Involvement of interferon gamma signaling in spinal trigeminal caudal subnucleus astrocyte in orofacial neuropathic pain in rats with infraorbital nerve injury

Abstract

Background: Trigeminal nerve injury causes orofacial pain that can interfere with activities of daily life. However, the underlying mechanism remains unknown, and the appropriate treatment has not been established yet. This study aimed to examine the involvement of interferon gamma (IFN-γ) signaling in the spinal trigeminal caudal subnucleus (Vc) in orofacial neuropathic pain. Methods: Infraorbital nerve (ION) injury (IONI) was performed in rats by partial ION ligation. The head-withdrawal reflex threshold (HWT) to mechanical stimulation of the whisker pad skin was measured in IONI or sham rats, as well as following a continuous intracisterna magna administration of IFN-γ and a mixture of IFN-γ and fluorocitrate (inhibitor of astrocytes activation) in naïve rats, or an IFN-γ antagonist in IONI rats. The IFN-γ receptor immunohistochemistry and IFN-γ Western blotting were analyzed in the Vc after IONI or sham treatment. The glial fibrillary acid protein (GFAP) immunohistochemistry and Western blotting were also analyzed after administration of IFN-γ and the mixture of IFN-γ and fluorocitrate. Moreover, the change in single neuronal activity in the Vc was examined in the IONI, sham, and IONI group administered IFN-γ antagonist. Results: The HWT decreased after IONI. The IFN-γ and IFN-γ receptor were upregulated after IONI, and the IFN-γ receptor was expressed in Vc astrocytes. IFN-γ administration decreased the HWT, whereas the mixture of IFN-γ and fluorocitrate recovered the decrement of HWT. IFN-γ administration upregulated GFAP expression, while the mixture of IFN-γ and fluorocitrate recovered the upregulation of GFAP expression. IONI significantly enhanced the neuronal activity of the mechanical-evoked responses, and administration of an IFN-γ antagonist significantly inhibited these enhancements. Conclusions: IFN-γ signaling through the receptor in astrocytes is a key mechanism underlying orofacial neuropathic pain associated with trigeminal nerve injury. These findings will aid in the development of therapeutics for orofacial neuropathic pain.

Keywords

Astrocyte involvement of interferon gamma infraorbital nerve injury orofacial neuropathic pain

Introduction

Orofacial neuropathic pain occurs due to peripheral nerve injuries,¹ caused not only by orofacial trauma but also by routine dental treatments, such as tooth extraction, pulpectomy, and dental implant placement. This pain is intractable and debilitating, and the therapeutic approach is limited.² It particularly affects the quality of life because of disturbances in food intake, tooth brushing, and speaking. Therefore, elucidation of the mechanism of orofacial neuropathic pain and development of a suitable treatment are urgently needed.

Recently, abundant evidence has indicated that non-neuronal cells in the central nervous system (CNS), such as T cells and glial cells, play essential roles in neuropathic or inflammatory pain.^3,4 Microglia and astrocytes are abundantly activated in the spinal cord or spinal trigeminal caudal subnucleus and upper cervical spinal cord (Vc/C1−2) in neuropathic pain models.^5–8 Activated astrocytes are thought to play an important role in the maintenance of neuropathic pain after peripheral nerve injury, and microglial activation may induce astrocyte activation.^9,10 T cells in the spinal cord also activate astrocytes, which contribute to neuropathic pain maintenance.^11–13 However, details of the non-neuronal involvement in neuropathic pain remain controversial.

Interferon-gamma (IFN-γ), a cytokine released by T cells and natural killer cells, play important role in the immune, endocrine, and nervous system.^14–16 Spinal cord IFN-γ levels were persistently increased in patients with acute and chronic pain^17–19 and in animal models of neuropathic pain.^20,21 In addition, spinal IFN-γ blockade depressed the mechanical allodynia and astrocyte activation.¹³ This evidence indicates that IFN-γ in the spinal cord contributes to the pathogenesis of neuropathic pain. Furthermore, IFN-γ and its receptor were present on murine astrocytes in a cerebral cortex culture.^22,23 The expression of IFN-γ and its receptor could also be detected in astrocytes but did not colocalize with microglia and oligodendrocyte in the spinal cord ipsilateral to the peripheral nerve injury in animal models of neuropathic pain or amyotrophic lateral sclerosis.^24–26 In contrast, it was reported that IFN-γ receptors exist in microglia, but not in astrocytes and neurons and that its activation transforms resting spinal microglia into the activated state, which induces tactile allodynia.²⁷ Another study reported that the IFN-γ receptor was not expressed in microglia and astrocytes, although it was expressed in axon terminals of the spinal nucleus.²⁸

