Protection of Ischemic/Traumatic Brain Injruy in SOD-Transgenic Mouse

氧自由基与缺血引起的血管源性水肿和梗死的发病机制有关。脂质体包埋铜锌超氧化物歧化酶（SOD）可减轻大鼠缺血性脑水肿和脑梗死。虽然这些研究为脑损伤的治疗提供了潜在的先例，但尼德替代的实验方法来解决氧自由基在缺血性脑损伤中的作用和SOD的作用方式。为了直接研究增加的脑CuZn-SOD在推测涉及超氧化物自由基的脑损伤的发病机制中的作用，我们使用了携带人CuZn-SOD基因的转基因（Tg）小鼠。本研究比较了Tg小鼠和正常小鼠c-fos原癌基因和70 kDa热休克蛋白（hsp 70）mRNA的表达，（3.1倍增加的SOD活性）和nonTg小鼠大脑中动脉（MCA）闭塞10分钟和60分钟后。并检测了一氧化氮合酶（NOS）对心肌细胞凋亡的影响。 ...更多信息 本研究观察了一氧化氮（NO）与超氧阴离子自由基（O2）反应生成过氧亚硝酸根（过氧亚硝基阴离子）的作用，并与L-NAME（神经元和内皮NOS抑制剂）、7-NI（选择性神经元NOS抑制剂）和7-NI（选择性神经元NOS抑制剂）进行了比较。缺血60 min时，Tg小鼠c-fos和hsp 70 mRNA的诱导表达面积明显小于nonTg小鼠。然而，与nonTg小鼠相比，在MCA阻断10分钟后再循环后，Tg小鼠中c-fos和hsp 70 mRNA的表达延长。在NOS抑制剂实验中，L-NAME处理的Tg小鼠表现出明显更大的梗死体积比盐水处理的，而7-NI显示出减少Tg小鼠的梗死的趋势。这些结果表明，超氧化物歧化酶（SOD）可以保护大脑免受严重缺血性损伤而导致梗死，神经元NOS产生的NO会导致脑损伤，这取决于超氧化物含量。其次，我们还研究了创伤后脑水肿的形成以及hsp 70和c-fos mRNA的诱导。使用非Tg小鼠以及杂合和纯合Tg小鼠（1.5，3.1，SOD活性提高5.0倍。SOD剂量依赖性地降低Tg小鼠顶叶皮质含水量和Evans蓝染料渗漏。损伤后Tg小鼠hsp 70和c-fos mRNA的诱导区域小于nonTg小鼠。在慢性期，Tg小鼠的神经功能较nonTg小鼠有明显改善。最后，我们用超氧阴离子产生剂甲萘醌（menadion）处理Tg和nonTg小鼠的单层脑内皮细胞培养物，测定其电阻，以评价超氧阴离子对血脑屏障通透性的影响。贴壁时，Tg小鼠的通透性显著高于nonTg小鼠。这一增加可能是由于过氧化氢清除系统的耗竭和通过超氧化物驱动的芬顿型Haber-Weise反应产生羟自由基所致，而去铁胺可拮抗这种增加，由此可见，SOD活性的增加可能是由于在特殊环境下产生羟自由基所致，尽管SOD在体内对缺血和创伤性脑损伤具有很强的保护作用。SOD通过清除超氧阴离子自由基而发挥保护作用，其过氧化氢清除系统是必需的。少

Oxygen-derived free radicals have been implicated in the pathogenesis of vasogenic edema and infarction caused by ischemia. Liposome-entrapped CuZn superoxide dismutase (SOD) ameliorated ischemic brain edema and infarction in rats. Although these studies provide potential therapeutic precedents for the management of brain injury, alternative experimental approaches are neede to address the issues of the role of oxygen free radicals and the mode of action of SOD in ischemic brain injury. To investigate directly the increased brain CuZn-SOD in the pathogenesis of brain injuries presumed to involve superoxide radicals, we have used trangenic (Tg) mice carrying the human CuZn-SOD gene.First, We have compared the c-fos protooncogene and 70kDa-heat shock protein (hsp70) mRNA induction between Tg mouse (3.1 fold increased SOD activity) and nonTg mouse following 10min and 60 min of middle cerebral artery (MCA) occlusion. And also we have examined the effect of nitric oxide synthase (NOS) inhib … More itors, L-NAME (neuronal and endothelial NOS inhibitor) and 7-NI (selective neuronal NOS inhibitor) and 7-NI (selective neuronal NOS inhibitor) on brain infarction in both mice, since nitric oxide (NO) has been considered to react with superoxide radicals and form peroxynitrite, a strong oxidant. In 60min ischemia the area of c-fos and hsp70 mRNA induction was significantly attenuated in Tg mice than nonTg mice. However, the prolonged expression of c-fos and hsp70 mRNA was demonstrated in Tg mice compared to nonTg mice after the recircualtion following 10min MCA occlusion. In the NOS inhibitor experiment, L-NAME treated-Tg mice showed significantly bigger infarct volume than saline-treated ones, whereas 7-NI showed the tendency to decrease the infarction in Tg mice. These resuts suggest that SOD could protect brain from severe ischemic insults causing infarction and that the NO generated by neuronal NOS results in the brain injury depending the superoxide content.Secondarily, it was also investigated that the brain edema formation and the induction of hsp70 and c-fos mRNA following traumatic injury using nonTg mice, and heterozygous and homozygous Tg mice (1.5,3.1, and 5.0 fold increased SOD activityies. The water content of injured parietal cortex and Evans Blue dye leakage were attenuated in Tg mice in a SOD dose dependent manner. The areas of hsp70 and c-fos mRNA induction were smaller in Tg mice than in nonTg mice following injury. In chronic stage, the neurological function was significantly improved in Tg mice compared to nonTg mice.Finally, we have measured the electrical resistance through the single layr of the brain endothelial cell culture from Tg and/or nonTg mice following the treatment with superoxide generating agent, menadion, in order to evaluate the effect of superoxide on the BBB permeability. Adversely, the permeability was significantly increased in Tg than nonTg mice. This increase is likely due to the depletion of scavenging system of hydrogen peroxide and forming hydroxyl radical through superoxide-dirven Fenton type Haber-Weise reaction since this increase was antagonized by adding desferroxamine.From these results, the increase of SOD activity could be toxic by generating hydroxyl radicals under the special circumstance although SOD has a strong brain protective effect against the ischemic and traumatic insult in vivo. And it is also suggested that the scavenging system of hydrogen peroxide is essential for SOD to perform the protective effect by eliminating superoxide radicals. Less