The Treatment of Transcranial Alternating Current Stimulation（tACS）on Patients With Cerebellar Ataxia: A Randomized, Triple-blind, Parallel-controlled Study
Phase: NA
Conditions: Spinocerebellar Ataxia Type 3, Multiple System Atrophy-cerebellar Type
Sponsor: First Affiliated Hospital of Fujian Medical University

Official Title: Effect of transcranial alternating current stimulation in patients with 
cerebellar ataxia (spinocerebellar ataxia type 3 and multiple system atrophy): a 
randomized proof-of-concept clinical trial. 
NCT number: NCT05557786 
Date of the document: June 6, 2023 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

STUDY PROTOCOL FOR SCA3 
1. Project title 
Effect of transcranial alternating current stimulation in spinocerebellar ataxia type 3: a 
randomized proof-of-concept clinical trial.  
2. Abstract of project 
Background/Objective: Spinocerebellar ataxia type 3 (SCA3) is the most common SCA 
worldwide. Currently, no specific pharmacological and treatments can alter natural course of 
the disease. Clinical practice guidelines had confirmed the safety of transcranial alternating 
current stimulation (tACS) in treating neuropsychiatric diseases, but to our knowledge, no 
randomized clinical trials have investigated whether tACS is safety and efficacy during 
improve symptoms in SCA3.The objectives of this study are to identify the efficacy and 
safety of tACS during improving ataxia symptoms and non-ataxia symptoms in adult patients 
with SCA3. 
Participants: Individual with SCA3 
Design: A triple-blind, randomized sham-controlled trial with two groups. At least 80 subjects 
will be recruited and randomized into either active tACS group or sham tACS group. 
Interventions: Cerebellar tACS (40 min, 2 mA, ramp-up and down periods of 10s each) will 
be delivered over 10 sessions, distributed in two groups of five consecutive days with a two-
day break in between. The active tACS group will receive whole course current stimulation, 
while the control group will receive a daily 40–seconds current stimulation for two weeks. 
Primary outcomes: The primary outcome was the proportion of participants whose Scale for 
the Assessment and Rating of Ataxia (SARA) score improved by at least 1.5 points compared 
with baseline (T0) on assessments immediately after treatment (T1), at 1-month (T2) and 3-
month (T3) follow-up visits. 
Secondary outcomes: Ataxia symptoms measured by SARA and The International 
Cooperative Ataxia Rating Scale (ICARS); health–related quality of life (HRQoL) measured 
by the EuroQol Five-dimensional questionnaire (EQ-5D); and objective quantitative outcome 
for gait measured by the wearable sensors. 
Impact of the project: With the promising preliminary benefits of tACS on improving 
cerebellar motor functions and is safe and effective in treating neurological diseases, however, 

the efficacy of tACS in cerebellar disorders with SCA3 has not been clinically investigated. 
Therefore, we performed a triple-blind, parallel-group, sham-controlled, randomized clinical 
trial to investigate the efficacy and safety of tACS in a large cohort of patients with SCA3. 
The primary outcome assessed ataxia severity, and secondary outcomes evaluated quality of 
life, and adverse events (AEs). 
Introduction 
Spinocerebellar ataxia type 3 (SCA3) is the most common autosomal dominantly inherited 
ataxia worldwide, also called Machado-Joseph disease (MJD), caused by an over-repetition of 
the trinucleotide CAG within the ATXN3 gene, which confers toxic properties to ataxin-3 
(ATXN3) species1. It is a devastating neurodegenerative disorder that principally affects the 
deep cerebellar and pontine nuclei, basal ganglia, and spinal cord, and leading to severe 
disability and premature death. Clinical symptoms in SCA3 are not restricted to the 
deterioration in the motor domain, but non-motor symptoms (NMS), such as sleep 
disturbance, fatigue, neuropathy, cognitive decline, depression, and bladder disturbance. 2.  
Currently, no specific pharmacological and treatments can alter natural course of the disease. 
Cerebellar transcranial alternating current stimulation (tACS) is an increasingly easy-used, 
safe, cheap, and noninvasive tool in neuroscience that is able to modulate cerebellar 
excitability3-5. Computational modeling studies have demonstrated the biophysical feasibility 
of modulating cerebellar structures using tACS with only negligible spreading effects to 
neighboring regions6. Initially, this technique has been successfully applied in healthy 
volunteers to investigate the neural correlates of motor learning and cognitive and emotional 
processes7-17. More recently, studies also started to explore its therapeutic potential in a 
variety of neurological conditions, such as Parkinson's disease 18,19, Alzheimer's disease20,21, 
epilepsy22, schizophrenia23, depression24, insomnia25, and stroke26. No serious adverse events 
of cerebellar tACS have been reported in these and other studies and it is therefore considered 
a safe and tolerable method 27. Possible side effects mainly include a transient optical illusion, 
itching, tingling, or mild burning sensation underneath the electrodes27. Inspired by these 
promising findings, we designed the clinically-oriented SCA3-tACS, randomized, parallel 
sham-controlled clinical trial to explore whether cerebellar tACS decreases ataxia severity 
and a variety of non-motor symptoms in a homogeneous cohort of SCA3 patients and if so, 

what the duration of this beneficial effect is. 
Methods 
Ethics and dissemination 
This study was carried out according to the Declaration of Helsinki and got the approval of 
the local ethics committee of The First Affiliated Hospital of Fujian Medical University, 
Fuzhou, China (MRCTA, ECFAH of FMU [2022]399) on Aug 5, 2022. The contacts of the 
ethics committee are: 0086–0591-87,981,028, fykyll@163.com, and No.20, Chazhong Road, 
Fuzhou, Fujian Province, China. Participants need to sign an informed consent form before 
this study and receive medical care after the study. The research results will be published 
through articles or conferences. Thus, this study will benefit the treatment of SCA3 patients 
and hopefully supply an effective non-pharmacological intervention soon. 
Study participants 
Participants were enrolled from the clinical Observation Cohort of the Organization in South-
East China for Cerebellar Ataxia Research (OSCCAR) (NCT04010214) in the Department of 
Neurology, the First Affiliated Hospital of Fujian Medical University from August 2022 to 
March 2023. All eligible participants will get access to the specific treatment plans and they 
can register directly to attend the trial for randomization. All potential risks will be informed 
in the consent. The withdrawal reason will be carefully recorded if any participant quits 
halfway. To maximize trial compliance, potential risks, requirements, the study schedule, and 
benefits will be fully explained to all recruited participants. 
Participants will be instructed to avoid any anti-rehabilitation treatment for SCA3 during the 
study. All participants will have freedom to quit at any time and choose other medical therapy 
strategies. 
Inclusion criteria 
We will include eligible participants: 1) detectable clinical symptoms in molecularly 
confirmed SCA3; 2) SARA score between 3–30 at a recent pre-study visit; 3) between 18–80 
years of age; 4) informed consent was provided by the participant or their family members. 
Exclusion Criteria: 
Participants will be excluded who: 1) medical history of stroke, encephalitis, or epilepsy; 2) 
presence of metallic particles in the eye, medical pumps such as an implanted cardiac 

