慢性间歇低氧诱导小鼠学习记忆障碍的实验研究

Experimental study on the impairment of spatial learning and memory in the mice exposed to chronic intermittent hypoxia

目的 探讨慢性间歇低氧（CIH）诱导小鼠空间学习记忆能力的变化及CIH对认知功能损伤的可能机制.方法 60只ICR雄性幼鼠随机分为常氧对照组（UC组）和间歇低氧组（CIH组）,每组30只;CIH组小鼠置于低氧舱内,通过吹入氮气及压缩空气,每60s做一次缺氧/再氧合循环,使氧浓度波动在6％～8％和20％～ 21％之间,每天8h.CIH组根据低氧造模时间分为3d（A组）、1周（B组）、2周（C组）、4周（E组）、6周（F组）,以及造模结束后常氧饲养1月的10周组（G组）,共6组,每组5只小鼠,同期设6组对照,每组5只小鼠.在慢性间歇低氧3d、1周、2周及6周终点,采用Morris水迷宫的方法测定CIH组和UC组空间学习记忆功能的变化;Western blot法分别测各组海马N-甲基-D-天冬氨酸受体（NMDAR）亚单位1（NR1）蛋白表达的变化;采用免疫荧光标记测定海马Caspase-3表达情况.结果 ①CIH组小鼠在间歇低氧3d时,水迷宫逃避潜伏期较对照组有所延长,但无统计学差异（P＞0.05）;随着间歇低氧时间的延长,逃避潜伏期较对照组逐渐延长（P＜0.05）;低氧6周后,CIH组逃避潜伏期[（68.64±26.52）s]较对照组[（21.36±14.14）s]明显延长（P＜0.01）.低氧6周后,CIH组穿越平台次数[（4.58±2.58）次]较对照组[（8.06±2.74）次]明显减少（P＜0.01）;②与对照组比较,CIH组B、C、E、F及G组小鼠海马神经元NR1蛋白表达均降低（P＜0.叭）,C、E组降至最低水平（P＜0.01）;复氧1月后的G组NR1蛋白与F组相仿,仍较对照组降低（P＜0.01）.CIH各组海马Caspase-3表达均明显升高,并成时间-效应关系,除A组外,其余各组与对照组相比,差异均有统计学意义（P均＜0.01）,CIH组组内指标比较无显著差异（P＞0.05）.结论 慢性间歇低氧诱导小鼠空间学习记忆障碍,且随低氧暴露时间延长学习记忆功能损害加重,这可能与海马神经元NR1蛋白表达下调以及与Caspase-3介导的海马神经元细胞凋亡的发生相关.

Objective To investigate the possible mechanism on spatial learning and memory impairment in the mice model of chronic intermittent hypoxia （CIH）.Methods Male ICR mice （n =60）were randomly assigned to two treatments：normoxic control （UC） and CIH （CIH）,CIH exposure was generated by cycling O2 concentration between 6％ ～ 8％ and 20％ ～ 21％ every 60 s during 8 h of day time.Each group were divided into 3 d （group A）,1 week （group B）,2 weeks（group C）,4weeks （group E） and 6weeks （group F）,after 6 weeks,animals were kept in room air for additional 4 weeks of recovery （group G）.Neurobehavioral assessments （Morris water maze） were performed at 3 d,1 week,2 weeks and 6 weeks.Hippocampal expression levels of Caspase-3 expression were evaluated by immunofluorescence,N-methyl-D-aspartate receptor （NMDAR） subunit 1 （NR1） protein expression was detected by Western blotting.Results ①Compared with the time-matched control animals in UC group,the CIH mice displayed longer mean escape latency in place navigation test starting as early as 3 d （group A）,followed by statistically significant progressive prolongation in group B,C and F.At 6 week,the mean ± SD escape latency was （68.64 ± 26.52）s in CIH mice compared to （21.36 ± 14.14） s in UC mice （P ＜ 0.01）.The number of times of crossing the platform in spatial probe test was also significantly reduced in CIH mice （CIH 4.58 ± 2.58 vs.UC 8.06 ± 2.74） at 6 weeks,（P ＜0.01）; ②Compared with the UC animals,CIH group B,C,E,and F,the hippocampal expression of NR1 proteins decreased significantly （P ＜ 0.01）,group C and E dropped to the lowest level （P ＜0.01）.NR1 expression persisted in group G mice after 4 weeks normoxic recovery （P ＜0.01）.The expression levels of caspase-3 in hippocampus in CIH mice were significantly higher than UC mice at all time points during the 6 weeks exposure of CIH （P ＜ 0.01）,except group A in 3 d.Conclusions Experimental mice CIH model demonstrated a time-dependent progressive impairment of spatial learning-memory ability,which may be related to the down regulation of the expressions of NR1 proteins,as well as the hippocampal neuron apoptosis associated with Caspase-3.