Wilson's disease (WD) was first systematically described by Kinnear Wilson in 1912; it is an autosomal recessive neurological disease characterized by chronic and progressive perturbations in copper ion metabolism (1, 2). Symptoms related to the nervous system usually appear after 11 years of age. Typical symptoms include juvenile Parkinson's and dystonia; indeed, Upper limb dystonia is one of the most common symptoms (3). Currently, no recognized standard objective evaluation of muscle tension or effective treatment methods are available. WD seriously affects patients' quality of life and results in social and economic burdens. Therefore, an urgent need exists for an objective evaluation system to monitor patients' muscle tone as well as alternative therapies that relieve dystonia.

Dystonia is a neurological disorder that consists of abnormal involuntary movements or posture due to continuous or intermittent muscle contractions (4). Although its anatomical basis is controversial, recent studies have indicated that dystonia involves basal ganglia circuits, the cerebellum, and multiple cortical regions including the primary motor cortex, the premotor cortex, the supplementary motor areas, the anterior cingulate cortex, and the cerebellar cortex (5, 6). Altogether, this data has led to the network pathophysiological model hypothesis (7). Critically, related studies have reported that the motor cortex is strongly plastic when under transcranial magnetic stimulation (TMS) (8–10).

TMS is a non-invasive neuromodulation technique in which repetitive magnetic impulses are delivered to specific brain regions for short periods of time via stimulation coils placed over the scalp (11). TMS was invented by Barker and his colleagues in 1985 (12). Since then, it has been widely used both experimentally and clinically to study cortical function in the brains of healthy subjects as well as in the brains of patients with psychiatric and neurological disorders. The repeated magnetic pulses alter the excitability of the stimulated site while also affecting areas of the brain that are anatomically connected to it (13). Most of the TMS work has been carried out on the human primary motor cortex (M1) and has provided significant clinical and pathophysiological insights into movement disorders (14). While the evidence showing that repeated TMS (rTMS) affects the strength of synaptic connections mainly comes from experiments in the motor cortex, it is generally believed that similar effects may be observed in all regions of the neocortex (15). Notably, one large sample, double-blind, randomized study reported that high-frequency 10 Hz rTMS on M1 improved the muscle tone and stiffness of patients (16). However, to date, no studies have specifically examined the effects of high-frequency rTMS on muscle tension and muscle stiffness in WD patients.

At present, no internationally recognized objective method for detecting muscle tension exists. MyotonPRO (Myoton Muscle Diagnostics Tallinn), a digital muscle function assessment system developed by Estonia in collaboration with the European Space Center, was used in this study. The device is a non-invasive portable and reliable tonometer that can provide an objective, quantitative value of the muscle oscillation frequency (F), which in turn describes muscle tension and muscle stiffness (S) (17). The MyotonPRO device does not have a normal reference range for muscle tone, depending on race, gender, age, fat thickness, etc., but several teams are working on the project. Currently, this instrument is widely used in the fields of aerospace and sports and medical rehabilitation. Before this study, few studies have applied this instrument to detect muscle tension in WD patients.

In our study, we used an alternative therapy, rTMS, to treat WD patients with upper limb dystonia, and applied MyotonPRO device to accurately assess changes in muscle tension and stiffness. The purpose is to seek an effective alternative therapy for the treatment of WD muscle tension. We hope that our results can provide an evidence-based basis for rTMS in the treatment of dystonia in WD patients as well as provide a new objective method for evaluating muscle tension and stiffness.

This study is the first controlled clinical trial that used the MyotonPRO to objectively evaluate dystonia and the efficacy of rTMS in the treatment of upper limb dystonia in WD patients. In this study, we found that high-frequency rTMS targeting M1 alleviated biceps brachii dystonic disorders in WD patients and significantly improved the biomechanical properties (F/S values) of the bicep muscles as well as MAS, UWDRS, BFM, and ADL scores. These results suggest that high-frequency rTMS targeting the M1 can reduce muscle tone and muscle spasms in WD patients as well as significantly improve their quality of life.

The standard treatment for WD is oral chelating substances (e.g., D-penicillamine and trientine) to enhance urinary copper excretion. Unfortunately, not all neurological symptoms respond equally well to this therapy, and sometimes D-penicillamine can even make them worse (21). Neurological symptoms may persist or even progress after treatment with chelates in up to 65% of WD patients. Neurological WD patients may develop Parkinson's symptoms, such as slow movement, rigidity, weakness, gait and postural disorders, dysarthria and dysphagia, at which time symptomatic treatment is required (22, 23). Anticholinergic drugs, baclofen, levodopa, trihexaphenyl, benzodiazepine, carbamazepine/oxamamazepine, and Botulinum neurotoxin have been recommended for symptomatic treatment of WD (24, 25). Multisegmental or systemic dystonia may be treated with the above recommended oral medications, while focal dystonia may be treated with Botulinum toxin (BTX) injections (26, 27). However, cell and animal experiments suggest that BTX may be less effective in Wilson's disease (28). Hefter et al. (29) reported that out of 156 WD patients regularly seen at the outpatient department of Dusseldorf University Hospital (Germany), only 6 patients had received botulinum neurotoxin A (BoNT/A) in the past 5 years. BoNT/A injection therapy is A rare (<4%) for symptomatic treatment of WD, which is needed only in exceptional circumstances and is usually used only temporarily. Holscher reported that 4 cases of dystonia were treated with anticholinergic drugs with good efficacy and 1 case was ineffective (30). Anticholinergic drugs can cause adverse events, mostly in older patients, while younger patients can tolerate higher doses, which is typical in WD cases (25). Poujois et al. (31) reported one patient with a severe dysarthria of WD who experienced a real and prolonged improvement of her voice after treatment by zolpidem. Relevant studies have shown that Chinese herbal medicine may alleviate neurological symptoms in WD animal models (32). There is no specific consensus on the treatment of WD neurological dysfunction (33). Dystonia can be treated with neurosurgery when medication fails, including deep-brain stimulation of globus pallidus internus, pallidotomy, or thalamotomy (34).

