Phenotypic Characterization of Corneal Endothelial Dystrophy in German Shorthaired and Wirehaired Pointers Using In Vivo Advanced Corneal Imaging and Histopathology

Abstract

Purpose: To evaluate corneal morphology using ultrasonic pachymetry (USP), Fourier-domain optical coherence tomography (FD-OCT), and in vivo confocal microscopy (IVCM) in 2 related canine breeds-German shorthaired pointers (GSHPs) and German wirehaired pointers (GWHPs)-with and without corneal endothelial dystrophy (CED). This condition is characterized by premature endothelial cell degeneration leading to concomitant corneal edema and is similar to Fuchs endothelial corneal dystrophy.

Methods: Corneas of 10 CED-affected (4 GSHP and 6 GWHP) and 19 unaffected, age-matched (15 GSHP and 4 GWHP) dogs were examined using USP, FD-OCT, and IVCM. A 2-sample t test or Mann-Whitney rank-sum test was used to statistically compare parameters between both groups. Data are presented as mean ± SD or median (range).

Results: Central corneal thickness determined using USP was significantly greater in CED-affected than in unaffected dogs at 1179 (953-1959) and 646 (497-737) μm, respectively (P < 0.001). Central epithelial thickness was found to be significantly decreased in CED-affected versus unaffected dogs at 47 ± 7.1 and 55 ± 7.1 μm, respectively (P = 0.011), using FD-OCT. With IVCM, corneal endothelial density was significantly less (P < 0.001) in 5 dogs with CED versus 19 unaffected controls at 499 ± 315 versus 1805 ± 298 cells/mm, respectively. CED-affected dogs exhibited endothelial pleomorphism and polymegethism, whereas CED-unaffected dogs had regular hexagonal arrangement of cells.

Conclusions: GSHPs and GWHPs with CED exhibit marked differences in corneal morphology when compared with age-matched control dogs. These 2 CED-affected breeds represent spontaneous, large animal models for human Fuchs endothelial corneal dystrophy.

Fig. 1. Corneal thickness in 10 CED-affected dogs as measured by USP markedly differed in comparison to 19 unaffected controls

At all 5 locations measured, CED-affected dogs had significantly greater corneal thickness in comparison to CED-unaffected dogs. Corneal thickness significantly differed between all locations in the CED-unaffected dogs but the inferior and nasal locations (P = 0.224). Box plots depict median (solid line), mean (dashed line), 25^th and 75^th percentiles, while whiskers show 10^th and 90^th percentiles; black circles indicate outliers. The P values were determined by a Kruskal Wallis ANOVA on ranks, ***P < 0.001 between CED-unaffected and affected dogs. ^a,b,c,dP < 0.005 between the different locations of CED-unaffected dogs.

Fig. 2. Corneal structure and endothelial morphology as determined by color photography, digital slit lamp biomicroscopy, FD-OCT and IVCM dramatically differed between control and CED-affected dogs with variable disease severity

A 14 year old spayed female GSHP with a clear cornea (A1-2), normal corneal thickness and lamellar arrangement of the collagen fibrils (A3) and regular, hexagonal arrangement of corneal endothelium (A4); this dog was categorized as a control. An 11 year old intact male with mild diffuse corneal edema (B1-2), mildly thickened cornea (B3) and moderate pleomorphism and polymegathism of the corneal endothelium (B4); an 8 year old spayed female GSHP with moderate diffuse edema of the cornea (C1-2), increased corneal thickness (C3) and moderate pleomorphism and polymegathism of the corneal endothelium (C4); a 13 year old spayed female GSHP with marked, diffuse corneal edema (E1-2), marked increase in corneal thickness (E3), and loss of the orderly arrangement of collagen fibrils within the anterior stroma (E3-4) such that the corneal endothelium and keratocytes could not be visualized (E4).

Fig. 3. Central corneal full, stromal, epithelial and DM-endothelium complex thickness significantly differed between dogs without and with CED as measured by FD-OCT

Central corneal thickness (CCT) was significantly greater in 9 CED-affected versus 19 unaffected dogs with a median (range) of 1100 (910–1755) and 614 (506–677) μm, respectively, primarily due to a significant increase in stromal thickness in CED-affected versus unaffected dogs at 1009 (831–1705) and 539 (442–591) μm, respectively (A). Epithelial thickness in the central cornea was significantly less in dogs with versus without CED at 47 ± 7.1 and 55 ± 7.1 μm, respectively (B). In the central cornea, DM-endothelium complex thickness was significantly greater in dogs with versus without CED at 36 ± 6.3 and 29 ± 4.5 μm, respectively. Box plots depict median (solid line), mean (dashed line), 25^th and 75^th percentiles, while whiskers show 10^th and 90^th percentiles; black circles indicate outliers. The P values were determined by a Mann-Whitney rank sum test, *P < 0.05, **P < 0.01, and ***P < 0.001. (C) A significant inverse relationship was identified between epithelial thickness and CCT as measured by FD-OCT using least squares linear regression (P = 0.049, R² = 0.141).

