【作者】 王玲；

【导师】 官大威；

【作者基本信息】 中国医科大学 ， 法医病理学， 2008， 硕士

【摘要】 前言曲马多(tramadol)化学名为(±)-2-(二甲氨甲基)-1-(间-甲氧苯基)-环已醇,是20世纪70年代末由原西德研究开发的中枢作用镇痛药。通过作用于阿片受体和抑制单胺类神经递质的再摄取两方面机制镇痛。与阿片类药物比较,曲马多具有呼吸抑制作用轻,成瘾性小等特点,被主要用于中、重度急、慢性疼痛的治疗。由于曲马多的广泛应用,其不良反应也日渐出现。国际上对曲马多上市后的不良反应和滥用监测表明,正常医疗目的使用情况下,曲马多滥用病例(相对于曲马多使用人群)的发生率很低。曲马多在临床上广泛使用和作为其它毒品的替代品是发生其滥用的主要原因。20世纪90年代末曲马多滥用问题基本得到控制,但近年来曲马多滥用又在我国许多地区开始流行,据媒体报道,在广东、吉林和北京等地区都出现了曲马多滥用成瘾的病例,造成不良社会影响。药物依赖是神经系统对反复使用成瘾药物后产生的适应性变化,是一类复杂的精神疾病,主要包括精神依赖性和躯体依赖性。精神依赖性是指药物对中枢神经系统作用后所产生的一种特殊的奖赏效应,表现为心理渴求和持续强迫觅药行为。躯体依赖性是指药物依赖后一旦停药或使用药物受体拮抗剂,将发生一系列生理功能紊乱,并出现戒断综合症。研究认为,药物依赖的持续状态在于成瘾性药物诱导了长期的神经适应性变化,尤其是转录过程的改变以及转录因子参与的基因表达在其中发挥了重要的作用。额叶皮质牵涉到高级认知功能和动机功能,依赖患者都存在认知功能的损害,说明额叶皮质是药物成瘾重要的脑区之一。目前环腺苷酸反应序列结合蛋白(cAMP-responsive element binding protein,CREB)是在药物滥用研究机制探讨较多的转录因子,吗啡、可卡因等阿片类药物发生依赖和戒断时,CREB在额叶皮质中表达增加,但海马及纹状体未发生改变。CREB在不同脑区表达量的不同与阿片受体的分布和脑组织的差异有关。CREB是首先被发现与药物依赖密切相关的转录因子,是脑内介导多个信号通路调节基因转录的主要细胞核因子,为多种受体后信号通路的归结点,是药物依赖形成长期记忆的分子开关。许多药物依赖的发生都与cAMP-蛋白激酶A(protein kinase A,PKA)-磷酸化环腺苷酸反应序列结合蛋白(phophorylated cAMP-responsive element bindingprotein,pCREB)通路激活有关。胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)是星形胶质细胞(astrocyte,AS)特有的标志物。生理情况下脑内少有表达,在病理情况下出现反应性表达上调,脑损伤及其他药物诱导细胞变性、增生、肥大。GFAP的高表达表明AS的活化,也增强了清除兴奋性神经递质和离子,包括谷氨酸(glutamic acid,Glu)、H~+、K~+及自由基等的能力,且贮存糖原、合成细胞因子和神经营养因子增多,有利于重建神经元的完整性,修复神经功能。本研究应用免疫组织化学染色和Western blot检测盐酸曲马多依赖大鼠额叶皮质pCREB和GFAP表达情况。目前国内外尚无有关盐酸曲马多药物依赖pCREB和GFAP的变化情况的研究。实验方法30日龄健康、雄性Wistar大鼠30只,体重100±10g,由中国医科大学实验动物部提供。动物饲养温度维持在18-26℃,光暗周期为12小时。实验前在上述条件下喂养5天,以适应环境。动物实验过程中遵循实验动物使用伦理手册相关规定执行。将30只大鼠随机分为对照组、依赖组及戒断组。依赖组每日以盐酸曲马多60mg/kg灌胃给药,共115天。戒断组每日以60mg/kg灌胃给药,于第113日给予生理盐水戒断。对照组每目给予生理盐水灌胃,共115天。给药期间,大鼠分笼饲养,自由摄取食物和水,每日观察各实验组大鼠行为表现并称重。末次灌胃后2小时立即以过量3%戊巴比妥钠麻醉处死,取额叶皮质,4%多聚甲醛溶液/0.01mol/LPBS(pH7.4)固定48小时,经水洗,脱水,透明,石蜡包埋,制作5gm连续切片。留取部分额叶皮质组织用冷生理盐水清洗后加蛋白裂解液匀浆,用于Western blot检测。应用免疫组织化学染色和Western blot检测曲马多依赖大鼠额叶皮质中pCREB和GFAP表达情况,以对照组作对照,显微镜×400倍下,随机选取10个视野,计数阳性细胞数(个/视野)和阳性细胞灰度值。以标准分子量Marker作指示,在PVDF膜上阳性表达显示为蓝色谱带,蓝色谱带的浓淡和大小表示蛋白表达的强弱。应用FluochemV2.0软件测各片段的平均光密度值,测得数据用均数士标准差((?)±s)表示。用SPSS11.0 for Windows进行单因素方差分析,以P＜0.05为差异有统计学意义。实验结果一、曲马多依赖和戒断大鼠行为学变化与对照组大鼠相比较,依赖组大鼠自给药后一周开始出现对声音及触摸刺激反应性增高,出现奔跑、跳跃、竖尾(照片2)、头部侧偏、轻度共济失调以及原地转圈行走等现象,上述症状可于给药后即出现,多持续1至2小时后逐渐消失。戒断组大鼠于停药36-48h逐渐出现明显的戒断症状:烦躁不安、高度激惹、蹦跳、尖叫、异常姿势(照片3)、叩齿、咬牙、湿狗样颤抖、甩头、上睑下垂、腹泻、清理皮毛、站立等,体重增长速度也明显下降。戒断24-36h最为明显。以后逐渐减弱,72h后戒断症状基本消失。二、曲马多依赖和戒断大鼠脑组织病理形态学改变1、脑宏观检查未见异常改变。2、HE染色额叶皮质神经元与胶质细胞周围间隙增宽,可见胶质细胞增生和胶质结节形成。三、曲马多依赖和戒断大鼠体重的变化1、依赖大鼠体重的变化与对照组相比较,依赖组大鼠自给药后第1-3周体重平均增加28g,对照组大鼠体重平均增加23g,增加速度高于对照组。第4周以后依赖组大鼠体重增加明显比对照组缓慢。利用统计学软件SPSS采用NPar Tests中的Wilcoxon Signed Ranks Test分析方法,P＜0.01,结果具有统计学意义。药物依赖大鼠体重的增长速度明显低于对照组大鼠体重增长的速度。2、戒断大鼠体重的变化各组在停药后至处死前体重变化情况:对照组体重仍呈上升趋势,平均体重由380g增长到401g,增长了19g;依赖组平均体重由223g增长到232g,增长了9g;戒断组平均体重由226g降低到212g,体重减少了14g。利用统计学软件SPSS采用单因素方差分析,P＜0.01,结果具有统计学意义。戒断组大鼠体重增长速度明显低于对照组和依赖组大鼠体重增长速度。四、pCREB和GFAP免疫组织化学染色对照组大鼠pCREB阳性染色主要表达在神经元胞核中,少数胶质细胞胞核也呈阳性表达,血管内皮细胞未见阳性表达。经阳性细胞计数,对照组大鼠额叶皮质pCREB阳性细胞数为40.9±1.89个/视野,依赖组78.5±3.79个/视野,戒断组130.5±2.53个/视野,依赖组和戒断组pCREB阳性细胞数均增多,经统计学分析,P＜0.05,有统计学意义。对照组大鼠额叶皮质pCREB阳性细胞灰度值为70.87±4.85,依赖组43.02±3.12,戒断组34.09±3.53,依赖组和戒断组pCREB阳性细胞灰度值均减少,经统计学分析,P＜0.05,有统计学意义。对照组GFAP阳性染色主要表达在AS胞质及突起中。经阳性细胞计数,对照组大鼠额叶皮质阳性细胞数12.56±6.43个/视野,依赖组22.23±9.34个/视野,戒断组34.91±8.36个/视野,依赖组和戒断组GFAP阳性细胞数均增多,经统计学分析,P＜0.05,有统计学意义。对照组大鼠额叶皮质阳性细胞灰度值为65.87±3.21,依赖组40.09±3.56,戒断组30.02±3.11,依赖组和戒断组GFAP阳性细胞灰度值均减少,经统计学分析,P＜0.05,有统计学意义。