Spreading Depolarization Activates the cGAS–STING Pathway and Drives Cranial Nociception: Therapeutic Potential of STING Modulation

Spreading Depolarization Activates the cGAS–STING Pathway and Drives Cranial Nociception: Therapeutic Potential of STING Modulation | 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 Spreading Depolarization Activates the cGAS–STING Pathway and Drives Cranial Nociception: Therapeutic Potential of STING Modulation Kadir Oguzhan Soylu, Buket Donmez-Demir, Hasan Basri Kilic, Melike Sever-Bahcekapili, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7160141/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Jan, 2026 Read the published version in The Journal of Headache and Pain → Version 1 posted 9 You are reading this latest preprint version Abstract Background Spreading depolarization (SD) is a transient wave of near-complete neuronal and glial depolarization in the cortex which underlies migraine aura. Beyond its electrophysiological effects, SD has been shown to trigger a cascade of sterile neuroinflammatory responses which might contribute to trigeminal activation and pain sensitization observed in migraine. Recent studies have highlighted the involvement of innate immune system pathways in SD-associated inflammation. The cyclic GMP-AMP synthase (cGAS)-Stimulator of interferon genes (STING) pathway induces the expression of type 1 interferons and pro-inflammatory cytokines in response to cellular stress. Although this pathway is increasingly recognized for its role in neuroinflammation and nociception, its specific contribution to SD-induced mechanisms remains poorly understood. In this study, we investigated whether SD triggers activation of the cGAS-STING pathway in the mouse cerebral cortex and evaluated the functional consequences of this pathway activation on SD associated cranial nociception. Methods SD was induced non-invasively with optogenetic stimulation. Animals were subjected to either a single or six SDs and the expression of cGAS-STING pathway proteins in the cortex were assessed by immunohistochemistry and capillary Western blotting. Sham-treated animals served as controls. The cellular localization of this pathway proteins in the cortex was also determined. Pharmacological modulation of the pathway was achieved via intraperitoneal administration of the STING inhibitor C-176 (20 mg/kg) or intranasal delivery of the STING agonist 2’3’-cGAMP (1 mg/kg). SD threshold was determined with potasium chloride application, and periorbital nociceptive responses were measured using the manual von Frey test. Furthermore, periorbital mechanical allodynia was assessed at 2 and 24 hours following six optogenetically induced SDs in animals treated with either 2’3’-cGAMP or vehicle. Results SD induced cGAS-STING signaling and IFN-β expression in the mouse cerebral cortex with prominent expression observed in neurons and downstream microglial activation. Pathway activation with 2’3’ cGAMP decreased SD susceptibility and significantly alleviated the development of periorbital mechanical allodynia following SD. Conclusions Our findings suggest that SD activates the cGAS-STING pathway, extending the scope of SD-induced neuroinflammation. These results also highlight the therapeutic potential of modulating STING to mitigate SD-related nociception and neuroinflammatory consequences associated with headache disorders such as migraine. Spreading depolarization migraine cGAS STING interferon neuroinflammation cGAMP optogenetics Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Spreading depolarization (SD) is a slowly spreading wave of neuronal and glial depolarization at a velocity of approximately 2–5 mm/min, which causes a temporary silencing of spontaneous cortical activity and alterations in cerebral blood flow [ 1 , 2 ]. SD has been detected in patients with ischemic stroke, subarachnoid hemorrhage, or traumatic brain injury under continuous monitoring in the intensive care unit [ 3 ]. It is also considered an electrophysiological correlate of migraine aura and contributes to the pathophysiology of migraine pain by activating the trigeminovascular system [ 4 – 7 ]. Hence, SD is used as a relevant experimental model to study migraine pathophysiology, as several clinically used and effective anti-migraine drugs have been shown to suppress SD [ 8 , 9 ]. SD imposes considerable metabolic burden on brain cells because of disruptions in ion and neurotransmitter homeostasis which requires substantial energy to restore [ 10 ]. This can lead to cellular stress in brain cells. While SD induced cellular stress does not cause cellular death in metabolically intact brain (in the absence of ischemia or trauma) it triggers a non-lethal but robust inflammatory response known as neuro-parainflammation, which can persist for hours to days [ 11 – 14 ]. This inflammatory response involves the release of pro-inflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor-α (TNF-α), and mediators such as cyclooxygenase-2 (COX-2) and high mobility group box-1 (HMGB1), including key mechanisms of activation of the nucleotide-binding domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome following the opening of pannexin-1 channels [ 12 , 15 – 18 ]. One of the key mediators of innate immunity involved in sterile inflammation is the cyclic GMP-AMP synthase–stimulator of interferon genes (cGAS–STING) pathway, where the cGAS enzyme detects free cytosolic double-stranded DNA (dsDNA), and leads to the production of 2'3'-cyclic GMP-AMP (2'3'-cGAMP) [ 19 ]. This secondary messenger then activates the adaptor protein STING located on the endoplasmic reticulum (ER) [ 20 – 22 ].Upon activation, STING subsequently leads to downstream effects, including the phosphorylation of interferon regulatory factor 3 (IRF3) to form p-IRF3 and the induction of type 1 interferon (IFN) signaling. Additionally, this pathway can lead to the translocation of nuclear factor-κB (NF-κB) to the nucleus, thereby enhancing the expression of pro-inflammatory cytokines such as IL-6 and TNF-α [ 23 ]. While initially studied in the context of antiviral defense and cancer immunology, cGAS-STING signaling has been implicated in a wide range of neurological conditions such as neurodegenerative diseases, ischemic stroke, and traumatic brain injury, in which overactivation of the pathway led to secondary tissue injury [ 24 – 30 ]. Moreover, the cGAS-STING pathway has been found to play a role in pain modulation, with preclinical evidence suggesting contrasting roles of the cGAS–STING pathway in nociception [ 31 ]. While STING agonists have been found to be anti-nociceptive in some experimental models, long term hyperactivation of the pathway has been shown to contribute to pain sensitization and maintenance in chronic pain conditions, while inhibition reduced nociception [ 32 – 34 ]. Despite its established roles in both inflammation and nociception, the cGAS–STING pathway has not been studied in experimental migraine models such as SD. As migraine is a neuroinflammatory disorder associated with pain, investigation of the involvement of this pathway in SD-associated inflammatory response and migraine mechanisms could provide novel insights into migraine pathophysiology and therapeutic interventions [ 35 ]. In this study, we aimed to investigate the activation and functional relevance of the cGAS-STING pathway following SD in the cerebral cortex. Both a non-invasive optogenetic model using Thy1-ChR2-YFP transgenic mice and the conventional potassium chloride (KCl) model were used to induce SD, followed by immunohistochemistry, WES capillary nano-immunoassay, and behavioral assays for analysis. We demonstrated that SD activates cGAS-STING signaling predominantly in neurons, with downstream effects on microglial activation. We determined the protein levels and cellular localization of pathway components, including cGAS, STING, p-IRF3, and IFN-β within the cerebral cortex. We further explored the consequences of pharmacological modulation of STING activity on microglial activation, susceptibility to SD, and development of periorbital mechanical allodynia following SD, as a surrogate for migraine-like pain. Our findings revealed that cGAS-STING pathway modulates cortical excitability and cranial nociception; thus, it may represent a novel mechanistic link between cortical hyperexcitability, neuroinflammation, migraine, and related disorders. Materials and Methods Animals All experimental procedures performed on animals were approved by Hacettepe University Animal Experimentations Local Ethics Board (Approval number: 2021/67). For animal care, in vivo procedures, and reporting, the ARRIVE guidelines were followed. Mice were housed under 12-hours light-dark cycle at 22 ± 2 ºC temperature and 50–60% humidity with ad-libitum access to food and water. We used 25–35 gr, 8–16 weeks old female and male Thy1-ChR2-YFP mice (Jackson Laboratories, Bar Harbor, ME) (n = 68) and wild-type C57BL6/J mice (n = 64). The animals were randomly assigned to experimental groups. Before surgery, the mice were anesthetized with 4% isoflurane for induction and 1–2% isoflurane for maintenance in 100% oxygen at 2 L/min. Anesthesia depth was regularly monitored using a paw pinch. Body temperature was maintained between 36.5–37.2 ºC during surgery using a rectal probe connected to a homeothermic blanket with a control unit (Harvard Apparatus, USA). Pulse rate and oxygen saturation were determined using a pulse oximeter from the right lower limb and maintained within homeostatic limits during the procedures. Following surgery, the mice were allowed to recover on a heating blanket until they resumed their normal activities and then returned to their standard housing conditions until sacrifice. The number of mice used in each group for the in vivo experiments is indicated in the respective figure legends. For all experiments, data from a single animal were used as the experimental unit. Optogenetic SD Induction SD induction with optogenetic stimulation was performed as described previously [ 36 ]. Thy1-ChR2-YFP mice, which express channelrhodopsin-2 in cortical layer 5 neurons, were used for optogenetic stimulation experiments. Briefly, after anesthesia, the mice were placed in a stereotaxic frame. After shaving and disinfecting the scalp, a midline incision was performed to expose the intact skull. To detect SDs, electrophysiological recordings were obtained using a silver/silver chloride (Ag/AgCl) pellet electrode to monitor direct current (DC) potential shifts. A small part of the right parietal bone was focally thinned using a dental drill (World Precision Instruments, USA), and recording electrodes were placed at this site. During drilling, the parietal bone was regularly flushed with cold saline to prevent heating and thermal damage. An optical fiber (Thorlabs Inc, USA) was placed on the right frontal bone (overlying the motor cortex) for transcranial light stimulation. Blue laser (470 nm, 4 mW power) stimulation was performed for 10 s to induce SD. Successful induction was confirmed by a DC potential shift of at least 5 mV amplitude on electrophysiological recordings. SD was induced once or six times within 1 h in different experimental groups. After SD induction was completed and the DC potential returned to baseline, the scalp was sutured, and the mice were returned to their standard housing conditions. For the sham controls, the same steps were followed except for the light stimulation, and the experiments were terminated after an equivalent duration as the induction groups. Determination of SD Threshold To assess the SD threshold, the conventional method of topical KCl application on the intact dura was performed on wild-type C57BL6/J mice. Although optogenetic stimulation in Thy1-ChR2-YFP mice provides a reliable and reproducible method for inducing SD, the optogenetic SD threshold is influenced by channelrhodopsin expression in the cortex and skull thickness, rather than reflecting the brain’s innate SD susceptibility [ 36 , 37 ]. To ensure comparability with the established literature on SD threshold determination, we used conventional KCl induction, which enables a well-known measurement of cortical susceptibility to SD [ 2 , 38 , 39 ]. Under anesthesia, the mice were secured in a stereotaxic frame, and following scalp shaving and disinfection, a midline incision was made to expose the skull. For SD induction via KCl application, a burr-hole of 1 mm diameter was opened into the right frontal bone (1 mm anterior, 1 mm lateral to the bregma). During drilling, the skull was regularly irrigated with cold saline to prevent thermal damage. The dura was kept intact, and care was taken to avoid bleeding and tissue injury. Also, a portion of the right parietal bone was focally thinned, for the placement of recording electrodes. Cotton balls (< 1 mm in diameter) soaked in gradually increasing KCl concentrations (12.5, 25, 50, 75, 100, 150, and 300 mM) were sequentially placed on the dura. Each concentration was applied for 5 min, and the occurrence of SD was monitored. If no SD occurred within 5 min, the cotton ball was removed, and the burr hole was rinsed with 0.9% sodium chloride (NaCl) solution. After 5 min of waiting, the next higher concentration was applied. This procedure was repeated until the first SD occurred. The lowest KCl concentration that induced SD was recorded as the SD threshold. The burr hole was closed with bone wax, the scalp was sutured, and the mice were returned to their cages for recovery. Assessment of Periorbital Allodynia Thy1-ChR2-YFP mice were anesthetized with isoflurane and placed in a stereotaxic frame. After a single midline incision on the scalp, the intact skull was exposed. Two plastic tubes (1 mm and 3 mm in diameter) were affixed to the frontal and parietal bones using cyanoacrylate and Superbond dental adhesive (Sun Medical, Japan), to allow light stimulation and electrophysiological recording, respectively. The tube openings were sealed, and the tubes were kept moist until SD induction. Mice were allowed to recover for one week after surgery to minimize the potential effects of surgical stress or inflammation on the baseline mechanical sensitivity measurements. On the day of the experiment, an LED fiber optical cable was inserted through the plastic tube on the frontal bone, and transcranial light stimulation with 470 nm blue laser (4 mW power) was performed for 15 s to induce SD. SDs were confirmed by electrophysiological recordings using Ag/AgCl pellet electrodes inserted through a plastic tube on the intact parietal bone. A total of six SDs were induced per mouse. After the procedure, the mice were returned to their home cages to recover and establish baseline behavior before allodynia testing. Periorbital mechanical sensitivity was assessed at predefined time points (2 and 24 h) following the last SD induction using the manual von Frey test to determine periorbital withdrawal thresholds. Periorbital allodynia was defined as a reduction in the periorbital withdrawal threshold. Mice were placed in custom-made chambers (3 × 3 × 7 cm) to prevent escape during testing while allowing free movement of the head and forepaws. They were acclimated to the chamber in three separate 30-minute sessions prior to testing. Calibrated von Frey monofilaments (Ugo Basile, Italy) with increasing forces (0.008, 0.02, 0.04, 0.07, 0.16, 0.40, 0.60, 1.0, and 1.4 g) were applied perpendicularly to the periorbital area (superior-medial to the eye) with slight bending for 3 s or until a positive withdrawal response was observed. Brisk head withdrawal, head shaking, or facial grooming with forepaws on the stimulated side were accepted as positive responses. Mechanical head withdrawal thresholds (HWT) were determined as the force that elicited a positive response in at least three of five consecutive applications. Baseline thresholds were established before the administration of pharmacological agents and/or SD inductions. Behavioral tests were performed in the same part of the day in the afternoon. The evaluators were blinded to the group allocations and drug treatments. Pharmacological Agents The STING inhibitor C-176 (20 mg/kg, Cayman Chemicals 25859) was dissolved in 5% dimethyl sulfoxide (DMSO) and 95% isotonic saline and delivered intraperitoneally 4 h before SD threshold assessment. The STING agonist 2’3’-cGAMP (1 mg/kg, Cayman Chemicals 19887) was dissolved in phospahte buffered saline (PBS) and delivered intranasally 4 h before the SD threshold assessment. Working solutions of the agents were freshly prepared before each experiment. The delivery methods and timings were determined according to previous studies [ 27 , 32 , 40 ]. To assess the effect of drug administration on steady-state nociceptive behavior, the periorbital von Frey test was performed before and 4 hours after drug or vehicle administration. To assess periorbital allodynia following SD, 2’3’-cGAMP (or its vehicle) was administered 4 h before SD induction. Immunofluorescent staining At 5 or 24 h after optogenetic SD induction, mice were anesthetized with urethane/xylazine (1.5 g/kg and 10 mg/kg, respectively, i.p.) and transcardially perfused with cold saline followed by cold 4% paraformaldehyde. Brains were harvested and immersed in 4% paraformaldehyde (Sigma Aldrich, Germany) solution at 4°C for 24 h. The brains were then transferred to 30% (w/v) sucrose in PBS and incubated for 48 h for cryoprotection. Brains were embedded in optimal cutting temperature (O.C.T.) compound (Tissue-Tek, Sakura) and stored at -80°C until sectioning. Frozen coronal brain sections (20 µm thick) were obtained using a cryostat (Leica-CM1100). For immunofluorescent staining, antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) at 85°C for 15 min in a water bath. The slides were then allowed to cool to room temperature for 30 min. The sections were washed with TBS and blocked with 10% normal goat serum (Jackson ImmunoResearch, 005-000-121) at room temperature for 1 h. Next, sections were incubated overnight at 4 ºC with primary antibodies: rabbit anti-STING antibody (1:200, monoclonal D2P2F, 13647S, Cell Signaling Technology), rabbit anti-cGAS antibody (1:200, polyclonal, MBS9143559, MyBioSource), rabbit anti-p-IRF3 antibody (1:300, monoclonal, 29047S, Cell Signaling Technology), rabbit anti-IFN beta antibody (1:300, polyclonal, NBP1-77288, Novus), mouse anti-NeuN antibody (1:300, monoclonal MAB377 clone A60, Merck Millipore), goat anti-Iba1 antibody (1:250, polyclonal, NB100-1028, Novus), mouse anti-S100β