The Effect Of NBP On Bcl-2/bax And Bim Proteins In Oxygen-glucose Deprivation Model Of Organotypic Brain Slices

Objective：to establish the oxygen and glucose deprivation(OGD) modelof organotypic brain slices of rat cerebral cortex,and observe the effects ofn-butyl phthalide(NBP) on bcl-2、bax、bim protein in apoptosis.Method：we choose the post-natal7days rats(SD) to prepare the brainslices which were cultured on the membrane insert,and then observe themorphological changes.after two weeks,brain slices were divided into fourgroups:the control group,the model group,the solvent control group and thetest group,nine slices each group. Replaced of the medium with propidiumiodide(PI,1ug/ml),the cultures were incubated in the CO2incubator20min37℃.then,we took pictures for the brain slices though fluorescencemicroscope(4*objective, the exposure time1.038ms,ISO1600). the modelgroup,the solvent control group and the test group were treated with OGD30min,then turned into normal conditions,and the model group with normalmedium,the solvent control group with normal medium includingDMSO(same as the test group),the test group with normal medium includingNBP(10μM,NBP was dissolved in DMSO). Medium of the control groupwould be changed at the same time with the normal. Pictures shoule be takenby the same parameters at different time points after OGD(1h,24h,72h).repeatthe above conditions of control group,solvent control group and test groupwithout PI staining.fix the brain slices with glutaraldehyde,made the paraffinsections and then make the immunohistochemical staining of bcl-2,bax andbim proteins.Results：1brain slices grew well in vitro,no obvious necrosis were found.after2weeks we fixed the brain slices with glutaraldehyde,made the paraffin sectionsand did the HE dying.through the microscope,we could easily find the vast majority of cells with normal structure.2PI staining shows no statistical difference of fluorescence intensity indifferent time points of the control group(P>0.05).the model group and thesolvent control group show the increase of fluorescence intensity,there wereno statistical difference between the two groups(P>0.05).3the test group also shows the increase fluorescence intensity,but underthe model group at24h、72h time points.4the following shows the immunohistochemistry staining results ofcontrol group,solvent control group and test group at6h time point: Bcl-2protein:model group and test group show more positive cells than controlgroup,the test group is the most of all.statistical analysis(P<0.05).Bax、bim:positive cells of the three groups are different,statisticalanalysis(P<0.05),control group is the lowest, model group is the highest.Conclusion:1SD rat post-natal7days could be used as the donor of brain slicesculture in vitro2brain slice OGD model can be used to result in cell injury,whichshows acute cell death and delayed apoptisis.3NBP has protective effect on cell injury after OGD,the mechanismmay be that,it can change the amount of bcl-2,bax,bim protein in the injuredcells.