Connexin 43 contributes to ectopic orofacial pain following inferior alveolar nerve injury

Abstract

Background: Clinically, it is well known that injury of mandibular nerve fiber induces persistent ectopic pain which can spread to a wide area of the orofacial region innervated by the uninjured trigeminal nerve branches. However, the exact mechanism of such persistent ectopic orofacial pain is not still known. The present study was undertaken to determine the role of connexin 43 in the trigeminal ganglion on mechanical hypersensitivity in rat whisker pad skin induced by inferior alveolar nerve injury. Here, we examined changes in orofacial mechanical sensitivity following inferior alveolar nerve injury. Furthermore, changes in connexin 43 expression in the trigeminal ganglion and its localization in the trigeminal ganglion were also examined. In addition, we investigated the functional significance of connexin 43 in relation to mechanical allodynia by using a selective gap junction blocker (Gap27).

Results: Long-lasting mechanical allodynia in the whisker pad skin and the upper eyelid skin, and activation of satellite glial cells in the trigeminal ganglion, were induced after inferior alveolar nerve injury. Connexin 43 was expressed in the activated satellite glial cells encircling trigeminal ganglion neurons innervating the whisker pad skin, and the connexin 43 protein expression was significantly increased after inferior alveolar nerve injury. Administration of Gap27 in the trigeminal ganglion significantly reduced satellite glial cell activation and mechanical hypersensitivity in the whisker pad skin. Moreover, the marked activation of satellite glial cells encircling trigeminal ganglion neurons innervating the whisker pad skin following inferior alveolar nerve injury implies that the satellite glial cell activation exerts a major influence on the excitability of nociceptive trigeminal ganglion neurons.

Conclusions: These findings indicate that the propagation of satellite glial cell activation throughout the trigeminal ganglion via gap junctions, which are composed of connexin 43, plays a pivotal role in ectopic mechanical hypersensitivity in whisker pad skin following inferior alveolar nerve injury.

Keywords: Trigeminal ganglion; connexin 43; ectopic mechanical allodynia; gap junction; inferior alveolar nerve injury; satellite glial cell.

Figure 1.

Changes in mechanical sensitivity following IANX. Bilateral MHWTs were measured in upper eyelid skin (a) and whisker pad skin (b) following IANX or sham operation. Data are expressed as percentage (mean ± SEM) of MHWT for each group, normalized to preoperation MHWT (100%). ^$p < 0.05, ^$$p < 0.01, *p < 0.05, **p < 0.01; compared to sham-operated rats. ^#p < 0.05, ^##p < 0.01; compared to contralateral side. (n = 8 in each group; two-way ANOVA with repeated measures, followed by Bonferroni’s multiple-comparison tests.)

Figure 2.

Cx43 and GFAP expression in TG following IANX. Photomicrographs of Cx43-IR cells (a), GFAP-IR cells (b), Cx43-IR and GFAP-IR cells (c) in TG on day 8 following IANX. Arrows denote double-IR cells. Scale bars: 50 µm. Relative amount of Cx43 (d) and GFAP protein (e) in the TG of naive rats and on days 1, 8, and 14 after IANX or sham operation. β-actin protein was used as loading control. Data represent mean ± SEM. **p < 0.01: compared to sham-operated rats; ^#p < 0.05, ^##p < 0.01: compared to naive rats. (n = 8 in each group; one-way ANOVA followed by Newman-Keuls’s multiple-comparison tests.)

Figure 3.

Cx43 and GFAP expression in TG of V2 and V3 following IANX. (a) Photomicrographs of Cx43-IR/GFAP-IR cells in TG of V3 on day 8 following IANX operation. Arrows denote double-IR cells. Scale bars: 50 µm. (b) Mean percentage of FG-labeled TG neurons encircled with GFAP-IR or Cx43-IR/GFAP-IR cells in TG of V2 and V3 on day 8 after IANX or sham operation. **p < 0.01 (n = 4 in each group; one-way analysis of ANOVA followed by Tukey’s multiple-comparison tests.)

Figure 4.

Effects of TG continuous-administration of Gap 27 on mechanical sensitivity in the orofacial region following IANX. Time course of changes in MHWT in upper eyelid skin (a) and whisker pad skin (b) in sham-operated or IANX rats with intra-TG Gap 27 or vehicle administration. Error bars indicate SEM. ^##p < 0.01 vs. prevalue; **p < 0.01 vs. value of IANX group with TG continuous-administration of Gap 27. (n = 8 in each group; two-way ANOVA with repeated measures followed by Bonferroni’s multiple-comparison tests.)

Figure 5.

Effects of TG single-administration of Gap 27 on mechanical sensitivity in the orofacial region following IANX. Time course of changes in MHWT in upper eyelid skin (a) and whisker pad skin (b) in sham-operated or IANX rats with intra-TG Gap 27 or vehicle administration. Error bars indicate SEM. **p < 0.01 (n = 8 in each group; two-way ANOVA with repeated measures followed by Bonferroni’s multiple-comparison tests).

Figure 6.

Effect of continuous-Gap 27 administration on SGC activation in TG after IANX. Photomicrographs of GFAP-IR and Cx43-IR cells encircled with FG-labeled TG neurons in naive rats (a), and GFAP-IR cells encircling FG-labeled TG neurons innervating the whisker pad skin on day 8 following sham operation (b) or IANX (c) with continuous administration of vehicle or Gap 27. Arrows denote FG-labeled TG neurons. Scale bars: 50 µm. Mean percentages (d) and size-frequency histograms illustrating distribution (e) of FG-labeled TG neurons encircled with GFAP-IR cells on day 8 after sham operation or IANX with continuous administration of vehicle or Gap 27. *p < 0.05, **p < 0.01 (n = 4 in each group; one-way analysis of ANOVA followed by Tukey’s multiple-comparison tests).

Figure 7.

Effects of TG continuous-administration of Gap 27 on Cx43 and GFAP expression in TG following IANX. Relative amount of Cx43 protein in the V1-V2 (a) and V3 (b) of TG, and relative amount of GFAP protein in the V1-V2 (c) and V3 (d) of TG on day 8 after IANX. β-actin protein was used as loading control. Data represent mean ± SEM. *p < 0.05, **p < 0.01 (n = 8 in each group; Student’s t-test).