Trace Heavy Metals During Post–Natal Development and Aging in Normal and Pathological Human and Rat Retina

Abstract

Abstract: : Purpose: To compare the localization of trace metals (Iron, Cupper, Zinc) in normal and pathological human and rat retina by two different quantitative techniques: PIXE (Proton Induced X–Ray Emission) and FEGTEM (Field Emission Gun Transmission Electron Microscopy). Methods: Iron and trace metals concentrations and their tissue distribution were determined in the retina of normal and RCS rats at 35, 45 and 55 days and of human 43 to 95 years old. Cryo sections of 20µm were collected on Formvar films prepared on aluminium holders and freeze–dried , before analysis. Then we used PIXE method with a proton microprobe to analyse the metal contents of different retinal layers especially the photoreceptors (PR). A post–irradiation staining of the tissue sections allows us to identify precisely the retinal layers . For the FEGTEM analysis, retina from 55 day old normal and RCS rats were fixed with glutaraldehyde 2.5%, and embedded in epon. Ultra thin sections were stained with uranyl acetate and lead citrate, vaporized with carbon and analyzed with a Jeol 2019 Feg Field emission electron microscope. At high magnification, the different parts of the photoreceptor outer segments were targeted with a 1nm probe. The signal was processed to give the amounts of Fe and Zn. Results: Pixe analysis (resolution: 10µm) shows that Iron is unevenly distributed throughout the rat retina. The highest concentration is observed in the choroid, the retinal pigmented epithelium (RPE) and the inner segments of PR. Iron content is lower in the outer segments but still significant. It increases during both the development and the disease at the level of the segments. The distribution of other elements (Cu, Zn) revealed interesting temporal progressions. FEGTEM analysis at the nanometric level reveals a higher amount of Fe associated to the membranes of the discs than outside or inside the discs. In the human retina analysed by PIXE, iron is similarly distributed as in rat retina. Cupper content is lower than iron content in all the layers and is higher in the segments of PR than in choroid and RPE. A tendency of increase with age is observed in each layer. Conclusions: PIXE and FEGTEM can be used to detect trace metals concentrations in the different retinal layers and at the subcellular level during pathology and aging. The abnormal accumulation of these elements may be associated with neurodegenerative processes.

Keywords: retina • degenerations/dystrophies • metabolism