EXTRACELLULAR OXYGEN RADICALS AND ISCHEMIC BRAIN INJURY

描述：（逐字来自申请人的摘要） 缺血性脑损伤是反应性O2物质的产生，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 歧化酶（SOD）。CuZnSOD和MnSOD已被证明是重要的 清除细胞内产生的O2-。剩下的同工酶， 胞外SOD（EC-SOD）仅存在于胞外（EC）空间中。 转基因（EC-SOD过表达）和敲除（EC-SOD缺陷）小鼠以及 金属卟啉化合物Mn-TM-2-PyP（和EC-SOD模拟物）提供了新的 有机会隔离和检查在EC空间中产生的O2的影响。我们 已经表明EC-SOD过表达减少了整体和局部缺血， 损伤，而EC-SOD缺乏增加局灶性缺血损伤。的目标 本研究旨在明确EC-SOD改善缺血性结局的机制 并确定这些性质是否可以通过使用EC-SOD模拟物来模拟 化合物.我们的基本假设是，EC-SOD提供了一个有益的 通过清除EC空间中的O2-对缺血脑的作用， 由活化的嗜中性粒细胞/小胶质细胞或膜结合氧化酶产生。 我们将研究EC-SOD是否需要再灌注才能影响 组织学/行为学结果。然后我们将研究EC-SOD缺乏是否 的全球缺血性损伤，以及这种缺陷是否可以纠正， 给予Mn-TM-2-PyP。O2敏感微电极，OH微透析， 和硝基酪氨酸测定将用于确定是否操作EC-SOD Mn-TM-2-PyP的表达和使用改变了缺血脑中的O2-浓度。 将通过比较来检查细胞外O2-的来源。 中性粒细胞耗竭EC-SOD组织学/行为学缺血结局 转基因/敲除小鼠以及EC-SOD药理学模拟物是否具有 在缺乏呼吸爆发活性的NADPH氧化酶敲除小鼠中的功效。 将进行长期恢复研究，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表达在缺血后脑中是否上调， 表达的EC-SOD是否完整或其肝素结合结构域被切割 这可能有助于扩散到炎症部位。我们相信这项工作 将提供重要的机制洞察如何缺血的结果是 受O2-在细胞外空间形成的影响，这项工作将 为缺血性脑损伤的治疗提供了新的研究途径。

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.