Trigeminal ganglion interferon-γ signaling drives orofacial neuropathic pain in rats

Trigeminal ganglion interferon-γ signaling drives orofacial neuropathic pain in rats | 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 Trigeminal ganglion interferon-γ signaling drives orofacial neuropathic pain in rats Momoyo Kobayashi, Akiko Okada-Ogawa, Yukinori Tanaka, Yoshinori Hayashi, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7739576/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Dec, 2025 Read the published version in The Journal of Headache and Pain → Version 1 posted 11 You are reading this latest preprint version Abstract Background Chronic neuropathic pain, particularly in the orofacial region, markedly reduces quality of life. Peripheral trigeminal nerve injury activates satellite glial cells (SGCs) in the trigeminal ganglion (TG), which contributes to orofacial neuropathic pain. However, the upstream signal responsible for SGC activation remains unclear. This study investigated the role and cellular sources of interferon gamma (IFN-γ) signaling in the TG following infraorbital nerve injury (IONI) in rats. Methods Mechanical sensitivity of the whisker pad skin was assessed after IONI. Changes in IFN-γ levels, IFN-γ receptor expression, and glial fibrillary acidic protein (GFAP; a marker of SGC activation) were examined in the TG by immunohistochemistry. The effects of intra-TG administration of IFN-γ, an IFN-γ receptor antagonist, and isolated CD8⁺ T cells on mechanical hypersensitivity were evaluated. GFAP expression after intra-TG administration of IFN-γ or the receptor antagonist was also quantified. Flow cytometry and immunohistochemistry were used to identify IFN-γ–producing cells. In primary SGC cultures, IFN-γ–induced interleukin-1β (IL-1β) release was measured, and the impact of IL-1 receptor antagonism on mechanical hypersensitivity was tested. IL-1 receptor localization and expression in TG neurons were further evaluated after IONI. Results IONI induced persistent mechanical hypersensitivity and upregulated IFN-γ, IFN-γ receptor, and GFAP expression in the TG. CD8⁺ T cells were the primary source of IFN-γ after IONI, and intra-TG transfer of isolated CD8⁺ T cells transiently induced mechanical hypersensitivity. IFN-γ receptors were localized to SGCs, with expression levels increasing after IONI. Intra-TG IFN-γ administration triggered mechanical hypersensitivity and SGC activation, whereas receptor antagonism attenuated IONI-induced hypersensitivity. IFN-γ stimulation of cultured SGCs enhanced IL-1β release. Co-administration of an IL-1 receptor antagonist prevented IFN-γ–induced mechanical hypersensitivity. IL-1β receptors were localized on TG neurons and were upregulated following IONI. Conclusions CD8⁺ T cell–derived IFN-γ activates SGCs in the TG, leading to IL-1β release that promotes neuronal hyperactivity and orofacial neuropathic pain following IONI. Targeting the IFN-γ–SGC–IL-1β signaling axis may represent a novel therapeutic strategy for orofacial neuropathic pain. Orofacial neuropathic pain Interferon-gamma (IFN-γ) CD8-positive T lymphocytes Satellite glial cells Interleukin-1 beta (IL-1β) Trigeminal ganglion Pain hypersensitivity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Chronic pain affects 11–50% of people worldwide and imposes substantial physical, emotional, and socioeconomic burdens [ 1 , 2 ]. Although many studies have examined the transition from acute to chronic pain, the underlying mechanisms remain incompletely understood [ 3 – 5 ]. Neuropathic pain, caused by peripheral nerve injury, is a major contributor to chronic pain. Chronic orofacial neuropathic pain is particularly debilitating, as it interferes with daily activities such as eating, speaking, and washing one’s face [ 6 ]. Peripheral nerve injury resulting from fractures, tooth extractions, dental implants, or root canal treatments can cause axonal degeneration, abnormal neuronal firing, and altered signal transmission [ 7 , 8 ]. Increasing evidence indicates an interaction between neuronal and non-neuronal systems such as satellite glial cells (SGCs) and immune cells play a central role in the development of neuropathic pain within the trigeminal ganglion (TG) and dorsal root ganglion (DRG) [ 9 – 11 ]. SGC activation has been associated with increased gap junction coupling among SGCs and between SGCs and primary sensory neurons, heightened sensitivity to adenosine triphosphate (ATP), release of proinflammatory cytokines, and upregulation of glial fibrillary acidic protein (GFAP) [ 12 , 13 ]. However, the upstream signals responsible for SGC activation after peripheral nerve injury and the mechanisms sustaining their hyperactivity remain poorly defined. In the DRG, TG, spinal cord, and spinal trigeminal caudal subnucleus of patients with neuropathic pain and in animal models, the proinflammatory cytokine interferon gamma (IFN-γ)—primarily produced by T lymphocytes and natural killer cells—has been identified as an important mediator of neuropathic pain development [ 14 – 18 ]. We recently demonstrated that IFN-γ receptor signaling in astrocytes of the spinal trigeminal nucleus caudalis (Vc) is essential for the development of orofacial neuropathic pain following infraorbital nerve injury (IONI) in rodents [ 15 ]. Nevertheless, the contribution of IFN-γ signaling within the TG itself to neuropathic pain remains insufficiently characterized. Recent studies have further emphasized the role of T lymphocytes in neuropathic pain [ 19 – 21 ]. Following peripheral nerve injury, T cells infiltrate the damaged nerve [ 22 , 23 ], where they can support regeneration but also release pronociceptive mediators such as proinflammatory cytokines [ 24 ]. T cell infiltration has also been observed in the TG after IONI, in addition to accumulation at the injury site [ 25 ]. Both CD8-positive (CD8 + ) and CD4-positive (CD + ) T lymphocytes are capable of producing IFN-γ after the development of antigen-specific immunity [ 26 – 29 ], suggesting their potential involvement in neuropathic pain. However, the precise role of these T cell subsets in the TG is not yet known. In this study, we investigated whether T cell-derived IFN-γ activates SGCs and whether this signaling contributes to the pathogenesis of chronic orofacial neuropathic pain in a rat model of IONI. Methods Animals Male Sprague–Dawley rats (n = 152; Japan SLC, Hamamatsu, Japan), weighing 150–250 g, were used. Animals were housed under a 12-h light/dark cycle at a constant temperature of 23 ± 1 °C, with ad libitum access to food and water. All experimental procedures adhered to the ethical guidelines of the International Association for the Study of Pain and were approved by the Animal Experimentation Committee of Nihon University (Approval No. AP23DEN007). Infraorbital nerve injury (IONI) IONI was performed as previously described [30]. Briefly, rats were anesthetized intraperitoneally with midazolam (2.0 mg/kg; Sandoz, Tokyo, Japan), butorphanol (2.5 mg/kg; Meiji Seika Pharma, Tokyo, Japan), and medetomidine (0.15 mg/kg; Zenoaq, Koriyama, Japan). A ~10 mm intraoral incision was made in the left buccal mucosa along the gingival margin proximal to the first molar. The left infraorbital nerve (ION) bundle was exposed and separated from surrounding tissue, after which one-third of the bundle was tightly ligated with 6-0 silk suture. For sham-operated controls, the ION was exposed but not ligated. All behavioral assessments were performed by an experimenter blinded to treatment groups. Magnetic separation of CD8 + T lymphocytes CD8 + T lymphocytes were purified using magnetic bead separation as previously reported [31]. Rats were deeply anesthetized with 5% isoflurane and perfused transcardially with isotonic saline. The ipsilateral TG was rapidly dissected and incubated in Hanks’ balanced salt solution (HBSS) containing collagenase D and DNase at 37 °C for 45 min. The dissociated cell suspension was then incubated on ice for 15 min with anti-CD8 + microbeads (80 μL/10 7 total cells; Miltenyi Biotec, Germany). The suspension was passed through a magnetic separation column, retaining CD8 + T lymphocytes, were subsequently eluted in culture medium. The purity of the CD8 + T lymphocyte fraction was confirmed by flow cytometry. Intra-TG administration of IFN-γ, IFN-γ receptor antagonist, CD8 + T lymphocytes, and IL-1β receptor antagonist The rat skull was exposed by removal of the overlying skin, and a small hole (diameter: 1 mm) was drilled immediately above the ipsilateral TG (2.7 mm lateral to the sagittal suture; 2.8 mm anterior to the posterior fontanelle). The skull was stabilized in a stereotaxic apparatus under deep intraperitoneal anesthesia, as described above. A guide cannula was inserted into the TG through the hole and secured to the skull using dental resin and two stainless-steel screws, positioned at a depth of 9 mm below the cranial surface, as previously described [32]. For continuous intra-TG infusion, a polyethylene tube (SP10, 0.61 mm diameter; Natsume, Tokyo, Japan) was inserted through the guide cannula and connected to an osmotic mini-pump (Alzet model 2001; Durect, Cupertino, CA, USA) 7 days after cannulation. The tube and pump were implanted subcutaneously in the back. The pump had a total volume of 200 μL and an infusion rate of 1 μL/h for 7 days. Pumps and cannulas were pre-filled with one of the following solutions: recombinant IFN-γ (100 ng/mL; Biotechne, Minneapolis, MN, USA) in vehicle (0.01 M phosphate-buffered saline [PBS]); IFN-γ receptor antagonist (IFNG ANTAG; 1 mg/mL; Bachem, Torrance, CA, USA) in vehicle (20% acetic acid); or recombinant IFN-γ mixed with an interleukin-1β receptor type 1 (IL-1R1) antagonist (IL-1R1 ANTAG; 0.5 mg/mL; Fujifilm Wako, Tokyo, Japan). For single administrations, a suspension of CD8 + T lymphocytes (10 μL), isolated from rats 7 days after IONI as described above, was directly injected into the TG of naïve rats through the guide cannula 7 days after cannulation. Injections were performed under deep anesthesia using a 31-gauge needle. Mechanical sensitivity assessment in the whisker pad skin Mechanical sensitivity of the whisker pad skin was assessed as previously described [33]. In brief, rats were trained for 7 days to protrude their perioral region, including the whisker pad skin, through a small hole in a plastic cage for a few minutes while mechanical stimulation was applied. IONI or sham surgery was performed after completion of training. Mechanical stimulation with von Frey filaments (0.16, 0.4, 0.6, 1.0, 1.4, 2.0, 4.0, 6.0, 8.0, 10, 15, 26, and 60 g; Touch-Test Sensory Evaluator, North Coast Medical, Morgan Hill, CA, USA) was applied to the whisker pad skin ipsilateral to IONI, sham, and naïve rats. Each filament was applied five times at 1-min intervals before and 1–7 days after IONI or sham surgery. A cut-off of 60 g was set to avoid tissue damage. The mechanical head-withdrawal reflex threshold (MHWT) was defined as the lowest filament force that evoked head withdrawal in at least three of five trials. In additional experiments, an intra-TG catheter was implanted in naïve rats, and IFN-γ, vehicle, or IFN-γ combined with IL-1R1 ANTAG was administered continuously for 7 days. MHWT was measured before and 1–7 days after drug administration. To test the effect of IFNG ANTAG on IONI-induced mechanical sensitivity, the intra-TG catheter was implanted at the time of IONI surgery. IFNG ANTAG or vehicle was infused for 7 days, and MHWT was measured before and 1–7 days after treatment. Administration of retrograde neurotracer Fluoro-Gold (FG) Five days before IONI surgery, 10 μL of 4% hydroxystilbamidine (Fluoro-Gold; FG; Fluorochrome, Denver, CO, USA) was injected into the left whisker pad skin using a 27-gauge needle to label TG neurons innervating this region. Injections were performed under 2.0% isoflurane anesthesia. Immunohistochemistry Immunohistochemical analyses were performed in naïve rats and in rats 7 days after IONI or sham surgery. Animals were deeply anesthetized with 5% isoflurane and perfused transcardially with saline followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4, 4 °C). TGs were dissected, post-fixed in 4% PFA for 24 h at 4 °C, and cryoprotected in 30% sucrose (w/v) in PBS for 24 h at 4 °C. Ganglia were sectioned horizontally at 14 mm thickness on a cryostat (Tissue-Tek Polar, Sakura Finetek, Tokyo, Japan) and mounted on MAS-coated glass slides (Matsunami, Osaka, Japan). Sections were rinsed in PBS, blocked for 1 h at room temperature in 0.01 M PBS containing 10% normal donkey or goat serum in 1% bovine serum albumin (BSA; Proliant Biologicals, Boone, IA, USA) and 0.05% azide, and incubated overnight at 4°C with one of the following primary antibodies: Armenian hamster monoclonal anti-IFN-γ receptor (1:500, sc-12753, Santa Cruz, Dallas, TX, USA); rabbit polyclonal anti-Kir4.1 (1:1,000, APC-035, Thermo Fisher Scientific, Waltham, MA, USA); rabbit polyclonal anti-GFAP (1:1,000, ab7260, Abcam, Cambridge, UK); rabbit polyclonal anti-CD8 (1:1,000, ab4680, Abcam); rabbit polyclonal anti-IL-1R1 (1:1,000, sc689, Santa Cruz); or mouse monoclonal anti-NeuN (neuronal marker; 1:1,000, MAB377, Merck Millipore). After washing in PBS, sections were incubated with Alexa Fluor 488- or 568-conjugated secondary antibodies (1:1,000, Thermo Fisher Scientific) for 2 h at room