Blood-Brain Barrier Ion Transport in Cerebral Ischemia

描述（由申请人提供）：该项目的长期目标是鉴定介导缺血性脑水肿的血脑屏障（BBB）离子转运体。在缺血性中风的早期，在血脑屏障存在的情况下，通过血脑屏障将Na和Cl从血液转运到脑的过程形成水肿。我们之前的研究表明，存在于腔血脑屏障膜的Na- k - cl共转运（NKCC）和Na/H交换（NHE）受到缺氧、血糖和血管加压素（AVP）等缺血因素的刺激，抑制血脑屏障NKCC和/或NHE活性可减少脑水肿大鼠脑中动脉闭塞（MCAO）模型。在初步研究中，我们已经发现血脑屏障Na- hco3共转运体（NBC）构成了第三个主要的血脑屏障Na转运体，在缺血期间也受到刺激，实质性地参与脑水肿的形成。这为我们提供了一个重要的额外治疗靶点，以减少缺血性卒中中高度有害的早期水肿形成。虽然我们的研究表明，NKCC、NHE和NBC似乎是减少其他健康脑卒中患者水肿的有希望的治疗靶点，但必须认识到，高血糖是约30%的脑卒中患者常见的合并症，会导致更严重的脑水肿和恶化的预后。人们对其潜在机制知之甚少。然而，在最近的研究中，我们发现高血糖增加了血脑屏障NKCC、NHE和NBC的表达和活性，并增加了缺血因子对它们的刺激。此外，我们还发现有证据表明，抑制血脑屏障NKCC、NHE和NBC可以减轻MCAO患者脑水肿的高血糖加重。我们的假设是血脑屏障NKCC、NHE和/或NBC参与了高血糖诱导的脑水肿形成的增加，并构成高血糖卒中的有效治疗靶点。第一个目的是确定NBC蛋白是否存在于管腔血脑屏障膜上，并受到缺血因素的刺激。我们将采用Western blot和免疫电镜技术评估CMEC和原位血脑屏障中血脑屏障NBC蛋白，并采用显微荧光法评估缺血性因子对CMEC NBC活性的影响。第二个目的是确定高血糖状况是否会增加血脑屏障NKCC、NHE和/或NBC的表达和活性，并增加缺血性因子对这些Na转运体的刺激。我们将使用Western blot，显微荧光光谱法和放射性同位素通量法检测高血糖对CMEC NKCC， NHE和NBC蛋白和活性的影响。我们还将采用免疫电镜法评估高血糖大鼠血脑屏障原位NKCC、NHE和NBC。第三个目的是确定抑制血脑屏障NKCC、NHE和/或NBC是否能减轻高血糖动物的缺血诱导水肿。本研究将采用核磁共振方法和stz诱导的高血糖大鼠，研究抑制BBB NKCC、NHE和NBC对缺血大鼠脑Na和水变化的影响。我们还将评估NKCC、NHE和NBC抑制剂在缺血发作后减少水肿的功效。

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. In preliminary studies we have now found evidence that a BBB Na-HCO3 cotransporter (NBC) constitutes a 3rd prominent BBB Na transporter that is also stimulated during ischemia to substantially participate in cerebral edema formation. This gives us an important additional therapeutic target for reducing the highly injurious early edema formation in ischemic stroke. While our studies indicate that NKCC, NHE and NBC appear to be promising therapeutic targets for reducing edema in otherwise healthy stroke patients, it must be recognized that hyperglycemia, a common co-morbidity in stroke present in ~30% of patients, causes greater cerebral edema and worsened outcome. The underlying mechanisms for this are poorly understood. However, in recent studies we have found that hyperglycemia increases expression and activity of BBB NKCC, NHE and NBC and augments their stimulation by ischemic factors. Further, we have also found evidence that the hyperglycemia exacerbation of brain edema in MCAO is reduced by inhibition of BBB NKCC, NHE and NBC. Our hypothesis is that BBB NKCC, NHE and/or NBC participate in the hyperglycemia-induced augmentation of cerebral edema formation and constitute effective therapeutic targets in hyperglycemic stroke. The first aim is to determine whether 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 effects on CMEC NBC activity. The second aim is to determine whether hyperglycemic conditions increase expression and activity of BBB NKCC, NHE and/or NBC and augment stimulation of these Na transporters by ischemic factors. We will use Western blot, microspectrofluorometry and radioisotopic flux assays to test the effects of hyperglycemia on CMEC NKCC, NHE and NBC protein and activity. We will also use immunoEM to evaluate NKCC, NHE and NBC in BBB in situ of hyperglycemic rats. The third aim is to determine whether inhibition of BBB NKCC, NHE and/or NBC attenuates ischemia-induced edema in hyperglycemic animals. Here, we will examine the effects of inhibiting BBB NKCC, NHE and NBC 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, NHE and NBC inhibitors for reduction of edema when administered after the onset of ischemia.