【作者】 史慧勤；

【导师】 彭双清；

【作者基本信息】 中国人民解放军军事医学科学院 ， 卫生毒理学， 2012， 博士

【摘要】 毒性化合物暴露下，机体往往会出现包括神经传递、突触连接和细胞存活在内的神经系统功能障碍或器质性损伤，从而损害健康甚至对生命造成威胁。许多已经上市的药物正是由于存在不同程度的神经毒副作用，严重影响了药物的使用，甚至最终导致药品退出市场。因此，早期神经毒性评价成为药物风险性评估的重要内容。做为神经毒性评价的主要动物模型，鼠、兔、狗、猴等哺乳动物存在着实验周期长、费用高、低通量等诸多弊端。发展新的实验动物模型应用于药物早期、灵敏、高通量神经毒性筛查势在必行。斑马鱼具有典型的脊椎动物脑部特征，神经行为遵循顺序发育原则。与啮齿动物模型相比，它具有饲养经济；产卵量大、发育快速，容易获得足够样本量；胚胎透明、易于观察；体型小，空间占用少，具备高通量筛查的条件；水体观察，动物产生的应激少，实验结果比较客观等优点。目前模式动物斑马鱼已广泛应用于神经发育、神经损伤和神经行为学等神经科学研究。同样在神经毒性评价研究领域，斑马鱼的应用也逐渐增多，但由于检测方法不一致、评价指标不统一，大多数实验研究还只停留在表型观察阶段。鉴于行为活动能够从宏观上早期反映神经功能障碍，比病理学改变更为直接、敏感，依据幼鱼体型小的优势，在引入计算机和相关参数分析软件基础上，近年来研究人员开发出幼鱼神经行为学检测方法，本研究我们将主要采用这些技术，建立和优化斑马鱼幼鱼神经行为学高通量检测方法，提高化合物神经毒性检测效率，进而验证幼鱼模型的效能。氯胺酮是一种临床儿科用麻醉药和新型致幻剂，对胎儿和哺乳期婴幼儿具有潜在的神经发育毒性。目前哺乳动物实验研究表明，麻醉剂量的氯胺酮能够导致中枢神经元退行性改变。但对于亚麻剂量氯胺酮暴露能否引起神经系统发育的毒性作用及作用机理尚不明确。本课题主要采用Noldus幼鱼行为视频跟踪系统，对药物暴露后幼鱼的自发运动、惊恐逃避反射等多种行为活动实时跟踪记录，采用Ethovision XT7.0软件将运动轨迹可视化，导出运动参数用于后期统计分析。通过对已知阳性或阴性化合物神经毒性的高通量检测与评价，验证了斑马鱼模型的有效性。结合一般毒性检测和组织病理学检查，建立了化合物神经发育毒性评价方法。本研究还对斑马鱼高级神经活动检测方法进行优化。通过检测亚麻剂量氯胺酮早期持续暴露后幼鱼神经行为毒性变化以及较大幼鱼的远期高级神经活动损害效应，明确了亚麻剂量氯胺酮的神经发育毒性特征。随后分别从组织病理学凋亡检测和行为相关基因表达变化，探索亚麻剂量氯胺酮引发的剂量-行为效应发生机制。通过对无神经毒性的酵母以及有明确神经毒性的乙醇、氯胺酮、氯化镉、醋酸铅、氯雷他定等化合物的神经毒性作用检测，验证了幼鱼模型的有效性。结果表明，在神经毒物暴露后，6dpf幼鱼的自发行为活动能力和/或行为方式发生改变，可产生兴奋性、抑制性或倒“U”型运动效应。对外界刺激的反应能力降低、恢复能力或适应能力减弱，神经毒性表现同文献报道相一致，并具有一定的剂量-效应关系。同时验证了斑马鱼模型具有较好的准确性和实验可重复性。通过鉴别临床成药氯雷他定、酵母的神经毒性，还证明了该模型在成药毒性评价中具有一定的实用性。本研究还建立了胚胎和幼鱼神经发育毒性评价方法，检测了氯丙嗪、异烟肼的神经发育毒性，结果类似临床神经毒副作用表现。应用建立的斑马鱼幼鱼模型，本研究检测了亚麻剂量氯胺酮的神经发育毒性作用及剂量-行为效应机制。结果发现，氯胺酮暴露后幼鱼兴奋性运行效应与抑制性运动效应并存。应用TUNEL法凋亡细胞检测发现，出现抑制性效应的暴露组幼鱼中枢神经细胞凋亡明显增多，而出现兴奋性效应的暴露组神经细胞凋亡不明显。进一步应用RT-PCR法检测中枢神经NMDA受体亚基表达情况，发现出现兴奋性效应的暴露组NMDA受体结构亚基和部分调节亚基表达代偿性上调。提示中枢神经细胞凋亡和NMDA受体表达代偿性上调可能是引发抑制或兴奋运动效应的原因。通过光惊恐实验和较大幼鱼环境适应性、社会交互性及学习记忆等高级神经活动检测发现，未发生神经细胞凋亡的剂量组对外界刺激的反应能力和适应能力降低，并且发育至较大幼鱼时高级神经活动能力受损。RT-PCR法检测发现，这些剂量组幼鱼体内与神经突触可塑性密切相关的基因bcl-2、c-fos表达呈下调趋势。提示bcl-2、c-fos下调与高级神经行为发育障碍具有一定的相关性。综上所述，本课题建立了斑马鱼幼鱼神经行为毒性和神经发育毒性检测评价方法，优化了较大幼鱼高级神经活动的检测方法，通过验证斑马鱼幼鱼模型的效能，为应用斑马鱼模型进行早期、快速、高通量的药物神经毒性评价提供了实验基础。同时阐明了亚麻剂量氯胺酮早期持续暴露对斑马鱼幼鱼神经发育的毒性特征，并从神经细胞凋亡和NMDA受体亚基及行为相关基因bcl-2、c-fos表达调控方面研究氯胺酮引发的剂量-行为毒性效应机制，为进一步深入研究氯胺酮的神经毒性机理提供了研究新思路。更多还原

【Abstract】 Some toxicants can alter the normal activities of the nervous system, including neuraltransmission, connection and survival. Sometimes neurotoxicity result from exposure tothese toxicants can damage to health and threaten life. Numerous approved drugs haveneurotoxicity and have been limited to use or withdraw market. Thus the neurotoxicityassessment on early stage is the important content of drug riskassessment.The main evaluation mammals model, such as rodents, dogs, monkeys, raising costs ishigh, and take up more space because of they large size, time-and labor-consuming. There-fore, it is urgent to develop new complement or alternative models to early, fast andsensitively discover or evaluate the neurotoxicity of medicine. As a popular