Defining the Spatiotemporal Underpinnings of Neutrophil Recruitment, Microvascular Flow, and Oxygenation in Ischemic Stroke

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Project Summary: While mechanical thrombectomy has advanced the treatment of large vessel occlusion (LVO) stroke, over 50% of patients still suffer from significant disability or death. Ischemia/reperfusion injury (I/RI), the result of restoring blood flow to deeply ischemic tissue, accelerates recruitment of polymorphonuclear neutrophils (PMNs). PMNs exert poor outcomes in two ways. First, PMNs physically obstruct cerebral microvessels in the stroke bed despite macrovascular reperfusion – a condition known as microvascular ‘no-reflow’. Second, PMNs exert toxic effects regionally once recruited at postcapillary venules and extravasated into the infarcted brain. Accordingly, preclinical studies that block PMN recruitment have had success in reducing stroke burden and improving neurologic outcome. Unfortunately, these preclinical studies have not been successful in human trials. These translational roadblocks may be addressed by investigating the spatiotemporal determinants of PMN recruitment as it relates to the in vivo cerebrovasculature during stroke. Using a mouse stroke model to simulate the LVO population and novel histopathological and imaging techniques, my preliminary data have found that PMN recruitment throughout I/RI is non-uniform up to 72 hours after infarction. PMNs were also found to progress cortically to subcortically throughout I/RI over the course of 72 hours, with partial restriction to the cortical surface by administration of an antibody that blocks transendothelial migration (TEM). These results support the concept of stroke microenvironments – highly regionalized areas within an infarct where inflammation and impaired microcirculation interface with each other. I hypothesize that these stroke microenvironments within an infarct are due to feedback loops between 1) microvascular flow and oxygenation; and 2) PMN recruitment. To test this hypothesis, I will investigate two aims: 1) Defining and physiologically manipulating the stroke microenvironment 2) Determine how PMN infiltration and position regulates I/RI over time. I will test these aims using techniques of multimodal in vivo animal imaging, advanced microscopy, and targeted manipulation of both leukocyte biology and stroke physiology. These studies will ultimately be used to identify molecular similarities of PMNs in particularly toxic stroke microenvironments, facilitating the investigation and creation of novel leukocyte-based therapies. To complete these long-term goals, I will incorporate a multidisciplinary mentorship team and short-term goals of developing expertise in live-animal imaging, advanced microscopy, and leukocyte biology. With this K08 proposal, I will build a unique translational stroke program that defines the interplay of stroke physiology and pathology to develop more precise and translatable therapies for stroke patients.