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Research Article
This study aims to investigate the effects of scalp acupuncture combined with exercise on motor cortex synaptic plasticity in a rat model of post-stroke limb spasticity and to explore the association between PI3K/Akt pathway activity and the therapeutic effects.
Effective treatments for post-stroke spasticity (PSS) remain limited, and the mechanism by which scalp acupuncture combined with exercise (SAE) alleviates PSS is not fully understood. This study investigated the effects of SAE in rats with PSS and explored whether SAE regulates synaptic plasticity through the PI3K/Akt pathway. A middle cerebral artery occlusion (MCAO) rat model was established, and rats were randomly assigned to five groups: Blank, Sham, MCAO, SAE, and Baclofen. All interventions were administered for 7 consecutive days. Neurological function, cerebral infarct volume, histopathology, synaptic ultrastructure, and related protein expression were then evaluated. Compared with the MCAO group, SAE improved neurological deficits, reduced muscle hypertonia, decreased infarct volume, alleviated neuronal injury, increased synapse number, and improved synaptic morphology. SAE also upregulated glial cell line-derived neurotrophic factor (GDNF), phosphorylated PI3K (p-PI3K), phosphorylated Akt (p-AKT), and synaptic proteins, including synaptophysin (SYN), postsynaptic density protein 95 (PSD-95), and growth-associated protein 43 (GAP-43). Immunofluorescence staining further suggested enhanced expression of SYN and p-Akt in the motor cortex after SAE treatment. These findings suggest that SAE may be correlated with the activation of PI3K/Akt signaling via upregulation of GDNF, thereby improving synaptic plasticity and alleviating PSS symptoms. This study investigated the effects of SAE in rats with PSS and explored the potential association between SAE intervention, PI3K/Akt pathway activity, and synaptic plasticity.
Cerebral infarction remains a leading cause of death and disability worldwide. Ischemia and hypoxia damage neurons, disrupt the excitation–inhibition balance in motor circuits, and lead to motor dysfunction1. Post-stroke spasticity is manifested as increased muscle tone and reduced joint mobility, which impairs activities of daily living and increases caregiver burden2. Although antispastic agents such as baclofen can provide temporary symptomatic relief, long-term use may lead to drug tolerance and adverse effects, and does not restore impaired neural function3. Conventional motor rehabilit....
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All procedures were approved by the Animal Ethics Committee of Changchun University of Chinese Medicine (Approval No. 20251038) and were conducted in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE 2.0) guidelines as well as the NIH Guide for the Care and Use of Laboratory Animals. All materials used in this study are shown in the Table of Materials.
Experimental procedures
Experimental animals and grouping
Eighty specific pathogen-free (SPF) adult male Sprague–Dawley rats (220–250 g) were purchased from Hongda Animal Breeding Farm (Kuancheng District, C....
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SAE improves neurological function and reduces muscle spasticity in PSS rats
The therapeutic effects of SAE on behavioral dysfunction in rats with post-stroke spasticity were evaluated using the Zea Longa score, modified Neurological Severity Score, and modified Ashworth Scale on Days 1, 3, 5, and 7 after model validation (n = 12 per group). The Zea Longa score and mNSS were used to assess neurological deficits, whereas the MAS score was used to evaluate muscle tone/spasticity. After modeling, rats i.......
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Previous clinical studies have shown that scalp acupuncture combined with exercise can relieve spastic symptoms in patients with post-stroke spasticity, while improving motor function and activities of daily living (ADL)14,24. These findings support its clinical value. The present study further confirmed the therapeutic benefit of SAE in a rat model using three behavioral measures, including the Zea Longa score, mNSS, and MAS. Repeated behavioral assessment showe.......
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The authors declare that they have no competing financial interests.
AUTHOR’S CONTRIBUTIONS:
Sainan Zhao: Conceptualization, Methodology, Formal analysis, Investigation, Project administration, Writing – original draft, Writing – review & editing. Lingxu Li: Investigation, Data curation, Validation. Wenna Li: Data curation, Formal analysis, Visualization. Chunmeng Wang: Investigation, Resources. Yanlong Jin: Software, Validation, Writing – review & editing. Guangcheng Ji: Supervision, Funding acquisition, Resources, Writing – review & editing.