temperature. Slides were mounted with PermaFluor Aqueous Mounting Medium (Thermo Fisher Scientific) and imaged using a BZ-X800 fluorescence microscope (Keyence, Osaka, Japan) or an Andor BC43 confocal microscope (Andor Technology, Belfast, UK). Images were analyzed with Imaris Viewer (Oxford Instruments, Abingdon, UK). Fluorescence intensity greater than twice the mean background was defined as immunoreactive (IR). Omission of primary antibodies resulted in no specific IR. The density of GFAP-IR cells and IFN-γ receptor (IFNGR)-IR cells localized with Kir4.1-IR SGCs was quantified within a 320 × 320 μm 2 region of the TG area innervating the whisker pad skin using ImageJ software (version 1.54, NIH). Western blotting Seven days after IONI or sham surgery, rats were deeply anesthetized with 5% isoflurane and perfused transcardially with isotonic saline. The ipsilateral TG was rapidly removed and homogenized in ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% Triton X-100; 0.5% NP-40) supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Homogenates were centrifuged at 2,0142 g for 10 min at 4 °C, and the protein concentration of the supernatant was determined using a protein assay kit (Bio-Rad, Hercules, CA, USA). Samples were heat-denatured at 95 °C in Laemmli sample buffer (Bio-Rad). Equal amounts of protein were separated on 10% sodium dodecyl sulfate–polyacrylamide gels (4–20% gradient, Bio-Rad) and transferred to polyvinylidene fluoride membranes (Trans-Blot Turbo Transfer Pack; Bio-Rad) using the Trans-Blot Turbo rapid transfer system (Bio-Rad) for 7 min. Membranes were washed in Tris-buffered saline containing 0.1% Tween 20 (TBST), blocked in TBST with 5% Blocking-One (Nacalai Tesque, Kyoto, Japan) for 1 h at room temperature, and incubated overnight at 4 °C with one of the following primary antibodies: anti-IFN-γ (1:2,000, Pure Chemicals Corporation, Osaka, Japan); anti-IL-1R1 (1:500); or mouse monoclonal anti-β-actin (1:5,000, Santa Cruz Biotechnology). After washing, membranes were incubated with horseradish peroxidase–conjugated anti-rabbit or anti-mouse secondary antibodies (1:2,000, Cytiva, Marlborough, MA, USA) for 2 h at room temperature. Protein bands were detected using Western Lightning ECL Pro (PerkinElmer, Waltham, MA, USA), visualized with Immobilon ECL Ultra Western HRP Substrate (Merck Millipore), and imaged on a ChemiDoc XRS system (Bio-Rad). Band intensities were quantified using ImageJ software (NIH, version 1.54) and normalized to β-actin. Cultured satellite glial cells SGCs were dissociated as previously described [34]. Briefly, naïve rats were decapitated under 5% isoflurane, and TGs were immediately dissected. Samples were digested with 0.125% collagenase P (Roche, Indianapolis, IN, USA), 0.02% DNase (Sigma-Aldrich), and 0.25% trypsin–ethylenediaminetetraacetic acid (Thermo Fisher Scientific), then mechanically triturated in dissociation solution consisting of HBSS (5 mL; Nacalai Tesque) containing 0.295% MgSO 4 and 0.02% DNase. The dissociated cells were then cultured in Dulbecco’s Modified Eagle Medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Nichirei, Tokyo, Japan), streptomycin (100 μg/mL; Thermo Fisher Scientific), penicillin (100 U/mL; Thermo Fisher Scientific), and L-glutamine (2 mmol/L; Fujifilm Wako Pure Chemical, Osaka, Japan). After incubation in feeding medium, cells were seeded onto uncoated 24-well plates and maintained for 7 days as mixed primary neuron glia cultures. SGC-enriched cultures were prepared on day 7 by detaching the mixed cultures with Accutase (Nacalai Tesque) for 5 min at 37 °C, followed by reseeding into uncoated 24-well plates for an additional 48-h. A subset of cells was fixed in 4% PFA for 10 min and processed for immunohistochemistry as described above. The remaining cells were used for enzyme-linked immunosorbent assay (ELISA). Enzyme-linked immunosorbent assay (ELISA) Recombinant IFN-γ was adjusted to 100 ng/mL in culture medium and applied to SGC cultures for 24 h. The concentration of IL-1β in the culture supernatant was quantified using a rat IL-1β ELISA kit (Quantikine, RLB00, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. IL-1β concentrations were normalized to the total protein concentration of the supernatant, determined using a Bio-Rad protein assay (Bio-Rad). Control cultures were treated with medium or IFN-γ diluent alone, and IL-1β concentrations were measured using the same procedure. Isolation of TG cells for flow cytometry Sham- or IONI-treated rats were perfused with ice-cold saline, and ipsilateral TGs were collected in RPMI 1640 medium (Fujifilm Wako Pure Chemical) supplemented with 10% heat-inactivated FBS (Gibco, Waltham, MA, USA) and antibiotics (complete RPMI). TGs were minced into small fragments and digested in complete RPMI containing collagenase type IV (1 mg/mL, #C5138, Sigma-Aldrich), dispase II (1 mg/mL, #D4693, Sigma-Aldrich), and DNase I (50 μg/mL, #DN25, Sigma-Aldrich) for 30 min at 37 °C with shaking. The digested tissue was passed through a 70-μm nylon mesh, centrifuged at 700 g for 3 min, and resuspended in 30% Percoll solution (MP Biomedicals, Santa Ana, CA, USA). After centrifugation at 1,000 g for 20 min, the pellet was resuspended in complete RPMI. Cell viability was assessed by trypan blue exclusion (Nacalai Tesque), and viable cells were counted manually using a hemocytometer under a light microscope. Flow cytometry TG cells were isolated as described above and stimulated with phorbol 12-myristate 13-acetate (PMA, 100 ng/mL) and ionomycin (1 μg/mL; both from Cayman Chemical, Ann Arbor, MI, USA) in the presence of brefeldin A (5 μg/mL; BioLegend, San Diego, CA, USA) for 5 h at 37 °C. After stimulation, cells were stained with Hoechst 33342 (Dojindo, Kumamoto, Japan), Zombie Yellow fixable viability dye (BioLegend), and the following fluorochrome-conjugated antibodies (BioLegend): APC–anti-CD3 (clone 1F4), PE-Cy7–anti-CD8a (clone OX-8), PerCP-Cy5.5–anti-CD11b/c (clone OX-42), and PE–anti-CD45 (clone OX-1). Cells were then fixed with 4% paraformaldehyde in PBS for 10 min at 4 °C, permeabilized with Intracellular Staining Permeabilization Wash Buffer (BioLegend), and stained with FITC–anti-IFN-γ (clone DB-1, BioLegend). Fc receptor blocking was performed with anti-CD32 (clone D34-485, BD Biosciences, Franklin Lakes, NJ, USA). Data were acquired on an LSRFortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software, version 10.7.1 (BD Biosciences). Absolute numbers of each cell population were calculated by multiplying the viable cell count by the frequency of the respective population obtained from flow cytometry. Statistical analyses Normality of data distribution was assessed using the Shapiro–Wilk test, and homoscedasticity was evaluated using the Brown–Forsythe test. Depending on data distribution, comparisons were performed using the unpaired Student’s t -test or Mann–Whitney test. For multiple group comparisons, two-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test was applied. All statistical analyses were conducted using GraphPad Prism 9 (GraphPad Software, Boston, MA, USA). Differences were considered statistically significant at p < 0.05. MHWT data are presented as median with interquartile range (25–75%), and whisker plots represent minimum and maximum values. Results IONI induces orofacial nocifensive behavior and elevates IFN-γ expression in the TG The MHWT of the left whisker pad skin was measured to assess the development of mechanical hypersensitivity following IONI. In the IONI group, MHWT values were near the cutoff threshold (60 g) before surgery but were significantly reduced from day 1 to day 7 after IONI compared with baseline. MHWT was also significantly lower at days 4 and 7 after IONI compared with sham-operated controls. In the sham group, MHWT remained close to 60 g throughout the observation period, showing no significant change (Fig. 1 A; n = 6 per group). Western blot analysis demonstrated that IFN-γ protein levels in the TG were significantly increased in the IONI group compared with the sham group on day 7 (Fig. 1 B; n = 13 per group). Together, these findings indicate that orofacial mechanical hypersensitivity develops from day 1 after IONI and becomes pronounced by days 4–7, coinciding with elevated IFN-γ expression in the TG. CD8⁺ T cells are the major source of IFN-γ in the TG following IONI To identify the cellular source of IFN-γ, cells were isolated from ipsilateral TGs 7 days after IONI, stimulated with PMA and ionomycin in the presence of brefeldin A, and analyzed by flow cytometry (Fig. 2 ; Supplementary Fig. 1; n = 8). TG cells were classified based on surface marker expression into non-hematopoietic cells (CD45 − ), myeloid cells (CD45⁺CD11b/c + ), CD8⁺ T cells (CD45⁺CD3⁺CD8a + ), CD8⁻ T cells (CD45⁺CD3⁺CD8a⁻; presumed CD4⁺ T cells), and other hematopoietic cells (CD45 + CD11b/c − CD3 − ) (Fig. 2 A; Supplementary Fig. 1A). The absolute numbers of total cells, myeloid cells, and CD8⁺ T cells were significantly higher in the IONI group compared with sham controls (Fig. 2 B; n = 7–8). Notably, ~ 60% of IFN-γ⁺ cells in TGs from IONI-treated rats were CD8⁺ T cells (Supplementary Fig. 1B, C). IONI also significantly increased both the frequency and absolute number of IFN-γ⁺ CD8⁺ T cells (Fig. 2 C–E). These results indicate that CD8⁺ T cells are the predominant cellular source of IFN-γ in the TG after IONI. CD8 + T cells infiltrate TG neurons after IONI and transiently induce orofacial mechanical hypersensitivity To assess the distribution of CD8⁺ T cells in the TG on day 7 after IONI, immunohistochemical staining was performed In IONI rats, numerous small CD8⁺ T cells were observed infiltrating around FG-labeled TG neurons innervating the whisker pad skin, whereas sham rats showed markedly fewer CD8⁺ T cells in the same region (Fig. 3 A).We next examined the functional effect of intra-TG administration of CD8⁺ T cells isolated from IONI rats on MHWT in naïve rats. MHWT values were significantly reduced in the CD8⁺ T cell–treated group compared with vehicle controls at day 3 post-administration, but returned to baseline by day 4 (Fig. 3 B; n = 6 per group). These findings suggest that CD8⁺ T cells infiltrate TG neurons after IONI and contribute to orofacial mechanical hypersensitivity, although their direct effects in naïve TG are transient. IFN-γ receptors localize to SGCs and are upregulated in the TG after IONI Given the observed changes in mechanical sensitivity and IFN-γ expression after IONI, we examined IFNGR distribution in the TG. Immunohistochemistry showed that IFNGR immunoreactivity (IR) was localized to Kir4.1-positive cells, indicating expression in SGCs (Fig. 4 A). Many IFNGR-IR cells were found encircling FG-labeled TG neurons, confirming that SGCs surrounding neurons innervating the whisker pad skin express IFNGR (Fig. 4 B). In IONI rats, the number of IFNGR-IR cells was markedly greater than in sham controls (Fig. 4 A). Quantitative analysis revealed that the percentage area of IFNGR-IR signal colocalized with Kir4.1-IR SGCs was significantly larger in the IONI group than in the sham group (Fig. 4 C; n = 5 per group). These results demonstrate that IFNGR is predominantly expressed by SGCs and is significantly upregulated in the TG following IONI. SGCs surrounding TG neurons are activated after IONI To determine whether SGCs are activated following IONI, GFAP immunoreactivity (IR), a marker of SGC activation, was assessed in the TG on day 7. Numerous GFAP-IR cells were observed in the TG of IONI rats, whereas only a few were detected in sham controls (Fig. 5 A). Most GFAP-IR cells encircled FG-labeled TG neurons, indicating that activated SGCs surrounded neurons innervating the whisker pad skin (Fig. 5 B). Quantitative analysis showed that the percentage area occupied by GFAP-IR cells in the TG was significantly greater in IONI rats compared with sham rats (Fig. 5 C; n = 5 per group). These findings indicate that SGCs surrounding TG neurons are robustly activated following IONI. IFN-γ induces mechanical hypersensitivity and SGC activation, while IFNGR antagonism attenuates these effects after IONI We first examined the effect of continuous IFN-γ administration into the TG of naïve rats for 7 days on mechanical sensitivity of the ipsilateral whisker pad skin. IFN-γ infusion significantly reduced MHWT from days 2–7 compared with both baseline and vehicle-treated controls, whereas vehicle administration had no effect (Fig. 6 A; n = 6 per group). In parallel, IFN-γ administration markedly increased GFAP immunoreactivity, with numerous GFAP-IR cells observed in TG, whereas only a few were present in vehicle-treated rats (Fig. 6 B). Quantitative analysis confirmed that the percentage area occupied by GFAP-IR cells was significantly larger in the IFN-γ–treated group (Fig. 6 C; n = 5 per group). To further assess the role of IFN-γ signaling via the IFNGR, IFNGR ANTAG was continuously administered into the TG of IONI rats for 7 days. In vehicle-treated IONI rats, MHWT significantly decreased from days 1–7. This reduction was significantly attenuated by IFNGR ANTAG administration from days 3–7 (Fig. 6 D; n = 6 per group). Robust GFAP-IR expression was observed in the TG of vehicle-treated IONI rats, whereas lower GFAP-IR levels were detected in IFNGR ANTAG-treated rats (Fig. 6 E). Quantitative analysis confirmed that GFAP-IR