RhoA And Cytoskeleton Proteins In Oxygen/glucose Deprived Model Of Neonatal And Immature SD Rat's Oligodendrocyte Precursor Cells In Vitro

ObjectivesTo establish a culture system of rat's oligodendrocyte precursor lineage cells and obtain highly purified oligodendrocyte precursor lineage cells from neonatal and immature SD rat's brain in vitro, so that we can get enough oligodendrocyte precursor cells(OPCs) which are in the given stage of differentiation to investigate the expression of RhoA and cytoskeleton proteins in an oxygen/glucose deprived model.MethodsNeonatal SD rats born less than 72 hours of age were sacrificed by disconnecting neck under aseptic conditions, brains were taken out the brain and brain stems and hippocampus were discarded, then meninges and vessels were removed. The unicellular suspension was prepared by trypsogen and then the cells were mixed cultured for 7 to 9 days. The oligodendrocyte precursors were separated from astrocyte by orbital shaker, further purified by differential adhesion, and finally cultured in chemically defined serum-free medium with N2 supplement. Immunofluorescence assay was applied to identify the specific antigens A2B5,04 and 01 which expressed in order during the development of oligodendrocyte precursors. GFAP which is the astrocyte-specific antigen was also detected by immunofluorescence assay to evaluate the purity of oligodendrocyte precursors obtained.Results1. The percentage of cultured oligodendrocyte precursor cells was obtained above 95%.2. The ratio of astrocyte-stained cells was less than 5%.3. The stage-specific antigens expressed in order in OPCs cells.ConclusionsSeparation and purification by developed shaking and differential adhesion and chemically defined medium with N2 are suitable and effective to obtain oligodendrocyte precursor cells, and the purity of oligodendrocyte precursor cells can be high. The need for oligodendrocyte precursors at specific stage can be met. ObjectivesTo establish an oxygen/glucose deprived(OGD) model of SD rat's oligodendrocyte precursor cells(OPCs) in a nomaxic environment in vitro with dithionite sodium and DMEM without glucose, and evaluate the model by investigating to the changes of cellular shapes, survival and ultrastructure caused by OGD.MethodsDithionite was applied into DMEM without glucose in a normaxic environment and the pressure of O₂ and CO₂ was detected. To establish the OGD model of neonatal SD rat's oligodendrocyte precursors which are predominently O4 positive with 10mM sodium dithionite and DMEM without glucose. The persistence of 04 positive oligodendrocyte precursors in OGD model and normal control which were cultured in DMEM-F12 in normoxic environment was detected with MTT at 0min, 10min, 30min, 60min and 90min after the oxygen/glucose deprivation. The degree of injury was evaluated by observing the morphologic changes of oligodendrocyte precursors at different time spots under inverted phasecontrast microscope, survival, nuclear changes by DAPI staining, and ultrastructural changes by transmission electron microscope.Results 1. Sodium dithionite eliminated the oxygen in culture medium rapidly and keep the medium in anoxia for 60minutes at least when the medium is placed in a normoxic incubator.2. When the O4 positive OPCs were cultured with sodium dithionite and DMEM without glucose, the morphologic changes were obvious: cell body swelling and the processes swelling, deformed and collapsed gradually.3. When the O4 positive oligodendrocyte precursor cells were cultured with sodium dithionite and DMEM without glucose, the survival rate decreases gradually with the time and the rate was about 50% at 60min while it decreased to a level lower than 50% at 90min.4. Necrosis occured in the cells which had been oxygen/glucose deprived for 60 min while apoptosis took place one day later after reoxygenation.Conclusions1. Sodium dithionite eliminated the oxygen in culture medium rapidly and kept it in anoxia for 60minutes at least when the medium was placed in a normoxic incubator.2. DMEM without glucose and sodium dithionite can be applied to the construction of OGD model in O4 positive oligodendrocytes in a normoxic incubator in vitro.3. The damages of oligodendrocyte precursor cells caused by hypoxia-ischemia in vivo can be analogized with this model, and similar damages occur in the oligodendrocyte precursor cells in vitro. ObjectivesTo observe the expressions and locations of RhoA, phosphorylated ezrin(p-ezrin) and F-actin in the oxygen/glucose deprived model of immature neonatal SD rat's oligodendrocyte precursor cells and normally developing OPCs, and investigate their changes induced by OGD damage ,the roles of the three proteins and the relationship among them during the damage process.MethodsFirst, immunofluorescence assay and laser scanning Confocal microscope were used to observe the expressions and locations of RhoA, p-ezrin(Thr567) and F-actin in normally developing OPCs when they had been cultured for 1d, 2d and 4d. Then the OPCs which had been cultured for 2d and were mostly O4 positive were divided into two groups, normal control and oxygen/glucose deprived(OGD). The OGD model was esstablished by sodium dithionite and DMEM without glucose first, then the expressions and locations of RhoA, p-ezrin(Thr567) and F-actin were observed at 0min, 10min, 30min and 60min after oxygen/glucose deprivation by immunofluorescence assay, laser scanning Confocal microscope and western blotting. The mean fluorescence densities were analyzed by SPSS 11.0.Results1. In developing OPCs, RhoA located in cytoplasm and processes which branches stretch out, and the location didn't change obviously.2. P-ezrin(Thr567) in OPCs were in cell body first, then transfered to submembrane and processes, and concentrated in tips of process at last during developing.3. The distribution and alteration of F-actin coincided with p-ezrin.4. After OGD, the expressions of RhoA increased and reached to the peak at 10min, then decreased gradually, but the levels were higher than the control group's at all the time spots(P<0.05). But the distribution didn't change obviously.5. The fluorescence density of p-ezrin(Thr567) increased to the peak and was higher than the control's level, then descentd rapidly, kept decreasing to a level persistently lower than the control group's(P<0.05), but the tendency didn't coincide with RhoA's. And the distribution of p-ezrin changed to a nonuniform state after OGD damage.6. F-actin's expression decreased and was always lower than the control group's too(P<0.05), and its distribution also becomes nonuniform which is similar to p-ezrin's.Conclusions1. Expressions and locations of RhoA, p-ezrin and F-actin regulate the morphology of OPCs during developing, and all of them play an important role in differentiation and maturation of OPCs.2. Oxygen/glucose deprivation in OGD model of rats' OPCs induces the changes of expression and distribution of RhoA, p-ezrin(Thr567) and F-actin significantly, and change the cytoskeleton and morphology of OPCs ultimately.3. OGD increase expression and activity of RhoA which cause changes of p-ezrin and F-actin in their expressions and distributions sequently. But there may be some other mechanisms which affect the expressions and distributions of RhoA, p-ezrin and F-actin in rat's OPCs during the OGD damage.