antibody (1:200, polyclonal, Atlas Antibodies). The next day, the sections were washed and incubated with the appropriate secondary antibodies at room temperature for 1 h. Secondary antibodies used were as follows: Cy3- or Cy5-conjugated secondary antibodies of IgG (H + L) were used (all from Jackson ImmunoResearch): goat anti-rabbit IgG (Cy3, 111-165-144; Cy5, 111-175-144), goat anti-mouse IgG (Cy3, 115-165-003), donkey anti-mouse IgG (Cy5, 715-175-151), and donkey anti-goat IgG (Cy3, 705-165-003). For double labeling, the sections were washed and incubated with the second primary antibody overnight at 4°C. The following day, the sections were incubated with the appropriate secondary antibodies and mounted with PBS/glycerol mounting medium containing Hoechst 33258 (Invitrogen, 1:1000 dilution for nuclear staining). Imaging and Analysis Images of stained brain sections were acquired using a confocal laser scanning microscope (Leica TCS SP8) equipped with a diode (405, 638 nm) and OPSL (488, 552 nm) lasers, with X, Y, and Z-movement controllers, and high-resolution PMT (Zeiss, Oberkochen, Germany) and HyD (Leica) detectors. Three images were obtained from the ipsilateral parietal cortex region (corresponding to the region used for electrophysiological recordings) for each animal at 40x magnification with identical acquisition settings between the experimental and control groups. Images were captured in Z-stack mode with 0.50 µm wide steps along the Z-axis, and mid-plane images from each Z-stack were used for analysis. For quantification, the number of positively stained cells for each protein (cGAS, STING, p-IRF3, IFN-β) was counted and expressed as a ratio to the total number of Hoechst, NeuN, Iba1, and S100β positive cells. Positive cell counting and colocalization analyses were performed using ImageJ 1.54 software (National Institutes of Health, USA). Microglia Morphology Analysis To assess microglial activation, microglial morphology was analyzed using FIJI software (version 1.54). Z-stack images of Iba1 stained sections were examined for microglial cells, and the slices containing microglia were processed as maximal intensity projections while avoiding overlapping cells from different focal planes. Maximum intensity projection images were processed to remove the background and thresholded to generate binary images using consistent settings across all samples. In these binary images, microglial cells were identified, and their areas and perimeters were calculated from the binary silhouettes. The ramification index (RI), a metric reflecting the degree of process extension and morphological complexity of microglia, was calculated for each cell using the following formula: [perimeter of cell (µm)] 2 /4π [cell area (µm 2 )] [ 41 , 42 ]. Three non-overlapping images from the parietal cortex ipsilateral to SD were used per animal, and the mean RI was used for comparisons between groups. Detection of protein levels with the sensitive automated capillary-based Western blot technique WES (Western Electrophoresis System) At 5 or 24 h after SD induction, the mice were anesthetized and sacrificed by decapitation. Brains were harvested, and the ipsilateral and contralateral parietal cortex regions were isolated. Brain tissues were frozen in liquid nitrogen and stored at -80°C until use. Tissues were lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer containing 1X HALT Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific), sonicated, and homogenized on ice. Lysates were centrifuged at 10,000×g for 20 min at + 4°C. Supernatants were transferred to new tubes, and the protein concentrations were determined using a BCA protein assay kit (Thermo Scientific™, 23225). Protein levels were assessed using a WES Automated Western Blot System (ProteinSimple, Bio-Techne) with the 12–230 kDa Separation Module (ProteinSimple, SM-W004) and Anti-Rabbit Detection Module (ProteinSimple DM-001) according to the manufacturer’s instructions. The WES system was selected to enable the quantitative, highly sensitive, and automated detection of target proteins from limited tissue samples [ 43 ]. Briefly, samples were diluted to 1 mg/ml in sample buffer, mixed with a master mix containing the internal standard and DTT, and heated at 95°C for 5 min. Samples, antibody diluents, primary antibodies, HRP-conjugated secondary antibodies, and chemiluminescent substrates were loaded into the plate that was part of the separation module. The following primary antibodies were used: rabbit anti-STING antibody (13647S, Cell Signaling Technology), rabbit anti-IRF3 antibody (MA5-32348, Invitrogen), rabbit anti-p-IRF3 antibody (29047S, Cell Signaling Technology), rabbit anti-IFN beta antibody (NBP1-77288, Novus), and anti-vinculin (E1E9V, XP® Rabbit mAb #13901). Capillary electrophoresis was performed in the WES instrument using default settings: stacking and separation at 475 V for 30 min; blocking reagent for 5 min; primary and secondary antibody incubations for 30 min; and Luminol/peroxide chemiluminescence detection for ~ 15 min (exposures of 1, 2, 4, 8, 16, 32, 64, 128, and 512 s). Compass for Simple Western (Bio-Techne) was used to generate lane view plots, and the peak area data were plotted as the ratio of the target protein peak to the loading control. Statistical Analysis Statistical analyses were performed using the Statistical Package for Social Sciences (SPSS for Windows, version 26.0) and GraphPad Prism 9.0 (GraphPad, San Diego, CA). Data are presented as mean ± standard error of the mean or median ± interquartile range, depending on the data distribution. Data were analyzed using the Shapiro-Wilk test, and if data were distributed normally, the Student’s t-test was used for paired or unpaired 2 group comparisons, and one-way analysis of variance (ANOVA) or repeated measures ANOVA was used for multiple group comparisons. For non-normally distributed data, the Mann-Whitney U and Kruskal-Wallis tests were used for unpaired group, and the Wilcoxon and Friedman tests were used for paired group comparisons. Statistical significance was set at p < 0.05. Results Spreading Depolarization Increases cGAS-STING Pathway Activity in the Cortex To elucidate whether the cGAS-STING pathway is activated in the mouse cerebral cortex following SD, we performed immunohistochemistry to determine the levels of pathway proteins in the ipsilateral cortices of mice subjected to six SDs, a single SD, or sham surgery (n = 6/group). SDs were optogenetically induced to prevent injury. Brain samples were collected 5 and 24 h after SD induction (Fig. 1 A). First, we performed immunohistochemistry for cGAS and STING, the primary mediators of pathway activation. Immunohistochemical analysis revealed a significant increase in the percentage of cGAS-immunopositive and STING-immunopositive cells in the ipsilateral cortex 5 h after multiple SDs compared to the sham group (Fig. 1 B; Supplementary Fig. 1B, 2A, D). This increase persisted for 24 h (Supplementary Fig. 1B, 2D). Since the increase in cGAS and STING immunopositivity suggests pathway activation, we further examined the expression of the downstream effectors, p-IRF3 and IFN-β, which play key roles in cGAS-STING-mediated inflammation. In line with this, the percentage of p-IRF3 and IFN-β-immunopositive cells was significantly increased at both 5 h and 24 h following SDs compared to sham (Fig. 1 B; Supplementary Fig. 1C, 1D). To assess whether a single SD was sufficient to activate the pathway, we also examined cGAS-STING pathway immunopositivity after a single SD. We observed a notable increase in the number of cGAS-, STING-, p-IRF3-, and IFN-β-positive cells compared to the sham group, with the effect lasting up to 24 h (Fig. 1 B; Supplementary Fig. 1B-D, 2D). These findings suggest that even a single SD can activate the cGAS-STING pathway in the cortex. To quantitatively measure cGAS-STING pathway protein levels in the ipsilateral cerebral cortex after SD, we used the WES capillary nanoimmunoassay technique in mice subjected to six SDs, a single SD, or sham surgery (n = 4/group) (Fig. 1 A). Following both single and multiple SDs, STING protein levels decreased significantly (Fig. 1 C, D). While immunohistochemistry showed increased STING immunoreactivity, the reduction observed in WES is likely due to the detection being limited to the unphosphorylated form of STING. Since immunohistochemistry is not always reliable to distinguish between phosphorylated and unphosphorylated forms, and the change in staining pattern reflects STING oligomerization and translocation from the ER to vesicles after activation, the overall STING signal in immunostaining might show both active and inactive forms (Supplementary Fig. 1A) [ 44 , 45 ]. This methodological distinction may suggest that the apparent reduction in STING levels in WES reflects STING activation and post-translational modification following SD, rather than true downregulation [ 46 ]. Confirming this, the levels of downstream effectors of STING, p-IRF3 and IFN-β, were significantly increased following multiple SDs. Notably, even a single SD robustly increased p-IRF3 and IFN-β levels in the ipsilateral cortex (Fig. 1 C, F, G). Together, these findings indicate that the cGAS-STING pathway is activated in the brain cortex following SD. SD Increases cGAS-STING Pathway Activity Predominantly in Neurons To identify whether cGAS-STING pathway activation following SD occurs in a cell type–specific manner, we performed double immunostaining for cGAS, STING, p-IRF3, and IFN-β alongside cell type–specific markers for neurons, microglia, and astrocytes. We then quantified the proportion of cGAS-STING pathway-immunopositive cells within each cell type (Fig. 2 A). At baseline, neurons displayed considerable cGAS and STING immunopositivity (Fig. 2 B, C; Supplementary Fig. 2B, E). Following both multiple SDs and a single SD, the percentage of cGAS, STING, p-IRF3, and IFN-β immunopositive neurons in the ipsilateral cortex significantly increased compared to that in the sham group, suggesting pathway activation in neurons following SD (Fig. 2 B, C, D, F; Supplementary Fig. 2B, E). Microglia, the brain’s primary resident immune cells, exhibited high baseline STING immunopositivity (Fig. 2 B, E). Although both single and multiple SDs significantly increased STING immunopositivity in microglia, there were no significant changes in cGAS, p-IRF3, or IFN-β immunopositivity in microglia compared to the sham group (Fig. 2 B, D, E, F; Supplementary Fig. 2B, F). These findings suggest that neurons are the main cellular sites of cGAS-STING pathway activation following SD. In contrast, although microglia showed high baseline STING expression and further upregulation following SD, the lack of corresponding increases in cGAS, p-IRF3, or IFN-β suggests following SD, the pathway activation is limited in these cells within the studied timeframe. Besides, astrocytes, which are also known to play roles in cGAS-STING mediated neuroinflammation, exhibited baseline cGAS and STING immunopositivity (Fig. 2 B, G; Supplementary Fig. 2B, G). However, neither single nor multiple SDs significantly increased the immunopositivity of cGAS, STING, p-IRF3 or IFN-β in astrocytes compared to sham (Fig. 2 B, D, F, G; Supplementary Fig. 2B, G). Together, these findings suggest that SD activates the cGAS-STING pathway predominantly in neurons, with limited activation in microglia. cGAS-STING Pathway Inhibition Prevents Microglial Morphological Changes Following SD It has been shown that multiple SDs activate microglia, as evidenced by distinct morphological alterations [ 47 ]. This activation is characterized by a transition from a highly ramified, surveillant morphology to a more amoeboid, reactive state, reflecting a shift toward an inflammatory or phagocytic phenotype. Consistent with these reports, our observations also revealed significant microglial morphological changes following SD. To evaluate microglial activation following SD, we examined microglial morphology in optogenetic SD induced brains and quantified the ramification index for microglia, as described in methods section (Fig. 3 A). At 24 h after multiple SDs, microglia in the ipsilateral cortex displayed distinct morphological changes, with more amoeboid morphology and retracted processes compared to sham group, accompanied by a significantly reduced ramification index (Fig. 3 B, C). However, no significant morphological changes were observed at 5 h after multiple SDs, and a single SD also failed to induce significant morphological alterations in microglia (Fig. 3 B, C). Our findings indicate that microglia are activated 24 h after multiple SDs, but not after a single SD, supporting the findings in previous literature [ 18 , 47 ]. We also hypothesized whether cGAS-STING pathway could have a role in SD-induced microglial morphological changes. To determine whether STING signaling is required for SD-induced microglial morphological changes, we pharmacologically inhibited STING using the selective antagonist, C-176 (Fig. 3 A). Immunohistochemistry and WES capillary nanoimmunoassay analysis confirmed effective inhibition, as the SD-induced increases in pathway proteins were significantly suppressed in C-176–treated mice compared to those in vehicle controls (Supplementary Fig. 3A-G). Importantly, STING inhibition with C-176 also effectively prevented SD-induced morphological changes in microglia, as reflected by a more ramified morphology and significantly higher ramification indices in C-176-treated mice, compared to vehicle treated mice, 24 h after multiple SDs (Fig. 3 D, E). These findings indicate that STING activity contributes to SD-induced microglial activation, possibly through a non-canonical, interferon-independent mechanism in microglia [ 48 ]. It is also possible that SD-induced microglial activation is downstream to increased neuronal cGAS-STING signaling, as it was shown that release of inflammatory mediators from neurons after SD is critical for microglial activation [ 47 ]. cGAS-STING Pathway Modulates Susceptibility to SD and Cranial Nociception The cGAS-STING pathway has been implicated in the regulation of neuronal excitability and nociception through enhanced type 1 interferon signaling in neurons [ 32 ]. To determine whether this pathway also modulates susceptibility to SD and cranial nociception, we pharmacologically manipulated STING activity using its endogenous agonist 2’3’-cGAMP and its specific antagonist C-176. Activation of the cGAS-STING pathway following intranasal administration of 2’3’ cGAMP was confirmed by immunohistochemistry and WES capillary nanoimmunoassay analysis, in which we observed significantly increased p-IRF3 and IFN-β expression in the 2’3’ cGAMP treated group compared to vehicle treatment (Supplementary Fig. 4A-F). Four hours after drug administration, we assessed the SD thresholds by KCl induction (Fig. 4 A). STING inhibition with C-176 significantly decreased the SD threshold compared to vehicle, suggesting increased vulnerability to SD (Fig. 4 B). Conversely, STING activation with 2’3’-cGAMP significantly increased the SD threshold compared to that in vehicle-treated mice, indicating reduced susceptibility to SD (Fig. 4 B). Together, these findings demonstrate that cGAS-STING pathway activity can affect the brain’s susceptibility to SD, pointing to a potential role in SD-related pathophysiology, such as migraine aura and cortical injury. To explore the role of the cGAS-STING pathway in craniofacial nociception, we investigated its effects on periorbital sensitivity and trigeminal nociception. Periorbital HWT was assessed at baseline and 4 h after treatment with either the STING inhibitor C-176 (or vehicle) or the STING agonist 2’3’ cGAMP (or vehicle) using manual von-Frey testing (Fig. 4 A). We observed no change in periorbital HWT following vehicle treatment, confirming test stability (Fig. 4 C, D). Notably, C-176 administration significantly decreased periorbital HWT compared to baseline, indicating heightened trigeminal nociception (Fig. 4 C). Conversely, administration of 2’3’ cGAMP significantly increased periorbital HWT compared to baseline, suggesting reduced cranial nociceptive sensitivity (Fig. 4 D). These findings indicate that cGAS-STING pathway activity modulates basal cranial pain sensitivity and may influence the steady-state trigeminal nociceptive processing. Based on our findings that cGAS-STING pathway activation suppresses SD susceptibility and reduces periorbital mechanical sensitivity, we next investigated if the pathway activation prior to SD could prevent the SD-induced development of periorbital allodynia. Hence, 2’3’ cGAMP or vehicle was intranasally administered to mice 4 h before optogenetic induction of six SDs. Periorbital HWT was assessed using the manual von Frey test at 2 and 24 h after the final SD wave (Fig. 4 A). In the vehicle-treated group, SDs induced significant periorbital mechanical allodynia, as evidenced by significantly decreased periorbital HWT at 2 and 24 hours compared to baseline (Fig. 4 E, F). Remarkably, 2’3’ cGAMP administration prior to SDs prevented this decrease in periorbital HWT, with no significant difference from baseline values (Fig. 4 E, F), indicating a protective effect. There was no significant effect of sex on periorbital HWT either at baseline or after SDs, regardless of the intervention. Together, these findings suggest that the cGAS-STING pathway modulates both SD susceptibility and trigeminal nociception and may serve as a potential therapeutic target for headache disorders. Discussion Our findings demonstrate that optogenetically induced SD activates the cGAS-STING innate immune pathway in the cerebral cortex predominantly in neurons and contributes to microglial morphological changes. Although it is established that SD can trigger sterile neuroinflammation in the brain parenchyma, we suggest a previously unrecognized mechanism linking SD to neuroinflammatory signaling and pain processing. Modulation of the cGAS-STING pathway activity influenced both cortical susceptibility to SD and periorbital nociceptive responses. Activation of this pathway suppressed SD generation and effectively prevented the SD-induced development of periorbital allodynia. These findings highlight the cGAS-STING pathway as a potential therapeutic target for SD-induced neuroinflammation and nociception, with implications to headache and other SD-related neurological disorders. Using both immunohistochemistry and capillary-based Western blot (WES) analysis, we showed