Effects of Motion-Style Scalp Acupuncture on External Capsule Microstructure and Spinal Reflex Excitability in a Rat Model of Post-Stroke Spasticity

Summary

This protocol describes motion-style scalp acupuncture in a rat model of post-stroke spasticity and demonstrates its evaluation using behavioral, magnetic resonance imaging, histological, and electrophysiological assessments to investigate changes in external capsule microstructure and spinal reflex excitability.

Abstract

Post-stroke spasticity (PSS) severely impairs motor function and quality of life after stroke. Disruption of the cortico-reticulospinal pathway and impaired supraspinal descending inhibition are considered important pathological mechanisms; however, the neural and microstructural mechanisms underlying the therapeutic effects of motion-style scalp acupuncture (MSSA) remain incompletely understood. This study aimed to investigate the effects of MSSA on PSS using a multimodal assessment integrating neuroimaging, electrophysiology, and histology in a rat model of middle cerebral artery occlusion. T2-weighted magnetic resonance imaging was performed to quantify cerebral infarct volume, and diffusion tensor imaging was used to evaluate the microstructural integrity of the external capsule white matter. Luxol Fast Blue (LFB) staining assessed myelin integrity within the external capsule, while H-reflex recordings evaluated spinal reflex excitability. Correlation analyses were performed to examine the relationships among infarct volume, white matter microstructure, spinal reflex excitability, and spasticity severity. MSSA reduced cerebral infarct volume, alleviated external capsule microstructural damage, and attenuated demyelination. In addition, MSSA improved abnormal H-reflex parameters, normalized spinal reflex excitability, and reduced spasticity. Correlation analyses showed that larger infarcts were associated with greater external capsule injury, whereas better-preserved white matter integrity was associated with improved spinal reflex regulation and less severe spasticity. These findings suggest that MSSA may alleviate PSS by reducing ischemic brain injury, preserving external capsule microstructural integrity, and improving cortico-reticulospinal pathway-associated descending inhibitory function, providing preclinical evidence supporting its therapeutic application.

Introduction

Post-stroke spasticity (PSS) is a prevalent and disabling sequela of ischemic stroke, clinically defined by velocity- and length-dependent increases in muscle tone. This condition often leads to abnormal limb posture and dysfunctional movement patterns, severely compromising patients’ motor independence and quality of life while imposing substantial economic and caregiver burdens worldwide^1,2. As a typical manifestation of upper motor neuron syndrome, the pathogenesis of PSS is primarily attributed to exaggerated hyperexcitability of spinal stretch reflexes. Accumulating neuroanatomical and neurophysiological studies have demonstrated that the loss of descending inhibitory modulation from the extrapyramidal system to spinal reflex circuits is a key pathological mechanism underlying the initiation and progression of PSS, with the cortico-reticulospinal pathway serving as the primary neural circuit mediating this descending regulatory control^3,4,5.

Needling technique is a key determinant of acupuncture efficacy and occupies a pivotal position in clinical acupuncture practice. Motion-style scalp acupuncture (MSSA) is a distinctive needling technique specifically developed for PSS intervention that combines scalp acupuncture with simultaneous active or passive limb exercise³. Numerous clinical trials have shown that MSSA produces superior outcomes compared with scalp acupuncture alone or exercise rehabilitation alone in reducing spasticity severity, restoring locomotor function, and improving activities of daily living in stroke survivors^6,7,8,9. Our previous animal studies further demonstrated that MSSA exerts more pronounced antispastic effects than conventional scalp acupuncture in rat models of PSS. Mechanistically, these therapeutic effects have been associated with improved cerebral perfusion in the ischemic penumbra, reduced spinal stretch reflex excitability, and increased expression of potassium-chloride cotransporter 2 (KCC2) in spinal anterior horn motoneurons. This molecular modulation may further enhance spinal γ-aminobutyric acid (GABA)ergic inhibitory transmission, thereby contributing to spasticity relief^10,11.

Existing mechanistic investigations of MSSA remain limited, with no prior studies specifically examining how upstream intracerebral white matter within extrapyramidal descending pathways contributes to PSS. The external capsule forms a critical white matter segment of the cortico-reticulospinal pathway; it contains cortico-tegmental fibers that convey cortical signals to the brainstem reticular formation, thereby contributing to supraspinal inhibitory control of spinal reflex circuits^12,13. Consistent with this anatomical role, multiple voxel-based lesion-symptom mapping studies have shown that external capsule damage is associated with upper- and lower-limb spasticity after stroke^12,13,14. These anatomical and clinical observations support the selection of the external capsule as the primary region of interest (ROI). To date, no in vivo evidence has demonstrated whether MSSA preserves external capsule microstructural integrity or whether such alterations are associated with changes in spinal reflex excitability. Clinical evaluations based on behavioral assessments, conventional magnetic resonance imaging (MRI), and electrophysiological testing permit only correlational analyses and may be influenced by patient heterogeneity, disease duration, and other clinical confounding factors. In contrast, standardized rat models of PSS enable rigorous control of experimental variables and uniform intervention protocols, thereby providing a complementary platform for mechanistic investigation.

Focusing on cerebral white matter microstructure and neurophysiological function, this study integrated multimodal approaches, including T2-weighted imaging (T2WI), diffusion tensor imaging (DTI), H-reflex electrophysiological recording, targeted Luxol Fast Blue (LFB) staining of the external capsule, and behavioral assessments. By establishing an integrated structure-function-behavior correlation framework, this study aimed to investigate the relationships among cerebral infarction, external capsule microstructure, spinal reflex excitability, and spasticity following MSSA treatment in a rat model of PSS. The findings are intended to provide preclinical evidence supporting the association between preserved external capsule integrity and reduced spinal reflex hyperexcitability following MSSA, while providing a theoretical basis for the translational application of MSSA in PSS rehabilitation.