This study aimed to clarify whether IFN-γ in the Vc is involved in the mechanism of orofacial neuropathic pain by analyzing the effect of IFN-γ antagonism on orofacial pain hypersensitivity and the expression of IFN-γ and its receptor in the Vc of a rat model of neuropathic pain in the orofacial region.^29,30

Materials and methods

Animals

Male Sprague–Dawley rats (n = 100, Japan SLC, Shizuoka, Japan) weighing 200–280 g were used in all experiments. This study was approved by the Animal Experimentation Committee of Nihon University (approval no. AP19DEN018). Animal use and procedures were performed according to the guidelines of the International Association for the Study of Pain.³¹ Animals were kept in a temperature-controlled room (23°C) with a 12-h light-dark cycle. All efforts were made to minimize animal suffering and reduce the number of animals used. This manuscript adheres to the applicable ARRIVE guidelines.

Infraorbital nerve injury

Infraorbital nerve (ION) injury (IONI) was established 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), and a small incision was made intraorally in the left buccal mucosa. The ION was freed from the surrounding tissue, and the half of ION was tightly ligated using 6-0 silk. For the sham-treated control rats, a small incision was made in the left buccal mucosa, and the ION was freed from the surrounding tissue; however, ION ligation was not performed. The experimenter was blinded to the treatment of the animals.

Drugs

For drug dissolution, IFN-γ (Biotechne, Minneapolis, MN, USA) was dissolved with phosphate-buffered saline (PBS), and the IFN-γ antagonist (Bachem, Torrance, CA, USA) was dissolved with 20% acetic acid. Fluorocitrate (FC; Merck Millipore, Darmstadt, Germany), a metabolic inhibitor of astrocyte activation, was dissolved with PBS. These vehicles were used as the control.

Intracisterna magna administration of IFN-γ, IFN-γ antagonist, and FC

IFN-γ, FC, and FC with IFN-γ were administered to naïve rats, and IFN-γ antagonist was administered to the IONI group intracisternally as previously described.³² Briefly, rats were anesthetized, as described above, and placed in a stereotaxic frame. A small hole was drilled into the most caudal part of the occipital bone to insert a polyethylene tube (0.8 mm diameter, Natsume, Tokyo, Japan). The tip of the tube was placed at a depth of 4 mm in the cisterna magna, and the tube was fixed to the skull. The other tip of the tube was connected to an osmotic mini pump (Figure 1). The total volume of the pump was 100 μL; moreover, it was used to inject drugs at a rate of 1 μL/h for 3 days (Alzet model 1003D, Durect, Cupertino, CA, USA). The tube connected to the pump was implanted subcutaneously on the back. The pump and tube were filled with IFN-γ (10 ng/100 μL), FC (100 fmol/100 μL), IFN-γ mixed with FC, or vehicle (PBS); or IFN-γ antagonist (0.5 mg/500 μL) or vehicle (acetic acid).

Figure 1.

Intracisternal catheter fixed to the occipital bone. The catheter is connected to an osmotic mini pump and drug was administered into the cisterna magna.

Nocifensive behavior test

To evaluate mechanical sensitivity in whisker pad skin of IONI and sham rats, the behavioral test was performed as described previously.³² In brief, the animals were trained daily to be able to stick out their perioral region, including the maxillary whisker pad skin, through a small window in the plastic cage for a few minutes and stay still for long periods in the plastic cage for 7 days. Mechanical stimuli were applied to the center of the left whisker pad skin (territory of maxillary divisions of the trigeminal nerve) and increased in stimulus intensity according to von-Frey filaments (Touch-Test Sensory Evaluator, North Coast Medical, CA, USA) number until the rat escaped from the stimulus. When the rats exhibited a head-withdrawal response to the stimulation at least three out of five times, the stimulus intensity was defined as a head-withdrawal reflex threshold (HWT). The HWT was measured before and 1−7, 10, 14, and 21 days after the IONI or sham treatment.

