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Purpose: Many older dogs (Canis lupus familiaris) develop cognitive impairment. Dog owners often describe impairments in multiple sensory functions, yet the relationships between sensory and cognitive function in older dogs is not well understood., Methods: We performed assessments of dog vision and hearing, both clinically (n = 91, electroretinography and brainstem auditory evoked potential) and via validated questionnaire (n = 238). We determined prevalence of sole and dual hearing/vision impairments in younger (<8 years) and older (≥8 years) dogs. Impairment cutoffs were determined using data from young dogs. We assessed the relationships between questionnaire-assessed vision and/or hearing impairments and cognitive impairment using logistic regression., Results: Younger and older dog groups had similar distributions of sex and purebred/mixed breed status. Sex had no relationship to prevalence of sensory impairments. Older dogs had higher prevalence of hearing, vision, and dual sensory impairments, assessed both clinically and by questionnaire (P<0.001), and cognitive impairment assessed by questionnaire (P<0.001). Dogs had higher prevalence of reported cognitive impairment when owners reported dual vision and hearing impairments (79-94%, versus 25-27% in dogs with no sensory impairments), which was most consistent in dogs aged ≥8 years. In these older dogs, dual vision/hearing impairments were associated with a significantly increased risk of cognitive impairment (1.8-2.0 odds ratio)., Conclusion: Dogs aged ≥8 years are at higher risk for dual hearing/vision impairments and associated cognitive impairments. The causal relationship between these impairments is not defined, but clinical consideration of these multimorbidity risks should be made in older dogs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hopper et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The dog retina contains a central macula-like region, and there are reports of central retinal disorders in dogs with shared genetic etiologies with humans. Defining central/peripheral gene expression profiles may provide insight into the suitability of dogs as models for human disorders. We determined central/peripheral posterior eye gene expression profiles in dogs and interrogated inherited retinal and macular disease-associated genes for differential expression between central and peripheral regions. Bulk tissue RNA sequencing was performed on 8 mm samples of the dog central and superior peripheral regions, sampling retina and retinal pigmented epithelium/choroid separately. Reads were mapped to CanFam3.1, read counts were analyzed to determine significantly differentially expressed genes (DEGs). A similar analytic pipeline was used with a published bulk-tissue RNA sequencing human dataset. Pathways and processes involved in significantly DEGs were identified (Database for Annotation, Visualization and Integrated Discovery). Dogs and humans shared the extent and direction of central retinal differential gene expression, with multiple shared biological pathways implicated in differential expression. Many genes implicated in heritable retinal disorders in dogs and humans were differentially expressed between central and periphery. Approximately half of genes associated with human age-related macular degeneration were differentially expressed in human and dog tissues. We have identified similarities and differences in central/peripheral gene expression profiles between dogs and humans which can be applied to further define the relevance of dogs as models for human retinal disorders., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Companion animals, namely dogs, cats, and horses, can be affected with many forms of hereditary retinal disease. The number of such diseases characterized in the last decade has increased substantially, and nomenclature is nonstandardized, heterogenous, and confusing. We provide in this viewpoint article consensus guidelines for naming of companion animal hereditary retinal diseases, either prospectively or retrospectively. These consensus guidelines have been developed with the purpose of standardizing nomenclature. We provide examples for the iterative nomenclature process and a comprehensive File S1 on proposed renaming of previously described diseases., (© 2024 The Authors. Veterinary Ophthalmology published by Wiley Periodicals LLC on behalf of American College of Veterinary Ophthalmologists.)

Despite availability of video content marketed for dog ( Canis familiaris ) entertainment, there is little information on dog behaviors when viewing content, nor describing which content is engaging. The aims of this study were to define demographics of dogs that engage with screens, owner observed behaviors, and perceived content interest. A digital survey was distributed to dog owners (03/2022-03/2023). We collected demographics, home environment, owner-rated behaviors, content interest, and interest in 4 presented videos. We compared the representation of dogs from different purebred dog groups (categorized by job/purpose by the American Kennel Club) with the estimated general purebred dog population. Most respondents (total n=1,246) lived in the USA (89%). Median age was 4 years, 54% were purebred, 51% were female. Most (86%, n=1,077) stated their dog watched screen content. Excitement behaviors were often described: 78% of dogs approached the screen, 76% vocalized. Many owners played videos for their dogs when left alone. Dogs most frequently engaged with animal content; dogs were the most popular animal. Age and visual status influenced the frequency of perceived interaction; age and breed influenced content interest. Within purebred dogs that were stated to watch content, there was a relative over-representation of "sporting" and "herding"-type breeds. A dog's age, visual status, and breed type may influence their interest in video content at home. Because many owners reported excitement in their dogs in reaction to screen content, owners may wish to determine whether video content would be suitable for use when their dogs are left alone., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Objective: To evaluate the relationship between nuclear sclerosis (NS) and refractive error in companion dogs., Animals Studied: One hundred and eighteen companion dogs., Procedures: Dogs were examined and found to be free of significant ocular abnormalities aside from NS. NS was graded from 0 (absent) to 3 (severe) using a scale developed by the investigators. Manual refraction was performed. The effect of NS grade on refractive error was measured using a linear mixed effects analysis adjusted for age. The proportion of eyes with >1.5 D myopia in each NS grade was evaluated using a chi-square test. Visual impairment score (VIS) was obtained for a subset of dogs and compared against age, refractive error, and NS grade., Results: Age was strongly correlated with NS grade (p < .0001). Age-adjusted analysis of NS grade relative to refraction showed a mild but not statistically significant increase in myopia with increasing NS grade, with eyes with grade 3 NS averaging 0.58-0.88 D greater myopia than eyes without NS. However, the myopia of >1.5 D was documented in 4/58 (6.9%) eyes with grade 0 NS, 12/91 (13.2%) eyes with grade 1 NS, 13/57 (22.8%) eyes with grade 2 NS, and 7/23 (30.4%) eyes with grade 3 NS. Risk of myopia >1.5 D was significantly associated with increasing NS grade (p = .02). VIS was associated weakly with refractive error, moderately with age, and significantly with NS grade., Conclusions: NS is associated with visual deficits in some dogs but is only weakly associated with myopia. More work is needed to characterize vision in aging dogs., (© 2023 American College of Veterinary Ophthalmologists.)

