【作者】 于洪鸾；

【导师】 潘芳；

【作者基本信息】 山东大学 ， 医学心理学， 2012， 博士

【摘要】 研究发现,发育期大脑受到损伤可导致不可逆改变,且对药物滥用敏感。氯胺酮是近年迅速发展起来的滥用物质,作为非竞争性N-甲基-D-天门冬氨酸(N-methyl-D-aspartate, NMDA)受体拮抗剂,氯胺酮可导致幼龄大鼠和小鼠发生广泛的神经细胞凋亡,引起神经细胞死亡,导致脑结构发生不可逆改变。另外,有研究提示,青少年期是多巴胺(dopamine, DA)系统神经元重组、皮层突触重塑、受体消减的关键期,对药物滥用敏感。物质滥用可影响多巴胺(DA)神经系统的传递功能,导致生长发育和奖赏敏感性改变。致幻作用是氯胺酮滥用的基本因素。有研究发现,长期滥用者出现社会功能受损、行为和人格改变、记忆力下降和身体多器官损害,其中,最明显的改变是中枢神经系统损伤。现有研究发现,氯胺酮滥用导致大脑皮质、脊髓、丘脑、海马和后扣带皮质等部位神经元调亡。氯胺酮作为NMDA受体阻断剂,通过降低γ-氨基丁酸(gamma-Aminobutyric Acid, GABA)能神经元活性,导致γ-氨基丁酸(GABA)神经元抑制,引起广泛分布在皮层区域的谷氨酸能神经元脱抑制,引发谷氨酸过度释放和皮层兴奋,引起类似精神疾病的行为和认知功能异常。既往研究已经证实,急性氯胺酮应用主要作用于前额叶(prefrontal cortex, PFC)和周边皮层,在这些脑区可检测到谷氨酸和多巴胺(DA)水平升高。但慢性低剂量氯胺酮重复应用对前额叶(PFC)多巴胺(DA)神经元、工作记忆和功能执行的影响及分子机制尚不够明晰。大多数成瘾药物是通过激活中脑-边缘多巴胺系统(mesolimbic dopaminesystem, MLDS)的多巴胺(DA)神经通路,实现成瘾药物的奖赏和强化作用。动物实验发现,在反复给予成瘾物质后,功能核磁共振成像(functional magnetic resonance imaging, fMRI)显示多巴胺(DA)分布区域包括纹状体、伏隔核及背侧丘脑出现明显激活。边缘叶多巴胺(DA)系统的激活,特别是纹状体多巴胺(DA)活性的增强,可破坏感觉运动控制功能。但随着新型成瘾物质的不断出现,不同成瘾物质对脑功能的作用部位不同,其作用方式具有区域选择性和作用靶点特异性。氯胺酮成瘾作用的靶点和神经通路尚不清楚。麻醉剂量、急性氯胺酮使用可致成人工作记忆和执行功能的缺陷,使用者表现为面部情绪的处理加工、工作记忆、回忆、言语流畅和学习障碍。而慢性氯胺酮滥用研究报告较少,少有研究是关注长期低剂量氯胺酮滥用对青少年期相关脑区功能的影响及其生理机制。本实验将建立青少年期食蟹猴氯胺酮长期滥用模型,观察低剂量长期使用氯胺酮对动物体重生长、自主行为、脑功能和脑组织代谢的影响,并探讨可能的分子机制。目的1.观察长期低剂量氯胺酮滥用对青少年期食蟹猴体重、自发行为的影响。2.探讨长期低剂量氯胺酮滥用对青少年期食蟹猴不同脑区功能的影响。3.探讨长期低剂量氯胺酮滥用对额叶、纹状体脑组织代谢的影响。4.观察长期低剂量氯胺酮滥用对豆状核(lentiform nucleus, LN)神经元凋亡的影响。5.观察长期低剂量氯胺酮滥用对前额叶(PFC)皮质酪氨酸羟化酶(tyrosine hydroxylase, TH)的表达的影响。材料和方法1.实验模型制备12只食蟹猴随机分为两组,8只实验组,4只对照组,实验时间为6个月。实验组每天给予盐酸氯胺酮1mg/kg静脉注射,对照组给予生理盐水1mg/kg。2.体重测量实验开始之前测量食蟹猴基础体重,之后每个月末测量并记录体重变化情况,观察长期氯胺酮滥用是否会影响食蟹猴体重增长。3.行为学观察在实验的第1、3、7、14、56、112、183、184、185天给药后录像观察食蟹猴自发行为(行为量表)15min。观察内容包括移动、行走、攀爬、跳跃四种行为。4.功能磁共振成像(fMRI)检查实验的第186天开始做放射影像学检查。功能磁共振成像(fMRI)扫描过程中外部闭路检测仪监控呼吸状态,由麻醉师全程检测麻醉状态和生命体征,采用轴位扫描。功能磁共振成像(fMRI)中,抬高食蟹猴右后肢(60°)频率1次/秒,诱发相应脑区激活,扫描时间12min48s。5.核磁共振波谱检查(Magnetic Resonance Spectroscopy, MRS)核磁共振波谱(MRS)扫描采用single voxel PROBE PRESS点解析波谱成像(point resolved spectroscopy),以轴位扫描,分别选取额叶、纹状体两个脑区域,尽量避开颅骨和脑脊液,扫描时间3mmin,每个部位扫描3次。6.神经元凋亡检测用核糖核酸末端转移酶介导的缺口末端标记法(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, TUNEL)来检测豆状核(LN)中神经元凋亡情况。取LN石蜡切片蛋白酶K孵育,平衡液处理,TUNEL反应混合液孵育,DAB显色液,观察神经元凋亡数目。7.酪氨酸羟化酶(TH)检测影像学检查结束后,随机取4只食蟹猴(实验组3只对照组1只)留取脑组织豆状核(LN)及前额皮质(PFC),制成石蜡切片备用。用免疫组织化学方法检测前额叶(PFC)酪氨酸羟化酶(TH)的表达。前额叶(PFC)石蜡切片抗原修复,1%的牛血清白蛋白和10%兔血清处理1h,多克隆羊抗人酪氨酸羟化酶过夜,辣根过氧化物酶结合的兔抗羊IgG室温下抚育,DAB试剂盒显像,观察酪氨酸羟化酶(TH)阳性细胞数目。结果1.长期低剂量氯胺酮滥用抑制青少年期食蟹猴体重的增加速度实验过程中两组食蟹猴体重均增加,差异无统计学意义。但与对照组比较,氯胺酮组体重增加缓慢,对照组在第2个月末时体重增加10%,在后来的4个月里体重增加10%,而氯胺酮组在第4个月末时体重增加10%,6个月末时两组体重平均差为10%。2.长期低剂量氯胺酮滥用抑制食蟹猴自发运动与对照组比较,氯胺酮组的自发运动有下降趋势。与对照组比较,氯胺酮组在第183、184、185天自发行为包括移动、行走、攀爬下降明显,有统计学意义(p<0.05)。3.长期低剂量氯胺酮滥用导致食蟹猴广泛性脑功能失调功能磁共振成像(fMRI)检查结果中,氯胺酮组右侧大脑半球豆状核(LN)、梭状回(fusiform gyrus, FG)和内嗅球(entorhinal cortex, Ent)可观察到明显活跃脑区,其中豆状核(LN)激活最明显,梭状回(FG)明显激活,内嗅球(Ent)的激活次之。对照组左侧大脑半球的黑质(substantial nigra in midbrain, SN)、腹侧被盖核(VTA)、视皮质(visual cortex, VC)及右侧大脑半球的后扣带皮质(posterior cingulate cortex, CGp)及躯体感觉区(somatosensory cortex, SC)明显活跃。