Sequential Modulation of the cGAS-STING Pathway Promotes Spinal Cord Injury Repair

Sequential Modulation of the cGAS-STING Pathway Promotes Spinal Cord Injury Repair | 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 Sequential Modulation of the cGAS-STING Pathway Promotes Spinal Cord Injury Repair Qianqian Peng, Miao Gu, Hui Li, Lü Chen, Xin Wu, Min Yuan, Ling Luo, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8596952/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Spinal cord injury (SCI) is a devastating condition with limited treatment options, where dysregulated neuroinflammation critically impedes repair. The cGAS-STING pathway, a central cytosolic DNA-sensing axis of innate immunity, is implicated in neuroinflammatory disorders, yet its precise spatiotemporal role and therapeutic potential in SCI remain undefined. Given the complex, phase-specific nature of post-SCI immune responses, we hypothesized that a time-dependent modulation of this pathway, rather than continuous intervention, could optimally coordinate inflammation for repair. Methods Using a mouse model of thoracic compressive SCI, we assessed pathway activation via transcriptomics, western blot, and immunofluorescence. Motor recovery was evaluated longitudinally using the Basso Mouse Scale (BMS) and footprint analysis. Pharmacological agonists and antagonists of STING were administered either continuously or in a sequential regimen. Histological and ultrastructural analyses evaluated axonal regeneration, myelination, and glial scarring. RNA sequencing elucidated molecular mechanisms. Microglia-specific depletion using PLX5622 and in vitro neuron-microglia co-cultures were employed to determine cellular mechanisms. Results The cGAS-STING pathway was significantly activated post-SCI, primarily within microglia. Continuous pathway activation or inhibition failed to improve recovery. In contrast, sequential treatment (early agonist, late antagonist) significantly enhanced functional recovery, axonal regeneration, and remyelination while limiting glial scarring. Mechanistically, upregulated genes associated with microglial phagocytosis and chemotaxis, promoting debris clearance. Subsequent inhibition relieved inflammation, elevated anti-inflammatory cytokine and pro-regenerative programs. Microglial depletion completely abolished the therapeutic benefits of the sequential strategy, confirming their role as essential effector cells. Conclusion This study establishes the critical importance of timing in targeting the cGAS-STING pathway after SCI. We demonstrate that a phase-specific strategy that sequentially activates then inhibits this pathway optimally harnesses microglial functions for repair, offering a novel precision medicine approach for SCI. Spinal cord injury cGAS-STING Sequential modulation Microglia Neuroinflammation Regeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Globally, hundreds of thousands of individuals are diagnosed with spinal cord injuries (SCI) annually, with 90% of these cases attributed to traumatic events such as traffic accidents, falls, sports-related injuries, and acts of violence [ 1 ]. SCIs sever the neural communication between the brain and the spinal cord, resulting in severe and often permanent neurological deficits, including sensory and motor impairments, abnormal reflex arcs, and autonomic dysregulation. These deficits ultimately lead to a significant reduction in quality of life and may even shorten the patient's lifespan. [ 2 ]. Currently, the clinical management of SCI primarily involves early surgical decompression and stabilization, enhancement of spinal cord perfusion, intravenous administration of high-dose corticosteroids for anti-inflammatory therapy, and long-term neuro-rehabilitation training. Regrettably, the efficacy of these interventions remains limited [ 3 ]. Despite remarkable progress in modern medicine, recovery following SCI remains suboptimal and insufficient, with a continued paucity of effective therapeutic approaches and strategies for SCI management [ 4 ]. The pathophysiological process of SCI involves two phases: primary injury and secondary injury. Primary injury arises from acute mechanical trauma, causing neuronal, glial, and endothelial cell death, axonal disruption, blood-spinal cord barrier breakdown, vascular rupture, and other pathological alterations. These initial injuries release signaling molecules that trigger a cascade of events, including oxidative stress, excitotoxicity, metabolic disturbances, neuroinflammation, gliosis, extracellular matrix remodeling, demyelination, and scar formation, collectively referred to as secondary injury. Since primary injury is irreversible, the overall severity of SCI largely depends on the extent of secondary injury. Current therapeutic focus for SCI involves preserving the structural integrity of the spinal cord to the greatest extent possible while effectively mitigating secondary injury through targeted interventions [ 5 ]. Research has demonstrated that excessive activation of both adaptive and innate immune responses following SCI, along with the associated neuroinflammation, are the critical mechanisms underlying secondary injury, which significantly influence the severity of the injury [ 6 ]. Consequently, selectively modulating immune responses to minimize secondary injury has emerged as the principal therapeutic strategy for promoting SCI repair and improving functional recovery. Stimulator of interferon genes (STING), also known as MITA and MPYS, is an endoplasmic reticulum (ER)-associated signaling molecule. It collaborates with cyclic GMP-AMP synthase (cGAS) to detect abnormal double-stranded DNA (dsDNA) derived from pathogens, cellular damage, or stress responses through a complex and precise regulatory mechanism. In mammals, cGAS functions as a pattern recognition receptor (PRR) that senses dsDNA in the cytoplasm. Both microbial DNA (a pathogen-associated molecular pattern, PAMP) and endogenous DNA (a damage-associated molecular pattern, DAMP) can activate cGAS to produce the second messenger 2'3'-cyclic GMP-AMP (cGAMP). Upon binding to STING, cGAMP induces a conformational change in STING, leading to its autophosphorylation. Phosphorylated STING subsequently activates the downstream kinase TBK1, which in turn phosphorylates interferon regulatory factor 3 (IRF3) and nuclear factor-κB (NF-κB). The activated IRF3 and NF-κB translocate to the nucleus, promoting the secretion of type I interferons (Type I IFN) and other inflammatory factors, thereby initiating the transcription of innate immune genes [ 7 – 9 ]. As a core component of the innate immune system, the cGAS-STING pathway plays a critical role not only in microbial defense but also in various physiological and pathological processes, including autophagy, inflammation, aging, metabolic homeostasis, DNA damage response, autoimmunity, cancer biology, and cell death [ 10 ]. Additionally, in the nervous system, the cGAS-STING pathway has been implicated in the development of brain injury, neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, and neuropathic pain [ 11 – 13 ]. Recent studies have highlighted the significance of the cGAS-STING signaling pathway in the pathological process of SCI. Wang et al. demonstrated that STING expression is significantly upregulated after SCI, contributing to neuroinflammatory responses in secondary injury via the activation of mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways [ 14 ]. Wei et al. further revealed that the upregulation of mitofusin 2 (Mfn2) after SCI reduced the release of mitochondrial DNA (mtDNA) from microglia, thereby restricting the activation of the cGAS-STING pathway and alleviating neuroinflammation post-injury [ 15 ]. Fan et al. utilized the small molecule compound tryptanthrin to inhibit the cGAS-STING pathway, promoting the polarization of microglia toward the anti-inflammatory M2 phenotype and significantly enhancing functional recovery in SCI mice [ 16 ]. These findings collectively indicate that targeting the cGAS-STING signaling pathway may offer promising therapeutic strategies for promoting repair and functional recovery in SCI. In this study, using a mouse model of spinal cord compressive injury, we investigated the role of the cGAS-STING pathway in SCI. Our results demonstrated that the cGAS-STING pathway is predominantly activated in phagocytes, particularly microglia, following SCI. By employing pharmacological interventions with STING-specific antagonists (e.g., C-176 and H-151) or agonists (e.g., ADU-S100 and 2',3'-cGAMP) at distinct time windows post-SCI, we found that sequential activation followed by inhibition of the cGAS-STING pathway significantly enhances functional recovery after SCI. Further analysis revealed that the cGAS-STING pathway primarily exerts its effects through the regulation of microglial function. Specifically, during the early phase of SCI, activation of this pathway boosts the phagocytic capacity of microglia towards cellular debris; whereas in the later stages, inhibition of this pathway induces microglia to polarize toward an anti-inflammatory phenotype, thereby mitigating neuroinflammatory responses and promoting axonal regeneration and myelin formation. Notably, upon depletion of microglia, these effects were entirely abolished, confirming that microglia serve as the key effector cells for cGAS-STING pathway-mediated functions. Moreover, intervention in the cGAS-STING pathway effectively reduces glial scar expansion, further enhancing functional recovery after SCI. Importantly, based on the pathological progression of SCI, this study proposes a novel strategy involving precisely timed modulation of the cGAS-STING pathway to improve functional recovery following SCI. Materials and Methods Mice and SCI models Adult male C57BL/6 mice (6-8-weeks old, 20–22 g) were obtained from the Laboratory Animal Center of Nantong University and housed under specific pathogen-free (SPF) conditions in a controlled environment (25°C, 30–35% humidity, 12 h light/dark cycle) with free access to standard rodent chow and water. All experimental procedures were performed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Experimental Animal Ethics Committee of Nantong University. Investigators were blinded to the treatment groups throughout the experiments. For the SCI model, mice were anesthetized by inhalation of 5% isoflurane for 3–4 min and maintained under 2.5% isoflurane during surgery. Body temperature was maintained using a heating pad. Following skin preparation and disinfection, a laminectomy was performed at the T9–11 level to expose the T10 spinal cord. A compression injury was induced using a 5-gauge Dumont forceps (tip width 1 mm) applied bilaterally to the spinal cord for 5 s, producing a consistent 1 mm lesion. The ventral side of the spinal canal was gently scraped to prevent residual ventral cord tissue. After injury, muscle and skin layers were sutured with 6 − 0 absorbable sutures. This standard crush model resulted in complete spinal cord injury and hindlimb motor loss. Postoperatively, mice were kept in a temperature-controlled environment with free access to food and water, and manual bladder expression was performed twice daily. Sham-operated mice underwent laminectomy only, without compression. Behavioral/Locomotor Assessment Motor function was evaluated blindly by two independent observers using the Basso Mouse Scale (BMS) and footprint analysis one day before SCI and on days 1, 3, 7, 14, 21, and 28 post-injury (dpi). For BMS scoring, mice were allowed to walk freely in an open field for 5 min and were assessed based on gait, ankle movement, plantar stepping, trunk stability, coordination, and tail position, with scores ranging from 0 (no movement) to 9 (normal locomotion). Footprint analysis was used to objectively evaluate hindlimb function during quadrupedal locomotion. Hind paws were painted with red ink and forepaws with blue ink. Mice were then allowed to walk along a 50-cm-long straight runway lined with white paper. Footprints were collected and analyzed for placement, stride length, stride width, and overall gait characteristics. Immunofluorescent Staining For tissue staining, mice were transcardially perfused with 4% paraformaldehyde (PFA). Spinal cord segments extending approximately 0.5 cm rostral and caudal to the lesion epicenter were collected, post-fixed, dehydrated, embedded in OCT compound, and cut into serial transverse (20–40 µm) or horizontal (15 µm) cryosections using a cryostat. Sections were blocked for 1 h at room temperature in 5% bovine serum albumin (BSA) with 0.3% Triton X-100, followed by incubation overnight at 4°C with primary antibodies: anti-cGAS (79978, CST, 1:200), anti-STING (340061, CST, 1:400), anti-NeuN (ab177487, Abcam, 1:400), anti-GFAP (MAB360, Merck Millipore, 1:500), anti-Iba1 (016-20001, Wako, 1:1000), anti–β3-tubulin (5568, CST, 1:500), and anti-F4/80 (ab6640, Abcam, 1:200). After washing with PBS, sections were incubated for 1 h at room temperature with appropriate secondary antibodies (Jackson ImmunoResearch, 1:1000). Nuclei were counterstained with Hoechst 33342 (14533, Sigma, 1:5000). For cell staining, cultures were fixed with 4% PFA for 20 min at room temperature, washed with PBST, and blocked in 5% BSA with 0.1% Triton X-100 for 1 h. Subsequent steps were identical to tissue staining. Images were acquired using a confocal microscope (Leica, Germany) and analyzed with ImageJ and Photoshop (Adobe, USA). Specific color channels were separated, and total area was quantified for comparison. Luxol Fast Blue Staining Resin-embedded spinal cord tissues were sectioned at 2 µm thickness, with one section collected every 20 µm. The injury epicenter, identified as the region with the least myelinated axons, was examined under a light microscope. Sections were immersed in preheated 0.1% Luxol fast blue (LFB) solution for 4 h to stain myelin. After staining, sections were cooled to room temperature, rinsed sequentially in 95% ethanol and deionized water (3 min each), differentiated in a differentiation solution for 30 s, rinsed in 70% ethanol for 45 s, dehydrated, and mounted. Demyelinated areas were quantified from microscopic images using Image-Pro Plus software. Transmission Electron Microscopy Myelin regeneration was assessed by transmission electron microscopy (TEM). Tissue samples from the lesion core were rapidly dissected from three groups: sham, SCI (28 days post-injury), and compound-intervention (28 days post-injury). Tissues were cut into 1 mm³ blocks on ice and fixed in 2.5% glutaraldehyde for 4 h, followed by post-fixation in 1% osmium tetroxide for 1 h and embedding in Epon 812 epoxy resin. Ultrathin sections (50 nm) were stained with lead citrate and 1% uranyl acetate for 2 h and examined under a TEM (HT7700, Hitachi, Japan). Images were acquired under standardized brightness and contrast settings. Axon diameter, myelin thickness, number of myelin layers, and G-ratio were quantified blindly by two independent examiners using Image-Pro Plus software. A total of 252 axons from the control group, 204 from DMSO-treated mice, 326 from ADU-S100 + C176-treated mice, and 430 from C176 + ADU-S100-treated mice were analyzed. Quantitative Real‑Time PCR Total RNA was extracted from cells or spinal cords using TRIzol™ reagent (15596018CN, Invitrogen) according to the manufacturer's instructions. cDNA was synthesized using the SuperScript™ One-Step RT-PCR Kit with Platinum™ Taq DNA Polymerase (11732020, Invitrogen). Quantitative Real‑Time PCR (qRT‑PCR) was performed on A StepOne™ Real-Time PCR System. Gene expression levels were quantified using the 2−∆∆Ct method with Gapdh as the internal control. Primer sequences are listed in supplementary table 1 (Table S1 ). Each sample was assayed in triplicate. Western Blot Fresh spinal cord tissues or cells were lysed in the ice-cold RIPA buffer (P0013B, Beyotime) containing 100 mM PMSF (a protease inhibitor cocktail), incubated at 4°C for 30 min, and further disrupted by sonication. Protein concentrations were determined using BCA assay. The protein samples were separated by 10% or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Merck Millipore). After blocking with 5% skim milk for 2 h at room temperature, membranes were incubated overnight at 4°C with the primary antibodies: anti-cGAS (79978, CST, 1:1000), anti-STING (340061, CST, 1:1000), anti-TBK (3504, CST, 1:1000), anti-p-TBK1 (5483, CST, 1:1000), and GAPDH (p-TBK1, Proteintech, 1:8000). Subsequently, the membranes were washed three times with PBST and incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies (Pierce, 1:10000) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL, PerkinElmer) and quantified using Quantity One software (Bio-Rad). Pharmacological intervention Agonists (2',3'-cGAMP and ADU-S100) and inhibitors (H-151 and C-176) of cGAS-STING signaling were purchased from MedChemExpress (MCE). In vivo treatments were divided into “continuous intervention” and “time‑window intervention”, as outlined in Fig. 3 A and 4 A. For continuous intervention, 3 µL of compound (10 µM for agonists, 1 µM for inhibitors) injected at the lesion site immediately after SCI. Starting on day 1 post-injury, 100 µL of the same compound was administered daily via intraperitoneal (i.p.) injection to maintain sustained pathway modulation. For time‑window intervention, agonist (ADU-S100) and inhibitor (C-176) was injected at the lesion site after SCI; from day 1 to day 10, 100 µL was given daily via i.p. injection. On day 11, the compound was switched (agonist to inhibitor or vice versa), and administration continued until day 28. Control animals received vehicle only. For in vitro studies, agonists (2',3'-cGAMP and ADU-S100) and inhibitors (H-151 and C-176) were dissolved in sterile DMSO. Primary microglia, motor neurons, microglia–motor neuron cocultures were treated with agonists (10 µM) or inhibitors (1 µM). Microglia Depletion with PLX5622 PLX5622 (MCE, HY-114153), a brain-penetrant CSF1R inhibitor, enable long-term and specific depletion of microglia prior to and during pathological progression [ 17 ]. Mice received i.p. injections of PLX5622 (50 mg/kg, formulated in saline containing 10% DMSO, 40% PEG300, and 5% Tween‑80 as per manufacturer’s instructions) or vehicle twice daily for > 10 days. Detailed treatment schedules are shown in Figs. 6 A and 7 A. Primary Microglia Culture Microglia were separated from spinal cords as described before. Briefly, the spinal cords of 1- to 2-day-old mice were dissected, minced, and digested with 0.125% trypsin. The cell suspension was plated onto poly-D-lysine (PDL)-coated T-flasks and cultured in DMEM supplemented with 10% FBS, 1% penicillin, and 1% streptomycin. After 8–10 days, mixed glial cultures were shaken at 200 rpm for 30 min at 37°C to detach microglia. The supernatant was collected, centrifuged at 1200 rpm for 6 min, resuspended in complete medium, and used for subsequent experiments. Motor Neuron Culture and Axonal Injury Model Motor neurons from embryonic day 15 (E15) rat spinal cords as described [ 18 ]. Spinal cords were digested with 0.125% trypsin, gently triturated, and centrifuged