Scalp Acupuncture Combined with Exercise Induces PI3K/Akt Activation and Improves Synaptic Plasticity in Post-Stroke Spastic Rats

Summary

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.

Abstract

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.

Introduction

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 rehabilitation is also limited by a slow onset of benefit and prolonged treatment duration. Therefore, safer and more effective therapeutic strategies are urgently needed, and the underlying mechanisms warrant further investigation. Neuroplasticity is a central mechanism of post-stroke functional recovery and involves synaptic remodeling, axonal regeneration, and neuronal survival⁴. After ischemic stroke, neurons in the infarct core undergo rapid death, while local inflammatory responses exacerbate tissue injury in the ischemic penumbra; together, these processes drive brain injury^5,6. Synaptic plasticity, a key component of neuroplasticity, can be evaluated by synapse-related proteins, including presynaptic markers such as synaptophysin (SYN) and growth-associated protein 43 (GAP-43), as well as postsynaptic density protein 95 (PSD-95)^7,8,9.

Neurotrophic factors provide a molecular basis for synaptic plasticity. Glial cell line-derived neurotrophic factor (GDNF) is particularly important because it promotes neuronal survival and repair, suppresses inflammation, and improves the synaptic microenvironment¹⁰. By binding to the Ret receptor, GDNF can activate PI3K/Akt signaling¹¹. The PI3K/Akt cascade is a major pro-survival pathway in the nervous system, and its activation inhibits apoptosis while supporting protein synthesis required for synaptic remodeling^12,13. Scalp acupuncture combined with exercise (SAE) is an integrative rehabilitation strategy that combines scalp acupuncture stimulation with active motor training. The overall goal of this method is to simultaneously modulate central neural activity and promote task-related motor recovery, thereby reducing muscle hypertonia and improving functional outcomes after stroke. SAE is widely used in China for the treatment of post-stroke spasticity (PSS). The rationale for this combined approach is that scalp acupuncture may modulate cortical excitability and cerebral perfusion, whereas exercise therapy provides activity-dependent motor training that promotes functional recovery and synaptic remodeling. Compared with exercise therapy alone, previous clinical evidence has shown that scalp acupuncture at scalp points combined with exercise therapy further reduces limb spasticity and improves motor function and activities of daily living in patients with post-stroke limb spasm¹⁴.

A recent systematic review and meta-analysis also suggested that motion-style scalp acupuncture or related concurrent scalp acupuncture-exercise interventions provide greater improvements in spasticity, motor function, balance, and activities of daily living than single-component or sequential interventions, supporting the potential advantage of concurrent neuromodulation and motor training¹⁵. Preclinical studies have shown that SAE or related motion-style scalp acupuncture protocols can improve cerebral blood flow and attenuate spasticity through 5-HT₂A receptor-mediated spinal KCC2 reactivation^16,17. A recent study further suggests that acupuncture promotes neuroplasticity via PI3K/Akt signaling¹⁸. Therefore, SAE may be particularly suitable for experimental and clinical contexts in which modulation of post-stroke neural plasticity, motor recovery, and spasticity reduction are key therapeutic goals. However, the potential association between SAE, GDNF/PI3K/Akt signaling, and synaptic plasticity in relieving PSS remains poorly understood. The present study established a rat model of PSS to evaluate the effects of SAE on neurological deficits and muscle spasticity, and to characterize changes in GDNF expression, PI3K/Akt pathway activity, and synaptic plasticity-related proteins after intervention. This study may provide experimental evidence for determining whether SAE is an appropriate rehabilitative strategy for PSS and for clarifying its potential mechanism of action.

Protocol

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, Changchun, China). Animals were housed at the Experimental Animal Center of Changchun University of Chinese Medicine under controlled conditions of 22–25 °C, 50–60% relative humidity, and a 12-h light/dark cycle, with free access to food and water. After 1 week of acclimatization, rats were randomly assigned to a blank group (n = 12), a Sham group (n = 12), and a model-reserve group (n = 56). The blank group was included as a normal, untreated control to provide baseline values for behavioral performance, histopathological features, and molecular marker expression in healthy rats. The Sham group underwent the same anesthesia and surgical exposure as the MCAO group, except that arterial occlusion was not performed; therefore, this group served as a control for the potential effects of anesthesia and surgical manipulation. The inclusion of both Blank and Sham groups allowed the effects of ischemic injury to be distinguished from those related to the surgical procedure itself.

