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. Author manuscript; available in PMC: 2023 Jul 1.
Published in final edited form as: Exp Eye Res. 2022 May 16;220:109106. doi: 10.1016/j.exer.2022.109106

Quantitative and Qualitative Characterization of Retinal Dystrophies in Canine Models of Inherited Retinal Diseases Using Spectral Domain Optical Coherence Tomography (SD-OCT)

Shin Ae Park 1,2,*,†, Jamie Rhodes 3,*, Simone Iwabe 3, Gui-Shuang Ying 4, Wei Pan 4, Jiayan Huang 4, András M Komáromy 1,3,†
PMCID: PMC9789526  NIHMSID: NIHMS1854348  PMID: 35588783

Abstract

The purpose of this study was to establish spectral domain optical coherence tomography (SD-OCT) assessment data in well-established canine models of inherited retinal dystrophies: PDE6B-rod-cone dysplasia 1 (RCD1: early onset retinitis pigmentosa), PRCD-progressive rod-cone degeneration (PRCD: late onset retinitis pigmentosa), CNGB3-achromatopsia, and RPE65-Leber congenital amaurosis (LCA). High resolution SD-OCT images of the retina were acquired from both eyes in 5 planes: temporal; superotemporal; superior; nasal; and inferior in adult dogs with: RCD1 (n = 4 dogs, median age: 1.5 yrs); PRCD (n = 2, 4.3 yrs); LCA (n = 3, 5.2 yrs); achromatopsia (n = 3, 4.2 yrs); and wild types (wt, n=6, 5.5 yrs). Total, inner and outer retinal thicknesses and ellipsoid zone were analyzed. In selected animals, histomorphometric evaluations were performed. In dogs with RCD1, PRCD, and LCA, the thickness of the outer retina was, compared to wt, significantly decreased (p ≤ 0.02) in all OCT imaging planes, and in superotemporal and inferior imaging planes in dogs with achromatopsia. No significant thinning was observed in inner retina thickness in any disease model except in the inferior imaging plane in dogs with RCD1. Dogs with RCD1, PRCD, and LCA had significantly more areas with disrupted ellipsoid zone in the presumed area centralis than wt (p ≤ 0.001). OCT findings provide baseline information for research of retinal dystrophies using these canine models.

Keywords: Dog, RCD1, PRCD, LCA, achromatopsia, PDE6B, RPE65, CNGB3B

Introduction

Inherited retinal dystrophies are a group of hereditary disorders affecting the retina such as retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), achromatopsia, cone-rod dystrophies, and macular dystrophies. These diseases are major causes of incurable visual impairment. RP, for example—the most common retinal dystrophy, which affects 1 in 3000 individuals—causes severe visual impairment and blindness (Megaw et al., 2015). Canine models for inherited retinal dystrophies have been established and extensively studied for characterizing the phenotypes, investigating the pathophysiology, identifying the gene mutations, and developing DNA tests and novel therapeutics (Annear et al., 2013; Marinho et al., 2021; Pasmanter et al., 2021; Petersen-Jones and Komaromy, 2015; Tuntivanich et al., 2009; Winkler et al., 2020). Spontaneously occurring retinal dystrophy models in canine species are highly valuable particularly because (1) the mutations occur in the homologous gene in both dogs and humans, (2) the disease phenotype in both species are comparable, (3) the size and morphology of dog eyes are similar to human eyes, and (4) density of photoreceptor distribution in both species is similar (Petersen-Jones and Komaromy, 2015).

Spectral domain optical coherence tomography (SD-OCT) is a noninvasive in vivo imaging method that provides high-resolution cross-sectional imaging of the anterior and posterior segments of the eye (Hernandez-Merino et al., 2011). OCT has been widely used for qualitative assessment of retinal pathologies such as edema, degeneration, cyst, telangiectasia, and holes in the macula, serous retinal detachment, vitreomacular traction, and epiretinal membrane (Murthy et al., 2016). This imaging method has contributed significantly to the clinical assessment of patients with retinal dystrophies including RP (Battaglia Parodi et al., 2015; Dvir et al., 2010; Mitamura et al., 2012), LCA (Wen and Birch, 2015), achromatopsia (Yu et al., 2014), cone dystrophy (Michaelides et al., 2006), and birdshot chorioretinopathy (Symes et al., 2015). Normative data have been reported for canine whole retinal thickness, nerve fiber layer thickness, photoreceptor layer thickness, and outer nuclear thickness measured with OCT (Hernandez-Merino et al., 2011). However, despite the importance of OCT as a parameter of the disease status and as an outcome measure of novel therapeutic approaches in studies using animal models, OCT remains underutilized in canine models of retinal diseases.

