The Expression Of14-3-3ε Protein In Astrocyte In Spinal Cord Injury

The disability caused by spinal cord injury (SCI) was remaining a worldwide challenging problem. Gliar scar and the inhibitory microenviroment were the key reasons. Abundant of14-3-3protein was existed in CNS, which had ability of protecting neurons from ischemic injury was confirmed. But there’s few rechearch in spinal cord especially in SCI. A significant increasing of14-3-3s protein was reported in the early stage of SCI, while which had no significant change in ischemic neurons. So we hypothesized the increasing expression of14-3-3ε protein was related to astrocytes (AS). We build injury models in vivo and in vitro to find their relationship, which we suppose could give a new view to treatment of SCI.Section IThe expression of14-3-3ε protein in primary cultured reactive astrocytes in central nerve system injuryOBJECTIVE:To find out the expression trend of14-3-3ε protein in reactive astrocytes, and the relationship between14-3-3ε protein and GFAP in central nerve system injuryMETHODS:Primary cultures of cerebral cortical astrocytes of newborn SD rats. Matured AS had been treated with oxygen-glucose deprivation4h then refused with normal culture conditionto create ischemia injury model of AS. Another matured AS had been scratched for the mechanical injury. The expression of GFAP and14-3-3ε was detected at different time point (4h,8h,12/16h,24h and72h) with Western blots (WB) and immunofluorescence (IF).RESULTS ONE (with OGD/IR):①The expression of14-3-3s protein and GFAP after injury:both14-3-3ε and GFAP were unregulated in the acute phase (4h after injury) after OGD/IR injury. The upregulation of14-3-3ε protein was earlier than GFAP, which was increased gradually to later stage, while14-3-3s was stopped increasing since OGD/IR24h.②. There was a significant linear correlation between the expressions of14-3-3ε protein and GFAP in the first24hours (R2=0.3060, P=0.017), theres no statistical significant for the last48hours. p. IF showed14-3-3s protein and GFAP had the same subcellular location after OGD/IR.RESULTS TWO (Scratch):①.Different with OGD/IR, in scratch model, WB didn’t show significant change of14-3-3ε in AS, and GFAP was only showed obviously increasing after48h after scratch.②. Linear correlation analysis showed significant relationship between14-3-3s protein and GFAP (R2=0.3119, P=0.009). p. IF showed they both had intensive immunoreactivity around injury area.CONCLUSIONS:14-3-3sprotein and GFAP were increased in the injury-induced reactive AS.They had the same expression trend and subcellular location. We thought the increasing of14-3-3ε protein in the early stage was due to the reactivation of AS, which made the14-3-3ε protein fuctional after injury. Section IIThe expression of14-3-3sproteinin reactive astrocytes in spinal cord injuryOBJECTIVE:To find out the expression trend and relationship of14-3-3ε protein in reactived astrocytes in spinal cord injury (SCI)METHODS:A contusion injury was produced by Allen’s SCI model with a10g impactor dropped from40mm hight. Shame was only did laminectomy. After4h,8h,16h,24h,3d,7d,14d,28d of injury, a1cm long segment with injury site in the centre was removed quickly in the ice for WB. And at the same time point after4%PFA perfusion, the same spinal cord segment was removed for frozen section for IF.RESULTS ONE:①. Similar with OGD/IR, both14-3-3s proteinand GFAP were significantly upregulated in the acute phase (4h/8h) of SCI. The14-3-3s protein increased significantly in the first24hours after SCI, and maintains a high level of expression and activity during the7to28days,14-3-3s protein had earlier lift platform compared to GFAP.②. There was a significant linear correlation between the expressions of14-3-3ε protein and GFAP in SCI in vivo (linear regression coefficient was0.6478, R2=0.7100, P<0.001). p.14-3-3εprotein and GFAP showed matched intensive immunoreactivity around injury areas.CONCLUSIONS:14-3-3sprotein and GFAP were upregulated aroud injuried spinal cord tissures from early stage, and14-3-3εprotein had a quicker flatform than GFAP. But both of them had significant linear correlation, similar trend and the same tissure location after SCI. We speculated the upregulating of14-3-3sprotein after SCI was due to astrocyte activation, which may took part in or effected the reactive AS in the early stage after SCI.