| Background IntroductionIt generally held that the failure of spinal cord axons to regrow and functional recovery are primarily due to the following factors: (1) the secondary phase of tissue loss in the vicinity of the injury following the first phase of mechanical destruction, which is principally caused by the release of excitatory amino acid , formation of free radicals, and activation of the immune system, and neuronal cell apoptosis; (2) gliotic scar tissue and fluid-filled cavity formation at the lesion site, which act as a physical barrier for regenerative regrowth of injured neuritis; (3) severe local disturbance of blood supply; (4) lack a permissive environment for the regeneration of injured axons, unsufficient neurotrophic factors and the release of large amount inhibitory proteins such as Nogo-A, myelin-associated glycoprotein(MAG), chondroitin sulfate proteoglycans(CSPGs) and so on.Cell transplantation is at present considered to be the most effective way to repair function of injured spinal cord. Various types of cellular implants have been developed, including embryonic stem cells(ESC), neural stem cells(NSC) or progenitor cells, olfactory ensheating glia(OEG), Schwann cells, fibroblast, and bone marrow stroma cells(BMSC), These transplanted cells can not only intended to provide a bridge for axonal connectivity between the proximal and distal lesion sties but also reconstruct the cytoarchitecture and anatomy of the damaged site to inhibit the formation of scar formation. And at some time these cells can differenciate into oliogodendrocye ,astrocyte cells and neurons to help the recovery of signal transduction. At same time, the transplanted cells can secrete extracellular matrix and various neurotrophic factors and cell adhesion molecules, which are essential for the regrowth and extending of neurite. Compared with other cells, BMSC has it's own advantage: BMSC are available in limitless numbers from adult self-donor from a simple bone marrow biopsy; BMSC can be readily cultivated and growth more rapidly in a minimum average doubling time of 10h; BMSC can undergo over 25 passages(over 50 cell doublings)under optimal culture conditions , producing billions cells in a relative short time; BMSC has no ethically controversial and immunal rejection problems; and, BMSC harbor pluripotent stem cells capable of neural differentiation. So, we can state that BMSC is an optimal cell for implantation in the treatment of SCI.FK506, a kind of antibiotics, has strong ability to reduce immune system- mediated swelling of injured spinal cord , and has been applied in several rat spinal cord imjury model and indeed suppress the secondary neuronal necrosis. We applied BMSC and combined with FK506 to treat spinal cord injury rat model, and to examine whether BMSC and FK506 would enhance functional recovery of injured spinal cord, and whether such strategy would reduce the formation of gliotic scar and volume of cavity, and inhibit the cascade of apoptosis of injured spinal cord cells. We also use the CSF collected from spinal cord treated by BMSC implantation(CSF-BMSC)to cultivate neural stem cells and cocultured Schwann cells-neural stem cells to observe the effect of CSF-BMSC on the defferentiation of neural stem cells.ExperimentsExperiment one Effect of BMSC and FK506 on functional recovery of spinal cordMethods: The models of acute spinal cord injury were made by clamping method. 30 Sprague-Dawley(SD) rats were randomly divided into three groups, each of which comprised ten rats: groupA , the control group without any treatment; groupB, the injured animas were treated with BMSC implantation; groupC, experimental rats were co-treated by BMSC transplantation and FK506 injection. Behavioral evaluation was performed using the open-field BBB scoring systeem and oblaque plane test. Scores were recorded at time point of 1w, 2w, 4w, 6w, 8w after operation respectively.Results: Transplanted BMSCsurvived within the injured tissue. Treated animals in groupB and C showed better performance of gait than control animals at each time point. They could extensively bend hindlimbs 2 weeks after operation. These recovery are remarkable at 4 weeks and even showed weight-supproting. At 8 weeks after operatin ,forelimbs and hindlimbs movements are consistent coordination(BBB score 14).In contrast, in the control group, the animal could move the hindlimb joints from 2 weeks postoperation. No weight support or coordination movement was observed. The outcome of C group surpass the B group's, the highest BBB scores were 14. There was significant difference between B and C group(P <0.05).Experimen two Effect of BMSC and FK506 on apoptosis of spinal cord cellsMethods: The SCI models were induced by clamping method. 