EXTRACELLULAR OXYGEN RADICALS AND ISCHEMIC BRAIN INJURY

细胞外氧自由基和缺血性脑损伤

• 批准号: 6130629
• 负责人: DAVID WARNER
• 金额: $ 29.48万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-07 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6130629
• 关键词: NAD(P)H dehydrogenase; cerebral ischemia /hypoxia; enzyme activity; extracellular; free radical oxygen; gene expression; gene targeting; genetically modified animals; histology; laboratory mouse; neutrophil; pathologic process; superoxide dismutase

DESCRIPTION: (Verbatim from the Applicant's Abstract) An important mechanism of ischemic brain damage is production of reactive O2 species, including superoxide (O2-). Natural defenses against O2- include 3 isozymes of superoxide dismutase (SOD). CuZnSOD and MnSOD have been shown to be important in scavenging O2- produced in the intracellular space. The remaining isozyme, extracellular SOD (EC-SOD), is found only in the extracellular (EC) space. Transgenic (EC-SOD overexpressing) and knockout (EC-SOD deficient) mice and the metalloporphyrin compound Mn-TM-2-PyP (and EC-SOD mimetic) provide novel opportunity to isolate and examine effects of O2- produced in the EC space. We have shown that EC-SOD overexpression reduces both global and focal ischemic injury while EC-SOD deficiency increases focal ischemic injury. The goal of this research is to define mechanisms by which EC-SOD improves ischemic outcome and determine if these properties can be emulated by use of EC-SOD mimetic compounds. Our fundamental postulate is that EC-SOD provides a beneficial effect on ischemic brain by scavenging O2- in the EC space which has been generated by either activated neutrophils/microglia or membrane bound oxidases. We will examine whether reperfusion is required for EC-SOD to affect histologic/behavioral outcome. We will then examine whether EC-SOD deficiency worsens global ischemic injury and whether this deficiency can be corrected by administration of Mn-TM-2-PyP. O2- sensitive microelectrodes, OH microdialysis, and nitrotyrosine assays will be used to determine if manipulation of EC-SOD expression and use of Mn-TM-2-PyP alters O2- concentrations in ischemic brain. The source of extracellular O2- will be examined by comparing histologic/behavioral ischemic outcome in neutrophil depleted EC-SOD transgenic/knockout mice and whether EC-SOD pharmacologic mimetics have efficacy in NADPH oxidase knock-out mice lacking respiratory burst activity. Long-term recovery studies will be performed to assure that neuroprotective effects of EC-SOD are permanent. Immunoblotting techniques will be used to determine if EC-SOD expression is upregulated in post-ischemic brain and whether expressed EC-SOD is intact of cleaved of its heparin binding domain which might facilitate diffusion to sites of inflammation. We believe this work will provide important mechanistic insight into how ischemic outcome is affected by O2- formed in the extracellular space and that this work will provide novel routes of investigation for therapy of ischemic brain injury.

描述：(一字不差地摘自申请人的摘要)一个重要的机制 缺血性脑损伤是指产生活性氧物种，包括 超氧阴离子(O2-)。对O2-的天然防御包括3种超氧化物同工酶 超氧化物歧化酶(SOD)。铜锌超氧化物歧化酶和锰超氧化物歧化酶已被证明在 清除细胞内产生的O2-。剩下的同工酶， 细胞外超氧化物歧化酶(EC-SOD)，仅存在于细胞外(EC)间隙。 转基因(EC-SOD过表达)和基因敲除(EC-SOD缺陷)小鼠 金属卟啉化合物Mn-TM-2-PYP(和EC-SOD模拟物)提供新的 有机会分离和检查EC空间中产生的O2-的影响。我们 已经表明EC-SOD的过度表达可以减少全局性和局灶性缺血 EC-SOD缺乏时的损伤会增加局灶性缺血性损伤。的目标是 本研究旨在明确EC-SOD改善缺血结局的机制 并确定是否可以通过使用EC-SOD模拟物来模拟这些特性 化合物。我们的基本假设是EC-SOD提供了一种有益的 清除EC空间中O2-对缺血脑的影响 由激活的中性粒细胞/小胶质细胞或膜结合的氧化酶产生。 我们将检查EC-SOD是否需要再灌流才能影响 组织学/行为结局。然后我们将检查EC-SOD缺乏 加重全脑缺血损伤以及这一缺陷是否可以通过 Mn-TM-2-PYP的给药。氧气敏感微电极，羟基微渗析， 硝基酪氨酸测定将被用来确定EC-SOD的操纵 Mn-TM-2-PYP的表达和使用改变缺血脑组织中的O2-浓度。 胞外O2-的来源将通过比较 中性粒细胞耗竭EC-SOD的组织学/行为学缺血结局 转基因/基因敲除小鼠以及EC-SOD药理模拟物是否有 对呼吸爆发活性缺失的NADPH氧化酶基因敲除小鼠的疗效。 将进行长期康复研究，以确保神经保护 EC-SOD的作用是永久性的。免疫印迹技术将用于 确定EC-SOD在缺血后脑内的表达是否上调 表达的EC-SOD是否完整地切割其肝素结合区 这可能会促进扩散到炎症部位。我们相信这是可行的 将提供重要的机制洞察缺血的结果是如何的 受到在细胞外空间形成的O2-的影响，这项工作将 为缺血性脑损伤的治疗提供新的研究途径。