 12 ] and different cortical activity and functional connectivity of brain networks in EEG[ 13 ]. Therefore, the location of lesions would be a critical factor to be considered when exploring the neurological mechanisms of clinical effects of rTMS in stroke patients[ 14 ]. Currently, a number of studies have demonstrated the potential clinical effectiveness of rTMS in patients with stroke. Based on the interhemispheric competition theory, some clinical trials have selected HF-rTMS to stimulate the affected hemisphere to increase cortical excitability[ 15 ]. In terms of the guidelines for clinical application, rTMS has a relatively high recommendation grade for recovery of motor function in the post-acute phase after stroke[ 7 ]. The evidence quality of the application of LF-rTMS on the contralateral motor cortex is regarded as an A grade for recovery of hand function whilst that of the application of HF-rTMS on the ipsilateral M1 is regarded as a B grade for upper limb function improvement[ 7 ]. On the other hand, although there is no consensus about the standardized guideline to suggest the optimal frequency of HF-rTMS at the affected hemisphere to promote upper limb recovery among the stroke patients, accumulative evidence suggests that rTMS with 10 Hz, which is considered to be a high frequency, acting on the affected hemisphere M1 is beneficial for motor recovery among stroke patients[ 10 , 16 – 18 ]. Besides, motor evoked potentials (MEPs) ha been commonly used to evaluate the neural effects of LF and HF-rTMS with the use of the single pulse TMS[ 19 ]. However, the findings about MEPs in exploring cortical regions of the brain beyond M1 appear to be limited in the literature[ 20 ]. Hence, resting-state EEG, which is one of the common and economical tools, would be an appropriate measure to examine the neurological processes underlying the changes in upper limb motor functions induced by HF/LF-rTMS among people with based on the interhemispheric competition theory[ 21 ]. Normalized power (NP) characterizes cortical activity by quantifying the power of brain oscillations, and while balanced cortical activity is characteristic of a healthy brain, Electrode directional asymmetry (EDA) can visualize it[ 22 , 23 ]. At the same time, rich and strong brain network function connectivity facilitates the brain's ability to perform physiological functions[ 24 – 26 ]. This series of hierarchical resting-state EEG indicators can provide us with a panoramic view of the neurophysiology of the brain[ 22 , 23 , 26 , 27 ]. Previous studies showed that rTMS with different frequency combined with other rehabilitation training could modulate power spectral and functional connectivity in healthy participants to enhance motor function[ 28 – 32 ]. The stroke population suffers from dysfunction, the cortical oscillatory activity and network functional connectivity differ from the healthy population[ 22 , 26 ]. Similarly, resting-state EEG indicators as biomarkers also can be used to explore neurological mechanisms in stroke patients after rTMS interventions. Ding found that iTBS can increase brain oscillatory power and network connectivity, while the efficiency of brain information transfer is improved[ 21 ]. However, the study did not confirm whether the improvement in these EEG indicators was a neurophysiologic mechanism for the improvement in clinical function, as no correlation was found between them[ 21 ]. Besides, Zhong and colleagues also reported that an intervention protocol with 10-Hz rTMS reduced oscillation activity in the affected hemisphere, resulting an improvement in interhemispheric imbalance[ 33 ]. But, the study also did not confirm whether improved cortical activity balance was associated with improved clinical functioning[ 33 ]. In addition, rTMS was applied on the vertex of the brain, which is different from the conventional protocol in which HF-rTMS was applied on M1 of the affected hemisphere[ 33 ]. Thus, whether rTMS stimulation on cortical motor areas of stroke patients can modulate the cortical activity balance characterized by EDA has not been answered. Moreover, all of these studies only explored immediate changes in resting-state EEG after the intervention and did not reflect changes after a period of intervention. Thus, there is need to examine the long-term effect of the rTMS on motor recovery among stroke patients. In conclusion, while rTMS has been shown to stimulate cortical motor areas in healthy individuals can modulate brain oscillatory power and brain network functional connectivity to enhance limb function[ 28 – 32 ]. However, this was not confirmed in a series of pre-studies of rTMS interventions in stroke patients, and variation existed in rTMS protocols[ 21 ] and stimulation areas[ 33 ]. Meanwhile, reports of changes in resting-state EEG indicators after LF-rTMS treatment of stroke patients are scarce. Therefore, the neurophysiological mechanisms underlying the clinical effects of LF- and HF-rTMS stimulation of cortical motor areas as a traditional therapeutic protocol for improving function of stroke patients need to be clarified. Objectives {7} Thus, the objective of this study was to investigate the neurophysiological mechanisms underlying the clinical effects of rTMS in stroke patients at different levels by measuring changes in cortical oscillatory power, EDA, and functional connectivity in stroke patients after receiving LF- and HF-rTMS, and whether these changes correlate with improvements in clinical function. Therefore, we hypothesized that HF- and LF-rTTMS stimulation of cortical motor areas in stroke patients could modulate cortical oscillatory power thereby balancing the EDA while enhancing the brain network functional connectivity. Meanwhile, changes in resting-state indicators after rTMS intervention correlated with changes in clinical scales. Trial design {8} This study protocol is based on the “Standard Protocol Items: Recommendations for Interventional Trials” (SPIRIT) 2013 statement[ 34 ]. A design of randomized double-blind controlled trial (RCT) with pre-post measurement time points will be adopted for this study. The outcome measures will be accessed at the day before the first intervention (T0) baseline and post 10 sessions intervention (T1) by an assessor who is unaware of the participant's allocation information (Fig. 1). Methods: Participants, interventions and outcomes Study setting {9} Participants will be recruited from both inpatient wards and outpatient clinics at the Affiliated Hospital of Southwest Medical University. Who will provide informed consent? {26a} Prior to the start of the trial, the investigator will explain the details and steps of the trial in detail to the participants, and obtain their consent and sign an informed consent form before the trial begins. Confidentiality and anonymity of all participants will be guaranteed, in accordance with the requirements of the university where the research team is based and the ethical requirements of the trial. Additional consent provisions for collection and use of participant data and biological specimens {26b} The informed consent form will provide participants with a detailed explanation of the medical records collected, biospecimens and personal data that will be analyzed as part of the research. Eligibility criteria {10} Participants to be recruited in this study will be required to fulfill the following inclusion criteria: (1) the first episode of a unilateral ischemic stroke with stable condition. (2) the infarct lesion location determined by imaging and classified as cortical, subcortical, or cortico-subcortical[ 14 , 29 ]. (3) at the age ≥ 18 years. (4) right-handedness. (5) the Functional Test of the Upper Extremity in Hemiplegia (FTHUE-HK) being ≤ 4[ 35 ]. (6) Having intact comprehension and expression functions. (7) being able to understand and voluntarily sign a written consent form to participate in the study. Participants will be excluded if they will meet one of the following conditions: (1) hemorrhagic stroke. (2) previous diagnosis of a neurological disorder besides stroke. (3) presence of cognitive impairment or aphasia that affects the comprehension ability, Mini-Mental State Examination(MMSE)≤21[ 36 , 37 ]. (4) presence of pain that prevents completion of functional assessment. (5) impaired upper limb injury functions due to neurological diseases rather than stroke. Interventions Explanation for the choice of comparators {6b} In this research, the no sham-rTMS group will be used as a control group. Intervention description {11a} The rTMS intervention will be implemented at a public hospital in the Mainland China. The rTMS instrument model is the Magpro R30 stimulator (MagVenture, Lucernemarken, Denmark), with a figure-of-eight coil connected to emit magnetic stimulations. rTMS stimulations with 10 Hz or 1 Hz at the contralateral primary motor cortex (M1) are expected to elicit contraction of the first interosseous muscle of the affected hand. If muscle activity cannot be observed by the above method, the corresponding position for the M1 of the ipsilateral hemisphere will be selected as the stimulation location. The minimum transcranial magnetic stimulation intensity will be set based on a resting motor threshold (RMT) that elicits motor evoked potentials with an amplitude of 50µV for at least half of the 10 consecutive stimulations at the primary motor area[ 39 ]. The rTMS stimulation parameters for each of groups will be performed as follows: (1) The HF-rTMS stimulation protocol will follow the previous study[ 38 ], with 10 Hz-rTMS applied to ipsilateral hemisphere M1 with a stimulation intensity of 90% of the RMT. Each treatment session including 30 trains, with 50 pulses per trains session which will last 25 seconds, leading to a total 1500 pulses a session. Each participant will receive 10 consecutive sessions of treatment. (2) The LF-rTMS group will receive the same parameters as HF-rTMS at the contralesional primary motor area, except that 1 Hz will be be used. The rTMS operations will all be performed by the same experienced clinician, who will be responsible for managing and documenting adverse events for each stimulation. Criteria for discontinuing or modifying allocated interventions {11b} If a participant develops any discomfort while participating in the study, we will stop the trial immediately. The trial will be restarted only after the participant's discomfort has been appropriately managed by the medical team and we have received a signal from the participant that he or she agrees to continue to the next step. Also, we will terminate the trial at any time, once the participant signals termination or refuses to participate in the next step of the process. Strategies to improve adherence to interventions {11c} The investigator will coordinate the timing of the participant's receipt of the rTMS and the timing of the outcome assessment to ensure that the participant is able to complete the full trial process as planned. If necessary, the investigator will again explain in detail to the participant the benefits and risks of participating in the study to ensure that the participant fully understands the study. Relevant concomitant care permitted or prohibited during the trial {11d} Participants in both the HF- and LF-rTMS groups will be able to receive traditional rehabilitation treatments such as physical therapy and occupational therapy at the same time. Provisions for post-trial care {30} All participants receiving the rTMS intervention who experience a serious adverse reaction will be referred to the medical team for continued monitoring and treatment. Outcomes {12} Clinical Outcome Measures The National Institutes of Health Stroke Scale (NIHSS) [ 41 ]. The NIHSS is an established tool utilized to evaluate the severity of stroke. It encompasses 15 items, generating a cumulative score ranging from 0 to 42. A lower score represents a reduced degree of neurological impairment. The scale assesses various aspects, including