Figure 1.
Orientation in the canine eye.
(A) Funduscopy of a RPE65−/− dog. (B) Extracted eye after euthanasia. The superior part of the eye determines the tapetum lucidum (t). The red marked area corresponds to the retinal flatmount used in the cone opsin expression analysis. The grey marked areas are used for cyrosections used in the bipolar cell sprouting analysis and the numbers correspond to regions shown in Figure 3 und 4. (C) Retinal flatmount for cone analysis. Every single quadrat stands for one microscope image (see material and methods). The retina was divided into 4 different regions. The numbers correspond to the regions shown in Figure 2. p = papilla, t = tapetum lucidum.
Table 1.
List of antibodies used in this study.
Figure 2.
Analysis of sprouting events of rod bipolar cells.
(A) Bar diagrams of the 4 different analyzed regions (see Figure 1B) compared wild type (n = 12 images) and RPE65−/− (n = 24 images). In regions 1–3 there are no essential differences between wild type and mutated dogs. Region 4 displays a statistical significant discrepancy between wild type and RPE65−/− dogs (* p<0,001, student t-test). (B) High resolution confocal images for the analysis. The images are projections of the 4 considered images of a z-stack. The ribbon synapses are marked with CtBP2 (red) and the rod bipolar cells are marked with PKC α (green). The counted sprouting events are marked with white circles. Wild type (WT), outer nuclear layer (ONL), outer plexiform layer (OPL), scale 20 µm.
Figure 3.
Scatter plot of correlation between sprouting events and ONL thickness.
In RPE65−/− dogs, the highest number of sprouting events as well as the thinnest ONL are present in region 4. There is a strong correlation between sprouting events and ONL thickness (correlation coefficient R = 0.8333 for wild type and R = 0.8374 for RPE65−/− dogs).
Figure 4.
Cone distribution in healthy and affected animals.
(A) Absolute mean S-cone distribution [103 cones per mm2] in wild-type retina (n = 2). (B) Absolute mean S-cone distribution [103 cones per mm2] in RPE65−/− dogs (n = 4). (C) Relative mean S-cone loss [%] in the affected animals compared to healthy tissue. (D) Absolute mean L/M-cone distribution [104 cones per mm2] in wild-type retina (n = 2). (E) Absolute mean L/M-cone distribution [104 cones per mm2] in RPE65−/− dogs (n = 4). (F) Relative mean LM-cone [%] loss in the affected animals compared to health tissue. p = papilla.
Figure 5.
Quantification of cone distribution.
(A–D) S-cone (green) and L/M-cone (red) distribution and quantification in wild-type and mutated canine RPE65−/− retinae. Box blots display the respective distributions of S-cones and L/M-cones in the 4 regions defined in Figure 1. Statistical analysis was done by a Mann Whitney rank sum test, significant values are marked with asterisks * p<0.001. Bar diagrams show the percentage loss of both cone types compared healthy and affected animal medians in the analyzed regions. Both types of cones are significantly reduced in all analyzed regions. L/M-cones in RPE65−/− retinae are not only reduced but look different in shape, thus appearing worm-like. Wild type (WT), scale 25 µm.