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 intac...

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 ...

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