consciousness, eye movement, visual fields, facial palsy, motor function, sensory function, language, and neglect. Trained evaluators will administer the NIHSS before and after the rTMS to determine changes in stroke severity. Fugl-Meyer Assessment upper Extremity (FMA-UE) [ 42 ]. The FMA-UE will be employed in this study as a validated tool recommended for clinical application in assessing upper limb impairment in patients after stroke[ 42 ]. It assesses the movement, coordination and reflexes of each joint of the upper limb of the hemiplegic side through 33 items. Each item is divided into three scoring levels (0,1,2) with a total score of 66. In addition, there are 6 items to assess upper limb sensation, including light touch and position sense, with a total of 12 points. Trained evaluators will administer the FMA-UE before and after the rTMS to monitor changes in upper limb function. Modified Barthel Index (MBI) [ 43 ]. The MBI is a scale that assesses the ability to perform self-care activities of daily living and contains assessments ranging from feeding, self-care, and mobility. Each item can be rated at five levels, with different levels representing different levels of independence. The total score is 0 to 100, with higher scores indicating higher independence in daily living. In this study, the MBI will be administered pre- and post-rTMS to measure any changes in participants' functional independence. EEG data measures EEG data acquisition Each participant will have their hair scalp cleansed before the rTMS experiment. Each EEG data collection will be conducted in a semi-isolation using the same set of equipment. During resting-state EEG signal collection, participants will be seated comfortably on chair. Each of them was required to keep his or her eyes closed and mind clear throughout the experiment process. The acquisition of EEG signals will be recorded via a 19-channel analog recorder according to the international 10–20 system (EB Neuro, Firenze, Italy). The placement of the 19 electrodes (FP1, FP2, F3, F4, F7, F8, T3, T4, T5, T6, C3, C4, P3, P4, O1, O2, Fz, Cz, and Pz) on the scalp will be determined by the aid of a quantitative ruler. The impedance of each electrode will be kept 5 kΩ or below, and the sampling rate will be set to 256 Hz. The data collection process will be performed by an experienced experimenter to ensure the data quality during the data acquisition. EEG data preprocessing The raw EEG signal will be band-passed between 1 and 50 Hz using a fourth-order zero-phase Butterworth filter. The data of each trial will be extracted by segmenting from the beginning to the end into 2-second contiguous but non-overlapping segments. Then, the bad channels will be checked and deleted manually, and the value of the deleted channels will be replaced by the interpolation of the surrounding electrode channels. The EEG data of participants with stroke will be flipped so that the ipsilateral hemispheres are always presented on the same side for easy observation. In addition, artifacts caused by eye movements, muscle movements, or other factors will be removed manually. The EEG data will be re-referenced using the full average reference method and finally, the processed data from each channel will be decomposed by finite impulse response (FIR) bandpass filtering into δ (0.5-4 Hz), θ (4–7 Hz), α1 (8–10 Hz), α2 (10–12 Hz), β (13–30 Hz) and γ (30–40 Hz). To reduce bias, a normalized method will be applied to process EEG power and use the processed power values as an indicator of network activity. Functional connectivity between cortical regions will be measured using orthogonal correlations of EEG activity. Power spectrum analysis Spectrum analysis changes the time domain signal into the frequency domain and provides a distribution curve of the power, amplitude or phase along the frequency, i.e., power spectrum[ 44 , 45 ]. To examine the spatial characteristics of resting-state EEG power in each frequency band, first to calculate the power spectrum for each electrode using the method of welch[ 46 ] and then perform power spectrum analysis at the electrode level. The frequency bands will then be extracted from the power spectrum and normalized at each electrode using Eq. 1, $$\:NP=100\times\:\frac{\sum\:F}{\sum\:Total}$$ 1 , NP means normalized power, i.e., the ratio between the power of a band and the total power of the whole frequency range. ∑F means the sum of power within a frequency band, ∑Total means the sum of power across the frequency spectrum. This power normalization method not only allows to observe whether the cortical areas change in function by determining the power distribution across the spectrum, but also eliminates the effects of biasing factors, such as electrode impedance and the size of the neuronal population. To further clarify the true cause of the normalized power difference between the real rTMS and sham rTMS groups, we will calculate and plot the mean absolute power of each electrode in the frequency band. If, similarly, the two groups also differ in absolute power, the possible bias introduced to the power normalization is further excluded. Electrode directional asymmetry Based on the normalized power, Eq. 2 is further used to calculate the electrode direction asymmetry and thus determine whether there is a difference in power in frequency bands between the two hemispheres. $$\:EDA=100\times\:\frac{1}{n}\sum\:\frac{{NP}_{L}-{NP}_{R}}{\left|{NP}_{L}\right|+\left|{NP}_{R}\right|}$$ 2 , EDA means the whole head electrode directional asymmetry, NPL means the normalized power of the homologous electrode in the left hemisphere, NPR is the normalized power of the homologous electrode in the right hemisphere, and n is the total number of electrode pairs. Functional connectivity analysis Volume source localization will be performed on the EEG data before applying the eeglab toolbox for functional connectivity analysis. In order to determine whether there is a difference in the connection of the frequency bands between the two hemispheres, the indicator of connection direction asymmetry between analogue connections of the two hemispheres will be calculated using Eq. 3, $$\:CDA=100\times\:\frac{1}{n}\sum\:\frac{{C}_{L}-{C}_{R}}{\left|{C}_{L}\right|+\left|{C}_{R}\right|}$$ 3 , CDA means the whole brain connectivity directional asymmetry metric, CL means the connectivity of the homologous connections in the left hemisphere, CR means the connectivity of the homologous connections in the right hemisphere, and n means the total number of homologous connection pairs. Deviations in the shape of the control group connection spectrum will be quantified by correlating the connection spectrum of all participants with the average connection spectrum of the control group. Participant timeline {13} Data collection will take place from November 2022 through November 2025, with participant recruitment continuing through November 2024. Ethical monitoring will continue throughout the data collection period. Sample size {14} Previous related studies utilizing EEG to gauge rTMS treatment effects in stroke patients did not provide effect size values for sample size estimation[ 21 , 28 , 29 , 31 , 33 ]. These studies, however, with sample sizes ranging from 15 to 30, reported significant statistical differences, supporting our sample size determination. The study also aimed to evaluate improvement in post-stroke upper limb dysfunction, assessed by the FMA-UE. Based on a previous study that reported altered FMA-UE values post-intervention[ 38 ], we calculated an effect size of 0.57. Using G*power 3.1 software, with α set to 0.05 and power to 0.9 for a paired samples t-test, we determined that a total of 35 samples were needed. To account for potential drop-outs, with a projected 20% drop-out rate, we decided to include at least 42 total participants, or 21 in each group. Recruitment {15} Participants will be recruited from both inpatient wards and outpatient clinics at the Affiliated Hospital of Southwest Medical University. The investigator will provide potential participants with a detailed description of the research, the possible benefits, and the risks. Assignment of interventions: allocation Sequence generation {16a} All participants will be assigned randomly to the higher rTMS frequency (10 Hz rTMS) and lower rTMS frequency (1 Hz rTMS) groups in a 1:1 ratio using a random assignment tool with the lesion sites at cortical, subcortical and cortico-subcortical levels matched between the two interventions group. Randomized serial lists will be produced with the help of statistical software and the randomly generated serial numbers will be placed in sealed, opaque envelopes to ensure that they are not compromised. Implementation {16c} Sequence numbers will be sealed in opaque numbered envelopes, and participants will draw envelopes according to the number, and the envelopes that have been drawn will be marked with the basic information of the participants and then strictly stored for inspection and prevent confusion. Assignment of interventions: Blinding Who will be blinded {17a} The allocation information will be blinded from participants and assessors. Data collection and management Plans for assessment and collection of outcomes {18a} After passing the screening, the investigator will determine to which group the participant will be allocated by drawing lots from a sealed envelope, but the results will be blinded from the assessor and the participant. At the baseline, the demographic information of each participant, who fulfils the inclusion criteria, will be obtained and the participants will be assessed using the NIHSS, The motor deficit (FMA-UE), MBI, and resting-state EEG before the rTMS treatment. Then, ten sessions of rTMS will be given to each participant with 5 times a week for two weeks. The participants will be assessed by the same outcome measures after the 10-session rTMS intervention. The entire experimental procedure that each participant will go through is illustrated in Table 1. Plans to promote participant retention and complete follow-up {18b} There will be no process for follow-up in this research. Data management {19} Paper forms will be kept in a secure place and transcribed data will be collected by individuals using password-protected computers. Research materials and data will be kept by the principal investigator for the appropriate number of years as required by the supervisory authority. Confidentiality {27} The Principal Investigator will ensure that all study data are stored securely and that confidentiality and privacy are protected in accordance with applicable laws and regulations. Participants will remain anonymous throughout the research. Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use {33} N/A; Biological samples, such as blood, will not be collected for this research. Statistical methods Statistical methods for primary and secondary outcomes {20a} Demographic and baseline data will be compared using SPSS 19, with variance analysis for continuous data and x 2 test for categorical data. The independent t test with false discovery rate (FDR) will be employed to investigate difference changes in normalized power distribution, absolute power distribution, EDA between HF-and LF rTMS group after intervention. In addition, paired t -tests with FDR correction will employ for clinical outcomes and rs-EEG indicators in the comparison of post-rTMS and baseline outcomes in each group. Correlation of resting-state EEG indicators will be analyzed with clinical functional changes. The type I error rate for statistical tests is α = 0.05. Interim analyses {21b} There are no interim analyses planned for this research. Methods for additional analyses (e.g. subgroup analyses) {20b} There are no additional analyses planned for this research. Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c} This analysis is not planned for this research. Plans to give access to the full protocol, participant level-data and statistical code {31c} There are no plans to provide public access to datasets or statistical codes at the participant level. However, all results, both positive and negative, will be published. Funding sources will not affect the reporting of results. Oversight and monitoring Composition of the coordinating center and trial steering committee {5d} The research team consists of physicians, rehabilitation therapists, senior researchers, and doctoral students with clinical experience in rehabilitation. The senior investigator has extensive experience in clinical trials. All team members will be trained prior to the start of the study and regular meetings will be held throughout the research process to ensure quality control and prevent errors. Composition of the data monitoring committee, its role and reporting structure {21a} The Principal Investigator will meet with the team monthly to audit monitoring activities and adverse events. Based on these audits, recommendations for study continuation, modification, or termination will be made.Adverse event reporting and harms {22} Although rTMS may cause some adverse effects such as seizures, temporary hearing discomfort, localized pain, syncope, and minor cognitive changes, these discomforts have been rarely reported[ 40 ]. For the safety reasons, participants meeting the following contraindications to rTMS will be excluded: (1) unstable disease status; (2) history of epilepsy, impaired consciousness or intracranial hypertension; (3) history of heart disease; (4) pregnancy; (5) metal implants in the body, such as an artificial cochlear, pacemaker; (6) underwent a craniotomy; (7) taking any centrally acting medication within the recent 3 months. TMS operators will complete operational and safety knowledge training. All participants will be screened for safety risks and those unsuitable for TMS intervention will be excluded. First aid facilities will be equipped in the EEG acquisition room and the rTMS intervention room. During the EEG acquisition and rTMS intervention, the experimenter will observe the status of the participant closely and will halt the operation of the rTMS immediately if he or she will report any discomfort. In case of sudden illnesses such as coma or epilepsy, immediately take necessary measures to protect the safety of the participant and transfer to the emergency room by medical personnel for further treatment. An adverse event survey will be conducted by the experimenter for each participant after the completion of the rTMS intervention. Frequency and plans for auditing trial conduct {23} Audits and inspections may be conducted by the Ethics Committee of the Hong Kong Polytechnic University. Auditors have the right to access all data, raw materials, informed consent forms and any other necessary clinical trial documentation. Plans for communicating important protocol amendments to relevant parties (e.g. trial participants, ethical committees) {25} At present, no modifications to this study are planned. If modifications are required in the future, the researchers will inform the ethics committee. Dissemination plans {31a} The results of this study will be sent to a peer-reviewed professional journal for publication. Discussion This study protocol outlines a randomized controlled trial designed to investigate the effects of HF- and LF-rTMS on upper limb dysfunction in post-stroke patients. The primary aim is to elucidate the neurophysiological mechanisms underlying the clinical efficacy of rTMS, focusing on changes in cortical oscillatory activity and the effectiveness of the complex brain network. One of the key strengths of this study is its rigorous design, including randomization and the use of both HF- and LF-rTMS protocols, which allows for a comprehensive comparison of their effects. Additionally, the use of EEG to measure resting-state brain activity provides valuable insights into the neurophysiological changes associated with rTMS. However, the study also has limitations. The relatively small sample size (n=42) may limit the generalizability of the findings. Furthermore, the short follow-up period may not capture long-term effects of rTMS on motor recovery. Previous studies have demonstrated the potential of rTMS in improving motor function in stroke patients, but the optimal parameters and underlying mechanisms remain unclear. This study aims to address these gaps by comparing the effects of HF- and LF-rTMS on both clinical outcomes and neurophysiological measures. The use of a well-defined protocol and standardized outcome measures, such as the NIHSS and the FMA-UE, ensures the reliability and validity of the results. The findings from this study could have significant implications for clinical practice. If HF- or LF-rTMS is shown to be effective in enhancing motor recovery and modulating brain activity, it could be incorporated into rehabilitation programs for stroke patients. This would provide a non-invasive, adjunctive treatment option to improve upper limb function, potentially reducing the burden on healthcare systems and improving the quality of life for stroke survivors. Future studies should consider larger sample sizes and longer follow-up periods to validate and extend the findings of this trial. Additionally, exploring the combination of rTMS with other rehabilitation interventions, such as physical therapy or occupational therapy, could provide insights into synergistic effects and optimize treatment protocols. Investigating the differential effects of rTMS on various subtypes of stroke and patient characteristics could also help tailor interventions to individual needs. This study will contribute to the understanding of the neurophysiological changes in the brain corresponding to the clinical effects of HF- and LF-rTMS in patients with stroke. The results could pave the way for more effective and personalized rehabilitation strategies, ultimately enhancing the recovery process for stroke survivors. Trial status Start date for recruiting participants: November 2022; End of data collection: expected to end in December 2024. Abbreviations rTMS Repetitive transcranial magnetism stimulation LF Low frequency HF High frequency NIHSS National Institutes of Health Stroke Scale FMA-UE Fugl–Meyer Assessment upper Extremity MBI Modified Barthel Index EEG Electroencephalogram NP Normalized power EDA Electrode directional asymmetry Declarations Acknowledgements EEG equipment and rTMS equipment from the Affiliated Hospital of Southwest Medical University will be used in this study, for which we would like to express our gratitude. Authors’ contributions {31b} Sam C. C. Chan (SCC) and Chen Bo (CB) conceived and designed this study collaboratively. CB, Wu Hengming (WHM) wrote the first draft of this protocol, and CCC reviewed and revised the manuscript. Funding {4} CB's work was supported by Program of Doctor of Health Science of the Hong Kong Polytechnic University, Sichuan Provincial Innovative Scientific Research Program for Medical Youth (Q21100) (An additional file shows this in more detail [see Additional file 1]), Sichuan Science and Technology Program (2023NSFSC1496) (An additional file shows this in more detail [see Additional file 2]). Availability of data and materials {29} No data was used for the research described in the article. Ethics approval and consent to participate {24}This study was registered in November 2022 (https://www.chictr.org.cn). Actual recruitment for this trial begins in November 2022 and is expected to last one year. This proposed study will be conducted in compliance with the Declaration of Helsinki. The participant's consent and sign an informed consent form will be obtained before starting the trial. Modifications to this study protocol will be performed only after review and consent by the Ethics Committee (HSEARS20220303002). Consent for publication {32} All authors have approved the publication of this protocol. Competing interests {28} All authors declare that they have no competing interests. References Murray CJL, Atkinson C, Bhalla K, Birbeck G, Burstein R, Chou D, et al. The state of US health, 1990–2010: burden of diseases, injuries, and risk factors. JAMA. 2013;310:591–608. Virani SS, Alonso A, Benjamin EJ, Bittencourt MS, Callaway CW, Carson AP, et al. Heart Disease and Stroke Statistics-2020 Update: A Report From the American Heart Association. Circulation. 2020;141:e139–596. Liu Y, Popescu M, Longo S, Gao M, Wang D, McGillis S, et al. Fibrinogen Reduction and Motor Function Improvement by Hematopoietic Growth Factor Treatment in Chronic Stroke in Aged Mice: A Treatment Frequency Study. Cell Transpl. 2016;25:729–34. Barker AT, Jalinous R, Freeston IL. Non-invasive magnetic stimulation of human motor cortex. Lancet. 1985;1:1106–7. Chen R, Classen J, Gerloff C, Celnik P, Wassermann EM, Hallett M, et al. Depression of motor cortex excitability by low-frequency transcranial magnetic stimulation. Neurology. 1997;48:1398–403. Post RM, Kimbrell TA, McCann UD, Dunn RT, Osuch EA, Speer AM, et al. Repetitive transcranial magnetic stimulation as a neuropsychiatric tool: present status and future potential. J ECT. 1999;15:39–59. Lefaucheur J-P, Aleman A, Baeken C, Benninger DH, Brunelin J, Di Lazzaro V, et al. Evidence-based guidelines on the therapeutic use of repetitive transcranial magnetic stimulation (rTMS): An update (2014–2018). Clin Neurophysiol. 2020;131:474–528. Xiang H, Sun J, Tang X, Zeng K, Wu X. 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Effects of High-Frequency (HF) Repetitive Transcranial Magnetic Stimulation (rTMS) on Upper Extremity Motor Function in Stroke Patients: A Systematic Review. Med (Kaunas). 2021;57:1215. Khedr EM, Etraby AE, Hemeda M, Nasef AM, Razek A. a. E. Long-term effect of repetitive transcranial magnetic stimulation on motor function recovery after acute ischemic stroke. Acta Neurol Scand. 2010;121:30–7. Lefaucheur J-P. Transcranial magnetic stimulation. Handb Clin Neurol. 2019;160:559–80. Suppa A, Huang Y-Z, Funke K, Ridding MC, Cheeran B, Di Lazzaro V et al. Ten Years of Theta Burst Stimulation in Humans: Established Knowledge, Unknowns and Prospects. Brain Stimulation [Internet]. 2016 [cited 2022 Nov 13];9:323–35. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1935861X16300018 Ding Q, Zhang S, Chen S, Chen J, Li X, Chen J et al. The Effects of Intermittent Theta Burst Stimulation on Functional Brain Network Following Stroke: An Electroencephalography Study. Front NeuroSci. 2021;15. Snyder DB, Schmit BD, Hyngstrom AS, Beardsley SA. Electroencephalography resting-state networks in people with Stroke. Brain Behav. 2021;11. Paolo, Manganotti. Miloš Ajčević, Alex Buoite Stella. EEG as a marker of brain plasticity in clinical applications. Handb Clin Neurol. 2022;91–104. Penalver-Andres JAA, Buetler KAA, Koenig T, Mueri RM, Marchal-Crespo L. Resting-State Functional Networks Correlate with Motor Performance in a Complex Visuomotor Task: An EEG Microstate Pilot Study on Healthy Individuals. Brain Topogr [Internet]. [cited 2023 Mar 13]; Available from: https://link.springer.com/article/ 10.1007/s10548-022-00934-9 Bistriceanu CE, Danciu FA, Cuciureanu DI. Cortical connectivity in stroke using signals from resting-state EEG: a review of current literature. Acta Neurol Belg. 2022. Yoshimichi Sato O, Schmitt Z, Ip G, Rabiller S, Omodaka T, Tominaga et al. Pathological changes of brain oscillations following ischemic stroke. Journal of Cerebral Blood Flow and Metabolism. 2022;0271678X2211056-0271678X2211056. Miraglia F, Vecchio F, Pappalettera C, Nucci L, Cotelli M, Judica E, et al. Brain Connectivity and Graph Theory Analysis in Alzheimer’s and Parkinson’s Disease: The Contribution of Electrophysiological Techniques. Brain Sci. 2022;12:402. Qiu S, Yi W, Wang S, Zhang C, He H. The Lasting Effects of Low-Frequency Repetitive Transcranial Magnetic Stimulation on Resting State EEG in Healthy Subjects. IEEE Trans Neural Syst Rehabil Eng [Internet]. 