A Study on Precise Cerebral Localization and Network Mechanisms of Repetitive Transcranial Magnetic Stimulation in Treating Chronic Ankle Instability Based on High-resolution Functional Magnetic Resonance Imaging
Phase: NA
Conditions: Chronic Ankle Instability, CAI, Functional Magnetic Resonance Imaging (fMRI)
Sponsor: Wuhan University

Research Protocol 
Project Source and Number: Joint Fund Cultivation Project supported by Natural Science 
Foundation of Hubei Province, China (2025AFD635) 
 
 
A study on precise cerebral localization and 
network mechanisms of repetitive transcranial 
magnetic stimulation in treating chronic ankle 
instability based on high-resolution functional 
magnetic resonance imaging 
 
 
 
Lead Institution: Zhongnan Hospital of Wuhan University, Wuhan Univesity 
Principal Investigator: Qing Shu 
Department: Department of Rehabilitation Medicine 
Contact Number: +86 13971081682 
Participating Institution: Hubei Provincial Institute of Sports Science 
Study Duration: March 2025 – December 2026 
Version: V1.0 0126 
Version Date: January 26, 2025 
 
 
 
 
 
 

1. Research Background 
Chronic ankle instability (CAI) is a prevalent sports injury encountered in clinical 
rehabilitation settings. Approximately 60% of individuals have experienced ankle sprains. 
Even with rehabilitation, nearly half of these patients develop recurrent injuries and progress 
to CAI. Although the direct treatment costs for CAI are relatively low, the indirect costs 
associated with subsequent rehabilitation and time loss prove to be substantial. Notably, 
current treatment strategies for CAI patients, including therapeutic exercises and surgical 
interventions, can effectively restore ankle joint stability. However, for a non-life-threatening 
condition that is often overlooked, surgery is rarely the first-line option for patients. Even 
with therapeutic exercises, managing ankle dysfunction and achieving functional recovery 
still requires a prolonged duration. Consequently, elucidating the pathological mechanisms of 
CAI and identifying its contributing factors are critical for developing targeted interventions 
and preventing long-term symptomatic progression. 
Abnormal central adaptive alterations may predispose CAI individuals to injury-prone 
movement patterns. The neurophysiological mechanisms underlying these central nervous 
system abnormalities remain poorly understood in rehabilitation medicine, warranting urgent 
investigation. Current consensus posits that CAI-related functional deficits involve not only 
peripheral systems like ligamentous and muscular components but also exhibit potential 
neural control dysregulation within central pathways. Emerging evidence demonstrates 
moderate negative correlations between ankle disability indices and motor cortical excitability 
of the peroneus longus muscle in CAI populations, accompanied by bilateral elevation of 
resting motor thresholds. These findings suggest that cortical excitability modulations directly 
influence ankle functional capacity, with unilateral injury potentially inducing bilateral 
cortical excitability alterations. Furthermore, compared to healthy controls, CAI patients 
exhibit diminished tibialis anterior cortical excitability during single-leg stance, correlating 
with impaired postural control performance. While conventional stability-focused 
rehabilitation protocols can improve biomechanical parameters, CAI patients demonstrate 
functional deterioration when executing superimposed motor-cognitive tasks, likely 
attributable to aberrant cortical activation patterns that precipitate maladaptive movement 
strategies and reinjury risks. Therefore, integrating cortical excitability modulation and 

enhancing central nervous system plasticity should be prioritized as novel therapeutic 
paradigms in CAI rehabilitation. 
Repetitive transcranial magnetic stimulation (rTMS), a safe non-invasive neuromodulation 
technique, employs high-intensity magnetic pulses through induction coils to regulate cortical 
excitability, and has gained increasing application in sports injury rehabilitation in recent 
years. Over the past three decades, blood-oxygen-level-dependent functional magnetic 
resonance imaging (fMRI) has provided unprecedented opportunities for non-invasive 
investigation of human brain functional localization and large-scale neural network 
interactions. Recent discoveries concerning the geometric characteristics and integrative 
functions of the sensorimotor cortex have challenged traditional textbook concepts of 
somatotopic organization within this brain region. Parti cularly noteworthy is the development 
and clinical implementation of Chinese domestically innovated high-end MRI systems with 
proprietary intellectual property, which has created novel prospects for elucidating 
multi-modal and multi-scale fine-grained neural architecture. Consequently, utilizing fMRI to 
validate CAI-specific neural signatures in critical cortical regions, combined with mechanistic 
validation of rTMS-induced cortical modulation effects on ankle biomechanical adaptations, 
holds transformative potential for advancing neurorehabilitation protocols in chronic ankle 
instability management. 
 