pacemaker or neurostimulator, or surgical clips (above the shoulder line); 3) serious cognitive 
or behavioral disorders or mental illness; 4) had taken investigational products within 4 weeks 
prior enrollment into this study; 5) pregnant or breastfeeding.  
This is a single-centre, triple-blind, randomised, sham-controlled intervention trial. A total of 
at least 80 recruited participants with SCA3 will be randomly and equally assigned to the 
active or sham treatment group. The randomization was created by a computer program in 
permuted blocks of four and managed by an offsite statistician who does not participate in this 
study. Every participant gets an unknown-beforehand number from a sealed opaque envelope, 
which determines the trial he/she will participate in. The envelopes for the two tACS plans 
(sham and active) have the same characteristics regarding size, color, appearance, weight, and 
odor, and different plans are numbered by the statistician. All participants were blinded to the 
specific assignment during the triple-blind treatment period until the end of the follow-up, 
except for any emergency when the blinding will be stopped. Participants will receive a 2-
week intervention, followed by a 4-week follow-up (conducted at the 6th week, T2) and a 12-
week follow-up (conducted at the 14th week, T3). The entire process will strictly follow the 
consolidated standards of reporting trials guidelines (Figure 1) and standard protocol items: 
recommendations for interventional trials checklist28,29. 

 
FIGURE 1 Design and flow of participants through the study. 
Termination of the trial will be decided by the principal investigators, according to the 
following criteria: (1) lack of two consecutive tACS sessions, (2) severe adverse events 
(AEs), and (3) affected tACS-Assessment by treatment of other diseases. If a patient is lost to 
follow-up at T2 or T3, the data collected up to that point will still be used in the statistical 
analysis at T2/T3. 
Sample size calculation 
Positive effect is supposed to occur in both groups according to previous report30. The 
primary clinical remission criterion defined a ≥ 1.5 points SARA score decrease caused by 2 
weeks of treatment. The SARA scores were expected to increase in 25% of placebo patients 
and 60% of the treated patients. 80% power was tested for 36 patients in each group, with a 
35% δ between the groups, by using a 2-sided test and at the 5% level. Taking 10% attrition 
rate in consideration, 40 patients for each group will be needed to reach a minimum total 
sample size of 72. Based on these calculations, we estimate that at least 80 SCA3 participants 
will enroll in the study. 

Transcranial alternating current stimulation intervention 
We applied 3 saline-soaked (0.9% NaCl) surface sponge electrodes (all rectangular-shaped in 
two sizes of 5×5 and 5×7 cm) to the scalp and used the battery-driven current stimulator 
(Neustim NSS18, Neuracle, Changzhou, China) to administer tACS. Five-digit codes were 
used to ensure blinding of the study coordinators with regard to the stimulation conditions. 
The Neustim NSS18 device does not display any information that would provide insights into 
whether active or sham stimulation is applied. The return electrode (5×7 cm) was placed 2 cm 
below the inion and the two active electrodes were over the bilateral buccinator muscles. 
After computational modeling of electric field distribution, we chose this CB-tACS particular 
montage with an extracephalic electrode, which can lead to significant entrainment of brain 
oscillations 6. Electrodes are fixed with elastic gauze and coated with conductive gel to reduce 
contact resistance (<5 kΩ for all sessions). 
The two stimulation electrodes each delivered an alternating sinusoidal current waveform 
with 2mA peak-to-peak amplitude at a frequency of 70 Hz. Each stimulation ramped up and 
down for 10 s respectively. For active 70Hz-tACS, the stimulation lasted for 40 mins. Sham 
stimulation was identical to active, except that stimulation only lasted for 40 seconds. All 
participants completed forty-minute daily sessions of 5 days/week for 2 consecutive weeks at 
approximately the same time of day (±90 min), combined with either Active-tACS or sham 
tACS. The treatment type (A-tACS vs. S-tACS) was encoded in the in-house software and 
was masked for both patients and the researchers. An independent technician to set the 
position of an internal switch on the active or sham controller. No one was aware of the 
switch position (T or B) was the sham position and treatment type (A-tACS vs. S-tACS) 
representative for sham, except for the independent technician. Researchers only needed to 
start stimulation according to the treatment type corresponding to the random number chosen 
by the participant, without knowing whether those specific numbers were assigned to A-tACS 
or S-tACS. The technician finally confirmed whether an A-tACS or S-tACS was delivered 
after each treatment was done.  
Participants were asked to sit in a bright and quiet room during all stimulation period, with 
their eyes open but no speaking and no large movement. To examine differences between 
simulated perceptions, participants need to answer whether they feel they were treated with 

real or sham stimulation, and whether they experienced tingling skin sensations or 
phosphenes/light flickers. Sensations were rated from 0 (no sensation) to 4 (very strong 
sensations). 
Computational modelling of electric field distribution 
Stimulation montage and modeling of electric field distribution were simulated using 
SimNIBS (version 3.2.431). The same standard MNI152 head model was used and 
conductivities of various tissues were assigned according to Wagner and colleagues32. 
Procedures   
The initial screening visit occurred at least 7 days before enrollment. Participants were 
interviewed to obtain demographic information and general clinical history by telephone or in 
person. After a screening visit to assess eligibility, participants were invited to attend in-
person visits at T0, T1, T2, and T3. Treatment fidelity was monitored by the principal 
investigator and the lead clinician in charge of the study through periodic observations of 
assessment and treatment sessions.  
The following procedures were repeated at different timepoints (T0, T1, T2, T3) over the 
study period. 
Assessment of symptom of ataxia 
SARA is a validated clinical scale to measure the severity of ataxia comprising eight items 
assessing the gait, posture, speech, and limb kinetic functions. Higher SARA scores indicate 
worse performance, and the scores range from 0 (no ataxia) to 40 (most severe ataxia). SARA 
scores were obtained by experienced, blinded investigators, and the assessments were video 
recorded. To also used the International Cooperative Ataxia Rating Scale (ICARS), which 
widely used to measure ataxia severity. The ICARS comprises 19 items from four subscales, 
including 7 posture and gait disturbances, 7 kinetic functions, 3 oculomotor disorders, and 2 
speech disorders. Higher ICARS scores indicate worse performance, and the total score of all 
subscores ranges from 0 to 100. 
Assessment of life ability and quality of life 
Health-related quality of life (HRQoL) was measured using the European Five Dimensions 
Questionnaire (EQ-5D-3L), which includes the EQ-5D descriptive system, the European Five 
Dimensions Questionnaire Visual Analogue Scale (EQ-VAS), and the Utility Index. The EQ-