The intracisternal catheter was implanted in naïve rats, and IFN-γ, IFN-γ mixed with FC, and FC were intracisternally administered for 3 days. These HWTs were measured before and at 1, 2, and 3 days after the administration (Figure 2a). Furthermore, IFN-γ antagonist or acetic acid began being administered intracisternally while the intracisternal catheter was implanted following IONI treatment. These drugs were administered for 3 days and the HWTs were measured before and at 1, 2, and 3 days after IONI treatment and drug administration (Figure 2b).

Figure 2.

A schematic illustration of the time course of the present experiment. (a) Time course of implantation of intracisternal catheter, drugs administration and measurement of mechanical sensitivity in naïve rat. (b) Time course of IONI treatment, implantation of intracisternal catheter, drugs administration and measurement of mechanical sensitivity in IONI rat.

Immunohistochemistry

The naïve, sham, and IONI groups were deeply anesthetized as described above and perfused with saline followed by 4% paraformaldehyde (PFA) for immunohistochemistry on day 3 after the treatment. The medulla, including the trigeminal subnucleus caudalis, was removed and postfixed in a 4% PFA for 24 h at 4°C. Next, the tissues were transferred to 20% sucrose (w/v) in PBS for 24 h to prevent cryolesion and sliced into 30 µm-thick sections using a freezing microtome (Leica, Wetzlar, Germany). The slices were blocked with 0.01 M PBS containing 3% normal goat serum diluted in 0.3% Triton-X-100 for 1 h at room temperature after being rinsed with 0.01 M PBS. Subsequently, they were incubated with Armenian hamster monoclonal anti-IFN-γ receptor (1:50, Santa Cruz Biotech, Dallas, TX), rabbit polyclonal anti-glial fibrillary acidic protein (GFAP; astrocyte marker) antibody (1:1,000, Dako, Glostrup, Denmark), rabbit polyclonal anti-Iba1 antibody (microglia marker, 1:1,000, FUJIFILM Wako, Osaka, Japan), or mouse monoclonal anti-NeuN antibody (neuron marker, 1:1,000, Merck Millipore) for 3 days at 4°C. After washing with 0.01 M PBS, the slices were incubated with either Alexa Fluor 568-conjugated goat anti-rabbit IgG (1:200; Abcam, Cambridge, MA, USA) or Alexa Fluor 488-conjugated goat anti-Armenian hamster IgG (1:200; Abcam) for 2 h at room temperature. The sections were rinsed with 0.01 M PBS and mounted on gelatin-coated slides with mounting medium (Thermo Fisher Scientific, Waltham, MA, USA). The double immunofluorescent-stained specimens were analyzed using confocal microscope (Keyence, Osaka, Japan).

Measurements for the immunoreactive product density of IFN-γ receptor or GFAP were performed in a 100 × 100 μm² area of the fluorescence images, which represented the superficial lamina of the caudal Vc (0.7 mm caudal to the obex) receiving afferent inputs from the 2^nd branch of the trigeminal nerve using a computer-assisted imaging analysis system (Image J, 8-bit). We also performed immunohistochemical staining without primary antibody for the IFN-γ receptor in the naïve, sham, and IONI groups, and could not confirm any immune products (data not shown).

Western blotting

On day 3 after the IONI and sham treatments, rats were anesthetized with 2% isoflurane inhalation and perfused with saline (500 mL). In each group, the medulla in the left Vc was removed and homogenized in ice-cold lysis buffer (400 μL, 150 mM NaCl, 10 mM Tris-HCl, 0.5% NP40, pH 7.4 and 1% Triton X-100) at 4°C. A protein assay kit (Bio-Rad, Hercules, CA) was used to determine the protein concentration of the supernatant after centrifuging at 15,000 r/min for 10 min at 4°C. Next, Laemmli sample buffer solution (Bio-Rad) was used to heat-denature (95°C) the protein sample. Proteins (30 μg) were separated using 10% sodium dodecyl sulfate–polyacrylamidegel electrophoresis (4%-20%, Bio-Rad) and transferred onto polyvinylidene difluoride membranes using a Trans-Blot Turbo rapid transfer system (Bio-Rad) for 7 min (2.5, 25 V). The membrane was incubated in 3% bovine serum albumin (BSA, Bovogen, Australia), and then incubated with antibodies using anti-IFN-γ antibody (1:1,000, rabbit polyclonal anti-IFN-γ antibody, Proteintech, IL, USA), anti-GFAP antibody (1:1000), or mouse monoclonal anti-β-actin antibody (1:200, Santa Cruz) diluted in Tris-buffered saline containing 0.1% Tween 20 (TBST) and 3% BSA overnight at 4°C. Afterwards, the sections were incubated with horseradish peroxidase-conjugated anti-rabbit or mouse antibody (1:3,000, Cytiva, Marlborough, MA, USA) for 2 h. The bound antibody was visualized using a Western Lightning ELC Pro (PerkinElmer, Waltham, MA, USA) and image analyzer, ChemiDocXRS system (Bio-Rad). The intensity of the band was quantified using ImageJ software and normalized by β-actin.