Protocol

Forty adult male Specific Pathogen-Free (SPF) Sprague–Dawley (SD) rats, weighing 240–280 g, were purchased from Beijing Vital River Laboratory Animal Technology [License No.: SCXK (Jing) 2021-0011]. All rats were housed in the Laboratory Animal Center of Beijing University of Chinese Medicine under controlled conditions, with a room temperature of 23°C ± 1°C, relative humidity of 50% ± 5%, and a 12 h light/12 h dark cycle, with free access to food and water. The rats were acclimatized for 5 days, during which treadmill exercise acclimation training was performed simultaneously. Animals underwent treadmill acclimation once daily at a speed of 10 m/min for 15 min per session without electrical stimulation to reduce handling stress.

Based on our previous study, the success rate of PSS induction via the middle cerebral artery occlusion (MCAO) method is approximately 60%¹⁵. Using a random number table, 9 rats were randomly selected in advance as the sham-operated group; the remaining 31 rats were used for model establishment. After successful modeling, these rats were randomly divided into the model group and the MSSA group. Ultimately, nine rats were included in each group. This experiment was conducted in accordance with the 3R principles of laboratory animal welfare and was approved by the Animal Ethics Committee of Beijing University of Chinese Medicine (BUCM 20250915-008).

Model Establishment

The MCAO model was established in rats using a modified intraluminal filament technique. Rats were anesthetized with 3% isoflurane for induction, and anesthesia was maintained with 1.5% isoflurane delivered in oxygen at a constant flow rate of 1 L/min throughout the surgical procedure. A midline cervical incision was made, followed by blunt dissection to expose the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). A nylon monofilament suture (diameter, 0.36 ± 0.02 mm) was introduced into the ICA through the ECA and advanced approximately 18–20 mm to the origin of the middle cerebral artery (MCA) to achieve arterial occlusion. The vessels were ligated, and the incision was closed. Rats were then placed on a heating pad until recovery from anesthesia. Rats in the sham-operated group underwent identical surgical exposure of the CCA, ICA, and ECA without insertion of the intraluminal filament.

After surgery, all MCAO rats received standardized postoperative care. Animals were maintained on a 37°C heating pad for the first 12 h to prevent hypothermia. Surgical incisions were cleaned daily for three consecutive days, ampicillin (100 mg/kg) was administered by intraperitoneal injection for infection prophylaxis, and softened food was provided to maintain adequate nutrition. General activity, food intake, wound condition, and limb motor function were monitored daily from postoperative days 1 to 10.

Neurological deficits were evaluated by investigators blinded to group allocation using the Zea Longa neurological deficit score, and muscle tone was assessed using the Modified Ashworth Scale (MAS). Assessments were performed 1 day before surgery (day −1), on the day of surgery before MCAO (day 0), and on postoperative days 1, 3, 5, 7, and 10. Rats with Zea Longa and MAS scores ≥1 on postoperative day 3 were considered to have established a successful PSS model.

MSSA Treatment

Interventions commenced on postoperative day 3. The scalp acupuncture stimulation site was the ipsilesional MS 6 line (anterior parietotemporal oblique line). In accordance with the National Standard for Standardized Manipulations of Scalp Acupuncture (GB/T 21709.2-2021), the MS 6 line extends from GV 21 (Qianding) to GB 6 (Xuanli).

Disposable sterile acupuncture needles (0.25 × 13 mm) were inserted beneath the galea aponeurotica at GV 21 at an approximately 15° oblique angle relative to the scalp surface and advanced toward GB 6 along the anterior parietotemporal oblique line to a depth of approximately 10 mm. Rapid twirling manipulation (200 revolutions/min) was applied continuously for 1 min. After 15 min of needle retention, the same twirling manipulation was repeated once, resulting in a total needle retention time of 30 min. During the entire needle retention period, rats simultaneously underwent treadmill exercise at a constant speed of 10 m/min.

Rats in the sham-operated and model groups received neither acupuncture nor treadmill exercise. To control for nonspecific handling and restraint stress, they were gently handled and restrained in non-compressive stocking holders for 30 min/day, matching the total daily intervention duration of the MSSA group.

Behavioral Assessments

Zea Longa Score for Neurological Deficits:

Neurological deficits in rats were assessed using the Zea Longa scoring system¹⁶. The following criteria were applied: a score of 0 indicated no neurological impairment and normal unconstrained movement; a score of 1 indicated incomplete extension of the forelimb or hindlimb contralateral to the affected side; a score of 2 indicated circling toward the contralateral side during locomotion; a score of 3 indicated falling to the contralateral side while walking; and a score of 4 indicated loss of consciousness with an absence of spontaneous ambulation.

MAS for Muscle Tone:

Muscle tone was assessed using the MAS¹⁷. The criteria were defined as follows: grade 0, no increase in muscle tone; grade 1, slight increase in muscle tone, with a transient catch or minimal resistance at the end of passive flexion and extension; grade 1+, marked increase in muscle tone, with a transient catch in the mid-range of motion and persistent resistance during the latter half of movement; grade 2, pronounced increase in muscle tone, with resistance evident throughout most of the movement range, although the limb remained easily movable; grade 3, substantial increase in muscle tone that rendered passive flexion and extension difficult; and grade 4, limited passive movement due to joint rigidity. Grade 1+ was assigned a score of 2 points, yielding a maximum MAS score of 5 points in this study¹⁰.

