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Figure 1.
Partial repopulation of the retina with rod photoreceptors in bP23H animals. A, A′, Wild-type retina. B, B′, bP23H transgenic retina reared in cyclic light since fertilization. C, C′, P23H transgenic retina reared in complete darkness for 3 weeks after initial (21 d) exposure to light. Retinal cryosections were stained with WGA (green) and Hoechst (blue). IS, Inner segment; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars: top, 100 μm; bottom, 20 μm. D, Relative thickness of tadpole retinas under normal cyclic light or dark-recovered rearing conditions as measured from histological sections (n > 6, mean ± SD). The asterisk indicates that the relative thickness of the dark-recovered bP23H retina is significantly thicker than the cyclic light-reared bP23H retina (Student's t test, p < 0.0002). WT, Wild-type.
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Figure 2.
Dysmorphic bP23H rod photoreceptors with truncated outer segments identified via GFP fluorescence. Representative confocal micrographs of two different models of retinal degeneration before (top) and after (bottom) degeneration. A, C, bP23H/GFP double-transgenic retinas. GFP was coexpressed in the rods of bP23H tadpoles to label rod photoreceptors. A, Dark-reared bP23H/GFP retina. C, Degenerating bP23H retina after 6 weeks in cyclic light. Note the presence of GFP-positive rod cell bodies with severely truncated outer segments. B, D, iCasp9/GFP double-transgenic retinas. B, iCasp9/GFP retina before induction of apoptosis. D, iCasp9/GFP retina 4 d after administrating 10 mM AP20187. Rod photoreceptors are permanently lost and noticeably absent after drug-induced apoptosis. Sections were stained with Alexa Fluor 555-conjugated WGA to visualize membranes (red) merged with Hoechst nuclear stain (blue). Rod photoreceptors were visualized by GFP expression (green). IS, Inner segment;
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Figure 3.
Dysmorphic rhoP23H rod cells are able to regenerate in the dark. A, Dysmorphic rod cells can be observed in the degenerating bP23H retina for up to 6 weeks in cyclic light-rearing conditions. Sections were stained with Alexa Fluor 555-conjugated WGA to visualize membranes (red) merged with Hoechst nuclear stain (blue). Rod photoreceptors were visualized by GFP expression (green). B, Relative density of GFP-positive rods in the central retina over time (n = 3, mean ± SEM). C, bP23H rod cells are able to regenerate outer segments lost after 6 weeks in cyclic light. Cessation of degenerating light signal reverses ROS degeneration. Sections were stained with WGA to visualize membranes (green) merged with Hoechst nuclear stain (blue). Scale bars, 20 μm. WT, Wild type.
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Figure 4.
Apoptosis in bP23H retinas reared in cyclic light is comparable with WT retinas in cyclic light. Comparison of wild-type retina (left) versus bP23H retina (right) after 9 weeks in cyclic light. The arrowheads point to TUNEL-positive (red) ONL cells. Insets show enlargements of respective boxed areas. The number of TUNEL-positive cells in the ONL were counted over a 9 week period. The highest number of TUNEL-positive ONL cells per section per retina at week 9 is shown (n > 3, mean ± SEM). WGA, Green; Hoechst nuclear stain, blue. Scale bars: 100 μm; inset, 10 μm.
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Figure 5.
Short-term degeneration of bP23H retinas over a 3 week period as monitored by TUNEL. Tadpoles were allowed to develop for 14 d in complete darkness. At 14 dpf, the animals were moved to cyclic light (12 h light/dark cycle) rearing condition. A, TUNEL-stained (red) retinal sections of animals at 2, 5, 13, and 19 d in cyclic light. TUNEL-positive cells in the ONL are indicated by arrowheads. WGA, Green; Hoechst nuclear stain, blue. Scale bar, 100 μm. B, The number of TUNEL-positive cells in the ONL were counted over the 3 week cyclic light period. Graph represents the number of TUNEL-positive ONL cells per cryosection, averaged over at least three samples per time point (n > 3, mean ± SEM). For each sample, TUNEL-positive cells were counted in three sections obtained from the center of the tissue block. WT, Wild type.
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Figure 6.
Short-term metabolic changes in bP23H retinas. A–I, Visualization of metabolic content and distribution by quantitative metabolite mapping on 90 nm sections probed with Igs specific to each metabolite and visualized with silver intensification. J–L, Red–green–blue channels represent taurine–glutathione–glutamate mapping. The pink and red compartments in the photoreceptor outer segments (OS) and inner segments (IS) contain distinct taurine–glutamate–glutathione mixtures, whereas various blue-to-azure cells in the interneuron layer (INL) and ganglion cell layer (GCL) are neurons with distinctive glutamate content. The green–yellow background of RPE reflect high levels of glutathione. M–O, Theme maps display results of multispectral analyses and clustering analyses to extract all distinct molecular phenotypes in the photoreceptor layer. Signals from glutamate, glutathione, taurine, aspartate, GABA, glycine, arginine, alanine, and CRALBP were used for multispectral analyses and clustering analyses. M, N, Theme maps show that the metabolic profiles of rods and cones in control and degenerating retina were similar; whereas in recovering retina, rods displayed variability in their metabolic profile because of variability in their taurine, glutamate, and glutathione content. Arrows and arrowheads indicate Müller glia and regenerating rods, respectively.
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