2020 [cited 2022 Oct 9];28:832–41. Available from: https://ieeexplore.ieee.org/document/9022937/ Jin J, Wang X, Li Y, Wang H, Liu Z, Yin T. rTMS combined with motor training changed the inter-hemispheric lateralization. Exp Brain Res [Internet]. 2019 [cited 2022 Oct 9];237:2735–46. Available from: http://link.springer.com/ 10.1007/s00221-019-05621-z Zrenner C, Desideri D, Belardinelli P, Ziemann U. Real-time EEG-defined excitability states determine efficacy of TMS-induced plasticity in human motor cortex. Brain Stimulation [Internet]. 2018 [cited 2022 Oct 9];11:374–89. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1935861X17309725 Zhang JJ, Fong KNK. The Modulatory Effects of Intermittent Theta Burst Stimulation in Combination With Mirror Hand Motor Training on Functional Connectivity: A Proof-of-Concept Study. Front Neural Circuits. 2021;15:548299. Jin J-N, Wang X, Li Y, Jin F, Liu Z-P, Yin T. The Effects of rTMS Combined with Motor Training on Functional Connectivity in Alpha Frequency Band. Front Behav Neurosci [Internet]. 2017 [cited 2022 Oct 9];11:234. Available from: http://journal.frontiersin.org/article/ 10.3389/fnbeh.2017.00234/full Zhong Y, Fan J, Wang H, He R. Simultaneously stimulating both brain hemispheres by rTMS in patients with unilateral brain lesions decreases interhemispheric asymmetry. RNN [Internet]. 2021 [cited 2022 Dec 5];39:409–18. Available from: https://www.medra.org/servlet/aliasResolver?alias=iospress&doi=10.3233/RNN-211172 Chan A-W, Tetzlaff JM, Altman DG, Laupacis A, Gøtzsche PC, Krleža-Jerić K, et al. SPIRIT 2013 statement: defining standard protocol items for clinical trials. Ann Intern Med. 2013;158:200–7. Fong K, Ng B, Chan D, Chan E, Ma D, Au B et al. Development of the Hong Kong Version of the Functional Test for the Hemiplegic Upper Extremity (FTHUE-HK). Hong Kong Journal of Occupational Therapy [Internet]. 2004 [cited 2022 Nov 21];14:21–9. Available from: http://linkinghub.elsevier.com/retrieve/pii/S1569186109700257 Feng L, Chong MS, Lim WS, Ng TP. The Modified Mini-Mental State Examination test: normative data for Singapore Chinese older adults and its performance in detecting early cognitive impairment. Singap Med J. 2012;53:458–62. Folstein MF, Folstein SE, McHugh PR. Mini-mental state. A practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res. 1975;12:189–98. Guo Z, Jin Y, Bai X, Jiang B, He L, McClure MA et al. Distinction of High- and Low-Frequency Repetitive Transcranial Magnetic Stimulation on the Functional Reorganization of the Motor Network in Stroke Patients. Cheung VCK, editor. Neural Plasticity [Internet]. 2021 [cited 2022 Dec 3];2021:1–11. Available from: https://www.hindawi.com/journals/np/2021/8873221/ Groppa S, Oliviero A, Eisen A, Quartarone A, Cohen LG, Mall V et al. A practical guide to diagnostic transcranial magnetic stimulation: Report of an IFCN committee. Clinical Neurophysiology [Internet]. 2012 [cited 2022 Dec 3];123:858–82. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1388245712000569 Rossi S, Hallett M, Rossini PM, Pascual-Leone A. Safety, ethical considerations, and application guidelines for the use of transcranial magnetic stimulation in clinical practice and research. Clinical Neurophysiology [Internet]. 2009 [cited 2022 Nov 21];120:2008–39. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1388245709005197 Kwah LK, Diong J. National Institutes of Health Stroke Scale (NIHSS). Journal of Physiotherapy [Internet]. 2014 [cited 2022 Dec 5];60:61. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1836955314000137 Kwakkel G, Lannin NA, Borschmann K, English C, Ali M, Churilov L et al. Standardized measurement of sensorimotor recovery in stroke trials: Consensus-based core recommendations from the Stroke Recovery and Rehabilitation Roundtable. International Journal of Stroke [Internet]. 2017 [cited 2022 Dec 3];12:451–61. Available from: http://journals.sagepub.com/doi/ 10.1177/1747493017711813 Shah S, Vanclay F, Cooper B. Improving the sensitivity of the Barthel Index for stroke rehabilitation. Journal of Clinical Epidemiology [Internet]. 1989 [cited 2022 Dec 3];42:703–9. Available from: https://linkinghub.elsevier.com/retrieve/pii/0895435689900656 Kay SM. Modern spectral estimation: theory and application. Englewood Cliffs, N.J: Prentice Hall; 1988. Rao KD, Swamy MNS. Spectral Analysis of Signals. Digital Signal Processing. Singapore: Springer Singapore; 2018. pp. 721–51. Welch P. The use of fast Fourier transform for the estimation of power spectra: A method based on time averaging over short, modified periodograms. IEEE Trans Audio Electroacoust. 1967;15:70–3. Table Table 1 is available in the Supplementary Files section. Supplementary Files Trialstable1.docx TrialsStructuredStudyPotocolTemplatev01.docx SPIRITFillablechecklist15Aug2013filled.docx Cite Share Download PDF Status: Published Journal Publication published 16 Mar, 2026 Read the published version in Trials → Version 1 posted Editorial decision: Major revision 20 Jan, 2026 Reviewers agreed at journal 05 Sep, 2025 Reviewers invited by journal 02 Sep, 2025 Editor invited by journal 04 Mar, 2025 Editor assigned by journal 17 Feb, 2025 First submitted to journal 16 Feb, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5345711","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":509302489,"identity":"11fc3bb2-05b0-4896-abc4-939228c33cdf","order_by":0,"name":"Sam Chi Chung Chan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYBCDBAYG5gMMDAVgjgEDY0MCMVrYEkCKSdLCY0CcFvn204kffjDY5fHP7vn24IPB4cQG9uZtEow70nBqMTiTu1myhyG5WOLO2e2GM0BaeI6VSTCeycGthSF3GzMDw4HEhhu526R5QFokcswkGNsqcDus/y1Ey/wbOc8gWuTf4NfCcANqy4YbOWxQW3hAWvA47MZboF8MkhM33kgzB/ol3biNJ63YIrENt/fl+3M3fvhRYZc470byswcfKqxl+9kPb7zxsS0Zt8OggQACbEDcDCZB0UQUACmuI1LtKBgFo2AUjCQAALX4VbbrmnZLAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-7109-0697","institution":"The Hong Kong Polytechnic University","correspondingAuthor":true,"prefix":"","firstName":"Sam","middleName":"Chi Chung","lastName":"Chan","suffix":""},{"id":509302490,"identity":"66346adc-3da1-4060-9635-20533ec5134b","order_by":1,"name":"Bo Chen","email":"","orcid":"","institution":"first affiliated hospital of southwest medical University","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Chen","suffix":""},{"id":509302491,"identity":"5be8232f-6828-4e62-ad36-ae936c7120ac","order_by":2,"name":"Heng Ming Wu","email":"","orcid":"","institution":"first affiliated hospital of xinjiang Medical University","correspondingAuthor":false,"prefix":"","firstName":"Heng","middleName":"Ming","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2024-10-28 09:17:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5345711/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5345711/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13063-026-09627-1","type":"published","date":"2026-03-16T15:57:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90908229,"identity":"c6d7f90c-c785-4232-8de4-2888e0a272dd","added_by":"auto","created_at":"2025-09-09 13:24:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":32225,"visible":true,"origin":"","legend":"\u003cp\u003eTrial flow chart\u003c/p\u003e\n\u003cp\u003eHF-rTMS, high frequency repetitive transcranial magnetism stimulation; LF-rTMS, low frequency repetitive transcranial magnetism stimulation; EEG, electroencephalography; NIHSSS: national institutes of health stroke scale; FMA-UE, Fugl-Meyer Assessment-Upper Extremity scores; MBI, Modified Barthel Index.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5345711/v1/b123f3f75f9eac8852fe4859.png"},{"id":105223276,"identity":"7fb1078e-8b04-4e2f-a9af-60f5ba25ed90","added_by":"auto","created_at":"2026-03-23 16:01:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1858689,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5345711/v1/6a6f18a9-2ab5-49e9-922b-845df2aaa79d.pdf"},{"id":90909836,"identity":"be9cc3e5-cd76-478a-8ff5-fca787be4186","added_by":"auto","created_at":"2025-09-09 13:32:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":20935,"visible":true,"origin":"","legend":"","description":"","filename":"Trialstable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5345711/v1/af2dea178bfcc6d253fb58c5.docx"},{"id":90908232,"identity":"47b7054c-0564-4188-9edd-1c4ec3e74ea1","added_by":"auto","created_at":"2025-09-09 13:24:17","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":39157,"visible":true,"origin":"","legend":"","description":"","filename":"TrialsStructuredStudyPotocolTemplatev01.docx","url":"https://assets-eu.researchsquare.com/files/rs-5345711/v1/4d9b76e0d74334857ba8c930.docx"},{"id":90908233,"identity":"46a68497-d5be-461c-8cbc-a8183cc259e9","added_by":"auto","created_at":"2025-09-09 13:24:17","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":63368,"visible":true,"origin":"","legend":"","description":"","filename":"SPIRITFillablechecklist15Aug2013filled.docx","url":"https://assets-eu.researchsquare.com/files/rs-5345711/v1/581c4dd70bcebf7e8c195d59.docx"}],"financialInterests":"","formattedTitle":"Effects of repetitive transcranial magnetic stimulation on electroencephalographical measures of post-stroke upper limb dysfunction: study protocol for a randomized controlled trial","fulltext":[{"header":"Administrative information","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"639\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 34.1158%;\"\u003e\n \u003cp\u003eTitle {1}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65.8842%;\"\u003e\n \u003cp\u003eEffects of repetitive transcranial magnetic stimulation on electroencephalographical measures of post-stroke upper limb dysfunction: study protocol for a randomized controlled trial\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 34.1158%;\"\u003e\n \u003cp\u003eTrial registration {2a and 2b}.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65.8842%;\"\u003e\n \u003cp\u003eThis study was registered in November 2022 (https://www.chictr.org.cn). Clinical trial registration number is ChiCTR2200065639\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 34.1158%;\"\u003e\n \u003cp\u003eProtocol version {3}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65.8842%;\"\u003e\n \u003cp\u003eDate:11/01/2022, version 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 34.1158%;\"\u003e\n \u003cp\u003eFunding {4}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65.8842%;\"\u003e\n \u003col\u003e\n \u003cli\u003eSichuan Provincial Innovative Scientific Research Program for Medical Youth (Q21100)\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSichuan Science and Technology Program (2023NSFSC1496)\u003c/li\u003e\n \u003c/ol\u003e\n \u003cp\u003eOther: Program of Doctor of Health Science of the Hong Kong Polytechnic University\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 34.1158%;\"\u003e\n \u003cp\u003eAuthor details {5a}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65.8842%;\"\u003e\n \u003cp\u003eBo Chen \u003csup\u003e1,2,3\u003c/sup\u003e, Heng Ming Wu Heng \u003csup\u003e4\u003c/sup\u003e ,Sam C.C. Chan\u003csup\u003e1\u003c/sup\u003e*.\u003c/p\u003e\n \u003cp\u003e1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Department of Rehabilitation Sciences, The Hong Kong Polytechnic University\u003c/p\u003e\n \u003cp\u003e2 \u0026nbsp; \u0026nbsp; Department of Rehabilitation, The Affiliated Hospital of Southwest Medical University\u003c/p\u003e\n \u003cp\u003e3\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Department of Rehabilitation, The Affiliated Hospital of Southwest Medical University Laboratory of Neurological Diseases and Brain Function\u003c/p\u003e\n \u003cp\u003e4\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Department of Rehabilitation, The First Affiliated Hospital of Xinjiang Medical University\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 34.1158%;\"\u003e\n \u003cp\u003eName and contact information for the trial sponsor {5b}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65.8842%;\"\u003e\n \u003col\u003e\n \u003cli\u003eDepartment of Rehabilitation Sciences, The Hong Kong Polytechnic University\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDepartment of Rehabilitation, The Affiliated Hospital of Southwest Medical University\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 34.1158%;\"\u003e\n \u003cp\u003eRole of sponsor {5c}\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 65.8842%;\"\u003e\n \u003cp\u003eThe study sponsor and funder have approved the study design but do not participate in data collection, management, analysis, or interpretation of results, writing the report, or deciding whether to submit the report for publication.