| The treatment of Spinal cord injury (SCI) has always been a big difficulty to all of humanity in a long period of time. Especially with the rapid development of modern society and the ever-accelerating pace of life, the incidence of all kinds of traumatic SCI is increasing year by year. According to the statistics, there is about 50,000 SCI patients each year in our country, which is increasing on a rate of 10%. SCI brings a heavy burden to family and society because of high rate of deformity, long-term hospitalization and rehabilitation. People has tried numerous methods to treat it, but no one is ideal. On recent, following development of study of neuro-pathophysiology and auxology, the conventional idea that nervous tissue do not regenerative had challenged. The discover and confirm of Neural stem cells have brought a new way for intractable diseases of center neural system including SCI.Neural stem cells(NSCs) are a kind of cells that have the ability to differentiate into neurons, astrocytes and oligodendrocytes. They have self-renewal activity and multi-potential proliferation, which is important for theory research and clinical applications. The transplantation of a variety of cells, particularly the neural stem cells or NPCs, in multiple central nervous system (CNS) injury paradigms has provided to a certain degree encouraging results for functional recovery. While induction and recruitment of endogenous adult NSCs, has received relatively little attention. Compared with transplantation of cells,the activation of endogenous NSCs is use self-resource. It not only get rid of the limit and danger of acquiring NSCs from tissue, but also avoid the exiting questions of ethics , immunological and limited source. It will have a promising future in the treatment of head trauma and degenerative diseases of the central nervous system.Although we have got some progress on neural stem cells, little is knowen about the proliferation, migration, and differentiation of adult NSCs in the mammalian spinal cord, especially the mechanism of regulation in vivo. So used bromodeoxyuridine(BrdU)/Nestin to label the endogenous NSCs in this study, we study the proliferation and differentiation of endogenous neural stem and the expression of Wnt-1 after spinal cord injury in rat for the aim to explore the self-duplication regularity of endogenous NSCs. In addition, Wnt/β—Catenin signaling pathway has been recently shown to be an important role in the development of central nervous system and in vivo proliferation of NSCs. Therefore, we examined whether exogenous administration of Lithium chloride(LiCl ) would further increase the numbers of NSCs after SCI in vivo.Part I : Experimental study on the expression of wnt-1 in the proliferation process of endogenous neural stem cells after spinalcord injury in ratObjective: To investigate the Expression of wnt-1 in the earlier proliferationprocess of endogenous neural stem after spinal cord injury(SCI) in rats.Methods:(1)Fifty Wister rats were randomly divided into two groups: the normal control group (group A) and the spinal cord injury group (group B). (2) A group only exposed the vertebral plate, B and C group SCI models were established by Allen method.(3) The rats were killed at 1d, 3d, 1,2 and 4 weeks after injury, the expression of wnt-1 and the proliferation of ENSCs were analyzed in the samples which were got from the two levels of the spinal cord with the length of 5mm harvested both rostral and caudal region to the injury center by immunoreactivity. The linear regression analysis was used to evaluate the correlation between the expression of wnt-1 and the proliferation of ENSCs.Results: Group A shows that there is a very small number of Brdu/Nestin positive cells in the peripheral and ependymal region of spinal cord , and with sparse in the gray matter and white matter. Twenty four hours after SCI there is visible portion of Brdu/Nestin positive cells,seen mainly in the surrounding of injured spinal cord and ependymal areas in Group B, which appeared in scattered spots flake and filament expression in the gray matter and white matter. It begins increasing in three days, and reaching the peak in one week after SCI. BrdU / Nestin-positive cells were seen mainly in ependymal and meninx vasculosa around the spinal cord, and extent to the centre of the spinal cord. The number of Brdu/Nestin positive cells Starts to decline two weeks, and show up significant decrease in four weeks after SCI.Almost no expression of Nestin exists in group A. One days after SCI, a small number of Nestin expression begin showing up, which can be seen more in seven days after SCI, mainly distribute in the ependyma area and gray matter. It gets the beginning of decline gradually in two weeks after SCI, and decreases significantly after four weeks. The dendrite with expression of Nestin extend to the gray matter, the expression in gray matter is mainly distributed in neurons and blood vessels. Some cells have the similar morphology with astrocytes. Occasionally a small amount of neuron-like Nestin-positive cells appear in the gray matter. There is no obvious Wnt-1 expression in Group A, and Group B haves the contradictory phenomenon. one day after SCI expression of Wnt-1-positive region can be seen around the central canal and adventitia of spinal cord, little appears in the white and gray matter,which is quite dispersed. In three days the expression reach a peak, and stay at a relatively high level till one week after SCI, which followed by a gradual decreasing process, in four weeks only a very small number of positive expression of Wnt-1 can be seen.Conclusions: Normal rat spinal cord has the presence of endogenous neural stem cells, which own proliferative capacity and maintain a state of proliferative activity. These cells can be obviously activated by the stimulation of injury through one mechanism that may be associated with Wnt signaling pathway.PartⅡ: Effect of Lithium chloride on endogenous neural stem cellsafter spinal cord injury in ratsObjective: To investigate the effect of Lithium chloride on endogenous stem cells after spinal cord injury in rats.Methods: Seventy five female Wister rats were divided into three groups randomly: the control group (group A); spinal cord injury group(group B); lithium group(groupC). A group only exposed the vertebral plate, B and C group were made a injury at T10 level by Aliens method. The rats in C group were injected with lithium 3mmol/Kg every day after SCI, while the rats in B group received same amount of 0.9% normal saline by the same way. All rats were labeled three times with Brdu at 1d, 3d, 7d, 14d and 28d. The equivalent rats were killed four hours after the last label in all group. The spinal cord with the length of 5mm to the injury center were the samples were analyzed by Brdu and p—Catenin immunohistochemical detection to study the response of endogenous stem cells.Results: There was few Brdu positive cells and less expression of p—Catenin in the center canal and adventitia of spinal cord in group A. many Brdu positive cells and little expression ofβ—Catenin were found in gray matter and ependyma of injury area in group B 1d after SCI, which reached the peak at 7d, and declined gradually 14d postinjury. Just a few Brdu positive cells and little expression ofβ—Catenin existed 28d after SCI. There was no difference between B and C group on the Brdu positive cells and the expression ofβ—Catenin 1d after SCI. Group C had more Brdu positive cells and the expression ofβ—Catenin in the center canal and adventitia of spinal cord, which were also at the high level 14d and 28d after SCI. Conclusions: Lithium could activate the endogenous stem cells after SCI, the mechanism may be relate with the Wnt signal pathway.Conclusion:1. Endogenous neural stem cells exist in normal rat spinal cord, which are staying in "static"or "hibernation" state. In the role of specific factors such as injury, their potential of division and differentiation is activated, and thus give rise to proliferation.2. After SCI the capacity of endogenous neural stem cells increase by degrees, which is accompanied by the gradual mult of expression of Wnt-1, all of this prompt that Wnt-1 or Wnt signaling pathway may be involved in proliferation process of endogenous neural stem cells after SCI.3. The application of lithium chloride can promote proliferation of endogenous neural stem cells in rat, which reveals that lithium chloride may be playing an active role in the treatment of SCI.
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