5D-3L covers five dimensions: mobility, self-care, usual activities, pain/discomfort, and 
anxiety/depression, for which a three-level response (no, moderate, or extreme problems) is 
given. The EQ-5D-3L describes a total of 243 (=35) health states, wherein 11,111 represents 
the best health state and 33,333 represents the worst health state.  
Quantitative evaluation indices  
Gait variability   
Gait variability (GV) was defined as the percent range of variance (regularity and 
consistency) of a step cycle that is directly linked to dynamic postural control.34 GV may 
reflect subtle changes in neurodegenerative diseases, has been observed in pre-symptomatic 
SCA, is correlated with clinical disease severity scores, increases with progression of ataxia, 
and is associated with fall risk.35-38 Participants were instructed to walk in a comfortable and 
natural state on an overground 22-m distance in a well-lit flat room without any obstacles. 
Participants walked from 1 meter before the walkway and through 1 meter past the walkway 
to prevent acceleration and deceleration effects contributing to assessment of gait 
performance. Participant instructions were as follows: “When I say go, walk across the mat 
and then continue walking until I tell you to stop. Walk at your normal speed, as if you were 
walking down the street to go to the store.” Gait parameters were measured using two nine-
dimensional (three-axis accelerometers + three-axis gyroscopes + three-axis magnetometers) 
Inertial Measurement Unit (BWT901CL, WIT-MOTION, Shenzhen, China), as well as two 
flexible pressure sensor insoles. Use Visual Studio software (Visual Studio 2017, Microsoft, 
the USA) was used to synchronize timestamp and task data and to process the raw data. 
Participants completed the walk three times at their selected speed and the mean and standard 
deviation of gait parameters, including stride time variability, double support time variability, 
toe-out angle variability, and toe-off angle variability were determined. A detailed description 
of the technical devices is provided elsewhere. 39  
Adverse Events 
Participants were assessed at the end of each treatment session for adverse events (AEs), vital 
signs, and discomfort ratings (for potential scalp sensations associated with CB-tACS) using 
the commonly used questionnaire in CB-tACS trials, with telephone call every 2 weeks to 
record AEs during follow-up. Participants were instructed to report any additional adverse 

events experienced during or after treatment that were not included in the questionnaire. 
Outcomes 
Primary outcome measurement  
Efficacy outcome. The primary outcome measurement was the proportion of participates with 
clinical remission at the completion of treatment (defined as the reduction of SARA score at 
least 1.5 point from baseline). 
The secondary outcome measures were proportion of patients with clinical remission at T2 
and T3, and the changes at different timepoints (T1, T2, T3) from T0, compared to sham 
stimulation, including SARA, ICARS, EQ-5D, and gait variability. The safety outcomes 
included vital signs (blood pressure, body temperature, heart rate, pulse), adverse effects 
linked to tACS. 
Data management And Quality Control 
To ensure the rigor of this study, the whole process will be carried out strictly according to 
Guidelines for Good Clinical Practice of the International Conference on Harmonisation 
(ICH). Data will be collected from T0, T1, T2, and T3, respectively. All investigators, trial 
supervisors, and raters involved in the study must be trained before they participate in 
communicating and instructing participants, assessing, collecting data, and completing the 
CRF. Double entries of data into the software Yidu Research platform (Yiducloud (Beijing) 
Technology Ltd.) will be finished by two independent investigators, and these electronic data 
will be stored on a secure university server with regularly back-up and password protection. 
Yidu Research platform has an authority management mechanism associated with personnel 
and roles and a strict data audit mechanism to fully protect the security of data utilisation. 
Double-check will be performed to correct inconsistent entries or typos. The details of the 
quit participants, including reasons, date, AEs, and duration of the treatment, will be recorded. 
The final trial dataset, including the intent-to-treat and per-protocol dataset will be analyzed 
by an independent statistician. 
Data analysis 
Baseline characteristics will be summarized and presented using appropriate descriptive 
statistics. Normality of the variables will be assessed by using the skewness statistic and 
normal probability plot. Baseline characteristics between two groups will be compared using 

independent–t test for continuous variables and the χ² analysis or Fisher’s Exact test for 
categorical variables. Generalized estimating equations (GEE) 40 analysis will be adopted to 
adjust for any significant differences in baseline characteristics. Baseline characteristics of the 
participants who completed the study will be compared with those who did not finish the 
study. All primary and secondary outcomes will be compared between groups on the basis of 
intention–to–treat (ITT) principle. GEE models will be used to assess differential change of 
each primary and secondary outcomes across different time–points between two groups and 
within group from baseline. IBM SPSS 26.0 software will be used for all statistical analysis. 
Two–tailed test will be used and statistical significance will be set at a p–value < 0.05. 
Intention-to-treat (ITT) refers to all eligible patients who were randomized to treatment, 
include drop-out cases.  
Per-protocol (PP) refers to the set of cases that have completed the overall study protocol, a 
subset of the FAS in which each subject in the dataset is a valid case or sample with good 
adherence, no protocol violations, and complete baseline values for key indicators.  
Missing data 
Due to the long duration of intervention and the long follow-up period, so there may be a lot 
of missing data. If missing data were found, the percentage of missing data will be reported, 
the potential patterns of missing data should be examined, and appropriate method should be 
used for multiple imputation of missing data. The multiple imputation method will be 
preferred for analyzing the missing data, and the complete-case data should be reported in the 
manuscript as sensitivity analysis.    
Analysis of primary outcome 
The Type I error rate (α-level) used in the assessment of χ² analysis or Fisher’s Exact test for 
the primary efficacy outcome is 5%. The assessment of significance for the A-tACS versus S-
tACS contrasts will use χ² analysis or Fisher’s Exact test, based on ITT, PP, and sensitivity 
analysis (multiple assumptions for the missing value). 
Analysis of secondary outcomes 
The Type I error rate (α-level) used in the assessment of pair-wise treatment comparisons for 
the primary efficacy outcome is 5%. All secondary outcomes will be compared between 
groups on the basis of intention–to–treat (ITT) principle. GEE models will be used to assess 

differential change of each secondary outcomes across different time–points between two 
groups and within group from baseline.  
ALFF, ReHo, and FC Analysis 
Statistical analysis of MRI data was performed using Matlab2013b, SPM12 
(https://www.filion.ucl.ac.uk/spm/) (Sidhu et al. 2016). The Flexible factorial of second-order 
analysis was used to design the statistical matrix and to set the three factors of subjects, 
groups, and time. Groups and time were fixed factors, and the serial number of subjects was a 
random factor. Then, the interaction matrix of groups × time to analysis was set to determine 
the intergroup interaction effect. The resulting statistical map was set at p<0.05 (AlphaSim 
correction) with a combined voxel p<0.001. Use RestPlus toolbox 
(https://github.com/topics/restplus, version 1.2) was used to extract the values of the above 
abnormal brain regions, and Bonferroni tests were performed for all results with significant 
differences to verify between-group differences. Statistical significance was set at p<0.05. 
Image presentation used the xjview toolbox in Matlab 2013b. 
Analysis of intra-network functional connectivity 
A one-sample Student’s t-test was applied to the spatial maps of all participants for each IC 
using the SPM12 toolbox, and regions surviving multiple comparison corrections with voxel-
level family-wise error (FWE) correction (p<0.05) were saved as the specific group-level 
mask for each group. These four masks for each IC were then integrated into a union mask, 
which was used for the group comparison. A single model using a flexible-factorial design 
(2×2) was performed to obtain the ICs showing significant between-group differences. 
Regions showing significant differences (voxel-level FWE correction, p<0.05) were saved as 
the mask for subsequent post-hoc analyses, and the significance level was set at p<0.0125 
(0.05/4) for Bonferroni correction across 4 tests. 
Software and significant level of the statistical analyses 
IBM SPSS 26.0 (IBM, Armonk, NY, USA) software and R, version 4.2.1 software (The R 
Project for Statistical Computing, www.r-project.org) will be used for all statistical analysis. 
Statistical significance was defined as P <0.05 with 2-sided testing. 
REFERENCES 

