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Bogale Aredo, xiao chen, Kaiyan Zhang, Rafael Ufret-Vincenty; Change in the distribution and phenotype of subretinal macrophages with aging in C57BL/6 mice. Invest. Ophthalmol. Vis. Sci. 2013;54(15):162. doi: https://doi.org/.
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There is significant debate surrounding the role of macrophages-microglia in age-related macular degeneration (AMD) and also in retinal dystrophies. In order to better understand the behavior of subretinal microglia, we explored the natural history of their distribution and phenotype in wild type B6 mice vs. RD8 mutant mice.
Naïve C57BL/6 mice wild type (WT), heterozygous or homozygous for the RD8 mutation were used. Mice were divided into two age groups: young/adult (1-9 months) and old (14-21 months). Retinal images were taken using a Micron III rodent fundus camera (Phoenix Research Labs). Yellow spots in the central fundus (5 disc radius from the center of optic disc) were counted. Eyes were enucleated and fixed in 4% paraformaldehyde. Posterior segment flat mounts (sclera-choroid-RPE complex) were single or triple stained for ionized calcium binding adaptor molecule 1 (Iba-1), mouse Macrophage Mannose Receptor (MMR) or mouse FcγIII/II Receptor (CD16/CD32). Stained microglia on flat mounts were counted for each of the markers, for both the central region (within a 5 disc diameter radius from the optic nerve), and for the total RPE. RPE cells were isolated from either the central or peripheral retina, using a new technique we developed, and the expression of inflammatory and chemotactic genes was analyzed by RT-PCR.
The number of yellow spots in the central fundus of RD8 mutant mouse was higher than in WT and highly correlated with Iba-1+ cells. The differences increased with aging; in the young/adult mice, mutants had more central Iba-1+ cells than WT (p=0.002) and this difference was more dramatic in the old group (p<0.001). MMR+ and CD16/32+ staining cells were significantly increased in the RD8 mutant mouse vs. WT and the ratio of CD16+/MMR+CD16- increased with age in RD8 mutants. The total and peripheral microglia counts in the whole RPE FM also increased in the RD8 mutant mouse vs. WT. We will also present gene expression levels of microglia activation markers and chemoattractants in central vs. peripheral RPE.
Both aging and the RD8 mutation affect the distribution and phenotype of subretinal microglia.
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