Introduction: Dim light vision as assessed by proxy and clinical tools is commonly impaired in older humans and impacts quality of life. Although proxy visual assessment tools have been developed for dogs, it is unclear if they are sensitive enough to detect subtle visual dysfunction in older dogs. We sought to determine if a newly designed proxy visual function questionnaire could detect age-associated differences in visual behaviors in varying lighting conditions in dogs., Methods: A 27-item questionnaire (the dog variable lighting questionnaire, dogVLQ) was designed to assess visual behavior in dogs in different lighting settings. We conducted the dogVLQ, a previously validated visual function questionnaire the dog vision impairment score and performed light- and dark-adapted electroretinography (ERG) on a subset of dogs. Questionnaire scores were analyzed for dog age associations using correlation analysis., Results: Questionnaire responses from 235 dog owners were obtained (122 female, 112 male dogs), 79 of which underwent ERG (43 female, 36 male dogs). Bright light visual behavior was significantly associated with light-adapted bright flash ERG amplitudes, visual behavior in near darkness was associated with dark-adapted ERG amplitudes. The dogVLQ identified worse vision in older dogs in bright light, dim light, and darkness; predicted onset was younger for vision in near darkness. Older dogs had more difficulty navigating transitions between lighting conditions., Discussion: Subjective dog owner assessment of visual function associates with objective measurement of retinal function in dogs and supports reduced vision-mediated behaviors in older dogs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rogers, Salzman, Li, Merten, Russell, Lillesand and Mowat.)

Purpose: To determine the association between age and retinal full-field electroretinographic (ERG) measures in companion (pet) dogs, an important translational model species for human neurologic aging., Methods: Healthy adult dogs with no significant ophthalmic abnormalities were included. Unilateral full-field light- and dark-adapted electroretinography was performed using a handheld device, with mydriasis and topical anaesthesia. Partial least squares effect screening analysis was performed to determine the effect of age, sex, body weight and use of anxiolytic medication on log-transformed ERG peak times and amplitudes; age and anxiolytic usage had significant effects on multiple ERG outcomes. Mixed model analysis was performed on data from dogs not receiving anxiolytic medications., Results: In dogs not receiving anxiolytics, median age was 118 months (interquartile range 72-140 months, n = 77, 44 purebred, 33 mixed breed dogs). Age was significantly associated with prolonged peak times of a-waves (dark-adapted 3 and 10 cds/m 2 flash p < 0.0001) and b-waves (cone flicker p = 0.03, dark-adapted 0.01 cds/m 2 flash p = 0.001). Age was also significantly associated with reduced amplitudes of a-waves (dark-adapted 3 cds/m 2 flash p < 0.0001, 10 cds/m 2 flash p = 0.005) and b-waves (light-adapted 3 cds/m 2 flash p < 0.0001, dark-adapted 0.01 cds/m 2 flash p = 0.0004, 3 cds/m 2 flash p < 0.0001, 10 cds/m 2 flash p = 0.007) and flicker (light-adapted 30 Hz 3 cds/m 2 p = 0.0004). Within the Golden Retriever breed, these trends were matched in a cross-sectional analysis of 6 individuals that received no anxiolytic medication., Conclusions: Aged companion dogs have slower and reduced amplitude responses in both rod- and cone-mediated ERG. Consideration of anxiolytic medication use should be made when conducting ERG studies in dogs., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Sudden acquired retinal degeneration syndrome (SARDS) is a common cause of irreversible blindness in dogs. It bears clinical resemblance to hypercortisolism, which can be associated with hypercoagulability. The role of hypercoagulability in dogs with SARDS is unknown., Objective: Determine hemostatic profiles in dogs with SARDS., Animals: Prospective pilot study: Dogs with a history of SARDS (n = 12). Prospective case-control study: Dogs with recent onset of SARDS (n = 7) and age-, breed-, and sex-matched controls (n = 7)., Methods: Prospective pilot study: We performed thromboelastography (TEG). Prospective case-control study: Dogs had CBC, serum biochemistry, urinalysis, TEG, fibrinogen concentration, antithrombin activity, D-dimers, thrombin-antithrombin complexes, and optical platelet aggregometry performed., Results: Prospective pilot study: 9/12 dogs with a history of SARDS were hypercoagulable with increased TEG G value and 2/3 had hyperfibrinogenemia. Case-control study: All dogs with SARDS and 5/7 controls were hypercoagulable based on TEG G value. Dogs with SARDS had significantly higher G values (median, 12.7 kdynes/s; range, 11.2-25.4; P = .04) and plasma fibrinogen concentration (median, 463 mg/dL; range, 391-680; P < .001) compared to controls., Conclusions and Clinical Importance: Hypercoagulability was common in both dogs with SARDS and controls, but dogs with SARDS were significantly more hypercoagulable on TEG. The role of hypercoagulability in the pathogenesis of SARDS remains to be determined., (© 2023 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Human age-related macular degeneration (AMD) is a prevalent age-related disease which causes retinal dysfunction and disability. Genetic and cell culture studies from AMD patients have implicated impaired activity of peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1α). PGC-1α is a transcriptional co-regulator that acts to control a plethora of metabolic processes relevant to AMD pathophysiology including gluconeogenesis, oxidative phosphorylation, and response to oxidative injury. Perturbation of PGC-1α activity in mice causes AMD-like RPE and retinal pathology. There is potential for therapeutic modulation of the PGC-1α pathway in AMD treatment., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

