Inhibition Effects Of Propofol On Hydrogen Peroxide Induced Damage In Astrocytes From Spinal Cord

Part I The establishment of method for culturing in vitro of astrocytes from spinal cord of adult ratObjective:To study methods for dissociation, purification and culturing in vitro of primary astrocytes from spinal cord of adult rat, providing an available experimental model for further research on astrocytes and spinal cord injury (SCI).Methods:Primary culture and passage of astrocytes in vitro. Under sterile environment, thoracic segments of the spinal cord was obtained from adult rat and made into cell suspension by digestion with the use of trypsin and mechanical digestion, then transferred to culture flasks without any stratum, and cultured in a incubator (37℃,5%CO2). Fibroblasts were removed by using differential velocity adherent technique. The oligodentrocytes and microglial cells were reduced with orbital shaker when the cells were confluent by90%on days10through14.The primary culture was passaged when it was confluent. Finally the secondary culture was observed and the astrocytes were identified by immunofluorescence staining.Results:By differential velocity adherent technique,orbital shaker in constant temperature and passage, the purified astrocytes with typical shapes were obtained. Astrocytes grew in a good condition through culture. Immunocytochemical stain showed:while the positive rate of vimentin stain was up to100%. The positive rate of GFAP stain was low very much.Conclusion:The study provided one method to culture astrocytes from spinal cord of adult rat successfully. The method could be a model for study astrocytes or could be an ideal model to study some impaired or protective mechanisms of astrocytes in SCI. The positive rate of GFAP stain was low very much.Part II Inhibition effects of propofol on hydrogen peroxide induced damage in astrocytesObjective:To study the effect and mechanism of propofol on hydrogen peroxide (H2O2) induced damage in astrocytes.Methods:Based on the culture of primary astrocytes from spinal cord of adult rat, the third generation of astrocytes was used in our experiment. By measuring cellular vitality with MTT assay, the suitable H2O2concentration was determined. The dependence of astrocytes vitality on the concentration of propofol and corresponding intralipid were studied.The astrocytes were randomly divided into CD normal control:the DMEM-F12culture medium;②Intralipid group;②H2O2damaged group:hatched for30min with400μmol/L H2O2;④ropofol group: hatched for4h with100μmol/L propofol;⑤propofol and H2O2treated group:the different concentration of25μM、50μM、100μM propofol was added to the culture medium30min before H2O2exposure. The cells were further cultured for30min with the addition of H2O2(400μM) into the medium. Then continue to cultivate4h;⑥ntralipid and H2O2treated group:after treatment with Intralipid, the cells were further cultured for30min with the addition of H2O2(400μM) into the medium. Then continue to cultivate4h.As the experiments were completed, nutrient solution and cells of each group were collected respectively for further testing. The cell morphology and structure of each group were observed and photographed by inverted microscope. The cell viability was tested by MTT assay. The cell level of MDA was detected by assay Kit and the cell culture medium level of LDH was measured by ELISA.Results:The H2O2concentration of400μmol/L has obvious cell mediated cytotoxicity. The cell vitality decreased, LDH and MDA increased, which has statistical significance (P<0.05) with comparison to normal control. By comparing with H2O2group, the cell vitality of propofol and H2O2treated group increased, the LDH and MDA were suppressed (P<0.05), also showing a dependence on the dose (P<0.01), which has statistical significance. This results indicates that propofol has a protective effect on hydrogen peroxide induced oxidative damage in astrocytes and is dose dependent. Intralipid didn’t show this effect. The results observed by inverted microscope were demonstrated as follows:normal control group:Cells form normal; H2O2group:astrocytes swelling, cytoplasm diminish and become hyaline, nuclear enrichment, some of which fell off, floating and dead; propofol pretreatment group morphological changes was not obvious.Conclusion:H2O2decreased cell vitality by damaging AS, increased LDH and MDA. Propofol has a protective effect on hydrogen peroxide induced oxidative damage in astrocytes and is dose dependent. The protective effect of propofol on hydrogen peroxide induced oxidative damage in astrocytes may be due to the suppression of oxidative stress.