that even a single, injury-free optogenetically induced SD was sufficient to initiate the neuroinflammatory cGAS-STING pathway activation in neurons in the cerebral cortex, and that multiple SDs amplified this activation. Activation of the cGAS-STING pathway after SD induced the expression of cGAS, STING, p-IRF3, and IFN-β in the ipsilateral cortical neurons. Our findings showed increased STING immunoreactivity after SD, whereas WES analysis revealed a reduction in unphosphorylated STING levels. STING activation involves phosphorylation, oligomerization and translocation to vesicles [ 19 ]. Our WES analysis might have detected only the unphosphorylated form because the STING antibody we used is not specific to phosphorylated STING; therefore, this apparent reduction does not indicate true downregulation [ 46 ]. On the contrary, immunohistochemistry, which can show total STING regardless of phosphorylation status, revealed an overall increase, supporting the notion that SD induces STING activation rather than true downregulation [ 44 , 45 ]. The persistence of type I interferon signaling up to 24-h following SD suggests that cGAS-STING activation may contribute to sustained neuroinflammatory and excitability-related alterations in the cortex. These findings align with the established concept that SD triggers sterile inflammatory responses in brain parenchyma. SD leads to elevated expression of pro-inflammatory cytokines and mediators in brain parenchyma, such as IL-1α, IL-1β, IL-6, IL-13, TNF-α, COX-2 and inducible nitric oxide synthase (iNOS), starting approximately at 3-h post-SD, reaching maximal level 24-h post-SD and resolving at 48-, 72-h post-SD [ 12 , 14 , 15 , 49 – 51 ]. Importantly, even a single optogenetically induced SD -despite the absence of tissue injury- was sufficient to trigger the upregulation of pro-inflammatory mediators in the cerebral cortex, confirming an intrinsic consequence of SD itself [ 16 , 18 , 52 ]. Although some inflammatory mediators, such as HMGB-1, exhibited increased expression after multiple SDs, but not after a single SD, with a dose-response relationship [ 53 ]. Indeed, in our study we observed that a single SD can robustly increase type 1 interferon expression through the cGAS-STING pathway in mouse cerebral cortex and it persisted up to 24-h post-SD. Conventionally the inflammatory response induced by SD was thought to affect only the ipsilateral hemisphere, so the contralateral hemisphere was used as control [ 54 ]. However recent studies have shown that neuroinflammation induced by SD can be bilateral, regardless of the induction method, and local N -methyl-D-aspartate receptor (NMDAR) and purinergic P2X7 receptor (P2X7R) antagonism in ipsilateral hemisphere was able to attenuate neuroinflammatory changes in the contralateral hemisphere [ 52 , 55 ]. Taking this into consideration we did not use contralateral hemisphere as control in our study and compared SD induced ipsilateral hemisphere to sham controls. Notably, cGAS-STING pathway activation and increased type 1 interferon expression following SD was neuron specific in brain cortex. Neurons emerging as the primary responders, displaying early and robust upregulation of pathway aligns with earlier studies identifying neurons as key initiators of SD-induced sterile inflammation through mechanisms involving the opening of Panx1 megachannels, caspase-1 activation, and the release of IL-1β and alarmins such as HMGB1 [ 12 ]. HMGB1 release from neurons in turn causes to astrocyte and microglia activation and pro-inflammatory cytokine release via NF-kB signaling [ 12 ]. Additionally, another innate immune system pathway NLRP3 inflammasome was also shown to be predominantly assembled in neurons after SD. NLRP3 inflammasome activation in neurons leads to the release of pro-inflammatory cytokines and has been shown to play a role in trigeminovascular system activation after SD, while microglia and astrocytes do not show NLRP3 inflammasome assembly following SD [ 18 ]. We did not observe activation of the canonical cGAS-STING pathway following SD in astrocytes, and microglia while an increased immunoreactivity constrained to STING was exhibited in microglia. However, this was not accompanied by p-IRF3/IFN-β induction. This raises the possibility that STING signaling in microglia may proceed through alternative, non-canonical pathways, such as NF-κB signaling [ 19 , 56 ]. While we did not directly assess this mechanism, such a route could underlie the morphological changes we observed in microglia 24 h after repeated SDs. The fact that these changes were abolished by STING inhibition suggests a potential contribution of STING signaling to the later stages of the microglial inflammatory response. It is possible that cGAS-STING pathway activation and associated type 1 interferon signaling can be induced in microglia beyond the timeframe we studied. In ischemia and trauma models cGAS–STING pathway activation in microglia was typically determined approximately 3 days after the injury [ 30 , 57 ]. Full-blown pathway activation and induction of type 1 interferon expression in microglia might happen in later time-points following SD. Furthermore, neuronal cGAS-STING pathway activity can also be responsible for the changes observed in microglia following SDs as it was shown that following SD, especially multiple SDs, inflammatory mediators released from neurons play critical roles in later microglial activation [ 18 , 47 ]. Earlier cGAS-STING pathway activation in neurons following SD can play a similar role for microglial morphological changes in this context. Additional studies are needed to determine whether neuronal STING activity is responsible for microglial morphological changes following multiple SDs or whether microglial STING activity plays a delayed, non-canonical role in SD-induced neuroinflammation. Beyond its antiviral role, the cGAS–STING pathway has been implicated in various neuroinflammatory and neurodegenerative disorders, and its over-activation leads to increased tissue injury in ischemic stroke, subarachnoid hemorrhage, and traumatic brain injury, whereas pharmacological inhibition mitigates damage [ 27 , 58 , 59 ]. In microglia, cGAS–STING pathway triggers NF-κB and NLRP3 signaling, leading to pro-inflammatory cytokine release, M1 polarization, and pyroptosis [ 30 , 56 , 57 , 60 , 61 ]. Considering that optogenetically induced SD does not cause any tissue injury in the brain but can cause a significant metabolic stress to neurons, it is plausible that inflammatory response initiates in neurons and microglial response emerges later. In line with this we observed microglial morphological changes associated with microglial activation starting at 24-h following multiple SDs, as reported previously [ 47 ]. In our study, these morphological alterations were quantified using the ramification index, a well-established measure of microglial complexity in the literature, which reflects soma size and process extension. Thus, a lower index indicates a transition to an activated, amoeboid morphology [ 41 , 62 ]. Notably, treatment with the STING inhibitor C-176 preserved the ramified microglial morphology and prevented the decrease in ramification index following multiple SDs, highlighting the role of the cGAS–STING pathway in SD-induced microglial activation. This aligns with previous reports showing that enhanced cGAS–STING signaling in microglia promotes a pro-inflammatory phenotype, whereas its inhibition reduces M1 polarization and supports M2 transition, particularly in models of brain ischemia and neuropathic pain [ 34 , 60 , 63 , 64 ]. Consistent with this evidence, our findings add SD to the growing list of neurological conditions in which cGAS–STING signaling contributes to microglial activation. Astrocytes express notable cGAS and STING [ 65 ] and contribute to neuroinflammation in several central nervous system (CNS) diseases, including SD. However, astrocytes do not exhibit the full spectrum of inflammatory responses observed in neurons following SD [ 12 , 66 – 70 ]. This has been reported for NLRP3 inflammasome activation, which occurs predominantly in neurons rather than in astrocytes or microglia following SD [ 18 ]. In a similar manner, we did not detect SD-induced cGAS–STING pathway activation in astrocytes. Our findings suggest that key innate immune responses induced by SD are primarily initiated in neurons, positioning them as the main drivers of the SD-induced inflammatory cascade. Beyond inflammation, our study demonstrates that cGAS-STING pathway activation can modulate cortical excitability, thereby influencing susceptibility to SD and associated pain sensitivity. Pharmacological activation of STING with 2’3’-cGAMP decreased SD susceptibility and attenuated periorbital mechanical sensitivity, whereas inhibition with C-176 increased SD susceptibility and exacerbated nociceptive responses. Notably, STING activation by an agonist prior to multiple SDs prevented the development of periorbital allodynia at 2 and 24 h later, indicating a protective role in SD-induced pain responses. These findings suggest that cGAS–STING activation exerts a protective effect against SD-induced craniofacial pain. SD susceptibility is the ease of brain tissue to develop SD waves. It is affected by various factors such as excitation/inhibition balance in the brain and physiological parameters [ 39 ]. SD susceptibility is the most relevant SD attribute, and it is used as a therapeutic target for SD-associated disorders [ 8 , 39 ]. We used the conventional KCl induction method for SD threshold assessment. Optogenetic stimulation provides a reliable and reproducible method for inducing SD, which overcomes the confounders resulting from tissue injury. Even though electrophysiological characteristics of SD induced with optogenetic stimulation are consistent with those of conventional induction methods, properties of optogenetic SD susceptibility differs from conventional methods [ 36 ]. Optogenetic SD susceptibility is strongly correlated with relative channel rhodopsin expression in the cortex rather than the innate SD susceptibility of the brain [ 37 ]. Since light stimulation is done through the intact skull, differences in skull thickness can also affect optogenetic SD thresholds [ 37 ]. Burr hole opening for KCl application was shown not to affect SD thresholds when it is done without damaging the cortex or dura [ 36 ]. Moreover, threshold KCl concentration for SD induction shows good concordance with other conventional methods such as direct electrical stimulation [ 39 , 71 ]. Thus, in our study KCl-induced SD thresholds were deemed more accurate for reflecting brains innate susceptibility to SD. The link between SD and cephalic allodynia has been well established. Optogenetically induced SD was determined to cause periorbital allodynia within an hour, resolving in two days, while multiple SDs induced prolonged pain responses lasting up to 14 days [ 7 , 72 , 73 ]. Since our findings revealed a role for cGAS–STING signaling in SD, we extended our investigation to examine its influence on SD-induced periorbital pain. Recent studies on pain models where it was observed that STING activation induced type I interferon signaling in sensory neurons, which reduced sodium and calcium channel activity, and suppressed pain behaviors in cancer induced pain and neuropathic pain models [ 32 , 74 ]. It is worth noting that, STING agonists produced acute analgesic effects, alleviating mechanical allodynia within 4 h of administration, with an effect that lasted 24 h [ 32 , 74 , 75 ]. Analgesia level was comparable to morphine and is shown to be STING- and IFN-α/β-dependent through direct suppression of cancer-induced nociceptor hyperexcitability in dorsal root ganglia [ 75 ]. However, the role of cGAS–STING signaling in nociception remains controversial, as conflicting evidence also points to pro-nociceptive effects of pathway activation in chronic pain models. Some studies have reported that prolonged cGAS–STING activation in microglia promotes neuropathic pain via microglial M1 polarization, NF-κB signaling and pro-inflammatory cytokine release [ 34 , 63 , 76 ]. Inhibition of this pathway was shown to reverse hyperalgesia in chronic pain models, underscoring the dual role of cGAS–STING signaling depending on the cellular context [ 31 ]. Thus, the dual role of cGAS–STING signaling appears to depend on cell type -particularly neurons and microglia- as well as timing, and experimental design. In this study, we focused on the acute effects of STING activation (up to 24 h) on SD-induced periorbital allodynia and SD susceptibility, both of which reflect changes in neuronal excitability. The absence of microglial activation within this timeframe suggests that the observed anti-nociceptive effects are likely mediated by neuronal mechanisms. The protective role of early cGAS-STING activation highlights its context-dependent functions in pain modulation. Future studies assessing long-term pain responses in chronic migraine models are warranted to better understand the role of microglial cGAS–STING signaling in the pathogenesis of headache disorders. Our study has a couple of limitations. First, we used Thy1-ChR2-YFP transgenic mice to investigate cGAS–STING pathway activity and nociceptive responses after SD. While this optogenetic model avoids the confounding effects of brain injury during SD induction, inbred transgenic mice may exhibit different inflammatory and nociceptive responses compared to outbred mice or humans, who display greater genetic variability [ 77 ]. Nonetheless, previous studies have shown that the inflammatory response to SD is comparable, whether induced optogenetically in transgenic mice or by topical KCl application in wild-type mice [ 16 ]. Second, although we pharmacologically manipulated cGAS–STING activity using specific agonists and antagonists, genetic validation would further strengthen these findings. Previous studies have demonstrated that both global and sensory neuron-specific STING knockout increases mechanical and cold sensitivity, an effect also observed with the deletion of type I interferon receptor (Ifnar1), confirming the role of STING–IFN signaling in nociception [ 32 ]. Applying similar conditional knockout strategies targeting trigeminal neurons could help elucidate the specific contribution of STING and type I interferon signaling to craniofacial nociception. Third, while we used equal numbers of male and female mice to assess periorbital allodynia after SD, we did not monitor the estrous cycle in females, which can influence nociceptive behaviors. Previous studies have shown that females exhibit lower pain thresholds, with estrous stages characterized by high estradiol levels further lowering these thresholds. Interestingly, SD induction appears to override the influence of the estrous cycle by reducing periorbital pain thresholds, regardless of the hormonal stage [ 78 ]. In line with this, we did not observe any sex-based differences in baseline or post-SD nociceptive responses. Conclusion In conclusion, our study highlights the significant role of the cGAS-STING pathway in neuroinflammation, and nociception associated with SD. We demonstrated that optogenetically induced SD activates the cGAS–STING signaling cascade primarily in cortical neurons. We also showed that STING signaling can play a role in microglial morphological changes observed after multiple SDs. Moreover, our findings demonstrated that activation of the cGAS-STING pathway decreases SD susceptibility and cranial nociceptive responses. These results offer novel insights into the relationship between SD and neuroinflammation and suggest that the cGAS–STING pathway may function as a potential protective mechanism and therapeutic target for migraine and other SD-associated disorders. These results reveal that a novel innate immune pathway, previously implicated in various neuroinflammatory conditions, also serves as a neuroimmune interface that links SD to neuroinflammation and pain. Abbreviations SD Spreading depolarization IL Interleukin TNF-α Tumor necrosis factor-α COX-2 Cyclooxygenase-2 HMGB1 High mobility group box-1 NLRP3 Nucleotide-binding domain (NOD)-like receptor family pyrin domain containing 3 cGAS Cyclic GMP-AMP synthase STING Stimulator of interferon genes dsDNA double-stranded DNA 2’3’ cGAMP 2'3'-cyclic GMP-AMP ER Endoplasmic reticulum IRF3 Interferon regulatory factor 3 IFN Interferon NF-κB Nuclear factor-κB KCl Potassium chloride Ag/AgCl Silver/silver chloride DC Direct current NaCl Sodium chloride HWT Head withdrawal thresholds DMSO dimethyl sulfoxide PBS Phospahte buffered saline RI Ramification index ANOVA Analysis of variance iNOS Inducible nitric oxide synthase NMDAR N -methyl-D-aspartate receptor P2X7R purinergic P2X7 receptor CNS Central nervous system Ifnar1 Type I interferon receptor Declarations Ethics approval and consent to participate All experimental procedures performed on animals were approved by Hacettepe University Animal Experimentations Local Ethics Board (Approval number: 2021/67). Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This study is supported by Hacettepe University No: TSA-2022-19749 Authors' contributions Design and conceptualization of the study: MY, HK, KOS ; acquisition and analysis of data: KOS, BDD, HBK, MSB, CCA, HK, MY ; scientific discussions and interpretation of data: KOS, BDD, HBK, MSB, CCA, YCK HK, MY; Preparing figures: KOS; drafting the manuscript: KOS, BDD, HBK, MSB, CCA, YCK HK, MY. Acknowledgements We thank Mesut Fırat for his expert help with technical issues, Dilan Bozanoglu MD, PhD candidate for her help with analysis of WES capillary nano-immunoassay data, Gokce Gurler MD, PhD for her help with analysis of immunohistochemistry and imaging data. Schematic diagrams were prepared at Biorender.com. 