慢性脑低灌注对大鼠学习记忆及脑海马组织中周期素依赖性激酶5表达的影响

Effect of chronic cerebral hypoperfusion on the learning and memory abilities and the expression of cyclin-dependent kinase 5 in hippocampus of rats

目的:慢性脑低灌注大鼠的学习记忆能力下降,观察此过程中脑海马组织中周期素依赖性激酶5mRNA及其蛋白表达的动态变化。方法:实验于2004-03/12在解放军第二军医大学实验动物中心进行。取雄性SD大鼠60只,随机分为空白对照组18只,假手术组18只,慢性脑低灌注组24只。空白对照不进行干预,慢性脑低灌注组采用双侧颈总动脉结扎建立大鼠慢性脑低灌注模型,假手术组仅行颈前切开,不结扎颈总动脉。分别于术前、术后2,4,8周应用Y迷宫评价错误次数和全天总反应时间,苏木精-伊红染色观察脑海马组织病理变化,脱氧尿嘧啶缺口末端标记染色检测凋亡细胞数目,免疫组化法观察周期素依赖性激酶5蛋白含量的变化,反转录-聚合酶链反应法检测周期素依赖性激酶5mRNA的表达水平。结果:慢性脑低灌注组大鼠死亡3只,57只大鼠进入结果分析。①Y迷宫测试结果:与空白对照组和假手术组相比较,从术后2周开始,慢性脑低灌注组大鼠的迷宫作业错误反应次数增多犤(1.00±0.59),(1.06±0.54),(2.05±0.72)次;F=4.89,P<0.05犦,全天总反应时间延长犤(88.28±17.41),(90.38±15.57),(142.51±21.72)s,F=4.56,P<0.05犦,且随术后时间的延长错误次数和总反应时间增加(P<0.01)。②周期素依赖性激酶5表达:与正常大鼠和假手术组大鼠比较,慢性脑低灌注组大鼠2周时周期素依赖性激酶5阳性表达开始减少(21.68±1.22,21.32±1.38,17.32±1.11,F=3.84,P<0.05),至术后4,8周其表达继续减少(11.59±0.76,8.49±0.42)。结论:慢性脑低灌注可诱导脑海马组织中周期素依赖性激酶5表达下调,同时伴随着大鼠学习、记忆能力下降,提示该酶可能参与了学习记忆过程。

AIM：The learning and memory abilities decline in rats after chronic cerebral hypoperfusion.The purpose of this paper is to observe the dynamic changes of the expressions of cyclin-dependent kinase 5 mRNA and protein in hip-pocampus during the process.METHODS：The experiment was carried out in the Experimental Animal Center of the Second Military Medical University of Chinese PLA between March and December 2004.Sixty male SD rats were selected and randomly divided into blank control group （n=18）,sham-operated group（n=18）,and chronic cerebral hypoperfusion group（n=24）.Rats in the blank control group received no intervention;In the chronic cerebral hypoperfusion group,rat models of chronic cerebral hypoperfusion were made by bilateral common carotid arteries occlusion;In the sham-operated group,rats only received incision of anterior neck,and common carotid artery was not occluded.At 0,2,4 and 8 weeks after ischemia,the incorrect time and the whole day total reaction time were evaluated with the Y-maze,the pathological changes of hippocampus were observed with hematoxylin and eosin staining,the number of apoptotic cells was detected with TdT-mediated dUTP-biotin nick end-labeling（TUNEL）,the changes of the content of cyclin-dependent kinase 5 protein were observed with immunohistochemistry,and the expression level of eyelin-dependent kinase 5 mRNA was determined with reverse transcription-polymerase chain reaction（RT-PCR）.RESULTS：Three .rats died in the chronic cerebral hypoperfusion group,and 57 rats were involved in the analysis of resuhs.①Resuhs of Y-maze test：Compared with the blank control group and sham-operated group,from the 2^nd weeks after ischemia in the chronic cerebral hypoperfusion,the number of error reactions was increased [（1.00±0.59）, （1.06±0.54）,（2.05±0.72） times; F=4.89, P 〈 0.05],the whole day total reaction time was prolonged[（88.28±17.41）,（90.38±15.57）,（142.51±21.72） s,F=4.56,P〈 0.05]. ②The expression of cyclin-dependent kinase 5：Compared with the blank control group and sham-operated group,the positive expression of cyclin-dependent kinase 5 hegan to decrease at 2 weeks（21.68±1.22,21.32±1.38, 17.32±1.11, F=3.84, P 〈 0.05）,and continued to decrease at 4 and 8 weeks（11.59±0.76, 8.49±0.42）.CONCLUSION：Chronic cerebral hypoperfusion can down-regulate the expression of cyclin-dependent kinase 5 in the hippocampus accompanied by the decrease of learning and memory abilities,which indicates that cyclin-dependent kinase 5 may be involved in the learning and memory process.