Fig 1.
CD59a expression in mouse retina and mRNA levels of CD59a in NSR and isolated RPE.
Photomicrograph showing CD59a immunolabeling in all layers of mouse retina, RPE and choroid (B), but not in CD59aKO eyes (C). Graph of qPCR results showing that mRNA levels of CD59a in NSR were more than 60-fold higher than that in isolated RPE cells in WT eyes (**P<0.001) (D). Data are expressed as means ± SD. N = 4. RPE, retinal pigment epithelium; OS, photoreceptor outer segment; IS, photoreceptor inner segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.
Fig 2.
Photomicrographs of plastic sections of WT and CD59aKO retinas, and quantification of ONL nuclei.
Histology of mouse retinas showing thinning of the ONL (white brackets) and IS/OS in post-LD WT retinas, while CD59aKO retinas showed less thinning of the ONL and IS/OS (A). Plot of the number of nuclei per column in the ONL. In WT retinas on both the Balb/c and C57BL/6 background, there was a significant decrease of ONL thickness across all sampled retinal regions (a-d) comparing non-light- damaged (NLD) (black line in B and C) and LD (green line in B and C). However, there was a smaller reduction of ONL thickness comparing NLD (blue line in B and C) and LD CD59aKO retinas (red line in B and C). Data are expressed as means ± SD. N = 4. *P<0.01, and significance markings refer to overall differences between groups across all four retinal regions (a-d).
Fig 3.
ERG responses before and after LD.
Before LD, rod-a (P<0.05) and rod-b (P<0.05) waves were decreased in CD59aKO Balb/c mice compared to WT (A). At day 7 after LD, however, rod-a (P = 0.04) and rod-b (P = 0.01) waves were all larger in CD59aKO compared to WT (A). Data are expressed as means ± SD. N = 4. Similarly, rod-a (P<0.05) and rod-b (P<0.01) wave amplitudes were smaller in CD59aKO C57BL/6J mice compared to C57BL/6J mice at baseline, and while C57BL/6J mice have diminished ERG amplitudes 10 days after LD, amplitudes in CD59aKO mice remain unchanged (B). Data are expressed as means ± SD. N = 6–7.
Fig 4.
11-cis retinal regeneration and rhodopsin levels in CD59aKO retinas.
11-cis retinal regeneration analysis (A). Before bleaching, there was no difference in the percentage of 11-cis retinal of total retinoid. Also, no differences were observed in regenerated 11-cis retinal at 10 m intervals after bleaching as indicated. Data are expressed as means ± SD. N = 8. Western blotting analysis of rhodopsin protein in WT and CD59aKO-Balb/c mice (B and C). There was significantly lower rhodopsin protein in CD59aKO mice compare to WT-Balb/c mice (P = 0.03). N = 3. Microspectrophotometric rhodopsin measurement in NSR of C57BL/6 WT and CD59aKO (D). There was significantly lower rhodopsin concentration per retina in CD59aKO mice compared to WT C57BL/6 mice (P = 0.001). N = 5.
Fig 5.
Quantification of RPE phagocytosis of shed outer segment fragments via immunostaining with anti-rhodopsin antibody.
CD59aKO RPE showed maximal phagosome content 1 h after light onset as characteristic for normal outer segment renewal. Representative retinal cross sections (A) showing opsin immunolabeling (green) and cell nuclei (blue) of CD59aKO outer retina of mice sacrificed at 1 h, 2.5 h, and 13 h after light onset as indicated. In each section, two phagosomes in the RPE are indicated by arrows. Scale bar = 10 μm. Quantification of phagosome counts (B). Eyes from 5 different mice for each time point, and 6 sections of each eye were imaged for each mouse. Data are expressed as mean ± SD. N = 5.
Fig 6.
Immunofluorescence microscopy of GFAP in NLD CD59aKO and WT retinas.
Compared to the labeling limited to the innermost retina in WT mice (C), the GFAP signal was up-regulated in Müller cells of CD59aKO retinas (D). Quantification of fluorescence intensity showed that GFAP was significantly increased in CD59aKO retinas compared to controls (B) (P = 0.03). Data are expressed as means ± SD. N = 4.
Fig 7.
The mRNA levels of ER-stress genes and neurotrophic factor genes in NLD CD59aKO retinas versus WT retinas.
Two ER-stress genes, Grp78 (P = 0.01) and Grp94 (P = 0.01), were significantly up-regulated in CD59aKO retinas (A). Neurotrophic factor genes, Fgf2 (P<0.01) and Vegf (P = 0.03), also were significantly up-regulated in CD59aKO retinas (B). mRNA levels were also measured for three members of the neuropoietic cytokine family known to have retinal neuroprotective effects: ciliary neurotrophic factor (CNTF), interleukin-6 (IL-6), and leukemia inhibitory factor (LIF). mRNA levels for all three factors appeared lower in CD59aKO relative to WT retinas, with significant differences in IL-6 (P<0.01) and LIF (P<0.001) expression (C). Data are expressed as means ± SD. N = 4.
Fig 8.
Western blotting analysis of heat shock proteins in NLD CD59aKO versus WT retinas.
The anti-KDEL antibody recognizes both GRP78 and GRP94, and they were both increased in retinas of CD59aKO-Balb/c mice (A). Comparison of the levels of GRP78 (P = 0.02) and GRP94 (P = 0.01) showed higher levels in CD59aKO (B). Similarly, both GRP78 (P = 0.03) and GRP94 (P<0.01) were increased in retinas of CD59aKO-C57BL/6 mice (C and D). Data are expressed as means ± SD. N = 3.