Session Title: Imaging II
Session Date/Time: Sunday 29/09/2013 | 08:00-10:00
Paper Time: 08:40
Venue: Hall G1 (Level 2)
First Author: T.Ach USA
Co Author(s): J. Messinger M. Bentley F. Delori R. Smith
To provide a digital map of human RPE cell number and total AF as a function of position relative to the fovea, using RPE-only flat-mounts of young and older adult donors.
University of Alabama at Birmingham, Department of Ophthalmology, Birmingham, AL, USA
1) Preserved chorioretinal tissues with healthy maculas were dissected and photo-documented with retina on, retina off, and retina + choroid off. Manually assigned landmarks were used to align all images and localize the fovea on resulting 18-mm square RPE-only flat mounts. 2) RPE cytoskeleton labeled with AlexaFluor647-phalloidin enabled semi-automatic location of cell centers. 3) Total AF (excitation 460-490 nm, emission > 505 nm) in projections of z-stacks through the RPE layer was evaluated relative to a fluorescence standard (Delori et al, PMID 22016060) using spinning disk confocal fluorescence microscopy. Voronoi Diagram analysis controlled gathering of AF intensities. 4) Locations for analysis (~90 per flat-mount) were chosen using an unbiased sampling grid that was more closely spaced at the fovea than in the perifovea.
Initial observations from 4 donors (34F, 47F, 83F, and 90F, white) indicate that cell geometry is hexagonal at 34 yr and variable with some large or binucleate cells at 90 yr. The Table shows data from 2 regions with different photoreceptor content - foveal center (all cones) and perifovea (ring of high rod density, 2 mm superior to fovea). RPE cell density is highest under the fovea and exhibits a shallow central-to-peripheral gradient. At all ages AF is low in the fovea, and highest 2-4 mm from the fovea, with quadrant varying. Normalized AF in the 3 older eyes is 2.0-2.7X higher than in the youngest eye. RPE/mm2 Fovea, perifovea 34F: 6380, 4810; 47F: 6420, 4360; 83F: 5690, 5120; 90F: 5808, 5311. AF (normalized; standard = 0.5) Fovea, perifovea 34F: 0.25, 0.37; 47F: 0.54, 0.65; 83F: 0.69, 0.89; 90F: 0.50, 0.61.
An RPE-only flat-mount tissue preparation, confocal microscopy, and new computational methods allow a precise mapping of RPE cell number and AF properties as a function of location relative to the fovea. AF intensities superficially follow the distribution of rod photoreceptors, yet are higher nasal to the disk than predicted by rod topography (Curcio et al, PMID 2324310). RPE cell number is more tightly regulated than AF. Additional maps, in progress, will provide a comparison for AMD eyes and a histological reference for clinical fundus AF."