T2WI Neuroimaging for Quantification of Cerebral Infarct Volume

All T2WI scans were acquired using a PharmaScan MRI scanner operated with ParaVision 360 software (version 3.5). A T2-weighted turbo rapid acquisition with relaxation enhancement (T2_TurboRARE, also termed RARE/fast spin echo [FSE]) pulse sequence was applied to MCAO rats for the quantification of cerebral infarct volume. The imaging parameters were configured as follows: repetition time (TR) = 3000.0 ms, echo time (TE) = 33.0 ms, field of view (FOV) = 35.00 × 35.00 mm², matrix size = 250 × 250, slice thickness = 0.80 mm, number of slices = 35, number of excitations (NEX) = 3.0, and flip angle (FA) = 180.0°. Cerebral infarct regions were manually delineated on a slice-by-slice basis in ITK-SNAP software (version 4.4.0) by a single investigator blinded to group assignment using signal intensity changes and morphological characteristics consistent with ischemic infarction. Cerebral infarct volume was then calculated automatically by the software.

DTI Neuroimaging for Evaluation of White Matter Microstructural Integrity

MRI data were acquired using a 7.0 T PharmaScan magnetic resonance scanner. Anesthesia was induced with 3% isoflurane delivered in compressed air (0.4–0.6 L/min) and maintained with 1% isoflurane throughout DTI acquisition. Core body temperature was maintained using a thermostatically controlled water bath throughout the imaging procedure.

During MRI scanning, a three-plane localization sequence was first applied to position the rat brain within the magnet. DTI was performed using an axial single-shot spin-echo echo-planar imaging sequence with the following parameters: TR/TE = 3000/27 ms, matrix = 90 × 75, FOV = 20 × 15 mm², slice thickness = 0.7 mm, 30 diffusion gradient directions, and b-values of 0 and 1000 s/mm². For DTI analysis, regions of interest were drawn in the external capsule on brain slices 1.3 mm posterior to bregma. Parametric maps of fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) were reconstructed using ParaVision 360 software. Data are expressed as the ratio of the affected hemisphere to the contralateral healthy hemisphere.

LFB Staining for Assessment of Myelin Injury and Remyelination

LFB staining was performed to evaluate structural damage to myelinated axons in the external capsule. Myelin injury, characterized by myelin rarefaction and vacuolation, was quantified by measuring the integrated optical density (IOD) of LFB-stained myelinated fibers within the ROI in the external capsule using ImageJ software (version 6.0; grayscale threshold, 35–225). Based on the rat brain stereotaxic atlas¹⁸, three coronal sections were analyzed for each animal at bregma +1.0 mm, −1.3 mm, and −1.6 mm, corresponding to the anterior, middle, and mid-posterior portions of the external capsule. The mean IOD across the three sections was calculated to represent each animal (n = 4 per group). Ipsilateral IOD values were normalized to the contralateral side and expressed as a percentage¹⁹.

Electrophysiological Recordings for Assessment of Spinal Reflex Excitability

The spinal monosynaptic H-reflex was recorded using a biological function experiment system. Rats were lightly anesthetized with α-chloralose (50 mg/kg, intraperitoneally) to preserve spinal reflex excitability during electrophysiological recordings. The left sciatic nerve was exposed by blunt dissection and placed on bipolar hook electrodes for electrical stimulation. An oily vitamin solution was applied intermittently to the exposed nerve to maintain tissue hydration and neural viability. A pair of stainless-steel recording electrodes was inserted into the interosseous muscles of the left hind paw, and a ground electrode was placed in the tail. The H-reflex was evoked using a monopolar-bipolar pulse stimulator with a pulse duration of 100 μs. The H-reflex threshold and motor threshold (MT) were determined using incremental electrical stimulation, with the current increased in 0.05 mA steps to a maximum intensity of 3.0 mA to avoid irreversible peripheral nerve injury.

Using the same stimulation intensity that elicited the maximal H-reflex (H_max), pulse stimuli of varying frequencies (0.3, 5, and 10 Hz) were delivered to obtain H-reflex amplitudes at different stimulation frequencies. Repeated pulse stimulation at 0.3 Hz was applied to verify that the M-wave amplitude remained within 95% of its initial value; otherwise, the dataset was discarded. H-reflex frequency-dependent depression (FDD) was calculated as the percentage of the H-wave/M-wave amplitude ratio (H/M ratio) at 5 Hz and 10 Hz relative to that at 0.3 Hz, with the H/M ratio at 0.3 Hz serving as the baseline reference¹⁰.

Data Analysis

Statistical analyses were performed using SPSS software (version 20.0). Continuous data are presented as the mean ± standard deviation (SD). Data normality was assessed using the Shapiro–Wilk test. One-way analysis of variance (ANOVA) was used to compare conventional imaging outcomes and other quantitative variables among groups. When homogeneity of variance was satisfied, Bonferroni post hoc tests were performed; otherwise, Dunnett’s T3 test was applied. Behavioral data were analyzed using two-way ANOVA with experimental group and postoperative time point as fixed factors. Spearman correlation analysis was used to evaluate associations among study variables. A value of p < 0.05 was considered statistically significant.