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., (© 2022 The Authors. Veterinary Ophthalmology published by Wiley Periodicals LLC on behalf of American College of Veterinary Ophthalmologists.)

Background: Aging dogs may suffer from canine cognitive dysfunction syndrome (CCDS), a condition in which cognitive decline is associated with amyloid pathology and cortical atrophy. Presumptive diagnosis is made through physical examination, exclusion of systemic/metabolic conditions, and completion of screening questionnaires by owners., Objective: This study aimed to determine whether cognitive function could be quantified in aging pet dogs, and to correlate cognitive testing with validated questionnaires and plasma neurofilament light chain (pNfL) concentration., Methods: Thirty-nine dogs from fifteen breeds were recruited (9.3 to 15.3 years). Owners completed the Canine Dementia Scale (CADES) and Canine Cognitive Dysfunction Rating scale (CCDR). Executive control and social cues were tested, and pNfL was measured with single molecule array assay. Comparisons were made between cognitive testing scores, CADES, CCDR scores, and pNfL., Results: CADES scoring classified five dogs as severe CCDS, six as moderate, ten as mild, and eighteen as normal. CCDR identified seven dogs at risk of CCDS and thirty-two as normal. Cognitive testing was possible in the majority of dogs, although severely affected dogs were unable to learn tasks. CADES score correlated with sustained attention duration (r = -0.47, p = 0.002), inhibitory control (r = -0.51, p = 0.002), detour (r = -0.43, p = 0.001), and pNfL (r = 0.41, p = 0.025). Concentration of pNfL correlated with inhibitory control (r = -0.7, p≤0.001). The CCDR scale correlated with performance on inhibitory control (r = -0.46, p = 0.005)., Conclusion: Our findings suggest that a multi-dimensional approach using a combination of questionnaires, specific cognitive tests, and pNfL concentration can be used to quantify cognitive decline in aging pet dogs.

The distribution of cone photoreceptor subtypes (important for color vision and vision quality) varies widely in different carnivore species, but there have been limited studies on bear (ursid) cone distribution. A previous behavioral study suggests that American black bears (Ursus americanus) are dichromatic, indicating that they possess two cone subtypes, although the retinal distribution of cones is unknown. The purpose of this study was to examine the subtype and topography of cones in American black bear retinas to further predict the nature of their color vision and image resolution. We studied 10 eyes from seven individual legally hunted black bears in northeastern North Carolina. Cryosections and retinal wholemounts were labeled using antibodies targeting two cone opsin subtypes: long/medium (L/M) wavelength sensitive and short (S) wavelength sensitive. Cones in fluorescent microscopy images were counted and density maps were created for retinal wholemounts. The black bear retina contains both cone subtypes and L/M cones outnumber S cones by at least 3:1, a finding confirmed in retinal frozen sections. There are higher concentrations of S cones present than typically seen in other carnivores with some evidence for co-expression of L/M and S cones. A cone-dense area centralis is present dorsotemporal to the optic nerve, similar to other carnivores. These results confirm that American black bears are predicted to have a dichromatic vision with high acuity indicated by the presence of a dorsotemporally located area centralis., (© 2020 American Association for Anatomy.)

Background: Canine sudden acquired retinal degeneration syndrome (SARDS) causes blindness for which there are no proven effective treatments. We aimed to clarify the time to vision loss, treatment response/side effects, and prognosis for life in dogs with SARDS., Methods: An online questionnaire was administered to owners of dogs with a historical diagnosis of SARDS. Mortality data were compared with a published purebred reference population. Select parameters were analyzed statistically using general linear model with least square means, two-sample t tests, and chi-squared or Fisher's exact tests., Results: Responses from owners that stated that their dog visited an ophthalmologist and had electroretinography performed (n = 434) were analyzed. The majority of owners (65.4%) reported the time from vision disturbance to complete vision loss as <2 weeks; 19.4% reported >4 weeks. Onset of systemic clinical signs to complete vision loss was >4 weeks in 44.5% of responses. A higher proportion of owners reported some vision recovery with combination treatment (14.4%) compared with monotherapy (3.2%, P = .0004). Side effects of treatment were commonly reported. Dogs with SARDS did not have a shorter lifespan than the reference population but had higher incidence of kidney disease (P = .0001) and respiratory disease (P = .0004) at death., Conclusions: Dogs with SARDS have a rapid onset of vision loss. In the owner's opinion, treatment is unlikely to restore vision and is associated with systemic side effects. The potential for systemic pathologies that arise after SARDS diagnosis warrants further study., (© 2020 American College of Veterinary Ophthalmologists.)

