Neurotoxicity Of Norfloxacin In Zebrafish Embryos(Danio Rerio)

Objective:Fluoroquinolones(FQs)are a group of commonly used antimicrobials.The central neurotoxicity is one of the most common adverse reactions seen in the clinical application of FQs.However,the mechanism of FQs-induced central neurotoxicity has not been clarified.In this study,we aimed to elucidate the developmental neurotoxic effects of norfloxacin(NOR)on zebrafish embryos and the mechanisms underneath them.We hope our research results could contribute to the further understanding of the developmental neurotoxicity of FQs especially in mammals.Methods:(1)Zebrafish embryos were exposed to different concentrations of norfloxacin(NOR)from 2 hpf to 14 dpf,and the morphology of the embryos was observed,and the rates of hatching,survival and malformation were recorded during the exposure of NOR.(2)After the exposure to NOR,the whole embryos were collected at 2 hpf,12 hpf,24 hpf,48 hpf and 72 hpf for the extraction of total RNA,and the transcription level of neurodevelopmental markers(sox2,nestin,gfap,olig2,map2 and eno2)and apoptosis-related genes(bax and bcl-2)were measured by quantitative real-time PCR(qRT-PCR).To analyze the spatial expression patterns of above-mentioned neurodevelopmental markers,whole mount in situ hybridization was performed in 24 hpf and 48 hpf embryos.To examine the effects of NOR on apoptosis and neuronal differentiation in the developing neural system of zebrafish embryos,the immunostaining of activated caspase 3 and HuC was conducted.(3)To antagonize the effects of NOR,MK-801,a NMD A receptor antagonist,was microinjected into 1 hpf embryos before the exposure of NOR.To analyze the antagonistic effects of MK801 to NOR on apoptosis and neuronal differentiation,the expression levels of bax,bcl2,activated Caspase 3 and HuC were measured.Result:(1)The exposure of NOR caused the decreasing of hatching rate,and increasing of mortality rate and malformation rate in zebrafish embryos in a dose-dependent manner.As the exposure time became longer,the effects of NOR on the rate of embryonic malformations and mortality was stronger.(2)After the exposure to NOR,the mRNA level of sox2,a stem cell marker,was significantly up-regulated at 12 hpf and 24 hpf;At 24 hpf,the neural stem cell marker nestin,glial cell marker gfap and oligodendrocyte marker olig2 were markedly down-regulated;At 48 hpf,neuron cell marker map2,neuron-specific RNA binding protein HuC and mature neuron marker eno2 were dramatically up-regulated,and the upregulation of eno2 was sustained till 72 hpf.(3)The NOR exposure induced obvious up-regulation of apoptosis in developing central neural system of zebrafish embryos,but this effect could be blocked by advanced microinjection of MK-801 to embryos.Meanwhile,MK-801 could also reverse the abnormal up-regulation of neuronal differentiation induced by NOR treatment.Conclusion:The early neurodevelopment of zebrafish embryos could be affected by NOR exposure,possibly due to the disrupted balance between neuronal differentiation and glial differentiation.The neurotoxicity of NOR might be closely related to the abnormal levels of apoptosis in the developing central nervous system,and the overactivation of NMDA receptor is a possible reason for the abnormal changes in apoptosis.