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Journal of Neuroimmune Pharmacology 17 (3):453-469. doi:10.1007/s11481-021-10031-6 Mogil JS (1999) The genetic mediation of individual differences in sensitivity to pain and its inhibition. Proceedings of the National Academy of Sciences 96 (14):7744-7751. doi:doi:10.1073/pnas.96.14.7744 Harriott AM, Waruinge A, Appiah-Danquah V, Berhanu L, Morais A, Ayata C (2023) The effect of sex and estrus cycle stage on optogenetic spreading depression induced migraine-like pain phenotypes. The Journal of Headache and Pain 24 (1):85. doi:10.1186/s10194-023-01621-1 Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract.png Supplementaryfigure1.tif Supplementary Fig. 1: a. Representative immunofluorescence images showing changes in STING staining pattern following SD. Upon activation, STING undergoes oligomerization and translocation which appears as more condensed and intense staining (white arrows), in contrast to the diffuse and weaker signals observed in the non-activated state (arrow heads). To ensure specificity, only cells displaying the distinct condensed STING staining pattern (white arrows) were classified as STING-positive. b-d. Quantification of STING-, p-IRF3-, and IFN-β-positive cells as a percentage of total cells (Hoechst positive) at 5- and 24-h time points following one or six SDs (n=6/group). Data points in the graphs represent individual animals, with each value corresponding to the mean of three non-overlapping images from the ipsilateral parietal cortex. Values are expressed as mean ± SEM. Statistical comparisons were performed using Student’s t-test with Welch’s correction. ns: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001 Supplementaryfigure2.tif Supplementary Fig. 2: a.Representative immunofluorescence images showing cGAS staining in the ipsilateral parietal cortex of mice following SD. Scale bars: 50 µm. b.Representative immunofluorescence images showing co-staining of cGAS with NeuN, Iba1 and S100β in the ipsilateral parietal cortex following SD. Scale bars: 50 µm. c. Representative immunofluorescence images showing co-staining of cGAS with NeuN in the ipsilateral parietal cortex of mice, treated with C-176 (STING inhibitor) or vehicle, following six SDs. Scale bars: 50 µm. White arrows inside insets indicate cells immunopositive for the corresponding protein (red). Arrow heads indicate cells that are immunonegative for the corresponding protein. d. Quantification of cGAS-positive cells as a percentage of total cells (Hoechst positive) at two time points following one or six SDs (n=6/group). e-g. Quantification of the proportion of cGAS-positive cells within neurons, microglia and astrocytes at 5-h and 24-h time points following one or six SDs (n=6/group) h. Percentage of cGAS-positive neurons in the ipsilateral parietal cortex of mice treated with C-176 or vehicle following six SDs (n=6/group). Data points in the graphs represent individual animals, with each value corresponding to the mean of three non-overlapping images from the ipsilateral parietal cortex. Values are presented as mean ± SEM. Statistical comparisons were performed using Student’s t-test with Welch’s correction. ns: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001 Supplementaryfigure3.tif Supplementary Fig. 3: a. Representative immunofluorescence images showing co-staining of STING, p-IRF3, and IFN-β with NeuN in the ipsilateral parietal cortex of mice treated with C-176 (STING inhibitor) or vehicle following six SDs. Scale bars: 50 µm. White arrows inside insets indicate cells immunopositive for the corresponding protein (red). Arrow heads indicate cells that are immunonegative for the corresponding protein. b.Quantification of the percentage of STING-positive, p-IRF3-positive, and IFN-β-positive neurons in the ipsilateral parietal cortex of mice treated with C-176 or vehicle following six SDs. Data points represent individual animals each calculated as the mean of three non-overlapping images from the ipsilateral parietal cortex. Values are presented as mean ± SEM. c. Representative lane view images of capillary-based nanoimmunoassay (WES) analysis of STING, IRF3, p-IRF3, and IFN-β protein levels in the ipsilateral parietal cortex of mice treated with C-176 or vehicle following six SDs d–g.Capillary-based nanoimmunoassay (WES) analysis of STING, IRF3, p-IRF3, and IFN-β protein levels in the ipsilateral parietal cortex of mice treated with C-176 or vehicle following six SDs (n=3/group). Protein levels measured by WES were normalized to vinculin and data are presented as fold change relative to the sham group (mean ± SEM). Statistical comparisons were performed using Student’s t-test with Welch’s correction. ns: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001 Supplementaryfigure4.tif Supplementary Fig. 4: a. Representative immunofluorescence images showing STING, p-IRF3 and IFN-β staining in the parietal cortex of mice which are treated with 2’3’ cGAMP (STING agonist) or vehicle. Scale bars: 50 µm. b. Representative lane view images of capillary-based nanoimmunoassay (WES) analysis of STING, IRF3, p-IRF3, and IFN-β protein levels in the parietal cortex of mice treated with 2’3’ cGAMP or vehicle (n=3/group). c-f. Capillary-based nanoimmunoassay (WES) analysis of STING, IRF3, p-IRF3, and IFN-β protein levels in the parietal cortex of mice treated with 2’3’ cGAMP or vehicle (n=3/group). Protein levels measured by WES were normalized to vinculin and data are presented as fold change relative to the sham group (mean ± SEM). Statistical comparisons were performed using Student’s t-test with Welch’s correction. ns: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001 Cite Share Download PDF Status: Published Journal Publication published 22 Jan, 2026 Read the published version in The Journal of Headache and Pain → Version 1 posted Editorial decision: Revision requested 23 Oct, 2025 Reviews received at journal 03 Oct, 2025 Reviewers agreed at journal 18 Sep, 2025 Reviews received at journal 19 Aug, 2025 Reviewers agreed at journal 31 Jul, 2025 Reviewers invited by journal 29 Jul, 2025 Editor assigned by journal 21 Jul, 2025 Submission checks completed at journal 21 Jul, 2025 First submitted to journal 18 Jul, 2025 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7160141","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":493917441,"identity":"17739782-f6a8-4226-acde-4e608d9c56c7","order_by":0,"name":"Kadir Oguzhan Soylu","email":"","orcid":"","institution":"1-\tInstitute of Neurological Sciences and Psychiatry, Hacettepe University","correspondingAuthor":false,"prefix":"","firstName":"Kadir","middleName":"Oguzhan","lastName":"Soylu","suffix":""},{"id":493917442,"identity":"4887d1b5-07da-4563-8149-f0cfd7cb33e6","order_by":1,"name":"Buket Donmez-Demir","email":"","orcid":"","institution":"1-\tInstitute of Neurological Sciences and Psychiatry, Hacettepe University","correspondingAuthor":false,"prefix":"","firstName":"Buket","middleName":"","lastName":"Donmez-Demir","suffix":""},{"id":493917443,"identity":"414b603d-58f2-4d66-bd98-489b7d772712","order_by":2,"name":"Hasan Basri Kilic","email":"","orcid":"","institution":"2-\tDepartment of Medical Biology, School of Medicine, Hacettepe University","correspondingAuthor":false,"prefix":"","firstName":"Hasan","middleName":"Basri","lastName":"Kilic","suffix":""},{"id":493917444,"identity":"6ed37497-7526-4405-8c7a-acdd74d4f811","order_by":3,"name":"Melike Sever-Bahcekapili","email":"","orcid":"","institution":"1-\tInstitute of Neurological Sciences and Psychiatry, Hacettepe University","correspondingAuthor":false,"prefix":"","firstName":"Melike","middleName":"","lastName":"Sever-Bahcekapili","suffix":""},{"id":493917445,"identity":"c42a5e9c-e85a-4e5e-ac0b-56d12fbf5b79","order_by":4,"name":"Canan Cakir-Aktas","email":"","orcid":"","institution":"1-\tInstitute of Neurological Sciences and Psychiatry, Hacettepe University","correspondingAuthor":false,"prefix":"","firstName":"Canan","middleName":"","lastName":"Cakir-Aktas","suffix":""},{"id":493917446,"identity":"4b2fc9af-2f7b-4a07-b9d6-420021cf8d38","order_by":5,"name":"Yusuf Cetin Kocaefe","email":"","orcid":"","institution":"2-\tDepartment of Medical Biology, School of Medicine, Hacettepe University","correspondingAuthor":false,"prefix":"","firstName":"Yusuf","middleName":"Cetin","lastName":"Kocaefe","suffix":""},{"id":493917447,"identity":"c4616415-7ff7-4928-8317-ee277282022c","order_by":6,"name":"Hulya Karatas","email":"","orcid":"","institution":"1-\tInstitute of Neurological Sciences and Psychiatry, Hacettepe University","correspondingAuthor":false,"prefix":"","firstName":"Hulya","middleName":"","lastName":"Karatas","suffix":""},{"id":493917448,"identity":"cf60ce3f-7ef6-4389-8e83-c87a7e11014a","order_by":7,"name":"Muge Yemisci","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIiWNgGAWjYLCCBAYGHgYGNiCrAoiZmRtI0XIGpIWRCC0QANTC2AZiENCiOyP56IYHf+xk+PmPJX4unFcbzd8O1PKjYhtOLWY30tJuJLYl80g2HDssPXPb8dwZhxkbGHvO3MajJcfsRmLDAR6Dg+0N0rzbjuU2ALUwM7bh05L/7UbCH6CWw+zNv3nnHMudT1hLDtuNBDaglmNsx6R5G2pyNxDUcuaZGcQvPWxp1jzHDuRuBGo5iNcvx5Of3fzxx84eGGLGt3lq6nLnnT988MGPCtxaGAQSULiHweQB3OqBgB9Vug6v4lEwCkbBKBiZAAAZtV7FyNE7HwAAAABJRU5ErkJggg==","orcid":"","institution":"1-\tInstitute of Neurological Sciences and Psychiatry, Hacettepe University","correspondingAuthor":true,"prefix":"","firstName":"Muge","middleName":"","lastName":"Yemisci","suffix":""}],"badges":[],"createdAt":"2025-07-18 18:38:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7160141/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7160141/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s10194-026-02267-5","type":"published","date":"2026-01-22T15:57:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88233077,"identity":"57d9cb87-73f9-4251-8b7f-53811962d900","added_by":"auto","created_at":"2025-08-04 09:48:03","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2584935,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of cGAS-STING pathway proteins in the ipsilateral parietal cortex increases following SD. \u003cstrong\u003ea. \u003c/strong\u003eSchematic representation of the experimental design. \u003cstrong\u003eb. \u003c/strong\u003eRepresentative\u003cstrong\u003e \u003c/strong\u003eimmunofluorescence images showing STING, p-IRF3, and IFN-β expression in the ipsilateral parietal cortex of mice which underwent sham surgery, single or six SDs. Scale bars: 50 µm. \u003cstrong\u003ec. \u003c/strong\u003eRepresentative lane view images of capillary-based nanoimmunoassay (WES) analysis of STING, IRF3, p-IRF3, and IFN-β protein levels in the ipsilateral parietal cortex following SD (n=4/group).\u003cstrong\u003e d-g. \u003c/strong\u003eCapillary-based nanoimmunoassay (WES) analysis of STING, IRF3, p-IRF3, and IFN-β protein levels in the ipsilateral parietal cortex following SD (n=4/group). Protein levels measured by WES were normalized to vinculin, a commonly used housekeeping protein, and the data are presented as fold changes relative to the sham group. Values are mean ± SEM. Statistical comparisons were made using Student’s t test with Welch’s correction. \u003cem\u003ens: p \u0026gt; 0.05, *: p \u0026lt; 0.05, **: p \u0026lt; 0.01, ***: p \u0026lt; 0.001. \u003c/em\u003ePanel \u003cstrong\u003ea. \u003c/strong\u003ewas created in https://BioRender.com.\u003c/p\u003e","description":"","filename":"Figure1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7160141/v1/90977650e1bb21b8d1a0ae44.jpg"},{"id":88233084,"identity":"503b9da0-3a38-400c-8edf-3ad3a9bdda90","added_by":"auto","created_at":"2025-08-04 09:48:03","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5262420,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of cGAS-STING pathway proteins increase predominantly in neurons, but not in microglia or astrocytes, in the ipsilateral cerebral cortex following SD. \u003cstrong\u003ea. \u003c/strong\u003eSchematic representation of the experimental design. \u003cstrong\u003eb-d-f. \u003c/strong\u003eRepresentative immunofluorescence images showing co-staining of STING, p-IRF3 and IFN-β with NeuN (neuronal marker), Iba1 (microglial marker), and S100β (astrocytic marker) in the ipsilateral parietal cortex of mice which underwent sham surgery, single or six SDs. Scale bars: 50 µm. White arrows inside insets indicate cells immunopositive for the corresponding protein (red). Arrow heads indicate cells that are immunonegative for the corresponding protein. \u0026nbsp;\u003cstrong\u003ec-e-g. \u003c/strong\u003eQuantification of the proportion of STING, p-IRF3, or IFN-β positive cells within neurons, microglia, and astrocytes at 5-h and 24-h time points following one or six SDs (n=6/group). Data points in the graphs represent individual animals, with each value corresponding to the mean of three non-overlapping images from the ipsilateral parietal cortex. Values are presented as the mean ± SEM. Statistical comparisons were made using Student’s t test with Welch’s correction. \u003cem\u003ens: p \u0026gt; 0.05, *: p \u0026lt; 0.05, **: p \u0026lt; 0.01, ***: p \u0026lt; 0.001. \u003c/em\u003ePanel a. was created in https://BioRender.com.\u003c/p\u003e","description":"","filename":"Figure2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7160141/v1/770f39b86ac9f6258fa0449b.jpg"},{"id":88233076,"identity":"fc4d4bef-7523-4419-817d-86bba2889555","added_by":"auto","created_at":"2025-08-04 09:48:03","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2355053,"visible":true,"origin":"","legend":"\u003cp\u003eMicroglia exhibit morphological changes associated with activation 24 h after six SDs, and inhibition of STING abrogates microglial activation following SDs. \u003cstrong\u003ea. \u003c/strong\u003eSchematic representation of the experimental design. \u003cstrong\u003eb. \u003c/strong\u003eRepresentative immunofluorescence images of Iba1 staining in the ipsilateral parietal cortex of mice at 5-h and 24-h time points following one or six SDs. Scale bars: 50 µm. \u003cstrong\u003ec. \u003c/strong\u003eQuantification of microglial activation using ramification index (RI) scores in the ipsilateral parietal cortex region following SDs. \u003cstrong\u003ed. \u003c/strong\u003eRepresentative immunofluorescence images of Iba1 staining in the ipsilateral parietal cortex of mice, treated with C-176 or vehicle, following six SDs. Scale bars: 50 µm\u003cstrong\u003e. e.\u003c/strong\u003e RI scores of microglia in the ipsilateral parietal cortex of mice, treated with C-176 or vehicle, following six SDs. Data points in the graphs represent individual animals, each showing the mean of RI score of microglia analyzed per animal (n=6/group). Values are presented as mean ± SEM. Statistical comparisons were made using Student’s t test with Welch’s correction. \u003cem\u003ens: p \u0026gt; 0.05, *: p \u0026lt; 0.05, **: p \u0026lt; 0.01, ***: p \u0026lt; 0.001. \u003c/em\u003ePanel a.\u003cstrong\u003e \u003c/strong\u003ewas created in https://BioRender.com.\u003c/p\u003e","description":"","filename":"Figure3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7160141/v1/397fa92bcfadedbdf3824aa8.jpg"},{"id":88233080,"identity":"5a0c8363-246f-401b-802b-25635515004c","added_by":"auto","created_at":"2025-08-04 09:48:03","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2375480,"visible":true,"origin":"","legend":"\u003cp\u003ecGAS-STING pathway modulates susceptibility to SD and periorbital mechanical allodynia. \u003cstrong\u003ea. \u003c/strong\u003eSchematic representation of the experimental design. \u003cstrong\u003eb. \u003c/strong\u003eChange in SD thresholds of C57BL/6J mice following administration of C-176 (or vehicle) and 2’3’ cGAMP (or vehicle). The lines represent the median ± interquartile range, with individual data points showing the SD thresholds in mM KCl concentration values for each mouse (n=6/group). Triangle symbols indicate male mice, and circle symbols indicate female mice. \u003cstrong\u003ec.\u003c/strong\u003e HWT were assessed using the periorbital von Frey test in C57BL/6J mice before and after treatment with C-176 or vehicle (n=7/group) \u003cstrong\u003ed.\u003c/strong\u003e HWT was assessed using the periorbital von Frey test in C57BL/6J mice before and after treatment with 2’3’ cGAMP or vehicle (n=8/group). \u003cstrong\u003ee.\u003c/strong\u003e Assessment of HWT following optogenetically induced six SDs in Thy1-ChR2-YFP mice which are treated with 2’3’ cGAMP or vehicle prior to SD induction. HWTs were measured at 2- and 24-h after the last SD. The lines represent the mean ± SEM, and individual data points show withdrawal thresholds in grams (g) per mouse. Male and female animals are indicated by triangles and circles, respectively. \u003cstrong\u003ef.\u003c/strong\u003e Group averages of HWT data at each time point illustrating differences between 2’3’ cGAMP and vehicle-treated groups. Values are presented as mean ± SEM. For statistical comparisons, Mann-Whitney U test was used for SD threshold comparisons due to non-normal data distribution, while paired or unpaired Student’s t test with Welch’s correction and one-way ANOVA was used for other comparisons. \u003cem\u003ens: p \u0026gt; 0.05, *: p \u0026lt; 0.05, **: p \u0026lt; 0.01, ***: p \u0026lt; 0.001. \u003c/em\u003ePanel a.\u003cstrong\u003e \u003c/strong\u003ewas created in https://BioRender.com.\u003c/p\u003e","description":"","filename":"Figure4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7160141/v1/99fa52fb866006ba178ab91f.jpg"},{"id":101151690,"identity":"daf514c5-8ece-440e-a727-128110ffe095","added_by":"auto","created_at":"2026-01-26 16:01:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13547658,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7160141/v1/8c830c3f-84ce-4b16-80c9-82dde79aa77f.pdf"},{"id":88234143,"identity":"da487355-14a4-4404-a603-50e65ee9af81","added_by":"auto","created_at":"2025-08-04 09:56:03","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":226315,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-7160141/v1/5ddaf8bfcfe21897ac4ddf7a.png"},{"id":88233085,"identity":"ef5cdea9-35a0-44db-acb6-f4685b2676b1","added_by":"auto","created_at":"2025-08-04 09:48:03","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1149064,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 1: a. \u003c/strong\u003eRepresentative immunofluorescence images showing changes in STING staining pattern following SD. Upon activation, STING undergoes oligomerization and translocation which appears as more condensed and intense staining (white arrows), in contrast to the diffuse and weaker signals observed in the non-activated state (arrow heads). To ensure specificity, only cells displaying the distinct condensed STING staining pattern (white arrows) were classified as STING-positive. \u003cstrong\u003eb-d.