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Peter Kayatz, Klaus Heimann, Ulrich Schraermeyer; Ultrastructural Localization of Light-Induced Lipid Peroxides in the Rat Retina. Invest. Ophthalmol. Vis. Sci. 1999;40(10):2314-2321.
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purpose. Localization of light-induced lipid peroxides in the rat retina at an
ultrastructural level as benzidine-reactive substances.
methods. Long–Evans rats with nondilated pupils were exposed to intense light
of 6000 lux for 12 or 24 hours. Control animals were kept under
physiological light conditions. Rats with dilated pupils were exposed
to a light intensity of 50 lux or 150,000 lux for 1 hour. For
ultrastructural localization the enucleated eyes were fixed in a 0.1-M
cacodylate buffer (pH 7.4) containing 2% glutaraldehyde for 2 hours.
Pieces of the superior part of the central eyecup were incubated
overnight with tetramethylbenzidine (TMB; pH 3.0) at 4°C, postfixed
with 1.5% OsO4, and embedded for electron microscopy.
results. In animals exposed to intense light, electron-dense structures appeared
exclusively throughout the rod outer segments after an irradiation of
6000 lux for 24 hours or 150,000 lux for 1 hour and were absent in
animals with nondilated pupils kept at physiological light conditions.
Dilation of the pupils leads to the appearance of electron-dense
structures after just 1 hour of 50 lux, whereas rats with nondilated
pupils withstand even a 12-hour irradiation with 6000 lux. No
electron-dense structures were found when no TMB was used in
conclusions. The appearance of electron-dense structures in the rod outer segments
depends on the incubation with TMB and intensive light exposure of the
rat. Dilation of the pupils lowers the threshold for the emergence of
electron-dense structures significantly. This strongly supports the
view that light-induced lipid peroxides in the rat retina are localized
at an ultrastructural level as benzidine-reactive substances. This
protocol presents a tool for the generation and ultrastructural
localization of lipid peroxides in rat
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