ASCORBATE AND GLUTATHIONE IN CNS INJURY

抗坏血酸和谷胱甘肽在中枢神经系统损伤中的作用

• 批准号: 6336719
• 负责人: Margaret E Rice
• 金额: $ 19.33万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6336719
• 关键词: age difference; antioxidants; ascorbate; biomarker; brain cell; cellular pathology; cerebral ischemia /hypoxia; electrophysiology; gender difference; glutathione; guinea pigs; hippocampus; histopathology; homeostasis; laboratory rat; neuroprotectants; nitric oxide; sectioning; spreading cortical depression; western blottings

Cerebral antioxidants are neuroprotective agents that prevent oxidative injury during normal aerobic metabolism. Ascorbic acid (ascorbate) and glutathione (GSH) are the most abundant antioxidants of low molecular weight in the CNS. At the onset of ischemic injury, energy-dependent mechanisms that maintain cellular levels of ascorbate and GSH fail, so that they and other small molecules are lost brain cells. The underlying premise of this proposal is that loss of intracellular ascorbate and GSH during ischemia leaves brain cells vulnerable to oxidative damage. Despite the central role of ascorbate and GSH in the antioxidant network, surprising little is known about the mechanisms that regulate their homeostasis in the CNS. The long-term objective of the studies is to determine the mechanisms that regulate antioxidant homeostasis in the CNS, which is a necessary first step in the development of appropriate therapies. Preliminary data indicate ascorbate and GSH levels decrease with age and vary with gender. These data are consistent with human epidemiology, which indicate that stroke incidence and severity increase with age and are greater in males than in females. Proposed studies, therefore, will necessarily address factors that govern age and sex differences. Experiments will use HPLC to determine total tissue ascorbate and GSH, and histological methods to evaluate tissue damage and antioxidant neuroprotection. In addition, tools are available to monitor the extracellular levels of ascorbate ([Asc]o). Using voltammetry with carbon fiber microelectrodes, we will detect [Asc]o in brain slice in vitro and after middle cerebral artery occlusion (MCAO) in vivo, then combine these data with total tissue ascorbate content to calculate [Asc]i. Specific aims are: 1. To determine the differential localization of ascorbate and GSH in the CNS, with emphasis on the hypothesis that ascorbate is in neurons and GSH is in glia. We will confirm antioxidant loss in the aging brain and extend gender studies to include antioxidant enzymes and iron. II. To evaluate the mechanism of intra-and extracellular ascorbate homeostasis. In vitro experiments will test the hypothesis that intra- and extracellular ascorbate concentrations ([Asc]i and [Asc]o) are regulated homeostatically and that the dynamics of this process change with age. III. To evaluate where and how ascorbate and GSH protect. In vitro experiments will test the hypotheses that ascorbate and GSH act intracellularly. IV. To determine mechanisms of ascorbate and GSH loss in ischemia. We will extend preliminary data indicating that loss of antioxidants during ischemia is greater in male rats than in females. V. To determine the relationship between loss of intracellular ascorbate and in focal ischemia. Experiments will test the hypothesis that regions with the greatest loss of [Asc]i during ischemia have the greatest extent of damage.

大脑抗氧化剂是防止氧化的神经保护剂 在正常有氧代谢过程中的损伤。抗坏血酸(抗坏血酸)和 谷胱甘肽(GSH)是含量最丰富的低分子抗氧化剂 中枢神经系统中的重量。在缺血性损伤开始时，能量依赖 维持细胞内抗坏血酸和谷胱甘肽水平的机制失效，因此 它们和其他小分子是丢失的脑细胞。潜在的 这一提议的前提是细胞内抗坏血酸和谷胱甘肽的损失 在缺血期间，脑细胞容易受到氧化损伤。尽管 抗坏血酸和谷胱甘肽在抗氧化网络中的中心作用， 令人惊讶的是，人们对其调节机制知之甚少 中枢神经系统的动态平衡。这项研究的长远目标是 确定调节中枢神经系统中抗氧化剂动态平衡的机制， 这是制定适当的 治疗。初步数据显示抗坏血酸和谷胱甘肽水平下降 随着年龄和性别的不同而不同。这些数据与人类的数据一致 流行病学，这表明中风的发病率和严重性增加 随着年龄的增长，男性比女性更大。拟议的研究， 因此，必须解决支配年龄和性别的因素 不同之处。实验将使用高效液相色谱法来测定总组织中的抗坏血酸 和GSH，以及组织学方法评估组织损伤和抗氧化剂 神经保护。此外，还可以使用工具来监控 细胞外抗坏血酸水平([ASC]o)。碳的伏安法 纤维微电极，我们将在体外检测脑片中的[ASC]o和 在体大脑中动脉闭塞(MCAO)后，将它们联合 用组织总抗坏血酸含量来计算[ASC]的数据i.特定 目标是： 1.确定抗坏血酸和谷胱甘肽在小鼠脑内的差异定位 CNS，强调抗坏血酸存在于神经元和GSH中的假设 在神经胶质细胞中。我们将确认老化大脑中的抗氧化剂损失，并延长 性别研究，包括抗氧化酶和铁。二、评估 细胞内外抗坏血酸动态平衡的机制。离体 实验将检验细胞内和细胞外的假设 抗坏血酸浓度([Asc]i和[Asc]o)是恒定调节的 这个过程的动态会随着年龄的增长而变化。三、评估 抗坏血酸和谷胱甘肽在哪里以及如何保护。体外实验将测试 抗坏血酸和谷胱甘肽在细胞内起作用的假说。IV.至 确定缺血时抗坏血酸和谷胱甘肽丢失的机制。我们将延长 初步数据表明，在缺血期间抗氧化剂的损失是 雄性大鼠大于雌性大鼠。五、确定关系 在细胞内抗坏血酸丢失和局灶性缺血之间。实验 将检验[ASC]I损失最大的区域 在缺血期间有最大程度的损害。