The main focus of research in the Weinstein lab is to elucidate the role of microglia in the neuroimmune response associated with ischemic preconditioning and stroke. Ischemic preconditioning (IPC) in the brain is a robust neuroprotective phenomenon in which a brief ischemic exposure increases resistance to the injurious effects of subsequent prolonged ischemia. Microglia, the brain's resident tissue macrophages, are primary mediators of neuroinflammation and are critical in the pathophysiology of stroke. Mechanistic information on the function of microglia in ischemia is limited and the role of microglia in IPC is unknown.
Stroke is the leading cause of serious long-term disability in the United States and few stroke patients qualify for the only FDA approved therapy, IV tPA. Thus, our ultimate goal is to improve the understanding of the mechanisms of neuroinflammation in stroke and identify possible molecular targets for therapeutic intervention.
The Weinstein Lab employs both in vivo and in vitro experimental models of ischemia to study microglial responses. For our in vivo studies, we couple the mouse middle cerebral artery occlusion (MCAO) stroke model with ex vivo flow cytometric isolation of microglia (and macrophage subsets as well) from cortex. To characterize the neuroimmune response, we carry out cell targeted microarray analyses and qRT-PCR on the sorted cortical microglia. We also use flow cytometry to assess changes in cell surface antigen expression and phosphorylation of key intracellular signaling kinases. We use immunofluorescent microscopy coupled with quantitative stereology to further assess microglial morphology, cell number and phenotype. In order to determine infarct volume in our stroked mice we use both standard histological methods and multiparametric MRI. In addition, we evaluate neurobehavioral outcomes. Using this multi-faceted approach, we are able to compare the microglial response to IPC/ischemia in wild-type mice with that of microglia in selected innate immune signaling-related knockout and cell type specific knockdown lines. Correspondingly, in parallel studies we are able to elucidate the effects of experimental treatments that modulate the neuroimmune response on a variety of outcome parameters.
For our in vitro experimental paradigms, we expose cultured primary mouse microglia or astrocytes to a variety of specific stimuli and/or ischemia/reperfusion-like conditions and then monitor an array of experimental parameters including cell viability, surface antigen expression, gene expression and intracellular phosphorylation/signaling. Past studies have included characterizing the cellular consequences of microglial activation by the blood coagulation proteinase thrombin -- an important factor in stroke pathophysiology. Our more recent work has implicated both Toll-like receptor-4 (TLR4) and type 1 interferon (IFN)-stimulated genes as key mediators of the microglial response to ischemia/IPC. Both TLR4 and the IFN family of cytokines are recognized as key components of the innate immune response. Ongoing projects in our laboratory are examining how disruption of the TLR and/or type 1 IFN signaling pathways, specifically in microglia, can affect both IPC and stroke.