In this study, we demonstrate quantification data of the total, inner, and outer retina thickness, and assess the ellipsoid zone in the presumed area centralis in well-established canine models of four inherited retinal dystrophies including PDE6B-RCD1 (early onset RP), PRCD (late onset RP), CNGB3-achromatopsia, and RPE65-LCA using OCT. All four disease models had various degrees of outer retinal thinning with the severest occurring in dogs with RCD1 as well as the disrupted ellipsoid zone. We also present validation data by histological measurements of the total, outer, and inner retinal thicknesses in selected animals.

Materials and methods

Animals

Thirty-five eyes of adult dogs (18 dogs, n = 35 eyes) with or without inherited retinal dystrophy were included in this study. All study dogs were beagle-derived crossbred dogs with similar sizes only differing in the genes of interest. Both males (n = 5) and females (n = 13) were included, with their ages ranging from 8 months to 9 years (Table 1). The disease models included (1) early onset, fast-progressing RCD1 due to PDE6B mutation (Petersen-Jones et al., 1995) (4 dogs, 7 eyes); (2) late onset, slow progressing PRCD due to PRCD mutation (Zangerl et al., 2006) (2 dogs, 4 eyes); (3) LCA due to RPE65 mutation (Veske et al., 1999) (3 dogs, 6 eyes); and (4) achromatopsia due to CNGB3 mutation (Sidjanin et al., 2002) (3 dogs, 6 eyes). Also included as controls were wild types (wt) without any systemic or ocular diseases (6 dogs, n = 12). Table 1 details the animal identification, phenotype, genotype, gender, ages at the time of imaging and onset of disease, cells affected, and the imaging or histology performed. All animals were bred and housed at the Retinal Disease Studies Facility of the University of Pennsylvania (Kennett Square, PA, USA). All procedures adhered to recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The protocols were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania (IACUC Protocol #s 801870, 803422).

Table 1.

The animal identification, phenotype, genotype, gender, ages at which imaging was performed and onset of disease, cells affected (if disease is present), and the imaging or histology performed.

PHENOTYPE EQUIVALENT DISEASE IN HUMANS MUTATED GENE CELLS AFFECTED ONSET DOG ID GENDER AGE (years) SD-OCT HISTOLOGY
Wild type N/A None Not applicable Not applicable N284 F 0.7 OU
GS126 F 2.2 OU OD
BR223 M 5.5 OU OD
N241 F 5.6 OU OD
E946 M 8.1 OU
M479 F 9.1 OU OD
RCD1 RP PDE6B Rods Early 2003 M 2.1 OU
2013 M 1.5 OU
2016 F 1.5 OD OU
1841 F 7.3 OD
PRCD RP PRCD Rods, then cones Late P1450 F 4.3 OU
P1451 F 4.2 OU
LCA LCA RPE65 RPE Early BR304 F 5.2 OU OS
BR318 F 5.3 OU OS
BR361 M 4.4 OU
Achromatopsia Achromatopsia CNGB3 Cones Early M501 F 7.3 OU OD
M586 F 4.2 OU
M648 F 2.1 OU
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OU: both eyes, OD: right and OS: left eye included. F: female and M: male dogs.

In vivo SD-OCT imaging and retinal thickness measurements

High resolution SD-OCT images (Spectralis HRA/OCT, ver. 5.1.3.0, Heidelberg Engineering, Germany) of the ocular fundus were acquired from each dog under general anesthesia. OCT images were not able to be obtained from dogs with dense cataracts due to advanced retinal degeneration (Table 1). Dogs were premedicated with intravenous administration of acepromazine (0.02 mg/kg, AceproJect; Henry Schein, Dublin, OH, USA). Anesthesia was induced with intravenous administration of propofol (to effect, starting with 4 mg/kg, Propoflo28; Abbott, Abbott Park, IL, USA) and maintained with isoflurane (Isothesia; Henry Schein, Melville, NY, USA) at the concentration of 2–3% in oxygen. Dogs were positioned in sternal recumbency. The palpebral fissures were kept open with Barraquer wire lid specula, and sterile saline was administered topically as needed to maintain the cornea surface moist. SD-OCT scans were acquired at five planes from the optic nerve head for each eye; the planes were at the following angles: 0° (temporal), 45° (superotemporal), 90° (superior), 180° (nasal), and 270° (inferior; Fig. 1A).

Figure 1.

Figure 1.