The authors thank all participants who participated in this study. This work was supported by the Science and Technology Development Project of Jilin Province [No. YDZJ202401122ZYTS].
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 2,3,5-triphenyltetrazolium chloride staining solution | Beyotime | C0651 | TTC staining |
| 3MM Filter Paper | Whatman, UK | 3030861 | Western blot transfer |
| AKT Antibody | Affinity | AF0836 | Primary antibody |
| Acetone | Sinopharm Chemical Reagent Co., Ltd. | 10000418 | Infiltration of electron microscopy specimens |
| Adobe Illustrator | Adobe Inc. | Version 29.5 (64-bit), https://www.adobe.com/products/illustrator.html | 64-bit version, used for graphic editing and scientific figure preparation |
| Anhydrous ethanol | Sinopharm Chemical Reagent Co., Ltd. | 100092683 | Deparaffinization of paraffin sections |
| Anti-fade mounting medium | Wuhan Baiqiandu Biotechnology Co., Ltd. | C1020 | coverslipping |
| BCA Protein Assay Kit | MDL | MD913053 | Protein concentration determination |
| Bluishing solution | Wuhan Baiqiandu Biotechnology Co., Ltd. | C2104 | Hematoxylin staining |
| Chemiluminescence Imaging System | CLINX, China | ChemiScope6100 | System Detect target protein |
| Concentrated normal goat serum | Boster Biological Technology | AR1009 | serum blocking |
| DAPI | Wuhan Baiqiandu Biotechnology Co., Ltd. | C1030 | nuclear staining |
| Diamond Knife | Daitome | Ultra 45° | Slice ultrathin specimens |
| Differentiation solution | Wuhan Baiqiandu Biotechnology Co., Ltd. | C2104 | HE staining |
| Drying Oven | Tianjin Labory Instruments Equipment Co., Ltd. | GFL-230 | Bake fixed tissue slices |
| EDTA (pH 9.0) antigen retrieval solution | Wuhan Baiqiandu Biotechnology Co., Ltd. | C1052 | Antigen retrieval |
| Electron microscopy fixative | ASPEN | AS1063 | Fixation of electron microscopy specimens |
| Electrophoresis Power Supply | Longfang | LF-2000 | Provide electrophoresis voltage |
| Embed 812 embedding medium | SPI | 90529-77-4 | Infiltration of electron microscopy specimens |
| EndNote X9 | Clarivate Analytics (US) LLC | Version X9, https://endnote.com/ | Used for reference management and academic citation formatting |
| Freezing Stage | Wuhan Junjie Electronics Co., Ltd. | JB-L5 | Freeze frozen tissue blocks |
| GAP-43 Antibody | Affinity | Df7766 | Primary antibody |
| GAPDH Antibody | Affinity | AF7021 | Primary antibody |
| GDNF Antibody | Biosynthesis | Bs-1024r | Primary antibody |
| Glacial acetic acid | Wuhan Baiqiandu Biotechnology Co., Ltd. | C2138 | Nissl staining |
| Glass Slides and Coverslips | Sinopharm Chemical Reagent Co., Ltd. | 10212432C | Mount tissue sections |
| GraphPad Prism | GraphPad Software, LLC | Version 10.1.2 (324) for Windows 64-bit, https://www.graphpad.com/ | 64-bit version, used for statistical analysis and scientific plotting |
| Grinding machine | Beijing Herde | N.9548 | Grind tissue samples |
| HE staining solution | Wuhan Baiqiandu Biotechnology Co., Ltd. | C2103 | HE staining |
| HRP-Conjugated Goat Anti-Mouse Secondary Antibody | Servicebio | C030205 | Secondary Antibody |
