Electroacupuncture Alleviates Brain Injury Through Vagus Nerve Activation and Gut Microbiota in a Rat Model of Ischemic Stroke

Abstract

Background

Emerging evidence implicates gut microbiota dysbiosis in exacerbating stroke pathogenesis via the gut‐brain axis, suggesting novel therapeutic targets. While electroacupuncture (EA) demonstrates anti‐inflammatory effects through vagus nerve activation, its neuroprotective mechanisms via vagus nerve–microbiota crosstalk remain unexplored.

Methods

Rats with middle cerebral artery occlusion received daily ST36 (Acupoint Zusanli) EA for 1 to 7 days postischemia. Subdiaphragmatic vagotomy and fecal microbiota transplant were implemented to validate pathway specificity. Multimodal assessments included longitudinal neurological scoring, infarct volume, systemic/neuroinflammatory profiling (enzyme‐linked immunosorbent assay, immunohistochemistry), intestinal fucosylation dynamics (quantitative polymerase chain reaction, lectin staining), and 16S ribosomal RNA sequencing of gut microbiota.

Results

EA significantly improved neurological outcomes and reduced infarct volumes at 3 to 7 days after middle cerebral artery occlusion (versus controls), which was abolished by vagotomy. Mechanistically, EA restored gut barrier integrity through vagus‐dependent upregulation of fucosyltransferase 2 (Fut2)‐driven epithelial α1,2‐fucosylation, enhancing mucin 2+ goblet cell density and tight junction protein expression (ZO‐1/occludin/claudin‐1). Concurrent microbiota shifts included Lactobacillales/Bacteroidales enrichment (linear discriminant analysis >4.0) and pathobiont suppression, which was reversed by vagotomy. Crucially, fecal microbiota transplant from EA‐treated donors replicated neuroprotection in germ‐free recipients, achieving 33% infarct reduction and 30% survival improvement (P=0.012), whereas fecal microbiota transplant from vagotomized donors showed no therapeutic benefits.

Conclusions

EA at ST36 produced neuroprotection through activating vagal efferent pathways to orchestrate intestinal mucosal repair via Fut2‐mediated fucosylation, which reshape microbial ecosystems and attenuate neuroinflammation. These findings establish a previously unrecognized vagus nerve‐gut‐brain axis mechanism for stroke recovery, positioning microbiota‐directed neuromodulation by EA as a translatable therapeutic strategy.

Nonstandard Abbreviations and Acronyms

EA

electroacupuncture

Fut2

fucosyltransferase 2

IEC

intestinal epithelial cell

I/R

ischemia–reperfusion

LEfSe

linear discriminant analysis effect size

MCAO

middle cerebral artery occlusion

Muc2

mucin 2

OTU

operational taxonomic unit

SDV

subdiaphragmatic vagotomy

ST36

Acupoint Zusanli

Research Perspective.

What Is New?

• Our study reveals that electroacupuncture at the ST36 (Acupoint Zusanli) acupoint alleviates ischemic brain injury by activating the vagus nerve to restore gut barrier integrity and remodel gut microbiota.

What Question Should Be Addressed Next?

• Future studies should investigate whether combining electroacupuncture with microbiota‐targeted therapies (eg, probiotics or prebiotics) synergistically enhances neuroprotection in stroke.

Ischemic stroke is globally recognized as a leading cause of mortality and disability, with accelerating disease burden. ¹ , ² Current standard reperfusion therapies for acute ischemic stroke primarily include intravenous thrombolysis and endovascular mechanical thrombectomy. ³ , ⁴ However, these interventions face 2 critical clinical limitations: limited optimal population and unsatisfied treatment effect. ⁵ , ⁶ Regarding neuroprotective strategies, despite encouraging preclinical findings, phase III clinical trials have encountered repeated setbacks, with few pharmacological agents demonstrating definitive therapeutic efficacy. ⁷ This underscores an urgent need for multidimensional intervention strategies targeting cascade injury pathways.

Recent years have witnessed a paradigm shift in stroke research with the emergence of the “gut‐brain axis” concept. ⁸ , ⁹ , ¹⁰ Breakthrough studies revealed that stroke rapidly induces gut dysbiosis, which reciprocally exacerbates cerebral infarction severity. ¹¹ Mechanistic investigations demonstrated that gut microbiota disturbances inhibit effector T‐cell trafficking from intestines to leptomeninges, confirming that intestinal commensal microbiota significantly influence ischemic stroke pathogenesis through gut γδ T‐cell regulation. ¹² Additional studies identified gut microbial modulation of stroke severity via the trimethylamine N‐oxide pathway. ¹³ These pivotal findings not only validate bidirectional regulation between gut microbiota and stroke but also provide novel therapeutic targets.

As a pivotal component of traditional Chinese medicine, acupuncture has gained clinical validation from the National Institutes of Health and World Health Organization. ¹⁴ Electroacupuncture (EA), an advanced form of modern acupuncture therapy, has accumulated substantial evidence supporting its efficacy in postoperative and stroke rehabilitation. ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ Recent mechanistic studies elucidate that EA exerts systemic anti‐inflammatory effects through vagus‐adrenal axis activation. ¹⁹ , ²⁰ , ²¹ Notably, vagus nerve stimulation modulates intestinal VIPergic neuronal function, influencing gut mucosal fucosylation processes to maintain microbial community homeostasis. ²² Building on these groundbreaking discoveries, we propose an innovative scientific hypothesis: EA may alleviate brain injury through restoring poststroke gut microbiota homeostasis via vagal nerve pathway activation. This hypothesis not only provides novel perspectives for understanding EA‐mediated neuroprotection in stroke but also establishes theoretical foundations for developing innovative stroke therapeutics targeting gut‐brain axis modulation.

Methods

Data Availability

Data are available on reasonable request to the corresponding author.

Animals

Male Sprague–Dawley rats (280–330 g, 8–10 weeks old; supplied by a Beijing biotechnology company [license: SCXK (Jing) 2019‐0008]) were housed under a 12‐hour light/dark cycle with free access to food and water. All procedures followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care Committee of the General Hospital of Northern Theater Command (ethics number: 2025‐17).

Animal Models

Cerebral ischemia–reperfusion (I/R) injury was induced using established middle cerebral artery occlusion (MCAO)/reperfusion protocols. ²³ Anesthesia was continuously maintained with 1.5% to 2% isoflurane during the MCAO period via nose cone delivery, with physiological parameters including respiration rate and pedal reflex monitored at 15‐minute intervals to ensure stable anesthesia depth. Following a midline cervical incision, the external carotid artery and internal carotid artery were exposed under microscopy. A 0.26‐mm nylon monofilament was advanced from the external carotid artery into the middle cerebral artery origin. The right middle cerebral artery was occluded for 2 hours before filament removal to allow reperfusion. Sham‐operated controls underwent identical procedures without occlusion.