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Imaging planes of 0° (temporal); 45° (superior-temporal); 90° (superior); 180° (nasal); and 270° (inferior) (A) and an SD-OCT image with discernible retinal layers of the right eye in a wild type dog (B). Yellow line on the 45° line in (A) indicate the approximate area that we assessed the ellipsoid zone within presumed area centralis. Red arrows with numbers 1–3 indicate internal limiting membrane (1), inner plexiform layer – inner nuclear layer interface (2), and retinal pigment epithelium (RPE) – choroid interface (3). The black arrow (e) indicates the ellipsoid zone.

Built-in software for analysis (Eye Explorer software ver. 1.6.2.0, Heidelberg Engineering) was used for all imaging and measurements. The automated segmentation lines were manually corrected by the same investigator (JR). As the software parameters are calibrated for human retinal scan measurements, in dogs the automatic delineation of the layers is not always accurate (Hernandez-Merino et al., 2011). The total retinal thickness was measured as the distance between the inner limiting membrane (ILM) and retinal pigment epithelium (RPE)-choroid interface by using the automated segmentation lines within the software (Fig. 1B and Fig. 2A). In order to measure the inner retinal thickness, an automated segmentation line was manually moved to the interface of the inner plexiform layer (IPL) and inner nuclear layer (INL). The inner retinal thickness was measured as the distance between the ILM and IPL-INL interface (Fig. 1B and Fig. 2B). The IPL-INL interface was selected for measurement due to the reliability of discerning its demarcation in images of variable contrast and disease status. For each high-resolution image, retinal thickness measurements were initiated at the edge of the optic nerve head (ONH) and repeated at 250-μm intervals towards the peripheral retina. Measurements from the areas containing retinal blood vessels were excluded from the data analysis. Outer retinal thickness was calculated by subtracting inner retinal thickness from total retinal thickness; this is equivalent to the distance between IPL-INL interface and RPE-choroid interface for each location. The length of the discernible ellipsoid zone (inner/outer segment junction; Fig. 1B) was measured in the superotemporal imaging plane (45°). In addition, the ellipsoid zone of the presumed area centralis, the area of superotemporal retina located 3–4 mm from the ONH (Beltran et al., 2014; Donovan et al., 1974), was qualitatively assessed and recorded as intact or disrupted.

Figure 2.

Figure 2.

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Total (A) and inner (B) retinal thicknesses measured at 0° (temporal area) using SD-OCT in a wild type dog. Shown in red are the manually corrected segmentation lines for total retinal thickness (A) and inner retinal thickness (B). Histological measurements of total (red lines) and inner retina thicknesses (blue lines) at temporal area in the same wild type dog (C). Bar in (C) = 50 μm

Histological retinal thickness measurements

For histological evaluation, the dogs (n = 9) were euthanized with an overdose of sodium pentobarbital (200 mg/kg intravenous injection, Euthasol, Virbac, Fort Worth, TX, USA). Eyes were enucleated immediately post-mortem and fixed in paraformaldehyde, then processed and embedded in optimal cutting temperature compound as previously described (Beltran et al., 2006). Cryo-sections (in 7 μm thickness) of the posterior segment were made in four planes—0° (temporal), 90° (superior), 180° (nasal), and 270° (inferior)—relative to the ONH. These sections were stained with hematoxylin and eosin.

Slides were digitally scanned (ScanScope™, Aperio, Vista, CA) with a 40X magnification for histological analysis. The Ruler function within the built-in software (Aperio’s ImageScope™, Aperio, Vista, CA) was utilized to measure total and inner retinal thicknesses using the same landmarks for OCT image analysis (Fig. 2C). Measurements were initiated near the edge of the ONH and continued towards the peripheral retina until 70 measurements were taken to approximate OCT measurements as closely as possible. The standard distance between each measurement was 100 μm. Measurements from the areas containing retinal blood vessels were excluded from the data analysis.

Statistics

Results are presented as mean ± standard error of mean (SEM). In each OCT imaging plane of each eye, the thicknesses were analyzed and compared at the previously defined locations, starting from the edge of the ONH and progressing towards the peripheral retina; those locations are as follows: the first 22 (area of 0–5.5 mm from the ONH), the first 4 (area of 0–1 mm from the ONH), the second 4 (area of 1–2 mm from the ONH), the third 4 (area of 2–3 mm from the ONH), the fourth 4 (3–4 mm from the ONH), the fifth 4 (4–5 mm from the ONH) and the last 2 measurements (area of 5–5.5 mm from the ON). A linear mixed effects model was used to compare the SD-OCT and histological measurements of total, inner and outer retinal thicknesses, as well as the length of ellipsoid zone among phenotype groups; the model permitted adjusting for the inter-eye correlation arising from the same animal (Ying et al., 2017). Post-hoc pairwise comparisons were done for each canine model of inherited retinal dystrophy to wt group. Age and gender were included as covariates for the linear mixed effect model. All the data were analyzed in SAS v9.4 (SAS Institute Inc., Cary, NC). P ≤ 0.05 for a two-tailed test was considered considered statistically significant.