 
1. 
Kuo SH. Ataxia. Continuum (Minneap Minn). 2019;25(4):1036-1054. 
doi:10.1212/CON.0000000000000753 
2. 
Manto M, Gandini J, Feil K, Strupp M. Cerebellar ataxias: an update. Curr Opin Neurol. 
2020;33(1):150-160. doi:10.1097/WCO.0000000000000774 
3. 
Antal A, Alekseichuk I, Bikson M, et al. Low intensity transcranial electric stimulation: Safety, 
ethical, legal regulatory and application guidelines. Clin Neurophysiol. 2017;128(9):1774-1809. 
doi:10.1016/j.clinph.2017.06.001 
4. 
Wessel MJ, Draaisma LR, Hummel FC. Mini-review: Transcranial Alternating Current 
Stimulation and the Cerebellum. Cerebellum. 2023;22(1):120-128. doi:10.1007/s12311-021-
01362-4 
5. 
Yavari F, Jamil A, Mosayebi Samani M, Vidor LP, Nitsche MA. Basic and functional effects of 
transcranial Electrical Stimulation (tES)-An introduction. Neurosci Biobehav Rev. 2018;85:81-
92. doi:10.1016/j.neubiorev.2017.06.015 
6. 
Sadeghihassanabadi F, Misselhorn J, Gerloff C, Zittel S. Optimizing the montage for cerebellar 
transcranial alternating current stimulation (tACS): a combined computational and experimental 
study. J Neural Eng. 2022;19(2). doi:10.1088/1741-2552/ac676f 
7. 
Hu P, He Y, Liu X, Ren Z, Liu S. Modulating emotion processing using transcranial alternating 
current stimulation (tACS) - A sham-controlled study in healthy human participants. Annu Int 
Conf IEEE Eng Med Biol Soc. 2021;2021:6667-6670. doi:10.1109/EMBC46164.2021.9630564 
8. 
Naro A, Milardi D, Cacciola A, et al. What Do We Know About the Influence of the Cerebellum 
on Walking Ability? Promising Findings from Transcranial Alternating Current Stimulation. 
Cerebellum. 2017;16(4):859-867. doi:10.1007/s12311-017-0859-4 
9. 
Miyaguchi S, Otsuru N, Kojima S, et al. Transcranial Alternating Current Stimulation With 
Gamma Oscillations Over the Primary Motor Cortex and Cerebellar Hemisphere Improved 
Visuomotor Performance. Front Behav Neurosci. 2018;12:132. doi:10.3389/fnbeh.2018.00132 
10. 
Antonino N, Alessia B, Antonino L, et al. Effects of cerebellar transcranial alternating current 
stimulation on motor cortex excitability and motor function. Brain Structure & Function. 
2017;222(6).  
11. 
Naro A, Leo A, Russo M, et al. Does Transcranial Alternating Current Stimulation Induce 
Cerebellum Plasticity? Feasibility, Safety and Efficacy of a Novel Electrophysiological 
Approach. Brain Stimul. 2016;9(3):388-395. doi:10.1016/j.brs.2016.02.005 
12. 
Koganemaru S, Mikami Y, Matsuhashi M, et al. Cerebellar transcranial alternating current 
stimulation modulates human gait rhythm. Neurosci Res. 2020;156:265-270. 
doi:10.1016/j.neures.2019.12.003 
13. 
Schubert C, Dabbagh A, Classen J, Krämer UM, Tzvi E. Alpha oscillations modulate premotor-
cerebellar connectivity in motor learning: Insights from transcranial alternating current 
stimulation. Neuroimage. 2021;241:118410. doi:10.1016/j.neuroimage.2021.118410 
14. 
Giustiniani A, Tarantino V, Bracco M, Bonaventura RE, Oliveri M. Functional Role of 
Cerebellar Gamma Frequency in Motor Sequences Learning: a tACS Study. Cerebellum. 
2021;20(6):913-921. doi:10.1007/s12311-021-01255-6 
15. 
Wessel MJ, Draaisma LR, de Boer A, et al. Cerebellar transcranial alternating current 
stimulation in the gamma range applied during the acquisition of a novel motor skill. Sci Rep. 
2020;10(1):11217. doi:10.1038/s41598-020-68028-9 

