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

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 (IL-1R1) antagonism on mechanical hypersensitivity was tested. IL-1R1 localization and expression in TG neurons was 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, and its receptor antagonism attenuated the hypersensitivity. IFN-γ stimulation of cultured SGCs enhanced IL-1β release. Co-administration of an IL-1R1 antagonist prevented IFN-γ–induced mechanical hypersensitivity. IL-1R1 was 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.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s10194-025-02242-6.

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 = 179; 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/10⁷ 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-1 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 (IFNGR ANTAG; 1 mg/mL; Bachem, Torrance, CA, USA) in vehicle (20% acetic acid); or recombinant IFN-γ mixed with an interleukin-1 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 IFNGR ANTAG on IONI-induced mechanical sensitivity, the intra-TG catheter was implanted at the time of IONI surgery. IFNGR 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 3 and 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 μm 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, RRID: AB_673490, Santa Cruz, Dallas, TX, USA); rabbit polyclonal anti-Kir4.1 (1:1,000, RRID: AB_2040120, Thermo Fisher Scientific, Waltham, MA, USA); rabbit polyclonal anti-GFAP (1:1,000, RRID: AB_305808, Abcam, Cambridge, UK); rabbit polyclonal anti-CD8 (1:1,000, RRID: AB_304560, Abcam); rabbit polyclonal anti-IL-1R1 (1:1,000, RRID: AB_631807, Santa Cruz); or mouse monoclonal anti-NeuN (neuronal marker; 1:1,000, RRID: AB_2298772, MAB377, Merck Millipore). After washing in PBS, sections were incubated with secondary antibodies conjugated with Alexa Fluor 488 (1:1,000, Host: Armenian hamster; RRID: AB_2936402, Host: mouse; RRID: AB_2534088, Host: rabbit; RRID: AB_143165, Thermo Fisher Scientific) or Alexa Fluor 568 (1:1,000, Host: mouse; RRID: AB_2534072, Host: rabbit; RRID: AB_143157, Thermo Fisher Scientific) for 2 h at roo m temperature. Slides were mounted with PermaFluor Aqueous Mounting Medium (Thermo Fisher Scientific) and imaged using BZ-X800 fluorescence microscope (Keyence, Osaka, Japan) or 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 µm² 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, RRID: AB_10857066, Pure Chemicals Corporation, Osaka, Japan); anti-IL-1R1 (1:500); or mouse monoclonal anti-β-actin (1:5,000, RRID: AB_306371, Santa Cruz Biotechnology). After washing, membranes were incubated with horseradish peroxidase–conjugated anti-rabbit (1:2,000, RRID: AB_772206, Cytiva, Marlborough, MA, USA) or anti-mouse (1:2,000, RRID: AB_772210, Cytiva) secondary antibodies 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% MgSO₄ 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).

To confirm that the cultured cells are SGCs, they were transferred onto ε-Poly-L-lysine-coated (Cosmo Bio, Tokyo, Japan) glass slides and immunostained with an anti-Kir4.1 antibody (1:500). Furthermore, to ensure that the cultured cells did not contain neurons, fibroblasts, or macrophages, immunostaining with NeuN (1:100), α-smooth muscle actin (α-SMA, 1:100, a marker of fibroblast, RRID: AB_262054, Sigma-Aldrich, Darmstadt, Germany) or Iba1 (1:500, a marker of macrophage, RRID: AB_839504,Fujifilm Wako Pure Chemical, Osaka, Japan) was also performed. For nuclear staining, 4’, 6-Diamidino-2-phenylindole dihydrochloride (DAPI, 1:100, Nacalai Tesque, Kyoto, Japan) was used.

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 or tumor necrosis factor-α (TNF-α) in the culture supernatant was quantified using a rat IL-1β ELISA kit (Quantikine, RLB00, R&D Systems, Minneapolis, MN, USA) or TNF-α ELISA kit (Quantikine, RTA00, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. IL-1β or TNF-α 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 or TNF-α 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 33,342 (Dojindo, Kumamoto, Japan), Zombie Yellow fixable viability dye (BioLegend), and the following fluorochrome-conjugated antibodies (BioLegend): APC–anti-CD3 (RRID: AB_2494815, clone 1F4), PE-Cy7–anti-CD8a (clone OX-8, RRID: AB_2814099), PerCP-Cy5.5–anti-CD11b/c (clone OX-42, RRID: AB_2565948), and PE–anti-CD45 (RRID: AB_10683172, 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, RRID: AB_315495, BioLegend). Fc receptor blocking was performed with anti-CD32 (clone D34-485, RRID: AB_393568, 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 analysis

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. Pearson’s correlation coefficients were used to evaluate correlations between the percentage area occupied by GFAP-IR cells and MHWT values during the linear regression analysis.

