| Experimental purposeTo study the changes of motor function after spinal cord injury in rats and the therapeutic effect of hyperbaric oxygen combined with erythropoietin.To explore the regulation mechanism of combined therapy on myelin regeneration and cell apoptosis in order to provide new strategies and targets for the treatment of spinal cord injury.Experimental methodClean Sprague-Dawley(SD)rats,female,weighing about 250g.Ninety rats were randomly divided into sham group,SCI group,HBO group,EPO group and HBO+EPO group,18 in each group.In sham group,only laminectomy was performed after anesthesia in rats,but the spinal cord was not injured.After operation,rats were treated with ordinary air and injected with saline intraperitoneally.The spinal cord was injured after laminectomy in rats of SCI group.Normal air treatment and intraperitoneal injection of saline were performed after operation.Rats in HBO group were treated with hyperbaric oxygen and intraperitoneal injection of saline after laminectomy and spinal cord injury.Rats in EPO group were treated with normal air and intraperitoneal injection of erythropoietin after laminectomy and spinal cord injury.In HBO+EPO group,the spinal cord was injured after laminectomy,hyperbaric oxygen was performed and erythropoietin was injected intraperitoneally after operation.According to the modified Allen's method,a metal cylinder with a diameter of 2.5 mm and a weight of about 8 g was directly impacted on the exposed spinal cord from a height of 8 cm through a glass tube,resulting in moderate contusion.Paralysis of both hind limbs in rats after spinal cord injury suggested that the model was successful.To prevent bacterial infections,penicillin was injected once a day and bladder massage was given twice a day in the morning and evening to assist in urination.The motor function of all rats was recorded by BBB score,inclined plane test and footprint analysis on 1,3,7,14 and 21 days after operation.At 21 days after operation,all rats were executed and the injured spinal cord(about 2 cm long)was collected for relevant detection.HE staining was used to assess the degree of spinal cord injury,Nissl staining was used to detect the survival of spinal cord neurons in each group,Hoechst staining was used to detect apoptosis of spinal cord,immunohistochemistry was used to detect apoptosis-related genes Bcl-2 and Bax,and myelin sheath-related genes MBP,immunofluorescence was used to detect p53 signaling pathway,and Western blot was used to detect GPR17,apoptosis-related genes Cleaved-Caspase 3,Bcl-2,Bax and MBP.Experimental resultBBB score,inclined plane test and footprint analysis showed that the motor function of rats in sham group was normal at all time points,the motor function of rats in SCI group was significantly limited,and that of rats in HBO group and EPO group was also limited,but the motor function recovery was better than that of rats in SCI group at 7 days after operation,and was most obvious at 21 days after operation.The recovery of motor function in HBO+EPO group was better than that in HBO group and EPO group.HE staining results showed that the structure of spinal cord in sham group was basically normal,and a large number of syringomyelia were formed in SCI group after necrosis,while the syringomyelia in HBO group and EPO group were less than that in SCI group,and the reduction of syringomyelia in HBO+EPO group was more obvious.Nissl staining results showed that the number of neurons in SCI group was significantly lower than that in sham group,while the number of neurons in HBO group and EPO group increased,and the number of neurons in HBO+EPO group increased significantly.Hoechst staining showed that there were a lot of apoptotic cells in SCI group.Hyperbaric oxygen and erythropoietin could effectively inhibit the apoptosis,while combined treatment could further inhibit the apoptosis.Immunohistochemical results showed that Bcl-2 positive cells decreased and Bax positive cells increased after spinal cord injury,Bcl-2 positive cells increased significantly and Bax positive cells decreased significantly after hyperbaric oxygen and erythropoietin treatment,while MBP gene expression decreased significantly after spinal cord injury,and increased significantly after hyperbaric oxygen and erythropoietin treatment.Immunofluorescence results showed that p53 pathway was activated after spinal cord injury,while hyperbaric oxygen and erythropoietin could effectively inhibit the activation of p53 pathway after treatment.Western blot results showed that the expression of Bcl-2,Bax and MBP protein was similar to that of immunohistochemistry,while the expression of GPR17 and Cleaved-Caspase 3 protein increased after spinal cord injury,and decreased significantly after hyperbaric oxygen and erythropoietin treatment.ConclusionSevere demyelinating lesions occurred after spinal cord injury in rats,and obvious apoptotic phenomena appeared in spinal cord tissue.The combination of hyperbaric oxygen and erythropoietin may induce oligodendrocyte precursor cells to differentiate into mature oligodendrocyte through GPR17 molecular target and express the corresponding myelin-related gene MBP,thus forming a new myelin sheath.The protective barrier formed by the regeneration of myelin sheath may inhibit cell apoptosis by regulating p53 signaling pathway,so that neurons can survive and promote the recovery of motor function. |
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