Experimental Protocols

Our study included 3 parts, and 385 male Sprague–Dawley rats were randomly allocated into different groups. Of the 385 animals undergoing experimental protocols, 82 were excluded from statistical analysis: 29 due to mortality during surgical procedures and 53 owing to unsuccessful surgical procedures. Surgical failure was rigorously defined by the absence of significant neurological deficits at 24 hours after reperfusion, assessed through a validated multiscale approach requiring failure in at least 2 of the following criteria: modified Garcia score <8 (maximum 18), modified Petullo score <2 (maximum 5), or Bederson score <1 (maximum 3).

The experimental sequence proceeded sequentially with part 1 (n=160) completed first, followed by part 2 (n=100), and finally part 3 (n=125). Within each part, rats were stratified by body weight (280–330 g) and randomized using computer‐generated sequences (GraphPad), while post‐MCAO single housing prevented coprophagy‐induced microbiota transfer and daily cage rotation within environmental cabinets minimized location‐based bias.

From the initial cohort of 385 male Sprague–Dawley rats, allocation was stratified as follows: part 1 included sham (n=32), MCAO (n=48), MCAO + EA (n=64), and MCAO + non‐EA (n=16); parts 2 and 3 maintained n=25 per group. Exclusions (total n=82: 29 in part 1, 23 in part 2, 30 in part 3) were proportionally distributed among all surgical groups and resulted from either intraoperative mortality (29 cases) or failure to meet neurological deficit criteria (53 cases), with no evidence of group‐specific exclusion bias. Detailed animal numbers at each time point are shown in Table S1. All outcome measures were assessed under rigorous blinding protocols: as to behavioral testing, all assessors received unified training and were permanently assigned to single‐scale evaluation groups, with a rigorous operator‐data analyst double‐blind design maintained throughout the process; histology/2,3,5‐triphenyltetrazolium chloride used coded slides imaged by one technician and quantified by another; enzyme‐linked immunosorbent assay/quantitative polymerase chain reaction (PCR) masked sample identities during processing; and microbiome analysis blinded bioinformaticians to group assignments.

Part 1

To evaluate the therapeutic efficacy and acupoint specificity of EA, rats received daily ST36 (Acupoint Zusanli) EA postreperfusion for 1, 3, or 7 days. An additional nonacupoint EA control group (adjacent to ST36) was included for 7‐day intervention. Rats (n=160) were randomized into 3 experimental cohorts: sham, MCAO 1d, MCAO + EA 1d; sham, MCAO 3d, MCAO + EA 3d; and sham, MCAO 7d, MCAO + EA 7d, MCAO + non‐EA 7d (n=16 per subgroup).

Part 2

To investigate whether the association of EA‐mediated neuroprotection with gut is mediated via vagus‐dependent intestinal fucosylation, we performed subdiaphragmatic vagotomy (SDV) 7 days before MCAO. SDV involved bilateral transection of vagal trunks under aseptic conditions (for the detailed surgical procedure, see Data S1), with sham controls receiving identical exposure without neural transection. Based on prior screening that identified 7 days as the optimal EA intervention duration, we assessed therapeutic outcomes at this end point. Rats (n=100) were randomized into 4 groups: sham; MCAO; MCAO + EA; and MCAO + EA + SDV (n=25 per subgroup).

Part 3

To further validate vagus‐dependent microbiota mechanisms in EA‐mediated neuroprotection, we conducted fecal microbiota transplant (FMT) in cerebral I/R models. Male Sprague–Dawley rats (8–10 weeks) received a 7‐day antibiotic cocktail (ampicillin 1 g/L, metronidazole 1 g/L, neomycin 1 g/L, vancomycin 0.5 g/L; ABX) via oral gavage to ablate gut microbiota. Postdepletion, these rats underwent MCAO as FMT recipients. Fresh fecal microbiota from MCAO + EA or MCAO + EA + SDV donors was transplanted via oral gavage on poststroke days 1 to 3. Rats (n=125) were allocated into FMT donor groups (MCAO + EA and MCAO + EA + SDV) and FMT recipient groups (ABX + MCAO + FMT(EA) and ABX + MCAO + FMT(SDV)) (n=25 per subgroup).

EA Treatment

EA intervention was initiated immediately following successful establishment of the MCAO/reperfusion model. Unipolar stainless‐steel acupuncture needles (0.25 × 25 mm) were vertically inserted into the unilateral acupoint region at a depth of 7.0 ± 0.5 mm with a 2.0‐mm interneedle spacing. Stimulation parameters were configured as follows: baseline frequency 10 Hz, intensity 0.5 mA, administered once daily for 20 minutes. Notably, this focal electrical stimulation paradigm—featuring a 2.0‐mm separation between anode and cathode needles—differs fundamentally from conventional diffuse stimulation modalities (eg, current entry via left limb and exit through contralateral limb), ensuring precise acupoint targeting and localized current confinement. EA was administered to awake rats restrained in specialized holders without anesthesia or sedation to eliminate pharmacological confounding of behavioral outcomes, with sham groups receiving identical 20‐minute restraint handling daily without needle insertion or electrical stimulation.

Behavioral Testing

This study implemented a multidimensional behavioral assessment system. Before testing, experimental animals completed 1‐hour environmental acclimatization in standardized observation chambers. Neurological functions were then evaluated using 3 validated scales: modified Garcia score, ²⁴ modified Petullo score, ²⁵ and Bederson score. ²⁶

Fecal Microbiota Transplantation

FMT was conducted in specific pathogen‐free rats maintained on sterile purified water. Before FMT, animals received a quadruple antibiotic cocktail (ampicillin 1 g/L, metronidazole 1 g/L, neomycin 1 g/L, and vancomycin 0.5 g/L) via daily oral gavage for 7 consecutive days to deplete endogenous gut microbiota. FMT was administered via oral gavage using 0.4 mL supernatant per 100 g body weight at a standardized biota concentration of 100 mg feces/mL PBS, translating to 1.2 mL containing 120 mg fecal equivalents for a 300‐g rat. All antibiotics were procured from Wanlei Biotech. Following a 1‐day recovery period postantibiotic treatment, I/R models were established as FMT recipients. Fresh fecal samples collected from MCAO with EA (MCAO + EA) and MCAO + EA with vagotomy (MCAO + EA + SDV) groups were homogenized in PBS, filtered through 100‐μm cell strainers, and centrifuged at 7000g for 10 minutes to obtain supernatant‐derived microbial suspensions. Recipient rats underwent daily oral administration of donor microbiota for 3 consecutive days.

