Effect of auricular acupuncture on neuroplasticity of stroke patients with motor dysfunction: A fNIRS study

Cerebral Stroke is an acute cerebrovascular disease, a disease of brain tissue damage caused by the sudden rupture or blockage of blood vessels in the brain that prevents blood flow to the brain. Acupuncture has become a popular treatment for stroke, with auricular acupuncture providing a new idea for stroke treatment. However, the neuromodulatory mechanism of auricular acupuncture in the brain is still unclear. The aim of this study was to investigate the effect of auricular acupuncture in the treatment of upper limb dysfunction and the activation of specific brain regions in stroke patients. Forty patients with stroke hemiplegia who met the nerf criteria were included in the experiment and randomly assigned into two groups (20 patients in each group): the auricular acupuncture group and the control group. Fugl-Meyer score (FMA) assessment of upper limb motor function, motor evoked potential (MEP) measurement, and functional near-infrared brain function imaging (fNIRS) data acquisition in the primary motor M1 area of the brain at rest were performed before and after treatment, respectively. It was found that: 1) after auricular acupuncture treatment, the patients in the auricular acupuncture group showed significantly greater peak MEP and significantly higher oxyhemoglobin content in the M1 region of the brain compared with the control group, with a significant activation effect (MEP: P-value = 0.032, t = -2.22; HbO2; f = 4.225, p = 0.046); 2) in the clinical efficacy assessment, the FMA score in the auricular acupuncture group after treatment (p = 0.0122, t = 2.769). The results suggest that auricular acupuncture has an ameliorative effect on upper limb motor deficits after stroke and that activation of the M1 region of the brain may be a key node in auricular acupuncture for treating upper limb dysfunction in stroke patients, a finding that emphasizes the potential for clinical application of auricular acupuncture therapy for stroke patients with potential mechanisms influencing the outcome.


Introduction
Stroke is a cerebrovascular disease with multifactorial etiology, which is associated with high morbidity, mortality, recurrence, and disability [1]. It is the leading cause of mortality among Chinese residents and a major cause of long-term disability in adults worldwide. Upper limb dysfunction is one of the main functional impairments that stroke patients suffer from [2]. Hemiplegia, a common clinical manifestation of stroke, usually occurs in the early stages of the disease and can result in lifelong disability if left untreated or improperly managed [4].
Recent research on stroke has revealed the potential for recovery of damaged nerves and the relationship between central nervous system plasticity and post-stroke rehabilitation [5]. Several treatments have been used to address upper limb dysfunction resulting from stroke, including restraint-induced movement therapy [6], robot-assisted therapy [7], mirror therapy [8], and body acupuncture [9], each with limitations such as slow efficacy, reliance on instruments and environment, and cost. Additionally, these high-tech therapies often require specialized equipment and resources, making them less generalizable to lower-level hospitals.
Traditional Chinese medicine, particularly acupuncture, is a costeffective and widely accessible therapy for upper limb movement disorders in China. Auricular acupuncture, a form of acupuncture that stimulates specific ear acupuncture points, has been shown to be effective in treating post-stroke dyskinesia. Despite the lack of a clear understanding of the underlying mechanisms of auricular acupuncture, the bioholographic rhythm theory is currently the predominant theory explaining its therapeutic principles. This theory suggests that ear acupuncture points treat diseases in the body through holographic reflex pathways, with the distribution of ear acupuncture points resembling an "inverted fetus". Studies have demonstrated that acupuncture of the upper limb motor area of auricular points significantly improves upper limb motor function in stroke patients, as measured by the FMA, and that acupuncture of the cortical motor area projection points in the ear has superior efficacy in treating post-stroke limb numbness compared to conventional body acupuncture. Additionally, electroacupuncture of the "subcortical" and "margin middle" auricular points in rats effectively improves behavioral disorders. While the exact mechanism of improvement remains unclear and unsupported by objective data, these findings suggest that auricular acupuncture can elicit changes in motor ability.
The neural mechanisms underlying the therapeutic effects of auricular acupuncture in treating patients with brain injury remain unclear, limiting its implementation and development as a potentially valuable therapy. However, functional near-infrared spectroscopy (fNIRS) offers a powerful approach for investigating brain function in a non-invasive manner by measuring changes in brain tissue concentrations of oxyhemoglobin (HbO2) and deoxyhemoglobin (HbR) in response to neuronal activation. This enables the recording of real-time hemodynamic responses to cortical activation and the identification of differences in the brain's responses to various complex activities. Compared to fMRI, fNIRS is both more convenient and safer, and more sensitive to changes in the subject's cerebral cortex during sensory-motor tasks, thereby facilitating the study of functional specialization in the sensory-motor system. In an auricular acupuncture experiment, fNIRS can be utilized to measure the excitability of the subject's cerebral cortex, observe brain activity, and investigate the differences in cerebral cortex activation before and after auricular acupuncture treatment, with the aim of elucidating the underlying mechanisms of auricular acupuncture's therapeutic effects. This may provide valuable clinical guidance for the treatment selection of stroke patients. Auricular acupuncture is a promising approach for the treatment of upper limb dysfunction in stroke patients, yet its efficacy and neural mechanisms remain largely unexplored. While some studies have investigated the neurophysiological aspects of auricular acupuncture in stroke patients, clinical applications of this therapy have been limited. Our research team previously demonstrated that specific ear acupuncture points can increase the degree of brain activation in healthy subjects. However, it is unclear whether this effect can be replicated in the cerebral cortex of stroke patients, who have pre-existing brain damage. To address this gap in knowledge, we conducted an fNIRS study to investigate the correlation between the degree of upper limb dysfunction recovery and cortical activation following auricular acupuncture intervention in stroke patients. This research will provide insights into the clinical efficacy and neural mechanisms of auricular acupuncture therapy for stroke patients, thereby contributing to the development and promotion of this promising therapy.

