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

项目总结：虽然机械血栓切除术推进了大血管闭塞的治疗 (LVO)中风后，超过50%的患者仍然遭受严重残疾或死亡。缺血/再灌注损伤 （I/RI），恢复血流到深度缺血组织的结果，加速多形核细胞的募集， 中性粒细胞（PMNs）。PMNs在两个方面产生不良结果。首先，PMNs在物理上阻碍 脑微血管在中风床尽管大血管再灌注-一种条件称为微血管 “无回流”。第二，PMNs一旦在毛细血管后小静脉聚集并外渗， 注入梗塞的大脑因此，阻断PMN募集的临床前研究已经成功地减少了 卒中负担和改善神经功能结局。不幸的是，这些临床前研究还没有被 在人体试验中取得成功。这些翻译障碍可以通过调查时空 中性粒细胞募集的决定因素，因为它涉及在中风期间体内血管。使用鼠标 中风模型，以模拟LVO人口和新的组织病理学和成像技术，我的初步 数据已经发现，在梗塞后72小时内，I/RI期间的PMN募集是不均匀的。PMNs是 还发现在72小时的过程中，在整个I/RI中从皮质到皮质下进展，部分限制 通过给予阻断跨内皮迁移（TEM）的抗体将其转移到皮质表面。这些 结果支持中风微环境的概念--梗塞内高度区域化的区域， 炎症和微循环受损相互作用。我假设这些中风 梗塞内的微环境是由于1）微血管流动和氧合之间的反馈回路; （2）招聘PMN。为了验证这一假设，我将研究两个目标：1）定义和生理学 2）确定PMN浸润和位置如何调节I/RI， 时间我将使用多模式体内动物成像技术，先进的显微镜， 白细胞生物学和中风生理学的靶向操纵。这些研究最终将用于 确定在特别有毒的中风微环境中中性粒细胞的分子相似性，促进调查 和创造新的基于白细胞的疗法。为了实现这些长期目标，我将把一个 多学科指导团队和发展活体动物成像专业知识的短期目标 显微镜和白细胞生物学。有了这个K 08提案，我将建立一个独特的平移笔画程序， 定义了中风生理学和病理学的相互作用，以开发更精确和可翻译的治疗方法， 中风患者

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.