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Michael Sulewski, Delu Song, Jiantao Song, Chenguang Wang, Esther Clark, Jacob Sterling, Joshua L Dunaief; Complement component 5 facilitates the influx of microglia/macrophages into the photoreceptor layer of light damaged mouse retina. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3554.
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© ARVO (1962-2015); The Authors (2016-present)
Although the precise cellular mechanism is unknown, previous work has demonstrated that complement components 3a and 5a (C3a and C5a) contribute to retinal damage in a light-damage model for retinal degeneration. Because C3a and C5a are chemotactic and macrophages express receptors for these proteins, we tested the hypothesis that migration of retinal microglia/macrophages may be an integral component of complement-dependent immune damage.
Wild-type (WT) as well as C3aR-/- and C5aR-/- mice, all on a Balb/c background, were exposed to bright white fluorescent light for 3h. At 48h following exposure, animals were sacrificed and eyes were enucleated. The neurosensory retina was dissected from one eye from each animal for quantification of Iba1 mRNA expression by RT-qPCR. Contralateral eyes were cryo-sectioned or flat-mounted and immunolabeled with Iba1 specific antibodies. Retinal flat-mount samples were imaged with a Zeiss confocal imaging system. Iba1 positive cells in cryo-sections were counted by a masked observer in three zones (zone 1: ganglion layer to outer plexiform layer, zone 2: outer nuclear layer, zone 3: inner and outer segment layers). Statistical differences among groups were analyzed by one-way ANOVA.
Relative mRNA levels of Iba1, a marker of microglia/macrophages, in the neurosensory retina were significantly less in C5aR-/- mice exposed to LD (n= 3, P=0.02) but not in C3aR-/- mice (n=4, P=0.70) when compared to WT-LD mice (n=3). Confocal images showed fewer Iba1 positive cells in retinal flat mounts from C5aR-/- compared to WT and C3aR-/- mice. In cryosections, the number of Iba1 positive cells was significantly increased in WT retinas exposed to LD compared to non-LD wild-type retinas (WT-LD vs NLD, zone 1: 28.2 vs 10.3 , zone 2: 24.5 vs 3.3, zone 3: 15.4 vs 4.7 P<0.001 ). Following LD, the number of Iba1 positive cells was not significantly different in C3aR-/- retinas (zone 1: 24.7, zone 2: 21.4, zone 3: 14.0) when compared to WT-LD. In C5aR-/- mice, the number of Iba1 positive cells were significantly reduced (zone 1: 14.1, zone 2: 11.6, zone 3: 8.4; P<0.01) when compared to WT-LD mice.
C5aR but not C3aR is important for microglia/macrophage migration to all layers of the retina following LD. The C5aR pathway may be important in microglia-mediated retinal degeneration including age-related macular degeneration.
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