Participants
This investigation was carried out within the inpatient department of rehabilitation medicine at the Third Affiliated Hospital of Sun Yat-sen University. In order to participate, individuals had to satisfy the following inclusion criteria: (1) presenting with unilateral hemiparesis secondary to stroke, as confirmed by MRI or computed tomography; (2) demonstrating stable vital signs and consciousness during examination; (3) experiencing onset to 6 months of disease; and (4) exhibiting Brunnstrom stage I-IV of the upper extremity. Patients were excluded if they (1) presented with a severe or clinically unstable condition; (2) displayed a neurological condition other than stroke; (3) were unable to understand the examination instructions and complete the examination tasks due to severe cognitive and communication impairment (MMSE < 23 points); (4) had a pacemaker, metal implant, or cranial defect; or (5) were excluded from the guidelines for use published in 2020 of additional contraindications to rTMS.
This study was conducted in accordance with the ethical standards of the 1975 Declaration of Helsinki and was approved by the internal review committee of the Third Affiliated Hospital of Zhongshan University. Each participant provided written informed consent. The trial was registered with the Chinese Clinical Trials Registry (ChiCTR2100051608) and was reported in line with the comprehensive criteria of the Trial Reporting Group guidelines.
The present study involved the inclusion of 40 S patients. Following the initial assessment, these participants were randomly divided into two groups: the auricular acupuncture group and the control group, with each group consisting of 20 patients. No significant differences were noted in the general characteristics of the two groups (p > 0.05).

