Spatiotemporal Expression Of Ski After Rat Spinal Cord Injury

Part 1 Behavioral and Pathological Changes after Rat Spinal Cord InjuryObjective To establish a scientific and reliable spinal cord injury(SCI)model and to identify the behavioral and pathological changes of rats after SCI,so as to lay the foundation for further study.Methods 30 adult female Sprague-Dawley rats were randomly divided into sham group(n=15)and spinal cord injury(SCI)group(n=15),each group had five time points(1 weeks,2 weeks,4 weeks,8 weeks,12 weeks),3 rats per time point.Spinal cord injury model at T10 was established with modified Allen’s technique(10 g×25 mm).The hindlimbs behavior of rats was rated with Basso-Beattie-Bresnahan(BBB)scores at 1day,3days,1weeks,2weeks,4weeks,8weeks and 12 weeks after SCI.3 rats in each group were detected with HE staining at 1 weeks,2 weeks,4 weeks,8 weeks and 12 weeks after injury to observe the pathological changes of the spinal cord and the formation of cavity.Results 1.The BBB scores of each time point in the injury group were lower than those in the sham group(n=3,P < 0.05).2.Compared with the sham group,necrosis in 1 week and 2 weeks post-injury groups was the major pathological change;at 4 weeks,cystic cavity completely formed,with dense scar tissue around cavity;There was no significant change in the cavity and scar in 8 weeks and 12 weeks post-injury groups,but the injury center and the adjacent spinal cord were obviously thinner.3.The results of BBB scores and HE staining were compared with the results of previous studies,and the success of spinal cord model was fully established.Conclusion In this study,a scientific and reliable SCI model was successfully established by behavioral and pathological evaluation,and the behavioral and pathological changes of spinal cord after SCI were determined,which laid the foundation for the next experiment.Part 2 Changes of Ski Expression after Rat Spinal Cord InjuryObjective A preliminary study on the changes of Ski expression in rats after SCI.Methods 60 adult female Sprague-Dawley rats were randomly divided into sham group(n=30)and SCI group(n=30)each group had five time points(1 weeks,2 weeks,4 weeks,8 weeks,12 weeks),6 rats per time point.SCI model was established and BBB score was performed with the same method like part one.3 rats were analyzed by immunofluorescence staining and semi quantitative analysis and the other 3 were detected by Western Blot to observe the changes of Ski expression in rats after SCI.Results 1.The result of BBB scores was the same as the part one.2.Immunofluorescence staining and semi quantitative analysis showed that ski was low expression in normal spinal cord,after injury,ski expression gradually increased,reached the peak at 8 weeks and decreased again at 12 weeks;Compared with sham group,the increase of ski at 1 weeks was not statistically significant(n=3,P > 0.05),ski expression of the other groups were significantly higher(n=3,P < 0.001);At the same time,ski was mainly distributed in white matter in sham and 12-weeks post-injury groups,while there was a clear ski expression in gray matter at 2,4 and 8 weeks after injury.3.Immunofluorescence staining of injury center showed that ski was highly expressed around the cavity and obviously formed high expression band.4.Western Blot detection demonstrated that the Ski expression at 1 week after injury was not increased,but it began to increase from 2 weeks and significantly increased at 4 weeks.At 8 and 12 weeks,the Ski expression reached the peak,and showed a stable trend.Conclusion From this study,we firstly defined the distribution pattern in the gray and white matter and changes of Ski after SCI.And we also found that Ski was intensively high expressed around damaged cavity,suggesting that Ski may have a certain relationship with the formation of glial scar.Part 3 Cellular Localization of Ski Expression after Rat Spinal Cord InjuryObjective To further clarify the cellular localization of Ski expression after SCI,and to analyze the possible role of Ski in the process of SCI in rats.Methods 60 adult female Sprague-Dawley rats were randomly divided into sham group(n=30)and SCI group(n=30)each group had five time points(1 weeks,2 weeks,4 weeks,8 weeks,12 weeks),6 rats per time point.SCI model was established and BBB score was performed with the same method like part one.3 rats were analyzed by double immunofluorescence staining and the other 3 were detected by Western Blot to observe the changes of GFAP expression in rats after SCI.Correlation analysis was performed between relative expression level of Ski,GFAP and BBB scores.Results 1.The result of BBB scores was the same as the part one.2.No clear co-expression signals in double immunofluorescence staining of Ski and Neu N were observed in sham group and SCI group at each time points.The same situation was found in double immunofluorescence staining of Ski and Nestin.While clear co-expression signals in double immunofluorescence staining of Ski and GFAP were found in both sham and SCI group at each time points,and this kind of co-expression signals markedly enhanced around the cystic cavity of injured spinal cord and stabilized from fourth weeks.3.Western blot detection of GFAP showed that the expression of GFAP was not significantly increased at 1 and 2 weeks after injury,but was significantly higher at 4 weeks and tended to stabilize at 8 and 12 weeks after injury compared with sham group.4.Correlation analysis between Ski,GFAP and BBB score showed that Ski expression was positively correlated with the expression of GFAP both in the sham group and the injury group at each time point.Moreover,in the SCI group,the relative expression level of GFAP was also positively correlated with the BBB scores.Conclusion In this part of study,we found Ski was mainly expressed in astrocytes but not in neurons and neural stem cells after SCI for the first time.Meanwhile,the clear correlation was found between Ski expression and astrocyte proliferation and glial scar formation.Combined with the former two part of the study,we speculated that Ski may be a new molecule that regulates the process of reactive astrogliosis and glial scar formation.