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)破坏是中风期间组织损伤的主要因素。了解调节这一过程的机制将导致减轻脑损伤和延长溶栓治疗窗口的新方法。周细胞对于血脑屏障的发育和维持是必不可少的。然而，在成人脑缺血损伤过程中，它们对血脑屏障完整性的影响知之甚少。使用活体双光子显微镜，我们发现周细胞在其体细胞上引起点状的血脑屏障破坏。周细胞体细胞仅占整个毛细血管表面的7%，而其细小而广泛的突起覆盖了其余的部分。然而，有害的血液传播分子可以扩散到远远超出渗出点的地方，这加强了我们了解这种未知的渗漏途径的必要性。我们的中心假设是，周细胞迅速上调基质金属蛋白酶活性，导致内皮紧密连接的局部解体(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，我们将使用体内双光子显微镜，在周细胞标记的小鼠卒中诱导后，使用荧光明胶探针直接观察MMP9的活性。在……里面 将进行体内药理学和周细胞特异性缺失实验，以测试可能导致MMP9快速激活的信号级联反应。将进行体外生化研究，以确认这些信号级联的作用。在目标2中，我们将使用3-D系列块面电子显微镜来检查周细胞胞体内皮细胞破坏的性质。我们的发现将与邻近未被体细胞覆盖的毛细血管区域进行比较。这项拟议的研究具有重要意义，因为它有望将周细胞定义为缺血期间血脑屏障损伤的诱导者，这与它们在发育和正常脑功能期间作为血脑屏障完整性养育者的新角色形成了鲜明对比。

 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.