Sciatic Nerve Stimulation Attenuates Intracranial Inflammation and Neuronal Injury in Acute Ischemic Stroke

Sciatic Nerve Stimulation Attenuates Intracranial Inflammation and Neuronal Injury in Acute Ischemic Stroke | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Sciatic Nerve Stimulation Attenuates Intracranial Inflammation and Neuronal Injury in Acute Ischemic Stroke RAN RAN, Yuan Zhao, Jin Zhao, Linlin Hu, Shuang Zhang, Changxiong Gong, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9107953/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Ischemic stroke, a leading cause of neurological disability, occurs due to cerebrovascular occlusion, resulting in cerebral ischemia and hypoxia that lead to neurological deficits. In the acute phase, neuronal injury and exacerbated intracranial inflammation contribute to disease progression. Effective modulation of inflammation and reduction of neuronal damage during this critical period are essential for improving stroke outcomes.A middle cerebral artery occlusion (MCAO) mouse model was established. The effects of sciatic nerve electrical stimulation on intracranial inflammation and neural injury in the acute phase after stroke were evaluated using immunostaining, enzyme-linked immunosorbent assay (ELISA), and 2,3,5-triphenyltetrazolium chloride (TTC) staining. Furthermore, transcriptomic sequencing, immunostaining, ELISA, and TTC staining were employed to assess the outcomes of optogenetic activation of Prokr2-positive neuronal subpopulations within the sciatic nerve on intracranial inflammation and neural injury. Sciatic nerve stimulation was found to ameliorate intracranial inflammation and neuronal damage, as evidenced by reduced microglial activation, decreased pro-inflammatory cytokine levels, smaller cerebral infarct volume, and attenuated neuronal injury. Optogenetic activation of Prokr2-positive somatosensory neurons in the sciatic nerve induced distinct transcriptomic changes in the brain, attenuated acute inflammation, and promoted neuronal homeostasis. This study demonstrates that sciatic nerve stimulation alleviates intracranial inflammation and neuronal injury during the acute phase of ischemic stroke. Electrical Stimulation Optogenetics Ischemic Stroke Transcriptome Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Ischemic stroke, a leading neurological disorder responsible for death and disability worldwide, is characterized by persistently high incidence, disability, and recurrence rates, placing a heavy burden on global healthcare systems and affected families[ 1 ]. Ischemic stroke is mainly caused by cerebrovascular occlusion, which leads to local cerebral ischemia and hypoxia, thereby inducing neuronal death and neuroinflammation[ 1 – 4 ]. Responding ischemic stroke, a major public health challenge, demands the development of novel therapeutic strategies to optimize patient outcomes[ 5 ]. In the acute phase of ischemic stroke, a rapid disruption of energy metabolism and ion homeostasis triggers neuronal damage and degeneration in the ischemic region, resulting in impaired neurological function[ 6 ]. Within hours of onset, a robust inflammatory cascade is initiated, primarily driven by the activation of microglia[ 7 ]. As the resident predominant immune cells of the brain, microglia play a critical role in monitoring and maintaining cerebral homeostasis[ 8 ]. It is well-established that inflammatory responses strongly influence functional outcomes after stroke, and suppressing acute-phase inflammation can exert beneficial effects in ameliorating stroke-related deficits[ 9 , 10 ]. Recent studies have revealed that activating specific neural circuits through peripheral nerve electrical stimulation can exert profound modulatory effects on immune system[ 11 – 15 ]. A typical case is stimulation of the sciatic nerve can modulate systemic immune response[ 16 ]. Notably, peripheral nerve modulation also offers unique advantages for acute ischemic stroke management—including its minimally invasive nature, targeted regulatory capacity over neuroinflammation, and potential for non-pharmacological intervention—that render it highly appealing as a novel therapeutic strategy with substantial clinical potential. In this study, we evaluated the effects of sciatic nerve stimulation on systemic inflammation and neural injury in the MCAO model. Intracranial inflammation was first assessed based on microglial activation and inflammatory cytokine levels. Subsequently, neural injury was evaluated by TTC staining and microtubule-associated protein 2 (MAP2) immunostaining, respectively. Additionally, using optogenetics, we found specifically activate the Prokr2-positive nerves within sciatic nerve also can affected neural injury and Intracranial inflammation. Collectively, our findings provide a novel thought for stroke therapy and may inform the development of improved therapeutic strategies. 2. Materials and methods 2.1 Animals C57BL/6N mice were provided by GemPharmatech Co., Ltd. (Chengdu, China). The Prokr2-Cre[ 12 ] (Stock No. 043846) and Ai32[ 17 ] (Stock No. 012569) mice were purchased from the Jackson Laboratory. Prokr2-ChR2-EYFP mice were generated by crossing Prokr2-cre mice with Ai32 mice. All mice were housed under a 12 h/12 h light–dark cycle at a controlled temperature of 20 ± 2°C with free access to food and water. Adult mice of both sexes were used in this study, with sexes randomly selected. All animal procedures in accordance with the National Institutes of Health Guidelines on the Care and Use of Laboratory Animals, and were approved by the Animal Management Committee of the Third Military Medical University, Chongqing, China. 2.2 Transient middle cerebral artery occlusion Transient focal cerebral ischemia was induced in adult mice by intraluminal occlusion of the left middle cerebral artery, following an established protocol[ 18 ]. Before surgery, the mice were fasted overnight. Anesthesia was induced with 1% pentobarbital sodium, and the animals were placed in a supine position on a heating pad to maintain body temperature at approximately 37°C. The surgical area was shaved and disinfected, after which a midline skin incision was made. Under a stereo microscope, the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were carefully isolated. MCAO was achieved by introducing a silicone rubber-coated monofilament suture (L1800, Jialing, Shanghai, China) into the common carotid artery and advancing it 9–10 mm into the internal carotid artery until mild resistance was felt. Regional cerebral blood flow (rCBF) was monitored using a laser speckle imaging system (PFLSI Pro+, RWD Life Science). Only mice exhibiting a reduction in rCBF greater than 70% of the pre-MCAO baseline were included in subsequent procedures. After 90 minutes of occlusion, the suture was withdrawn, the ligature on the common carotid artery was released to allow reperfusion. Sutured the cervical skin of the mice, placed them on a heating pad until full recovery of their consciousness. The ligature on the common carotid artery was released to allow reperfusion. 2.3 ES Under guidance of a stereomicroscope, the sciatic nerve was carefully exposed through a small incision in the thigh region. The overlying connective tissue and muscle layers were gently separated to visualize the nerve. Electrical stimuli were then delivered to the exposed sciatic nerve via a parallel double electrode (wire diameter: 0.1 mm; inter-electrode distance: 1.0 mm). Square-wave stimuli parameters were set to 1.0 mA constant current, 0.05 ms pulse width, and 10 Hz frequency, controlled by PowerLab system (ADInstruments, New South Wales, Australia). The stimulation lasted for 30 seconds and was repeated at 20-minute intervals during the middle cerebral artery occlusion. Subsequently, a daily 90-second stimulation session was administered using the same parameters. The control mice underwent the same surgical procedure, including exposure of the sciatic nerve, but without electrical stimulation. 2.4 Optogenetics Anesthetized Prokr2-ChR2-EYFP mice were maintained on a heating pad. The sciatic nerve was exposed using the aforementioned procedure. Pulsed blue light (wavelength: 465 nm; power: 1.5 mW; pulse width: 5 ms; frequency: 10 Hz) was delivered to the sciatic nerve via an optical fiber (core diameter: 200 µm; NA: 0.22) connected to an optogenetic light source system (Inper, Hangzhou, PR China). During MCAO, each mouse received light stimulation for 30 seconds at 20-minute intervals. Following MCAO, a daily stimulation session of 90 seconds was applied using identical parameters. As a control, the same optogenetic stimulation procedure was performed on Prokr2-Cre mice. 2.5 TTC staining TTC staining was employed to assess the infarct volume. Briefly, mice were deeply anesthetized with chloral hydrate and decapitated. The brains were rapidly dissected and chilled at − 20°C. Each brain was then placed in a rodent brain matrix (68707, RWD Life Science) and coronally sliced into 1 mm-thick slices. These slices were incubated in 2% TTC solution at 37.0°C for 15 minutes. Subsequently, sections were fixed in 4% paraformaldehyde for 3 hours. The percentage infarct volume was calculated as follows: (total contralateral hemispheric volume − total non-infarcted ipsilateral hemispheric volume) / total contralateral hemispheric volume × 2 × 100%. 