Topographical characterization of cone photoreceptors and the area centralis of the canine retina

Abstract

Purpose: The canine is an important large animal model of human retinal genetic disorders. Studies of ganglion cell distribution in the canine retina have identified a visual streak of high density superior to the optic disc with a temporal area of peak density known as the area centralis. The topography of cone photoreceptors in the canine retina has not been characterized in detail, and in contrast to the macula in humans, the position of the area centralis in dogs is not apparent on clinical funduscopic examination. The purpose of this study was to define the location of the area centralis in the dog and to characterize in detail the topography of rod and cone photoreceptors within the area centralis. This will facilitate the investigation and treatment of retinal disease in the canine.

Methods: We used peanut agglutinin, which labels cone matrix sheaths and antibodies against long/medium wavelength (L/M)- and short wavelength (S)-cone opsins, to stain retinal cryosections and flatmounts from beagle dogs. Retinas were imaged using differential interference contrast imaging, fluorescence, and confocal microscopy. Within the area centralis, rod and cone size and density were quantified, and the proportion of cones expressing each cone opsin subtype was calculated. Using a grid pattern of sampling in 9 retinal flatmounts, we investigated the distribution of cones throughout the retina to predict the location of the area centralis.

Results: We identified the area centralis as the site of maximal density of rod and cone photoreceptor cells, which have a smaller inner segment cross-sectional area in this region. L/M opsin was expressed by the majority of cones in the retina, both within the area centralis and in the peripheral retina. Using the mean of cone density distribution from 9 retinas, we calculated that the area centralis is likely to be centered at a point 1.5 mm temporal and 0.6 mm superior to the optic disc. For clinical funduscopic examination, this represents 1.2 disc diameters temporal and 0.4 disc diameters superior to the optic disc.

Conclusions: We have described the distribution of rods and cone subtypes within the canine retina and calculated a predictable location for the area centralis. These findings will facilitate the characterization and treatment of cone photoreceptor dystrophies in the dog.

Figure 1

Comparison between rod and cone inner segment size, density, and ratio in the area centralis versus the inferior periphery. Six eyes from 3 animals were examined. On retinal flatmounts stained with peanut agglutinin (PNA; green) we identified a clear visual streak superior to the optic nerve, with a temporal area centralis. A representative retinal flatmount is shown in A illustrating a clear visual streak superior to the optic nerve head (ONH). Orientation is depicted: nasal (N), temporal (T), superior (S), and inferior (I), and representative magnified images from the highlighted areas of the retina are shown. The area centralis contained a higher density of smaller rods and cones than the inferior periphery. B shows representative differential interference contrast (DIC) images of flatmounts from the area centralis and the inferior periphery. PNA was used as a positive marker of the cone inner segment (green). Six areas per retina were examined (3 area centralis, 3 inferior periphery) and average calculations for the rod and cone inner segment cross sectional area and number were made. C shows that the inner segment area of both cones and rods was significantly smaller in the area centralis (cone: p=0.0028, rod: p=0.0034). The density of both rods and cones was significantly higher in the area centralis than in the periphery (D, cone: p<0.0001, rod: p<0.0005). Note the difference in scale of each y-axis. E shows the ratio between rods and cones in the areas of the retina; the ratio was significantly lower in the area centralis (p<0.0001). Using an unpaired t test, significant values are marked with asterisks **** p<0.0001, *** p<0.001, ** p<0.01. In all graphs, the inferior periphery bars are marked in red.

Figure 2

Comparison of opsin subtype distribution in the beagle retina. Nine eyes from 5 animals were examined. A-C: retinal flatmounts stained with peanut agglutinin (PNA; blue) and either long wavelength (L/M) or short wavelength (S) cone opsin (red) were analyzed. Representative images are shown in A. Absolute L/M cone density was significantly higher in the area centralis (B; p=0.0225), there was no difference in S cone density between the 2 areas examined (B; p=0.49). Quantification of L/M and S cones as a percentage of total cone number showed no statistical difference between the two areas examined (C; S cones p=0.16, L/M cones p=0.12). D-E: Serial 10 μm retinal cryosections were stained with PNA (green) and either L/M or S opsin (red) in a similar manner. Representative images are shown in D. The following areas in cross section are identified in D: outer nuclear layer (ONL), inner/outer segments (OS), retinal pigment epithelium (RPE), tapetum lucidum (TL) Nuclei are identified with Hoechst 33342 in blue. Analysis of cryosections for L/M and S cone percentage in the two areas examined showed no significant difference, supporting the flatmount data (E; S cones p=0.18, L/M cones p=0.11). Using an unpaired t-test, significant values are marked with asterisks **** p<0.0001, * p<0.05. In all graphs, the inferior periphery bars are marked in red.

Figure 3

Cone density maps with correlation to predicted area centralis. Nine eyes from 5 animals were examined. A shows all 9 retinas used in analysis, demonstrating a marked visual streak of high cone density with an area centralis temporal to the optic nerve head. Data sets were reflected as necessary to represent all eyes as right. Orientation is depicted: nasal (N), temporal (T), superior (S), and inferior (I). B shows the average calculation from all 9 retinas demonstrating the mean density in the visual streak and a predictable location of the area centralis 0.4 optic nerve head (ONH) diameters superior and 1.1 ONH diameters temporal to the optic disc.