其中,视皮质(VC)、躯体感觉区(SC)激活最明显,黑质(SN)与腹侧被盖核(VTA)有明显激活,后扣带皮质(CGp)的激活次之。4.长期低剂量氯胺酮滥用导致食蟹猴脑组织代谢异常与对照组比较,氯胺酮组食蟹猴额叶、纹状体中N-天门冬氨酸(NAA)、肌酸(Cr)、NAA/Cr数值降低,但差异无统计学意义；与对照组比较,氯胺酮组食蟹猴额叶、纹状体中胆碱(Cho)的水平明显降低(p<0.05)。5.未观察到明显神经元凋亡与对照组比较,氯胺酮组豆状核(LN)神经元凋亡数目未见显著性增加。6.长期低剂量氯胺酮滥用降低食蟹猴前额叶(PFC)酪氨酸羟化酶(TH)表达酪氨酸羟化酶(TH)检测结果发现,氯胺酮组酪氨酸羟化酶(TH)阳性细胞数目明显少于对照组(p<0.01)。结论1.长期低剂量氯胺酮滥用可抑制生长发育过程中体重增加。2.长期低剂量氯胺酮滥用产生可导致运动抑制,白发行为减少,提示产生行为耐受性。3.长期低剂量氯胺酮滥用明显抑制左侧大脑半球黑质(SN)与腹侧被盖核(VTA),可能为实验动物未出现觅药行为与成瘾性的主要原因。主要通过激活纹状体中豆状核(LN)而导致感觉运动控制障碍,导致运动失调,可能是自发行为减少、运动抑制的原因；激活梭状回(FG)可引起视幻觉,而致幻作用为氯胺酮滥用最基本因素；激活内嗅球(Ent),使内嗅球(Ent)-纹状体通路活化,从而在精神分裂样症状的出现及物质滥用过程中起重要作用。除了上述脑区外,与以往的研究不同,我们还发现躯体感觉区(SC)活性降低,提示长期低剂量氯胺酮滥用损害躯体感觉功能,首次明确了氯胺酮滥用对躯体感觉区(SC)的损害作用,补充了中枢作用机制,为研究青少年物质滥用及戒毒提供了实验依据。4.长期低剂量氯胺酮滥用主要通过影响神经胶质细胞膜的合成、周转和代谢,影响青少年期食蟹猴额叶、纹状体代谢。5.豆状核(LN)未见明显的神经元凋亡。可能引起不同于经典细胞凋亡的其他形式的细胞死亡,虽然最终发生以胞核变化为特征的细胞凋亡,但凋亡进程会明显延迟,提示随着用药时间的延长可能导致细胞凋亡6.长期低剂量氯胺酮滥用可损害前额叶(PFC)多巴胺(DA)系统功能,进而导致食蟹猴认识功能和执行功能的损害,自发运动减少。更多还原

【Abstract】 Studies indicate that developing brain is sensitive to drug abuse, in which damage may induce inreversible changes. Ketamine is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist that was abused rapidly recent years, with profound socioeconomic and health impacts. It was reported that even exposed little dose of ketamine to infant or adolescence can trigger widespread apoptotic neurodegeneration in brain tissue. Moreover, adolescence is a biologically critical period in neurodevelopment where reorganization, synaptic remodeling and receptor pruning occur in the dopaminergic system. Drug abuse can influence transmission of dopaminergic neurons, and then induce changes of growth and sensibility of reward system.Ketamine abuse can induce hallucinations, which is regarded as the main reason of drug addicts. Lots of reports demonstrated that ketamine abuser showed up objection of social ability, behavior and personality changes, damage of memory and organs, and especial evident damage of central nervous system. Present studies indicate that ketamine abuse can induce neuron apoptosis in many encephalic regions, including cerebral cortex, spinal cord, thalamus, hippocampus cingulate gyrus cortex and so on.Ketamine induces hypofunction of NMDAR including excessive glutamate release through decreased activity of GABAergic interneurons, and subsequent exciteds cortex which may induce psychosis-like behavior and cognitive anomalies. Previous studies have demonstrated that acute ketamine administration mainly influenced prefrontal lobe and periphery cortex. There are evidently increased levels of glutamic acid and dopamine in these different encephalic regions. However, the mechanism of chronic ketamine administration on prefrontal lobe, dopamine neurons is still unclear. The dopaminergic neurons play an important