Neuronal recording

On day 3 after the IONI, the IONI with IFN-γ antagonist administration was performed for 3 days, and sham treatment, a single-neuron recording experiment, was conducted as described previously.³³ Briefly, continuous intracisternal administration of IFN-γ antagonist was performed for 3 days before neural recording on day 3 after IONI. Rats were placed in a stereotaxic instrument to expose the spinal trigeminal nucleus under deep anesthesia with butorphanol, midazolam, and medetomidine, as mentioned above. The trachea and left jugular vein were cannulated to supply 2% isoflurane mixed with oxygen and allow intravenous administration of rocuronium bromide (0.6 mg/kg, MSD, Tokyo, Japan), respectively. Rats were artificially ventilated after being immobilized. Exhaled CO₂, heart rates, and body temperatures were monitored during neuronal recordings and maintained at 3−4%, 250−300 bpm, and 37°C, respectively. The single neuronal activity was recorded extracellularly via neurons of the Vc with receptive fields in the whisker pad skin innervated by the second branch of the trigeminal nerve using tungsten microelectrodes (impedance = 13 MΩ, 1000 Hz, FHC Instruments, Bowdoin, ME, USA) after excision of the dura and soft membrane. The neurons were functionally identified as wide dynamic range (WDR) neurons that responded with small responses to non-noxious and graded and stronger responses to noxious mechanical stimuli, or nociceptive-specific (NS) neurons that responded to noxious and not non-noxious stimuli of their receptive fields.^33,34 The spike frequencies were analyzed using Spike two software (CED1401; CED, Cambridge, UK). After the identification of neurons, background activity was recorded for 30 s, and mechanical stimuli were applied to the left whisker pad skin. In mechanical stimulation, non-noxious brushing, noxious pinch stimulation, and graded pressure stimulation via von Frey filaments (1, 6, 15, 26, and 60 g) were applied for 5 s at 10 s intervals.

Statistical analysis

Data were presented as the median ± interquartile range. HWT data were shown as median and interquartile range (25% and 75%), and the upper and lower whiskers indicated the maximum and minimum values, respectively. Data normality was assessed using the Shapiro–Wilk test. Using GraphPad Prism nine software (GraphPad Software, Boston, MA), the one-way repeated measurement analysis of variance (ANOVA) followed by Tukey or Sidak multiple comparison test or unpaired Student’s t test was used in immunohistochemistry, Western blotting, and neuronal recording test. The Mann–Whitney and the Kruskal-Wallis tests were used as nonparametric tests for the analysis of changes in HWT because they were not considered to be normally distributed and satisfy homoscedasticity. Statistical significance was set at p < 0.05. These data are shown as standard error of the mean.

Results

Changes in orofacial nocifensive behavior and quantification of IFN-γ in the Vc after IONI

The mechanical HWT of the left maxillary whisker pad skin significantly decreased after IONI compared with that before IONI and that of the sham group at 1 day after IONI. The decrease lasted for at least 14 days after IONI. The decrease recovered slightly at 21 days after IONI, but it was still significantly different from before IONI and that of the sham group (Figure 3) (n = 13 in each group). On day 3 after IONI or sham treatment, the relative protein level of IFN-γ in the Vc ipsilateral to IONI was significantly greater than that in the sham group (Figure 4) (n = 6 in each group).

Figure 3.

Mechanical sensitivity of the ipsilateral whisker pad skin both before and after IONI or sham treatment (IONI group, n = 13; sham group, n = 13). ****: p < 0.0001, IONI vs. Sham group; ###: p < 0.001, vs. Head withdrawal threshold on day pre (Mann–Whitney test).

Figure 4.

