吡拉西坦影响幼年大鼠慢性癫痫与学习记忆复合动物模型脑海马乙酰胆碱含量和胆碱乙酰转移酶活性变化的量效作用(英文)

Effects of piracetam on chronic epilepsy in childhood rats and quantitative effects on content of acetylcholine and activity of cholinacetyltranslase in hippocampus in learning-memory complex animal model

背景:脑内投射至海马结构的胆碱能系统与学习记忆有关。吡拉西坦具有保护和修复大脑神经细胞的作用,可抵抗因物理因素、化学因素所致的脑功能损伤,改善学习记忆能力。目的:制备幼年慢性癫痫与学习记忆复合动物模型,观察大鼠脑海马乙酰胆碱含量、胆碱乙酰转移酶活性变化以及吡拉西坦的干预效应。设计:随机对照实验,非盲法评估。单位:河北医科大学第二医院儿科,河北医科大学中医药研究院。材料:实验于2004-07/12在河北医科大学及河北师范大学生命科学学院完成。选择Wistar幼年大鼠50只,清洁级,雌雄各半。方法:肌注马桑内脂注射液复制大鼠慢性癫痫大发作模型。每间隔3天重复肌注1次,在造模期间连续3次出现后肢站立的全身阵挛性惊厥或站立伴摔倒或全身强直-阵挛性发作的动物改为每隔14天肌注1次。选取10只大鼠为正常对照组,不进行造模,其余40只大鼠造模3个月时随机分为4组:吡拉西坦2.4g/L组、吡拉西坦4.8g/L组、苯妥英钠6g/L+吡拉西坦4.8g/L组、造模组,每组10只。各组于造模3个月开始连续灌胃给药,1次/d,10mL/kg。吡拉西坦2.4g/L,4.8g/L组分别灌服吡拉西坦混悬液2.4g/L,4.8g/L;苯妥英钠6g/L+吡拉西坦4.8g/L组灌服苯妥英钠6g/L及吡拉西坦悬浮液4.8g/L。造模组、正常对照组灌服10mL/kg生理盐水。用药1个月后进行相关指标检测。Morris水迷宫测试癫痫大鼠发现平台时间及搜索距离,连续测试3d,2次/d。水迷宫实验结束后,断头取脑,测定双侧海马乙酰胆碱含量,胆碱乙酰转移酶、乙酰胆碱脂酶活性用放射免疫法。主要观察指标:①Morris水迷宫测试各组大鼠发现平台时间及搜索距离。②各组大鼠海马乙酰胆碱含量,胆碱乙酰转移酶、乙酰胆碱脂酶活性。结果:50只大鼠全部进入结果分析。①各组大鼠搜索平台时间比较:造模组各组次相应平均搜索时间比正常对照组有不同程度的增加(63±11)s,(40±8)s;(61±9)s,(38±7)s;(57±8)s,(36±9)s;(55±11)s,(33±10)s;(52±7)s,(30±9)s;(49±9)s,(27±6)s,P<0.01。苯妥英钠6g/L+吡拉西坦4.8g/L组,吡拉西坦4.8g/L组6次搜索时间均比造模组有不同程度的减少(44±9)s,(45±9)s;(43±9)s,(42±8)s;(42±7)s,(42±7)s;(40±9)s,(39±9)s;(38±7)s,(35±9)s;(35±6)s,(34±8)s,t=2.352～4.029,P<0.05～0.01。各给药组内随训练次数增加平均搜索时间逐渐减少。②各组大鼠平均搜索距离比较:造模组各组次相应平均搜索距离较正常对照组明显增加(793±74)cm,(420±81)cm;(763±89)cm,(418±57)cm;(690±67)cm,(382±69)cm;(623±81)cm,(356±71)cm;(592±98)cm,(330±69)cm;(550±54)cm,(301±97)cm,P<0.01。苯妥英钠6g/L+吡拉西坦4.8g/L组,吡拉西坦4.8g/L组6次平均搜索距离均比造模组有不同程度的减少(586±91)cm,(510±89)cm;(566±70)cm,(497±76)cm;(521±84)cm,(455±56)cm;(480±74)cm,(421±63)cm;(437±51)cm,(396±79)cm;(392±79)cm,(385±48)cm,t=2.364～4.230,P<0.05～0.01。各给药组随训练次数增加平均搜索时间逐渐减少。③各组大鼠脑海马乙酰胆碱含量及胆碱乙酰转移酶和乙酰胆碱脂酶活性:造模组均较正常对照组明显降低(2.2±0.7)nmol/g,(3.8±0.9)nmol/g;(503.3±103.3)pkat/g,(778.3±125.0)pkat/g;(190.0±51.7)μkat/g,(368.3±86.7)μkat/g,P<0.01。苯妥英钠6g/L+吡拉西坦4.8g/L组,吡拉西坦4.8g/L组脑海马乙酰胆碱含量和乙酰胆碱脂酶活性明显高于造模组(2.7±0.6)nmol/g,(2.9±0.6)nmol/g;(256.7±58.3)μkat/g,(306.7±88.3)μkat/g,t=3.445～4.148,P<0.01。吡拉西坦4.8g/L组脑海马胆碱乙酰转移酶活性(668.3±118.3)pkat/g明显高于造模组(P<0.01)。吡拉西坦2.4g/L组各指标与造模组基本一致。结论:慢性癫痫大鼠大发作模型具有空间学习、记忆能力下降的特点,同时伴有脑海马乙酰胆碱含量及胆碱乙酰转移酶、乙酰胆碱脂酶活性降低,说明已是学习功能障碍的良好复合模型,应用4.8g/L吡拉西坦后可增加模型大鼠脑海马乙酰胆碱含量及胆碱乙酰转移酶、乙酰胆碱脂酶活性,改善学习和记忆能力,但2.4g/L吡拉西坦效果不明显。