Longevity-associated neurological disorders have been observed across human and canine aging populations. Alzheimer's disease (AD) and canine cognitive dysfunction syndrome (CDS) represent comparable diseases affecting both species as they age. Translational diagnostic and therapeutic research is needed for these incurable diseases. The amyloid β (Aβ) peptide family are AD-associated peptides with identical amino acid sequences between dogs and humans. Plasma Aβ42 concentration increases with age and decreases with AD in humans, and cerebrospinal fluid (CSF) concentration decreases in AD and correlates inversely with the amyloid load within the brain. Similarly, CSF Aβ42 concentrations decrease in dogs with CDS but there is limited and conflicting information on plasma Aβ42 concentrations in aging dogs and dogs with CDS. We measured plasma concentrations of Aβ42 and Aβ40 with an ultrasensitive single-molecule array assay (SIMOA) in a population of healthy aging dogs of different life stages (n = 36) and dogs affected with CDS (n = 11). In addition, the ratio of Aβ42/β40 was calculated. The mean plasma concentrations of Aβ42 and Aβ40 increased significantly with age (r 2 = 0.27, p = 0.001; and r 2 = 0.42, p < 0.001, respectively) and with life stage: puppy/junior group (0.43-2 years): 1.23 ± 0.95 and 38.26 ± 49.43 pg/mL; adult/mature group (2.1-9 years): 10.99 ± 5.45 and 131.05 ± 80.17 pg/mL; geriatric/senior group (9.3-14.5 years): 18.65 ± 16.65 and 192.88 ± 146.38 pg/mL, respectively. Concentrations of Aβ42 and Aβ40 in dogs with CDS (11.0-15.6 years) were significantly lower than age-matched healthy dogs at 11.61 ± 6.39 and 150.23 ± 98.2 pg/mL (p = 0.0048 and p = 0.001), respectively. Our findings suggest the dynamics of canine plasma amyloid concentrations are analogous to that found in aging humans with and without AD.

The Rpe65-deficient dog has been important for development of translational therapies of Leber congenital amaurosis type 2 (LCA2). The purpose of this study was to provide a comprehensive report of the natural history of retinal changes in this dog model. Rpe65-deficient dogs from 2 months to 10 years of age were assessed by fundus imaging, electroretinography (ERG) and vision testing (VT). Changes in retinal layer thickness were assessed by optical coherence tomography and on plastic retinal sections. ERG showed marked loss of retinal sensitivity, with amplitudes declining with age. Retinal thinning initially developed in the area centralis , with a slower thinning of the outer retina in other areas starting with the inferior retina. VT showed that dogs of all ages performed well in bright light, while at lower light levels they were blind. Retinal pigment epithelial (RPE) inclusions developed and in younger dogs and increased in size with age. The loss of photoreceptors was mirrored by a decline in ERG amplitudes. The slow degeneration meant that sufficient photoreceptors, albeit very desensitized, remained to allow for residual bright light vision in older dogs. This study shows the natural history of the Rpe65-deficient dog model of LCA2.

Objective: A previously published study successfully isolated photoreceptor responses from canine rods, long/medium-wavelength (L/M) cones, and short-wavelength (S) cones using silent substitution electroretinography (ERG) performed under general anesthesia. We hypothesized that responses would be similar in dogs under sedation and that a curtailed protocol suitable for use in clinical patients could effectively isolate responses from all three photoreceptor subtypes., Animals Studied: Three normal adult purpose-bred beagles (2 females and 1 male)., Methods: Dogs were dark-adapted for 1 hour. Sine wave color stimuli were delivered using LEDs in a Ganzfeld dome. The ERG protocol under anesthesia was performed as previously published; sedated ERG protocols were performed after a 3-day washout period. Intravenous sedation (dexmedetomidine 1.25 mcg/kg, butorphanol 0.1 mg/kg) was administered for sedation. Statistical analysis was performed using two-way repeated-measures ANOVA and linear regression., Results: In both anesthetized and sedated animals, rod-derived responses peaked at low frequency (4-12 Hz), L/M-cone responses peaked at high frequency (32-38 Hz), and S-cone responses peaked at low frequency (4-12 Hz). The frequencies eliciting maximal responses were similar in sedated and anesthetized protocols, although rod amplitudes were significantly higher in the sedated protocols compared with anesthetized (P < .001)., Conclusion: We present a clinically applicable method to consistently isolate rod and cone subclass function in sedated dogs. This may allow detailed evaluation of photoreceptor function in clinical patients with rod or cone subclass deficits without the need for general anesthesia or protracted adaptation times., (© 2020 American College of Veterinary Ophthalmologists.)

The red wolf ( Canis rufus ) is a critically endangered North American canid, with surviving conspecifics divided between a captive breeding population and a reintroduced free-ranging population. The goal of this study was to assess the prevalence of selected vector-borne pathogens in captive red wolves. Whole blood samples were collected from 35 captive red wolves. Quantitative polymerase chain reaction (PCR) assays were performed on extracted DNA to identify infection by Trypanosoma cruzi and vector-borne organisms within the following genera: Anaplasma , Babesia , Bartonella , Ehrlichia , Mycoplasma , Neoehrlichia , Neorickettsia, and Rickettsia . All red wolves sampled were PCR-negative for all tested organisms. These pathogens are unlikely to constitute threats to red wolf conservation and breeding efforts under current captive management conditions. The results of this study establish a baseline that may facilitate ongoing disease monitoring in this species.

