Effect Of EphA4 Receptor On Bdnf Expression In Hippocampal Neurons Of Rats With Oxygen Glucose Deprivation/Reoxygenation

Objective:1.Establish a suitable in vitro culture method of hippocampal neurons,and oxygen glucose deprivation/reoxygenation model of hippocampal neuron.2.Establish a glucose and oxygen deprivation/reoxygenation model to explore the effects of EphA4 receptors on the expression of brain-derived neurotrophic factor(BDNF)and precursor BDNF(pro-BDNF).Methods:1.In vitro primary hippocampal neurons culture.1-3d SD suckling rats with no gender limit were used to isolate hippocampal tissue and culture primary hippocampal neurons.The cultured rat hippocampal neurons were randomly divided into a complete inhibition group,an incomplete inhibition group and a non-inhibition group.After 7d of culture,according to the growth of neurons,the most suitable culture conditions was selected for subsequent neuron culture.2.Preparation of oxygen glucose deprivation/reoxygenation(OGD/R)model.The normal control group was cultured under conventional conditions;the OGD/R group was further divided into 9 groups,according to the oxygen glucose deprivation for 0.5,1.0 and1.5h,and then reoxygenation for 24h,36h and 48h respectively.They are OGD0.5h/R24h group,OGD0.5h/R36h group,OGD0.5h/R48h group,OGD1h/R24h group,OGD1h/R36h group,OGD1h/R48h group,OGD1.5h/R24h group,OGD1.5h/R36h group and OGD1.5h/R48h group.After 7d of culture,the most appropriate OGD/R conditions were selected for subsequent experiments according to the degree of neuronal damage and survival rate.3.CCK-8 method to verify the cell viability of cultured neurons.Inoculate hippocampal neurons on a 96-well culture plate for culture and divide them into OGD0.5h/R24h group,OGD0.5h/R36h group,OGD0.5h/R48h group,OGD1h/R24h group,OGD1h/R36h group,OGD1h/R48h group,OGD1.5h/R24h group,OGD1.5h/R36h group and OGD1.5h/R48h group,each group is equipped with 3 multiple wells,each well is 100μl culture system,and the cells are about 6×10~3/well.On the 7th day of incubation,perform OGD/R treatment on each group.Add 10μl of CCK-8 solution to each well in the dark,incubate at 37°C for 4h.Measure the corresponding absorbance value of each group at 450nm wavelength with a microplate reader.4.Lactate dehydrogenase(LDH)measures neuronal activity.The cell culture fluid of each group of neuronal cells was collected at different timepoints after neuronal cell OGD/R.Corresponding to each absorbance measured at 450nm with an enzyme label.5.According to the experimental results,select OGD1h/R36h as the model group in the subsequent experiments.6.EphA4-Fc antagonizes EphA4 receptor function.Add the EphA4-Fc solution into the cell culture medium,keep the EphA4-Fc concentration in the cell culture medium at4μg/ml.Every time the medium is changed,re-add the EphA4-Fc solution to ensure a constant EphA4-Fc concentration in the cell culture medium.7.Use Western blot technology to detect pro-BDNF protein and BDNF protein.The experiment detects the changes of pro-BDNF protein and BDNF protein after the control group,OGD/R treatment,and administration of EphA4-Fc to antagonize the function of EphA4 receptor.8.Apply RT-qPCR to discover the BDNF m RNA expression.The primary hippocampal neurons cultured to maturity were grouped according to the needs of the experiment.After10 days,total RNA was extracted,multiple BDNF promoter primers were designed,and RT-qPCR amplification program was run.Calculate the relative expression of Bdnf gene transcripts.Results:1.The results show:if not inhibited,the glial cells are cultured and divide rapidly.After7 days,it is difficult to observe the growth and state of hippocampal neurons under the microscope,and the number of cultured neurons is relatively small,the purity of neurons is low.When the glial cells are completely inhibited,the glial cells basically do not grow.At this time,the number of neurons is large,and the purity can reach more than 95%.However,the number of glial cells under this culture condition is too small,which is more suitable for patch clamps,etc.When the glial cells are partially inhibited,the cultured hippocampal neurons are good uniformity,the cell are round and triangular,with smooth edges,and the number of glial cells is close to 1:1 with the number of hippocampal neurons.The information exchange between glial cells and neurons is preserved.This culture condition is more practical for this experiment.2.When culturing hippocampal neurons by conventional methods,observe their growth state and morphology.It is found that neurons grow well under these conditions and have strong adherence.The dendrites of neurons are thick and long,widely distributed,and cross-linked to each other to form a dense network structure.The neuron presents a vertebral body shape with strong refractive index.After 0.5h of deprivation of the neuron’s glucose and oxygen,the neuron cell body was swollen and enlarged under an optical microscope.After depriving the neuron of glucose and oxygen for 1 hour,it was observed that the dendritic structure of the neuron became thinner and shorter,and the distribution range began to shrink.The neuron is more enlarged,and the refractive index is weakened.After 1.5 hours of glucose and oxygen deprivation,neurons began to appear as balls under the microscope,which was due to the rupture of the dendrites of the neurons.The ability of neurons to adhere to the wall is weakened and clustering begins to occur.At this time,neurons are easily detached from the cell plate,and some cells begin to fall off.3.LDH and CCK-8 experiment results show that:the LDH content and cell survival rate of the OGD0.5h/R24h group,OGD0.5h/R36h group and OGD0.5h/R48h group,compared with the normal control group,did not distinct diffience.OGD1h/R24h group and OGD1h/R36h no significant discrepancy in cell viability and LDH content.The LDH content in the OGD1h/R48h group was significantly increased,the cell viability was obvious reduction.The content of LDH in the OGD1.5h/R24h group and the OGD1.5h/R48h group was obviously increase,and the survival rate of cell was obviously declined.OGD1.5h/R36h group LDH has no distinct change,and the cell survival rate was significantly reduced.4.Western blot results:Compared with the control group,the pro-BDNF protein and BDNF protein in the OGD1h/R36h group were distinctly reduced.The pro-BDNF protein and BDNF protein expression in the OGD1h/R36h+EphA4-Fc group,compared with the OGD1h/R36 group,were distinctly increased.5.The RT-qPCR results showed:after administration of EphA4-Fc,the expression of Bdnf transcripts containing exons I,IV and VI,compared with the control group,was significantly reduced.Compared with the control group,the expression of BDNF gene transcripts containing exons I,IV and VI in the OGD1h/R36h group was significantly increased.Compared with the OGD1h/R36h group,the OGD1h/R36h+EphA4-Fc group contains significantly lower expression of Bdnf transcripts containing exons I,IV and VI.The expression of the transcripts of the Bdnf containing exon IIc did not change significantly in each group.Conclusion:1.When the growth of glial cells is not completely inhibited,the primary cultured hippocampal neurons grow well,and the results are easy to observe.2.After OGD1h/R36h,the dendrites of neurons are shortened and thinned,the network structure between neurons is destroyed and became sparse.The vitality of neurons is reduced,but the survival rate is higher,which is conducive to the subsequent experiments.3.After OGD1h/R36h,the decreased expression of pro-BDNF protein and BDNF protein in hippocampal neurons was correlated with the increased expression of BDNF transcripts containing exons I,IV and VI.The increased expression of pro-BDNF and BDNF proteins caused by antagonizing EphA4 receptor function is related to the decreased expression of Bdnf transcripts containing exons I,IV and VI.