This proposal for a NIH Small Research Grant Program (R03) focuses on determining the effect of ischemic preconditioning (IPC) on the microglial cell transcriptome. IPC is a robust neuroprotective phenomenon in which a brief period of cerebral ischemia confers transient tolerance to subsequent ischemic challenge. Characterization of the cellular and molecular mechanisms that underlie IPC is an important and active area of investigation in stroke research. Microarray analyses on RNA isolated from whole brain tissue that has undergone ischemia with or without prior IPC has demonstrated that both IPC and ischemia (alone or in sequential combination) induce large scale genomic reprogramming responses that are each distinct (and even disparate) in nature. However, little is known about the genomic changes that occur at the cellular level. Inflammatory responses in the brain are critical in the pathophysiology of stroke. Microglia, the brain's resident tissue macrophages, are critical in this process. The role of microglia in the IPC phenomenon is unknown. Here we will combine a well-established in vivo model of stroke (the mouse middle cerebral artery occlusion/reperfusion (MCAO/R) paradigm) with a novel form of cellular analysis (genomic microarray characterization of ex vivo flow cytometrically sorted microglia). The project requires that we establish and optimize two methods: (A) mouse MCAO/R model with the IPC paradigm and (B) ex vivo flow cytometric technique for acute isolation of resident microglia from adult mouse brain tissue. We will then perform microarray genomic analysis on RNA extracted from microglia isolated from mice that have undergone IPC and/or stroke (via MCAO/R). Establishment of this cell-type- and disease- specific genomic dataset will allow for ontological analysis as well as identification of promoter elements, transcription factors, signaling pathways and individual genes that are activated/regulated specifically in microglia following IPC and/or stroke. Establishment of the dataset will also allow for direct comparison between the microglia-specific and (the previously published) whole-brain derived ischemic preconditioned and/or """"""""stroked"""""""" mouse transcriptomes. We anticipate that the dataset, which will be made available to the scientific community through posting on the Gene Expression Omnibus, will be valuable to researchers trying to elucidate the cellular and molecular mechanisms of IPC, neuroinflammation and stroke. It should provide key insights into the pathophysiologic state of microglia in the ischemic penumbra and enhance our ability to generate novel hypotheses about the role of microglia in IPC. It may also prove effective in identifying molecular targets for therapeutic intervention in acute stroke.
Stroke is the leading cause of serious long-term disability and the third leading cause of death in the United States. Currently available pharmacologic therapies for acute stroke are few in number and limited by temporal restrictions on their use, modest efficacy and potential for serious side effects. A major goal of this research project is to increase our mechanistic understanding of stroke pathophysiology. By doing so, we hope to identify novel cellular and molecular targets for therapeutic intervention in acute stroke.