Fig. 3. Central corneal full, stromal, epithelial and DM-endothelium complex thickness significantly differed between dogs without and with CED as measured by FD-OCT

Central corneal thickness (CCT) was significantly greater in 9 CED-affected versus 19 unaffected dogs with a median (range) of 1100 (910–1755) and 614 (506–677) μm, respectively, primarily due to a significant increase in stromal thickness in CED-affected versus unaffected dogs at 1009 (831–1705) and 539 (442–591) μm, respectively (A). Epithelial thickness in the central cornea was significantly less in dogs with versus without CED at 47 ± 7.1 and 55 ± 7.1 μm, respectively (B). In the central cornea, DM-endothelium complex thickness was significantly greater in dogs with versus without CED at 36 ± 6.3 and 29 ± 4.5 μm, respectively. Box plots depict median (solid line), mean (dashed line), 25^th and 75^th percentiles, while whiskers show 10^th and 90^th percentiles; black circles indicate outliers. The P values were determined by a Mann-Whitney rank sum test, *P < 0.05, **P < 0.01, and ***P < 0.001. (C) A significant inverse relationship was identified between epithelial thickness and CCT as measured by FD-OCT using least squares linear regression (P = 0.049, R² = 0.141).

Fig. 3. Central corneal full, stromal, epithelial and DM-endothelium complex thickness significantly differed between dogs without and with CED as measured by FD-OCT

Central corneal thickness (CCT) was significantly greater in 9 CED-affected versus 19 unaffected dogs with a median (range) of 1100 (910–1755) and 614 (506–677) μm, respectively, primarily due to a significant increase in stromal thickness in CED-affected versus unaffected dogs at 1009 (831–1705) and 539 (442–591) μm, respectively (A). Epithelial thickness in the central cornea was significantly less in dogs with versus without CED at 47 ± 7.1 and 55 ± 7.1 μm, respectively (B). In the central cornea, DM-endothelium complex thickness was significantly greater in dogs with versus without CED at 36 ± 6.3 and 29 ± 4.5 μm, respectively. Box plots depict median (solid line), mean (dashed line), 25^th and 75^th percentiles, while whiskers show 10^th and 90^th percentiles; black circles indicate outliers. The P values were determined by a Mann-Whitney rank sum test, *P < 0.05, **P < 0.01, and ***P < 0.001. (C) A significant inverse relationship was identified between epithelial thickness and CCT as measured by FD-OCT using least squares linear regression (P = 0.049, R² = 0.141).

Fig. 4. Endothelial cell density as measured by IVCM was markedly less in dogs with CED

Corneal endothelial density was significantly less in 5 dogs with CED (499 ± 315 cells/mm²) versus 19 unaffected controls (1805 ± 298 cells/mm²). Endothelial cell density could not be assessed in 5 dogs with CED due to severe, bilateral corneal edema. Box plots depict median (solid line), mean (dashed line), 25^th and 75^th percentiles, while whiskers show 10^th and 90^th percentiles; black circles indicate outliers. The P value was determined by a Students’s t-test, ***P < 0.001.

Figure 5. Histology morphology of the corneal endothelium and DM markedly differed between a dog with and without CED

Photomicrographs of sections of the inner cornea from the right eye of a 15 year old intact male GSHP (A & B) unaffected with CED and a 14 year old intact female CED-affected GSHP (C & D) stained with hematoxylin and eosin (200x magnification) or Periodic acid-Schiff (400x magnification) where DM is bright magenta, respectively. In A & B, note the smooth contour of the inner aspect of DM (*), and the frequent nuclei in the cuboidal endothelial cells (example at arrowhead) in a dog unaffected with CED. In the CED-affected dog (C), note the irregularities (arrows) on the inner surface of DM (*) and the slight increase in thickness of DM in comparison to the CED-unaffected dog (A). The endothelium is more sparsely cellular, and the cells are attenuated (example at arrowhead) in the CED-affected (C) versus unaffected dog (A). In D, note the pale-staining guttae-like excrescences of loose, fibrillar ECM (arrows) on the inner surface of DM (magenta) in the CED-affected dog; two of these excrescences are associated with flattened endothelial cells (examples at arrowheads). In D, DM (*) is also slightly thicker than in B (CED-unaffected).