at 400 × g for 10 min in 1.06 g/L Optiprep™ (Sigma) to isolate motor neurons. A total of 3 × 10 5 motor neurons were seeded into PDL-coated 24-well plates or the proximal chamber of microfluidic devices (SND450, Xona Microfluidics). Cultures were maintained in neurobasal medium (Gibco) supplemented with 10% FBS, 2% B27 (Invitrogen), 1% antibiotic-antimycotic solution, 0.5 mM L-glutamine, and 25 mM L-glutamate. Axonal injury models were constructed using two methods: vigorous pipetting and microfluidic technology. For the former, motor neurons were subjected to 50 vigorous pipetting cycles to induce axonal damage. For the latter, neurons were cultured on one side of the device for 48–72 h until processes crossed the microgroove barrier. Axons extending into the opposite chamber were then severed using a vacuum pump. Immunofluorescence confirmed abundant axons before transection and their absence afterward. In Vitro Coculture of Motor Neurons (Injured or Uninjured) with Microglia Microglia were cultured to 80% confluence, detached, and seeded at 1.5 × 10⁶ cells per well onto pre-plated motor neurons (injured or uninjured) in 24-well plates. Motor neurons had been allowed to adhere for 4 h prior to microglia addition. Microglia were adhered for 30 min in complete medium (DMEM/F12 + 20% FBS), which was then replaced with neuron-specific medium. This protocol maintained an optimal neuron-to-microglia ratio and supported axonal growth for subsequent assays. Cocultures were maintained for an additional 24 h before drug treatment to assess axonal outgrowth and inflammatory responses. Fluorescence Microsphere Phagocytosis Assay To evaluate the effect of STING pathway modulation on the phagocytic capacity of microglia, a fluorescence-based microsphere uptake assay was performed in microglia–injured motor neuron cocultures. After pretreatment with STING agonist or inhibitor for 12 h, fluorescent microspheres (Sigma, L3030-1ML) were added to the culture medium. Cells were incubated at 37°C for 1.5 h to allow phagocytosis. Subsequently, the medium containing unbound microspheres was carefully removed, and cells were gently washed with PBS to remove non-internalized beads. For quantification, 10 random fields per well were imaged under a fluorescence microscope (Zeiss). The percentage of microglia that had engulfed fluorescent microspheres was determined manually or using ImageJ software. The experiment was repeated three times independently, and a total of 30 fields were analyzed per treatment group. RNA Sequencing RNA sequencing was performed on an Illumina Novaseq X Plus platform by Genedenovo Biotechnology Co., Ltd (Guangzhou, China). Total RNA was isolated from spinal cords with TRIzol reagents (Invitrogen). RNA quality was evaluated with an Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit) assessing concentration, RIN, 28S/18S ratio, and fragment distribution. Raw reads were processed using SOAPnuke software to filter out low-quality reads. Clean reads were aligned to the reference genome using HISAT2, and gene expression level was calculated with RSEM. Differentially expressed genes (DEGs) were identified by the DEGseq with thresholds of |fold‑change| ≥2 and a q-value (FDR) < 0.05. Gene Ontology (GO) classification and functional enrichment analysis were performed based on DEGs. Bioinformatic analysis was conducted using the Omicsmart online platform ( http://www.omicsmart.com ) Statistical analysis Graphs and statistical analyses were made using Prism 8 software (GraphPad, CA, USA). Comparisons between two groups were performed using Student's t-test. For comparisons among three or more groups, one-way ANOVA followed by Tukey's multiple comparisons test was applied. Longitudinal data were analyzed by two-way repeated‑measures ANOVA with Tukey's post-hoc test. Data are presented as mean ± SEM. Significance levels were set as *p < 0.05, **p < 0.01, ***p < 0.001. All experiments were independently repeated at least three times.. Results Upregulation of cGAS/STING Pathway-Related Genes in the Spinal Cord following SCI Traumatic SCI causes motor and sensory dysfunction but is often followed by partial spontaneous recovery. Using a mouse model of thoracic SCI, we observed progressive motor recovery over a three-week period ( Fig. S1 ). Time-course transcriptome analysis of injured spinal cords at 3 and 14 days post-injury (dpi) versus sham controls revealed four distinct gene expression patterns: continuously downregulated (down), downregulated then upregulated (down-up), upregulated then downregulated (up-down), and continuously upregulated (up). Gene ontology analysis linked these patterns to specific biological processes: axonogenesis and neurotransmitter secretion (down), ion transport and nervous system development (down-up), neutrophil chemotaxis and angiogenesis (up-down), inflammation and innate immune regulation (up). Notably, genes related to neural regeneration or repair were minimally expressed during this period. Instead, inflammatory and immune responses were strongly upregulated during both acute and subacute phases after SCI (Fig. 1A and Table S1 ). Cross-group comparisons identified 2,425 differentially expressed genes (DEGs) commonly upregulated at both time points, primarily involved in immunity and inflammation, and 2,335 commonly downregulated DEGs, largely associated with ion transport and synaptic transmission (Fig. 1B-C and Table S2-3 ). Among these, key genes of the cGAS-STING pathway ( Sting1 , cGAS , IRF1 , IRF3 , IRF8 , TNF , and IL6 ) showed consistent and pronounced upregulation (Fig. 1D), which was confirmed by qPCR (Fig. 1E). Given the established role of inflammation in SCI recovery and the central function of the cGAS-STING pathway in immune regulation, we focused subsequent investigation on its involvement in the SCI recovery process. Activation and Microglial Predominance of the cGAS-STING Signaling Pathway after SCI We assessed the expression and activation of the cGAS-STING signaling pathway after SCI by Western blot. The results showed that protein levels of cGAS and STING were significantly upregulated post-SCI, peaking at day 7 and day 14, respectively, accompanied by enhanced phosphorylation of TBK1 (Fig. 2A), indicating sustained activation of the pathway during the injury phase. Double immunofluorescence labeling was used to determine the cellular localization of STING and cGAS relative to spinal cord cell markers (Fig. 2B-C and Fig. S2-7 ). In uninjured tissue, STING strongly colocalized with NeuN + neurons, partially present in F4/80 + microglia, but was not detected in GFAP + astrocytes. After SCI, STING expression increased significantly in F4/80 + cells and remained detectable in NeuN + cells (Fig. 2B and Fig. S2-4 ), suggesting a shift from neuronal to microglial expression. cGAS exhibited a similar localization pattern to STING: it was present in both NeuN + and F4/80 + cells under baseline conditions, but after injury, it became predominantly colocalized with F4/80 + microglia, with a partial retention in NeuN + cells (Fig. 2C and Fig. S5-7 ). Together, these findings indicate that SCI activates the cGAS-STING pathway, primarily within activated microglia and spared neurons. Continuous Activation or Inhibition of the cGAS-STING Pathway Fails to Promote SCI Repair Small-molecule compounds are widely utilized for modulating specific signaling pathways owing to their advantages in standardization and potential for clinical translation [19]. To explore the therapeutic role of the cGAS‑STING pathway in SCI, we employed well‑characterized pharmacological agents: the agonists ADU-S100 [20, 21] and 2',3'-cGAMP [22, 23], along with the antagonists C-176 [24] and H-151 [25]. We first examined the consequences of sustained pathway modulation. As outlined in Fig. 3A, compounds were administered intrathecally at the injury site immediately after SCI, followed by daily intraperitoneal injections from day 1 onward to maintain continuous activation or inhibition, with vehicle alone as control. Motor recovery was evaluated at multiple time points using the BMS scoring system and automatedfootprint analysis. BMS scores indicated that only ADU‑S100 elicited a transient improvement at 3 dpi, with no sustained benefit thereafter (Fig. 3B). Footprint analysis further confirmed that gait parameterssuch as stride length and base width did not differ significantly among groups (Fig. 3C-E). Consistent with these functional outcomes, immunostaining for the axonal marker Tuj1 in spinal cord tissues harvested at 28 dpi showed detectable axonal labeling in all groups, but with no significant differences in fluorescence intensity was observed (Fig. 3F), indicating that prolonged activation or inhibition of the cGAS-STING pathway has limited effects on axonal regeneration. Together, these data demonstrate that continuous pharmacological activation or inhibition of the cGAS-STING pathway fails to promote meaningful functional or structural recovery after SCI. The absence of a therapeutic effect may reflect a dual-phase role of the pathway: early activation could support repair through regulated inflammatory responses, whereas sustained signaling may become detrimental. Conversely, early inhibition might compromise beneficial innate immune functions and delay the clearance of damage-associated debris, thereby impeding tissue repair. Based on these observations, we hypothesized that time-restricted modulation of the cGAS-STING pathway —tailored to distinct pathological phases after SCI—could offer a more effective therapeutic benefits. Sequential Activation Followed by Inhibition of the cGAS-STING Pathway Enhances SCI Repair Building on the understanding that acute-phase inflammation can be neuroprotective but becomes harmful if dysregulated in the subacute phase [26], we devised a time-dependent therapeutic strategy modulating the cGAS-STING pathway with agonists and inhibitors during specific post-injury windows (Fig. 4A ) . Immediately after SCI, either the agonist ADU-S100 or the inhibitor C-176 was administered at the injury site. From day 1 to day 10, mice received daily intraperitoneal injections of the same compound. On day 11, treatment was switched: the agonist group received the inhibitor (designated ADU-S100→C-176), and vice versa (designated C-176→ADU-S100), continuing until day 28. BMS scores showed that from 14 dpi onward, the ADU-S100→C-176 group exhibited significantly better hindlimb motor recovery than the vehicle control group, whereas the C-176→ADU-S100 group showed no improvement (Fig. 4B). Footprint analysis revealed that stride length was significantly longer in the ADU-S100→C-176 group compared to both the control and C-176→ADU-S100 groups between 14 and 28 dpi (Fig. 4C-E). As stride length is a sensitive indicator of locomotor recovery, its increase supports the conclusion that the ADU-S100→C-176 treatment enhances motor function. Double immunostaining with Tuj1 and the glial‑scar component CSPG on spinal cord tissues collected at 28 dpi revealed that the ADU-S100→C-176 group possessed more Tuj1-positive fibers and smaller glial scars than the other groups. No significant differences were found between the C-176→ADU-S100 and control groups (Fig. 4F). These findings suggest that early activation followed by later inhibition of the pathway mitigates secondary injury and facilitates axonal regeneration. We further assessed axonal regeneration and myelination using LFB staining and TEM. LFB staining showed severe demyelination in all groups at 28 dpi, however, the ADU-S100→C-176 group displayed a smaller demyelinated area and visible remyelination at the lesion center (Fig. 5A). TEM confirmed a greater number of myelinated axons in this group ( Fig. 5B). Based on the classification proposed by Du et al.[27], axons with a g ratio 0.8 as remyelinated, and = 1 as unmyelinated (Fig. 5C). Quantitative analysis indicated that 81.3% of axons in the sham group were myelinated (g ratio < 0.8). After SCI, this proportion decreased across all groups, but the ADU-S100→C-176 group exhibited the lowest proportion of unmyelinated axons and the highest proportion of myelinated axons (Fig. 5D-E), indicating its superior capacity to promote remyelination. In conclusion, sequential modulation of the cGAS-STING pathway—activation followed by inhibition—significantly enhances repair after SCI. Microglial Depletion Abolishes the Beneficial Effects of Sequential cGAS-STING Pathway Modulation Microglia, key components of the innate immune system, play essential roles in structural remodeling and functional homeostasis of central nervous system [28]. Our findings demonstrated that cGAS and STING expression following SCI was primarily localized to activated microglia (Fig. 2), suggesting the pathway influences SCI pathology via microglial regulation. To test this, we used the CSF1R-specific inhibitor PLX5622 [17] to deplete microglia while targeting the cGAS-STING pathway within defined temporal windows. We first confirmed the efficacy of PLX5622, finding that intraperitoneal administration of three daily doses reduced microglial numbers by 90.3 ± 0.89% ( Fig. S8 ). Based on post-SCI microglial dynamics and pathway intervention timing, we established two depletion protocols: (1) Early depletion (Fig. 6A): PLX5622 administered from 3 days before SCI until 7 dpi (every 12 h); and (2) Late depletion (Fig. 7A): treatment began on day 9 post-SCI and continued until day 14. IBA1 staining confirmed a marked reduction in microglial numbers in all PLX5622-treated groups ( Fig. S9-10 ). BMS scores revealed that both early and late microglial depletion significantly impaired motor recovery in the ADU-S100→C-176, C-176→ADU-S100, and vehicle groups compared with their non-depleted counterparts. No significant differences in recovery were observed among the depleted treatment groups (Fig. 6B and 7B), indicating that microglial depletion abolished the functional benefits conferred by sequential cGAS-STING modulation. Notably, ADU-S100→C-176 treatment reduced neuronal apoptosis after SCI, as confirmed by caspase activation staining, however, this protective effect was lost upon microglial depletion (Fig. 6C). Consistent with these findings, microglial depletion led to increased demyelination areas (Fig. 7C) and decreased remyelination levels across all treatment groups (Fig. 7D). Given potential astrocyte-microglia crosstalk, we assessed whether astrocytes might compensate for microglial loss by engaging the cGAS-STING pathway. However, no cGAS or STING expression was detected in astrocytes ( Fig. S11 ), suggesting that astrocytes do not directly contribute to this regulatory mechanism. Collectively, these data demonstrate that the cGAS-STING pathway influences SCI recovery through a microglia-dependent mechanism. RNA Sequencing Reveals a Dual‑Phase Mechanism Underlying the Beneficial Effects of Sequential cGAS–STING Pathway Modulation To investigate the mechanism by which sequential modulation of the cGAS-STING signaling pathway promotes SCI repair, we performed RNA sequencing on spinal cord lesion tissues collected 3 and 14 days after treatment with ADU-S100→C-176, C-176→ADU-S100, or vehicle control. DEG analysis (screening criteria: |Fold Change| ≥ 2, Q value < 0.05) revealed that, compared to the vehicle group, early STING inhibition (C-176→ADU-S100) at 3 days post-treatment resulted in the significant upregulation of only one gene: MT3 (Metallothionein-3), also known as growth inhibitory factor (Fig. 8A and Table S4 ). MT3 is a CNS-enriched metalloprotein that maintains neuronal zinc and redox balance, thereby supporting neuronal survival and suppressing aberrant growth [29, 30]. This finding suggests that early STING inhibition after SCI may confer neuroprotective effects. Unexpectedly, MT3 was also significantly upregulated following early STING activation (ADU-S100→C-176 group), indicating that both activation and inhibition of STING can enhance MT3 expression (Fig. 8B and Table S4 ). This shared upregulation pattern suggests that MT3 may act as a common mediator bridging different immune states, promoting repair during SCI recovery by maintaining intracellular metal ion homeostasis and exerting neuroprotective functions. Notably, the expression levels of key inflammatory cytokines (such as TNF-α , IL-6 , and IL-1β ) showed no significant differences among the three groups, indicating that early intervention targeting the cGAS-STING pathway did not markedly modulate the intensity of the inflammatory response at this stage. To elucidate the downstream mechanisms regulated by STING activation, we ranked the DEGs from the ADU-S100→C-176 group at 3 days by expression level (Fig. 8B and Table S4 ). This analysis revealed strong upregulation of granzyme family members ( Gzma , Gzmb , Gzmc , Gzmf , Gzmd , etc.) and multiple chemokines (e.g., CXCL10 , Ccl5 , Ccl8 , Ccl2 , Ccl7 ). Granzymes are serine proteases that play a central role in immune clearance by inducing apoptosis in abnormal cells. They also participate in regulating inflammation and remodeling the extracellular matrix, thereby influencing cell migration and tissue repair [31]. We thus hypothesize that the significant upregulation of granzymes upon STING activation during the acute phase of SCI may accelerate the apoptosis and clearance of damaged cells. Concurrent upregulation of chemokines such as CXCL10 is closely associated with the recruitment of monocytes, macrophages, and neutrophils to the injury site [32]. The increased expression of Ly6C2, a key marker expressed on murine myeloid cells, particularly monocytes and newly recruited macrophages, further supports that STING activation promotes monocytes/macrophages accumulation at the lesion site. In summary, early cGAS-STING activation may facilitate subsequent repair by enhancing immune cell infiltration and accelerating the clearance of damaged cells. At 14 days post‑treatment (after 3 days of switched agonist/antagonist administration), the elevated expression levels of granzymes (e.g., Gzma , Gzmb , Gzmc ) and chemokines (e.g., CXCL10 , Ccl5 , Ccl8 , Ccl2 , Ccl7 ) observed in the ADU-S100→C-176 group had returned to baseline, showing no difference from the vehicle group. In contrast, the C-176→ADU-S100 group exhibited significantly increased expression of chemokines ( CXCL1 , Ccl5 , Ccl2 , Ccl7 ) and pro-inflammatory cytokines ( IL-6 and IL-1β ), suggesting that late-stage STING activation exacerbates the subacute inflammatory response. High expression of the neutrophil markers S100a8 and S100a9 indicated persistent neutrophil retention, further supporting this conclusion (Fig. 8C and Table S4 ). Notably, expression of the anti-inflammatory cytokine IL-10 was significantly increased in the ADU-S100→C-176 group. IL-10 is a key anti-inflammatory cytokine that potently inhibits the production of pro-inflammatory cytokines such as TNF-α, IL-1, IL-6, and IL-12. Its primary role is to limit and terminate immune responses, preventing excessive inflammation and subsequent tissue damage [33, 34]. Therefore, late-stage inhibition of the cGAS-STING pathway may alleviate subacute inflammation and create a favorable environment for neural