Relative IFN-γ protein level in sham and IONI group on day 3 in the spinal trigeminal caudal subnucleus (Vc) Typical example of western blotting protein (a) and relative protein amount of IFN-γ (b) in sham and IONI group. The amount of IFN-γ protein in the IONI group was significantly larger than that in the sham group (n = 6 in each group). **: p < 0.01. (Student’s t test).

Expression of IFN-γ receptor in the Vc after IONI

Based on the changes in mechanical sensitivity and IFN-γ amount in the Vc after IONI, we hypothesized that the IFN-γ receptor is expressed in the Vc. To evaluate its distribution, we performed immunohistochemical analysis on day 3 following IONI or sham treatment. The IFN-γ receptor immunoreactive cells had a large soma with thick processes in the IONI group, but a small soma with thin processes in the sham group (Figure 5a). The area occupied by the IFN-γ immunoreactive products in the IONI group was significantly larger than that in the sham group (Figure 5b) (n = 5 in each group). Furthermore, IFN-γ receptor immunoreactive cells expressed GFAP, but not Iba1 and NeuN, indicating that the IFN-γ receptor was expressed in astrocytes, but not in microglia and neurons (Figure 5c).

Figure 5.

IFN-γ receptor-immunoreactive cells in Vc of IONI and sham group on day 3. (a) Expression of IFN-γ receptor in Vc of IONI (a) and sham group (b). c, (d) High magnification of boxes in a and b, respectively (scale bar in b = 100 μm, scale bar in d = 20 μm). (b) Area occupied by IFN-γ receptor immuno-products in the Vc of IONI and sham group (n = 5 in each group). **: p < 0.01 (Student’s t test). (c) Expression of IFN-γ receptor (a) and GFAP- immunoreactive cells (b), merged image of a and b (c), and IFN-γ receptor (d) and Iba1-immunoreactive cells (e), merged image of d and e (f), and IFN-γ receptor (g) and NeuN- immunoreactive cells (h), merged image of g and h (i) in IONI group. (scale bar in i = 20 μm). Arrows indicate the IFN-γ receptor-immunoreactive cells and GFAP-immunoreactive cells and their co-immunoreactive cells.

Effect of IFN-γ and inhibition of astrocyte activation in the Vc on orofacial nocifensive behavior and GFAP expression

Next, we examined the effect of continuous IFN-γ intracisternal administration for 3 days on the mechanical sensitivity of the left whisker pad skin in naïve. IFN-γ administration significantly decreased the HWT compared with vehicle administration on days 2 and 3. However, the significant decrement of HWT on days 2 and 3 by IFN-γ administration was completely suppressed by continuous FC co-administration (Figure 6a). The administration of FC in the Vc did not change the HWT (n = 5 in each group) (Figure 6a).

Figure 6.

Effect of intracisternal administration of IFN-γ and inhibitor administration of astrocyte activation on orofacial nocifensive behavior and expression of glial fibrillary acidic protein (GFAP) in Vc in naïve rats (a) Mechanical sensitivity of the ipsilateral whisker pad skin following intracisterna magna administration of IFN-γ, fluorocitrate (FC, an inhibitor of astrocyte activation), IFN-γ mixed with FC, and vehicles (PBS) in naïve rats (n = 5 in each group). **: vs. vehicle, p < 0.01; ***: vs. vehicle, p < 0.001; ###: vs. day 0, p < 0.001 (Kruskal-Wallis with Dunn’s multiple comparisons tests). (b) Expression of GFAP in Vc after administration of IFN-γ, IFN-γ mixed with FC, FC, and vehicle for 3 days in naïve rats. GFAP-immunoreactive cells are observed following vehicle (a), IFN-γ (b), FC (c) and IFN-γ with FC (d) administration (scale bar = 100 μm, Scale bar in insert = 10 μm). (c) Typical example of GFAP protein bands in IFN-γ, IFN-γ mixed with FC, FC, or vehicle-administrated rats. (d) These relative protein amount of GFAP protein. ****: p < 0.0001 (n = 5 in each group; one-way ANOVA with Tukey’s multiple comparison test).

We also examined the effect of continuous IFN-γ intracisternal administration for 3 days on GFAP expression in the Vc. GFAP immunoreactive cells had a large soma with thick processes on day 3 after IFN-γ administration, and the morphological changes were depressed by concurrent administration of FC (Figure 6b). FC administration did not induce the morphological change in GFAP immunoreactive cells. The GFAP protein level was significantly higher on day 3 after continuous IFN-γ administration compared with that after vehicle administration. The significant increases were suppressed by continuous FC co-administration and the FC single administration did not change GFAP protein level in Vc (n = 5 in each group) (Figure 6C and D).

