PROJECT SUMMARY/ABSTRACT Neonatal stroke is an important cause of death and disability, and diagnosis is often delayed. There is insufficient knowledge regarding repair mechanisms that occur in response to focal ischemia-reperfusion injury that is the most common cause of early stroke. Angiogenesis, fibrosis, and perivascular cell repopulation occur in close proximity, with paracrine signaling supporting endothelial cell interactions that are vital for repair. Modulating this neurovascular niche may be a potential target for enhancing outcomes after ischemic injury in the developing brain. Erythropoietin and cell-based therapies have emerged as promising delayed treatment strategies for stroke, although the mechanism of their benefit is still not entirely clear. It is likely that dynamic release of pro-angiogenic growth factors and activation of signaling pathways downstream of erythropoietin receptor have differential effects on endothelial cell subtypes in distinct brain regions and at different time points after injury. In addition, the defined role of local fibrosis in injury progression and repair following early focal brain injury is unknown. Effectively inducing long-term, functional angiogenesis requires understanding and mimicking mechanisms that occur in the developing brain. Our objectives are to understand local angiogenesis and fibrosis in ischemic and peri-infarct regions following focal ischemia-reperfusion injury in the developing brain, and to determine the mechanisms of regeneration and repair with delayed erythropoietin by focusing on the vascular response. In Aim 1, we will test the hypothesis that endothelial tip cells at the vascular front are critical for angiogenesis following neonatal stroke, and that delayed erythropoietin will enhance angiogenesis and alter endothelial cell-subtype gene expression profiles to promote tip cell programs. In Aim 2, we will quantify fibroblasts and perivascular cells in the ischemic core and peri-infarct penumbra in the acute, subacute, and chronic stages after stroke and determine how erythropoietin signaling impacts local fibrosis and repair. Finally, in Aim 3, we will determine and modify specific signaling pathways to test the hypothesis that dynamic endothelial cell signaling modulated by erythropoietin is crucial for promoting local angiogenesis following focal brain injury. This will determine critical, modifiable pathways important for injury progression and repair following neonatal stroke. Our primary hypothesis is that delayed erythropoietin treatment will promote vascular growth and remodeling, reduce subacute fibrosis and astrocytic proliferation in the ischemic core, and enhance perivascular signaling to improve histological and functional outcomes after neonatal stroke. Together, these three aims will explore the roles of specific cellular subtypes and pathways in recovery after focal brain injury, with the broader goal of optimizing therapeutic strategies to improve long-ter...