Enzyme‐Linked Immunosorbent Assay

Serum and tissue lysate concentrations of lipopolysaccharide (WLE03), interleukin (IL)‐1β (WLE03), IL‐6 (WLE04), tumor necrosis factor α (WLE03), IL‐10 (EK310), and IL‐17 (EK317) were quantified using commercially available enzyme‐linked immunosorbent assay kits (Wanlei Biotech) following manufacturer protocols. Serum samples were obtained from subclavian venous blood of rats, centrifuged at 3000 rpm for 15 minutes, with the supernatant serum collected. Small intestinal tissues were processed by rinsing intestinal luminal contents with precooled PBS, scraping mucosal layers, and homogenizing in radioimmunoprecipitation assay lysis buffer containing 1% sodium deoxycholate (1:10 w/v) on ice, followed by centrifugation at 10000g for 15 minutes at 4°C. Cerebral tissues were pulverized in liquid nitrogen, lysed in CHAPS‐supplemented radioimmunoprecipitation assay buffer (1:8 w/v) and centrifuged at 12000g for 15 minutes at 4°C, with supernatants filtered through 0.22‐μm membranes. Absorbance measurements were performed at 450 nm using a microplate reader (model specified). All kits were procured from Wanlei Biotech.

Quantitative Reverse‐Transcription PCR

The total rna of tissue and cells was extracted using TRIpure reagent (RP1001, BioTeke) according to the manufacturer’s instructions. RNA Concentration and quality were measured by Nanodrop One Spectrophotometer (Thermo Fisher Scientific). RNA was reverse‐transcribed to complementary DNA using All‐in‐One First‐Strand SuperMix (MD80101, Magen Biotech), and quantitative reverse‐transcription PCR was performed on an Exicycler 96 (version 4) Real‐Time Quantitative Thermal Block (Exicycler 96, Bioneer) using 2 × Taq PCR MasterMix (PC1150, Solarbio). The expression level was normalized to the housekeeping Actb gene, and data were analyzed using the 2^−ΔΔCT method. The primers used in quantitative reverse‐transcription PCR are shown in Table S2.

Immunofluorescence Staining

Small intestinal and cerebral tissues were cryopreserved for immunofluorescence localization studies. Intestinal sections were incubated with rabbit anti‐Muc2 antibody (1:100, ab272692, Abcam) and UEA‐1 lectin (1:100, L32476, Thermo Fisher Scientific), while brain sections were treated with rabbit anti‐Fos antibody (1:100, NBP2‐50057SS, Novus Biologicals), all at 4°C overnight. Following primary antibody incubation, sections were exposed to goat anti‐rabbit secondary antibody (1:200, A27039, Invitrogen) at room temperature (22–25°C) for 90 minutes. Nuclear counterstaining was uniformly performed using DAPI (1 ng/μL, D106471‐5mg, Aladdin) for 5 minutes at room temperature. Fluorescent signals were visualized with a confocal fluorescence microscope (Olympus), with intestinal samples targeting mucin 2 (Muc2)/UEA‐1 colocalization and cerebral samples focusing on Fos/ChAT interactions.

Hematoxylin and Eosin Staining

Brain and small intestinal tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 4 μm. Following hematoxylin and eosin staining, histopathological alterations were evaluated through brightfield microscopy, with cerebral sections focusing on ischemic penumbra and intestinal sections targeting mucosal architecture.

16S Ribosomal RNA Microbiome Sequencing and Microbial Community Analysis

Genomic DNA was extracted from fecal samples using the TIANamp Soil DNA Kit (DP336, Tiangen Biotech) following the manufacturer’s protocol. DNA purity and concentration were verified by spectrophotometry (OD260/280=1.7–1.9). Quantitative PCR amplification was performed using universal 16S ribosomal RNA (rRNA) primers targeting the V4 region (341F: 5′‐CCTAYGGGRBGCASCAG‐3′; 806R: 5′‐GGACTACNNGGGTATCTAAT‐3′). The PCR products with proper size were selected by 2% agarose gel electrophoresis. The same amount of PCR product from each sample was pooled, end‐repaired, A‐tailed, and further ligated with Illumina adapters. Libraries were sequenced on a paired‐end Illumina platform to generate 250 bp paired‐end raw reads. Subsequently, library quality was assessed and quantified by quantitative PCR. Quantified libraries were pooled and sequenced on Illumina platforms, according to the effective library concentration and data amount required (Illumina Novaseq6000, Illumina). Paired‐end reads were assigned to samples based on their unique barcode and truncated by cutting off the barcode and primer sequence. Paired‐end reads were merged using FLASH (http://ccb.jhu.edu/software/FLASH/), ²⁷ and the spliced sequences were termed raw tags. Quality filtering on the raw tags was performed using fastp software to obtain high‐quality Clean Tags. ²⁸ The tags were compared with the reference database using the UCHIME algorithm to detect chimera sequences, and then the chimera sequences were removed. ²⁹ The Effective Tags were then finally obtained.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 9.0 and R software (version 4.3.0). For data sets with small sample sizes (n<5 per group), robust nonparametric permutation tests were conducted using the ‘coin’ package in R to ensure validity without reliance on large‐sample approximations or distributional assumptions. For other data, group comparisons were performed using 1‐way ANOVA or Kruskal‐Wallis test as appropriate according to the scientific question. All post hoc tests incorporated appropriate corrections for multiple comparisons (Tukey test for ANOVA, Dunn test for Kruskal‐Wallis, single‐step min‐p adjustment for permutation tests). Survival data were analyzed using Cox proportional hazards regression with Firth penalized likelihood correction to address small sample size limitations. A threshold of P<0.05 was defined as statistical significance.

Results

EA Confers Neuroprotection in a Time‐ and Acupoint‐Dependent Manner

To investigate the therapeutic effects of EA, rats in each group received once‐daily ST36 EA stimulation after reperfusion for 1, 3, or 7 days. Neurological function was objectively evaluated using a multidimensional behavioral assessment system comprising the modified Garcia score, ²⁴ modified Petullo score, ²⁵ and Bederson score, ²⁶ with cerebral infarct volume quantified via 2,3,5‐triphenyltetrazolium chloride staining (Figure 1A). At 1 day post‐MCAO, no significant differences were observed in infarct volumes (Figure 1B and 1C) and neurological scores (Figure 1D through 1F) between the MCAO and MCAO + EA groups. However, the MCAO + EA group exhibited marked infarct volumes at 3 and 7 days post‐MCAO (Figure 1B and 1C), accompanied by significantly reduced neurological improvement (Figure 1D through 1F). To confirm the specificity of EA, nonacupoint regions adjacent to ST36—specifically the gastrocnemius muscles (hindlegs) and semitendinosus muscles (thigh regions) that lack traditional acupoint designation and are conventionally employed as nonacupoint controls—were subjected to electrical stimulation. ¹⁹ , ³⁰ Critically, this intervention failed to reduce infarct volumes (Figure 1B and 1C) or improve neurological scores (Figure 1D through 1F) in the MCAO + non‐EA group compared with MCAO controls at 7 days. Taken together, these results indicate that EA confers time‐dependent neuroprotection against cerebral ischemia–reperfusion injury, with therapeutic efficacy strictly contingent on ST36 acupoint stimulation.

