Abstract
Purpose :
The biochemical and histological examination of choroid and the status of its cellular components have been very challenging due to the presence of the retinal pigmented epithelium (RPE) layer between the choroid and retina. Here we developed methods for labeling and visualization of choroidal macrophages, mast cells, and vasculature in pigmented mice.
Methods :
Eyes from three albino lines (FVBN, CD-1, and BALB/c) and one pigmented line (C57BL/6J) of mice were collected, fixed for 1 h in 4% paraformaldehyde, washed 3 times in PBS, and kept in cold PBS until dissected. The wholemount of choroid-sclera complex from the albino and pigmented mice were independently stained with anti-Iba1 and anti-podocalyxin to label the choroidal macrophages and vasculature, respectively. Mast cells were labeled with Avidin-conjugated rhodamine. To eliminate the pigment from RPE cells of C57BL/6J eyes, the stained tissues were fixed again before bleaching; 4-5 h in a 55°C water bath using 1% H2O2. Tissues were then washed and incubated with secondary antibodies and Avidin-conjugated rhodamine. Choroidal macrophages, mast cells, and the vasculature were visualized using confocal microscopy.
Results :
The novel staining methods described here allowed the successful visualization of choroidal macrophages, mast cells, and the vasculature in both albino and pigmented mice. Choroidal macrophages in all mouse lines were denser around the optic nerve compared with the equator and periphery. Choroidal mast cells were relatively dense around the optic nerve of all mouse lines examined. However, their distribution in the equator and the periphery were different. The FVBN, CD-1 and C57BL/6J mice had fewer mast cells in the equator compared to the periphery. BALB/c mice had more mast cells, which were almost uniformly distributed in the equator as compared to the periphery. The total number of choroidal mast cells was highest in BALB/c and FVBN mice. The structure of the choroid in all four lines of mice was roughly comparable, except albino mice showed signs of hypotrophy.
Conclusions :
Staining and bleaching of pigmented eyes allowed the visualization of choroidal macrophages, mast cells, and vasculature in the eyes of pigmented mice. These methods should allow the evaluation of choroid and its various cellular components in transgenic and preclinical mouse models of eye diseases.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.