An unusual inherited electroretinogram feature with an exaggerated negative component in dogs.

OBJECTIVES: To assess an inherited abnormal negative response electroretinogram (NRE) that originated in a family of Papillon dogs. ANIMALS STUDIED: Thirty-eight dogs (Papillons, or Papillon cross Beagles or Beagles). PROCEDURES: Dogs underwent routine ophthalmic examination and a detailed dark-adapted, light-adapted and On-Off electroretinographic study. Vision was assessed using a four-choice exit device. Spectral-domain optical coherence tomography (SD-OCT) was performed on a subset of dogs. Two affected males were outcrossed to investigate the mode of inheritance of the phenotype. RESULTS: The affected dogs had an increased underlying negative component to the ERG. This was most pronounced in the light-adapted ERG, resulting in a reduced b-wave and an exaggerated photopic negative response (PhNR). Changes were more pronounced with stronger flashes. Similarly, the On-response of the On-Off ERG had a reduced b-wave and a large post-b-wave negative component. The dark-adapted ERG had a significant increase in the scotopic threshold response (STR) and a significant reduction in the b:a-wave ratio. Significant changes could be detected at 2 months of age but became more pronounced with age. Vision testing using a four-choice device showed affected dogs had reduced visual performance under the brightest light condition. There was no evidence of a degenerative process in the affected dogs up to 8.5 years of age. Test breeding results suggested the NRE phenotype had an autosomal dominant mode of inheritance. CONCLUSIONS: We describe an inherited ERG phenotype in Papillon dogs characterized by an underlying negative component affecting both dark- and light-adapted ERG responses.

Phenotypic characterization of complete CSNB in the inbred research beagle: how common is CSNB in research and companion dogs?

PURPOSE: Although congenital stationary night blindness (CSNB) has been described in a Japanese beagle dog research colony, certain clinical correlates with human CSNB have not yet been described, nor has an estimate of frequency of the condition been made in inbred and outbred beagle populations. METHODS: A beagle with CSNB obtained from a commercial research dog supplier in the USA and matched control dogs (n = 3) underwent examination, refraction, ocular imaging, assessment of visual navigation ability and detailed electroretinography (ERG). Retrospective review of ERGs in two independent groups of inbred (n = 15 and 537, respectively) and one group of outbred dogs (n = 36) was used to estimate CSNB frequency in these populations. RESULTS: In the affected dog, there were absent dark-adapted b-waves in response to dim-light flashes, severely reduced dark-adapted b-waves in response to bright-light flashes, and normal light-adapted b-waves with a-waves that had broadened troughs. Long-flash ERGs confirmed a markedly reduced b-wave with a preserved d-wave, consistent with cone ON-bipolar cell dysfunction. There was evidence of normal rod photoreceptor a-wave dark adaptation, and rapid light adaptation. In the wider beagle populations, five inbred beagles had a b/a wave ratio of < 1 in dark-adapted bright-flash ERG, whereas no outbred beagles had ERGs consistent with CSNB. CONCLUSIONS: The identified dog had clinical findings consistent with complete type CSNB, similar to that described in the Japanese colony. CSNB appears to be a rare disorder in the wider beagle population, although its detection could confound studies that use retinal function as an outcome measure in research dogs, necessitating careful baseline studies to be performed prior to experimentation.

Gene Therapy in a Large Animal Model of PDE6A-Retinitis Pigmentosa.

Despite mutations in the rod phosphodiesterase 6-alpha (PDE6A) gene being well-recognized as a cause of human retinitis pigmentosa, no definitive treatments have been developed to treat this blinding disease. We performed a trial of retinal gene augmentation in the Pde6a mutant dog using Pde6a delivery by capsid-mutant adeno-associated virus serotype 8, previously shown to have a rapid onset of transgene expression in the canine retina. Subretinal injections were performed in 10 dogs at 29-44 days of age, and electroretinography and vision testing were performed to assess functional outcome. Retinal structure was assessed using color fundus photography, spectral domain optical coherence tomography, and histology. Immunohistochemistry was performed to examine transgene expression and expression of other retinal genes. Treatment resulted in improvement in dim light vision and evidence of rod function on electroretinographic examination. Photoreceptor layer thickness in the treated area was preserved compared with the contralateral control vector treated or uninjected eye. Improved rod and cone photoreceptor survival, rhodopsin localization, cyclic GMP levels and bipolar cell dendrite distribution was observed in treated areas. Some adverse effects including foci of retinal separation, foci of retinal degeneration and rosette formation were identified in both AAV-Pde6a and control vector injected regions. This is the first description of successful gene augmentation for Pde6a retinitis pigmentosa in a large animal model. Further studies will be necessary to optimize visual outcomes and minimize complications before translation to human studies.

Early-Onset Progressive Degeneration of the Area Centralis in RPE65-Deficient Dogs.