五、Western blot检测平均光密度检测,对照组大鼠额叶皮质pCREB平均光密度为40.26±3.01,依赖组60.36±2.14,戒断组105.24±2.04,依赖组和戒断组pCREB平均光密度增多,经统计学分析,P＜0.05,有统计学意义。对照组大鼠额叶皮质GFAP平均光密度为12.22±3.29,依赖组22.34±2.04,戒断组34.61±3.01,依赖组和戒断组GFAP平均光密度增多,经统计学分析,P＜0.05,有统计学意义。讨论本研究中,依赖组和戒断组大鼠额叶皮质pCREB蛋白表达与对照组比较明显增加,变化有统计学意义。曲马多使大鼠额叶皮质中pCREB蛋白表达发生改变。提示pCREB参与曲马多精神依赖及躯体依赖中的形成。pCREB与增强神经元的存活与保护机制有关,pCREB是药物依赖过程中最关键的转录因子之一,是多种受体后信号通路的归结点,pCREB促使许多基因的表达发生了改变,影响细胞内信号转导途径引起神经元细胞核功能的改变,从而导致部分靶基因的转录速率发生变化,继而改变了神经元的活动,最终会使这些神经元相互作用的环路发生改变,结果表现为动物行为持久的改变。本研究还发现,依赖组和戒断组大鼠额叶皮质中GFAP蛋白表达与对照组比较明显增加,变化有统计学意义。GFAP蛋白表达量的增加表明AS的增生和发育,是AS对神经元损伤的适应性改变,在曲马多依赖及戒断时GFAP蛋白表达增多,有利于增加AS骨架的稳定性,AS的激活有利于满足其代谢活动的增加,减轻药物对神经元的损伤,本研究表明AS在曲马多药物依赖和戒断后可能具有保护作用。结论1、曲马多(60mg/kg)慢性给药引起实验大鼠依赖症状,戒断组大鼠出现戒断症状。本实验研究成功复制了曲马多依赖与戒断的大鼠动物模型。2、依赖和戒断大鼠额叶皮质区pCREB表达增加,提示大鼠额叶皮质是产生曲马多药物依赖的主要部位之一,并可能通过pCREB而产生。3、大鼠曲马多依赖及戒断后额叶皮质GFAP表达增多,有利于增加AS骨架的稳定性,AS的激活有利于满足其代谢活动的增加,减轻曲马多对神经元的损伤,提示AS在曲马多药物依赖和戒断后可能具有保护作用。更多还原

【Abstract】 IntroductionTramadol is chemically(±)- 2 -(dimethyl methyl ammonia)- 1-(intermethoxyphenyl)-cyclohexanol, and a typital analgesic which was initially approved in Germany in 1977 for pain treatment.In fact,tramadol is both an agonist of opioid receptors with selectivity for theμ-and less affinity for theδ-andκ-receptors,and inhibitor of the reuptake of noradrenaline and serotonine.Moreover,tramadol shows a lower abuse potential and respiratory inhibition in comparision with morphine.More and more adverse effects are increasingly emerged.Tramadol abuse are mainly associated with both widely clinical prescription and the possible effect of addiction and substitute of other illegal drugs.The problem of tramadol abuse was basically under control during the late 1990s.However,abuse and dependence of tramadol,which has produced untoward impact on social community,are increasingly realized.Drug dependence including psychological and physical dependence,is a kind of complex mental illnesses of the nervous system.The mental dependence is a special reward effect generated by central nervous system,characterized by continued craving for forced seeking behavior.Physical dependence refers to a series of physiological disorders after withdrawal or use of the antagonist.Research shows that drug addiction is a psychological adaptation with long-term drug intake,which may inflect gene transcription.Currently,cAMP response element binding protein(CREB)is the transcriptive factor involved in the mechanism of drug abuse.The level of CREB is found to be increased in the frontal cortex after morphine dependence and withdrawa1 without changes in hippocampus and nucleus accumbens,which may be attributed to different distribution of opioid receptors in the brain.It is reported that CREB is the first transcriptive factor found to be related closely with drug dependence.CREB mediates signaling pathway by regulating gene transcription.Glial fibrillary acidic protein(GFAP)is astrocyte(AS)specific marker.Its expression is reportedly increased in response to cell degeneration or death because of injury and drugs which activates of AS,for enhancing the clearance of excitatory neurotransmitters and ions including glutamate,H~+,K~+ and free radicals.GFAP is also responsible for glycogen storge,synthesis of cytokines and