加藤 一郎 東北大学, 医学部, 助手 (50250741)
冨永 悌二 東北大学, 医学部, 助手 (00217548)
CHAN PaK H. カリフオルニア大学, サンフランシスコ校・医学部, 教授
木内 博之 東北大学, 医学部, 助手 (30241623)
TOMINAGA Teiji Tohoku University School of Medicine
KATO Ichiro Tohoku University School of Medicine
KINOUCHI Hiroyuki Tohoku University School of Medicine
CHAN Pak h. University oF California San Francisco
|Budget Amount *help
¥4,400,000 (Direct Cost : ¥4,400,000)
Fiscal Year 1994 : ¥4,400,000 (Direct Cost : ¥4,400,000)
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
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