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Background and ObjectiveWith the structural and functional damage, Spinal cord injury resulted from a variety of harmful factors can lead to dyskinesia, disfunction of sphincter, loss of feelings, and disorder of autonomic nerves away from the damage plane. This disease has high morbidity and many complications without effective methods to cure. The bad microenvironment of the central nervous system is not benefit for the self-regeneration of damaged axons. Therefore, the key to cure spinal cord injury is to change the bad microenvironment and improve nerve regeneration in spinal cord. Many researches have demonstrated that low frequency electromagnetic fields (LFEMF) could promote cell proliferation, development and differentiation and also play a role in anti-inflammating, easing swelling, alleviating pain, and increasing tissue regeneration and decreasing apoptosis. Recent studies have found that LFEMF can obviously improve the motor function of cats or rats with spinal cord injury, because it can speed up the damaged nerve regeneration, and influence the activity and expression level of growth factor, and thus LFEMF is conducive to axonal transportation and neurite regeneration. Along with the development of stem cells research, they are thought to play an important role in the process of tissue repair. Among them, Bone Mesenchymal Stem Cells (BMSC), a kind of pluripotential stem cells, can differentiate into bone cells, cartilage cells, fat cells and so on. Studies have shown that bone marrow cells can transform into neural cell line in vivo and in vitro. Moreover, BMSC used in spinal cord injury suggested that it is helpful to improve the recovery of limb motor function. Our research proved that LFEMF could not only promote proliferation of BMSC but also enhance differentiation of BMSC into functional neurons in vitro. But the study on whether LFEMF can promote recovery of motor function in rat spinal cord injury model through influencing micro-environment and BMSC transplantation or not has not been reported. So our experiment established incomplete spinal cord injury model to observe the effects of LFEMF on locomotor functions of the spinal cord injured rats after BMSC transplantation.MethodsBMSC were isolated and purified with the fast adherence method. We observed the proliferation and morpholgy of primary or passage cells. We used flow cytometry to analyze cell phenotype and cell cycle. The third to fifth generation BMSC were chosen as seed cells, and labelled with BrdU before transplantation. We constructed T10incomplete spinal cord injury model by the spinal cord compression method.64SD rats were randomly divided into control group, BMSC group, EMF group and EMF+BMSC group. There were16rats in each group. According to the different time points, every group were redivided into3,7,14and21d subgroups,4rats for each subgroup. BMSC labelled with BrdU were injected with micro syringe into the cranial and caudal part of injured spinal cord in BMSC group and EMF+BMSC group within30mins after spinal cord injury. Rats in EMF group and EMF+BMSC group were explosed to uniform electromagnetic field (50Hz,5mT) on24hours after spinal cord injury,60mins per day for21days. Rats in control group and BMSC group with the same condition without electromagnetic fields were observed. Locomotor function was assessed by the Basso, Beattie and Brenahan (BBB) score. The test score was performed by two trained persons under blind conditions and the mean value was recorded for statistical analysis at preoperation and postoperation3,7,14and21d in an open field (90cm x120cm). At3,7,14, and21d,4rats in each group were sacrificed respectively to get spinal cord samples. The pathology of spinal cord tissue was tested with HE staining. We observed the BrdU-positive BMSC in spinal cord injured area by immune histochemistry staining. GFAP, MMP-2and BrdU positive expression of BMSC in injured spinal cord area were detected by immunofluorescence.Results1. The model was successful, because tail spastically swinged and lower limbs twitched immediately after operation, and BBB score was0-2after awaking up for one hour after anesthesia sober; and the extension of wave peak latency (Lat) in P1, N and P2, amplitude was almost2times more than normal showed by the motor evoked potentials.2. BMSC isolated from bone marrow were cultured, formed cell clusters appeared more processes and gave fusiform appearance. The results of flow cytometry demonstrated that BMSC had high expression of CD90, CD29, CD44and CD73with low expression of CD45. In the3rd generation, G0/G1phase