model, zebrafish(Danio rerio) are used in neurotoxicity of exogenous compounds in recent years. Comparedto rodent models, zebrafish have many advantages: low cost, easy raising; large numbers ofprogeny and rapid development apt to obtain sufficient sample size; small size permit tohigh-throughput screening; transparent embryos easy to observe toxic endpoints andconvenient drug delivery be able to improve objectivity of results. At present model animalszebrafish has been widely used in neuroscience researches including neural development,nerve damage and neural behavior studies. Also in neurotoxicity assessment fields, zebrafishapplication is increasing. However, because of unformed detection methods and inconsistentevaluation indexes, the most of neurotoxicity studies stayed only in the phenotypeobservation phase. Behaviors can early reflect disorders of nervous system functions onmacroscopic, which more direct and sensitive than pathology changes. By introducingcomputers and related parameters analysis software, researchers have developed the youngfish neural behaviors detection methods in recent years. The activities can early reflectdisorders of nervous system functions on macroscopic, which more direct and sensitive thanpathology changes. Thus, by introducing computers and related parameters analysissoftware, researchers have developed the young fish neural behaviors detection methods inrecent years. In this study we will mainly adopt these technologies, and establish andoptimize zebrafish larvae neural behavior high throughput detection methods in order toimprove efficiency of neurotoxicity research. And then we use some chemical compounds to validate larvae model efficiency.As a non-competitive glutamatergic antagonist, Ketamine is used to induce sedation andanalgesia in clinic. At present the research results from rodents test showed that anestheticdoses Ketamine exposure could induce neuron apoptosis. However, the toxic effects of sub-anesthetic Ketamine on nervous system development and mechanism are still undefined.By using the Noldus larvae behavior video tracking system and Ethovision XT7.0software, we detected larvae spontaneous movement, panic escape reflection after chemicalcompounds treatment. Many kinds of activities were recorded by real-time tracking, andsome motion parameters were exported by used Ethovision XT7.0for later statisticalanalysis. According to the research results of neurotoxicity of known toxin ornon-neurotoxin compounds, such as, ethanol, ketamine, cadmium chloride, lead acetate, andmodel validity were verified, we observed that movement and behavioral pattern of larvaespontaneous behavior changed after neurotoxic substance exposure, and produced differenteffects of motion, such as excitabity, inhibitive or reverse“U”type. Moreover, the reactionand habituation of