Age is a primary risk factor for multiple comorbidities including neurodegenerative diseases. Pet dogs and humans represent two populations that have experienced a significant increase in average life expectancy over the last century. A higher prevalence of age-related neurodegenerative diseases has been observed across both species, and human diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), have canine analogs, canine cognitive dysfunction (CCD), and degenerative myelopathy (DM) respectively. In humans, protein biomarkers have proved useful in the prediction and diagnosis of neurodegeneration. Molecular signatures of many proteins are highly conserved across species. In this study, we explored the potential of the neuronal cytoskeletal protein neurofilament light chain (NfL) as a biomarker of neuro-aging in dogs using an ultrasensitive single-molecule array assay to measure plasma concentrations. Healthy dogs of different ages and dogs affected with CCD and DM were evaluated. The mean plasma NfL concentrations in the different age groups of the healthy population were as follows: 4.55 ± 1.70 pg/mL in puppy/junior group (0.43-2 years), 13.51 ± 6.8 pg/mL in adult/mature group (2.1-9 years), and 47.1 ± 12.68 pg/mL in geriatric/senior group (9.3-14.5 years). Concentrations in dogs with DM (7.5-12.6 years) and CCD (11.0-15.6 years) were 84.17 ± 53.57 pg/mL and 100.73 ± 83.72 pg/mL, respectively. Plasma NfL increases in an age-dependent manner and is significantly elevated in dogs diagnosed with neurodegenerative disease. This work identified plasma NfL as a key clinical index of neuro-aging and neurodegeneration in pet dogs. Our findings mirror recent reports from human neurodegenerative diseases.

Sudden acquired retinal degeneration syndrome (SARDS) in dogs is proposed to have an immune-mediated etiology. However, there is conflicting evidence regarding the presence of antiretinal antibodies, as assessed by western blotting, in the serum of SARDS patients. Because of the possibility that antibodies recognize only conformational epitopes, we hypothesized that a more sensitive method to investigate circulating retinal autoantibodies in SARDS is immunofluorescence. Sera from 14 dogs with early SARDS, and 14 age- and breed-matched healthy control dogs were screened for circulating antiretinal IgG, IgM, IgE and IgA using indirect immunofluorescence on lightly fixed frozen sections of normal canine retina. Controls without canine serum were also performed. A nuclear counterstain was used to identify cellular retinal layers. Images were obtained using a fluorescence microscope, and 2-3 separate masked observers graded retinal layers for fluorescence staining intensity using a 0-3 scale. Total circulating IgG and IgM was assessed by radial immunodiffusion. Statistical analysis was performed using 2-way ANOVA, paired 2-tailed student's t-test and correlation analysis. Intensity of IgG staining of photoreceptor outer segments was significantly higher using serum from dogs with SARDS compared with healthy controls in 2/3 observers (P < 0.05). Intensity of IgM staining throughout the retina was higher in SARDS dogs compared to matched healthy controls (P < 0.0001), although no specific retinal layer was statistically significant. There were no differences in staining intensity for IgE or IgA. Dogs with SARDS had a comparably lower circulating IgG and higher IgM than healthy controls (P = 0.01 and 0.001 respectively) and IgG and IgM were negatively correlated (r = -0.69, P = 0.007). Despite having decreased serum IgG compared with healthy controls, circulating IgG in dogs with SARDS binds photoreceptor outer segments to a greater extent. Dogs with SARDS have a relatively higher circulating IgM than matched healthy controls. The pathogenic nature of these antibodies is unknown., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ophthalmoscopy is an important examination technique in the diagnosis of disease. Although it is difficult to learn, practice increases confidence and proficiency. Practicing ophthalmoscopy on live animals presents an additional level of complexity, so we sought to evaluate how students would respond to practicing ophthalmoscopy on an ocular fundus model. We constructed a simple and inexpensive model and allowed half of the students (49/100) in a first-year veterinary medicine class to practice ophthalmoscopy (direct, PanOptic, and indirect) for 20 minutes using the model. Students completed a questionnaire regarding ease of use, enjoyment, and recommendations for future use of the model immediately after the practice session. Six weeks later, we tested students' ability to correctly match a fundus to a photograph using indirect ophthalmoscopy. All students who used the model rated it as 'easy' or 'somewhat easy' to use. All students reported that they 'enjoyed' (93.9%) or 'somewhat enjoyed' (6.1%) using the model. Also, all students who used the model stated the models should continue to be used to aid student learning. Students who used the model were significantly more likely ( p = .013) to correctly match a fundus photograph to the fundus being observed than students who had not used the model. These findings demonstrate that the model used in this study is well received by students and results in discernible gains in proficiency.

