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确定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，水肿是在完整 BBB 存在的情况下通过 BBB 将 Na 和 Cl 从血液转运到大脑的过程形成的。我们的研究表明，存在于管腔 BBB 膜中的 Na-K-Cl 协同转运蛋白受到缺血因素的刺激，包括缺氧、血糖、加压素 (AVP)，并且抑制协同转运蛋白可减少中风大鼠模型中的水肿。因此，BBB Na-K-Cl 协同转运蛋白似乎是缺血引起的水肿的主要贡献者。我们现在在初步研究中发现，BBB Na/H 交换器似乎也参与了缺血引起的水肿形成，这表明在中风早期减少水肿的额外 BBB 目标具有令人兴奋的可能性。我们的假设是，除了协同转运蛋白外，缺血期间还会刺激管腔 BBB Na/H 交换器，以增加 Na 从血液到大脑的转运。第一个目的是确定管腔 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原位评估 BBB Na/H 交换蛋白，并使用显微分光荧光法评估缺血因子对 CMEC Na/H 交换活性的影响。第二个目的是确定 BBB Na/H 交换体的抑制是否可以减轻缺血引起的水肿。在这里，我们将使用核磁共振方法检查 BBB Na/H 交换抑制对缺血引起的大鼠脑 Na 和水变化的影响。我们还将评估在缺血发作后施用 Na/H 交换和 Na-K-Cl 共转运抑制剂减轻脑水肿的功效。该项目的第三个目标是评估缺血刺激 BBB Na-K-Cl 协同转运蛋白和 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 诱导的共转运蛋白和交换蛋白刺激中的作用，原因在申请中详述。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，我们将使用脑微血管内皮细胞和灌注固定的大鼠大脑，分别通过蛋白质印迹和共聚焦免疫荧光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、血糖不足和 AVP）刺激的协同转运蛋白活性（放射性同位素通量）和 Na/H 交换活性（荧光光谱和放射性同位素通量）的影响。公共卫生相关性：缺血引起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 BBB Na-K-Cl 协同转运蛋白和/或缺血刺激的治疗方法是否有效。 Na/H 交换活动可能对减轻中风引起的脑水肿有价值。事实上，面向管腔的 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药物所接近，这使得它们成为我们必须研究的治疗靶点。