建立新生大鼠吸入麻醉模型及异氟醚对其海马凋亡的影响

目的:建立新生大鼠吸入麻醉模型并探讨吸入麻醉药异氟醚对其海马凋亡的影响。方法:Penlon Prima SP麻醉机、异氟醚挥发罐及自制带进出气口的麻醉小室。共55只7日龄的SD大鼠用于实验。将其中35只大鼠随机分为7组(n=5)。实验组(Ⅰ-Ⅵ组)异氟醚挥发罐刻度分别为0.125%,0.25%,0.5%,1%,1.5%,2%;新生大鼠置于自制密封麻醉小室内,分别通入含上述异氟醚浓度的混合气体。对照组(第Ⅶ组)给予未混合异氟醚的30%的氧气。将小室安放于37℃恒温箱内。调节气体流量2L/min。实验组于通入气体5,10,15,30,90,180,360 min(T1-7)时于小室出口处抽取10mL气体,采用气相色谱法测定麻醉小室内异氟醚浓度。于通入气体360 min(T7)自新生大鼠左心室采血行血气分析;另取SD大鼠20只,随机分为对照组(C组,n=10),1.5%异氟醚组(I组,n=10),按上述方法建立异氟醚吸入麻醉模型,麻醉结束后2h处死大鼠,采用免疫组织化学法观察C组和I组大鼠大脑海马区Active caspase-3的表达。结果:①麻醉小室出口异氟醚浓度(Y)与麻醉机挥发罐异氟醚浓度(X)的直线回归方程为Y=1.5472X-0.0575(r=0.9993)。②血气分析结果显示:Ⅰ-Ⅵ组与Ⅶ组血气分析组间差异无统计学意义(P0.05)。③免疫组化结果显示:与C组相比,I组大鼠海马Active caspase-3明显增加,差异有统计学意义(P0.05)。结论:通过麻醉机、异氟醚挥发罐及自制密封带进出气口的麻醉小室成功建立了新生大鼠异氟醚麻醉模型;为进一步研究异氟醚及相关吸入麻醉药对突触发生期的神经毒性提供了实验基础。

Establish a Model for Evaluating the Effects of Isoflurane on the Apotosis in Hippocampal Cells of Neonatal Rats

Objective: To establish a model of applying inhalation anesthesia for neonatal rats and evaluate the reliability of this model,then to investigate the apotosis of isoflurane on the hippocampal cells of neonatal rats.Methods: Connected the anaesthesia mechine equipped with a calibrated vaporizer of isoflurane,to the domestic air-tight chamber with an air-scoop and an air-out to form anesthesia pipeline.55 seven-day-old male and female Sprague Dawley rats were used for the experiment.35 SD rats were divided into seven randomly selected groups(n=5).According to the different concentrations of the isoflurane in the air stream,the mixture gas were divided into six groups as follows:(1)Experiment groups: 0.125%(groupⅠ),0.25%(groupⅡ),0.5%(group Ⅲ),1%(group Ⅳ),1.5%(group Ⅴ),2%(groupⅥ);(2) Control groups(group Ⅶ): air stream composed only of 70% nitrogen mixed 30% oxygen.The experimental rats were exposed to isoflurane in the air-tight chamber,which was placed inside a constant temperature incubator,setting up at 37℃.Isoflurane was continuously delivered into chamber through inlet with an air stream(70% nitrogen +30% oxygen) containing desired anesthetic concentration using a calibrated vaporizer(before each test,the mixture gas was flow through the chamber several minutes).The mixture gas samples in chamber of each experiment group were taken at 5min,10min,15min,30min,90min,180min and 360min after isoflurane intervention.Isoflurane concentrations in the chamber were measure by gas chromatograph.The samples of both the experiment groups and the control group were taken at the end of anesthesia for blood gas analysis.The other 20 seven-day-old SD rats were divided into two randomly selected groups(n=10),control group and 1.5%isoflurane group,to establish the isoflurane inhalation model as above-mentioned,and using immunohistochemistry to detect the expression of Active caspase-3.Results: ①Whereafter the isoflurane concentrations in them became linearly dependented,the linear dependence equation of them is Y=1.5472X-0.0575(r=0.9993).②The values of pH,PO2,PCO2,BE,HCO3-and SaO2 between experiment groups and control groups had no significant difference at the end of anesthesia time point(P>0.05).③Compared with C group,the Active caspase-3 immono-stain positive cells were significantly increased in the I group in the hippocampal areas(P<0.05).Conclusion: The model for evaluating the effects of isoflurane on neonatal rats is successfully established using an anaesthesia apparatus,a calibrated vaporizer of isoflurane,a domestic air-tight chamber with an air-scoop and an air-out,which may provided experimental basis for further studies of inhalation anesthetics such as isoflurane on the triggering of synaptic neurotoxicity.