The Proteomics Study In Primary Cultured Astrocytes Following Fluid Percussion Injury

Objective: To establish the model of mechanical brain injury of primary cultured astrocytes , and to study the alteration of the morphology and protein expression pattern in post-injury astrocytes, and to find biomarkers of brain injury.Methods: Primary cultures of astrocytes were prepared from cerebral hemispheres of 24h-old rat as previously described (Ciccarelli et al., 1994). and were randomly divided into control group and injury groups which were subjected to Scott's fluid percussion injury and then subdivided into 12h,24h and 48h group after injury. The abnormalities of the morphology of astrocytes after injury was observed. The changes of protein expression pattern in astrocytes were monitored with two dimensional gel electrophoresis.Results: 1 Isolation and purification of Primary cultured astrocytes. We isolated and purified astrocytes from newborn rat cerebral cortex. The isolated cerebral tissue was cut into pieces with scissor. And the whole cells of cerebral cortex were mixed to cultivate into tissue culture flasks. when cultures were confluent astrocytes were isolated by shaking mixed glial cultures overnight, non-adhesive cells were discarded. The remaining adhesive cells were detached by exposure to 0.25% trypsin and replated at 0.8×105cells/ml denstity into 60 mm Petri dishes. Base on the different properties of adhesions, developmental time-courses and growth pattern of astroglial cells,astrocytes were cultured and purified.The purification of astrocytes was identified by immunocytochemical staining of glial fribrillary acidic protein(GFAP).2 The evaluation of fluid percussion injury: The pressure of FPI was reflected through the pressure of the chamber and plexiglass cylinder. The relation was that: Y=4.4763X+0.2986. The pressure of the cylinder and chamber increased when the angle of incidence raising.3 Morphological study of astrocytes following fluid percu- ssion injury. Primary cultured astrocytes were randomly divided into control group and injury groups subdivided into 0.05Mpa,0.1Mpa,0.2Mpa,0.4Mpa,0.8Mpa injury group. Dyeing the separating cells by 0.4% Trypan blue.We have investigated sequential morphological changes of astrocytes after different fluid percussion pressure. Typical morphological features of injury cells were detected by Microscopy.:swollen,necrosis,turning round and scattered,cytomembrane ruptured,junction among cells contracted. morphological change was Pressure- depended. Similar results were obtained in the experiment for the apoptotic analysis by Hoechst-PI fluorescence staining. Different rate of cells were necrosis after different pressure injury and apoptosis was observed in 0.2MPa pressure group. However necrosis rate was obviously declined in 0.8 MPa pressure group. The problem has several explanations and the most probable is that the pressure was too big and the damaged astrocytes were disintegration and can not be observed in microscope.Based on the result of the experiments mentioned before,we have found that cultured astrocytes were moderate damaged in 0.2MPa groups and the cell state in this group is suitable for the experiments next. Primary cultured astrocytes were randomly divided into control group and injury groups subdivided into 2h,4h,8h,12h,24h,48h injury group. Primary cultured astrocytes change culturing solution after 0.2MPa pressure injury. Morphological study of astrocytes at 2h,4h,8h,12h,24h,48h after injury.The morphological change was apparent and time-dependence is also obviously:swollen,turning round and scattered,Shrinkage,necrosis,restoration.We have observed that the damaged degree of astrocytes was heavy in 2h,4h,astrocytes begin restoration in 8h,12h,The morphology of the injury group almost the same with control group in 24h,48h.4 The results of 2-DE: Primary cultured astrocytes were randomly divided into control group and injury groups subdivided into12h,24h and 48h groups after injury.All cultured cells were harvested for protein sample preparation in 2D electrophoresis study.The proteins was preserved in liquid nitrogen and used for 2DE electrophoresis after quantitated by Bradford measurement method.1179±89,1279±113,1233±73 and 1256±132 protein spots were detedted respectively in control,12h,24h,48h groups via PDquest software. Dynamic changes were identified and total different 32 spots were detected in this study from the 2DE gels.Total 24 spots were up-regulated and 8 spots were down-regulated at different period after injury.5 The different displayed spots were identified via MALDI -TOF: cofilin 1, destrin, Phosphoglycerate mutase 1, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 10, hypothetical protein, annexin 1.Conclusion: 1 The obvious alteration of morphology in primary cultured astrocytes could be induced after fluid percussion injury.2 Damaged degree of primary cultured astrocytes is increasing with the pressure's increase. Morphological change was apparent and time-dependence is also obviously.3 The alteration of protein expression patterns in primary cultured astrocytes could be induced after mechanical brain injury.4 The distinct protein detected at different time-points in primary cultured astrocytes may be the specific expression biomarkers of brain injury. The further study of the injury mechanism from protein molecular level by 2-DE could be useful for the dianogsis or therapy of brain injury.