Experimental Study On Situ Mobilization Of Neural Stem Cells And Its Regulation By Notch Signaling Pathway After Traumatic Brain Injury In Adult Rats

BackgroundWith the development of social industrialization, traumatic brain injury (TBI) is the most common acquired injury in traumatic injury with high morbidity and mortality rates. The molecular mechanisms of restoration in the adult central nervous system (CNS), after TBI. were not clear completely. Recent studies demonstrated the neural function deficit and neuron death are inevitable for most TBI patients. Activation of endogenous neural stem cells (NSCs) under TBI in situ, that exist in the central nervous system of adult mammals, and most of them were at resting stage in vivo in normal. NSCs have the potential of self renewal and can proliferate then differentiate into neurons and astrocytes under some condition, NSCs may be involved in neural function restoration. Recent studies in vivo and in vivto demonstrated NSCs existed in the CNS of adult mammals, and activation of NSCs could further proliferate differentiate and be capable of regenerating function after TBI. The molecular mechanisms underlying increased proliferation differentiation and migration of endogenous NSCs after TBI , still remain unclear and need to be elucidated to aid development of a novel strategy for enhancing neurogenesis after TBI. Although studies demonstrated that activation of a lot of NSCs in the hippocampus dentate gyrus (DG) and subventricular zone (SVZ) could proliferate differentiate into neurons and glial cells after TBI., there are controversial studies on NSCs proliferating and differentiating in the adult CNS after TBI. Notch signaling pathway has been shown to play a fundamental role in regulating the fate of NSCs proliferating and differentiating, however, the complex intrinsic mechanisms of Notch signaling that controls and coordinates NSCs proliferation and differentiation are still poorly understood.In this study, three parts were designed in the experiment. Part one, we developed the brain injury model described by Feeney with some modification, and identified the model on cellar level at the aspect of nerve behavior grade, general and pathology. Part two, we observed the change process of endogenous NSCs proliferating and differentiating in situ by the method immunohistochemistry and immunofluochemistry, found out the rules of endogenous NSCs proliferating and differentiating in the process of self renewal; Part three, we observed the change of Notch1 protein expression in Notch signaling pathway by the means of immunofluochemistry and Proteomics (western-blot), analyzed the relation between Notch1 signal protein expression and NSCs proliferation and differentiation in situ. It is in order to discover the endogenous NSCs proliferating and differentiating in situ by the method immunohistochemistry and immunofluochemistry, elucidate the molecular mechanisms of NSCs proliferation in situ after cerebral injury, and to provide the theorical and experimental foundation on neural function restoration by NSCs proliferating and differentiating.PartⅠObjective: To developing the brain injury model described by Feeney with some modification, that is easy to observe, control, repeat and classify. To identify the model on cellar level from the aspect of nerve behavior grade, general and pathology, etc.Method:The brain contusion device was adopted from the impact method described by Feeney, Moderate brain trauma was made by dropping 20g weight from 30cm. Therefore, the injury wallop is 600g.cm. A series of general and pathology observation were among the different groups. And then, nerve behavior scale was evaluated at 1～14d after TBI, upon Wayne Clark's standard. The results were compared among the difference groups. These model animals were sacrificed according to the time point of divided groups.Results:Developing the brain injury model by Feeney with some modification, that easy to be observe, controlled, repeated and classify. This experiment showed that there were remarkabl differences among the control, sham and experimental groups on nerve behavior grade, pathology, and so on.Conclusion:A stabilized Moderate brain trauma model was established. The model has practical value in traumatic brain injury experimental and clinical traunatic studies, provides the experimental model foundation for exploring the NSCs in situ activating following TBI. PartⅡObjective:To explore the possible rules of proliferation, differentiation, migration and self-renew of endogenous NSCs in the different regions including SVZ, DG and injury site, at different time points after TBI model was established, by mainly immunohistochemistry and immunofluorescence.Method:At different time points, sections obtained from SVZ, DG and injury site of different experimental groups were stained by anti-Nestin or ant-BrdU antibodies. Double immunofluorescent labeling was performed by using Nestin/ GFAP, Nestin/NSE, BrdU/GFAP or BrdU/NSE antbodies. Immunohistochemical and immunofluorescent staining was photographed with a light microscope and laser confocal microscope. By the help of special software (Leica Q Win), we counted the cell numbers with double labeling in the SVZ, DG and injury site.Results:Under light microscope, Nestin and BrdU immunopositive products with different intensities could be observed in the cells of SVZ and DG in different experimental groups. Nestin-positive staining mainly localized in the cytoplasm, while BrdU immunoreactivity (IR) mainly expressed in the cellular nuclei. The cells numbers with BrdU IR in the experimental group was significant more than the control and sham-operative groups, which was most obvious at 3 days post operation.Under laser confocal microscope, Nestin- and BrdU-positive products with different intensities could be observed in the cells of SVZ and DG in different experimental groups. In the TBI group, the Nestin/GFAP, Nestin/NSE, BrdU/GFAP and BrdU/NSE double labeled positive cells numbers were elevated at 1 day post injury, peaked at 3 days, and then decline at 7 days, recovery to the normal level at 14 days. The changes were most obvious in the SVZ. The glia with abnormal morphologies could be found around the injury site. However, the Nestin-positive cells numbers had no significant changes.ConclusionAccording to the present data, TBI induced in situ the endogenous NSCs that localized in the DG and SVZ proliferation and differentiation. TBI lead to the glia numbers increase abnormally and formed glia scar in the cerebral cortex around the injury cites. Largely Nestin immunoreactive products were observed in the SVZ and DG, which implied many neuronal precursors were mobilized in these regions. Largely BrdU immunoreactive products were observed in the SVZ and DG,, it suggested NSCs began to proliferation and differentiation at 3 days post operation.PartⅢObjective:To observe the change of Notch1 protein expression in Notch signaling pathway by the means of immunofluochemistry and Proteomics (western-blot), analyse the relation between Notch1 signal protein expression and NSCs proliferation and differentiation in situ. And to discover the NSCs proliferating and differentiating in situ by the method of Immunohistochemistry and immunofluochemistry. To elucidate the molecular mechanisms of NSCs proliferation in situ after cerebral jury.Method: The temporal changes of Notch1 (the signal maker of Notch signal pathway) were checked with the ways of qualitative and semi-quantitative investigating, by immunohistochemistry and Western blot respectively. At different time points, sections obtained from SVZ, DG and injury site in different experimental groups were doubly labeled by using Notch1/GFAP and Notch1/NSE antibodies. The cells numbers with double labeling were counted and the OD levels of proteins was detected. Statistics was analyzed by the help of SPSS10.0 software.Results:Under laser confocal microscope, the double labeled cells with Notchl/GFAP,Notch1/NSE at different intensities could be observed in the cells of SVZ and DG in different experimental groups. In the TBI group, the Nestin/GFAP, Nestin/ NSE, BrdU/GFAP and BrdU/NSE double labeled positive cells numbers were elevated at 1 day post injury, remaining high levels at 7 days, and began to decline at 14 days. The immunopostive cells numbers had significant statistics differences between TBI group and control or sham operative groups. Western blot analysis showed that each antibody might specifically recognized the appropriate bands at a molecular weight of approximately 110 KDa (active part of NICD-Notch), and actin control antibody was recognized the bands located at 130 KDa, the Notch content was abundant in the SVZ and DG, remaining high levels a t 7 days, and began to decline at 14 days.Conclusion:Notch1 play a potential regulation role during the course of the in situ activation, differentiation and migration of endogenous NSCs located in the SVZ and DG after TBI.