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Background and rationale {6a}","content":"\u003cp\u003eStroke is one of the common non-communicable diseases that causes long-term disability in people[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The prevalence of stroke in adults is expected to increase significantly by 2030, compared to data reported in 2012[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite various approaches of rehabilitation interventions, a high proportion of patients with stroke still have residual functional impairment[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Those patients with stroke manifest limited potential for functional recovery and present as a challenge for rehabilitation interventions. With the advent of an aging society, stroke, especially with persistent physical dysfunction, is bound to place a heavier burden on society in the future. Thus, there is a need to explore better treatment modalities and to understand the underlying neurological mechanisms[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSince the 80s in the last century, transcranial magnetic stimulation (TMS) machines were introduced in rehabilitation field to serve a non-pharmaceutical treatment[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Through continuous developments, repetitive TMS (rTMS) has gradually been applied in the rehabilitation of stroke. Accumulative evidence suggested that high-frequency-TMS (HF-rTMS) (\u0026ge;\u0026thinsp;3 Hz) would induce an excitatory effect of the synaptic transmission at the cortical level while low-frequency-rTMS (LF-rTMS) (\u0026le;\u0026thinsp;3 Hz) would lead to an inhibitory effect[\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Stroke patients with different lesions in the brain respond differently to rTMS. Previous studies have shown that stroke patients with subcortical lesions have better clinical outcomes than those with cortical lesions alone after rTMS[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This may be explained that better clinical outcomes may be attributed to more preserved and intact cortical network [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and more hyperactive contralateral motor cortex in stroke patients with subcortical lesions[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Besides, some observational studies have found that stroke patients with subcortical and cortical lesions show different activated brain areas in fMRI[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and different cortical activity and functional connectivity of brain networks in EEG[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Therefore, the location of lesions would be a critical factor to be considered when exploring the neurological mechanisms of clinical effects of rTMS in stroke patients[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Currently, a number of studies have demonstrated the potential clinical effectiveness of rTMS in patients with stroke. Based on the interhemispheric competition theory, some clinical trials have selected HF-rTMS to stimulate the affected hemisphere to increase cortical excitability[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In terms of the guidelines for clinical application, rTMS has a relatively high recommendation grade for recovery of motor function in the post-acute phase after stroke[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The evidence quality of the application of LF-rTMS on the contralateral motor cortex is regarded as an A grade for recovery of hand function whilst that of the application of HF-rTMS on the ipsilateral M1 is regarded as a B grade for upper limb function improvement[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. On the other hand, although there is no consensus about the standardized guideline to suggest the optimal frequency of HF-rTMS at the affected hemisphere to promote upper limb recovery among the stroke patients, accumulative evidence suggests that rTMS with 10 Hz, which is considered to be a high frequency, acting on the affected hemisphere M1 is beneficial for motor recovery among stroke patients[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Besides, motor evoked potentials (MEPs) ha been commonly used to evaluate the neural effects of LF and HF-rTMS with the use of the single pulse TMS[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, the findings about MEPs in exploring cortical regions of the brain beyond M1 appear to be limited in the literature[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Hence, resting-state EEG, which is one of the common and economical tools, would be an appropriate measure to examine the neurological processes underlying the changes in upper limb motor functions induced by HF/LF-rTMS among people with based on the interhemispheric competition theory[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNormalized power (NP) characterizes cortical activity by quantifying the power of brain oscillations, and while balanced cortical activity is characteristic of a healthy brain, Electrode directional asymmetry (EDA) can visualize it[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. At the same time, rich and strong brain network function connectivity facilitates the brain's ability to perform physiological functions[\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This series of hierarchical resting-state EEG indicators can provide us with a panoramic view of the neurophysiology of the brain[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Previous studies showed that rTMS with different frequency combined with other rehabilitation training could modulate power spectral and functional connectivity in healthy participants to enhance motor function[\u003cspan additionalcitationids=\"CR29 CR30 CR31\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The stroke population suffers from dysfunction, the cortical oscillatory activity and network functional connectivity differ from the healthy population[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Similarly, resting-state EEG indicators as biomarkers also can be used to explore neurological mechanisms in stroke patients after rTMS interventions. Ding found that iTBS can increase brain oscillatory power and network connectivity, while the efficiency of brain information transfer is improved[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, the study did not confirm whether the improvement in these EEG indicators was a neurophysiologic mechanism for the improvement in clinical function, as no correlation was found between them[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Besides, Zhong and colleagues also reported that an intervention protocol with 10-Hz rTMS reduced oscillation activity in the affected hemisphere, resulting an improvement in interhemispheric imbalance[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. But, the study also did not confirm whether improved cortical activity balance was associated with improved clinical functioning[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In addition, rTMS was applied on the vertex of the brain, which is different from the conventional protocol in which HF-rTMS was applied on M1 of the affected hemisphere[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Thus, whether rTMS stimulation on cortical motor areas of stroke patients can modulate the cortical activity balance characterized by EDA has not been answered. Moreover, all of these studies only explored immediate changes in resting-state EEG after the intervention and did not reflect changes after a period of intervention. Thus, there is need to examine the long-term effect of the rTMS on motor recovery among stroke patients. In conclusion, while rTMS has been shown to stimulate cortical motor areas in healthy individuals can modulate brain oscillatory power and brain network functional connectivity to enhance limb function[\u003cspan additionalcitationids=\"CR29 CR30 CR31\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. However, this was not confirmed in a series of pre-studies of rTMS interventions in stroke patients, and variation existed in rTMS protocols[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and stimulation areas[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Meanwhile, reports of changes in resting-state EEG indicators after LF-rTMS treatment of stroke patients are scarce. Therefore, the neurophysiological mechanisms underlying the clinical effects of LF- and HF-rTMS stimulation of cortical motor areas as a traditional therapeutic protocol for improving function of stroke patients need to be clarified.\u003c/p\u003e\n\u003ch3\u003eObjectives {7}\u003c/h3\u003e\n\u003cp\u003e\u003cb\u003eThus, the objective of this study was to investigate the neurophysiological mechanisms underlying the clinical effects of rTMS in stroke patients at different levels by measuring changes in cortical oscillatory power, EDA, and functional connectivity in stroke patients after receiving LF- and HF-rTMS, and whether these changes correlate with improvements in clinical function. Therefore, we hypothesized that HF- and LF-rTTMS stimulation of cortical motor areas in stroke patients could modulate cortical oscillatory power thereby balancing the EDA while enhancing the brain network functional connectivity. Meanwhile, changes in resting-state indicators after rTMS intervention correlated with changes in clinical scales.\u003c/b\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eTrial design {8}\u003c/h2\u003e\u003cp\u003eThis study protocol is based on the \u0026ldquo;Standard Protocol Items: Recommendations for Interventional Trials\u0026rdquo; (SPIRIT) 2013 statement[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. A design of randomized double-blind controlled trial (RCT) with pre-post measurement time points will be adopted for this study. The outcome measures will be accessed at the day before the first intervention (T0) baseline and post 10 sessions intervention (T1) by an assessor who is unaware of the participant's allocation information (Fig.\u0026nbsp;1).\u003c/p\u003e\u003c/div\u003e"},{"header":"Methods: Participants, interventions and outcomes","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eStudy setting {9}\u003c/h2\u003e\u003cp\u003eParticipants will be recruited from both inpatient wards and outpatient clinics at the Affiliated Hospital of Southwest Medical University.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eWho will provide informed consent? {26a}\u003c/h3\u003e\n\u003cp\u003ePrior to the start of the trial, the investigator will explain the details and steps of the trial in detail to the participants, and obtain their consent and sign an informed consent form before the trial begins. Confidentiality and anonymity of all participants will be guaranteed, in accordance with the requirements of the university where the research team is based and the ethical requirements of the trial.\u003c/p\u003e\n\u003ch3\u003eAdditional consent provisions for collection and use of participant data and biological specimens {26b}\u003c/h3\u003e\n\u003cp\u003e The informed consent form will provide participants with a detailed explanation of the medical records collected, biospecimens and personal data that will be analyzed as part of the research.