2.6 Immunofluorescence staining As described in detail in our previous article[ 18 , 19 ], following anesthesia, mice were transcardially perfused with 0.01 M PBS, followed by 4% PFA. The brains were harvested and post-fixed overnight in 4% PFA at 4°C, then dehydrated. Coronal sections (40 µm) were prepared using a cryostat (Leica CM1950). After washing with PBS, the sections were blocked in 10% goat serum containing 0.2% Triton X-100 for 1 hour at room temperature, and then incubated overnight at 4°C with the following primary antibodies: rabbit anti-IBA1 (1:500; Fujifilm Wako, 019-19741) and rabbit anti-MAP2 (1:300; Sigma-Aldrich, ab5622). The sections were subsequently rewarmed, washed, and incubated for 2 hours at room temperature with an Alexa Fluor 555-conjugated goat anti-mouse secondary antibody. After a final series of PBS washes, the sections were mounted for imaging. For dorsal root ganglion (DRG) immunohistochemistry, DRGs from spinal levels L3–L5 were dissected under a stereomicroscope. The isolated DRGs were fixed overnight in 4% PFA, rinsed three times in PBS, and blocked with 10% goat serum. They were then incubated overnight at 4°C with the primary antibody NeuN (1:500; Abcam), followed by incubation with an appropriate secondary antibody. After four additional PBS washes, the samples were mounted. Images were acquired using a laser scanning confocal microscope (FV3000; Olympus, Tokyo, Japan) and analyzed with ImageJ software ( https://imagej.net/ij/ ). 2.8 Biochemical Assays For serum preparation, mice were deeply anesthetized and secured on a surgical board. The thoracic cavity was opened to expose the heart, and blood was promptly collected from the right ventricle using a syringe. After clotting at room temperature, the samples were centrifuged at 3000 rpm for 15 minutes at 4°C to isolate serum. then, the serum was collected and stored at -80°C until analysis. For brain tissue extracts preparation, anesthetized mice were transcardially perfused with chilled PBS. The infarcted cerebral hemispheres were dissected, weighed, and homogenized in a corresponding volume of sterile PBS containing protease inhibitors using a tissue homogenizer. The homogenates were centrifuged at 12,000 × g for 20 minutes at 4°C. The resulting supernatants were collected and stored at -80°C for subsequent analysis. The concentrations of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were quantified using ELISA kits (M6000B, R&D Systems; BMS607-3, Thermo Fisher Scientific) in accordance with the manufacturer's protocols[ 20 ]. Absorbance was measured at 450 nm using a microplate reader (Flash Varioskan, Thermo Fisher Scientific). 2.9 Transcriptome sequencing Mice were deeply anesthetized and transcardially perfused with chilled PBS. The infarcted cerebral hemispheres and spleens were then harvested and immediately flash-frozen in liquid nitrogen. Total RNA extraction, library preparation, RNA sequencing, and downstream transcriptomic analyses were performed by a commercial service provider (Annoroad Gene Technology (Beijing) Co., Ltd). The qualified libraries were then sequenced on the Illumina NovaSeq 6000 platform, with 150 bp paired-end reads. The bases with low quality and sequences from adapters in raw data were removed using fastp (version 0.20.0)[ 21 ]. The filtered reads were aligned to the reference genomes by using HISAT2 (version 2.1.0). FeatureCounts (version 2.0.1) in the Subread package was applied for gene abundance quantification[ 22 ]. Transcriptomic analyses included Principal Component Analysis (PCA) for dimensionality reduction. Differential expression analysis was carried out with the limma package (threshold: |log 2 FC| ≥ 1.5, P ≤ 0.05). Gene Ontology (GO) enrichment analysis for Biological Processes (BP) was performed on the list of differentially expressed genes using the clusterProfiler. Inflammatory cytokine expression (IL1b, TNF-α, IL4, IL6) was visualized using GraphPad Prism. Gene Set Enrichment Analysis (GSEA) was implemented evaluate functional pathways including "Neuron Cellular Homeostasis" and "Acute Inflammatory Response" based on MSigDB gene sets. 2.10 Statistical analysis Bar graphs show the mean ± SEM., with error bars indicating the SEM. The sample size (n) denotes the number of independent biological replicates conducted in the study. To assess normality, a Kolmogorov–Smirnov test was performed. Statistical significance was set at p < 0.05. Data analysis and graph generation were conducted using GraphPad Prism software (version 8; San Diego, CA) and Adobe Illustrator 2024 (Adobe, San Jose, USA). More details of statistical tests can be found in the figure legends and Supplemental Source Data. 3. Results 3.1 Sciatic Nerve Electrical Stimulation Attenuates Intracranial Inflammation in Acute Ischemic Stroke. During the acute phase of stroke, with the progression of injury, microglia become activated and the intracranial concentrations of inflammatory factors gradually increase. To evaluate the impact of sciatic nerve stimulation on neuroinflammation, MCAO mice were randomized into MCAO-Sham and MCAO-ES groups. The success of the MCAO model was assessed by monitoring cerebral blood flow perfusion via laser speckle contrast imaging (Fig. 1 A–C). The effects on neuroinflammation were evaluate by quantifying microglial activation in the infarct region and measuring inflammatory factor levels in the hemispheric infarction. We performed IBA1 immunostaining to assess the activation of microglia in the infarct region at 24 and 72 hours after stroke. (Fig. 1 D, E). The results showed that the density of IBA1 + cells was higher in the ES group than in the sham group at both time points (Fig. 1 F). Analysis of microglial morphology further revealed that more intact branches were preserved in the ES group at 24 h, with this difference becoming more evident at 72 h (Fig. 1 G), These results indicate that sciatic nerve stimulation modulates the recruitment and activation of microglia. The levels of intracranial inflammatory factors were then examined. The infarcted hemispheres were collected for ELISA. The data showed, at 24 h, no significant difference in IL-6 concentration was found between the two groups (Fig. 1 H). At 72 hour, IL-6 levels were significantly lower in the ES group compared to the Sham group (Fig. 1 H). For TNF-α, the results showed no difference at 24 h (Fig. 1 I), whereas a profound decrease was detected in the ES group at 72 h. (Fig. 1 I). These results suggesting that sciatic nerve electrical stimulation significantly alleviated intracranial inflammation at the 72-hour. 3.2 Sciatic Nerve Stimulation Ameliorates Acute Ischemic Neural Damage To assess the impact of sciatic nerve stimulation on neural injury, infarct volume was evaluated by TTC staining, and neuronal damage was examined using MAP2 immunofluorescence. For infarct volume, result showed that electrical stimulation significantly reduced infarct volume at both 24 and 72 hour compared to the sham group (Fig. 2 B). We evaluated neuronal injury using immunofluorescence staining for MAP2, a protein primarily distributed in neuronal cell bodies and dendrites. In healthy neurons, MAP2 exhibits a continuous filamentous pattern, but it becomes fragmented or beaded upon injury and is entirely ablated in severe cases. The results showed, at 24 h, neuronal injury was apparent in both groups; however, the density of MAP2-positive signals was higher in the ES group (Fig. 2 C, D). At 72 h, MAP2 loss and neuronal damage were further exacerbated (Fig. 2 E, F). These findings consistently indicated that ES effectively attenuates neural injury during the acute phase of ischemic stroke. 