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The exact mechanism underlying chronic masseter muscle pain, a conspicuous symptom in temporomandibular disorder, remains unclear. We investigated whether expression of P2X3 receptor (P2X3R) is involved in mechanical hyperalgesia after contraction of masseter muscle (CMM). As compared with sham rats, the head-withdrawal threshold (HWT) to mechanical pressure stimulation of masseter muscle (MM) (but not after similar stimulation of facial skin) was significantly lower, and IL-1β level was significantly higher, in CMM rats on day 7 after CMM. The mean percentage of FG-labeled P2X3R-positive neurons was significantly increased in TG following successive IL-1β injections into the MM for 7 days. Successive administration of an IL-1β receptor-antagonist into the MM attenuated the increase of P2X3-IR cells in the TG. ATP release from MM after 300-g pressure stimulation of MM was also significantly enhanced after CMM. Administration into MM of the selective P2X3,2/3 receptor antagonist A-317491 attenuated the decrement of HWT in CMM rats. A significant increase in HWT was also observed at 30 min after A-317491 (60 µg) injection in IL-1β-injected rats. These findings suggest that P2X3R expression associated with enhanced IL-1β expression and ATP release in MM has a possible important role in MM mechanical hyperalgesia after excessive muscular contraction.

It is well known that exposure to maternal separation (MS) in early life causes plastic changes in the nervous system in adulthood, occasionally resulting in ubiquitous chronic pain. However, the pathogenic mechanisms of pain hypersensitivity remain unclear. Here, the authors examined the involvement of corticosterone in orofacial mechanical hypersensitivity induced by MS. To establish a rat model of MS, pups were placed in isolated cages 180 min/d and kept in a temperature-controlled environment at 22 ± 2 °C for 14 d. Mechanical allodynia in the whisker pad skin in adulthood was induced by MS and was significantly suppressed by successive postnatal subcutaneous administration of the glucocorticoid receptor antagonist mifepristone. Corticosterone levels were increased in the serum of MS rats, and successive postnatal administration of subcutaneous corticosterone to naive rats induced mechanical allodynia in the whisker pad skin. The number of P2X3 receptor-immunoreactive (P2X3R-IR) trigeminal ganglion (TG) neurons innervating the whisker pad skin was significantly increased in MS rats and decreased following subcutaneous administration of mifepristone. The number of P2X3R-IR TG neurons innervating the whisker pad skin was also significantly increased following successive postnatal administration of subcutaneous corticosterone in naive rats. Moreover, the mechanical allodynia was suppressed 30 min after administration of the P2X3R antagonist A317491 to the whisker pad skin in MS rats. These findings suggest that the increase in P2X3R-IR TG neurons innervating the whisker pad skin via enhanced neonatal corticosterone signaling by MS plays an important role in orofacial mechanical allodynia in adulthood.

To elucidate if microglial P2Y12 receptor (P2Y12R) mechanisms are involved in the trigeminal spinal subnucleus caudalis (Vc; also known as the medullary dorsal horn) in intraoral cancer pain, we developed a rat model of tongue cancer pain. Squamous cell carcinoma (SCC) cells were inoculated into the tongue of rats; sham control rats received the vehicle instead. Nociceptive behavior was measured as the head-withdrawal reflex threshold (HWRT) to mechanical or heat stimulation applied to the tongue under light anesthesia. On day 14 after the SCC inoculation, activated microglia and P2Y12R expression were examined immunohistochemically in the Vc. The HWRT was also studied in SCC-inoculated rats with successive intra–cisterna magna (i.c.m.) administration of specific P2Y12R antagonist (MRS2395) or intraperitoneal administration of minocycline, a microglial activation inhibitor. Tongue cancer was histologically verified in SCC-inoculated rats, within which the HWRT to mechanical stimulation of the tongue was significantly decreased, as compared with that of vehicle-inoculated rats, although the HWRT to heat stimulation was not. Microglia was strongly activated on day 14, and the administration of MRS2395 or minocycline reversed associated nocifensive behavior and microglial activation in SCC-inoculated rats for 14 d. The activity of Vc wide dynamic range nociceptive neurons was also recorded electrophysiologically in SCC-inoculated and sham rats. Background activity and noxious mechanically evoked responses of wide dynamic range neurons were significantly increased in SCC-inoculated rats versus sham rats, and background activity and mechanically evoked responses were significantly suppressed following i.c.m. administration of MRS2395 in SCC-inoculated rats as compared with sham. The present findings suggest that SCC inoculation that produces tongue cancer results in strong activation of microglia via P2Y12 signaling in the Vc, in association with increased excitability of Vc nociceptive neurons, reflecting central sensitization and resulting in tongue mechanical allodynia.

The aim of the present study was to clarify the involvement of protein kinase Cγ (PKCγ) in the facial neuropathic pain following infraorbital nerve injury. We analyzed the change in PKCγ expression in the trigeminal spinal subnucleus caudalis (Vc) and upper cervical spinal cord (C1/C2) following chronic constriction injury of the infraorbital nerve (ION-CCI). We also studied ION-CCI-mediated mechanical nocifensive behavior in rats. The mechanical head-withdrawal threshold significantly decreased 1 to 14 days after ION-CCI compared with that before ION-CCI and in sham rats. The expression of PKCγ was significantly larger in the ipsilateral Vc compared with the contralateral side in ION-CCI rats 3, 7, and 14 days after ION-CCI. Intrathecal (i.t.) administration of the PKCγ inhibitor chelerythrine prevented an increase in the PKCγ expression in the ipsilateral Vc. Moreover, i.t. administration of chelerythrine annulled ION-CCI-mediated reduction in the head-withdrawal threshold. Taken together, these findings suggest that PKCγ expression in the Vc played an important role in the mechanism of orofacial static mechanical allodynia following trigeminal nerve injury.