付有娟, 刘志宏, 赵枫, 关素珍. 母鼠孕期慢性应激对子代学习记忆功能及海马脑源性神经营养因子表达的影响[J]. 环境与职业医学, 2020, 37(3): 267-273. DOI: 10.13213/j.cnki.jeom.2020.19662
引用本文: 付有娟, 刘志宏, 赵枫, 关素珍. 母鼠孕期慢性应激对子代学习记忆功能及海马脑源性神经营养因子表达的影响[J]. 环境与职业医学, 2020, 37(3): 267-273. DOI: 10.13213/j.cnki.jeom.2020.19662
FU You-juan, LIU Zhi-hong, ZHAO Feng, GUAN Su-zhen. Effects of maternal chronic stress during pregnancy on learning and memory function and expression of hippocampal brain-derived neurotrophic factor in offspring rats[J]. Journal of Environmental and Occupational Medicine, 2020, 37(3): 267-273. DOI: 10.13213/j.cnki.jeom.2020.19662
Citation: FU You-juan, LIU Zhi-hong, ZHAO Feng, GUAN Su-zhen. Effects of maternal chronic stress during pregnancy on learning and memory function and expression of hippocampal brain-derived neurotrophic factor in offspring rats[J]. Journal of Environmental and Occupational Medicine, 2020, 37(3): 267-273. DOI: 10.13213/j.cnki.jeom.2020.19662

母鼠孕期慢性应激对子代学习记忆功能及海马脑源性神经营养因子表达的影响

Effects of maternal chronic stress during pregnancy on learning and memory function and expression of hippocampal brain-derived neurotrophic factor in offspring rats

  • 摘要: 背景

    孕期慢性应激刺激会对子代的生长发育产生长远的影响,其中最严重的损害是引起子代学习记忆能力下降。研究发现孕早期和孕晚期母源性应激对胎儿大脑发育的影响最明显,可引起海马组织中单胺类神经递质及胰岛素样生长因子Ⅱ表达异常,但突触相关蛋白脑源性神经营养因子(BDNF)的表达及其与学习记忆功能之间的关联较少报道。

    目的

    观察孕期慢性应激后子鼠学习记忆功能的变化,检测海马组织突触相关蛋白BDNF的表达情况,并分析其与子代学习记忆功能之间的关联。

    方法

    选择SPF级未曾受孕、80~90日龄的Wistar雌鼠20只,随机分为模型组和对照组,每组10只;SPF级90~100日龄的Wistar雄鼠15只,随机分为模型组10只和对照组5只。适应性饲养一周后,通过慢性不可预知温和应激(CUMS)建立母鼠孕期应激模型,模型组母鼠每天给予刺激,连续21 d不间断;对照组母鼠正常环境饲养。应激刺激开始的第3天,将模型组雌鼠与雄鼠按照1:1合笼,对照组按2:1合笼。母鼠应激前1 d和应激后1、7、14 d分别进行内眦静脉取血,采用125I皮质醇放射免疫试剂盒对血浆皮质醇的含量进行测定,根据公式计算皮质酮浓度,当模型组皮质酮浓度高于对照组表示建模成功。子鼠出生后21 d(记为PND21)时断乳,两组均从不同窝别中随机抽取16只子鼠(雌、雄各8只),分别为对照子鼠组和模型子鼠组,编号后每笼4只正常环境饲养。PND28时,测量子鼠体重。PND42时测量子鼠体重,利用Morris水迷宫(主要指标为逃避潜伏期和跨越平台次数)和Y迷宫(主要指标为达标所需训练的次数和正确反应率)对子代学习记忆能力进行测定,通过HE染色及电镜对子鼠海马组织形态学进行观察,采用实时荧光定量PCR法和Western blotting法测定海马组织突触相关蛋白BDNF mRNA和蛋白的表达水平。

    结果

    在应激第7天,模型组母鼠血浆皮质酮水平(348.50±13.24)μg·L-1高于对照组(224.00±39.84)μg·L-1且达到高峰(P < 0.05),在应激第14天,模型组大鼠皮质酮水平(258.38±22.77)μg·L-1仍高于对照组(218.78±16.68)μg·L-1,提示模型组大鼠处于应激状态。Morris水迷宫实验结果显示:模型子鼠组逃避潜伏期时间(32.00±3.61)s长于对照子鼠组(20.01±4.12)s(F=7.30,P < 0.05);模型子鼠组跨越平台次数(5.54±2.11)次少于对照子鼠组(7.38±1.38)次(t=2.26,P < 0.05)。在Y迷宫试验中,模型子鼠组达标所需的训练次数(31.32 ±17.12)次多于正常子鼠组(19.38 ±11.22)次,而记忆正确反应率(45%±15%)低于对照子鼠组(89%±25%),且差异均有统计学意义(t=6.77,P < 0.05)。模型子鼠组海马组织结构清晰度弱于对照子鼠组,细胞形态不规则,胞体缩小,细胞间距明显增大,细胞排列松散。模型子鼠组的海马突触相关蛋白BDNF mRNA相对表达(0.61±0.15)低于对照子鼠组(1.00±0.22)(t=3.79,P < 0.05);与对照子鼠组(0.50±0.02)相比,模型子鼠组BDNF蛋白相对表达(0.15±0.10)也降低(t=28.11,P < 0.05)。子鼠的逃避潜伏期(r=-0.59,P < 0.05)和训练次数(r=-0.59,P < 0.05)与突触相关蛋白BDNF相对表达存在负相关关系,其正确反应率与BDNF蛋白相对表达存在正相关关系(r=0.66,P < 0.05)。