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eEligibility criteria {10}\u003c/h2\u003e\u003cp\u003eParticipants to be recruited in this study will be required to fulfill the following inclusion criteria:\u003c/p\u003e\u003cp\u003e(1) the first episode of a unilateral ischemic stroke with stable condition.\u003c/p\u003e\u003cp\u003e(2) the infarct lesion location determined by imaging and classified as cortical, subcortical, or cortico-subcortical[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e(3) at the age\u0026thinsp;\u0026ge;\u0026thinsp;18 years.\u003c/p\u003e\u003cp\u003e(4) right-handedness.\u003c/p\u003e\u003cp\u003e(5) the Functional Test of the Upper Extremity in Hemiplegia (FTHUE-HK) being \u0026le;\u0026thinsp;4[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e(6) Having intact comprehension and expression functions.\u003c/p\u003e\u003cp\u003e(7) being able to understand and voluntarily sign a written consent form to participate in the study.\u003c/p\u003e\u003cp\u003eParticipants will be excluded if they will meet one of the following conditions:\u003c/p\u003e\u003cp\u003e(1) hemorrhagic stroke.\u003c/p\u003e\u003cp\u003e(2) previous diagnosis of a neurological disorder besides stroke.\u003c/p\u003e\u003cp\u003e(3) presence of cognitive impairment or aphasia that affects the comprehension ability, Mini-Mental State Examination(MMSE)\u0026le;21[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e(4) presence of pain that prevents completion of functional assessment.\u003c/p\u003e\u003cp\u003e(5) impaired upper limb injury functions due to neurological diseases rather than stroke.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eInterventions\u003c/h3\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eExplanation for the choice of comparators {6b}\u003c/h2\u003e\u003cp\u003eIn this research, the no sham-rTMS group will be used as a control group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eIntervention description {11a}\u003c/h2\u003e\u003cp\u003eThe rTMS intervention will be implemented at a public hospital in the Mainland China. The rTMS instrument model is the Magpro R30 stimulator (MagVenture, Lucernemarken, Denmark), with a figure-of-eight coil connected to emit magnetic stimulations. rTMS stimulations with 10 Hz or 1 Hz at the contralateral primary motor cortex (M1) are expected to elicit contraction of the first interosseous muscle of the affected hand. If muscle activity cannot be observed by the above method, the corresponding position for the M1 of the ipsilateral hemisphere will be selected as the stimulation location. The minimum transcranial magnetic stimulation intensity will be set based on a resting motor threshold (RMT) that elicits motor evoked potentials with an amplitude of 50\u0026micro;V for at least half of the 10 consecutive stimulations at the primary motor area[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The rTMS stimulation parameters for each of groups will be performed as follows: (1) The HF-rTMS stimulation protocol will follow the previous study[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], with 10 Hz-rTMS applied to ipsilateral hemisphere M1 with a stimulation intensity of 90% of the RMT. Each treatment session including 30 trains, with 50 pulses per trains session which will last 25 seconds, leading to a total 1500 pulses a session. Each participant will receive 10 consecutive sessions of treatment. (2) The LF-rTMS group will receive the same parameters as HF-rTMS at the contralesional primary motor area, except that 1 Hz will be be used. The rTMS operations will all be performed by the same experienced clinician, who will be responsible for managing and documenting adverse events for each stimulation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eCriteria for discontinuing or modifying allocated interventions {11b}\u003c/h2\u003e\u003cp\u003eIf a participant develops any discomfort while participating in the study, we will stop the trial immediately. The trial will be restarted only after the participant's discomfort has been appropriately managed by the medical team and we have received a signal from the participant that he or she agrees to continue to the next step. Also, we will terminate the trial at any time, once the participant signals termination or refuses to participate in the next step of the process.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eStrategies to improve adherence to interventions {11c}\u003c/h2\u003e\u003cp\u003eThe investigator will coordinate the timing of the participant's receipt of the rTMS and the timing of the outcome assessment to ensure that the participant is able to complete the full trial process as planned. If necessary, the investigator will again explain in detail to the participant the benefits and risks of participating in the study to ensure that the participant fully understands the study.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eRelevant concomitant care permitted or prohibited during the trial {11d}\u003c/h2\u003e\u003cp\u003eParticipants in both the HF- and LF-rTMS groups will be able to receive traditional rehabilitation treatments such as physical therapy and occupational therapy at the same time.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eProvisions for post-trial care {30}\u003c/h2\u003e\u003cp\u003eAll participants receiving the rTMS intervention who experience a serious adverse reaction will be referred to the medical team for continued monitoring and treatment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eOutcomes {12}\u003c/h2\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003eClinical Outcome Measures\u003c/h2\u003e\u003cp\u003e\u003cem\u003eThe National Institutes of Health Stroke Scale (NIHSS)\u003c/em\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The NIHSS is an established tool utilized to evaluate the severity of stroke. It encompasses 15 items, generating a cumulative score ranging from 0 to 42. A lower score represents a reduced degree of neurological impairment. The scale assesses various aspects, including consciousness, eye movement, visual fields, facial palsy, motor function, sensory function, language, and neglect. Trained evaluators will administer the NIHSS before and after the rTMS to determine changes in stroke severity.\u003c/p\u003e\u003cp\u003e\u003cem\u003eFugl-Meyer Assessment upper Extremity (FMA-UE)\u003c/em\u003e[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The FMA-UE will be employed in this study as a validated tool recommended for clinical application in assessing upper limb impairment in patients after stroke[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. It assesses the movement, coordination and reflexes of each joint of the upper limb of the hemiplegic side through 33 items. Each item is divided into three scoring levels (0,1,2) with a total score of 66. In addition, there are 6 items to assess upper limb sensation, including light touch and position sense, with a total of 12 points. Trained evaluators will administer the FMA-UE before and after the rTMS to monitor changes in upper limb function.\u003c/p\u003e\u003cp\u003e\u003cem\u003eModified Barthel Index (MBI)\u003c/em\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The MBI is a scale that assesses the ability to perform self-care activities of daily living and contains assessments ranging from feeding, self-care, and mobility. Each item can be rated at five levels, with different levels representing different levels of independence. The total score is 0 to 100, with higher scores indicating higher independence in daily living. In this study, the MBI will be administered pre- and post-rTMS to measure any changes in participants' functional independence.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eEEG data measures\u003c/h2\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003eEEG data acquisition\u003c/h2\u003e\u003cp\u003eEach participant will have their hair scalp cleansed before the rTMS experiment. Each EEG data collection will be conducted in a semi-isolation using the same set of equipment. During resting-state EEG signal collection, participants will be seated comfortably on chair. Each of them was required to keep his or her eyes closed and mind clear throughout the experiment process. The acquisition of EEG signals will be recorded via a 19-channel analog recorder according to the international 10\u0026ndash;20 system (EB Neuro, Firenze, Italy). The placement of the 19 electrodes (FP1, FP2, F3, F4, F7, F8, T3, T4, T5, T6, C3, C4, P3, P4, O1, O2, Fz, Cz, and Pz) on the scalp will be determined by the aid of a quantitative ruler. The impedance of each electrode will be kept 5 kΩ or below, and the sampling rate will be set to 256 Hz. The data collection process will be performed by an experienced experimenter to ensure the data quality during the data acquisition.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eEEG data preprocessing\u003c/h2\u003e\u003cp\u003eThe raw EEG signal will be band-passed between 1 and 50 Hz using a fourth-order zero-phase Butterworth filter. The data of each trial will be extracted by segmenting from the beginning to the end into 2-second contiguous but non-overlapping segments. Then, the bad channels will be checked and deleted manually, and the value of the deleted channels will be replaced by the interpolation of the surrounding electrode channels. The EEG data of participants with stroke will be flipped so that the ipsilateral hemispheres are always presented on the same side for easy observation. In addition, artifacts caused by eye movements, muscle movements, or other factors will be removed manually. The EEG data will be re-referenced using the full average reference method and finally, the processed data from each channel will be decomposed by finite impulse response (FIR) bandpass filtering into δ (0.5-4 Hz), θ (4\u0026ndash;7 Hz), α1 (8\u0026ndash;10 Hz), α2 (10\u0026ndash;12 Hz), β (13\u0026ndash;30 Hz) and γ (30\u0026ndash;40 Hz). To reduce bias, a normalized method will be applied to process EEG power and use the processed power values as an indicator of network activity. Functional connectivity between cortical regions will be measured using orthogonal correlations of EEG activity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003ePower spectrum analysis\u003c/h2\u003e\u003cp\u003eSpectrum analysis changes the time domain signal into the frequency domain and provides a distribution curve of the power, amplitude or phase along the frequency, i.e., power spectrum[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. To examine the spatial characteristics of resting-state EEG power in each frequency band, first to calculate the power spectrum for each electrode using the method of welch[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and then perform power spectrum analysis at the electrode level. The frequency bands will then be extracted from the power spectrum and normalized at each electrode using Eq.\u0026nbsp;1,\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:NP=100\\times\\:\\frac{\\sum\\:F}{\\sum\\:Total}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e,\u003c/p\u003e\u003cp\u003eNP means normalized power, i.e., the ratio between the power of a band and the total power of the whole frequency range. \u0026sum;F means the sum of power within a frequency band, \u0026sum;Total means the sum of power across the frequency spectrum. This power normalization method not only allows to observe whether the cortical areas change in function by determining the power distribution across the spectrum, but also eliminates the effects of biasing factors, such as electrode impedance and the size of the neuronal population. To further clarify the true cause of the normalized power difference between the real rTMS and sham rTMS groups, we will calculate and plot the mean absolute power of each electrode in the frequency band. If, similarly, the two groups also differ in absolute power, the possible bias introduced to the power normalization is further excluded.