3.3 Transcriptomic Responses to Optogenetic Stimulation in the MCAO Mouse We generated Prokr2-Ai32 mice by cross Prokr2-Cre mice and Ai32 mice, which express ChR2 and EYFP in Prokr2 expression neurons (Fig. 3 A). Anatomical examination of the L3—L5 DRG revealed that this neuronal population was predominantly localized to afferent nerves, (Fig. 3 B), and constituted 36 ± 4.8% of the total neuronal population in the DRG (Fig. 3 C, D). Whole nerve electrophysiological revealed robust light-induced action potentials[ 23 ], suggesting that the Prokr2-positive nerves was activated by optical stimulation (Fig. 3 F). To investigate the transcriptomic effects of optogenetic activation of Prokr2-positive somatosensory nerve on the brain in mice, we collected brain samples from MCAO mice and performed RNA sequencing. Principal component analysis (PCA) revealed clear separation between the MCAO-Sham and MCAO-Light groups, indicating distinct global transcriptomic profiles (Fig. 3 G). Differential expression analysis identified numerous significantly regulated genes, compared to the MCAO-sham group, the MCAO-light group exhibited upregulation of 667 genes and downregulation of 2417 genes (Fig. 3 H). Gene Ontology (GO) enrichment analysis for Biological Processes (BP) revealed that the differentially expressed genes were significantly enriched in three primary categories: immune-related processes (e.g., leukocyte cell adhesion, regulation of leukocyte migration), inflammatory pathways (including IκB kinase/NF-κB signaling and regulation of inflammatory response), and developmental processes (such as aorta development and epithelial tube morphogenesis). These results suggest these genes are predominantly involved in immune regulation, inflammatory responses, and specific aspects of development (Fig. 3 I). Among the regulated genes, several key pro-inflammatory cytokines (IL1β, TNF-α, IL4, IL6) were down-regulated in the MCAO-Light group (Fig. 3 J). Consistent with the results from electrical stimulation, these data further support the anti-inflammatory action of the intervention. Further GSEA indicated significant up-regulation of the "neuronal cell homeostasis" (Fig. 3 K) and down-regulation of the "acute inflammatory response" (Fig. 3 L) in the MCAO-Light group. These findings suggested that selective active Prokr2 positive nerves in sciatic nerve could promotes transcriptional programs related to neuronal homeostasis while suppressing neuroinflammatory. 3.4 Optogenetic Stimulation of Prokr2-positive sciatic nerve influence Neural Damage and acute inflammatory reaction. We further evaluated neuroinflammation and neuronal damage in MCAO model after optogenetic stimulation. Infarct volume was evaluated by TTC staining, which revealed a significant reduction in the MCAO-Light group compared with the sham group (Fig. 4 A, B). We next performed immunostaining for IBA1 to assess microglial responses in the ischemic core (Fig. 4 C). The result showed the density of Iba1 cells was higher in the MCAO-light group (Fig. 4 D), while their branching complexity was significantly lower (Fig. 4 E), collectively indicating attenuated microglial activation. We then measured the concentrations of IL-6 and TNF-α in the infarcted hemisphere. The data showed both cytokines were significantly lower in the MCAO-Light stimulation group than in sham (Fig. 4 F, G). Furthermore, the result of MAP2 immunostaining demonstrated a higher level of MAP2 expression in the MCAO-light group (Fig. 4 H, I), suggesting a preservation of neuronal structure and mitigation of injury. In summary, optogenetic activation of Prokr2-positive somatosensory nerves attenuates intracranial inflammation and reduces neural damage during the acute phase of ischemic stroke. 4. Discussion This study aimed to explore a novel therapeutic strategy for acute ischemic stroke by sciatic nerve stimulation. Our results suggested that both electrical stimulation of the sciatic nerve and optogenetic activation of its Prokr2-positive somatosensory nerves attenuated neuroinflammation and reduced neural damage after stroke. Specifically, our interventions led to a marked reduction in pro-inflammatory cytokines and morphological changes of microglia, which was accompanied by a decreased infarct volume and a higher preservation of MAP2, a key marker of neuronal integrity[ 24 ]. We first applied sciatic nerve electrical stimulation to investigate neural injury and neuroinflammation in acute ischemic stroke. The observed anti-inflammatory effect of sciatic nerve electrical stimulation in acute ischemic stroke aligns with previous findings that demonstrated its efficacy in mitigating inflammation across other pathological model[ 25 ]. The observed neuroprotection, evidenced by reduced infarct volume and higher preservation of MAP2, is likely a direct downstream consequence of the attenuated neuroinflammation. An exacerbated inflammatory response is a key driver of secondary brain injury after ischemia. By dampening the activation of microglia and the production of detrimental cytokines, our interventions presumably created a more permissive environment for neuronal survival and synaptic stability, thereby limiting the final extent of the damage. However, the conclusions of this study must be interpreted within its limitations. Firstly, our investigation into the mechanism remains at a phenotypic level. This study establishes the therapeutic potential of nerve electrical stimulation in acute stroke but also highlights key areas for future investigation. Three major questions remain: first, what are the specific molecular pathways, such as the role of the cholinergic anti-inflammatory pathway (CAP) or other neuroimmune axes remain to be further elucidated[ 26 , 27 ]. Secondly, does nerve electrical stimulation remain effective in the subacute and chronic phases of stroke? [ 28 ] Thirdly, how do peripheral immune cells, such as T lymphocytes and neutrophils, contribute to the immunomodulatory response?[ 26 ] Expanding our analysis beyond microglia and a narrow cytokine profile to a full immunophenotypic study will be essential to completely unravel the mechanisms of nerve electrical stimulation. Despite these limitations, our findings nonetheless underscore the considerable translational potential of sciatic nerve stimulation. Targeting severe central nervous system disorders such as stroke through sciatic nerve stimulation presents a therapeutically attractive strategy, potentially circumventing the challenges of direct cerebral interventions[ 29 ]. Future studies should prioritize elucidating the precise mechanisms of action, including the identification of key neuroimmune messengers. Moreover, leveraging the accessible anatomy of the sciatic nerve, it is worthwhile to explore minimally or non-invasive approaches that achieve comparable efficacy by transcutaneously stimulating its downstream branches. Translating this neuromodulatory strategy whether via non-invasive electrical stimulation or advanced bioelectronic medicine into an adjuvant therapy for acute stroke patients represents a highly promising future direction. Declarations Competing Interests: The authors declare that they have no competing interests. Ethics approval : All animal experiments were approved by the Institutional Animal Committee of Army Medical University University (Approval No. AMUWEC2023, AMUWEC20234922). Data sharing information Data that support the findings of this study are available from the corresponding author upon reasonable request. Funding: This work was supported by grants from the National Natural Science Foundation of China (Grant number: 82300503), Natural Science Foundation of Chongqing, China (Grant number: CSTB2025NSCQ-GPX0647, CSTB2024NSCQ-MSX1121) and Young PhD Talents Cultivation Project of Xinqiao Hospital (2022YQB069, 2023YQB014). Author Contribution Qingwu Yang, Xiaofeng Cheng, and Shilun Zuo conceived the study, Ran Ran and Xiaofeng Cheng designed the experiments and wrote the manuscript. Ran Ran and Yuan Zhao performed animal studies and prepared illustrations. Ran Ran and Jin Zhao performed data analysis and statistics, Shuang Zhang, Changxiong Gong, and Changwei Guo participated in the project discussions and provided valuable revision suggestions for this work, Linlin Hu, Mengya Shan, Rong Wang and Chenhao Zhao provided valuable assistance during the preliminary electrical stimulation exploration process of this study, Zhaouyou Meng, Qi Xie, Yiliang Fang, Sen Lin, Qingwu Yang, Xiaofeng Cheng, and Shilun Zuo assisted in reviewing this manuscript. Acknowledgement We thank the staff of the Department of Neurology, Xinqiao Hospital for their assistance in the initiation of this research project. Data Availability Data that support the findings of this study are available from the corresponding author upon reasonable request. References Feigin VL, Abate MD, Abate YH, Abd ElHafeez S, Abd-Allah F, Abdelalim A, et al. 