| Background:Spinal cord injury (SCI) is a major cause of disability, and at present, there is no universally accepted treatment in Adult mammalian.The functional decline following SCI is contributed to both direct mechanical injury and secondary pathophysiological mechanisms that are induced by the initial trauma.Marcia published a "new concept of spinal cord injury," in 1996 on the authoritative magazine-"Science", he reported the " The secondary cell death of spinal cord injury was not a direct result of injury but because of apoptosis." This new view about spinal cord injury has greatly made a long step towards success. Primary tissue damage of SCI is considered irreversible, whereas secondary damage has been considered to be reversible. Accordingly this view provides the possibility of researching the regeneration of spinal cord after SCI for us. Microglia are derived from myeloid cells in the periphery and comprise approximately 10-20 percent of glial cells in the central nervous system.In the event of the central nervous system injury, the activated MG shows the effect of " double-edged sword": the action of protecting cells and the action of damaging cells.Microglia likely plays a critical role in the secondary damage process of SCI. In view of the above, on the basis of animal experiments, we try to culture activated microglia, transplante activated MG to the SCI model, evaluate spinal cord function, and observe the changes of histomorphology of MG. We intend to test espression of TGF-β1 mRNA and protein of damaged spinal cord,explore the treatment mechanism of spinal cord injury after transplanting activated MG.Objective:1.Optimizing and accessing actived microglia from rats, purifying and identifying the cells, studying their biological characteristics and providing seed cells for transplantation in SCI model;2.Evaluating the spinal cord function after transplanting activated MG to SCI model; 3 . Observing morphology changes in the treatment of spinal cord injury by transplanting activated microglia;4.In the SCI model, testing TGF-β1 mRNA and protein expression after transplanting activated MG to SCI model and discussing mechanisms of activated MG restoring SCI.Method:1.We chosed new life-SPF Wistar rats-suckling mice, under anesthesia after surgical sterile conditions, cut its brain hemisphere, the brain and the corpus callosum, divided into four groups, Group A - conventional control group, Group B - mechanical oscillations group, Group C - trypsin digestion Group, Group D -lidocaine hydrochloride-induced group. By the McCarthy classical methods combined with the loss of nutrition, mechanical oscillations, and moderate digestion method,we purified microglia from mixed glial cells under the condition of lidocaine hydrochloride mixed-induced. We subcultured activated microglial, observed cell morphology and the expression of surface marker, thereby identified them. We have observed the proliferation of the second generation, 4th generation, 6th generation, 8th generation microglial, and drawn a growth curve of them.2.Adult female Wistar rats were randomly divided into two groups, each with 10, Group A: the experimental group, Group B: control group, established the animal model of moderate spinal cord injury by the method of modified Allen. We used the indirect immunofluorescence jointing FCM to test the purity of the second generation of purified glial cells. After the first seven days we used a microsyringe to transplant purified microglial into animals of group A model in spinal cord injury site, of group B 0.9% saline injection as a control. At the different points of time, we had adopted a BBB Rating System,Sloping Rats Experimental Observation and motor evoked potentials test (MEPT) to survey the function of rats lower limb of SCI for evaluating its motor function and judging the curative effects of actived MG transplantation.3.We chosed adult female Wistar rats 52, established animal models of moderate spinal cord injury.After the success of model, we randomly killed 2 rats of 22 at the various points of time, selected segments of spinal cord injury as tissue samples of frozen section, observed microglia's activity marked with OX42 by indirect immunofluorescence staining. Another 30 would be divided into two groups, Group A: transplantation group; B groups: control group; each of 15. After the successful model in the first seven days we used microsyringes to transplant purified microglia to the segment of spinal cord injury in Group A, while in group B, we injected 0.9% saline as a control. After transplantation at the various points of time, we selected segments of spinal cord injury specimens to observe the histological changes by HE staining, indirect immunofluorescence staining (purified microglia were marked by CD68 and OX42) and Naounenko-Feigin Baptist silver staining.4.By RT-PCR and Western blot technique, we detected the expression of TGF-β1 mRNA and protein of spinal cord tissue of SCI rats after MG transplanting at different time slots.Results:1.After using McCarthy classical cultivation methods combined with the loss of nutrition, mechanical oscillations, moderate digestion method, induced by lidocaine hydrochloride, the activated microglia were very pure. Expression of CD68 and OX42 of the isolated cells from the cultured were positive, purity to 94.92±7.05%, significantly higher than (conventional control group, 84.72±6.30%, mechanical oscillations group 88.14±6.11% trypsin digestion group 90.63±7.04%). 