Results

MSSA Ameliorates Neurological Functional Deficits

Two-way repeated-measures ANOVA revealed significant main effects of time (F[2.613, 62.71] = 315.3, p < 0.001) and group (F[2, 24] = 142.9, p < 0.001), as well as a significant time × group interaction (F[12, 144] = 99.94, p < 0.001) for the Zea Longa neurological scores. One day before surgery, all rats in the sham-operated, model, and MSSA groups had a Zea Longa score of 0, indicating intact baseline neurological function without neurological deficits. Beginning on postoperative day 1, Zea Longa scores gradually increased in both the model and MSSA groups. No significant difference was observed between these groups from postoperative day 1 to day 5 (p > 0.05). With continued intervention, Zea Longa scores in the MSSA group were significantly lower than those in the model group on postoperative days 7 and 10 (p < 0.001), indicating improved neurological function (Figure 1A).

Figure 1. Motion-style scalp acupuncture improves neurological deficits and reduces muscle tone in rats with post-stroke spasticity. (A) Zea Longa neurological deficit scores measured before middle cerebral artery occlusion (MCAO; day −1), immediately before MCAO (day 0), and on postoperative days 1, 3, 5, 7, and 10. (B) Modified Ashworth Scale scores measured at the corresponding time points. The dashed vertical line indicates MCAO on day 0. Data are presented as the mean ± standard deviation (SD). Error bars represent SD. ***p < 0.001 for the comparisons indicated by brackets. Please click here to view a larger version of this figure.

MSSA Reduces Cerebral Infarct Volume

Sham-operated rats exhibited normal T2WI findings, with homogeneous parenchymal signal intensity throughout the brain. No pathological hyperintensity, cerebral infarction, morphological abnormalities, or ventricular dilation were observed (Figure 2A). In contrast, the model group exhibited prominent T2 hyperintensity in the ipsilateral cerebral hemisphere, corresponding to the ischemic infarct region, together with evident ventricular enlargement (Figure 2A). Quantitative morphometric analysis demonstrated that cerebral infarct volume was significantly reduced in the MSSA group compared with the model group (p < 0.001; Figure 2B). The MSSA intervention is illustrated in Figure 2C.

Figure 2. Motion-style scalp acupuncture reduces cerebral infarct volume in rats with post-stroke spasticity. (A) Representative T2-weighted imaging (T2WI) brain images from the sham-operated, model, and motion-style scalp acupuncture (MSSA) groups at 1.00, 0.20, −0.92, and −1.88 mm relative to bregma. (B) Quantification of cerebral infarct volume from T2WI. Individual symbols represent individual animals. (C) Photograph of the MSSA intervention performed concurrently with treadmill exercise. Data are presented as the mean ± SD. Error bars represent SD. ***p < 0.001 versus the model group. Please click here to view a larger version of this figure.

MSSA Attenuates Post-Stroke Muscle Spasticity

For the MAS scores, two-way repeated-measures ANOVA revealed significant main effects of time (F[2.158, 51.79] = 183.7, p < 0.001) and group (F[2, 24] = 57.68, p < 0.001), together with a significant time × group interaction (F[10, 120] = 58.70, p < 0.001). All rats exhibited an MAS score of 0 one day before surgery and on postoperative day 1, reflecting normal baseline muscle tone without spasticity. From postoperative day 3 onward, MAS scores progressively increased in the model and MSSA groups, indicating the development of post-stroke limb spasticity, with no significant difference observed between the two groups at this time point. From postoperative day 5 to day 10, MAS scores in the MSSA group were significantly lower than those in the model group (p < 0.01 to p < 0.001), indicating that MSSA alleviated PSS (Figure 1B).

MSSA Preserves White Matter Microstructural Integrity in the External Capsule

Representative DTI parameter maps are shown in Figure 3A. On postoperative day 10, compared with the sham-operated group, the model group exhibited a marked reduction in the ratio of fractional anisotropy (rFA) within the external capsule (p < 0.001; Figure 3B), accompanied by significant increases in the ratios of mean diffusivity (rMD) and radial diffusivity (rRD) (both p < 0.001; Figure 3C,D). Compared with the model group, MSSA significantly increased rFA (p < 0.001; Figure 3B) and significantly decreased rMD and rRD (both p < 0.01; Figure 3C,D). No significant difference in the ratio of axial diffusivity (rAD) was observed among the groups (p > 0.05; Figure 3E). The positioning of rats during DTI acquisition and the anatomical location of the external capsule ROI are shown in Figure 3F,G.

Figure 3. Motion-style scalp acupuncture alters diffusion tensor imaging parameters in rats with post-stroke spasticity. (A) Representative fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) maps from the sham-operated, model, and motion-style scalp acupuncture (MSSA) groups at −1.30 mm relative to bregma. (B–E) Quantification of the ratios of ipsilesional to contralateral FA (rFA), MD (rMD), RD (rRD), and AD (rAD), respectively. Individual symbols represent individual animals. (F) Photograph of a rat positioned for diffusion tensor imaging (DTI). (G) Schematic and corresponding magnetic resonance image showing the contralateral and ipsilesional external capsule regions of interest. Data are presented as the mean ± SD. Error bars represent SD. #p < 0.05 and ###p < 0.001 versus the sham-operated group; **p < 0.01 and ***p < 0.001 versus the model group. Please click here to view a larger version of this figure.