Effect of IFN-γ antagonist on orofacial nocifensive behavior and Vc neuronal activity after IONI

To investigate the involvement of IFN-γ in the Vc in orofacial mechanical hypersensitivity associated with IONI, we examined the change in the HWT to mechanical stimulation and neuronal activities in the Vc after intracisternal administration of IFN-γ antagonist in the IONI group.

The HWT significantly decreased on days 1 to 3 after IONI, and the decrease in HWT was significantly suppressed by continuous intracisternal administration of IFN-γ antagonist (n = 5 in each group) (Figure 7).

Figure 7.

The effect of IFN-γ antagonism in Vc on mechanical hypersensitivity in whisker pad skin after IONI. The HWT on days 1, 2, and 3 after the beginning of IFN-γ antagonist administration in IONI group (n = 7 in each). ***: p < 0.001, IFN-γ antagonist vs. vehicle; ###: p < 0.001 vs. pre-treatment value (day 0) (Mann–Whitney test).

The effect of intracisternal administration of IFN-γ antagonist on neuronal excitability in the Vc was examined on day 3 after IONI. Eight WDR and 4 NS neurons in the IONI group (n = 6), eight WDR and 3 NS neurons in the sham group (n = 6), and eight WDR and 3 NS neurons in the IFN-γ antagonist-administrated IONI group (n = 6) were assessed. The recording sites of the three groups, marked by applying direct currents, were plotted in the horizontal plane of the brainstem image (Figure 8a). These neurons analyzed in the present study were located in the superficial laminae of the Vc receiving inputs from the 2^nd branch of the trigeminal nerve. Typical mechanical evoked responses of these neurons are demonstrated in Figure 8b. The neuronal firing frequency in the three groups increased corresponding to an increase in mechanical stimulus intensities (brush, mechanical pressure (1−60 g), and pinch). Sixty-gram mechanical stimulation, pinch, and brush-evoked neuronal firings were significantly increased in the IONI group than in the sham and IONI groups with IFN-γ antagonism (Figure 8c and d), suggesting that IONI-induced Vc neuronal hyperexcitability was dependent on IFN-γ signaling in Vc.

Figure 8.

Effect of IFN-γ antagonist on mechanical-evoked firing of Vc neurons on day 3 after IONI. (a) Recording sites of sham, IONI, IFN-γ antagonist-administered IONI group. ○ indicates sham group, ● indicates IONI group and indicates IFN-γ antagonist-administered IONI group. (b) Typical response of Vc neurons for von Frey filaments (1, 6, 15, 26 and 60 g), brush, and pinch stimulation on whisker pad skin in sham, IONI, and IFN-γ antagonist-administered IONI group. (c) Response to 1, 6, 15, 26 and 60 g pressure stimulation in each group. **: p < 0.01, sham vs. IONI; ††: p < 0.01, IONI vs. IFN-γ antagonist-administered IONI, (Kruskal-Wallis with Dunn’s multiple comparisons tests) (d) Neuronal response to brush, and pinch stimulation on whisker pad skin in sham, IONI, and IFN-γ antagonist-administered IONI group. *: p < 0.05, **: p < 0.01 (one-way ANOVA followed by Sidak multiple comparison test). White circle and bar indicate sham group. Black circle and bar indicate IONI group. Grey triangle and bar indicate IFN-γ antagonist-administered IONI group. BG: background neuronal firing ( 8 WDR and 3 NS neurons from 6 sham rats; 8 WDR and 4 NS neurons from 6 IONI rats; 8 WDR and 3 NS neurons from 6 IFN-γ antagonist-administered IONI).

Discussion

Mounting evidence has indicated that astrocyte activation plays a critical role in maintaining neuropathic pain; however, little is known about the trigger for astrocyte activation. In the present study, administration of IFN-γ to the Vc-activated astrocytes, which induced orofacial nocifensive behavior, and simultaneous administration of an astrocyte activation inhibitor suppressed the enhancement of nocifensive behavior and astrocyte activation following IONI, suggesting that IFN-γ is a critical component of trigger for astrocyte activation in the Vc following trigeminal nerve injury and the IFN-γ-triggered astrocyte activation is implicated in the manifestation of trigeminal neuropathic pain.