| HRP-conjugated goat anti-rabbit antibody | SeraCare | 5220-0336 | Secondary antibody |
| Hydrogen peroxide (H2O2) | Wuhan Baiqiandu Biotechnology Co., Ltd. | C5101 | preparation of TSA reagents |
| IBM SPSS Statistics | IBM Corp. | Version 25, 64-bit, https://www.ibm.com/products/spss-statistics | 64-bit version, used for statistical data analysis and processing |
| Imaging System | Nikon, Japan | Nikon DS-U3 | Capture microscopic images |
| Metal Bath | COYOTE | H203-H | Constant temperature incubation |
| Microwave Oven | Galanz Microwave Electrical Appliance Co., Ltd. | P70D20TL-P4 | Antigen retrieval |
| NC Membrane (0.22 μm) | GVS, USA | ISEQ00010 | Protein blot transfer |
| Neutral balsam | Sinopharm Chemical Reagent Co., Ltd. | 10004160 | dehydration and coverslipping |
| PBS buffer | Wuhan Baiqiandu Biotechnology Co., Ltd. | C0003 | decolorization and washing |
| PI3K Antibody | Affinity | AF6241 | Primary antibody |
| PSD-95 Antibody | Affinity | Af5283 | Primary antibody |
| Pathological Microtome | Leica Instruments (Shanghai) Co., Ltd. | RM2016 | Cut paraffin sections |
| Phospho-AKT Antibody | Affinity | Af0832 | Primary antibody |
| Phospho-PI3K Antibody | Affinity | Af3241 | Primary antibody |
| Pipette | Dragonlab | KE0003087/KA0056573 | Quantitative liquid transfer |
| Protease Inhibitor Cocktail | Beyotime | P1005 | Protease inhibition |
| RIPA Lysis Buffer (Medium) | Beyotime | P0013C | Tissue lysis |
| Refrigerated Centrifuge | SEILOGEX, China | CF1524R | Low-speed sample separation |
| SDS-PAGE Electrophoresis System | Beyotime | MiniProGel™ Gel Casting, Electrophoresis and Western Blot Transfer System | Protein electrophoretic separation |
| SDS-PAGE Precast Gel Kit | MDL | MD911919 | SDS-PAGE electrophoresis |
| SYN (Synapsin) Antibody | Biosynthesis | bs-3501R | Primary antibody |
| Shaker | Qilinbeier | TS-8 | Sample oscillation washing |
| Tissue Dehydrator | Wuhan Junjie Electronics Co., Ltd. | JJ-12J | Dehydrate paraffin tissue |
| Tissue Embedding Machine | Wuhan Junjie Electronics Co., Ltd. | JB-P5 | Embed tissue in paraffin |
| Tissue Spreading Machine | Wuhan Junjie Electronics Co., Ltd. | KD-P | Unfold paraffin slices |
| Toluidine blue staining solution | Wuhan Baiqiandu Biotechnology Co., Ltd. | C2131 | Nissl staining |
| Transmission Electron Microscope | FEI | Tecnai G2 20 TWIN | Observe ultrastructure |
| Tri-Color Pre-Stained Broad Range Protein Molecular Weight Marker (10–180 kDa) | Servicebio | G2091 | SDS-PAGE electrophoresis |
| Tyramide-488 | Wuhan Baiqiandu Biotechnology Co., Ltd. | C6002 | TSA reagents |
| Tyramide-CY3 | Wuhan Baiqiandu Biotechnology Co., Ltd. | C6003 | TSA reagents |
| Ultramicrotome | Leica | Leica UC7 | Cut ultrathin EM sections |
| Ultrasonic Cell Disruptor | Xianchang | XC-CD | Lyse cells |
| Upright Optical Microscope | Nikon, Japan | Nikon Eclipse ci | Observe stained slices |
| Vortex Mixer | Tianyue Electronics | TYXH-II | Mix liquid samples |
| Western Blot Transfer Tank | Junyi Dongfang | JY-ZY6 | Protein membrane transfer |
| Xylene | Sinopharm Chemical Reagent Co., Ltd. | 10023418 | Deparaffinization of paraffin sections |