Changes Of Phosphorylated Cell Cycle Related Proteins In Developing And Ischemic Rats

Objective:Cell cycle is the basic process of cell life. It has been reported that several cell-cycle related proteins expressed in the postmitotic neurons. These studies indicate that cell-cycle related proteins play a physiological role beyond the regulation of cell cycle. The activity of cyclin-dependent kinases (CDKs) is the key step in the cell cycle regulation and the phosphorylation of CDKs and Retinoblastoma Tumor Suppressor Protein (Rb) can stand for the activation of cell cycle. To observe the expression of phosphorylated CDKs and Rb in the developing and aging rat cortex and hippocampus is the main purpose of this study.Methods: We used Immunofluorescent staining for anti-phospho-CDK2, anti-phospho-CDC2, anti-phospho-Rb and anti-NeuN antibodies to determine their expression and distribution in Wistar rat cortex and hippocampal CA1 area at different ages including post natal 1d, post natal 11d, post natal 1m, post natal 3m and post natal 15m. The expression of phosphorylated cell cycle related proteins in hippocampus at different ages were also assessed by Western-blot analysis.Results: 1) the number of neurons of cortex and CA1 area decreased during the period from PN l day to PN 15 months.2) The positive expression of phospho- CDK2 and phospho-Rb increased while aging in both cortex and hippocampal CA1 area, especially in the PN15m group.3) There was also increasing positive expression with age determined by Western-blot. The positive expression in the PN15m group increased greatly and there was a significant differnce between PN15m group and other groups. 4) There was no apparent change in the expression of phospho- CDC2 in the cortex and hippocampus of all the five groups.Conclusion: The expression of phospho- CDK2 and phospho-Rb suggests that differentiated neuron can re-enter the cell cycle and more neurons enter the cell cycle in the aged rat brain. But low expression of phospho-CDC2 indicates that postmitotic neurons can't complete the whole cell cycle. Re-entering the cell cycle of terminal differential neurons probably results in apoptosis. Objective:The responses to the focal cerebral ischemia between neurons and astrocytes are different. The mechanisms involved in the process are complex. The activity of cell cyclin-dependent kinases (CDKs) is the key step of cell cycle regulation and phosphorylated CDKs stand for the activity of CDKs. In some previous studies some cyclins and CDKI were found in the neurons after cerebral ischemia. But there are few studies about the activity of CDKs. In this study we aimed to observe the expression of phosphorylated CDKs and the activity of the retinoblastoma tumor suppressor protein (Rb) after focal cerebral ischemia in rats.Methods: Ischemia/reperfusion injury was induced by temporary middle cerebral artery occlusion (MCAO). For rats induced by MCAO, experimental rats were sacrificed and the brains were taken on day1, 3, 7 and 14 of reperfusion. We used Immunofluorescent staining for anti-phospho-CDK2, anti-phospho-CDC2, anti-phospho-Rb and anti-NeuN or anti-GFAP antibodies to determine their expressions in the cortex around the infarction of rats 1d,3d,7d,14d after focal cerebral ischemia.Results: (1) In the core of the infarction, most neurons died while neurons around the infarction showed irregular morphology. (2) Few positive expression of phospho-CDK2 and phospho-Rb in the controlled group. The expression of phospho-CDK2 and phospho-Rb increased in the neurons near the infarction in the groups of 1d and 3d after focal cerebral ischemia compared to the controlled group. In the groups of 7d and 14d, there was no significant change of expression. In all groups, there was no expression of phospho-CDC2. (3) The astrocytes around the infarction became active soon after ischemia. (4) Low positive expression of phospho-CDK2, phospho-CDC2 and phospho-Rb in the controlled group. The expression of phospho-CDK2, phospho-CDC2 and phospho-Rb increased in the astrocytes near the infarction in the groups of 1d, 3d and 7d after focal cerebral ischemia and the peak appeared in the group of 3d after focal cerebral ischemia.Conclusion: The expression of phospho- CDK2 and phospho-Rb suggests that differentiated neurons can re-enter the cell cycle and more neurons enter the cell cycle in the early stage after ischemia. Re-entering the cell cycle of differential neurons probably results in apoptosis. In the early stage of ischemia, astrocytes were activated and the expression of phospho-CDK2, phospho-CDC2 and phospho-Rb in the astrocytes suggests astrocytes re-enter the cell cycle after cerebral ischemia. The cell cycle events are related with proliferation and activation of astrocyte closely.