repair, potentially through the induction of IL-10. We further analyzed the expression of genes associated with axonal regeneration. In the ADU-S100 → C-176 group, axon growth-related genes including Tubb3 , Map2 , Nefl , Nefh , Nefm , and Doublecortin ( DCX ) were significantly upregulated compared with the other groups, indicating that late STING inhibition promotes axonal regeneration. DCX is a microtubule-associated protein expressed in migrating neuroblasts and is an established marker for nascent axonal growth [35, 36]. Immunohistochemistry confirmed that DCX-positive signals were markedly stronger in the ADU-S100→C-176 group than that in the other groups (Fig. 8D), providing additional support for the conclusion that inhibiting the cGAS-STING pathway during the late phase facilitates axonal regeneration. In summary, sequential modulation of the cGAS-STING signaling pathway (ADU-S100→C-176) promotes repair through a temporally coordinated mechanism: early STING activation promotes the clearance of cellular debris and initiates necessary immune infiltration, while subsequent inhibition resolves inflammation and directly enables axonal regeneration. Activation of the cGAS–STING Pathway Promotes Axon Growth and Regeneration in Motor Neurons To investigate the role of the cGAS–STING pathway in axon growth and regeneration, we treated motor neurons for 24 h with its agonists (ADU‑S100 or 2',3'‑cGAMP), inhibitors (H‑151 or C-176), or vehicle control (Fig. 9A). CCK-8 assay showed no significant difference in cell viability, indicating no cytotoxicity of the compounds (Fig. 9B). qPCR analysis revealed that agonist significantly upregulated expression of the axonal growth marker GAP43, whereas inhibitor downregulated it (Fig. 9C). Consistent with this, immunocytochemistry revealed that agonists enhanced axon outgrowth, while inhibitors reduced it (Fig. 9D). For quantitative morphological assessment, axons were categorized by length as short (0–30 µm), medium (30–60 µm), long (60–100 µm), or very long (> 100 µm). Distribution analysis indicated a significantly higher proportion of neurons with long and very long axons in agonist-treated cultures, whereas inhibitor-treated groups exhibited increased proportions of neurons with short axons (Fig. 9D). Together, these results indicate that cGAS–STING activation promotes axon growth in motor neurons, whereas its inhibition impairs this process. We next evaluated the role of this pathway in axon regeneration using a microfluidic axotomy model. Neurons cultured in the left chamber extended axons into the right chamber, which were then severed by vacuum aspiration. Immunocytochemistry confirmed complete axonal removal in the right chamber, validating model integrity (Fig. 9E). After axotomy, application of agonists, inhibitors, or DMSO to neuronal sonata for 24 h revealed that agonist treatment significantly enhanced axon regeneration relative to the DMSO, whereas inhibitors did not exert a significant effect (Fig. 9F). In summary, these findings demonstrate that cGAS–STING pathway activation promotes both developmental axon growth and post-injury regeneration in motor neurons. The cGAS–STING Pathway in Microglia Regulates Neuroinflammation and Axonal Regeneration through Neuronal Interaction In vivo experiments demonstrated that depleting microglia significantly attenuates the beneficial effect of modulating the cGAS–STING pathway on functional recovery after SCI, suggesting that this pathway participate in repair processes by regulating microglia-mediated neuroinflammation. To further investigate its role in inflammation modulation and axonal regeneration, we established an in vitro coculture system consisting of microglia and motor neurons (injured or uninjured). Scanning electron microscopy revealed that after 24 h of coculture, microglia migrated and adhered to the neuronal surface, confirming the suitability of this system (Fig. 10A). Stimulation of the uninjured coculture system with IFN-γ plus LPS significantly upregulated the expression of the pro-inflammatory cytokines TNF-α and IL-6, but did not markedly alter the expression or activation of STING, indicating that such stimulation induces inflammation independently of cGAS–STING pathway (Fig. 10B). In contrast, coculture with injured neurons not only elevated inflammatory cytokine levels but also activated the STING pathway, suggesting that factors released from damaged neurons, such as dsDNA, may serve as a key trigger in microglia (Fig. 10C). Immunocytochemical analysis revealed that microglia cocultured with uninjured neurons retained a ramified, resting morphology, whereas those exposed to injured neurons adopted a spindle‑like shape with reduced branching, indicating a shift toward a polarized state. This morphological shift was further corroborated by the upregulation of established microglial polarization markers, such as iNOS , CCL2 and F/480 . Moreover, these polarized microglia were found to co-localize with neuronal markers, implying their potential involvement in phagocytosing cellular debris from injured neurons (Fig. 10D). Notably, while STING expression in uninjured cocultures was largely neuronal, it was detected in both neurons and microglia when cocultured with injured neurons (Fig. 10D), consistent with in vivo observations. We next treated the injured neuron–microglia coculture with STING agonists, inhibitors, or DMSO to evaluate their effects on axonal regeneration, inflammatory response, and microglial phagocytosis. Agonist treatment significantly upregulated the expression of the axonal growth-related genes GAP43 and Tubulin and promoted axonal regeneration, whereas inhibitors suppressed these effects (Fig. 10E). Furthermore, the proportion of microglia engulfing fluorescent microspheres was markedly higher in the agonist-treated group compared with the DMSO control, whereas the C-176-treated group showed significantly reduced phagocytic ability (Fig. 10F), indicating that activation of the cGAS–STING pathway enhances the capacity of microglia to clear injury-associated debris. In summary, although IFN-γ plus LPS stimulation induces inflammation in the uninjured neuron–microglia co-culture system, it does not activate the cGAS–STING pathway. In contrast, injured neurons trigger both activation of this pathway and associated inflammatory responses. Activation of cGAS–STING signaling in this co-culture system promotes axonal regeneration and enhances the phagocytic function of microglia. Discussion This study found that the cGAS-STING signaling pathway, a core sensor of innate immunity, is significantly activated after SCI, with its expression predominantly enriched in microglia. More importantly, we innovatively proposed and validated a temporal regulation strategy based on the pathological progression of the injury: activating the pathway in the early (acute) phase and inhibiting it in the later (subacute) phase. This sequential intervention of "activation followed by inhibition," compared to sustained activation or inhibition alone, more effectively coordinates microglia-mediated debris clearance and inflammation resolution, ultimately leading to significantly enhanced axonal regeneration, myelination, and motor functional recovery. This research not only reveals the complex time-dependent functions of the cGAS-STING pathway in SCI but also provides a new theoretical foundation and practical approach for developing precise phase-specific therapeutic strategies centered on immune modulation. 1. The Activation Pattern of the cGAS-STING Pathway Determines Its Biphasic Role in SCI The core function of the cGAS-STING pathway is to sense abnormal double-stranded DNA in the cytoplasm, whether derived from pathogens or endogenous DNA released due to tissue damage, cellular stress, or genomic instability [ 8 ]. In the context of SCI, damage leads to the rupture of neurons and glial cells, releasing mitochondrial DNA and genomic DNA, which serve as critical DAMPs. Our transcriptomic and protein analyses consistently showed significant upregulation of cGAS, STING expression, and phosphorylation of their downstream effector TBK1 in spinal cord tissue post-SCI, aligning with previous reports [ 37 ]. A key contribution of this study is the identification, via cellular co-localization analysis, that the activation of this pathway occurs primarily in phagocytic cells, especially microglia. Under physiological conditions, STING is basally expressed in neurons, possibly involved in DNA surveillance and homeostasis maintenance. After injury, microglia rapidly activate and upregulate cGAS/STING, becoming the primary effector cells for this pathway. This spatial distribution suggests that the cGAS-STING pathway likely serves as a crucial molecular switch for microglia to sense "danger signals" (e.g., dsDNA from disintegrating neurons) and initiate innate immune responses post-SCI. The sustained upregulation pattern implies that this pathway may play different roles at different stages of SCI: participating in necessary immune clearance during the acute phase, while prolonged activation may exacerbate secondary injury. Indeed, our pharmacological experiments showed that neither sustained agonism nor inhibition of the pathway improved functional recovery, indicating its phase-dependent functionality, a phenomenon analogous to the bidirectional effects reported for other inflammation-related pathways, such as NF-κB and NLRP3 [ 38 – 40 ]. 2. The "Activation-Followed-by-Inhibition" Strategy Promotes SCI Repair by Coordinating Immune Clearance and Inflammation Resolution Initial experiments revealed that neither continuous activation nor continuous inhibition of the cGAS-STING pathway significantly improved long-term motor function or axonal regeneration. This outcome underscores the functional complexity of this pathway in SCI repair and highlights the necessity for temporal intervention. The drawback of sustained activation lies in the fact that while the early robust inflammatory response aids in initiating clearance, its prolonged persistence leads to destructive chronic neuroinflammation, aggravating the loss of neurons and oligodendrocytes, potentially promoting the transformation of astrocytes into the regeneration-inhibitory A1 phenotype, and exacerbating glial scar formation. Conversely, the strategy of sustained inhibition, although aimed at controlling inflammation, may impair the essential phagocytic function required during the acute phase. This can result in the accumulation of injury debris, particularly inhibitory myelin debris, hindering the differentiation of oligodendrocyte precursor cells and remyelination, and delaying the initiation of tissue repair signals. Therefore, based on the concept that "inflammatory responses have a temporal window," we designed a sequential modulation regimen (ADU-S100→C-176). SCI involves distinct inflammatory phases: the acute phase (approximately days 1–10), where inflammation is primarily clearance-oriented and a prerequisite for repair; and the subacute phase (approximately days 10–28), where persistent inflammation becomes an obstacle to regeneration if not resolved. Our intervention matched these phases: administering the agonist ADU-S100 early (days 1–10) and switching to the inhibitor C-176 later (days 11–28). This strategy yielded results significantly superior to both the control group and the reverse sequential group (C-176→ADU-S100), strongly demonstrating that intervention targeting the cGAS-STING pathway must be "time-specific," precisely matching its function at different pathological stages. Mechanistically, RNA-seq data provided key insights: During the "early activation" phase, ADU-S100 treatment significantly upregulated the expression of granzyme family members ( Gzma , Gzmb , etc.) and chemokines (e.g., CXCL10 , CCL5 ). Granzymes may enhance the clearance efficiency of microglia towards apoptotic/necrotic cells, while chemokines help recruit peripheral immune cells to assist in debris cleanup. In vitro experiments directly confirmed that STING agonists enhance the phagocytic capacity of microglia. Thus, the core significance of early activation is "accelerating cleanup." During the "late inhibition" phase (day 14), the early activation group, after switching to the inhibitor, showed reduced levels of pro-inflammatory factors and a significant upregulation of the anti-inflammatory cytokine IL-10. IL-10 can promote the polarization of macrophages/microglia towards the reparative M2-like phenotype and support neuronal survival and axonal growth [ 34 , 41 ]. Concurrently, this group exhibited enhanced expression of axonal growth-related genes ( Tubb3 , Map2 , DCX , etc.). This indicates that the core role of late inhibition is "switching the mode," transforming the immune microenvironment from a pro-inflammatory state to an anti-inflammatory, pro-repair state, thereby lifting the inhibition on regeneration and activating regenerative programs. In summary, this strategy dynamically optimizes the immune response: activation in the acute phase accelerates clearance, removing obstacles for repair; inhibition in the subacute phase mitigates inflammation, creating a favorable environment for regeneration. 3. Microglia Are the Key Effectors Mediating cGAS–STING-Regulated Repair Following SCI, the interplay between microglia and neurons is central to the ensuing pathological cascade. This bidirectional communication critically regulates the intensity and persistence of neuroinflammation, with direct implications for neuronal survival, axonal regeneration, and functional recovery [ 42 , 43 ]. Notably, our findings reveal that cGAS and STING are expressed in both cell types after SCI, indicating a shared engagement in innate immune signaling during the injury response. To define the specific contribution of microglial cGAS-STING signaling to repair, we employed a combined strategy of temporal microglia depletion using PLX5622 and phased pathway modulation. The results were definitive: microglial depletion completely abolished the functional improvement, neuroprotection, and enhanced remyelination conferred by the sequential modulation strategy. This establishes microglia as the indispensable effector cells through which the timed modulation of the cGAS-STING pathway exerts its benefits. This conclusion aligns with our protein localization data, which showed predominant enrichment of cGAS and STING in activated microglia post-injury, with no compensatory upregulation in astrocytes. Supporting this in vivo mechanism, further in vitro coculture experiments demonstrated that injured neurons release factors which specifically activate the STING pathway in microglia, driving their polarization toward a phagocytic phenotype. This indicates that neuron-to-microglia “danger signaling” is a pivotal event initiating a reparative immune response. Therefore, the cGAS-STING pathway promotes recovery by dynamically modulating microglial function, thereby shaping a “permissive microenvironment” that supports axonal regeneration and tissue repair. 4. MT3: A Potential Downstream Hub Molecule Mediating Common Protective Effects Across Immune States An interesting finding was that both early activation and early inhibition of STING significantly upregulated the expression of MT3. MT3 is a central nervous system-enriched metal-binding protein widely recognized for for its neuroprotective properties [ 29 , 44 – 46 ]. It maintains neuronal homeostasis by chelating excess zinc ions and scavenging reactive oxygen species, thereby countering excitotoxicity and oxidative stress damage [ 30 , 47 ]. Recent advances have established MT3 as a compelling therapeutic target in neurological disorders due to its critical role in axonal integrity and neuroinflammatory regulation [ 48 – 51 ]. Given the pronounced excitotoxic and oxidative insults during the acute phase of SCI, we hypothesized that the upregulation of MT3 may represent an intrinsic neuroprotective response regulated by the cGAS-STING pathway (regardless of its activation or suppression), potentially helping neurons survive the initial inflammatory fluctuations. This suggests that distinct modes of cGAS-STING modulation may converge on shared protective mechanisms through common downstream effectors such as MT3. These results reveal a potential uncoupling between inflammatory phenotype dynamics and neuroprotection, raising the possibility that MT3 could function as a universal protective node. Whether MT3's protective roleoperates independently of inflammatory phenotype switching warrants future investigation. In summary, this study elucidates the dynamic and phase-dependent functions of the cGAS-STING pathway in the pathological progression of spinal cord injury. We demonstrate that while sustained activation or inhibition of this innate immune sensor fails to promote repair, a precisely timed sequential strategy—activating the pathway in the acute phase to enhance microglia-mediated debris clearance, followed by its inhibition in the subacute phase to resolve inflammation and foster a pro-regenerative milieu—significantly improves functional and structural recovery. Our findings establish microglia as the indispensable cellular effector through which temporal cGAS-STING modulation coordinates the immune response. Furthermore, the identification of MT3 as a convergent protective molecule highlights a potential shared axis of neuroprotection downstream of divergent immune signals (Fig. 11 ). Conclusion This study establishes the cGAS-STING pathway as a dynamically regulated, phase-dependent modulator of repair following SCI. It demonstrates that a time-sequenced therapeutic strategy can effectively coordinate immune clearance and inflammation resolution, SCI improving functional recovery. Collectively, our findings provide a novel conceptual framework for “chrono-immunomodulation” in CNS trauma, moving beyond static pathway manipulation towards interventions that are synchronized with the evolving pathology. This work not only advances the mechanistic understanding of neuro-immune crosstalk in SCI but also presents a translationally viable strategy with promising therapeutic implications for SCI and potentially other neuroinflammatory conditions. The illustration depicts the spatiotemporally specific and cell type-dependent roles of the cGAS-STING signaling pathway during post-SCI repair. Microglia, as the central effector cells of this sequential strategy, coordinate the transition from inflammatory clearance to regenerative repair through dynamic interactions with neurons. In the acute and early subacute phases, activation of cGAS-STING in neurons and microglia responds to damage-associated molecular patterns (e.g., dsDNA), initiates immune responses, and enhances the phagocytic capacity of microglia to clear cellular debris, thereby preparing the tissue for repair. In the late subacute and chronic phases, inhibition of this pathway alleviates persistent neuroinflammation, promotes microglial polarization toward an anti-inflammatory phenotype, and induces the expression of axonal regeneration-related genes, ultimately fostering a microenvironment conducive to functional recovery. Declarations Ethics approval and consent to participate All experimental procedures were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals, the institutional guidelines of Nantong University, and the Regulations for the Administration of Affairs Concerning Experimental Animals of China. The study was approved by the Institutional Review Board and the Ethics Committee of Nantong University. All animal experiments were performed in compliance with the relevant guidelines and regulations. Consent for publication Not applicable. Competing interests The authors declare no conflicts of interest. Funding This work was sponsored by the National Natural Science Foundation of China (grant No. 82272169, 32271418), the Natural Science Foundation of Hebei Province (grant No. C2024406010), and the Science Research Project of Hebei Education Department (grant No. QN2025285). Author Contribution YG conceived the overall study. All authors contributed to the study design. QQP, MG, HL, and XW performed the experiments including cell culture, cell biology experiments, molecular biology experiments, animal behavior experiments and morphology experiments. LC, MY, XW, and MLH analyzed the data. YG and CLZ provided methodology guidance. LL, and SRW performed and analyzed additional IHC. CLZ performed LFB staining and QC. QQP and MG prepared the manuscript and figures. YG, QQP, MG, CLZ oversaw the analysis and edited the manuscript. 