| BackgroundSpinal cord injury (SCI) is one of the most difficult problems of the medical domain in the world. SCI cause the interruption of connection between the brain and the part of the body under the injury level,including the sensory,movement, sphincter function impaired permanently. There is no effective treatment up to now. In the past viewpoint,the regeneration and functional recovery of the central nervous system after injury was absolutely impossible. Since 1981 the neuroscientist confirmed that the repair of the central nervous system structure after injury was possible,the study on repair of SCI has been becoming hot spot in many acdemic institute. The factors that interfering axon regeneration after SCI were extremely complicated.First,primary and secondary injury on spinal cord caused a great of spinal cord functional neurons apoptosis and necrosis,which making the regeneration of axons difficulty.Second,a great quantity secretion and release of myelin-associated inhibitor and axon growth inhibition protein after SCI. The secondary glial scar impeded the axons growth and properly connected.Third,the decreased secretion of many neurotrophic factors resulting from partial cells apoptosis destroied the microenvironment benefiting for neurons repair and axon regeneration. The mechanism of SCI recovery is very complicated. SCI priminged various inhibit genes and regulate genes which interfering the regeneration of the spinal cord. Thus, there was rarely effection when aiming at single factor obstracting spinal cord regeneration. The measures of repairing SCI currently are mainly the following aspects:drug treatment,cells transplantation,tissue transplantation, improve the local microenvironment and union treatment. The traditional high-dose corticosteroids ( methylprednisolone ) stosstherapy could effectively reduce the secondary inflammation after SCI,and protect the residual damaging neurons and prevent the further apoptosis. At present,the studies claimed that lithium chloride (LiCl) and the immunosuppressant FK506,and so on,could protect neurons from injury and could effectively promote axon regeneration. Cells transplantation's principle is to use some seed cells secreting a variety of neurotrophic and adhesion agents to nutrite and protecte the neuron after injury,and then induced axon regeneration and re-myelinization to repair SCI. Previous studies have provided a more mature seeds cells for SCI,such as:embryonic stem cells (ESCs) ,schwann cells (SCs) ,olfactory ensheathing cells (OECs) ,neural stem cells (NSCs),bone marrow mesenchymal stem cells (BMSCs) ,the umbilical cord blood stem cells (UCBCs) .Many studies have confirmed that cell transplantation facilitates the restoration of spinal cord function. With the rapid development of tissue engineering, a more advanced concept appeared gradually that repair of SCI must include the organization levels reconstruction. Integrating effective treatments of SCI repair strategy,we could reduce glial scar formation at the same time to induce the directional axon regeneration. At present,there were two engineering scaffolds applicating in spinal cord tissue,one was biodegradable polymer synthetic biological material,such as: polylactic acid,and polyglycolic acid copolymer,another was autologous or allogeneic tissue transplantation such as allogeneic embryonic spinal cord tissue,autologous muscle fiber frame,free of peripheral nerve (FPN) ,or the free vascularized peripheral nerve (VPN) tissue transplantation to repair spinal cord injury,the therapeutic effect indicates these ways is very effective. The obstacle for axon regenerate after SCI is the regeneration micro-environment has been damaged and myelin-derived protein (myelin,MAG,Nogo-A,Omgp) which inhibited the axon regeneration were secreted. Another factor is lacking of various neurotrophic factor (BDNF and GDNF,NGF,NT3 ) and the glial scar formation hindered axon regeneration.Recently,a new therapy strategy combining with different mechanism was used,which may have a better synergy to promote nerve repairation after SCI. For example,cells transplantition combinated with drug application ; compounding seed cells to synthetic polymer materials or peripheral nerve tissue material stent transplanted into injuried spinal cord; Seed cells tansfected varies neurotrophic factor genes by gene engineering to repair SCI,which have received satisfactory results.Neurons of central nervous system is terminal differentiate and can not regenerate,but in recent studys,there are neural stem cells in the areas of hippocampus,subependymal region and spinal cord could proliferate and multi-directional differentiate,it is very significate for the function recovery after central nervous system injury. The neural stem