\u003c/strong\u003e Quantification of STING-, p-IRF3-, and IFN-β-positive cells as a percentage of total cells (Hoechst positive) at 5- and 24-h time points following one or six SDs (n=6/group). Data points in the graphs represent individual animals, with each value corresponding to the mean of three non-overlapping images from the ipsilateral parietal cortex. Values are expressed as mean ± SEM. Statistical comparisons were performed using Student’s t-test with Welch’s correction. \u003cem\u003ens: p \u0026gt; 0.05, *: p \u0026lt; 0.05, **: p \u0026lt; 0.01, ***: p \u0026lt; 0.001\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Supplementaryfigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7160141/v1/e6271943a2d599f6e34f6965.tif"},{"id":88233092,"identity":"4733d59a-c651-42cf-8f31-288dde258c5f","added_by":"auto","created_at":"2025-08-04 09:48:04","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":9013988,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 2: a.\u003c/strong\u003eRepresentative immunofluorescence images showing cGAS staining in the ipsilateral parietal cortex of mice following SD. Scale bars: 50 µm. \u003cstrong\u003eb.\u003c/strong\u003eRepresentative immunofluorescence images showing co-staining of cGAS with NeuN, Iba1 and S100β in the ipsilateral parietal cortex following SD. Scale bars: 50 µm. \u003cstrong\u003ec. \u003c/strong\u003eRepresentative immunofluorescence images showing co-staining of cGAS with NeuN in the ipsilateral parietal cortex of mice, treated with C-176 (STING inhibitor) or vehicle, following six SDs. Scale bars: 50 µm. White arrows inside insets indicate cells immunopositive for the corresponding protein (red). Arrow heads indicate cells that are immunonegative for the corresponding protein. \u003cstrong\u003ed.\u003c/strong\u003e Quantification of cGAS-positive cells as a percentage of total cells (Hoechst positive) at two time points following one or six SDs (n=6/group). \u003cstrong\u003ee-g.\u003c/strong\u003e Quantification of the proportion of cGAS-positive cells within neurons, microglia and astrocytes at 5-h and 24-h time points following one or six SDs (n=6/group) \u003cstrong\u003eh.\u003c/strong\u003e Percentage of cGAS-positive neurons in the ipsilateral parietal cortex of mice treated with C-176 or vehicle following six SDs (n=6/group). Data points in the graphs represent individual animals, with each value corresponding to the mean of three non-overlapping images from the ipsilateral parietal cortex. Values are presented as mean ± SEM. Statistical comparisons were performed using Student’s t-test with Welch’s correction.\u003cem\u003e ns: p \u0026gt; 0.05, *: p \u0026lt; 0.05, **: p \u0026lt; 0.01, ***: p \u0026lt; 0.001\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Supplementaryfigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7160141/v1/8476fbdd9d3be87740ed2d72.tif"},{"id":88234150,"identity":"3c410c76-971d-4b02-8624-cc4d9927a7af","added_by":"auto","created_at":"2025-08-04 09:56:04","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":4089888,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 3:\u003c/strong\u003e \u003cstrong\u003ea.\u003c/strong\u003e Representative immunofluorescence images showing co-staining of STING, p-IRF3, and IFN-β with NeuN in the ipsilateral parietal cortex of mice treated with C-176 (STING inhibitor) or vehicle following six SDs. Scale bars: 50 µm. White arrows inside insets indicate cells immunopositive for the corresponding protein (red). Arrow heads indicate cells that are immunonegative for the corresponding protein. \u0026nbsp;\u003cstrong\u003eb.\u003c/strong\u003eQuantification of the percentage of STING-positive, p-IRF3-positive, and IFN-β-positive neurons in the ipsilateral parietal cortex of mice treated with C-176 or vehicle following six SDs. Data points represent individual animals each calculated as the mean of three non-overlapping images from the ipsilateral parietal cortex. Values are presented as mean ± SEM. \u003cstrong\u003ec. \u003c/strong\u003eRepresentative lane view images of\u003cstrong\u003e \u003c/strong\u003ecapillary-based nanoimmunoassay (WES) analysis of STING, IRF3, p-IRF3, and IFN-β protein levels in the ipsilateral parietal cortex of mice treated with C-176 or vehicle following six SDs \u003cstrong\u003ed–g.\u003c/strong\u003eCapillary-based nanoimmunoassay (WES) analysis of STING, IRF3, p-IRF3, and IFN-β protein levels in the ipsilateral parietal cortex of mice treated with C-176 or vehicle following six SDs (n=3/group). Protein levels measured by WES were normalized to vinculin and data are presented as fold change relative to the sham group (mean ± SEM). Statistical comparisons were performed using Student’s t-test with Welch’s correction.\u003cem\u003e ns: p \u0026gt; 0.05, *: p \u0026lt; 0.05, **: p \u0026lt; 0.01, ***: p \u0026lt; 0.001\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Supplementaryfigure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7160141/v1/d2f44aefcfb975d55538f011.tif"},{"id":88234146,"identity":"38270b62-85e7-4de5-86ab-8eae71da6e91","added_by":"auto","created_at":"2025-08-04 09:56:03","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":4624364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 4: a.\u003c/strong\u003e Representative immunofluorescence images showing STING, p-IRF3 and IFN-β staining in the parietal cortex of mice which are treated with 2’3’ cGAMP (STING agonist) or vehicle. Scale bars: 50 µm. \u003cstrong\u003eb. \u003c/strong\u003eRepresentative lane view images of\u003cstrong\u003e \u003c/strong\u003ecapillary-based nanoimmunoassay (WES) analysis of STING, IRF3, p-IRF3, and IFN-β protein levels in the parietal cortex of mice treated with 2’3’ cGAMP or vehicle (n=3/group). \u003cstrong\u003ec-f. \u003c/strong\u003eCapillary-based nanoimmunoassay (WES) analysis of STING, IRF3, p-IRF3, and IFN-β protein levels in the parietal cortex of mice treated with 2’3’ cGAMP or vehicle (n=3/group). Protein levels measured by WES were normalized to vinculin and data are presented as fold change relative to the sham group (mean ± SEM). Statistical comparisons were performed using Student’s t-test with Welch’s correction.\u003cem\u003e ns: p \u0026gt; 0.05, *: p \u0026lt; 0.05, **: p \u0026lt; 0.01, ***: p \u0026lt; 0.001\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Supplementaryfigure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7160141/v1/04ab34e0d239d61f72743f45.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Spreading Depolarization Activates the cGAS–STING Pathway and Drives Cranial Nociception: Therapeutic Potential of STING Modulation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSpreading depolarization (SD) is a slowly spreading wave of neuronal and glial depolarization at a velocity of approximately 2\u0026ndash;5 mm/min, which causes a temporary silencing of spontaneous cortical activity and alterations in cerebral blood flow [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. SD has been detected in patients with ischemic stroke, subarachnoid hemorrhage, or traumatic brain injury under continuous monitoring in the intensive care unit [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It is also considered an electrophysiological correlate of migraine aura and contributes to the pathophysiology of migraine pain by activating the trigeminovascular system [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Hence, SD is used as a relevant experimental model to study migraine pathophysiology, as several clinically used and effective anti-migraine drugs have been shown to suppress SD [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. SD imposes considerable metabolic burden on brain cells because of disruptions in ion and neurotransmitter homeostasis which requires substantial energy to restore [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This can lead to cellular stress in brain cells. While SD induced cellular stress does not cause cellular death in metabolically intact brain (in the absence of ischemia or trauma) it triggers a non-lethal but robust inflammatory response known as neuro-parainflammation, which can persist for hours to days [\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This inflammatory response involves the release of pro-inflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor-α (TNF-α), and mediators such as cyclooxygenase-2 (COX-2) and high mobility group box-1 (HMGB1), including key mechanisms of activation of the nucleotide-binding domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome following the opening of pannexin-1 channels [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOne of the key mediators of innate immunity involved in sterile inflammation is the cyclic GMP-AMP synthase\u0026ndash;stimulator of interferon genes (cGAS\u0026ndash;STING) pathway, where the cGAS enzyme detects free cytosolic double-stranded DNA (dsDNA), and leads to the production of 2'3'-cyclic GMP-AMP (2'3'-cGAMP) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This secondary messenger then activates the adaptor protein STING located on the endoplasmic reticulum (ER) [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].Upon activation, STING subsequently leads to downstream effects, including the phosphorylation of interferon regulatory factor 3 (IRF3) to form p-IRF3 and the induction of type 1 interferon (IFN) signaling. Additionally, this pathway can lead to the translocation of nuclear factor-κB (NF-κB) to the nucleus, thereby enhancing the expression of pro-inflammatory cytokines such as IL-6 and TNF-α [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile initially studied in the context of antiviral defense and cancer immunology, cGAS-STING signaling has been implicated in a wide range of neurological conditions such as neurodegenerative diseases, ischemic stroke, and traumatic brain injury, in which overactivation of the pathway led to secondary tissue injury [\u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28 CR29\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Moreover, the cGAS-STING pathway has been found to play a role in pain modulation, with preclinical evidence suggesting contrasting roles of the cGAS\u0026ndash;STING pathway in nociception [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. While STING agonists have been found to be anti-nociceptive in some experimental models, long term hyperactivation of the pathway has been shown to contribute to pain sensitization and maintenance in chronic pain conditions, while inhibition reduced nociception [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Despite its established roles in both inflammation and nociception, the cGAS\u0026ndash;STING pathway has not been studied in experimental migraine models such as SD. As migraine is a neuroinflammatory disorder associated with pain, investigation of the involvement of this pathway in SD-associated inflammatory response and migraine mechanisms could provide novel insights into migraine pathophysiology and therapeutic interventions [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, we aimed to investigate the activation and functional relevance of the cGAS-STING pathway following SD in the cerebral cortex. Both a non-invasive optogenetic model using Thy1-ChR2-YFP transgenic mice and the conventional potassium chloride (KCl) model were used to induce SD, followed by immunohistochemistry, WES capillary nano-immunoassay, and behavioral assays for analysis. We demonstrated that SD activates cGAS-STING signaling predominantly in neurons, with downstream effects on microglial activation. We determined the protein levels and cellular localization of pathway components, including cGAS, STING, p-IRF3, and IFN-β within the cerebral cortex. We further explored the consequences of pharmacological modulation of STING activity on microglial activation, susceptibility to SD, and development of periorbital mechanical allodynia following SD, as a surrogate for migraine-like pain. Our findings revealed that cGAS-STING pathway modulates cortical excitability and cranial nociception; thus, it may represent a novel mechanistic link between cortical hyperexcitability, neuroinflammation, migraine, and related disorders.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eAnimals\u003c/b\u003e\u003c/p\u003e\u003cp\u003e All experimental procedures performed on animals were approved by Hacettepe University Animal Experimentations Local Ethics Board (Approval number: 2021/67). For animal care, \u003cem\u003ein vivo\u003c/em\u003e procedures, and reporting, the ARRIVE guidelines were followed. Mice were housed under 12-hours light-dark cycle at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026ordm;C temperature and 50\u0026ndash;60% humidity with ad-libitum access to food and water. We used 25\u0026ndash;35 gr, 8\u0026ndash;16 weeks old female and male Thy1-ChR2-YFP mice (Jackson Laboratories, Bar Harbor, ME) (n\u0026thinsp;=\u0026thinsp;68) and wild-type C57BL6/J mice (n\u0026thinsp;=\u0026thinsp;64). The animals were randomly assigned to experimental groups.\u003c/p\u003e\u003cp\u003eBefore surgery, the mice were anesthetized with 4% isoflurane for induction and 1\u0026ndash;2% isoflurane for maintenance in 100% oxygen at 2 L/min. Anesthesia depth was regularly monitored using a paw pinch. Body temperature was maintained between 36.5\u0026ndash;37.2 \u0026ordm;C during surgery using a rectal probe connected to a homeothermic blanket with a control unit (Harvard Apparatus, USA). Pulse rate and oxygen saturation were determined using a pulse oximeter from the right lower limb and maintained within homeostatic limits during the procedures. Following surgery, the mice were allowed to recover on a heating blanket until they resumed their normal activities and then returned to their standard housing conditions until sacrifice.\u003c/p\u003e\u003cp\u003eThe number of mice used in each group for the \u003cem\u003ein vivo\u003c/em\u003e experiments is indicated in the respective figure legends. For all experiments, data from a single animal were used as the experimental unit.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOptogenetic SD Induction\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSD induction with optogenetic stimulation was performed as described previously [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Thy1-ChR2-YFP mice, which express channelrhodopsin-2 in cortical layer 5 neurons, were used for optogenetic stimulation experiments. Briefly, after anesthesia, the mice were placed in a stereotaxic frame. After shaving and disinfecting the scalp, a midline incision was performed to expose the intact skull. To detect SDs, electrophysiological recordings were obtained using a silver/silver chloride (Ag/AgCl) pellet electrode to monitor direct current (DC) potential shifts. A small part of the right parietal bone was focally thinned using a dental drill (World Precision Instruments, USA), and recording electrodes were placed at this site. During drilling, the parietal bone was regularly flushed with cold saline to prevent heating and thermal damage. An optical fiber (Thorlabs Inc, USA) was placed on the right frontal bone (overlying the motor cortex) for transcranial light stimulation. Blue laser (470 nm, 4 mW power) stimulation was performed for 10 s to induce SD. Successful induction was confirmed by a DC potential shift of at least 5 mV amplitude on electrophysiological recordings. SD was induced once or six times within 1 h in different experimental groups. After SD induction was completed and the DC potential returned to baseline, the scalp was sutured, and the mice were returned to their standard housing conditions. For the sham controls, the same steps were followed except for the light stimulation, and the experiments were terminated after an equivalent duration as the induction groups.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetermination of SD Threshold\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess the SD threshold, the conventional method of topical KCl application on the intact dura was performed on wild-type C57BL6/J mice. Although optogenetic stimulation in Thy1-ChR2-YFP mice provides a reliable and reproducible method for inducing SD, the optogenetic SD threshold is influenced by channelrhodopsin expression in the cortex and skull thickness, rather than reflecting the brain\u0026rsquo;s innate SD susceptibility [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. To ensure comparability with the established literature on SD threshold determination, we used conventional KCl induction, which enables a well-known measurement of cortical susceptibility to SD [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Under anesthesia, the mice were secured in a stereotaxic frame, and following scalp shaving and disinfection, a midline incision was made to expose the skull. For SD induction via KCl application, a burr-hole of 1 mm diameter was opened into the right frontal bone (1 mm anterior, 1 mm lateral to the bregma). During drilling, the skull was regularly irrigated with cold saline to prevent thermal damage. The dura was kept intact, and care was taken to avoid bleeding and tissue injury. Also, a portion of the right parietal bone was focally thinned, for the placement of recording electrodes. Cotton balls (\u0026lt;\u0026thinsp;1 mm in diameter) soaked in gradually increasing KCl concentrations (12.5, 25, 50, 75, 100, 150, and 300 mM) were sequentially placed on the dura. Each concentration was applied for 5 min, and the occurrence of SD was monitored. If no SD occurred within 5 min, the cotton ball was removed, and the burr hole was rinsed with 0.9% sodium chloride (NaCl) solution. After 5 min of waiting, the next higher concentration was applied. This procedure was repeated until the first SD occurred. The lowest KCl concentration that induced SD was recorded as the SD threshold. The burr hole was closed with bone wax, the scalp was sutured, and the mice were returned to their cages for recovery.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAssessment of Periorbital Allodynia\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThy1-ChR2-YFP mice were anesthetized with isoflurane and placed in a stereotaxic frame. After a single midline incision on the scalp, the intact skull was exposed. Two plastic tubes (1 mm and 3 mm in diameter) were affixed to the frontal and parietal bones using cyanoacrylate and Superbond dental adhesive (Sun Medical, Japan), to allow light stimulation and electrophysiological recording, respectively. The tube openings were sealed, and the tubes were kept moist until SD induction. Mice were allowed to recover for one week after surgery to minimize the potential effects of surgical stress or inflammation on the baseline mechanical sensitivity measurements. On the day of the experiment, an LED fiber optical cable was inserted through the plastic tube on the frontal bone, and transcranial light stimulation with 470 nm blue laser (4 mW power) was performed for 15 s to induce SD. SDs were confirmed by electrophysiological recordings using Ag/AgCl pellet electrodes inserted through a plastic tube on the intact parietal bone. A total of six SDs were induced per mouse. After the procedure, the mice were returned to their home cages to recover and establish baseline behavior before allodynia testing.