Figure 1. EA reduced infarct volume and improved neurological function in MCAO rats.

A, Study flow chart. B and C TTC staining and analysis of infarct volume at 1, 3, and 7 days after MCAO. D through F, Neurological deficit scores based on the modified Garcia score, modified Petullo score, and Bederson score, at 1, 3, and 7 days after MCAO. (n=7 per group.) EA indicates electroacupuncture; MCAO, middle cerebral artery occlusion; Non, nonacupoint electroacupuncture; and TTC, 2,3,5‐triphenyltetrazolium chloride.

Vagotomy Abolishes EA‐Induced Neuroprotection

The clinical efficacy of EA in stroke treatment has been supported by evidence‐based medicine. ³¹ , ³² While the gut‐brain axis notion has long been proposed, ³³ , ³⁴ , ³⁵ recently, studies further confirm that EA specifically activates vagal pathways. ²¹ Given the key role of vagal nerve in gut‐brain axis, we hypothesize that EA produces neuroprotection through the vagus nerve–dependent bidirectional gut‐brain axis. Based on prior screening that identified 7 days as the optimal EA intervention duration, we assessed therapeutic outcomes at this time point (Figure 2A). As expected, results showed that EA‐induced neuroprotection was nearly abolished in the MCAO + EA + SDV group, evidenced by no effect on infarct volumes (Figure 2B and 2C) and neurological scores (Figure 2D through 2F) compared with MCAO controls (F _3,24=77.35, P=0.45). Intriguingly, however, EA induced Fos expression (a neuronal activation marker) in ChAT‐positive vagal efferent neurons located in the dorsal motor nucleus of the vagus in both the MCAO + EA and MCAO + EA + SDV groups (as determined by permutation test; Figure 2G and 2H). Furthermore, Nissl staining revealed a marked reduction in Nissl bodies in the MCAO group, accompanied by a morphological shift from well‐defined round structures to irregular fragments. Importantly, EA treatment significantly restored both the number and morphology of Nissl bodies, whereas this restorative effect was abolished by SDV (Figure 2I). Similarly, hematoxylin and eosin staining showed neuronal loss, disorganized architecture, and vacuolar changes in the cerebral infarction area of MCAO rats. These pathological alterations were robustly attenuated by EA, eventually reaching levels comparable to sham‐operated controls. Notably, vagotomy completely abolished EA’s ameliorative effects (Figure 2I).

Figure 2. SDV abrogates EA‐induced neuroprotection.

A, Study flow chart. B and C, TTC staining and analysis of infarct volume at 7 days after MCAO. (n=7 per group.) D through F, Neurological deficit scores based on the modified Garcia score, modified Petullo score, and Bederson score at 7 days after MCAO. (n=7 per group.) G and H, Images and quantification of Fos expression (red) induced by EA (0.5 mA) at the ST36 acupoint in ChAT+ (green) DMV neurons. Scale bars, 100 μm. (n=3 per group.) Data in panel H were analyzed by permutation test. (I) Nissl and HE staining at 7 days after MCAO. (n=3 per group). Scale bars, 400 μm. J, Changes of body weight at 3 and 7 days after MCAO. (n=7 per group). K, Percent survival at 1 to 7 days after MCAO (n=15 per group.) Survival data were analyzed using Cox proportional hazards regression with Firth penalized likelihood correction. Data in panel H are presented as median and interquartile range. 16sRNA indicates 16S ribosomal RNA; ChAT, choline acetyltransferase; DMV, dorsal motor nucleus of the vagus; EA, electroacupuncture; ELISA, enzyme‐linked immunosorbent assay; HE, hematoxylin and eosin; IHC, immunohistochemistry; MCAO, middle cerebral artery occlusion; SDV, subdiaphragmatic vagotomy; ST36, Acupoint Zusanli; and TTC, 2,3,5‐triphenyltetrazolium chloride.

To explore systemic impacts, longitudinal monitoring indicated that all groups exhibited ≈17% body weight loss by post‐MCAO day 3. By day 7, the MCAO + EA group regained 97.54% of baseline weight (versus 89.62% in the MCAO and 88.22% in the MCAO + EA + SDV groups; F _3,24=46.75), with no significant difference between the latter 2 groups (P=0.29; Figure 2J). In parallel, survival analysis revealed that the survival rate at day 7 was significantly higher in the MCAO + EA group (80%) compared with the MCAO group (50%), representing an absolute improvement of ≈30 percentage points (hazard ratio [HR], 0.32 [95% CI, 0.11–0.89], P=0.029) (Figure 2K). Both per‐protocol and intention‐to‐treat analyses were conducted for neurological outcomes. In the intention‐to‐treat analysis, deceased animals were assigned the worst possible scores. The results consistently demonstrated significant improvement in neurological function and survival rates in the MCAO + EA group compared with MCAO controls, effects that were abolished by SDV.

Given that IL‐1β, IL‐6, tumor necrosis factor α, IL‐17, and IL‐10 represent the core inflammatory axis in stroke‐induced gut barrier disruption, ¹² we investigated the effect of EA on them. The results showed that EA significantly reduced gut‐derived endotoxin (lipopolysaccharide) and proinflammatory cytokines (IL‐17, IL‐6, IL‐1β, tumor necrosis factor α) while elevating anti‐inflammatory IL‐10 levels in peripheral blood, intestinal tissue, and ischemic penumbra (1‐way ANOVA with post hoc Tukey test), whereas these effects were attenuated or abolished by SDV (Figure 3A through 3C). This implies that EA alleviates systemic inflammation and neuroinflammation in stroke rats through vagal‐dependent gut‐brain axis modulation. The above data demonstrate that the cerebroprotective effects were fully reversed by SDV (Figure 2B through 2F, 2I through 2K, 3A through 3C). Synthesizing these results, this suggests that EA‐mediated vagal activation likely exerts neuroprotection primarily through peripheral gut‐brain axis interactions rather than central vagal circuit modulation alone.

Figure 3. EA mitigates systemic and neuroinflammation while reducing gut‐derived LPS in stroke rats.

