Transgenic animal injury paradigms

在动物和人类中，短暂的全脑缺血在再灌注后数天引起海马CA 1锥体神经元的延迟性细胞死亡是公认的。虽然许多因素已表明在这一现象，这种迟发性神经元细胞死亡的机制仍然知之甚少。我们已经证明，脑梗死和神经功能缺损显着减少在转基因小鼠过度表达铜锌超氧化物歧化酶（SOD 1）活性急性局灶性中风后，而血管源性水肿，梗死和神经功能缺损的突变小鼠SOD 1或线粒体锰SOD（SOD 2）活动的缺陷加剧。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，轻度缺血和再灌注引起的氧化应激导致迟发性海马神经元损伤和死亡，通过涉及坏死和凋亡的途径，后者是加剧线粒体功能障碍时发生再灌注。我们的目的是使用过表达Sod 1和Sod 2活性的转基因小鼠和不含SOD 1-/-（纯合子）、半（杂合子，Sod 1 +/-）或SOD 2 +/-活性的敲除突变小鼠来测试我们的假设。为了深入研究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浆分布，即缺血性脑组织中线粒体蛋白细胞色素c和细胞色素c氧化酶的胞浆分布。线粒体中细胞色素c的释放归因于外源性凋亡刺激后细胞中caspase 3的激活和随后的细胞凋亡。为了阐明亚细胞区室化的氧化作用（即，细胞溶质对线粒体）在坏死和凋亡中的作用，我们将产生含有Sod 1表达增加和Sod 2 +/-敲除突变体组合的基因型的小鼠。我们将研究是否增加胞质CuZnSOD（SOD 1）的活性将减少神经元凋亡的Sod 2 +/-敲除小鼠，容易受到短暂的前脑缺血。我们相信这些是独特的和新鲜的方法，将提供深入了解线粒体的氧化机制，在全脑缺血和再灌注后延迟海马细胞死亡的细胞凋亡的基础。

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.