Experimental Study On The Effects Of Valproate Sodium Combined With Methylprednisolone On Spinal Cord Injury In Rats

To explore the role and mechanism of sodium valproate（VPA）combined with methylprednisolone（MP）in the recovery of neurological function after spinal cord injury（SCI）in rats.Sixty male healthy SD rats aged 8-10 weeks were selected and randomly divided into sham operation group,SCI group,VPA group,MP group and VPA+MP group according to the digital table method,with 12 rats in each group;rats in each group were then randomized Divided into 2subgroups,group A and group B,with 6 animals in each group.In the sham operation group,only the spinal cord was exposed and the SCI model was not made,and the other 4 groups were used to prepare the rat SCI model by the modified Allen method.In the sham operation group and the SCI group,normal saline was injected into the abdominal cavity at 30 min,6 h,8 h and24 h after the operation(a dose of 30 mg kg^-1).After 24 h,the same dose of normal saline was injected into the abdominal cavity every day for 28 days;the VPA group:Saline(30 mg kg^-1)was injected into the abdominal cavity at 30 min,6 h,and 24 h after the operation,and VPA(30 mg kg^-1)was injected into the abdominal cavity at 8 h after the operation.After 24 hours,the same dose of VPA was injected into the abdominal cavity every day for28 days.;MP group:MP(30 mg kg^-1)was injected into the abdominal cavity at 30 min,6 h,and 24 h after the operation,and physiological saline(30 mg kg^-1)was injected into the abdominal cavity at 8 h after the operation.The same dose was injected into the abdominal cavity every day after 24 h Normal saline for 28 days.VPA+MP group:MP(30 mg kg^-1)was injected intraperitoneally at 30 min,6 h,and 24 h after surgery,VPA(30 mg kg^-1)was injected intraperitoneally at 8 h after surgery,and the same dose of VPA was injected intraperitoneally every day after 24 h,Lasting 28 d.Select the rats of each group A subgroup:1,3,7,14,28 days after operation,the spinal cord injury behavioral motor function（BBB）scoring method and the modified Rivlin inclined plate test were used to evaluate the limb movement of each group after SCI.Recovery of functions.Select subgroup B rats of each group:7 days after operation,the spinal cord tissue in the SCI area was surgically harvested,the morphological changes of the spinal cord tissue of each group were observed by HE staining,the spinal cord motor neurons were observed by Nissl staining,and the number of apoptotic motor neurons was calculated.Histochemical staining was used to semi-quantitatively analyze the relative expression of tumor necrosis factor（TNF-α）and interleukin-1β（IL-1β）protein,and Western blot was used to detect apoptosis-related protein B lymphoma-2（Bcl-2）,The relative expression of Bcl-2 related factor X（Bax）and Caspase-3.（1）Intra-group comparison:Compared with preoperatively,there was no significant difference in BBB scores and Rivlin inclined plate test results in the sham operation group at 1,3,7,14,and 28 days after surgery（all P values>0.05）;SCI group,VPA group,MP group,VPA+MP group BBB score and Rivlin oblique plate test results at different time points after surgery were significantly reduced,but with time,the BBB score and Rivlin oblique plate test results gradually increased,and there was a difference All were statistically significant（all P values<0.05）.Comparison between groups:SCI group,VPA group,MP group,VPA+MP group BBB score after SCI and Rivlin inclined plate test results were significantly lower than the sham operation group,VPA group,MP group,VPA+MP group BBB score and Rivlin The results of the inclined plate test were higher than those of the SCI group.The BBB score and the Rivlin inclined plate test results of the VPA+MP group were higher than those of the VPA group and the MP group,and the differences were statistically significant（all P values<0.05）.（2）On the 7th day after modeling in the sham operation group,the histological characteristics of HE staining were observed:the sham operation group spinal cord tissue was normal;the spinal cord tissue of the SCI group showed a large amount of inflammatory cytokine infiltration,cell hemorrhage,and edema.Neuron necrosis and apoptosis are obvious;compared with the SCI group,the rats in the VPA group,MP group,and VPA+MP group have significantly reduced hemorrhagic and edema,significantly reduced inflammatory factor infiltration,and reduced motor neuron dissolution and apoptosis.Among them,VPA+The treatment effect of MP group was more significant.The area of syringomyelia in the SCI group,VPA group,MP group,and VPA+MP group was significantly larger than that of the sham operation group.The area of syringomyelia in the VPA group,MP group,and VPA+MP group was significantly smaller than that of the SCI group.The area of syringomyelia in the VPA+MP group was all Less than the VPA group and MP group,the difference is statistically significant（P value<0.05）.（3）On the 7th day after sham operation and modeling,Nissl staining observed the number of normal neurons in the spinal cord:the number of normal neurons in the SCI group,VPA group,MP group,and VPA+MP group was significantly less than that in the sham operation group and VPA group The number of normal neurons in the MP,MP,and VPA+MP groups were all higher than those in the SCI group,and the number of normal neurons in the VPA+MP group was higher than that in the VPA and MP groups,and the differences were statistically significant（all P values<0.05）.（4）On the 7th day after sham operation and modeling,immunohistochemical detection of TNF-αand IL-1βexpression:SCI group,VPA group,MP group,VPA+M group TNF-α,IL-1βexpression was obvious Higher than the sham operation group,the expression levels of TNF-αand IL-1βin the VPA group,MP group,and VPA+MP group were significantly lower than those in the SCI group,and the expression levels of TNF-αand IL-1βin the VPA+MP group were significantly lower than those in the VPA group and MP Groups,the differences were statistically significant（all P values<0.05）.（5）On the 7th day after sham operation and model building,Western blot was used to test the expression of apoptotic protein in the spinal cord tissue of rats:Compared with the sham operation group,the SCI group,VPA group,MP group,and VPA+MP group decreased.The relative expression of death-related proteins Bcl-2,Bax and Caspase-3 increased significantly;compared with the SCI group,the relative expression of Bcl-2 in the VPA group,MP group,and VPA+M group increased significantly,Bax,Caspase-3 The relative expression of Bcl-2 in the VPA+MP group was significantly lower than that of the VPA and MP groups,and the relative expression of Bax and Caspase-3 was significantly reduced lower than that of the VPA and MP groups.The differences were statistically significant.Academic significance（all P values<0.05）.VPA combined with MP can significantly improve the neuromotor function of rats after SCI.The mechanism may be related to the inhibition of local inflammatory response,the promotion of inhibiting the production of the apoptotic protein Bcl-2,and the reduction of the expression of the apoptotic protein Bax and Caspase-3.