| Background and AimsSpinal cord injury (SCI) is a trauma with high incidence in the central nervous system (CNS), which usually results in severe impairment of sensary, motor and autonomic function below the level of injury. The misfortune brings a lot of trouble and huge burden to the patients themself, their family and the whole society. Although there have been many inspiriting findings from laboratory experiments of animals, none of current treatments or therapies is able to effectively cure SCI in the clinical setting. Thus, the researches to treat SCI are of great scientific and humanity significance.With the better knowledge of the pathological processes following SCI, the axonal demyelination is recently realized as a common pathological change in the lesioned spinal cord experimentally and clinically. There are usually some mild-impaired white matter area spared across the lesion site, however several secondary injury mechanisms further cause the axonal demyelination of these preserved nerve fibers. The related researches indicate that the demyelination of axons after SCI has a close relationship with the death of oligodendrocytes in the lesion area. Not only die a lot of neurons after SCI, but a number of oligodendrocytes are also impaired and the demyelination of axons are brought about consequently. The normal function of myelin sheaths and myelinating cells is vital to maintain function of axons, thus, the death of oligodendrocytes and axonal demyelination after SCI further hinder the neural function of the spared nerve fibers. From above mentioned findings, it is demonstrated that the loss of oligodendrocytes and the secondary injuries cause the demyelination of spared axon in the lesioned spinal cord, which further hampers the neural function of the preserved nerve fibers. So, we speculate that amelioration of axonal myelination after SCI will be favorable for the functional recovery of the injured spinal cord.Since the axonal demyelination in the lesion area after SCI is primarily attributed to the death of oligodendrocytes, the supplement of oligodendrocytes and amelioration of the spared axons will maximize the function of the preserved and demyelinated nerve fibers and consequently enhance the functional recovery of injured spinal cord. Oligodendrocyte precursor cells (OPCs) are immature oligodendrocytes which are able to proliferate, migrate, and finally differentiate into mature oligodendrocytes and produce myelin. As immature cells, OPCs have more advantages over mature oligodendrocytes to be a transplant, and OPCs are also less influenced by the hostile microenviroment of the lesion areas as compared with stem cells. In this experiment, OPCs are intendedly transplanted to improve axonal myelination of lesioned cord and maximize the function of spared nerve fibers after SCI, and hence to enhance the functional recovery of injured spinal cord. From this investigation we will better realize the biological significance of axonal demyelination in SCI, and the study will provide a novel strategy for the treatment of SCI.Methods1. The cerebral cortices of 48-hour neonate Sprague-Dawle rats were harvested for the OPCs primary culture. The growth of OPCs in vitro was observed consecutively under the contrast phase microscope and scanning electron microscope.2. After the primary culture in vitro, the OPCs were further dissociated by the shaking process and differential adhesion. Then, the separated OPCs were also identified and analyzed by the immunocytochemical technique.3. While the OPCs further grew in the chemical defined condition medium, the differentiation and maturation of OPCs in vitro were intensively examined by contrast phase microscopy and immunocytochemistry. Then, the proliferative ability of OPCs in vitro was also investigated with the MTT assay.4. The contusive injury of spinal cord in a rat model was produced by the means of the weight-drop impact of Allen's method. The experimental animals were randomly divided into 4 groups, i.e. OPCs transplant group, transplant control group, plain injury group and sham operation group. The transplantation of the labelled OPCs was performed 7 days after injury to treat SCI of rats.5. The recovery of hindlimb locomotive function of SCI rats was consecutively examined by the BBB open-field locomotion scoring. Furthermore, the recovery of the conductive ability of the injured spinal cord was also assessed electrophysiologically.6. Eight weeks after the OPCs transplantation, the gross pathological changes of spinal cord tissue was observed and compared in all animals of 4 groups, and the spared white matter in the lesion area was further studied after OPCs transplantation. In this study, the survival, distribution, differentiation and maturation of the transplanted OPCs in vivo were investigated by immunohistochemistry and immunofluorescent technique. The effects of OPCs transplantation on the myelin at injury site, on the experssion of myelin-associated gene and on the ultrastructure of axonal myelination were further investigated by immunohistochemistry, special staining of myelin, RT-PCR and transmission electron microscopy. The influence of OPCs transplantation on the survival of neurons and the axons, and on the glial reaction in the lesion area after SCI were also studied intensively.Results1. The OPCs extensively grew and proliferated in the primary culture, showing the typical appearance of precursor cells. Under the observation of microscope, the OPCs were seen with round soma and bipolar processes of their bodies. The observation of scanning electron microscopy shew that OPCs grew close adhesive onto the surface of astrocytes, which had a small and round soma, 6-10μm in diameter, with two or three fine processes around the bodies.2. Around 9-10 days in vitro, the distinct stratification of glial cells formed in the primary culture. The upper layer consisted of clustered-growing OPCs and the bottom layer was mostly constituted of confluent astrocytes. A large number of OPCs was able to be harvested through the procedures of shaking separation and