Behavior associated with formalin injection in the masseter and TMJ

Objective: To develop a quantitative behavioral measure of TMJ/masseter muscle pain. Methods: Forty nine C57BL/6J male mice (mean weight 27.54 gm, mean age 15 weeks) were randomly assigned to 4 groups: single injection into the right TMJ or masseter muscle of (a) formalin (TMJ injection: 1.25%/50µl or masseter muscle: 0.625%/50µl), (b) formalin + morphine (60µl IP, 20 min before formalin injection), (c) saline (50µl), and (d) no injection control. Following injection each mouse was videotaped for 45 minutes to quantitatively evaluate rubbing of the face and flinching of the head. For each 3 min period, the time the animal exhibited rubbing of the face and number of head flinches was counted by one investigator, blind to the animal group assignment. Two hours after the injection, the mice were sacrificed and perfused transcardially. The brain stem and trigeminal ganglia were harvested. The levels of c-Fos, CGRP and COX-2 expression were quantitated with immunocytochemistry and analyzed by one-way ANOVA. Results: There was a 5 fold increase (P<0.001) in c-fos expression in the trigeminal ganglia of formalin injected mice compared to other animal groups, an 8 fold increase in COX-2 and 2.5 increase in CGRP. Significant behavioral increase in rubbing and flinching (formalin group increased 75% over controls) in the formalin group compared to other animals (p<0.001). Resistance to jaw opening was reduced in the formalin group (TMJ: 58% decrease; masseter group: 45% decrease) compared to other groups (p<0.05). Systemic morphine administration attenuated the behavioral and neurotransmitter changes following formalin injections. Conclusion: Formalin injection into the TMJ and masseter muscle significantly increased behavioral activity and decreased the resistance to jaw opening, increased c-fos, CGRP and COX-2 expression in the trigeminal ganglia and brain stem. This suggests induced pain in the masseter and TMJ produce behavioral changes and dysfunction (decrease in muscle force).