TRPV1 channel-mediated bilateral allodynia induced by unilateral masseter muscle inflammation in rats

Abstract

Pain in masticatory muscles is among the most prominent symptoms of temperomandibular disorders (TMDs) that have diverse and complex etiology. A common complaint of TMD is that unilateral pain of craniofacial muscle can cause a widespread of bilateral pain sensation, although the underlying mechanism remains unknown. To investigate whether unilateral inflammation of masseter muscle can cause a bilateral allodynia, we generated masseter muscle inflammation induced by unilateral injection of complete Freund's adjuvant (CFA) in rats, and measured the bilateral head withdrawal threshold at different time points using a von Frey anesthesiometer. After behavioral assessment, both right and left trigeminal ganglia (TRG) were dissected and examined for histopathology and transient receptor potential vanilloid 1 (TRPV1) mRNA expression using quantitative real-time PCR analysis. A significant increase in TRPV1 mRNA expression occurred in TRG ipsilateral to CFA injected masseter muscle, whereas no significant alteration in TRPV1 occurred in the contralateral TRG. Interestingly, central injection of TRPV1 antagonist 5-iodoresiniferatoxin into the hippocampus significantly attenuated the head withdrawal response of both CFA injected and non-CFA injected contralateral masseter muscle. Our findings show that unilateral inflammation of masseter muscle is capable of inducing bilateral allodynia in rats. Upregulation of TRPV1 at the TRG level is due to nociception caused by inflammation, whereas contralateral nocifensive behavior in masticatory muscle nociception is likely mediated by central TRPV1, pointing to the involvement of altered information processing in higher centers.

Figure 1

Morphological indications of inflammation occurred in the masseter muscle following injection of CFA. (A) Haematoxylin and eosin staining 4 days following injection of CFA in masseter muscle; note the extensive infiltration of inflammatory cells (indicated in arrows) and vacuoles (indicated in asterisks). (B) Masseter muscle contralateral to CFA injected side. (C) Masseter muscle 4 days after injection of saline, and no signs of inflammation were observed in saline injected and contralateral masseter muscle. The scale bar represents 20 μm.

Figure 2

The time course of head withdrawal thresholds following injection of CFA and saline into the masseter muscle. Unilateral injection of CFA into the masseter muscle induces allodynia in ipsilateral masseter at 4 h, day 1, day 4 and day 7 after injection. (A) Head withdrawal thresholds for the ipsilateral masseter (two-way ANOVA followed by Bonferroni post-hoc, n = 6). Allodynia of contralateral masseter was observed at time point of day 1and day 4. (B) Contralateral masseter (two-way ANOVA followed by Bonferroni post hoc, n = 6). Results are presented as means ± s.e.m. *p < 0.05; ** p < 0.01;*** p < 0.001 versus saline group at same time point. Note the lowest head withdrawal thresholds at bilateral masseter muscle 4 days after CFA injection.

Figure 3

TRPV1 mRNA expressions in the trigeminal ganglion. (A) Real-time PCR revealed up-regulation of TRPV1 expression after masseter inflammation in ipsilateral TRG; (B) There was no difference in TRPV1 expression in contralateral TRG. Data are presented as means ± s.e.m., n = 8. **p < 0.01 versus control group, using t-test.

Figure 4

Confirmation of cannula placement in the hippocampus. (A) Reconstruction of serial coronal sections of the brain illustrates the bilateral injection sites of the cannulas. The black dots on the schematic illustration of the coronal section of rat brain indicate the placements of cannulas in the brain. (B) Representative microphotograph of the coronal section shows the placements (indicated by circles and arrows) of a pair of cannulas in the CA1 region of the hippocampus.

Figure 5

Attenuation of bilateral masseter muscle allodynia by TRPV1 antagonist. Experimental design of intrahippocampal injection of TRPV1 antagonists (A). The head withdrawal threshold was not different between groups with intrahippocampal injection of 5-iodo-resiniferatoxin (0.1 or 0.5 nmol) and vehicle injected group before induction of masseter muscle inflammation (p > 0.05). Significant decrease in head withdrawal threshold 4 days after CFA injection in both ipsilateral and contralateral masseter confirmed the development of allodynia (B, C). The attenuation of allodynia of ipsilateral masseter was observed 30 min, 45 min and 60 min after intrahippocampal injections of 5-iodo-resiniferatoxin (B) and in all time points in contralateral masseter (C). Data are presented as means ± s.e.m., n = 6. * p < 0.05; ** p < 0.01; *** p < 0.001 versus vehicle group at the same time point, using two-way ANOVA followed by Bonferroni post- hoc.