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

氧源性自由基参与血管源性水肿和缺血引起的梗死的发病机制。脂质体包裹CuZn超氧化物歧化酶（SOD）改善大鼠缺血性脑水肿和梗死。尽管这些研究为脑损伤的治疗提供了潜在的治疗先例，但还需要其他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的作用模式。为了直接研究脑CuZn-SOD升高在推测与超氧自由基有关的脑损伤发病机制中的作用，我们使用了携带人类CuZn-SOD基因的转基因（Tg）小鼠。首先，我们比较了Tg小鼠（SOD活性增加3.1倍）和非Tg小鼠在大脑中动脉（MCA）闭塞10min和60min后c-fos原癌基因和70kda -热休克蛋白（hsp70） mRNA的诱导。我们还研究了一氧化氮合酶（NOS）抑制剂的作用。更多的研究表明，L-NAME（神经元和内皮细胞NOS抑制剂）和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）对两种小鼠脑梗死的影响，因为一氧化氮（NO）被认为与超氧自由基反应并形成过氧亚硝酸盐，一种强氧化剂。在缺血60min时，Tg小鼠的c-fos和hsp70 mRNA诱导面积明显低于非Tg小鼠。然而，与非Tg小鼠相比，Tg小鼠在MCA闭塞10min后再循环后c-fos和hsp70 mRNA的表达时间延长。在NOS抑制剂实验中，L-NAME处理的Tg小鼠梗死体积明显大于盐水处理的Tg小鼠，而7-NI有减小Tg小鼠梗死体积的趋势。这些结果表明，SOD可保护脑免受严重缺血损伤引起的脑梗死，神经元NOS产生的NO与超氧化物含量有关，导致脑损伤。其次，我们还研究了非Tg小鼠、杂合和纯合Tg小鼠（1.5倍、3.1倍和5.0倍）创伤后脑水肿的形成和hsp70和c-fos mRNA的诱导。Tg小鼠损伤顶叶皮层含水量和Evans蓝染色渗漏呈SOD剂量依赖性减弱。Tg小鼠损伤后hsp70和c-fos mRNA诱导面积小于非Tg小鼠。在慢性期，Tg小鼠的神经功能较非Tg小鼠有明显改善。最后，我们测量了Tg和/或非Tg小鼠脑内皮细胞单层培养后的电阻，以评估超氧化物对血脑屏障通透性的影响。相反，Tg组的通透性明显高于非Tg组。这种增加可能是由于过氧化氢清除系统的耗尽和通过超氧化物驱动的Fenton型Haber-Weise反应形成羟基自由基，因为这种增加可以通过添加去铁胺来对抗。从这些结果可以看出，SOD在体内对缺血和创伤性脑损伤具有较强的保护作用，但在特殊情况下，SOD活性的增加可能通过产生羟基自由基而产生毒性。过氧化氢清除系统对SOD发挥清除超氧自由基的保护作用至关重要。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