The Protective Effects Of Methane And Hypoxic Preconditioning BMSCs On Rats Acute Spinal Cord Injury And Related Underlying Mechanism

Part 1 The Protective Effects of Methane on Rats Acute Spinal Cord Injury and Related Underlying MechanismObjective.We investigated the hypothesis that methane-rich saline(MS)can be used to repair spinal cord injury(SCI)in a rat model through suppressing oxidative,inflammatory and apoptotic injury.Methods.MS was injected intraperitoneal to the rats at doses of 0.5,5,20 ml/kg every 12 h after SCI to tested the MS dosage effective.Hematoxylin-eosin(HE)staining,oxidative stress,inflammatory parameters and cell apoptosis were detected at 72 h after SCI to determine the optimal dose.Then we investigated the protective mechanisms and the long-term effect of MS(20 ml/kg/12h)on SCI.HE and glial activation were observed at 72 h and 14 days after SCI.Neurological function was evaluated by Basso,Beattie and Bresnahan scale(BBB).Results.MS can significantly decrease infarct area,inhibit oxidative stress,inflammation,and cell apoptosis at 72 h following SCI.The protective effect in dose 20 ml/kg was better.Moreover,MS can significantly suppress microglia and astrocytes activation after SCI,and improve hind limb neurological function.Conclusion.MS could repair SCI and reduce the release of oxidative stress,inflammatory cytokines and cell apoptosis.MS provides a novel and promising strategy for the treatment of SCI.Part 2 Shake,an ideal way for whole bone marrow adherent method improvement.Objectives: To study the potential effects of shaking on the purity,activity,differentiation,and possible apoptosis of rats' bone marrow mesenchymal stem cells(BMSCs).Method: Based on the whole bone marrow adhesion method,different lengths and frequencies of shaking were used additionally to purify primary cells,then biomarkers of CD29,CD90,CD45,CD31 as well as the apoptosis labeled by Annexin V-FITC and PI were investigated by flow-cytometric analysis.Then differentiation ability after purification were evaluated.Results: After shaking treatment,the purification of adherent cells increased differently.Meanwhile the apoptosis rates increased along with the increasing of shaking length and frequencies.Cells after shaking could be induced to differentiate into osteoblasts and adipocytes.Conclusion: Shaking can obtain mesenchymal stem cells with higher positive rates of CD29 and CD90.Besides horizontal shaking has few influence to cell activity or differentiation and the apoptosis due to shaking is acceptable.Part 3 The Protective Effects of Hypoxic Preconditioning BMSCs on Rats Acute Spinal Cord Injury and Related Underlying MechanismObjective.Due to the special hypoxic,inflammatory symptoms microenvironment in spinal cord lesions after SCI limits the survival and efficacy of cells transplantation.The aim of this study was to determine whether hypoxia preconditioning(HP)of BMSCs(H-BMSCs)could increase the therapeutic effect on SCI.Methods: BMSCs were pretreated by cobalt chloride(Co Cl2)in vitro before transplantation.BMSCs and H-BMSCs proliferative activity,apoptosis and migration were tested.RT-PCR were used to detect apoptosis-related gene m RNA(caspase-3 and bcl-2)and the migration-related gene m RNA(HIF-1? and CXCR4).BMSCs and H-BMSCs were transplanted into SCI rat in vivo,and hindlimb motor function,histopathology,inflammatory after SCI were detected.Migration and neurogenesis of transplanted BMSCs and H-BMSCs were tested.Results.H-BMSC could significantly enhanced the tolerance of BMSC to apoptotic injure(reduced caspase-3 and increased bcl-2 expression)and the migration(increased expression of HIF-1? and CXCR4)in vitro.Experiments in vivo showed that H-BMSC transplantation can significantly improve neurological function,reduce spinal cord damage,and suppress the inflammatory response related to microglia activation than BMSC.The number of Neu N-positive cells in SCI core and peripheral region of H-BMSC group was higher than those in BMSC group.Some Neu N-positive showed GFP reactivity,suggesting BMSCs can differentiate into neuronal cells.Conclusions.HP can enhance migration and neurogenesis capacity of BMSCs and promote BMSCs therapeutic potential for SCI.