A through C, EA attenuates gut‐derived LPS and proinflammatory cytokines (IL‐17/IL‐6/IL‐1β/TNF‐α) while elevating IL‐10 in peripheral (A), cerebral compartments (B), and intestinal (C) tissue. (n=7 per group.) EA indicates electroacupuncture; IL, interleukin; LPS, lipopolysaccharide; MCAO, middle cerebral artery occlusion; SDV, subdiaphragmatic vagotomy; and TNF‐α, tumor necrosis factor α.

EA Confers a Protective Phenotype in Intestinal Epithelial Cell Fucosylation and Enhances Gut Integrity

An evolving concept posits that gut glycosylation is a key force in maintaining the homeostatic relationship between the intestinal epithelium and microbiota, particularly intestinal epithelial fucosylation. ³⁶ , ³⁷ The α1,2‐fucosylation of intestinal epithelial cells (IECs) is mechanistically driven by elevated fucosyltransferase 2 (Fut2) expression. ³⁸ , ³⁹ Emerging evidence highlights that central neural circuits regulate IEC α1,2‐fucosylation via vagal afferent signaling to preserve intestinal epithelial integrity. ²² Importantly, we have previously demonstrated that EA elicits neuroprotective effects via vagal nerve activation. However, how vagal activation by EA modulates intestinal regulation and impacts gut function remains unclear. Building on this finding, we hypothesize that EA may influence gut glycosylation through vagal pathways to restore intestinal homeostasis poststroke. To address this hypothesis, we first performed hematoxylin and eosin staining to evaluate structural damage to the intestinal barrier following cerebral ischemia (MCAO). The results demonstrated that compared with the sham group, MCAO rats exhibited severe pathological alterations in the small intestinal epithelium, including crypt architectural disruption, epithelial necrosis, and inflammatory infiltration in the lamina propria (versus sham), indicating significant impairment of the intestinal mucosal barrier postischemia. The MCAO + EA group effectively attenuated these injuries, characterized by crypt regeneration, reduced inflammation, and restored mucosal thickness (versus MCAO). However, SDV entirely abolished protective effects of EA, with the MCAO + EA + SDV group exhibiting pathological alterations comparable to the MCAO group (Figure 4A). Subsequently, immunohistochemical analysis further corroborated these findings: Muc2 + goblet cell density was significantly reduced in MCAO versus sham controls (H₃=9.46), whereas EA treatment reversed this depletion and restored mucosal integrity (P<0.0001 versus MCAO). Notably, this restoration was completely reversed by SDV (P=0.58, versus MCAO; Figure 4B). We next assessed epithelial fucosylation by immunofluorescence, which showed diminished UEA‐1 (α1,2‐fucose lectin) and Muc2 colocalization in MCAO rats (H₃=10.385, P<0.0001 versus sham), reflecting impaired fucosylation. EA enhanced colocalization signals (P=0.0049, versus MCAO), whereas SDV reversed this restoration (P=0.35, versus MCAO; Figure 4C and 4D). Critically, Fut2 messenger RNA expression mirrored this pattern: MCAO group showed a significant reduction in Fut2 levels compared with sham controls (H₃=9.359, P<0.0001), which was restored by EA treatment (P=0.019, versus MCAO), while EA effect was fully reversed by SDV (P=0.79, versus MCAO; Figure 4E), suggesting direct linking of vagal signaling to glycosylation regulation. To further elucidate mechanical barrier repair mechanisms, RT‐quantitative PCR analysis demonstrated that MCAO rats exhibited significant downregulation of tight junction protein genes (ZO‐1, occludin, claudin‐1) at the messenger RNA level compared with the sham group (Kruskal‐Wallis test with Dunn post hoc correction). EA intervention upregulated their expression (versus MCAO), while SDV abolished this regulatory effect (versus MCAO; Figure 4F). Collectively, histological, immunohistochemical, and molecular evidence demonstrates that EA restores intestinal barrier function and reinstates epithelial homeostasis by augmenting IEC glycosylation through vagus nerve–dependent mechanisms.

Figure 4. EA restores gut barrier integrity via vagus nerve‐mediated Fut2 fucosylation and mucosal defense.

A, SI histopathology: HE staining (top; scale bar=400 μm) and Muc2 IHC (bottom; scale bar=200 μm); red arrow: crypt disruption/yellow arrow: epithelial necrosis/blue arrow: lamina propria inflammation. B, Quantitative analysis of Muc2+ goblet cells. C and D, Dual IF imaging of Muc2 (red) and UEA‐1 (green) in SI with co‐localization quantification. Scale bars, 100 μm. E, Real‐time PCR analysis of Fut2 mRNA in the SI. F, Real‐time PCR analysis of Cldn2/Ocln/ZO‐1 mRNA in SI. Data in panels B, D, E, and F are presented as median and interquartile range. (n=3 per group). Cldn2 indicates claudin‐2; EA, electroacupuncture; Fut2, fucosyltransferase 2; H&E, hematoxylin and eosin; IHC, immunohistochemistry; IF, immunofluorescence; MCAO, middle cerebral artery occlusion; mRNA, messenger RNA; Muc2, mucin 2; Ocln, occludin; PCR, polymerase chain reaction; SDV, subdiaphragmatic vagotomy; SI, small intestine; UEA‐1, Ulex europaeus agglutinin 1; and ZO‐1, zonula occludens‐1.

