Empirical Study Of The Cell Proliferating Effect After Central Nervous System Injury

Glia scar formed in adult central nervous system(CNS) injury has been considered as the major facor of the faliure of regeneration.Traditionally, mature glia scar include some extral celluar ingredients(such as type-IV collagan, laminin) and celluar ingredients(such as astrocytes, fibroblasts, macrophages).Reactive astrocytes are the major components of glia scar. Oligodendrocyte precursor cells ,briefly OPCs, are the fifth element in the CNS, in addition to neurons, astrocytes, oligodendrocytes and microglia, which has been recently detected[1]. However,there has not been much research on the reaction of OPCs in CNS injury.In 1990s neural stem cells(NSC) has been confirmed to exsit in various regions of CNS[2] .Those stem cells are mostly dormant ,but they react to CNS injury and other exogenous signals by activating and proliferating. Subventricular zone(SVZ) of the lateral ventricle and subgranular zone(SGZ) of the dentate gyrus are the two neurogenic regions of the adult mamalian central nervous system. Parent's researches confirmed that many experimental brain injury can arouse the proliferation of NSC in distant SVZ and SGZ, and these cells has the ability to develop into astrocyes as well as neurons[3],[4],[5],[6].So,adult CNS react to injury not only by reactive gliosis,but by the proliferation of distant NSCs.Recently more and more researches indicate that ,adult mamalian NSC can differentiate into neurons in special niches [7].BMP4 and Noggin has important roles in modulate the proliferation and differentiation of NSCs.Lim et. al found that BMP4 and Noggin cooperate to affect the differentiation of the stem cells in SVZ,BMPs promote the generation of astrocytes ,concurrently inhibit neurogenesis[8]. Reasearches into adult hippocampaus also find that BMP4 has the ability of promoting the generation of astrocytes[9].Whether BMP4 and Noggin has the same function in regions other than SVZ and SGZ and How it affect the differentiation of the proliferating cells after CNS injury,needs further study.This research use unilateral eye ball enucleation as the CNS injury model. And then detectting the change of GFAP, NG2 IR cells in contralateral superior colliculus at different time points to confirm that after unilateral eye ball enucleation there has been injury reactions in contralateral superior colliculus in the brain . In addition we try to investigate the significance of the proliferation of GFAP positive astrocytes and NG2 positive OPCs.In the second part, we use BrdU labelling method to detect the proliferation of BrdU positive cells in the injury place, dentate gyrus and subventricular zone. Combined with BrdU/GFAP, BrdU/DCX immunofluorescence double labelling ,we further investigate the differentiation of the proliferating cells.In the last part ,to investigate the factors affecting the differtiation of the proliferating cells in the injury place ,we use in situ hybridization and westen-blot to detect the expression of BMP4 mRNA ,Noggin mRNA and Noggin protein.The main results are as follows:1. 24 hours after unilateral eye ball enucleation the immunostaining of GFAP in contralateral superior colliculus increaded compared with control group, this staining progressively increased during 24h to 1 week and reached the top at the time point of 1 week. But in the time span of 1 week to 3 week there has been no increase.2. 24 hours after unilateral eye ball enucleation the immunostaining of NG2 in contralateral superior colliculus increaded obviously. The number of the NG2 IR cells reached the top amount at 1 week, and then decrease slowly in the following two weeks.3. Use BrdU labelling method,we find that 24h after unilateral eye ball enucleation some BrdU IR cells was detected in contralateral superior colliculus .There has been most BrdU IR cells at 1 week, and decrease obviously in 2 week group and 3 week group.4. 24 hours after unilateral eye ball enucleation the number of BrdU IR cells in dentate gyrus and SVZ increased slightly,reached the top at 1 week ,and then gradually decreased in the following two weeks. Additionally we find that 2 weeks after the injury the number of BrdU IR cells in the dorsalateral SVZ has been obviously increased, and the distance it extends also increased. 2 weeks after the injury the number of BrdU IR cells in corpus callosum increased.5. 1 week after unilateral eye ball enucleation the number of DCX IR cells in both dentate gyrus and SVZ increased obviously compared with control group, decreased in the following 2 weeks and then reach the control group level at 3 week.6. Immunofluorescence double labelling indicate that, the BrdU IR cells in contralateral superior colliculus include some BrdU/GFAP double labelling cells, without BrdU/DCX double labelling cells. The BrdU IR cells in dentate gyrus and SVZ has both BrdU/GFAP and BrdU/DCX double labelling cells.7. In situ hybridization indicate that , 24 hours after unilateral eye ball enucleation the number of BMP4 mRNA IR cells increased obviously compared with the control group, 1 week group decreased compared with 24 hour group, and decreased gradually in 2 week and 3 week groups.8. However, the expression of Noggin mRNA in each group has no obvious change, the expression of Noggin protein indicated by western-blot also hasn't any change in each group.The main conclusions:1. Both GFAP IR astrocytes and NG2 IR OPCs react to unilateral eye ball enucleation, which indicate that contralateral superior colliculus did undergo an injury after unilateral eye ball enucleation. We established a new, easy CNS injury animal model by enucleate the eye ball of the animal. At the same time we confirm that except reactive astrogliosis, OPCs are activated in CNS injury.2. Besides reactive gliosis (including astrocytes and OPCs), CNS injury can also initiate the proliferating of BrdU IR cells in the injury place ,dendrate gyrus and SVZ.The proliferating cells in the injury place can only differertiate into astrocytes, while the proliferating cells in dendrate gyrus and SVZ can develop into both astrocytes and neuroprecursors.3. The expression of BMP4 and Noggin may be important for the differentiating of the proliferating BrdU IR cells in the injury place.