area in the TG was significantly reduced by IFNG ANTAG administration compared with vehicle (Fig. 6 F; n = 5 per group). These results demonstrate that IFN-γ signaling through the IFNGR in SGCs contributes to orofacial mechanical hypersensitivity and SGC activation following IONI. IL-1β released from IFN-γ–stimulated SGCs activates TG neurons and promotes mechanical hypersensitivity We next investigated how IFN-γ–activated SGCs influence TG neurons. ELISA showed that IL-1β concentration in the supernatant of cultured SGCs stimulated by IFN-γ was significantly higher than that of control cultures (Fig. 7 A; n = 5–6). Immunohistochemistry confirmed that all cultured 4′,6-diamidino-2-phenylindole (DAPI)-positive cells were also Kir4.1-positive, validating that the cultures consisted entirely of SGCs (Fig. 7 B). To assess the functional role of IL-1β, we examined the effect of IL-1R1 ANTAG on IFN-γ–induced mechanical hypersensitivity. The reduction in MHWT caused by IFN-γ administration was significantly suppressed by co-administration of the IL-1R1 ANTAG in naïve rats (Fig. 7 C; n = 6 per group). We next investigated the localization of IL-1R1 in the TG after IONI. Immunohistochemistry revealed numerous IL-1R1-IR cells colocalized with NeuN-positive neurons in IONI rats, whereas sham rats showed fewer IL-IR1-IR neurons (Fig. 7 D). Some IL-1R1-IR cells were also FG-labeled, indicating that TG neurons innervating the whisker pad skin express IL-1R1 (Fig. 7 E). Western blot analysis further demonstrated thatIL-1R1 protein levels in the TG were significantly higher in IONI rats compared with sham controls on day 7 (Fig. 7 F; n = 7 per group). Taken together, these findings suggest that IL-1β released from IFN-γ–stimulated SGCs acts on IL-1R1expressed by TG neurons, enhancing neuronal excitability and promoting the mechanical hypersensitivity observed after IONI. Discussion Peripheral nerve injury induces hyperactivation of damaged nerves, which contributes to neuropathic pain. Activation of SGCs surrounding neuronal somata is also increasingly recognized as a critical factor in this process. Although several candidates have been proposed as initiators of SGC activation, the mechanisms remain incompletely understood. In the present study, we demonstrate for the first time that IFNGRs are expressed on SGCs and that IFN-γ signaling in the TG is a key driver of SGC activation following IONI, leading to orofacial mechanical hypersensitivity. Furthermore, our results identify CD8⁺ T cells as the primary source of IFN-γ in the TG after IONI. After peripheral nerve injury, both macrophages and T lymphocytes are activated. Resident macrophages proliferate and initiate phagocytic activity within 24–48 h of injury [ 35 , 36 ]. While macrophages contribute to axonal degeneration and regeneration, they also release inflammatory mediators during the repair process. T lymphocytes, in contrast, play an especially important role in the transition from acute to chronic pain [ 37 ]. For example, T cell infiltration occurs 7–28 days after sciatic nerve injury, during which T cells release cytokines such as IFN-γ, IL2, IL-4, IL-10, and IL-13 [ 22 ]. T cells can also infiltrate the dorsal root ganglion, where they release leukocyte elastase, a mediator that promotes analgesia but paradoxically can induce mechanical allodynia [ 38 ]. In our study, intra-TG administration of CD8⁺ T cells or IFN-γ did not immediately alter the MHWT; instead, hypersensitivity emerged after a 2–3 day delay. Similarly, suppression of IFN-γ–induced MHWT reduction by co-administration of an IL-1R1 antagonist appeared only after 2 days. In IONI rats, suppression of mechanical hypersensitivity by an IFN-γ antagonist compared with vehicle was evident starting on day 3. Taken together, these findings suggest that CD8⁺ T cells infiltrate the TG and release IFN-γ, which activates SGCs to produce IL-1β. IL-1β then acts on TG neurons, enhancing their excitability and leading to mechanical hypersensitivity within 2–3 days of injury. Overall, our findings support a model in which IFN-γ signaling in the TG contributes to the transition from acute to chronic stages of orofacial neuropathic pain. IFN-γ is produced by both CD8 + and CD4 + T cells after the induction of antigen-specific immunity [ 39 ]. CD4 + T cells can also infiltrate the spinal cord and activate microglia and astrocytes, thereby contributing to neuropathic pain [ 40 – 42 ]. Thus, both CD8 + and CD4 + T cells were considered potential sources of IFN-γ following IONI. Flow cytometric analysis revealed that approximately 60% of IFN-γ + cells in the TG after IONI were CD8 + T cells, whereas ~ 25% were CD4 + T cells (CD45 + CD3 + CD8a − ). Moreover, the frequency and absolute number of IFN-γ + CD8 + T cells were significantly higher in IONI rats compared with sham controls, whereas no significant differences were observed for CD4 + T cells. These results suggest that CD8 + T cells are the predominant source of IFN-γ after INOI. Astrocytes and SGCs share many critical functions, and GFAP is upregulated in both cell types after peripheral nerve injury and inflammation [ 43 ]. We previously demonstrated that IFNGRs are expressed in Vc astrocytes and that the number of GFAP-IR astrocytes expressing IFNGRs increased after IONI [ 15 ]. Consistent with these findings, the present study confirmed enhanced GFAP and IFNGR immunostaining in SGCs after IONI, supporting the notion that IFN-γ signaling plays an important role in SGC-mediated neuropathic pain. However, while SGC activation has been reported to last only hours to days after peripheral nerve injury, astrocyte activation in the ipsilateral dorsal horn can persist for days to weeks [ 44 ]. In our study, significant reductions in MHWT and robust SGC activation accompanied by IFN-γ expression were still evident 7 days after IONI, suggesting that additional factors may contribute to sustaining SGC activation. Consistent with this idea, intra-TG injection of CD8⁺ T cells isolated from IONI rats transiently reduced MHWT in naïve rats, but the effect was not maintained. Damage-associated molecules may contribute to sustaining T cell activation. For example, high-mobility group box 1 (HMGB1), a nuclear DNA-binding protein released from damaged cells, has been shown to enhance CD8⁺ T cell proliferation and IFN-γ expression more potently than in CD4⁺ T cells [ 45 ]. In addition, the C-C motif chemokine ligand 2(CCL2) and thymic stromal lymphopoietin (TSLP) have been identified as T cell migratory factors in the DRG, both of which are upregulated after peripheral nerve injury [ 46 , 47 ]. Further studies are needed to determine whether HMGB1, CCL2, TSLP, or related signals are required for sustained IFN-γ release by CD8 + T cells and prolonged SGC activation in orofacial neuropathic pain. We further investigated the mediator responsible for TG neuronal hyperactivity following SGC activation. IL-1β is a potent hyperalgesic cytokine [ 48 ] that increases the frequency of action potentials in primary sensory neurons via IL-1 receptor signaling. Elevated IL-1β expression has also been reported in injured peripheral nerves, DRG, and spinal cord in animal models of neuropathic pain [ 49 ]. Increased neuronal spike frequency mediated by IL-1 receptor signaling has been directly linked to neuropathic pain induction [ 50 , 51 ]. In our study, IL-1β was released from cultured SGCs in response to IFN-γ stimulation. Furthermore, IL-1β receptors were expressed in TG neurons, and their levels were upregulated after IONI. These findings suggest that IL-1β released from IFN-γ–stimulated SGCs binds to the IL-1 receptor on TG neurons, thereby enhancing neuronal excitability and contributing to orofacial hypersensitivity after IONI. Beyond its role in pain, IFN-γ has antiviral, antitumor, and immunomodulatory properties and is used clinically to treat conditions such as hepatitis C, non-Hodgkin’s lymphoma, and multiple sclerosis [ 52 ]. Although IFN-γ has been implicated in neuropathic pain [ 16 , 53 , 54 ], its precise role remains incompletely defined, and clinical applications in this context have not yet been established. Previous studies demonstrated that IFN-γ signaling contributes to neuropathic pain in the Vc and spinal cord [ 15 , 16 ], and the present study extends this mechanism to the TG. Thus, our findings suggest that not only systemic IFN-γ antagonism but also local suppression of IFN-γ signaling at the level of primary sensory neurons could represent a promising therapeutic strategy for neuropathic pain. Currently, tricyclic antidepressants such as amitriptyline and nortriptyline are the most frequently prescribed drugs for chronic neuropathic pain worldwide [ 55 , 56 ]. Interestingly, IFN-γ–producing CD8 + T cells have been reported to decrease significantly following treatment with these agents [ 57 ]. However, their therapeutic use is often limited by off-target effects, as they also block postsynaptic H1 histamine, α1-adrenergic and muscarinic receptors, causing side effects such as dry mouth, dizziness, urinary retention, and even life-threatening arrhythmias [ 58 ]. Therefore, strategies that specifically target IFN-γ signaling, IFN-γ-producing CD8 + T cells, or IFN-γ–activated SGCs may provide effective treatments for chronic neuropathic pain while minimizing adverse effects. This study has several limitations. First, it was recently reported that IFN-γ is induced in TG neurons after IONI, particularly in nociceptive C-fiber neurons [ 17 ]. Although we demonstrated that IFN-γ signaling plays an essential role in the TG, we did not investigate which neuronal subtypes are specifically involved in this pathway. Second, although we focused on IL-1β released from activated SGCs, other mediators such as ATP and tumor necrosis factor-α are also known to be released from SGCs and to contribute to orofacial neuropathic pain [ 59 ]. Their involvement was not addressed in this study. Third, while IFN-γ antagonists suppressed IONI-induced mechanical hypersensitivity, the degree of recovery was only ~ 15–30% compared with pre-injury baseline, suggesting that mechanisms beyond IFN-γ signaling contribute to chronic neuropathic pain after IONI but remain to be identified. Additionally, neuropathic pain exhibits both sex-specific differences and shared mechanisms [ 60 ]; however, our experiments were conducted exclusively in male rats. Finally, we examined the effect of fresh CD8 + T cells isolated from IONI rats by injecting them into naïve TGs. Although this reduced MHWT, the effect was transient. Continuous infusion of CD8 + T cells may provide further insight, but this approach is technically challenging and was not tested in the present study. Conclusions In summary, this study demonstrates that following infraorbital nerve injury, IFN-γ is primarily produced by CD8⁺ T cells and activates SGCs via IFN-γ receptor signaling. Activated SGCs subsequently release IL-1β, which binds to IL-1 receptors on TG neurons, enhancing neuronal excitability and contributing to orofacial mechanical hypersensitivity. Targeting IFN-γ signaling—at the level of CD8⁺ T cells, SGC activation, or IFN-γ receptor signaling—represents a promising therapeutic approach for the treatment of orofacial neuropathic pain. Abbreviations ANOVA analysis of variance ANTAG antagonist ATP adenosine triphosphate BSA bovine serum albumin CCL2 C-C motif chemokine ligand 2 CD8 + T cellsCD8-positive T cells CD4 + T cells CD4-positive T cells DMEM Dulbecco’s Modified Eagle Medium DNase deoxyribonuclease DRG dorsal root ganglion ELISA enzyme-linked immunosorbent assay FBS fetal bovine serum FG Fluoro-Gold GFAP glial fibrillary acidic protein HBSS Hanks’ balanced salt solution IFN-γ interferon-γ IFNGR interferon-γ receptor IL interleukin IL-1β interleukin-1β IL-1R1 interleukin-1 receptor 1 IR immunoreactive ION infraorbital nerve IONI infraorbital nerve injury LE leukocyte elastase MHWT mechanical head-withdrawal reflex threshold NeuN neuronal nuclei NP-40 Nonidet P-40 PBS phosphate-buffered saline PFA paraformaldehyde PMA phorbol 12-myristate 13-acetate Rpm revolutions per minute SGCs satellite glial cells SEM standard error of the mean SD standard deviation TBST Tris-buffered saline containing 0.1% Tween 20 TG trigeminal ganglion TNF-α tumor necrosis factor-α TSLP thymic stromal lymphopoietin Vc spinal trigeminal caudal subnucleus Declarations Ethics approval All experimental procedures adhered to the ethical guidelines of the International Association for the Study of Pain and were approved by the Animal Experimentation Committee of Nihon University (Approval No. AP23DEN007). Availability of data and material No datasets were generated or analyzed during the current study. Declaration of competing interests The authors declare no conflicts of interest relevant to this article. Consent for publication Not applicable. Funding This study was supported by the Dental Research Center, Nihon University School of Dentistry (DRC(A)-2025-5), the Sato Fund (SATO-2024-11), and JSPS KAKENHI Grant-in-Aid for Scientific Research (C) (24K13164, A.O.; 25K12959, K.I.; 23K09405, Y.H.; 23K09130, S.H.) and for Scientific Research (B) (JP 23K27798, M.S.). Authors’ contributions MK: data collection and methodology. AO: study design, data analysis, and original draft preparation. YT, AK, HT, NN, KM: data collection and methodology. YH, KI, SH: manuscript review and editing. MS: supervision, study design, and manuscript review and editing. Acknowledgments Flow cytometry was performed at the Biomedical Research Unit of Tohoku University Hospital (Sendai, Japan). 