Results

Normal OCT and histology findings in wild type dogs

The mean (± SEM) total retinal thicknesses measured with OCT were thickest in the superior area (243.9 ± 9.4 μm), followed by superotemporal (234.1 ± 4.5 μm), temporal (228.2 ± 5.7 μm), nasal (221.3 ± 6.5 μm ), and inferior (182.8 ± 7.0 μm) areas (p ≤ 0.02; Fig. 3A). The central retina including the first and second 4 measurements which correspond to 0–1 mm and 1–2 mm from the ONH, respectively, was significantly thicker than the peripheral retina, third ( and fourth 4 and last 2 measurements: 2–5.5 mm from the ONH in each imaging plane. (p ≤ 0.04; Fig. 4A1–5). No significant effect of age (0.7 – 9.1 years) on total retinal thickness was observed in any imaging or section plane with OCT or histology in wt (p ≥ 0.26). The female group had significantly decreased total retinal thickness in the superior imaging plane (90°; 230.0 ± 10.6 μm) compared to the male group (273.47 ± 14.98 mm, p = 0.02). For the rest of the imaging or section planes, no significant sex effect was observed (p ≥ 0.18). Representative funduscopic, OCT, and histology images of wt are presented in Figure 5E, J, O, S, and W.

Figure 3.

Figure 3.

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Canine models of RCD1, PRCD, and LCA exhibit outer and total retinal atrophy in most planes assessed with SD-OCT (A and B). Inner retina is mostly well preserved in models of RCD1, PRCD, LCA, and achromatopsia. Each column and error bar indicate mean and SEM, respectively, of the retinal thickeness.’*’ indicates p ≤ 0.05 compared to wild type.

Figure 4.

Figure 4.

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Total (A1 – A5), outer (B1 – B5) and inner (C1 – C5) mean ± SEM retinal thicknesses of canine wild type and inherited retinal dystrophy models and wild types measured at various locations from the ONH in the temporal, superotemporal, superior, nasal, and inferior imaging planes. Images were obtained with SD-OCT. Each letter (a: achromatopsia; l: LCA; p: PRCD; and r: RCD1) indicates statistical difference with wild type for each imaging location. Data points with statistical differences are also shown as filled circle (p ≤ 0.05).

Figure 5.

Figure 5.

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Canine models of RCD1 and PRCD exhibit funduscopic changes of the retina and ONH including tapetal hyperreflectivity (a sign of retinal thinning in dogs, red arrows) retinal vascular attenuation (black arrows), and ONH atrophy (A and B). SD-OCT (F, G, K and L) and histology images (P and T) show thin atrophied total and outer retina. Eyes with PRCD were not available for histology. Bars in P-W = 100 μm.

Severe Outer Retinal Atrophy and Disruption of the Ellipsoid Zone in Dogs with RCD1

The temporal, superotemporal, superior, nasal, and inferior total retinal thicknesses measured with OCT were 181.2 ± 10.1, 170.0 ± 8.0, 162.9 ± 17.0, 137.0 ± 11.4, and 110.6 ± 12.6 μm, respectively (Fig. 3A). The thicknesses of the total and outer retina in the temporal and superotemporal imaging planes were significantly higher than those in the nasal and inferior retina (p ≤ 0.03; Fig. 3A and B). Dogs with RCD1 had significantly decreased total and outer retinal thicknesses in all imaging planes compared to wt (p ≤ 0.001, Fig. 3A and B and supplementary Fig. 1A and B). When the retina was analyzed at different locations from the center to the periphery in each imaging plane, total retinal thickness was significantly decreased compared to wt in all locations (p ≤ 0.009) except for the central area (0 – 1 mm from the ONH) in the superotemporal imaging plane (p = 0.06; Fig. 4A, D, G, J, and M).