The model-reserve group underwent MCAO modeling. The first 3 days after surgery were defined as the model-induction period. During this period, 8 of 56 rats died, corresponding to a mortality rate of 14.29%, and 12 rats were excluded because modeling was unsuccessful. The remaining 36 rats with successful model establishment were randomly allocated, using a random-number table, to the MCAO, SAE, and Baclofen groups, with 12 rats in each group. Group interventions were as follows: (I) Blank group: routine feeding only, with no intervention. (II) Sham group: surgical exposure of the common carotid artery, internal and external carotid arteries, and vagus nerve, without ligation or filament insertion. (III) MCAO group: MCAO was established under intraperitoneal anesthesia with 3% sodium pentobarbital, with no subsequent treatment. (IV) SAE group: after successful MCAO modeling and validation, rats received scalp acupuncture combined with exercise. Isoflurane inhalation anesthesia at 1.5%–2.0% was used only during the brief needle-insertion phase. Treadmill training was initiated only after full recovery of consciousness and restoration of spontaneous walking ability. Acupuncture was performed at GV20 and bilateral EX-HN1. Needles were inserted to a depth of 1 mm and retained during the treadmill-training period. The standardized 30-min needle-retention/training period was defined as the treadmill-training period and consisted of 6 m/min for 10 min followed by 12 m/min for 20 min at 0° incline. The intervention was administered once daily for 7 consecutive days. (V) Baclofen group: after successful MCAO modeling and validation, rats received baclofen by intragastric gavage at 0.4 mg/kg once daily for 7 consecutive days.

Establishment of the MCAO model
The rat MCAO model was established using a modified intraluminal filament method¹⁹. After 12 h of fasting and water deprivation, rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital and placed in the supine position. After skin disinfection, a midline cervical incision was made to expose the right common carotid artery (CCA) and adjacent nerves and muscles. The external carotid artery (ECA) was identified and ligated, and the internal carotid artery (ICA) was isolated. Ligatures were placed at the proximal ICA and CCA, and the distal CCA was temporarily clamped. A small arteriotomy was then made in the CCA proximal to the clamp. A 0.26 mm nylon filament was inserted and advanced 18–20 mm through the ICA to occlude the origin of the middle cerebral artery. The ICA and CCA were subsequently ligated, and the incision was sutured. After surgery, rats were kept warm during recovery and received an intraperitoneal injection of penicillin (40,000 U) to prevent infection. In the Sham group, only vascular exposure was performed, without filament insertion; wound closure and postoperative care were identical to those in the MCAO group. Successful model establishment was defined as a Zea Longa score ≥ 2 at 3 days after surgery¹⁹ and a modified Ashworth Scale (MAS) muscle tone score ≥ Grade 1²⁰.

Intervntion methods
Scalp acupuncture combines with exercise intervention
Previous studies show that in MCAO rats, neurological deficits peak on day 3 and remain elevated within 10 days after surgery, while peripheral muscle resistance progressively increases from day 6 to day 9²¹. Therefore, treatment was initiated on postoperative day 3 and continued once daily for 7 consecutive days to minimize the impact of spontaneous recovery. Before modeling, rats assigned to the SAE group underwent 3 days of adaptive treadmill training at 5 m/min for 10 min/day²². This adaptive training was used to familiarize rats with treadmill running and to reduce the likelihood of struggling, falling, refusal to run, or stopping during the formal intervention period. During adaptive training and formal intervention, abnormal treadmill-related events were defined as persistent struggling, falling from the running lane, refusal to run for more than 1 min, or stopping for more than 1 min during treadmill training. These events were recorded in experimental logs when observed. Brief hesitation or transient stopping did not lead to exclusion if treadmill running could be resumed after gentle prompting. Data were excluded only if an animal was unable to complete the assigned daily treadmill session because of repeated falling, persistent refusal to run, or severe distress. After successful model validation on Day 3, the SAE intervention was administered once daily for 7 consecutive days.

For each treatment session, rats were anesthetized with 1.5%–2.0% isoflurane. The anesthesia was discontinued immediately after needle insertion. In experimental practice, rats typically regained full consciousness within approximately 1–2 min after isoflurane withdrawal. Treadmill training was not started while rats were anesthetized. Acupoints were located by experienced animal acupuncturists according to the Nomenclature and Localization of Common Acupoints in Experimental Animals issued by the China Association of Acupuncture-Moxibustion in 2020. GV20 was defined as the midpoint of the line connecting the anterior and posterior fontanelles on the cranial midline. Bilateral EX-HN1 points were located 2 mm lateral to GV20. Disposable needles measuring 0.25 mm × 13 mm were inserted vertically to a depth of 1 mm and retained throughout the standardized 30 min treadmill-training period. Needles were secured with breathable medical tape to prevent displacement, and skull penetration was excluded by palpation to ensure procedural consistency. Needle position and tape fixation were checked before treadmill training, during brief pauses if abnormal running behavior occurred, and immediately after completion of training. In addition, needle position was visually checked every 5 min during treadmill training, regardless of animal behavior. Needle displacement was defined as visible loosening, a change in needle angle, partial withdrawal, or complete dislodgement during treadmill running. If mild loosening occurred without loss of needle position, the tape was gently reinforced while the treadmill was paused briefly. If a needle was partially withdrawn or completely dislodged, the needle was not reinserted during that session; the rat completed the remaining treadmill training without additional needle manipulation, and the event was documented in the experimental log. Animals were not excluded solely because of a single needle-displacement event unless the event was accompanied by an inability to complete treadmill training or signs of severe distress.

