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

Relation of clustered anatomical variation to cross-modal response and fractional anisotropy.

<p>A: For each of 52 subjects (blind and sighted), we obtained the BOLD fMRI response in V1 while subjects listened to auditory sentences played forwards and in reverse, as compared to white noise. We modeled the ability of individual variation in the three anatomical clusters to account for variation in cross-modal BOLD fMRI response. For each subject, the x-axis gives the prediction of the model for BOLD fMRI response, and the y-axis the observed response. There was a significant model fit (p = 0.00051). B: Model weights for the fit to the cross-modal response data. Shown are the mean and standard error of weights upon each of the clusters of anatomical variation in their prediction of V1 BOLD fMRI response. Only the first cluster of anatomical variation (V1 cortical <i>thinness</i>) had a fitting weight significantly different from zero. The loading on this weight is negative, indicating that <i>thicker</i> V1 cortex predicts greater cross-modal responses. C: For each of 59 subjects, we measured fractional anisotropy within the optic radiations and splenium of the corpus callosum. We modeled the ability of individual variation in the three anatomical clusters to account for variation in FA. For each subject, the x-axis gives the prediction of the model for FA, and the y-axis the observed measure. The entire model fits the data above chance (p = 0.016). D: Model weights for the fit to the FA data. Shown are the mean and standard error of weights upon each of the clusters of anatomical variation in their prediction of the FA measure. Only the third cluster of anatomical variation (chiasm and LGN volume) had a fitting weight significantly different from zero.</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

Patterns of shared variation in visual pathway anatomy.

<p>A: The eight measures of visual pathway anatomy are illustrated on an axial schematic of the human brain. The groupings of the measures are to assist subsequent interpretation of the data. B: The Euclidean distance matrix and dendrogram for the 8 measures across the sighted population. <i>Left</i>. The square-root, sum-squared difference in values between two measures across subjects provides a measure of Euclidean distance. Darker shades indicate pairings of measures that have similar variation across subjects, and thus lower distance values. <i>Right</i>. The distance matrix was subjected to hierarchical clustering, yielding a dendrogram. The length of each branch reflects the distance between the paired measures. The three primary clusters of anatomical variation are colored green, blue, and red. C: <i>Left</i>. The distance matrix across the 8 measures for the blind population. A similar overall structure is seen as compared to the sighted. <i>Right</i>. The dendrogram derived from measures from the blind subjects. The same overall cluster structure is seen. Note that there is some rearrangement in the measurements assigned to cluster #2 in the blind as compared to the sighted.</p

Mean group differences in the anatomical measures.

<p>Mean group differences in the anatomical measures.</p

Relation of clustered anatomical variation to cross-modal response and fractional anisotropy.

<p>A: For each of 52 subjects (blind and sighted), we obtained the BOLD fMRI response in V1 while subjects listened to auditory sentences played forwards and in reverse, as compared to white noise. We modeled the ability of individual variation in the three anatomical clusters to account for variation in cross-modal BOLD fMRI response. For each subject, the x-axis gives the prediction of the model for BOLD fMRI response, and the y-axis the observed response. There was a significant model fit (p = 0.00051). B: Model weights for the fit to the cross-modal response data. Shown are the mean and standard error of weights upon each of the clusters of anatomical variation in their prediction of V1 BOLD fMRI response. Only the first cluster of anatomical variation (V1 cortical <i>thinness</i>) had a fitting weight significantly different from zero. The loading on this weight is negative, indicating that <i>thicker</i> V1 cortex predicts greater cross-modal responses. C: For each of 59 subjects, we measured fractional anisotropy within the optic radiations and splenium of the corpus callosum. We modeled the ability of individual variation in the three anatomical clusters to account for variation in FA. For each subject, the x-axis gives the prediction of the model for FA, and the y-axis the observed measure. The entire model fits the data above chance (p = 0.016). D: Model weights for the fit to the FA data. Shown are the mean and standard error of weights upon each of the clusters of anatomical variation in their prediction of the FA measure. Only the third cluster of anatomical variation (chiasm and LGN volume) had a fitting weight significantly different from zero.</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

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

Blind subject group details.

<p>Blind subject group details.</p