大鼠脑组织缺血再灌注后促红细胞生成素对神经元的保护

Erythropoietin for protecting neurons in rats with cerebral ischemia-reperfusion injury

目的:观察促红细胞生成素对缺血再灌注大鼠脑组织海马CA1区神经元数量、凋亡细胞数量变化、脑组织匀浆一氧化氮含量、丙二醛含量及超氧化物歧化酶活性改变的影响。方法:实验于2003-03/2004-12在解放军第三军医大学附属新桥医院中心实验室完成。选择健康Wistar大鼠66只,随机分为正常对照组6只,缺血再灌注生理盐水组30只(生理盐水对照组),缺血再灌注促红细胞生成素组30只(促红细胞生成素治疗组)。除正常对照组线栓法制备大鼠大脑中动脉阻断局灶性脑缺血模型,缺血2h后再灌注。促红细胞生成素组于再灌注开始时经腹腔按2000U/kg注入生理盐水稀释的重组人红细胞生成素(200U/mL),生理盐水对照组经腹腔注入等量的生理盐水。再灌注后观察时相点为2,6,12,24,48h,每个时间点6只。应用苏木精-伊红染色观察脑海马CA1区细胞数量情况,应用末端脱氧核苷酸转移酶缺口标记法检测海马CA1区神经元凋亡情况,应用硝酸还原酶法测定脑组织一氧化氮含量,羟胺法测定脑组织超氧化物歧化酶活性,硫代巴比妥酸法测定脑组织中丙二醛含量。结果:66只大鼠全部进入结果分析。①脑海马CA1区细胞计数:促红细胞生成素治疗组再灌注后12,24,48h比生理盐水对照组细胞数明显增加犤(286.7±33.8),(268.6±44.5)个/视野;(271.9±30.4),(240.8±22.5)

AIM:To study the effect of erythropoietin on the number of neurons and apoptotic cells in hippocampus CA1 region,the content of nitric oxide and malondialdehyde,and the activity of superoxide dismutase in rats with ischemia reperfusion injury.METHODS:The experiment was finished in the Central Laboratory,Xinqiao Hospital of Third Military Medical University of Chinese PLA from March 2003 to December 2004.A total of 66 healthy Wistar rats were divided randomly into control group(n=60),ischemia reperfusion saline group(n=30,saline group) and ischermia reperfusion erythropoietin group(n=30,erythropoietin group).The models of focal cerebral ischemia were established with middle cerebral artery occlusion in saline and erythropoietin groups,and reperfusion was performed after 2 hour ischema.The rats in erythropoietin group were injected with recombinant erythropoietin(200 U/mL) diluted with saline according to the standard of 2 000 U/kg into abdominal cavity at the beginning of reperfusion,and saline group received the same dose of saline.Every six rats were observed at hours 2,6, 12,24 and 48(n=6) following reperfusion.The number of cells in hippocampus CA1 region was observed with hematoxylin and eosin staining;the neuronal apoptosis was detected by using terminal deoxynucleotidyl transferase mediated nick end labeling;the content of nitric oxide was detected by using nitrate reductase method;the activity of superoxide dismutase was detected with hydroxylamine;the contend of malondialdehyde was detected with thiobarbitric acid.RESULTS:All 66 rats were involved in the result analysis.① The cell number in hippocampus CA1 region increased significantly in erythropoietin group at hours 12,24 and 48 following reperfusion as compared with saline group (286.7± 33.8,268.6± 44.5;271.9± 30.4,240.8± 22.5;258.3± 29.5,201.8± 30.7)(t=3.189- 5.589,P < 0.01).② The number of apoptotic neurons decreased significantly in erythropoietin group at hours 6,12,24 and 48 following reperfusion as compared with saline group(21.5± 5.8,28.4± 6.5;32.7± 4.6,48.8± 7.6;42.8± 6.6,60.7± 10.2;51.8± 9.5,81.6± 12.3)(t=3.457- 6.347,P< 0.01).③ The content of nitric oxide in brain tissue decreased significantly in erythropoietin group at hours 2,6, 12,24 and 48 following reperfusion as compared with saline group [(48.2± 5.1),(59.4± 5.5) mmol/L;(51.4± 6.3),(66.3± 9.8) mmol/L;(59.7± 6.6),(80.7± 10.2) mmol/L;(55.7± 8.1),(77.8± 9.2)mmol/L;(49.3± 6.7),(67.3± 7.1)mmol/L;(t=3.258- 5.624,P< 0.01)].④ The activity of superoxide dismutase increased significantly in erythropoietin group at hours 2,6, 12,24 and 48 following reperfusion as compared with saline group[(3.78± 0.74),(3.21± 0.31) μ kat /L;(3.41± 0.43),(2.92± 0.31) μ kat/L;(3.21 ± 0.35), (2.51± 0.27) μ kat /L;(3.64± 0.55),(3.01± 0.32) μ kat/L;(4.10± 0.56),(3.55± 0.41)μ kat /L];(t=3.485- 6.457,P< 0.01). ⑤ The content of malondialdehyde decreased significantly in erythropoietin group at hours 2,6, 12,24 and 48 following reperfusion as compared with saline group[(40.7± 4.4),(54.6± 6.7) μ mol/L;(51.3± 5.4),(65.7± 8.8) μ mol/L;(61.7± 7.2),(77.4± 7.5) μ mol/L;(53.8± 6.4),(67.8± 9.1) μ mol/L;(39.7± 4.0),(53.3± 4.9)μ mol/L](t=3.541 to 6.045,PCONCLUSION:Erythropoietin can increase the number of survival cells in hippocampus CA1 region in rats with cerebral ischemia reperfusion injury,inhibit the apoptosis,decrease the content of nitric oxide and lipid peroxidation,which plays an important role in protecting nerve cells.