MSSA Improves Myelin Integrity in the External Capsule

LFB staining was performed to evaluate myelin integrity in the external capsule. Histologically, myelinated fibers exhibited intact morphology and regular arrangement in the sham-operated group, whereas evident myelin loss and structural disorganization were observed in the external capsule of MCAO model rats (Figure 4A). Quantitative analysis showed that the relative IOD of LFB staining was significantly lower in the model group than in the sham-operated group (p < 0.001; Figure 4B). Compared with the model group, the MSSA group exhibited significantly higher relative IOD values (p < 0.001; Figure 4B), indicating improved myelin integrity in the external capsule following MSSA treatment.

Figure 4. Motion-style scalp acupuncture attenuates external capsule myelin injury in rats with post-stroke spasticity. (A) Representative panoramic Luxol Fast Blue (LFB)-stained brain sections and magnified views of the contralateral and ipsilesional external capsule regions of interest (ROIs) from the sham-operated, model, and motion-style scalp acupuncture (MSSA) groups. Blue and red boxes in the panoramic images indicate the contralateral and ipsilesional ROIs, respectively. Scale bars in the magnified images = 50 µm. (B) Quantification of the relative integrated optical density (IOD) of LFB staining in the ipsilesional external capsule normalized to the contralateral side. Individual symbols represent individual animals; n = 4 per group. Data are presented as the mean ± SD. Error bars represent SD. ###p < 0.001 versus the sham-operated group; ***p < 0.001 versus the model group; △p < 0.05 for the comparison indicated by the bracket. Please click here to view a larger version of this figure.

MSSA Inhibits Spinal H-Reflex Hyperexcitability

Representative M-wave and H-wave recordings are shown in Figure 5A, and the electrophysiological recording setup is shown in Figure 5B. On postoperative day 10, electrophysiological assessment demonstrated that, compared with the sham-operated group, the model group exhibited significantly increased motor threshold (MT; p < 0.001) and maximum H-wave/maximum M-wave amplitude (H_max/M_max) ratio (p < 0.001), a modest increase in M-wave latency (p < 0.05), and a corresponding decrease in H-reflex latency (p < 0.05; Figure 5C–H). Compared with the model group, the MSSA group exhibited significantly lower MT (p < 0.001) and H_max/M_max ratio (p < 0.001), together with a significantly higher H-reflex threshold (p < 0.001; Figure 5C–H). No significant difference in the stimulation intensity required to elicit H_max expressed as a multiple of MT, was observed between the model and MSSA groups (Figure 5H). Furthermore, when normalized to the H/M ratio at 0.3 Hz, H-reflex FDD progressively decreased with increasing stimulation frequency in all three groups (Figure 5I). At both 5 Hz and 10 Hz, the H/M ratio was significantly lower in the MSSA group than in the model group (both p < 0.001), indicating partial restoration of H-reflex FDD toward the physiological pattern (Figure 5J; Table 1).

Figure 5. Motion-style scalp acupuncture reduces spinal reflex hyperexcitability in rats with post-stroke spasticity. (A) Representative superimposed M-wave and H-wave traces from the sham-operated, model, and motion-style scalp acupuncture (MSSA) groups. (B) Photograph of the electrophysiological recording setup showing the sciatic nerve stimulation site, recording electrodes, and ground electrode. (C–H) Quantification of motor threshold (MT), maximum H-wave/maximum M-wave amplitude ratio (H_max/M_max), H-wave latency, H-reflex threshold, M-wave latency, and the stimulation intensity eliciting H_max expressed as a multiple of MT, respectively. Individual symbols represent individual animals. (I) Representative H-reflex traces obtained at stimulation frequencies of 0.3 Hz, 5 Hz, and 10 Hz in each group. (J) Frequency-dependent depression of the H-reflex, expressed as the H/M ratio at 5 Hz and 10 Hz normalized to the H/M ratio at 0.3 Hz. Shaded regions represent the SD of the mean values. Data are presented as the mean ± SD. Error bars represent SD. #p < 0.05 and ###p < 0.001 versus the sham-operated group; ***p < 0.001 versus the model group. Please click here to view a larger version of this figure.

Group	MT (mA)	Mmax (mV)	Hmax (mV)	M latency (ms)	H latency (ms)	H-reflex threshold (×MT)	Hmax (×MT)	H/M ratio (0.3 Hz)	H/M ratio (5 Hz)	H/M ratio (10 Hz)
Sham (n = 9)	0.808 ± 0.218	11.119 ± 7.802	2.804 ± 2.896	4.567 ± 0.731	11.311 ± 0.849	1.296 ± 0.251	1.554 ± 0.264	0.452 ± 0.264	0.304 ± 0.155	0.176 ± 0.137
Model (n = 9)	1.855 ± 0.203###	3.282 ± 2.040##	2.307 ± 1.296	5.333 ± 0.893	10.456 ± 1.056	0.729 ± 0.299	1.517 ± 0.303	0.826 ± 0.120##	0.741 ± 0.129###	0.684 ± 0.131###
MSSA (n = 9)	0.663 ± 0.236***	5.908 ± 2.751	1.497 ± 0.688	4.744 ± 0.590	10.978 ± 0.602	1.939 ± 0.819***	1.832 ± 1.557	0.556 ± 0.238*	0.390 ± 0.193**	0.142 ± 0.134***

Table 1: Electrophysiological parameters of the H-reflex in rats with post-stroke spasticity. Electrophysiological measurements obtained from the sham-operated, model, and motion-style scalp acupuncture (MSSA) groups, including motor threshold (MT), maximum M-wave amplitude (M_max), maximum H-wave amplitude (H_max), M-wave latency, H-wave latency, H-reflex threshold expressed as a multiple of MT (×MT), stimulation intensity eliciting H_max expressed as a multiple of MT (×MT), and H/M ratios measured at stimulation frequencies of 0.3, 5, and 10 Hz. Data are presented as mean ± SD. ##p < 0.01 and ###p < 0.001 versus the sham-operated group; *p < 0.05, **p < 0.01, and ***p < 0.001 versus the model group.

