The Effeetions On Glial Scar Formation By LY294002after Spinal Cord Injury

Objective: LY294002as the specificity inhibitor of PI3K-Akt signal pathway were used to intervene spinal cord injury model in vitro and in vivo. Detect the expression of CSPGs and GFAP by Western Blot to explicit LY294002whether or not has influence on glial scar formation after SCI.Methods:(1)60SD rats which weight200～250g (200±8g), male and female are not limited,were divided into3groups randomly,each group has20rats,A group as the sham-operation one,and B group as the LY294002treatment one,C group as control one.Clamp method to build SD rats thoracic10spinal cord injury model, the A group had only laminectomy without spinal cord injury, was intrathecal injected with the DMSO in6h after operation; B group was intrathecal injected with LY294002in the first6h after SCI; The C group was intrathecal injected with DMSO.Using BBB score to assess SD rats hind legs movement function of different groups in the1,2, and4weeks.(2) Extraction the protein of Spinal cord tissue in the1,2, and4weeks, detect the different expression of CSPGs by Western Blot.(3) Extract brain tissue of1-2days newborn SD infant rats,using the method of continuous differential passage of cultured astrocytes, take the third generation astrocytes identificated of more than95%by immunohistochemistry and immunofluorescence,used for following experiment. Plants the astrocytes in board,A group joins LY294002and complete growth substrate; B group joins DMSO and complete growth substrate; C group was induced by TGF-β1,then join the complete growth substrate including the LY294002; D group was induced by TGF-β1,then join the complete growth substrate including the DMSO. We observed the morphology of AST by inverted microscope and take pictures. Extraction the protein of astrocytes, detect the different expression of CSPGs and GFAP by Western Blot.Results:(1) The vivo experimental spinal cord injury model T10in SD rats were successful.Pathological detection demonstrated:the spinal cord tissue had hyperemia, and the neuron had the denaturation. The sham-operation group rats’BBB scores were restore normally in the short time after the damage. The LY294002treatment group had restoration,and the damage control group had the worst restoration. Western Blot results showed that surgical control group had no or only a low level expression of CSPGs, LY294002treatment group had few expressions of CSPGs, the damage control group had the highest expression of CSPGs(P<0.01).(2) The astrocytes which were cultured by the method of continuous differential passage had normal morphology, high-purity and strong activity. The purity of astrocytes was97.3%identificated by immunohistochemistry and immunofluorescence. TGF-β1induced neurotrosis model successfully in vitro, caused AS to have the morphology changes, had increasing degree of glioma. Western Blot results showed blank control group had no or only a low level expression of CSPGs and GFAP, the LY294002treatment group had a lower level in CSPGs and GFAP than TGF-β1injury group(P<0.01).Conclusion:(1) LY294002reduces the formation of CSPGs after spinal cord injury in SD rats, improves their hind legs BBB grading effectively, it conducive to the spinal cord function recovery.(2) LY294002can effectively inhibit of GFAP expression after nerve injury in vitro, and inhibit the expression of CSPGs.(3) PI3K-Akt signaling pathway mediated and promote GFAP and CSPGs expression increases of astrocytes after nerve injury,and cause glial scar finally.