Compared to wt dogs, the outer retinal thickness was significantly decreased in all locations (p ≤ 0.001; Fig. 4B, E, H, K, and N). No significant difference was observed in inner retinal thickness between RCD1-affected and wt in the superotemporal, superior, nasal, and inferior imaging planes (p ≥ 0.16, Fig. 3C). Compared to the wt dogs, however, RCD1 dogs had a significantly thicker inner retina in the temporal imaging plane (p ≤ 0.001, Fig. 3C). In the RCD1-affected eyes, the discernible ellipsoid zone in the superotemporal imaging plane was significantly shorter (0.94 ± 0.70 mm) compared to wt dogs (5.19 ± 0.36 mm, p ≤ 0.001; Fig. 6). The presence of the discernible ellipsoid zone in the presumed area centralis was significantly lower in the RCD1-affected eyes (0%) compared to the wt (100%, P ≤ 0.001; Fig. 6). Histological measurements agreed with OCT measurements: Dogs with RCD1 had significantly decreased total and outer retinal thicknesses (p ≤ 0.003; Fig. 7A and B). Dogs with RCD1 had a significantly increased inner retinal thickness in the superior plane (p = 0.002; Fig. 7C). Similar to the OCT results, inner retinal thickness in the temporal plane was thicker, though not statistically significant (p = 0.13; Fig. 7C). Figures 5A, F, K, P, and T show representative funduscopic, OCT, and histology images of a canine eye with RCD1.

Figure 6.

Figure 6.

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Canine models of RCD1, PRCD, and LCA had significantly more disrupted/absent ellipsoid zone in the presumed area centralis compared to wild type while achromatopsia model had normal ellipsoid zone in SD-OCT imaging (A-E). (F) and (G) show representative images of normal ellipsoid (red arrow) and disrupted/absent ellipsoid zone, respectively. (H) RCD1 and LCA model had significantly shorter discernible ellipsoid zone in the superotemporal imaging plane (p ≤ 0.001). Each column and the error bar indicates mean and SEM, respectively, length of the discernible ellipsoid zone in the imaging planes.

Figure 7. Canine RCD1 model shows significant total and outer retinal thinning in all section planes in histology.

Figure 7.

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(A and B). (C) Inner retina is well preserved in models of RCD1, LCA, and achromatopsia. Each column and the error bar indicates mean and SEM, respectively, length of the discernible ellipsoid zone in the imaging planes. ‘*’ indicates p ≤ 0.05 compared to wild type.

Moderate Outer Retinal Atrophy and Preserved Ellipsoid Zone in Dogs with PRCD

In PRCD-affected dogs, the temporal, superotemporal, superior, nasal, and inferior total retinal thicknesses measured 187.1 ± 10.0, 219.2 ± 7.8, 226.3 ± 16.3, 160.2 ± 11.3, and 114.7 ± 12.2 μm with OCT, respectively. The thicknesses of the total and outer retina in the temporal, superotemporal, and superior imaging planes were significantly higher than those in the nasal and inferior retina (p ≤ 0.001; Fig. 3A and B).

Dogs with PRCD had significantly decreased total retinal thickness in temporal, nasal, and inferior imaging planes (p ≤ 0.001) but not in the superotemporal (p = 0.1) and superior imaging planes (p = 0.54) compared to wt (Fig. 3A and supplementary Fig. 1A). In all imaging planes of the PRCD-affected dogs, the outer retinal thickness decreased significantly compared to wt dogs (p ≤ 0.001 Fig. 3B and supplementary Fig. 1B). When the retina was analyzed at different locations from central to peripheral in each imaging plane, the outer retinal thickness was significantly thinner in all locations compared to that of the wt (p ≤ 0.05; Fig. 4B, E, H, K, and N). The thickness of the inner retina in the PRCD-affected dogs was significantly increased in the temporal imaging plane (p = 0.03) and decreased in the nasal plane (p ≤ 0.001) compared to the inner retinal thickness of the wt (Fig. 3C). The percentage decreases in thickness of total, outer, and inner retina in each imaging plane is presented in supplement figure 1.

In a comparison of the PRCD-affected eyes with those of the wt (see Figure 6), no significant difference was observed in the length of the discernible ellipsoid zone in the superotemporal imaging plane (4.25 ± 0.63 mm in PRCD-affected versus 5.19 ± 0.36 mm in wt, p = 0.14). The presence of the discernible ellipsoid zone in the presumed area centralis was significantly lower in the PRCD-affected eyes (0%) than in the wt (100%, p ≤ 0.001; Fig. 6). Tissue samples of the PRCD-affected group were not available for histology. Representative funduscopic and OCT images of a canine eye with PRCD are presented in Figures 5B, G, and L.