\u003c/p\u003e\u003cp\u003ePeriorbital mechanical sensitivity was assessed at predefined time points (2 and 24 h) following the last SD induction using the manual von Frey test to determine periorbital withdrawal thresholds. Periorbital allodynia was defined as a reduction in the periorbital withdrawal threshold. Mice were placed in custom-made chambers (3 \u0026times; 3 \u0026times; 7 cm) to prevent escape during testing while allowing free movement of the head and forepaws. They were acclimated to the chamber in three separate 30-minute sessions prior to testing. Calibrated von Frey monofilaments (Ugo Basile, Italy) with increasing forces (0.008, 0.02, 0.04, 0.07, 0.16, 0.40, 0.60, 1.0, and 1.4 g) were applied perpendicularly to the periorbital area (superior-medial to the eye) with slight bending for 3 s or until a positive withdrawal response was observed. Brisk head withdrawal, head shaking, or facial grooming with forepaws on the stimulated side were accepted as positive responses. Mechanical head withdrawal thresholds (HWT) were determined as the force that elicited a positive response in at least three of five consecutive applications. Baseline thresholds were established before the administration of pharmacological agents and/or SD inductions.\u003c/p\u003e\u003cp\u003eBehavioral tests were performed in the same part of the day in the afternoon. The evaluators were blinded to the group allocations and drug treatments.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePharmacological Agents\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe STING inhibitor C-176 (20 mg/kg, Cayman Chemicals 25859) was dissolved in 5% dimethyl sulfoxide (DMSO) and 95% isotonic saline and delivered intraperitoneally 4 h before SD threshold assessment. The STING agonist 2\u0026rsquo;3\u0026rsquo;-cGAMP (1 mg/kg, Cayman Chemicals 19887) was dissolved in phospahte buffered saline (PBS) and delivered intranasally 4 h before the SD threshold assessment. Working solutions of the agents were freshly prepared before each experiment. The delivery methods and timings were determined according to previous studies [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo assess the effect of drug administration on steady-state nociceptive behavior, the periorbital von Frey test was performed before and 4 hours after drug or vehicle administration. To assess periorbital allodynia following SD, 2\u0026rsquo;3\u0026rsquo;-cGAMP (or its vehicle) was administered 4 h before SD induction.\u003c/p\u003e\u003cp\u003e\u003cb\u003eImmunofluorescent staining\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt 5 or 24 h after optogenetic SD induction, mice were anesthetized with urethane/xylazine (1.5 g/kg and 10 mg/kg, respectively, i.p.) and transcardially perfused with cold saline followed by cold 4% paraformaldehyde. Brains were harvested and immersed in 4% paraformaldehyde (Sigma Aldrich, Germany) solution at 4\u0026deg;C for 24 h. The brains were then transferred to 30% (w/v) sucrose in PBS and incubated for 48 h for cryoprotection. Brains were embedded in optimal cutting temperature (O.C.T.) compound (Tissue-Tek, Sakura) and stored at -80\u0026deg;C until sectioning. Frozen coronal brain sections (20 \u0026micro;m thick) were obtained using a cryostat (Leica-CM1100).\u003c/p\u003e\u003cp\u003eFor immunofluorescent staining, antigen retrieval was performed in 10 mM citrate buffer (pH 6.0) at 85\u0026deg;C for 15 min in a water bath. The slides were then allowed to cool to room temperature for 30 min. The sections were washed with TBS and blocked with 10% normal goat serum (Jackson ImmunoResearch, 005-000-121) at room temperature for 1 h. Next, sections were incubated overnight at 4 \u0026ordm;C with primary antibodies: rabbit anti-STING antibody (1:200, monoclonal D2P2F, 13647S, Cell Signaling Technology), rabbit anti-cGAS antibody (1:200, polyclonal, MBS9143559, MyBioSource), rabbit anti-p-IRF3 antibody (1:300, monoclonal, 29047S, Cell Signaling Technology), rabbit anti-IFN beta antibody (1:300, polyclonal, NBP1-77288, Novus), mouse anti-NeuN antibody (1:300, monoclonal MAB377 clone A60, Merck Millipore), goat anti-Iba1 antibody (1:250, polyclonal, NB100-1028, Novus), mouse anti-S100β antibody (1:200, polyclonal, Atlas Antibodies). The next day, the sections were washed and incubated with the appropriate secondary antibodies at room temperature for 1 h. Secondary antibodies used were as follows: Cy3- or Cy5-conjugated secondary antibodies of IgG (H\u0026thinsp;+\u0026thinsp;L) were used (all from Jackson ImmunoResearch): goat anti-rabbit IgG (Cy3, 111-165-144; Cy5, 111-175-144), goat anti-mouse IgG (Cy3, 115-165-003), donkey anti-mouse IgG (Cy5, 715-175-151), and donkey anti-goat IgG (Cy3, 705-165-003). For double labeling, the sections were washed and incubated with the second primary antibody overnight at 4\u0026deg;C. The following day, the sections were incubated with the appropriate secondary antibodies and mounted with PBS/glycerol mounting medium containing Hoechst 33258 (Invitrogen, 1:1000 dilution for nuclear staining).\u003c/p\u003e\u003cp\u003e\u003cb\u003eImaging and Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eImages of stained brain sections were acquired using a confocal laser scanning microscope (Leica TCS SP8) equipped with a diode (405, 638 nm) and OPSL (488, 552 nm) lasers, with X, Y, and Z-movement controllers, and high-resolution PMT (Zeiss, Oberkochen, Germany) and HyD (Leica) detectors. Three images were obtained from the ipsilateral parietal cortex region (corresponding to the region used for electrophysiological recordings) for each animal at 40x magnification with identical acquisition settings between the experimental and control groups. Images were captured in Z-stack mode with 0.50 \u0026micro;m wide steps along the Z-axis, and mid-plane images from each Z-stack were used for analysis. For quantification, the number of positively stained cells for each protein (cGAS, STING, p-IRF3, IFN-β) was counted and expressed as a ratio to the total number of Hoechst, NeuN, Iba1, and S100β positive cells. Positive cell counting and colocalization analyses were performed using ImageJ 1.54 software (National Institutes of Health, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMicroglia Morphology Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess microglial activation, microglial morphology was analyzed using FIJI software (version 1.54). Z-stack images of Iba1 stained sections were examined for microglial cells, and the slices containing microglia were processed as maximal intensity projections while avoiding overlapping cells from different focal planes. Maximum intensity projection images were processed to remove the background and thresholded to generate binary images using consistent settings across all samples. In these binary images, microglial cells were identified, and their areas and perimeters were calculated from the binary silhouettes. The ramification index (RI), a metric reflecting the degree of process extension and morphological complexity of microglia, was calculated for each cell using the following formula: [perimeter of cell (\u0026micro;m)]\u003csup\u003e2\u003c/sup\u003e/4π [cell area (\u0026micro;m\u003csup\u003e2\u003c/sup\u003e)] [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Three non-overlapping images from the parietal cortex ipsilateral to SD were used per animal, and the mean RI was used for comparisons between groups.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetection of protein levels with the sensitive automated capillary-based Western blot technique WES (Western Electrophoresis System)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAt 5 or 24 h after SD induction, the mice were anesthetized and sacrificed by decapitation. Brains were harvested, and the ipsilateral and contralateral parietal cortex regions were isolated. Brain tissues were frozen in liquid nitrogen and stored at -80\u0026deg;C until use. Tissues were lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer containing 1X HALT Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific), sonicated, and homogenized on ice. Lysates were centrifuged at 10,000\u0026times;g for 20 min at +\u0026thinsp;4\u0026deg;C. Supernatants were transferred to new tubes, and the protein concentrations were determined using a BCA protein assay kit (Thermo Scientific\u0026trade;, 23225). Protein levels were assessed using a WES Automated Western Blot System (ProteinSimple, Bio-Techne) with the 12\u0026ndash;230 kDa Separation Module (ProteinSimple, SM-W004) and Anti-Rabbit Detection Module (ProteinSimple DM-001) according to the manufacturer\u0026rsquo;s instructions. The WES system was selected to enable the quantitative, highly sensitive, and automated detection of target proteins from limited tissue samples [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Briefly, samples were diluted to 1 mg/ml in sample buffer, mixed with a master mix containing the internal standard and DTT, and heated at 95\u0026deg;C for 5 min. Samples, antibody diluents, primary antibodies, HRP-conjugated secondary antibodies, and chemiluminescent substrates were loaded into the plate that was part of the separation module. The following primary antibodies were used: rabbit anti-STING antibody (13647S, Cell Signaling Technology), rabbit anti-IRF3 antibody (MA5-32348, Invitrogen), rabbit anti-p-IRF3 antibody (29047S, Cell Signaling Technology), rabbit anti-IFN beta antibody (NBP1-77288, Novus), and anti-vinculin (E1E9V, XP\u0026reg; Rabbit mAb #13901). Capillary electrophoresis was performed in the WES instrument using default settings: stacking and separation at 475 V for 30 min; blocking reagent for 5 min; primary and secondary antibody incubations for 30 min; and Luminol/peroxide chemiluminescence detection for ~\u0026thinsp;15 min (exposures of 1, 2, 4, 8, 16, 32, 64, 128, and 512 s). Compass for Simple Western (Bio-Techne) was used to generate lane view plots, and the peak area data were plotted as the ratio of the target protein peak to the loading control.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using the Statistical Package for Social Sciences (SPSS for Windows, version 26.0) and GraphPad Prism 9.0 (GraphPad, San Diego, CA). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean or median\u0026thinsp;\u0026plusmn;\u0026thinsp;interquartile range, depending on the data distribution. Data were analyzed using the Shapiro-Wilk test, and if data were distributed normally, the Student\u0026rsquo;s t-test was used for paired or unpaired 2 group comparisons, and one-way analysis of variance (ANOVA) or repeated measures ANOVA was used for multiple group comparisons. For non-normally distributed data, the Mann-Whitney U and Kruskal-Wallis tests were used for unpaired group, and the Wilcoxon and Friedman tests were used for paired group comparisons. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eSpreading Depolarization Increases cGAS-STING Pathway Activity in the Cortex\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo elucidate whether the cGAS-STING pathway is activated in the mouse cerebral cortex following SD, we performed immunohistochemistry to determine the levels of pathway proteins in the ipsilateral cortices of mice subjected to six SDs, a single SD, or sham surgery (n\u0026thinsp;=\u0026thinsp;6/group). SDs were optogenetically induced to prevent injury. Brain samples were collected 5 and 24 h after SD induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). First, we performed immunohistochemistry for cGAS and STING, the primary mediators of pathway activation. Immunohistochemical analysis revealed a significant increase in the percentage of cGAS-immunopositive and STING-immunopositive cells in the ipsilateral cortex 5 h after multiple SDs compared to the sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB; Supplementary Fig.\u0026nbsp;1B, 2A, D). This increase persisted for 24 h (Supplementary Fig.\u0026nbsp;1B, 2D). Since the increase in cGAS and STING immunopositivity suggests pathway activation, we further examined the expression of the downstream effectors, p-IRF3 and IFN-β, which play key roles in cGAS-STING-mediated inflammation. In line with this, the percentage of p-IRF3 and IFN-β-immunopositive cells was significantly increased at both 5 h and 24 h following SDs compared to sham (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB; Supplementary Fig.\u0026nbsp;1C, 1D). To assess whether a single SD was sufficient to activate the pathway, we also examined cGAS-STING pathway immunopositivity after a single SD. We observed a notable increase in the number of cGAS-, STING-, p-IRF3-, and IFN-β-positive cells compared to the sham group, with the effect lasting up to 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB; Supplementary Fig.\u0026nbsp;1B-D, 2D). These findings suggest that even a single SD can activate the cGAS-STING pathway in the cortex.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo quantitatively measure cGAS-STING pathway protein levels in the ipsilateral cerebral cortex after SD, we used the WES capillary nanoimmunoassay technique in mice subjected to six SDs, a single SD, or sham surgery (n\u0026thinsp;=\u0026thinsp;4/group) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Following both single and multiple SDs, STING protein levels decreased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). While immunohistochemistry showed increased STING immunoreactivity, the reduction observed in WES is likely due to the detection being limited to the unphosphorylated form of STING. Since immunohistochemistry is not always reliable to distinguish between phosphorylated and unphosphorylated forms, and the change in staining pattern reflects STING oligomerization and translocation from the ER to vesicles after activation, the overall STING signal in immunostaining might show both active and inactive forms (Supplementary Fig.\u0026nbsp;1A) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. This methodological distinction may suggest that the apparent reduction in STING levels in WES reflects STING activation and post-translational modification following SD, rather than true downregulation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Confirming this, the levels of downstream effectors of STING, p-IRF3 and IFN-β, were significantly increased following multiple SDs. Notably, even a single SD robustly increased p-IRF3 and IFN-β levels in the ipsilateral cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, F, G). Together, these findings indicate that the cGAS-STING pathway is activated in the brain cortex following SD.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSD Increases cGAS-STING Pathway Activity Predominantly in Neurons\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo identify whether cGAS-STING pathway activation following SD occurs in a cell type\u0026ndash;specific manner, we performed double immunostaining for cGAS, STING, p-IRF3, and IFN-β alongside cell type\u0026ndash;specific markers for neurons, microglia, and astrocytes. We then quantified the proportion of cGAS-STING pathway-immunopositive cells within each cell type (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). At baseline, neurons displayed considerable cGAS and STING immunopositivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C; Supplementary Fig.\u0026nbsp;2B, E). Following both multiple SDs and a single SD, the percentage of cGAS, STING, p-IRF3, and IFN-β immunopositive neurons in the ipsilateral cortex significantly increased compared to that in the sham group, suggesting pathway activation in neurons following SD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C, D, F; Supplementary Fig.\u0026nbsp;2B, E). Microglia, the brain\u0026rsquo;s primary resident immune cells, exhibited high baseline STING immunopositivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, E). Although both single and multiple SDs significantly increased STING immunopositivity in microglia, there were no significant changes in cGAS, p-IRF3, or IFN-β immunopositivity in microglia compared to the sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, D, E, F; Supplementary Fig.\u0026nbsp;2B, F). These findings suggest that neurons are the main cellular sites of cGAS-STING pathway activation following SD. In contrast, although microglia showed high baseline STING expression and further upregulation following SD, the lack of corresponding increases in cGAS, p-IRF3, or IFN-β suggests following SD, the pathway activation is limited in these cells within the studied timeframe. Besides, astrocytes, which are also known to play roles in cGAS-STING mediated neuroinflammation, exhibited baseline cGAS and STING immunopositivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, G; Supplementary Fig.\u0026nbsp;2B, G). However, neither single nor multiple SDs significantly increased the immunopositivity of cGAS, STING, p-IRF3 or IFN-β in astrocytes compared to sham (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, D, F, G; Supplementary Fig.\u0026nbsp;2B, G). Together, these findings suggest that SD activates the cGAS-STING pathway predominantly in neurons, with limited activation in microglia.