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K. Koo, J. I. Matsui, J. E. Dowling; Post-Exposure Reduction of Ethanol-Induced Microphthalmia in the Developing Zebrafish Rescues Retinal Morphology and Behavior. Invest. Ophthalmol. Vis. Sci. 2007;48(13):65.
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© ARVO (1962-2015); The Authors (2016-present)
Zebrafish embryos exposed to ethanol exhibit microphthalmia, which is often observed in children with fetal alcohol syndrome. The reduction in eye size is caused by dose-dependent changes in cell proliferation and apoptosis (Matsui JI, et al. IOVS 2005;46:ARVO E-Abstract 583). Chronic exposure to ethanol also causes a decrease in visual performance as measured by the optokinetic reflex (OKR). This study examines whether allowing zebrafish to recover after ethanol exposure can reverse the changes underlying the phenotypes.
Zebrafish embryos were treated with ethanol (1.5% or 1.75% by volume) at 2 days post-fertilization (dpf) and fixed at 3, 4, or 5 dpf. Some embryos were treated with ethanol for 24 or 48 hours and then allowed to recover in fresh fish water before being fixed at 5 dpf or raised to 14 dpf to test the OKR. To assess relative eye size, data comparing eye diameter (ED) to body length (BL) were collected. Changes in proliferation were evaluated by cell density and BrdU immunohistochemistry with a pulse/fix paradigm. To confirm that caspase-dependent apoptosis contributes to the microphthalmic phenotype, the pan-caspase inhibitor, z-VAD-fmk, was used with appropriate apoptotic-marker immunostaining.
Ethanol treatment caused a reduction in cell density 24 hours post-exposure. Fish that were allowed to recover after ethanol exposure had increased eye size relative to body length. Cell densities rebounded to normal levels in 5 dpf fish with a 48-hour recovery. Rescue of the OKR was observed in 14 dpf fish with 24 or 48, but not 72, hours of ethanol treatment. A partial reestablishment of cell proliferation in the marginal zone and an absence of cytoplasmic cytochrome c or active caspase-3 in the central retina were observed. Fish exposed to both ethanol and a caspase inhibitor showed similar eye size and cell density as the controls and had normal OKRs.
These results indicate that caspase-dependent apoptosis can be reduced and cell proliferation reestablished following acute treatment of ethanol, leading to a complete rescue of the morphological phenotype and a partial rescue of the behavioral phenotype.
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