Procedures
All participants, regardless of group assignment, received daily treatment, five times per week, for a total of 10 sessions. Both the auricular acupuncture group and the control group received regular rehabilitation training on a daily basis. Regular rehabilitation training included physiotherapy and task-oriented occupational therapy utilizing Bobath and Rood techniques, and lasted for a duration of 60 min per day.
In this study, the auricular acupuncture group received auricular acupuncture therapy in addition to conventional rehabilitation training. The auricular acupuncture points chosen for this intervention were SF1 (finger), SF3 (elbow), and SF5 (shoulder), as specified by the International Standards of Acupuncture Trade (Fig. 1). These points are located on the ear and were selected based on their known therapeutic effects. The patient assumed a seated or lying position and the auricular points were routinely disinfected prior to treatment. The therapist used three milli-needles to pierce each of the three auricular points, being careful not to penetrate the cartilage. The patient reported experiencing a range of sensations during treatment, such as soreness, numbness, swelling, pain, heat or conduction. The acupuncture was retained for a duration of 20 min and was administered once daily for a total of 10 treatments. For the control group, we used sham auricular acupuncture in the same location. We removed the tip of needle and only retained the circular needle handle. The circular needle handle was taped to the skin of same location (SF1, SF3, SF5) for 20 min [43].

Fugl-Meyer assessment scale
The current study aimed to investigate the impact of a specific experimental intervention on the upper extremity and hand function of patients. To assess this, the upper extremity portion of the Fugl-Meyer Movement Assessment Scale (FMA) was employed to evaluate patients before and after the experiment. The FMA is a widely used and validated assessment tool for measuring motor function in stroke patients, consisting of 33 items that are scored on a three-level scale (0-2). The scoring system follows specific guidelines, whereby a score of 0 indicates the inability to complete a task, 1 indicates partial completion, and 2 denotes full completion. The maximum possible score is 66, which reflects the individual's upper limb motor ability. By utilizing this scale, we were able to quantify the improvement in upper extremity and hand function of the patients, thus providing objective measures of the intervention's effectiveness.

Motor evoked potential
In this study, the efficacy of the treatment was monitored by measuring the motor evoked potential (MEP), which is a measure of the excitability generated by stimulating the cortical motor area and downstream motor conduction pathway to the spinal motor neurons. The MEP reflects the transmissibility of the corticospinal tract and is recorded as a compound muscle action potential using surface electrodes. Specifically, the compound action potentials of the thumb short adductor muscles of the hand were recorded before and after the treatment session using a transcranial magnetic stimulation system and a figure-of-eight coil. The recording electrode was placed on the normal side of the thumb short adductor muscle belly of the patient, while the reference electrode was positioned at the tendon approximately 2 cm from the recording electrode. A rod was placed on the proximal end of the ipsilateral forearm, and the coil was placed in the motor cortex of the healthy hemisphere with the coil tangential to the scalp. The minimum stimulus intensity at which a motor evoked potential of 50 μV was recorded on the electromyogram (EMG) was determined by systematically moving the coil in the primary motor cortex (M1) while the patient was in a relaxed seated position.

fNIRS acquisition
The acquisition of resting-state data was conducted using a nearinfrared brain function imaging monitor (NirSmart, Danyang Huichuang Medical Devices Co., Ltd, China) with wavelengths of 740 and 850 nm, as suggested by Nieuwhof et al. (2016). Each sensor of the instrument consisted of a light-emitting diode and a detector optode, which were positioned at a distance of 30 mm and operated at a sampling frequency of 10 Hz. The instrument was equipped with a total of 38 measurement channels, including 18 light-emitting probes and 16 detector probes, which were symmetrically positioned in various regions of the brain. The regions of interest (ROI) included the ipsilateral and contralateral premotor cortex (IPMC/CPMC), lesioned ipsilateral and lesioned contralateral primary motor cortex (IM1/CM1), and ipsilateral and contralateral primary somatosensory cortex (IS1/CS1) (Fig. 2).
The HbO2 concentration changes in the cerebral cortex were recorded using fNIRS before and after the session in 40 subjects. The instrument was operated by a skilled researcher to ensure the smooth running of the experiment. The participants were instructed to sit still in a dimly lit and noise-free room for 10 min after wearing the fNIRS probe cap. During the procedure, they were asked to remain rested, relaxed, and still as much as possible.

