氯胺酮诱导不同年龄大鼠脑海马区热休克蛋白70表达的量效依赖性

背景研究证实,热休克蛋白70家族的蛋白质对细胞有保护作用.氯胺酮可导致患者出现幻觉、谵妄等精神症状,可对大鼠边缘系统神经元产生损害,在这些受损的神经元内用免疫组化染色方法可检测到热休克蛋白70的表达.目的观察不同剂量的氯胺酮在不同年龄大鼠海马中诱导热休克蛋白70的表达,探讨氯胺酮对神经损害的作用.设计随机对照实验.单位四川大学华西医院麻醉学与危重医学教研室.材料实验于2000-01/05在四川大学华西医院麻醉学与危重医学教研室实验室完成.选择SD大鼠70只,雌雄不限,清洁级.方法35只成年SD大鼠随机分为对照组和氯胺酮20.0,40.0,60.0,80.0,100.0,120.0 mg/kg 6个实验组,每组5只,分别给予生理盐水和氯胺酮20.0,40.0,60.0,80.0,100.0,120.0 mg/kg腹腔内注射.另取0～10,11～20,21～30,31～45,46～60,61～90,91～120 d年龄阶段大鼠35只,每个年龄段5只,分别腹腔内注射氯胺酮80.0 mg/kg.给药后正常喂养24 h后,在麻醉状态下快速断头取脑,于海马取5 μm冠状切面切片,应用免疫组化染色检测大鼠海马中热休克蛋白70的表达.主要观察指标大鼠海马热休克蛋白70阳性细胞百分率、密度和灰度值.结果70只大鼠均进入结果分析.①不同剂量氯胺酮对大鼠脑海马区热休克蛋白70表达对照组,20.0,40.0,60.0,80.0,100.0,120.0mg/kg剂量氯胺酮组诱导热休克蛋白70表达的阳性细胞密度分别为0,8.12±1.82,27.07±5.98,45.35±5.84,78.51±7.34,74.16±8.17,60.84±6.27.可见氯胺酮剂量在80.0 mg/kg以内,随剂量的增加,热休克蛋白70阳性细胞密度显著增加(P＜0.01);氯胺酮剂量在80.0mg/kg以上,随剂量的增加,热休克蛋白70阳性细胞密度显著降低(P＜0.01).②氯胺酮对不同年龄段大鼠脑海马区热休克蛋白70表达20 d以下的幼鼠神经细胞热休克蛋白70阳性细胞密度为0;在21～30 d,31～45 d,46～60 d,61～90 d大鼠海马中热休克蛋白70阳性细胞密度分别为34.17±6.18,55.42±4.80,78.51±7.34,83.16±11.10,与前一年龄组相比,随着年龄的增加,热休克蛋白70阳性细胞密度显著增加(P＜0.01);61～90 d和91～120 d大鼠海马中热休克蛋白70阳性细胞密度分别为83.16±11.10和85.83±9.33(P＞0.05).结论氯胺酮可诱导热休克蛋白70在大鼠脑海马区表达,提示海马区的神经元可能受到损害;随剂量的增加,其损害作用加强;氯胺酮对成年大鼠的脑损害作用大于幼鼠.

Dose-effect dependence of ketamine in inducing the expression of heat shock protein 70 in the hippocampus of rats of different ages

BACKGROUND: It is proved that protein of heat shock protein 70 family has protective effects on cells. Ketamine can cause psychiatric symptoms such as illusion and delirium in patients, which can damage neurons of the limbic system in rats. The expression of heat shock protein 70 can be detected in the damaged neurons with immunohistochemical staining.OBJECTIVE: To observe the expression of heat shock protein 70 induced by ketamine in the hippocampus of rats of different ages and probe into the damaging effect on nerves.DESIGN: Randomized controlled trial.SETTING: Department of Anesthesiology and Critical Care Medicine,Huaxi Hospital of Sichuan University.MATERIALS: The experiment was conducted in the laboratory of the Department of Anesthesiology and Critical Care Medicine, Huaxi Hospital of Sichuan University, from January to May 2001. Totally 70 SD rats,weighing 25 to 285 g, of either gender and clean grade, were recruited.Thirty-five adult SD rats were randomly divided into control group and ketamine groups of 20.0, 40.0, 60.0, 80.0, 100.0 and 120.0 mg/kg with 5 rats in each group. The rats in each group were injected intraperitoneally with normal saline and 20.0, 40.0, 60.0, 80.0, 100.0 and 120.0 mg/kg of ketamine, respectively. Another 35 rats aged 0-10, 11-20, 21-30, 31-45,46-60, 61-90 and 91-120 days, 5 rats in each age stage, were given intraperitoneal injection of 80.0 mg/kg ketamine. After 24-hour survival time, the animals were put to death and their brains were removed. 5 μm-thick coronal sections of the hippocampus were cut on a vibratome. The expression of heat shock protein 70 was detected in the hippocampus of rats of different ages with immunohistochemical staining.MAIN OUTCOME MEASURES: Positive cellular percentage, density and grayscale of heat shock protein 70 in the rats' hippocampus.of different doses of ketamine on the expression of heat shock protein in rat hippocampus: In control group, the cellular density of heat shock protein 70 expression induced by 20.0, 40.0, 60.0, 80.0, 100.0 and 120.0 mg/kg of ketaminewas0, 8.12±1.82, 27.07±5.98, 45.35±5.84, 78.51±7.34,74.16±8.17 and 60.84±6.27, respectively. It indicated that when ketamine was under 80.0 mg/kg, the cellular density of heat shock protein 70 in creased significantly with the dose increase (P ＜ 0.01). When ketamine was over 80.0 mg/kg, the cellular density significantly decreased with the dose tamine in the hippocampus of rats of different ages: The density of positive nerve cells of heat shock protein 70 in young rats aged under 20 days was 0; the density of young rats aged 21-30 days, 31-45 days, 46-60 days and 61-90 days was 34.17±6.18, 55.42±4.80, 78.51±7.34 and 83.16±11.10,respectively. Compared with that of the first age group, with the increased age, the density of positive cells of heat shock protein 70 was significantly increased (P ＜ 0.01); it was 83.16±11.10 and 85.83±9.33 in the hippocampus of rats aged 61-90 days and 91-120 days, re spectively (P ＞ 0.05).CONCLUSION: Ketamine can induce the expression of heat shock protein 70 in the hippocampus of rats, indicating that neurons of the hippocampus may be damaged; with the increase of dose, its damaging effect is enhanced.The damage of ketamine is greater in adult rats than in young rats.