重组人促红细胞生成素对缺氧缺血新生鼠脑损伤学习记忆能力的保护

Protective effects of recombinant human erythropoietin on the learning and memory performances in neonatal rats with hypoxic-ischeimc brain damage

目的:观察重组人促红细胞生成素对缺氧缺血性脑损伤新生鼠海马CA1区半胱氨酸天冬氨酸蛋白酶3的影响及对学习、记忆能力的保护作用。方法:实验于2002/2003在南京医科大学儿科医学研究所完成。选用7d龄SD大鼠62只,随机分为缺氧缺血模型组(n=22),重组人促红细胞生成素治疗组(n=20)和假手术组(n=20)。缺氧缺血模型组和治疗组结扎左侧颈总动脉,并吸入体积分数0.08的O2。假手术组不结扎颈总动脉,不缺氧。①缺氧缺血模型组和假手术组缺氧前腹腔注射生理盐水,治疗组腹腔注射生理盐水稀释的重组人红细胞生成素(500U/mL),剂量均为0.01mL/g。观察模型组和治疗组大鼠在缺氧过程中的躁动和抽搐情况;记录缺氧后0,6,12,24h时间点两组大鼠发生抽搐情况(自发左旋、夹尾左旋及夹尾尖叫)只数。②缺氧24h后随机取缺氧缺血模型组9只,治疗组10只,假手术组9只,取脑组织,在海马及小脑部位进行冠状切片,免疫组织化学法检测各组海马CA1区活化的半胱氨酸天冬氨酸蛋白酶3的表达。③取缺氧缺血模型组,治疗组和假手术组各10只,模型组与假手术组再行腹腔注射生理盐水,治疗组注射重组人红细胞生成素,剂量均为0.01mL/g,48h各组再注射一次。利用跳台实验观察各组大鼠生后75d学习、记忆潜伏期和错误次数。结果:模型组动物在缺氧缺血模型制作过程中死亡3只,治疗组及假手术组动物无死亡,均进入结果分析。①行为学结果:治疗组缺氧后24h自发左旋、夹尾左旋、夹尾尖叫的发生率与模型组比较明显降低大鼠[(16.7%、50%、50%),(66.7%、83.3%、91.7%),P<0.05]。②缺氧缺血后大鼠脑组织内活化半胱氨酸天冬氨酸蛋白酶3阳性细胞明显增多,在海马、大脑皮质分布较为密集。模型组、治疗组、假手术组海马CA1区单位面积内阳性细胞数差异有显著性意义[(41.38±2.09),(22.63±3.17),(10.52±2.70)个/mm2,F=296.20,P<0.01]。模型组明显高于假手术组,治疗组较模型组明显减少,但仍高于假手术组,差异非常显著(q=21.28,P<0.01)。③跳台试验:模型组学习潜伏期与假手术组相比明显延长;错误次数明显增多;记忆潜伏期明显缩短;错误次数显著增加。而治疗组较模型组学习、记忆能力明显改善,表现为学习潜伏期明显缩短;错误次数明显减少;记忆潜伏期明显延长;错误次数显著减少。结论:缺氧缺血性脑损伤新生大鼠海马半胱氨酸天冬氨酸蛋白酶3活性显著增高,重组人红细胞生成素能抑制半胱氨酸天冬氨酸蛋白酶3激活,减轻对新生鼠缺氧缺血的脑损伤,改善动物学习、记忆能力。

AIM： To investigate the effects of recombinant human erythropoietin （rhEPO） on the caspase-3 in hippocampal CA 1 region and the in protecting leaning and memory abilities in neonatal rats with hypoxic-ischemic brain damage. METHODS： The study was completed in the Institute of Paediatrics, Nanjing Medical University between 2002 and 2003. Sixty-two even-day- old SD rats were randomly divided into hypoxic-ischemic model group （n =19）, rhEPO treated group （n=20） and sham-operated group （n=20）. Rats in the bypoxic-ischemic model group and rhEPO treated group were treated with ligation of left common carotid artery, and inhaled oxygen with the volume fraction of 0.08, and those in the sham-operated greup were not given ligation of common carotid artery and hypoxia. （1） Rats in the hypoxic-ischemic model group and sham-operated group were treated with intraperitoneal injection of saline, and those in the rhEPO treated group were injected intraperitoneally with rhEPO diluted with saline （500 U/mL）, the dosage was 0.01 mL/g. The restlessness and twitches during hypoxia were observed in the hypoxic-ischemic model group and rhEPO treated group; the number of the rats with twitches （autonomous left-handed rotation, left rotation and yelping after clipping its tail） at 0, 6, 12 and 24 h after hypoxia were. recorded. （2） At 24 hours after hypoxia, 9, 10 and 9 rats were randomly selected from the hypoxic-ischemic model group, rhEPO treated group and sham-operated group respectively, the brain tissues were taken, the hippocampus and cerebrum were prepared into sections, and the expression of activated Caspase-3 in hippocampal CA1 region was detected with immunohistochemistry. （3） Ten rats were selected from the hypoxic- ischemic model group, rhEPO treated group and sham-operated group respectively, and those in the model group and sham-operated group were treated with intraperitoneal injection of saline again, and those in the rhEPO treatedgroup were injected with rhEPO, the dosages were all 0.01 mL/g, and the injections were given again at 48 hours, Step down test was used to determine the escape latency, step down latency and both error times of learning and memory in the three groups at 75 days. RESULTS： Three rats died in the process of model establishment, and no one died in the rhEPO treated group and sham-operated group, and all the rest rats were involved in the analysis of results. （1） Behavioral results： The incidences of autonomous left-handed rotation, left rotation and yelping after clipping its tail at 24 hour after hypoxia in the rbEPO treated group were significantly decreased as compared with those in the model group （16.7%, 50%, 50%; 66.7%, 83.3%, 91,7%; P 〈 0.05）. （2） The activated Caspase-3 positive stained ceils in brain tissue were obviously increased after hypoxia-ischemia, and distributed more densely in hippoeampus and cerebral cortex. The numbers of positive ceils in unit area of CA1 region in the model group, rhEPO treated group and sham-operated group were （41.38±2.09）, （22.63±3.17）, （10.52±2.70） cells/mm^2 （F=296.20, P 〈 0.01）, respectively, it was obviously higher in the model group than in the sham- operated group, obviously decreased in the rhEPO treated group as compared with that in the model group, but still significantly higher than that in the sham-operated group （q=21.28, P 〈 0.01）. （3） As compared with the sham-operated group, learning latencies obviously prolonged and the error times were obviously increased in the model group; the memory latencies were obviously shortened and the error times were significantly increased. The learning and memory performances in the rhEPO treated group were obviously ameliorated as compared with those in the model group, manifested by the obviously shortened learning latencies, markedly reduced error times, obviously prolonged memory latencies and significantly decreased error times. CONCLUSION： The activity of Caspase-3 is significantly increased in the hippocampus of newborn rats with hypoxic-ischemic brain damage, rhEPO might alleviate the hypoxic-ischemic brain damage in newborn rats though inhibiting the activation of Caspase-3, and improve the learning and memory performances.