The standardized 30 min needle-retention/training period began when the rat started treadmill running after full recovery from isoflurane anesthesia and ended immediately after completion of treadmill training, when the needles were removed. The brief recovery interval after isoflurane discontinuation was not included in the standardized 30 min training period. Because needles were inserted before recovery, the actual physical indwelling time was approximately 1–2 min longer than the 30-min treadmill-training period. This definition was applied consistently across all rats in the SAE group. A rat was considered sufficiently recovered to begin treadmill training when the following criteria were met: full recovery of consciousness, ability to stand independently, ability to walk steadily on a flat surface, and normal response to gentle external stimulation. These criteria were checked before placement on the treadmill.

During needle retention, rats performed treadmill exercise at 0° incline: 6 m/min for 10 min, followed by 12 m/min for 20 min. Electrical shock was not used at any point during the study. If a rat stopped running or refused to run for more than 1 min during formal training, gentle non-aversive auditory stimulation, such as lightly tapping the treadmill wall, was used to encourage movement. The use of such prompting, as well as any struggling, falling, stopping, refusal to run, or needle displacement, was documented in the experimental log when observed. These events were monitored descriptively and documented in the experimental log when observed. The exercise intensity was comprehensively adjusted based on previously reported protocols combining scalp acupuncture with treadmill training^16,23. The SAE intervention was administered once daily for 7 consecutive days (Figure 1A).

Baclofen intervention
After successful model validation, rats in the Baclofen group received baclofen solution by intragastric gavage at a dose of 0.4 mg/kg. The drug was administered once daily at 9:00 AM for 7 consecutive days.

Outcome meansure
General observation
General condition was monitored daily in all groups, including body weight, mental status, food and water intake, and spontaneous activity.

Neurological function and muscle tone assessment
Neurological deficits and limb spasticity were assessed on Days 1, 3, 5, and 7 after model validation using the Zea Longa score, modified neurological severity score (mNSS), and modified Ashworth Scale. All behavioral assessments at all time points were performed independently by two investigators who were blinded to group allocation throughout the observation period. Detailed scoring criteria are shown (Tables 1–3).

Determination of cerebral infarct volume
Cerebral infarct volume was assessed using 2,3,5-triphenyltetrazolium chloride (TTC) staining. After 7 days of intervention, three rats from each group were randomly selected and euthanized by intraperitoneal overdose of sodium pentobarbital, followed by rapid decapitation for whole-brain collection. The brains were first frozen at −20 °C and then cut into 2-mm-thick coronal sections. The sections were incubated in 2% TTC solution at 37 °C for 30 min in the dark, then fixed overnight in 4% paraformaldehyde at 4 °C. The following day, all sections were photographed with a digital camera. Normal brain tissue was stained dark red, whereas infarcted tissue appeared pale white. Infarct areas were quantified with ImageJ, and infarct volume was expressed as a percentage using the formula:

Infarct volume (%) = infarct area / total brain section area X 100%.   (1)

Histopathological examination of brain tissue
Hematoxylin–eosin (HE) staining
Hematoxylin and eosin staining was used to evaluate histopathological changes in the peri-infarct cortex. After 7 days of intervention, rats were euthanized by intraperitoneal overdose of sodium pentobarbital. Rapid transcardial perfusion was then performed with 0.01 M phosphate-buffered saline (PBS) to remove blood, followed by 250 mL of cold 4% paraformaldehyde (4 °C) for fixation. Whole brains were collected, and peri-infarct cortical tissue was isolated and fixed in 4% paraformaldehyde at 4 °C for 24 h. Tissues were paraffin-embedded and sectioned coronally at 5 µm. Sections were mounted on slides, baked at 60 °C for 30 min, and deparaffinized in xylene I–III (10 min each). HE staining was then performed, followed by dehydration and clearing. Finally, sections were coverslipped with neutral balsam, and images were acquired using a Nikon light microscope.

