Monocular retinopathy of prematurity-like retinal vasculopathy in a dog

Abstract

Purpose:

To describe a case of monocular retinopathy of prematurity (ROP)-like vasculopathy without oxygen supplementation in the dog.

Methods:

Fundus photographs (RetCam), spectral-domain optical coherence tomography (sdOCT), confocal scanning laser ophthalmoscopy (cSLO), and fluorescein angiography (FA), as well as postmortem histology and immunohistochemistry (Collagen IV and anti-vWF antibodies), were carried out to characterize the vascular abnormalities.

Results:

Ophthalmic examination showed peripheral and mid-temporal avascular areas in the tapetal region, neovascularization and abnormally dilated and tortuous retinal vessels in the left eye. sdOCT demonstrated not only cross-sectional views of preretinal fibrovascular proliferation but also extensive proliferation extraretinally into the vitreous. FA emphasized demarcation of vascular and avascular zones with neovascular tufts “popcorns.” Histology and immunohistochemistry confirmed presence of abnormally dilated vessels and the intravitreal blood vessels.

Conclusions:

ROP is a disease of abnormally developed retinal vascularization associated with oxygen supplementation therapy, potentially causing blindness in premature infants. Although the mechanism of ROP-like vasculopathy in our case is unclear, it is important to appreciate that the abnormal vascular pattern seen in ROP in premature infants can occur in canines without oxygen administration.

1 |. INTRODUCTION

Retinopathy of prematurity (ROP) is a disease of abnormally developed retinal vascularization associated with oxygen supplementation therapy, potentially causing blindness in premature infants. ROP is a biphasic disease, vaso-obliteration (vessel regression) followed by vasoproliferation (neovascularization). The term, ROP, previously known as retrolental fibroplasia, was first described in 1942 by Terry who discovered the association between retinal neovascularization and prematurity (See¹ for review, and²). Major risk factors for developing ROP are prematurity and oxygen supplementation. In human patients, there is standardized classification regarding location, stage, and extent of disease, determined by the International Classification for ROP (ICROP) based on ophthalmoscopic findings.³

In man, the retinal vasculature normally develops fully in utero, originating first from the center of the optic nerve head then extending peripherally. In contrast, full-term new born puppies have only about 66% of retinal vascularization at birth, which reaches the periphery by Day 14.⁴ Taking advantage of the similarity of the incomplete retinal vasculature seen in both premature infants and newborn animals, several animal models, for example, rats, mice, cats, and dogs, have contributed to the current understanding of human ROP.^5–9 These newborn animals are experimentally exposed to high oxygen concentrations in order to reproduce ROP-like retinal vasculopathy, termed oxygen-induced retinopathy (OIR). The success of these experiments has demonstrated that the incomplete vasculature of neonates in a variety of species, like premature infants, is highly susceptible to oxygen toxicity. McLeod et al. (1996) established a canine-specific model of OIR, which consistently produced robust responses closely mimicking the pattern of vaso-obliteration and vasoproliferation seen in human ROP.^7,8

Here, we report a case of unilateral ROP-like vasculopathy in a dog characterized by an avascular mid-peripheral retinal zone, neovascularization and dilated and tortuous vessels with vitreal extension, using non-invasive retinal imaging methods, as well as postmortem histology and immunohistochemistry. To our knowledge, this is the first naturally occurring canine case of ROP-like vasculopathy. Most intriguing is the fact that while oxygen supplementation is apparently imperative for OIR in dogs, the dog in the current case report did not receive any therapeutic oxygen at any time in its life and the findings were monocular.

2 |. CASE REPORT

2.1 |. Clinical history

A male intact miniature long-haired dachshund cross was bred at the Retinal Disease Studies Facility (RDSF, Kennett Square, PA) as part of studies to investigate cone-rod dystrophy in homozygous RPGRIP1-mutant dogs.¹⁰ All 8 puppies from the litter were born at full-term with an uneventful labor and parturition, and did not receive any oxygen supplementation. There had not been any obvious difference in intrinsic or extrinsic factors between this pup and the rest of the litter, yet, this pup was the only one affected with abnormal retinal vasculature. The study was conducted in full compliance with the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) approval, adhered to the Association for Research in Vision and Ophthalmology (ARVO) Resolution for the Use of Animals in Ophthalmic and Vision Research, and followed the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

