Canine Retina Has a Primate Fovea-Like Bouquet of Cone Photoreceptors Which Is Affected by Inherited Macular Degenerations

Retinal areas of specialization confer vertebrates with the ability to scrutinize corresponding regions of their visual field with greater resolution. A highly specialized area found in haplorhine primates (including humans) is the fovea centralis which is defined by a high density of cone photoreceptors connected individually to interneurons, and retinal ganglion cells (RGCs) that are offset to form a pit lacking retinal capillaries and inner retinal neurons at its center. In dogs, a local increase in RGC density is found in a topographically comparable retinal area defined as the area centralis. While the canine retina is devoid of a foveal pit, no detailed examination of the photoreceptors within the area centralis has been reported. Using both in vivo and ex vivo imaging, we identified a retinal region with a primate fovea-like cone photoreceptor density but without the excavation of the inner retina. Similar anatomical structure observed in rare human subjects has been named fovea-plana. In addition, dogs with mutations in two different genes, that cause macular degeneration in humans, developed earliest disease at the newly-identified canine fovea-like area. Our results challenge the dogma that within the phylogenetic tree of mammals, haplorhine primates with a fovea are the sole lineage in which the retina has a central bouquet of cones. Furthermore, a predilection for naturally-occurring retinal degenerations to alter this cone-enriched area fills the void for a clinically-relevant animal model of human macular degenerations

<i>SPATA7</i>: Evolving phenotype from cone-rod dystrophy to retinitis pigmentosa

<p><i>Background: SPATA7</i> mutation<i>s</i> have been associated with different autosomal recessive retinal degeneration phenotypes. Long-term follow-up has not been described in detail.</p> <p><i>Materials and methods</i>: A Hispanic patient with <i>SPATA7</i> mutations was evaluated serially over a 12-year period with kinetic and static chromatic perimetry, optical coherence tomography (OCT), and fundus autofluorescence (AF) imaging. Electroretinography (ERG) was performed at the initial visit.</p> <p><i>Results</i>: The patient was homozygous for a mutation in <i>SPATA7</i> (p.V458fs). At age 9, the ERG showed an abnormally reduced but preserved rod b-wave and no detectable cone signals. There were two islands of vision: a midperipheral island with greater cone than rod dysfunction and a central island with normal cone but no rod function. Serial measures of rod and cone vision and co-localized retinal structure showed that the midperipheral island slowly became undetectable. By age 21, only the central island and its cone function remained, but it had become more abnormal in structure and function.</p> <p><i>Conclusion</i>: The disease resulting from <i>SPATA7</i> mutations in this patient initially presented as a cone-rod dystrophy (CRD), but changed over time into a phenotype more reminiscent of late-stage retinitis pigmentosa (RP). The differential diagnosis for both CRD and RP should include this rare molecular cause of autosomal retinal degeneration. An evolving phenotype complicates not only clinical diagnosis and patient counselling but also future strategies aimed at treating specific retinal regions.</p

Nystagmus and foveal function in BCM.

<p>(A) Fixation locations in a normal subject and 3 BCM patients. For each subject, 10 s long epochs of eye movement data during fixation to a large visible red target (Target I) are shown in spatial (left) and spatio-temporal (right) coordinates. Spatial distribution of fixation clouds are shown on infrared SLO images of each macula with standard circles centered on the anatomical foveal depression. Spatio-temporal distribution of eye movements are shown on chart records for X and Y directions; up is nasal retina for X and superior retina for Y. All results are presented as equivalent right eyes for comparability. Horizontal dashed lines on the chart records depict the location of the anatomical fovea. (B) Fixation location and instability in BCM patients as a function of the bright red standard target (I), a green target (II) scotopically-matched to the standard target but expected to show greater visibility to S-cones, and a dim red target (III). N.S., not significant; *, P<0.05. (C) Distribution of fixation locations with the standard target in all patients. I = inferior, N = nasal, S = superior, and T = temporal retina. (D) Fixation location and instability as a function of best-corrected visual acuity. (E) Test pattern used with microperimetric stimuli to evaluate visual function under chromatic adaptation displayed on a normal near-infrared reflectance image. Stimulus locations are divided into 5 groups; f, foveal region, s, superior, i, inferior, t, temporal and n, nasal retina. (F,G) Sensitivities to blue stimuli on yellow background (BonY) and red stimuli on cyan background (RonC) in individual BCM patients (bars left to right; P2, P3, P4, P6, P8, P9, P10, P15, P16, P17, P18, P20, P25, P26, P28, and P29) compared to normal results (symbols; mean ±1sd) at the five regions shown in panel E. BCM results plotted below the zero line in Panel G represent those cases where the brightest available stimulus was not seen.</p

High density of cone photoreceptors at the wildtype canine fovea-like area.