of cell was82.0%, G2was1.46%and S was16.5%. Majority were in the quiescent phase, and minority in the active proliferative phase, which was in accordance with stem cell characteristics.3. Prior to the experiment, the BBB score of spinal cord injury model had=no statistical difference. With repeated measures ANOVA analysis, the results showed that BBB score of four groups (F=36.755, P=0.000) was different and this difference was statistically significant. At different time points the difference was significant (F=374.025, P=0.000), the BBB scores was relevant with the time. Interaction existed between treatment and time (F=7.471, P=0.000), suggesting that the BBB scores of different treatments was not consistent with the increasing time, and the EMF+BMSC group changed obviously. Further analysis of the separate effect, the results showed that the BBB score of each group was not statistically significant (F=1.941, P=0.177) in3d, but groups showed a significant difference (P=0.000) at other any time. At7d, the four groups BBB score were improved, and the difference was statistically significant (F=13.918, P=0.000). But there was not statistically significant difference between control group and EMF group (P=0.240), also BMSC group and EMF+BMSC group (P=0.472). At14d, difference was statistically significant (P=0.000) between control group and BMSC group, control group with EMF group (P=0.001), control group with BMSC+EMF group (P=0.000), EMF group with BMSC group (P=0.035) and EMF group with BMSC+EMF group (P=0.004), except BMSC group and EMF+BMSC group (P=0.258). At21d, difference was statistically significant (P=0.000) between control group and BMSC group, control group with EMF group (P=0.000), control group with BMSC+EMF group (P=0.000), EMF group and BMSC group (P=0.009), EMF group with BMSC+EMF group (P=0.000) and BMSC group and EMF+BMSC group (P=0.005). It was suggested that both BMSC transplantation and EMF treatment can improve the motor function of spinal cord injured rats, effect in BMSC transplantation group is more obvious. But when both LFEMF and BMSC transplantation in spinal cord injury model, compared with other experimental group, spinal cord injured rats hind limbs motor function in the EMF+BMSC transplantation group got recovered faster and better.4. At3d, the structure was disorder in rat spinal cord damaged tissue with hemorrhage, edema and necrosis cells. At7d, in control group and EMF group, tissue structure is badly destroyed, including bigger cystic cavity after necrosis and liquid absorption and significantly decreased, degenerated and swelled nerve cells, surrounded by inflammatory cells and swelling tissue and led to blood flow further reduce. But in BMSC groups and EMF+BMSC, cystic cavity area became smaller, swelling slightly, glial cells and macrophages hypertrophy and the organizational structure was still vague. As time changes, spinal cord tissue structure of each group was gradually improving. At14d, in addition to the control group, cystic empty in the other three groups almost closed and replaced by other organizations. Tissue edema disappeared with a small amount of inflammatory cells infiltration in BMSC group. And in EMF+BMSC group, structure of spinal cord injury were mostly repaired, including arranged orderly cells, disappeared basically inflammatory cells and numerous nerve cells. After21d, the damaged structure were basicly repaired except of control group.5. At14d and21d, a large amount of GFAP protein was seen in spinal cord injury area in control group; in EMF group and BMSC group, much GFAP protein distributed around the damaged area; but GFAP protein became the least in EMF+BMSC group.7d after BMSC transplantation in the BMSC and EMF+BMSC group, the expression of MMP-2in local tissue began to rise, quantity was the most obvious at14d. From3d to21d after surgery, BrdU positive cells can be observed in the spinal cord injured tissue in BMSC and EMF+BMSC group, and grow to mix together with the organization. Its growth condition was better in EMF+BMSC group, and there were no BrdU positive cells in control group and EMF group.Conclusions 1. Spinal cord injured model is established successfully by compression method which has a good repeatability, and practise simply.2. Cells isolated from rat bone marrow are pure rat BMSC. They can survive after transplantation in spinal cord injury model.3. The low frequency electromagnetic fields can not only improve the locomotor function of rats with spinal cord injury but also promote the recovery of spinal cord injured rats after BMSC transplantation.
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