BACKGROUND： Cholinergic system projected in brain and hippocampal structure is relevant with learning and memory. Piracetam acts on protecting and repairing cerebral neural cell, resisting cerebral functional injury due to physical and chemical factors and improving learning-memory capacity. OBJECTIVE： Chronic epilepsy in childhood animal anti learning-memory complex animal model were self-prepared to observe the changes in content of acetylcholine and activity of cholinacetyltranslase in cerebral hippocampus and the intervention of piraeetam. DESIGN： Randomized control experiment and non-blind evaluation were designed. SETTING： Department of Pediatries of Second Hospital of Hebei Medical University and Institute of Traditional Chinese Medicine of Hebei Medical University MATERIALS： The experiment was performed in Hebei Medical University and College of Life Sciences of Hebei Normal University from July to December 2004, in which, 50 Wistar childhood rats of clean grade and either sex were employed. METHODS： Coriamyrthin injection was administrated muscularly to duplicated chronic epibeptic grand real model in rats. Muscular injection was repeated once every three days. During modeling, those with general paroxysmal convulsion with posterior extremities standing or falling with standing or general stiffness-paroxysmal attack continuously for 3 times, the injection was changed to be once every 14 days. Ten rats were selected to be in normal control without modeling. The rest 40 rats after 3 months of modeling were rarldomized into 4 groups, named piracetam of 2.4 g/L group （Group A）, piracetam of 4.8 g/L group （Group B）, dilantin 6 g/L ＋piracetanl 4.8 g/L group （Group C） and model group （Group D）, 10 rats in each. In each group, gastric infusion was performed continuously in 3 months after modeling, onee per day, 10 mL/kg. In Group A and Group B , piracetam mixed solution of 2.4 g/l, and 4.8 g/L was administrated for infusion respectively. In Group C, dilantin 6 g/L and piracetam 4.8 g/L were infused. In group D and the eontro] group, normal saline 10 mL/kg was administrated. Relevant index determination was done 1 month after medication. Morris water maze test was performed to discover platform time and searching distance of epileptic rats, continuously for 3 days, twice per day. After test, the rats were sacrificed to collect brains to determine the content of acetylcholine in bilateral hippocampus. The activities of cholinacetyltranslase and acetylcholinesterase were determined with radinimmunity method. MAIN OUTCOME MEASURES： ①Platform time and searching distane of epileptic rats discovered in Morris water maze test. ②The content of acetycholine in bilateral hippoeampus and the activities of cholinacetyltranslase and acetylcholinesterase of rats in each group. RESULTS： Fifty rats all entered result analysis. ① Comparison of searching platform time of rats in every group： the corresponding average searching time in Group 1） was increased compared with the control group [（63±11） s, （40±8） s; （61±9） s, （38±7） s; （57±8） s, （36±9） s; （55±11） s, （33±10） s; （52±7） s, （30±9） s; （49±9） s, （27±6） s, P 〈 0.01]. In Group C and Group B, the searching time of 6 tests was decreased of various degrees compared with Group D [（44±9） s,（45±9） s;（43±9） s, （42±8） s; （42±7） s, （42±7） s; （40±9） s, （39±9） s; （38±7） s, （35±9） s; （35±6） s, （34±8） s, t=2.352-4.029, P 〈 0.05-0.01]. In every medication group, the average searching time was decreased gradually by the increased frequency of training. ② Comparison average searching distance in every group： the average searching distance in Group D was remarkably increased compared with the control [（793±74） cm, （420±81） cm;（763±89）cm, （418±57） cm; （690±67） cm, （382±69） cm; （623±81） cm, （356±71） cm; （592±98） cm, （330±69） cm;（550±54） cm,（301±97） cm,P 〈 0.01]. In Group C and Group B, the average searching distance of 6 tests was decreased of various degrees compared with Group D [（586±91） cm, （510±89） cm ;（566±70） cm, （497±76） cm;（521±84） cm, （455±56） cm; （480±74） cm, （421±63） cm; （437±51） cm, （396±79） cm;（392±79） cm, （385±48） cm, t=2.364-4.230, P 〈 0.05-0.01]. In every medication group, the average searching distanee was decreased gradually by the increased frequeney of training. ③ The content of acetylcholine in brain hippocampus and the activities of cholinacetyltranslase and acetylcholinesterase of rats in each group： those in Group D were all remarkably reduced compared with the control [（2.2±0.7） nmol/g, （3.8±0.9） nmol/g;（503.3±103.3） pkat/g, （778.3±125.0） pkat/g; （190.0±51.7）μkat/g, （368.3±86.7） μkat/g, P 〈 0.01]. In mixed group and Group B, the content of acetylcholine and activity of acetylcholinesterase were remarkably higher than the Group D [（2.7±0.6） nmol/g, （2.9±0.6） nmol/g; （256.7±58.3） μkat/g, （306.7±88.3） μkat/g, t=3.445-4.148, P 〈 0.01]. In Group B, the activity of cholinacetyltranslase l（668.3±118.3） kat/g] was remarkably higher than those in the Group D（P 〈 0.01）. Every index in group A was basically same as model .group. CONCLUSION： Grand mal of chronic epileptic rat model is characterized as declined capacity of spatial learning and memory and associated with decreased content of acetylcholine and the activities of cholinanetyltranslase and acetylcholinesterase in brain hippoeampus, explaining the successful complex model of learning and memory disturbance. Piracetam 4.8 g/L may increase content of acetylcholine and the activities of cholinacetyltranslase and acetylcholinesterase in brain hippocampus and improve learning-memory capacity, but its effect at 2.4 g/L was not remarkable.