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. 2023 Jan-Dec:19:17448069231222403.
doi: 10.1177/17448069231222403.

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

Sayaka Asano  1   2 , Akiko Okada-Ogawa  3   4 , Momoyo Kobayashi  3 , Mamiko Yonemoto  2 , Yasushi Hojo  2 , Ikuko Shibuta  2 , Noboru Noma  3   4 , Koichi Iwata  2 , Suzuro Hitomi  2 , Masamichi Shinoda  2
Affiliations

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

Sayaka Asano et al. Mol Pain. 2023 Jan-Dec.

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; infraorbital nerve injury; involvement of interferon gamma; orofacial neuropathic pain.

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Figures

Figure 1.
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.
Figure 2.
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.
Figure 3.
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.
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).
Figure 5.
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.
Figure 6.
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).
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).
Figure 8.
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 formula image 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).
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