Blood-Brain Barrier Ion Transport in Cerebral Ischemia

脑缺血中的血脑屏障离子转运

• 批准号: 7871318
• 负责人: Martha E O'Donnell
• 金额: $ 32.92万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7871318
• 关键词: Address; Attenuated; Blood; Blood - brain barrier anatomy; Brain; Brain Edema; Brain Hypoxia-Ischemia; Brain Injuries; Bumetanide; CME-Carbodiimide; Cause of Death; Cerebral Edema; Cerebral Ischemia; Cerebrum; Confocal Microscopy; Data; Edema; Electrolytes; Endothelial Cells; Evaluation; Fluorescence Spectrometry; Goals; Homeostasis; Hour; Hypoxia; Immunoelectron Microscopy; Immunofluorescence Immunologic; In Situ; Investigation; Ion Transport; Ions; Ischemia; Ischemic Stroke; MAPK8 gene; Mediating; Membrane; Methods; Middle Cerebral Artery Occlusion; Mitogen Activated Protein Kinase 1; Mitogen-Activated Protein Kinases; Modeling; NHE1; NHE2; Nuclear Magnetic Resonance; Patients; Perfusion; Pharmaceutical Preparations; Phosphotransferases; Process; Protein Isoforms; Protein Kinase C; Proteins; Radioisotopes; Rattus; Risk; Role; Signal Pathway; Stroke; Testing; Therapeutic; Vasopressins; Water; Western Blotting; adenylate kinase; chloride-cotransporter potassium; in vivo; inhibitor/antagonist; kinase inhibitor; mitogen-activated protein kinase p38; prevent; public health relevance; research study; therapeutic target; therapy development; uptake

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.

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。我们的研究表明，存在于腔血脑屏障膜中的钠-钾-氯共转运蛋白受到缺血性因素的刺激，包括缺氧、无血糖、加压素（AVP），并且抑制共转运蛋白可减少大鼠中风模型中的水肿。因此，BBB Na-K-Cl协同转运蛋白似乎是缺血诱导的水肿的主要原因。我们现在已经在初步研究中发现，BBB Na/H交换剂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BBB靶点的令人兴奋的可能性。我们的假设是，除了协同转运蛋白，一个管腔血脑屏障Na/H交换器在缺血期间被刺激，以增加Na从血液到大脑的运输。第一个目的是确定Na/H交换是否存在于腔BBB膜上，并受到缺血因子的刺激。我们将使用免疫电镜原位评估BBB Na/H交换蛋白，并使用显微荧光分光光度法评估缺血因素对CMEC Na/H交换活性的影响。第二个目的是确定BBB Na/H交换器的抑制是否减弱缺血诱导的水肿。在这里，我们将研究BBB Na/H交换抑制对缺血诱导的大鼠脑Na和水的变化的影响，使用核磁共振方法。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给药时减少脑水肿的疗效。本项目的第三个目的是评估缺血刺激血脑屏障Na-K-Cl协同转运体和Na/H交换体活性的信号通路。我们将首先评估AMP激酶、p38 MAP激酶和细胞内[Ca]在缺氧、血糖缺乏和AVP诱导的协同转运蛋白和交换蛋白刺激中的作用，原因详见本申请。然而，我们的研究还将包括对蛋白激酶C、ERK 1/2 MAP激酶和JNK MAP激酶的初步评估。在这些研究中，我们将使用脑微血管内皮细胞和灌注固定的大鼠脑，分别通过Western印迹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）刺激的协同转运蛋白活性（放射性同位素通量）和Na/H交换剂活性（荧光光谱法和放射性同位素通量）的影响。公共卫生相关性：缺血诱导的水肿是中风脑损伤的主要原因，中风是美国死亡的主要原因。拟议的研究将揭示旨在预防缺血刺激BBB Na-K-Cl协同转运蛋白和/或Na/H交换活性的治疗方法是否可能对减轻中风诱导的脑水肿有价值。事实上，面向腔血脑屏障钠转运蛋白似乎有助于水肿的形成，并很容易获得静脉给药的药物，使他们成为一个治疗目标，我们必须调查。