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Metallothionein-3-mediated intracellular zinc mediates antioxidant and anti-inflammatory responses in the complete Freund's adjuvant-induced inflammatory pain mouse model. Cell Death Discov. 2025;11:45. Additional Declarations No competing interests reported. Supplementary Files Supplementaryfigureandlegends.docx TableS1.xlsx TableS2.xlsx TableS3.xlsx TableS4.xlsx TableS5.xlsx Cite Share Download PDF Status: Posted Version 1 posted 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. 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07:11:05","extension":"html","order_by":87,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":243609,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8596952/v1/0c4a0205156df16f35e0ff97.html"},{"id":100951675,"identity":"486ebbff-4d08-47d1-9dbb-b015ec0b1028","added_by":"auto","created_at":"2026-01-23 07:11:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5998558,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic profiling implicates the cGAS–STING pathway in the subacute phase of spinal cord injury.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Heatmap of gene expression changes in the injured spinal cord at 3 (SCI-3D) and 14 (SCI-14D) days post-injury (dpi) versus sham controls, identifying four distinct clusters (C1–C4). Gene Ontology (GO) enrichment analysis was performed for each cluster, with the top five enriched terms (by P value) displayed.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB\u003c/strong\u003e) \u003cem\u003eLeft\u003c/em\u003e: MA plot of differentially expressed genes (DEGs) identified from pairwise comparisons among Sham, SCI-3D, and SCI-14D groups (criteria: |log\u003csub\u003e2\u003c/sub\u003eFC| \u0026gt; 1 and \u003cem\u003eQ\u003c/em\u003e value \u0026lt; 0.05). \u003cem\u003eRight\u003c/em\u003e: Bar chart of representative DEGs.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eC\u003c/strong\u003e) Venn diagram of unique and shared up- or down-regulated DEGs at 7 and 14 dpi compared to the sham group. Representative enriched GO terms are shown.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eD\u003c/strong\u003e) Heatmap of expression levels for cGAS–STING pathway genes at 7 and 14 dpi and in sham controls (n=3 per group).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eE\u003c/strong\u003e) qPCR validation of mRNA expression for cGAS–STING pathway genes at 7 and 14 dpi relative to sham (n=3 per group). Data are mean ± SEM. Student’s t-test; **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001 vs. sham; ns, not significant.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8596952/v1/9551ffadcc879fe944bae25c.png"},{"id":100917890,"identity":"65a39a2d-62e5-4e92-95c0-e0728baa2250","added_by":"auto","created_at":"2026-01-22 19:10:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":28495508,"visible":true,"origin":"","legend":"\u003cp\u003eSpatiotemporal expression and cellular localization of the cGAS-STING pathway after SCI.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Western blot analysis of cGAS-STING pathway expression and activation in the injured spinal cord at the indicated time points. Uninjured tissue served as control (Ctrl), with GAPDH as a loading control. \u003cem\u003eLeft\u003c/em\u003e: Representative blots. \u003cem\u003eRight\u003c/em\u003e: Quantification (mean ± SEM; n=3). One-way ANOVA, *, **, ***, **** indicate p \u0026lt; 0.05, 0.01, 0.001, 0.0001 vs. Ctrl.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB\u003c/strong\u003e,\u003cstrong\u003eC\u003c/strong\u003e) Cellular localization of STING (B) and cGAS (C) shown by double immunofluorescence with spinal cord cell markers: NeuN (neurons), F4/80 (activated microglia), and GFAP (astrocytes). White arrowheads indicate co-localization. Scale bar: 200 μm (overview); 50 μm (magnified insets).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8596952/v1/11a69d26fbdfa2d85d68b2c4.png"},{"id":100917988,"identity":"3490496e-b2b3-44b6-89d7-025216245b6f","added_by":"auto","created_at":"2026-01-22 19:11:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":20786517,"visible":true,"origin":"","legend":"\u003cp\u003eSustained pharmacological modulation of the cGAS-STING pathway fails to promote functional recovery after SCI.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic of the drug regimen. Compounds were delivered intrathecally at the lesion site immediately after SCI, followed by daily intraperitoneal (i.p.) injections (days 1–28). Vehicle served as control.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB\u003c/strong\u003e) BMS locomotor scores over time after treated with STING agonists (2',3'-cGAMP, ADU-S100) or inhibitors (H-151, C-176). Data are mean ± SEM (n=8). Student's t-test; *p \u0026lt; 0.05 vs. vehicle; \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05, \u003csup\u003e###\u003c/sup\u003ep \u0026lt; 0.001 versus ADU-S100 group.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eC–E\u003c/strong\u003e) Footprint analysis (stride length and width) at 14 (\u003cstrong\u003eC\u003c/strong\u003e), 21 (\u003cstrong\u003eD\u003c/strong\u003e), and 28 (\u003cstrong\u003eE\u003c/strong\u003e) dpi. Forelimbs and hindlimbs were dyed blue and red, respectively. Data are mean ± SEM (n=5). Student’s t-test; *, **, *** indicate p \u0026lt; 0.05, 0.01, 0.001 vs. vehicle; ns, not significant.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eF\u003c/strong\u003e) \u003cem\u003eLeft\u003c/em\u003e: Axonal regeneration assessed by Tuj1 immunofluorescence at 28 dpi. Scale bars: 500 μm (overview); 100 μm (magnified insets). \u003cem\u003eRight\u003c/em\u003e: Quantification of Tuj1 fluorescence intensity in the lesion. Data are mean ± SEM (n=5); ns vs. vehicle.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8596952/v1/0ca6200222dad388930ab78d.png"},{"id":100917901,"identity":"0ebb388f-b30b-4b54-a45a-8278a5e826ef","added_by":"auto","created_at":"2026-01-22 19:10:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":22064008,"visible":true,"origin":"","legend":"\u003cp\u003eTime-switch modulation of the cGAS–STING pathway promotes functional and structural repair after SCI.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic of the sequential treatment. Mice received an intrathecal injection of agonist (ADU-S100) or inhibitor (C-176), then daily intraperitoneal injections (days 1–10). Treatments were crossed over (ADU-S100→C-176 or C-176→ADU-S100) from day 11 to 28. Vehicle served as control.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB\u003c/strong\u003e) BMS locomotor scores after time-switch treatment. Data are mean ± SEM (n=8). Student’s t-test; *, **, *** indicate p \u0026lt; 0.05, 0.01, 0.001 vs. vehicle; \u003csup\u003e##\u003c/sup\u003e \u003csup\u003e###\u003c/sup\u003e indicate p \u0026lt; 0.01, 0.001 vs. C-176+ADU-S100 group; ns, not significant.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eC–E\u003c/strong\u003e) Footprint analysis (stride length and width) at 14 (\u003cstrong\u003eC\u003c/strong\u003e), 21 (\u003cstrong\u003eD\u003c/strong\u003e), and 28 (\u003cstrong\u003eE\u003c/strong\u003e) dpi. Forelimbs (blue) and hindlimbs (red) were tracked. Data are mean ± SEM (n=6–8). Statistics as in (\u003cstrong\u003eB\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eF\u003c/strong\u003e) \u003cem\u003eLeft\u003c/em\u003e: Axonal regeneration and glial scar assessed by co-immunofluorescence for Tuj1 (axons, green) and CSPG (scar, red) at day 28. Scale bars: 500 μm (overview); 100 μm (magnified insets). \u003cem\u003eRight\u003c/em\u003e: Quantification of the Tuj1\u003csup\u003e+\u003c/sup\u003e and the CSPG\u003csup\u003e+ \u003c/sup\u003eareas. Data are mean ± SEM (n=3). *p \u0026lt; 0.05 vs. vehicle; #p \u0026lt; 0.05 vs. the C-176+ADU-S100 group; ns, not significant.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8596952/v1/0d8a7ec578c5ac160806d685.png"},{"id":100917953,"identity":"ba2cf196-89c7-477a-9ba5-1f2787f8467b","added_by":"auto","created_at":"2026-01-22 19:10:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":27129140,"visible":true,"origin":"","legend":"\u003cp\u003eSequential modulation of the cGAS–STING pathway enhances myelin preservation and remyelination after SCI.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Representative Luxol Fast Blue (LFB)-stained sections at 28 dpi after indicated treatment. Scale bar: 500 μm. Bar graph quantifies the demyelinated area. Data are mean ± SEM (n=3). Student’s t-test; **p \u0026lt; 0.01 vs. vehicle; \u003csup\u003e##\u003c/sup\u003ep \u0026lt; 0.01 vs. C-176+ADU-S100 group; ns, not significant.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB\u003c/strong\u003e) Representative transmission electron microscopy (TEM) images of the lesion epicenter.\u0026nbsp; Pseudocolor: green, thick myelinated axons; blue, thin remyelinated axons; pink, demyelinated axons. The ADU-S100+C-176 group had fewer demyelinated and more myelinated axons. Scale bar: 5 μm.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eC\u003c/strong\u003e) Schematic of axon categories identified in (\u003cstrong\u003eB\u003c/strong\u003e): myelinated (green), remyelinated (blue), and demyelinated (pink).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eD\u003c/strong\u003e) Scatter plot of g-ratio vs. axon diameter. The ADU-S100+C-176 group (red) distribution resembles sham (black), indicating favorable myelination vs. vehicle (blue) and C-176+ADU-S100 (green).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eE\u003c/strong\u003e) G-ratio frequency distribution. The ADU-S100+C-176 group has the highest proportion of axons with low g-ratios (thick myelin) and the fewest unmyelinated axons.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8596952/v1/51cb50620ead2b1966df0daf.png"},{"id":100917873,"identity":"2616ff2b-61a2-4107-8f58-c71208597fde","added_by":"auto","created_at":"2026-01-22 19:10:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":20356408,"visible":true,"origin":"","legend":"\u003cp\u003eEarly microglial depletion abolishes the therapeutic benefit of sequential cGAS–STING modulation.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic combining early microglial depletion (PLX5622, i.p., from 3 days pre- to 7 days post-SCI) with sequential pharmacological treatment (intrathecal then i.p. injections, crossover on day 11–14). Vehicle-treated mice served as controls.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB\u003c/strong\u003e) BMS locomotor scores at 3, 7, and 14 dpi. Data are mean ± SEM (n=6). **p \u0026lt; 0.01, ***p \u0026lt; 0.001 vs. SCI no treatment or vehicle-treated SCI group (Student’s t-test).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eC\u003c/strong\u003e) Neuronal apoptosis at 7 dpi assessed by co-immunofluorescence for NeuN (red) and cleaved caspase-3 (green). White arrowheads indicate apoptotic neurons. Scale bars: 200 μm (overview), 50 μm (magnified insets). Bar graph quantifies apoptotic neurons per unit area. Data are mean ± SEM (n=5). *, ** indicate p \u0026lt; 0.05, 0.01 vs. the corresponding therapeutic group without microglial depletion; \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05 versus vehicle-treated SCI group (Student’s t-test).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8596952/v1/4658bd0fbe0ca14ae5ae8126.png"},{"id":100917891,"identity":"ab8d0b97-7305-4ef2-a54c-a57c16b756f9","added_by":"auto","created_at":"2026-01-22 19:10:25","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":30567020,"visible":true,"origin":"","legend":"\u003cp\u003eLate-phase microglial depletion impairs the sustained therapeutic effects of sequential cGAS–STING modulation.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic combining sequential drug treatment (days 0–28) with late microglial depletion (PLX5622, i.p., days 9–14 post-SCI).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB\u003c/strong\u003e) BMS locomotor scores at 14, 21, and 28 dpi. Data are mean ± SEM (n=6). *, **, *** indicate p \u0026lt; 0.05, 0.01, 0.001 vs. vehicle-treated SCI group (Student’s t-test).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eC\u003c/strong\u003e) Myelin preservation by LFB staining at 28 dpi. Dashed box outlines demyelinated area. Scale bar: 500 μm. Bar graph quantifies the area. Data are mean ± SEM (n=3). *, **indicate p \u0026lt; 0.05, 0.01 vs. the corresponding group without microglial depletion; \u003csup\u003e#\u003c/sup\u003ep \u0026lt; 0.05 vs. vehicle-treated SCI group (Student’s t-test).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eD\u003c/strong\u003e) Ultrastructural analysis by TEM at 28 dpi. Scale bar: 5 μm. Histogram shows g-ratio frequency distribution. Late microglial depletion impaired remyelination similarly across groups.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8596952/v1/20b3f4e3ccebbc4e63f68e0f.png"},{"id":100917909,"identity":"2b700cce-bd7d-4c1f-8266-7761a66d6d31","added_by":"auto","created_at":"2026-01-22 19:10:26","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":10933968,"visible":true,"origin":"","legend":"\u003cp\u003eSequential cGAS–STING modulation orchestrates a biphasic transcriptional program, enabling a regenerative niche for SCI repair.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) RNA-seq of spinal cord lesions 3 and 14 days after indicated treatment. DEGs were identified (threshold: |Fold Change| ≥ 2, Q \u0026lt; 0.05). Plot shows the number of DEGs; y-axis, Log\u003csub\u003e2\u003c/sub\u003eFC (red: up-regulated; green: down-regulated)\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB\u003c/strong\u003e) Early-phase transcriptome (3 days). Venn diagram of up-regulated DEGs vs. vehicle. MT3 was the sole common gene; 924 DEGs were unique to the ADU‑S100+C‑176 group. Heatmap (Z-score) and table of mean TPM values for MT3 and selected genes related to immune clearance, recruitment, and neuronal processes (criteria: |Fold change| ≥ 2, Q \u0026lt; 0.05). Bar graphs are qPCR validation. Data are mean ± SEM (n=3). *, **, ***, **** indicate p \u0026lt; 0.05, 0.01, 0.001, 0.0001 vs. vehicle; \u003csup\u003e#\u003c/sup\u003e, \u003csup\u003e##\u003c/sup\u003e, \u003csup\u003e###\u003c/sup\u003e, \u003csup\u003e####\u003c/sup\u003e indicate p \u0026lt; 0.05, 0.01, 0.001, 0.0001 vs. C‑176+ADU‑S100 group (Student’s t-test).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eC\u003c/strong\u003e) Late-phase transcriptome (14 days). \u003cem\u003eLeft\u003c/em\u003e: Heatmap (Z-score) of selected up-regulated DEGs related to immune regulation, axon regeneration, myelin, cytokines, and cell markers. \u003cem\u003eMiddle\u003c/em\u003e: Table of TPM values and Log\u003csub\u003e2\u003c/sub\u003eFC vs. vehicle (significance: *, **, ***, **** for Q \u0026lt; 0.05, 0.01, 0.001, 0.0001). \u003cem\u003eRight\u003c/em\u003e: qPCR validation. Data and statistics as in (\u003cstrong\u003eB\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eD\u003c/strong\u003e) \u003cem\u003eLeft\u003c/em\u003e: Axonal regeneration assessed by immunofluorescence for DCX (a nascent axonal marker) at day 14. Scale bars: 500 μm (overview); 200 μm (magnified insets). \u003cem\u003eRight\u003c/em\u003e: Quantification of the DCX\u003csup\u003e+\u003c/sup\u003e area. Data are mean ± SEM (n=4). **p \u0026lt; 0.01 vs. vehicle; \u003csup\u003e##\u003c/sup\u003ep \u0026lt; 0.01 vs. the C-176+ADU-S100 group; ns, not significant (Student’s t-test).\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-8596952/v1/203728069ad45e3b0625008b.png"},{"id":100917913,"identity":"b764c254-1e02-4380-9d80-bb56cc51c76f","added_by":"auto","created_at":"2026-01-22 19:10:26","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":15523636,"visible":true,"origin":"","legend":"\u003cp\u003e. cGAS–STING modulation directly controls axonal growth and regeneration in spinal motor neurons in vitro\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic for testing the STING agonist/inhibitor effects on the axonal growth of cultured spinal motor neurons.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB\u003c/strong\u003e) CCK‑8 assay shows no cytotoxic effect on motor neuron viability. Data are mean ± SEM (n=5); ns vs. vehicle (Student’s t-test).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eC\u003c/strong\u003e) qPCR of GAP43 expression. Agonist increased, inhibitor decreased expression vs. vehicle (DMSO). Data are mean ± SEM (n=4). **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eD\u003c/strong\u003e) (\u003cem\u003ed1\u003c/em\u003e) Representative TUJ1 immunocytochemistry. Agonist increased, inhibitor decreased neurite length. Scale bars: 50 μm. (\u003cem\u003ed2\u003c/em\u003e) Axon length distribution (short: 0–30 μm; medium: 30–60 μm; long: 60–100 μm; very long: \u0026gt;100 μm). (\u003cem\u003ed3-d4\u003c/em\u003e). Quantification of longest axon per neuron (350–480 cells) and average length per field (40 fields). Data are mean ± SEM. ****p \u0026lt; 0.0001 versus vehicle.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eE\u003c/strong\u003e) \u003cem\u003eTop:\u003c/em\u003e Schematic of the microfluidic axotomy model. \u003cem\u003eBottom\u003c/em\u003e: Representative image post-axotomy (vacuum aspiration in the right chamber) Scale bars: 100 μm.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eF\u003c/strong\u003e) Representative images of axon regeneration in the microfluidic model. Agonist promoted robust regrowth. Scale bars: 100 μm. Bar graph showing the quantification of total axonal length (fold change vs. vehicle). Data are mean ± SEM (n=4). **p \u0026lt; 0.01, ****p \u0026lt; 0.0001 vs. vehicle; ns, not significant (Student’s t-test).