Cortical Spreading Depression, Proteases and Ischemia

描述(由申请人提供)：皮质扩散性抑制(CSD)在实验性中风期间以及在正常和创伤后的人脑内发展。近年来，基质金属蛋白酶(MMPs)通过降解基质蛋白、增加血脑屏障(BBB)通透性和脑水肿，以及在tPA应用后促进出血，参与了卒中的发病机制和神经血管单位的完整性。虽然脑损伤或血管机制提供了触发因素，但我们最近发现，CSD期间强烈的神经元胶质去极化至少会激活MMP9 72小时。并促进血管周围血浆蛋白渗漏。我们提出的4个目的是为了探索这一新的假设，即CSD期间MMPs的激活部分是由神经元和神经胶质细胞的激活直接引起的，并参与了正常脑缺血时的水肿和血脑屏障的破坏。目标1将确认和扩展初步数据，确定CSD是触发基质金属蛋白酶激活的因素，并将确定相关的异构体和起源细胞。目的2验证CSD伴有基质金属蛋白酶(MMPs)激活引起的血浆蛋白泄漏的假说，并对初步数据进行扩展，结果表明：(1)CSD后同侧血管内伊文思蓝通透性增加，(2)内皮细胞屏障抗原和层粘连蛋白的抗原性通过依赖于MMPs的机制降低。AIM 3将研究在慢性阻塞性肺疾病期间触发基质金属蛋白酶激活的上游机制。由于一氧化氮S通过激活基质金属蛋白酶活性部位来促进基质金属蛋白酶的活化，因此我们将通过选择性一氧化氮合酶基因敲除小鼠来检测慢性阻塞性肺疾病诱导的一氧化氮的产生是否触发基质金属蛋白酶的活化，如果是的话，检测相关的一氧化氮合酶亚型促进基质金属蛋白酶的活化。我们还将研究NFkB和其他途径在基质金属蛋白酶-9转录调控中的重要性。目的4研究CSD诱导的基质金属蛋白酶在缺血时激活的后果，并探讨基质金属蛋白酶在缺血损伤和远隔脑区血管通透性改变中的作用。利用缺乏表达在白细胞上的基质金属蛋白酶-8的突变小鼠，我们建议确定基质金属蛋白酶-8在多大程度上引起缺血和非缺血组织中基质金属蛋白酶诱导的血脑屏障破坏，并通过白细胞中基质金属蛋白酶-8依赖的机制检测CSD诱导基质金属蛋白酶-9激活的可能性。通过这样做，我们打算探索脑缺血期间神经元活动调节细胞外基质和神经血管单位并促进组织损伤的方式。

DESCRIPTION (provided by applicant): Cortical spreading depression (CSD) develops during experimental stroke and within normal and traumatically injured human brain. Recently, (MMP) have been implicated in stroke pathogenesis and in the integrity of the neurovascular unit by degrading matrix proteins, enhancing blood brain barrier (BBB) permeability and brain edema, and promoting hemorrhage after tPA administration. Although brain injury or vascular mechanisms provide triggers, we recently found that intense neuronal glial depolarization during CSD activated MMP-9 for at least 72 hrs. and promotes perivascular plasma protein leakage. We propose 4 Aims to explore the novel hypothesis that MMP activation during CSD is caused in part by neuronal and glial activation directly and contributes to the development of edema in ischemia and BBB disruption in normal brain. Aim 1 will confirm and extend preliminary data establishing CSD as a trigger for MMP activation and will identify relevant isoforms and cells of origin. Aim 2 will test the hypothesis that CSD is accompanied by leakage of plasma protein caused by MMP activation and will extend preliminary data showing that, (1) permeability of Evans blue increases within ipsilateral vessels and that, (2) antigenicity of endothelial barrier antigen and laminin is decreased by MMP-dependent mechanisms after CSD. Aim 3 will examine upstream mechanisms triggering MMP activation during CSD. Because nitric oxide s-nitrosylates the MMP active site to promote activation, we will examine whether CSD-induced NO generation triggers MMP activation and if so, examine the relevant NOS isoform promoting MMP activation by using selective NOS knockout mice. We will also examine the importance of NFkB and other pathways in the transcriptional regulation of MMP-9. Aim 4 will examine the consequences of CSD-induced MMP activation during ischemia and explore the contributions of MMPs to vascular permeability changes both within the ischemic lesion and in remote brain areas. Using mutant mice lacking MMP-8 expressed on leucocytes, we propose to determine the extent to which MMP-8 causes MMP-induced BBB disruption in ischemic and non-ischemic tissue, and examine the potential for CSD-induced MMP-9 activation by MMP-8 dependent mechanisms in leucocytes. By doing so, we intend to explore ways in which neuronal activity during cerebral ischemia modulates the extracellular matrix and neurovascular unit and contribute to tissue injury.