<p>(a,b) Central areas of specialization (white) are avoided by retinal blood vessels in human and canine eyes. (c) Retinal ganglion cell (RGC) density map across the canine retina and peak density at the center of the area of specialization. Brn3a: brain-specific homeobox/POU domain protein 3A (d) Retinal cross-section (H&E stained) through the fovea-like area of a 6 week-old dog shows a focal elevation on the retinal surface, thickening of the ganglion cell layer (GCL), and thinning of the outer nuclear layer (ONL). Immunohistochemistry at 7 weeks shows focal high density of cones (red), markedly reduced density of rods (green), elongated inner segments (IS), outer segments (OS) and multiple layers of RGCs in GCL. CA: cone arrestin; Rho: Rhodopsin. ONL is stained with DAPI (blue). (e) Abrupt increase of cone density associated with an abrupt decrease of rod density in 4 eyes (at different postnatal ages for later comparison with mutant dogs). Rod and cone nuclei are highlighted in enlarged insets with blue and yellow, respectively, for visibility. (f) Two-photon microscopy imaging of the fovea-like area and immediate surrounding region. <i>En face</i> view (XY) is an overlay of two Z scans taken at different depths (shown as dotted lines on the orthogonal XZ view) to illustrate the cone IS densities at the fovea-like area and surrounding regions. Insets illustrate the abrupt increase in central peak cone IS density within the canine fovea-like area. vWF: von Willebrand factor VIII; (g) Comparison of peak cone densities in dogs to that reported for macaques and humans measured by adaptive optics imaging, or histology Filled symbols are mean±sd.</p

FST results with two colors under dark-adapted conditions and on a range of white and chromatic backgrounds.

<p>(A,B) FST-TVI with blue (A) and red (B) stimuli on white backgrounds in BCM patients and normals. Gray region defines the expected desensitization of the rod system. Rectangles define the data further explored in panels C-F. (C-F) Comparison of thresholds with chromatic and white backgrounds; blue stimuli on white (BonW) or yellow (BonY), and red stimuli on white (RonW) or on blue (RonB) backgrounds are shown. Different panels show the effectiveness of the background for the scotopic (C,D), S-cone (E) and photopic (F) systems. Lines with unity slope are fit to the data, and offset between the lines is shown in log units. (G,H) Sensitivity loss and predicted photoreceptor mediation using a pair of RonW and RonB FST thresholds. Both individual results and group averages (Avg) are shown. Error bars, when visible, are ±1SD.</p

Histolopathology at the fovea-like area in two canine models of inherited macular degeneration.

<p>(a) Fovea-like area in a 112 week-old <i>BEST1</i>-mutant dog. (a₁) Epifluorescence microscopy image (with DIC/Nomarski optics) showing at the fovea-like area increased autofluorescence (yellow) in the retinal pigment epithelium (RPE). (a₂) Immunohistochemistry on the same section as (b₁) shows focal separation (diamond) of cone (red) outer segments (OS) from the underlying RPE. Note: Red fluorescent signal originating from the RPE is endogenous autofluorescence (see a₁). (a₃) Immunohistochemistry shows focal separation (diamond) of rod OS (green) from hypertrophied RPE cells (red; arrowheads), and (a₄) extension of RPE apical processes (arrowheads). (b) Fovea-like areas in <i>RPGR</i> mutant dogs. (b₁) Horizontal retinal cross-section (H&E stained) shows the abrupt ONL thinning and shortened inner segments (IS) at the fovea-like area of a 4 week-old mutant dog while ONL thickness and structure of photoreceptors is preserved in the immediate peri-foveal regions. (b₂) Horizontal retinal cross-section (H&E stained) shows more prominent ONL thinning at the fovea-like area which has now extended peri-foveally in a 22 week-old dog. (b3) Early reduction in the number of cones is seen at the fovea-like area on retinal cross sections (left; cone nuclei are highlighted in yellow for visibility) and quantitative comparison to wildtype results (right). CA: cone arrestin; Rho: rhodopsin.</p

Visual fields of BCM patients evaluated with kinetic and static perimetry.

<p>(A) Light-adapted (LA) vertical sensitivity profiles from a normal subject and a BCM patient using achromatic (black line) and 600-nm (orange line) stimuli on a 10 cd.m^-2 white background, or 440-nm (blue line) stimuli on a yellow background (YB). (B) S-cone sensitivity profiles (filled circles) of the BCM patients using a 440-nm stimulus on YB compared to normal limits (gray = ±2SD). (C) LA white vertical sensitivity profiles of BCM patients (filled circles) compared to normal (gray). Blue line is the S-cone sensitivities from Panel C shifted according to the difference in effectiveness between the white and 440-nm stimuli. (D) LA 600-nm vertical sensitivity profiles of BCM patients (filled circles) compared to normal (gray). (E) Sensitivity differences between LA white and LA 600-nm stimuli are shown for the BCM patients (filled circles) and normal (unfilled circles). Predicted differences for rod (green dashes) and L/M cone (orange dashes) mediation are shown. (F) Dark-adapted (DA) vertical sensitivity profiles from a normal subject and a BCM patient using 500-nm (green line) and 650-nm (red line) stimuli. Above the results it is shown whether there is rod (R) or mixed (M) mediation, as determined by the differences between sensitivities to the stimuli. (G) DA 500-nm vertical sensitivity profiles of BCM patients (filled circles) compared to normal (gray). (H) DA 650 nm vertical sensitivity profiles of BCM patients (filled circles) compared to normal (gray). (I) Sensitivity differences between DA 500- and DA 650-nm stimuli are consistent with rod mediation (gray) at all locations except for the normal results with 650 nm at fixation. S, superior; I, inferior. (J) DA 650-nm sensitivities at fixation in normal and BCM. Normal 650-nm sensitivities are mediated by the L/M cones (C) whereas BCM sensitivities are mediated by the rods (R). Error bars are ±1SD.</p

Spectral sensitivity functions in normal subjects and BCM patients recorded at 14° superior field.

