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）：该项目的长期目标是识别介导缺血引起的脑水肿的血脑屏障（BBB）离子转运蛋白。在缺血性中风的早期，通过涉及Na和Cl从血液转移到大脑的BBB转运的过程中，在完整的BBB存在下形成了水肿。我们的研究表明，腔内BBB膜中存在的Na-k-CL共同运动受到缺血因素的刺激，包括缺氧，aglycemia，加压素，加压素（AVP），并抑制共移孢子的抑制作用会减少中风大鼠模型中的水肿。因此，BBB Na-k-Cl共转运蛋白似乎是缺血诱导的水肿的主要因素。现在，我们在初步研究中发现，BBB Na/H交换器似乎也参与缺血诱导的水肿形成，这表明在中风的较早时间内，有其他BBB靶标可减少水肿的令人兴奋的可能性。我们的假设是，除了共转运蛋白外，在缺血期间还刺激了腔内BBB Na/h交换器，以增加Na从血液进入大脑的转运。第一个目的是确定在腔内BBB膜上是否存在Na/H交换，并受到缺血因素的刺激。我们将使用免疫电子显微镜来评估BBB NA/H交换蛋白原位和微光谱荧光测定法来评估缺血因子对CMEC NA/H交换活性的影响。第二个目的是确定抑制BBB Na/H交换器是否减弱缺血诱导的水肿。在这里，我们将使用核磁共振方法研究BBB 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施用时降低脑水肿的功效。该项目的第三个目的是评估局部缺血刺激BBB Na-k-Cl共转运蛋白和Na/H交换器活性的信号通路。我们将首先评估AMP激酶，p38 MAP激酶和细胞内[CA]在缺氧，aglycemia和AVP引起的刺激中的刺激和交换剂的作用。但是，我们的研究还将包括对蛋白激酶C，ERK1/2 MAP激酶和JNK MAP激酶的初步评估。在这些研究中，我们将分别使用脑膜印迹和共聚焦免疫荧光分别使用脑微血管内皮细胞和灌注固定大鼠脑来评估缺血诱导的激酶。我们还将评估激酶抑制剂对缺血（低氧，agLACEMIA和AVP）刺激的共转运蛋白活性（放射性同位素通量）和Na/H交换器活性（Spectrofluuoremetry和Releisotope Flux）的作用。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，这是美国的主要死亡原因，拟议的研究将揭示旨在防止BBB Na-k-Cl cotransporter和/或Na/H Exchanse的治疗方法是否具有降低衰减的Stroke troke诱导的脑部或Na-k-Cl cotransporter和Na-k-Cl cotroke诱导的脑部或Na-k-cl cot诱导的方法。面向腔的BBB NA转运蛋白似乎有助于水肿形成，并且很容易被静脉注射的药物进入，这一事实使它们成为我们必须研究的治疗靶标。