Abstract
Purpose :
To investigate age-dependent changes in mouse retinal epithelium (RPE) cells, including cell number, cell size, and the concentration of the pigmented granules and their emission spectra, using multicolor confocal fluorescent microscopy (MCFM). These changes were compared between a mouse model of Stargardt disease (Abca4-/- [129S4]) and agouti wild-type (WT) controls.
Methods :
We performed in situ live tissue imaging of RPE flat-mounts from agouti Abca4-/- and agouti WT (129S1/SvlmJ) controls at multiple ages (1.5 months, 4 months, 8 months and 13 months) using a Nikon A1 confocal microscope. High-resolution confocal image z-stacks of the RPE cell mosaic were acquired with four different excitation wavelengths (405nm, 488nm, 561nm, and 640nm). The autofluorescence images of RPE, including voxel-by-voxel emission spectra, were acquired and processed with Nikon NIS-AR Elements software.
Results :
An enhanced visualization of the RPE cell mosaic as well as pigmented granules (melanosomes and lipofuscin) with an unprecedented resolution and contrast were generated using MCFM for both mouse strains at all ages. The changes in RPE cell number, cell volume, concentration of the lipofuscin and melanosomes (per cells), the emission spectra from the lipofuscin granules, and the total emission spectra from the RPE volume were quantified and compared over time between strains. We found the concentration of lipofuscin consistently increased with age whereas the number of melanosomes decreased proportionally in both mouse strains. At all ages there was an increased number of lipofuscin granules in Abca4-/- compared to WT.
Conclusions :
A systematic and comprehensive investigation of the age-dependent changes in RPE cells and pigmented granules was possible with MCFM. An increased number of lipofuscin and decreased number of melanosomes with age is common in both mouse strain, albeit, more rapid in Abca4-/-. The reduction in melanosomes could be linked with the formation of melanolipofuscin, which requires additional investigations. The changes in the RPE with age and Abca4-/- deficiency could be linked the pathological condition associated with a Stargardt disease.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.