P2X3 Receptor In Central Nervous System Is Involved In Masticatoty Muscle Hyperalgesia Induced By Occlusal Interference In Rats

ObjectivesA number of researchers have explored the etiology and related pathophysiology of masticatory muscle pain and suggested that improper occlusion was a contributing factor. However, the mechanisms of the initiation and perpetuation of masticatory muscle hyperalgesia caused by occlusal interference were not elucidated. P2X3 receptor plays a role in nociception transmission of orofacial pain in patients. A previous study found that P2X3 receptors in masseter muscle afferent neurons and the trigeminal ganglia were involved in masseter muscle pain induced by inflammation caused by chemical agents or eccentric muscle contraction. In this study, to explore the relationship between occlusal interference and masticatory muscle hyperalgesia, we created an occlusal interference animal model and observed the changes in nociceptive thresholds of masseter muscles. To evaluate whether the P2X3 receptor in subnuclei caudalis (Vc), midbrain periaqueductal gray (PAG) and dorsal raphe nucleus (DR) neurons is involved in masticatory muscle hyperalgesia associated with occlusal interference, we attempted to investigate changes in P2X3 receptors in the Vc, PAG and DR in relation to the hyperalgesia of masseter muscles induced by occlusal interferenceMethods1.54 rats were randomly divided into 1 sham-control groups and 5 groups with occlusal interference, and 9 rats in group. A metallic crown (1.0 mm, uniform thickness) was bonded to the right mandibular first molar using resin cement in occlusal interference group. Sham-treated rats in the control groups were anesthetized and their mouths were forced opened for approximately five minutes using a protocol similar to the occlusal interference groups; however, no crowns were cemented.2. On day 1,3,7,14,28 after crown-application, the mechanical pain threshold score of masseter muscles was examined by von-Frey filaments. The expression of P2X3 receptor in Vc, PAG and DR was investigated by immunohistochemistry, quantitative-PCR and Western Blot. Statistical analysis of the collected data was performed by repeated measures one-way analysis of variance (ANOVA) comparison tests followed by least significant difference (LSD) comparison tests. Calculations were carried out using SPSS 17.0 (Polar Engineering and Consulting). A P-value <0.05 was considered statistically significant.Results1. The mechanical pain threshold score of bilateral masseter muscles significantly increased after occlusal interference, and the hyperalgesia persisted for the whole experimental period. The mechanical pain threshold score of both masseter muscles in experimental group reached the peak on day 7 (p＜0.05), and no statistical significance had been found in both sides of masseter muscles of experimental group (P＞0.05)2. The results of immunohistochemistry showed that abundant small-sized neurons in the Vc, PAG and DR of rats were P2X3 receptor positive. In the Vc, P2X3 receptor-positive neurons were found mainly in the superficial laminae throughout the dorsoventral aspect. Compared to sham-treated rats in the control groups, a significant increase in P2X3 receptor-positive neurons took place in both the ipsilateral and contralateral Vc of rats during the whole experiment; and peaked on day seven(p＜0.05). Meanwhile, there were no significant differences between the ipsilateral and contralateral Vc of rats in all occlusal interference groups (p＞0.05). In the PAG, P2X3 receptor-positive neurons were found mainly in the lateral PAG After wearing crowns, the number of P2X3 receptor-positive neurons in the PAG of rats in the occlusal interference groups significantly increased on day seven compared to sham-treated rats in the control group (p＜0.05). The number of P2X3 receptor-positive cells in DR significantly increased on day, and peaked on day seyen (P＜0.05).3. The results of quantitative-PCR showed that P2X3 receptor mRNA expressions in Vc of rats that underwent crown application significantly increased on day one, three, seven and fourteen, which peaked on day one after occlusal interference. Moreover, there was no statistical significance between the ipsilateral and contralateral Vc of rats in the occlusal interference groups. Compared with sham-treated rats in the control group, P2X3 receptor mRNA expressions in the PAG and DR of rats that underwent crown application significantly increased on day one (P＜0.05).4. The results of Western blot showed that the expression levels of P2X3 receptor protein in Vc, increased significantly in occlusal interference groups, peaked on day seven (p＜0.05). No statistical significance had been found in both sides of Vc of occlusal interference group (p＞0.05). And in PAG and DR, the expression levels of P2X3 receptor protein significantly increased only on day seven (p＜0.05).Conclusions1) Occlusal interference is directly related to masticatory muscle pain.2) P2X3 receptor positive was expressed mainly in small-sized neurons in the superficial laminae of Vc, the lateral PAG and DR of rats.3) The up-regulated expression of P2X3 receptor in Vc, PAG and DR may contribute to the development of orofacial pain induced by occlusal interference. The central sensitization mechanism and endogenous pain modulation system are involved in the maintenance of the occlusal interference-induced masticatory muscle mechanical hyperalgesia.