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eElectrode directional asymmetry\u003c/h2\u003e\u003cp\u003eBased on the normalized power, Eq.\u0026nbsp;2 is further used to calculate the electrode direction asymmetry and thus determine whether there is a difference in power in frequency bands between the two hemispheres.\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:EDA=100\\times\\:\\frac{1}{n}\\sum\\:\\frac{{NP}_{L}-{NP}_{R}}{\\left|{NP}_{L}\\right|+\\left|{NP}_{R}\\right|}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e,\u003c/p\u003e\u003cp\u003eEDA means the whole head electrode directional asymmetry, NPL means the normalized power of the homologous electrode in the left hemisphere, NPR is the normalized power of the homologous electrode in the right hemisphere, and n is the total number of electrode pairs.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eFunctional connectivity analysis\u003c/h2\u003e\u003cp\u003eVolume source localization will be performed on the EEG data before applying the eeglab toolbox for functional connectivity analysis. In order to determine whether there is a difference in the connection of the frequency bands between the two hemispheres, the indicator of connection direction asymmetry between analogue connections of the two hemispheres will be calculated using Eq.\u0026nbsp;3,\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:CDA=100\\times\\:\\frac{1}{n}\\sum\\:\\frac{{C}_{L}-{C}_{R}}{\\left|{C}_{L}\\right|+\\left|{C}_{R}\\right|}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e,\u003c/p\u003e\u003cp\u003eCDA means the whole brain connectivity directional asymmetry metric, CL means the connectivity of the homologous connections in the left hemisphere, CR means the connectivity of the homologous connections in the right hemisphere, and n means the total number of homologous connection pairs. Deviations in the shape of the control group connection spectrum will be quantified by correlating the connection spectrum of all participants with the average connection spectrum of the control group.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eParticipant timeline {13}\u003c/h2\u003e\u003cp\u003eData collection will take place from November 2022 through November 2025, with participant recruitment continuing through November 2024. Ethical monitoring will continue throughout the data collection period.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eSample size {14}\u003c/h2\u003e\u003cp\u003ePrevious related studies utilizing EEG to gauge rTMS treatment effects in stroke patients did not provide effect size values for sample size estimation[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. These studies, however, with sample sizes ranging from 15 to 30, reported significant statistical differences, supporting our sample size determination.\u003c/p\u003e\u003cp\u003eThe study also aimed to evaluate improvement in post-stroke upper limb dysfunction, assessed by the FMA-UE. Based on a previous study that reported altered FMA-UE values post-intervention[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], we calculated an effect size of 0.57. Using G*power 3.1 software, with α set to 0.05 and power to 0.9 for a paired samples t-test, we determined that a total of 35 samples were needed. To account for potential drop-outs, with a projected 20% drop-out rate, we decided to include at least 42 total participants, or 21 in each group.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eRecruitment {15}\u003c/h2\u003e\u003cp\u003eParticipants will be recruited from both inpatient wards and outpatient clinics at the Affiliated Hospital of Southwest Medical University. The investigator will provide potential participants with a detailed description of the research, the possible benefits, and the risks.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eAssignment of interventions: allocation\u003c/h2\u003e\u003cdiv id=\"Sec28\" class=\"Section4\"\u003e\u003ch2\u003eSequence generation {16a}\u003c/h2\u003e\u003cp\u003eAll participants will be assigned randomly to the higher rTMS frequency (10 Hz rTMS) and lower rTMS frequency (1 Hz rTMS) groups in a 1:1 ratio using a random assignment tool with the lesion sites at cortical, subcortical and cortico-subcortical levels matched between the two interventions group. Randomized serial lists will be produced with the help of statistical software and the randomly generated serial numbers will be placed in sealed, opaque envelopes to ensure that they are not compromised.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eImplementation {16c}\u003c/h2\u003e\u003cp\u003eSequence numbers will be sealed in opaque numbered envelopes, and participants will draw envelopes according to the number, and the envelopes that have been drawn will be marked with the basic information of the participants and then strictly stored for inspection and prevent confusion.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAssignment of interventions: Blinding\u003c/h3\u003e\n\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003eWho will be blinded {17a}\u003c/h2\u003e\u003cp\u003eThe allocation information will be blinded from participants and assessors.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\u003ch2\u003eData collection and management\u003c/h2\u003e\u003cdiv id=\"Sec33\" class=\"Section3\"\u003e\u003ch2\u003ePlans for assessment and collection of outcomes {18a}\u003c/h2\u003e\u003cp\u003eAfter passing the screening, the investigator will determine to which group the participant will be allocated by drawing lots from a sealed envelope, but the results will be blinded from the assessor and the participant. At the baseline, the demographic information of each participant, who fulfils the inclusion criteria, will be obtained and the participants will be assessed using the NIHSS, The motor deficit (FMA-UE), MBI, and resting-state EEG before the rTMS treatment. Then, ten sessions of rTMS will be given to each participant with 5 times a week for two weeks. The participants will be assessed by the same outcome measures after the 10-session rTMS intervention. The entire experimental procedure that each participant will go through is illustrated in Table\u0026nbsp;1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section3\"\u003e\u003ch2\u003ePlans to promote participant retention and complete follow-up {18b}\u003c/h2\u003e\u003cp\u003eThere will be no process for follow-up in this research.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eData management {19}\u003c/h3\u003e\n\u003cp\u003ePaper forms will be kept in a secure place and transcribed data will be collected by individuals using password-protected computers. Research materials and data will be kept by the principal investigator for the appropriate number of years as required by the supervisory authority.\u003c/p\u003e\n\u003ch3\u003eConfidentiality {27}\u003c/h3\u003e\n\u003cp\u003eThe Principal Investigator will ensure that all study data are stored securely and that confidentiality and privacy are protected in accordance with applicable laws and regulations. Participants will remain anonymous throughout the research.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePlans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use {33}\u003c/b\u003e\u003c/p\u003e\u003cp\u003eN/A; Biological samples, such as blood, will not be collected for this research.\u003c/p\u003e\u003cdiv id=\"Sec37\" class=\"Section2\"\u003e\u003ch2\u003eStatistical methods\u003c/h2\u003e\u003cdiv id=\"Sec38\" class=\"Section3\"\u003e\u003ch2\u003eStatistical methods for primary and secondary outcomes {20a}\u003c/h2\u003e\u003cp\u003eDemographic and baseline data will be compared using SPSS 19, with variance analysis for continuous data and \u003cem\u003ex\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e test for categorical data. The independent \u003cem\u003et\u003c/em\u003e test with false discovery rate (FDR) will be employed to investigate difference changes in normalized power distribution, absolute power distribution, EDA between HF-and LF rTMS group after intervention. In addition, paired \u003cem\u003et\u003c/em\u003e-tests with FDR correction will employ for clinical outcomes and rs-EEG indicators in the comparison of post-rTMS and baseline outcomes in each group. Correlation of resting-state EEG indicators will be analyzed with clinical functional changes. The type I error rate for statistical tests is α\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec39\" class=\"Section2\"\u003e\u003ch2\u003eInterim analyses {21b}\u003c/h2\u003e\u003cp\u003eThere are no interim analyses planned for this research.\u003c/p\u003e\u003cdiv id=\"Sec40\" class=\"Section3\"\u003e\u003ch2\u003eMethods for additional analyses (e.g. subgroup analyses) {20b}\u003c/h2\u003e\u003cp\u003eThere are no additional analyses planned for this research.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c}\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis analysis is not planned for this research.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePlans to give access to the full protocol, participant level-data and statistical code {31c}\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThere are no plans to provide public access to datasets or statistical codes at the participant level. However, all results, both positive and negative, will be published. Funding sources will not affect the reporting of results.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOversight and monitoring\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eComposition of the coordinating center and trial steering committee {5d}\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe research team consists of physicians, rehabilitation therapists, senior researchers, and doctoral students with clinical experience in rehabilitation.\u003c/p\u003e\u003cp\u003eThe senior investigator has extensive experience in clinical trials. All team members will be trained prior to the start of the study and regular meetings will be held throughout the research process to ensure quality control and prevent errors.\u003c/p\u003e\u003cp\u003e\u003cb\u003eComposition of the data monitoring committee, its role and reporting structure {21a}\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe Principal Investigator will meet with the team monthly to audit monitoring activities and adverse events.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBased on these audits, recommendations for study continuation, modification, or termination will be made.Adverse event reporting and harms {22}\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAlthough rTMS may cause some adverse effects such as seizures, temporary hearing discomfort, localized pain, syncope, and minor cognitive changes, these discomforts have been rarely reported[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. For the safety reasons, participants meeting the following contraindications to rTMS will be excluded: (1) unstable disease status; (2) history of epilepsy, impaired consciousness or intracranial hypertension; (3) history of heart disease; (4) pregnancy; (5) metal implants in the body, such as an artificial cochlear, pacemaker; (6) underwent a craniotomy; (7) taking any centrally acting medication within the recent 3 months. TMS operators will complete operational and safety knowledge training. All participants will be screened for safety risks and those unsuitable for TMS intervention will be excluded.\u003c/p\u003e\u003cp\u003eFirst aid facilities will be equipped in the EEG acquisition room and the rTMS intervention room. During the EEG acquisition and rTMS intervention, the experimenter will observe the status of the participant closely and will halt the operation of the rTMS immediately if he or she will report any discomfort. In case of sudden illnesses such as coma or epilepsy, immediately take necessary measures to protect the safety of the participant and transfer to the emergency room by medical personnel for further treatment. An adverse event survey will be conducted by the experimenter for each participant after the completion of the rTMS intervention.