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Natalie Pankova, Hai Wang, Xu Zhao, Huiyuan Liang, Shelley R Boyd; Macrophages contribute to the two-dimensional patterns observed using Fundus Autofluorescence (FAF) in a rodent model of retinal damage.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2214.
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© ARVO (1962-2015); The Authors (2016-present)
Phagocytic macrophages (MΦs) are reported in diseases including Age-related Macular Degeneration (AMD), but it is not possible to identify MΦ populations clinically in vivo. Eyes at high risk of geographic atrophy (GA) or expanding GA demonstrate complex 2-dimensional (2D) patterns of hyperfluorescent fundus autofluorescence (FAF), the source of which is not fully understood. We previously reported a novel technique known as Immuno-Delayed Near-Infrared Analysis (Immuno-DNIRA), an in vivo imaging method using indocyanine green dye to label MΦs and view them by confocal Scanning Laser Ophthalmoscopy (cSLO) in the rodent eye. The purpose of this study was to 1) combine DNIRA with FAF to correlate the spatial distribution of labelled MΦs with patterns of toxin-induced tissue damage, and 2) compare the 2D en face DNIRA patterns against 2D patterns of MΦs in excised wholemount tissue samples following retinal damage.
RPE/retinal damage was induced in Sprague Dawley rats by systemic injection of sodium iodate (vs saline control), and FAF and DNIRA imaging was performed in the subsequent days and weeks using a commercial cSLO. At corresponding timepoints, posterior globes were harvested, and retinal and RPE/eyecup tissue was dissected and examined using an epifluorescent microscope with 488 nm excitation. Immunofluorescence was performed using antibodies against MΦ markers (CD68, Iba1), compared to IgG controls.
In vivo imaging using DNIRA demonstrates migration of labelled MΦs into the damaged outer retina following retinal/RPE damage in toxin-treated animals compared to controls. In vivo comparison between the FAF and DNIRA signal suggests co-localization of MΦs within areas of tissue damage. Over time, labeled cells accumulate and appear to move spatially in areas of ongoing damage. Autofluorescent microscopy of excised tissue from toxin-treated animals shows bright mononuclear hyperfluorescent cells arrayed in 2D patterns representative of those seen in vivo. Immunofluorescent microscopy confirms these cells stain positively for CD68 and Iba1.
These data demonstrate that acute RPE injury induces corresponding spatiotemporal changes using both FAF and DNIRA. Autofluorescent and immunofluorescent microscopy of excised tissue suggests that MΦs contribute to the 2D patterns observed in vivo at both wavelengths.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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