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J.I. Matsui, A.L. Egana, K. Koo, J.E. Dowling; Ethanol–Induced Microphthalmia Is Mediated by Changes in Cell Death and Proliferation in the Developing Zebrafish Retina . Invest. Ophthalmol. Vis. Sci. 2005;46(13):583.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Microphthalmia is a component of fetal alcohol syndrome (FAS), a collection of morphological and cognitive abnormalities exhibited by children born to mothers that consumed alcohol during their pregnancy. The mechanisms that underlie the decrease in eye size identified in FAS children remain largely unexplored. Recently, the reduction in the eye size phenotype has been recapitulated in zebrafish (Bilotta et al., Neurotoxicology Teratology, 46:737–43). The goal of this study is to determine if the microphthalmia observed in ethanol–exposed zebrafish embryos was caused by an increase in apoptotic cell death in the retina and/or a down regulation in cell proliferation in the ciliary marginal zone. Methods: To study the effect of ethanol on cell proliferation and death independently from the initial process of retinal cell specification and differentiation, zebrafish embryos were raised in fish water supplemented with ethanol (1%–1.75% per volume) from 2 days post–fertilization (dpf) and fixed on 3, 4, or 5 dpf. The area and diameter of eye and length of body from nose to tail were measured. TUNEL staining and antibodies to detect activated caspase–3 were used to determine if the ethanol triggered apoptosis in the retina. BrdU immunohistochemistry using a pulse/fix paradigm and transmission electron microscopy (TEM) were used to study the patterns of cell proliferation. Results: Analysis of the ratio between eye diameter and body length indicated that ethanol treatment caused a decrease in eye size that was not proportional to the concomitant reduction in body length observed. Based on TEM morphological data, we hypothesized that the ethanol activated an apoptotic–signaling pathway. A concentration dependent increase in the number of apoptotic cells was observed in the retina by TUNEL staining and activated caspase–3 immunohistochemistry. TEM and BrdU analysis of proliferation at the ciliary marginal zone indicated a decrease in cell proliferation as a consequence of ethanol treatment. Conclusions: These results indicate that the decrease in eye size observed in ethanol–treated embryos is not proportional to the decrease in body length, and thus there are ethanol–dependent mechanisms at play in the retina that underlie the microphthalmia phenotype. Studies with TUNEL and activated caspase–3 immunohistochemistry show that caspase–dependent apoptosis may be one factor regulating the size. A second factor is the inhibition in cell proliferation at the ciliary marginal zone based on the TEM and BrdU studies.
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