Blood-Brain Barrier in Cerebral Ischemia

描述(申请人提供)：该项目的长期目标是确定血脑屏障(BBB)离子转运体，以介导脑缺血引起的脑水肿，这是中风脑损伤的主要原因。在脑缺血的早期，在完整的血脑屏障存在的情况下会形成脑水肿。在这个过程中，血脑屏障内皮细胞将钠和氯从血液输送到脑间质，随后是渗透压作用的水。具体负责的离子转运体尚不清楚，但血脑屏障腔内的钠和氯转运体似乎发挥了关键作用。大量证据表明，缺血时迅速发展的低氧、葡萄糖耗竭时发生的血糖升高以及中枢释放的加压素都是缺血诱导脑水肿形成的介质。这个项目的一个新的方面是，初步发现Na-K-Cl共转运体似乎定位于脑微血管内皮细胞的管腔膜上，并且血管加压素、低氧和血糖刺激了该共转运体的活性。这导致了一个中心假设，即位于血脑屏障管腔膜上的Na-K-Cl协转运体在缺血期间被刺激，以增加Na和Cl与渗透压水从血液到脑的运输，导致水肿形成。本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)管腔膜上存在Na-K-Cl共转运的假设。这些研究将通过放射性同位素流量分析评估牛脑微血管腔和腔膜制剂的共转运活性。此外，还将通过脑切片的免疫电子显微镜检查共转运体的原位分布。第二个目的是验证一种假说，即血脑屏障内皮细胞的Na-K-CI共转运是由介导缺血引起的脑水肿的药物刺激的。在这里，我们将在培养的人和牛的CMEC和新鲜分离的牛脑微血管上检测低氧、血糖和加压素对共转运活性的影响。第三个目的是验证抑制Na-K-Cl共转运蛋白活性可减轻脑缺血所致脑水肿的假说。要做到这一点，将通过核磁共振方法检测抑制共转运体对缺血诱导的大鼠脑组织钠和水变化的影响，从而实时跟踪体内脑组织钠和水的变化。拟议的研究应该揭示旨在阻断血脑屏障Na-K-Cl共转运体活性的治疗方法是否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.