| Objective: To establish a rat spinal cord injury model and use multiphoton microscopy to observe whether rapamycin promotes neuronal survival and axonal elongation in injured regions by early activation of autophagy after SCI injury,revealing autophagy activation after spinal cord injury.Effects on neuronal cells and axons and protective effects on hind limbs motor function in rats.Method:(1)Clean-grade SD female rats were randomly divided into 4 groups: normal control group,placebo group,rapamycin group and 3-MA group.The rat model of spinal cord injury was established by Allen method(T10 was striking).The BBB scores were used to evaluate the changes of hindlimb motor function in rats at different time points after injury(1h,6h,1d,3d,5d,7d,10d).(2)The rats were sacrificed at different time points after injury,and the spinal cord tissue samples were taken.Western blot was used to detect the expression of autophagy-related proteins LC3 II and Beclin1,and the expression of autophagy protein in different groups and at different time points after injury was observed.(3)The collected spinal cord specimens were sectioned,and the changes of spinal cord tissue were observed by HE staining under normal light microscope and compared with the images observed under multiphoton microscopy.(4)Observed under a multiphoton microscope,using advanced image analysis and spectral analysis techniques to reflect the morphological and structural characteristics of nerve fibers at different stages from the microstructure and spectral angles,and obtain axons that can quantitatively reflect different stages of axonal injury repair process.Optical parameter indicators of trends in necrosis and survival.Result:(1)From the 5th day after injury,the BBB score of the rapamycin group was significantly higher than that of the placebo group and the 3-MA group.The difference was statistically significant,and the difference between the groups was also statistically significant(p< 0.05);(2)It was confirmed that autophagy was induced after spinal cord injury in rats.The results of Western blot showed that the autophagy-related protein was observed increased 6h after spinal cord injury(p<0.05),and reached the peak at 3d(p<0.05).After autophagy,the autophagy activity of rapamycin group was significantly higher than that of placebo group,and decreased in 3-MA group.The difference between groups was statistically significant(p<0.05).(3)Microscopic observation results:1.HE staining: Under normal light microscope,the morphology of spinal cord neurons in normal control group was normal,the cell structure was intact and clear,the arrangement was neat and tight,the nucleus was round or elliptical,the cytoplasm was transparent,the chromatin was evenly distributed,and the nucleolus was clear.The axons were intact and there was no break or disorder.The neurons in the placebo group were incomplete,arranged disorderly,the cells were swollen and ruptured,the cell spacing was increased,the contours were blurred,the boundaries were unclear,and there were a large number of degenerated,necrotic neurons,and nuclei.Concentration,nuclear pyknosis,unclear nucleoli,loss of normal structure;the degree of injury from the 3rd day in the rapamycin group was significantly reduced compared with the placebo group: the degree of cell edema was reduced,the staining was uniform,and the degree of cell edema was small,and 3 The degree of-MA damage was worse than in the placebo group.2.Multiphoton microscopy quantifies damage parameters:ECM content: The ECM content in the placebo group continued to decrease after injury,reached the lowest at the 3d time point,and then increased slowly.The 3-MA group had the lowest ECM content in the inter-group comparison at the 3d time point.The ECM content in the group was higher than that in the placebo group,and the difference between the groups was statistically significant(p<0.05).Cavity density: The density of the cavity increased significantly at the time of spinal cord injury and stabilized on the 3rd day.In the detection of the cavity density at the 3d time point,we found that the rapamycin group had more comparison with the placebo group and the 3-MA group.The low cavity density value indicated that the ECM morphology was relatively complete,and the difference between the three experimental groups was statistically significant(p<0.05).Bleeding: Since the strong TPEF signal of hemoglobin in red blood cells emits red fluorescence,by calculating the difference in spectral intensity,we found that there were bleeding signals at various time points of spinal cord injury,and reached the peak at 1d,but the hemoglobin TPEF signal began to decrease at the 3d time point.As the active bleeding of autophagy was gradually absorbed,the intensity of the spectrum was reduced.The comparison of the spectral signals of each group at 3d time point showed that the rapamycin group showed the lowest bleeding signal,indicating that the bleeding was less or absorbed faster,3-MA The group showed a higher bleeding signal,and the difference between the groups was statistically significant(p<0.05).Conclusion:(1)It is confirmed that autophagy can be induced after spinal cord injury;(2)Promoting the survival of rat neuronal cells and axons and the recovery of hindlimb motor function by promoting autophagy at an early stage,inhibiting autophagy promotes the death of neuronal cells and axons,and restores the motor function of rats.Unfavorable effect.(3)ECM content,cavity density and hemoglobin spectral intensity changes may be useful indicators for quantitative monitoring of SCI development by multiphoton microscopy. |
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