\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-8596952/v1/4ebf549aaeebd5b9c7b8a4e6.png"},{"id":100917915,"identity":"a8bdfaa4-e358-43d1-af2f-2429f20395cf","added_by":"auto","created_at":"2026-01-22 19:10:26","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":12349597,"visible":true,"origin":"","legend":"\u003cp\u003eMicroglia sense neuronal injury via the cGAS–STING pathway, regulating neuroinflammation and phagocytosis to influence axon regeneration\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Microglia–motor neuron coculture establishment. (\u003cem\u003ea1\u003c/em\u003e) Schematic. (\u003cem\u003ea2\u003c/em\u003e) Scanning electron microscopy images. Red/green arrows indicate motor neurons/microglia. Scale bar: 5 µm.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eB\u003c/strong\u003e) Induction of inflammation in coculture with intact neurons. (\u003cem\u003eb1\u003c/em\u003e) Schematic of IFNγ + LPS induction. (\u003cem\u003eb2\u003c/em\u003e) qPCR of \u003cem\u003eTNFα\u003c/em\u003e, I\u003cem\u003eL-6\u003c/em\u003e, and \u003cem\u003eSTING\u003c/em\u003e. Data are mean ± SEM (n=5–6). ****p \u0026lt; 0.0001 vs. vehicle; ns (Student’s t-test). (\u003cem\u003eb3\u003c/em\u003e) Western blot of STING pathway. \u003cem\u003eLeft\u003c/em\u003e: Representative blots. \u003cem\u003eRight\u003c/em\u003e: Quantification (mean ± SEM; n=3). ns vs. vehicle (Student’s t-test).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eC\u003c/strong\u003e) Enhanced response in coculture with injured neurons. (\u003cem\u003ec1\u003c/em\u003e) Schematic. (\u003cem\u003ec2\u003c/em\u003e) qPCR of \u003cem\u003eTNFα\u003c/em\u003e and \u003cem\u003eIL-6\u003c/em\u003e vs. controls (microglia alone, neurons alone, or coculture with intact neurons). Data are mean ± SEM (n=6). ****P\u0026lt;0.0001 vs. each control (Student’s t-test). (\u003cem\u003ec3\u003c/em\u003e) Western blot of STING pathway in injured cocultures. \u003cem\u003eLeft\u003c/em\u003e: Representative blots. \u003cem\u003eRight\u003c/em\u003e: Quantification (mean ± SEM; n=3). *, **, ***, **** indicate p \u0026lt; 0.05, 0.01, 0.001, 0.0001 vs. vehicle (Student’s t-test).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eD\u003c/strong\u003e) Microglial polarization and STING upregulation in response to neuronal injury. (\u003cem\u003ed1\u003c/em\u003e) Double immunofluorescence for ChAT (motor neurons) and IBA1 (microglia). White arrowheads indicate polarized, potentially phagocytic microglia. Scale bar: 50 µm. (\u003cem\u003ed2\u003c/em\u003e) qPCR of microglial polarization markers (\u003cem\u003eiNOS\u003c/em\u003e, \u003cem\u003eCCL2\u003c/em\u003e, \u003cem\u003eF/480\u003c/em\u003e, and \u003cem\u003eCD68\u003c/em\u003e). Data are mean ± SEM (n=6). **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p\u0026lt;0.0001 (Student’s t-test). (\u003cem\u003ed3\u003c/em\u003e) STING and IBA1 double staining shows increased STING in microglia within injured cocultures. Scale bar: 50 µm.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eE\u003c/strong\u003e) STING modulation regulates axonal regrowth in injured coculture. (\u003cem\u003ee1\u003c/em\u003e) Schematic. (\u003cem\u003ee2\u003c/em\u003e) \u003cem\u003eLeft\u003c/em\u003e: TUJ1 immunocytochemistry. Scale bar: 50 µm. \u003cem\u003eRight\u003c/em\u003e: Quantification of longest axon (300–350 cells) and average length (20–30 fields). Data are mean ± SEM. **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001 vs. vehicle (Student’s t-test). (\u003cem\u003ee3\u003c/em\u003e) qPCR of \u003cem\u003eGAP43\u003c/em\u003e and \u003cem\u003eTubulin\u003c/em\u003e. Data are mean ± SEM (n=5). *, **, ***, **** indicate p \u0026lt; 0.05, 0.01, 0.001, 0.0001 vs. vehicle (Student’s t-test).\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eF\u003c/strong\u003e) STING activation enhances microglial phagocytosis. (\u003cem\u003ef1\u003c/em\u003e) Schematic of the fluorescent bead phagocytosis assay. (\u003cem\u003ef2\u003c/em\u003e) Representative images. White arrowheads indicate bead-engulfing microglia. Scale bars: 50 μm. Bar graph is percentage of phagocytic microglia (30 fields). Data are mean ± SEM. *p \u0026lt; 0.05, ****p \u0026lt; 0.0001 versus vehicle (Student’s t-test).\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-8596952/v1/1f42b1109b0acf8544316630.png"},{"id":100917883,"identity":"7f23fcf4-b895-480c-b1e9-127be53f538f","added_by":"auto","created_at":"2026-01-22 19:10:24","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":15871769,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic summary of the time-dependent modulation mechanism of the cGAS-STING pathway after SCI\u003c/p\u003e\n\u003cp\u003eThe illustration depicts the spatiotemporally specific and cell type-dependent roles of the cGAS-STING signaling pathway during post-SCI repair. Microglia, as the central effector cells of this sequential strategy, coordinate the transition from inflammatory clearance to regenerative repair through dynamic interactions with neurons. In the acute and early subacute phases , activation of cGAS-STING in neurons and microglia responds to damage-associated molecular patterns (e.g., dsDNA), initiates immune responses, and enhances the phagocytic capacity of microglia to clear cellular debris, thereby preparing the tissue for repair. In the late subacute and chronic phases , inhibition of this pathway alleviates persistent neuroinflammation, promotes microglial polarization toward an anti-inflammatory phenotype, and induces the expression of axonal regeneration-related genes, ultimately fostering a microenvironment conducive to functional recovery.\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-8596952/v1/f4f7f4c246d504f4654c53b2.png"},{"id":100917986,"identity":"be4b0021-5db9-430d-9b3b-9dfc3773c60b","added_by":"auto","created_at":"2026-01-22 19:11:13","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15109160,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigureandlegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-8596952/v1/db5bcd08e495764ad34abde1.docx"},{"id":100917932,"identity":"f31491d2-506c-4dd9-bbcc-16aff5aeb88f","added_by":"auto","created_at":"2026-01-22 19:10:28","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18936,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8596952/v1/2c70a07100b533419cd278cf.xlsx"},{"id":100917987,"identity":"6791e902-dc3d-40be-a37a-9dd90f6cae41","added_by":"auto","created_at":"2026-01-22 19:11:13","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1639474,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8596952/v1/962ed5acb5cf313cb059f85d.xlsx"},{"id":100951754,"identity":"52d16a90-6675-428a-9c2b-f338319aa2d7","added_by":"auto","created_at":"2026-01-23 07:11:11","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":24529,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8596952/v1/69857a9cc24508bde94e1c2d.xlsx"},{"id":100917907,"identity":"a4b2788b-3367-4c7e-9d2b-48fbf875069a","added_by":"auto","created_at":"2026-01-22 19:10:26","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":379311,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8596952/v1/8597c1e8eba249c79a70abde.xlsx"},{"id":100951823,"identity":"7abf2bd2-83b0-4b11-b148-3b2fa05be98c","added_by":"auto","created_at":"2026-01-23 07:11:19","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":15384,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8596952/v1/df6d4493a1c6e46c172ecbca.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sequential Modulation of the cGAS-STING Pathway Promotes Spinal Cord Injury Repair","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlobally, hundreds of thousands of individuals are diagnosed with spinal cord injuries (SCI) annually, with 90% of these cases attributed to traumatic events such as traffic accidents, falls, sports-related injuries, and acts of violence [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. SCIs sever the neural communication between the brain and the spinal cord, resulting in severe and often permanent neurological deficits, including sensory and motor impairments, abnormal reflex arcs, and autonomic dysregulation. These deficits ultimately lead to a significant reduction in quality of life and may even shorten the patient's lifespan. [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Currently, the clinical management of SCI primarily involves early surgical decompression and stabilization, enhancement of spinal cord perfusion, intravenous administration of high-dose corticosteroids for anti-inflammatory therapy, and long-term neuro-rehabilitation training. Regrettably, the efficacy of these interventions remains limited [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Despite remarkable progress in modern medicine, recovery following SCI remains suboptimal and insufficient, with a continued paucity of effective therapeutic approaches and strategies for SCI management [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe pathophysiological process of SCI involves two phases: primary injury and secondary injury. Primary injury arises from acute mechanical trauma, causing neuronal, glial, and endothelial cell death, axonal disruption, blood-spinal cord barrier breakdown, vascular rupture, and other pathological alterations. These initial injuries release signaling molecules that trigger a cascade of events, including oxidative stress, excitotoxicity, metabolic disturbances, neuroinflammation, gliosis, extracellular matrix remodeling, demyelination, and scar formation, collectively referred to as secondary injury. Since primary injury is irreversible, the overall severity of SCI largely depends on the extent of secondary injury. Current therapeutic focus for SCI involves preserving the structural integrity of the spinal cord to the greatest extent possible while effectively mitigating secondary injury through targeted interventions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Research has demonstrated that excessive activation of both adaptive and innate immune responses following SCI, along with the associated neuroinflammation, are the critical mechanisms underlying secondary injury, which significantly influence the severity of the injury [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Consequently, selectively modulating immune responses to minimize secondary injury has emerged as the principal therapeutic strategy for promoting SCI repair and improving functional recovery.\u003c/p\u003e \u003cp\u003eStimulator of interferon genes (STING), also known as MITA and MPYS, is an endoplasmic reticulum (ER)-associated signaling molecule. It collaborates with cyclic GMP-AMP synthase (cGAS) to detect abnormal double-stranded DNA (dsDNA) derived from pathogens, cellular damage, or stress responses through a complex and precise regulatory mechanism. In mammals, cGAS functions as a pattern recognition receptor (PRR) that senses dsDNA in the cytoplasm. Both microbial DNA (a pathogen-associated molecular pattern, PAMP) and endogenous DNA (a damage-associated molecular pattern, DAMP) can activate cGAS to produce the second messenger 2'3'-cyclic GMP-AMP (cGAMP). Upon binding to STING, cGAMP induces a conformational change in STING, leading to its autophosphorylation. Phosphorylated STING subsequently activates the downstream kinase TBK1, which in turn phosphorylates interferon regulatory factor 3 (IRF3) and nuclear factor-κB (NF-κB). The activated IRF3 and NF-κB translocate to the nucleus, promoting the secretion of type I interferons (Type I IFN) and other inflammatory factors, thereby initiating the transcription of innate immune genes [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. As a core component of the innate immune system, the cGAS-STING pathway plays a critical role not only in microbial defense but also in various physiological and pathological processes, including autophagy, inflammation, aging, metabolic homeostasis, DNA damage response, autoimmunity, cancer biology, and cell death [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Additionally, in the nervous system, the cGAS-STING pathway has been implicated in the development of brain injury, neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, and neuropathic pain [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent studies have highlighted the significance of the cGAS-STING signaling pathway in the pathological process of SCI. Wang et al. demonstrated that STING expression is significantly upregulated after SCI, contributing to neuroinflammatory responses in secondary injury via the activation of mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Wei et al. further revealed that the upregulation of mitofusin 2 (Mfn2) after SCI reduced the release of mitochondrial DNA (mtDNA) from microglia, thereby restricting the activation of the cGAS-STING pathway and alleviating neuroinflammation post-injury [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Fan et al. utilized the small molecule compound tryptanthrin to inhibit the cGAS-STING pathway, promoting the polarization of microglia toward the anti-inflammatory M2 phenotype and significantly enhancing functional recovery in SCI mice [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These findings collectively indicate that targeting the cGAS-STING signaling pathway may offer promising therapeutic strategies for promoting repair and functional recovery in SCI.\u003c/p\u003e \u003cp\u003eIn this study, using a mouse model of spinal cord compressive injury, we investigated the role of the cGAS-STING pathway in SCI. Our results demonstrated that the cGAS-STING pathway is predominantly activated in phagocytes, particularly microglia, following SCI. By employing pharmacological interventions with STING-specific antagonists (e.g., C-176 and H-151) or agonists (e.g., ADU-S100 and 2',3'-cGAMP) at distinct time windows post-SCI, we found that sequential activation followed by inhibition of the cGAS-STING pathway significantly enhances functional recovery after SCI. Further analysis revealed that the cGAS-STING pathway primarily exerts its effects through the regulation of microglial function. Specifically, during the early phase of SCI, activation of this pathway boosts the phagocytic capacity of microglia towards cellular debris; whereas in the later stages, inhibition of this pathway induces microglia to polarize toward an anti-inflammatory phenotype, thereby mitigating neuroinflammatory responses and promoting axonal regeneration and myelin formation. Notably, upon depletion of microglia, these effects were entirely abolished, confirming that microglia serve as the key effector cells for cGAS-STING pathway-mediated functions. Moreover, intervention in the cGAS-STING pathway effectively reduces glial scar expansion, further enhancing functional recovery after SCI. Importantly, based on the pathological progression of SCI, this study proposes a novel strategy involving precisely timed modulation of the cGAS-STING pathway to improve functional recovery following SCI.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMice and SCI models\u003c/h2\u003e \u003cp\u003eAdult male C57BL/6 mice (6-8-weeks old, 20\u0026ndash;22 g) were obtained from the Laboratory Animal Center of Nantong University and housed under specific pathogen-free (SPF) conditions in a controlled environment (25\u0026deg;C, 30\u0026ndash;35% humidity, 12 h light/dark cycle) with free access to standard rodent chow and water. All experimental procedures were performed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Experimental Animal Ethics Committee of Nantong University. Investigators were blinded to the treatment groups throughout the experiments.\u003c/p\u003e \u003cp\u003eFor the SCI model, mice were anesthetized by inhalation of 5% isoflurane for 3\u0026ndash;4 min and maintained under 2.5% isoflurane during surgery. Body temperature was maintained using a heating pad. Following skin preparation and disinfection, a laminectomy was performed at the T9\u0026ndash;11 level to expose the T10 spinal cord. A compression injury was induced using a 5-gauge Dumont forceps (tip width 1 mm) applied bilaterally to the spinal cord for 5 s, producing a consistent 1 mm lesion. The ventral side of the spinal canal was gently scraped to prevent residual ventral cord tissue. After injury, muscle and skin layers were sutured with 6\u0026thinsp;\u0026minus;\u0026thinsp;0 absorbable sutures. This standard crush model resulted in complete spinal cord injury and hindlimb motor loss. Postoperatively, mice were kept in a temperature-controlled environment with free access to food and water, and manual bladder expression was performed twice daily. Sham-operated mice underwent laminectomy only, without compression.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBehavioral/Locomotor Assessment\u003c/h3\u003e\n\u003cp\u003eMotor function was evaluated blindly by two independent observers using the Basso Mouse Scale (BMS) and footprint analysis one day before SCI and on days 1, 3, 7, 14, 21, and 28 post-injury (dpi). For BMS scoring, mice were allowed to walk freely in an open field for 5 min and were assessed based on gait, ankle movement, plantar stepping, trunk stability, coordination, and tail position, with scores ranging from 0 (no movement) to 9 (normal locomotion). Footprint analysis was used to objectively evaluate hindlimb function during quadrupedal locomotion. Hind paws were painted with red ink and forepaws with blue ink. Mice were then allowed to walk along a 50-cm-long straight runway lined with white paper. Footprints were collected and analyzed for placement, stride length, stride width, and overall gait characteristics.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescent Staining\u003c/h3\u003e\n\u003cp\u003eFor tissue staining, mice were transcardially perfused with 4% paraformaldehyde (PFA). Spinal cord segments extending approximately 0.5 cm rostral and caudal to the lesion epicenter were collected, post-fixed, dehydrated, embedded in OCT compound, and cut into serial transverse (20\u0026ndash;40 \u0026micro;m) or horizontal (15 \u0026micro;m) cryosections using a cryostat. Sections were blocked for 1 h at room temperature in 5% bovine serum albumin (BSA) with 0.3% Triton X-100, followed by incubation overnight at 4\u0026deg;C with primary antibodies: anti-cGAS (79978, CST, 1:200), anti-STING (340061, CST, 1:400), anti-NeuN (ab177487, Abcam, 1:400), anti-GFAP (MAB360, Merck Millipore, 1:500), anti-Iba1 (016-20001, Wako, 1:1000), anti\u0026ndash;β3-tubulin (5568, CST, 1:500), and anti-F4/80 (ab6640, Abcam, 1:200). After washing with PBS, sections were incubated for 1 h at room temperature with appropriate secondary antibodies (Jackson ImmunoResearch, 1:1000). Nuclei were counterstained with Hoechst 33342 (14533, Sigma, 1:5000).\u003c/p\u003e \u003cp\u003eFor cell staining, cultures were fixed with 4% PFA for 20 min at room temperature, washed with PBST, and blocked in 5% BSA with 0.1% Triton X-100 for 1 h. Subsequent steps were identical to tissue staining. Images were acquired using a confocal microscope (Leica, Germany) and analyzed with ImageJ and Photoshop (Adobe, USA). Specific color channels were separated, and total area was quantified for comparison.