Project Summary/Abstract This project (begun in 1984) continues its long-term goal to enhance understanding of stroke by novel mechanistic investigations. Our principal interest is cerebral microhemorrhage, the pathological substrate of cerebral microbleeds and the major hemorrhagic component of microvascular cerebral disease. The proposed project will utilize a combination of novel animal models of cerebral microbleeds, new cell culture models of microbleeds that address red blood cell (RBC)-endothelial interactions, and cutting edge analytic techniques. Our objective is to identify mechanisms that broadly impact development of cerebral microbleeds as well as those more specifically related to individual vascular risk factors for microbleeds, The project investigators are a unique group of investigators with expertise in stroke neurology, vascular neurobiology, nephrology, bioengineering, and biostatistics, all with capabilities relevant to animal and cell culture studies. We specifically propose the following aims: Specific Aim 1: To determine mechanisms of cerebral microhemorrhage in vivo. Specific Aim 2: To delineate paracellular mechanisms of RBC passage across brain microvascular endothelium in vitro. Specific Aim 3: To delineate transcellular mechanisms of RBC passage across brain microvascular endothelium. The proposed studies are designed as a series of synergistic experiments that combine in vivo and in vitro studies to provide novel insights into microvascular brain disease. We will use a combination of studies of microvascular physiology and neuropathology to identify the vascular source of cerebral microbleeds and to delineate those interactions between RBC and brain microvascular endothelium that immediately precede establishment of cerebral microbleeds. The project is a direct extension of work we completed during the most recent funding period of this grant and incorporate new animal models of cerebral microbleeds and novel conceptual models describing microbleed pathogenesis. Our in vivo studies will address mechanisms of aging, hypertension, and chronic kidney disease in microbleed development, along with the roles of microglia and matrix metalloproteinase-9. This project directly addresses the highly prevalent problem of hemorrhagic microvascular disease of the brain. Our work and that of others have shown the near-ubiquitous presence of microhemorrhagic changes in aging human brain. Our multi-disciplinary team of investigators are uniquely capable of taking this project to completion, directly testing our microbleed conceptual models in the expectation that robust insights will emerge helping to transform the management of microvascular disorders of the brain which, along with Alzheimer’s disease, represents the most common cause of neurological morbidity of aging.