A new RPE flatmount technique for comparative morphometric analysis using Alizarin red staining, confocal and electron microscopy in different species

Abstract

Abstract: : Purpose: To visualize, delineate and compare the retinal pigment epithelium (RPE) by using a new flatmount technique in several species. Methods: A new technique was designed to prepare the freshly enucleated eye by radial cuts in the cornea. Subsequently the iris, lens, vitreous and retina were removed by microscopic technique. The radial incisions were extended beyond the equator and the specimen was mounted on glass. In human eyes the sclera was replaced by Millipore membrane support (Del Priore et al IOVS 2002; 43:3312–3318). Cohesive flatmounts of corneal endothelium and RPE in albino mice, rats, rabbits and caucasian human tissue were examined by 0.2 % Alizarin Red S staining in light microscopy and phase contrast microscopy. Specimens were also studied by confocal scanning laser microscopy (Nidek Confoscan 3) by propidium iodine counterstain to CK–18, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). After digital photography the images were analyzed using Konan KSS–300 software to determine retinal pigment epithelial cell density (RPECD). Age influence was studied in rats of different maturity (6 wks vs.1 year). RPE damage was assessed in rats after two weeks exposure to subretinal perfluorooctane (PFO). Results: Dye–uptake of Alizarin Red S at the hexagonal intercellular borders was more thin and tight in RPE than in corneal endothelium in all species. Cell loss was indicated by staining of sub cellular supporting basal membrane area. The RPECD in human was 1805+– 198, in rabbit 1355 +– 322, in mouse 2308 +– 142, in 6 wk rat 1159 +– 317 and in 1 year old rat 1195+– 360. Many RPE cells showed two nuclei or more. PFO exposure caused extensive localized damage to the RPE. Human tissue analysis was dependent on a time limit of less than 35 hours between death and tissue dissection. Highly pigmented tissue was less suitable for staining. Conclusions: Highly undisturbed micro dissected flatmounts of the RPE offer excellent monitoring of disease processes in these tissues in many species including humans. The RPE cell density is less than the normal endothelial cell density in each species. Alizarin Red S staining is a valuable tool in delineating the morphometric parameter of the RPE.

Keywords: retinal pigment epithelium • pathology techniques • pathobiology