促红细胞生成素对中年小鼠脑缺血后神经新生的影响

Effect of Erythropoietin on Neurogenesis after Cerebral Ischemia in Middle Aged Mice

目的探究促红细胞生成素(erythropoietin,EPO)对中年小鼠脑缺血后神经新生的影响;方法将24只健康8月龄雄性C57BL/6小鼠随机均分为假手术(Sham)组、大脑中动脉闭塞缺血模型组(Middle Cerebral Artery Occlusion,MCAO)和模型后EPO处理组,每组7只。制作小鼠大脑中动脉闭塞缺血(MCAO)模型,EPO组小鼠在再灌注即刻腹腔注入EPO(5000IU/kg),以后隔日注射1次。脑缺血后第2天开始每天以50mg/kg的剂量腹腔注射Brdu,1次/d,持续1周。第1、3、5、7天采用转棒试验(Rota-rod test)对小鼠神经功能进行评估。在脑缺血后第7天,提取脑组织蛋白、制作冰冻切片,进行western blot检测及免疫荧光染色。结果 EPO组小鼠生存率较MCAO组相比显著提高;EPO组小鼠脑缺血后第5天及第7天神经功能较MCAO组有明显改善,差异有统计学意义(P<0.05);EPO组小鼠脑缺血后第7天BrdU+/DCX+双阳性细胞及BrdU+/NeuN+双阳性细胞数量均显著高于Sham组小鼠和MCAO组小鼠,差异有统计学意义(P<0.05)。EPO组小鼠脑缺血后第7天脑组织中酪氨酸蛋白激酶受体B4(Eph B4)的蛋白水平较MCAO组显著升高(P<0.05)。结论 EPO可促进中年小鼠脑缺血再灌注后神经新生,从而改善神经功能,提高生存率。神经保护药向临床转化时应该考虑到年龄因素的影响。

Object To explore the effect of erythropoietin（EPO） on neurogenesis in middle-aged mice after cerebral ischemia. Methods 24 8-month-old male C57 BL/6 mice were randomly divided into sham group, middle cerebral artery occlusion（MCAO） group and EPO group, with 7 mice in each group. Middle cerebral artery occlusion model was made. The EPO group was injected intraperitoneally with EPO（5000 IU/kg） immediately after reperfusion and was injected every other day. Bromodeoxyuridine（Brd U） was injected intraperitoneally daily to analyze the number of newly proliferating cells. The neurological function of mice was evaluated by rota-rod test at 1, 3, 5 and 7 days after MCAO. Mice were euthanized 7 days after MCAO and brain tissue was extrated for western blot detection and immunofluorescence staining. Results The survival rate of mice in the EPO group was significantly improved compared with that in the MCAO group（P〈0.05）; neurological function improved significantly in 5 d and 7 d after MCAO（P〈0.05）; the number of Brd U＋/DCX＋ and Brd U＋/Neu N＋ double positive cells positive cells were significantly higher than that of sham group and MCAO group（P〈0.05）; the protein level of Eph B4 in brain tissue of EPO group 7 d after MCAO was significantly higher than that in MCAO group（P〈0.05）. Conclusion EPO can promote neurogenesis after cerebral ischemia and reperfusion in middle aged mice, thus improving the nerve function and the survival rate. The factor of age should be taken into account in the clinical transformation of EPO.