PROJECT SUMMARY Ischemic stroke, characterized by acute loss of blood flow to the brain caused by thrombus or embolus, accounts for 80% of all stroke cases and is a leading cause of disability in adults in the U.S. The loss of blood flow creates a zone of complete infarction (the core) and a surrounding zone of surviving tissue, where much of the limited repair after stroke occurs (peri-infarct tissue). In this repair, angiogenesis and the formation of new connections (axonal sprouting) are two key components. The proposed predoctoral NRSA aims to examine innate cellular responses to a potential stroke therapeutic, a biocompatible hydrogel scaffold with nanoparticle-immobilized vascular endothelial growth factor, that promotes infiltration of endothelial cells and axons from the surrounding peri-infarct. I hypothesize that introduction of this hydrogel into a cortical stroke infarct will revascularize the infarct and promote axonal sprouting and migration of glial cells and neural progenitors into the hydrogel scaffold, and that neuronal responses, particularly axonal sprouting and synaptogenesis, will be enhanced by rehabilitation of the affected forelimb in combination with the hydrogel. I will induce cortical stroke in the mouse forelimb motor cortex and inject the novel multi-component hydrogel into the stroke cavity. In the studies proposed in Aim 1, I will use 2-photon microscopic live imaging to longitudinally record and quantify the rate of vessel growth, perfusion, and permeability within the infarct and peri-infarct. To assess vascular-guided migration, tissue will be analyzed using immunofluorescence images of cell-type-specific markers, which will enable further studies investigating post-stroke signaling, blood brain barrier, and glial biology in and without the context of a hydrogel. The second aim of the project is to virally label axons that have been shown to project into the hydrogel, allowing for identification of the parent neurons and quantification of the axonal sprouting response to the hydrogel in both the stroke core and peri-infarct. Finally, to mimic a dual therapeutic approach that would be likely in a clinical setting, a third aim will investigate the effect of motor rehabilitation on axonal sprouting and connectivity with the recovering tissue stimulated by the hydrogel. Together, these aims will elucidate fundamental biological mechanisms underlying neural repair after ischemic stroke, while also establishing a paradigm to assess longitudinal revascularization and virally labeled axonal sprouting within this novel multi-component hydrogel.