Oxidative Stress and Pathological Glutamate Release in Stroke

描述（由申请人提供）：中风是美国和其他工业化国家的第三大死亡原因和长期残疾的主要原因。每年有75万美国人遭受痛苦，超过15万人死于新发和复发性中风。尽管进行了广泛的研究工作，但迄今为止只有一种药物，即溶栓剂tPA，已被FDA批准用于中风治疗。已经在动物缺血模型中确定了许多替代的神经保护方法，但在临床试验中失败了。因此，持续需要开发和改进新的中风疗法。在这项研究中，我们提出了一个新的假设，即氧化应激通过激活氧化还原敏感的谷氨酸转运途径，增强神经胶质细胞谷氨酸的释放，从而有效地增强和传播缺血性脑损伤。该实验室和其他实验室的研究已经确定了三种氧化还原敏感的谷氨酸释放途径：体积调节阴离子通道（VRAC），胱氨酸/谷氨酸反向转运蛋白（xCT）和连接蛋白半通道（Cxs）。在拟议的工作中，我们将使用大鼠星形胶质细胞和小胶质细胞的纯和混合原代培养物，以找到减少神经胶质细胞病理性谷氨酸释放的最佳方法。随后将在大鼠短暂局灶性缺血模型中测试体内新的药理学治疗。提出了以下具体目的：（目的1）使用药理学工具和siRNA方法阐明VRAC、xCT和Cxs对原代神经胶质培养物中响应于氧化应激的谷氨酸释放的相对贡献;（目的2）在大鼠短暂性局灶性脑缺血模型中，使用微透析方法和透析液内药物递送;（AIM 3）通过测量对照缺血组和神经保护剂治疗后的脑梗死体积，测试AIMS 1和2中验证的药理学治疗的神经保护潜力。本研究的主要目的是建立一个新的组织生产的活性氧和病理性谷氨酸从神经胶质细胞释放之间的基本关系，并确定新的药理学目标中风和其他神经系统疾病（如多发性硬化症，阿尔茨海默氏病，创伤性脑损伤），其中涉及氧化应激。公共卫生相关性：中风是一种毁灭性的脑部疾病，每年影响75万美国人，造成超过15万人死亡，并使许多人永久衰弱。目前，只有一种药物，组织纤溶酶原激活剂（tPA），已被批准用于急性中风的治疗。由于一些临床限制，tPA仅用于5%至8%的中风患者。本项目将研究以前未被认识到的脑损伤机制，并寻求开发新的中风治疗药物。

DESCRIPTION (provided by applicant): Stroke is the third leading cause of death and the leading cause for long-term disability in the U.S. and other industrialized nations. Each year 750,000 Americans suffer, and more than 150,000 die as a result of new and recurrent strokes. In spite of extensive research efforts, to date only one drug, the thrombolytic agent tPA, has been approved by the FDA for stroke treatment. A number of alternative neuroprotective approaches have been identified in animal ischemia models, but failed in clinical trials. Therefore there is continuous need for development and improvement of novel stroke therapies. In this study we propose to test the novel HYPOTHESIS that oxidative stress potently augments and propagates ischemic brain damage by enhancing glial glutamate release via activation of redox-sensitive glutamate transport pathways. Studies from this and other laboratories has identified three redox-sensitive glutamate release pathways: volume-regulated anion channels (VRAC), the cystine/glutamate antiporter (xCT), and the connexin hemichannels (Cxs). In the proposed work we will use pure and mixed primary cultures of rat astrocytes and microglial cells to find the optimal approaches for reducing pathological glutamate release from glia. This will be followed by testing novel pharmacological treatments in vivo, in a rat model of transient focal ischemia. The following SPECIFIC AIMS are proposed: (AIM 1) to elucidate relative contribution of VRAC, xCT, and Cxs to glutamate release in response to oxidative stress in primary glial cultures, using pharmacological tools and an siRNA approach; (AIM 2) to identify the relationship between oxidative stress and pathological glutamate release in vivo in a rat model of transient focal ischemia, using a microdialysis approach and the intra-dialysate drug delivery; (AIM 3) to test the neuroprotective potential of the pharmacological treatments validated in AIMS 1 and 2, by measuring brain infarction volumes in the control ischemia group and after treatment with neuroprotectants. The major objective of this study is to establish a new fundamental relationship between tissue production of reactive oxygen species and pathological glutamate release from glial cells, and to identify novel pharmacological targets in stroke and other neurological disorders (such as multiple sclerosis, Alzheimer's disease, and traumatic brain injury), which involve oxidative stress. PUBLIC HEALTH RELEVANCE: Stroke is a devastating brain disorder that affects 750,000 Americans every year, killing more than 150,000 people and leaving many others permanently debilitated. Presently, only one pharmacological agent, tissue plasminogen activator (tPA), has been approved for acute stroke treatment. Because of several clinical limitations, tPA is used in only 5 to 8% of stroke patients. The present project will study previously unrecognized mechanisms of brain damage and seek to develop new pharmacological agents for stroke treatment.