Zebrafish Model For Drug Neural Toxicity Assessment And Mechanism Of Developmental Neurotoxicity Of Ketamine

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.