Studies Of Influence On Astrocyte Injuries Induced By Hypoxia/Reoxygenation In Vitro And Protective Effects Of SHENMAI Injection

Ischemic cerebrovascular diseases has been the main topic of the scientists all the time as an important disease in neurology in the world．The morbidity and disability iS still on high level.Nowadays．the focuus on ischemia/reperfusion has transferred from neurons to astrocytes．Astrocytes are the most numerous nonneuronalCeIl Type in the central nervous system．The regeneration and functional recovery of the CNS rely on the reactivity of astrocytes to some extent．Amout of astrocytes congregation affect the volume ofinfarction after injuries of the central nervous system．The mechanism of pathophysiology on the topic which the ischemic penumbra transform into ischemic infarction ls quite unknown．There are a great of evidence on the pathophysiology of cerebral ischemia involved in NO．NO may be one of the most important molecular in adjusting changing from the salvage ischemic penumbra to ischemic infarcion．【objective】:This study examined the cultured astrocytes imitatingthe model of ischemia/reperfusion in Vivo through establishing the model of hypoxia/reoxygenation in Vitro,to observe the changes of morphology of astrocytes and the expression of iNOS by immunocytochemical examinations and the protective effectof SHENMAI injection． 【methods】:The cultured astrocytes were prepared from cerebral cortices of newborn Wistar rats(1ess than 24 hours)．Trypsinized and dissociated cells were plated in culture flakes containing high glucose Dulbecco'S modifided Eagle,s medium supplemented with 20％fetal bovine serum．After incubation for 7～10 days．the cells were trypsinized and subcultured．It would take 4 progress of subculture to get the stable cells．Homogeneity of the cell populations was confirmed by immunosytochemical examinations with anti-gliafibrillary acid protein(GFAP)．the number of GFAP positive cells exceed 95％． The cultured cortical astrocyte are divided into 3 groups:normalgroup；hypoxia/reoxygenation group；treatmeat with SHENMAI injection group．The confluent astrocytes were placed in a controlled atmosphere culture chamber,a humidified airtight apparatus with inflow and outflow valves,into which a mixture of 95％N2and 5％ C02 was flushed into the apparatus for the appointed time．The chamber was sealed to maintain the gas Composition and kept at 37。C．During the exposure to hypoxia and reoxygenation,the cultured cells were collected at the indicated times from 0 to 24 hours for monitoring the lactate dehydrogenase(LDH ) and MTT to estimate cellular damage．After exposure to hypoxia for l2 hours the culturedishes were transfefred to an ordinary incubator with 95％air and 5%C02 from the cells reoxygenation．The culture medium was notreplaced during hypoxia and reoxygenation.At the same time,the cultured cells were collected for immunocytochemistry about GFAP and iNOS in the different time point． Meanwhile.the adoption of optium concentration on SHENMAI iniection iS going on in 5％,1O％,l5％,20％．On the basis of the effect of the study．the 20％ SHENMAI injection iS the most adaptable concentration for treatment． 【results】:the cell mophology,LDH, MTT werenot impacted by hypoxia for l2 hours．however,reoxyenation make a damage obviously．The longer time,the geater damage to the shape of astrocytes．Correspondingly,the LDH changed little in hypoxia and made great transform in reoxyenation．The MTT was observed as the same．So we could deduce that hypoxia and reoxyenation induce the damage of the cultured astrocytes．In addition．the expression 0f iNOSis dynamic,its expression appears after hypoxia for l2 hours．The max point is hypoxia for 12 hours then reoxygenation for 48 hours．The decline point is hypoxia for 12 hours then reoxygenation for 72 hours． The 20％SHENMAI iniection protective effect on cultured astrocytes induced by hypoxia/reoxygeantion．the possible mechanism of the protective effect on SHENMAI injection decrease the expression of iNOS during the time of hypoxia/reoxygenation．【Conclusions...