Effects Of Bone Marrow-derived Mesenchymal Stem Cells On The Axonal Outgrowth And Expression Of AKT/GSK-3β/CRMP-2in Rat Cortical Neurons Followed OGD Injury

Object: To investigate the effects of bone marrow-derived mesenchymal stem cells (BMSCs) on the axonal outgrowth and expression of AKT/GSK-3β/CRMP-2signaling pathway in rat cortical neurons followed OGD injury. We sought to potentialmechanisms that BMSCs effect on axonal outgrowth in non-contact system.Methods:1. BMSCs and primary cortical neurons from rats were cultured andidentified in vitro.2. To establish OGD-injured model, after cultured5days, primary corticalneurons were cultured in anaerobic chamber for90min. After12h later, morphologyof OGD-injured cortical neurons were observed by light microscope and electronmicroscope.3. OGD-injured neurons were cocultured with P3BMSCs in non-contactTranswell system. Then experiments were randomly divided into4groups: Normalgroup (Control group), OGD-treated group (OGD group), Co-cultured group(OGD+BMSCs group), LY294002treated group (OGD+BMSCs+LY294002group).4. All the groups were cultured for48h later, immunofluorescent stainingidentified cell bodies and neurites. Images were captured by ZEISS LSM710confocal microscope and the morphology of neurites was analyzed with ZEN2009software.5. All the groups were cultured for24h later, protein expression level of AKT、pAKT、GSK-3β、pGSK-3β、CRMP-2、pCRMP-2and GAP-43were detected byWestern-bolt.6. Statistical analysis. Results:1. After OGD injury, we observed that neurite of neurons were shortenedand partially disappeared. Subcellular organelle of neurons, such as mitochondrionand endoplasmic reticulum, were swollened and partially disappeared.2. As shown in the length of axon assay, the average length of axon wassignificantly shortened in OGD group compared with the Control group (P<0.01).And compared with the OGD group and the OGD+BMSCs+LY294002group, theaverage length of axon was increased in OGD+BMSCs group (P<0.05).3. Western blot analysis revealed that protein expression level of pAKT、pGSK-3β and GAP-43were increased in OGD+BMSCs group compared with theOGD group and the OGD+BMSCs+LY294002group (P<0.05), while expression ofpCRMP-2were decreased in OGD+BMSCs group (P<0.05).Conclusion: BMSCs might promote repair and outgrowth of injured axon bysecretion, which may be related to activation of AKT/GSK-3β/CRMP-2signalingpathway.