Influence Of Microenvironment Of Neuron Expressing Seizure-like Discharge On The Differentiation Of Oligodendrocyte Precursor Cells

PARTⅠ:THE CHANGES IN GLUTAMIC ACID CONCENTRATION OF MICROENVIRONMENT OF NEURON EXPRESSING SEIZURE-LIKE DISCHARGE INDUCED BY FREE-MAGNESIUM EXTRACELLULAR FLUID,AND THE INFLUENCE OF THOSE ON THE DIFFERENTIATION OF OLIGODENDROCYTE PRECURSOR CELLSObjective:To investigate the effect of microenvironment of normal neuron and neuron expressing seizure-like discharge induced by free-magnesium extracellular fluid on the differentiation of oligodendrocyte precursor cells(OPCs),and to explore the influence of epileptic seizure on the remyelination.Methods: The media after neuron expressing seizure-like discharge induced by free-magnesium extracellular fluid 1h、2h、4h、6h、12h、24h、48h、72h were collected,and the media after neuron cultured by normal extracellular fluid 1h、2h、4h、6h、12h、24h、48h、72h were collected.The glutamic acid concentration of those media were detected by High Performance Liquid Chromatography(HPLC).OPCs were purified on the third day or fourth day of proliferating phase.According to the proportion of additional neuronal medium in which neurons survived in the total medium,objects were divided into three levels: 1/8,1/4,1/2.Every level was divided into three groups: OPC differentiation medium(ODM)group,OPC differentiation medium + corresponding proportion of induced seizure discharge neuron medium(ODM+INM)group,OPC differentiation medium + corresponding proportion of normal neuron medium(ODM+NNM)group.Myelin basic protein(MBP)transcription expressions of oligodendrocyte cells on the third day,fifth day and seventh day of differentiation phase were detected by RT-q PCR.Every Mehod was repeated three times.Results: There were significant differences in glutamic acid concentration between seizure-like discharge neuron microenvironment group(91.79±0.230μM)and normal neuron microenvironment group(90.71±0.478μM)(P<0.05)at the 72 h time point.There were significant differences in MBP transcription expressions between ODM group and ODM+1/2NNM group on the differentiation phase,whereas MBP transcription expressions of ODM+1/2NNM group on the third day and fifth day of differentiation higher than ODM group(P<0.05),on the seventh day of differentiation lower than ODM group(P<0.05).There were also significant differences in MBP transcription expressions between ODM+1/2NNM group and ODM+1/2INM group on the differentiation phase,whereas MBP transcription expressions of ODM+1/2INM group on the third day of differentiation higher than ODM+1/2NNM group(P<0.05),on the fifth day and seventh day of differentiation lower than ODM+1/2NNM group(P<0.05).Conclusion: The neuron microenvironment has a significant effect on the differentiation of OPCs.The normal neuron microenvironment can promote OPC differentiation,and the induced seizure-like discharge neuron microenvironment can inhibit OPC differentiation.PARTⅡ:THE ROLE OF AMPA RECEPTOR IN THE INFLUENCE OF GLUMATIC ACID OF SEIZURE-LIKE DISCHARGE NEURON MICROENVIRONMENT ON THE DIFFERENTIATION OF OLIGODENDROCYTE PRECURSOR CELLSObjective : To investigate whether α-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid(AMPA)receptor had the function when glutamic acid affected the differentiation of oligodendrocyte precursor cells.Method: Objects were divided into four groups on the basis of OPC differentiation microenvironment composition: normal control group(normal neuron microenvironment),glutamic acid group(glutamic acid was added to normal neuron microenvironment),AMPA receptor antagonist group(AMPA receptor antagonist was added to seizure-like discharge neuron microenvironment),seizure-like discharge group(seizure-like discharge neuron microenvironment).MBP expressions were detected by RT-q PCR,immumofluorescence and Westernblotting.Glu R2 subnits fluorescence intensitys were detected by immumofluorescence.Every method was repeated three times.Results: MBP expressions of normal control group were higher than glutamic acid group and seizure-like discharge group(P<0.05).MBP expressions of seizure-like group were lower than AMPA receptor antagonist group(P<0.05).Glu R2 subnits fluorescence intensitys of normal control group were lower than glutamic acid group and seizure-like discharge group(P<0.05).Glu R2 subnits fluorescence intensitys of seizure-like group were higher than AMPA receptor antagonist group(P<0.05).Conclusion: Neuron expressing seizure-like discharge released more glutamic acid.Glutamic acid integrated with AMPA receptor,which leaded to Glu R2 subnits increase and celluer calcium infux reduction.Finally,seizure-like discharge group inhibited OPC differentiation.