Pericytes as Inducers of Blood-brain Barrier Injury During Stroke

 描述（由申请人提供）：缺血性血脑屏障（BBB）破坏是卒中期间组织损伤的主要原因。了解调节这一过程的机制将导致新的方法来减轻脑损伤和延长溶栓治疗的窗口。周细胞对于BBB的发育和维持至关重要。然而，很少有人知道他们对血脑屏障完整性的影响在成人脑缺血损伤。使用体内双光子显微镜，我们已经发现，周细胞引起点状血脑屏障中断，特别是在他们的胞体。周细胞胞体仅占毛细血管总表面积的7%，其余部分则由其精细而广泛的突起所覆盖。然而，有害的血液传播分子可以扩散到远远超出外渗点的地方，这加强了我们了解这种未表征的泄漏途径的必要性。我们的中心假设是，周细胞迅速上调基质金属蛋白酶的活性，导致局部解体的内皮紧密连接（细胞旁渗漏）。这一假设将使用最先进的方法进行检验。在目标1中，我们将使用荧光明胶探针，在周细胞标记的小鼠中诱导中风后，使用体内双光子显微镜直接观察MMP 9活性。在 将进行体内药理学和周细胞特异性缺失实验，以测试可导致快速MMP 9活化的推定信号级联。将进行离体生物化学研究以确认这些信号级联的作用。在目标2中，我们将使用3-D连续块面电子显微镜检查的性质，在周细胞胞体的内皮细胞破坏。我们的研究结果将与邻近的毛细血管区不包括胞体。拟议的研究是重要的，因为它预计将周细胞定义为缺血期间BBB损伤的诱导物，这与它们在发育和正常脑功能期间作为BBB完整性的养育者的新兴作用形成对比。

 DESCRIPTION (provided by applicant): Ischemic blood-brain barrier (BBB) disruption is a major contributor to tissue injury during stroke. Understanding the mechanisms that regulate this process will lead to new approaches to mitigate brain damage and lengthen the window for thrombolytic treatment. Pericytes are essential for development and maintenance of the BBB. However, little is known about their impact on BBB integrity during ischemic injury in the adult brain. Using in vivo two-photon microscopy, we have found that pericytes elicit punctate BBB disruptions specifically at their somata. Pericyte somata cover only 7% of the total capillary surface, while their fine and extensive processes cover the rest. However, harmful blood-borne molecules can diffuse far beyond the point of extravasation, strengthening our need to understand this uncharacterized leakage route. Our central hypothesis is that pericytes rapidly upregulate matrix metalloproteinase activity, leading to local disassembly of endothelial tight junctions (paracellular leakage). This hypothesis will be tested using state-of- the-art approaches. In Aim 1, we will use in vivo two-photon microscopy to directly visualize MMP9 activity using a fluorescent gelatin probe, following stroke induction in pericyte-labeled mice. In vivo pharmacological and pericyte-specific deletion experiments will be performed to test putative signaling cascades that can lead to rapid MMP9 activation. Ex vivo biochemical studies will be performed to confirm the role of these signaling cascades. In Aim 2, we will use 3-D serial block-face electron microscopy to examine the nature of endothelial disruption at pericyte somata. Our findings will be compared with neighboring capillary regions not covered by somata. The proposed research is significant because it is expected to define pericytes as inducers of BBB injury during ischemia, which contrasts their emerging role as nurturers of BBB integrity during development and normal brain function.