| Ischemia often results in human death and serious disability. There is still no effective therapy to solve the problem of the lost neurons caused by ischemia injury up to now. So many disease caused by ischemia may lead to hemiplegia and cognitive declining disorder. The regeneration of new neurons from neural stem cells (NSCs) is restricted to two regions of the adult mammalian CNS: the subventricular zone of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus. NSCs in both regions are capable of migration and differentiation under ischemia condition. These two regions play an important role in brain repair and neuron regeneration. Endogeneous NSCs provides an ideal approach for repair after ischemia injury.Ischemia only induces insufficient NSCs proliferation spontaneously and little is understood about how ischemia induces quiescent stem cells to proliferate, which is the major barrier for utilizing the endogenous NSCs to repair nervous system injury. Previous researches indicate that Wnt/β-catenin signaling plays an important role in regulating NSCs proliferation and tumorigenesis. So in this study ischemia reperfusion models in mice were employed in order to investigate the effect of Wnt/β-catenin signaling in regulating the proliferation of NSCs after ischemia reperfusion.The study consisted of two parts:Part I The establishment and identification of the ischemia reperfusion model in miceThe model of ischemia reperfusion in mice was made by clamping bilateral common carotid arteries half an hour.The different neuronal population in the hippocampus were differently vulnerable to ischemic injury; the CA1 pyramidal neurons were the most vunerable to this injury. The expression of BrdU and Nestin were detected by immunohistochemistry and immunofluorescence staining. BrdU labelled were the newborn cells and the Nestin labelled were the NSCs. The number of BrdU+ cells began to increase in hippocampus 1 day after ischemia reperfusion, and the BrdU+ cells peaked at 7 days. The number of BrdU+ cells returned to normal level at 28 days. The change of the number of Nestin+ cells was same as the BrdU+ cells. The number of BrdU+ /Nestin+ cells also peaked at 7 days after ischemia operation.PartⅡThe effect of Wnt/β-catenin signaling in regulating the proliferation of NSCs after ischemia reperfusion.Axin is an axis inhibitor. As a scaffold protein, it plays a critical role in Wnt/β-catenin signaling patyway. It facilitatesβ-catenin degradation by assembling aβ-catenin destruction complex. Beta-catenin in brain was overexpressed or inhibited through objective gene transfection, and then it was detected the influence ofβ-catenin on the proliferation of hippocampal NSCs. Recently gene transfection techniques is to use viral vectors and nonviral vectors. Nonviral vectors are safer than the viral vectors in vivo experiment. So in this study liposome-mediatedβ-catenin and Axin plasmid were injected into lateral ventricle. The results showed that in Axin plasmid treatment group the number of BrdU+ cells, Nestin+ cells, BrdU+ /Nestin+ cells were less than ischemia control group(P<0.05) but more than normal group, while inβ-catenin plasmid treatment group the number of BrdU+ cells, Nestin+ cells, BrdU+ /Nestin+ cells were more than ischemia control group(P<0.05). These results suggested thatβ-catenin plays an important role in the hippocampal NSCs proliferation.In conclusion, clamping bilateral common carotid arteries could result in ischemia reperfusion. The ischemia reperfusion injury can promote the proliferation of endogenous neural stem cells. Overexpressedβ-catenin or decreasedβ-catenin could obviously promote or reduce the proliferation of hippocampal NSCs. This study may hint highlight for regulating endogenous NSCs proliferation in clinical practice. |