2.2 |. Ophthalmic examination

Complete ophthalmic examinations were performed by board-certified veterinary ophthalmologists at the age of 6, 13, and 23 months and included fundus photography using RetCam wide-field retinal imaging system (Clarity Medical System). In the initial examination, the dog had bilateral hyaloid artery remnants, which were less extensive in the right eye. In the left eye, there was a fibrotic plaque at the posterior lens capsule that increased in density over time. These are incidental findings that occur sporadically in normal or affected dogs with different retinal diseases in our research colony (GDA, KM, unpublished data). The most striking findings were in the left eye, which showed an unusual retinal vasculopathy with a more peripheral and mid-temporal avascular areas (Figure 1*). Additionally, neovascularization and abnormally dilated and tortuous retinal vessels were present (Figure 1A–C). These abnormal vessels were visible from around the optic disc, extended outward, and abruptly terminated prematurely before reaching the periphery. These vessels were so engorged that it was not possible to distinguish the major retinal arterioles from venules during the clinical examination or in the fundus photographs (Figure 1A–C). In contrast, the right fundus was normal (Figure 1D).

FIGURE 1.

RetCam fundus photography. Fundus photograph of the affected eye at 26 (A) and 30 (B) weeks age. (C) Near-infrared imaging composite by confocal Scanning Laser Ophthalmoscopy of the affected eye at 31 weeks of age. Note the avascular zone in the peripheral and mid-temporal retina (asterisks *), dilated and tortuous retinal vessels (selected arteriole and venule identified with yellow and blue arrows, respectively), and neovascular tufts at the vascular-avascular border. (D) RetCam fundus photograph of unaffected right eye at 30 weeks of age

Fluorescein angiography (FA) with an IV injection of 1.5 ml of fluorescein 10% (A-K Fluor 10%, Akorn) was done to characterize the vascular abnormalities after OCT and cSLO (see below). Video images were taken by cSLO using the autofluorescence settings (488 nm wavelength) with a 55º lens at ~55%–95% intensities in the affected left eye and subsequently in the normal right eye.

Figure 2 shows snapshots of the left eye from the FA video at different stages. After choroidal flush, the arterioles filled first (A white arrows, B), then without distinct capillary bed filling, the dye from arterioles join into venules (C, D) indicating presence of arteriovenous (A–V) anastomoses. As these abnormal vessels end abruptly in the mid-periphery, the demarcation of vascular and avascular zones was apparent. At the tip of abnormal vessels, there was unique shape of vascular proliferation which looked like glomeruli or “popcorns”, demonstrating a classic appearance of neovascular tufts as in human ROP³ (Figure 2D, red arrow heads). Rarely, was there a frond-like configuration of retinal neovascularization in rather small single isolated clumps (Figure 2D, red circle). It is reminiscent of sea-fan lesions described as atypical findings in human ROP and more commonly seen in sickle-cell retinopathy.¹¹ As the dye at the tip of vessels appears the brightest after the venous phase (D), it suggests these “sea-fan” and “popcorn” neovascularizations are arising from the venous side of the A-V anastomoses. Video S1 shows the FA taken from the affected eye.

FIGURE 2.

Fluorescein angiography. Snapshots of fluorescein angiography video of the affected eye at 31 weeks of age; time in the images are in seconds after the IV injection. (A) Choroidal flush and beginning of arterial phase (0:05–0:07): Dye is seen in the background resulting in patchy choroidal flush followed by filling of two retinal arterioles (white arrows). (B) Arterial phase (0:07–0:11): (Dye completely fills all the arterioles toward the periphery (yellow arrow) and stops at the vascular and non-vascular junction. (C) Arteriovenous phase (0:11–0:16): Small arterioles and less distinct capillary beds are completely filled, and retinal venules begin to be filled with the dye (blue arrow). (D) Venous phase (0:16-): Retinal veins are homogeneously filled and neovascular tufts are visible (red arrow heads “popcorn,” red circle “sea-fan” lesion). The yellow and blue arrows mark the same arteriole and venule in all the images

From the fundus photographs, it was not possible to differentiate retinal arterioles from venules due to vascular tortuosity and dilation. However, FA (Figure 2) allowed us to determine retinal arterioles (yellow arrow) filled during arterial phase (B) and retinal venules (blue arrow) which began to be filled during arteriovenous phase (C), and showed that they were equal in size or even larger than venules.

