Oxidative Stress and Pathological Glutamate Release in Stroke

描述(申请人提供)：在美国和其他工业化国家，中风是第三大致死原因，也是导致长期残疾的首要原因。每年有75万美国人罹患中风，超过15万人死于新发和反复发作的中风。尽管进行了广泛的研究，但到目前为止，只有一种药物--溶栓剂tPA--被FDA批准用于中风治疗。在动物缺血模型中已经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，不断需要开发和改进新的卒中疗法。在这项研究中，我们建议检验这一新的假设，即氧化应激通过激活氧化还原敏感的谷氨酸运输途径来促进神经胶质谷氨酸的释放，从而有效地增加和传播缺血性脑损伤。来自该实验室和其他实验室的研究已经确定了三个对氧化还原敏感的谷氨酸释放途径：容量调节阴离子通道(Vrac)、胱氨酸/谷氨酸逆向转运体(XCT)和连接蛋白半通道(CXS)。在这项拟议的工作中，我们将使用大鼠星形胶质细胞和小胶质细胞的纯原代培养和混合原代培养，寻找减少胶质细胞病理性谷氨酸释放的最佳方法。随后，将在短暂性局灶性脑缺血的大鼠模型中，在体内测试新的药物治疗方法。提出了以下具体目标：(目的1)利用药理学工具和siRNA方法，阐明vrac、XCT和CXS对原代培养的胶质细胞氧化应激释放谷氨酸的相对贡献；(目的2)使用微透析法和透析液中的药物释放方法，确定氧化应激与病理性谷氨酸释放之间的关系；(目的3)通过测量对照组缺血组和神经保护剂治疗后的脑梗塞体积，测试目标1和2中验证的药物治疗的神经保护潜力。本研究的主要目的是建立组织产生的活性氧与神经胶质细胞的病理性谷氨酸释放之间的新的基本关系，并确定与氧化应激有关的中风和其他神经疾病(如多发性硬化症、阿尔茨海默病和创伤性脑损伤)的新的药理靶点。与公共卫生相关：中风是一种毁灭性的大脑疾病，每年影响75万美国人，导致超过15万人死亡，其他许多人永久虚弱。目前，只有一种药物，组织纤溶酶原激活剂(TPA)被批准用于急性卒中的治疗。由于几个临床限制，tPA仅在5%至8%的中风患者中使用。本项目将研究以前未知的脑损伤机制，并寻求开发治疗中风的新药理药物。

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.