EXTRACELLULAR OXYGEN RADICALS AND ISCHEMIC BRAIN INJURY

细胞外氧自由基和缺血性脑损伤

• 批准号: 6351889
• 负责人: DAVID WARNER
• 金额: $ 33.76万
• 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/6351889
• 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：（逐字研究申请人的摘要） 缺血性脑损伤是反应性O2物种的产生，包括 超氧化物（O2-）。反对O2的天然防御包括3个超氧化物的同工酶 歧义酶（SOD）。 Cuznsod和Mnsod在 在细胞内空间中产生的清除O2。其余的同工酶， 细胞外SOD（EC-SOD）仅在细胞外（EC）空间中发现。 转基因（EC-SOD过表达）和基因敲除（EC-SOD缺乏）小鼠和 金属卟啉化合物MN-TM-2-PYP（和EC-SOD MIMETIC）提供了新颖的 有机会分离和检查在EC空间中产生的O2的影响。我们 已经表明，EC-SOD过表达降低了全球和局灶性缺血 损伤而EC-SOD缺乏会增加局灶性缺血性损伤。目标 这项研究是为了定义EC-SOD改善缺血结果的机制 并确定是否可以通过使用EC-SOD模拟来模拟这些特性 化合物。我们的基本假设是EC-SOD提供了有益的 通过在EC空间中清除O2-对缺血性大脑的影响 由活化的嗜中性粒细胞/小胶质细胞或膜结合氧化酶产生。 我们将检查EC-SOD是否需要再灌注以影响 组织学/行为结果。然后，我们将检查EC-SOD缺乏症是否 恶化了全球性缺血性伤害，是否可以通过 MN-TM-2-PYP的给药。 O2-敏感的微电极，OH微透析， 硝基酪氨酸测定法将用于确定是否操纵EC-SOD MN-TM-2-pyp的表达和使用改变了缺血性脑中的O2浓度。 细胞外O2-的来源将通过比较 中性粒细胞耗尽的EC-SOD的组织学/行为缺血结果 转基因/基因敲除小鼠以及EC-SOD药理学模拟物是否具有 缺乏呼吸爆发活性的NADPH氧化酶敲除小鼠的功效。 将进行长期恢复研究以确保神经保护 EC-SOD的影响是永久性的。免疫印迹技术将用于 确定在缺血后大脑中EC-SOD表达是否上调 表达的EC-SOD是否完整地裂解其肝素结合结构域 这可能有助于扩散到炎症部位。我们相信这项工作 将提供重要的机械洞察力，以了解缺血结果的方式 受细胞外空间中形成的O2的影响，这项工作将 为缺血性脑损伤治疗提供新的调查途径。