EA Modulated Gut Microbiota Profiles

Fucosylation serves as a protective glycosylation mechanism for maintaining host‐microbial symbiosis. α1,2‐fucosylated carbohydrates on IECs and luminal contents can act as substrates for adhesion receptors of symbiotic beneficial bacteria. ³⁷ , ⁴⁰ To investigate whether EA‐enhanced fucosylation modulates gut microbiota structure, we performed 16S rRNA sequencing on fecal samples from the MCAO, MCAO + EA, and MCAO + EA + SDV groups. The species accumulation curve reached asymptotes among all groups, indicating sufficient sequencing depth and adequate sample representation (Figure 5A). Further analysis of operational taxonomic units (OTUs) revealed distinct microbial partitioning among groups. Analysis of shared and unique OTUs using Venn diagrams demonstrated a notable increase in unique OTUs specific to the MCAO + EA group compared with both MCAO and MCAO + EA + SDV groups (Figure 5B), suggesting that EA selectively enriched microbial taxa. α‐Diversity indices (abundance‐based coverage estimator, Chao1 richness estimator, and observed species) corroborated this observation, showing significantly higher bacterial richness in the MCAO + EA group versus MCAO (1‐way ANOVA with post hoc Tukey test). Vagotomy abolished this effect, as α‐diversity in the MCAO + EA + SDV group reverted to levels comparable to the MCAO group (Figure 5C). Beta diversity analysis via principal coordinates analysis and component analysis demonstrated that the MCAO and MCAO + EA + SDV groups shared highly similar microbial compositions, whereas both groups diverged significantly from the MCAO + EA group (Figure 5D). Linear discriminant analysis effect size (LEfSe) analysis identified hierarchical microbial shifts among groups. The MCAO group exhibited dysbiosis dominated by Actinobacteria, Enterococcus, and opportunistic pathogens, whereas the MCAO + EA group showed marked enrichment of beneficial taxa, including Prevotellaceae and Butyrivibrio, indicating EA‐driven microbial restoration. In contrast, the MCAO + EA + SDV group reverted to a profile enriched with Peptostreptococcaceae and pathogens (eg, Enterococcus faecalis), suggesting that SDV abrogated EA’s regulatory effects (Figure 5E). Notably, at the order level, EA significantly elevated Lactobacillales (beneficial lactic acid producers) and Bacteroidales (polysaccharide‐degrading bacteria with immunomodulatory functions), both of which were suppressed in the MCAO and MCAO + EA + SDV groups (Figure 5F). These findings collectively demonstrate that EA restores gut microbiota homeostasis through vagus nerve–dependent pathways, likely by enhancing epithelial fucosylation to favor colonization of Lactobacillales and Bacteroidales, thereby counteracting stroke‐induced dysbiosis.

Figure 5. Regulatory effect of EA on the intestinal microbiota.

A, Species accumulation curves demonstrating sampling sufficiency among experimental groups. B, Venn diagrams of gut microbiota OTU distribution. C, α‐Diversity indices (ACE, Chao 1, and observed species). D, Gut microbiota β‐diversity: PCoA using unweighted UniFrac distances (PERMANOVA, R ²=0.19573, P=0.014) and PCA. E, Overall representation of bacterial profiles by LEfSe analysis. F, Bacterial composition at the order level. (n=6 per group.) ACE indicates abundance‐based coverage estimator; Chao 1, Chao1 richness estimator; EA, electroacupuncture; LEfSe, linear discriminant analysis effect size; MCAO, middle cerebral artery occlusion; OTU, operational taxonomic unit; PCA, principal component analysis; PCoA, principal coordinates analysis; PERMANOVA, permutational multivariate analysis of variance; SDV, subdiaphragmatic vagotomy; and UniFrac, unique fraction metric.

Poststroke Restoration of EA Group Microbiota Improves Neurological Recovery in Germ‐Free Stroke Rats

To further validate whether EA exerts neuroprotective effects against cerebral I/R injury through vagus nerve–dependent modulation of gut microbiota, we performed FMT experiments. First, male Sprague–Dawley rats (8–10 weeks old) underwent gut microbiota ablation via a 7‐day antibiotic cocktail regimen (ampicillin 1 g/L, metronidazole 1 g/L, neomycin 1 g/L, and vancomycin 0.5 g/L) administered via daily oral gavage. Following microbiota depletion, these antibiotic‐treated rats were then used to establish I/R models as FMT recipients. On days 1 to 3 after stroke, fresh fecal microbiota from either the MCAO + EA group or the MCAO + EA + SDV group was transplanted into the recipients via oral gavage (Figure 6A). Interestingly, recipient groups ABX + MCAO + FMT(EA) and ABX + MCAO + FMT(SDV) exhibited neuroprotective effects closely mirroring their respective donor profiles. Compared with the MCAO group, the ABX + MCAO + FMT(EA) group demonstrated a marked reduction in cerebral infarct volume (F _4,30=77.92, all P<0.0001; Figure 6B and 6C) and significant improvements in neurological function (1‐way ANOVA with post hoc Tukey test; Figure 6D), but there were no statistical differences compared with the MCAO + EA groups. In contrast, the ABX + MCAO + FMT(SDV) group showed no significant differences in neurological scores (Figure 6D) or infarct volume compared with both the MCAO and MCAO + EA + SDV groups (Figure 6B and 6C). To explore systemic impacts, longitudinal monitoring revealed that all groups experienced ≈17% body weight loss by poststroke day 3. By day 7, the ABX + MCAO + FMT(EA) group regained 97.73% of baseline weight, significantly higher than the recovery observed in the MCAO (89.62%), MCAO + EA + SDV (88.22%), and ABX + MCAO + FMT(SDV) (88.38%) groups (F _5,36=30.15, P=0.0021), while no significant differences were detected among the latter 3 groups (Figure 6E). In parallel, survival analysis revealed that the survival rate at day 7 was significantly higher in the ABX + MCAO + FMT(EA) group (80%) compared with the MCAO, MCAO + EA + SDV, and ABX + MCAO + FMT(SDV) groups (≈50% when pooled), representing an absolute improvement of ≈30 percentage points (HR, 0.35 [95% CI, 0.13–0.94]; P=0.038), while the latter 3 groups remained statistically indistinguishable (Figure 6F). Taken together, these findings confirm striking consistency between FMT donors and recipients among neurological outcomes, infarct volume, survival rates, and weight recovery profiles. The transfer of neuroprotection by EA‐modulated microbiota and the complete reversal of this effect by microbiota from MCAO + EA + SDV donors strongly support that EA optimizes gut microbiota through vagus nerve–dependent mechanisms to mediate neuroprotection in cerebral I/R injury.

Figure 6. EA‐modulated gut microbiota improves poststroke recovery via FMT in GF rats.

A, Study flow chart. B and C, TTC staining and analysis of infarct volume at 7 days after MCAO. D, Neurological deficit scores based on the modified Garcia score, modified Petullo score, and Bederson score, at 7 days after MCAO. E, Changes of body weight at 3 and 7 days after MCAO. F, Percent survival at 1 to 7 days after MCAO (n=15 per group.) Survival data were analyzed using Cox proportional hazards regression with Firth penalized likelihood correction. 16sRNA indicates 16S ribosomal RNA; ABX, antibiotic therapy; EA, electroacupuncture; FMT, fecal microbiota transplant; GF, germ‐free; MCAO, middle cerebral artery occlusion; ns, not significant; SDV, subdiaphragmatic vagotomy; and TTC, 2,3,5‐triphenyltetrazolium chloride.

