Figure 7.
Figure 7. Pathology in retinas of 18–20-mo-old Klc1−/− mice. (A and B) Semithin sections of WT (A) and Klc1−/− (B) central retinas. The ONL (black bar) is thinner in the mutant. (C) Number (mean ± SEM) of aligned photoreceptor nuclei across the ONL (i.e., per column) in WT (n = 5; closed circles) and Klc1−/− (n = 6; open squares) mice. Note the significant loss of nuclei in the central retina of mutant mice (Student’s t test; *, P < 0.05; **, P < 0.01). (D and E) Retinal sections showing autofluorescence (488-nm excitation, 500–600-nm emission) of RPE in WT (D) and Klc1−/− (E) mice. (F and G) EMs of RPE of control (F) and Klc1−/− (G) retinas showing that Bruch’s membrane (BM) is thicker in the mutant than in the control. (H) Quantification of Bruch’s membrane thickness in retinas of 4–6 (n = 4 WT and 4 Klc1−/−; P = 0.33)-, 17–18 (n = 7 WT and 7 Klc1−/−; ***, P = 0.0005)-, and 21–22 (n = 4 WT and 7 Klc1−/−; **, P = 0.003)-month old animals. (I) Quantification of the sub-RPE basal laminar deposits (BlamD) and membranous (Mb) debris in retinas of 4–6 (P = 0.21 for BlamD), 17–18 (**, P = 0.001 for BlamD; **, P = 0.004 for Mb debris) and 21–22 (**, P = 0.002 for BlamD; *, P = 0.008 for Mb debris) months old WT and Klc1−/− animals. (J and K) WT (J) and Klc1−/− (K) retinas immunolabeled for fibronectin, showing that the enlargement of BM is mainly caused by an expansion of the inner collagen layer (which contains fibronectin). (L) EM of Klc1−/− RPE, showing electron-dense web-like material (arrowhead) and individual profiles in a sub-RPE aggregate of membranous vesicles (arrows) in the inner collagen layer of the BM (similar to the deposits shown in Fig. 3 of Curcio et al., 2005). (M) EM section of the aggregates, fixed by the OTAP method, which preserves neutral lipids. Solid particles are evident among the BlamD, rather than membrane vesicles with aqueous interiors. (Inset) A membrane surrounding the neutral lipids is evident. BM, Bruch’s membrane; RPE, retinal pigment epithelium; OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; OCL, outer collagen layer; EL, elastic layer; ICL, inner collagen layer. Bars: (A, B, D, E, J, and K) 10 µm; (F and G) 2 µm; (F and G insets) 1 µm; (L) 300 nm; (M) 500 nm; (M inset) 250 nM. Error bars are means ± SEM.

Pathology in retinas of 18–20-mo-old Klc1−/− mice. (A and B) Semithin sections of WT (A) and Klc1−/− (B) central retinas. The ONL (black bar) is thinner in the mutant. (C) Number (mean ± SEM) of aligned photoreceptor nuclei across the ONL (i.e., per column) in WT (n = 5; closed circles) and Klc1−/− (n = 6; open squares) mice. Note the significant loss of nuclei in the central retina of mutant mice (Student’s t test; *, P < 0.05; **, P < 0.01). (D and E) Retinal sections showing autofluorescence (488-nm excitation, 500–600-nm emission) of RPE in WT (D) and Klc1−/− (E) mice. (F and G) EMs of RPE of control (F) and Klc1−/− (G) retinas showing that Bruch’s membrane (BM) is thicker in the mutant than in the control. (H) Quantification of Bruch’s membrane thickness in retinas of 4–6 (n = 4 WT and 4 Klc1−/−; P = 0.33)-, 17–18 (n = 7 WT and 7 Klc1−/−; ***, P = 0.0005)-, and 21–22 (n = 4 WT and 7 Klc1−/−; **, P = 0.003)-month old animals. (I) Quantification of the sub-RPE basal laminar deposits (BlamD) and membranous (Mb) debris in retinas of 4–6 (P = 0.21 for BlamD), 17–18 (**, P = 0.001 for BlamD; **, P = 0.004 for Mb debris) and 21–22 (**, P = 0.002 for BlamD; *, P = 0.008 for Mb debris) months old WT and Klc1−/− animals. (J and K) WT (J) and Klc1−/− (K) retinas immunolabeled for fibronectin, showing that the enlargement of BM is mainly caused by an expansion of the inner collagen layer (which contains fibronectin). (L) EM of Klc1−/− RPE, showing electron-dense web-like material (arrowhead) and individual profiles in a sub-RPE aggregate of membranous vesicles (arrows) in the inner collagen layer of the BM (similar to the deposits shown in Fig. 3 of Curcio et al., 2005). (M) EM section of the aggregates, fixed by the OTAP method, which preserves neutral lipids. Solid particles are evident among the BlamD, rather than membrane vesicles with aqueous interiors. (Inset) A membrane surrounding the neutral lipids is evident. BM, Bruch’s membrane; RPE, retinal pigment epithelium; OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; OCL, outer collagen layer; EL, elastic layer; ICL, inner collagen layer. Bars: (A, B, D, E, J, and K) 10 µm; (F and G) 2 µm; (F and G insets) 1 µm; (L) 300 nm; (M) 500 nm; (M inset) 250 nM. Error bars are means ± SEM.

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