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. 1A; 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. 1B; 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.

Fig. 1.

IONI induces orofacial mechanical hypersensitivity and increases IFN-γ in TG. (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 of all replicates 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), #: p < 0.05, ##: p < 0.01, ###: p < 0.001 by Mann–Whitney test; vs. sham, **: p < 0.01 (A). *: p < 0.05 by unpaired Student’s t-test (B). Abbreviations: IFN-γ, interferon-γ; IONI, infraorbital nerve injury; MHWT, mechanical head-withdrawal reflex threshold; SEM, standard error of the mean; TG, trigeminal ganglion

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. 2A; 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. 2B; 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. 2C–E). These results indicate that CD8⁺ T cells are the predominant cellular source of IFN-γ in the TG after IONI.

Fig. 2.

CD8⁺ T cells are the major source of IFN-γ in TG after IONI. Cells 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. (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⁻) 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: *: p < 0.05, **: p < 0.01 by Welch’s t-test (B); *: p < 0.05, ***: p < 0.001 by two-way ANOVA with Bonferroni’s post hoc test (D, E). ns, not significant. Abbreviations: ANOVA, analysis of variance; IFN-γ, interferon-γ; IONI, infraorbital nerve injury; SD, standard deviation; TG, trigeminal ganglion

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. 3A).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. 3B; n = 6 per group).

Fig. 3.

CD8⁺ T cells infiltrate TG after IONI and transiently reduce MHWT. (A) Immunohistochemical analysis of FG-labeled TG neurons and CD8⁺ T cells 7 days after IONI or sham treatment. The images represent all replicates. (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: *: p < 0.05 by Mann–Whitney test (B). Abbreviations: FG, Fluoro-Gold; IONI, infraorbital nerve injury; MHWT, mechanical head-withdrawal reflex threshold; SEM, standard error of the mean; TG, trigeminal ganglion

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. 4A). Many IFNGR-IR cells were found encircling FG-labeled TG neurons, confirming that SGCs surrounding neurons innervating the whisker pad skin express IFNGR (Fig. 4B). In IONI rats, the number of IFNGR-IR cells was markedly greater than in sham controls (Fig. 4A). 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. 4C; n = 5 per group).

Fig. 4.

IFN-γ receptors colocalize with SGCs and increase in TG after IONI. (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. (B) Colocalization of FG-labeled neurons and IFNGR-IR cells in TG on day 7 after IONI. Panels A and B contain representative images of all replicates. (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: ***: p < 0.001 by unpaired Student’s t-test (C). Abbreviations: FG, 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

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. Furthermore, regarding SGC activation, it was also confirmed 3 days after IONI, which is the intermediate time point of the 7 days after IONI. Numerous GFAP-IR cells were observed on day 7, whereas they were scarcely observed on day 3 after IONI. In contrast, only a few GFAP-IR cells were detected on days 3 and 7 following sham treatment (Fig. 5A).

Fig. 5.

SGCs surrounding TG neurons are activated after IONI. (A) Immunohistochemistry showing GFAP-IR SGCs in TG on 3 and 7 days after IONI or sham treatment. (B) FG-labeled TG neurons and GFAP-IR cells with merged images in IONI rats. Arrowheads indicate GFAP-IR cells surrounding FG-labeled neurons. Panels A and B contain representative images of all replicates. (C) Quantification of percentage area occupied by GFAP-IR cells in TG on 3 and 7 days (n = 5/group). Data are presented as mean ± SEM. Statistical significance: vs. day 3 after IONI, ###: p < 0.001, vs. sham, ****: p < 0.0001 by two-way ANOVA with Bonferroni’s post hoc test (C). (D) Immunohistochemistry of IFNGR-IR and GFAP-IR cells in TG on day 3 or 7 after IONI or sham treatment. Arrowheads indicate co-immunoreactive cells. Abbreviations: ns, not significant; FG, 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; IFNGR, interferon-γ receptor

Most GFAP-IR cells encircled FG-labeled TG neurons, indicating that activated SGCs surrounded neurons innervating the whisker pad skin (Fig. 5B). Quantitative analysis showed that the percentage area occupied by GFAP-IR cells in the TG was significantly greater in rats on day 7 after IONI compared to rats on day 3 after IONI and rats on day 7 after sham treatment (Fig. 5C; n = 5 per group). Furthermore, the IFNGR-IR in GFAP-IR cells was examined on days 3 and 7 after IONI. The IFNGR-IR in GFAP-IR cells was minimal on day 3 but markedly increased on day 7(Fig. 5D). In addition, we examined the correlation between the percentage area occupied by GFAP-IR cells in TG and MHWT values on day 7 after IONI, revealing a strong negative correlation (Supplementary Fig. 2).

