异氟烷／丙泊酚配伍对轻度认知功能障碍大鼠术后认知功能的影响

Effect of sub-anesthetic doses of isoflurane and propofol on postoperative cognition and underlying mechanism in rats with mild cognitive impairment

目的研究异氟烷（Iso）／丙泊酚（Pro）亚麻醉剂量配伍对轻度认知功能障碍（MCI）大鼠骨科手术后认知功能的影响。方法随机选取健康雄性sD大鼠100只分为模型组和假手术组。假手术组只暴露双侧颈总动脉，模型组造成双侧颈总动脉狭窄。30d后行Morris水迷宫实验，在模型组挑选平均逃避潜伏期与假手术组逃避潜伏期之差占该鼠平均逃避潜伏期比例〈20％的大鼠，即为MCI大鼠。造模第36天，将MCI模型组大鼠随机分为4组（n=18），单纯麻醉组：Iso（1．9％）3h；复合麻醉组：Pro（20mg·kg^-1·h^-1）＋Iso（1％）3h；抑制剂加异氟烷组和抑制剂加复合麻醉组：分别于单纯麻醉和复合麻醉前15min静脉注射钾离子氯离子共转运体2（KCC2）抑制剂己酸二异辛酯（DIOA）30μg。随后行胫骨骨折切开复位内固定手术。假手术组仅切开皮肤后缝合。于术后7d，采用Y迷宫检测术后认知功能变化，尼式染色观察神经元凋亡，免疫荧光观察表达KCC2的γ-氨基丁酸（GABA）能中间神经元-GAD67的数量，采用免疫印迹实验（Western blot）检测海马KCC2蛋白的表达。结果复合麻醉组大鼠新异臂停留时间的比例、海马CA1区存活神经元数量、CA1区GAD67表达GABA能中间神经元的KCC2阳性细胞平均数目以及海马KCC2蛋白表达分别为（52．94±2．04）％、（176．81±13．07）个、（196．72±8．02）个、（80．67±3．46）％，假手术组分别为（53．44±2．45）％、（183．79±10．88）个、（207．45±13．17）个、（81．42±3．59）％，差异均无统计学意义（均P〉0．05），但各指标均显著高于单纯麻醉组的（36．10±1．57）％、（97．21±8．23）个、（158．94±13．47）个、（43．22±4．91）％，差异均有统计学意义（均P〈0．05）；抑制剂加单纯麻醉组、抑制剂加复合麻醉组分别与单纯麻醉组和复合麻醉组比较，上述指标均显著降低（均P〈0．05）。结论与单纯异氟烷相比，丙泊酚与异氟烷亚麻醉剂量配伍对MCI大鼠的术后认知功能无明显影响，维持海马KCC2表达是其重要机制。

Objective To study the effect of sub-anesthetic doses of isoflurane and propofol on the postoperative cognitive function of rats with mild cognitive impairment （MCI） following orthopedic operation. Methods Male sprague-dawley rats （ n = 100） were randomly divided into sham group and model group. Bilateral carotid artery was separated with no bilateral narrowing in the sham operation group while bilateral carotid artery was occluded in model group. After 30 days, Morris Water Maze test were used to select MCI rats. The proportion of the value that average escape latency in model group minused average escape latency in sham group less than 20% was marked as MCI rats. After 36 days, the rats with mild cognitive impairment were randomly divided into 4 groups （n = 18 ）. Iso group： Isoflurane （ 1.9% ） 3 h, combination group： propofol （20 mg· kg^-1 · h^-1） ＋ isofiurane （1%） 3 h. Inhibitor with these two groups were respectively used the selective KCC2 inhibitor-DIOA 30 μg 15 rain before anesthesia. Then rats received an open tibial fracture on the hind paw with an intramedullary fixation. The rats in sham group were only sutured skin incision. At the day 7 postoperatively, the Y maze test were used to test the change of cognition, nissl staining was used to observe the neurons apoptosis. Immunofluoreseence was adopted to observe the neurons expressing GAD67/KCC2 and Western blot were applied to observe the protein level of KCC2 in hippocampus. Results The rate of time spent in novel arm, the number of surviving neurons, the number of neurons expressing GAD67/KCC2 and protein expression levels of KCC2 in hippocampus in combination group were （52. 94 ± 2. 04） %, （ 176. 81 ±13.07 ）, （ 196. 72 ±8.02 ）, （ 80. 67 ±3.46 ） % respectively. The indexes in sham group were （53.44 ±2.45） % , （ 183.79 ± 10. 88 ） , （207.45 ±13.17）, （81.42 ± 3.59 ）% , respectively. There was no significant difference compared with the sham group （all P 〉 0. 05 ）. However these indexes in above two groups were higher than that in Iso group（ （ 36. 10 ±1.57 ） % , （97.21 ± 8.23 ）, （ 158.94 ± 13.47 ）, （43.22 ±4. 91 ） %, all P 〈 0. 05 ）. The indexes in inhibitor with Iso groups, combination group was lower compared with groups without inhibitor respectively （ all P 〈 0. 05 ）. Conclusion Compared with isoflurane, sub-anesthetic doses of isoflurane and propofol has no significant effect on postoperative cognition in rats with MCI, and its mechanism is to maintain KCC2 expression in hippocampus.