An Experimental Study On The Protective Effect Of Bone Marrow Mesenchymal Stem Cells On Motorneurons After Brachial Plexus Avulsion Injuries

Roots avulsion injury is the most severe injury type of the brachial plexus lesions characterized by the interruption of rootlets from cervical cord. About 65% of the adult and 75% of the obstetrical brachial plexus lesions are avulsion injuries. the cases of total roots avulsion account for one third of clinical operated cases . This type of avulsion is considered to be a problem of the central nervous system (CNS). To regain the function of limbs, current surgical modalities are based on nerve transposition and functional reestablishment.but the prognosis is not ideal. Therefore, the way of direct reimplantationof the avulsed rootlets into the cord was explored to gain functional restoration. because the injury is interruption of the axon near to neurons,not only to result in the interruption of nerve continuity, but also the damage to motoneurons. the motoneurons of spinal cord will loss the ability of regeneration after injury,so the prognosis is poor.Bone marrow mesenchymal stem cells (BMSCs) is a kind of multipotent stem cell of adult. thay are the cell mass that possess the ability of self-renewal and multi-directional differentiation. MSCs have been reported to be present in bone marrow ,peripheral blood ,bone, cartilage ,somatic muscles, subcutaneous fat, cord blood and placenta. MSCs can be readily isolated and easily expanded. when cultured under defined in vitro conditions, MSCs can be induced to differentiate into osteoblast, chondrocyte, adipocyte, islet-like cells. in vivo MSCs can also be induced to differentiate into neuronlike cells. MSCs make up ethical and lawful limitation of embryonic stem cell and neural stem cells. because of being easily obtained, autografting ,poor immunogenicity and being easily transfected by exogenous gene, moreover MSCs can secrete many kinds of neurotrophy factors. MSCs is an ideal seeding cells used to treat trauma and diseases of nerve system by cell and gene therapy.In this study, the model of nerve roots avulsion was made by posterior approach of cervicothoracic vertebra.in the part of study in vitro, we use supernatant fluid that was made from the model rat's myelohomogenate for different time after injury to simulat microenvironment in vivo.our aim is to probe the role of Induction and differentiation for MSCs. in the part of study in vivo, we delivered MSCs into the injured spinal cord at the same time of reimplanting nerve roots to study the fate of transplanted MSCs and possible effects on motoneuron in the anterior horn of the spinal cord.which will provide theoretical foundation for further study.1. The establishment and evaluation of the brachial plexus avulsion model in the rat.The rat model of C5~C7 nerve roots avulsion was setted up by posterior approach of cervicothoracic vertebra. The postoperative rat`s functional outcomes were measured by the Terzis grooming test. The achievement ratio of rat model is 90.3%.Conclusions: 1. The rat avulsion model used in this experiment is particularly worthwhile because of its common features with the corresponding injury in man. This established the groundwork for scientific study. 2. In this way we can set up experimental model conveniently without electrophysiology and get a High achievement ratio. 3. This model is suitable for the research of intraspinal implantation of avulsed nerve roots and stem cell transplantation.2. Isolating, culturing, expansion,identification and labeling of rat bone marrow MSCs.MSCs were isolated by wall sticking method, then cultured and amplified in condition of L-DMEM with 20% fetal calf serum , 37℃,5%CO2, saturated humidity, passaged with 0.25% trypsinization in vitro. MSCs were identified by being induced to differentiate into osteoblast, adipocyte, neuron-like cells and by FACS. MSCs were labeled at 48 hours before cellular transplantation.Conclusions: In this way,we can gain pure and uniform cells.the labeling ratio reached 58.2±6.8%.the motility rate is proved to be above 99% with trypan blue stain.3. The role of myelohomogenate supernatant fluid in induction and differentiation of MSCs.The induced cells were identified with NSE and GFAP Immuno- cytochemical stain.In this part MSCs were divided into 5 groups for induction and differentiation study: group control,group normal myelohomogenate, group 0 day postinjury myelohomogenate, group 7 days postinjury myelohomogenate, group 30 days postinjury myelohomogenate.There was not positive cells in control group. The group 7 days postinjury myelohomogenate has a higher expression rates of NSE and GFAP than other groups.RT-PCR technique was used to detect the NGFmRNA expression in the induced cells. MSCs were divided into 4 groups: group control,group normal myelohomogenate, group 0 day postinjury myelohomogenate, group 7 days postinjury myelohomogenate. all groups expressed NGFmRNA, but the group 7 days postinjury myelohomogenate has a higher expression level of NGFmRNA than others. but there was not significant difference between other groups. Conclusions:1. The myelohomogenate supernatant fluid of the brachial plexus avulsion model rat can induced MSCs differentiated into neuron-like cells in vitro.The morphous of induced cells looks like neuron and gliocyte. Moreover,the induced cells expressed cell surface marker-NSE or GFAP.the positive cell ratio of group-7 days postinjury myelohomogenate is the highest.2. The myelohomogenate supernatant fluid of the brachial plexus avulsion model rat 7 days postinjury can up-regulated the level of NGFmRNA expression in the induced cells.4. An experimental study on the protective effect of bone marrow mesenchymal stem cells on motorneurons after brachial plexus avulsion injuries.The model rats were randomly divided into 3 groups: group model, group experiment(the avulsed nerve roots were reimplanted one week after injury),group experimental control(the avulsed roots were reimplanted one week after injury, at the same time PBS was transplanted into spinal cord as control). The rats were perfused on 3 days, 1 week, 2 weeks, 4 weeks,6 weeks and 8 weeks postoperation.Histological view of spinal cord by HE stain : the pathological change of group experiment is better than other two groups. except the 3 days postoperation, at other time the survival ratio of motor neuron in anterior horn of group experiment and group experimental control is higher than group model. and the ratio of group experiment is higher than group experimental control.By TUNEL stain the death modality of motor neuron in spinal cord lesion is proved to be apoptosis post injury.BrdU immunohistochemistry stain result display of group experiment: at the third day after grafting, BrdU positive cells localized the injection site adjoining to anterior horn of spinal cord, And double staining positive cells were observed. Later, the cells migrated into anterior horn gradually.NGF immunohistochemistry stain result display:The NGF expression level of motorneuron in uninjured side of spinal cord is low and does not change with time postoperation. The NGF expression level of group experiment is higher than other groups before the 2nd week postoperation, but at the 4th week postoperation difference was not found between them. This level of group experimental control and group model is higher than uninjured side on the 3rd day postoperation, but at other time there isn`t difference. Difference was also not found in the two groups at any time.Conclusions:1. The model rats are suitable for this study, the method of nerve roots reimplantation and cell transplantation will establish the foundation to heal brachial plexus roots avulsed injury. 2. the cells being transplanted into spinal cord can survive well and migrate in spinal cord, In a certain degree the cells differentiated into neuron-like cells and presented NSE marker.3. BMSCs transplantation can enhance the NGF expression level of motorneuron in anterior horn of spinal cord, this will play a part in supporting, nutrition and protection for motorneuron. 4. BMSCs transplantation can increase the survival ratio of motorneuron in anterior horn of spinal cord.