Activation of voltage-gated KCNQ/Kv7 channels by anticonvulsant retigabine attenuates mechanical allodynia of inflammatory temporomandibular joint in rats

Abstract

Background: Temporomandibular disorders (TMDs) are characterized by persistent orofacial pain and have diverse etiologic factors that are not well understood. It is thought that central sensitization leads to neuronal hyperexcitability and contributes to hyperalgesia and spontaneous pain. Nonsteroidal anti-inflammatory drugs (NSAIDs) are currently the first choice of drug to relieve TMD pain. NSAIDS were shown to exhibit anticonvulsant properties and suppress cortical neuron activities by enhancing neuronal voltage-gated potassium KCNQ/Kv7 channels (M-current), suggesting that specific activation of M-current might be beneficial for TMD pain.

Results: In this study, we selected a new anticonvulsant drug retigabine that specifically activates M-current, and investigated the effect of retigabine on inflammation of the temporomandibular joint (TMJ) induced by complete Freund's adjuvant (CFA) in rats. The results show that the head withdrawal threshold for escape from mechanical stimulation applied to facial skin over the TMJ in inflamed rats was significantly lower than that in control rats. Administration of centrally acting M-channel opener retigabine (2.5 and 7.5 mg/kg) can dose-dependently raise the head withdrawal threshold of mechanical allodynia, and this analgesic effect can be reversed by the specific KCNQ channel blocker XE991 (3 mg/kg). Food intake is known to be negatively associated with TMJ inflammation. Food intake was increased significantly by the administration of retigabine (2.5 and 7.5 mg/kg), and this effect was reversed by XE991 (3 mg/kg). Furthermore, intracerebralventricular injection of retigabine further confirmed the analgesic effect of central retigabine on inflammatory TMJ.

Conclusions: Our findings indicate that central sensitization is involved in inflammatory TMJ pain and pharmacological intervention for controlling central hyperexcitability by activation of neuronal KCNQ/M-channels may have therapeutic potential for TMDs.

Figure 1

Histopathologic examination of inflammatory TMJ region induced by CFA in rats. A. Photomicrograph of TMJ 24 hours after injection of saline as a normal control. B. Photomicrograph of TMJ 24 hours after injection of CFA. Exuberant hypertrophy of the synovial tissue, angiogenesis in the enlarged area (indicated by arrowhead) and fibrin-like exudates (indicated by arrow) in the joint space were observed (Magnification × 4, scale bar = 200 μm).

Figure 2

Development of mechanical allodynia in TMJ induced by CFA in rats. Head withdrawal threshold was measured using an electric von Frey anesthesiometer over a 96-hour period for the saline control group (open squares) and the CFA group (filled triangles). CFA was injected at time 0 and the withdrawal threshold was measured at time points 6 h, 12 h, 24 h, 48 h, and 96 h. The lowest head withdrawal threshold occurred 12 h after CFA injection and was maintained for about 36 h (P < 0.001 for time period of 12 to 96 hours, compared with the saline group, two-way ANOVA).

Figure 3

Attenuation of mechanical TMJ allodynia by anticonvulsant retigabine in a dose-dependent manner. Drugs (Mor: morphine and RTG: retigabine) of different concentrations were administered to rats with TMJ inflammation by intraperitoneal (i.p) injection at time 0. The withdrawal threshold was measured at 15, 30, 60 and 120 min after drug treatment, with morphine as a positive control. Statistical significance of p < 0.05 (two-way ANOVA followed by Bonferroni post hoc) was obtained for the period 15-60 min for the groups CFA + morphine (5 mg/kg), and CFA + retigabine (7.5 mg/kg or 2.5 mg/kg), as compared with the group CFA + vehicle.

