| Part one:Study of hyperalgesia and histological change of rat masseter muscle and temporomandibular joint following occlusal interferenceObjectivesTo explore the relationship between occlusal interference and orofacial pain, we created an occlusal interference animal model and observed the changes of nociceptive thresholds and histological study of the masseter muscles and temporomandibular joints (TMJs) on both sides.Methods40 rats were randomly divided into 5 experimental groups and 5 sham-control groups, and 4 rats in each group. The experimental interference was established by crown application in experimental. On day 1,3,7,14,28 after crown-application, the mechanical pain threshold score was examined by von-Frey filaments. The histological changes of the masseter muscles and temporomandibular joint (TMJ) synovial membranes on both sides were observed.Results1. The mechanical pain threshold score of bilateral TMJs significantly increased after occlusal interference and the hyperalgesia persisted for the whole experimental period. The mechanical pain threshold score of both masseter muscles and TMJs 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 and TMJs of experimental group.2. Most masseter muscle fibres in rats with occlusal interference remained intact, and no infammatory cell appeared in the muscle fibres. Partial histological changes were observed on day 7,14,28 after crown-application. Connective tissue was extended and some muscle fibres with central nuclei were observed. The expression levels of desmin in both sides of masseter muscles were lower in rats with occlusal interference. The ultrastructural changes at different stages of the masseter muscle: masseter muscle fiber disorderation, muscle fiber shrinked or broken, striated muscle blurred or disappeared, muscle internal vascular proliferation, expansion and so on.3. There are no infammatory cell appeared in the TMJ synovial membranes of rats with occlusal interference. On day 7,14,28 after crown-application, the proliferation of synovial membrane cells and vascular dilation was observed, glycogen particles in fibroblasts endocrine increased, and macrophage cell membranes lining is not clear.Conclusions1. Occlusal interference is directly related to masticatory muscle pain.2. Occlusal interference can not lead to inflammatory change of masseter muscle and TMJ, but can induce masseter muscle microdamage and TMJ synovial microtrauma, which are not the main cause of orofacial pain.Part two:P2X3 receptor in trigeminal ganglion and subnuclei caudalis involvemented in orofacial pain induced by occlussal interferences in ratsObjectivesTo evaluate whether the P2X3 receptor in trigeminal ganglion (TG) and subnuclei caudalis (Vc) neurons isinvolved in orofacial hyperalgesia associated with occlusal interference. In the present study, the functional significance of P2X3 receptor (P2X3R) in the TG and Vc in relation to the hyperalgesia of masseter muscles and TMJs induced by occlusal interference was investigated.Methods1.6 rats aged 3 weeks were randomly divided into 2groups, and 3 rats in each group. Expression, distribution and location of P2X3 receptor in the masseter muscles TMJs and TG were determined by FG retrogradeneural tracing technique combined with a double-labeled immunohistochemical technique.2.60 rats were randomly divided into 5 experimental groups and 5 sham-control groups, and 6 rats in each group. On day 1,3,7,14,28 after crown-application, the mechanical pain threshold score was examined. The expression of P2X3R in TGs and Vcs was investigated by immunohistochemistry and real-time PCR.3.60 rats were randomly divided into 4 groups, and 3 rats in each group.7 days after crown treatment, P2X3R antagonist A-317491 was administrated respectivly in rat masseter muscles and TMJs of 2 groups, and the saline solution was administrated respectivly in rat masseter muscles and TMJs in another 2 groups. The changes of the mechanical pain threshold score in masseter muscles and TMJs was testedResults1. There were about 14.4 ± 2.0% of all TG neurons controlling the TMJ afferent nerve, of which about 44.1 ± 7.0% showed P2X3 receptor position. There were about 18.1 ± 2.5% of all neurons controlling the masseter muscle afferent nerve in TG, of which about 48.5 ± 5.4% expressed P2X3 receptor.2. The result of immunohistochemistry showed that the proportion of P2X3R-positive cells increased in both sides of small neurons in TGs and Vcs in occlusal interference groups after crown-application. The proportion of P2X3 receptor-positive cells in TGs peaked on day 7 (p<0.05), while the proportion of P2X3 receptor -positive cells in Vcs peaked on day 3 and persisted to day 7 (p<0.05). No statistical significance had been found in both sides of TGs and Vcs of occlusal interference group.3. The results of real-time PCR showed that expression of P2X3R mRNA significantly increased on day 3 in both sides of TGs in occlusal interference groups (p<0.05). And in Vcs, the expression of P2X3R mRNA peaked on day 1, and statistical significance had been showed on day 1,3,7 (p<0.05).4. The result of Western blot showed that the expression levels of P2X3 receptor increased in TGs increased non-significantly in occlusal interference groups(p>0.05), and the expression levels of P2X3 receptor increased significantly in Vcs, peaked on day 7 (p<0.05). No statistical significance had been found in both sides of TGs and Vcs of experimental group. (p>0.05)5. The antagonist A-317491 could reverse hyperalgesia caused by occlusal interference.Conclusions1) The up-regulated expression of P2X3 receptor in TG and Vc may contribute to the development of orofacial pain induced by occlusal interference. The periphery and central sensitization mechanisms are involved in the maintenance of the occlusal interference-induced mechanical hyperalgesia.2) P2X3 receptor antagonist may probably be used in treatment of temporomandibular disorders related hyperalgesia. |
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