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18:13:09","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":176563,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7739576/v1/5c274f98c8bedb5427d8a922.html"},{"id":93620813,"identity":"f3924559-db75-4274-97fc-f999b399c934","added_by":"auto","created_at":"2025-10-15 17:57:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":66100,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIONI induces orofacial mechanical hypersensitivity and increases IFN-γ in TG.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Time course of MHWT in the ipsilateral whisker pad skin before and up to 7 days after IONI or sham treatment (n = 6/group). (B) Representative western blot showing IFN-γ protein in the ipsilateral TG on day 7 after IONI or sham treatment, and quantification of IFN-γ levels normalized to β-actin (n = 13/group). In panel A, MHWT was significantly reduced after IONI compared with baseline and sham. In panel B, IFN-γ expression was significantly increased after IONI compared with sham. Data are presented as mean ± SEM. Statistical significance: vs. pre-treatment (day 0), #: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ##: \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ###: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 by Mann–Whitney test; vs. sham, **: \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01 (A). *: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 by unpaired Student’s \u003cem\u003et\u003c/em\u003e-test (B).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAbbreviations\u003c/em\u003e: IFN-γ, interferon-γ; IONI, infraorbital nerve injury; MHWT, mechanical head-withdrawal reflex threshold; SEM, standard error of the mean; TG, trigeminal ganglion.\u003c/p\u003e","description":"","filename":"Binder11.png","url":"https://assets-eu.researchsquare.com/files/rs-7739576/v1/44a50c36f9c93426359f1f19.png"},{"id":93620822,"identity":"b312035e-5be3-441e-8cf8-c845b5d553a6","added_by":"auto","created_at":"2025-10-15 17:57:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":214133,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCD8⁺ T cells are the major source of IFN-γ in TG after IONI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were isolated from ipsilateral TGs 7 days after sham or IONI treatment, stimulated with PMA and ionomycin in the presence of brefeldin A, and analyzed by flow cytometry.\u003c/p\u003e\n\u003cp\u003e(A) Classification of TG cell populations; gating for live singlets is shown in Supplementary Fig. 1A. (B) Absolute numbers of indicated cell populations. (C, D) Frequencies of IFN-γ⁺ cells among each cell population. (E) Absolute numbers of IFN-γ⁺ cells within each cell population. Representative flow plots are shown in (A, C). CD8⁻ T cells (CD45⁺CD3⁺CD8a\u003csup\u003e−\u003c/sup\u003e) represent CD4⁺ T cells. CD8⁺ T cells were the predominant source of IFN-γ after IONI. Data are pooled from two independent experiments (n = 7–8/group) and presented as mean ± SD. Statistical significance: *: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 by Welch’s \u003cem\u003et\u003c/em\u003e-test (B); *: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ***: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 by two-way ANOVA with Bonferroni’s post hoc test (D, E). ns, not significant.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAbbreviations: \u003c/em\u003eANOVA, analysis of variance; IFN-γ, interferon-γ; IONI, infraorbital nerve injury; SD, standard deviation; TG, trigeminal ganglion.\u003c/p\u003e","description":"","filename":"Binder12.png","url":"https://assets-eu.researchsquare.com/files/rs-7739576/v1/8403ed7ef30aefa04efe5806.png"},{"id":93621550,"identity":"c948d3a2-24c2-4bd0-9f20-c04c0ce1dd31","added_by":"auto","created_at":"2025-10-15 18:05:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":222034,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCD8⁺ T cells infiltrate TG after IONI and transiently reduce MHWT.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Immunohistochemical analysis of FG-labeled TG neurons and CD8⁺ T cells 7 days after IONI or sham treatment. Scale bar = 50 μm. (B) MHWT in naïve rats after intra-TG administration of CD8⁺ T cells (isolated from IONI rats on day 7) or vehicle (n = 6/group). CD8⁺ T cell transfer significantly decreased MHWT at day 3 compared with vehicle, but the effect was transient. Data are presented as mean ± SEM. Statistical significance: *: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 by Mann–Whitney test (B).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAbbreviations\u003c/em\u003e: FG, Fluoro-Gold; IONI, infraorbital nerve injury; MHWT, mechanical head-withdrawal reflex threshold; SEM, standard error of the mean; TG, trigeminal ganglion.\u003c/p\u003e","description":"","filename":"Binder13.png","url":"https://assets-eu.researchsquare.com/files/rs-7739576/v1/6bed2c822c2257dd09e518d2.png"},{"id":93620824,"identity":"85469922-2089-4788-89da-188b47be2f51","added_by":"auto","created_at":"2025-10-15 17:57:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":225514,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIFN-γ receptors colocalize with SGCs and increase in TG after IONI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Immunohistochemistry of IFNGR-IR cells and Kir4.1 (SGC marker) in TG on day 7 after IONI or sham treatment. Arrowheads indicate double-positive cells. Scale bar = 50 μm. (B) Colocalization of FG-labeled neurons and IFNGR-IR cells in TG on day 7 after IONI. Scale bar = 50 μm. (C) Quantification of percentage area occupied by IFNGR-IR signal relative to Kir4.1-IR cells (n = 5/group). Data are presented as mean ± SEM. Statistical significance: ***:\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.001 by unpaired Student’s \u003cem\u003et\u003c/em\u003e-test (C).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAbbreviations: \u003c/em\u003eFG, Fluoro-Gold; IFNGR, interferon-γ receptor; IR, immunoreactive; IONI, infraorbital nerve injury; SEM, standard error of the mean; SGC, satellite glial cell; TG, trigeminal ganglion.\u003c/p\u003e","description":"","filename":"Binder14.png","url":"https://assets-eu.researchsquare.com/files/rs-7739576/v1/329e4377bff49458beb00d94.png"},{"id":93621554,"identity":"c4df0a72-a3fb-4eac-94f3-e50823783b44","added_by":"auto","created_at":"2025-10-15 18:05:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":427829,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSGCs surrounding TG neurons are activated after IONI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Immunohistochemistry showing GFAP-IR SGCs in TG 7 days after IONI or sham treatment. Scale bar = 50 μm. (B) FG-labeled TG neurons and GFAP-IR cells with merged images in IONI rats. Arrowheads indicate GFAP-IR cells surrounding FG-labeled neurons. Scale bar = 50 μm. (C) Quantification of percentage area occupied by GFAP-IR cells in TG (n = 5/group). Data are presented as mean ± SEM. Statistical significance: ***:\u003cem\u003e p\u003c/em\u003e\u0026lt; 0.001 by unpaired Student’s \u003cem\u003et\u003c/em\u003e-test (C).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAbbreviations: \u003c/em\u003eFG, Fluoro-Gold; GFAP, glial fibrillary acidic protein; IR, immunoreactive; IONI, infraorbital nerve injury; SEM, standard error of the mean; SGC, satellite glial cell; TG, trigeminal ganglion.\u003c/p\u003e","description":"","filename":"Binder15.png","url":"https://assets-eu.researchsquare.com/files/rs-7739576/v1/b32646f674eb12e1859bfea8.png"},{"id":93621551,"identity":"90091e62-edf8-441b-81db-b18cd0e95d72","added_by":"auto","created_at":"2025-10-15 18:05:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":192215,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIFN-γ induces hypersensitivity and SGC activation, while IFNGR antagonism suppresses these effects.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) MHWT of the ipsilateral whisker pad skin in naïve rats after intra-TG administration of IFN-γ or vehicle (PBS) for 7 days (n = 6/group). (B) GFAP-IR cells in TG after intra-TG administration of IFN-γ or vehicle for 7 days in naïve rats. Scale bar = 50 μm. (C) Quantification of GFAP-IR area in TG from panel B (n = 5/group). (D) MHWT of IONI rats after intra-TG administration of IFNGR ANTG or vehicle for 7 days (n = 6/group). (E) GFAP-IR cells in TG from IONI rats treated with IFNGR ANTG or vehicle for 7 days. Scale bar = 50 μm. (F) Quantification of GFAP-IR area in TG from panel E (n = 5/group). Data are presented as mean ± SEM. Statistical significance: *:\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, **:\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.01 by unpaired Student’s \u003cem\u003et\u003c/em\u003e-test (C, F).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAbbreviations\u003c/em\u003e: ANTG, antagonist; GFAP, glial fibrillary acidic protein; IFN-γ, interferon-γ; IFNGR, interferon-γ receptor; IR, immunoreactive; IONI, infraorbital nerve injury; MHWT, mechanical head-withdrawal reflex threshold; PBS, phosphate-buffered saline; SEM, standard error of the mean; SGC, satellite glial cell; TG, trigeminal ganglion.\u003c/p\u003e","description":"","filename":"Binder16.png","url":"https://assets-eu.researchsquare.com/files/rs-7739576/v1/005914f82172bf77f6964d25.png"},{"id":93620815,"identity":"e49642cf-5aa1-4e7b-908e-ed0cbe1c5658","added_by":"auto","created_at":"2025-10-15 17:57:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":405493,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-1β released from SGCs activates TG neurons after IONI.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) IL-1β concentrations in cultured SGCs stimulated with IFN-γ or control medium (n = 5–6). (B) Immunostaining of cultured SGCs showing DAPI and Kir4.1 with merged images. Scale bar = 100 μm. (C) MHWT in naïve rats after intra-TG administration of IFN-γ or IFN-γ combined with IL-1R1 ANTAG for 7 days (n = 6/group). (D) Immunohistochemistry of NeuN and IL-1R1 in TG on day 7 after IONI or sham treatment. Scale bar = 50 μm. (E) FG-labeled IL-1R1-IR cells in TG of IONI rats. Arrowheads indicate co-positive cells. Scale bar = 50 μm. (F) Representative western blot of IL-1R1 protein in TG on day 7 after IONI or sham treatment, and quantification normalized to β-actin (n = 7/group). Data are presented as mean ± SEM. Statistical significance: **: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 by unpaired Student’s \u003cem\u003et\u003c/em\u003e-test (A, F), vs. IFN-γ; **: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 by Mann–Whitney test (C).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAbbreviations: \u003c/em\u003eANTAG, antagonist; DAPI, 4′,6-diamidino-2-phenylindole; FG, Fluoro-Gold; IFN-γ, interferon-γ; IL-1β, interleukin-1β; IL-1R1, interleukin-1 receptor 1; IR, immunoreactive; IONI, infraorbital nerve injury; MHWT, mechanical head-withdrawal reflex threshold; NeuN, neuronal nuclei; SGC, satellite glial cell; SEM, standard error of the mean; TG, trigeminal ganglion.\u003c/p\u003e","description":"","filename":"Binder17.png","url":"https://assets-eu.researchsquare.com/files/rs-7739576/v1/183646dd1319f6da6c97affb.png"},{"id":98244104,"identity":"1fe78316-b682-4016-a319-58e3fb0937ab","added_by":"auto","created_at":"2025-12-15 16:13:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3045238,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7739576/v1/f5a4f84b-f5c0-496c-9651-f4fcc8b4c203.pdf"},{"id":93620814,"identity":"3d88c454-395d-4a38-84ba-ea34cf83b148","added_by":"auto","created_at":"2025-10-15 17:57:09","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":119896,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7739576/v1/77b4cb4b76dfb58207ed1389.jpg"},{"id":93622875,"identity":"5adbe049-ed78-44b0-99ce-15443eafefeb","added_by":"auto","created_at":"2025-10-15 18:21:09","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":242723,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.1.pptx","url":"https://assets-eu.researchsquare.com/files/rs-7739576/v1/1e1ab1bdbf2a9497ff6686a6.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Trigeminal ganglion interferon-γ signaling drives orofacial neuropathic pain in rats","fulltext":[{"header":"Background","content":"\u003cp\u003eChronic pain affects 11\u0026ndash;50% of people worldwide and imposes substantial physical, emotional, and socioeconomic burdens [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Although many studies have examined the transition from acute to chronic pain, the underlying mechanisms remain incompletely understood [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Neuropathic pain, caused by peripheral nerve injury, is a major contributor to chronic pain. Chronic orofacial neuropathic pain is particularly debilitating, as it interferes with daily activities such as eating, speaking, and washing one\u0026rsquo;s face [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Peripheral nerve injury resulting from fractures, tooth extractions, dental implants, or root canal treatments can cause axonal degeneration, abnormal neuronal firing, and altered signal transmission [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIncreasing evidence indicates an interaction between neuronal and non-neuronal systems such as satellite glial cells (SGCs) and immune cells play a central role in the development of neuropathic pain within the trigeminal ganglion (TG) and dorsal root ganglion (DRG) [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. SGC activation has been associated with increased gap junction coupling among SGCs and between SGCs and primary sensory neurons, heightened sensitivity to adenosine triphosphate (ATP), release of proinflammatory cytokines, and upregulation of glial fibrillary acidic protein (GFAP) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, the upstream signals responsible for SGC activation after peripheral nerve injury and the mechanisms sustaining their hyperactivity remain poorly defined.