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Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 24 Apr, 2026 Reviewers agreed at journal 04 Apr, 2026 Reviewers invited by journal 30 Mar, 2026 Editor assigned by journal 24 Mar, 2026 Submission checks completed at journal 15 Mar, 2026 First submitted to journal 12 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9107953","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":614728649,"identity":"b65afd10-9223-4453-95e9-503192b0270d","order_by":0,"name":"RAN RAN","email":"","orcid":"","institution":"Second Affiliated Hospital, Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"RAN","middleName":"","lastName":"RAN","suffix":""},{"id":614728650,"identity":"00eeb639-19c2-4932-8ad4-7ecd5eeeb6d3","order_by":1,"name":"Yuan Zhao","email":"","orcid":"","institution":"Second Affiliated Hospital, Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Zhao","suffix":""},{"id":614728651,"identity":"acbf8010-ec70-4024-9e09-cb653814b09f","order_by":2,"name":"Jin Zhao","email":"","orcid":"","institution":"Chongqing Institute for Brain and Intelligence, Guangyang Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Zhao","suffix":""},{"id":614728652,"identity":"a0897968-bda6-43ed-850e-f3e42655d60e","order_by":3,"name":"Linlin Hu","email":"","orcid":"","institution":"Second Affiliated Hospital, Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Linlin","middleName":"","lastName":"Hu","suffix":""},{"id":614728653,"identity":"68c134f7-206a-415a-afc4-9fb1e9ad1318","order_by":4,"name":"Shuang Zhang","email":"","orcid":"","institution":"Second Affiliated Hospital, Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shuang","middleName":"","lastName":"Zhang","suffix":""},{"id":614728654,"identity":"6e9982b0-f0b9-412d-86ef-0c6a2b43c2f5","order_by":5,"name":"Changxiong Gong","email":"","orcid":"","institution":"Chongqing Institute for Brain and Intelligence, Guangyang Bay Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Changxiong","middleName":"","lastName":"Gong","suffix":""},{"id":614728655,"identity":"d519bc39-4951-44da-bfb8-18fec61e816c","order_by":6,"name":"Changwei Guo","email":"","orcid":"","institution":"Second Affiliated Hospital, Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Changwei","middleName":"","lastName":"Guo","suffix":""},{"id":614728656,"identity":"57787847-d52f-421e-a3e1-2bc41233fbfc","order_by":7,"name":"Mengya Shan","email":"","orcid":"","institution":"Second Affiliated Hospital, Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mengya","middleName":"","lastName":"Shan","suffix":""},{"id":614728657,"identity":"d790b6d8-f144-4659-9765-cac0b7c05586","order_by":8,"name":"Rong Wang","email":"","orcid":"","institution":"Second Affiliated Hospital, Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"Wang","suffix":""},{"id":614728658,"identity":"bc23ac55-7873-4cbe-ad50-7473f45b3bd0","order_by":9,"name":"Qi Xie","email":"","orcid":"","institution":"Second Affiliated Hospital, Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Xie","suffix":""},{"id":614728659,"identity":"3e19fcc0-7e74-40a6-902e-ec7d2d34245d","order_by":10,"name":"Yiliang Fang","email":"","orcid":"","institution":"Second Affiliated Hospital, Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yiliang","middleName":"","lastName":"Fang","suffix":""},{"id":614728660,"identity":"dd0c6777-5b7f-4b0c-8f57-ec807b706707","order_by":11,"name":"Sen Lin","email":"","orcid":"","institution":"Second Affiliated Hospital, Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Sen","middleName":"","lastName":"Lin","suffix":""},{"id":614728661,"identity":"bf98f59c-4bf7-4ef5-960c-98c58638ac10","order_by":12,"name":"Chenhao Zhao","email":"","orcid":"","institution":"Second Affiliated Hospital, Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chenhao","middleName":"","lastName":"Zhao","suffix":""},{"id":614728662,"identity":"5aa1a85c-d30d-4f0e-9d69-a203ccdd4043","order_by":13,"name":"Zhaoyou Meng","email":"","orcid":"","institution":"Second Affiliated Hospital, Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhaoyou","middleName":"","lastName":"Meng","suffix":""},{"id":614728663,"identity":"115ea8c0-1ba1-434e-88d3-0643efda2ea3","order_by":14,"name":"Shilun Zuo","email":"","orcid":"","institution":"Second Affiliated Hospital, Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shilun","middleName":"","lastName":"Zuo","suffix":""},{"id":614728664,"identity":"4b8160d0-4ae8-4410-8847-d1c120c9d0c1","order_by":15,"name":"Xiaofeng Cheng","email":"","orcid":"","institution":"Second Affiliated Hospital, Army Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaofeng","middleName":"","lastName":"Cheng","suffix":""},{"id":614728665,"identity":"31d6a1e5-cc3e-4f82-a20e-bf565e2cb61d","order_by":16,"name":"Qingwu Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIie3NPwrCMByG4V8p6BLr2oKoR4gUBHHwKoqgSyoeQRDqoicQD+ESHCMdXCJZI138A+JQQXAQFxFbR426OeSdvuXhA9Dp/rgcgMmeu/4dQQCpRu9XgkrfEbxYBoejTxEW/LJdkQCyaYLhOlMQ3mlVJn6IsPSmfY8G4AwjbIy4gjBSdjP0QTIJwZJg0/AVRERPIvgmJrWPRBJ3FxNGjOTF/kAcGZWNyS1EjmyVxh5tI5vvu/ORgliCuKeIh3lLBOuzR6v57KA5XV8VpMggZaNkxD02sPcAoNAD84SSodPpdLrX3QHRT1sVOKiY/QAAAABJRU5ErkJggg==","orcid":"","institution":"Second Affiliated Hospital, Army Medical University","correspondingAuthor":true,"prefix":"","firstName":"Qingwu","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2026-03-12 19:38:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9107953/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9107953/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105923820,"identity":"39a61165-08a9-461a-8ddf-8ac43b356555","added_by":"auto","created_at":"2026-04-01 13:03:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":345487,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of sciatic nerve electrical stimulation on microglial activation and pro-inflammatory cytokine levels in the brains of MCAO mice (a) Schematic diagram of MCAO model establishment and electrical stimulation protocol. (b) Represent laser speckle imaging of rCBF in tMCAO mice before, during, and after occlusion. (c) Statistical results for (b). n= 5 male mice in each group, Fridman test, Dunn's multiple comparisons test. (d) Confocal imaging of microglia in the infarct region at day 1 (I/R 1D) after reperfusion. Scale bar, 40 μm. The microglia indicated by the red arrow are magnified, skeletonized, and shown below. Scale bar, 10 μm. (e) Confocal imaging of microglia in the infarct region at day 3 (I/R 3D) after reperfusion. Scale bar, 40 μm. The microglia indicated by the red arrow are magnified, skeletonized, and shown below. Scale bar, 10 μm. (f) Microglial density in the infarct region at day 1 and day 3 after reperfusion in sham and ES treated mice. n = 5 male mice in each group, unpaired two side student t test, mean ± SEM. (g) Microglial branch number in the infarct region at day 1 and day 3 after reperfusion in sham and ES treated mice. n = 5 male mice in each group, unpaired two side student t test, mean ± SEM. (h) Brain IL-6 levels at day 1 and day 3 after reperfusion in sham and ES treated mice. n = 5 male mice in each group, unpaired two side student t test for day 1, unpaired two side student t test with Welch's correction for day 3, mean ± SEM. (i) Brain TNF-α levels at day 1 and day 3 after reperfusion in sham and ES treated mice. n = 5 male mice in each group, unpaired two side student t test, mean ± SEM.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9107953/v1/9f1c0373f3d1e5a1aedf6448.png"},{"id":106093150,"identity":"835f6b3d-06e0-4632-b1c5-7efddd1a3f14","added_by":"auto","created_at":"2026-04-03 11:35:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":304545,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of sciatic nerve electrical stimulation on neural damage in the brains of MCAO mice (a) TTC-stained brain sections at day 1 and day 3 after reperfusion in sham and ES treated mice. (b) Quantitative analysis of cerebral infarct volume in sham and ES treated mice at day 1 and day 3. n = 5 male mice in each group. unpaired two side student t test with Welch's correction for day 1, unpaired two side student t test for day 3, mean ± SEM (c) Immunofluorescence staining of MAP2 in sham and ES treated mice at day 1. The dashed box encloses the infarct area, whereas the green box demarcates the imaging region, scar bar,100 μm. (d) Density quantification of MAP2 in sham and ES treated mice at day 1. n = 5 male mice in each group, Mann-Whitney test for day 1, unpaired two side student t test for day3. (e) Immunofluorescence staining of MAP2 in sham and ES treated mice at day 3. The dashed box encloses the infarct area, whereas the green box demarcates the imaging region, scar bar, 100 μm. (f) Density quantification of MAP2 in sham and ES treated mice at day3. n = 5 male mice in each group, unpaired two side student t test, mean ± SEM.