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFrequency and plans for auditing trial conduct {23}\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAudits and inspections may be conducted by the Ethics Committee of the Hong Kong Polytechnic University. Auditors have the right to access all data, raw materials, informed consent forms and any other necessary clinical trial documentation.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePlans for communicating important protocol amendments to relevant parties (e.g. trial participants, ethical committees) {25}\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt present, no modifications to this study are planned. If modifications are required in the future, the researchers will inform the ethics committee.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDissemination plans {31a}\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe results of this study will be sent to a peer-reviewed professional journal for publication.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study protocol outlines a randomized controlled trial designed to investigate the effects of HF- and LF-rTMS on upper limb dysfunction in post-stroke patients. The primary aim is to elucidate the neurophysiological mechanisms underlying the clinical efficacy of rTMS, focusing on changes in cortical oscillatory activity and the effectiveness of the complex brain network.\u003c/p\u003e\n\u003cp\u003eOne of the key strengths of this study is its rigorous design, including randomization and the use of both HF- and LF-rTMS protocols, which allows for a comprehensive comparison of their effects. Additionally, the use of EEG to measure resting-state brain activity provides valuable insights into the neurophysiological changes associated with rTMS. However, the study also has limitations. The relatively small sample size (n=42) may limit the generalizability of the findings. Furthermore, the short follow-up period may not capture long-term effects of rTMS on motor recovery.\u003c/p\u003e\n\u003cp\u003ePrevious studies have demonstrated the potential of rTMS in improving motor function in stroke patients, but the optimal parameters and underlying mechanisms remain unclear. This study aims to address these gaps by comparing the effects of HF- and LF-rTMS on both clinical outcomes and neurophysiological measures. The use of a well-defined protocol and standardized outcome measures, such as the NIHSS and the FMA-UE, ensures the reliability and validity of the results.\u003c/p\u003e\n\u003cp\u003eThe findings from this study could have significant implications for clinical practice. If HF- or LF-rTMS is shown to be effective in enhancing motor recovery and modulating brain activity, it could be incorporated into rehabilitation programs for stroke patients. This would provide a non-invasive, adjunctive treatment option to improve upper limb function, potentially reducing the burden on healthcare systems and improving the quality of life for stroke survivors.\u003c/p\u003e\n\u003cp\u003eFuture studies should consider larger sample sizes and longer follow-up periods to validate and extend the findings of this trial. Additionally, exploring the combination of rTMS with other rehabilitation interventions, such as physical therapy or occupational therapy, could provide insights into synergistic effects and optimize treatment protocols. Investigating the differential effects of rTMS on various subtypes of stroke and patient characteristics could also help tailor interventions to individual needs.\u003c/p\u003e\n\u003cp\u003eThis study will contribute to the understanding of the neurophysiological changes in the brain corresponding to the clinical effects of HF- and LF-rTMS in patients with stroke. The results could pave the way for more effective and personalized rehabilitation strategies, ultimately enhancing the recovery process for stroke survivors.\u003c/p\u003e\n\u003ch2\u003eTrial status\u003c/h2\u003e\n\u003cp\u003eStart date for recruiting participants: November 2022; End of data collection: expected to end in December 2024.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003erTMS \u0026nbsp; Repetitive transcranial magnetism stimulation\u003c/p\u003e\n\u003cp\u003eLF \u0026nbsp; \u0026nbsp; Low frequency\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHF \u0026nbsp; \u0026nbsp; High frequency\u003c/p\u003e\n\u003cp\u003eNIHSS \u0026nbsp; National Institutes of Health Stroke Scale\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFMA-UE \u0026nbsp;Fugl\u0026ndash;Meyer Assessment upper Extremity\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMBI \u0026nbsp; Modified Barthel Index\u003c/p\u003e\n\u003cp\u003eEEG \u0026nbsp;Electroencephalogram\u003c/p\u003e\n\u003cp\u003eNP \u0026nbsp;Normalized power\u003c/p\u003e\n\u003cp\u003eEDA \u0026nbsp;Electrode directional asymmetry\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eEEG equipment and rTMS equipment from the Affiliated Hospital of Southwest Medical University will be used in this study, for which we would like to express our gratitude.\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026rsquo;\u0026nbsp;contributions {31b}\u003c/h2\u003e\n\u003cp\u003eSam C. C. Chan (SCC) and Chen Bo (CB) conceived and designed this study collaboratively. CB, Wu Hengming (WHM) wrote the first draft of this protocol, and CCC reviewed and revised the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eFunding {4}\u003c/h2\u003e\n\u003cp\u003eCB\u0026apos;s work was supported by Program of Doctor of Health Science of the Hong Kong Polytechnic University, Sichuan Provincial Innovative Scientific Research Program for Medical Youth (Q21100) (An additional file shows this in more detail [see Additional file 1]), Sichuan Science and Technology Program (2023NSFSC1496) (An additional file shows this in more detail [see Additional file 2]).\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials {29}\u003c/h2\u003e\n\u003cp\u003eNo data was used for the research described in the article.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate {24}This study was registered in November 2022 (https://www.chictr.org.cn). Actual recruitment for this trial begins in November 2022 and is expected to last one year. This proposed study will be conducted in compliance with the Declaration of Helsinki. The participant\u0026apos;s consent and sign an informed consent form will be obtained before starting the trial. Modifications to this study protocol will be performed only after review and consent by the Ethics Committee (HSEARS20220303002).\u003c/p\u003e\n\u003ch2\u003eConsent for publication {32}\u003c/h2\u003e\n\u003cp\u003eAll authors have approved the publication of this protocol.\u003c/p\u003e\n\u003ch2\u003eCompeting interests {28}\u003c/h2\u003e\n\u003cp\u003eAll authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMurray CJL, Atkinson C, Bhalla K, Birbeck G, Burstein R, Chou D, et al. The state of US health, 1990\u0026ndash;2010: burden of diseases, injuries, and risk factors. 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IEEE Trans Audio Electroacoust. 1967;15:70\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"trials","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trls","sideBox":"Learn more about [Trials](http://trialsjournal.biomedcentral.com/)","snPcode":"13063","submissionUrl":"https://www.editorialmanager.com/trls","title":"Trials","twitterHandle":"MedicalEvidence","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Stroke, Resting-state electroencephalography, Brain network, Non-invasive brain stimulation","lastPublishedDoi":"10.21203/rs.3.rs-5345711/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5345711/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatients with stroke would experience upper limb dysfunction, leading to functional impairments in daily living and placing a burden on society and families. Repetitive transcranial magnetism stimulation (rTMS), a non-invasive neuromodulation tool, has been shown to upper limb dysfunction. However, the clinical efficacy and the underlying neurophysiological mechanisms of optimal intensity of rTMS to promote motor recovery of stroke patients with upper limb function need further investigations. This study aims to investigate the changes in the brain neurophysiological mechanisms ranging from cortical oscillatory activity to the effectiveness of the complex network after low frequency (LF)- and high frequency (HF)-rTMS on motor areas of patient post-stroke.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 42 stroke patients with upper limb dysfunction will be randomized into high frequency (HF) rTMS group, low frequency (LF) rTMS group (1:1 ratio). The HF-rTMS group will use 90% rest motor threshold (RMT), 10 Hz acting with the ipsilesional M1 for a total of 1500 pulses for 2 weeks, and the LF-rTMS group acting on the contralesional M1 with the same parameters, except that 1 Hz was used. The National Institutes of Health Stroke Scale (NIHSS), The motor deficit (the Fugl–Meyer Assessment upper Extremity (FMA-UE)), Modified Barthel Index (MBI), and resting-state electroencephalogram (EEG) signals will be obtained at the baseline and within one week after the rTMS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiscussion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study will contribute to the understanding of the neurophysiological changes in the brain corresponding to the clinical effects of HF- and LF-rTMS in patients with stroke.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial registration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of the Hong Kong Polytechnic University and the Ethics Committee of the Affiliated Hospital of Southwest Medical University (HSEARS20220303002). This study was registered in November 2022 (https://www.chictr.org.cn). Clinical trial registration number is ChiCTR2200065639.\u003c/p\u003e","manuscriptTitle":"Effects of repetitive transcranial magnetic stimulation on electroencephalographical measures of post-stroke upper limb dysfunction: study protocol for a randomized controlled trial","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-09 13:24:10","doi":"10.21203/rs.3.rs-5345711/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2026-01-21T04:38:33+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-09-05T11:10:05+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-02T20:36:33+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Trials","date":"2025-03-04T12:10:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-17T11:37:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Trials","date":"2025-02-16T23:59:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"trials","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trls","sideBox":"Learn more about [Trials](http://trialsjournal.biomedcentral.com/)","snPcode":"13063","submissionUrl":"https://www.editorialmanager.com/trls","title":"Trials","twitterHandle":"MedicalEvidence","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c2c1a378-f7b6-4485-896f-cac1e0bea106","owner":[],"postedDate":"September 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-23T16:00:31+00:00","versionOfRecord":{"articleIdentity":"rs-5345711","link":"https://doi.org/10.1186/s13063-026-09627-1","journal":{"identity":"trials","isVorOnly":false,"title":"Trials"},"publishedOn":"2026-03-16 15:57:58","publishedOnDateReadable":"March 16th, 2026"},"versionCreatedAt":"2025-09-09 13:24:10","video":"","vorDoi":"10.1186/s13063-026-09627-1","vorDoiUrl":"https://doi.org/10.1186/s13063-026-09627-1","workflowStages":[]},"version":"v1","identity":"rs-5345711","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5345711","identity":"rs-5345711","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}