Estimation of serine protease mRNA in glial cells after ischemic and hypoxic conditions.

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It is well-known that Caspase, kinds of Cistein proteases, are concerned in neuronal cell death. On the other hand, the roles of tPA, uPA and r-gsp, kinds of Serin proteases, for neuronal cells have not yet been fully clarified. We investigated changes in tPA and uPA mRNA expressions in the rat brain after middle cerebral artery (MCA) occlusion and PC12 cells under hypoxic condition.Focal cerebral ischemia was produced by MCA occlusion using an intraluminal suture inserted from right internal carotid artery in male Sprague-Dawley rats (280 to320 g) under anesthesia with a mixture of fluothane. After rats were decapitated at 6 hours of MCA occlusion, total RNA were extracted from each hippocampus of bilateral hemispheres.PC12 cell line was maintained in medium supplemented with 10% FBS, 5% horse serum in a humidified atmosphere containing 5% C02 at 37ﾟC.After 24h of incubation in the medium containing 2% FBS, cells were placed into hypoxic condition (multigas incubator ; set to 1.0% 02, with 5% C02 and the balance N2). Total RNA were extracted after exposure of hypoxia for 0, 3, 6, 12 and 24 hours. Extracted RNAs were analysed by RT-PCR technique using specific primer of tPA, uPA and r-gsp for investigation of changes in these mRNA expressions in each period.Up-regulation of tPA and uPA mRNA were revealed in the rat hippocampus following 6 hours ischemia. The level of tPA mRNA was increased from the period of 6 hours exposure of hypoxia in PC12 cell, rustled in apoptoic changes. There is a tendency of up-regulation of r-gsp expression in ischemic condition. However, the level of uPA mRNA was not revealed significant changes. These results suggest that some of Serine proteases, especially tPA or r-gsp may play a role in the neuronal damage after ischemia or hypoxia.