丙泊酚对大鼠海马长时程增强诱发的影响

Effect of propofol on induction of long-term potentiation in hippocampal area of rats

目的:长时程增强与学习记忆密切相关,分析丙泊酚对长时程增强的作用有助于了解丙泊酚是如何对认知功能产生影响的。方法:实验于2004-11/2005-03在徐州医学院江苏省麻醉学重点实验室完成。选取SD雄性大白鼠38只,随机分为6组:对照组8只、丙泊酚低浓度组6只、丙泊酚中浓度组6只、丙泊酚高浓度组6只、印防己毒素组6只、印防己毒素加丙泊酚组6只。①给药:对照组不给予任何药物,丙泊酚低、中、高浓度组分别给予丙泊酚10,20,50μmol/L,印防己毒素组给予印防己毒素25μmol/L,印防己毒素加丙泊酚组给予印防己毒素25μmol/L+丙泊酚20μmol/L。各药物加入人工脑脊液配成终浓度,用含有药物的人工脑脊液灌流脑片,给药10min时给予强直刺激,20min后去除药物,恢复正常人工脑脊液灌流。②海马脑片制备:动物麻醉后断头取脑,垂直于海马长轴切取三四片海马脑片,厚度400μm。将海马脑片置于人工脑脊液中孵育2.0～3.0h,然后再全部浸于(32.0±0.5)°C恒温灌流槽内,液面高出脑片表面2mm,不断通入经体积分数为0.95的O2和0.05的CO2混合气体饱和的人工脑脊液,气体流量为200mL/min,人工脑脊液的灌流速度为1.5～2.0mL/min。③群峰电位的记录和长时程增强的诱发:双极电刺激大鼠海马脑片CA3区锥体细胞的谢弗侧支,玻璃微电极置于CA1区锥体细胞层,记录CA1区群峰电位,然后施以100Hz,400串的强直刺激,诱发长时程增强产生。给予强直刺激后群峰电位幅度增加20%、并持续30min以上,即判定为长时程增强发生。整个实验过程中每5min观察记录一次,至少观察60min以上。结果:实验共纳入大鼠38只,全部进入结果分析。①对照组海马脑片长时程增强情况:以强直刺激前脑片的群峰电位幅度为100%计算,对照组脑片强直刺激后其群峰电位幅度显著增高,在60min时为刺激前的(135.35±3.9)%,形成了长时程增强。②丙泊酚各浓度组海马脑片长时程增强情况:预试验显示作为溶剂的脂肪乳剂对长时程增强产生无影响。在强直刺激后60min时,丙泊酚低、中、高浓度组群峰电位幅度分别为刺激前的(124.72±3.9)%,(116.77±2.2)%,(109.39±0.9)%,与对照组相比均有显著性差异(t=2.25,P<0.05;t=4.54,P<0.01;t=6.88,P<0.01)。③印防己毒素组、印防己毒素加丙泊酚组海马脑片长时程增强情况:印防己毒素组在强直刺激后60min时群峰电位幅度为刺激前的(137.57±3.4)%,对长时程增强产生无影响。而印防己毒素加丙泊酚组在强直刺激后60min时,群峰电位幅度为刺激前的(129.32±3.5)%,与丙泊酚中浓度组比较具有明显差异(t=3.02,P<0.05)。④丙泊酚对大鼠海马脑片突触基础传递的影响:丙泊酚低、中浓度组在测试刺激条件下加药20min后,脑片的群峰电位幅度分别为用药前的(98.86±1.5)%和(98.65±1.8)%,与用药前基本相近(t=0.75,P>0.05;t=0.73,P>0.05)。丙泊酚高浓度组脑片的群峰电位幅度明显减小,加药20min时为用药前的(82.37±2.9)%,与用药前相比具有明显差异(t=6.08,P<0.01),撤药后脑片的群峰电位幅度可恢复正常。⑤印防己毒素对大鼠海马脑片突触基础传递的影响:印防己毒素组在测试刺激条件下加入25μmol/L的印防己毒素20min后,脑片的突触基础传递兴奋性有所增高,表现为出现多个群峰电位波,但第一个群峰电位幅度为用药前的(102.35±2.4)%,与用药前相比基本相近(t=0.97,P>0.05)。结论:丙泊酚低、中、高浓度组在给药过程中给予强直刺激后,群峰电位幅度均明显降低,长时程增强的形成受到抑制,且呈明显的剂量相关性,而印防己毒素对长时程增强产生无影响。提示丙泊酚对长时程增强的诱发有阻断作用,可能与激活γ-氨基丁酸(GABA)受体有关。

