Blood-Brain Barrier Ion Transport 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的血脑屏障（BBB）离子转运蛋白。在缺血性卒中的早期，在存在完整BBB的情况下，通过涉及血脑屏障将Na和Cl从血液转运到脑中的过程形成水肿。我们之前的研究表明，存在于腔BBB膜中的Na-K-Cl共转运（NKCC）和Na/H交换（NHE）受到缺血因素（包括缺氧、血糖过低和加压素（AVP））的刺激，并且抑制BBB NKCC和/或NHE活动可以减少大鼠大脑中动脉闭塞（MCAO）中风模型中的脑水肿。因此，BBB、NKCC和NHE似乎是减少缺血诱导的脑水肿的有希望的治疗靶点。高血压是卒中中常见的合并症，约30%的患者存在，可导致更严重的脑水肿。然而，对潜在的机制知之甚少。近年来的研究发现，高血糖可增加血脑屏障NKCC和NHE的表达和活性，并增强缺血因素对它们的刺激作用。此外，我们已经发现证据表明，在MCAO中脑水肿的高血糖恶化通过抑制BBB NKCC和NHE而减少。我们的假设是，血脑屏障NKCC和NHE参与高血糖诱导的脑水肿形成的增加，并构成有效的治疗高血糖中风的目标。我们最近还发现了证据表明，Na-HCO 3协同转运蛋白（NBC）是第三个突出的BBB Na转运蛋白，它受缺血因素刺激，高血糖增加BBB NBC的表达和活性，这表明BBB NBC可能提供一个额外的治疗靶点，用于减少缺血性卒中（血糖正常和高血糖）中水肿的形成。因此，我们的研究也将探讨这种可能性。第一个目的是确定高血糖状况是否增加BBB NKCC和/或NHE的表达和活性，并增加缺血因子对NKCC和NHE的刺激。我们将采用Western blot、显微荧光分光光度法和放射性同位素通量法检测高血糖对脑微血管内皮细胞（CMEC）NKCC和NHE蛋白和活性的影响。我们将采用免疫电镜技术对高血糖大鼠血脑屏障中的NKCC和NHE进行原位观察。第二个目的是确定BBB NKCC和/或NHE的抑制是否减弱高血糖动物中缺血诱导的水肿。在这里，我们将研究BBB NKCC和NHE抑制对缺血诱导的大鼠脑钠和水的变化的影响，使用核磁共振方法和STZ诱导的高血糖大鼠。我们还将评估NKCC和NHE在缺血发作后给药时减少脑水肿的疗效。第三个目的是确定NBC蛋白是否存在于腔BBB膜上并受到缺血因子的刺激。我们将使用Western blot和免疫电镜来评估CMEC和BBB原位中的BBB NBC蛋白，并使用显微荧光光谱法来评估缺血因素和高血糖对CMEC NBC活性的影响。

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. During the early hours of ischemic stroke, edema forms in the presence of an intact BBB by a process involving BBB transport of Na and Cl from blood into brain. Our previous studies have shown that Na-K-Cl cotransport (NKCC) and Na/H exchange (NHE), present in the luminal BBB membrane, are stimulated by ischemic factors, including hypoxia, aglycemia and vasopressin (AVP) and that inhibiting BBB NKCC and/or NHE activity reduces brain edema in the rat middle cerebral artery occlusion (MCAO) model of stroke. Thus, BBB NKCC and NHE appear to be promising therapeutic targets for reducing ischemia- induced brain edema. Hyperglycemia, a common co-morbidity in stroke present in ~30% of patients, causes greater cerebral edema. However, the underlying mechanisms are poorly understood. In recent studies we have found that hyperglycemia increases expression and activity of BBB NKCC and NHE and augments their stimulation by ischemic factors. Further, we have found evidence that the hyperglycemia exacerbation of brain edema in MCAO is reduced by inhibition of BBB NKCC and NHE. Our hypothesis is that BBB NKCC and NHE participate in the hyperglycemia-induced augmentation of cerebral edema formation and constitute effective therapeutic targets in hyperglycemic stroke. We have recently also found evidence that a Na-HCO3 cotransporter (NBC) is a third prominent BBB Na transporter that is stimulated by ischemic factors and that hyperglycemia increases expression and activity of BBB NBC, suggesting the exciting possibility that BBB NBC may provide an additional therapeutic target for reducing edema formation in ischemic stroke, both normoglycemic and hyperglycemic. Our studies wil thus also examine this possibility. The first aim is to determine whether hyperglycemic conditions increase expression and activity of BBB NKCC and/or NHE and augment NKCC and NHE stimulation by ischemic factors. We will use Western blot, microspectrofluorometry and radioisotopic flux assays to test the efects of hyperglycemia on cerebral microvascular endothelial cell (CMEC) NKCC and NHE protein and activity. We will use immunoelectron microscopy to evaluate NKCC and NHE in BBB in situ of hyperglycemic rats. The second aim is to determine whether inhibition of BBB NKCC and/or NHE attenuates ischemia-induced edema in hyperglycemic animals. Here, we will examine the effects of BBB NKCC and NHE inhibition on ischemia-induced changes in rat brain Na and water, using nuclear magnetic resonance methods and rats with STZ-induced hyperglycemia. We will also assess the efficacy of NKCC and NHE for reduction of cerebral edema when administered after the onset of ischemia. The third aim is to determine whether an NBC protein is present at the luminal BBB membrane and is stimulated by ischemic factors. We will use Western blot and immunoEM to evaluate BBB NBC protein in CMEC and in BBB in situ, and microspectrofluorometry to assess ischemic factor and hyperglycemia effects on CMEC NBC activity.