兔脑海马c-fos基因表达与异丙酚不同麻醉深度影响的关系(英文)

Relationship between c-fos gene expression in hippocampus in rabbit and various depths of propofol anesthesia

背景:脑海马作为边缘系统的重要组成部分,参与情绪、感觉活动、学习记忆功能,麻醉状态下对其产生影响。目的:利用异丙酚靶控输注精确控制麻醉深度,观察不同麻醉深度下兔脑海马不同区域c-fos基因的表达水平,了解异丙酚发挥中枢抑制的作用位点。设计:随机对照观察。单位:深圳市第二人民医院麻醉科,华中科技大学同济医学院附属协和医院麻醉科。材料:实验于2000-05/2001-06在华中科技大学同济医学院神经生物学实验室完成。选取日本大耳兔30只,随机分为空白对照组、浅麻醉组、深麻醉组,10只/组。方法:全部动物行颈外静脉、股动脉穿刺置管。以相应靶控血药浓度进行靶浓度控制输注,严格控制各组麻醉深度。浅麻醉组给予异丙酚靶控血药浓度为(9.28±0.12)m g/L,深麻醉组给予异丙酚靶控血药浓度为(11.63±0.29)m g/L,达到所需麻醉状态30m in 后两组动物断头处死,空白对照组行耳缘静脉注射空气造成空气栓塞后处死。各组常规进行连续冠状切片,片厚7μm ,每隔100μm 取1张,进行原位杂交检测脑海马CA1区、CA3区和齿状回区的c-fos m RNA 表达水平。每只兔随机取5张切片,光镜下分别选取各区15～20个视野进行拍摄,计算平均吸光度与平均灰度值。灰度分为256个等级,阳性率越高,灰度值越低。主要观察指标:①不同麻醉深度下兔脑海马CA1区及齿状回区原位杂交检测结果。②不同麻醉深度下兔脑海马CA1区、CA3区和齿状回区的平均灰度值。结果:30只兔全部进入结果分析。①不同麻醉深度下兔脑海马CA1区原位杂交检测结果:空白对照组可见深浅不一、疏密不等的c-fos阳性细胞,染色呈棕色,分布稀疏;浅麻醉组可见中等着色的c-fos阳性神经元,分布密集;深麻醉组锥体细胞胞浆染色明显加深,分布也更密集。②不同麻醉深度下兔脑海马齿状回区原位杂交检测结果:浅麻醉组阳性细胞染色呈深棕褐色,染色强烈,细胞核无色透明,呈空泡状;深麻醉组出现大量密集的c-fos阳性表达。③不同麻醉深度下兔脑海马各区的平均灰度值:与空白对照组CA1区及齿状回区比较,浅麻醉组和深麻醉组均明显降低犤(168±5),(80±7),(59±5)%,P 均<0.05;(163±8),(103±15),(67±6)%,P <0.05,P <0.01犦,并且深麻醉组均比浅麻醉组下降显著(P <0.01);各组CA3区基本相似。结论:①异丙酚麻醉随深度的增加,其兔脑海马c-fos基因表达升高。②麻醉后兔脑海马CA1区及齿状回区的平均灰度值明显下降,深麻醉时更为显著,但CA3区无明显变化。提示异丙酚的中枢抑制作用部位存在区域性差异,由此推断脑海马CA3区可能不是异丙酚中枢作用的位点。

BACKGROUND： As an important part of systemalimbica, hippocampus involves in emotion, perceiving and learning memory and can be affected by anesthesia. OBJECTIVE： With target controlled infusion of propofol, the depth of anesthesia was well controlled. And under anesthesia in various depths, c- fos gene expressions in different regions of hippocampus in rabbits were detected to find the target site for central nervous inhihition by propofol. DESIGN ：It was a randomized controlled study. SETTING：Department of Anesthesiology ,Shenzhen Second People＇s Hospital; Department of Anesthesiology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology. MATERIALS： The experiment was conducted in the Neurobiological Laboratory of Tongji Medical College, Huazhong University of Science and Technology from May 2000 to June 2001. Thirty Japanese white rabbits were selected and randomly divided into control group, light anesthesia group and deep anesthesia group, with 10 rabbits in each group. METHODS：Intravenous cannulas were placed in external jugular vein （EJV） and femoral artery in all animals. According to the propofol plasma concentration, the infusion of propofol and the depths of anesthesia were well controlled. In light anesthesia group, the plasma concentration of propofol was （9.28±0.12）mg/L. In deep anesthesia group, the plasma concentration of propofol was （ 11.63 ±0.29）mg/L. Thirty minutes after being anesthetized, the animals in the two experimental groups were decapitated and the animals in control group were killed by air embolism through ear vein. Coronal sections were sliced continuously, in thickness of 7 μm and 1 slice in 100 μm tissue was selected. In situ hybridization was performed to detect the c-fos mRNA in Area CA1, CA3 and dentate gyrus of the hippocampus. In each rabbit, 5 sections were selected randomly. Under a light microscope, photos were taken in 15-20 fields. And then average absorbency and average grayscale were calculated. The grayscale scores were classified as 256 scales. A lower grayscale score indicated a higher positive rate. MAIN OUTCOME MEASURES： ①Under various depths of anesthesia, in situ hybridization results of Area CA1, CA2 and dentate gyrus of the hippocampus in rabbits were assessed. ②Under various depths of anesthesia, average grayscale scores of Area CA1, CA2 and dentate gyrus of the hippocampus in rabbits were calculated. RESULTS：Thirty rabbits entered the statistical analysis procedure. ①Under various depths of anesthesia, in situ hybridization results of Area CA 1 of the hippocampus in rabbits： In control group, brown, sparse or dense, light-stained or deep-stained c-fos positive cells could be observed. In light anesthesia group, dense, moderately stained c-fos positive neurons could be observed. In deep anesthesia group, cells were denser with deeper stained cytoplasma. ②Under various depths of anesthesia, in situ hybridization results of dentate gyms of the hippocampus in rabbits： In light anesthesia group, positive cells were strongly stained in deep brown with transparent and vacuolar nuclei. In deep anesthesia group, a large number of c-fos positive cells in great dense could be observed. ③Under various depths of anesthesia, grayscale scores of different regions of the hippocampus in rabbits： Compared with control group, grayscale scores of Area CA1 and dentate gyms of the hippocampus were significantly decreased in both light and deep anesthesia groups [（168±5）,（80±7）,（59±5）%,P 〈 0.05;（163±8）, （103±15）,（67±6）%,P 〈 0.05,P 〈 0.011. This was more significant in deep anesthesia group than in light anesthesia ,group （P 〈 0.01 ）. For Area CA3, the grayscale scores in each group were similar. CONCLUSION： ①With the increasing depth of propofol anesthesia, e-fos gene expression is increased in hippocampus in rabbits. ②After anesthesia, the average grayscale score of Area CA1 and dentate gyms of the hippocampus are significantly decreased, and this is more significant after deep anesthesia. However, there is no significant change in Area CA3. This indicates that the central inhibitory receptor sites of propofol are various in different brain regions, which supposes that the Area CA3 is not the central receptor sites of propofol.