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ecGAS-STING Pathway Inhibition Prevents Microglial Morphological Changes Following SD\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIt has been shown that multiple SDs activate microglia, as evidenced by distinct morphological alterations [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. This activation is characterized by a transition from a highly ramified, surveillant morphology to a more amoeboid, reactive state, reflecting a shift toward an inflammatory or phagocytic phenotype. Consistent with these reports, our observations also revealed significant microglial morphological changes following SD. To evaluate microglial activation following SD, we examined microglial morphology in optogenetic SD induced brains and quantified the ramification index for microglia, as described in methods section (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). At 24 h after multiple SDs, microglia in the ipsilateral cortex displayed distinct morphological changes, with more amoeboid morphology and retracted processes compared to sham group, accompanied by a significantly reduced ramification index (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). However, no significant morphological changes were observed at 5 h after multiple SDs, and a single SD also failed to induce significant morphological alterations in microglia (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). Our findings indicate that microglia are activated 24 h after multiple SDs, but not after a single SD, supporting the findings in previous literature [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe also hypothesized whether cGAS-STING pathway could have a role in SD-induced microglial morphological changes. To determine whether STING signaling is required for SD-induced microglial morphological changes, we pharmacologically inhibited STING using the selective antagonist, C-176 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Immunohistochemistry and WES capillary nanoimmunoassay analysis confirmed effective inhibition, as the SD-induced increases in pathway proteins were significantly suppressed in C-176\u0026ndash;treated mice compared to those in vehicle controls (Supplementary Fig.\u0026nbsp;3A-G). Importantly, STING inhibition with C-176 also effectively prevented SD-induced morphological changes in microglia, as reflected by a more ramified morphology and significantly higher ramification indices in C-176-treated mice, compared to vehicle treated mice, 24 h after multiple SDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E). These findings indicate that STING activity contributes to SD-induced microglial activation, possibly through a non-canonical, interferon-independent mechanism in microglia [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. It is also possible that SD-induced microglial activation is downstream to increased neuronal cGAS-STING signaling, as it was shown that release of inflammatory mediators from neurons after SD is critical for microglial activation [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003ecGAS-STING Pathway Modulates Susceptibility to SD and Cranial Nociception\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe cGAS-STING pathway has been implicated in the regulation of neuronal excitability and nociception through enhanced type 1 interferon signaling in neurons [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. To determine whether this pathway also modulates susceptibility to SD and cranial nociception, we pharmacologically manipulated STING activity using its endogenous agonist 2\u0026rsquo;3\u0026rsquo;-cGAMP and its specific antagonist C-176. Activation of the cGAS-STING pathway following intranasal administration of 2\u0026rsquo;3\u0026rsquo; cGAMP was confirmed by immunohistochemistry and WES capillary nanoimmunoassay analysis, in which we observed significantly increased p-IRF3 and IFN-β expression in the 2\u0026rsquo;3\u0026rsquo; cGAMP treated group compared to vehicle treatment (Supplementary Fig.\u0026nbsp;4A-F). Four hours after drug administration, we assessed the SD thresholds by KCl induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). STING inhibition with C-176 significantly decreased the SD threshold compared to vehicle, suggesting increased vulnerability to SD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Conversely, STING activation with 2\u0026rsquo;3\u0026rsquo;-cGAMP significantly increased the SD threshold compared to that in vehicle-treated mice, indicating reduced susceptibility to SD (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Together, these findings demonstrate that cGAS-STING pathway activity can affect the brain\u0026rsquo;s susceptibility to SD, pointing to a potential role in SD-related pathophysiology, such as migraine aura and cortical injury.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo explore the role of the cGAS-STING pathway in craniofacial nociception, we investigated its effects on periorbital sensitivity and trigeminal nociception. Periorbital HWT was assessed at baseline and 4 h after treatment with either the STING inhibitor C-176 (or vehicle) or the STING agonist 2\u0026rsquo;3\u0026rsquo; cGAMP (or vehicle) using manual von-Frey testing (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We observed no change in periorbital HWT following vehicle treatment, confirming test stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). Notably, C-176 administration significantly decreased periorbital HWT compared to baseline, indicating heightened trigeminal nociception (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Conversely, administration of 2\u0026rsquo;3\u0026rsquo; cGAMP significantly increased periorbital HWT compared to baseline, suggesting reduced cranial nociceptive sensitivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These findings indicate that cGAS-STING pathway activity modulates basal cranial pain sensitivity and may influence the steady-state trigeminal nociceptive processing.\u003c/p\u003e\u003cp\u003eBased on our findings that cGAS-STING pathway activation suppresses SD susceptibility and reduces periorbital mechanical sensitivity, we next investigated if the pathway activation prior to SD could prevent the SD-induced development of periorbital allodynia. Hence, 2\u0026rsquo;3\u0026rsquo; cGAMP or vehicle was intranasally administered to mice 4 h before optogenetic induction of six SDs. Periorbital HWT was assessed using the manual von Frey test at 2 and 24 h after the final SD wave (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In the vehicle-treated group, SDs induced significant periorbital mechanical allodynia, as evidenced by significantly decreased periorbital HWT at 2 and 24 hours compared to baseline (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F). Remarkably, 2\u0026rsquo;3\u0026rsquo; cGAMP administration prior to SDs prevented this decrease in periorbital HWT, with no significant difference from baseline values (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F), indicating a protective effect. There was no significant effect of sex on periorbital HWT either at baseline or after SDs, regardless of the intervention. Together, these findings suggest that the cGAS-STING pathway modulates both SD susceptibility and trigeminal nociception and may serve as a potential therapeutic target for headache disorders.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur findings demonstrate that optogenetically induced SD activates the cGAS-STING innate immune pathway in the cerebral cortex predominantly in neurons and contributes to microglial morphological changes. Although it is established that SD can trigger sterile neuroinflammation in the brain parenchyma, we suggest a previously unrecognized mechanism linking SD to neuroinflammatory signaling and pain processing. Modulation of the cGAS-STING pathway activity influenced both cortical susceptibility to SD and periorbital nociceptive responses. Activation of this pathway suppressed SD generation and effectively prevented the SD-induced development of periorbital allodynia. These findings highlight the cGAS-STING pathway as a potential therapeutic target for SD-induced neuroinflammation and nociception, with implications to headache and other SD-related neurological disorders.\u003c/p\u003e\u003cp\u003eUsing both immunohistochemistry and capillary-based Western blot (WES) analysis, we showed that even a single, injury-free optogenetically induced SD was sufficient to initiate the neuroinflammatory cGAS-STING pathway activation in neurons in the cerebral cortex, and that multiple SDs amplified this activation. Activation of the cGAS-STING pathway after SD induced the expression of cGAS, STING, p-IRF3, and IFN-β in the ipsilateral cortical neurons. Our findings showed increased STING immunoreactivity after SD, whereas WES analysis revealed a reduction in unphosphorylated STING levels. STING activation involves phosphorylation, oligomerization and translocation to vesicles [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Our WES analysis might have detected only the unphosphorylated form because the STING antibody we used is not specific to phosphorylated STING; therefore, this apparent reduction does not indicate true downregulation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. On the contrary, immunohistochemistry, which can show total STING regardless of phosphorylation status, revealed an overall increase, supporting the notion that SD induces STING activation rather than true downregulation [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The persistence of type I interferon signaling up to 24-h following SD suggests that cGAS-STING activation may contribute to sustained neuroinflammatory and excitability-related alterations in the cortex. These findings align with the established concept that SD triggers sterile inflammatory responses in brain parenchyma.\u003c/p\u003e\u003cp\u003eSD leads to elevated expression of pro-inflammatory cytokines and mediators in brain parenchyma, such as IL-1α, IL-1β, IL-6, IL-13, TNF-α, COX-2 and inducible nitric oxide synthase (iNOS), starting approximately at 3-h post-SD, reaching maximal level 24-h post-SD and resolving at 48-, 72-h post-SD [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Importantly, even a single optogenetically induced SD -despite the absence of tissue injury- was sufficient to trigger the upregulation of pro-inflammatory mediators in the cerebral cortex, confirming an intrinsic consequence of SD itself [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Although some inflammatory mediators, such as HMGB-1, exhibited increased expression after multiple SDs, but not after a single SD, with a dose-response relationship [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Indeed, in our study we observed that a single SD can robustly increase type 1 interferon expression through the cGAS-STING pathway in mouse cerebral cortex and it persisted up to 24-h post-SD. Conventionally the inflammatory response induced by SD was thought to affect only the ipsilateral hemisphere, so the contralateral hemisphere was used as control [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. However recent studies have shown that neuroinflammation induced by SD can be bilateral, regardless of the induction method, and local \u003cem\u003eN\u003c/em\u003e-methyl-D-aspartate receptor (NMDAR) and purinergic P2X7 receptor (P2X7R) antagonism in ipsilateral hemisphere was able to attenuate neuroinflammatory changes in the contralateral hemisphere [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Taking this into consideration we did not use contralateral hemisphere as control in our study and compared SD induced ipsilateral hemisphere to sham controls.\u003c/p\u003e\u003cp\u003eNotably, cGAS-STING pathway activation and increased type 1 interferon expression following SD was neuron specific in brain cortex. Neurons emerging as the primary responders, displaying early and robust upregulation of pathway aligns with earlier studies identifying neurons as key initiators of SD-induced sterile inflammation through mechanisms involving the opening of Panx1 megachannels, caspase-1 activation, and the release of IL-1β and alarmins such as HMGB1 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. HMGB1 release from neurons in turn causes to astrocyte and microglia activation and pro-inflammatory cytokine release via NF-kB signaling [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Additionally, another innate immune system pathway NLRP3 inflammasome was also shown to be predominantly assembled in neurons after SD. NLRP3 inflammasome activation in neurons leads to the release of pro-inflammatory cytokines and has been shown to play a role in trigeminovascular system activation after SD, while microglia and astrocytes do not show NLRP3 inflammasome assembly following SD [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWe did not observe activation of the canonical cGAS-STING pathway following SD in astrocytes, and microglia while an increased immunoreactivity constrained to STING was exhibited in microglia. However, this was not accompanied by p-IRF3/IFN-β induction. This raises the possibility that STING signaling in microglia may proceed through alternative, non-canonical pathways, such as NF-κB signaling [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. While we did not directly assess this mechanism, such a route could underlie the morphological changes we observed in microglia 24 h after repeated SDs. The fact that these changes were abolished by STING inhibition suggests a potential contribution of STING signaling to the later stages of the microglial inflammatory response. It is possible that cGAS-STING pathway activation and associated type 1 interferon signaling can be induced in microglia beyond the timeframe we studied. In ischemia and trauma models cGAS\u0026ndash;STING pathway activation in microglia was typically determined approximately 3 days after the injury [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Full-blown pathway activation and induction of type 1 interferon expression in microglia might happen in later time-points following SD. Furthermore, neuronal cGAS-STING pathway activity can also be responsible for the changes observed in microglia following SDs as it was shown that following SD, especially multiple SDs, inflammatory mediators released from neurons play critical roles in later microglial activation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Earlier cGAS-STING pathway activation in neurons following SD can play a similar role for microglial morphological changes in this context. Additional studies are needed to determine whether neuronal STING activity is responsible for microglial morphological changes following multiple SDs or whether microglial STING activity plays a delayed, non-canonical role in SD-induced neuroinflammation.\u003c/p\u003e\u003cp\u003eBeyond its antiviral role, the cGAS\u0026ndash;STING pathway has been implicated in various neuroinflammatory and neurodegenerative disorders, and its over-activation leads to increased tissue injury in ischemic stroke, subarachnoid hemorrhage, and traumatic brain injury, whereas pharmacological inhibition mitigates damage [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In microglia, cGAS\u0026ndash;STING pathway triggers NF-κB and NLRP3 signaling, leading to pro-inflammatory cytokine release, M1 polarization, and pyroptosis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eConsidering that optogenetically induced SD does not cause any tissue injury in the brain but can cause a significant metabolic stress to neurons, it is plausible that inflammatory response initiates in neurons and microglial response emerges later. In line with this we observed microglial morphological changes associated with microglial activation starting at 24-h following multiple SDs, as reported previously [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In our study, these morphological alterations were quantified using the ramification index, a well-established measure of microglial complexity in the literature, which reflects soma size and process extension. Thus, a lower index indicates a transition to an activated, amoeboid morphology [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Notably, treatment with the STING inhibitor C-176 preserved the ramified microglial morphology and prevented the decrease in ramification index following multiple SDs, highlighting the role of the cGAS\u0026ndash;STING pathway in SD-induced microglial activation. This aligns with previous reports showing that enhanced cGAS\u0026ndash;STING signaling in microglia promotes a pro-inflammatory phenotype, whereas its inhibition reduces M1 polarization and supports M2 transition, particularly in models of brain ischemia and neuropathic pain [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Consistent with this evidence, our findings add SD to the growing list of neurological conditions in which cGAS\u0026ndash;STING signaling contributes to microglial activation.\u003c/p\u003e\u003cp\u003eAstrocytes express notable cGAS and STING [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e] and contribute to neuroinflammation in several central nervous system (CNS) diseases, including SD. However, astrocytes do not exhibit the full spectrum of inflammatory responses observed in neurons following SD [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR67 CR68 CR69\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. This has been reported for NLRP3 inflammasome activation, which occurs predominantly in neurons rather than in astrocytes or microglia following SD [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In a similar manner, we did not detect SD-induced cGAS\u0026ndash;STING pathway activation in astrocytes. Our findings suggest that key innate immune responses induced by SD are primarily initiated in neurons, positioning them as the main drivers of the SD-induced inflammatory cascade.\u003c/p\u003e\u003cp\u003eBeyond inflammation, our study demonstrates that cGAS-STING pathway activation can modulate cortical excitability, thereby influencing susceptibility to SD and associated pain sensitivity. Pharmacological activation of STING with 2\u0026rsquo;3\u0026rsquo;-cGAMP decreased SD susceptibility and attenuated periorbital mechanical sensitivity, whereas inhibition with C-176 increased SD susceptibility and exacerbated nociceptive responses. Notably, STING activation by an agonist prior to multiple SDs prevented the development of periorbital allodynia at 2 and 24 h later, indicating a protective role in SD-induced pain responses. These findings suggest that cGAS\u0026ndash;STING activation exerts a protective effect against SD-induced craniofacial pain.