In this study, we observed a significant increase in IFN-γ protein on day 3 following IONI and a significant decrease in HWT on day 3 following IFN-γ administration to the Vc. Since it has been reported that microglia are involved in the early phase of neuropathic pain and astrocytes in the late phase,³⁴ we hypothesized that IFN-γ receptors might be amplified in microglia in Vc following IONI and the IFN-γ signaling would activate microglia. However, surprisingly, we observed the amplification of IFN-γ receptors on astrocytes but not on microglia and neuron on day 3 after IONI. We showed for the first time that the IFN-γ receptor is expressed in astrocytes in the Vc, although several studies reported that they were present in astrocytes in the cerebrum.²² Furthermore, continuous administration of IFN-γ to Vc induced astrocyte activation. Astrocyte activation is known to be involved in the transition from acute to chronic pain and the maintenance of chronic pain,³⁴ as it begins on day 7 and is maintained until day 150 after neuropathy.^8,35 Considering these results and reports, it may take as long as 3 days for a significant increase of IFN-γ in the Vc after peripheral nerve injury, followed by 3 days for astrocytes activation in Vc by the enhancement of IFN-γ signaling.

Activated astrocytes have been reported to release interleukin (IL)-1β, IL-18, glutamate, and chemokines, such as C-X-C motif chemokine ligand one and C-C motif chemokine ligand 2, all of which excite spinal dorsal horn nociceptive neurons, and these molecules are involved in the maintenance of neuropathic pain.³⁴ Although the IFN-γ receptor was expressed on astrocytes on day 3 after IONI, the same pattern of expression was observed on day 21 (Supplemental Figure 1). The present study also showed a decrease in HWT until day 21 after IONI. Therefore, it is also possible that IFN-γ-induced astrocyte activation may be involved in the maintenance of neuropathic pain. However, this study did not examine changes in astrocyte activation over time for a longer period, which is needed in future studies.

We could not detect IFN-γ expression in Vc in this study (Supplemental Figure 2), although a previous study reported that IFN-γ mRNA was expressed in microglia.³⁶ Since this study showed that IFN-γ receptors are present in astrocytes, it is suggested that IFN-γ is one of the regulative factors between microglia and astrocytes. Many studies indicated that the interaction between spinal microglia and astrocytes was accompanied by the upregulation of various molecules in glial cells. However, it is still unclear whether the activation of astrocytes depends on activated microglia following peripheral nerve injury. Astrocytes were converted to reactive astrocytes by activated microglia with the secretion of IL-1α, tumor necrosis factor (TNF)-α, and C1q in vitro and in vivo.^37,38 Moreover, peripheral nerve injury has been reported to upregulate IL-18 and IL-18 receptor expression in the spinal microglia and astrocytes, respectively, which is crucial for tactile allodynia.³⁹ This implies that the interaction of microglia and astrocytes is important for the neuropathic pain mechanism. Contrarily, other studies have suggested that the inhibition of microglial activation did not suppress the maintenance of chronic pain correlated to astrocyte activation. For example, minocycline, a selective inhibitor of microglial activation, attenuated the induction of ATP-producing allodynia but not the maintenance of allodynia.⁴⁰ Suppression of microglial activation attenuated the development of neuropathic pain but failed to inhibit existing or established neuropathic pain.^41,42 As mentioned above, IFN-γ is reported to be produced by microglia,³⁶ but most IFN-γ are known to be produced by T cells and natural T cells.¹⁴ These reports and the present results indicate that astrocytes may be activated not only by microglia but also by T cells through IFN-γ signaling. Further experiments are desired to investigate the source of IFN-γ that activates Vc astrocytes and to extract IFN-γ from cell cultures of T cells, neurons and microglia in Vc of IONI rats.

IFN-γ stimulates the Janus-activated kinase/signal transducer and activator of the transcription (JAK/STAT) pathway,^14,43 which is involved in the mechanism of neuropathic pain.^44–46 One of the molecules related to astrocyte activation is STAT3, which is thought to be an essential molecule in the neuropathic pain mechanism.^47,48 Activated STAT3, which accumulates in spinal microglia after spinal nerve ligation, is also an essential signal transducer and activator of astrocytes and contributes to neuropathic pain. STAT3 activation drives the proliferation of spinal astrocytes following peripheral nerve injury, and the inhibition of STAT3 activation suppresses the proliferation of astrocytes and recovers from neuropathic pain.⁴⁸ Therefore, IFN-γ may activate astrocytes through the JAK/STAT pathway, particularly through STAT3, which contributes to neuropathic pain.