fNIRS data analyses
The hemodynamic response index used in this study was the HbO2 signal, as it is more sensitive to local blood flow than HbR. Preprocessing of the raw data collected from fNIRS was performed using the NIR-SPARK software, which included artifact processing, filtering, segmentation, and baseline comparison. The raw NIRS optical intensity was converted to bituminous density signal and then to blood oxygen concentration data. Statistical analysis was performed on the data to calculate the average value of the region of interest (ROI).
The preprocessing module of the NirSpark software was utilized to preprocess the fNIRS data collected in this study. The threshold standard deviation was set to 6.0 and the magnitude threshold was set to 0.5. Motion artifacts were removed by a shift of the standard deviation combined with three spline interpolations. Interference signals caused by heart rate, respiratory rate, and Mayer waves were removed using 0.01 to 0.1 Hz bandpass filtering. The differential path length factors were set to 6.0, and the Delta HbO concentration was calculated for each channel in the rest/task state according to the modified Beer-Lambert law.
The resting state data were analyzed using NirSpark software in block mode. The baseline was set to [-2s, 0 s], and the average oxyhemoglobin value for each channel was calculated. The fNIRS data of patients with right hemisphere infarction were flipped using the mirror transformation technique and transformed into left hemisphere infarction to facilitate data processing. The mean oxyhemoglobin for each ROI at rest was obtained by dividing the concentration of oxyhemoglobin in all channels in each ROI by the number of channels in each ROI. The normality of between-group differences was tested using the Shapiro-Wilk method, and data expressed as mean ± standard deviation were considered normally distributed. The paired t-test was used to compare HbO2 concentrations at rest before and after the intervention to detect activation of ROIs in each hemisphere.

Statistical analysis
The statistical analyses for both FMA and MEP were performed using the Statistical Package for Social Sciences (SPSS) for Windows (V.25) by statisticians who were blind to the allocation and intervention process. Histograms and normal probability plots were examined, and Shapiro-Wilk tests were conducted to assess the normality of the data. Continuous variables were expressed as means and standard deviations (SD), along with 95% confidence intervals (CI). Categorical variables were presented as numbers and percentages. For demographic and clinical data at baseline, dichotomous data were compared using chi-square tests or Fisher exact tests. Independent two-sample t-tests were used for normally distributed continuous data, whereas Wilcoxon rank sum tests were used for non-normally distributed data. The independent twosample t-tests were used to compare changes in scores before and after treatment between the groups. A two-tailed analysis was performed, and the level of statistical significance was set at p < 0.05.

Participants and baseline demographics
A total of 44 patients with stroke were included in this study. Of these, 4 patients did not complete the treatment (auricular acupuncture group: did not complete 10 treatments, n = 3; control group: shoulder pain too pronounced, n = 1) (Fig. 3). Finally, 40 subjects participated in the final analysis (Table 1). Baseline demographics were not significantly different between the auricular acupuncture group and the control group (all P > 0.05). At baseline, the mean age of the patients was 57.87 years. The time since stroke was 1.58 months and the majority of patients were patients with cerebral hemorrhage, about 77.5%. There were more males than females among the patients, approximately 62.5%.
For the auricular acupuncture group, we conducted a paired t-test to compare the mean FMA score before and after the auricular acupuncture treatment. The results showed a statistically significant difference (t = -5.222, p < 0.001) ( Table 2). For the control group, the results didn't show a statistically significant difference (t = 1.4050, p = 0.176). To compare the difference between the two groups after treatment, the results show a statistically significant improvement in auricular acupuncture group (t = 2.27, p = 0.028).
Based on the results, there was a significant difference in the auricular acupuncture group before and after the treatment in the peak value of MEP, with a P-value<0.001 (t = 5.24). For the control group, there was not a significant difference before and after the treatment, with a Pvalue of 0.18 (t = 1.41).When comparing the two groups, there was a significant difference in the mean values before the treatment (P-value = 0.158, t = -1.44), but there was a significant difference after the treatment (P-value = 0.032, t = -2.22).
In the evaluation of fNIRS, we used M1, PMC and S1 bilaterally as the regions of interest. The reason is that the activation of these regions is directly correlated with the movement of the upper limbs. A more pronounced activation in these regions, especially in the affected brain region, would indicate a more excitable nerve in the cerebral cortex and   would be more helpful for motor recovery. In our experiment, there was no difference in the activation of brain areas between the two groups before the experiment (Table 3). However, in the ipsilateral M1 area, the mean value of blood oxygen was significantly higher in the auricular acupuncture group after the experiment than in the control group after the experiment (f = 4.225, p = 0.046) (Fig. 4). Also, comparing the mean blood oxygen values before and after the experiment in the ear acupuncture group also showed a significant increase (f = 4.416, p = 0.046).