2.3 |. Spectral-domain optical coherence tomography (sdOCT) and confocal scanning laser ophthalmoscopy (cSLO)

Evaluation of the OCT images provided additional insight of the retinal vasculopathy. At 31 weeks of age, retinal imaging was performed under general anesthesia using confocal scanning laser ophthalmoscopy (cSLO) and high resolution retinal spectral-domain optical coherence tomography (sdOCT) (Spectralis™ HRA/OCT, Heidelberg Engineering) using methods previously described.¹² Overlapping (30° × 20°) raster scans were obtained with a 30° lens to cover the visible areas of the retina whose view was not obstructed by the fibrotic plaque at the posterior lens capsule in the affected eye.

The cSLO near-infrared composite image of the left eye demonstrates similar findings to the fundus photographs, such as dilated and tortuous retinal vessels, avascular zone, and the neovascular tufts (Figure 1C). OCT B-scans precisely capture cross-sectional views of the abnormal retinal vasculature. The features of stage 2 and stage 3 ROP in man are demonstrated in Figure 3A and 3B, respectively. The major difference between these stages is in the extent of proliferation.³ The proliferative tissues are preretinal (Figure 3A, white solid arrow), and the characteristics of “popcorn” neovascular tufts are commonly seen in stage 2 ROP. More extensive fibrovascular proliferation, stage 3 ROP in man, is demonstrated in Figure 3A and 3B (white dashed arrows) where it extends extraretinally into the vitreous; this appearance somewhat resembles sea fan, the marine invertebrate Gorgonia flabellum. Cross-sectional views of dilated and tortuous vessels, Plus disease in ROP, highlight the marked dilation of retinal vessels and their extension into the outer retina (Figure 3C, white arrows). In the contralateral unaffected eye, normal retinal vessels are present at the level of GCL/IPL (Figure 3D, white arrows). Lastly, a clear transition of avascular zone to vascular zone is confirmed with the absence of retinal vessels and presence of fibrovascular tissues, on the left and right sides of the arrow, respectively (Figure 3E). The rest of the retinal structure in the avascular zone is normal.

FIGURE 3.

OCT B-scans of the affected eye (A, B, C, E) and the contralateral unaffected eye (D) at 31 weeks of age (right panels). Corresponding images indicating the location of the B-scan displayed in the right panel (left panels, green arrow). Fibrovascular tufts are not limited to preretinal region (white solid arrow), but extend intravitreally (A and B, white dashed arrows). Marked dilation of retinal vessels taking up almost the full retinal thickness (C) in contrast to the contralateral unaffected eye (D, white arrow). A clear transition of vascular-avascular zone (E, white arrow) of peripheral retina is shown, left side of the arrow is avascular, and the right side is vascular with presence of preretinal and extraretinal fibrovascularization

2.4 |. Histology and immunohistochemistry

The ROP-like affected globe was collected following euthanasia (Euthasol^®, Virbac) at 23 months of age for an unrelated study, and fixed in alcoholic Bouin’s solution and transferred to 70% ethanol after 72 h before sectioning by Excalibur Pathology Inc. Sections were stained with H&E, or unstained sections were deparaffinized and rehydrated for immunohistochemistry using anti-Collagen IV (Milipore, cat# AB769; dilution 1:100) and anti-vWF (Agilent, cat# A0082, dilution 1:400) antibodies. Because alcoholic Bouin’s solution is not optimal for immunohistochemistry and works with only a few antibodies, we limited the immunoreactions to anti-vWF and anti-Collagen IV in order to show vessels in the vitreous, but not to do an exhaustive analysis of the neovascularization.

The conventional H&E-stained sections showed that abnormalities were limited to the retinal vasculature. Abnormal vessels were present at multiple sites intraretinally at the level of the nerve fiber layer compressing and distorting the inner nuclear layer (Figure 4A–C), and ectopic vessel-like structures were present within the vitreous (Figure 4D,E). Immunohistochemistry using basement membrane and endothelial vascular markers confirmed the intravitreal structures to be blood vessels (Figure 4F–K). Immunolabeling of retinal stress marker GFAP revealed inactive GFAP labeling along the inner retina (Figure 4L,N) with occasional areas of GFAP-negative segments in both the superior and inferior hemispheres (Figure 4O). Abnormal intraretinal vessels (Figure 4M) consistent with those observed histologically (Figure 4A–C) were found to compress the GFAP-positive layer. The histologic and immunohistochemical findings across the posterior eye cup support a diffuse vascular anomaly. The striking intravitreal neovascularization could not be demonstrated by histology as efffectively as by sdOCT, possibly due to globe processing artifacts and the orientation of sectioning.

FIGURE 4.