2. Aim of this study 
This study aims to investigate the therapeutic efficacy of rTMS targeting key brain regions 
in sports rehabilitation for CAI populations and to evaluate its impact on the activation levels 
of ankle-stabilizing muscles. Focusing on athletes with CAI, the study integrates rTMS into 
conventional ankle stability training protocols. A comparative analysis of post-intervention 
differences in ankle functional scores, muscle activation patterns of ankle stabilizers, and 
cerebral cortex excitability between the two groups will clarify the superior efficacy of key 
brain region-targeted rTMS within ankle stability training protocols. 
 
3. Methods and design 
3.1 Study setting and design 

 
(1) Study Design 
This study is a single-center randomized single-blind parallel-controlled trial. 
(2) Generation of Allocation Sequence 
The randomization sequence for subject grouping will be generated by research members 
uninvolved in trial operations and assessments using the SPSS 22.0 statistical software 
program. 
(3) 3.3 Allocation Concealment 
Allocation concealment will be implemented by using the sealed envelope method. An 
opaque envelope containing the randomized grouping scheme will be prepared, and envelopes 
will be sequentially opened according to the enrollment order to determine subject allocation. 
(4) 3.4 Allocation Implementation 
The allocation sequence will be generated by personnel not participating in the experiment. 
Research staff will recruit subjects, and the personnel responsible for generating the allocation 
sequence will assign the interventions to the subjects based on the allocation sequence. 
(5) Blinding 
Subjects will be blinded following intervention allocation. This study will employ a 
randomized single-blind parallel-controlled design. Real rTMS will involve 10 minutes of 
magnetic stimulation delivered to the target brain region, while sham stimulation will mimick 
the sensory experience of real stimulation without inducing physiological effects. Unblinding 
can be performed by researchers at study conclusion or if a subject requires trial 
discontinuation due to severe adverse reactions. 
(6) Sample Size Calculation 
This study aims to compare the therapeutic efficacy of two interventions for chronic ankle 
instability, with the Cumberland Ankle Instability Tool (CAIT) as the primary outcome. Given 
the limited population size of athletes and anticipated heterogeneous data variance, sample 
size estimation was performed using the "Two-Sample T-Tests Allowing Unequal Variance" 
module in PASS 15 software. Based on existing literature, the control group was assumed to 
have a mean ± standard deviation of 20 ± 4, while the treatment group was projected to 
demonstrate 24 ± 4. With a power of 0.8 and α= 0.05 (two-tailed), the calculation indicated a 