Correlations Among T2WI Infarct Volume, DTI Metrics of the External Capsule, Electrophysiological Indices, and MAS Scores

Correlations between cerebral infarct volume measured by T2WI and DTI metrics of the external capsule were first examined. In the pooled analysis, infarct volume was strongly negatively correlated with the rFA (ρ = −0.902, p < 0.001) and positively correlated with the rMD and rRD (ρ = 0.511, p = 0.03; ρ = 0.661, p = 0.003; Figure 6A–D). No significant correlations between infarct volume and DTI parameters were observed in the subgroup analyses. The relationships among DTI metrics of the external capsule, electrophysiological indices of spinal reflex excitability, and muscle tone assessed by the MAS were then evaluated. In the pooled analysis, lower rFA was associated with a higher H_max/M_max ratio (ρ = −0.895, p < 0.001), whereas higher rRD was associated with a lower H-reflex threshold (ρ = −0.504, p = 0.03; Figure 6E,F). In addition, higher H_max/M_max ratios and lower H-reflex thresholds were associated with higher MAS scores (ρ = 0.760, p < 0.001; ρ = −0.479, p = 0.01; Figure 6G,H). Subgroup analyses demonstrated a significant negative correlation between H-reflex threshold and MAS score only in the model group (ρ = −0.736, p = 0.03), whereas no significant correlations were observed in the sham-operated or MSSA groups.

Figure 6. Associations among cerebral infarct volume, diffusion tensor imaging parameters, H-reflex indices, and muscle tone in rats with post-stroke spasticity. (A–D) Associations of T2-weighted imaging (T2WI)-derived cerebral infarct volume with the ipsilesional-to-contralateral ratios of fractional anisotropy (rFA), mean diffusivity (rMD), radial diffusivity (rRD), and axial diffusivity (rAD), respectively. (E) Association between rFA and the maximum H-wave/maximum M-wave amplitude ratio (H_max/M_max). (F) Association between rRD and H-reflex threshold. (G) Association between H_max/M_max and Modified Ashworth Scale (MAS) score. (H) Association between H-reflex threshold and MAS score. Circles, squares, and triangles represent the sham-operated, model, and motion-style scalp acupuncture (MSSA) groups, respectively. Dashed lines correspond to the linear regression fitting curve generated from the measured data. Overall and group-specific Spearman correlation coefficients (ρ) and corresponding p values are displayed in each panel. Please click here to view a larger version of this figure.

Data Availability:

All data generated or analyzed during this study are included in this published article and in the Supplementary Material. The raw datasets supporting the findings of this study are provided in Supplementary Table 1.

Supplementary Table 1. Raw datasets supporting the quantitative analyses presented in this study. The workbook contains the raw experimental data used for the behavioral assessments, T2-weighted imaging (T2WI) infarct volume analysis, diffusion tensor imaging (DTI) metrics, Luxol Fast Blue (LFB) staining quantification, and H-reflex electrophysiological measurements for all experimental groups.Please click here to download this file.

Discussion

From the perspective of traditional Chinese medicine, PSS falls into the category of muscle-tendon disorders, with a core pathogenesis described as cerebral injury being the root cause and limb spasticity the superficial manifestation²⁰. Therapeutic strategies should therefore address both the primary cerebral lesion and the secondary peripheral manifestations²¹. The rationale underlying MSSA emphasizes holistic regulation from the ischemic brain to the spastic limbs, with the therapeutic goals of promoting blood circulation, unblocking the meridians, relaxing tendons, and alleviating spasticity. Needling technique is central to acupuncture efficacy, and selecting an appropriate technique tailored to the disease pattern is critical for achieving favorable clinical outcomes. MSSA is a characteristic scalp acupuncture modality for PSS that combines scalp acupuncture with active or passive limb movement during needle retention³. A recent systematic review and meta-analysis demonstrated that MSSA produces superior effects in reducing muscle spasticity, improving motor function, and enhancing activities of daily living in patients with PSS compared with conventional scalp acupuncture or exercise rehabilitation alone⁹. Notably, concurrent administration of scalp acupuncture and exercise rehabilitation, as implemented in MSSA, produces better outcomes than asynchronous intervention, as supported by moderate-quality clinical evidence⁹. MS 6, as defined in GB/T 21709.2-2021, is an anterior parietotemporal oblique scalp acupuncture line selected for its anatomical relevance and established clinical utility⁹. It overlies the primary and secondary motor cortices, which are the principal sources of descending corticofugal projections that shape brainstem reticular output and modulate spinal reflex excitability. Clinically, MS 6 is the most commonly used scalp acupuncture line for PSS, as confirmed by the aforementioned meta-analysis, making it an optimal target for modulating motor pathways in the present study. These findings highlight the distinctive procedural characteristics and potential clinical advantages of MSSA in the management of PSS. Preclinical studies have further shown that MSSA improves cerebral perfusion in the ischemic penumbra and modulates the excitability of spinal anterior horn motoneurons, providing preliminary mechanistic support for its therapeutic application^10,11.