neurotrophic factors,contributing to the reconstruction of the integrity of neurons and neuronal repair.In the present study, pCREB and GFAP expression were studied for the first time in the frontal cortex in rats with tramadol dependence by immunohistochemical staining and Western blotting.Materials and MethodsA total of male 30 rats,weighing 90～110g,were randomly divided into three groups, one control group and two experiment groups which include tramado- dependent group and withdrawal group.Rats were supplied by the Animal Laboratory of China Medical University.Rats were bred for 5 days at 18-26℃to adapt to the environment.Under the permission of Ethical Committee for Animal Experiment of China Medical University. Rats were intragastrically given hydrochloride tramadol 60mg/kg per day for 115 days in tramadol-dependent group.Rats were intragastrically administrated hydrochloride tramadol 60mg/kg per day for 112 days and then given saline instead in withdrawal group. Rats were given saline per day for 115 days in control group.The rats got chow and water freely and housed individually in cage.The behaviors of the drug dependence and withdrawal were observed and recorded and body weight was measured.Two hours after the last administration,the rats were executed by intragastrically,administration of overdose of sodium pentobarbital.Frontal cortex was collected and fixed in 4% parafonmaldehyde/0.01MPBS(pH7.4)for 48h.and then washed,dehydrated,and embedded in paraffin.5μm-thick consecutive paraffin sections were prepared.For Western blotting,fontal cortex was sampled on a wax plate cooled with ice.The immunostaining sections were observed at 400 times magnification under microscope at randomly selected 10 fields in frontal cortex,pCREB- or GFAP-positive cells were counted by two staff members independent of the experiment.Gray density was analyzed by computerized imaging system for Western blotting results,bands that means expression of strength.Band color intensity was analyzed by Fluochem V2.0 software. The data were expressed as mean±deviation.All data were statistically analyzed by SPSS11.0 for Windows with a single factor analysis of variance.P＜0.05 was statistically significant.Results1.Behavior changes in tramadol dependent and withdrawal ratsAs compared with control rats,rats in tramadol-dependent group showed the symptoms of exciting,hyper-reactivity to sound and touching stimulation,running,tail uprighting as well as run in circle.But 36-48 hours after the withdrawal of tramadol, symptoms were characterized by agitating,restlessness,outraging,jumps,screaming, abnormal postures,teeth clicking,the wet dog-like shivering,flings,ptosis,and the diarrhea.2.Morphological changes of the brain(1)Gross examinationNo abnormal changes were found.(2)Microscopic examinationMild cerebral edema and gliosis were detected in the frontal cortext in the rats with tramadol dependence and withdrawal.3.Body weight measurements(1)Tramadol-dependent groupIn comparison with those in the control group,increase in body weight was greater than in the tramadol-dependent rats during the first 1-3 weeks.However,the increase in body weight became slower than in the control group since the 4th week after drug delivery,which was statistically significant(P＜0.01).