<p>(A) Sensitivities (mean±1 SD) to six spectrally distinct stimuli in normal subjects (n = 3) under dark-adapted (left), and on 1 (middle) and 10 cd.m^-2 (right) white backgrounds. (B) Sensitivities to the spectrally distinct stimuli in BCM patients for the same three adaptation conditions as in Panel A. Results from P8 are shown at the correct ordinate location; results from remaining patients have been adjusted by 1 log increments for visibility. Theoretical functions describing rod (green), S cone (blue), L/M cone (orange) sensitivities are shown after vertical shifts to fit relevant normal and BCM data in Panels A and B. (C) Comparison of individual normal and BCM sensitivities at 500 nm. (D,E) Comparison of individual normal and BCM sensitivities at 440, 500 and 560 nm. Symbols in Panels C, D, and E are painted by colors derived from the fit of theoretical functions to the spectral data. N = normal, B = BCM.</p

Photoreceptor layer lamination in wildtype dogs and in naturally-occuring genetic diseases primarily affecting the canine fovea-like area.

<p>(a,d,f) <i>En face</i> infrared view of representative wildtype (a), <i>BEST1</i>-mutant (d) and <i>RPGR</i>-mutant (f) dogs. *, fovea-like area. Arrows, locations of cross-sectional OCT scans shown below each panel. Outer photoreceptor nuclear layer (ONL) and retinal pigment epithelium (RPE) are highlighted for visibility on OCT scans. (b) ONL thickness topography in a 22-wk-old wildtype dog displayed in pseudo-color. There is a distinct localized region of ONL thinning supero-temporal (ST) to the optic nerve head (black circle) corresponding to the fovea-like area. Major blood vessels are overlaid. (c) Diagonal profiles of ONL thickness (along arrow shown in b) in individual wildtype dogs (lines; ages: 7 wks –8 yrs; n = 13). 95% confidence interval shown (gray area). The break in the axis corresponds to the optic nerve head which lacks photoreceptors. F, fovea-like area. (e) Topographic localization of the sites (*) of the early macular lesions in <i>BEST1</i>-mutant dogs (ages: 10–62 wks; n = 7, left) correspond to the localization of the fovea-like area in wildtype dogs (ages: 7 wks –8 yrs; n = 13, right). (g) ONL thickness topography in an 11-wk-old <i>RPGR</i>-mutant dog displayed in pseudo-color. *, fovea-like area. (h) Diagonal profiles of ONL thickness in young <i>RPGR</i>-mutant dogs (lines; age: 11 wks; n = 6 eyes of 3 dogs) shows abnormal thinning corresponding to the fovea-like area and its immediate surrounds compared to WT (gray area). All eyes are shown as left eyes (temporal retina to the right).</p

OCT abnormalities in <i>rd16;Nrl^−/−</i> mice.

<p>(A) Upper panels: Representative OCT scans vertically across ∼2 mm of retina (centered at the ONH, optic nerve head) in a WT mouse and in two <i>rd16;Nrl^−/−</i> mice of different ages. Lower panels: Magnified parts of the superior region of the retinal sections with overlaid longitudinal reflectivity profiles (LRPs) to demonstrate the reflective abnormalities in the outer retinal region in <i>rd16;Nrl^−/−</i> mice (b and c) compared with C57BL6 WT (a). (B) Upper two panels: Vertical OCT sections quantified for ONL+ thickness in two age groups of <i>rd16;Nrl^−/−</i> mice. Regions of outer retina with pseudorosettes were excluded in the measurement. ONL+ profiles in the older (P83–89, n = 12 eyes) age group were thinner than those in younger (P31–41, n = 35 eyes) mice; gray bands in the P83–89 plot represent mean±2 SD for ONL+ thickness of the P31–41 mice. For reference, insets at lower right of the upper two plots show original raw data before suppression of pseudorosette regions. Third panel from top: Means of ONL+ data across the vertical meridian in two age groups (error bar: ± SD; P31–41, open circles; P83–89, filled triangles). Lowest panel: Histograms showing average ONL+ fraction across vertical meridian of two age groups (*represents <i>p</i><0.001). (C) Histological sections of <i>rd16;Nrl^−/−</i> retina at 4 different ages from P21 to P80, compared with a WT retinal section. Histograms show ONL fraction (based on the earlier age group) in <i>rd16;Nrl^−/−</i> mice from peripheral retina (n = 6 eyes in each of the two age groups, *represents <i>p</i> = 0.01).</p