Study Of Cell Cycle Machinery Between Astrocytes And Neurons

Objective: Astrocytes and neurons are the predominant cell types found in brain. Recent findings suggest that aberrant reentry of neuronal cells into the cell cycle occurs after cerebral ischemia. But the characteristics of the cell cycle remain unknown, especially the difference controls of the cell cycle between astrocytes and neurons are unclear. In the present study, we investigated the basic characteristics of the cell cycle between astrocytes and neurons following focal cerebral ischemia in rats in vivo, which gives experiment proves for the study of cell cycle control.Materials and Methods: Ischemia/reperfusion injury was induced by temporary middle cerebral artery occlusion (MCAO). Experimental rats were sacrificed on day1, 3, 7, and 14 of reperfusion, then the brain tissues from cortex and hippocampus areas were taked. And the cell cycle of astrocytes and neurons was detected by flow cytometry (FCM).Results: There was a significant difference in each phases of cell cycle between astrocytes and neurons in normal animals in vivo (P<0.01). After MCAO, in the cortex the ratios of neurons re-entering cell cycle significantly increased at day1 of reperfusion, but at day3 astrocytes were activated to enter into new cell cycle events. While in the ipsilateral hippocampus of rats at day1 of reperfusion, astrocytes proceeded to enter into cell cycle, and a statistically significant increase was observed compared with the normal groups (P<0.01). After MCAO, the difference of cell ratios in G0/G1 phase between astrocytes and neurons was also significant (P<0.01), and the percentages of astrocytes in S phase and G2/M phase increased remarkably at day14 compared with those of neurons (P<0.01).Conclusion: There are different characteristics of the cell cycle between astrocytes and neurons in normal rats in vivo. In addition to a differential sensitivity between astrocytes and neurons to ischemic injury, there is also a differential sensitivity among astrocytes from different brain regions. And re-entering into cell cycle occurred in both astrocytes and neurons after MCAO, some neurons are more likely to repair DNA and even lead to apoptosis, but astrocytes have active cell cycle and may enter into the cell cycle to proliferate. Objective: In recent years, apoptosis is increasing attracted following cerebral ischemia. There were many reports about the apoptosis of neurons after cerebral ischemia/reperfusion, but few studies have shown that astrocytes can be induced to undergo apoptosis due to some factors relevant to ischemia. There is no direct conclusive evidence that the mechanism of cell cycle may involve apoptosis. In this study, we investigate the characteristics of apoptosis during the different phase of the cell cycle between astrocytes and neurons, to find the different apoptotic mechemisms.Materials and Methods: Ischemia/reperfusion injury was induced by temporary middle cerebral artery occlusion (MCAO). For rats induced by MCAO, experimental rats were sacrificed on day1, 3, 7 and 14 of reperfusion. And the brain was taken at 1, 3, 7, 14 day after injury, then the ratios and the dispositions of apoptosis during the different phase of the cell cycle between astrocytes and neurons was detected by immunofluorescence, flow cytometry (FCM) and teminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) methods.Results: After MCAO, in the hippocampus, the apoptosis ratios of astrocytes were more than neurons, the apoptosis astrocytes reached its peak at 3 days, neurons at 7 days. But in the cortex the number of apoptosis cells in neurons were more than astrocytes, they all reached its apoptosis peak at 7 days. There are few BrdU with TUNEL positive cells showing co-localization in ipsilateral cortex following MCAO when compared to the normal controls. The most apoptotic neurons and astrocytes were in G0/G1 phase, the apoptotic ratios of neurons were less than those of astrocytes in G0/G1 phase, but the apoptotic ratios of neurons were more than those of astrocytes in S phase.Conclusion: Both neurons and astrocytes appeared apoptosis after MCAO in cortex and hippocampus areas of rats. There are different characteristics of apoptosis during the different phase of the cell cycle between astrocytes and neurons. The apoptotic astrocytes may be occurred during the different phase of the cell cycle following MCAO, and the most apoptotic neurons were in G0/G1 phase, and they may re-entry S phase and G2 phase of cell cycle to occur apoptosis. It suggests that the apoptosis between astrocytes and neurons was induced at different check-points. Objective: Recent increasing evidences suggest that aberrant cell cycle reentry occurred after cerebral ischemia. In our previous study, we observed that neurons can re-entry into the cell cycle and repair the DNA damage or initiate apoptosis, but astrocytes can rapidly enter into the cell cycle to proliferate. In this study, we will investigate the gene and protein expressions of cyclins between astrocytes and neurons.Materials and Methods: Ischemia/reperfusion injury was induced by temporary middle cerebral artery occlusion (MCAO), and the brain was taken at 3 days after injury. To investigate the gene and protein level expressions of Cyclin D1, Cyclin E and Cyclin B1 between astrocytes and neurons after focal cerebral ischemia in the adult rats brain in vivo, the methods of flow cytometry sorting and post-sorting RT-PCR and Western Blot were used.Results: In the normal groups, the genes and proteins of Cyclin D1, Cyclin E and Cyclin B1 are expressed mildly in astrocytes in vivo, and there are the genes and proteins of Cyclin D1 mildly and Cyclin E distinctly expressed in neurons in vivo, but the gene of Cyclin B1 is less expressed and the protein of Cyclin B1 is not detected in neurons. After MCAO, in neurons the genes and proteins levels of Cyclin D1 and Cyclin B1 increased, but the genes and proteins levels of Cyclin E fall. But in astrocytes, the genes and proteins of Cyclin D1 and Cyclin E increased distinctly, while the genes and proteins levels of Cyclin B1 increased slightly.Conclusion: It was suggested that the abortive activated genes and proteins level expression of Cyclin D1 , Cyclin E and Cyclin B1 may be not only related to the proliferation of astrocytes, but also participate in the apoptosis process in postmitotic neurons after focal cerebral ischemia. And the expressions of Cyclin D1 and Cyclin E in neurons in the normal adult rats brain in vivo, which might play a solo roles that were not related to cell cycle mechanisms.