Mild Outer Retinal Atrophy and Disruption of the Ellipsoid Zone in Dogs with LCA

The temporal, superotemporal, superior, nasal, and inferior total retinal thicknesses measured with OCT were 211.2 ± 8.0, 225.9 ± 6.2, 232.4 ± 13.6, 198.6 ± 9.0, and 170.1 ± 9.7 μm, respectively. The thicknesses of total and outer retina in the temporal, superotemporal, and superior imaging planes were significantly higher than those in nasal and inferior retina (p ≤ 0.002). Dogs with LCA had significantly decreased total retinal thickness in the temporal and nasal imaging planes compared to wt (p = 0.03; Fig. 3A and supplementary Fig. 1A). The outer retinal thickness was, in all imaging planes, significantly decreased in the LCA-affected dogs (p ≤ 0.04; Fig. 3B and supplementary Fig. 1B). Central outer retina tended to be more preserved in most imaging planes with no significant difference between LCA-affected dogs and wt in the areas 0–3 mm from the ONH in superotemporal, 0–2 mm in superior, and 0–1 mm in nasal and inferior imaging planes (p ≥ 0.07; Fig. 4B, E, H, K, and N). The length of the discernible ellipsoid zone in the superotemporal imaging plane was significantly lower in the LCA-affected eyes (3.49 ± 0.50 mm) than in the wt (5.19 ± 0.36 mm, p = 0.03; Fig. 6). The presence of the discernible ellipsoid zone in the presumed area centralis was significantly lower in the LCA-affected eyes (16.7%) than in the wt (100%, p ≤ 0.001; Fig. 6). Histology failed to reveal a significant difference in any retinal thicknesses between the LCA-affected dogs and wt (p ≥ 0.10, Fig. 7). Representative funduscopic, OCT, and histology images of a canine eye with LCA are presented in Fig. 5C, H, M, Q and U.

Mild Outer Retinal Atrophy in the Inferior and Nasal Imaging Planes in Dogs with Achromatopsia

The temporal, superotemporal, superior, nasal, and inferior total retinal thicknesses measured 224.7 ± 8.3, 228.2 ± 6.5, 254.2 ± 13.6, 207.1 ± 9.4, and 170.7 ± 10.1 μm with OCT in achromatopsia-affected dogs, respectively. The total and outer retinas were significantly thicker in the temporal and superotemporal and superior imaging planes than in the inferior plane (p ≤ 0.01). Dogs with achromatopsia had significantly thinner total retinas in the nasal and inferior imaging planes than did the wt (p ≤ 0.03; Fig. 3A and supplementary Fig. 1A). The achromatopsia-affected dogs had significantly thinner outer retinas in the superotemporal (p = 0.02) and inferior imaging planes (p ≤ 0.001) than did the wt (Fig. 3B and supplementary Fig. 1B). The peripheral outer retina was significantly decreased in the superotemporal (area 3 – 5.5 mm from ONH; p ≤ 0.03) and inferior (area 2 – 5.5 mm from ONH; p ≤ 0.007) imaging planes compared to wt dogs (Fig. 4B, E, H, K, and N). No significant difference was observed in the length of the discernible ellipsoid zone in the superotemporal imaging plane between the achromatopsia-affected eyes (5.4 ± 0.6 mm) compared to wt (5.2 ± 0.4 mm, p = 0.14; Fig. 6). The presence of the discernable ellipsoid zone in the presumed area centralis was not significantly different between the achromatopsia-affected eye (100%) and the wt (100%, p = 1.00; Fig. 6). Histology revealed a significantly increased total retinal thickness in the nasal plane of achromatopsia-affected eye (p ≤ 0.001) and increased inner retinal thickness in the superior section plane compared to wt (p ≤ 0.001, Fig. 7). Representative funduscopic, OCT, and histology images of a canine eye with achromatopsia are presented in Fig. 5D, I, N, R, and V.

Discussion

The present study characterizes changes in the thickness of total, outer, and inner retina and ellipsoid zone assessed with in vivo OCT in four canine models of inherited retinal diseases, PDE6B-RCD1, PRCD-PRCD, RPE65-LCA, and CNGB3-achromatopsia. All affected dogs had significantly decreased total and outer retinal thickness to various degrees of severity, while the inner retina showed a significant increase in some areas of the eyes with RCD1 and PRCD. While OCT has been used as an outcome measure of gene therapy in several canine disease models including RPE65-LCA (Annear et al., 2021; Bainbridge et al., 2015; Cideciyan et al., 2013; Gardiner et al., 2020; Mowat et al., 2013) and PDE6B-RCD1 (Petit et al., 2012), this is the first, to the authors’ knowledge, report presenting comparative data of detailed OCT assessment of retinal thicknesses and ellipsoid zone in representative canine retinal disease models with identified gene mutations including early and late onset RPs as well as a cone specific disease.