part in the reward system, in which glutamatergic-dopaminergic pathway interaction within the mesolimbic dopamine system are thought to contribute to addiction-related behavior in ketamine abuse. Functional magnetic resonance imaging (fMRI) about animal experiments showed that drug abuse enhanced activity of dopamine system in the mesolimbic, especially in striatum, robustly disrupting sensorimotor gating. With the emerging of new abused drugs successively, the modes of action and target areas of them are specificity differently. At present, little is known about the target areas and brain circuits of chronic ketamine administration.Anaesthetic dose and acute ketamine administration in adult lead to impaired working memory and executive function. Disorders of face emotion processing, working memory, language fluency and learning were observed. There is little report about chronic low dose of ketamine administration, especially the effects on brain function and physiological mechanism in adolescent. This study was designed to determine the consequences of chronic ketamine administration in adolescent cynomolgus monkeys following an exposure pattern that mimics adolescence drug abuse. The chronic drug effects on monkey’s weight, behavior, brain functions and brain metabolism were investigated and the molecule mechanisms were explored.1. Objectives1.1. To observe body weight and behavioral changes in adolescent cynomolgus monkey following chronic low dose of ketamine exposure.1.2. To investigate the effects of chronic low dose of ketamine on brain function in adolescent cynomolgus monkey.1.3. To investigate the brain metabolism in striatum and frontal lobe following chronic low dose of ketamine exposure in adolescent cynomolgus monkey.1.4. To assay apoptosis in lentiform nucleus (LN) in adolescent cynomolgus monkey following chronic low dose of ketamine exposure.1.5. To examine expression of tyrosine hydroxylase (TH) in the prefrontal cortex (PFC) following chronic low dose of ketamine administration in adolescent cynomolgus monkey.2. Materials and methods2.1. Animal model establishedTwelve adolescent male cynomolgus monkeys were randomly divided into2 groups:eight to be administered with ketamine and four with saline as controls. A ketamine dose of1mg/kg in an injection volume of1ml (in saline) freshly prepared on the day of injection was given daily intravenously via arm vein under mild physical constraints for6months. Control monkeys were given sterile saline (1ml) daily.2.2. Body weight measurementAnimal body weights were recorded monthly to monitor the monkeys’well-being throughout the whole experiment.2.3. Behavioral observation15-min video recordings were made for each monkey after ketamine or saline injection at day1, day3, day7, day14, day56, day112, day183, day184and day185. Behavior observation