75 SD rats were randomly divided into 3 groups: each of which comprised 25 rats: groupA , the control group without any treatment; groupB, the injured animas were treated with BMSC implantation; groupC, experimental rats were co-treated by BMSC transplantation and FK506 injection. Animals were sacrificed and the spinal cords in the lesion were cut at 1, 2, 3, 7, 14 day after injury. The level of neuronal cell apoptosis was detected by the terminal deoxynucleotidyl transferase mediated DUTP nick end labeling (TUNEL) methods.Results: Compared with groupB and C, much more TUNEL positive cells were observed respectively at each time point in groupA. TUNEL positive cells appeared both in gray matter and white matter 1 day after operation. The positive cells peaked at 3d, and began to decrease 7 days postoperation. Compared with group B, TUNEL positive cells in group C decreased statistically significantly. Experimen three Effect of BMSC and FKS06 on reactive gliosis in rats following spinal cord injuryMethods: The SCI models were induced by clamping method. 30 SD rats were randomly divided into 3 groups, each of which comprised 10 rats: groupA , the control group without any treatment; groupB, the injured animals were treated with BMSC implantation; groupC, experimental rats were co-treated by BMSC transplantation and FK506 injection. After 8 weeks of treatment, rats were sacrificed and the spinal cords at the injury site were collected. Immunohistochemical study was used to analysis the immunoreactivity to glial fibrillary acidic protein (GFAP).the number and the average area of reactive astrocytes was quantified using BioQuant image analysis systemwere detected by immunohistochemical technique.Results: In groupA, a significant increase in the number of reactive astrocytes in both gray and white matters was observed. The average size of the reactive astrocytes in groupA is much greater. Treatment with BMSC and FK506 significantly reduced both the number and the size of reactive astrocytes in injured spinal cord. As for Nogo-A positive cells, groupB and C decrease much more significantly compared with groupA in number.Experimen four Effect of BMSC and FK506 on volume of cavity in injured spinal cordMethods: We made the SCI models in 30 SD rats. The animals were randomly assigned into 3 experimental groups. In groupA, animals conducted no treatment,; in groupB, the injured animals were treated with BMSC; in groupC, experimental rats were co-treated by BMSC transplantation and FK506 injection. 8 weeks postoperatin,rats were sacrificed and the spinal cords at the injury site were collected. Cryostat horizontal sections were stained with HE, and the area of the cavity in the spinal cord was measured with an image-processing and -analysis program (NIH Image 1.61). The areas of cavity were measured from consecutive sectin at an interval of 50μm. The cavity volume was then calculated by multiplying the average area by the depth of the spinal cord.Results:The average volume of cavity of groupA,B,C are 23.3Am~3,14.5Am~3 and 39.8 Am~3 respectively. Compared with the case in control, the cavity at the lesion of treatment groups become significantly smaller.Experimen five Effects of CSF-BMSC on the differentiation of neural stem cellsMethods: 60 SD rats were randomly assigned into 6 groups, each of which comprised 10 rats. In groupA1, the spinal cord were exposed and keep the cord intact, and CSF was collected from the cisterna magna 3 days after operation. In groupA2, spinal cord keep intact as group1, but the CSF was collected 5 weeks post operation. In groupB1 and B2, SCI models were made by clamping method, and CSF was collected 3days and 5 weeks postoperation respectively. In groupC1 and C2, SCI models were treated with BMSC implant, and CSF was collected 3days and 5 weeks postoperation respectively. Neurosphere cells were plated in serum-free medium in noncoated culture dishes. 3-5 days after plating, 250 microliters of CSF was added to the culture medium. The change of neurospheres including adhesion to the culture dish and differentiation including cellular process extension were observed using a phase contrasr microscope 3 days after CSF added. Immunohistochemical study was used to analysis the immunoreactivity to NF.Results: In groupA1,A2,B1,B2, neurosphere cells were still in floating condition, and no protruding cellular process were observed. NF staining was negative. In groupC1 ,C2, the neurosphere cells adhered to the dish bottom and extended cellular processes toward the periphery, and NF staining was positive. But in groupC2, the cellular processes were rather shorter and NF positive cells were less compared with group C1.Experimen six Effects of CSF-BMSC on the differentiation of neural stem cells co-cultured with Schwann cellsMethods: 30 SD rats were randomly assigned into 3 groups, each of which comprised 10 rats. In groupA, the spinal cord were exposed and keep the cord intact, and CSF was collected from the cisterna magna 3 days after operation. In groupB, SCI models were made by clamping method, and CSF was collected 3days postoperation. In groupC, SCI models were treated with BMSC implant, and CSF was collected 3days postoperation. Neurosphere cells were plated in serum-free medium in noncoated culture dishes. 