Discussion
The present study provides evidence that auricular acupuncture therapy may be beneficial in promoting upper limb motor function recovery following stroke. The findings indicate that the Fugl-Meyer Motor Assessment Scale (FMA) upper extremity partial score showed significant improvement after treatment in the auricular acupuncture group (P < 0.05), compared to the control group. It was suggested that auricular acupuncture has a positive effect on the recovery of upper extremity function.
Neuroimaging results demonstrated that the peak motor evoked potential (MEP) was significantly lower in the auricular acupuncture group after treatment compared to the control group. This indicates that auricular acupuncture treatment stimulates the motor area of the healthy side of the cerebral cortex, leading to a lower brain signal. The reduced neurosensitivity in the healthy brain relative to the affected brain may suggest that auricular acupuncture therapy could improve the excitatory imbalance between the two hemispheres, which could alleviate the inhibited condition of the affected side of the brain, thereby facilitating the expression of the motor cortex of the affected side. Besides, the results of the fNIRS found significant changes in HbO2 concentrations in ipsilateral M1 brain regions before and after treatment in the control group. The M1 region of the affected brain in the auricular acupuncture group showed better activation after treatment than before treatment, indicating that auricular acupuncture therapy may have positive effects on the functional rehabilitation of upper limbs in stroke patients.
In recent years, stroke has become more prevalent among younger individuals and is associated with a higher recurrence rate, which can significantly impact patients' quality of life [25]. Upper limb dysfunction is a common consequence of stroke, and the large area of upper limb projection in the cerebral cortex makes restoring its function a challenging task. Specifically, the recovery of hand function remains a major issue in stroke rehabilitation. The restoration of motor function after stroke is closely linked to the reconstruction of the motor system in both the contralateral and ipsilateral hemispheres [26]. The primary motor cortex (M1) plays a critical role in locomotion by improving control of the muscles in the contralateral distal arm [27]. Consequently, unilateral damage to M1 or the corresponding projection area after stroke may lead to motor dysfunction in the corresponding area [28]. Studies have found that the connectivity between M1 in both hemispheres is significantly associated with manual dexterity in upper limb dysfunction patients [29]. Enhanced activation of the corresponding region in the contralateral hemisphere or increased connectivity between the bilateral hemispheres is generally considered a mechanism of recovery following brain injury [30]. fNIRS has been used to evaluate stroke patients undergoing interventions, and this has produced significant effects in motor-related brain regions on the affected side. This effect may help the brain to better express itself in the motor cortex and facilitate motor function recovery. This suggests that positive effects on motor function in post-stroke hemiplegic patients can be achieved by inducing excitation in the affected M1 area. Our experimental results confirmed this hypothesis. Following auricular acupuncture stimulation, the patient produced more pronounced activation in the affected brain area, and concurrently, the patient's upper limb motor function scores improved. Thus, there is a clear relationship between the expression of the patient's cerebral cortex and the improvement of the motor function of the upper limbs.
Auricular acupuncture is a therapeutic modality whereby acupuncture stimulation is applied to specific points on the ear, with the aim of regulating the balance of yin and yang, unblocking qi and blood, and enhancing the resistance of corresponding tissues to disease through the meridian-ear connection [31]. In accordance with international standards for ear acupuncture points [32], the present study utilized ear points corresponding to the body projection points of the upper extremities on the ear-cord, which are linked to the motor areas of the cerebral cortex. By consistently stimulating these ear points, the excitation level of the brainstem reticular formation and cerebral cortex is influenced, leading to improved blood circulation in the injured areas and amelioration of unfavorable limb movement symptoms, ultimately improving motor function in the upper limbs [33,34].