\u003c/p\u003e\u003cp\u003eIn the DRG, TG, spinal cord, and spinal trigeminal caudal subnucleus of patients with neuropathic pain and in animal models, the proinflammatory cytokine interferon gamma (IFN-γ)\u0026mdash;primarily produced by T lymphocytes and natural killer cells\u0026mdash;has been identified as an important mediator of neuropathic pain development [\u003cspan additionalcitationids=\"CR15 CR16 CR17\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. We recently demonstrated that IFN-γ receptor signaling in astrocytes of the spinal trigeminal nucleus caudalis (Vc) is essential for the development of orofacial neuropathic pain following infraorbital nerve injury (IONI) in rodents [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Nevertheless, the contribution of IFN-γ signaling within the TG itself to neuropathic pain remains insufficiently characterized.\u003c/p\u003e\u003cp\u003eRecent studies have further emphasized the role of T lymphocytes in neuropathic pain [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Following peripheral nerve injury, T cells infiltrate the damaged nerve [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], where they can support regeneration but also release pronociceptive mediators such as proinflammatory cytokines [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. T cell infiltration has also been observed in the TG after IONI, in addition to accumulation at the injury site [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Both CD8-positive (CD8\u003csup\u003e+\u003c/sup\u003e) and CD4-positive (CD\u003csup\u003e+\u003c/sup\u003e) T lymphocytes are capable of producing IFN-γ after the development of antigen-specific immunity [\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], suggesting their potential involvement in neuropathic pain. However, the precise role of these T cell subsets in the TG is not yet known.\u003c/p\u003e\u003cp\u003eIn this study, we investigated whether T cell-derived IFN-γ activates SGCs and whether this signaling contributes to the pathogenesis of chronic orofacial neuropathic pain in a rat model of IONI.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale Sprague\u0026ndash;Dawley rats (n = 152; Japan SLC, Hamamatsu, Japan), weighing 150\u0026ndash;250 g, were used. Animals were housed under a 12-h light/dark cycle at a constant temperature of 23 \u0026plusmn; 1 \u0026deg;C, with ad libitum access to food and water. All experimental procedures adhered to the ethical guidelines of the International Association for the Study of Pain and were approved by the Animal Experimentation Committee of Nihon University (Approval No. AP23DEN007).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInfraorbital nerve injury (IONI)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIONI was performed as previously described [30]. Briefly, rats were anesthetized intraperitoneally with midazolam (2.0 mg/kg; Sandoz, Tokyo, Japan), butorphanol (2.5 mg/kg; Meiji Seika Pharma, Tokyo, Japan), and medetomidine (0.15 mg/kg; Zenoaq, Koriyama, Japan). A ~10 mm intraoral incision was made in the left buccal mucosa along the gingival margin proximal to the first molar. The left infraorbital nerve (ION) bundle was exposed and separated from surrounding tissue, after which one-third of the bundle was tightly ligated with 6-0 silk suture. For sham-operated controls, the ION was exposed but not ligated. All behavioral assessments were performed by an experimenter blinded to treatment groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMagnetic separation of CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes were purified using magnetic bead separation as previously reported [31]. Rats were deeply anesthetized with 5% isoflurane and perfused transcardially with isotonic saline. The ipsilateral TG was rapidly dissected and incubated in Hanks\u0026rsquo; balanced salt solution (HBSS) containing collagenase D and DNase at 37 \u0026deg;C for 45 min. The dissociated cell suspension was then incubated on ice for 15 min with anti-CD8\u003csup\u003e+\u003c/sup\u003e microbeads (80 \u0026mu;L/10\u003csup\u003e7\u003c/sup\u003e total cells; Miltenyi Biotec,\u0026nbsp;Germany). The suspension was passed through a magnetic separation column, retaining CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes, were subsequently eluted in culture medium. The purity of the CD8\u003csup\u003e+\u003c/sup\u003e T lymphocyte fraction was confirmed by flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIntra-TG administration of IFN-\u0026gamma;, IFN-\u0026gamma; receptor\u0026nbsp;antagonist, CD8\u003cem\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/em\u003e T lymphocytes, and IL-1\u0026beta; receptor antagonist\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rat skull was exposed by removal of the overlying skin, and a small hole (diameter: 1 mm) was drilled immediately above the ipsilateral TG (2.7 mm lateral to the sagittal suture; 2.8 mm anterior to the posterior fontanelle). The skull was stabilized in a stereotaxic apparatus under deep intraperitoneal anesthesia, as described above. A guide cannula was inserted into the TG through the hole and secured to the skull using dental resin and two stainless-steel screws, positioned at a depth of 9 mm below the cranial surface, as previously described [32]. For continuous intra-TG infusion, a polyethylene tube (SP10, 0.61 mm diameter; Natsume, Tokyo, Japan) was inserted through the guide cannula and connected to an osmotic mini-pump (Alzet model 2001; Durect, Cupertino, CA, USA) 7 days after cannulation. The tube and pump were implanted subcutaneously in the back. The pump had a total volume of 200 \u0026mu;L and an infusion rate of 1 \u0026mu;L/h for 7 days. Pumps and cannulas were pre-filled with one of the following solutions: recombinant IFN-\u0026gamma; (100 ng/mL; Biotechne, Minneapolis, MN, USA) in vehicle (0.01 M phosphate-buffered saline [PBS]); IFN-\u0026gamma; receptor antagonist (IFNG ANTAG; 1\u0026nbsp;mg/mL; Bachem, Torrance, CA, USA) in vehicle (20% acetic acid); or recombinant IFN-\u0026gamma; mixed with an interleukin-1\u0026beta; receptor type 1 (IL-1R1) antagonist (IL-1R1 ANTAG; 0.5 mg/mL; Fujifilm Wako, Tokyo, Japan). For single administrations, a suspension of CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes (10 \u0026mu;L), isolated from rats 7 days after IONI as described above, was directly injected into the TG of na\u0026iuml;ve rats through the guide cannula 7 days after cannulation. Injections were performed under deep anesthesia using a 31-gauge needle.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMechanical sensitivity assessment in the whisker pad skin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMechanical sensitivity of the whisker pad skin was assessed as previously described [33]. In brief, rats were trained for 7 days to protrude their perioral region, including the whisker pad skin, through a small hole in a plastic cage for a few minutes while mechanical stimulation was applied. IONI or sham surgery was performed after completion of training. Mechanical stimulation with von Frey filaments (0.16, 0.4, 0.6, 1.0, 1.4, 2.0, 4.0, 6.0, 8.0, 10, 15, 26, and 60 g; Touch-Test Sensory Evaluator, North Coast Medical, Morgan Hill, CA, USA) was applied to the whisker pad skin ipsilateral to IONI, sham, and na\u0026iuml;ve rats. Each filament was applied five times at 1-min intervals before and 1\u0026ndash;7 days after IONI or sham surgery. A cut-off of 60 g was set to avoid tissue damage. The mechanical head-withdrawal reflex threshold (MHWT) was defined as the lowest filament force that evoked head withdrawal in at least three of five trials.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn additional experiments, an intra-TG catheter was implanted in na\u0026iuml;ve rats, and IFN-\u0026gamma;, vehicle, or IFN-\u0026gamma; combined with IL-1R1 ANTAG was administered continuously for 7 days. MHWT was measured before and 1\u0026ndash;7 days after drug administration. To test the effect of IFNG ANTAG on IONI-induced mechanical sensitivity, the intra-TG catheter was implanted at the time of IONI surgery. IFNG ANTAG or vehicle was infused for 7 days, and MHWT was measured before and 1\u0026ndash;7 days after treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdministration of retrograde neurotracer\u0026nbsp;Fluoro-Gold (FG)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFive days before IONI surgery, 10 \u0026mu;L of 4% hydroxystilbamidine (Fluoro-Gold; FG; Fluorochrome, Denver, CO, USA) was injected into the left whisker pad skin using a 27-gauge needle to label TG neurons innervating this region. Injections were performed under 2.0% isoflurane anesthesia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemical analyses were performed in na\u0026iuml;ve rats and in rats 7 days after IONI or sham surgery. Animals were deeply anesthetized with 5% isoflurane and perfused transcardially with saline followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4, 4 \u0026deg;C). TGs were dissected, post-fixed in 4% PFA for 24 h at 4 \u0026deg;C, and cryoprotected in 30% sucrose (w/v) in PBS for 24 h at 4 \u0026deg;C. Ganglia were sectioned horizontally at 14 mm thickness on a cryostat (Tissue-Tek Polar, Sakura Finetek, Tokyo, Japan) and mounted on MAS-coated glass slides (Matsunami, Osaka, Japan). Sections were rinsed in PBS, blocked for 1 h at room temperature in 0.01 M PBS containing 10% normal donkey or goat serum in 1% bovine serum albumin (BSA; Proliant Biologicals, Boone, IA, USA) and 0.05% azide, and incubated overnight at 4\u0026deg;C with one of the following primary antibodies: Armenian hamster monoclonal anti-IFN-\u0026gamma; receptor (1:500, sc-12753, Santa Cruz, Dallas, TX, USA); rabbit polyclonal anti-Kir4.1 (1:1,000, APC-035, Thermo Fisher Scientific, Waltham, MA, USA); rabbit polyclonal anti-GFAP (1:1,000, ab7260, Abcam, Cambridge, UK); rabbit polyclonal anti-CD8 (1:1,000, ab4680, Abcam); rabbit polyclonal anti-IL-1R1 (1:1,000, sc689, Santa Cruz); or mouse monoclonal anti-NeuN (neuronal marker; 1:1,000, MAB377, Merck Millipore). After washing in PBS, sections were incubated with Alexa Fluor 488- or 568-conjugated secondary antibodies (1:1,000, Thermo Fisher Scientific) for 2 h at room temperature. Slides were mounted with PermaFluor Aqueous Mounting Medium (Thermo Fisher Scientific) and imaged using a BZ-X800 fluorescence microscope (Keyence, Osaka, Japan) or an Andor BC43 confocal microscope (Andor Technology, Belfast, UK). Images were analyzed with Imaris Viewer (Oxford Instruments, Abingdon, UK). Fluorescence intensity greater than twice the mean background was defined as immunoreactive (IR). Omission of primary antibodies resulted in no specific IR. The density of GFAP-IR cells and IFN-\u0026gamma; receptor (IFNGR)-IR cells localized with Kir4.1-IR SGCs was quantified within a 320 \u0026times; 320 \u0026mu;m\u003csup\u003e2\u003c/sup\u003e region of the TG area innervating the whisker pad skin using ImageJ software (version 1.54, NIH).