Protective Effects Of Endothelial Progenitor Cells On Neurons After Oxygen Glucose Deprivation / Reoxygenation

PartⅠPrimary culture of mice hippocampal neurons and establishment of oxygen glucose deprivation/reoxygenation injured modelObjective To establish oxygen glucose deprivation/reoxygenation(OGD/R) injured model of mice hippocampal neurons with a hypoxia chamber(Anaero Pack Pouch CX-31) and glucose-free culture medium, and to determine the optimal length of time for OGD and whether reoxygenation has further damage to neurons.Methods Hippocampal neurons were isolated and cultured from neonatal C57BL/6 mice. After culturing for 8 days, neurons were assigned randomly to the control group and OGD/R groups. There were there time points for OGD(4h,8h,12h) and two for reoxygenation(Oh,24h). The OGD/R injured model of neurons was performed by replacing the Neurobasal/B27 medium with the glucose-free Earle’s balanced salt solution and placing the neurons into a hypoxia chamber for 4h,8h and 12h respectively, and then normal culture condition was provided for Oh or 24h. MTT assay was used to detect cell viability and neuronal injury was assessed by LDH level in the culture medium.Results With longer OGD duration, cell survival rate was decreased (P<0.001) and LDH level was increased (P<0.001), and the differences were statistically significant compared with the control group. Compared with the OGD4h/R0h group, the OGD4h/R24h group had lower cell survival rate (P<0.01) and higher LDH level (P<0.01). The OGD4h/R24h group provided an optimum cell survival rate (59.96%) and could be the most appropriate OGD/R model.Conclusion OGD/R injured model of neurons could be established with a hypoxia chamber and the glucose-free Earle’s balanced salt solution. Reoxygenation could produce further damage to neurons after oxygen glucose deprivation for 4h. OGD4h/R24h was proposed as an optimal model.Part ⅡProtective effects of endothelial progenitor cells on neurons after oxygen glucose deprivation/reoxygenationObjective To investigate whether endothelial progenitor cells (EPCs) co-culture has protective effect on oxygen and glucose deprivation/reoxygenation (OGD/R) injured neurons.Methods Hippocampal neurons were isolated and cultured from newborn C57BL/6 mice, and OGD/R injured model of neurons was performed by using a hypoxia chamber. Bone marrow derived EPCs of C57BL/6 mice were isolated and cultured by density gradient centrifugation. Transwell was used to build the co-culture system of EPCs and the OGD/R injured neurons. Neurons were assigned randomly to the control group, the OGD/R group, the EPCs co-culture group or the EPCs co-culture along with nitric oxide synthase inhibitor (Nω-nitro-L-arginine methyl ester, L-NAME) group. Neuronal apoptosis rate of each group was detected by TUNEL assay. Western blot was used to detect neuronal caspase 3 and endothelial nitric oxide synthase (eNOS) activity of each group.Results Neuronal apoptosis rate and caspase 3 activity were significantly increased in the OGD/R group compared with the control group. Neuronal apoptosis was significantly decreased (P<0.05) and eNOS phosphorylation was increased (P<0.05) in the EPCs co-culture group compared with the OGD/R group. However the EPCs+L-NAME group showed increased neuronal apoptosis (P<0.05) and decreased eNOS phosphorylation (P<0.05) compared with the EPCs co-culture group.Conclusion EPCs co-culture can protect OGD/R injured neurons by promoting the activation of eNOS. The protective effect of EPCs co-culture on OGD/R injured neurons could be suppressed by L-NAME.