Cortical spreading depression, proteases and ischemia

皮质扩散性抑制（CSD）在实验性中风期间和正常范围内发展， 受创伤的人脑最近，基质金属蛋白酶（MMP）已涉及中风的发病机制和神经血管单位的完整性，通过降解基质蛋白，增强血脑屏障（BBB）的通透性和脑水肿，并促进tPA给药后出血。虽然脑损伤或血管机制提供触发器，我们最近发现，强烈的神经元和神经胶质细胞去极化在一个单一的CSD激活MMP-9至少72小时，并促进血管周围血浆蛋白渗漏。我们提出4的目的是探索新的假设，即CSD期间MMP活化部分是由神经元和胶质细胞活化直接引起的，并有助于缺血水肿的发展和正常脑中BBB破坏。目标1将确认和扩展初步数据建立 CSD作为MMP活化的触发剂，并将鉴定相关的同种型和起源细胞。目的2将检验CSD伴随着MMP激活引起的血浆蛋白渗漏的假设，并将扩展初步数据，这些数据表明：（1）CSD后，同侧血管内8 kD葡聚糖和Evans蓝的渗透性增加，（2）MMP依赖性机制降低了内皮硬抗原和层粘连蛋白的抗原性。目的3将研究在CSD过程中触发MMP激活的上游机制。由于一氧化氮（NO）S-亚硝基化MMP活性位点，以促进激活，我们将研究是否CSD诱导的NO生成触发S-亚硝基化MMP激活，如果是这样，研究相关的NOS亚型促进MMP激活使用选择性NOS敲除小鼠。我们还将 研究NF κ B通路在CSD诱导的MMP转录调节和TIMP-1表达中的重要性，TIMP-1是CSD后MMP-9活性的调节剂。目的4将检查CSD诱导的MMP激活在缺血期间的后果，并探讨MMP的缺血性病变内的血管通透性的变化，并在远程脑区的贡献。使用突变小鼠缺乏MMP-8表达的白细胞，我们建议确定MMP-8在缺血和非缺血组织中引起CSD诱导的BBB破坏的程度，并检查CSD诱导的MMP-9激活的可能性MMP-8依赖性机制在白细胞。通过这样做，我们打算探讨如何 脑缺血期间的神经元活动调节细胞外基质和神经血管单位并导致组织损伤。

Cortical spreading depression (CSD) develops during experimental stroke and within normal and traumatically-injured human brain. Recently, matrix metalloproteinases (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 and glial depolarization during a single CSD activates MMP-9 for at least 72 hr 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 8kD dextran and Evans blue increases within ipsilateral vessels and that, (2) antigenicity of endothelial harder 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 (NO) S-nitrosylates the MMP active site to promote activation, we will examine whether CSD-induced NO generation triggers S-nitrosylated 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 NFkappaB pathway in the transcriptional regulation of CSD-induced MMP and the expression of TIMP-1, as a regulator of MMP-9 activity after CSD. 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 leukocytes, we propose to determine the extent to which MMP-8 causes CSD-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 leukocytes. By so doing, we intend to explore ways in which neuronal activity during cerebral ischemia modulates the extracellular matrix and neurovascular unit and contribute to tissue injury.