Roles of TRPV1 and TRPA1 in Spontaneous Pain from Inflamed Masseter Muscle

Abstract

Craniofacial muscle pain, such as spontaneous pain and bite-evoked pain, are major symptoms in patients with temporomandibular disorders and infection. However, the underlying mechanisms of muscle pain, especially mechanisms of highly prevalent spontaneous pain, are poorly understood. Recently, we reported that transient receptor potential vanilloid 1 (TRPV1) contributes to spontaneous pain but only marginally contributes to bite-evoked pain during masseter inflammation. Here, we investigated the role of transient receptor potential ankyrin 1 (TRPA1) in spontaneous and bite-evoked pain during masseter inflammation, and dissected the relative contributions of TRPA1 and TRPV1. Masseter inflammation increased mouse grimace scale (MGS) scores and face wiping behaviors. Pharmacological or genetic inhibition of TRPA1 significantly attenuated MGS but not face wiping behaviors. MGS scores were also attenuated by scavenging putative endogenous ligands for TRPV1 or TRPA1. Simultaneous inhibition of TRPA1 by AP18 and TRPV1 by AMG9810 in masseter muscle resulted in robust inhibition of both MGS and face wiping behaviors. Administration of AP18 or AMG9810 to masseter muscle induced conditioned place preference (CPP). The extent of CPP following simultaneous administration of AP18 and AMG9810 was greater than that induced by the individual antagonists. In contrast, inflammation-induced reduction of bite force was not affected by the inhibition of TRPA1 alone or in combination with TRPV1. These results suggest that simultaneous inhibition of TRPV1 and TRPA1 produces additive relief of spontaneous pain, but does not ameliorate bite-evoked pain during masseter inflammation. Our results provide further evidence that distinct mechanisms underlie spontaneous and bite-evoked pain from inflamed masseter muscle.

Keywords: TRP channels; inflammation; muscle pain; trigeminal pain.

Figure 1. Effects of pharmacological inhibition of TRPA1 on MGS and face wiping behaviors during masseter inflammation in mice

A. Time line of mouse grimace scale (MGS) and wiping experiment. Day 0 is the time when baseline behaviors were measured and CFA (20 μl) or vehicle (saline) was injected to one side of masseter muscle. M, 30 min video recording session of MGS; W, 30 min video recording session of face wiping. B-C. Averaged mouse grimace scale (MGS) scores (B) and number of face wiping behaviors (C) following time course indicated in panel A. AP18 (TRPA1 antagonist; 800 nmol in 20 μl) or vehicle was injected into the ipsilateral masseter muscle 1 day after CFA injection. MGS and wiping behaviors were measured before and 1 h after drug or vehicle injection. ****p<0.0001; NS, not significant; Bonferroni post-hoc test following two-way ANOVA with repeated measures. Mean±SEM. N=10 in each group.

Figure 2. Dose-dependent inhibition MGS scores by TRPA1 inhibitor on different days during masseter inflammation in mice

A. Time line of MGS assay. M, 30 min video recording session of MGS. B. Effects AP18 on MGS. Masseter muscle was injected with AP18 (three different doses) 1 day after CFA (day 1) and 2 days after CFA (day 2). MGS was assayed 1 h after drug or vehicle injection. **p<0.01; ****p<0.0001; Bonferroni post-hoc test following two-way ANOVA with repeated measures. Mean±SEM. N=5 in each group.

Fig. 3. Effects of genetic knockout of TRPA1 on MGS and face wiping behaviors during masseter inflammation in mice

MGS scores (A) and face wiping behaviors (B) following unilateral masseter injection of CFA in TRPA1 knockout (KO) or littermate wildtype (WT) mice. ****p<0.0001; NS, not significant; Bonferroni post-hoc test following two-way ANOVA with repeated measures. Mean±SEM. N=12 in each group.

