| Experiment one:The establishment of a new model of complete spinal cord transection in ratsObjectiveTo establish a complete spinal cord transection model in rats with double edged scissors, and to explore the feasibility of the new model by comparing with the conventional method.MethodsA total of 42 adult female Sprague-Dawley rats were divided randomly into group A (n=6), group B (n=18) and group C (n=18). Group A only received laminectomy. In the group B, the spinal cord was transected with a sharp-pointed knife. Knife point should touch anterior wall of spinal canal and sidewall bone surface. Complete spinal cord transection models were prepared by repeated cutting. In group C, complete spinal cord transection models were established by using self-made double edged micro scissors. The homogeneity of the model was evaluated by the methods of BBB score, histology and fluorescence tracing.ResultsThere was no significant change in BBB score before and after operation in group A. At 1 week after model establishment, in the groups B and C, complete paralysis of the hind limbs was found, and BBB scores were similar. However, significant differences in the spacing of broken end were detected. At 4 weeks after model establishment, hind limb functions could restore to different degrees in both groups, but no significant difference in BBB scores was found.At 8 weeks after model establishment, significant differences in hindlimb motor function scores were detectable between both groups. Biotin glucosamine tracer display:In group B, a few labeled axon fibers were observed at the caudal side of the injured spinal cord. In group C, spinal cord was completely transected, and labeled axon fibers cannot be found at the caudal side.ConclusionThe new method can effectively eliminate the individual differences, which is beneficial to the quantitative analysis and research of the therapeutic effect.Experiment Two:Effect of tanshinone ⅡA on the microvascular area in transected spinal cord in ratsObjectiveTo observe the protective effect of TⅡA on microvascular area in injured spinal cord, as well as on the nerve tissue protection.MethodsA total of 63 adult female SPF rats were randomly divided into 4 groups. Group A(sham): n=9, All rats received laminectomy solely. Group B(SCI):n=18, complete transection of the spinal cord was performed in all rats. Group C(SCI/MP):n=18, intraperitoneal injection of methylprednisolone prednisone (30mg/kg) after transection was conducted. Group D (SCI/TIIA):n=18, intraperitoneal injection of TIIA (20mg/kg) was performed from 1 to 7 days post-injury on a daily basis. Specimens of the injured spinal cord were collected at 1w, 2w, and 4w after cardiac perfusion. Specimen was prepared to 15μm thickness slice with serial sagittal frozen sections. HE stain was conducted to observe hemorrhage, edema and inflammatory reaction. Nissl stain was carried out to observe the neuron damage. Vascular endothelial cell immune fluorescence staining was applied to observe the microvascular area in the injury area of spinal cord. Sagittal frozen sections was observed ar low power of laser confocal microscopy. Then pictures were taken at 100 times lens. Image-Pro Plus 6 software was employed to calculate the microvascular area. Statistical analysis was conducted with SPSS22.0 statistical software package.Results1. Tissue structure was damaged after spinal cord injury. A large number of infiltrating inflammatory cells were observed in the damaged region. Neuronal condensation, necrosis and cytolysis in gray matter was found with enlarged inter-fiber spaces. TIIA can reduce the inflammation cells infiltration, improve axon degeneration..2. The number of neurons in the gray matter of the anterior horn significantly decreased after the spinal cord injury. The neuron morphology was damaged, with nuclear membrane crack, loss of circle shape and shrinkage or loss of Nissl body. TIIA can protect neurons and amileorate secondary injuries.3. Local microvascular number decreased obviously after the injury with spastic morphology. TIIA can relieve vasospasm, inhibit the apoptosis of the endothelial cells and promote the regeneration of blood vessels in the injury region.Conclusions1. Local microvascular network structure was damaged after injury. Ischemic changes was found in injured area.2. TIIA can improve the morphology of blood vessels and promote the angiogenesis in injured spinal cord.3. TIIA can alleviate secondary spinal cord injury.Experiment Three:Restoration of tanshinone IIA combined with fibrin hydrogel to spinal cord injury in ratsObjectiveTo observe the effects of tanshinone IIA combined with fibrin hydrogel on the motor function and axon growth in rats with spinal cord injury.MethodsA total of 40 adult female SD rats were randomly divided into 4 groups. Group A (sham): n=10, all rats received laminectomy solely. Group B(SCI):n=10, complete transection of the spinal cord were conducted. Group C(SCI/Fibrin):n=10, directional fibrin gel was implanted to defect region. Grroup D(SCI/Fibrin/TIIA):n=10, directional fibrin gel^was implanted to defect region and intraperitoneal injection of TIIA (20mg/kg) was carried out from 1 to 7 days after the operation on a daily basis. Hind limb performance was evaluated using the Basso,Beattie and Bresnahan (BBB) 3d, 1w,4w,8w and 12w after operation.12 weeks after operation,5 rats were randomly taken from each group and the MEP was measured by EMG. The remaining rats received cardiac perfusion, then, spinal cord specimens were collected. Specimens were made to sagittal frozen section with the thickness of 15 μm per slice. NF and GAP-43 immunofluorescence staining was performed.Results1. BBB score:BBB scores were 21 in all groups prior to operation. BBB scores in group B, C, D reduced to 0 after operation, while that in group A restore to normal at 3d. Rats in all operation groups displayed lower BBB score during 12w period. Group D showed higher BBB score at 8w compared with group B and C.12w after operation, BBB score in group D was 12 while those in group B and C were 8-9.2. Results of motor evoked potentials 12 weeks after the operation:In group A, normal wave could be elicited, with stable and regular waveform. MEP amplitude in operation groups reduced remarkably. The latency of motor evoked potentials in group B was longer than that in group A, with decreased amplitude and disorganized waveform. Compared with group B, the latency of group C was shorter with significant difference. Group C showed a relatively higher amplitude compared with group B without significant difference.. Group D displayed a more regular, stable, shorter latency and higher amplitude wave compared with group B and C with significant difference3. Immunofluorescence results:(1)12 weeks after operation, immunofluorescence staining was performed with GAP-43, positive nerve fibers showed red color. In group B, positive staining could be observed only in the cephalic area in a disorganized manner. In group C, positive staining could be observed in the cephalic and center area, with the fibers parallel with long axis of spinal cord. While in group D, the number of stained nerve fibers could be observed in all areas of injured region, and was significantly higher than those in group B and C. The fibers in damaged area were parallel with long axis of spinal cord and arranged in an orderly manner.(2) 12 weeks after operation, immunofluorescence staining was performed with NF, positive nerve fibers showed green color. Positive staining nerve fiber could be observed in normal spinal cord tissues in all rats. In group B, positive staining could be observed only in the cephalic area in a disorganized manner. In group C, positive staining could be seen in the cephalic and center area, with the fibers parallel with long axis of spinal cord. While in group D, the number of stained nerve fibers could be observed in cephalic, center and caudally areas of injured region, with the fibers parallel with long axis of spinal cord.Conclusions1. The combination of TIIA with directional fibrin hydrogel can improve the motor function of rats with spinal cord transection and restore the electrophysiological properties of the motor pathway.2. TIIA could protmote expression of GAP-43. The combination of TIIA with directional fibrin hydrogel could guide the orderly growth of axons.3. "The combination of traditional Chinese medicine and tissue engineering technology" may be a meaningful therapeutic strategy for SCI treatment.
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