Blood-Brain Barrier in Cerebral Ischemia

描述（由申请人提供）：本项目的长期目标是鉴定介导缺血诱导的脑水肿（卒中脑损伤的主要原因）的血脑屏障（BBB）离子转运蛋白。在脑缺血的早期，在存在完整的BBB的情况下发生脑水肿形成。在这个过程中，血脑屏障内皮细胞将Na和Cl从血液转运到脑屏障，随后是水。具体负责离子转运蛋白是未知的，但血脑屏障管腔钠和氯转运蛋白似乎发挥了关键作用。 许多证据表明，在缺血期间迅速发展的缺氧、随着葡萄糖耗尽而发生的血糖缺乏以及中枢释放的加压素是缺血诱导的脑水肿形成的介质。这个项目的一个新的方面是初步发现，钠-钾-氯协同转运蛋白似乎是本地化的脑微血管内皮细胞的管腔膜和加压素，缺氧和血糖刺激活性的协同转运蛋白。这导致了一个中心假设，即位于BBB腔膜的Na-K-Cl协同转运蛋白在缺血期间受到刺激，以增加Na和Cl从血液到脑的转运，从而导致水肿形成。本项目有三个具体目标。第一个目的是检验脑微血管内皮细胞（CMEC）腔膜中存在钠-钾-氯共转运的假设。这些研究将通过放射性同位素通量分析评价牛脑微血管腔和近腔膜制剂的共转运活性。此外，将通过脑切片的免疫电子显微镜检查协同转运蛋白的原位分布。第二个目的是检验BBB内皮细胞的Na-K-Cl共转运由介导缺血诱导的脑水肿的试剂刺激的假设。在这里，将在培养的人和牛CMEC和新鲜分离的牛脑微血管中检查缺氧，血糖和加压素对共转运活性的影响。第三个目的是检验抑制Na-K-Cl共转运活性减轻缺血诱导的脑水肿的假设。为此，将通过核磁共振方法检查抑制协同转运蛋白对缺血诱导的大鼠脑Na和水变化的影响，该方法允许在真实的时间内跟踪脑Na和水的体内变化。拟议的研究应揭示是否旨在阻断血脑屏障钠钾氯协同转运活性的治疗方法可能是有价值的衰减缺血诱导的脑水肿。

DESCRIPTION (provided by applicant): The long term goal of this project is to identify blood-brain barrier (BBB) ion transporters that mediate ischemia induced brain edema, a major cause of brain damage in stroke. During the early hours of cerebral ischemia, brain edema formation occurs in the presence of an intact BBB. In this process, BBB endothelial cells transport Na and Cl from blood into brain interstitium, with osmotically obliged water following. The specific ion transporters responsible are unknown, however BBB luminal Na and Cl transporters appear to play a key role. Much evidence indicates that hypoxia, which rapidly develops during ischemia, aglycemia occurring as glucose is depleted, and also centrally-released vasopressin are mediators of ischemia-induced brain edema formation. A novel aspect of this proposed project is the preliminary finding that a Na-K-Cl cotransporter appears to be localized in the luminal membrane of brain microvessel endothelial cells and that vasopressin, hypoxia and aglycemia stimulate activity of the cotransporter. This has led to the central hypothesis that a Na-K-Cl cotransporter, located at the luminal membrane of the BBB, is stimulated during ischemia to increase transport of Na and Cl with osmotically obliged water from blood to brain, causing edema formation. The present project has three specific aims. The first aim is to test the hypothesis that Na-K-Cl cotransport is present in luminal membranes of cerebral microvascular endothelial cells (CMEC). These studies will evaluate bovine brain microvessel luminal and abluminal membrane preparations for cotransport activity by radioisotopic flux analyses. Also, the in situ distribution of the cotransporter will be examined by immunoelectron microscopy of brain sections. The second aim is to test the hypothesis that Na-K-CI cotransport of BBB endothelial cells is stimulated by agents that mediate ischemia-induced cerebral edema. Here, the effects of hypoxia, aglycemia and vasopressin on cotransport activity will be examined in cultured human and bovine CMEC and freshly isolated bovine cerebral microvessels. The third aim is to test the hypothesis that inhibition of Na-K-Cl cotransport activity attenuates ischemia-induced brain edema. To do this, the effect of inhibiting the cotransporter on ischemia-induced changes in rat brain Na and water will be examined by nuclear magnetic resonance methods, which allow in vivo changes in brain Na and water to be followed in real time. The proposed studies should reveal whether therapeutic approaches aimed at blocking BBB Na-K-Cl cotransporter activity may be of value for attenuating ischemia-induced brain edema.