Establish Oxygen-glucose Deprivation Model Of Organotypic Brain Slices And Observe The Effect Of NBP On Apoptosis In The Model

Objective： To establish the oxygen and glucose deprivation (OGD)model of organotypic brain slices of rat cerebral cortex and observe the effectsof n-butyl phthalide (NBP) on apoptosis and mitochondrial membranepotential.Method： The brain slices were prepared from SD(Sprague Dawley) ratspost-natal7days, cultured on the membrane insert and observed themorphological changes. We characterized pyramidal cells of the cultures bycombining SMI-32stain with immunohistochemistry at1w,2w,3w,4wrespectively, observed the number and the development of SMI-32positivepyramidal cells.Following two weeks in vitro, brain slices were divided intosix groups: the control group(group C),the test group(group T：T0,T0.01,T0.1,T1,T10),nine slices each group. Replaced of the medium withpropidium iodide (PI,1μg/ml),the cultures were incubated in the CO2incubator20min,37℃。Then, we took pictures for the brain slices though fluorescencemicroscope(4*objective, the exposure time1.038ms,ISO1600). Group T wastreated with OGD30min,turned into the normal conditions with differentconcentrations of NBP(0,0.01,0.1,1,10μM).Medium of group C would bechanged at the same time with the normal. After one hour, pictures should betaken by the same parameters. According to the above data,we took photos forslices of T0and T10at different points(OGD30min,NBP1h,24h,72h,7d).Another group C and each subgroups of group T,OGD andNBP intervention(no PI),were detected the mitochondrial membrane potentialby JC-1kits.Results：1Brain slices grew well in vitro, we did not observe obvious necrosis. Through the microscope, we found the slices growing bigger, thinner and thetransparency increased. Meanwhile, the dividing line of the cortex andsubcortical structures is clear. By SMI-32immunohistochemical staining,there are several SMI-32positive cells in brain slices into a layer. The numberof pyramidal cells maintain stable at1,2,3,4w time points, and each cell has along top dendritic to the cortex.2Before and after OGD treatment, the fluorescence intensity of brainslices have change obviously, further more the value are still increasing afterslices returned to normal oxygen-glucose supply, especially in the72h and7d.It is statistically significant that difference between the intensity value ofthe group C and the group T10at each time point(p<0.05).3After OGD30min Organotypic brain slices treated, it showed differentbrightness of red fluorescence that slices of the control group and theexperimental group (T0,T0.01,T0.1,T1,T10) combined with PI staining. Comparedwith control group,the fluorescence intensity of each experimental subgroupare greater, but negatively related to the concentration of NBP(0-10μM).Thatdifference are dramatic (p<0.05).4Observing the slices of the group T0and T10at different timepoints(OGD30min,NBP1h,24h,72h,7d),we found there are apoptosis of twogroups. The number of cell death has no significant different at OGD30mim.With the time prolonged, the value of fluorescence intensity of T0wasincreasing, especial at72h and7d.Meanwhile, the value of T10decreasedgradually. The number was statistically significant difference between eachtime points (p<0.05).5Using the mitochondrial membrane potential detection kit(JC-1kit)after deprived oxygen-glucose30min and returned to the normal1h, the valueof red/green fluorescence of each group (C, T0,T0.01,T0.1,T1,T10)dramaticreduced correlated positively with NBP dose, and the value of each subgroupof experiment were much smaller than the control. The differences werestatistically significant. Conclusion：1SD rat post-natal5-7days could be used as the donor of brain slicesculture in vitro.2The cerebral cortex organotypic brain slices cultured in vitro could bea reliable method to study the effect of a variety of stimulate, which haveparallel cell structure and pyramidal cells distribution with in vivo.3OGD-induced brain slice injury shows similarly the pathologicaldamage, acute cell death and delayed apoptosis. This model operate easily andgood reproducibility.4NBP has protective effect on cerebral ischemic injury, both acute anddelayed cell death, but not block apoptosis completely. There is a dose-effectrelationship with NBP and cell death.5Dissipation of mitochondrial membrane potential in brain slices afterOGD injury has occurred in the early, related to delayed apoptosis, NBP couldprevent the decline of mitochondrial potential in dose-effect dependent way.