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeven days after IONI or sham surgery, rats were deeply anesthetized with 5% isoflurane and perfused transcardially with isotonic saline. The ipsilateral TG was rapidly removed and homogenized in ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% Triton X-100; 0.5% NP-40) supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Homogenates were centrifuged at 2,0142 \u003cem\u003eg\u003c/em\u003e for 10 min at 4 \u0026deg;C, and the protein concentration of the supernatant was determined using a protein assay kit (Bio-Rad, Hercules, CA, USA). Samples were heat-denatured at 95 \u0026deg;C in Laemmli sample buffer (Bio-Rad). Equal amounts of protein were separated on 10% sodium dodecyl sulfate\u0026ndash;polyacrylamide gels (4\u0026ndash;20% gradient, Bio-Rad) and transferred to polyvinylidene fluoride membranes (Trans-Blot Turbo Transfer Pack; Bio-Rad) using the Trans-Blot Turbo rapid transfer system (Bio-Rad) for 7 min. Membranes were washed in Tris-buffered saline containing 0.1% Tween 20 (TBST), blocked in TBST with 5% Blocking-One (Nacalai Tesque, Kyoto, Japan) for 1 h at room temperature, and incubated overnight at 4 \u0026deg;C with one of the following primary antibodies: anti-IFN-\u0026gamma; (1:2,000, Pure Chemicals Corporation, Osaka, Japan); anti-IL-1R1 (1:500); or mouse monoclonal anti-\u0026beta;-actin (1:5,000, Santa Cruz Biotechnology). After washing, membranes were incubated with horseradish peroxidase\u0026ndash;conjugated anti-rabbit or anti-mouse secondary antibodies (1:2,000, Cytiva, Marlborough, MA, USA) for 2 h at room temperature. Protein bands were detected using Western Lightning ECL Pro (PerkinElmer, Waltham, MA, USA), visualized with Immobilon ECL Ultra Western HRP Substrate (Merck Millipore), and imaged on a ChemiDoc XRS system (Bio-Rad). Band intensities were quantified using ImageJ software (NIH, version 1.54) and normalized to \u0026beta;-actin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCultured\u003c/strong\u003e\u003cstrong\u003esatellite glial cells\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSGCs were dissociated as previously described [34]. Briefly, na\u0026iuml;ve rats were decapitated under 5% isoflurane, and TGs were immediately dissected. Samples were digested with 0.125% collagenase P (Roche, Indianapolis, IN, USA), 0.02% DNase (Sigma-Aldrich), and 0.25% trypsin\u0026ndash;ethylenediaminetetraacetic acid (Thermo Fisher Scientific), then mechanically triturated in dissociation solution consisting of HBSS (5 mL; Nacalai Tesque) containing 0.295% MgSO\u003csub\u003e4\u003c/sub\u003e and 0.02% DNase. The dissociated cells were then cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Nichirei, Tokyo, Japan), streptomycin (100 \u0026mu;g/mL; Thermo Fisher Scientific), penicillin (100 U/mL; Thermo Fisher Scientific), and L-glutamine (2 mmol/L; Fujifilm Wako Pure Chemical, Osaka, Japan). After incubation in feeding medium, cells were seeded onto uncoated 24-well plates and maintained for 7 days as mixed primary neuron glia cultures. SGC-enriched cultures were prepared on day 7 by detaching the mixed cultures with Accutase (Nacalai Tesque) for 5 min at 37 \u0026deg;C, followed by reseeding into uncoated 24-well plates for an additional 48-h. A subset of cells was fixed in 4% PFA for 10 min and processed for immunohistochemistry as described above. The remaining cells were used for\u0026nbsp;enzyme-linked immunosorbent assay (ELISA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp skip=\"true\"\u003eRecombinant IFN-\u0026gamma; was adjusted to 100 ng/mL in culture medium and applied to SGC cultures for 24 h. The concentration of IL-1\u0026beta; in the culture supernatant was quantified using a rat IL-1\u0026beta; ELISA kit (Quantikine, RLB00, R\u0026amp;D Systems, Minneapolis, MN, USA) according to the manufacturer\u0026rsquo;s instructions. IL-1\u0026beta; concentrations were normalized to the total protein concentration of the supernatant, determined using a Bio-Rad protein assay (Bio-Rad). Control cultures were treated with medium or IFN-\u0026gamma; diluent alone, and IL-1\u0026beta; concentrations were measured using the same procedure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of TG\u0026nbsp;cells for flow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSham- or IONI-treated rats were perfused with ice-cold saline, and ipsilateral TGs were collected in RPMI 1640 medium (Fujifilm Wako Pure Chemical) supplemented with 10% heat-inactivated FBS (Gibco, Waltham, MA, USA) and antibiotics (complete RPMI). TGs were minced into small fragments and digested in complete RPMI containing collagenase type IV (1 mg/mL, #C5138, Sigma-Aldrich), dispase II (1 mg/mL, #D4693, Sigma-Aldrich), and DNase I (50 \u0026mu;g/mL, #DN25, Sigma-Aldrich) for 30 min at 37 \u0026deg;C with shaking. The digested tissue was passed through a 70-\u0026mu;m nylon mesh, centrifuged at 700 \u003cem\u003eg\u003c/em\u003e for 3 min, and resuspended in 30% Percoll solution (MP Biomedicals, Santa Ana, CA, USA). After centrifugation at 1,000 \u003cem\u003eg\u003c/em\u003e for 20 min, the pellet was resuspended in complete RPMI. Cell viability was assessed by trypan blue exclusion (Nacalai Tesque), and viable cells were counted manually using a hemocytometer under a light microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTG cells were isolated as described above and stimulated with phorbol 12-myristate 13-acetate (PMA, 100 ng/mL) and ionomycin (1 \u0026mu;g/mL; both from Cayman Chemical, Ann Arbor, MI, USA) in the presence of brefeldin A (5 \u0026mu;g/mL; BioLegend, San Diego, CA, USA) for 5 h at 37 \u0026deg;C. After stimulation, cells were stained with Hoechst 33342 (Dojindo, Kumamoto, Japan), Zombie Yellow fixable viability dye (BioLegend), and the following fluorochrome-conjugated antibodies (BioLegend): APC\u0026ndash;anti-CD3 (clone 1F4), PE-Cy7\u0026ndash;anti-CD8a (clone OX-8), PerCP-Cy5.5\u0026ndash;anti-CD11b/c (clone OX-42), and PE\u0026ndash;anti-CD45 (clone OX-1). Cells were then fixed with 4% paraformaldehyde in PBS for 10 min at 4 \u0026deg;C, permeabilized with Intracellular Staining Permeabilization Wash Buffer (BioLegend), and stained with FITC\u0026ndash;anti-IFN-\u0026gamma; (clone DB-1, BioLegend). Fc receptor blocking was performed with anti-CD32 (clone D34-485, BD Biosciences, Franklin Lakes, NJ, USA). Data were acquired on an LSRFortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software, version 10.7.1 (BD Biosciences). Absolute numbers of each cell population were calculated by multiplying the viable cell count by the frequency of the respective population obtained from flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNormality of data distribution was assessed using the Shapiro\u0026ndash;Wilk test, and homoscedasticity was evaluated using the Brown\u0026ndash;Forsythe test. Depending on data distribution, comparisons were performed using the unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test or Mann\u0026ndash;Whitney test. For multiple group comparisons, two-way analysis of variance (ANOVA) followed by Bonferroni\u0026rsquo;s post hoc test was applied.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were conducted using GraphPad Prism 9 (GraphPad Software, Boston, MA, USA). Differences were considered statistically significant at \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05. MHWT data are presented as median with interquartile range (25\u0026ndash;75%), and whisker plots represent minimum and maximum values.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eIONI induces orofacial nocifensive behavior and elevates IFN-γ expression in the TG\u003c/h2\u003e\u003cp\u003eThe MHWT of the left whisker pad skin was measured to assess the development of mechanical hypersensitivity following IONI. In the IONI group, MHWT values were near the cutoff threshold (60 g) before surgery but were significantly reduced from day 1 to day 7 after IONI compared with baseline. MHWT was also significantly lower at days 4 and 7 after IONI compared with sham-operated controls. In the sham group, MHWT remained close to 60 g throughout the observation period, showing no significant change (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA; n\u0026thinsp;=\u0026thinsp;6 per group). Western blot analysis demonstrated that IFN-γ protein levels in the TG were significantly increased in the IONI group compared with the sham group on day 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB; n\u0026thinsp;=\u0026thinsp;13 per group). Together, these findings indicate that orofacial mechanical hypersensitivity develops from day 1 after IONI and becomes pronounced by days 4\u0026ndash;7, coinciding with elevated IFN-γ expression in the TG.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eCD8⁺ T cells are the major source of IFN-γ in the TG following IONI\u003c/h2\u003e\u003cp\u003eTo identify the cellular source of IFN-γ, cells were isolated from ipsilateral TGs 7 days after IONI, stimulated with PMA and ionomycin in the presence of brefeldin A, and analyzed by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Supplementary Fig.\u0026nbsp;1; n\u0026thinsp;=\u0026thinsp;8). TG cells were classified based on surface marker expression into non-hematopoietic cells (CD45\u003csup\u003e\u0026minus;\u003c/sup\u003e), myeloid cells (CD45⁺CD11b/c\u003csup\u003e+\u003c/sup\u003e), CD8⁺ T cells (CD45⁺CD3⁺CD8a\u003csup\u003e+\u003c/sup\u003e), CD8⁻ T cells (CD45⁺CD3⁺CD8a⁻; presumed CD4⁺ T cells), and other hematopoietic cells (CD45\u003csup\u003e+\u003c/sup\u003eCD11b/c\u003csup\u003e\u0026minus;\u003c/sup\u003eCD3\u003csup\u003e\u0026minus;\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA; Supplementary Fig.\u0026nbsp;1A). The absolute numbers of total cells, myeloid cells, and CD8⁺ T cells were significantly higher in the IONI group compared with sham controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB; n\u0026thinsp;=\u0026thinsp;7\u0026ndash;8). Notably, ~\u0026thinsp;60% of IFN-γ⁺ cells in TGs from IONI-treated rats were CD8⁺ T cells (Supplementary Fig.\u0026nbsp;1B, C). IONI also significantly increased both the frequency and absolute number of IFN-γ⁺ CD8⁺ T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u0026ndash;E). These results indicate that CD8⁺ T cells are the predominant cellular source of IFN-γ in the TG after IONI.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003eCD8\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eT cells infiltrate TG neurons after IONI and transiently induce orofacial mechanical hypersensitivity\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eTo assess the distribution of CD8⁺ T cells in the TG on day 7 after IONI, immunohistochemical staining was performed In IONI rats, numerous small CD8⁺ T cells were observed infiltrating around FG-labeled TG neurons innervating the whisker pad skin, whereas sham rats showed markedly fewer CD8⁺ T cells in the same region (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).We next examined the functional effect of intra-TG administration of CD8⁺ T cells isolated from IONI rats on MHWT in na\u0026iuml;ve rats. MHWT values were significantly reduced in the CD8⁺ T cell\u0026ndash;treated group compared with vehicle controls at day 3 post-administration, but returned to baseline by day 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB; n\u0026thinsp;=\u0026thinsp;6 per group).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese findings suggest that CD8⁺ T cells infiltrate TG neurons after IONI and contribute to orofacial mechanical hypersensitivity, although their direct effects in na\u0026iuml;ve TG are transient.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eIFN-γ receptors localize to SGCs and are upregulated in the TG after IONI\u003c/h2\u003e\u003cp\u003eGiven the observed changes in mechanical sensitivity and IFN-γ expression after IONI, we examined IFNGR distribution in the TG. Immunohistochemistry showed that IFNGR immunoreactivity (IR) was localized to Kir4.1-positive cells, indicating expression in SGCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Many IFNGR-IR cells were found encircling FG-labeled TG neurons, confirming that SGCs surrounding neurons innervating the whisker pad skin express IFNGR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In IONI rats, the number of IFNGR-IR cells was markedly greater than in sham controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Quantitative analysis revealed that the percentage area of IFNGR-IR signal colocalized with Kir4.1-IR SGCs was significantly larger in the IONI group than in the sham group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC; n\u0026thinsp;=\u0026thinsp;5 per group).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese results demonstrate that IFNGR is predominantly expressed by SGCs and is significantly upregulated in the TG following IONI.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eSGCs surrounding TG neurons are activated after IONI\u003c/h2\u003e\u003cp\u003eTo determine whether SGCs are activated following IONI, GFAP immunoreactivity (IR), a marker of SGC activation, was assessed in the TG on day 7. Numerous GFAP-IR cells were observed in the TG of IONI rats, whereas only a few were detected in sham controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Most GFAP-IR cells encircled FG-labeled TG neurons, indicating that activated SGCs surrounded neurons innervating the whisker pad skin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Quantitative analysis showed that the percentage area occupied by GFAP-IR cells in the TG was significantly greater in IONI rats compared with sham rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC; n\u0026thinsp;=\u0026thinsp;5 per group).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese findings indicate that SGCs surrounding TG neurons are robustly activated following IONI.