\u003c/p\u003e\n\u003ch3\u003eLuxol Fast Blue Staining\u003c/h3\u003e\n\u003cp\u003eResin-embedded spinal cord tissues were sectioned at 2 \u0026micro;m thickness, with one section collected every 20 \u0026micro;m. The injury epicenter, identified as the region with the least myelinated axons, was examined under a light microscope. Sections were immersed in preheated 0.1% Luxol fast blue (LFB) solution for 4 h to stain myelin. After staining, sections were cooled to room temperature, rinsed sequentially in 95% ethanol and deionized water (3 min each), differentiated in a differentiation solution for 30 s, rinsed in 70% ethanol for 45 s, dehydrated, and mounted. Demyelinated areas were quantified from microscopic images using Image-Pro Plus software.\u003c/p\u003e\n\u003ch3\u003eTransmission Electron Microscopy\u003c/h3\u003e\n\u003cp\u003eMyelin regeneration was assessed by transmission electron microscopy (TEM). Tissue samples from the lesion core were rapidly dissected from three groups: sham, SCI (28 days post-injury), and compound-intervention (28 days post-injury). Tissues were cut into 1 mm\u0026sup3; blocks on ice and fixed in 2.5% glutaraldehyde for 4 h, followed by post-fixation in 1% osmium tetroxide for 1 h and embedding in Epon 812 epoxy resin. Ultrathin sections (50 nm) were stained with lead citrate and 1% uranyl acetate for 2 h and examined under a TEM (HT7700, Hitachi, Japan). Images were acquired under standardized brightness and contrast settings. Axon diameter, myelin thickness, number of myelin layers, and G-ratio were quantified blindly by two independent examiners using Image-Pro Plus software. A total of 252 axons from the control group, 204 from DMSO-treated mice, 326 from ADU-S100\u0026thinsp;+\u0026thinsp;C176-treated mice, and 430 from C176\u0026thinsp;+\u0026thinsp;ADU-S100-treated mice were analyzed.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative Real‑Time PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from cells or spinal cords using TRIzol\u0026trade; reagent (15596018CN, Invitrogen) according to the manufacturer's instructions. cDNA was synthesized using the SuperScript\u0026trade; One-Step RT-PCR Kit with Platinum\u0026trade; Taq DNA Polymerase (11732020, Invitrogen). Quantitative Real‑Time PCR (qRT‑PCR) was performed on A StepOne\u0026trade; Real-Time PCR System. Gene expression levels were quantified using the 2\u0026minus;∆∆Ct method with Gapdh as the internal control. Primer sequences are listed in supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Each sample was assayed in triplicate.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern Blot\u003c/h3\u003e\n\u003cp\u003eFresh spinal cord tissues or cells were lysed in the ice-cold RIPA buffer (P0013B, Beyotime) containing 100 mM PMSF (a protease inhibitor cocktail), incubated at 4\u0026deg;C for 30 min, and further disrupted by sonication. Protein concentrations were determined using BCA assay. The protein samples were separated by 10% or 12% sodium dodecyl sulfate\u0026ndash;polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Merck Millipore). After blocking with 5% skim milk for 2 h at room temperature, membranes were incubated overnight at 4\u0026deg;C with the primary antibodies: anti-cGAS (79978, CST, 1:1000), anti-STING (340061, CST, 1:1000), anti-TBK (3504, CST, 1:1000), anti-p-TBK1 (5483, CST, 1:1000), and GAPDH (p-TBK1, Proteintech, 1:8000). Subsequently, the membranes were washed three times with PBST and incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies (Pierce, 1:10000) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL, PerkinElmer) and quantified using Quantity One software (Bio-Rad).\u003c/p\u003e\n\u003ch3\u003ePharmacological intervention\u003c/h3\u003e\n\u003cp\u003eAgonists (2',3'-cGAMP and ADU-S100) and inhibitors (H-151 and C-176) of cGAS-STING signaling were purchased from MedChemExpress (MCE). In vivo treatments were divided into \u0026ldquo;continuous intervention\u0026rdquo; and \u0026ldquo;time‑window intervention\u0026rdquo;, as outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. For continuous intervention, 3 \u0026micro;L of compound (10 \u0026micro;M for agonists, 1 \u0026micro;M for inhibitors) injected at the lesion site immediately after SCI. Starting on day 1 post-injury, 100 \u0026micro;L of the same compound was administered daily via intraperitoneal (i.p.) injection to maintain sustained pathway modulation. For time‑window intervention, agonist (ADU-S100) and inhibitor (C-176) was injected at the lesion site after SCI; from day 1 to day 10, 100 \u0026micro;L was given daily via i.p. injection. On day 11, the compound was switched (agonist to inhibitor or vice versa), and administration continued until day 28. Control animals received vehicle only. For \u003cem\u003ein vitro\u003c/em\u003e studies, agonists (2',3'-cGAMP and ADU-S100) and inhibitors (H-151 and C-176) were dissolved in sterile DMSO. Primary microglia, motor neurons, microglia\u0026ndash;motor neuron cocultures were treated with agonists (10 \u0026micro;M) or inhibitors (1 \u0026micro;M).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMicroglia Depletion with PLX5622\u003c/h2\u003e \u003cp\u003ePLX5622 (MCE, HY-114153), a brain-penetrant CSF1R inhibitor, enable long-term and specific depletion of microglia prior to and during pathological progression [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Mice received i.p. injections of PLX5622 (50 mg/kg, formulated in saline containing 10% DMSO, 40% PEG300, and 5% Tween‑80 as per manufacturer\u0026rsquo;s instructions) or vehicle twice daily for \u0026gt;\u0026thinsp;10 days. Detailed treatment schedules are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePrimary Microglia Culture\u003c/h2\u003e \u003cp\u003eMicroglia were separated from spinal cords as described before. Briefly, the spinal cords of 1- to 2-day-old mice were dissected, minced, and digested with 0.125% trypsin. The cell suspension was plated onto poly-D-lysine (PDL)-coated T-flasks and cultured in DMEM supplemented with 10% FBS, 1% penicillin, and 1% streptomycin. After 8\u0026ndash;10 days, mixed glial cultures were shaken at 200 rpm for 30 min at 37\u0026deg;C to detach microglia. The supernatant was collected, centrifuged at 1200 rpm for 6 min, resuspended in complete medium, and used for subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMotor Neuron Culture and Axonal Injury Model\u003c/h2\u003e \u003cp\u003eMotor neurons from embryonic day 15 (E15) rat spinal cords as described [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Spinal cords were digested with 0.125% trypsin, gently triturated, and centrifuged at 400 \u0026times; g for 10 min in 1.06 g/L Optiprep\u0026trade; (Sigma) to isolate motor neurons. A total of 3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e motor neurons were seeded into PDL-coated 24-well plates or the proximal chamber of microfluidic devices (SND450, Xona Microfluidics). Cultures were maintained in neurobasal medium (Gibco) supplemented with 10% FBS, 2% B27 (Invitrogen), 1% antibiotic-antimycotic solution, 0.5 mM L-glutamine, and 25 mM L-glutamate.\u003c/p\u003e \u003cp\u003eAxonal injury models were constructed using two methods: vigorous pipetting and microfluidic technology. For the former, motor neurons were subjected to 50 vigorous pipetting cycles to induce axonal damage. For the latter, neurons were cultured on one side of the device for 48\u0026ndash;72 h until processes crossed the microgroove barrier. Axons extending into the opposite chamber were then severed using a vacuum pump. Immunofluorescence confirmed abundant axons before transection and their absence afterward.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003eCoculture of Motor Neurons (Injured or Uninjured) with Microglia\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMicroglia were cultured to 80% confluence, detached, and seeded at 1.5 \u0026times; 10⁶ cells per well onto pre-plated motor neurons (injured or uninjured) in 24-well plates. Motor neurons had been allowed to adhere for 4 h prior to microglia addition. Microglia were adhered for 30 min in complete medium (DMEM/F12\u0026thinsp;+\u0026thinsp;20% FBS), which was then replaced with neuron-specific medium. This protocol maintained an optimal neuron-to-microglia ratio and supported axonal growth for subsequent assays. Cocultures were maintained for an additional 24 h before drug treatment to assess axonal outgrowth and inflammatory responses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence Microsphere Phagocytosis Assay\u003c/h2\u003e \u003cp\u003eTo evaluate the effect of STING pathway modulation on the phagocytic capacity of microglia, a fluorescence-based microsphere uptake assay was performed in microglia\u0026ndash;injured motor neuron cocultures. After pretreatment with STING agonist or inhibitor for 12 h, fluorescent microspheres (Sigma, L3030-1ML) were added to the culture medium. Cells were incubated at 37\u0026deg;C for 1.5 h to allow phagocytosis. Subsequently, the medium containing unbound microspheres was carefully removed, and cells were gently washed with PBS to remove non-internalized beads. For quantification, 10 random fields per well were imaged under a fluorescence microscope (Zeiss). The percentage of microglia that had engulfed fluorescent microspheres was determined manually or using ImageJ software. The experiment was repeated three times independently, and a total of 30 fields were analyzed per treatment group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRNA Sequencing\u003c/h2\u003e \u003cp\u003eRNA sequencing was performed on an Illumina Novaseq X Plus platform by Genedenovo Biotechnology Co., Ltd (Guangzhou, China). Total RNA was isolated from spinal cords with TRIzol reagents (Invitrogen). RNA quality was evaluated with an Agilent 2100 Bioanalyzer (Agilent RNA 6000 Nano Kit) assessing concentration, RIN, 28S/18S ratio, and fragment distribution. Raw reads were processed using SOAPnuke software to filter out low-quality reads. Clean reads were aligned to the reference genome using HISAT2, and gene expression level was calculated with RSEM. Differentially expressed genes (DEGs) were identified by the DEGseq with thresholds of |fold‑change| \u0026ge;2 and a q-value (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Gene Ontology (GO) classification and functional enrichment analysis were performed based on DEGs. Bioinformatic analysis was conducted using the Omicsmart online platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.omicsmart.com\u003c/span\u003e\u003cspan address=\"http://www.omicsmart.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraphs and statistical analyses were made using Prism 8 software (GraphPad, CA, USA). Comparisons between two groups were performed using Student's t-test. For comparisons among three or more groups, one-way ANOVA followed by Tukey's multiple comparisons test was applied. Longitudinal data were analyzed by two-way repeated‑measures ANOVA with Tukey's post-hoc test. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Significance levels were set as *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001. All experiments were independently repeated at least three times..\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003eUpregulation of cGAS/STING Pathway-Related Genes in the Spinal Cord following SCI\u003c/h2\u003e\n \u003cp\u003eTraumatic SCI causes motor and sensory dysfunction but is often followed by partial spontaneous recovery. Using a mouse model of thoracic SCI, we observed progressive motor recovery over a three-week period (\u003cstrong\u003eFig. S1\u003c/strong\u003e). Time-course transcriptome analysis of injured spinal cords at 3 and 14 days post-injury (dpi) versus sham controls revealed four distinct gene expression patterns: continuously downregulated (down), downregulated then upregulated (down-up), upregulated then downregulated (up-down), and continuously upregulated (up). Gene ontology analysis linked these patterns to specific biological processes: axonogenesis and neurotransmitter secretion (down), ion transport and nervous system development (down-up), neutrophil chemotaxis and angiogenesis (up-down), inflammation and innate immune regulation (up). Notably, genes related to neural regeneration or repair were minimally expressed during this period. Instead, inflammatory and immune responses were strongly upregulated during both acute and subacute phases after SCI (Fig. 1A and \u003cstrong\u003eTable S1\u003c/strong\u003e). Cross-group comparisons identified 2,425 differentially expressed genes (DEGs) commonly upregulated at both time points, primarily involved in immunity and inflammation, and 2,335 commonly downregulated DEGs, largely associated with ion transport and synaptic transmission (Fig. 1B-C and \u003cstrong\u003eTable S2-3\u003c/strong\u003e). Among these, key genes of the cGAS-STING pathway (\u003cem\u003eSting1\u003c/em\u003e, \u003cem\u003ecGAS\u003c/em\u003e, \u003cem\u003eIRF1\u003c/em\u003e, \u003cem\u003eIRF3\u003c/em\u003e, \u003cem\u003eIRF8\u003c/em\u003e, \u003cem\u003eTNF\u003c/em\u003e, and \u003cem\u003eIL6\u003c/em\u003e) showed consistent and pronounced upregulation (Fig. 1D), which was confirmed by qPCR (Fig. 1E). Given the established role of inflammation in SCI recovery and the central function of the cGAS-STING pathway in immune regulation, we focused subsequent investigation on its involvement in the SCI recovery process.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003eActivation and Microglial Predominance of the cGAS-STING Signaling Pathway after SCI\u003c/h2\u003e\n \u003cp\u003eWe assessed the expression and activation of the cGAS-STING signaling pathway after SCI by Western blot. The results showed that protein levels of cGAS and STING were significantly upregulated post-SCI, peaking at day 7 and day 14, respectively, accompanied by enhanced phosphorylation of TBK1 (Fig. 2A), indicating sustained activation of the pathway during the injury phase. Double immunofluorescence labeling was used to determine the cellular localization of STING and cGAS relative to spinal cord cell markers (Fig. 2B-C and \u003cstrong\u003eFig. S2-7\u003c/strong\u003e). In uninjured tissue, STING strongly colocalized with NeuN\u0026thinsp;+\u0026thinsp;neurons, partially present in F4/80\u0026thinsp;+\u0026thinsp;microglia, but was not detected in GFAP\u0026thinsp;+\u0026thinsp;astrocytes. After SCI, STING expression increased significantly in F4/80\u0026thinsp;+\u0026thinsp;cells and remained detectable in NeuN\u0026thinsp;+\u0026thinsp;cells (Fig. 2B and \u003cstrong\u003eFig. S2-4\u003c/strong\u003e), suggesting a shift from neuronal to microglial expression. cGAS exhibited a similar localization pattern to STING: it was present in both NeuN\u0026thinsp;+\u0026thinsp;and F4/80\u0026thinsp;+\u0026thinsp;cells under baseline conditions, but after injury, it became predominantly colocalized with F4/80\u0026thinsp;+\u0026thinsp;microglia, with a partial retention in NeuN\u0026thinsp;+\u0026thinsp;cells (Fig. 2C and \u003cstrong\u003eFig. S5-7\u003c/strong\u003e). Together, these findings indicate that SCI activates the cGAS-STING pathway, primarily within activated microglia and spared neurons.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003eContinuous Activation or Inhibition of the cGAS-STING Pathway Fails to Promote SCI Repair\u003c/h2\u003e\n \u003cp\u003eSmall-molecule compounds are widely utilized for modulating specific signaling pathways owing to their advantages in standardization and potential for clinical translation [19]. To explore the therapeutic role of the cGAS‑STING pathway in SCI, we employed well‑characterized pharmacological agents: the agonists ADU-S100 [20, 21] and 2\u0026apos;,3\u0026apos;-cGAMP [22, 23], along with the antagonists C-176 [24] and H-151 [25]. We first examined the consequences of sustained pathway modulation. As outlined in Fig.\u0026nbsp;3A, compounds were administered intrathecally at the injury site immediately after SCI, followed by daily intraperitoneal injections from day 1 onward to maintain continuous activation or inhibition, with vehicle alone as control.\u003c/p\u003e\n \u003cp\u003eMotor recovery was evaluated at multiple time points using the BMS scoring system and automatedfootprint analysis. BMS scores indicated that only ADU‑S100 elicited a transient improvement at 3 dpi, with no sustained benefit thereafter (Fig.\u0026nbsp;3B). Footprint analysis further confirmed that gait parameterssuch as stride length and base width did not differ significantly among groups (Fig.\u0026nbsp;3C-E). Consistent with these functional outcomes, immunostaining for the axonal marker Tuj1 in spinal cord tissues harvested at 28 dpi showed detectable axonal labeling in all groups, but with no significant differences in fluorescence intensity was observed (Fig.\u0026nbsp;3F), indicating that prolonged activation or inhibition of the cGAS-STING pathway has limited effects on axonal regeneration.\u003c/p\u003e\n \u003cp\u003eTogether, these data demonstrate that continuous pharmacological activation or inhibition of the cGAS-STING pathway fails to promote meaningful functional or structural recovery after SCI. The absence of a therapeutic effect may reflect a dual-phase role of the pathway: early activation could support repair through regulated inflammatory responses, whereas sustained signaling may become detrimental. Conversely, early inhibition might compromise beneficial innate immune functions and delay the clearance of damage-associated debris, thereby impeding tissue repair. Based on these observations, we hypothesized that time-restricted modulation of the cGAS-STING pathway \u0026mdash;tailored to distinct pathological phases after SCI\u0026mdash;could offer a more effective therapeutic benefits.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\"\u003e\n \u003ch2\u003eSequential Activation Followed by Inhibition of the cGAS-STING Pathway Enhances SCI Repair\u003c/h2\u003e\n \u003cp\u003eBuilding on the understanding that acute-phase inflammation can be neuroprotective but becomes harmful if dysregulated in the subacute phase [26], we devised a time-dependent therapeutic strategy modulating the cGAS-STING pathway with agonists and inhibitors during specific post-injury windows (Fig.\u0026nbsp;4A\u003cstrong\u003e)\u003c/strong\u003e. Immediately after SCI, either the agonist ADU-S100 or the inhibitor C-176 was administered at the injury site. From day 1 to day 10, mice received daily intraperitoneal injections of the same compound. On day 11, treatment was switched: the agonist group received the inhibitor (designated ADU-S100\u0026rarr;C-176), and vice versa (designated C-176\u0026rarr;ADU-S100), continuing until day 28.\u003c/p\u003e\n \u003cp\u003eBMS scores showed that from 14 dpi onward, the ADU-S100\u0026rarr;C-176 group exhibited significantly better hindlimb motor recovery than the vehicle control group, whereas the C-176\u0026rarr;ADU-S100 group showed no improvement (Fig.