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(申请人提供)：该项目的长期目标是确定介导脑缺血引起的脑水肿的血脑屏障(BBB)离子转运体。在缺血性中风的早期，在完整的血脑屏障存在的情况下，通过血脑屏障将钠和氯从血液输送到脑中的过程而形成水肿。我们的研究表明，存在于管腔血脑屏障膜上的Na-K-Cl共转运蛋白受到缺血因素的刺激，包括缺氧、血糖、加压素(AVP)，抑制该共转运蛋白可减轻卒中大鼠的水肿。因此，血脑屏障钠-钾-氯共转运体似乎是导致缺血引起的水肿的主要因素。我们现在已经在初步研究中发现，血脑屏障Na/H交换器似乎也参与了缺血诱导的水肿的形成，这表明在卒中早期有可能增加一个额外的血脑屏障靶点来减轻水肿。我们的假设是，除了辅助转运体外，在缺血期间，还会刺激一个管腔内的血脑屏障Na/H交换器，以增加钠从血液到大脑的转运。第一个目的是确定管腔血脑屏障膜上是否存在Na/H交换，以及是否受到缺血因素的刺激。我们将用免疫电子显微镜原位检测血脑屏障Na/H交换蛋白，用微量荧光分光光度法检测缺血因子对CMEC Na/H交换活性的影响。第二个目的是确定抑制血脑屏障钠氢交换是否能减轻缺血引起的水肿。在这里，我们将利用核磁共振方法，研究血脑屏障钠氢交换抑制对缺血诱导的大鼠脑组织钠和水变化的影响。我们还将评估Na/H交换和Na-K-Cl共转运体抑制剂在脑缺血发作后应用时对减轻脑水肿的效果。本项目的第三个目的是评估缺血刺激血脑屏障、Na-K-Cl共转运体和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]在低氧、高血糖和AVP诱导的辅转运体和交换器刺激中的作用，具体原因将在申请中详细说明。然而，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，我们将使用大脑微血管内皮细胞和灌流固定的大鼠大脑，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)刺激的辅转运体活性(放射性同位素通量)和Na/H交换活性(荧光分光光度法和放射性同位素通量)的影响。公共卫生相关性：缺血性脑水肿是中风脑损伤的主要原因，中风是美国主要的死亡原因。拟议的研究将揭示旨在防止脑缺血刺激血脑屏障钠-钾-氯共转运体和/或钠/氢交换活性的治疗方法是否对减轻中风引起的脑水肿有价值。事实上，面向管腔的BBB钠转运体似乎有助于水肿的形成，并且很容易被静脉给药，这使得它们成为我们必须研究的治疗靶点。