Background: Sudden acquired retinal degeneration syndrome (SARDS) has clinical similarity to pituitary-dependent hypercortisolism (PDH) in dogs. Some studies have identified a greater frequency of SARDS in seasons with reduced daylight hours. Neurohormone imbalances contribute to retinal lesions in other species, warranting further study in dogs with SARDS., Hypothesis: Dysregulation of circulating melatonin concentration is present in dogs with SARDS but not in dogs with PDH., Animals: Fifteen client-owned dogs with spontaneous SARDS (median time of vision loss 18 days), 14 normal dogs, and 13 dogs with confirmed PDH., Procedures: Prospective case-control study. ELISA on samples (obtained in the morning) for measurement of plasma melatonin and dopamine, serum serotonin, urine 6-sulfatoxymelatonin (MT6s), and creatinine. Statistical analysis was performed using 1-way ANOVA, Spearman correlation and receiver operator characteristic area under the curve analysis., Results: There were no significant differences in circulating melatonin, serotonin or dopamine concentrations between the 3 groups, although the study was underpowered for detection of significant differences in serum serotonin. Urine MT6s:creatinine ratio was significantly higher in dogs with PDH (4.08 ± 2.15 urine [MT6s] ng/mL per mg of urine creatinine) compared with dogs with SARDS (2.37 ± .51, P < .01), but not compared with normal dogs., Conclusions and Clinical Relevance: We have identified neurohormone differences between dogs with SARDS and PDH., (© 2019 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Objective: To identify discriminating factors, using clinical ophthalmic examination findings and routine laboratory testing, that differentiate dogs with early sudden acquired retinal degeneration (SARDS; vision loss <6 weeks' duration), age- and breed-matched control dogs, and dogs with pituitary-dependent hyperadrenocorticism (PDH)., Animals: Client-owned dogs: 15 with SARDS with <6 weeks duration of vision loss, 14 age- and breed-matched control dogs, and 13 dogs with confirmed PDH., Procedures: Dogs underwent ophthalmic examination, electroretinography (ERG) fundus photography, and spectral-domain optical coherence tomography (SD-OCT) in addition to physical examination, urinalysis, serum biochemistry, complete blood count, and adrenocorticotrophic hormone (ACTH) stimulation testing. Statistical analysis was performed using receiver operating curve area under the curve analysis, principal component analysis with sparse partial least squares analysis, and one-way ANOVA., Results: Dogs with SARDS all had absent vision and ERG a- and b-waves. SD-OCT demonstrated that dogs with SARDS had significantly thicker inner retina, thinner outer nuclear layer, and thicker photoreceptor inner/outer segment measurements than either controls or dogs with PDH. Discriminating laboratory parameters between dogs with SARDS and PDH with high specificity included post-ACTH serum cortisol (<19.3 μg/dL), AST:ALT ratio (>0.343), and urine specific gravity (>1.030)., Conclusions and Clinical Relevance: We have identified significant discriminators between SARDS and PDH. This work provides the basis for future studies that could identify and examine dogs with SARDS prior to vision loss, which may extend the potential therapeutic window for SARDS., (© 2019 American College of Veterinary Ophthalmologists.)

The canine species has dichromatic color vision comprising short-wavelength (S-) and long/medium (L/M-) wavelength-sensitive cones with peak spectral sensitivity of 429-435 nm and 555 nm respectively. Although differentiation of rod- and cone-mediated responses by electroretinogram (ERG) in dogs is commonly performed, and standards have been developed based on standards for human observers, methods to differentiate S- and L/M-cone responses in dogs have not been described. We developed flicker protocols derived from previously published rod and cone spectral sensitivities. We used a double silent substitution paradigm to isolate responses from each of the 3 photoreceptor subclasses. ERG responses were measured to sine-wave modulation of photoreceptor excitation at different temporal frequencies (between 4 and 56 Hz) and mean luminance (between 3.25 and 130 cd/m 2 ) on 6 different normal dogs (3 adult female, and 3 adult male beagles) and one female beagle dog with suspected hereditary congenital stationary night blindness (CSNB). Peak rod driven response amplitudes were achieved with low frequency (4 Hz, maximal range 4-12 Hz) and low mean luminance (3.25 cd/m 2 ). In contrast, peak L/M-cone driven response amplitudes were achieved with high frequency (32 Hz, maximal range 28-44 Hz) and high mean luminance (32.5-130 cd/m 2 ). Maximal S-cone driven responses were obtained with low frequency stimuli (4 Hz, maximal range 4-12 Hz) and 32.5-130 cd/m 2 mean luminance. The dog with CSNB had reduced rod- and S-cone-driven responses, but normal/supernormal L/M cone-driven responses. We have developed methods to differentiate rod, S- and L/M-cone function in dogs using silent substitution methods. The influence of temporal frequency and mean luminance on the ERGs originating in each photoreceptor type can now be studied independently. Dogs and humans have similar L/M cone responses, whereas mice have significantly different L/M responses. This work will facilitate a greater understanding of canine retinal electrophysiology and will complement the study of canine models of human hereditary photoreceptor disorders., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The ability to noninvasively monitor retinal abnormalities using imaging and cognitive and electrophysiological assessment has made it possible to carefully characterize genetic influences on retinal health. Because genetic retinal traits in animal species are not commonly detrimental to survival beyond birth, it is possible to document the natural history of retinal disease. Human quality of life is greatly impacted by retinal disease, and blindness carries a significant financial burden to society. Because of these compelling reasons, there is an ongoing medical need to study the effect of genetic mutations on retinal health and to develop therapies to address them. Transgenic animal models have aided in these missions, but there are opportunities for novel gene discovery and a development of greater understanding of retinal physiology using animal models that develop naturally occurring heritable retinal disorders. In this chapter, the advantages and disadvantages of transgenic and spontaneous vertebrate animal models of human inherited retinal disease are debated, in particular those of carnivore species, and the potential resource of spontaneous heritable retinal disorders in inbred nondomestic carnivore species is discussed.

