| Objective:To establish the Wistar rats spinal cord hemisection injury model, and use the Nogo-A antagonist peptide (NEP1-40) after spinal cord injury through damage region injection.To analyze and evaluate NEP1-40 effectiveness for the repairment to spinal cord injury, and discuss the possibility mechanisms of NEP1-40 for the locomotor recovery on the animals of SCI. To observe the change in the expression of Nogo-A and in accordance with this change after spinal cord injury to explore the best time of NEP1-40 administration.Methods:1. Use self-made microblade to cut the spinal cord injury on the right half of the 10th thoracic segments,for the establishment of thoracic spinal cord hemisection injury model.2. Sixty adult female rats (200-220 grams) which were utilized and randomly allocated into sham-operated group, control (vehicle) and NEP1-40-treated groups. In all animals, the spinal cord was right hemi-transected at the 10th thoracic segments except of the sham-operated group。NEP1-40 solution 15μg/PBS 25μl was applied on the hemi-transected area in the treatment group for seven dayS。The same volume of phosphate-buffered saline solution was applied on the injured area in the control group. Each group was subdivided into four subgroups according to the postsurgical day of killing (7,14,21,28days). The BBB behavioral scores of the rats were evaluated at different time, while hematoxylin-eosinstain and immunohistochemical staining were used to observe and analyse the morphological transformation in the damaged spinal areas.3. Fifteen adult female rats (200-220 grams) which were utilized and randomly allocated into sham-operated group, control (vehicle) and NEP1-40-treated groups. The methods of therapy as above.2 weeks after SCI, put them on the stereotactic frame, then use micro-syringe to inject BDA anterograde tracer into the rats'brain sensorimotor areas, and 2 weeks after the injection of BDA,execute the animals and take the spinal cord, then observe the rapid refrigerated spinal cord section under the fluorescence microscope to analyse the axons running around the injured spinal cord area.4. The resulting images of the Nogo-A sections, NF200 sections and BDA sections were dealed with micro-image analysis software Image pro plus 6.0. Data analysis was performed with student's t-test and one-way ANOVA, p<0.05 was considered statistically different, p<0.01 was considered the difference among the data had significantly statistical difference.Results:1. The rat spinal cord hemisection model was successful and stable, it showed as a typical spinal cord hemi-transected syndrome, and the wound was clean, the modle was reproducible and the injury grade was constant, the anatomical location was accurate, the dysfunction was also confirmed. In a word, this modle was suitable for the basic research on spinal cord injury and for the neurobiological research.2. The BBB motor scores of all the animals preoperatively were 21 points,all of the SCI animals' right lower limbs were paralyzed within the first day after injury, and then all groups scores were gradually increased, but the recovery of different groups have different levels. BBB motor scores in the NEP1-40-treated groups were higher than those in the vehicle groups at 7days(P<0.05), and significantly higher at 14,21and 28 days post injury (P＜0.01). The sham-operated group animals'BBB scores nearly had no change after injury with the normal ones.3. Immunohistochemical staining for NF200 showed that there were not any positive staining cells in sham-operated group.The number of NF200 positive staining cells which were located in damage adjacent areas revealed a significant increase in the NEP1-40 treatment group than that in the vehicle group at 7,14,21 and 28 days post injury. (7d,14d,P<0.05;21d,28d,P<0.01) Immunohistochemical staining for Nogo-A showed that the positive staining oligodendrocytes and myelin were observed in sham-operated group, control (vehicle) and NEP1-40-treated groups. Immunohistochemical staining for Nogo-A on both sides of the spinal cord injurd areas were significantly increase in the vehicle and the treatment group than those in the sham-operated group at 7 and 14 days post injury (7d, P<0.01;14d the vehicle groups P＜0.05, the treatment group P<0.01), but the indexes of the treatment groups were not significantly different from those of the vehicle groups (P>0.05)4. BDA corticospinal tract anterograde tracing of nerve fibers showed that, the axons in the spinal cord of the sham-operated group had stained clearly and uniformly, they could smoothly cross downward through the 10th thoracic segments. From the injured spinal cord of vehicle group and NEP1-40-treated group, obvious signs of spinal cord hemisection in the right side of the spinal cord where there were fluorescent reagent concentrated could be seen. The nerve fibers in the injured spinal cord of vehicle group could rarely cross down through the damaged aera,.Very small amount of nerve fibers could descend through the injured aera.5. The BBB motor scores increased with the increase of the number of NF200 positive staining cells. There were linear positive relation between them (P＜0.01).Conclusions:1. The rat spinal cord hemisection model is successful and stable, and the wound is clean, the modle is reproducible and the injury grade is constant, the anatomical location is accurate, the dysfunction is also confirmed. In a word, this modle is suitable for the basic research on spinal cord injury and for the neurobiological research.2. Exogenous injection NEP1-40 can promote axonal regeneration and through the regeneration of nerve axons in rats with spinal cord injury improve lower extremity motor function.3. As a major myelin-related inhibitor, in the early stage after spinal cord injury the expression of Nogo-A significantly increases that determines time-dependent nature of its receptor antagonist NEP1-40 application, NEP1-40 should be applied within 2 weeks after injury, the best application begins from the first week after SCI. And NEP1-40 application will not affect the expression of Nogo-A, thus avoiding possible potential side effects.
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