cells could manifestate the proliferate capability and differentiate into neuron and neuroglia cells,but the endogenous NSCs is not enough,it's better combine with the exogenous NSCs. With the study,NSCs indicated the well prospect in repairing SCI.In recent studys,lithium chloride appearance the neuron protection power as a traditional anti-vesania drug. In 2004,Jin et al found LiCl could promote differentiate of the NSCs in the hippocampus of rat and limit the proliferation of glial cells; Yick et al found that combined use of LiCl together with chondroitinase ABC (ChABC) could be a novel treatment for spinal cord injury. LiCl could promotes the axonal regeneration of rubrospinal tract (RST) neurons following injury to the spinal cord. This study provide a new platform for treat the spinal cord injury.This study use the improved Allen method to establish the rat spinal cord injury models,culture the NSCs in vitro and observe the effect of LiCl on proliferation and differentiation of NSCs,we combined NSCs with LiCl to repair the acute spinal cord injury. From this study,the nerve regeneration and function recovery of SCI animal will be investigated,at the same time,the possible mechanism of recovery in SCI will be discussed.Objective:1. Use the improved Allen method to establish rat acute spinal cord injury model,behavior function test and histological examination were used to evaluate this model and provide the basic theory data for the study of SCI.2. Culture NSCs in vitro and identify it by Nestin,observe the effect of LiCl on proliferation and differentiation of NSCs and provide the experimental foundation of the combined treatment.3. Observe the repairing effect of NSCs transplantion combinated with LiCl to SCI and discuss the inner mechanism,provide the theory consideration for clinical study.Methods:1. Use the MASCIS Impactor to establish the T10 section spinal cord injury model,observe the regularity and feature of hind limb function spontaneous recover,observe the tissle damage by histological examination.2. Clture the neural stem cells in vitro by mechanical separate the hippocampus of the newborn Wistar rats and undertake the Nestin identification.3. Use the MTT method and BrdU immunofluorescence to test the effect of LiCl on proliferation and differentation of NSCs.4. Forty Wistar rats divided into 4 groups randomly: control group,simple lithium chloride treatment group (LiCl group),simple neural stem cells transplantation group (NSCs group) and neural stem cells associate with lithium chloride group(NSCs+LiCl group).The Basso-Beattie-Bresnahan (BBB) score were used to evaluate hindlimb motor function recovery; observe the damaged tissle and the persistence neurons by HE staining and Nissle staining; immunohistochemistry staining were used to indicate the distribution of nerve fiber and the remaining neurons and then evaluate the repair effect of this combination process generally.Results:1. The BBB score of spinal cord injury model established by improved Allen method have no significant difference in same groups,the histological examination indicate that the empty formed and the nerve fibers are raritas and disrupted,the numbers of neuron is decrease significantly.25mm×10g is the recommend style that could guarantee the injury degree and profit the post-operation attendance.2. Culture NSCs in vitro by mechanical separate the hippocampus of the newborn Wistar rats' brain could obtain the postive Nestin immunohistochemistry staining NSCs.3. 20mmol/L LiCl group have the high neuron differentiate rate,it shows that LiCl could promote the proliferation and differentation of NSCs.4. The BBB score showed that NSCs group and NSCs+LiCl group were more imprvoed than LiCl group and control group after 2 weeks following the SCI and have a Statistical significance; BBB score of NSCs+LiCl group after 4 weeks was significantly higher than NSCs group. Comparing to the LiCl group and the control group,the histological examination of NSCs group and NSCs+LiCl group was much better,NSCs+LiCl group was better than other 3 groups in promote the neuron regeneration.Conclusion:1. The improved Allen method has the advantages of that simple manipulation injury degree unification and actual clinical situation simulation 25mm×10g is the recommend style .2. Use the method of mechanical separate the hippocampus of the newborn Wistar rats'brain could obtain the high purity NSCs.3. LiCl could promote the proliferation and differentation of NSCs.4. Transplanting NSCs combinated with LiCl could protect the survival neuron,promote the regeneration of nerve axon of corticospinal tract and guide them though the injury area,this repair method could improve the hindlimb function partly.
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