Objective: Sudden acquired retinal degeneration syndrome (SARDS) is a leading cause of irreversible blindness in dogs, yet no treatment has been objectively evaluated, or proven to be effective. Consensus of opinion is that SARDS is immune-mediated, although corticosteroid medications may exacerbate associated systemic signs. We examined the effect of sole-agent treatment with mycophenolate mofetil (MMF), a potent immunosuppressive medication unlikely to exacerbate associated systemic signs., Animals Studied: Ten client-owned dogs with SARDS prospectively recruited within 6 weeks of vision loss., Procedures: Clinical history, findings of systemic and ophthalmic examinations, blood parameters, visual navigation ability, electroretinography, and optical coherence tomography (OCT) were collected at baseline and at recheck after approximately 6 weeks of treatment with 10 mg/kg q 12 h of oral MMF., Results: Twenty percent of dogs (2/10) experienced side effects (diarrhea, vomiting, lethargy), which resolved with reduction in dose to 8 mg/kg q12 h. No significant changes in systemic signs, physical examination findings, or laboratory test results were detected at the recheck examination. Compared with baseline, visual ability significantly declined at the recheck examination, and the amplitude of a slow-onset negative waveform noted on dark-adapted electroretinography was reduced at the recheck examination. The outer retinal layers were significantly thinner at the recheck examination as measured by OCT., Conclusions: Mycophenolate mofetil as a sole agent has no measureable positive effect on physical health, vision, or retinal structure following a 6-week trial period. Further studies are needed to evaluate other treatment options for SARDS., (© 2018 American College of Veterinary Ophthalmologists.)

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.

Competing Interests: Competing interests: None declared.

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.

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.

Background: Mutations in RPE65 cause Leber's congenital amaurosis, a progressive retinal degenerative disease that severely impairs sight in children. Gene therapy can result in modest improvements in night vision, but knowledge of its efficacy in humans is limited., Methods: We performed a phase 1-2 open-label trial involving 12 participants to evaluate the safety and efficacy of gene therapy with a recombinant adeno-associated virus 2/2 (rAAV2/2) vector carrying the RPE65 complementary DNA, and measured visual function over the course of 3 years. Four participants were administered a lower dose of the vector, and 8 were administered a higher dose. In a parallel study in dogs, we investigated the relationship among vector dose, visual function, and electroretinography (ERG) findings., Results: Improvements in retinal sensitivity were evident, to varying extents, in six participants for up to 3 years, peaking at 6 to 12 months after treatment and then declining. No associated improvement in retinal function was detected by means of ERG. Three participants had intraocular inflammation, and two had clinically significant deterioration of visual acuity. The reduction in central retinal thickness varied among participants. In dogs, RPE65 gene therapy with the same vector at lower doses improved vision-guided behavior, but only higher doses resulted in improvements in retinal function that were detectable with the use of ERG., Conclusions: Gene therapy with rAAV2/2 RPE65 vector improved retinal sensitivity, albeit modestly and temporarily. Comparison with the results obtained in the dog model indicates that there is a species difference in the amount of RPE65 required to drive the visual cycle and that the demand for RPE65 in affected persons was not met to the extent required for a durable, robust effect. (Funded by the National Institute for Health Research and others; ClinicalTrials.gov number, NCT00643747.).

The cat is emerging as a promising large animal model for preclinical testing of retinal dystrophy therapies, for example, by gene therapy. However, there is a paucity of studies investigating viral vector gene transfer to the feline retina. We therefore sought to study the tropism of recombinant adeno-associated viral (rAAV) vectors for the feline outer retina. We delivered four rAAV serotypes: rAAV2/2, rAAV2/5, rAAV2/8 and rAAV2/9, each expressing green fluorescent protein (GFP) under the control of a cytomegalovirus promoter, to the subretinal space in cats and, for comparison, mice. Cats were monitored for gene expression by in vivo imaging and cellular tropism was determined using immunohistochemistry. In cats, rAAV2/2, rAAV2/8 and rAAV2/9 vectors induced faster and stronger GFP expression than rAAV2/5 and all vectors transduced the retinal pigment epithelium (RPE) and photoreceptors. Unlike in mice, cone photoreceptors in the cat retina were more efficiently transduced than rod photoreceptors. In mice, rAAV2/2 only transduced the RPE whereas the other vectors also transduced rods and cones. These results highlight species differences in cellular tropism of rAAV vectors in the outer retina. We conclude that rAAV serotypes are suitable for use for retinal gene therapy in feline models, particularly when cone photoreceptors are the target cell.