3-5 days after plating, 250 microliters of CSF and 0.25ml Schwann cells were added to the culture medium. The change of neurospheres including adhesion to the culture dish and differentiation including cellular process extension were observed using a phase contrasr microscope 3 days after CSF added. Immunohistochemical study was used to analysis the immunoreactivity to NF.Results: In groupB,C, the neurosphere cells adhered to the dish bottom and long extended cellular processes toward the periphery. Differentiation of Neural stem cells is obvious. NF staining was positive in both group. But in groupC, the cellular processes were rather longer than group B.DiscussionIt generally believed that the failure of spinal cord axons to regrow and functional recovery are primarily due to the secondary damage caused by apoptosis, gliotic scar and cavity formation , severe local disturbance of blood supply, lack sufficient neurotrophic factors and large amount of inhibitory proteins production and so on.It is reported that BMSC can promote functional recovery of injured spinal cord or injured brain. However , the mechanism of these action are unclear. In the present study, BMSC-grafted animals show much smaller cavity than nontreated controls. Transplanted BMSC can live in the center of the injured site for a long time . These findings are different form those observed after transplantation of neural stem cells. Grafted neural stem cells migrated into tissue around the lesion site. So, it can be predicted that BMSC at the center of the injury pull the surrounding host tissue toward the center , preventing the formation of cavity. And, it is held that BMSC can secret various factors that reduce the degree of immune response in injured site. It maybe another reason that BMSC can decrease the volume of cavity.The Astrocytes undergo a process known as reactive gliosis in response to a CNS injury. Among the hallmarks of this process are an increase in immunoreactivity(IR) to glial fibrillary acidic protein(GFAP), hypertrophy of astrocytic cell bodies, and proliferation of astrocytes. Reactive astrocytes comprise the most abundant component of the "glial scar," which eventually forms within the CNS following injury. It is believed that this glial scar tissue inhibits regeneration in the CNS by forming a physical barrier impeding axon ingrowth. By treating rats with BMSC implantation, we significantly reduced reactive gliosis after spinal cord injury which stall the extending of neurite. and in the grafted animals. In the injured site, astrocytes and myelin sheaths of nervesnmay produce large amount of specific inhibitory proteins such as Nogo-A, MAG and CSPGs, that block neurite outgrowth. In our experiments we found that BMSC can reduce the expression of Nogo-A. It means that by decreasing the scar formation and production of inhibitory proteins, BMSC can help to improve the functional recovery of injured spinal cord.Apoptosis is believed to be a major mechanism of secondary injury in the model of spinal cord injury. We demonstrated that BMSC can reduce the level of cell apoptosisi near injured site. Nicola B. reported that BMSC mediate protection through stimulatin of P13-K/Akt and MAPK signaling in neurons by releasing neurotrophic fctors. Our study confirmed the fact that BMSC is great helpful in neuron protection in secondary phase of damage.Shortly after injury, the immune system is activated. This causes swelling of injured tissue within the vertebra resulting in secondary tissue loss. Macrophages initially predominated in the injured area and were gradually replaced by other cells,, including glia cells. Macrophage in the lesion are considered to play an important role in cavity formation. However, to the contrary, adequate macrophage recruitment and activation are essential for rapid clearance of myelin debris. Due to the immune-privilege status of the mammalian spinal cord , the beneficial effect of macrophages in the CNS recovery progress may be limited in lesions of untreated rats, thereby frustrated regeneration in spinal cord. Our research informed that FK506, an antibiotics that has strong ability to reduce immune system response, can not only relieve the immune system-mediated swelling of injured spinal cord ,but also have the ability of cooprating with BMSC in promoting the injured spinal cord repair through different mechanisms.It has been reported that BMSC and Schwann cells can enhance the differentiation of neurosphere cells in vitro by coculturing with neurosphere cells respectively. Our study informed that CSF collected from BMSC treated spinal cord can not only help enhance the differentiation of neurosphere cells, but also can enforce the effect of Schwann cells on inducing neurosphere cells differentiation. It means that BMSC enhances differentiation of neurosphere cells by secreting certain factors into CSF.Our experiments demonstrated that BMSC can overcome the problems above mentioned that interfere the functional recovery and neurites outgrowth by releasing neurotrophic factors to enhance trophic support of the injured neurons and improve neuronal plasticity, and maybe to induce spinal cord-derived neural stem cells differentiating into neurons or glia cells.CONCLUSION1. BMSC can survived well in the area of injury cord.2. BMSC possess promotion effects on injured spinal cord repair and functional recovery through various mechanisms.3. FK506 has synergism effects with BMSC on the repair of injured spinal cord.4. BMSC can not only enhance differentiation of neurosphere cells but also enforce the effect of Schwann cells on inducing neurosphere cells differentiation by releasing neurotrophic factors.
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