Transgenic animal injury paradigms

众所周知，在动物和人类中，短暂的全脑缺血会导致再灌流后数天的海马CA1区锥体神经元迟发性细胞死亡。虽然在这一现象中已经发现了许多因素，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，在急性局灶性卒中后，过表达铜锌超氧化物歧化酶(SOD1)活性的转基因小鼠的脑梗塞和神经功能障碍显著减少，而缺乏SOD1或线粒体锰超氧化物歧化酶(SOD2)活性的突变小鼠则加剧了血管源性水肿、脑梗塞和神经功能障碍。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，轻度缺血和再灌流诱导的氧化应激通过坏死和凋亡两条途径导致迟发性海马神经元损伤和死亡，而后者在再灌流期间发生线粒体功能障碍时加剧。我们的目标是使用过度表达SOD1和SOD2活性的转基因小鼠和不包含SOD1-/-(纯合)、一半(杂合，SOD1+/-)或SOD2+/-活性的敲除突变小鼠来验证我们的假设。为了探讨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浆分布，研究线粒体蛋白细胞色素c和细胞色素c氧化酶在缺血脑组织中的胞浆分布。细胞色素c从线粒体的释放归因于caspase 3的激活和细胞在外源凋亡刺激下的随后的凋亡。为了阐明亚细胞区隔(即胞浆和线粒体)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+/-基因敲除突变组合的基因型的小鼠。我们将研究胞质CuZnSOD(SOD1)活性增加是否会减少Sod2+/-基因敲除小鼠的神经细胞凋亡，这些小鼠容易受到短暂性前脑缺血的影响。我们相信，这些独特而新鲜的方法将为深入了解线粒体氧化机制提供洞察，线粒体氧化机制是全脑缺血和再灌流后迟发性海马细胞死亡的基础。

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.