16. 
Miyaguchi S, Inukai Y, Mitsumoto S, Otsuru N, Onishi H. Gamma-transcranial alternating 
current stimulation on the cerebellum and supplementary motor area improves bimanual motor 
skill. Behav Brain Res. 2022;424:113805. doi:10.1016/j.bbr.2022.113805 
17. 
Herzog R, Berger TM, Pauly MG, et al. Cerebellar transcranial current stimulation - An 
intraindividual comparison of different techniques. Front Neurosci. 2022;16:987472. 
doi:10.3389/fnins.2022.987472 
18. 
Guerra A, Colella D, Giangrosso M, et al. Driving motor cortex oscillations modulates 
bradykinesia in Parkinson's disease. Brain. 2022;145(1):224-236. doi:10.1093/brain/awab257 
19. 
Guerra A, Asci F, D'Onofrio V, et al. Enhancing Gamma Oscillations Restores Primary Motor 
Cortex Plasticity in Parkinson's Disease. J Neurosci. 2020;40(24):4788-4796. 
doi:10.1523/JNEUROSCI.0357-20.2020 
20. 
Zhou D, Li A, Li X, et al. Effects of 40 Hz transcranial alternating current stimulation (tACS) 
on cognitive functions of patients with Alzheimer's disease: a randomised, double-blind, sham-
controlled clinical trial. J Neurol Neurosurg Psychiatry. 2022;93(5):568-570. doi:10.1136/jnnp-
2021-326885 
21. 
Benussi A, Cantoni V, Grassi M, et al. Increasing Brain Gamma Activity Improves Episodic 
Memory and Restores Cholinergic Dysfunction in Alzheimer's Disease. Ann Neurol. 
2022;92(2):322-334. doi:10.1002/ana.26411 
22. 
San-Juan D, Espinoza-López DA, Vázquez-Gregorio R, et al. A pilot randomized controlled 
clinical trial of Transcranial Alternating Current Stimulation in patients with multifocal 
pharmaco-resistant epilepsy. Epilepsy Behav. 2022;130:108676. 
doi:10.1016/j.yebeh.2022.108676 
23. 
Mellin JM, Alagapan S, Lustenberger C, et al. Randomized trial of transcranial alternating 
current stimulation for treatment of auditory hallucinations in schizophrenia. Eur Psychiatry. 
2018;51:25-33. doi:10.1016/j.eurpsy.2018.01.004 
24. 
Wang H, Wang K, Xue Q, et al. Transcranial alternating current stimulation for treating 
depression: a randomized controlled trial. Brain. 2022;145(1):83-91. 
doi:10.1093/brain/awab252 
25. 
Wang HX, Wang L, Zhang WR, et al. Effect of Transcranial Alternating Current Stimulation for 
the Treatment of Chronic Insomnia: A Randomized, Double-Blind, Parallel-Group, Placebo-
Controlled Clinical Trial. Psychother Psychosom. 2020;89(1):38-47. doi:10.1159/000504609 
26. 
Schuhmann T, Duecker F, Middag-van Spanje M, et al. Transcranial alternating brain 
stimulation at alpha frequency reduces hemispatial neglect symptoms in stroke patients. Int J 
Clin Health Psychol. 2022;22(3):100326. doi:10.1016/j.ijchp.2022.100326 
27. 
Matsumoto H, Ugawa Y. Adverse events of tDCS and tACS: A review. Clin Neurophysiol 
Pract. 2017;2:19-25. doi:10.1016/j.cnp.2016.12.003 
28. 
Chan AW, Tetzlaff JM, Altman DG, et al. SPIRIT 2013 statement: defining standard protocol 
items for clinical trials. Ann Intern Med. 2013;158(3):200-207. doi:10.7326/0003-4819-158-3-
201302050-00583 
29. 
Chan AW, Tetzlaff JM, Gøtzsche PC, et al. SPIRIT 2013 explanation and elaboration: guidance 
for protocols of clinical trials. BMJ. 2013;346:e7586. doi:10.1136/bmj.e7586 
30. 
Romano S, Coarelli G, Marcotulli C, et al. Riluzole in patients with hereditary cerebellar ataxia: 
a randomised, double-blind, placebo-controlled trial. Lancet Neurol. 2015;14(10):985-991. 
doi:10.1016/S1474-4422(15)00201-X 

31. 
Thielscher A, Antunes A, Saturnino GB. Field modeling for transcranial magnetic stimulation: 
A useful tool to understand the physiological effects of TMS. Annu Int Conf IEEE Eng Med 
Biol Soc. 2015;2015:222-225. doi:10.1109/EMBC.2015.7318340 
32. 
Wagner TA, Zahn M, Grodzinsky AJ, Pascual-Leone A. Three-dimensional head model 
simulation of transcranial magnetic stimulation. IEEE Trans Biomed Eng. 2004;51(9):1586-
1598. doi:10.1109/TBME.2004.827925 
33. 
Wuyts FL, De Bodt MS, Molenberghs G, et al. The dysphonia severity index: an objective 
measure of vocal quality based on a multiparameter approach. J Speech Lang Hear Res. 
2000;43(3):796-809. doi:10.1044/jslhr.4303.796 
34. 
Hausdorff JM. Gait variability: methods, modeling and meaning. J Neuroeng Rehabil. 
2005;2:19. doi:10.1186/1743-0003-2-19 
35. 
Schniepp R, Wuehr M, Schlick C, et al. Increased gait variability is associated with the history 
of falls in patients with cerebellar ataxia. J Neurol. 2014;261(1):213-223. doi:10.1007/s00415-
013-7189-3 
36. 
Schniepp R, Huppert A, Decker J, et al. Fall prediction in neurological gait disorders: 
differential contributions from clinical assessment, gait analysis, and daily-life mobility 
monitoring. J Neurol. 2021;268(9):3421-3434. doi:10.1007/s00415-021-10504-x 
37. 
Zhou H, Nguyen H, Enriquez A, et al. Assessment of gait and balance impairment in people 
with spinocerebellar ataxia using wearable sensors. Neurol Sci. 2022;43(4):2589-2599. 
doi:10.1007/s10072-021-05657-6 
38. 
Shah VV, Rodriguez-Labrada R, Horak FB, et al. Gait Variability in Spinocerebellar Ataxia 
Assessed Using Wearable Inertial Sensors. Mov Disord. 2021;36(12):2922-2931. 
doi:10.1002/mds.28740 
39. 
Wanli Zhang, Zaiwei Zhou, Shiqi Chen, et al. Synergy of porous and air-gap structures for 
pressure sensing arrays with high sensitivity and wide detection range towards machine 
learning assisted gait analysis. Phys. Scr. 2023. 98. 055925.doi:10.1088/1402-4896/acc98c 
40. 
Salazar A, Ojeda B, Dueñas M, Fernández F, Failde I. Simple generalized estimating equations 
(GEEs) and weighted generalized estimating equations (WGEEs) in longitudinal studies with 
dropouts: guidelines and implementation in R. Stat Med. 2016;35(19):3424-3448. 
doi:10.1002/sim.6947 
 
 
 

STUDY PROTOCOL FOR MSA 
1. Project title 
Safety and efficacy of transcranial alternating current stimulation in multiple system atrophy– 
Cerebellar Type: a randomized proof-of-concept clinical trial.  
2. Abstract of project 
Background/Objective: Multiple system atrophy (MSA) is a rare, progressive 
neurodegenerative disorder characterized by a variable combination of autonomic failure, 
parkinsonism, cerebellar ataxia, and pyramidal signs. The etiology of MSA is unknown and 
there is no effective treatment available. Depending on the predominant motor symptom, 
MSA can be classified as MSA with predominant parkinsonism (MSA-P) or MSA with 
predominant cerebellar ataxia (MSA-C). In MSA-C, cerebellar dysfunction manifests as gait 
ataxia, limb ataxia, ataxic dysarthria, and eye movement abnormalities such as dysmetria, 
saccadic intrusion, and ocular dysmetria. Currently, no specific pharmacological and 
treatments can alter natural course of the disease. As well, we found that there are not enough 
clinical trials involving MSA-C and these studies usually had small samples. Clinical practice 
guidelines had confirmed the safety of transcranial alternating current stimulation (tACS) in 
treating neuropsychiatric diseases, but to our knowledge, no randomized clinical trials have 
investigated whether tACS is safety and efficacy during improve symptoms in MSA-C. The 
objectives of this study are to identify the efficacy and safety of tACS during improving 
ataxia symptoms and non-ataxia symptoms in adult patients with MSA-C. 
Participants: Individual with MSA-C 
Design: A triple-blind, randomized sham-controlled trial with two groups. At least 84 subjects 
will be recruited and randomized into either active tACS group or sham tACS group. 
Interventions: Cerebellar tACS (40 min, 2 mA, ramp-up and down periods of 10s each) will 
be delivered over 10 sessions, distributed in two groups of five consecutive days with a two-
day break in between. The active tACS group will receive whole course current stimulation, 
while the control group will receive a daily 40–seconds current stimulation for two weeks. 
Primary outcomes: The primary outcome was the difference in total Unified Multiple System 
Atrophy Rating Scale (UMSARS) score between the two groups being treated for 2 weeks 
(T1). 

