七氟烷对发育期小鼠海马低亲和力神经营养因子p75和远期认知功能的影响

Effects of sevoflurane on p75^(NTR) in hippocampus and long-term cognitive function in neonatal mice

目的探讨不同体积分数七氟烷麻醉对发育期小鼠海马脑源性营养因子和远期认知功能的影响。方法选取出生后7d的C57BL/6雄性小鼠(由南京大学模式动物研究所提供)182只。取60只小鼠按随机数字表法分为1.0最低肺泡有效浓度(MAC)组、0.5 MAC组和对照组,每组20只,分别给予体积分数为0.025 0(1.0 MAC)和0.012 5(0.5 MAC)七氟烷吸入麻醉或仅吸入体积分数为0.4的O_2,4h后采用Western印迹法测定海马组织天冬氨酸特异性半胱氨酸蛋白酶-3(Casp_ase-3)蛋白质含量,ELISA法测定外周血S100钙结合蛋白β(S-100β)和海马组织脑源性营养因子前体(pro-BDNF)、低亲和力神经营养因子p75(p75^(NTR))蛋白质含量。另取72只小鼠按随机数字表法分为1.0MAC组、0.5MAC组和对照组,每组24只,分别给予体积分数为0.025 0(1.0 MAC)和0.012 5(0.5 MAC)七氟烷吸入麻醉或仅吸入体积分数为0.4的O_2,4、12周后行Morris水迷宫实验,记录大鼠在90s内穿越原平台位置次数和平台所在象限停留时间。其余50只小鼠分别于麻醉前、吸入1.0 MAC七氟烷麻醉1或2h后、吸入0.5MAC七氟烷麻醉1或2h后5个时间点,每个时间点取10只小鼠测定动脉血pH值、动脉血氧分压(p_aO_2)、动脉血二氧化碳分压(p_aCO_2)和动脉血氧饱和度(S_aO_2)。结果1.0 MAC组和0.5 MAC组外周血S-100β水平,1.0 MAC组海马组织Casp_ase-3、pro-BDNF、p75^(NTR)蛋白质含量,以及0.5 MAC组海马组织Casp_ase-3、p75^(NTR)蛋白质含量均显著高于对照组(P值均<0.05);1.0 MAC组外周血S-100β水平显著高于0.5 MAC组(P<0.05),海马组织Casp_ase-3、pro-BDNF、p75^(NTR)蛋白质含量与0.5 MAC组间的差异均无统计学意义(P值均>0.05)。麻醉后4周,1.0 MAC组和0.5 MAC组小鼠的穿越原平台位置次数和平台所在象限停留时间均显著少于对照组同时间(P值均<0.05)。麻醉后12周,1.0 MAC组和0.5 MAC组小鼠的平台所在象限停留时间均显著少于对照组同时间(P值均<0.05),1.0 MAC组小鼠的穿越原平台位置次数显著少于0.5 MAC组和对照组同时间(P值均<0.05)。麻醉前、七氟烷1.0 MAC麻醉1或2h、七氟烷0.5MAC麻醉1或2h小鼠的动脉血pH值、p_aO_2、p_aCO_2、S_aO_2的差异均无统计学意义(P值均>0.05)。结论七氟烷可损伤发育期海马神经元,使海马p75^(NTR)含量发生改变。降低吸入七氟烷的体积分数可减轻成年后学习记忆障碍。

Objective To investigate whether brain-derived neurotrophic factor and its receptor biosynthesis in hippocampus can be regulated after different concentrations of sevoflurane anesthesia, and to observe the effect of sevoflurane on long-term cognitive function of mice. Methods A total of 182 C57BL/6 neonatal mice （7 days postnatal, supplied by Institute of Model Animal, Nanjing University） were used in this study. Of them, 132 mice were divided to 3 groups （n = 44 each） using a random number table which were exposed to sevoflurane （1.0 minimum alveolar concentration [MAC] or 0.5 MAC） or 40 % 02 for 2 h. Twenty mice in each group were sacrificed 4 h after anesthesia for the blood determination of S-100 calcium-binding protein [β （S-100β） using enzyme-linked immuno sorbent assay （ELISA）. Hippocampus were dissected for the measurement of protein contents of Caspase-3 （Western Blotting）, precursor of brain-derived neurotrophic factor （pro-brain- derived neurotrophic factor [BDNF], ELISA） and p75NTR （ELISA）. Morris water maze was performed 4 and 12 weeks after anesthesia in the remaining mice to evaluate their cognitive function. Other 50 mice were used for artery blood analysis （potential of hydrogen [pH], arterial partial pressure of oxygen E paO21, arterial partial pressure of carbon dioxide [PaCO2], and arterial oxygen saturation [SaO2]） during sevoflurane （1.0 MAC or 0.5 MAC） anesthesia （0, 1 h and 2 h）. Results The S-10015 in mouse peripheral blood and the protein contents of Caspase-3 and p75^NTR in hippocampus in 1.0 MAC group and 0.5 MAC group were significantly higher than those in control group （all P〈0.05）. Moreover, the pro-BDNF content in hippocampus in 1. 0 MAC group was significantly higher than that in control group （P〈0.05）. The S-100β in 1.0 MAC group was significantly higher than that in 0.5 MAC group （ P 〈 0.05）, but there was no statistical difference in the protein contents of Caspase-3, pro-BDNF or p75YNTR between the two groups （all P〈0.05）. The time on island and frequency across island in 1.0 MAC group and 0.5 MAC group were significantly decreased 4 weeks after anesthesia （all P〈0.05）. The time on island in 1.0 MAC group and 0.5 MAC group was significantly less than that in control group （both P〈0.05）. Latency in 1.0 MAC group was significantly longer than that in 0.5 MAC group and control group （both P〈0.05）. pH, p2O2, paCO2 and SaO2 were stable during anesthesia （all P 〈 0. 05）. Conclusion Sevoflurane can cause hippocampal neuronal damage during developmental phase and change the content of p75^NTR in hippocampus. Reducing the concentration of drugs can alleviate adult learning and memory disorders.