| Objective:To establish the Wistar rats spinal cord hemisection injury model, the damage region was applied recombinant human erythropoietin (rh-EPO) through subdural injection. Then to study the effection of rh-EPO on motor function recovery, pathological changes and neuronal apoptosis after spinal cord injury (SCI), and to discuss optimal treatment dose, the therapeutic effect and the dose-response relationship about EPO treatment of SCI, so as to provide experimental evidence for the clinical treatment of SCI.Methods:1. Spinal cord hemisection injury model:under the microscope, the right-half side of T10 spinal cord in rats was cut with microscopic blade and self-made injection tube was placed to the subdural.2.80 adult male Wistar rats were randomly divided into 5 groups (n=16):sham group, NS control group, low-dose EPO treatment group, middle-dose EPO treatment group and high-dose EPO treatment group. All groups underwent spinal cord hemisection except sham group. Sham operation group in which the line lamina removed, was no drug treatment; NS control group, according to different administration time points (within 30min after surgery, Id after surgery,3d after surgery and 7d post-operative), was injected slowly with saline 50μl to the subdural catheter every time; Small, medium and high doses EPO treatment group were given rh-EPO to the subdural catheter in accordance with the above delivery time points. Each time when the drug was given, 100IU/kg,500IU/kg, 1000IU/kg rh-EPO were respectively added sodium citrate buffer solution to 50μl, and then were injected slowly into the rats subdural of small, medium and high doses EPO treatment group accordingly. Rats in all groups were sacrificed after 14d.3. All the rats'behavial changes were valued regularly with the BBB scale before and after injury, and the motor functional recovery of rat hindlimb after SCI was observed continuously for 2 weeks. After sacrificed, the injuried spinal cord tissues of rats were paraffin-embedded and cutted into slices. Then the pathological changes were observed with hematoxylin-eosin staining, the apoptotic cells of the injuried spinal cord tissue were labelled with TUNEL method, the results were analyzed under the light microscope and the apoptotic index was calculated. Data analysis was performed with Student's t-test and one-way ANOVA, P<0.05 is considered statistically different, and P<0.01 is considered statistically significant different.Results:1. Hemisection model of spinal cord is successful, stable and repeatable. The wound is neat, degree of injury is constant, anatomical localization is accurate, and functional disorders are also identified.2. BBB score showed:①Compared with Id after surgery, BBB scores in all treatment groups increased significantly on 3d,7d and 14d after surgery (P< 0.05).②BBB scores of low-dose EPO treatment group were higher than the NS control group (P<0.05).③Medium and high dose EPO treatment group compared with the NS control group and low-dose EPO group, BBB scores increased significantly (P<0.01).④Comparing medium and high dose EPO treatment groups, BBB scores had no significant change (P>0.05).3. HE staining showed:①Sham group:Rat spinal cord tissue was normal, neuronal morphology were intact, nissl bodies were clearly visible, and axons of peripheral white matter was normal.②Injuried groups:There were spinal cord edema, diffuse hemorrhage, cysts and vacuoles formation, neuronal degeneration and even disappearance, confusion Nissl bodies, large proliferation of astrocytes, glial scar formation, axonal degeneration appeared. Neurons were surrounded by a large number of inflammatory cells and the gap between axon and myelin was larger.③The pathological damage of EPO treatment group was milder than the NS control group, and the pathological damage of medium and high dose group was lighter than smaller-dose EPO.4. Apoptotic cells results showed:Positive cells were seen occasional in the sham group, and there was a large number of positive cells in the white matter and around the lesion in injuried groups. Compared with the NS control group, the number of apoptotic cells and AI of low-dose EPO group were decreased (P< 0.05). Medium and high dose EPO treatment group compared with the NS control group and low-dose EPO group, the number of apoptotic cells and AI decreased significantly (P<0.01). Comparing medium and high dose EPO treatment groups, the number of apoptotic cells and AI had no significant difference (P>0.05).5. Correlation analysis showed:BBB score increased with the decreased number of apoptotic cells, and there was a negative correlation between the two.Conclusion:1. Rats spinal cord hemisection model is suitable to conduct fundamental research and neurobiology observation of SCI.2. rh-EPO helps rats after SCI with increasing BBB motor functional scores, and promotes recovery of motor function in rats.3. rh-EPO can improve the pathological changes of spinal cord injuried tissue in rats.4. rh-EPO can reduce neuronal apoptosis after spinal cord injury in rats, and reduce the apoptotic index.5. There was a trend that BBB score increased with the decreased number of apoptotic cells.6. Medium, high-dose rh-EPO treatment of spinal cord injury is better than low-dose rh-EPO; Medium-dose rh-EPO is optimal treatment dose of spinal cord injury. |
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