Transgenic animal injury paradigms

转基因动物损伤范例

• 批准号: 6396017
• 负责人: PAK H CHAN
• 金额: $ 18.53万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6396017
• 关键词: antioxidants; apoptosis; cerebral ischemia /hypoxia; cytochrome c; disease /disorder model; fluorescence microscopy; gene targeting; genetically modified animals; hippocampus; in situ hybridization; isozymes; laboratory mouse; laboratory rat; mitochondria; mutant; necrosis; neuropathology; oxidative stress; reperfusion; stainings; superoxide dismutase; western blottings

It is well-established that a brief period of global brain ischemia causes delayed cell death in hippocampal CA1 pyramidal neurons days after reperfusion in animals and humans. Although numerous factors have been indicated in this phenomenon, the mechanisms underlying this delayed neuronal cell death are still poorly understood. We have demonstrated that cerebral infarction and neurological deficits are significantly reduced in transgenic mice over-expressing CuZn-superoxide dismutase (Sod1) activity after acute focal stroke, whereas vasogenic edema, infarction and neurological deficits are exacerbated in mutant mice deficient in SOD1 or in mitochondrial manganese SOD (Sod2) activities. But the role of these antioxidant enzymes on the delayed hippocampal neuronal injury after global ischemia is still unknown. Our hypothesis is that oxidative stress induced by mild ischemia and reperfusion causes the delayed hippocampal neuronal injury and death through pathways involving both necrosis and apoptosis, and that the latter is exacerbated when mitochondrial dysfunction occurs during reperfusion. It is our aim to test our hypothesis using transgenic mice over-expressing Sod1 and Sod2 activities and knockout mutant mice that contain no SOD1 -/- (homozygous), half (heterozygous, Sod1 +/-) or SOD2 +/- activities. In order to dissect out the role of mitochondrial dysfunction in ischemic brain injury, we will study the cytosolic distribution of mitochondrial dysfunction in ischemic brain injury, we will study the cytosolic distribution of mitochondrial proteins cytochrome c and cytochrome c oxidase in ischemic brain tissue. Cytochrome c release from mitochondria has been attributed to the activation of caspase 3 and subsequent apoptosis in cells following exogenous apoptotic stimuli. In order to elucidate the oxidative role of subcellular compartmentation (i.e., cytosolic versus mitochondria) in necrosis and apoptosis, we will generate mice that contain genotypes with combinations of increased Sod1 expression and Sod2 +/- knockout mutants. We will investigate whether increased cytosolic CuZnSOD (SOD1) activity will reduce neuronal apoptosis in Sod2 +/- knockout mice that are vulnerable to transient forebrain ischemia. We believe these are unique and fresh approaches that will provide insights into the oxidative mechanism in mitochondria that underlies apoptosis in delayed hippocampal cell death after global cerebral ischemia and reperfusion.

众所周知，动物和人类再灌注几天后，短暂的全脑缺血会导致海马 CA1 锥体神经元延迟细胞死亡。尽管这种现象已经表明了许多因素，但这种延迟性神经细胞死亡的机制仍然知之甚少。我们已经证明，在急性局灶性中风后过度表达铜锌超氧化物歧化酶（Sod1）活性的转基因小鼠中，脑梗塞和神经功能缺损显着减少，而在SOD1或线粒体缺陷的突变小鼠中，血管源性水肿、梗塞和神经功能缺损加剧。锰SOD（Sod2）活性。但这些抗氧化酶对全脑缺血后迟发性海马神经元损伤的作用仍不清楚。我们的假设是，轻度缺血和再灌注引起的氧化应激通过涉及坏死和凋亡的途径导致迟发性海马神经元损伤和死亡，并且当再灌注期间发生线粒体功能障碍时，后者会加剧。我们的目的是使用过度表达 Sod1 和 Sod2 活性的转基因小鼠以及不含 SOD1 -/- （纯合）、半（杂合，Sod1 +/-）或 SOD2 +/- 活性的敲除突变小鼠来检验我们的假设。为了剖析线粒体功能障碍在缺血性脑损伤中的作用，我们将研究线粒体功能障碍在缺血性脑损伤中的胞质分布，我们将研究线粒体蛋白细胞色素c和细胞色素c氧化酶在缺血性脑组织中的胞质分布。线粒体释放细胞色素 c 归因于 caspase 3 的激活以及随后的细胞凋亡刺激后的细胞凋亡。为了阐明亚细胞区室（即胞质与线粒体）在坏死和凋亡中的氧化作用，我们将产生含有 Sod1 表达增加和 Sod2 +/- 敲除突变体组合的基因型的小鼠。我们将研究增加胞质 CuZnSOD (SOD1) 活性是否会减少易受短暂前脑缺血影响的 Sod2 +/- 敲除小鼠的神经元凋亡。我们相信这些是独特而新鲜的方法，将提供对线粒体氧化机制的见解，该机制是全脑缺血和再灌注后海马细胞延迟死亡的细胞凋亡的基础。