\u0026nbsp;4B). Footprint analysis revealed that stride length was significantly longer in the ADU-S100\u0026rarr;C-176 group compared to both the control and C-176\u0026rarr;ADU-S100 groups between 14 and 28 dpi (Fig.\u0026nbsp;4C-E). As stride length is a sensitive indicator of locomotor recovery, its increase supports the conclusion that the ADU-S100\u0026rarr;C-176 treatment enhances motor function. Double immunostaining with Tuj1 and the glial‑scar component CSPG on spinal cord tissues collected at 28 dpi revealed that the ADU-S100\u0026rarr;C-176 group possessed more Tuj1-positive fibers and smaller glial scars than the other groups. No significant differences were found between the C-176\u0026rarr;ADU-S100 and control groups (Fig.\u0026nbsp;4F). These findings suggest that early activation followed by later inhibition of the pathway mitigates secondary injury and facilitates axonal regeneration.\u003c/p\u003e\n \u003cp\u003eWe further assessed axonal regeneration and myelination using LFB staining and TEM. LFB staining showed severe demyelination in all groups at 28 dpi, however, the ADU-S100\u0026rarr;C-176 group displayed a smaller demyelinated area and visible remyelination at the lesion center (Fig. 5A). TEM confirmed a greater number of myelinated axons in this group \u003cstrong\u003e(\u003c/strong\u003eFig.\u0026nbsp;5B). Based on the classification proposed by Du et al.[27], axons with a g ratio\u0026thinsp;\u0026lt;\u0026thinsp;0.8 were considered myelinated, \u0026gt; 0.8 as remyelinated, and =\u0026thinsp;1 as unmyelinated (Fig.\u0026nbsp;5C). Quantitative analysis indicated that 81.3% of axons in the sham group were myelinated (g ratio\u0026thinsp;\u0026lt;\u0026thinsp;0.8). After SCI, this proportion decreased across all groups, but the ADU-S100\u0026rarr;C-176 group exhibited the lowest proportion of unmyelinated axons and the highest proportion of myelinated axons (Fig.\u0026nbsp;5D-E), indicating its superior capacity to promote remyelination.\u003c/p\u003e\n \u003cp\u003eIn conclusion, sequential modulation of the cGAS-STING pathway\u0026mdash;activation followed by inhibition\u0026mdash;significantly enhances repair after SCI.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\"\u003e\n \u003ch2\u003eMicroglial Depletion Abolishes the Beneficial Effects of Sequential cGAS-STING Pathway Modulation\u003c/h2\u003e\n \u003cp\u003eMicroglia, key components of the innate immune system, play essential roles in structural remodeling and functional homeostasis of central nervous system [28]. Our findings demonstrated that cGAS and STING expression following SCI was primarily localized to activated microglia (Fig.\u0026nbsp;2), suggesting the pathway influences SCI pathology via microglial regulation. To test this, we used the CSF1R-specific inhibitor PLX5622 [17] to deplete microglia while targeting the cGAS-STING pathway within defined temporal windows.\u003c/p\u003e\n \u003cp\u003eWe first confirmed the efficacy of PLX5622, finding that intraperitoneal administration of three daily doses reduced microglial numbers by 90.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89% (\u003cstrong\u003eFig. S8\u003c/strong\u003e). Based on post-SCI microglial dynamics and pathway intervention timing, we established two depletion protocols: (1) Early depletion (Fig.\u0026nbsp;6A): PLX5622 administered from 3 days before SCI until 7 dpi (every 12 h); and (2) Late depletion (Fig.\u0026nbsp;7A): treatment began on day 9 post-SCI and continued until day 14. IBA1 staining confirmed a marked reduction in microglial numbers in all PLX5622-treated groups (\u003cstrong\u003eFig. S9-10\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003eBMS scores revealed that both early and late microglial depletion significantly impaired motor recovery in the ADU-S100\u0026rarr;C-176, C-176\u0026rarr;ADU-S100, and vehicle groups compared with their non-depleted counterparts. No significant differences in recovery were observed among the depleted treatment groups (Fig.\u0026nbsp;6B and 7B), indicating that microglial depletion abolished the functional benefits conferred by sequential cGAS-STING modulation. Notably, ADU-S100\u0026rarr;C-176 treatment reduced neuronal apoptosis after SCI, as confirmed by caspase activation staining, however, this protective effect was lost upon microglial depletion (Fig.\u0026nbsp;6C). Consistent with these findings, microglial depletion led to increased demyelination areas (Fig.\u0026nbsp;7C) and decreased remyelination levels across all treatment groups (Fig.\u0026nbsp;7D). Given potential astrocyte-microglia crosstalk, we assessed whether astrocytes might compensate for microglial loss by engaging the cGAS-STING pathway. However, no cGAS or STING expression was detected in astrocytes (\u003cstrong\u003eFig. S11\u003c/strong\u003e), suggesting that astrocytes do not directly contribute to this regulatory mechanism.\u003c/p\u003e\n \u003cp\u003eCollectively, these data demonstrate that the cGAS-STING pathway influences SCI recovery through a microglia-dependent mechanism.\u003c/p\u003e\n \u003cdiv id=\"Sec23\"\u003e\n \u003ch2\u003eRNA Sequencing Reveals a Dual‑Phase Mechanism Underlying the Beneficial Effects of Sequential cGAS\u0026ndash;STING Pathway Modulation\u003c/h2\u003e\n \u003cp\u003eTo investigate the mechanism by which sequential modulation of the cGAS-STING signaling pathway promotes SCI repair, we performed RNA sequencing on spinal cord lesion tissues collected 3 and 14 days after treatment with ADU-S100\u0026rarr;C-176, C-176\u0026rarr;ADU-S100, or vehicle control. DEG analysis (screening criteria: |Fold Change| \u0026ge; 2, Q value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) revealed that, compared to the vehicle group, early STING inhibition (C-176\u0026rarr;ADU-S100) at 3 days post-treatment resulted in the significant upregulation of only one gene: MT3 (Metallothionein-3), also known as growth inhibitory factor (Fig. 8A and \u003cstrong\u003eTable S4\u003c/strong\u003e). MT3 is a CNS-enriched metalloprotein that maintains neuronal zinc and redox balance, thereby supporting neuronal survival and suppressing aberrant growth [29, 30]. This finding suggests that early STING inhibition after SCI may confer neuroprotective effects. Unexpectedly, MT3 was also significantly upregulated following early STING activation (ADU-S100\u0026rarr;C-176 group), indicating that both activation and inhibition of STING can enhance MT3 expression (Fig. 8B and \u003cstrong\u003eTable S4\u003c/strong\u003e). This shared upregulation pattern suggests that MT3 may act as a common mediator bridging different immune states, promoting repair during SCI recovery by maintaining intracellular metal ion homeostasis and exerting neuroprotective functions. Notably, the expression levels of key inflammatory cytokines (such as \u003cem\u003eTNF-\u0026alpha;\u003c/em\u003e, \u003cem\u003eIL-6\u003c/em\u003e, and \u003cem\u003eIL-1\u0026beta;\u003c/em\u003e) showed no significant differences among the three groups, indicating that early intervention targeting the cGAS-STING pathway did not markedly modulate the intensity of the inflammatory response at this stage.\u003c/p\u003e\n \u003cp\u003eTo elucidate the downstream mechanisms regulated by STING activation, we ranked the DEGs from the ADU-S100\u0026rarr;C-176 group at 3 days by expression level (Fig. 8B and \u003cstrong\u003eTable S4\u003c/strong\u003e). This analysis revealed strong upregulation of granzyme family members (\u003cem\u003eGzma\u003c/em\u003e, \u003cem\u003eGzmb\u003c/em\u003e, \u003cem\u003eGzmc\u003c/em\u003e, \u003cem\u003eGzmf\u003c/em\u003e, \u003cem\u003eGzmd\u003c/em\u003e, etc.) and multiple chemokines (e.g., \u003cem\u003eCXCL10\u003c/em\u003e, \u003cem\u003eCcl5\u003c/em\u003e, \u003cem\u003eCcl8\u003c/em\u003e, \u003cem\u003eCcl2\u003c/em\u003e, \u003cem\u003eCcl7\u003c/em\u003e). Granzymes are serine proteases that play a central role in immune clearance by inducing apoptosis in abnormal cells. They also participate in regulating inflammation and remodeling the extracellular matrix, thereby influencing cell migration and tissue repair [31]. We thus hypothesize that the significant upregulation of granzymes upon STING activation during the acute phase of SCI may accelerate the apoptosis and clearance of damaged cells. Concurrent upregulation of chemokines such as CXCL10 is closely associated with the recruitment of monocytes, macrophages, and neutrophils to the injury site [32]. The increased expression of Ly6C2, a key marker expressed on murine myeloid cells, particularly monocytes and newly recruited macrophages, further supports that STING activation promotes monocytes/macrophages accumulation at the lesion site. In summary, early cGAS-STING activation may facilitate subsequent repair by enhancing immune cell infiltration and accelerating the clearance of damaged cells.\u003c/p\u003e\n \u003cp\u003eAt 14 days post‑treatment (after 3 days of switched agonist/antagonist administration), the elevated expression levels of granzymes (e.g., \u003cem\u003eGzma\u003c/em\u003e, \u003cem\u003eGzmb\u003c/em\u003e, \u003cem\u003eGzmc\u003c/em\u003e) and chemokines (e.g., \u003cem\u003eCXCL10\u003c/em\u003e, \u003cem\u003eCcl5\u003c/em\u003e, \u003cem\u003eCcl8\u003c/em\u003e, \u003cem\u003eCcl2\u003c/em\u003e, \u003cem\u003eCcl7\u003c/em\u003e) observed in the ADU-S100\u0026rarr;C-176 group had returned to baseline, showing no difference from the vehicle group. In contrast, the C-176\u0026rarr;ADU-S100 group exhibited significantly increased expression of chemokines (\u003cem\u003eCXCL1\u003c/em\u003e, \u003cem\u003eCcl5\u003c/em\u003e, \u003cem\u003eCcl2\u003c/em\u003e, \u003cem\u003eCcl7\u003c/em\u003e) and pro-inflammatory cytokines (\u003cem\u003eIL-6\u003c/em\u003e and \u003cem\u003eIL-1\u0026beta;\u003c/em\u003e), suggesting that late-stage STING activation exacerbates the subacute inflammatory response. High expression of the neutrophil markers S100a8 and S100a9 indicated persistent neutrophil retention, further supporting this conclusion (Fig. 8C and \u003cstrong\u003eTable S4\u003c/strong\u003e). Notably, expression of the anti-inflammatory cytokine \u003cem\u003eIL-10\u003c/em\u003e was significantly increased in the ADU-S100\u0026rarr;C-176 group. IL-10 is a key anti-inflammatory cytokine that potently inhibits the production of pro-inflammatory cytokines such as TNF-\u0026alpha;, IL-1, IL-6, and IL-12. Its primary role is to limit and terminate immune responses, preventing excessive inflammation and subsequent tissue damage [33, 34]. Therefore, late-stage inhibition of the cGAS-STING pathway may alleviate subacute inflammation and create a favorable environment for neural repair, potentially through the induction of IL-10.\u003c/p\u003e\n \u003cp\u003eWe further analyzed the expression of genes associated with axonal regeneration. In the ADU-S100 \u0026rarr; C-176 group, axon growth-related genes including \u003cem\u003eTubb3\u003c/em\u003e, \u003cem\u003eMap2\u003c/em\u003e, \u003cem\u003eNefl\u003c/em\u003e, \u003cem\u003eNefh\u003c/em\u003e, \u003cem\u003eNefm\u003c/em\u003e, and \u003cem\u003eDoublecortin\u003c/em\u003e (\u003cem\u003eDCX\u003c/em\u003e) were significantly upregulated compared with the other groups, indicating that late STING inhibition promotes axonal regeneration. DCX is a microtubule-associated protein expressed in migrating neuroblasts and is an established marker for nascent axonal growth [35, 36]. Immunohistochemistry confirmed that DCX-positive signals were markedly stronger in the ADU-S100\u0026rarr;C-176 group than that in the other groups (Fig.\u0026nbsp;8D), providing additional support for the conclusion that inhibiting the cGAS-STING pathway during the late phase facilitates axonal regeneration.\u003c/p\u003e\n \u003cp\u003eIn summary, sequential modulation of the cGAS-STING signaling pathway (ADU-S100\u0026rarr;C-176) promotes repair through a temporally coordinated mechanism: early STING activation promotes the clearance of cellular debris and initiates necessary immune infiltration, while subsequent inhibition resolves inflammation and directly enables axonal regeneration.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\"\u003e\n \u003ch2\u003eActivation of the cGAS\u0026ndash;STING Pathway Promotes Axon Growth and Regeneration in Motor Neurons\u003c/h2\u003e\n \u003cp\u003eTo investigate the role of the cGAS\u0026ndash;STING pathway in axon growth and regeneration, we treated motor neurons for 24 h with its agonists (ADU‑S100 or 2\u0026apos;,3\u0026apos;‑cGAMP), inhibitors (H‑151 or C-176), or vehicle control (Fig.\u0026nbsp;9A). CCK-8 assay showed no significant difference in cell viability, indicating no cytotoxicity of the compounds (Fig.\u0026nbsp;9B). qPCR analysis revealed that agonist significantly upregulated expression of the axonal growth marker GAP43, whereas inhibitor downregulated it (Fig.\u0026nbsp;9C). Consistent with this, immunocytochemistry revealed that agonists enhanced axon outgrowth, while inhibitors reduced it (Fig.\u0026nbsp;9D). For quantitative morphological assessment, axons were categorized by length as short (0\u0026ndash;30 \u0026micro;m), medium (30\u0026ndash;60 \u0026micro;m), long (60\u0026ndash;100 \u0026micro;m), or very long (\u0026gt;\u0026thinsp;100 \u0026micro;m). Distribution analysis indicated a significantly higher proportion of neurons with long and very long axons in agonist-treated cultures, whereas inhibitor-treated groups exhibited increased proportions of neurons with short axons (Fig.\u0026nbsp;9D). Together, these results indicate that cGAS\u0026ndash;STING activation promotes axon growth in motor neurons, whereas its inhibition impairs this process.\u003c/p\u003e\n \u003cp\u003eWe next evaluated the role of this pathway in axon regeneration using a microfluidic axotomy model. Neurons cultured in the left chamber extended axons into the right chamber, which were then severed by vacuum aspiration. Immunocytochemistry confirmed complete axonal removal in the right chamber, validating model integrity (Fig.\u0026nbsp;9E). After axotomy, application of agonists, inhibitors, or DMSO to neuronal sonata for 24 h revealed that agonist treatment significantly enhanced axon regeneration relative to the DMSO, whereas inhibitors did not exert a significant effect (Fig.\u0026nbsp;9F).\u003c/p\u003e\n \u003cp\u003eIn summary, these findings demonstrate that cGAS\u0026ndash;STING pathway activation promotes both developmental axon growth and post-injury regeneration in motor neurons.\u003c/p\u003e\n \u003cdiv id=\"Sec25\"\u003e\n \u003ch2\u003eThe cGAS\u0026ndash;STING Pathway in Microglia Regulates Neuroinflammation and Axonal Regeneration through Neuronal Interaction\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e experiments demonstrated that depleting microglia significantly attenuates the beneficial effect of modulating the cGAS\u0026ndash;STING pathway on functional recovery after SCI, suggesting that this pathway participate in repair processes by regulating microglia-mediated neuroinflammation. To further investigate its role in inflammation modulation and axonal regeneration, we established an \u003cem\u003ein vitro\u003c/em\u003e coculture system consisting of microglia and motor neurons (injured or uninjured). Scanning electron microscopy revealed that after 24 h of coculture, microglia migrated and adhered to the neuronal surface, confirming the suitability of this system (Fig. 10A).\u003c/p\u003e\n \u003cp\u003eStimulation of the uninjured coculture system with IFN-\u0026gamma; plus LPS significantly upregulated the expression of the pro-inflammatory cytokines TNF-\u0026alpha; and IL-6, but did not markedly alter the expression or activation of STING, indicating that such stimulation induces inflammation independently of cGAS\u0026ndash;STING pathway (Fig. 10B). In contrast, coculture with injured neurons not only elevated inflammatory cytokine levels but also activated the STING pathway, suggesting that factors released from damaged neurons, such as dsDNA, may serve as a key trigger in microglia (Fig. 10C). Immunocytochemical analysis revealed that microglia cocultured with uninjured neurons retained a ramified, resting morphology, whereas those exposed to injured neurons adopted a spindle‑like shape with reduced branching, indicating a shift toward a polarized state. This morphological shift was further corroborated by the upregulation of established microglial polarization markers, such as \u003cem\u003eiNOS\u003c/em\u003e, \u003cem\u003eCCL2\u003c/em\u003e and \u003cem\u003eF/480\u003c/em\u003e. Moreover, these polarized microglia were found to co-localize with neuronal markers, implying their potential involvement in phagocytosing cellular debris from injured neurons (Fig. 10D). Notably, while STING expression in uninjured cocultures was largely neuronal, it was detected in both neurons and microglia when cocultured with injured neurons (Fig. 10D), consistent with \u003cem\u003ein vivo\u003c/em\u003e observations.\u003c/p\u003e\n \u003cp\u003eWe next treated the injured neuron\u0026ndash;microglia coculture with STING agonists, inhibitors, or DMSO to evaluate their effects on axonal regeneration, inflammatory response, and microglial phagocytosis. Agonist treatment significantly upregulated the expression of the axonal growth-related genes \u003cem\u003eGAP43\u003c/em\u003e and \u003cem\u003eTubulin\u003c/em\u003e and promoted axonal regeneration, whereas inhibitors suppressed these effects (Fig. 10E). Furthermore, the proportion of microglia engulfing fluorescent microspheres was markedly higher in the agonist-treated group compared with the DMSO control, whereas the C-176-treated group showed significantly reduced phagocytic ability (Fig. 10F), indicating that activation of the cGAS\u0026ndash;STING pathway enhances the capacity of microglia to clear injury-associated debris.\u003c/p\u003e\n \u003cp\u003eIn summary, although IFN-\u0026gamma; plus LPS stimulation induces inflammation in the uninjured neuron\u0026ndash;microglia co-culture system, it does not activate the cGAS\u0026ndash;STING pathway. In contrast, injured neurons trigger both activation of this pathway and associated inflammatory responses. Activation of cGAS\u0026ndash;STING signaling in this co-culture system promotes axonal regeneration and enhances the phagocytic function of microglia.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study found that the cGAS-STING signaling pathway, a core sensor of innate immunity, is significantly activated after SCI, with its expression predominantly enriched in microglia. More importantly, we innovatively proposed and validated a temporal regulation strategy based on the pathological progression of the injury: activating the pathway in the early (acute) phase and inhibiting it in the later (subacute) phase. This sequential intervention of \"activation followed by inhibition,\" compared to sustained activation or inhibition alone, more effectively coordinates microglia-mediated debris clearance and inflammation resolution, ultimately leading to significantly enhanced axonal regeneration, myelination, and motor functional recovery. This research not only reveals the complex time-dependent functions of the cGAS-STING pathway in SCI but also provides a new theoretical foundation and practical approach for developing precise phase-specific therapeutic strategies centered on immune modulation.