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T. Mizuno, T. Ohtsuka; Effect of Caspase Inhibitor on the Retinal Development of Adult Teleost. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5552.
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© ARVO (1962-2015); The Authors (2016-present)
The role of apoptosis in the development of retina is not fully known. In the marginal retina of adult goldfish, two types of apoptotic cells were found (Mizuno and Ohtsuka, 2008). We studied one of these apoptotic cells, caspase-3 positive cell, by intravitreal injection of caspase inhibitor.
Adult goldfish, about 7 cm body length, was used. Caspase inhibitor, zVAD-fmk (#627610, Calbiochem), 400 µM in PBS containing 0.2% DMSO was injected into the vitreous body of left eye; the final concentration was estimated as 40 µM. The vehicle was into the other eye. The eye from the decapitated fish was opened for incubation in a solution containing anti-cleaved caspase-3 polyclonal antibody (#9661, Cell Signaling Tec) 1:200 for overnight. The labeled cells visualized by AlexaFluor 488 anti-rabbit IgG were investigated in the whole mounted retina. Further the retinal cell density was separately studied in the serial radial section, 2µm thickness, stained by Toluidine blue.
In the retinal whole mount, the activated caspase-3 positive cells were found in the marginal retina from the retinal rim to 100 µm toward the central retina, mostly in the primary growth zone. At 12 hours after injection of caspase inhibitor, these labeled cells were decreased by about 60% in comparison to the control retina. This decrease was also observed in fish kept alive for 24 hours after injection. Furthermore, we studied the total number of retinal cells in the radial sections. At 12 hours after injection, a significant increase of cell density was found in the growth zone: about 20% in the primary and about 10% in the secondary growth zone. At 7 days, the increase was found only in the secondary growth zone. Fish kept alive more than 2 weeks after injection showed the increase of cell density in the inner nuclear layer of mature retina.
We confirmed the caspase inhibitor gave the hyperplasia of retinal cells in both the primary and secondary growth zones. It seemed that caspase-dependent cell death contributed to keep the total cell number in the developing retina.
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