Fig. 4. Effects of depletion of hydrogen peroxide or oxidized linoleic acid metabolites in masseter muscle on MGS during masseter inflammation

A. Catalase (300 units in 20 μl PBS; H₂O₂ detoxifying enzyme) or control (heat-inactivated catalase) was injected into ipsilateral masseter muscle 1 day after unilateral masseter injection of CFA or vehicle. Effects on MGS scores were measured 1 h after drug injection. *p<0.05; Bonferroni post-hoc test following two-way ANOVA with repeated measures. N=6 in each group. B. A mixture of antibodies against 9(S)-HODE and 13(S)-HODE (10 μg each in 20 μl PBS) or goat IgG (20 μg in 20 μl PBS) were injected into ipsilateral masseter muscle 1 day after unilateral masseter injection of saline or CFA. Effects on MGS scores were evaluated 1 h after drug injection. **p<0.01; Bonferroni post-hoc test following two-Way ANOVA with repeated measures. N=6 in each group.

Fig. 5. Effects of simultaneous pharmacological inhibition of TRPV1 and TRPA1 on MGS and wiping behaviors during masseter inflammation in mice

CFA was injected into the masseter muscle on day 0. Drugs or vehicle were injected into the ipsilateral (ipsi) or contralateral (contra) masseter muscle on day 1 (1 day after CFA). MGS scores (A) and numbers of face wiping (B) were evaluated 1 h (MGS) or 1.5 h (face wiping) after drug injection. AP18 (TRPA1 antagonist; 800 nmol in 20 μl) and AMG9810 (AMG, TRPV1 antagonist; 200 nmol in 20 μl) were injected simultaneously. *p<0.05; ****p<0.0001; Bonferroni post-hoc test following two-way ANOVA with repeated measures. N=10 in CFA/Veh/ipsi and CFA/AMG+AP18/ipsi; n=6 in CFA/Veh/contra and CFA/AMG+AP18/contra.

Fig. 6. Effects of combined pharmacological and genetic inhibition of TRPA1 and TRPV1 on MGS and wiping behaviors

CFA was injected unilaterally into masseter muscle on day 0. Drugs or vehicle were injected into the ipsilateral masseter muscle on day 1 (one day after CFA). AP18 was injected to ipsilateral masseter muscle in TRPV1 KO mice (A, B). AMG was injected to ipsilateral masseter muscle in TRPA1 KO mice (C, D). MGS scores (A, C) and numbers of face wiping (B, D) were evaluated 1 h (MGS) or 1.5 h (face wiping) after drug injection. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Bonferroni post-hoc test following two-way ANOVA with repeated measures. N=10 in each group.

Fig. 7. Effects of pharmacological inhibition of TRPV1 and TRPA1 on conditioned place preference (CPP) during masseter inflammation

A. Time line of CPP experiment. B-F. Time spent in saline- or drug-paired chambers were compared at baseline (Day 1), before conditioning (Day 2; 6 hours after unilateral injection of CFA), and after conditioning (Day 4). Mice were conditioned with vehicle (B; n=10 in each group), AMG9810 (C; n=11 in each group), AP18 (D; n=10 in each group), or AMG9810+AP18 (E; n=11 in each group) by injection of the drug into the ipsilateral masseter muscle. One group (F) was conditioned with AMG+AP18 by injection of the drugs into the contralateral masseter muscle. N=10 in each group. ***p<0.001, ****p<0.0001; Bonferroni post hoc test following two-way ANOVA with repeated measures. G. Difference scores calculated as increased time spent in the drug-paired chamber on test day compared to the day before conditioning. Increased time indicates increased preference for the drug treatment. *p<0.05; **p<0.01, ***p<0.001; Bonferroni post hoc test following one-way ANOVA.

Fig. 8. TRPA1 does not contribute to bite-evoked pain during masseter inflammation

A. Effects of AP18 (800 nmol/side) or vehicle injected bilaterally into the masseter muscles 1 day after bilateral CFA injections. NS, not significant. N=11 in AP18 and 7 in Veh. B. Bite force of TRPV1 KO or WT mice measured before and 1 day after bilateral injections of CFA (20 μl) into the masseter muscles. N=9 in WT and 10 in TRPA1 KO. C. Bilateral injection of drugs or vehicle into the masseter muscles 30 min prior to bilateral injections of CFA (10 μl per side). Bite force was measured 1, 2, and 4 hours after CFA injections, and was normalized to the baseline bite force of each animal. N=8 in Veh, 9 in AMG, 6 in AP18 and 7 in AMG+AP18.