Secondary outcomes: The difference in total UMSARS score between the two groups at 1-
month follow-up visits (T2) and 3-month follow-up visits (T3). Ataxia symptoms measured 
by SARA and The International Cooperative Ataxia Rating Scale (ICARS); health–related 
quality of life (HRQoL) measured by the the Multiple System Atrophy Quality of Life 
questionnaires (MSA-QoL) ; and objective quantitative outcome for gait measured by the 
wearable sensors. 
Impact of the project: With the promising preliminary benefits of tACS on improving 
cerebellar motor functions and is safe and effective in treating neurological diseases, however, 
the efficacy of tACS in cerebellar disorders with MSA-C has not been clinically investigated. 
Therefore, we performed a triple-blind, parallel-group, sham-controlled, randomized clinical 
trial to investigate the efficacy and safety of tACS in a large cohort of patients with MSA-C. 
The primary outcome assessed ataxia severity, and secondary outcomes evaluated quality of 
life, and adverse events (AEs). 
Introduction 
Multiple-system atrophy with predominant cerebellar ataxia (MSA-C) is a sporadic, adult-
onset, and rapidly progressive fatal neurodegenerative disease that presents with cerebellar 
ataxia and other associated symptoms of undetermined etiology with a mean life expectancy 
of 6–9 years after diagnosis, and increases functional disability, the likelihood of falls and the 
social economic burden of patients [ [1, 2]. Cerebellar ataxia, the main symptom of MSA-C, 
is the most disabling symptom because there is no satisfactory symptomatic treatment 
available; no curative or neuroprotective treatments for the disease have been identified to 
date [3]. Therefore, various pharmacological and non-pharmacological trials have been 
attempted, and non-invasive brain stimulation has been regarded as a possible treatment 
candidate for cerebellar ataxia.  
Cerebellar transcranial alternating current stimulation (tACS) is an increasingly easy-used, 
safe, cheap, and noninvasive tool in neuroscience that is able to modulate cerebellar 
excitability3-5. Computational modeling studies have demonstrated the biophysical feasibility 
of modulating cerebellar structures using tACS with only negligible spreading effects to 
neighboring regions6. Initially, this technique has been successfully applied in healthy 
volunteers to investigate the neural correlates of motor learning and cognitive and emotional 

processes7-17. More recently, studies also started to explore its therapeutic potential in a 
variety of neurological conditions, such as Parkinson's disease 18,19, Alzheimer's disease20,21, 
epilepsy22, schizophrenia23, depression24, insomnia25, and stroke26. No serious adverse events 
of cerebellar tACS have been reported in these and other studies and it is therefore considered 
a safe and tolerable method 27. Possible side effects mainly include a transient optical illusion, 
itching, tingling, or mild burning sensation underneath the electrodes27. Inspired by these 
promising findings, we designed the clinically-oriented MSA-tACS, randomized, parallel 
sham-controlled clinical trial to explore whether cerebellar tACS decreases ataxia severity 
and a variety of non-motor symptoms in a homogeneous cohort of MSA-C patients and if so, 
what the duration of this beneficial effect is. 
Methods 
Ethics and dissemination 
This study was carried out according to the Declaration of Helsinki and got the approval of 
the local ethics committee of The First Affiliated Hospital of Fujian Medical University, 
Fuzhou, China (MRCTA, ECFAH of FMU [2022]399) on Aug 5, 2022. The contacts of the 
ethics committee are: 0086–0591-87,981,028, fykyll@163.com, and No.20, Chazhong Road, 
Fuzhou, Fujian Province, China. Participants need to sign an informed consent form before 
this study and receive medical care after the study. The research results will be published 
through articles or conferences. Thus, this study will benefit the treatment of MSA-C patients 
and hopefully supply an effective non-pharmacological intervention soon. 
Study participants 
Participants were enrolled from the clinical Observation Cohort of the Organization in South-
East China for Cerebellar Ataxia Research (OSCCAR) (NCT04010214) in the Department of 
Neurology, the First Affiliated Hospital of Fujian Medical University from August 2022 to 
March 2024. All eligible participants will get access to the specific treatment plans and they 
can register directly to attend the trial for randomization. All potential risks will be informed 
in the consent. The withdrawal reason will be carefully recorded if any participant quits 
halfway. To maximize trial compliance, potential risks, requirements, the study schedule, and 
benefits will be fully explained to all recruited participants. 
Participants will be instructed to avoid any anti-rehabilitation treatment for MSA-C during the 

study. All participants will have freedom to quit at any time and choose other medical therapy 
strategies. 
Inclusion criteria 
We will include eligible participants: 1) 30–80 years old; 2) Clinically diagnose or probable 
MSA-C according to the latest MSA diagnostic criteria; 3) <8 years MSA-C medical history; 
4) Independent walk (or with assistance); 5) >3 years life expectancy; 6) Contraceptive 
measures for women of childbearing age; 7) informed consent was provided by the participant 
or their family members. 
Exclusion Criteria: 
Participants will be excluded who: 1) medical history of stroke, encephalitis, or epilepsy; 2) 
presence of metallic particles in the eye, medical pumps such as an implanted cardiac 
pacemaker or neurostimulator, or surgical clips (above the shoulder line); 3) serious cognitive 
or behavioral disorders or mental illness; 4) had taken investigational products within 4 weeks 
prior enrollment into this study; 5) pregnant or breastfeeding.  
This is a single-center, triple-blind, randomized, sham-controlled intervention trial. A total of 
at least 84 recruited participants with MSA-C will be randomly and equally assigned to the 
active or sham treatment group. The randomization was created by a computer program in 
permuted blocks of four and managed by an offsite statistician who does not participate in this 
study. Every participant gets an unknown-beforehand number from a sealed opaque envelope, 
which determines the trial he/she will participate in. The envelopes for the two tACS plans 
(sham and active) have the same characteristics regarding size, color, appearance, weight, and 
odor, and different plans are numbered by the statistician. All participants were blinded to the 
specific assignment during the triple-blind treatment period until the end of the follow-up, 
except for any emergency when the blinding will be stopped. Participants will receive a 2-
week intervention, followed by a 4-week follow-up (conducted at the 6th week, T2) and a 12-
week follow-up (conducted at the 14th week, T3). The entire process will strictly follow the 
consolidated standards of reporting trials guidelines (Figure 1) and standard protocol items: 
recommendations for interventional trials checklist28,29. 