RCD1- and PRCD-affected dogs are established canine models of RP (Petersen-Jones et al., 1995; Petersen-Jones and Komaromy, 2015; Winkler et al., 2020; Zangerl et al., 2006). RP encompasses a wide spectrum of inherited retinal dystrophies characterized by sequential degeneration of rod and cone photoreceptors (Petit et al., 2012). To date, more than 12 canine models of RP have been identified and characterized genetically and phenotypically (Petersen-Jones et al., 1995; Petersen-Jones and Komaromy, 2015; Tuntivanich et al., 2009; Winkler et al., 2013; Winkler et al., 2020). Histological studies have shown severely reduced photoreceptor layer with disorganized rods and cones in affected dogs (Aguirre et al., 1982; Aguirre and Acland, 1988). In the present study, both the RCD1- and PRCD-affected dogs had significantly decreased outer and total retinal thicknesses compared to wt; however, the percentage decrease was significantly higher in RCD1-affected dogs than in PRCD-affected ones. This is likely due to the more advanced stage of disease in RCD1-affected dogs. Our OCT findings on RCD1-affected dogs that were significantly decreased in all imaging planes, support the previously published OCT data on this disease model (Panzan et al., 2004). OCT data on PRCD-affected retinas have not been published.

This is the first report of increased thickness of the inner retinal layer in the eyes of dogs having RCD and PRCD. While this is a new observation in dogs, increased inner retinal thickness has been reported in humans with RPGR-X linked RP—one of the most common types of severe RP (Aleman et al., 2007; Jolly et al., 2020) and with CEP290-LCA (Cideciyan et al., 2007). Remodeling of the neuronal-glial inner retina secondary to photoreceptor loss has been suggested as a possible mechanism (Aleman et al., 2007; Jolly et al., 2020).

A histological study of canine PRCD has described that pathology is most severe in the inferior quadrants and the temporal quadrant is least affected (Aguirre and Acland, 1988). Our OCT measurements of the PRCD-affected dogs revealed that the inferior and nasal planes had significantly more thinning in total and outer retinal thickness than did to the superior and temporal planes; such findings are in agreement with the previously published histological data (Aguirre and Acland, 1988). In the present study, this variation in pathology was also observed in the RCD1-affected groups: similar to PRCD, the inferior plane was the most severely affected.

LCA is also a genetically heterogenous group associated with mutations in at least 19 different genes including RPE65 and CEP290 (Jacobson et al., 2016; Marlhens et al., 1997; Senechal et al., 2006). The canine LCA model with mutation of RPE65 has been successfully employed for establishing proof-of concept for the efficacy of gene therapy with a recombinant adeno-associated virus (AAV) carrying RPE65 (Acland et al., 2005; Acland et al., 2001; Winkler et al., 2020) which led to the development of the new, FDA-approved gene therapy. LCA patients with the RPE65 mutation have significantly decreased total retinal thickness in the central macular and perifoveal areas (Pasadhika et al., 2010). Dogs do not have a fovea. However, they do have an area of a high cone density, the area centralis, which is the dog semi-equivalent of fovea. In dogs, the area centralis is located 3–4 mm superotemporal to the ONH (Donovan et al., 1974). In the current study, a significant reduction in the outer retinal thickness was observed in all imaging planes including the presumed area centralis. Recent manuscripts on dogs with the RPE65 mutation showed a localized funduscopic lesion in conjunction with loss of photoreceptor layers with OCT in the area centralis (Annear et al., 2021; Mowat et al., 2017). While our data is supported by their findings of photoreceptor loss, the dogs included in the current study did not have a distinct lesion localized in the area centralis. Funduscopy images of dogs with LCA in the current study did not show those round to elongated lesions presented in the fundus and cSLO images reported in the previous study (Mowat et al., 2017). It is, however, possible that OCT findings of the area centralis lesions may not have been detected with single line OCT images obtained in the current study. Considering these colonies are from two different institutions—Michigan State University in the previous study (Mowat et al., 2017), and University of Pennsylvania in the current one—the discrepancy was likely due to a phenotypical variation of two different genetic traits.

Achromatopsia is a hereditary condition associated with the dysfunction or absence of cone photoreceptors (Hirji et al., 2018; Yu et al., 2014). Canine models of achromatopsia with the CNGB3 mutation have been reported (Sidjanin et al., 2002; Yeh et al., 2013) and utilized for gene therapy preclinical trials to restore vision (Komaromy et al., 2010; Komaromy et al., 2013). In the present study, dogs with advanced achromatopsia exhibited no significant change in total or outer retinal thickness in any retinal imaging plane of OCT except mild peripheral thinning of the outer retina in the superotemporal and inferior imaging planes. Histological measurements also showed no significant decrease in any retinal thickness. OCT data on the canine achromatopsia model has not been published.