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eIFN-γ induces mechanical hypersensitivity and SGC activation, while IFNGR antagonism attenuates these effects after IONI\u003c/h2\u003e\u003cp\u003eWe first examined the effect of continuous IFN-γ administration into the TG of na\u0026iuml;ve rats for 7 days on mechanical sensitivity of the ipsilateral whisker pad skin. IFN-γ infusion significantly reduced MHWT from days 2\u0026ndash;7 compared with both baseline and vehicle-treated controls, whereas vehicle administration had no effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA; n\u0026thinsp;=\u0026thinsp;6 per group). In parallel, IFN-γ administration markedly increased GFAP immunoreactivity, with numerous GFAP-IR cells observed in TG, whereas only a few were present in vehicle-treated rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Quantitative analysis confirmed that the percentage area occupied by GFAP-IR cells was significantly larger in the IFN-γ\u0026ndash;treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC; n\u0026thinsp;=\u0026thinsp;5 per group). To further assess the role of IFN-γ signaling via the IFNGR, IFNGR ANTAG was continuously administered into the TG of IONI rats for 7 days. In vehicle-treated IONI rats, MHWT significantly decreased from days 1\u0026ndash;7. This reduction was significantly attenuated by IFNGR ANTAG administration from days 3\u0026ndash;7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD; n\u0026thinsp;=\u0026thinsp;6 per group). Robust GFAP-IR expression was observed in the TG of vehicle-treated IONI rats, whereas lower GFAP-IR levels were detected in IFNGR ANTAG-treated rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Quantitative analysis confirmed that GFAP-IR area in the TG was significantly reduced by IFNG ANTAG administration compared with vehicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF; n\u0026thinsp;=\u0026thinsp;5 per group). These results demonstrate that IFN-γ signaling through the IFNGR in SGCs contributes to orofacial mechanical hypersensitivity and SGC activation following IONI.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eIL-1β released from IFN-γ\u0026ndash;stimulated SGCs activates TG neurons and promotes mechanical hypersensitivity\u003c/h2\u003e\u003cp\u003eWe next investigated how IFN-γ\u0026ndash;activated SGCs influence TG neurons. ELISA showed that IL-1β concentration in the supernatant of cultured SGCs stimulated by IFN-γ was significantly higher than that of control cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA; n\u0026thinsp;=\u0026thinsp;5\u0026ndash;6). Immunohistochemistry confirmed that all cultured 4\u0026prime;,6-diamidino-2-phenylindole (DAPI)-positive cells were also Kir4.1-positive, validating that the cultures consisted entirely of SGCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). To assess the functional role of IL-1β, we examined the effect of IL-1R1 ANTAG on IFN-γ\u0026ndash;induced mechanical hypersensitivity. The reduction in MHWT caused by IFN-γ administration was significantly suppressed by co-administration of the IL-1R1 ANTAG in na\u0026iuml;ve rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC; n\u0026thinsp;=\u0026thinsp;6 per group). We next investigated the localization of IL-1R1 in the TG after IONI. Immunohistochemistry revealed numerous IL-1R1-IR cells colocalized with NeuN-positive neurons in IONI rats, whereas sham rats showed fewer IL-IR1-IR neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Some IL-1R1-IR cells were also FG-labeled, indicating that TG neurons innervating the whisker pad skin express IL-1R1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Western blot analysis further demonstrated thatIL-1R1 protein levels in the TG were significantly higher in IONI rats compared with sham controls on day 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF; n\u0026thinsp;=\u0026thinsp;7 per group).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTaken together, these findings suggest that IL-1β released from IFN-γ\u0026ndash;stimulated SGCs acts on IL-1R1expressed by TG neurons, enhancing neuronal excitability and promoting the mechanical hypersensitivity observed after IONI.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePeripheral nerve injury induces hyperactivation of damaged nerves, which contributes to neuropathic pain. Activation of SGCs surrounding neuronal somata is also increasingly recognized as a critical factor in this process. Although several candidates have been proposed as initiators of SGC activation, the mechanisms remain incompletely understood. In the present study, we demonstrate for the first time that IFNGRs are expressed on SGCs and that IFN-γ signaling in the TG is a key driver of SGC activation following IONI, leading to orofacial mechanical hypersensitivity. Furthermore, our results identify CD8⁺ T cells as the primary source of IFN-γ in the TG after IONI.\u003c/p\u003e\u003cp\u003eAfter peripheral nerve injury, both macrophages and T lymphocytes are activated. Resident macrophages proliferate and initiate phagocytic activity within 24\u0026ndash;48 h of injury [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. While macrophages contribute to axonal degeneration and regeneration, they also release inflammatory mediators during the repair process. T lymphocytes, in contrast, play an especially important role in the transition from acute to chronic pain [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. For example, T cell infiltration occurs 7\u0026ndash;28 days after sciatic nerve injury, during which T cells release cytokines such as IFN-γ, IL2, IL-4, IL-10, and IL-13 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. T cells can also infiltrate the dorsal root ganglion, where they release leukocyte elastase, a mediator that promotes analgesia but paradoxically can induce mechanical allodynia [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In our study, intra-TG administration of CD8⁺ T cells or IFN-γ did not immediately alter the MHWT; instead, hypersensitivity emerged after a 2\u0026ndash;3 day delay. Similarly, suppression of IFN-γ\u0026ndash;induced MHWT reduction by co-administration of an IL-1R1 antagonist appeared only after 2 days. In IONI rats, suppression of mechanical hypersensitivity by an IFN-γ antagonist compared with vehicle was evident starting on day 3. Taken together, these findings suggest that CD8⁺ T cells infiltrate the TG and release IFN-γ, which activates SGCs to produce IL-1β. IL-1β then acts on TG neurons, enhancing their excitability and leading to mechanical hypersensitivity within 2\u0026ndash;3 days of injury. Overall, our findings support a model in which IFN-γ signaling in the TG contributes to the transition from acute to chronic stages of orofacial neuropathic pain.\u003c/p\u003e\u003cp\u003eIFN-γ is produced by both CD8\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003e T cells after the induction of antigen-specific immunity [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. CD4\u003csup\u003e+\u003c/sup\u003e T cells can also infiltrate the spinal cord and activate microglia and astrocytes, thereby contributing to neuropathic pain [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Thus, both CD8\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003e T cells were considered potential sources of IFN-γ following IONI. Flow cytometric analysis revealed that approximately 60% of IFN-γ\u003csup\u003e+\u003c/sup\u003e cells in the TG after IONI were CD8\u003csup\u003e+\u003c/sup\u003e T cells, whereas ~\u0026thinsp;25% were CD4\u003csup\u003e+\u003c/sup\u003e T cells (CD45\u003csup\u003e+\u003c/sup\u003eCD3\u003csup\u003e+\u003c/sup\u003eCD8a\u003csup\u003e\u0026minus;\u003c/sup\u003e). Moreover, the frequency and absolute number of IFN-γ\u003csup\u003e+\u003c/sup\u003e CD8\u003csup\u003e+\u003c/sup\u003e T cells were significantly higher in IONI rats compared with sham controls, whereas no significant differences were observed for CD4\u003csup\u003e+\u003c/sup\u003e T cells. These results suggest that CD8\u003csup\u003e+\u003c/sup\u003e T cells are the predominant source of IFN-γ after INOI.\u003c/p\u003e\u003cp\u003eAstrocytes and SGCs share many critical functions, and GFAP is upregulated in both cell types after peripheral nerve injury and inflammation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. We previously demonstrated that IFNGRs are expressed in Vc astrocytes and that the number of GFAP-IR astrocytes expressing IFNGRs increased after IONI [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Consistent with these findings, the present study confirmed enhanced GFAP and IFNGR immunostaining in SGCs after IONI, supporting the notion that IFN-γ signaling plays an important role in SGC-mediated neuropathic pain. However, while SGC activation has been reported to last only hours to days after peripheral nerve injury, astrocyte activation in the ipsilateral dorsal horn can persist for days to weeks [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In our study, significant reductions in MHWT and robust SGC activation accompanied by IFN-γ expression were still evident 7 days after IONI, suggesting that additional factors may contribute to sustaining SGC activation. Consistent with this idea, intra-TG injection of CD8⁺ T cells isolated from IONI rats transiently reduced MHWT in na\u0026iuml;ve rats, but the effect was not maintained. Damage-associated molecules may contribute to sustaining T cell activation. For example, high-mobility group box 1 (HMGB1), a nuclear DNA-binding protein released from damaged cells, has been shown to enhance CD8⁺ T cell proliferation and IFN-γ expression more potently than in CD4⁺ T cells [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In addition, the C-C motif chemokine ligand 2(CCL2) and thymic stromal lymphopoietin (TSLP) have been identified as T cell migratory factors in the DRG, both of which are upregulated after peripheral nerve injury [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Further studies are needed to determine whether HMGB1, CCL2, TSLP, or related signals are required for sustained IFN-γ release by CD8\u003csup\u003e+\u003c/sup\u003e T cells and prolonged SGC activation in orofacial neuropathic pain.\u003c/p\u003e\u003cp\u003eWe further investigated the mediator responsible for TG neuronal hyperactivity following SGC activation. IL-1β is a potent hyperalgesic cytokine [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] that increases the frequency of action potentials in primary sensory neurons via IL-1 receptor signaling. Elevated IL-1β expression has also been reported in injured peripheral nerves, DRG, and spinal cord in animal models of neuropathic pain [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Increased neuronal spike frequency mediated by IL-1 receptor signaling has been directly linked to neuropathic pain induction [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In our study, IL-1β was released from cultured SGCs in response to IFN-γ stimulation. Furthermore, IL-1β receptors were expressed in TG neurons, and their levels were upregulated after IONI. These findings suggest that IL-1β released from IFN-γ\u0026ndash;stimulated SGCs binds to the IL-1 receptor on TG neurons, thereby enhancing neuronal excitability and contributing to orofacial hypersensitivity after IONI.\u003c/p\u003e\u003cp\u003eBeyond its role in pain, IFN-γ has antiviral, antitumor, and immunomodulatory properties and is used clinically to treat conditions such as hepatitis C, non-Hodgkin\u0026rsquo;s lymphoma, and multiple sclerosis [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Although IFN-γ has been implicated in neuropathic pain [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], its precise role remains incompletely defined, and clinical applications in this context have not yet been established. Previous studies demonstrated that IFN-γ signaling contributes to neuropathic pain in the Vc and spinal cord [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and the present study extends this mechanism to the TG. Thus, our findings suggest that not only systemic IFN-γ antagonism but also local suppression of IFN-γ signaling at the level of primary sensory neurons could represent a promising therapeutic strategy for neuropathic pain.\u003c/p\u003e\u003cp\u003eCurrently, tricyclic antidepressants such as amitriptyline and nortriptyline are the most frequently prescribed drugs for chronic neuropathic pain worldwide [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Interestingly, IFN-γ\u0026ndash;producing CD8\u003csup\u003e+\u003c/sup\u003e T cells have been reported to decrease significantly following treatment with these agents [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. However, their therapeutic use is often limited by off-target effects, as they also block postsynaptic H1 histamine, α1-adrenergic and muscarinic receptors, causing side effects such as dry mouth, dizziness, urinary retention, and even life-threatening arrhythmias [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Therefore, strategies that specifically target IFN-γ signaling, IFN-γ-producing CD8\u003csup\u003e+\u003c/sup\u003e T cells, or IFN-γ\u0026ndash;activated SGCs may provide effective treatments for chronic neuropathic pain while minimizing adverse effects.