minimum requirement of 17 subjects per group. To account for potential attrition, each group 
will ultimately enroll 20 subjects. 
3.2 Participant recruitment 
3.2.1 
Inclusion Criteria 
(1) Age 18-60 years. 
(2) History of at least one significant ankle sprain within the past 12 months, accompanied by 
inflammatory response (including pain resulting in at least one day of restricted physical 
activity). 
(3) The most recent ankle sprain must have occurred more than 3 months prior to enrollment. 
(4) Self-reported symptoms of ankle instability, defined by one or more of the following: At 
least two episodes of "giving way" in the affected ankle within the past 6 months; 
Recurrent sprains (≥2 documented sprains in the same ankle); Subjective perception of 
instability confirmed by any one of the following validated instruments: Ankle Instability 
Instrument (AII): ≥5 affirmative responses; Cumberland Ankle Instability Tool (CAIT): 
Score <24; Identification of Functional Ankle Instability (IdFAI): Score >11. 
(5) Foot and Ankle Ability Measure (FAAM): Activities of Daily Living (ADL) subscale 
<90%; FAAM Sports subscale <80%; Foot and Ankle Outcome Score (FAOS): Scores 
<75% in ≥3 subcategories. 
3.2.2 
Exclusion Criteria 
(1) Chronic ankle instability caused by bilateral ankle sprains; 
(2) History of lower extremity musculoskeletal or neurological surgery; 
(3) History of lower extremity fractures; 
(4) Acute musculoskeletal injuries in other lower extremity joints within 3 months that 
compromised joint integrity and function, resulting in ≥1 day of interrupted daily or 
sports activities; 
(5) Concomitant severe systemic diseases (cardiac, pulmonary, hepatic, renal, etc.); 
(6) Severe psychiatric disorders or cognitive impairments; 
(7) Presence of metal implants or inability to tolerate MRI examinations. 
(8) Patient refusal to participate. 
3.2.3 
Withdrawal Criteria 

(1) Presence of cardiovascular diseases; 
(2) Signs of neurological impairment; 
(3) Central nervous system lesions;  
(4) Psychiatric or psychological disorders; 
(5) Diagnosis of malignant tumors or rheumatoid arthritis; 
(6) Current pregnancy or lactation; 
(7) Severe scalp trauma, extensive/open craniocerebral injuries contraindicating coil 
placement or posing infection risks; 
(8) Failure to obtain informed consent or concurrent enrollment in other clinical trials. 
3.2.4 
Study Termination Criteria 
Development of adverse events or medical conditions contraindicating continued 
participation; Voluntary withdrawal by the participant; Protocol non-compliance substantially 
compromising trial outcome assessments. 
3.3 Treatment Protocols 
Ankle Stability Training + Sham rTMS Group: Participants underwent once-daily 
ankle stability training combined with sham rTMS intervention. The ankle stability 
training protocol comprised three modalities: neuromuscular training, balance training, 
and proprioceptive training. Specific exercises included single-leg stance, balance pad 
training, heel raises, resistive ankle inversion/eversion exercises, MOBO board balance 
exercises, alphabet tracing (toe-writing), lateral walking, heel walking, toe walking, and 
jump and landing control drills. The training progressed from stable to unstable surfaces, 
starting at low intensity and gradually increasing in difficulty and intensity. The training 
was conducted three times per week for a total of four weeks. Before each session of ankle 
stability training, participants received sham rTMS intervention. The sham stimulation 
mimicked the sensory experience of real rTMS but did not produce any physiological 
effects. 
Ankle Stability Training + rTMS Group: Participants underwent rTMS intervention 
prior to daily ankle stability training. The interventional targets were the key cortical brain 
regions identified in preliminary studies, with stimulation applied to the contralateral 
cortical areas corresponding to the affected ankle joint. Parameters included a figure-of-8 

coil, 10 Hz frequency, 2-second stimulation duration, 8-second inter-train intervals, 60 
repetitions, totaling 1,200 pulses per session, with each session lasting 10 minutes. Ankle 
stability training commenced within 10 minutes post-rTMS, following the same duration 
and intensity as the Ankle Stability Training Group + Sham rTMS Group. The treatment 
was conducted three times per week for a total of 4 weeks. 
3.4 Outcomes 
3.4.1 
Primary parameter 
Cumberland Ankle Instability Tool Score (CAIT): A self-reported questionnaire 
designed to assess functional ankle instability. It consists of 9 items with a total score 
ranging from 0 to 30, where higher scores indicate better ankle stability. 
3.4.2 
Secondary parameters 
(1) Karlsson-Peterson Ankle Function Score (KPAFS) 
The KPAFS is a scoring system used to evaluate ankle dysfunction. It includes 10 items: 
pain, daily activities, walking ability, need for support, squatting ability, ankle range of 
motion, limb positioning, limb length, limb circumference, and limb appearance. Each 
item is scored based on the patient's condition, with a total possible score of 100. Higher 
scores indicate better ankle function. 
(2) American Orthopedic Foot and Ankle Society Score (AOFAS) 
The AOFAS is a widely used assessment tool for evaluating functional outcomes in foot 
and ankle disorders. It includes domains such as pain, function, gait, range of motion, 
stability, and foot alignment. The total score ranges from 0 to 100, with higher scores 
reflecting better foot and ankle function. 
(3) Surface Electromyography (sEMG) of Ankle-Stabilizing Muscles 
Based on existing literature, the following muscles are selected for sEMG testing: 
tibialis anterior, gastrocnemius, and peroneus longus. sEMG measurements are conducted 
during two conditions: single-leg stance and the Star Excursion Balance Test (SEBT). The 
following parameters are recorded: 
a) 
Time-domain analysis: Root Mean Square (RMS), Integrated Electromyography 
(iEMG), and Averaged Electromyography (AEM). 