    结论

    孕期慢性应激可使子代学习记忆功能和海马突触相关蛋白BDNF的相对表达下降,且学习记忆能力的下降与BDNF蛋白的相对表达下降有关。

     

    Abstract: Background

    Chronic stress stimulation during pregnancy will have a long-term impact on the growth and development of offspring, and the most serious damage is the decline of learning and memory ability. Previous studies have found that maternal stress in early or late pregnancy has the most significant effect on the development of fetal brain, such as abnormal expressions of monoamine neurotransmitter and insulin-like growth factor Ⅱ in hippocampus, but there are few reports about the expression of brain derived neurotrophic factor (BDNF) and its relationship with learning and memory function.

    Objective

    This experiment is designed to observe the changes of learning and memory function in offspring of pregnant rats with chronic stress, detect the expression of BDNF in hippocampus, and analyze the relationship between BDNF and learning and memory of offspring rats.

    Methods

    Twenty female Wistar rats of SPF grade aged 80-90 days and not pregnant were randomly divided into a model group and a control group, with 10 rats in each group. Fifteen male Wistar rats of SPF grade aged 90-100 days were randomly divided into a model group with 10 rats and a control group with 5 rats. After one week of adaptive feeding, a stress model of female rats was established by chronic unpredictable mild stress (CUMS) for 21 consecutive days, while the female rats in the control group were fed in normal environment. On the 3rd day of the stress stimulation, the female and male rats in the model group were caged according to 1:1 sex ratio, and the control rats were caged according to 2:1. One day before the stress and 1, 7, and 14 days after the stress, blood samples were collected from the angular vein of the female rats, plasma cortisol content was measured by 125I cortisol radioimmunoassay, and the concentration of corticosterone was calculated. If the concentration of corticosterone in the model group was higher than that in the control group, the model was considered successful. On postnatal day 21 (PND21), the offspring rats were weaned, and 16 pups (eight females and eight males) were randomly selected from different nests of the two groups, named correspondingly as model offspring group and control offspring group, and four pups were raised in normal environment in each cage after being grouped. On PND28, the body weight of the pups was recorded. On PND42, the body weight of the pups was recorded, the learning and memory ability of the pups was detected by Morris water maze (escape latency and the number of crossing platform) and Y maze (the training times for reaching learning criterion and correct response rate), the morphology of hippocampus was observed after HE staining using electron microscopy, and the mRNA and protein expressions of BDNF were measured by real-time fluorescence quantitative PCR and Western blotting respectively.

    Results

    On the 7th day of stress, the plasma corticosterone level of the model female rat group (348.50±13.24) μg·L-1 was higher than that of the control female rat group (224.00±39.84)μg·L-1 and reached a peak (P < 0.05). On the 14th day of stress, the corticosterone level of the model female rat group (258.38±22.77)μg·L-1 was still higher than that of the control female rat group (218.78±16.68) μg·L-1, suggesting that the model group was stressed. The results of Morris water maze experiment showed that the escape latency of the model offspring group (32.00±3.61) s was longer than that of the control offspring group (20.01±4.12) s (F=7.30, P < 0.05); the number of crossing platform of the model offspring group (5.54±2.11) was less than that of the control offspring group (7.38±1.38) (t=2.26, P < 0.05). In the Y maze test, the training times in the model offspring group (31.32±17.12) were significantly higher than that in the control offspring group (19.38±11.22), while the correct response rate in the model offspring group (45%±15%) was lower than that in the control offspring group (89%±25%) (t=6.77, P < 0.05). The hippocampal structure of the model offspring group was not as clear as that of the control offspring group, the cell morphology was irregular, the cell body was reduced, the cell spacing was significantly increased, and the cell arrangement was loose. The mRNA relative expression level of BDNF in the model offspring group (0.61±0.15) was lower than that in the control offspring group (1.00±0.22) (t=3.79, P < 0.05), and the protein relative expression of BDNF in the model offspring group (0.15±0.10) was also lower than that in the control offspring group (0.50±0.02) (t=28.11, P < 0.05). The escape latency (r=-0.59, P < 0.05) and training times (r=-0.59, P < 0.05) were negatively correlated with the protein relative expression of BDNF, and the correct response rate (r=0.66, P < 0.05) was positively correlated with the protein relative expression of BDNF in the offspring rats.

    Conclusion

    Chronic stress during pregnancy can decrease the learning and memory function and the relative expression of synaptic related protein BDNF in hippocampus of offspring rats, and the decline of learning and memory ability is related to the decline of BDNF protein relative expression.

     

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