Thrombin-induced microglial activation produces degeneration of nigral dopaminergic neurons in vivo.

The present study examined whether thrombin-induced microglial activation could contribute to death of dopaminergic neurons in the rat substantia nigra (SN) in vivo. Seven days after thrombin injection into the SN, tyrosine hydroxylase immunohistochemistry showed a significant loss of nigral dopaminergic neurons. In parallel, thrombin-activated microglia, visualized by immunohistochemical staining using antibodies against the complement receptor type 3 (OX-42) and the major histocompatibility complex class II antigens were also observed in the SN, where degeneration of nigral neurons was found. Reverse transcription PCR at various time points demonstrated that activated microglia in vivo exhibited an early and transient expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and several proinflammatory cytokines, including interleukin 1beta (IL-1beta), IL-6, and tumor necrosis factor alpha. Western blot analysis and double-label immunohistochemistry showed an increase in the expression of iNOS and COX-2 and the colocalization of these proteins within microglia. The thrombin-induced loss of SN dopaminergic neurons was partially inhibited by NG-nitro-L-arginine methyl ester hydrochloride, an NOS inhibitor, and by DuP-697, a COX-2 inhibitor. Additional studies demonstrated that extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) were activated in the SN as early as 30 min after thrombin injection, and that these kinases were localized within microglia. Inhibition of ERK1/2 and p38 MAPK reduced iNOS and COX-2 mRNA expression and rescued dopaminergic neurons in the SN. The present results strongly suggest that microglial activation triggered by endogenous compound(s) such as thrombin may be involved in the neuropathological processes of dopaminergic neuronal cell death that occur in Parkinson's disease.