Histology and immunohistochemistry (IHC) of the affected retina. Representative H&E images of abnormal intraretinal vessels in the superior retina in areas with poor tapetal development (A–C). Abnormal retinal vessels that distort the inner nuclear layer (arrowheads) were found in multiple areas across the retinal section. Abnormal vessels within the vitreous body (D–K). These abnormal intravitreal structures have a basement membrane containing collagen IV (F, G) and vWF-positive vascular endothelium (H, I) consistent with blood vessels. The IHC images were merged with bright field micrograph for localization (J, K). Retinal vascular location and the expression of GFAP based on IHC of the retinal cross-section of the posterior eye cup using anti-collagen IV (red) and anti-GFAP (green) antibodies (L–O). Representative images of each region: an avascular area in the superior peripheral retina (L); an abnormal retinal blood vessels that distort the retinal layer structure (M, *); GFAP is expressed in the innermost layers of the retina in the area just superior to the ONH (N); a few GFAP-negative area (O) are scattered in across the affected retinal cross-section. #Area of artificial tissue collapse. INL, inner nuclear layer; NFL, nerve fiber layer; ONH, optic nerve head; ONL, outer nuclear layer

3 |. DISCUSSION

This case possesses many features not seen in a healthy canine retina, most notable being: avascular mid- and far-peripheral zones, dilated and tortuous retinal vessels and “popcorn” or “sea-fan” like neovascular tufts at the most peripheral vascular border. These lesions are the hallmarks of ROP in infants and are also reproducible in animal models of OIR.^5–9 The canine OIR model provides a useful tool for evaluation of retinal angiogenesis as the resultant pathology closely resembles human ROP. However, what sets our case apart from OIR is the lack of oxygen supplementation and the bilateral abnormalities seen in the experimental models. Oxygen supplementation is one of the major risk factors for development of ROP in human infants, yet in our case, oxygen was not administered and the vascular lesions were unilateral.

In man, the normal development of retinal vasculature occurs in utero and is achieved by “physiologic hypoxia” inducing physiologic angiogenesis. During normal vascular development, vascular endothelial growth factor (VEGF) is upregulated by physiologic hypoxia and plays an important role in both the vaso-obliterative (phase 1) and vaso-proliferative (phase 2) phases in ROP. Phase 1 begins when an infant is born prematurely. In response to a sudden change of oxygen state from hypoxia in utero to hyperoxia after birth, VEGF is down-regulated inhibiting further retinal vessel growth, but also constriction and obliteration of previously formed vessels.¹ The addition of oxygen supplementation results in marked exacerbation of this process. In Phase 1, an avascular zone in the peripheral retina can be observed in affected infants.¹

The vaso-proliferative phase is induced by “relative hypoxia” when adequate levels of oxygen are required to meet metabolic demand within the retina and ensure normal development. However, due to the avascular zone of the retina in Phase 1, there are not enough vessels present to deliver the required oxygen, which leads to “relative hypoxia.” In this Phase 2, the induced hypoxic state upregulates VEGF leading to neovascularization at the vascular-avascular junction of the retina.¹³ As the process of neovascularization progresses, the new vessels become fibrotic and extend into the vitreous chamber in the leading edge of the vascularization, which can eventually lead to retinal detachment. Additionally, hypoxia-induced dilation and tortuous vessels seen in this case are the hallmarks of the severe form of ROP in people, called Plus disease.¹⁴

ROP in humans are known to cause varying degrees of visual impairment.^1,3 The case presented herein was an RPGRIP1-mutant dog affected with cone-rod dystrophy/progressive retinal atrophy, with expected cone-led decline in retinal function recorded by full-field ERG using methods previously described custom built Ganzfeld dome and LED stimuli of a ColorDome stimulator from Diagnosys).¹² Notably, there was no significant difference in scotopic or photopic ERG responses between the two eyes with or without ROP-like vasculopathy (Figure S1), indicating that the abnormal vasculature did not negatively impact overall retinal function. Since the ERG records responses from the photoreceptors and inner nuclear layer neurons, vascular abnormalities of the inner retina would not be expected to have an effect.

Unfortunately, there is no clear mechanism to support ROP-like vasculopathy in our case without the use of oxygen supplementation and lack of bilateral involvement. The unilateral nature of this case suspects local activity caused by, for example, anatomic local anomalies, rather systemic effect. “Sudden onset “of hypoxia is the key for development of vascular pathology in OIR.¹⁵ Nevertheless, it is important to appreciate that the abnormal vascular pattern seen in ROP in premature infants can occur in the dog even without oxygen administration and suggest there may be other factors at play.