In modern neurological theory, the pathogenesis of PSS is primarily attributed to three interrelated pathological processes: impaired supraspinal descending inhibition, abnormal intraspinal signal processing, and altered intrinsic muscle properties. Among these, the mechanisms by which supraspinal descending pathways regulate spinal reflex excitability remain incompletely understood². The reticulospinal tract (RST) plays an essential role in maintaining the balance of the stretch reflex arc. The lateral RST receives excitatory input from the contralateral premotor cortex and supplementary motor area through the cortico-reticular pathway, descends within the dorsolateral funiculus of the spinal cord, and exerts inhibitory control over spinal stretch reflex activity. In contrast, the medial RST is not regulated by the contralateral cortex, descends within the ventromedial funiculus, and facilitates stretch reflex activity. Cortical stroke disrupts the cortico-reticular pathway, reduces excitatory input to the medullary reticular formation, and results in the predominance of medial RST activity. This imbalance contributes to spinal stretch reflex hyperexcitability and the clinical manifestation of limb spasticity³. Clinical neuroimaging studies have linked PSS to lesions involving the premotor cortex, putamen, posterior limb of the internal capsule, and external capsule^12,14,22. Among these, the external capsule represents a key white matter conduit within the cortico-reticulospinal pathway, a major extrapyramidal tract that integrates cortical inputs, synapses in the brainstem reticular nuclei, and projects to the spinal cord to exert descending inhibitory control over α-motoneurons and interneurons²³. This anatomical framework is supported by neural tracing and DTI studies, which consistently identify the external capsule as a principal connection between the cerebral cortex, basal ganglia, and brainstem^23,24. Notably, spasticity is increasingly recognized as a network-level disorder resulting from disruption of the balance among multiple descending motor systems, including both corticospinal and extrapyramidal pathways, rather than from dysfunction of a single tract. In this context, the external capsule serves as a representative structure for probing extrapyramidal contributions to PSS, as its involvement has been consistently associated with exaggerated spinal reflex activity, whereas isolated corticospinal lesions more commonly produce weakness without proportionate spasticity. Accordingly, we selected the external capsule as the primary ROI for investigating white matter microstructural changes associated with spasticity severity. Consistent with this framework, T2WI revealed no obvious cerebral infarction in the sham-operated group, whereas prominent infarct lesions were observed in the model group. MSSA treatment significantly improved neurological function, as assessed by the Zea Longa scale, suggesting a neuroprotective effect against ischemic brain injury. The infarct-sparing effect of MSSA may involve multiple synergistic mechanisms. First, MSSA has been shown to enhance regional cerebral perfusion in the ischemic penumbra¹⁰, thereby creating a more favorable microenvironment for neuronal survival. In addition, although direct evidence for MSSA is currently limited, mechanistic studies of scalp acupuncture have suggested several other pathways that may also contribute, including the promotion of angiogenesis through activation of the MATN2/WNT3a/β-catenin signaling pathway²⁵ and attenuation of post-ischemic neuroinflammation through suppression of RORγt-mediated Th17 differentiation and restoration of the Th17/Treg balance²⁶. These findings raise the possibility that MSSA may engage similar neuroprotective mechanisms beyond its hemodynamic effects. However, the relative contribution of each pathway remains to be determined and warrants further investigation.

DTI enables sensitive quantitative assessment of white matter microstructure, with FA reflecting fiber integrity, RD serving as a marker of demyelination, and AD reflecting axonal integrity²³. In the present study, the external capsule was selected as the ROI to evaluate microstructural alterations associated with the cortico-reticulospinal pathway in PSS. Compared with the sham-operated group, model rats exhibited significant reductions in relative FA together with marked increases in relative MD and RD, indicative of disrupted white matter integrity, cerebral edema, and demyelination within the external capsule. LFB staining further demonstrated evident myelin loss and disorganization of white matter bundles, providing histological support for the DTI findings. Collectively, the neuroimaging and histological findings indicate that ischemic injury is associated with marked microstructural impairment of the external capsule, which was attenuated following MSSA treatment. The H-reflex is a well-established electrophysiological measure for evaluating spinal α-motoneuron pool excitability and supraspinal descending regulation²⁷. The H_max/M_max ratio reflects the overall excitability of spinal motoneurons, whereas the H-reflex threshold reflects presynaptic inhibition mediated by spinal interneurons together with integrated supraspinal descending inputs²⁷. Stroke-induced disruption of the cortico-reticulospinal pathway weakens descending inhibitory regulation, resulting in spinal motoneuron hyperexcitability characterized by an increased H_max/M_max ratio and a reduced H-reflex threshold²⁷. Our preliminary experiments explored the molecular mechanisms underlying the antispastic effects of MSSA, demonstrating that this therapy may upregulate spinal KCC2 expression and restore GABAergic inhibitory function¹⁰. Building upon these spinal molecular findings, the present study further elucidates the upstream supraspinal mechanisms responsible for exaggerated spinal reflexes by demonstrating that microstructural injury of the external capsule within the cortico-reticulospinal pathway is associated with markedly increased spinal excitability. Taken together, these findings suggest that MSSA may promote reconstruction of supraspinal descending inhibitory pathways and reverse spinal reflex hyperexcitability in rats with PSS.

