| Objective:Treat the rats with cerebral ischemia-reperfusion injury with various doses of erythropoietin and obeseve expressions of neuroglobin (NGB) and tumor necrosis factor-alpha (TNF-α) to study the potential its therapeutic mechanism and the most suitable dose.MethodsIn this study, ninety healthy male Sprague-Dawley rats, with weight from 180g to 220g, were divided into 3 groups based on completely randomized block design:normal control group(N group,30),non-treatment group (NT group,30),and EPO treatment group(T group,30). T group were randomly divided into 3 subgroups depending on different doses of EPO they were administrated ( EPO1: 1000u/Kg;EPO2: 3000u/Kg; EPO3: 5000u/Kg; 10 rats a subgroup.). Based on Longa suture method [1], an improved model of rats with focal cerebral ischemia was established. After two hours of ischemia, sutures of both treatment group and non-treatment group were pulled and reperfusion was performed.The non-treatment group was dosed saline intraperitoneally when ischemia occurred. All the objects were observed and rated by Longa's rEPOrt after 24 hours, and then were executed to obtain brains. All the brains were treated to form paraffin section before treated with HE staining, Nissl staining and immunohistochemical detection to observe pathological and morpholoical changes and the expression of NGB and TNF-αin cerebral cortex of ischemic side. Postive cells of ischemic side were counted, too. All the data were proceessed by SPSS13.0 system.Results1. The scores of neurologic impairmentAfter 24 hours, the normal group is scored 0±0.00, treatment group scored 1.70±0.79 and non treatment group 2.37±0.81. Compare non-treatment group with the treatment group, the result is P<0.01; compare the normal group with the non-treatment group, the result is P>0.05. Scores of each treatment subgroup was 2.30±0.82, 1.50±0.71 and 1.30±0.48, respectively; significant difference was observed between subgroup EPO3/EPO2 and EPO1 (P<0.05), while no significant difference was observed between subgroup EPO2 and EPO3(P>0.05).2 HE stainingArranged neatly, cortical cell membranes of the normal group were in normal shapes and quantities. For non-treatment group, there were sparse nerve cells, widened cells gaps, and a large number of cells degeneration and necrosis which represented as inclusions shrinkage, nuclear pyknosis deeply stained and nucleolus disappeared; while the number of survival nerve cells was significantly increased and the degree of injury wass reduced in EPO treated group.3. Nissl stainingNormal cortical membrane was blue-purple, light blue nucleus was full, we can see a lot of purple and blue Nissl body, the cells arranged in neat rows; Nissl body of non-treated rats were dissolved or disappearance or very few; treatment group Nissl body increased significantly, and as the dose increases.4. NGB expression in brain tissueNormal brain tissue in rats can be seen with the expression of NGB (normal group: 83.47±6.45, non-treatment group: 118.70±10.36; treatment group: 118.70±10.36); non-treatment group NGB expression was significantly increased compared with normal group, there were statistically significant (P <0.05); while the expression of treatment group was more significantly increase than that of normal group or the non-treatment group, and there were statistically significant (P <0.01); and there were also significant differences (P <0.05) between subgroup EPO1 and EPOE2 or subgroup EPO1 and EPOE3, while there was not significant difference(P>0.05) between subgroup EPO2 and EPOE3.5. TNF-αexpression in brain hippocampus The normal group did not found with the expression of TNF-α. TNF-αwas observed in the non-treatment group (104.37±13.17) and treatment group (70.27±7.75), and there was a statistical significance (P <0.01); compare the normal group with the non-treatment group or the treatment group, the result was P>0.05. Compare subgroup EPO1 (78.20±6.56) with EPO2 (68.30±5.12) or EPOE3 (64.30±3.23), there were also significant differences (P <0.05), while there was no significant difference (P>0.05) between subgroup EPO2 and EPOE3.Conclusions1. In cerebral ischemia-reperfusion injury model of rats, EPO plays a role in brain protection by inhibiting neuronal degeneration and necrosis and reducing cerebral ischemia-reperfusion injury. EPO's neuroprotective effect has something to do with the increase of NGB expression and the decrease of TNF-αexpression after the performance of cerebral ischemia-reperfusion.2. A dose of 3000U/kg of EPO might be the most suitable dose to treat cerebral ischemia-reperfusion injury on rats. |
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