The Establishment Of Neuronal Oxygen Glucose Deprivation/Reoxygenation Model And The Expression And Efftcs Of CX3CR1 In Neurons

ObjectiveTo improve the primary culture of mouse neurons and test the effect of cytosine arabinoside purification on neurite outgrowth,and to establish an efficient and simple model of primary neuronal oxygen glucose deprivation/reoxygenation.The expression of CX3CR1 in primary neurons was verified by vitro experiments,and the effects of CX3CR1 on(ischemia-reperfusion)OGD/R neurons was investigated.MethodsHippocampus and cortex neurons of postnatal C57/b16 mouse within 24h were separated,digested and made into cell suspention.After 48 hours,the primary neurons were randomly divided into 3 groups:Ara-C 2.5?M,Ara-C 5?M and control group.then cellular morphology was observed at different time,neuronal purity was identified with specific neurons maker MAP2 by immunofluorescence staining,neurites were measured and acquired Sholl Analysis.Analysising the effect of cytarabine purification on neuronal neurites by measurement of neurite length and Sholl Analysis.Preparation of neuronal oxygen glucose deprivation/reoxygenation model:the culture medium was changed to neurobasal-A medium without glucose,and neurons were placed into the hypoxia chamber for 15 min,30 min,45 min,60 min,the neuronal viability after 24-hour reoxygenation was deteted by DAPI staining to screen for the appropriate OGD time.The expression of CX3CR1 on neurons was detected by CX3CR1/MAP2 double immunofluorescence labeling method.According to whether the addition of CX3CR1 antibody and OGD time,the primary neurons were randomly divided into two groups:oxygen glucose deprivation 30min,antibody group and control group;oxygen glucose deprivation 15min/reoxygenation 24h,antibody group and control group,MTT was used to detect cell viability to analyze the effect of CX3CR1 on neurons after OGD/R.Results1.The morphological changes of primary neurons in different periods are consistent with the process of neuronal growth.the neuronal purity of Ara-C 2.5?M and Ara-C 5?M were 91.23%± 3.26%and 91.97%± 3.92%,which was significantly higher than that of the control group 62.04%± 19.87%(P<0.05).There was no significant difference between the longest neurites and the branching points in the three groups of neurons.2.With prolonged the time of OGD,neuronal survival rate decreased gradually,OGD 15 min,30 min,45 min,60 min survival rates were 65.73%±10.31%,24.25%± 12.43%,5.40%±3.99%±3.34%±1.69%,the survival rate of neurons decreased compared with 95.93%± 5.90%in the control group(p<0.05).There were statistical differences between OGD15min group and OGD30min group,OGD30min group and OGD45min group.3.The expression of CX3CR1 was found in mouse primary neurons by double immunofluorescence labeling.4.The OD of the Ab OGD30min group was 0.56±0.08,which was higher than it's OGD control group 0.38 ± 0.03,there was no statistical differences in OD between Ab OGD15min/R24h group and its OGD/R control group.ConclusionThe modified culture method can stably and efficiently obtain high purity mouse neurons,and addition of Ara-C did not affect the growth of neurite outgrowth.An effective OGD/R model was established on the basis of high purity primary neurons.Confirmed that expression of CX3CR1 in mouse primary neurons,and inhibition of CX3CR1 can reduce neuronal damage after ischemia injury.