These findings indicate that SGCs surrounding TG neurons are robustly activated following IONI, suggesting that SGC activation in TG might be associated with mechanical hypersensitivity 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. 6A; 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. 6B). Quantitative analysis confirmed that the percentage area occupied by GFAP-IR cells was significantly larger in the IFN-γ–treated group (Fig. 6C; 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. 6D; 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. 6E). Quantitative analysis confirmed that GFAP-IR area in the TG was significantly reduced by IFNGR ANTAG administration compared with vehicle (Fig. 6F; 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.

Fig. 6.

IFN-γ induces hypersensitivity and SGC activation, while IFNGR antagonism suppresses these effects. (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. (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. (F) Quantification of GFAP-IR area in TG from panel E (n = 5/group). Panels B and E contain representative images of all replicates. Data are presented as mean ± SEM. Statistical significance: *: p < 0.05, **: p < 0.01 by unpaired Student’s t-test (C, F). Abbreviations: 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

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. 7A; n = 5–6), although TNF-α concentration was scarcely detectable by IFN-γ stimulation. Immunohistochemistry confirmed that all cultured DAPI- positive cells were also Kir4.1-positive, validating that the cultures consisted entirely of SGCs (Fig. 7B). Furthermore, we confirmed that these cultured cells did not exhibit NeuN, α-SMA, and Iba1, indicating that they did not contain neuron, fibroblasts, or macrophages (Supplementary Fig. 3). In addition, immunohistochemical analysis confirmed the presence of IL-1β-IR cells in the SGCs (Supplementary Fig. 4).

Fig. 7.

IL-1β released from SGCs activates TG neurons after IONI. (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. (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. (E) FG-labeled IL-1R1-IR cells in TG of IONI rats. Arrowheads indicate co-positive cells. (F) Representative western blot of all replicates displaying IL-1R1 protein in TG on day 7 after IONI or sham treatment, and quantification normalized to β-actin (n = 7/group). Panels B and D contain representative images of all replicates. Data are presented as mean ± SEM. Statistical significance: **: p < 0.01, ***: p < 0.001 by unpaired Student’s t-test (A, F), vs. IFN-γ; **: p < 0.01 by Mann–Whitney test (C). Abbreviations: ANTAG, 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

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. 7C; 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. 7D). Some IL-1R1-IR cells were also FG-labeled, indicating that TG neurons innervating the whisker pad skin express IL-1R1 (Fig. 7E). 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. 7F; 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 and TNF-α are reportedly released from SGCs and contribute to orofacial neuropathic pain [48]. In this study, although IL-1β was released from cultured SGCs in response to IFN-γ stimulation, TNF-α was not. IL-1β is a potent hyperalgesic cytokine that increases the frequency of action potentials in primary sensory neurons via IL-1 receptor signaling [49]. Elevated IL-1β expression has also been reported in injured peripheral nerves, DRG, and spinal cord in animal models of neuropathic pain [50]. Increased neuronal spike frequency mediated by IL-1 receptor signaling has been directly linked to neuropathic pain induction [51, 52]. In our study, IL-1β was released from cultured SGCs in response to IFN-γ stimulation. Furthermore, IL-1R1s 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 [53]. Although IFN-γ has been implicated in neuropathic pain [16, 54, 55], 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 [56, 57]. Interestingly, IFN-γ–producing CD8⁺ T cells have been reported to decrease significantly following treatment with these agents [58]. 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 [59]. 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, 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. Third, cyclical fluctuations in sexual hormone, especially estrogen, levels are known to effect pain perception significantly [60, 61]. In addition, estrogen reportedly alleviates neuropathic pain by suppressing glial cell activation and influence T cell development [62, 63]. Estrogen receptors are widely distributed in the nervous system, regulating nociceptive processes, while estrogen receptor signaling in CD8⁺ T cells is also involved in its activation [64–66]. Therefore, in this study, male rats were used to rule out the effects of estrogen on pain development, glia, and T cell activation that vary cyclically. In the future, we will examine the impact of gender differences on IFN-γ–SGC–IL-1β signaling after trigeminal nerve injury. 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.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

ANOVA

Analysis of variance

ANTAG

Antagonist

ATP

Adenosine triphosphate

α-SMA

α-smooth muscle actin

BSA

Bovine serum albumin

CCL2

C-C motif chemokine ligand 2

CD8⁺ T

Cells CD8-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 type 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

Author 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.

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.).

Data availability

The data supporting the results in this study will be made available by the corresponding author on reasonable request.

Declarations

Ethical 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).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.