\u003c/p\u003e\u003cp\u003eThis study has several limitations. First, it was recently reported that IFN-γ is induced in TG neurons after IONI, particularly in nociceptive C-fiber neurons [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Although we demonstrated that IFN-γ signaling plays an essential role in the TG, we did not investigate which neuronal subtypes are specifically involved in this pathway. Second, although we focused on IL-1β released from activated SGCs, other mediators such as ATP and tumor necrosis factor-α are also known to be released from SGCs and to contribute to orofacial neuropathic pain [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Their involvement was not addressed in this study. Third, while IFN-γ antagonists suppressed IONI-induced mechanical hypersensitivity, the degree of recovery was only\u0026thinsp;~\u0026thinsp;15\u0026ndash;30% compared with pre-injury baseline, suggesting that mechanisms beyond IFN-γ signaling contribute to chronic neuropathic pain after IONI but remain to be identified. Additionally, neuropathic pain exhibits both sex-specific differences and shared mechanisms [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]; however, our experiments were conducted exclusively in male rats. Finally, we examined the effect of fresh CD8\u003csup\u003e+\u003c/sup\u003e T cells isolated from IONI rats by injecting them into na\u0026iuml;ve TGs. Although this reduced MHWT, the effect was transient. Continuous infusion of CD8\u003csup\u003e+\u003c/sup\u003e T cells may provide further insight, but this approach is technically challenging and was not tested in the present study.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, this study demonstrates that following infraorbital nerve injury, IFN-γ is primarily produced by CD8⁺ T cells and activates SGCs via IFN-γ receptor signaling. Activated SGCs subsequently release IL-1β, which binds to IL-1 receptors on TG neurons, enhancing neuronal excitability and contributing to orofacial mechanical hypersensitivity. Targeting IFN-γ signaling\u0026mdash;at the level of CD8⁺ T cells, SGC activation, or IFN-γ receptor signaling\u0026mdash;represents a promising therapeutic approach for the treatment of orofacial neuropathic pain.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eANOVA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;analysis of variance\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eANTAG\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;antagonist\u003c/p\u003e\n\u003cp\u003eATP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;adenosine triphosphate\u003c/p\u003e\n\u003cp\u003eBSA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;bovine serum albumin\u003c/p\u003e\n\u003cp\u003eCCL2\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;C-C motif chemokine ligand 2\u003c/p\u003e\n\u003cp\u003eCD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eT cellsCD8-positive T cells\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCD4\u003csup\u003e+\u003c/sup\u003e T\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;cells\u0026nbsp; \u0026nbsp; \u0026nbsp;CD4-positive T cells\u003c/p\u003e\n\u003cp\u003eDMEM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Dulbecco’s Modified Eagle Medium\u003c/p\u003e\n\u003cp\u003eDNase\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;deoxyribonuclease\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDRG\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;dorsal root ganglion\u003c/p\u003e\n\u003cp\u003eELISA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;enzyme-linked immunosorbent assay\u003c/p\u003e\n\u003cp\u003eFBS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;fetal bovine serum\u003c/p\u003e\n\u003cp\u003eFG\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Fluoro-Gold\u003c/p\u003e\n\u003cp\u003eGFAP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;glial fibrillary acidic protein\u003c/p\u003e\n\u003cp\u003eHBSS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Hanks’ balanced salt solution\u003c/p\u003e\n\u003cp\u003eIFN-γ\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;interferon-γ\u003c/p\u003e\n\u003cp\u003eIFNGR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;interferon-γ receptor\u003c/p\u003e\n\u003cp\u003eIL\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;interleukin\u003c/p\u003e\n\u003cp\u003eIL-1β\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;interleukin-1β\u003c/p\u003e\n\u003cp\u003eIL-1R1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;interleukin-1 receptor 1\u003c/p\u003e\n\u003cp\u003eIR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;immunoreactive\u003c/p\u003e\n\u003cp\u003eION\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;infraorbital nerve\u003c/p\u003e\n\u003cp\u003eIONI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;infraorbital nerve injury\u003c/p\u003e\n\u003cp\u003eLE\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;leukocyte elastase\u003c/p\u003e\n\u003cp\u003eMHWT\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;mechanical head-withdrawal reflex threshold\u003c/p\u003e\n\u003cp\u003eNeuN\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;neuronal nuclei\u003c/p\u003e\n\u003cp\u003eNP-40\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Nonidet P-40\u003c/p\u003e\n\u003cp\u003ePBS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;phosphate-buffered saline\u003c/p\u003e\n\u003cp\u003ePFA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;paraformaldehyde\u003c/p\u003e\n\u003cp\u003ePMA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;phorbol 12-myristate 13-acetate\u003c/p\u003e\n\u003cp\u003eRpm\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;revolutions per minute\u003c/p\u003e\n\u003cp\u003eSGCs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;satellite glial cells\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSEM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;standard error of the mean\u003c/p\u003e\n\u003cp\u003eSD\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;standard deviation\u003c/p\u003e\n\u003cp\u003eTBST\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Tris-buffered saline containing 0.1% Tween 20\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTG\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;trigeminal ganglion\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTNF-α\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;tumor necrosis factor-α\u003c/p\u003e\n\u003cp\u003eTSLP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;thymic stromal lymphopoietin\u003c/p\u003e\n\u003cp\u003eVc \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; spinal trigeminal caudal subnucleus\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures adhered to the ethical guidelines of the International Association for the Study of Pain and were approved by the Animal Experimentation Committee of Nihon University (Approval No. AP23DEN007).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analyzed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest relevant to this article.\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\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Dental Research Center, Nihon University School of Dentistry (DRC(A)-2025-5), the Sato Fund (SATO-2024-11), and JSPS KAKENHI Grant-in-Aid for Scientific Research (C) (24K13164, A.O.; 25K12959, K.I.; 23K09405, Y.H.; 23K09130, S.H.) and for Scientific Research (B) (JP 23K27798, M.S.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMK: data collection and methodology.\u003cbr\u003e\u0026nbsp;AO: study design, data analysis, and original draft preparation.\u003cbr\u003e\u0026nbsp;YT, AK, HT, NN, KM: data collection and methodology.\u003cbr\u003e\u0026nbsp;YH, KI, SH: manuscript review and editing.\u003cbr\u003e\u0026nbsp;MS: supervision, study design, and manuscript review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlow cytometry was performed at the Biomedical Research Unit of Tohoku University Hospital (Sendai, Japan). \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMills SEE, Nicolson KP, Smith BH (2019) Chronic pain: a review of its epidemiology and associated factors in population-based studies. 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In: Pratt VM, Scott SA, Pirmohamed M, Esquivel B, Kattman BL, Malheiro AJ (eds) Medical Genetics Summaries. Bethesda, MD\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGoto T, Kuramoto E, Iwai H, Yamanaka A (2024) Cytoarchitecture and intercellular interactions in the trigeminal ganglion: associations with neuropathic pain in the orofacial region. J Oral Biosci 66(3):485\u0026ndash;490. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.job.2024.07.003\u003c/span\u003e\u003cspan address=\"10.1016/j.job.2024.07.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGhazisaeidi S, Muley MM, Salter MW (2023) Neuropathic pain: mechanisms, sex differences, and potential therapies for a global problem. Annu Rev Pharmacol Toxicol 63:565\u0026ndash;583. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev-pharmtox-051421-112259\u003c/span\u003e\u003cspan address=\"10.1146/annurev-pharmtox-051421-112259\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\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":"Orofacial neuropathic pain, Interferon-gamma (IFN-γ), CD8-positive T lymphocytes, Satellite glial cells, Interleukin-1 beta (IL-1β), Trigeminal ganglion, Pain hypersensitivity ","lastPublishedDoi":"10.21203/rs.3.rs-7739576/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7739576/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eChronic neuropathic pain, particularly in the orofacial region, markedly reduces quality of life. Peripheral trigeminal nerve injury activates satellite glial cells (SGCs) in the trigeminal ganglion (TG), which contributes to orofacial neuropathic pain. However, the upstream signal responsible for SGC activation remains unclear. This study investigated the role and cellular sources of interferon gamma (IFN-γ) signaling in the TG following infraorbital nerve injury (IONI) in rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods \u003c/strong\u003eMechanical sensitivity of the whisker pad skin was assessed after IONI. Changes in IFN-γ levels, IFN-γ receptor expression, and glial fibrillary acidic protein (GFAP; a marker of SGC activation) were examined in the TG by immunohistochemistry. The effects of intra-TG administration of IFN-γ, an IFN-γ receptor antagonist, and isolated CD8⁺ T cells on mechanical hypersensitivity were evaluated. GFAP expression after intra-TG administration of IFN-γ or the receptor antagonist was also quantified. Flow cytometry and immunohistochemistry were used to identify IFN-γ–producing cells. In primary SGC cultures, IFN-γ–induced interleukin-1β (IL-1β) release was measured, and the impact of IL-1 receptor antagonism on mechanical hypersensitivity was tested. IL-1 receptor localization and expression in TG neurons were further evaluated after IONI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eIONI induced persistent mechanical hypersensitivity and upregulated IFN-γ, IFN-γ receptor, and GFAP expression in the TG. CD8⁺ T cells were the primary source of IFN-γ after IONI, and intra-TG transfer of isolated CD8⁺ T cells transiently induced mechanical hypersensitivity. IFN-γ receptors were localized to SGCs, with expression levels increasing after IONI. Intra-TG IFN-γ administration triggered mechanical hypersensitivity and SGC activation, whereas receptor antagonism attenuated IONI-induced hypersensitivity. IFN-γ stimulation of cultured SGCs enhanced IL-1β release. Co-administration of an IL-1 receptor antagonist prevented IFN-γ–induced mechanical hypersensitivity. IL-1β receptors were localized on TG neurons and were upregulated following IONI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions \u003c/strong\u003eCD8⁺ T cell–derived IFN-γ activates SGCs in the TG, leading to IL-1β release that promotes neuronal hyperactivity and orofacial neuropathic pain following IONI. Targeting the IFN-γ–SGC–IL-1β signaling axis may represent a novel therapeutic strategy for orofacial neuropathic pain.\u003c/p\u003e","manuscriptTitle":"Trigeminal ganglion interferon-γ signaling drives orofacial neuropathic pain in rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 17:57:04","doi":"10.21203/rs.3.rs-7739576/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-12T13:06:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-12T00:08:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-09T16:45:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"38581709571690898578562064596241603596","date":"2025-10-03T14:27:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32553147620170867939467666284801279466","date":"2025-10-03T14:05:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-03T01:38:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"74214399695431742468137714738186969508","date":"2025-10-02T14:29:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-02T09:42:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-02T08:52:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-02T08:51:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"The Journal of Headache and Pain","date":"2025-09-29T08:12:19+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":"b43f46a1-d82e-4662-8c4a-b8f23a595726","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-15T16:05:55+00:00","versionOfRecord":{"articleIdentity":"rs-7739576","link":"https://doi.org/10.1186/s10194-025-02242-6","journal":{"identity":"the-journal-of-headache-and-pain","isVorOnly":false,"title":"The Journal of Headache and Pain"},"publishedOn":"2025-12-11 15:57:42","publishedOnDateReadable":"December 11th, 2025"},"versionCreatedAt":"2025-10-15 17:57:04","video":"","vorDoi":"10.1186/s10194-025-02242-6","vorDoiUrl":"https://doi.org/10.1186/s10194-025-02242-6","workflowStages":[]},"version":"v1","identity":"rs-7739576","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7739576","identity":"rs-7739576","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}