A Study On Neuronal Cell Cycle Dysregulation And Potential Apoptotic Mechanisms Following Ischemia

Part One Cortical neurons re-enter cell cycle and undergo apoptosis Induced by oxygen-glucose deprivation in vitroObjective: To definite the relationship between cell cyler re-entery and neuronal apoptosis in cultured cortical neurons following oxygen-glucose deprivation (OGD) and to explore the potential mechanism of apoptosis in post-mitotic neurons induced by OGD.Methods: The model of OGD injury on cultured cortical neurons was established. The cultured neurons were randomly divided into 5 groups: control group, the group of 6h, 12h, 24h reoxygenation after 1h OGD and roscovitine treated group. Flow cytometry was used to detect the percent of neuronal apoptosis after 1h OGD. Immunofluorescence staining was used to detect uptake of BrdU and expression of phosphorylated Rb protein (p-Rb, ser 795) in neurons, and their co-localizated relationship with the TUNEL staining. Western Bolt was used to detect the expression of p-Rb and E2F1.Results: Compared with the control group, the percentage of neurons with BrdU uptake ,TUNEL staining and co-localization of both were increased in the 6h, 12h, 24h reoxygenation after 1h OGD. The expression of p-Rb in each group after OGD were significantly increased, in parallel with BrdU uptake. The co-localization of p-Rb and TUNEL in 6h and 12h after OGD was apparent, but the percent of co-localization between p-Rb and TUNEL was strikingly decreased in 24h after OGD. The expression of p-Rb and E2F1 and the percent of neuronal apoptosis were decreased in roscovitine treated group.Conclusion: Attempted cell cycle re-entry with phosphorylation of Rb in neurons induces neuronal apoptosis after OGD treatment. Inhibition of phosphorylation of Rb following OGD reduces neuronal apoptosis, phosphorylated Rb pathway is one of the mechanisms of neuronal apoptosis induced by OGD. Part Two The involvement of upregulation and translocation of phospho-Rb in early neuronal apoptosis following focal cerebral ischemia in ratsObjective: To investigate the expression and subcellular localization of phospho-Rb (ser 795), and temporal and spatial relationship between it and neuronal apoptotic death in rats subjected to transient focal cerebral ischemia.Methods: The model of middle cerebral artery occlusion (MCAO) was carried out. All animals were divided into five groups: sham-operated controls, 12 h, 1 d, 3 d, and 7 d after MCAO/reperfusion. By analyzing DNA fragmentation with a TUNEL assay, apoptotic cells were examined in the peri-infarct area of the ischemic cerebral cortex. Immunofluorescence staining was used to detect the expression and subcellular localization of phospho-Rb (ser 795) in the each time points after MCAO/reperfusion. Double-labeling analysis of TUNEL and phospho-Rb staining was used to detect the temporal and spatial relationship between phospho-Rb and neuronal apoptotic death.Results: Compared with sham-operated controls, at 12 h after MCAO/ reperfusion TUNEL-positive cells began to be detected, and the immunoreactivity of phospho-Rb in neurons was increased. One day after the transient ischemic injury, it was showed that subcellular localization of phospho-Rb translocated from the nucleus to the cytoplasm in many neurons in the peri-infarct area and the number of TUNEL-positive cells peaked at this time point. Most phospho-Rb appeared to be localized within TUNEL staining cells at 12 h and 1 d after MCAO/reperfusion in an overlapping and mosaic pattern. However, there was no apparent colocalization of phospho-Rb and TUNEL staining at 3 d and 7 d, although the number of phospho-Rb translocated neurons and TUNEL stained cells were still at high levels at these time points.Conclusion: phospho-Rb may be involved in the early stages of neuronal apoptosis after transient cerebral ischemia, and it may not be involved or in an indirect way in the late stages of neuronal apoptosis. Part Three Sorting of neurons in cortex and hippocampus from thy1-GFP-J transgenic mice by flow cytometryObjective: To establish an effective method fast getting purified neuronal populations from mammalian brain in vivo.Methods: Thy1-GFP-J transgenic mice, which expressed green fluorescent protein (GFP) in neurons, were confirmed by RT-PCR and confocal laser scan microscopy. The cortex and hippocampus of 2-months-old transgenic mice were dissected and then dissociated using the papin dissociation system. Single cells of brain tissue were treated with PI to label dead cells and sorted on a FACS-Aria cell sorter. Cells with high GFP signals and negative PI signals were selected, then RNA and protein were extracted from the sorted cells.Results: PCR amplification of DNA from tail of transgenic mice indicated that it appears two bands inculding transgene band (173bp) and internal control band (324bp), and the pictures of brain tissue captured by confocal microscopy showed that green fluorescent spots with similar round patterns can be seen in the cortex and hippocampus of these mice. After sorting, almost all cells seemed brightly fluorescent in their soma, and about 0.5-1×10~6 lived neurons were obtained in each sort. RNA and protein extracted from purified neurons were detected by RT-PCR and western blot.Conclusion: It is a reliable and practical method for study of neuronal cell type-specific gene expression profiling and quantitative analysis of proteins extracted from purified neuronal populations, which obtained from mammalian brain of adult thy1-GFP-J transgenic mice by FACS in vivo.