 
FIGURE 1 Design and flow of participants through the study. 
Termination of the trial will be decided by the principal investigators, according to the 
following criteria: (1) lack of two consecutive tACS sessions, (2) severe adverse events 
(AEs), and (3) affected tACS-Assessment by treatment of other diseases. If a patient is lost to 
follow-up at T2 or T3, the data collected up to that point will still be used in the statistical 
analysis at T2/T3. 
Sample size calculation 
Our sample size was calculated as previously described (PMID: 31278067). This study is 
expected to produce α of 0.05 at 80% power for a treatment effect of 50% on the annual 
progression of the movement examination score on the Unified Multiple System Atrophy 
Rating Scale (UMSARS); this contains 4 points from the active group and 8 points from the 
placebo group, which is above the minimal clinically detectable significant level of 3.8 points 
(PMID: 27474888). Therefore, we conducted this study to determine whether active tACS 
treatment would have a positive effect on MSA-C patients. The target total patients was 84, 
with an expected attrition rate of 10%.  

Transcranial alternating current stimulation intervention 
We applied 3 saline-soaked (0.9% NaCl) surface sponge electrodes (all rectangular-shaped in 
two sizes of 5×5 and 5×7 cm) to the scalp and used the battery-driven current stimulator 
(Neustim NSS18, Neuracle, Changzhou, China) to administer tACS. Five-digit codes were 
used to ensure blinding of the study coordinators with regard to the stimulation conditions. 
The Neustim NSS18 device does not display any information that would provide insights into 
whether active or sham stimulation is applied. The return electrode (5×7 cm) was placed 2 cm 
below the inion and the two active electrodes were over the bilateral buccinator muscles. 
After computational modeling of electric field distribution, we chose this CB-tACS particular 
montage with an extracephalic electrode, which can lead to significant entrainment of brain 
oscillations 6. Electrodes are fixed with elastic gauze and coated with conductive gel to reduce 
contact resistance (<5 kΩ for all sessions). 
The two stimulation electrodes each delivered an alternating sinusoidal current waveform 
with 2mA peak-to-peak amplitude at a frequency of 70 Hz. Each stimulation ramped up and 
down for 10 s respectively. For active 70Hz-tACS, the stimulation lasted for 40 mins. Sham 
stimulation was identical to active, except that stimulation only lasted for 40 seconds. All 
participants completed forty-minute daily sessions of 5 days/week for 2 consecutive weeks at 
approximately the same time of day (±90 min), combined with either Active-tACS or sham 
tACS. The treatment type (A-tACS vs. S-tACS) was encoded in the in-house software and 
was masked for both patients and the researchers. An independent technician to set the 
position of an internal switch on the active or sham controller. No one was aware of the 
switch position (T or B) was the sham position and treatment type (A-tACS vs. S-tACS) 
representative for sham, except for the independent technician. Researchers only needed to 
start stimulation according to the treatment type corresponding to the random number chosen 
by the participant, without knowing whether those specific numbers were assigned to A-tACS 
or S-tACS. The technician finally confirmed whether an A-tACS or S-tACS was delivered 
after each treatment was done.  
Participants were asked to sit in a bright and quiet room during all stimulation period, with 
their eyes open but no speaking and no large movement. To examine differences between 
simulated perceptions, participants need to answer whether they feel they were treated with 

real or sham stimulation, and whether they experienced tingling skin sensations or 
phosphenes/light flickers. Sensations were rated from 0 (no sensation) to 4 (very strong 
sensations). 
Computational modelling of electric field distribution 
Stimulation montage and modeling of electric field distribution were simulated using 
SimNIBS (version 3.2.431). The same standard MNI152 head model was used and 
conductivities of various tissues were assigned according to Wagner and colleagues32. 
Procedures   
The initial screening visit occurred at least 7 days before enrollment. Participants were 
interviewed to obtain demographic information and general clinical history by telephone or in 
person. After a screening visit to assess eligibility, participants were invited to attend in-
person visits at T0, T1, T2, and T3. Treatment fidelity was monitored by the principal 
investigator and the lead clinician in charge of the study through periodic observations of 
assessment and treatment sessions.  
The following procedures were repeated at different timepoints (T0, T1, T2, T3) over the 
study period. 
The primary outcome was the change from baseline to week 2 in the Unified Multiple System 
Atrophy Rating Scale (UMSARS) total score. Higher UMSARS scores denote poorer health. 
Severity of neurological deficits 
UMSARS is a clinician-scored rating scale to assess the severity of symptoms for patients 
with MSA. Disease severity was rated with part I (activities of daily living, 12 items), part II 
(motor examination, 14 items), part III (autonomic examination), and part IV (global 
disability) of the Unified Multiple System Atrophy Rating Scale (UMSARS). The total 
UMSARS score is the sum of parts I and II. UMSARS has also been demonstrated to be 
reliable and valid. Higher UMSARS scores denote poorer health. 
Assessment of symptom of ataxia 
SARA is a validated clinical scale to measure the severity of ataxia comprising eight items 
assessing the gait, posture, speech, and limb kinetic functions. Higher SARA scores indicate 
worse performance, and the scores range from 0 (no ataxia) to 40 (most severe ataxia). SARA 
scores were obtained by experienced, blinded investigators, and the assessments were video 

recorded. To also used the International Cooperative Ataxia Rating Scale (ICARS), which 
widely used to measure ataxia severity. The ICARS comprises 19 items from four subscales, 
including 7 posture and gait disturbances, 7 kinetic functions, 3 oculomotor disorders, and 2 
speech disorders. Higher ICARS scores indicate worse performance, and the total score of all 
subscores ranges from 0 to 100. 
Assessment of life ability and quality of life 
Health-related quality of life (HRQoL) was measured using the MSA-QoL questionnaire, 
developed in 2008. The MSA-QoL is composed of 40-item questionnaire that asks patients to 
rate their level of difficulty with specific mental or physical domains, as well as a question 
asking patients to mark overall life satisfaction on a scale from 0 to 100. The questionnaire 
was developed from physician and patient reports and psychometric data analysis and has 
been demonstrated to be reliable and valid. Items include questions about mobility, 
coordination, ability to complete self-care, other activities of daily living, symptoms of 
dysautonomia, sleep quality, cognitive function, and social/emotional impacts of the disease. 
In total, there are 14 motor items, 12 nonmotor items, and 14 emotional/social items. Higher 
total scores on the MSA-QoL indicate higher levels of impairment, and higher scores on the 
life satisfaction item correspond with higher life satisfaction. The original version was 
translated to adapt to the Chinese-speaking audience. Since 2008, patients have been asked to 
complete the MSA-QoL questionnaire during each annual consultation. 
Quantitative evaluation indices  
Gait variability   
Gait variability (GV) was defined as the percent range of variance (regularity and 
consistency) of a step cycle that is directly linked to dynamic postural control.34 GV may 
reflect subtle changes in neurodegenerative diseases, has been observed in pre-symptomatic 
SCA, is correlated with clinical disease severity scores, increases with progression of ataxia, 
and is associated with fall risk.35-38 Participants were instructed to walk in a comfortable and 
natural state on an overground 22-m distance in a well-lit flat room without any obstacles. 
Participants walked from 1 meter before the walkway and through 1 meter past the walkway 
to prevent acceleration and deceleration effects contributing to assessment of gait 
performance. Participant instructions were as follows: “When I say go, walk across the mat 