Purpose: Retinal epithelium-specific protein 65 kDa (RPE65)-deficient dogs are a valuable large animal model species that have been used to refine gene augmentation therapy for Leber congenital amaurosis type-2 (LCA2). Previous studies have suggested that retinal degeneration in the dog model is slower than that observed in humans. However, the area centralis of the dog retina is a cone and rod photoreceptor rich region comparable to the human macula, and the effect of RPE65 deficiency specifically on this retinal region, important for high acuity vision, has not previously been reported. Methods: Spectral-domain optical coherence tomography, fundus photography, and immunohistochemistry of retinal wholemounts and sagittal frozen sections were used to define the time-course and cell-types affected in degeneration of the area centralis in affected dogs. Results: Area centralis photoreceptor degeneration was evident from 6 weeks of age, and progressed to involve the inner retina. Immunohistochemistry showed that RPE65-deficient dogs developed early loss of S-cone outer segments, with slower loss of L/M-cone outer segments and rods. Conclusions: Early-onset severe photoreceptor degeneration in the area centralis of dogs with RPE65-deficiency offers a model of the early foveal/perifoveal degeneration in some patients with LCA2. This model could be used to refine interventions aiming to improve function and halt the progression of foveal/perifoveal photoreceptor degeneration.

Cyclosporine A prevents ex vivo PCO formation through induction of autophagy-mediated cell death.

The purpose of this study was to determine the Cyclosporine A (CsA) dose and minimum drug delivery time needed to prevent posterior capsule opacification (PCO) in an ex vivo canine model and evaluate the mechanism of CsA-induced cell death. Canine lens epithelial cells (LEC) were treated with CsA and changes in cell migration, proliferation, and density were monitored over time. CsA-treated LEC underwent transmission electron microscopy (TEM), immunofluorescence, and immunoblotting in the presence or absence of autophagy inhibitors to evaluate the mechanism of cell death. Lens capsules were harvested from canine cadaver eyes for an ex vivo model of PCO. Lens capsules were treated with CsA for 1, 2, 3, 4, 5, 6, or 7 days, and subsequently maintained in culture for a total of 28 days in the absence of drug. CsA reduced LEC viability in a dose dependent manner. Morphologically, CsA-treated LEC were swollen, had intact nuclei, lacked peripheral chromatin condensation, and demonstrated prominent vacuolization; TEM revealed autophagosomes. LC3-II protein expression and acridine orange fluorescence increased in CsA-treated cells. A small non-significant induction of cleaved caspase-3 was observed in CsA-treated LEC. Lens capsules treated with 5, 6, or 7 days of 10 µg/mL CsA showed a significant decrease in ex vivo PCO formation; 6 days of drug delivery prevented PCO. This study finds that morphologic changes, formation of acidic vesicles, and increased expression of LC3-II supports the hypothesis that CsA mediates LEC death via autophagy; this is a novel finding in the lens. Induction of CsA-induced apoptosis was minimal. Six days of intracapsular CsA drug delivery prevented ex vivo PCO formation.

Acute primary canine herpesvirus-1 dendritic ulcerative keratitis in an adult dog.

We present a report of dendritic ulcerative keratitis in a 4-year old locally immunosuppressed dog suspected to result from acute primary canine herpesvirus-1 (CHV-1) infection. The dog was presented for evaluation of mild blepharospasm and conjunctival hyperemia in the right eye (OD) shortly after attending a public boarding facility. For approximately 3 months, the dog had been receiving topical prednisolone acetate 1.0% and tacrolimus 0.02% in both eyes (OU) q12h for treatment of follicular conjunctivitis. Ophthalmic examination revealed three regions of corneal fluorescein retention OD. The lesions had a dendritic pattern, were approximately 2-3 mm in length, and were located at the dorsomedial, lateral, and ventromedial aspects of the cornea. No additional abnormalities were noted on complete ophthalmic and physical examinations. CHV-1 was identified in conjunctival samples OD by polymerase chain reaction, and paired CHV-1 serum virus neutralization antibody titers were positive and consistent with acute infection. Topical prednisolone acetate and tacrolimus were discontinued. The dog was treated with cidofovir 0.5% OU q12h for a period of 4 weeks, with resolution of corneal disease noted within 1 week of treatment. In conjunction with previous studies, this case report supports a central role for alterations in host immune status in the pathogenesis and clinical manifestations of CHV-1 ocular disease in dogs.

Environmental enrichment delays pup-induced maternal behavior in rats.

Adult, virgin rats do not spontaneously display maternal behavior when exposed to foster pups. However, continuous daily exposure of the female to foster pups for about 5-7 days can induce a set of maternal behaviors similar to those shown by postpartum dams. Induction latencies depend upon a number of factors, including the stress and anxiety levels of the female. The goal of this study was to attempt to mitigate the likely stressfulness of being singly housed during testing by enriching the rat's home cage environment and to determine if the concomitant environmental change would alter the latency to express maternal behavior. In addition, the effect of varying the number of test pups used for testing was examined. Two groups of virgin Sprague-Dawley rats were first tested on the elevated plus maze after 1 week of exposure to either control (standard housing) or enriched conditions. One week later, maternal behavior testing began using one or three pups. Upon completion of maternal behavior testing, plasma corticosterone concentrations were determined following a mild stressor. The data indicate that enrichment tends to increase anxiety-like behaviors in the elevated plus maze. In addition, enrichment delayed the onset of maternal behavior irrespective of the number of test pups. There were no effects of environmental enrichment on plasma corticosterone levels following exposure to a stressor. These results indicate that what is considered a modestly enriched environment delays the expression of pup-oriented responses and does not apparently reduce stress or improve performance on all behavioral tasks.