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.

在动物和人类中，短暂的全脑缺血可导致海马CA1锥体神经元在再灌注后数天的延迟细胞死亡。尽管在这一现象中已经发现了许多因素，但这种延迟神经元细胞死亡的机制仍然知之甚少。我们已经证明，急性局灶性中风后，过度表达cuzn -超氧化物歧化酶（Sod1）活性的转基因小鼠脑梗死和神经功能缺损显著减少，而Sod1或线粒体锰SOD （Sod2）活性缺乏的突变小鼠血管源性水肿、梗死和神经功能缺损加剧。但这些抗氧化酶在全脑缺血后迟发性海马神经元损伤中的作用尚不清楚。我们的假设是，轻度缺血再灌注引起的氧化应激通过涉及坏死和凋亡的途径导致海马神经元延迟损伤和死亡，再灌注过程中线粒体功能障碍加剧了后者。我们的目标是用过表达Sod1和Sod2活性的转基因小鼠和不含Sod1 -/-（纯合子）、一半（杂合子，Sod1 +/-）或Sod2 +/-活性的敲除突变小鼠来验证我们的假设。为了剖析线粒体功能障碍在缺血性脑损伤中的作用，我们将研究缺血性脑损伤中线粒体功能障碍的胞质分布，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。线粒体细胞色素c的释放归因于半胱天冬酶3的激活和外源性凋亡刺激后细胞的凋亡。为了阐明亚细胞区隔（即细胞质与线粒体）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 +/-敲除突变基因型组合的小鼠。我们将研究细胞内CuZnSOD （SOD1）活性的增加是否会减少Sod2 +/-敲除小鼠短暂性前脑缺血的神经元凋亡。我们相信，这些独特而新颖的方法将为全面脑缺血和再灌注后延迟海马细胞死亡的线粒体氧化机制提供深入的见解。