Blood-Brain Barrier in Cerebral Ischemia

描述（由申请人提供）：该项目的长期目标是确定介导缺血引起的脑水肿的血脑屏障（BBB）离子转运体，这是中风脑损伤的主要原因。在脑缺血早期，脑水肿的形成是在血脑屏障完好的情况下发生的。在这一过程中，血脑屏障内皮细胞将Na和Cl从血液中转运到脑间质，随后是渗透吸收的水。具体的离子转运蛋白是未知的，然而血脑屏障内钠和氯转运蛋白似乎起着关键作用。大量证据表明，缺血期间迅速发生的缺氧、葡萄糖耗竭时发生的血糖以及中央释放的加压素是缺血诱导脑水肿形成的介质。这个项目的一个新颖方面是初步发现Na-K-Cl共转运蛋白似乎定位于脑微血管内皮细胞的管腔膜，并且加压素、缺氧和血糖刺激了共转运蛋白的活性。这导致了一个中心假设，即位于血脑屏障管腔膜的Na- k -Cl共转运体在缺血期间受到刺激，增加Na和Cl随渗透性水从血液到脑的运输，导致水肿形成。目前的项目有三个具体目标。第一个目的是验证脑微血管内皮细胞（CMEC）管腔膜中存在Na-K-Cl共转运的假设。这些研究将通过放射性同位素通量分析来评价牛脑微血管管腔和管腔膜制剂的共转运活性。此外，将通过脑切片的免疫电镜检查共转运体的原位分布。第二个目的是验证血脑屏障内皮细胞的Na-K-CI共运输被介导缺血诱导脑水肿的药物刺激的假设。本文将在培养的人和牛CMEC以及新鲜分离的牛大脑微血管中研究缺氧、血糖和加压素对共转运活性的影响。第三个目的是验证Na-K-Cl共转运活性的抑制可以减轻缺血性脑水肿的假设。为了做到这一点，抑制共转运体对缺血诱导的大鼠脑钠和水变化的影响将通过核磁共振方法进行检测，该方法可以实时跟踪体内脑钠和水的变化。拟议的研究应揭示旨在阻断血脑屏障Na-K-Cl共转运蛋白活性的治疗方法是否可能对减轻缺血诱导的脑水肿有价值。

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.