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9107953/v1/596b79823935f5fa2bc0075e.png"},{"id":105923822,"identity":"958f5a9e-d718-4b35-b070-1439b6abd9fb","added_by":"auto","created_at":"2026-04-01 13:03:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":259972,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic effects of optogenetic stimulation of Prokr2-positive sciatic nerves in the MCAO mice brain (a) Schematic diagram of the construction strategy for Prokr2-Ai32 transgenic mice. (b) Distribution and magnified images of Prokr2-positive neurons in the L4 DRG, scar bar, 200 μm; the red box showed in right was dorsal root afferent nerves, the blue box showed in the lower right subpanel was ventral root efferent nerves, scar bar, 50 μm. (c) Immunofluorescence staining for colocalization of Prokr2-positive neurons with NeuN in the L4 DRG. (d) Quantitative analysis of the proportion of Prokr2-positive neurons among total neurons in the L3-5 DRG. (e) Schematic diagram of the optogenetic paradigm in MCAO mice. (f) Whole nerve electrophysiological recordings in Prokr2-Ai32 mice revealed light-induced action potentials. (g) Principal Component Analysis (PCA) plot of gene expression profiles. (h) Volcano plot showing up-(orange) and down-regulated (blue) genes. (i) GO BP enrichment analysis of differentially expressed genes. (j) Expression of cytokines in MCAO-Sham and MCAO-Light groups. (k) GSEA plot of the \"Neuron Cellular Homeostasis\" gene set (l) GSEA plot for the \"Acute Inflammatory Response\" gene set.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9107953/v1/c02558495021e8392a9d2ddc.png"},{"id":105923823,"identity":"24d11f38-397d-4bbe-b16e-200352a9bf36","added_by":"auto","created_at":"2026-04-01 13:03:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":231192,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of optogenetic stimulation of Prokr2-positive sciatic nerveson intracranial inflammation and neural damage in an MCAO mouse model\u003cstrong\u003e (\u003c/strong\u003ea) TTC-stained brain sections at day 3 after reperfusion in sham and light treated mice. (b) Quantitative analysis of cerebral infarct volume in sham and light treated mice at day 3, n = 5 male mice in each group, unpaired two side student \u003cem\u003et\u003c/em\u003e test, mean ± SEM. (c) Confocal imaging of microglia in the infarct region at day 3 after reperfusion. Scale bar, 40 μm. The microglia indicated by the red arrow are magnified, skeletonized, and shown below. Scale bar, 10 μm. (d) Microglial density in the infarct region at day 3 after reperfusion in sham and light treated mice. n = 5 male mice in each group, unpaired two side student \u003cem\u003et\u003c/em\u003etest, mean ± SEM. (e) Microglial branch number in the infarct region at day 3 after reperfusion in sham and light treated mice. n = 5 male mice in each group, unpaired two side student \u003cem\u003et\u003c/em\u003e test, mean ± SEM. (f) Brain IL-6 levels at day 3 after reperfusion in sham and light treated mice. n = 5 male mice in each group, unpaired two side student \u003cem\u003et\u003c/em\u003e test, mean ± SEM. (g) Brain TNF-α levels at day 3 after reperfusion in sham and light treated mice. n = 5 male mice in each group, unpaired two side student \u003cem\u003et\u003c/em\u003e test, mean ± SEM. (h) Immunofluorescence staining of MAP2 in sham and light treated mice at day 3. The dashed box encloses the infarct area, whereas the green box demarcates the imaging region, scar bar, 100 μm. (i) Density quantification of MAP2 in sham and light treated mice at day 3. n = 5 male mice in each group, unpaired two side student \u003cem\u003et\u003c/em\u003e test, mean ± SEM.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9107953/v1/bdfa97ae3f04c3129285f62a.png"},{"id":106723821,"identity":"c561c7c2-d9d2-4f9e-a61c-e54b467bf570","added_by":"auto","created_at":"2026-04-12 18:15:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1732364,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9107953/v1/419cf756-7588-48a2-9adf-a62b7d50ec67.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sciatic Nerve Stimulation Attenuates Intracranial Inflammation and Neuronal Injury in Acute Ischemic Stroke","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIschemic stroke, a leading neurological disorder responsible for death and disability worldwide, is characterized by persistently high incidence, disability, and recurrence rates, placing a heavy burden on global healthcare systems and affected families[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Ischemic stroke is mainly caused by cerebrovascular occlusion, which leads to local cerebral ischemia and hypoxia, thereby inducing neuronal death and neuroinflammation[\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Responding ischemic stroke, a major public health challenge, demands the development of novel therapeutic strategies to optimize patient outcomes[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the acute phase of ischemic stroke, a rapid disruption of energy metabolism and ion homeostasis triggers neuronal damage and degeneration in the ischemic region, resulting in impaired neurological function[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Within hours of onset, a robust inflammatory cascade is initiated, primarily driven by the activation of microglia[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. As the resident predominant immune cells of the brain, microglia play a critical role in monitoring and maintaining cerebral homeostasis[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. It is well-established that inflammatory responses strongly influence functional outcomes after stroke, and suppressing acute-phase inflammation can exert beneficial effects in ameliorating stroke-related deficits[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent studies have revealed that activating specific neural circuits through peripheral nerve electrical stimulation can exert profound modulatory effects on immune system[\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. A typical case is stimulation of the sciatic nerve can modulate systemic immune response[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Notably, peripheral nerve modulation also offers unique advantages for acute ischemic stroke management\u0026mdash;including its minimally invasive nature, targeted regulatory capacity over neuroinflammation, and potential for non-pharmacological intervention\u0026mdash;that render it highly appealing as a novel therapeutic strategy with substantial clinical potential.\u003c/p\u003e \u003cp\u003eIn this study, we evaluated the effects of sciatic nerve stimulation on systemic inflammation and neural injury in the MCAO model. Intracranial inflammation was first assessed based on microglial activation and inflammatory cytokine levels. Subsequently, neural injury was evaluated by TTC staining and microtubule-associated protein 2 (MAP2) immunostaining, respectively. Additionally, using optogenetics, we found specifically activate the Prokr2-positive nerves within sciatic nerve also can affected neural injury and Intracranial inflammation. Collectively, our findings provide a novel thought for stroke therapy and may inform the development of improved therapeutic strategies.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 \u003cb\u003eAnimals\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eC57BL/6N mice were provided by GemPharmatech Co., Ltd. (Chengdu, China). The Prokr2-Cre[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] (Stock No. 043846) and Ai32[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] (Stock No. 012569) mice were purchased from the Jackson Laboratory. Prokr2-ChR2-EYFP mice were generated by crossing Prokr2-cre mice with Ai32 mice.\u003c/p\u003e \u003cp\u003eAll mice were housed under a 12 h/12 h light\u0026ndash;dark cycle at a controlled temperature of 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C with free access to food and water. Adult mice of both sexes were used in this study, with sexes randomly selected. All animal procedures in accordance with the National Institutes of Health Guidelines on the Care and Use of Laboratory Animals, and were approved by the Animal Management Committee of the Third Military Medical University, Chongqing, China.