This Mentored Clinical Scientist Development (K08) Award proposal focuses on thrombin's induction of microglial cell activation in stroke. Inflammatory responses in the brain are critical in the pathophysiology of stroke. The coagulation proteinase thrombin, proteolytically activated receptors (PARs) and microglial cells have all been strongly implicated in the initial steps of this inflammatory response. In the setting of stroke, thrombin can extravasate across an ischemia-compromised blood-brain barrier, activate its receptors (PARs-1, -3 and -4) and elicit a wide range of cellular responses from neurons and glia. Recent data suggests that induction of thrombin/PAR signaling also stimulates activation of microglia - the immune cells of the brain. This activation in turn helps initiate/orchestrate the brain's inflammatory response to ischemia. For these proposed studies, our first Specific Aim will be to characterize the cellular consequences of microglial activation by thrombin in vitro. These experiments on cultured microglia will include quantifying changes in proliferation, viability, cytokine elaboration and antigenic profile in the absence or presence of ischemia-related conditions (hypoglycemia, hypoxia, oxygen-glucose deprivation). We will then use PCR and flow cytometric techniques to characterize PAR expression in quiescent and activated microglia at both the transcriptional and post-translational levels.
In Aim two, we will carry out in vivo studies focusing on the effects of systemic thrombin inhibitors and PAR antagonists on a variety of outcome parameters in an experimental animal paradigm for stroke: rat middle cerebral artery occlusion (MCAO)/reperfusion. Laser doppler flowmetry will be used to characterize changes in cerebral blood flow and confirm ischemic conditions. Standard immunohistochemical techniques and a newly established ex-vivo flow cytometric method will be used to determine infarct volume and extent of microglial activation/PAR expression, respectively following MCAO/reperfusion. Changes in neuro-behavioral outcomes will also be measured. In both Aims we will also address the impact of thrombin-induce cross-tolerance and ischemic pre-conditioning on microglial activation in stroke. By studying the impact of thrombin's PAR-mediated activation of microglia in ischemia we hope to both improve our understanding of the mechanism of neuro-inflammation in stroke and identify possible targets for therapeutic intervention. ? ?