Figure 4

Retigabine reduced allodynia reversed by the blocker XE991. RTG (retigabine) at 7.5mg/kg attenuated the inflamed TMJ allodynia, and the effect was reversed by XE991 (3 mg/kg or 1 mg/kg) in a dose- and time-dependent manner. XE991 alone (3 mg/kg) had no effect on the head withdrawal threshold in TMJ inflammation. Statistical significance of p < 0.05 (two-way ANOVA followed by Bonferroni post-test) was obtained for the period 15-60 min for the group CFA + RTG 7.5 mg/kg, as compared with CFA + vehicle; and was also obtained for the two groups CFA + RTG 7.5 mg/kg + XE 3 mg/kg, and CFA + RTG 7.5 mg/kg + XE 1 mg/kg, as compared with the group of CFA + RTG 7.5 mg/kg. XE: XE991, RTG: retigabine. The plus sign (+) in the legend indicates CFA treatment.

Figure 5

Head withdrawal threshold and food intake in normal rats was not affected by retigabine and XE991. (A), RTG (retigabine) at 7.5 mg/kg and XE991 at 3 mg/kg had no effect on head withdrawal threshold of normal rats without TMJ inflammation, as compared with the vehicle (two-way ANOVA followed by Bonferroni post-test). (B), Food intake values in normal rats without TMJ inflammation. During the 2 h period of food intake, retigabine (RTG) and XE991 had no effect on normal food intake in rats without TMJ inflammation. The food intake value in the baseline group (4.8 g) indicates the normal value obtained before drug or vehicle administration. The food intake values in the groups treated by retigabine (7.5 mg/kg, i.p.) and XE991 (3 mg/kg, i.p.) were 4.5 g and 4.8 g, respectively. The food intake value of the vehicle group was 5.5 g. There was no statistical significance between these four groups (one-way ANOVA followed by Dunnett's test).

Figure 6

Increased food intake with retigabine administration in rats with TMJ inflammation injected with CFA. The food intake in the baseline group was measured in normal rats without CFA injection. Twenty-four hours after CFA injection, the effect of retigabine or morphine as a positive control on food intake was measured, and compared with the vehicle (CFA alone) group. Retigabine enhanced the food intake in dose-dependent manner, as compared with the vehicle. Statistical significance was obtained between the vehicle group and the two groups RTG (7.5 mg/kg, i.p., p < 0.01; and 2.5 mg/kg, i.p., p < 0.05, one-way ANOVA followed by Dunnett's test), and the morphine group (Mor) (5 mg/kg, i.p., p < 0.05, one-way ANOVA followed by Dunnett's test).

Figure 7

Enhanced food intake by retigabine was reversed by the blocker XE991. The food intake in the baseline group represents the value for normal rats without TMJ inflammation. The effect of retigabine or retigabine and XE991 was tested 24 hours after CFA injection. The food intake in the vehicle group indicates the value for rats with TMJ inflammation induced by CFA. Retigabine at a dose of 7.5 mg/kg, i.p. significantly increased the food intake, as compared with the vehicle group (p < 0.005, one-way ANOVA followed by Dunnett's test). Co-injection (i.p.) of XE991 (at 3 mg/kg or 1 mg/kg) with RTG reversed the food intake which had been increased by RTG (7.5 mg/kg, p < 0.05, one-way ANOVA followed by Dunnett's test), as compared with the vehicle group. XE991 at 3 mg/kg had no effect on food intake in rats with TMJ inflammation (p = 0.98), compared with vehicle group. RTG: Retigabine; XE: XE991.

Figure 8

Allodynia was attenuated by central administration of retigabine in rats with TMJ inflammation. Intracerebroventricular injection of retigabine (45 μg or 15 μg) as well as morphine (1 μg) as positive control raised the head withdrawal threshold from 15 to 60 min, compared with the vehicle control (p < 0.05 two way ANOVA followed by Bonferroni post-test).

Figure 9

Increased food intake secondary to central administration of retigabine. Intracerebroventricular administration of retigabine at a dose of either 45 μg or 15 μg (p < 0.05), as well as the positive control morphine group 1 μg (p < 0.05, one-way ANOVA followed by Dunnett's test) raised the food intake in rats with TMJ inflammation.