Gut Bacterial Profiles in Recipient Germ‐Free Stroke Rats Replicate the Donor Profile

Building on our previous results demonstrating the regulatory effects of FMT on cerebral I/R outcomes—including neurological function, infarct volume, survival rate, and body weight dynamics—we subsequently conducted 16S rRNA sequencing to systematically profile gut microbial composition following FMT intervention. First, we validated sequencing data quality: the species accumulation curve reached asymptotes among all groups, confirming sufficient sequencing depth and unbiased sample representation (Figure 7A). Next, OTU analysis revealed distinct microbial partitioning: analysis of shared and unique OTUs using Venn diagrams demonstrated a significant increase in unique OTUs specific to the MCAO + EA and ABX + MCAO + FMT(EA) groups compared with the MCAO + EA + SDV and ABX + MCAO + FMT(SDV) groups (Figure 7B), indicating selective enrichment of beneficial taxa in ABX + MCAO + FMT(EA) and MCAO + EA groups. Consistent with these observations, α‐diversity indices (abundance‐based coverage estimator and Chao1 richness estimator) corroborated these findings, with significantly higher bacterial richness in the MCAO + EA and ABX + MCAO + FMT(EA) groups versus the MCAO + EA + SDV and ABX + MCAO + FMT(SDV) groups, while no significant differences were observed between donors and recipients (Figure 7C). β‐Diversity analysis via nonmetric multidimensional scaling showed that the MCAO + EA and ABX + MCAO + FMT(EA) groups shared highly similar microbial compositions, as did the MCAO + EA + SDV and ABX + MCAO + FMT(SDV) groups; however, the former 2 groups diverged significantly from the latter 2 groups (Figure 7D). LEfSe analysis revealed that the gut microbiota composition between MCAO + EA and MCAO + EA + SDV donor rats differed significantly, as illustrated in the cladogram. Specifically, we found that the MCAO + EA group was characterized by the proliferation of short‐chain fatty acid producers (eg, Lachnospiraceae) and anti‐inflammatory genera (eg, Eubacterium) compared with the MCAO + EA + SDV group (Figure 7E). Notably, recipient rats exhibited microbiota profiles reflecting those of their donors following FMT (Figure 7F). In aggregate, these results demonstrate that the microbiome of recipient rats closely mirrored their donors, providing robust evidence that EA exerts neuroprotective effects through vagus nerve–microbiota crosstalk.

Figure 7. Composition of the fecal microbiota of FMT donor rats and clustering in recipient stroke rats.

A, Species accumulation curves demonstrating sampling sufficiency among experimental groups. B, Venn diagrams of gut microbiota OTU distribution. C, α‐Diversity indices (ACE and Chao 1). D, Gut microbiota β‐diversity shifts in donor and recipient stroke rats post‐FMT: NMDS based on unweighted UniFrac distances (stress=0.052). E, Overall representation of bacterial profiles in donor rats at baseline, by linear discriminant effect size analysis. F, Bacterial composition at the family level in transplanted recipients cluster according to the donor profiles at day 7 post‐MCAO. (n=6 per group.) ACE indicates abundance‐based coverage estimator; Chao 1, Chao1 richness estimator; EA, electroacupuncture; FMT, fecal microbiota transplant; LEfSe, linear discriminant analysis effect size; MCAO, middle cerebral artery occlusion; NMDS, nonmetric multidimensional scaling; OTU, operational taxonomic unit; SDV, subdiaphragmatic vagotomy; and UniFrac, unique fraction metric.

Discussion

To our knowledge, this is the first study suggesting that EA may confer neuroprotection via vagus nerve–dependent regulation of gut dysbiosis. Significantly, we innovatively employed FMT to reveal a functional interaction between vagal activation and microbial remodeling in EA‐treated MCAO rats, thereby elucidating their interdependent causal relationship at the pathway level. This study elucidates a previously unrecognized vagus nerve‐gut‐brain axis mechanism through which EA at the ST36 site confers neuroprotection against cerebral ischemia–reperfusion injury. Our findings demonstrate that EA‐mediated vagal activation restores intestinal epithelial fucosylation, reinforces gut barrier integrity, and reprograms gut microbiota composition, ultimately mitigating neuroinflammation and improving stroke outcomes. EA’s neuroprotective effects primarily rely on vagal pathway integrity and ST36 acupoint specificity, positioning EA as a noninvasive neuromodulatory strategy to harness gut‐brain crosstalk for stroke recovery.

During the past decade, gut dysbiosis has been implicated in a spectrum of diseases spanning metabolic disorders to central nervous system pathologies. ⁴¹ , ⁴² , ⁴³ Accumulating studies reveal that EA not only ameliorates gastrointestinal dysfunction, but more importantly, demonstrates remarkable efficacy in neuropsychiatric disorders. ⁴⁴ , ⁴⁵ , ⁴⁶ Notably, it attenuates stroke‐induced neurological deficits through remodeling gut‐brain axis communication mediated by microbial metabolites. ⁴⁷ , ⁴⁸ However, the neurobiological mechanisms underlying EA‐mediated regulation of gut‐brain axis homeostasis and microbial ecosystem dynamics remain to be fully delineated. However, the neurobiological mechanisms underlying EA‐mediated regulation of gut‐brain axis homeostasis and microbial ecosystem dynamics remain to be fully delineated. Our data suggest that EA induced Fos expression (a neuronal activation marker) in ChAT‐positive vagal efferent neurons located in the dorsal motor nucleus of the vagus. Given that Fos is a well‐validated immediate‐early gene marker for neuronal activation, ²¹ the observed increase in Fos+/ChAT+ co‐expression provides direct evidence for EA‐mediated activation of vagal efferent pathways originating from the dorsal motor nucleus of the vagus. This central vagal engagement constitutes a mechanistic prerequisite for EA‐mediated gut‐brain communication. This finding is consistent with the observed multifaceted therapeutic outcomes in stroke rats, including improved neurological scores, reduced infarct volume, enhanced survival rates, attenuated systemic inflammation coupled with neuroinflammation, and restored gut microbiota homeostasis—all of which were abolished by SDV, underscoring the indispensable role of vagus‐dependent gut‐brain axis modulation. The abolition of EA’s therapeutic effects by SDV underscores the indispensable role of vagal signaling in mediating gut‐brain communication poststroke. While prior work established that EA activates the vagal‐adrenal axis to drive anti‐inflammatory responses in sepsis models, ¹⁹ , ²¹ our study extends this paradigm to cerebral ischemia: (1) efferent vagal activation upregulates intestinal Fut2‐dependent fucosylation to fortify mucosal integrity, and (2) afferent vagal signaling modulates systemic inflammation via gut microbiota remodeling. This bidirectional regulation aligns with emerging evidence that vagal circuits maintain gut homeostasis through epithelial glycosylation ²² and that stroke‐induced dysbiosis exacerbates brain injury via microbial metabolite pathways. ¹¹