including moving, walking, climbing and jumping was conducted.2.4. fMRIfMRI study was performed on186day. Respiration rate and body temperature were continuously monitored during the fMRI scan. For stimulation, the monkeys’ right lower limb was moved (raised leg to60°) once per second. Average scanning time of fMRI for one monkey was about12min48s. Images were acquired in axial level in order to localization accurately.2.5. MRSMRS use single voxel PROBE PRESS point resolved spectroscopy. We examined15x15x10mm area in striatum and frontal lobe regions, avoiding skull and cerebrospinal fluid. Average scanning time of MRS for one monkey was about3min, and3times were performed.2.6. Apoptosis assayAfter fMRI scanning,1control and3ketamine-abuse monkeys were randomly chosen to be sacrificed. Brains were dissected and embedded in paraffin wax. Sections of left PFC and LN were cut coronally from paraffin blocks at4-um thickness.TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was used to assay apoptosis. Brain tissues sections were incubated with Proteinase K, and TUNEL reaction mixture. Finally, the sections were developed with DAB kit. Apoptotic cells were assayed.2.7. Tyrosine hydroxylase (TH) For Immunohistochemistry TH positive cells in PFC was used to study morphological changes under light microscope. Brain tissues sections were treated with inactivate endogenous peroxidases. They were then blocked with1%bovine serum albuminand10%normal rabbit serum for1h, and then were incubated with horse radish peroxidase (HRP)-conjugated rabbit anti-goat IgG for1h. Finally, the sections were developed with a DAB kit. TH positive cells were assayed.3. Results3.1. Chronic low dose of ketamine administration inhibited the body weight increase in adolescent cynomolgus monkey following chronic ketamine exposure.There was no statistically significant difference between control and ketamine groups. But body weights of ketamine group increased more slowly compared with control group. Body weights increased for about10%in the control monkeys during the first2months, while the ketamine abuse monkeys took4months to gain the same10%increase in body weights. The mean difference at the end of6month was10%between the ketamine and control groups.3.2. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey depressed motor behaviorsFor behavior test, activities of moving, walking, climbing and jumping of ketamine group exhibited obvious decreased tendency as time went by. There were significant reduced activities of moving, walking and climbing of ketamine group in day183,184and185compared to control group (p<0.05).3.3. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey caused disturbance in the central nervous systemFirst, significantly higher levels of neural activities were observed in the right hemisphere of LN, fusiform gyrus (FG) and entorhinal cortex (Ent) in ketamine-challenged monkeys compared with controls. Second, monkeys in the control group had significantly higher neural activities in the substantia nigra (SN), ventral tegmental area (VTA), posterior cingulate cortex (CGp), visual cortex, and somatosensory cortex (SC) compared with ketamine-challenged monkeys.3.4. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey caused disturbance in brain metabolismIn MRS, Cho in striatum and frontal lobe in ketamine group are lower than those of control (p<0.05). Chronic ketamine abuse might affect cell metabolism such as membrane breakdown, turn over, synthesis, myelination and lipid metabolism in striatum and frontal lobe.3.5. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey did not cause obvious apoptosisThere were not significant differences of apoptotic cells between two groups in lentiform nucleus.3.6. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey decreased expression of TH protein in the prefrontal cortexTyrosine hydroxylase (TH) pilot experiment showed there was a qualitative reduction in the positively stained projective axon of dopaminergic neuron in the prefrontal cortex of monkeys with chronic ketamine abuse (p<0.01).4. Conclusion4.1. Chronic low dose of ketamine abuse inhibits the body weight increase in adolescent cynomolgus monkey following chronic ketamine exposure.4.2. Chronic low dose of ketamine abuse decreases locomotor activity, which suggesting induced tolerance after chronic ketamine administration in adolescent cynomolgus monkey.4.3. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey leads to depression in VTA and SN, which is correlated no drug seeking and behavior addiction. Significant brain activation in LN implicates a dysfunction of sensorimotor gating, which plausibly leading to motor inhibition. Activation in FG causes visual hallucination, which is regarded as the main reason of drug addicts. Activation in Ent-striatum may play an important role in schizophrenia and substance abuse. We identify the damage of SC firstly, which implicates injury function of somatosensory. This compensates the central action of ketamine, providing experiment proof and abstinence of ketamine abuse in adolescence.4.4. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey might affect neuroglial cell metabolism such as membrane turn over, synthesis and lipid metabolism in striatum and frontal lobe.4.5. There is no significant apoptosis. Perhaps chronic low dose of ketamine abuse in adolescent cynomolgus monkey induces other paths of apoptosis. Although apoptosis will take place at last, the procee of apoptosis was delayed.4.6. Chronic low dose of ketamine abuse impaires the function of prefrontal dopaminergic system, causing damage of cognitive function and decreased locomotor activity.更多还原