\u003c/p\u003e \u003cp\u003e \u003cb\u003e1. The Activation Pattern of the cGAS-STING Pathway Determines Its Biphasic Role in SCI\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe core function of the cGAS-STING pathway is to sense abnormal double-stranded DNA in the cytoplasm, whether derived from pathogens or endogenous DNA released due to tissue damage, cellular stress, or genomic instability [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In the context of SCI, damage leads to the rupture of neurons and glial cells, releasing mitochondrial DNA and genomic DNA, which serve as critical DAMPs. Our transcriptomic and protein analyses consistently showed significant upregulation of cGAS, STING expression, and phosphorylation of their downstream effector TBK1 in spinal cord tissue post-SCI, aligning with previous reports [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. A key contribution of this study is the identification, via cellular co-localization analysis, that the activation of this pathway occurs primarily in phagocytic cells, especially microglia. Under physiological conditions, STING is basally expressed in neurons, possibly involved in DNA surveillance and homeostasis maintenance. After injury, microglia rapidly activate and upregulate cGAS/STING, becoming the primary effector cells for this pathway. This spatial distribution suggests that the cGAS-STING pathway likely serves as a crucial molecular switch for microglia to sense \"danger signals\" (e.g., dsDNA from disintegrating neurons) and initiate innate immune responses post-SCI. The sustained upregulation pattern implies that this pathway may play different roles at different stages of SCI: participating in necessary immune clearance during the acute phase, while prolonged activation may exacerbate secondary injury. Indeed, our pharmacological experiments showed that neither sustained agonism nor inhibition of the pathway improved functional recovery, indicating its phase-dependent functionality, a phenomenon analogous to the bidirectional effects reported for other inflammation-related pathways, such as NF-κB and NLRP3 [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003e2. The \"Activation-Followed-by-Inhibition\" Strategy Promotes SCI Repair by Coordinating Immune Clearance and Inflammation Resolution\u003c/b\u003e \u003c/p\u003e \u003cp\u003eInitial experiments revealed that neither continuous activation nor continuous inhibition of the cGAS-STING pathway significantly improved long-term motor function or axonal regeneration. This outcome underscores the functional complexity of this pathway in SCI repair and highlights the necessity for temporal intervention.\u003c/p\u003e \u003cp\u003eThe drawback of sustained activation lies in the fact that while the early robust inflammatory response aids in initiating clearance, its prolonged persistence leads to destructive chronic neuroinflammation, aggravating the loss of neurons and oligodendrocytes, potentially promoting the transformation of astrocytes into the regeneration-inhibitory A1 phenotype, and exacerbating glial scar formation. Conversely, the strategy of sustained inhibition, although aimed at controlling inflammation, may impair the essential phagocytic function required during the acute phase. This can result in the accumulation of injury debris, particularly inhibitory myelin debris, hindering the differentiation of oligodendrocyte precursor cells and remyelination, and delaying the initiation of tissue repair signals.\u003c/p\u003e \u003cp\u003eTherefore, based on the concept that \"inflammatory responses have a temporal window,\" we designed a sequential modulation regimen (ADU-S100\u0026rarr;C-176). SCI involves distinct inflammatory phases: the acute phase (approximately days 1\u0026ndash;10), where inflammation is primarily clearance-oriented and a prerequisite for repair; and the subacute phase (approximately days 10\u0026ndash;28), where persistent inflammation becomes an obstacle to regeneration if not resolved. Our intervention matched these phases: administering the agonist ADU-S100 early (days 1\u0026ndash;10) and switching to the inhibitor C-176 later (days 11\u0026ndash;28). This strategy yielded results significantly superior to both the control group and the reverse sequential group (C-176\u0026rarr;ADU-S100), strongly demonstrating that intervention targeting the cGAS-STING pathway must be \"time-specific,\" precisely matching its function at different pathological stages.\u003c/p\u003e \u003cp\u003eMechanistically, RNA-seq data provided key insights: During the \"early activation\" phase, ADU-S100 treatment significantly upregulated the expression of granzyme family members (\u003cem\u003eGzma\u003c/em\u003e, \u003cem\u003eGzmb\u003c/em\u003e, etc.) and chemokines (e.g., \u003cem\u003eCXCL10\u003c/em\u003e, \u003cem\u003eCCL5\u003c/em\u003e). Granzymes may enhance the clearance efficiency of microglia towards apoptotic/necrotic cells, while chemokines help recruit peripheral immune cells to assist in debris cleanup. In vitro experiments directly confirmed that STING agonists enhance the phagocytic capacity of microglia. Thus, the core significance of early activation is \"accelerating cleanup.\" During the \"late inhibition\" phase (day 14), the early activation group, after switching to the inhibitor, showed reduced levels of pro-inflammatory factors and a significant upregulation of the anti-inflammatory cytokine IL-10. IL-10 can promote the polarization of macrophages/microglia towards the reparative M2-like phenotype and support neuronal survival and axonal growth [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Concurrently, this group exhibited enhanced expression of axonal growth-related genes (\u003cem\u003eTubb3\u003c/em\u003e, \u003cem\u003eMap2\u003c/em\u003e, \u003cem\u003eDCX\u003c/em\u003e, etc.). This indicates that the core role of late inhibition is \"switching the mode,\" transforming the immune microenvironment from a pro-inflammatory state to an anti-inflammatory, pro-repair state, thereby lifting the inhibition on regeneration and activating regenerative programs.\u003c/p\u003e \u003cp\u003eIn summary, this strategy dynamically optimizes the immune response: activation in the acute phase accelerates clearance, removing obstacles for repair; inhibition in the subacute phase mitigates inflammation, creating a favorable environment for regeneration.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3. Microglia Are the Key Effectors Mediating cGAS\u0026ndash;STING-Regulated Repair\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFollowing SCI, the interplay between microglia and neurons is central to the ensuing pathological cascade. This bidirectional communication critically regulates the intensity and persistence of neuroinflammation, with direct implications for neuronal survival, axonal regeneration, and functional recovery [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Notably, our findings reveal that cGAS and STING are expressed in both cell types after SCI, indicating a shared engagement in innate immune signaling during the injury response. To define the specific contribution of microglial cGAS-STING signaling to repair, we employed a combined strategy of temporal microglia depletion using PLX5622 and phased pathway modulation. The results were definitive: microglial depletion completely abolished the functional improvement, neuroprotection, and enhanced remyelination conferred by the sequential modulation strategy. This establishes microglia as the indispensable effector cells through which the timed modulation of the cGAS-STING pathway exerts its benefits. This conclusion aligns with our protein localization data, which showed predominant enrichment of cGAS and STING in activated microglia post-injury, with no compensatory upregulation in astrocytes.\u003c/p\u003e \u003cp\u003eSupporting this \u003cem\u003ein vivo\u003c/em\u003e mechanism, further \u003cem\u003ein vitro\u003c/em\u003e coculture experiments demonstrated that injured neurons release factors which specifically activate the STING pathway in microglia, driving their polarization toward a phagocytic phenotype. This indicates that neuron-to-microglia \u0026ldquo;danger signaling\u0026rdquo; is a pivotal event initiating a reparative immune response. Therefore, the cGAS-STING pathway promotes recovery by dynamically modulating microglial function, thereby shaping a \u0026ldquo;permissive microenvironment\u0026rdquo; that supports axonal regeneration and tissue repair.\u003c/p\u003e \u003cp\u003e \u003cb\u003e4. MT3: A Potential Downstream Hub Molecule Mediating Common Protective Effects Across Immune States\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAn interesting finding was that both early activation and early inhibition of STING significantly upregulated the expression of MT3. MT3 is a central nervous system-enriched metal-binding protein widely recognized for for its neuroprotective properties [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. It maintains neuronal homeostasis by chelating excess zinc ions and scavenging reactive oxygen species, thereby countering excitotoxicity and oxidative stress damage [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Recent advances have established MT3 as a compelling therapeutic target in neurological disorders due to its critical role in axonal integrity and neuroinflammatory regulation [\u003cspan additionalcitationids=\"CR49 CR50\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Given the pronounced excitotoxic and oxidative insults during the acute phase of SCI, we hypothesized that the upregulation of MT3 may represent an intrinsic neuroprotective response regulated by the cGAS-STING pathway (regardless of its activation or suppression), potentially helping neurons survive the initial inflammatory fluctuations. This suggests that distinct modes of cGAS-STING modulation may converge on shared protective mechanisms through common downstream effectors such as MT3. These results reveal a potential uncoupling between inflammatory phenotype dynamics and neuroprotection, raising the possibility that MT3 could function as a universal protective node. Whether MT3's protective roleoperates independently of inflammatory phenotype switching warrants future investigation.\u003c/p\u003e \u003cp\u003eIn summary, this study elucidates the dynamic and phase-dependent functions of the cGAS-STING pathway in the pathological progression of spinal cord injury. We demonstrate that while sustained activation or inhibition of this innate immune sensor fails to promote repair, a precisely timed sequential strategy\u0026mdash;activating the pathway in the acute phase to enhance microglia-mediated debris clearance, followed by its inhibition in the subacute phase to resolve inflammation and foster a pro-regenerative milieu\u0026mdash;significantly improves functional and structural recovery. Our findings establish microglia as the indispensable cellular effector through which temporal cGAS-STING modulation coordinates the immune response. Furthermore, the identification of MT3 as a convergent protective molecule highlights a potential shared axis of neuroprotection downstream of divergent immune signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e). \u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThis study establishes the cGAS-STING pathway as a dynamically regulated, phase-dependent modulator of repair following SCI. It demonstrates that a time-sequenced therapeutic strategy can effectively coordinate immune clearance and inflammation resolution, SCI improving functional recovery. Collectively, our findings provide a novel conceptual framework for \u0026ldquo;chrono-immunomodulation\u0026rdquo; in CNS trauma, moving beyond static pathway manipulation towards interventions that are synchronized with the evolving pathology. This work not only advances the mechanistic understanding of neuro-immune crosstalk in SCI but also presents a translationally viable strategy with promising therapeutic implications for SCI and potentially other neuroinflammatory conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe illustration depicts the spatiotemporally specific and cell type-dependent roles of the cGAS-STING signaling pathway during post-SCI repair. Microglia, as the central effector cells of this sequential strategy, coordinate the transition from inflammatory clearance to regenerative repair through dynamic interactions with neurons. In the acute and early subacute phases, activation of cGAS-STING in neurons and microglia responds to damage-associated molecular patterns (e.g., dsDNA), initiates immune responses, and enhances the phagocytic capacity of microglia to clear cellular debris, thereby preparing the tissue for repair. In the late subacute and chronic phases, inhibition of this pathway alleviates persistent neuroinflammation, promotes microglial polarization toward an anti-inflammatory phenotype, and induces the expression of axonal regeneration-related genes, ultimately fostering a microenvironment conducive to functional recovery.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e All experimental procedures were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals, the institutional guidelines of Nantong University, and the Regulations for the Administration of Affairs Concerning Experimental Animals of China. The study was approved by the Institutional Review Board and the Ethics Committee of Nantong University. All animal experiments were performed in compliance with the relevant guidelines and regulations.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was sponsored by the National Natural Science Foundation of China (grant No. 82272169, 32271418), the Natural Science Foundation of Hebei Province (grant No. C2024406010), and the Science Research Project of Hebei Education Department (grant No. QN2025285).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYG conceived the overall study. All authors contributed to the study design. QQP, MG, HL, and XW performed the experiments including cell culture, cell biology experiments, molecular biology experiments, animal behavior experiments and morphology experiments. LC, MY, XW, and MLH analyzed the data. YG and CLZ provided methodology guidance. LL, and SRW performed and analyzed additional IHC. CLZ performed LFB staining and QC. QQP and MG prepared the manuscript and figures. YG, QQP, MG, CLZ oversaw the analysis and edited the manuscript. All authors gave final approval of the manuscript\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eMost of the datasets supporting the conclusions of this article are included within this article and the additional files. The datasets used or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAshammakhi N, Kim HJ, Ehsanipour A, Bierman RD, Kaarela O, Xue C, Khademhosseini A, Seidlits SK. Regenerative Therapies for Spinal Cord Injury. Tissue Eng Part B Rev. 2019;25:471\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhuja CS, Wilson JR, Nori S, Kotter MRN, Druschel C, Curt A, Fehlings MG. Traumatic spinal cord injury. Nat Reviews Disease Primers 2017, 3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarsy M, Hawryluk G. Modern Medical Management of Spinal Cord Injury. Curr Neurol Neurosci Rep. 2019;19:65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian T, Zhang S, Yang M. Recent progress and challenges in the treatment of spinal cord injury. 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Cell Death Discov. 2025;11:45.\u003c/span\u003e\u003c/li\u003e \u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Spinal cord injury, cGAS-STING, Sequential modulation, Microglia, Neuroinflammation, Regeneration","lastPublishedDoi":"10.21203/rs.3.rs-8596952/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8596952/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSpinal cord injury (SCI) is a devastating condition with limited treatment options, where dysregulated neuroinflammation critically impedes repair. The cGAS-STING pathway, a central cytosolic DNA-sensing axis of innate immunity, is implicated in neuroinflammatory disorders, yet its precise spatiotemporal role and therapeutic potential in SCI remain undefined. Given the complex, phase-specific nature of post-SCI immune responses, we hypothesized that a time-dependent modulation of this pathway, rather than continuous intervention, could optimally coordinate inflammation for repair.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eUsing a mouse model of thoracic compressive SCI, we assessed pathway activation via transcriptomics, western blot, and immunofluorescence. Motor recovery was evaluated longitudinally using the Basso Mouse Scale (BMS) and footprint analysis. Pharmacological agonists and antagonists of STING were administered either continuously or in a sequential regimen. Histological and ultrastructural analyses evaluated axonal regeneration, myelination, and glial scarring. RNA sequencing elucidated molecular mechanisms. Microglia-specific depletion using PLX5622 and in vitro neuron-microglia co-cultures were employed to determine cellular mechanisms.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe cGAS-STING pathway was significantly activated post-SCI, primarily within microglia. Continuous pathway activation or inhibition failed to improve recovery. In contrast, sequential treatment (early agonist, late antagonist) significantly enhanced functional recovery, axonal regeneration, and remyelination while limiting glial scarring. Mechanistically, upregulated genes associated with microglial phagocytosis and chemotaxis, promoting debris clearance. Subsequent inhibition relieved inflammation, elevated anti-inflammatory cytokine and pro-regenerative programs. Microglial depletion completely abolished the therapeutic benefits of the sequential strategy, confirming their role as essential effector cells.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study establishes the critical importance of timing in targeting the cGAS-STING pathway after SCI. We demonstrate that a phase-specific strategy that sequentially activates then inhibits this pathway optimally harnesses microglial functions for repair, offering a novel precision medicine approach for SCI.\u003c/p\u003e","manuscriptTitle":"Sequential Modulation of the cGAS-STING Pathway Promotes Spinal Cord Injury Repair","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-22 19:10:13","doi":"10.21203/rs.3.rs-8596952/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6dce4b36-8180-41c8-a466-d9d2263d4633","owner":[],"postedDate":"January 22nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-05T17:10:21+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-22 19:10:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8596952","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8596952","identity":"rs-8596952","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}