| Neurotoxicity is a common toxic and side effect of many drugs, new drug lead compounds,environmental compounds, and pesticides. It should evaluate neurotoxicity in drug screening early stage and preclinical safety evaluation stage. In vivo animal experiments are commonly used to evaluate drug neurotoxicity in traditional preclinical safety study, but the disadvantage of animal experiment is consuming a lot of experimental animals, longer test cycle, higher cost and subjective judgment of experimental results. With the development of various technologies of in vitro cell culture in recent years, it is possible to achieve the evaluation of neurotoxicity induced by drug or compound through testing the multi-parameter end point of cell structure and function change in vitro. The advantage is being able to screen neurotoxicants by high-throughput and high-content technology and explain and evaluate the neurotoxic effects from the mechanism of action of drugs. However, a single in vitro alternative method and test endpoint can not cover all neurotoxic targets, therefore it is necessary to establish a neurotoxicity assessment method for various drugs or compounds. Based on the in vitro neurotoxicity study background, in the study, the rat primary neuronal culture was firstly used for evaluating the neurovirulence of live attenuated vaccine. The neural stem cells and their differentiated cells of in vitro culture were used to screen the neurotoxicity of drugs.In vitro neurotoxicity evaluation of live attenuated vaccine. Methods: The extracting cerebral parenchyma from 1 to 3-day old SD newborn rats was digested and separated into the mixed primary nerve cells in vitro with sterile conditions and the stably cultured primary nerve cell system would be used to evaluate the neurotoxicity of live attenuated vaccines. The neurons and astrocytes were identified by MAP2 polyclonal antibody and anti-GFAP monoclonal antibody in the primary nerve cell culture model, respectly. To evaluate the neurotoxic effect of measles attenuated live vaccine, mumps attenuated live vaccine, as well as Diphtheria-pertussis- tetanus (acellular), inactivated poliomyelitis(absorbed) and Haemophilus influenzae type b conjugate vaccine (i.e. conjugate vaccine) on neuronal cells. These endpoints of evaluation included CCK8 cell proliferation toxicity, neuronal cell morphology change and concentration changes of neuronal immune related factors TNFa, IL-1β and IL-6. The results showed no significant cytotoxicity in CCK8 assays, characteristic changes in neuronal morphology, and changes in cytokines TNFa, IL-1β and IL-6 in measles live attenuated vaccine and mumps live attenuated vaccine group. Results: It showed significant cytotoxicity in CCK8 assays,deformation of neuronal cell body, increased nuclear-cytoplasmic ratio, and TNFa, IL-1β cytokines significantly increased in conjugate vaccine group. In this study, the results of in vitro evaluation of these live attenuated vaccines were consistent with the results of histopathological evaluation in monkey neurotoxicity study and reports of vaccine-induced adverse effects.To evaluate neurotoxicity of drugs on rat neural stem cells and differentiated cells. Methods: About 14 days pregnant SD rat fetal brain tissue was cut take, digest it into single neural stem cell and form suspended neurospheres in SD rats neural stem cells complete culture medium. The second generation of neurospheres was obtained by subculture of cells. In vitro high-throughput and high-content screening method was used to evaluate the neurotoxicity and developmental neurotoxicity of eleven drugs and/or compounds through detecting toxic effects of drugs on neurosphere growth, proliferation toxicity of neural stem cells by EdU cell proliferation and cytotoxicity assay, neurite outgrowth of neural stem cell differentiation into neurons and the toxic effects of drug on neural stem cells differentiating into neurons and astrocytes. Results: It showed that cisplatin, iron-oxide nanoparticles(ION), sodium valproate, phenytoin and ethanol could cause growth inhibition of neurospheres at a given time and dose level. Vincristine, propofol, acrylamide and ION can cause neurospheres dissociated into single cells and cell broken at a given dose level. 9-cis-retinoic acid (9cRA) and remifentanil had no significant toxic effects of neurospheres. Virginine, cisplatin, remifentanil, ION,propofol, sodium valproate, phenytoin, acrylamide and ethanol could cause the toxic effects of neural stem cells in EdU cell proliferation and cytotoxicity assay with a dose-dependent manner. Cisplatin,ION, propofol, acrylamide and 9cRA were shown to be able to promote neurite outgrowth. Remifentanil and phenytoin had a tendency to inhibit neurite outgrowth. Cisplatin, remifentanil, ION, propofol,sodium valproate, phenytoin, acrylamide, ethanol could affect the differentiation of neural stem cells, in which remifentanil mainly affected neural stem cells differentiating into astrocytes and ethanol affected neural stem cells differentiating into astrocytes and neurons, and other drugs could mainly induce neuronal cytotoxicity. 9cRA didn’t show any toxic effect to neural stem cell differentiation.Conclusions: In vitro alternative method established in this study was carried out in the phase of preclinical drug safety assessment. In the study, rat primary nerve cells firstly were used to evaluate and screen neurovirulence of live attenuated vaccines and neurotoxicity of various anti-tumor drugs,anesthetics and other drugs or compounds acting on the nervous system through testing a battery of endpoints of neurotoxicity. The results of this research could provide data for promoting the standardization and regulation of neurotoxicity in vitro replacement technologies by Agency for the Evaluation of Medicinal Products around the globe, and provide technical support for studying new methods and new technologies of preclinical neurotoxicity evaluation of new drug in our country. |
PDF Full Text Request