Nissl staining
Nissl staining was used to assess neuronal survival and Nissl body density in the peri-infarct region. After 7 days of intervention, rats were euthanized and transcardially perfused with 0.01 M PBS (37 °C), followed by 250 mL of 4% paraformaldehyde (4 °C). Whole brains were removed, and peri-infarct and contralateral cortical tissues were dissected and fixed overnight in 4% paraformaldehyde at 4 °C. After paraffin embedding, 5 µm sections were prepared. Sections were deparaffinized in xylene for 10 min, rehydrated through a series of graded ethanol solutions (95%, 90%, 80%, 70%, and 50%; 5 min for each concentration), and stained with Nissl working solution for 30 min at room temperature. After a brief rinse with distilled water, sections were dehydrated through graded ethanol, cleared in xylene for 2 min, and finally mounted with coverslips. Images were captured and analyzed under a light microscope.

Western blot analysis of protein exprssion
Western blotting was performed to assess proteins involved in PI3K/Akt signaling and synaptic plasticity. After 7 days of intervention, rats were euthanized with an overdose of sodium pentobarbital, and peri-infarct motor cortex tissue was collected. Tissues were fully homogenized in Radioimmunoprecipitation assay (RIPA) lysis buffer on ice and centrifuged at 1610 × g. at 4 °C for 30 min to extract total protein. Protein concentration was quantified using a commercial bicinchoninic acid (BCA) protein assay kit. Equal amounts of protein (50 µg per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrically transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% bovine serum albumin (BSA) solution for 1 h at room temperature to eliminate non-specific binding, then incubated with primary antibodies overnight at 4 °C. After three rounds of washing with Tris-buffered saline with Tween-20 (TBST) buffer, membranes were incubated with secondary antibody (dilution ratio 1:1000) for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence (ECL), and band intensity was quantified with ImageJ. Relative protein expression was calculated as the ratio of the target protein to the internal control. Primary antibodies were as follows: GAPDH (1:1000), PI3K (1:1000), phospho-PI3K (1:1000), Akt (1:1000), phospho-Akt (1:1000), GAP-43 (1:1000), SYN (1:1000), PSD-95 (1:1000), and GDNF (1:1000).

Synaptic ultrastructure observation
Transmission electron microscopy (TEM) was used to assess synapse number and structural integrity. After 7 days of intervention, rats were anesthetized and transcardially perfused with mixed fixative containing 4% paraformaldehyde and 2.5% glutaraldehyde. Peri-infarct cortical tissue blocks (1 mm × 1 mm × 1 mm) were collected and fixed in 2.5% glutaraldehyde for 2 h, followed by post-fixation with 1% osmium tetroxide for 1 h. Samples were then processed with graded ethanol dehydration, epoxy resin embedding, ultrathin sectioning (50 nm thickness), and double staining with uranyl acetate and lead citrate. Synaptic ultrastructure was examined using a transmission electron microscope. Images were acquired at 8,000× for structural assessment, and synapses were counted at 5,000×. Three random neuropil fields per group were analyzed to calculate mean synapse number, and data were compared using one-way ANOVA.

Immunofluorescence double-labeling assay
Immunofluorescence double-labeling staining was performed to assess the expression and distribution of SYN and p-Akt in the peri-infarct cortex. After 7 days of intervention, rats were euthanized by intraperitoneal overdose of sodium pentobarbital, and peri-infarct cortical tissue was rapidly dissected. Samples were fixed in 4% paraformaldehyde for 24 h, embedded in optimal cutting temperature compound (OCT), and cut into 10 µm-thick sections. Sections were air-dried at room temperature for 1 h and washed three times with TBST buffer to remove residual OCT compound. Sections were permeabilized with 0.3% Triton X-100 for 20 min at room temperature, rinsed with PBS, and blocked with 5% BSA solution for 1 h at room temperature. Under dark conditions, sections were incubated overnight at 4 °C with rabbit anti-SYN (dilution 1:200) and rabbit anti-p-Akt (dilution 1:200) primary antibodies. The next day, sections were washed three times with PBS and incubated in the dark with secondary antibody (dilution 1:400) for 50 min at room temperature. After six further washes with TBST buffer, cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) working solution for 5 min at room temperature. Images were acquired under identical exposure and background settings using a fluorescence microscope. Representative images were used to evaluate SYN and p-Akt expression patterns in the motor cortex across groups.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 10.1.2 and IBM SPSS Statistics 25. Data are presented as mean ± standard deviation (SD). Behavioral data collected at multiple time points, including the Zea Longa score, modified Neurological Severity Score, and modified Ashworth Scale, were analyzed using a two-way repeated-measures ANOVA with group as the between-subjects factor and time as the within-subjects factor. When significant main effects or interactions were detected, Sidak’s multiple-comparisons test was used for post hoc analysis. Other data were analyzed using one-way ANOVA followed by Sidak’s multiple-comparisons test for post hoc comparisons. A two-tailed p. < 0.05 was considered statistically significant. In bar graphs, statistical significance is indicated by asterisks as defined in the corresponding figure legends.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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