and then continue walking until I tell you to stop. Walk at your normal speed, as if you were 
walking down the street to go to the store.” Gait parameters were measured using two nine-
dimensional (three-axis accelerometers + three-axis gyroscopes + three-axis magnetometers) 
Inertial Measurement Unit (BWT901CL, WIT-MOTION, Shenzhen, China), as well as two 
flexible pressure sensor insoles. Use Visual Studio software (Visual Studio 2017, Microsoft, 
the USA) was used to synchronize timestamp and task data and to process the raw data. 
Participants completed the walk three times at their selected speed and the mean and standard 
deviation of gait parameters, including stride time variability, double support time variability, 
toe-out angle variability, and toe-off angle variability were determined. A detailed description 
of the technical devices is provided elsewhere. 39  
Adverse Events 
Participants were assessed at the end of each treatment session for adverse events (AEs), vital 
signs, and discomfort ratings (for potential scalp sensations associated with CB-tACS) using 
the commonly used questionnaire in CB-tACS trials, with telephone call every 2 weeks to 
record AEs during follow-up. Participants were instructed to report any additional adverse 
events experienced during or after treatment that were not included in the questionnaire. 
Outcomes 
Primary outcome measurement  
The primary outcome was the change from baseline to week 2 in the Unified Multiple System 
Atrophy Rating Scale (UMSARS) total score (sum of parts I and II). PMID: 15452868. 
Higher UMSARS scores denote poorer health. 
The secondary outcomes included the change in UMSARS part II (UMSARS II; motor 
examination scores, on which scores range from 0 to 56), the changes at different timepoints 
(T1, T2, T3) from T0, compared to sham stimulation, including SARA, ICARS, MSA-QoL, 
and gait variability. The safety outcomes included vital signs (blood pressure, body 
temperature, heart rate, pulse), adverse effects linked to tACS. 
Data management And Quality Control 
To ensure the rigor of this study, the whole process will be carried out strictly according to 
Guidelines for Good Clinical Practice of the International Conference on Harmonisation 
(ICH). Data will be collected from T0, T1, T2, and T3, respectively. All investigators, trial 

supervisors, and raters involved in the study must be trained before they participate in 
communicating and instructing participants, assessing, collecting data, and completing the 
CRF. Double entries of data into the software Yidu Research platform (Yiducloud (Beijing) 
Technology Ltd.) will be finished by two independent investigators, and these electronic data 
will be stored on a secure university server with regularly back-up and password protection. 
Yidu Research platform has an authority management mechanism associated with personnel 
and roles and a strict data audit mechanism to fully protect the security of data utilisation. 
Double-check will be performed to correct inconsistent entries or typos. The details of the 
quit participants, including reasons, date, AEs, and duration of the treatment, will be recorded. 
The final trial dataset, including the intent-to-treat and per-protocol dataset will be analyzed 
by an independent statistician. 
Data analysis 
Baseline characteristics will be summarized and presented using appropriate descriptive 
statistics. Normality of the variables will be assessed by using the skewness statistic and 
normal probability plot. Baseline characteristics between two groups will be compared using 
independent–t test for continuous variables and the χ² analysis or Fisher’s Exact test for 
categorical variables. Generalized estimating equations (GEE) 40 analysis will be adopted to 
adjust for any significant differences in baseline characteristics. Baseline characteristics of the 
participants who completed the study will be compared with those who did not finish the 
study. All primary and secondary outcomes will be compared between groups on the basis of 
intention–to–treat (ITT) principle. GEE models will be used to assess differential change of 
each primary and secondary outcomes across different time–points between two groups and 
within group from baseline. IBM SPSS 26.0 software will be used for all statistical analysis. 
Two–tailed test will be used and statistical significance will be set at a p–value < 0.05. 
Intention-to-treat (ITT) refers to all eligible patients who were randomized to treatment, 
include drop-out cases.  
Per-protocol (PP) refers to the set of cases that have completed the overall study protocol, a 
subset of the FAS in which each subject in the dataset is a valid case or sample with good 
adherence, no protocol violations, and complete baseline values for key indicators.  
Missing data 

Due to the long duration of intervention and the long follow-up period, so there may be a lot 
of missing data. If missing data were found, the percentage of missing data will be reported, 
the potential patterns of missing data should be examined, and appropriate method should be 
used for multiple imputation of missing data. The multiple imputation method will be 
preferred for analyzing the missing data, and the complete-case data should be reported in the 
manuscript as sensitivity analysis.    
Analysis of primary outcome 
The Type I error rate (α-level) used in the assessment of χ² analysis or Fisher’s Exact test for 
the primary efficacy outcome is 5%. The assessment of significance for the A-tACS versus S-
tACS contrasts will use χ² analysis or Fisher’s Exact test, based on ITT, PP, and sensitivity 
analysis (multiple assumptions for the missing value). 
Analysis of secondary outcomes 
The Type I error rate (α-level) used in the assessment of pair-wise treatment comparisons for 
the primary efficacy outcome is 5%. All secondary outcomes will be compared between 
groups on the basis of intention–to–treat (ITT) principle. GEE models will be used to assess 
differential change of each secondary outcomes across different time–points between two 
groups and within group from baseline.  
ALFF, ReHo, and FC Analysis 
Statistical analysis of MRI data was performed using Matlab2013b, SPM12 
(https://www.filion.ucl.ac.uk/spm/) (Sidhu et al. 2016). The Flexible factorial of second-order 
analysis was used to design the statistical matrix and to set the three factors of subjects, 
groups, and time. Groups and time were fixed factors, and the serial number of subjects was a 
random factor. Then, the interaction matrix of groups × time to analysis was set to determine 
the intergroup interaction effect. The resulting statistical map was set at p<0.05 (AlphaSim 
correction) with a combined voxel p<0.001. Use RestPlus toolbox 
(https://github.com/topics/restplus, version 1.2) was used to extract the values of the above 
abnormal brain regions, and Bonferroni tests were performed for all results with significant 
differences to verify between-group differences. Statistical significance was set at p<0.05. 
Image presentation used the xjview toolbox in Matlab 2013b. 
Analysis of intra-network functional connectivity 

A one-sample Student’s t-test was applied to the spatial maps of all participants for each IC 
using the SPM12 toolbox, and regions surviving multiple comparison corrections with voxel-
level family-wise error (FWE) correction (p<0.05) were saved as the specific group-level 
mask for each group. These four masks for each IC were then integrated into a union mask, 
which was used for the group comparison. A single model using a flexible-factorial design 
(2×2) was performed to obtain the ICs showing significant between-group differences. 
Regions showing significant differences (voxel-level FWE correction, p<0.05) were saved as 
the mask for subsequent post-hoc analyses, and the significance level was set at p<0.0125 
(0.05/4) for Bonferroni correction across 4 tests. 
Software and significant level of the statistical analyses 
IBM SPSS 26.0 (IBM, Armonk, NY, USA) software and R, version 4.2.1 software (The R 
Project for Statistical Computing, www.r-project.org) will be used for all statistical analysis. 
Statistical significance was defined as P <0.05 with 2-sided testing.