Alteration And Significance Of Protein Expression Pattern In Astrocytes Following Fluid Percussion Injury In Vitro

Objective: To study the alteration of protein expression pattern in astrocytes in vitro following fluid percussion injury and to find biomarkers of brain injury.Methods: The astrocytes of cerebral cortex from SD rats born in 24 hours were cultured in DMEM-F12 medium primarily. The cells were identified by immunocytochemical staining with anti-GFAP and then were randomly divided into control group and injury groups which were subjected to fluid percussion injury and then subdivided into 4h,8h,12h,24h and 48h groups according to the time elapsed after injury. Extent of cell injury was qualitatively assesssed by trypan blue, PI/Hoechst3342 staining.The change of protein expression pattern in Astrocytes following fluid percussion injury were monitored with weak cationic exchanger chips(WCX-2) and strong anion exchange chips(SAX2) by surface-enhanced laser desorption ionization-time of flight-mass spectrometry.Results: 1 Culture of astrocytes in vitro: Almost pure cells were obtained after primary culture and subculture. The cells showed big cell body, abundant cytoplasm, many longer apophyses and round or ovi-round karyons which leaned to one side. It coincided with the form of astrocytes.2 Identification of astrocytes: Immunocytochemical staining demonstrated that nearly all of the cells were positive.3 Morphological changes of astrocytes following fluid percussion injury: Following the fluid percussion injury(0.2MPa), astrocytes showed hypertrophic cell body, widened intercellular space, and the number of dead cell obviously increased. Apoptosis cells can be detected by PI/Hoechst3342 staining at 4 hours post-injury.4 Changes of protein expression patterns: (1)A total of 688 protein peaks were detected on WCX-2 chips and a total of 549 protein peaks were detected on SAX2 chips. (2)Among the total of protein peaks detected, the relative intensity of 17 protein peaks was shown to be differentially altered on WCX-2 chips in astrocytes between injury groups and conrol group(P<0.05). The relative intensity of 15 protein peaks was shown to be differentially altered on SAX2 chips in astrocytes between injury groups and control group(P<0.05). There were 135 protein peaks in astrocytes of injury groups which couldn't be detected in sham operation group with WCX-2 chips. There were 195 protein peaks in astrocytes of injury groups which couldn't be detected in control group with SAX2 chips. (3)On WCX-2 chips, 14 protein peaks were detected at two time-points in astrocytes. On SAX2 chips, 33 protein peaks were detected at more than two time-points in astrocytes. (4)Compared with control groups, there were 8 protein peaks that showed the increase of protein expression and 24 protein peaks that showed the reduction of protein expression on two kinds of chips in astrocytes of injury groups.Conclusion: 1.The alteration of protein expression patterns in astrocytes could be induced after fluid percussion injury. 2.The distinct proteins detected in astrocytes included the proteins which were significant compared with control group and those which were not detected in control group. They could be biomarkers of brain injury and be useful for molecular mechanism, diagnosis or therapy of brain injury.