The core anatomy and dysfunction of WD is located in the basal ganglia, which is too deep to be easily stimulated by normal TMS coils. However, the basal ganglia are well-known to be part of a set of parallel closed circuits (basal ganglia-thalamic- cortical-basal ganglia) that originate in the cerebral cortex, cross the thalamus, and return to their respective frontal lobe origin (35). Notably, some WD patients have cerebral cortex damage (36). Thus, even though the direct effects of the TMS may be limited to the cerebral cortex, stimulation of this cortical region, if it is part of the basal ganglia circuitry, can affect the activity of these circuits and produce clinical effects. Confirming this idea, functional magnetic resonance imaging studies in humans have demonstrated that TMS of the presupplementary motor area and the left dorsal premotor cortex can increase the amount of signal in the striatum and thalamus (37). Moreover, positron emission tomography imaging has revealed that TMS can produce dopamine in the striatum (38). Although few studies have been carried out in WD models, this idea has been confirmed in animal studies of related movement disorders such as Parkinson's disease (PD). By interacting with a node in a complex circuit, the activity of a node some distance away can be changed. In an animal model of PD, optogenetic studies have indicated that the stimulation of afferent from the cortex to the subthalamic nucleus ameliorates hypokinesia and bradykinesia and leads to a broad increase in glutamate activity in the thalamus and other brain regions (16).

The mechanism by which rTMS alleviates WD dystonia remains unclear. Low or high frequency rTMS can induce lasting inhibitory or excitatory effects. Study regimens with extremely long stimulation times have positive or negative effects on cortical excitability and are associated with the prevention of cell death, brain-derived neurotrophic factor regulation, and γ-aminobutyric acid interneuron-mediated inhibition (39). rTMS has been used as a therapeutic strategy to modulate the brain regions involved in dystonia. Our results demonstrate that rTMS targeting M1 improves muscle tone and stiffness in WD patients and improves their ability to perform daily tasks, which is consistent with the findings of other related diseases (40). While a study has reported that low-frequency rTMS did not significantly improve the muscle tone of WD patients, these negative results could be due to the study's small sample size and short treatment time (41). Accordingly, perhaps only high frequency rTMS can obtain positive results for WD dystonia. However, note that results between studies can vary due to different rTMS frequencies and different locations of stimulation (42). Regardless, we hope that the effect of rTMS on WD dystonia can be systematically and comprehensively studied in the future.

In conclusion, TMS may affect the strength of synaptic connections in the human cortex and improve muscle tone. The evidence comes mainly from basic and clinical trials of the motor cortex, and it is widely believed that similar effects can be observed in all regions of the neocortex. For diseases of the basal ganglia, direct changes in the functioning of cortical areas via TMS may have secondary effects on the connective structure within the cortical ring of the basal ganglia. This effect is the rationale for attempting its use in the treatment of basal ganglia disease (11). Evidence suggests that rTMS is a useful treatment option for WD dystonia.

This study has some limitations that need to be considered. First, participants in this study were recruited from one center, with a small number of cases. Accordingly, the research results may not be highly generalizable. We hope to cooperate with other international hospitals in the future to conduct a multi-center, large-sample study. Second, we used the classical scheme of positioning the coil 90° as a sham strategy. Although several other ways to induce spurious stimuli exist, it has been demonstrated that turning the coil 90°, while not completely avoiding the stimulus, can induce much lower voltages than active rTMS (43). This scheme has been widely used in previous studies (44). Third, in clinical practice, WD patients can have various manifestations of dystonia. In this study, only patients with increased biceps muscle tension were selected. We hope to study more types of dystonia in WD patients in future studies. Finally, we only studied the short-term improvement of rTMS; the trial was evaluated over a period of 1 month, so our results may not reflect its long-term effects.

Our results suggest that 10 Hz rTMS can reduce the symptoms of biceps dystonia in WD patients in a short period of time and thereby improve their quality of life. Thus, our protocol can be used as a complementary or replacement therapy to relieve muscle spasm in WD patients.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by the First Affiliated Hospital of Anhui University of Chinese Medicine. The patients/participants provided their written informed consent to participate in this study.

WH and WY conceived and designed the study. WH, TW, YY, and TC participated in the recruitment of participants, data analysis, and management. XL, WD, HJ, and NQ implemented the treatment and collected the data. HW and MW contributed to the study conception, evaluation, study execution, and manuscript preparation. WH drafted the manuscript, while WY revised the manuscript. All authors read and approved the final manuscript.

This work was supported from the National Administration of Traditional Chinese Medicine: 2019 Project of building evidence based practice capacity for TCM (No. 2019XZZX-NB001), the National Natural Science Foundation of China (Grant No. 81973825), the Anhui Provincial Natural Science Foundation of China (Grant No. 2108085QH367), the Open Fund Project of Key Laboratory of Xin'An Medicine of Ministry of Education (No. 2020xayx12), and the University Synergy Innovation Program of Anhui Province (No. GXXT-2020-025).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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