Abstract
Purpose::
To understand the pathological changes of fundus autofluorescence (AF) in normal aged mice and in mice developing age-related macular degeneration (AMD)-like changes (CCL2 deficient mice).
Methods::
Fundus AF was evaluated in mice of different ages (2, 6, 12, 18, 24 months) both in vivo by scanning laser ophthalmoscopy (SLO) and in vitro by confocal microscopy. Retinal and choroid/retinal pigment epithelial (RPE) cell flatmounts were immunostained with anti Iba-1, F4/80 or CD45 antibodies to identify retinal microglia and Alexa Fluor 633-phalloidin to outline RPE cells. All samples were observed by confocal microscope (LSM510 META) using either the multi-channel scanning system or the META scanning system.
Results::
Confocal microscopic observation of retinal and choroid/RPE flatmounts revealed an increased number of Iba-1 positive microglia in the photoreceptor outer segment layer and on RPE cell surface with age. Autofluorescent granules were observed in microglia in the subretinal space of 6 months old mice. The number of autofluorescent granules per microglia and the number of autofluorescent granule-loaded microglia increase with age. In mice with CCL2 deficiency, many more AF microglia were observed in the subretinal space compared to wild-type controls of the same age. Autofluorescent granules were observed in RPE cells in mice older than 12 months and the number of AF-REP cell is very low (1.48/mm2 at 12 month and 5.02/mm2 at 24 month) compared to the number of AF-microglia (20.63/mm2 at 6 months and 76.36/mm2 at 24 months). The emission spectrums of autofluorescent granules in sub-retinal microglia, as detected by the LSM META scanning system, were the same as those in RPE cells. In SLO, fundus AF was observed in mice older than 12 months old. AF regions were characterized as small hyperfluorescent dots scattered in the fundus and corresponded with the confocal choroid/RPE flatmount images.
Conclusions::
Retinal microglia migrate to subretinal space with age. Early fundus AF is derived from subretinal microglia. Microglia may play an important role in retinal ageing and age-related macular degeneration.
Keywords: age-related macular degeneration • microglia • retinal pigment epithelium