differential adhesion. The PDGFR-αpositive OPCs accounted for up to 95 % of the isolated cells after the separation process.3. The OPCs were able to grow and continue to differentiate in the chemical defined condition medium. During the differentiation, the simple morphology of OPCs progressively evolved to more complex forms with profuse outgrowth of elongated processes and extensive secondary branching. After 5-7 days in the condition medium, the OPCs gradually differentiated into mature oligodendrocytes, which were characterized by the complex profile of"ramificated"or"cobweb-like"processes reticulating in their periphery. The differentiation of OPCs was further confirmed by immunocytochemistry with the specific expression of MBP, a marker of mature oligodendrocyte.4. This study demonstrated that the OPCs were able to still retain the proliferative ability in the culture. The number of OPCs in culture was detected by the MTT assay to constantly increase during the experiment (p < 0.05). At the beginning of the assay, the number of OPCs in culture was least, and later on the number of OPCs progressively increased and reached the peak on the 6th day in vitro.5. The recovery of hindlimb locomotor activity of the injured rats in the OPCs transplant group was much better than that of the transplant control group and plain injury group. On the 35th, 42nd, and 49th day after SCI, the results of BBB score of the hindlimb locomotion of the OPCs transplant group were better than that of the transplant control group (p < 0.05). On the 56th and 63rd day after SCI, the locomotor function of rat hindlimb in the OPCs transplant group was significantly better than that of the transplant control group (p < 0.01).6. The outcome of electrophysiological experiments indicated that the recovery of the conductive ability of injured spinal cord in the OPCs transplant group was also better than that of the transplant control group and plain injury group. At the 4th week after the transplantation, the results of the onset latency and amplitude of MEPs N1 wave in the OPCs transplant group were markedly better than that of the transplant control group (p < 0.05). At the 8th week after the transplantation, the recovery of both onset latency and amplitude of MEPs N1 wave in the OPCs transplant group was significantly better than that of the transplant control group (p < 0.01). At the 2nd and 4th weeks after the transplantation, the recovery of the amplitude of SSEPs N1 wave in the OPCs transplant group was better than that of the transplant control group (p < 0.05). At the 8th week after the transplantation, the outcomes of the onset latency and amplitude of SSEPs N1 wave in the OPCs transplant group were markedly better than that of the transplant control group (p < 0.01).7. At the 8th week after the transplantation, there were more white matter spared at the injury site of the OPCs transplant group as compared with that of the transplant control group and plain injury group (p < 0.05). The BrdU positive implant cells were able to be detected in the tissue of lesioned spinal cord, and the OPCs extensively distributed within the spinal cord and well integrated into the architecture of host tissue. The implanted OPCs continued to differentiate and maturate in vivo and express MBP as well. The number of Oligo-2 positive oligodendrocytes in the lesion area of the OPCs transplant group was much more than that of the transplant control group (p < 0.01). 8. The expression of MBP at the lesioned spinal cord was enhanced in the OPCs transplant group as compared to that of the transplant control group (p < 0.01). The specific staining of myelin by LFB also shew that the amount of myelin in the injured tissue of the OPCs transplant group was much more than that of the transplant control group (p < 0.01). The expression of the PLP gene in the spinal cord of the OPCs transplant group was significantly higher than that of the transplant control group (p < 0.01).9. The findings of transmission electron microscopy further demonstrated the improved axonal myelination in the lesioned spinal cord of the OPCs transplant group. In the transplant control group, a number of large swollen axons with broken myelin sheath were found across degeneration area. The thick and compact myelin sheaths had split and broke down, and many demyelinated axons were also seen present in the lesion area. Otherwise, the better profile of axonal myelination was seen across white matter area in the OPCs transplant group. The axonal myelination of the white matter was improved after OPCs transplantation. The broken myelin sheath and demyelinated axons were also found less across degenerating white matter area. 10. The moreβ-TubulinШpositive neurons and NF200 positive axons were preserved in the lesioned spinal cord in the OPCs transplant group than that of the transplant control group (p < 0.01). The expression of GFAP and the number of active astrocytes within the injury site of the OPCs transplant group were lower than that of the transplant control group (p < 0.05).Conclusion1. The OPCs culture is able to be well established in vitro. Timely primary culture, suitable shaking process and differential adhesion together do efficiently separate the OPCs in the primary culture.2. The isolated OPCs are found to be able to survive and continue to grow in vitro. These OPCs still retain the proliferative ability and further differentiate and maturate to oligodendrocytes.3. The transplantation of OPCs enhances the recovery of hindlimb locomotor activity and conductive ability of the lesioned spinal cord after SCI.4. The implanted OPCs can survive for a long term and differentiate and maturate in vivo. The transplanted cells are well integrated within the host tissue and supplement the oligodendrocytes in the lesioned spinal cord.5. The transplantation of OPCs is able to benefit the preservation of white matter, to ameliorate the axonal myelination of injured spinal cord, to improve the survival of neurons and axons and reduce the glial reaction as well.
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