(2)Tramadol-withdrawal groupBody weight of control rats gained continuosly from 380g to 401g with an increase of 19g in average within the 3 days.The weight of tramadol-dependent rats increased from 223g to 232g with an increase of 9g in average.The weight of tramadol- withdrawal rats decreased from 226 g to 212 g with a reduce 14g in average.The difference was statistically significant in body weight increase or decrease in tramadol-dependent or withdrawal group as compared with the control respectively(P＜0.01).4.pCREB and GFAP immunohistochemical stainingpCREB-positive cells were mainly detected in the nucli of the neurons,and in a small number of glial cells in the cortex in control rats.GFAP-immunoreactivity was found in the cytoplasm and dendrites of the astrocytes.An intensive immunoreactivity of pCREB and GFAP were detected in frontal cortex of rat in dependence and withdrawal rats.The number of pCREB-positive cells in tramadol-dependent rats(78.5±3.79)or withdrawal group(130.5±2.53)was increased in comparison with the control group (40.9±1.89),which was statistically significant(P＜0.05).Gray density of pCREB in positive cells in dependence group(43.02±3.12)and withdrawal group(34.09±3.53) was decreased in comparison with that in the control group(70.87±4.85),which was statistically significant(P＜0.05).Increase in number of GFAP-positive cells in dependence group(22.23±9.34)or withdrawal group(34.91±8.36)was noted in comparison with the control group(12.56±6.43),which was statistically significant(P＜0.05).Gray density of GFAP-positive cells in dependence group(40.09±3.56)or withdrawal group(30.02±3.11)was decreased in comparison with the control group(65.87±3.21),which was statistically significant(P＜0.05).5.Western blottingAverage optical density of pCREB bands in dependence group(60.36±2.14)or withdrawal group(105.24±2.04)was increased in comparison with the control group(40.26±3.01),which was statistically significant(P＜0.05).Average optical density of GFAP in dependence group(22.34±2.04)and withdrawal group(34.61±3.01)was increased in comparison with the control group(12.22±3.29),which was statistically significant(P＜0.05).Discussion and ConclusionsAs compared with the control group,pCREB expression was increased significantly in the frontal cortex in tramadol-dependent and withdrawal rats,suggesting that pCREB may take part in the development of tramadol psychlolgical and physical dependence. pCREB is considered to enhance the neuronal survival and protection.Recent studies indicate that pCREB is the most critical transcriptve factor responsible for development of drug dependence,pCREB may promote gene expressions which are associated with abnormal behaviors in drug addiction.GFAP expression was significantly enhanced in frontal cortex in tramadol -dependent and withdrawal rat.Up-regulated GFAP expression in AS indicates glial proliferation and development,indicates the neurons adaptation to damage.Enhanced GFAP expression indicates increased stability of AS skeleton in tramadol dependence and withdrawal. Activated AS may be helpful for meeting their the increased of metabolic activities and reducing drug-induced neuronal injury.Our study further implicates the protective roles of AS in the tramadol-induced neuronal damage.更多还原