A Study Of The Protection Of BDNF To Cortical Neurons Injured By Glutamate And The Impact On NMDA Receptor Expression

PurposeTo observe the injury function of glutamate on primary cultured rat corticalneuron in vitro, investigate the neuroprotective effects of BDNF on neurons injuredby glutamate, and explore the influence of BDNF on NMDAR of neurons injured byglutamate.Method1. The cortical neurons cultured in vitro were divided into Group A: normal control group;Group B: glutamate group, Group C: BDNF group. Group A: Primary neurons at days invitro (DIV)7were cultured by original medium for24hours. Group B: primaryneurons at days in vitro (DIV)7were added to50μmol/L glutamate, after30minutes, transformed the cell medium into the original medium to continue toculture24hours, Group C: primary neurons at days in vitro (DIV)6were added50ng/ml BDNF into cell medium for24hours, after24hours, transformed the cellmedium into original medium containing50μmol/L glutamate for30minutes,then cultured the cell using the virgin cell medium for24hours, the degree of theinjury neurons detected by MTT, the levels of intracellular calcium detected byconfocal microscopy.2. The cortical neurons were divided into A1group: normal control group, B1group:control group, C1group: glutamate group, D1group: BDNF group, E1group:BDNF antagonist group (K252a group). A1group: primary neurons at days invitro (DIV)7cultured by original medium, B1group: primary neurons at days invitro (DIV)6were added50ng/ml BDNF into cell medium for24hours, after24hours, cultured the cell using the virgin cell medium, C1group: primary neuronsat days in vitro (DIV)7were added to50μmol/L glutamate, after30minutes,transformed the cell medium into the original medium to continue to culture, D1 group: primary neurons at days in vitro (DIV)6were added50ng/ml BDNF intocell medium for24hours, after24hours, transformed the cell medium intooriginal medium containing50μmol/L glutamate for30minutes, then cultured thecell using the virgin cell medium, E1group: primary neurons at days in vitro (DIV)6were added50ng/ml BDNF and K252a into cell medium for24hours, after24hours, transformed the cell medium into original medium containing50μmol/Lglutamate for30minutes, then cultured the cell using the virgin cell medium. Wedetected the transcription of NMDAR1in each group after6,12and24hours,investigated the expression of NMDAR1by western blot in cortical neurons ofeach group, and explored the function of NMDAR receptors stimulated by NMDAunder confocal microscopy.Result1. Compared with the control group (0.8170±0.0323), the survival rate of glutamategroup (0.7126±0.0320) was significantly lower, compared with glutamate group,BDNF group (0.7858±0.0219) can improve cell survival rate.2. Under confocal microscope, the levels of intracellular calcium of glutamate group(128.1±2.5) and BDNF group (125.8±3.5) were higher than control group(112.4±2.0).3. Compared with the C1group, the transcription of NMDAR1in D1group and B1group Significantly increased after6h, the difference was statistically significant(p<0.05), the amount of transcription after12h and24h were not difference. Thetranscription of NMDAR1of E1group was not difference at each time (p>0.05).Compared with the B1group, the transcription of NMDAR1of D1group was notdifference at each time (p>0.05).4. Compared with the A1group (normal control group), the expression of NMDAR1protein in cortical neurons of B1group was significantly increased after6h,12h,decreased after24h, but still higher than the A1group (normal control group), thedifference was statistically significant (p <0.05). Compared with C1group and E1group, the expression of NMDAR1protein by western blot in cortical neurons ofB1group and D1group increased after6h,12h and24h, the difference was statistically significant (p<0.05), in D1group, the expression of NMDAR1proteindecreased after24h, the difference was statistically significant (p<0.05), theexpression level of NMDAR1protein in D1group and B1group at each timewere not different (p<0.05), the expression level of NMDAR1protein in C1groupand E1group at each time were not different (p>0.05). Under the stimulation ofNMDA, the levels of intracellular calcium in B1group and D1group increasehigher than in C1group and E1group.Conclusion1. BDNF has a protective effect on Neurons injured by Glutamate, which mechanismis not by reducing the level of its intracellular calcium.2. BDNF can influence the transcription and expression of NMDAR1in corticalneurons injured by Glutamate and can improve the function of its NMDAreceptor.