The Mechanism Of The Involvement Of NMDA Receptor NR2B Subunit In Central Sensitization Of Hypothalamic Arcuate Nucleus Following Inflammation Pain

Objective:To explore the mechanisms of central sensitization of hyperalgesia on supraspinal level by studying the glutamate receptor signal transduction in the hypothalamic arcuate nucleus following peripheral inflammation, and the role of NR2B subunit of NMD A receptor and Src protein tyrosine kinase in this phenomenon.Methods:The peripheral inflammation model was induced by sub-plantar injection of complete Freund’s adjuvant in rats. Pain threshold was measured by mechanical and thermal withdrawal methods. The experiment was performed on isolated mediobasal hypothalamus slice to observe the changes of spontaneous discharge of neurons in arcuate nucleus. The NMD A receptor antagonist MK-801, NR2B subunit antagonist Ro25-6981and Src protein tyrosine kinase inhibitor PP2were used to analyze the receptor mechanism and the signal transduction in the ARC neurons of normal rats and the CFA rats. The protein levels of phospho-NR1, phospho-NR2B and phospho-Src, as well as total NRl and NR2B in arcuate nuleus were detected using Western blot analysis method. The co-immunoprecipitation was used to study whether the NR2B subunit of the NMDAR is linked to Src protein tyrosine kinase in ARC. The Src inhibitor PP2was microinjected into the ARC before injection of CFA and the protein levels of phospho-NR2B and perception of painful sensation in rats was measuredResults:After adjuvant injection the pain threshold decreased significantly (P<0.01). The spontaneous discharge frequency of ARC neurons in CFA-inflamed rats was significantly higher than that in normal rats. The effect could be partially blocked by MK-801, Ro25-6981and PP2. Western blot analysis suggested that the NMDA receptor NR2B subunit phosphorylation was up-regulated; the level of Src phosphorylation was also up-regulated in ARC after peripheral inflammation. Co-immunoprecipitation indicated that NR2B closely associated with pSrc in ARC. Moreover, after PP2microinjection into the ARC in inflamed rats, the protein level of tyrosine-phosphorylated NR2B protein was decreased and the pain threshold was increased significantly.Conclusions:(1)The firing rate of spontaneous discharges of ARC neurons in inflamed rats was significantly higher and the pain threshold decreased significantly following peripheral inflammation (2)NMDA receptor and NR2B subunit are involved in the increase of the firing rate of ARC neurons in inflamed rats,(3) Src-dependent phosphorylation of NR2B might play a crucial role for producing central sensitization in the arcuate nucleus of mediobasal hypothalamus associated with peripheral inflammation.(4) In hypothalamic ARC, one of the key supraspinal centers for pain modulation, these results indicate that Src tyrosine kinase activation induced by CFA injected into sub-plantar contributes to the development and maintenance of inflammatory hyperalgesia by mediating NMDA receptor NR2B subunit.