单次短期七氟醚暴露联合母婴分离对新生大鼠远期认知功能的影响

Effects of maternal separation combined with a single short-term sevoflurane exposure on the long-term cognitive function of neonatal rats

中文关键词: 七氟醚  母婴分离  认知功能  Na⁺-K⁺-2Cl^-同向转运蛋白  边缘系统-下丘脑-垂体-肾上腺轴  大鼠

英文关键词: Developing brain  Sevoflurane  Material separation  Na⁺-K⁺-2Cl^- cotransporter  Limbic system-hypothalamus-pituitary-adrenal axis  Rats

中文摘要:

      
目的 探讨单次短期七氟醚暴露联合母婴分离对新生大鼠发育期大脑远期认知功能的影响及可能机制。
方法 选择出生后第6天的雄性SD新生大鼠64只。采用随机数字法将新生大鼠分为五组：对照组（C组，n=13）、七氟醚组（S组，n=13）、母婴分离组（M组，n=12）、七氟醚+母婴分离组（SM组，n=13）和布美他尼组（SMB组，n=13）。C组自由饮水饮食。S组于出生后第6天暴露于2.1%七氟醚1 h。M组于出生后第10天母婴分离3 h。SM组于出生后第6天暴露于2.1%七氟醚1 h，并于出生后第10天母婴分离3 h。SMB组于出生后第6天七氟醚暴露前腹腔注射Na⁺-K⁺-2Cl^-同向转运蛋白（NKCC1）抑制剂布美他尼1.82 mg/kg，后暴露于2.1%七氟醚1 h，于出生后第10天母婴分离3 h。于大鼠出生后第44天行旷场实验，记录运动总距离和穿越中央格次数。出生后第54或55天行新物体识别实验，记录大鼠对新物体和旧物体的探索时间，计算辨别指数。出生后第84～86天行场景性条件性恐惧实验，记录场景性恐惧测试阶段和条件性恐惧测试阶段的僵直时间。行为学测试结束后取大鼠下丘脑组织测定促肾上腺皮质激素释放激素（CRH）和海马组织测定盐皮质激素受体（MR）mRNA表达量。
结果 与C组比较，SM组辨别指数明显降低（P<0.05），场景性恐惧测试阶段僵直时间明显缩短（P<0.05），下丘脑组织CRH mRNA表达量明显升高（P<0.05），海马组织MR mRNA表达量明显降低（P<0.05）。与SM组比较，SMB组辨别指数明显升高（P<0.05），下丘脑CRH mRNA表达量明显降低（P<0.05），海马组织MR mRNA表达量明显升高（P<0.05）。C组、S组和M组新物体识别实验的辨别指数、场景性恐惧测试阶段的僵直时间、下丘脑组织CRH和海马组织MR mRNA表达量差异均无统计学意义。
结论 新生大鼠单次短期七氟醚暴露联合母婴分离可通过活化NKCC1，增加边缘系统-下丘脑-垂体-肾上腺轴的活性，导致大鼠的远期认知功能损伤。

英文摘要:

      
Ojective To investigate the effects and possible mechanisms of maternal separation combined with a single short-term exposure of sevoflurane on the cognitive function of the developing brain.
Methods Sixty-four male SD rats, postnatal day 6, were divided into 5 groups by random number method: control group (group C, n = 13), sevoflurane group (group S, n = 13), maternal separation group (group M, n = 12), sevoflurane + maternal separation group (group SM, n = 13) and bumetanide group (group SMB, n = 13). The rats of group C had free access to water and diet. The rats of group S were exposed to 2.1% sevoflurane for 1 hour on day 6. The rats of group M were subjected to maternal separation for 3 hours on day 10. The rats of group SM were exposed to 2.1% sevoflurane for 1 hour on day 6 and maternal separation for 3 hours on day 10. The rats of group SMB were intraperitoneally injected with bumetanide (Na⁺-K⁺-2Cl^-cotransporter blocker, 1.82 mg/kg) before 2.1 % sevoflurane exposure for 1 hour on day 6 and maternal separation for 3 hours on day 10. The rats were subjected to the open field test on day 44 after birth, the total travelled distance and the time of staying in the central area were recorded. The new object recognition test was performed on day 54 or day 55 after birth, the exploration time for new object and old object were recorded, and then the discrimination indexes were calculated. The fear conditioning test was performed on day 84-86 after birth, the freezing time of contextual fear conditioning test and cue fear conditioning test were recorded separately. After the behavioral experiments, the tissues of hypothalamus were taken to measure the expression of corticotropin-releasing hormone (CRH) mRNA, and tissues of hippocampus were taken to measure the expression of mineralocorticoid receptor (MR) mRNA.
Results Compared with group C, the discrimination index was significantly reduced in group SM (P < 0.05), the freezing time was significantly reduced (P < 0.05), the expression of CRH mRNA in the hypothalamus was significantly increased (P < 0.05), and the expression of MR mRNA in the hippocampus was significantly reduced (P < 0.05). Compared with group SM, the discrimination index was significantly increased in group SM (P < 0.05), the expression of CRH mRNA in the hypothalamus was significantly reduced (P < 0.05), and the expression of MR mRNA in the hippocampus was significantly increased (P < 0.05). There were no significant differences in the results of the discrimination index in the new object test recognition, the freezing time in the contextual fear conditioning test, the expression of CRH mRNA in the hypothalamus and MR mRNA in the hippocampus among groups C, S and M.
Conclusion A single short-term sevoflurane exposure of neonatal rats combined with maternal separation could cause the activation of NKCC1 and increased activity of the limbic system-hypothalamic-pituitary-adrenal axis, leading to the long-term neurocognitive impairment.