Recently, macrophages have been reported to contribute to the pathogenesis of neuropathic pain following peripheral nerve injury.^49–51 The depletion of macrophages in the dorsal root ganglion can prevent the development of mechanical hypersensitivity caused by peripheral nerve injury; however, the depletion of macrophages at the peripheral nerve injury site did not depress the mechanical hypersensitivity.⁵² In addition, macrophage activation has been found to occur mainly with IFN-γ.⁵³ It is known that astroglial morphology and function in the CNS are similar to that of satellite cells in the sensory ganglion.⁵⁴ We have shown that IFN-γ signaling in Vc astrocytes may be involved in the mechanism of orofacial neuropathic pain in the present study, speculating that IFN-γ signaling in the satellite cells in trigeminal ganglia is also involved in the orofacial neuropathic pain mechanism. However, further studies are necessary.

IFN-γ signaling is not implicated in only inflammatory and neuropathic pain, but it has also been thought to exert an anti-inflammatory and neuroprotective effect in the CNS.^55–57 These different effects of IFN-γ depend on the administered dosage, disease phase, and its targets.⁵⁸ Although we determined the appropriate dosages of IFN-γ and IFN-γ antagonists administered in this study by a preliminary experiment based on previous literature,^26,59 further research is necessary on the effect of IFN-γ in different duration after peripheral nerve injury and different disease conditions.

Although male rats were used in this study, several reports have implicated sex differences in neuropathic pain.^60,61 It is well known that microglia are closely involved in neuropathic pain in male mice, whereas the neuropathic pain in female mice does not require microglia, but adaptive immune cells such as T lymphocytes to produce the same pain.^62–64 On the other hand, in studies of pain in female animals, it is necessary to keep the sex cycle of all female animals constant because estrogen is often involved.^65,66 In addition, there have been several studies that have examined the relationship between IFN-γ and pain, and it is necessary to compare the results of these studies with those of this study, but all of these studies used male rats.^19,23,26 For these reasons, male rats were use in this study. However, future research should examine in detail the relationship between IFN-γ signaling in Vc and gender differences in orofacial neuropathic pain.

In conclusion, orofacial pain hypersensitivity is induced by IFN-γ administration to the Vc, whereas the IFN-γ antagonist inhibits the trigeminal nerve injury-induced orofacial pain hypersensitivity and Vc neuronal hyperexcitability. IFN-γ signaling in Vc is a target for the development of therapeutics for patients with orofacial neuropathic pain.

Supplemental Material

Supplemental Material - Involvement of interferon gamma signaling in spinal trigeminal caudal subnucleus astrocyte in orofacial neuropathic pain in rats with infraorbital nerve injury

Supplemental Material for Involvement of interferon gamma signaling in spinal trigeminal caudal subnucleus astrocyte in orofacial neuropathic pain in rats with infraorbital nerve injury by Sayaka Asano, Akiko Okada-Ogawa, Momoyo Kobayashi, Mamiko Yonemoto, Yasushi Hojo, Ikuko Shibuta, Noboru Noma, Koichi Iwata, Suzuro Hitomi and Masamichi Shinoda in Molecular Pain

Footnotes

Acknowledgements

We thank Editage () for English language editing.

Author contributions

S.A. contributed to the experiments, data acquisition and data analysis, and reviewed the manuscript. A.O. contributed to the study design, data acquisition and analysis, and manuscript preparation. M.K. contributed to the experiments and data analysis, and revised the manuscript. M.Y., Y.H., I.S., N.N., and S.H. contributed to the experiments and revised the manuscript. K.I. contributed to the study design, data analysis and interpretation, and reviewed the manuscript. M.S. contributed to the study design and detailed statistical analyses, and edited the manuscript.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported in part by research grants by the Sato Funds and the Dental Research Center of Nihon University School of Dentistry (DRC(B)-2023-11), and JSPS KAKENHI (Grant-in-Aid for Scientific Research [C] 20K10192 to A.O. and 21K10123 to I.S.). Nihon University Research Grant for M.S. (2021, 2022).

ORCID iD

Akiko Okada-Ogawa

Supplemental Material

Supplemental material for this article is available online.

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