Quantifying the Change in Pigment Position in Dark- and Light-Adapted Retinal Pigment Epithelium in Mouse Retinas
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Pigment granules change position in the retinal pigment epithelium (RPE) when light conditions change. In the light, pigment granules disperse into the apical extensions of the RPE, which contact photoreceptors. In the dark, pigment granules aggregate to the base of the RPE. The goal of this study is to quantify the position of pigment granules in light-adapted (LA) mice and dark-adapted (DA) mice and compare the pigment positions between the two treatment groups. To accomplish this, eyes from three LA mice and three DA mice were obtained from MD Anderson. The eyes were prepared for transmission electron microscopy (TEM). Thin (70 nm) sections were obtained and viewed using a TEM. The density of pigment granules was quantified by juxtaposing 2 x 2 μm boxes aligned with a feature known as Bruch's membrane and counting granules in each box. Average pigment density (pigment granules/μm²) was calculated for each mouse. We hypothesized that pigment density would be higher in the DA mice as compared to the LA mice. The average pigment density was 1.4 ± 0.1 pigment granules/μm² in DA mice and 0.7 ± 0.1 pigment granules/μm² in LA mice. The means were statistically significantly different (p value= 0.02). The higher pigment density calculated is consistent with previously reported observations that pigment granules aggregate towards the basal end of the RPE in the dark and disperse apically in the light. Having a reliable mean allows us to explore the signaling pathways that regulate pigment granule movement. The morphology in the sub-RPE (area between Bruch's membrane and RPE) appeared less voluminous in DA mice than the sub-RPE in LA mice. This study quantified the sub-RPE on the TEM images to compare the predicted difference in size. vii We hypothesized that size of the sub-RPE in LA mouse would be larger. Using PowerPoint tools, the sub-RPE on each TEM image was filled in with solid red and the length of the sub-RPE was measured in inches. Images were imported into Python to get a count of the number of pixels in the sub-RPE. An average pixel count/length of sub- RPE was calculated for each treatment group. The average pixel count was 10,000 ± 2,000 in LA mice and 14,000 ± 6,000 in DA mice. Overall the difference in the means was not significant.