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N. N. Dalal, C. Gardner, J. D. Wisard, C. J. Johnson, M. A. Chrenek, A. Agarwal, J. M. Nickerson; Quantitative Analysis of Mouse RPE Flat-Mounts. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2922.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigmented epithelial (RPE) cells play a central role in vision and disease, yet there is little quantitative data describing sheet structure in the mouse. We hypothesized there would be defects in the array of the RPE sheet in mutants that affect the function of RPE or adjacent photoreceptor cells. Quantitative measures of patterning and tiling should detect changes in the RPE monolayer during development, growth, and disease progression. We developed a high-throughput method for analyzing whole RPE mouse flat-mounts with several measures including cell shape, area, density, tiling and 2D organization of this sheet. We studied RPE sheets in Rpe65-/-, IRBP -/- and C57BL/6 (WT) mice.
Mice were sacrificed and eyes immediately enucleated and fixed prior to dissection. Six lateral cuts were made perpendicular to the limbus, originating from the center of the cornea and ending before the optic nerve. Lens, vitreous, and retina were removed to reveal the RPE monolayer. Tight junctions between RPE cells were illuminated through fluorescent staining of ZO-1 and subsequently imaged by confocal microscopy. Images were normalized for brightness and contrast and filtered to reduce noise through Adobe Photoshop CS3. CellProfiler (www.cellprofiler.org) was used to analyze specific regions of the sheet to yield quantitative measures for cell count, density, extent, eccentricity, form factor, and the number of neighbors per cell.
Comparing P720 IRBP-/- and Rpe65-/- mice with WT age-matched mice (n= 3 per genotype) significant differences in cell density were observed between IRBP-/- and WT, but not between Rpe65-/- and WT. Differences in cell patterning were observed in the number of neighboring cells. Rpe65-/- had the greatest variation in sidedness while both the IRBP -/- and the WT had similar variation. Rpe65-/- also had a significantly lower average number of neighbors per cell than WT or IRBP-/-. In measures of eccentricity, solidity, form factor and extent, Rpe65-/- differed significantly from IRBP -/- and WT.
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