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 \u003cb\u003eTransient middle cerebral artery occlusion\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTransient focal cerebral ischemia was induced in adult mice by intraluminal occlusion of the left middle cerebral artery, following an established protocol[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Before surgery, the mice were fasted overnight. Anesthesia was induced with 1% pentobarbital sodium, and the animals were placed in a supine position on a heating pad to maintain body temperature at approximately 37\u0026deg;C. The surgical area was shaved and disinfected, after which a midline skin incision was made. Under a stereo microscope, the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were carefully isolated. MCAO was achieved by introducing a silicone rubber-coated monofilament suture (L1800, Jialing, Shanghai, China) into the common carotid artery and advancing it 9\u0026ndash;10 mm into the internal carotid artery until mild resistance was felt. Regional cerebral blood flow (rCBF) was monitored using a laser speckle imaging system (PFLSI Pro+, RWD Life Science). Only mice exhibiting a reduction in rCBF greater than 70% of the pre-MCAO baseline were included in subsequent procedures. After 90 minutes of occlusion, the suture was withdrawn, the ligature on the common carotid artery was released to allow reperfusion. Sutured the cervical skin of the mice, placed them on a heating pad until full recovery of their consciousness. The ligature on the common carotid artery was released to allow reperfusion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 \u003cb\u003eES\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eUnder guidance of a stereomicroscope, the sciatic nerve was carefully exposed through a small incision in the thigh region. The overlying connective tissue and muscle layers were gently separated to visualize the nerve. Electrical stimuli were then delivered to the exposed sciatic nerve via a parallel double electrode (wire diameter: 0.1 mm; inter-electrode distance: 1.0 mm). Square-wave stimuli parameters were set to 1.0 mA constant current, 0.05 ms pulse width, and 10 Hz frequency, controlled by PowerLab system (ADInstruments, New South Wales, Australia). The stimulation lasted for 30 seconds and was repeated at 20-minute intervals during the middle cerebral artery occlusion. Subsequently, a daily 90-second stimulation session was administered using the same parameters. The control mice underwent the same surgical procedure, including exposure of the sciatic nerve, but without electrical stimulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 \u003cb\u003eOptogenetics\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eAnesthetized Prokr2-ChR2-EYFP mice were maintained on a heating pad. The sciatic nerve was exposed using the aforementioned procedure. Pulsed blue light (wavelength: 465 nm; power: 1.5 mW; pulse width: 5 ms; frequency: 10 Hz) was delivered to the sciatic nerve via an optical fiber (core diameter: 200 \u0026micro;m; NA: 0.22) connected to an optogenetic light source system (Inper, Hangzhou, PR China). During MCAO, each mouse received light stimulation for 30 seconds at 20-minute intervals. Following MCAO, a daily stimulation session of 90 seconds was applied using identical parameters. As a control, the same optogenetic stimulation procedure was performed on Prokr2-Cre mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 \u003cb\u003eTTC staining\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTTC staining was employed to assess the infarct volume. Briefly, mice were deeply anesthetized with chloral hydrate and decapitated. The brains were rapidly dissected and chilled at \u0026minus;\u0026thinsp;20\u0026deg;C. Each brain was then placed in a rodent brain matrix (68707, RWD Life Science) and coronally sliced into 1 mm-thick slices. These slices were incubated in 2% TTC solution at 37.0\u0026deg;C for 15 minutes. Subsequently, sections were fixed in 4% paraformaldehyde for 3 hours. The percentage infarct volume was calculated as follows: (total contralateral hemispheric volume\u0026thinsp;\u0026minus;\u0026thinsp;total non-infarcted ipsilateral hemispheric volume) / total contralateral hemispheric volume \u0026times; 2 \u0026times; 100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 \u003cb\u003eImmunofluorescence staining\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eAs described in detail in our previous article[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], following anesthesia, mice were transcardially perfused with 0.01 M PBS, followed by 4% PFA. The brains were harvested and post-fixed overnight in 4% PFA at 4\u0026deg;C, then dehydrated. Coronal sections (40 \u0026micro;m) were prepared using a cryostat (Leica CM1950). After washing with PBS, the sections were blocked in 10% goat serum containing 0.2% Triton X-100 for 1 hour at room temperature, and then incubated overnight at 4\u0026deg;C with the following primary antibodies: rabbit anti-IBA1 (1:500; Fujifilm Wako, 019-19741) and rabbit anti-MAP2 (1:300; Sigma-Aldrich, ab5622). The sections were subsequently rewarmed, washed, and incubated for 2 hours at room temperature with an Alexa Fluor 555-conjugated goat anti-mouse secondary antibody. After a final series of PBS washes, the sections were mounted for imaging.\u003c/p\u003e \u003cp\u003eFor dorsal root ganglion (DRG) immunohistochemistry, DRGs from spinal levels L3\u0026ndash;L5 were dissected under a stereomicroscope. The isolated DRGs were fixed overnight in 4% PFA, rinsed three times in PBS, and blocked with 10% goat serum. They were then incubated overnight at 4\u0026deg;C with the primary antibody NeuN (1:500; Abcam), followed by incubation with an appropriate secondary antibody. After four additional PBS washes, the samples were mounted.\u003c/p\u003e \u003cp\u003eImages were acquired using a laser scanning confocal microscope (FV3000; Olympus, Tokyo, Japan) and analyzed with ImageJ software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.net/ij/\u003c/span\u003e\u003cspan address=\"https://imagej.net/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.8 \u003cb\u003eBiochemical Assays\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eFor serum preparation, mice were deeply anesthetized and secured on a surgical board. The thoracic cavity was opened to expose the heart, and blood was promptly collected from the right ventricle using a syringe. After clotting at room temperature, the samples were centrifuged at 3000 rpm for 15 minutes at 4\u0026deg;C to isolate serum. then, the serum was collected and stored at -80\u0026deg;C until analysis.\u003c/p\u003e \u003cp\u003eFor brain tissue extracts preparation, anesthetized mice were transcardially perfused with chilled PBS. The infarcted cerebral hemispheres were dissected, weighed, and homogenized in a corresponding volume of sterile PBS containing protease inhibitors using a tissue homogenizer. The homogenates were centrifuged at 12,000 \u0026times; g for 20 minutes at 4\u0026deg;C. The resulting supernatants were collected and stored at -80\u0026deg;C for subsequent analysis.\u003c/p\u003e \u003cp\u003eThe concentrations of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were quantified using ELISA kits (M6000B, R\u0026amp;D Systems; BMS607-3, Thermo Fisher Scientific) in accordance with the manufacturer's protocols[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Absorbance was measured at 450 nm using a microplate reader (Flash Varioskan, Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.9 \u003cb\u003eTranscriptome sequencing\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eMice were deeply anesthetized and transcardially perfused with chilled PBS. The infarcted cerebral hemispheres and spleens were then harvested and immediately flash-frozen in liquid nitrogen. Total RNA extraction, library preparation, RNA sequencing, and downstream transcriptomic analyses were performed by a commercial service provider (Annoroad Gene Technology (Beijing) Co., Ltd).\u003c/p\u003e \u003cp\u003eThe qualified libraries were then sequenced on the Illumina NovaSeq 6000 platform, with 150 bp paired-end reads. The bases with low quality and sequences from adapters in raw data were removed using fastp (version 0.20.0)[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The filtered reads were aligned to the reference genomes by using HISAT2 (version 2.1.0). FeatureCounts (version 2.0.1) in the Subread package was applied for gene abundance quantification[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTranscriptomic analyses included Principal Component Analysis (PCA) for dimensionality reduction. Differential expression analysis was carried out with the limma package (threshold: |log\u003csub\u003e2\u003c/sub\u003eFC| \u0026ge; 1.5, P\u0026thinsp;\u0026le;\u0026thinsp;0.05). Gene Ontology (GO) enrichment analysis for Biological Processes (BP) was performed on the list of differentially expressed genes using the clusterProfiler. Inflammatory cytokine expression (IL1b, TNF-α, IL4, IL6) was visualized using GraphPad Prism. Gene Set Enrichment Analysis (GSEA) was implemented evaluate functional pathways including \"Neuron Cellular Homeostasis\" and \"Acute Inflammatory Response\" based on MSigDB gene sets.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e \u003cp\u003eBar graphs show the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM., with error bars indicating the SEM. The sample size (n) denotes the number of independent biological replicates conducted in the study. To assess normality, a Kolmogorov\u0026ndash;Smirnov test was performed. Statistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Data analysis and graph generation were conducted using GraphPad Prism software (version 8; San Diego, CA) and Adobe Illustrator 2024 (Adobe, San Jose, USA). More details of statistical tests can be found in the figure legends and Supplemental Source Data.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Sciatic Nerve Electrical Stimulation Attenuates Intracranial Inflammation in Acute Ischemic Stroke.