Related Experiment Video
Updated: May 25, 2026
Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures
Published on: April 14, 2023
Accumulation and autofluorescence of phagocytized rod outer segment material in macrophages and microglial cells
Lei Lei1, Radouil Tzekov, Shibo Tang
1The Department of Ophthalmology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Purpose:
To explore the ability of macrophages and microglial cells to phagocytize rod outer segments (ROSs) in a cell culture and characterize the resulting lipofuscin-like autofluorescence (LLAF).
Methods:
Either regular or modified ROSs or ROS components (11-cis-retinal, all-trans-retinal, lipids) were fed to macrophages and microglial cells for 4 days. Afterwards, autofluorescence was detected by fluorescence-activated cell sorting (FACS) at two different wavelengths (533 nm and 585 nm), and the cells were imaged by confocal and electron microscopy. Fluorescein isothiocyanate (FITC)-labeled ROSs were added to macrophage and microglial cell cultures for 1-24 h to determine the kinetics of phagocytosis in these cell lines.
Results:
Feeding with different ROSs or ROS components led to a significant increase in LLAF in both microglia and macrophages. The 4-hydroxynonenal (HNE)-modified ROSs gave rise to the highest increase in LLAF at both 533 nm and 585 nm. Application of 11-cis-retinal or all-trans-retinal resulted in higher LLAF at 585 nm, compared to application of 9-cis-retinal or liposomes. Fluorescein isothiocyanate-labeled ROSs co-localized well with lysosomes in both types of cells. HNE-modified ROSs were phagocytized more rapidly by both types of cells, compared to unmodified ROSs. Electron microscopy demonstrated inclusion bodies containing whorls of membranes in all types of cells fed with ROSs.
Conclusions:
Both macrophages and microglia have the ability to phagocytize ROSs, and this results in increased autofluorescence. Oxidation of ROSs results in faster phagocytosis, higher levels of LLAF, and the appearance of more inclusion bodies inside the cells. Results from the present study suggest that both types of cells accumulate lipofuscin-like material under physiologically relevant conditions. Such accumulation could interfere with their ability to clear cellular debris and could be part of the pathogenetic mechanism for age-related macular degeneration and other lipofuscinopathies.