\u003c/p\u003e\u003cp\u003eSD susceptibility is the ease of brain tissue to develop SD waves. It is affected by various factors such as excitation/inhibition balance in the brain and physiological parameters [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. SD susceptibility is the most relevant SD attribute, and it is used as a therapeutic target for SD-associated disorders [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. We used the conventional KCl induction method for SD threshold assessment. Optogenetic stimulation provides a reliable and reproducible method for inducing SD, which overcomes the confounders resulting from tissue injury. Even though electrophysiological characteristics of SD induced with optogenetic stimulation are consistent with those of conventional induction methods, properties of optogenetic SD susceptibility differs from conventional methods [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Optogenetic SD susceptibility is strongly correlated with relative channel rhodopsin expression in the cortex rather than the innate SD susceptibility of the brain [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Since light stimulation is done through the intact skull, differences in skull thickness can also affect optogenetic SD thresholds [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Burr hole opening for KCl application was shown not to affect SD thresholds when it is done without damaging the cortex or dura [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Moreover, threshold KCl concentration for SD induction shows good concordance with other conventional methods such as direct electrical stimulation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Thus, in our study KCl-induced SD thresholds were deemed more accurate for reflecting brains innate susceptibility to SD.\u003c/p\u003e\u003cp\u003eThe link between SD and cephalic allodynia has been well established. Optogenetically induced SD was determined to cause periorbital allodynia within an hour, resolving in two days, while multiple SDs induced prolonged pain responses lasting up to 14 days [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Since our findings revealed a role for cGAS\u0026ndash;STING signaling in SD, we extended our investigation to examine its influence on SD-induced periorbital pain. Recent studies on pain models where it was observed that STING activation induced type I interferon signaling in sensory neurons, which reduced sodium and calcium channel activity, and suppressed pain behaviors in cancer induced pain and neuropathic pain models [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. It is worth noting that, STING agonists produced acute analgesic effects, alleviating mechanical allodynia within 4 h of administration, with an effect that lasted 24 h [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Analgesia level was comparable to morphine and is shown to be STING- and IFN-α/β-dependent through direct suppression of cancer-induced nociceptor hyperexcitability in dorsal root ganglia [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHowever, the role of cGAS\u0026ndash;STING signaling in nociception remains controversial, as conflicting evidence also points to pro-nociceptive effects of pathway activation in chronic pain models. Some studies have reported that prolonged cGAS\u0026ndash;STING activation in microglia promotes neuropathic pain via microglial M1 polarization, NF-κB signaling and pro-inflammatory cytokine release [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Inhibition of this pathway was shown to reverse hyperalgesia in chronic pain models, underscoring the dual role of cGAS\u0026ndash;STING signaling depending on the cellular context [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Thus, the dual role of cGAS\u0026ndash;STING signaling appears to depend on cell type -particularly neurons and microglia- as well as timing, and experimental design. In this study, we focused on the acute effects of STING activation (up to 24 h) on SD-induced periorbital allodynia and SD susceptibility, both of which reflect changes in neuronal excitability. The absence of microglial activation within this timeframe suggests that the observed anti-nociceptive effects are likely mediated by neuronal mechanisms. The protective role of early cGAS-STING activation highlights its context-dependent functions in pain modulation. Future studies assessing long-term pain responses in chronic migraine models are warranted to better understand the role of microglial cGAS\u0026ndash;STING signaling in the pathogenesis of headache disorders.\u003c/p\u003e\u003cp\u003eOur study has a couple of limitations. First, we used Thy1-ChR2-YFP transgenic mice to investigate cGAS\u0026ndash;STING pathway activity and nociceptive responses after SD. While this optogenetic model avoids the confounding effects of brain injury during SD induction, inbred transgenic mice may exhibit different inflammatory and nociceptive responses compared to outbred mice or humans, who display greater genetic variability [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Nonetheless, previous studies have shown that the inflammatory response to SD is comparable, whether induced optogenetically in transgenic mice or by topical KCl application in wild-type mice [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSecond, although we pharmacologically manipulated cGAS\u0026ndash;STING activity using specific agonists and antagonists, genetic validation would further strengthen these findings. Previous studies have demonstrated that both global and sensory neuron-specific STING knockout increases mechanical and cold sensitivity, an effect also observed with the deletion of type I interferon receptor (Ifnar1), confirming the role of STING\u0026ndash;IFN signaling in nociception [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Applying similar conditional knockout strategies targeting trigeminal neurons could help elucidate the specific contribution of STING and type I interferon signaling to craniofacial nociception.\u003c/p\u003e\u003cp\u003eThird, while we used equal numbers of male and female mice to assess periorbital allodynia after SD, we did not monitor the estrous cycle in females, which can influence nociceptive behaviors. Previous studies have shown that females exhibit lower pain thresholds, with estrous stages characterized by high estradiol levels further lowering these thresholds. Interestingly, SD induction appears to override the influence of the estrous cycle by reducing periorbital pain thresholds, regardless of the hormonal stage [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. In line with this, we did not observe any sex-based differences in baseline or post-SD nociceptive responses.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our study highlights the significant role of the cGAS-STING pathway in neuroinflammation, and nociception associated with SD. We demonstrated that optogenetically induced SD activates the cGAS\u0026ndash;STING signaling cascade primarily in cortical neurons. We also showed that STING signaling can play a role in microglial morphological changes observed after multiple SDs. Moreover, our findings demonstrated that activation of the cGAS-STING pathway decreases SD susceptibility and cranial nociceptive responses. These results offer novel insights into the relationship between SD and neuroinflammation and suggest that the cGAS\u0026ndash;STING pathway may function as a potential protective mechanism and therapeutic target for migraine and other SD-associated disorders. These results reveal that a novel innate immune pathway, previously implicated in various neuroinflammatory conditions, also serves as a neuroimmune interface that links SD to neuroinflammation and pain.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSpreading depolarization\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInterleukin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTNF-α\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTumor necrosis factor-α\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCOX-2\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCyclooxygenase-2\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHMGB1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHigh mobility group box-1\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNLRP3\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNucleotide-binding domain (NOD)-like receptor family pyrin domain containing 3\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ecGAS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCyclic GMP-AMP synthase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSTING\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eStimulator of interferon genes\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003edsDNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003edouble-stranded DNA\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e2\u0026rsquo;3\u0026rsquo; cGAMP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e2'3'-cyclic GMP-AMP\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eER\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEndoplasmic reticulum\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIRF3\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInterferon regulatory factor 3\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIFN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInterferon\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNF-κB\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNuclear factor-κB\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eKCl\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePotassium chloride\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAg/AgCl\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSilver/silver chloride\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDirect current\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNaCl\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSodium chloride\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHWT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHead withdrawal thresholds\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDMSO\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003edimethyl sulfoxide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePhospahte buffered saline\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRamification index\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eANOVA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAnalysis of variance\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eiNOS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInducible nitric oxide synthase\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNMDAR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eN\u003c/em\u003e-methyl-D-aspartate receptor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eP2X7R\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003epurinergic P2X7 receptor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCNS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCentral nervous system\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIfnar1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eType I interferon receptor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures performed on animals were approved by Hacettepe University Animal Experimentations Local Ethics Board (Approval number: 2021/67).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is supported by Hacettepe University No: TSA-2022-19749\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDesign and conceptualization of the study: MY, HK, KOS ; acquisition and analysis of data: KOS, BDD, HBK, MSB, CCA, HK, MY ; scientific discussions and interpretation of data: KOS, BDD, HBK, MSB, CCA, YCK HK, MY; Preparing figures: KOS; drafting the manuscript: KOS, BDD, HBK, MSB, CCA, YCK HK, MY.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Mesut Fırat for his expert help with technical issues, Dilan Bozanoglu MD, PhD candidate for her help with analysis of WES capillary nano-immunoassay data, Gokce Gurler MD, PhD for her help with analysis of immunohistochemistry and imaging data. Schematic diagrams were prepared at Biorender.com. The manuscript was language-edited with the assistance of ChatGPT (OpenAI), an AI-based language model.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLeao AAP (1944) SPREADING DEPRESSION OF ACTIVITY IN THE CEREBRAL CORTEX. Journal of Neurophysiology 7 (6):359-390. doi:10.1152/jn.1944.7.6.359\u003c/li\u003e\n\u003cli\u003eSomjen GG (2001) Mechanisms of Spreading Depression and Hypoxic Spreading Depression-Like Depolarization. Physiological Reviews 81 (3):1065-1096. doi:10.1152/physrev.2001.81.3.1065\u003c/li\u003e\n\u003cli\u003eNedergaard M, Hansen AJ (1993) Characterization of cortical depolarizations evoked in focal cerebral ischemia. J Cereb Blood Flow Metab 13 (4):568-574. doi:10.1038/jcbfm.1993.74\u003c/li\u003e\n\u003cli\u003eAyata C (2010) Cortical spreading depression triggers migraine attack: pro. 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The Journal of Headache and Pain 24 (1):85. doi:10.1186/s10194-023-01621-1\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"the-journal-of-headache-and-pain","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tjhp","sideBox":"Learn more about [The Journal of Headache and Pain](https://thejournalofheadacheandpain.biomedcentral.com/)","snPcode":"10194","submissionUrl":"https://submission.nature.com/new-submission/10194/3","title":"The Journal of Headache and Pain","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Spreading depolarization, migraine, cGAS, STING, interferon, neuroinflammation, cGAMP, optogenetics","lastPublishedDoi":"10.21203/rs.3.rs-7160141/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7160141/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eSpreading depolarization (SD) is a transient wave of near-complete neuronal and glial depolarization in the cortex which underlies migraine aura. Beyond its electrophysiological effects, SD has been shown to trigger a cascade of sterile neuroinflammatory responses which might contribute to trigeminal activation and pain sensitization observed in migraine. Recent studies have highlighted the involvement of innate immune system pathways in SD-associated inflammation. The cyclic GMP-AMP synthase (cGAS)-Stimulator of interferon genes (STING) pathway induces the expression of type 1 interferons and pro-inflammatory cytokines in response to cellular stress. Although this pathway is increasingly recognized for its role in neuroinflammation and nociception, its specific contribution to SD-induced mechanisms remains poorly understood. In this study, we investigated whether SD triggers activation of the cGAS-STING pathway in the mouse cerebral cortex and evaluated the functional consequences of this pathway activation on SD associated cranial nociception.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eSD was induced non-invasively with optogenetic stimulation. Animals were subjected to either a single or six SDs and the expression of cGAS-STING pathway proteins in the cortex were assessed by immunohistochemistry and capillary Western blotting. Sham-treated animals served as controls. The cellular localization of this pathway proteins in the cortex was also determined. Pharmacological modulation of the pathway was achieved via intraperitoneal administration of the STING inhibitor C-176 (20 mg/kg) or intranasal delivery of the STING agonist 2\u0026rsquo;3\u0026rsquo;-cGAMP (1 mg/kg). SD threshold was determined with potasium chloride application, and periorbital nociceptive responses were measured using the manual von Frey test. Furthermore, periorbital mechanical allodynia was assessed at 2 and 24 hours following six optogenetically induced SDs in animals treated with either 2\u0026rsquo;3\u0026rsquo;-cGAMP or vehicle.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eSD induced cGAS-STING signaling and IFN-β expression in the mouse cerebral cortex with prominent expression observed in neurons and downstream microglial activation. Pathway activation with 2\u0026rsquo;3\u0026rsquo; cGAMP decreased SD susceptibility and significantly alleviated the development of periorbital mechanical allodynia following SD.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eOur findings suggest that SD activates the cGAS-STING pathway, extending the scope of SD-induced neuroinflammation. These results also highlight the therapeutic potential of modulating STING to mitigate SD-related nociception and neuroinflammatory consequences associated with headache disorders such as migraine.\u003c/p\u003e","manuscriptTitle":"Spreading Depolarization Activates the cGAS–STING Pathway and Drives Cranial Nociception: Therapeutic Potential of STING Modulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-04 09:47:58","doi":"10.21203/rs.3.rs-7160141/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-23T05:07:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-03T08:00:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72541076678347439494391588771029265377","date":"2025-09-18T05:57:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-19T13:37:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"241174443868763553482782291335540635123","date":"2025-07-31T18:07:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-29T14:24:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-21T12:50:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-21T12:34:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"The Journal of Headache and Pain","date":"2025-07-18T18:32:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"the-journal-of-headache-and-pain","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tjhp","sideBox":"Learn more about [The Journal of Headache and Pain](https://thejournalofheadacheandpain.biomedcentral.com/)","snPcode":"10194","submissionUrl":"https://submission.nature.com/new-submission/10194/3","title":"The Journal of Headache and Pain","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"27b678e4-258a-4a54-a6e2-89bacf3c88b4","owner":[],"postedDate":"August 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-26T15:59:59+00:00","versionOfRecord":{"articleIdentity":"rs-7160141","link":"https://doi.org/10.1186/s10194-026-02267-5","journal":{"identity":"the-journal-of-headache-and-pain","isVorOnly":false,"title":"The Journal of Headache and Pain"},"publishedOn":"2026-01-22 15:57:16","publishedOnDateReadable":"January 22nd, 2026"},"versionCreatedAt":"2025-08-04 09:47:58","video":"","vorDoi":"10.1186/s10194-026-02267-5","vorDoiUrl":"https://doi.org/10.1186/s10194-026-02267-5","workflowStages":[]},"version":"v1","identity":"rs-7160141","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7160141","identity":"rs-7160141","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}