Given the cross-sectional nature of the present data, all correlation analyses should be interpreted as exploratory and hypothesis-generating rather than as evidence of causal relationships. Correlation analyses were performed to investigate the relationships among infarct burden, external capsule microstructural integrity, spinal reflex excitability, and spasticity severity. Across all animals, larger infarct volumes were associated with more severe DTI-derived microstructural disruption within the external capsule, suggesting a link between primary ischemic injury and secondary white matter changes along the cortico-reticulospinal pathway. Notably, these overall correlations appeared to be driven primarily by intergroup differences rather than by within-group individual variation, as no significant correlations were observed within any individual group. Higher relative FA (rFA) was associated with a lower H_max/M_max ratio, whereas higher relative RD (rRD) was associated with a lower H-reflex threshold, consistent with the notion that preserved external capsule integrity is associated with more effective descending inhibitory regulation. In addition, higher H_max/M_max ratios and lower H-reflex thresholds were both associated with higher MAS scores, supporting an association between electrophysiological alterations and spasticity severity. Subgroup analyses further revealed that the overall correlation patterns were predominantly attributable to intergroup differences. Notably, a significant negative correlation between H-reflex threshold and MAS score was observed only in the model group, suggesting that impaired presynaptic inhibition may be associated with greater spasticity under untreated pathological conditions.

Several limitations should be acknowledged. First, the absence of acupuncture-only and exercise-only control groups precludes dissection of their independent and synergistic contributions. Future studies incorporating these comparison groups would help clarify the mechanisms underlying the combined intervention. Second, the observation period was limited to the acute phase (postoperative days 0–10), selected on the basis of the peak spasticity window in the permanent MCAO model. Whether MSSA confers sustained therapeutic benefits beyond the acute stage remains unknown, and validation in chronic PSS models will be important for determining its long-term translational potential. Third, although the MAS is widely used to evaluate muscle tone, it is inherently subjective and semi-quantitative. To complement this limitation, the H-reflex was simultaneously assessed as an objective electrophysiological measure of spinal reflex excitability. Fourth, the sample size was modest because of the technical demands associated with multimodal assessments, and studies including larger numbers of animals would improve statistical power and the robustness of the findings. Finally, all endpoints were evaluated only on postoperative day 10. Future studies incorporating longitudinal assessments at multiple time points would provide a more comprehensive understanding of the temporal trajectory of MSSA-induced neuroplastic changes.

Future studies may investigate the mechanisms of neurogenesis and remyelination following MSSA intervention. Because DTI cannot directly visualize neuronal regeneration, complementary approaches such as neural tracing and immunohistochemistry may further characterize neuronal plasticity and remyelination. From a translational perspective, DTI metrics of the external capsule, particularly FA and RD, may help stratify patients with stroke according to the severity of cortico-reticulospinal pathway injury and identify those most likely to benefit from MSSA. Such a biomarker-driven approach could facilitate patient selection and treatment stratification in future clinical trials, although its clinical utility remains to be validated in prospective stroke cohorts. In conclusion, this study investigated the neural mechanisms associated with MSSA treatment for PSS using an integrated framework comprising T2WI-based infarct assessment, DTI evaluation of white matter microstructure, H-reflex electrophysiology, and LFB histological analysis. The findings suggest that MSSA is associated with reduced cerebral infarct volume, preservation of external capsule microstructural integrity, and improved descending inhibitory regulation of the cortico-reticulospinal pathway. These changes were accompanied by normalization of spinal reflex excitability and reduced spasticity. Collectively, the findings provide mechanistic insights and preclinical evidence supporting the potential translational application of MSSA in the management of PSS.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
α-Chloralose	GoldBio	C-118-250	Anesthetic for H-reflex recordings
Absolute Ethanol	Beijing Tong Guang Fine Chemicals Co., Ltd.	VS104021-500 mL	Histological reagent
Acupuncture Needles (0.25 × 13 mm)	Beijing Zhongyan Taihe Acupuncture Needle Co., Ltd.	NA	Disposable sterile needles for scalp acupuncture
Ampicillin	Beijing Rui Da Heng Hui Technology Development Co., Ltd.	HD-3018-500g	Antibiotic for postoperative infection prophylaxis
Biological Function Experiment System (BL420N)	Chengdu Taimeng Technology Co., Ltd.	NA	Electrophysiological recording system
Diffusion Tensor Imaging Analysis Software (ParaVision 360)	Bruker	NA	MRI acquisition and DTI reconstruction
Eco-Friendly Dewaxing and Clearing Solution	Servicebio	G1128-500ML	Histological reagent
Fully Automatic Rotary Microtome	Leica Microsystems	RM2255	Tissue sectioning
Heating Pad	Suzhou Caiyang Electric Heating Technology Co., Ltd.	TT180×150-1XD	Postoperative temperature maintenance
ImageJ 6.0	National Institutes of Health	NA	Quantitative image analysis
Isoflurane	Beijing Solarbio Science & Technology Co., Ltd.	I8000-25g	Inhalation anesthetic
ITK-SNAP 4.4.0	University of Pennsylvania	NA	Cerebral infarct segmentation
Luxol Fast Blue Solution	Servicebio	G1030-100ML	Myelin staining
Monofilament Nylon Suture (0.36 ± 0.02 mm)	Beijing Cinontech Co., Ltd.	NA	MCAO induction
Paraformaldehyde (4% in PBS)	Servicebio	TBAB0102	Tissue fixation
PharmaScan 7.0 T Magnetic Resonance Scanner	Bruker	NA	MRI acquisition
SPSS Statistics 20.0	IBM	NA	Statistical analysis
Sprague–Dawley Rats (SPF, male)	Beijing Vital River Laboratory Animal Technology Co., Ltd.	NA	Experimental animals
Thermostatically Controlled Water Bath System	Shanghai Zhixin Experimental Instrument Technology Co., Ltd.	ZX-5A	Temperature maintenance during MRI
Treadmill for Rodents	Clever	TreadScan	Exercise training during acclimation and MSSA