AIM： It is widely accepted that long term potentiation is correlated with learning and memory. We aimed to analyze the effect of propofol on induction of long term potentiation in order to comprehend the influence of propofol on cognitive function. METHODS： The study was conducted in Jiangsu Provincial Key Laboratory Anesthesiology, Xuzhou Medical College, from November of 2004 to March of 2005. Thirty-eight male SD rats were randomly divided into control group（n=8）, low-concentration propofol group（n=6）, middle- concentration propofol group（n=6）, high-concentration propofol group（n=6）, picrotoxin group （n =6） and picrotoxin ＋propofol group （n =6）, ① Administration： No drugs were used in control group, propofol 10 μmol/L, 20 μmol/L, 50 μmol/L were used in low, middle, high concentration propofol groups respectively, picrotoxin 25 μmol/L was used in picrotoxin group, picrotoxin 25 μmol/L plus propofol 20 μmol/L were used in picrotoxin ＋propofol group. All drugs were dissolved in the artificial cerebrospinal fluid with final concentration and removed to recover the normal perfusion with artificial cerebrospinal fluid 20 minutes after tetanic stimulation was given after slices were perfused with artificial cerebrospinal fluid contained drugs for 10 minutes, ② Preparation of hippocampal slices： The animals were anaesthetized and decapitated, then the 3 or 4 slices （400-μm-thick） were cut from the hippocampus transversely. After 2.0 to 3.0 hours＇ preincubation in artificial cerebrospinal fluid, the hippocampal slices were immersed in instant the perfusion chamber at （32.0±0.5） ℃, and submerged 2 nun under fluid surface. The artificial cerebrospinal fluid was equilibrated with a 95% O2 mixed with 5% CO2 at the flow rate of 200 mL per minute, and the perfusion speed of the artificial cerebrospinal fluid was 1.5 to 2.0 mL per minute. ③Record of population spike potential and induction of long term potentiation： A bipolar tungsten electrode was placed on the Schaeffer collateral-commissural fibers of CA3 region to stimulate; a glass electrode was placed on CA1 pyramidal layer to record population spike potential. Long term potentiation was induced by tetanic stimulation （100 Hz, 400 pulses）, and it was defined successfully as an increase of 20% or greater in the initial population spike amplitude, which could last for more than 30 minutes, population spike potential were recorded every 5 minutes for more than 60 minutes after tetanie stimulation. RESULTS： Totally 38 male SD rats were involved in this experiment, and all of them were analyzed in the result. ① Induction of long term potentiation in the control group： The population spike amplitude increased to （135.35±3.9） % at 60 minutes after tetanic stimulation as population spike amplitude before tetanic stimulation was taken as 100%. Induction of long term potentiation was successful. ②Induction of long term potentiation in each propofol group： The dissolved solution intralipid had no effect on long term potentiation at preliminary test. In low, middle, high concentration groups, population spike amplitudes were （124.72±3.9）%, （116.77±2.2）%, （109.39±0.9）%, decreased significantly as compared with that of control group（t=2.25, P 〈 0.05; t=4.54, P 〈 0.01; t=6.88, P 〈 0.01）. ③lnduction of long term potentiation in picrotoxin group and picrotoxin ＋propofol group： In picrotoxin group, population spike amplitude was （137.57±3.4）% at 60 minutes after tetanic stimulation, and picrotoxin had no remarked role on long term potentiation. In picrotoxin＋propofol group, population spike amplitude was （129.32±3.5）%, decreased significantly as compared with that of middle concentration propofol group（t=3.02, P 〈 0.05）. ④The effect of propofol on baseline synaptic responds： In low and middle concentration propofol groups, population spike amplitudes were （ 98.86±1.5）% and （98.65±1.8）% at 20 minutes after drug administration, similar to that before drug administration（t=0.75, P 〉 0.05; t=0.73, P 〉 0.05）. In high concentration propofol group, population spike amplitude was （82.37±2.9）%, decreased significantly as compared with that before drug administration （t=6.08,P 〈 0.01）. In each propofol group, population spike amplitude could recover after the removal of drugs. ⑥The effect of picrotoxin on baseline synaptic responses： 25 μmol/L picrotoxin could increase synaptic excitability, manifesting as multi-population spike, but the first population spike amplitude was （102.35 ±2,4） % at 20 minutes after drug administration, similar to that before drug administration （t=0.97, P 〉 0.05）. CONCLUSION： In low, middle, high concentration propofol groups, long term potentiation which tetanic stimulation was induced could be inhibited during drug administratio in a dose-dependent manner. Picrotoxin had no remarked role on long term potentiation, indicating that propofol could inhibit the induction of long term potentiation via activating gamma- aminobutyric acid receptor in hippoeampal area of rats.