|摘要||目的 探讨褪黑素对异氟醚麻醉大鼠海马胆碱乙酰基转移酶(ChAT)的影响.方法雄性SD大鼠60只,体重390～440 g,采用随机数字表法,将大鼠随机分为5组(n=12):对照组(C组)、1%异氟醚组(Ⅰ组)、1%异氟醚+褪黑素组(IM组)、2%异氟醚组(J组)和2%异氟醚+褪黑素组(JM组).IM组和JM组腹腔注射褪黑素10 mg/kg,1次/d,连续7 d,C组、Ⅰ组和J组给予等容量生理盐水.Ⅰ组、IM组第7天吸入1%异氟醚4 h,J组、JM组第7天吸入2%异氟醚4 h.于麻醉次日行Morris水迷宫实验,测试逃避潜伏期及原平台象限探索时间;水迷宫实验结束后取血浆及脑组织,采用ELISA法测定血浆褪黑素浓度,Western blot法测定海马ChAT表达水平,采用比色法测定海马ChAT活性,采用免疫荧光法测定海马CA1区和齿状回的ChAT阳性神经元数量.结果 与C组比较,Ⅰ组血浆褪黑素浓度、ChAT表达水平和活性降低(P＜0.01);J组逃避潜伏期延长,原平台象限探索时间缩短,血浆褪黑素浓度、ChAT表达水平和活性降低(P＜0.05或0.01).与Ⅰ组比较,IM组逃避潜伏期缩短,原平台象限探索时间延长,褪黑素浓度升高,ChAT表达水平和活性升高(P＜0.05或0.01).与J组比较,JM组逃避潜伏期缩短,褪黑素浓度升高,ChAT活性升高(P＜0.05或0.01).海马CA1区和齿状回的ChAT阳性神经元数量与ChAT表达水平变化一致.结论 褪黑素可减轻异氟醚麻醉对ChAT表达水平及活性的抑制,从而改善异氟醚麻醉后大鼠的认知功能.
Objective To investigate the effects of melatonin on choline acetyltransferase (ChAT) in rat hippocampus after isoflurane anesthesia. Methods Sixty male SD rats weighing 390 - 440 g were randomized into 5 groups (n = 12 each): control group (group C), 1% isoflurane group (group Ⅰ), 1% isoflurane + melatonin group (group IM) , 2% isoflurane group (group J) and 2% isoflurane + melatonin group (group JM) . In IM and JM groups, melatonin 10 mg/kg was administered intraperitoneally once a day for 7 consecutive days, while equal volume of normal saline was given intraperitoneally instead of melatonin in C, I and J groups. Groups Ⅰ and IM inhaled 1% isoflurane and groups J and JM 2% isoflurane for 4 h on 7th day. All the rats underwent Morris water maze test on the day after anesthesia for assessment of learning and memory ability (escape latency and probe time) . The training test was performed 4 times a day for S days. Six rats randomly selected from each group were sacrificed the end of the test. The blood samples were collected for detection of plasma melatonin level by ELISA.The brain tissues were removed for determination of the expression and activity of ChAT in hippocampus by Western blot or colorimetric assay. The left rats were selected and sacrificed for determination of the number of ChAT positive neurons in hippocampal CA1 region and entate gyrus by immunofluorescence. Results The plasma melatonin level and expression and activity of ChAT were significantly lower in group I than in group C ( P ＜ 0.01) . The escape latency was significantly longer, the probe time was significantly shorter, and the plasma melatonin level and expression and activity of ChAT were significantly lower in group J than in group C ( P ＜ 0.05 or 0.01) . The escape latency was significantly shorter, the probe time was significantly longer, and the plasma melatonin level and expression and activity of ChAT were significantly higher in group IM than in group Ⅰ ( P ＜ 0.05 or 0.01). The escape latency was significantly shorter and the plasma melatonin level and ChAT activity were significantly higher in group JM than in group J ( P ＜ 0.05 or 0.01) . The results of immunofluorescent staining showed that the number of ChAT positive neurons in hippocampal CA1 region and dentate gyrus wag consistent with the changes in the measured ChAT expression. Conclusion Melatonin can reduce isoflurane-mediated inhibition of ChAT expression and activity and thus improve spatial memory impaired by isoflurane anesthesia in rats.|