The ellipsoid zone is a distinct, highly reflective line observed superficial to the RPE layer in retinal OCT images. Traditionally, the ellipsoid zone was considered to correspond to photoreceptor inner/outer segment (IS/OS) junction (Mitamura et al., 2012). Recent studies, though, suggest it may correlate with the outermost part of the inner segment, which is densely packed with mitochondria (Ha et al., 2018). Intact subfoveal ellipsoid zone is highly correlated with visual acuity (Mitamura et al., 2012), and disrupted or absent ellipsoid zone has been reported in patients with retinal dystrophies including RP and achromatopsia (Murthy et al., 2016; Petit et al., 2012). In the present study, moderate to severe disruption of ellipsoid zone was observed in the canine models of RP (RCD1 and PRCD) and LCA. This is the first report on the integrity of ellipsoid zone in canine models of retinal dystrophy.

One of the interesting findings in the wt dogs was a gender effect of the retinal thickness found in the superior OCT imaging plane: total retinal thickness was significantly thinner in females than males. While, to the best of the authors’ knowledge, this is the first report on the gender effect in dogs, in humans, the central retinal thickness has reported to be thinner in women compared to men.(Wong et al., 2005) Further studies with larger number of dogs will be needed to generalize this finding.

A limitation of this study is the small number of animals in each group due to the high value of these animal models. For the same reason, not all the animals included in the OCT study were available for histological evaluation. At the same time, not all the animals (eyes) that had histological evaluations were available for in vivo OCT imaging due to the progressed cataract formation secondary to the retinal disease. Since OCT imaging requires clear optics to obtain high-quality images, eyes with advanced cataracts had to be excluded from the OCT study.

Conclusions

Generalized retinal thinning, mainly in the outer retina, was observed in dogs with RCD1, PRCD, and LCA. In dogs with achromatopsia, mild peripheral thinning of the outer retina was observed in the superotemporal and inferior imaging planes. In dogs with advanced stage RCD1 and PRCD, the inner retina was mostly well preserved, showing an increase in thickness in the temporal imaging plane. This study provides the first documentation of disrupted ellipsoid zones in the models of RCD1, PRCD, and LCA. These findings, along with detailed quantitative and qualitative in vivo morphologic characterizations of the retina in various regions, provide baseline information for research of retinal dystrophies and development of novel therapeutics using these canine models.

Supplementary Material

Supplementary Table 1

Supplementary Table 1. Mean total, outer, and inner retinal thicknesses measured from the temporal OCT imaging plane and histology section in each eye. Measurements are in μm.

Supplementary figure 1

Supplement Figure 1. Percentage decrease in total (A), outer (B), and inner (C) mean ± SEM retinal thicknesses in canine models of RCD1, achromatopsia, and LCA compared to wild type controls analyzed from SD-OCT images of the temporal, superotemporal, superior, nasal, and inferior section planes. Inferior and nasal imaging planes tended to exhibit a more severe total and outer retinal thinning in the models of RCD1, PRCD, and LCA.

Highlights.

  • Thinning of the outer retina in dogs with RCD1, PRCD, and LCA with OCT imaging

  • No thinning of the inner retina in dogs with RCD1, PRCD, LCA, and achromatopsia

  • Disrupted Ellipsoid zone in dogs with RCD1, PRCD, and LCA

Acknowledgements

The authors thank Luis Felip Lima Pompeo Marinho for providing funduscopic images, as well as Dr. Gustavo D. Aguirre (University of Pennsylvania) and the staff at the Retinal Disease Studies Facility (University of Pennsylvania) for veterinary or technical support with the animals. Supported by U.S. National Institutes of Health Grants R01-EY006855, R01-EY019304, P30-EY001583, K08EY030950, and unrestricted grant from Research to Prevent Blindness.

Grant information:

The project described was supported by NIH Grants R01-EY006855, R01-EY019304, R01-EY025752, K12-EY015398, P30-EY001583, K08EY030950, and Foundation Fighting Blindness.

Footnotes

Disclosure: S.A. Park, None; J. Rhodes, None; S. Iwabe, None; G.S. Ying, None; W. Pan, None; J. Huang, None; A.M. Komáromy, None

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Table 1

Supplementary Table 1. Mean total, outer, and inner retinal thicknesses measured from the temporal OCT imaging plane and histology section in each eye. Measurements are in μm.

NIHMS1854348-supplement-Supplementary_Table_1.docx (39.5KB, docx)
Supplementary figure 1

Supplement Figure 1. Percentage decrease in total (A), outer (B), and inner (C) mean ± SEM retinal thicknesses in canine models of RCD1, achromatopsia, and LCA compared to wild type controls analyzed from SD-OCT images of the temporal, superotemporal, superior, nasal, and inferior section planes. Inferior and nasal imaging planes tended to exhibit a more severe total and outer retinal thinning in the models of RCD1, PRCD, and LCA.

NIHMS1854348-supplement-Supplementary_figure_1.tif (1.9MB, tif)

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