2th generation and 4th generation purified microglia have presented single cell morphology and characteristic adherent within 15-30 min. Its Rapid growth in the first period were about 3th-6th day, and eight hours had about 90% of the cells attached to the wall (but there was 80% adherent cells of 6th generation and 8th generation microglia ); The first generation, the second generation, 4th generation microglias have stronger proliferation ability.2.The results of indirect immunofluorescence jointing FCM showed that the second generation of cultured microglia had 97.8 percent of expressing CD68, and 89.4 percent expressed OX42, positive rate (96.2±2.8)%, which indicated that the purity of approximately (96.2±2.8)%.3.After activated MG were transplanted into SCI model, motor function score (BBB) of hindlimb motor function in group A(the experimental group) were significantly higher than that in group B(the control group) at 2 w,3w,4w,,6w,,8w ,the difference is statistically significant (p< 0.05). The results of sloping test show that angle of the group A were significantly higher than that in group B at 2 w,3w,4w,6w,8w, the difference is statistically significant (p <0.05). Motor evoked potential tests (MEPT) showed four weeks after the surgery we recorded waveform and found that low waveform blunt incubation period is extended, the values of peak -peak decreased, but the change was more obvious in group A than in group B, and the difference was statistically significant (p <0.05), eight weeks after the surgery I also recorded waveform obvious, but still extended incubation period, the values of peak-peak still reduced, but compared to waveforms in the first 4 week markedly improved, and group A resume obviously, the difference is statistically significant (p <0.05). The results were similar to those of motor function score.4.Purified microglia were in good condition after transplanting in rats of SCI within one week, recruitmented in the spinal cord injury area, and continued to survival for at least 14 days. In indirect fluorescence staining we found that activated microglia (marked with OX42) have two peaks after spinal cord injury within 1-3 h and 24-48h, this conditions holding on 5 days. We found the microglia activation gradually weakened after 5th day, to 7th day, the fluorescence intensity of activated microglia returned to the resting state. Transplanted microglia maintained high activity after 24th hour, and with the higher fluorescence intensity, the conditions continuing till 2th week after transplanted in spine cord injury areas. From 3th week to 4th week after the transplantation, the microglia remained active there, but the fluorescence intensity of them began to drop significantly. The differences of immunofluorescence intensity between transplant group and the control group were statistically significant (P <0.05); CD68 marked activated microglia were similar to the results of OX42 marked cells after transplanted in rats. There were statistically significant differences between transplantation group and control group (p <0.05). By the staining method of Naounenko-Feigin, we found that activated microglia transplanting in the area of SCI in 2th day, recruitmented to the damaged sites, in 7th day the count was up to, continued until 14th day. Activated microglia after transplanted in damaged sites of spine cord still existed, but the number has decreased. In various points in time, comparing with the control group,positive microglia with silver staining in transplantation group were statistically significant difference ( p <0.05).5.TGF-β1 mRNA and protein were clearly expressed in group A(transplantation group) at 48th,1th week,2th week,3th week after activated MG transplanted into SCI model. The high expression would hold on about 2 weeks.The expression of group B(control group) at 48th,1th week,2th week,3th week after NS transplanted into SCI model was significantly lower than its in group A (p <0.05). Conclusion:1.Isolated and cultured the cells were required activated microglia cell with its single components for us. The purity of the second generation, 4th generation microglia was pure and has a strong ability of proliferation, so it was suitable for the next study.2.Transplanting activated microglia to the region of impaired spinal cord in rats of SCI could be effective in restoring motor function of hind legs.3.The method of transplanting activated microglia to the region of spinal cord injury could significantly extend the survival time of microglia around the segments of spinal cord, likely playing a promoting role in the recovery of the spinal cord.4.Transplanting activated microglia to the region of impaired spinal cord in rats could increase the expression of TGF-β1mRNA and protein, and the up-regulation of TGF-β1 in spinal cord injury sites could play a promotive role in the recovery of SCI. Activated microglia might the main source of TGF-β1.
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