\u003c/h2\u003e \u003cp\u003eDuring the acute phase of stroke, with the progression of injury, microglia become activated and the intracranial concentrations of inflammatory factors gradually increase. To evaluate the impact of sciatic nerve stimulation on neuroinflammation, MCAO mice were randomized into MCAO-Sham and MCAO-ES groups. The success of the MCAO model was assessed by monitoring cerebral blood flow perfusion \u003cem\u003evia\u003c/em\u003e laser speckle contrast imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;C). The effects on neuroinflammation were evaluate by quantifying microglial activation in the infarct region and measuring inflammatory factor levels in the hemispheric infarction. We performed IBA1 immunostaining to assess the activation of microglia in the infarct region at 24 and 72 hours after stroke. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). The results showed that the density of IBA1\u003csup\u003e+\u003c/sup\u003e cells was higher in the ES group than in the sham group at both time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Analysis of microglial morphology further revealed that more intact branches were preserved in the ES group at 24 h, with this difference becoming more evident at 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), These results indicate that sciatic nerve stimulation modulates the recruitment and activation of microglia.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe levels of intracranial inflammatory factors were then examined. The infarcted hemispheres were collected for ELISA. The data showed, at 24 h, no significant difference in IL-6 concentration was found between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). At 72 hour, IL-6 levels were significantly lower in the ES group compared to the Sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). For TNF-α, the results showed no difference at 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI), whereas a profound decrease was detected in the ES group at 72 h. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). These results suggesting that sciatic nerve electrical stimulation significantly alleviated intracranial inflammation at the 72-hour.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Sciatic Nerve Stimulation Ameliorates Acute Ischemic Neural Damage\u003c/h2\u003e \u003cp\u003eTo assess the impact of sciatic nerve stimulation on neural injury, infarct volume was evaluated by TTC staining, and neuronal damage was examined using MAP2 immunofluorescence. For infarct volume, result showed that electrical stimulation significantly reduced infarct volume at both 24 and 72 hour compared to the sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). We evaluated neuronal injury using immunofluorescence staining for MAP2, a protein primarily distributed in neuronal cell bodies and dendrites. In healthy neurons, MAP2 exhibits a continuous filamentous pattern, but it becomes fragmented or beaded upon injury and is entirely ablated in severe cases. The results showed, at 24 h, neuronal injury was apparent in both groups; however, the density of MAP2-positive signals was higher in the ES group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). At 72 h, MAP2 loss and neuronal damage were further exacerbated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). These findings consistently indicated that ES effectively attenuates neural injury during the acute phase of ischemic stroke.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Transcriptomic Responses to Optogenetic Stimulation in the MCAO Mouse\u003c/h2\u003e \u003cp\u003eWe generated Prokr2-Ai32 mice by cross Prokr2-Cre mice and Ai32 mice, which express ChR2 and EYFP in \u003cem\u003eProkr2\u003c/em\u003e expression neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Anatomical examination of the L3\u0026mdash;L5 DRG revealed that this neuronal population was predominantly localized to afferent nerves, (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), and constituted 36\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8% of the total neuronal population in the DRG (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). Whole nerve electrophysiological revealed robust light-induced action potentials[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], suggesting that the Prokr2-positive nerves was activated by optical stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the transcriptomic effects of optogenetic activation of Prokr2-positive somatosensory nerve on the brain in mice, we collected brain samples from MCAO mice and performed RNA sequencing. Principal component analysis (PCA) revealed clear separation between the MCAO-Sham and MCAO-Light groups, indicating distinct global transcriptomic profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Differential expression analysis identified numerous significantly regulated genes, compared to the MCAO-sham group, the MCAO-light group exhibited upregulation of 667 genes and downregulation of 2417 genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). Gene Ontology (GO) enrichment analysis for Biological Processes (BP) revealed that the differentially expressed genes were significantly enriched in three primary categories: immune-related processes (e.g., leukocyte cell adhesion, regulation of leukocyte migration), inflammatory pathways (including IκB kinase/NF-κB signaling and regulation of inflammatory response), and developmental processes (such as aorta development and epithelial tube morphogenesis). These results suggest these genes are predominantly involved in immune regulation, inflammatory responses, and specific aspects of development (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003eAmong the regulated genes, several key pro-inflammatory cytokines (IL1β, TNF-α, IL4, IL6) were down-regulated in the MCAO-Light group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). Consistent with the results from electrical stimulation, these data further support the anti-inflammatory action of the intervention. Further GSEA indicated significant up-regulation of the \"neuronal cell homeostasis\" (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK) and down-regulation of the \"acute inflammatory response\" (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL) in the MCAO-Light group. These findings suggested that selective active Prokr2 positive nerves in sciatic nerve could promotes transcriptional programs related to neuronal homeostasis while suppressing neuroinflammatory.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Optogenetic Stimulation of Prokr2-positive sciatic nerve influence Neural Damage and acute inflammatory reaction.\u003c/h2\u003e \u003cp\u003eWe further evaluated neuroinflammation and neuronal damage in MCAO model after optogenetic stimulation. Infarct volume was evaluated by TTC staining, which revealed a significant reduction in the MCAO-Light group compared with the sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). We next performed immunostaining for IBA1 to assess microglial responses in the ischemic core (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The result showed the density of Iba1 cells was higher in the MCAO-light group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), while their branching complexity was significantly lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), collectively indicating attenuated microglial activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then measured the concentrations of IL-6 and TNF-α in the infarcted hemisphere. The data showed both cytokines were significantly lower in the MCAO-Light stimulation group than in sham (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, G). Furthermore, the result of MAP2 immunostaining demonstrated a higher level of MAP2 expression in the MCAO-light group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, I), suggesting a preservation of neuronal structure and mitigation of injury. In summary, optogenetic activation of Prokr2-positive somatosensory nerves attenuates intracranial inflammation and reduces neural damage during the acute phase of ischemic stroke.