b) 
Frequency-domain analysis: Mean Power Frequency (MPF) and Median Frequency 
(MF). 
(4) Task-Based fMRI Data Acquisition and Analysis 
Task-based fMRI data were acquired using a paradigm combining isometric contraction 
of the affected lower limb with motor imagery of the "single-leg stance" task on the 
paretic limb. A simplified protocol is as follows: One day prior to the fMRI scan, 
participants underwent training on MRI safety precautions and scanning procedures; 
during the formal scan session, localization and resting-state MRI scans were first 
performed, followed by the task-based scan, which comprised 12 blocks of 20-second 
lower-limb isometric contractions combined with "single-leg stance" motor imagery 
alternating with 11 blocks of 20-second rest periods, with participants keeping their eyes 
open throughout the experiment and following on-screen task cues. Imaging data were 
acquired using an echo-planar imaging (EPI) sequence with a slice thickness of 4 mm, 35 
slices total, TR = 2000 ms, TE = 30 ms, FA = 90°, Matrix = 64×64, FOV = 240×240 
mm. 
(5) Safety Evaluation 
Any adverse events during the treatment, such as mild headaches, were recorded and 
monitored, and participants completed an adverse reaction questionnaire following the 
intervention. 
 
3.5 Statistical Analysis 
In the fMRI data analysis, a T-contrast boxcar analysis was conducted for each participant, 
retaining only voxels with a significance level of P < 0.05 at the voxel-wise threshold. 
Family-wise error (FWE) correction was applied. Clusters were defined using a 
cluster-defining threshold of P = 0.001, with FWE-corrected cluster significance set at P < 
0.05, and only clusters exceeding 10 voxels were considered. For group-level analysis, a 
one-sample t-test was employed. Cohen’s d coefficient was used to quantify effect sizes 
between groups. Mean differences below 0.2 were disregarded, 0.2–0.5 were classified as 
small, 0.5–0.8 as moderate, and values > 0.8 as large. Statistical outputs from SPM software 
included cluster counts, voxel counts, peak voxel t-values, and Montreal Neurological 

Institute (MNI) coordinates. Contiguous voxels were aggregated into clusters, with peak 
voxel t-values representing the most statistically significant voxel within each cluster, and 
MNI coordinates indicating the location of the specific peak voxel in standardized brain space. 
Finally, activated regions were identified using the Automated Anatomical Labeling (AAL) 
atlas within the WFU PickAtlas toolbox. 
Statistical analyses for efficacy evaluation were performed using SPSS 22.0. Data are 
presented as mean ± standard deviation. Between-group differences were compared using 
independent samples t-tests. Overall differences across time points between groups were 
assessed via two-way repeated-measures ANOVA or mixed-effects models. If a significant 
overall effect was detected, pairwise comparisons were conducted using the Sidak method. A 
threshold of P < 0.05 was applied to determine statistical significance. 
 
4. Research Timeline 
March 2025 – May 2026: Participant recruitment, intervention implementation, and data 
collection. 
May 2026 – October 2026: Data analysis. 
May 2026 – December 2026: Continued data analysis and manuscript drafting.