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。我们的研究表明，存在于腔血脑屏障膜的Na-K-Cl共转运受到缺血因素的刺激，包括缺氧、血糖、血管加压素（AVP），抑制共转运蛋白可减少脑卒中大鼠模型中的水肿。因此，血脑屏障Na-K-Cl共转运蛋白似乎是缺血诱导水肿的主要因素。我们现在在初步研究中发现，血脑屏障Na/H交换器似乎也参与缺血诱导的水肿形成，这表明在中风早期减少水肿的额外血脑屏障靶点的可能性令人兴奋。我们的假设是，除了共转运体外，在缺血期间，管腔血脑屏障Na/H交换器受到刺激，以增加Na从血液到脑的运输。第一个目的是确定Na/H交换是否存在于腔血脑屏障膜上，并受到缺血因素的刺激。我们将使用免疫电镜原位评价血脑屏障Na/H交换蛋白，显微荧光法评估缺血性因子对CMEC Na/H交换活性的影响。第二个目的是确定血脑屏障Na/H交换器的抑制是否减轻缺血诱导的水肿。在此，我们将采用核磁共振方法研究血脑屏障Na/H交换抑制对缺血诱导的大鼠脑Na和水变化的影响。我们还将评估Na/H交换和Na- k - cl共转运抑制剂在缺血发作后减少脑水肿的疗效。本项目的第三个目的是评估缺血刺激血脑屏障Na- k - cl共转运蛋白和Na/H交换活性的信号通路。我们将首先评估AMP激酶、p38 MAP激酶和细胞内[Ca]在缺氧、血糖和avp诱导的共转运体和交换体刺激中的作用，原因在申请中详细说明。然而，我们的研究还将包括蛋白激酶C、ERK1/2 MAP激酶和JNK MAP激酶的初步评估。在这些研究中，我们将分别使用脑微血管内皮细胞和灌注固定大鼠脑，通过Western blot和共聚焦免疫荧光来评估缺血诱导的激酶激活。我们还将评估激酶抑制剂对缺血（缺氧、血糖和AVP）刺激的共转运体活性（放射性同位素通量）和Na/H交换体活性（荧光光谱法和放射性同位素通量）的影响。公共卫生相关性：缺血性水肿是脑卒中脑损伤的主要原因，在美国是导致死亡的主要原因。拟议的研究将揭示旨在防止脑卒中Na- k - cl共转运蛋白缺血刺激和/或Na/H交换活性的治疗方法是否可能对减轻脑卒中性脑水肿有价值。事实上，面向光的血脑屏障Na转运体似乎有助于水肿的形成，并且很容易被静脉给药，这使它们成为我们必须研究的治疗靶点。

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 studies have shown that Na-K-Cl cotransport, present in the luminal BBB membrane, is stimulated by ischemic factors, including hypoxia, aglycemia, vasopressin (AVP) and that inhibiting the cotransporter reduces edema in a rat model of stroke. Thus, the BBB Na-K-Cl cotransporter appears to be a major contributor to ischemia-induced edema. We have now found in preliminary studies that a BBB Na/H exchanger also appears to participate in ischemia- induced edema formation which suggests the exciting possibility of an additional BBB target for reduction of edema during the early hours of stroke. Our hypothesis is that, in addition to the cotransporter, a luminal BBB Na/H exchanger is stimulated during ischemia to increase transport of Na from blood into brain. The first aim is to determine whether Na/H exchange is present at the luminal BBB membrane and is stimulated by ischemic factors. We will use immunoelectron microscopy to evaluate BBB Na/H exchange protein in situ and microspectrofluorometry to assess ischemic factor effects on CMEC Na/H exchange activity. The second aim is to determine whether inhibition of the BBB Na/H exchanger attenuates ischemia-induced edema. Here, we will examine the effect of BBB Na/H exchange inhibition on ischemia-induced changes in rat brain Na and water, using nuclear magnetic resonance methods. We will also assess the efficacy of Na/H exchange and Na-K-Cl cotransport inhibitors for reduction of cerebral edema when administered after the onset of ischemia. The third aim of this project is to evaluate the signaling pathways by which ischemia stimulates BBB Na-K-Cl cotransporter and Na/H exchanger activities. We will start by evaluating the roles of AMP kinase, p38 MAP kinase and intracellular [Ca] in hypoxia, aglycemia and AVP-induced stimulation of the cotransporter and exchanger for reasons detailed in the application . However, our studies will also include an initial assessment of protein kinase C, ERK1/2 MAP kinase and JNK MAP kinase. For these studies we will use cerebral microvascular endothelial cells and perfusion-fixed rat brain to evaluate ischemia-induced activation of the kinases by Western blot and confocal immunofluorescence, respectively. We will also evaluate the effects of kinase inhibitors on ischemia (hypoxia, aglycemia and AVP) stimulated cotransporter activity (radioisotope flux) and Na/H exchanger activity (spectrofluorometry and radioisotope flux). PUBLIC HEALTH RELEVANCE: Ischemia-induced edema is a major cause of brain damage in stroke, a leading cause of death in the U.S. The proposed studies will reveal whether therapeutic approaches aimed at preventing ischemia stimulation of BBB Na-K-Cl cotransporter and/or Na/H exchange activity may be of value for attenuating stroke-induced brain edema. The fact that luminal-facing BBB Na transporters appear to contribute to edema formation and are readily accessible to intravenously administered drugs makes them a therapeutic target that we must investigate.