Our study has also demonstrated the viability of the overarching regulation principle of the "auricle-brain network-body function" [35]. The auricle contains the auriculotemporal nerve, facial nerve, glossopharyngeal nerve, and vagus nerve from the brain nerve, the auricular nerve and occipital nerve from the spinal nerve plexus, and the sympathetic nerve from the carotid artery [36]. The selected points in our study are mainly distributed by the occipital nerve and the auricular nerve. Ear acupuncture may also modulate neurological function through its effect on the autonomic nervous system, which may be particularly relevant to stroke patients, as autonomic dysfunction is common in this population and can interfere with motor recovery [37]. Our experimental results indicate that stimulation of acupuncture points on the ear affects the expression of the M1 area of the brain via the lesser occipital nerve and the greater auricular nerve on the ear. We postulate that the effectiveness of auricular stimulation is due to the transmission of the supratrochlear stimulation signal to the cerebral cortex, which in turn promotes the expression of the primary motor cortex. This, in turn, is transmitted to the affected hand via the corticospinal tract [38].
The observed increased blood oxygenation in the right M1 area among the auricular acupuncture group may be attributed to the phenomenon known as neurovascular coupling. Neurovascular coupling refers to the close coordination between neural activity and blood flow in the brain, where increased neural activity leads to increased blood flow to the activated areas of the brain [39]. In stroke patients, damage to the vascular system or neural tissue can impair neurovascular coupling, leading to reduced blood flow and limited oxygen and nutrient delivery to damaged neural tissue. This further exacerbates neurological damage and hinders recovery [40]. By increasing blood oxygenation in the right M1 region, auricular acupuncture may help restore neurovascular coupling and improve blood flow to damaged neural tissue in this region. Improved blood flow and metabolic activity in the brain can also facilitate neuroplasticity and neural repair, as oxygen and nutrients Table 3 Clinical outcomes of between auricular acupuncture group and sham acupuncture group. IPMC/CPMC: lesioned ipsilateral and lesioned contralateral premotor cortex, IM1/CM1: lesioned ipsilateral and lesioned contralateral primary motor cortex, IS1/CS1: lesioned ipsilateral and contralateral primary somatosensory cortex. are crucial for these processes [42]. Specifically, oxidative metabolism requires oxygen, which plays a critical role in energy production and cell signaling in the brain. Enhanced metabolic activity in the brain may further stimulate the production of growth factors and neurotrophic proteins, promoting neurogenesis and synaptic plasticity [43]. It is worth noting that there are some limitations to this study that should be taken into consideration. For example, the sample size was relatively small, which may limit the generalizability of the findings. In addition, the study did not include a sham acupuncture group, which makes it difficult to rule out the possibility that some of the observed effects were due to placebo or non-specific effects. Finally, the study only followed patients for a short period of time, so it is unclear whether the observed effects of auricular acupuncture treatment would persist in the long term.

Conclusion
In summary, the findings of this study suggest that auricular acupuncture therapy can promote the recovery of upper limb motor function after stroke. This may be attributed to its ability to stimulate the motor area of the healthy side of the cerebral cortex, thereby improving the excitatory imbalance between the two hemispheres and alleviating the inhibited condition of the affected side of the brain. These results may have important implications for the rehabilitation of stroke patients.

Funding
Mechanisms of synaptic plasticity mediated by Glu/GABA balance in prefrontal cortex in electroacupuncture to improve perimenopausal depression (No. 2021A515011464) and Introduction of Overseas Experts to Conduct Research on Improvement of Synaptic Plasticity in Mice by Combining Acupuncture with Active Nano Hydrogel and Exchange and Cooperation (No. 2020A414010231).

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability
Data will be made available on request.