Intestinal homeostasis is dynamically regulated through multilayered interactions involving neural, epithelial, and microbial components. The enteric nervous system, which forms an extensive network throughout the intestinal wall, plays a fundamental role in coordinating these processes. ⁴⁹ , ⁵⁰ The intestinal mucosal barrier maintains immunological equilibrium by simultaneously fostering noninflammatory commensal microbiota symbiosis and providing selective defense against enteric pathogens. ⁵¹ Recent advances have identified epithelial glycosylation patterns, particularly Fut2‐dependent α1,2‐fucosylation, as a central mechanism governing host‐microbiota crosstalk. ³⁶ , ⁵² Fut2‐mediated intestinal a1,2‐fucosylation enables expression of a1,2‐fucosylated carbohydrates in IECs and in luminal contents, which can serve as substrates for metabolites, energy source, and adhesion receptors for many symbiotic beneficial bacteria. ³⁷ , ⁴⁰ Disruption of this glycosylation circuitry creates an ecological niche favoring pathogenic expansion, ultimately leading to barrier dysfunction and inflammatory pathogenesis. ⁵³ Consistent with this, our investigation demonstrates that EA rescues Fut2 expression and α1,2‐fucosylation in IECs provides mechanistic clarity to the gut‐brain axis in stroke. Fucosylated glycans serve as ecological niches for symbiotic bacteria while excluding pathobionts—a phenomenon corroborated by our 16S rRNA data showing EA‐induced enrichment of Lactobacillales and Bacteroidales, taxa known to utilize fucose‐derived metabolites for colonization. This finding dovetails with recent work demonstrating that IEC fucosylation governs microbiota composition through VIPergic neuronal regulation. ²² This aligns with recent findings demonstrating that manual acupuncture can regulate blood‐brain barrier dysfunction in Alzheimer disease models through gut microbiota modulation, particularly by reducing lipopolysaccharide‐producing bacteria such as Escherichia‐Shigella. ⁵⁴ The vagotomy‐mediated reversal of both fucosylation and microbial shifts implies that EA engages a “top‐down” vagal circuit to sustain intestinal glycan‐bacteria symbiosis, thereby preventing endotoxin translocation and systemic inflammation—a critical determinant of stroke outcome. ¹¹ , ¹²

Increasing evidence demonstrates that gut microbiota–derived metabolites, particularly short‐chain fatty acids, exert potent immunomodulatory effects capable of suppressing both peripheral and central nervous system inflammation. ⁵⁵ , ⁵⁶ , ⁵⁷ , ⁵⁸ Stroke patients exhibit marked gut microbiota dysbiosis characterized by elevated lipopolysaccharide levels and reduced butyrate production, a metabolic imbalance that directly compromises intestinal barrier integrity. ⁵⁹ Subsequent translocation of lipopolysaccharide through the impaired gut barrier triggers systemic inflammatory cascades. ⁶⁰ Supporting this, Xian et al ⁶¹ showed that combined acupuncture and herbal medicine in stroke models produced synergistic effects by modulating gut microbiota (particularly Turicibacter) and plasma metabolites, highlighting the therapeutic potential of neuromodulation approaches targeting the gut‐brain axis. Notably, FMT from young donors to aged stroke rats significantly improves neurological outcomes, with short‐chain fatty acid–producing bacteria identified as critical neuroprotective mediators through mechanisms involving barrier preservation, immunoregulation, and neuroinflammation mitigation. ⁶² Our FMT experiments revealed that germ‐free rats colonized with microbiota from EA‐treated MCAO models fully recapitulated EA’s neuroprotective effects, demonstrating that gut microbiota actively modulates poststroke recovery rather than serving as passive bystanders. Striking donor‐recipient concordance in Lactobacillales/Bacteroidales enrichment and short‐chain fatty acid–producer expansion provided direct evidence for EA‐mediated microbial remodeling. Crucially, vagotomy completely abolished microbial transfer efficacy, suggesting that vagal signaling as the upstream regulator orchestrates neuroprotective microbiota profiles. These findings establish EA as a clinically translatable modulator of the gut‐brain axis—an emerging therapeutic frontier in stroke management. Furthermore, the target specificity and temporal precision of ST36 acupoint stimulation provide a mechanistic framework for optimizing EA protocols in clinical trials. Taken together with growing recognition of microbiota‐gut‐brain interactions in cerebrovascular diseases, the current study may pave the way for innovative therapeutic strategies leveraging neuro‐immune‐metabolic crosstalk.

However, this study has several limitations. Primarily, while the 7‐day postoperative evaluation window enabled acute‐phase analysis, it precludes definitive conclusions regarding potential enhanced neurorestorative outcomes from extended intervention protocols (eg, 14‐day regimens). In addition, although we have demonstrated EA‐induced augmentation of α1,2‐fucosylation in IECs, the precise molecular regulatory networks underlying this phenomenon—particularly the mechanistic interplay between vagal signaling and glycosylation machinery—remain to be fully elucidated. Third, the strategic implementation of a male‐exclusive cohort, while effectively controlling estrogen‐mediated endocrine confounders, inherently limits translational generalizability, necessitating caution in extrapolating findings to female‐specific neuropathophysiology. Fourth, the absence of key control groups represents an important constraint on mechanistic interpretation. Specifically, the lack of an antibiotic‐only MCAO control group prevents distinction between microbiota depletion effects and antibiotic‐mediated outcomes, ¹² while the absence of sham groups receiving EA or vagotomy alone makes it difficult to determine whether the observed microbial changes are stroke‐specific or reflect general neuromodulatory functions of vagal circuits. Future studies should systematically incorporate these critical control groups to clarify the specific role of the vagus nerve–microbiota axis in the stroke pathological process, which will provide a more precise mechanistic explanation for the neuroprotective effects of EA. Fifth, while this study established the vagus nerve–gut microbiota axis as a key conduit for EA’s neuroprotection, future investigations should include more detailed brain‐specific histological read‐outs, such as microglial activation (Iba1), neutrophil infiltration (Ly6G), and vascular integrity (CD31), to provide deeper mechanistic insights into neuroimmune modulation. Finally, it is important to note that the evidence supporting the involvement of central vagal circuitry, based on Fos expression data, is derived from a preliminary sample size (n=3). While the findings are mechanistically insightful and consistent with the overall study conclusions, they warrant further confirmation in future studies with larger cohorts designed specifically for neuronal activation mapping.

Conclusions

The current study demonstrates that EA stimulation at the ST36 site confers neuroprotection against ischemic stroke through vagus nerve–dependent restoration of gut barrier integrity and microbiota homeostasis. These findings may promote clinical translation of noninvasive neuromodulation strategies targeting microbial ecosystems.

Sources of Funding

This work was supported by the Shenyang Science and Technology Bureau (24‐214‐3‐152).

Disclosures

None.

Supporting information

Data S1

Table S1–S2