larvae to outside stimulus reduced. These changes had certaindoses-effect relations, and neurotoxicity performance was consistent with reported in rodenttests or clinical toxic and side effects on nervous system. For providing technical support forthe establishment of the zebrafish model used in the evaluation of drug neurotoxicity, weverified the reliability of the model using the clinical drugs with or without knownneurotoxic effects such as loratadine and yeast. Moreover, in the process of detectionchlorpromazine and isoniazid with unknown information of neurodevelopment toxicity，weset up a series of test methods and verified the reliability of our experiment by comparingwith face effect of the mammals or clinical nervous system symptoms as well as thesensitivity of the model.Based on the zebrafish larvae model, we investigated the effects of sub-anesthetic dosesof ketamine on neurodevelopment toxicity and the long-term damage of the advancednervous function of zebrafish larvae. The results indicated that the sub-anesthetic dose ofketamine can induce excited or inhibitory changes in spontaneous movement of zebrafishlarvae at the early exposure stages. The data of neuron apoptosis detection by TUNEL assayrevealed that high dose ketamine inhibiting locomotion can lead to neurodegeneration inbrain, while lower dose katmine improving locomotion failed to cause neuron apoptosis, butcan cause a significant up regulation in the level of NMDA receptors by RT-PCR. Inaddition, ketamine exposure failed to induce apoptosis could reduce adaptability toenvironment stimuli, and have long-term damage on3mpf juveniles in social interaction aswell as learning and memory ability. By RT-PCR detection, ketamine exposure could decrease c-fos, bcl-2expression in zebrafish larvae. These results demonstrated thatsub-anesthetic doses of ketamine can cause developmental neurotoxicity on zebrafish larvae,and neuron apoptosis, NMDA receptors up-regulation, behavior development relative genesc-fos, bcl-2down-regulation are important mechanism of developmental neurotoxicity ofkatmine.In conclusion, we established zebrafish larvae neurobehavioral and neurodevelopmenttoxicity detection and evaluation methods, and optimized higher nervous activity ofjuveniles detection methods. Through verification efficiency of zebrafish larvae model, weprovided the test foundation for using zebrafish model to early, fast, and high-throughputassess neurotoxicity of drug. Meanwhile, we clarified that sub-anesthetic doses of ketaminecontinue exposure has neurodevelopment toxicity to zebrafish larvae, and the nerve cellapoptosis and NMDA receptors, behavior related genes bcl-2, c-fos expression regulationmaybe the important dose-behavior mechanism of ketamine toxic effects, furtherprovided new ideas for ketamine neurotoxicity mechanism research.更多还原