Detecting Oxidative Stress in Zebrafish Embryos

Science Center, Bent Corridor

Autism Spectrum Disorder (ASD) is a category of diagnoses denoting cognitive, social, and behavioral abnormalities. Autism affects 1 out of every 68 children in the United States and is considered a neurodevelopmental disorder, mainly detected in childhood. The molecular mechanisms underlying ASD are not well known. Genetic analyses have recently associated oxidative stress pathways with ASD. To test oxidative stress in the context of neurodevelopment, we developed an assay to reliably measure levels of oxidative stress in zebrafish embryos. Oxidative stress was measured using a fluorescent dye, H2DCFDA, which fluoresces only after reacting with reactive oxygen species. Embryos were treated with the dye and then homogenized into lysates by sonication; oxidative stress in the samples were then quantified using a fluorescence plate reader. We optimized the assay for a number of variables, including dye concentration, time of exposure, and embryos per sample. The assay demonstrates a positive linear relationships between fluorescence output and dye concentration. This assay can assess the oxidative stress pathway in neurodevelopment in vivo, including the effects of environmental and genetic factors associated with disorders such as ASD.