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Blood-Brain Barrier Ion Transport in Cerebral Ischemia

脑缺血中的血脑屏障离子转运

基本信息

批准号：
9059450
负责人：
Martha E O'Donnell
金额：
$ 41.68万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-08-01 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9059450
关键词：
Animals Attenuated Bicarbonates Biological Assay Blood Blood - brain barrier anatomy Blood Glucose Brain Brain Edema Brain Injuries Bumetanide Cause of Death Cells Cerebral Edema Cerebral Infarction Cerebral Ischemia Comorbidity Diabetes Mellitus Edema Endothelial Cells Evaluation Goals Grant Health Hour Human Hyperglycemia Hypoxia In Situ In Vitro Individual Infarction Intravenous Ion Transport Ions Ischemia Ischemic Stroke Magnetic Resonance Imaging Mediating Membrane Methods Middle Cerebral Artery Occlusion Modeling Nuclear Magnetic Resonance Outcome Patients Perfusion Population Process Proteins Rattus Sodium Sodium-Potassium-Chloride Symporters Streptozocin Stroke Swelling Testing Therapeutic Translating Vasopressins Water Western Blotting Work brain endothelial cell diabetic effective therapy in vivo inhibitor/antagonist intravenous administration research study response targeted treatment therapeutic target uptake

项目摘要

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 previous studies have shown that Na-K-Cl cotransport (NKCC) and Na/H exchange (NHE), present in the luminal BBB membrane, are stimulated by ischemic factors, including hypoxia, aglycemia and vasopressin (AVP) and that inhibiting BBB NKCC and/or NHE activity reduces brain edema in the rat middle cerebral artery occlusion (MCAO) model of stroke. In preliminary studies we have now found evidence that a BBB Na-HCO3 cotransporter (NBC) constitutes a 3rd prominent BBB Na transporter that is also stimulated during ischemia to substantially participate in cerebral edema formation. This gives us an important additional therapeutic target for reducing the highly injurious early edema formation in ischemic stroke. While our studies indicate that NKCC, NHE and NBC appear to be promising therapeutic targets for reducing edema in otherwise healthy stroke patients, it must be recognized that hyperglycemia, a common co-morbidity in stroke present in ~30% of patients, causes greater cerebral edema and worsened outcome. The underlying mechanisms for this are poorly understood. However, in recent studies we have found that hyperglycemia increases expression and activity of BBB NKCC, NHE and NBC and augments their stimulation by ischemic factors. Further, we have also found evidence that the hyperglycemia exacerbation of brain edema in MCAO is reduced by inhibition of BBB NKCC, NHE and NBC. Our hypothesis is that BBB NKCC, NHE and/or NBC participate in the hyperglycemia-induced augmentation of cerebral edema formation and constitute effective therapeutic targets in hyperglycemic stroke. The first aim is to determine whether NBC protein is present at the luminal BBB membrane and is stimulated by ischemic factors. We will use Western blot and immunoEM to evaluate BBB NBC protein in CMEC and in BBB in situ, and microspectrofluorometry to assess ischemic factor effects on CMEC NBC activity. The second aim is to determine whether hyperglycemic conditions increase expression and activity of BBB NKCC, NHE and/or NBC and augment stimulation of these Na transporters by ischemic factors. We will use Western blot, microspectrofluorometry and radioisotopic flux assays to test the effects of hyperglycemia on CMEC NKCC, NHE and NBC protein and activity. We will also use immunoEM to evaluate NKCC, NHE and NBC in BBB in situ of hyperglycemic rats. The third aim is to determine whether inhibition of BBB NKCC, NHE and/or NBC attenuates ischemia-induced edema in hyperglycemic animals. Here, we will examine the effects of inhibiting BBB NKCC, NHE and NBC on ischemia-induced changes in rat brain Na and water, using nuclear magnetic resonance methods and rats with STZ-induced hyperglycemia. We will also assess the efficacy of NKCC, NHE and NBC inhibitors for reduction of edema when administered after the onset of ischemia.

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。我们以前的研究表明，在大鼠大脑中动脉闭塞(MCAO)模型中，存在于管腔血脑屏障膜上的钠-钾-氯共转运(NKCC)和钠/氢交换(NHE)受到包括缺氧、血糖和加压素(AVP)在内的缺血因素的刺激，抑制BBB、NKCC和/或NHE活性可减轻脑水肿。在初步研究中，我们现在已经发现证据表明，血脑屏障钠-HCO3共转运体(NBC)构成了第三个重要的血脑屏障钠转运体，在脑缺血时也受到刺激，实质上参与了脑水肿的形成。这为减少缺血性卒中高损伤性早期水肿的形成提供了一个重要的额外治疗靶点。虽然我们的研究表明，NKCC、NHE和NBC似乎是减轻其他健康的中风患者水肿的有希望的治疗靶点，但必须认识到，高血糖是中风中常见的共同发病率，约30%的患者存在，导致更大的脑水肿和恶化的预后。人们对此的基本机制知之甚少。然而，在最近的研究中，我们发现高血糖增加了BBB、NKCC、NHE和NBC的表达和活性，并增强了它们对缺血因子的刺激。此外，我们还发现有证据表明，抑制BBB、NKCC、NHE和NBC可以减轻MCAO患者高血糖加重脑水肿的程度。我们的假设是，BBB、NKCC、NHE和/或NBC参与了高血糖引起的脑水肿形成，是高血糖卒中有效的治疗靶点。第一个目的是确定NBC蛋白是否存在于管腔血脑屏障膜上，并受到缺血因素的刺激。我们将用免疫印迹和免疫电子显微镜原位检测CMEC和BBB中的BBB-NBC蛋白，并用显微荧光分光光度法检测缺血因子对CMEC-NBC活性的影响。第二个目的是确定高血糖条件是否增加BBB、NKCC、NHE和/或NBC的表达和活性，并增强缺血因子对这些钠转运体的刺激。我们将使用蛋白质印迹、微量荧光分光光度和放射性同位素流量分析来检测高血糖对CMEC NKCC、NHE和NBC蛋白和活性的影响。用免疫电子显微镜检测高血糖大鼠血脑屏障中NKCC、NHE和NBC的含量。第三个目的是确定抑制BBB、NKCC、NHE和/或NBC是否能减轻高血糖动物的缺血所致的水肿。在这里，我们将利用核磁共振方法和STZ诱导的高血糖大鼠，观察抑制BBB、NKCC、NHE和NBC对缺血诱导的大鼠脑钠水变化的影响。我们还将评估NKCC、NHE和NBC抑制剂在缺血发作后应用时减轻水肿的疗效。