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T. Mizuno, T. Ohtsuka; Diurnal Variation of Apoptosis in the Adult Teleost Retina. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4951.
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© ARVO (1962-2015); The Authors (2016-present)
In the adult teleost, germinal cells in the retinal margin proliferate, and some of the daughter cells die before retinal maturation. The underlying mechanism of cell death in this case is not fully known. We identified and counted apoptotic cells in the adult goldfish retina by immunohistochemical labeling of activated caspase-3.
Adult goldfish, ~7 cm body length, were sacrificed during daytime and nighttime. Eyes were removed and opened, the whole retina was incubated overnight in polyclonal antibodies to cleaved caspase-3 (#9661, Cell Signaling Tec), and labeled cells were visualized by AlexaFluor 488 anti-rabbit IgG. The whole retina was mounted flat on a glass slide, caspase-3-positive cells were counted, and the whole retina was stained by 2% Toluidine Blue for further histological analysis. We also investigated the localization and morphology of labeled cells in serial 15µm cryosections.
By Toluidine Blue staining, the marginal retina was divided into three regions (from periphery to center): the proliferating zone, the growth zone, and mature retina. In whole-mounts, labeled cells were found from the retinal rim to 100 µm toward the central retina, mostly in the growth zone; only a few labeled cells were found in the central retina. The cell density (cells/mm2) in the retinal margin of the dorsal quadrant was 158±21.9 in three goldfish sacrificed at midday, but only 120±2.63 in three goldfish at midnight; there was a statistical significant difference (P>0.05, t-test). In cross-sections, labeled cells of the growth zone were localized distal to the immature inner plexiform layer. In about half of these cells, the non-fluorescent nucleus was visible as a dark spot, whereas in the remaining half no nucleus could be detected; these two cell types may represent, respectively, early and late stage of apoptosis.
In the retinal margin of adult teleosts, apoptosis (caspase-dependent cell death) was found mainly in the circumferential growth zone. The increase of apoptotic cell numbers during daytime indicates that caspase-3 activation may depend on environmental light conditions or an endogenous circadian rhythm.
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