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study aimed to explore a novel therapeutic strategy for acute ischemic stroke by sciatic nerve stimulation. Our results suggested that both electrical stimulation of the sciatic nerve and optogenetic activation of its Prokr2-positive somatosensory nerves attenuated neuroinflammation and reduced neural damage after stroke. Specifically, our interventions led to a marked reduction in pro-inflammatory cytokines and morphological changes of microglia, which was accompanied by a decreased infarct volume and a higher preservation of MAP2, a key marker of neuronal integrity[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe first applied sciatic nerve electrical stimulation to investigate neural injury and neuroinflammation in acute ischemic stroke. The observed anti-inflammatory effect of sciatic nerve electrical stimulation in acute ischemic stroke aligns with previous findings that demonstrated its efficacy in mitigating inflammation across other pathological model[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe observed neuroprotection, evidenced by reduced infarct volume and higher preservation of MAP2, is likely a direct downstream consequence of the attenuated neuroinflammation. An exacerbated inflammatory response is a key driver of secondary brain injury after ischemia. By dampening the activation of microglia and the production of detrimental cytokines, our interventions presumably created a more permissive environment for neuronal survival and synaptic stability, thereby limiting the final extent of the damage.\u003c/p\u003e \u003cp\u003eHowever, the conclusions of this study must be interpreted within its limitations. Firstly, our investigation into the mechanism remains at a phenotypic level. This study establishes the therapeutic potential of nerve electrical stimulation in acute stroke but also highlights key areas for future investigation. Three major questions remain: first, what are the specific molecular pathways, such as the role of the cholinergic anti-inflammatory pathway (CAP) or other neuroimmune axes remain to be further elucidated[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Secondly, does nerve electrical stimulation remain effective in the subacute and chronic phases of stroke? [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] Thirdly, how do peripheral immune cells, such as T lymphocytes and neutrophils, contribute to the immunomodulatory response?[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] Expanding our analysis beyond microglia and a narrow cytokine profile to a full immunophenotypic study will be essential to completely unravel the mechanisms of nerve electrical stimulation.\u003c/p\u003e \u003cp\u003eDespite these limitations, our findings nonetheless underscore the considerable translational potential of sciatic nerve stimulation. Targeting severe central nervous system disorders such as stroke through sciatic nerve stimulation presents a therapeutically attractive strategy, potentially circumventing the challenges of direct cerebral interventions[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Future studies should prioritize elucidating the precise mechanisms of action, including the identification of key neuroimmune messengers. Moreover, leveraging the accessible anatomy of the sciatic nerve, it is worthwhile to explore minimally or non-invasive approaches that achieve comparable efficacy by transcutaneously stimulating its downstream branches. Translating this neuromodulatory strategy whether via non-invasive electrical stimulation or advanced bioelectronic medicine into an adjuvant therapy for acute stroke patients represents a highly promising future direction.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the Institutional Animal Committee of Army Medical University University (Approval No. AMUWEC2023, AMUWEC20234922).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData sharing information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (Grant number: 82300503), Natural Science Foundation of Chongqing, China (Grant number: CSTB2025NSCQ-GPX0647, CSTB2024NSCQ-MSX1121) and Young PhD Talents Cultivation Project of Xinqiao Hospital (2022YQB069, 2023YQB014).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQingwu Yang, Xiaofeng Cheng, and Shilun Zuo conceived the study, Ran Ran and Xiaofeng Cheng designed the experiments and wrote the manuscript. Ran Ran and Yuan Zhao performed animal studies and prepared illustrations. Ran Ran and Jin Zhao performed data analysis and statistics, Shuang Zhang, Changxiong Gong, and Changwei Guo participated in the project discussions and provided valuable revision suggestions for this work, Linlin Hu, Mengya Shan, Rong Wang and Chenhao Zhao provided valuable assistance during the preliminary electrical stimulation exploration process of this study, Zhaouyou Meng, Qi Xie, Yiliang Fang, Sen Lin, Qingwu Yang, Xiaofeng Cheng, and Shilun Zuo assisted in reviewing this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the staff of the Department of Neurology, Xinqiao Hospital for their assistance in the initiation of this research project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFeigin VL, Abate MD, Abate YH, Abd ElHafeez S, Abd-Allah F, Abdelalim A, et al. 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Neurorehabil Neural Repair 2010; 24: 263\u0026ndash;72. https: //doi.org/10.1177/1545968309349946.\u003c/li\u003e\n\u003cli\u003eGoyal M, Yu AYX, Menon BK, Dippel DWJ, Hacke W, Davis SM, et al. Endovascular Therapy in Acute Ischemic Stroke: Challenges and Transition From Trials to Bedside. Stroke 2016; 47: 548\u0026ndash;53. https: //doi. org/10. 1161/STROKEAHA. 115. 011426.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"translational-stroke-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trsr","sideBox":"Learn more about [Translational Stroke Research](http://jcmr-online.biomedcentral.com)","snPcode":"12975","submissionUrl":"https://submission.nature.com/new-submission/12975/3","title":"Translational Stroke Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Electrical Stimulation, Optogenetics, Ischemic Stroke, Transcriptome","lastPublishedDoi":"10.21203/rs.3.rs-9107953/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9107953/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIschemic stroke, a leading cause of neurological disability, occurs due to cerebrovascular occlusion, resulting in cerebral ischemia and hypoxia that lead to neurological deficits. In the acute phase, neuronal injury and exacerbated intracranial inflammation contribute to disease progression. Effective modulation of inflammation and reduction of neuronal damage during this critical period are essential for improving stroke outcomes.A middle cerebral artery occlusion (MCAO) mouse model was established. The effects of sciatic nerve electrical stimulation on intracranial inflammation and neural injury in the acute phase after stroke were evaluated using immunostaining, enzyme-linked immunosorbent assay (ELISA), and 2,3,5-triphenyltetrazolium chloride (TTC) staining. Furthermore, transcriptomic sequencing, immunostaining, ELISA, and TTC staining were employed to assess the outcomes of optogenetic activation of Prokr2-positive neuronal subpopulations within the sciatic nerve on intracranial inflammation and neural injury. Sciatic nerve stimulation was found to ameliorate intracranial inflammation and neuronal damage, as evidenced by reduced microglial activation, decreased pro-inflammatory cytokine levels, smaller cerebral infarct volume, and attenuated neuronal injury. Optogenetic activation of Prokr2-positive somatosensory neurons in the sciatic nerve induced distinct transcriptomic changes in the brain, attenuated acute inflammation, and promoted neuronal homeostasis. This study demonstrates that sciatic nerve stimulation alleviates intracranial inflammation and neuronal injury during the acute phase of ischemic stroke.\u003c/p\u003e","manuscriptTitle":"Sciatic Nerve Stimulation Attenuates Intracranial Inflammation and Neuronal Injury in Acute Ischemic Stroke","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-01 13:03:38","doi":"10.21203/rs.3.rs-9107953/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-24T20:11:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"289473615313861730617578492610838707806","date":"2026-04-04T23:49:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-30T13:52:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-24T15:09:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-16T01:17:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Translational Stroke Research","date":"2026-03-12T19:34:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"translational-stroke-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trsr","sideBox":"Learn more about [Translational Stroke Research](http://jcmr-online.biomedcentral.com)","snPcode":"12975","submissionUrl":"https://submission.nature.com/new-submission/12975/3","title":"Translational Stroke Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b6eb2945-d202-4fab-b412-2d5b40a80aae","owner":[],"postedDate":"April 1st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-01T13:03:38+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-01 13:03:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9107953","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9107953","identity":"rs-9107953","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}