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。在初步的研究中，我们现在已经发现证据表明，BBB Na-HCO 3协同转运蛋白（NBC）构成了第三个突出的BBB Na转运蛋白，其在缺血期间也被刺激以实质上参与脑水肿形成。这为我们提供了一个重要的额外的治疗目标，以减少缺血性卒中中高度损伤性的早期水肿形成。虽然我们的研究表明，NKCC、NHE和NBC似乎是减少健康卒中患者水肿的有前景的治疗靶点，但必须认识到，高血糖症（约30%的患者存在的卒中常见合并症）会导致脑水肿加重和结局恶化。对此的潜在机制知之甚少。然而，在最近的研究中，我们发现，高血糖增加BBB NKCC，NHE和NBC的表达和活性，并增强缺血因素对它们的刺激。此外，我们还发现证据表明，在MCAO中脑水肿的高血糖恶化通过抑制BBB NKCC、NHE和NBC而减少。我们的假设是，血脑屏障NKCC，NHE和/或NBC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。第一个目的是确定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对CMEC NBC活性的影响。第二个目的是确定高血糖状况是否增加BBB NKCC、NHE和/或NBC的表达和活性，并增加缺血因子对这些Na转运蛋白的刺激。我们将使用Western blot、显微荧光分光光度法和放射性同位素通量测定来检测高血糖对CMEC NKCC、NHE和NBC蛋白和活性的影响。我们还将采用免疫电镜技术对高血糖大鼠血脑屏障中的NKCC、NHE和NBC进行原位检测。第三个目的是确定BBB NKCC、NHE和/或NBC的抑制是否减弱高血糖动物中缺血诱导的水肿。在这里，我们将研究的影响，抑制血脑屏障NKCC，NHE和NBC对缺血引起的变化，大鼠脑钠和水，使用核磁共振方法和大鼠STZ诱导的高血糖症。我们还将评估NKCC、NHE和NBC抑制剂在缺血发作后给药时减轻水肿的疗效。

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.