Recombinant adeno-associated viruses are important vectors for retinal gene delivery. Currently utilized vectors have relatively slow onset, and for efficient transduction it is necessary to deliver treatment subretinally, with the potential for damage to the retina. Amino-acid substitutions in the viral capsid improve efficiency in rodent eyes by evading host responses. As dogs are important large animal models for human retinitis pigmentosa, we evaluated the speed and efficiency of retinal transduction using capsid-mutant vectors injected both subretinally and intravitreally. We evaluated AAV serotypes 2 and 8 with amino-acid substitutions of surface-exposed capsid tyrosine residues. The chicken beta-actin promoter was used to drive green fluorescent protein expression. Twelve normal adult beagles were injected; four dogs received intravitreal injections and eight dogs received subretinal injections. Capsid-mutant viruses tested included AAV2(quad Y-F) (intravitreal and subretinal) and self-complementary scAAV8(Y733F) (subretinal only). Contralateral control eyes received injections of scAAV5 (subretinal) or scAAV2 (intravitreal). Subretinally delivered vectors had a faster expression onset than intravitreally delivered vectors. Subretinally delivered scAAV8(Y733F) had a faster onset of expression than scAAV5. All subretinally injected vector types transduced the outer retina with high efficiency and the inner retina with moderate efficiency. Intravitreally delivered AAV2(quad Y-F) had a marginally higher efficiency of transduction of both outer retinal and inner retinal cells than scAAV2. Because of their rapid expression onset and efficient transduction, subretinally delivered capsid-mutant AAV8 vectors may increase the efficacy of gene therapy treatment for rapid photoreceptor degenerative diseases. With further refinement, capsid-mutant AAV2 vectors show promise for retinal gene delivery from an intravitreal approach.

Young Rpe65-deficient dogs have been used as a model for human RPE65 Leber congenital amaurosis (RPE65-LCA) in proof-of-concept trials of recombinant adeno-associated virus (rAAV) gene therapy. However, there are relatively few reports of the outcome of rAAV gene therapy in Rpe65-deficient dogs older than 2 years of age. The purpose of this study was to investigate the success of this therapy in older Rpe65-deficient dogs. Thirteen eyes were treated in dogs between 2 and 6 years old. An rAAV2 vector expressing the human RPE65 cDNA driven by the human RPE65 promoter was delivered by subretinal injection. Twelve of the 13 eyes had improved retinal function as assessed by electroretinography, and all showed improvement in vision at low lighting intensities. Histologic examination of five of the eyes was performed but found no correlation between electroretinogram (ERG) rescue and numbers of remaining photoreceptors. We conclude that functional rescue is still possible in older dogs and that the use of older Rpe65-deficient dogs, rather than young Rpe65-deficient dogs that have very little loss of photoreceptors, more accurately models the situation when treating human RPE65-LCA patients.

Recent clinical trials of retinal pigment epithelium gene (RPE65) supplementation therapy in Leber congenital amaurosis type 2 patients have demonstrated improvements in rod and cone function, but it may be some years before the effects of therapy on photoreceptor survival become apparent. The Rpe65-deficient dog is a very useful pre-clinical model in which to test efficacy of therapies, because the dog has a retina with a high degree of similarity to that of humans. In this study, we evaluated the effect of RPE65 gene therapy on photoreceptor survival in order to predict the potential benefit and limitations of therapy in patients. We examined the retinas of Rpe65-deficient dogs after RPE65 gene therapy to evaluate the preservation of rods and cone photoreceptor subtypes. We found that gene therapy preserves both rods and cones. While the moderate loss of rods in the Rpe65-deficient dog retina is slowed by gene therapy, S-cones are lost extensively and gene therapy can prevent that loss, although only within the treated area. Although LM-cones are not lost extensively, cone opsin mislocalization indicates that they are stressed, and this can be partially reversed by gene therapy. Our results suggest that gene therapy may be able to slow cone degeneration in patients if intervention is sufficiently early and also that it is probably important to treat the macula in order to preserve central function.

Dog models with spontaneously occurring mutations in retinal dystrophy genes are an invaluable resource for preclinical development of retinal gene therapy. Adeno-associated virus (AAV) vectors have been most successful; to target the outer retina and RPE they are delivered by subretinal injection, causing a temporary retinal detachment with some potential for retinal morbidity. A recent reporter gene study using an AAV2/8 vector in dogs reported transgene expression beyond the boundary of the subretinal bleb. This could be a desirable feature which increases the area of retina treated while minimizing the retinal detachment and any associated morbidity. We performed a detailed study of the lateral spread of transgene expression beyond the subretinal injection site following subretinally delivered AAV vectors in normal dogs. Vectors expressed green fluorescent protein (GFP) using a small chicken beta-actin promoter. AAV2/2 (quadruple tyrosine to phenylalanine (Y-F) capsid mutant), self-complementary (sc) AAV2/8 (single Y-F capsid mutant) and a scAAV2/5 were used. We found that in all eyes GFP expression involved retina beyond the initial post-injection subretinal bleb boundary. In all eyes there was post-injection spread of the retinal detachment within the first 3 days post procedure and prior to retinal reattachment. In 11/16 eyes this accounted for the entire "lateral spread" of GFP expression while in 5/16 eyes a very slight extension of GFP expression beyond the final boundary of the subretinal bleb could be detected. All 3 AAV constructs induced GFP expression in the nerve fiber layer with spread to the optic nerve. Patients treated by subretinal injection should be monitored for possible expansion of the subretinal injection bleb prior to reattachment. Injections in the para-foveal region may expand to lead to a foveal detachment that may be undesirable. Cell-specific promoters may be required to limit spread of expressed transgene to the brain with these AAV serotypes.

A 6-year-old neutered male domestic short-haired cat was presented to the Comparative Ophthalmology service at Michigan State University with a 3-week history of decreased appetite and redness of the left eye. The left forelimb had been removed 15 months previously because of the presence of a subcutaneous fibrosarcoma. In the left globe, a large iridal mass was associated with increased intraocular pressure and retinal detachment. A smaller mass involving the right iris was also present. Imaging revealed a 2-cm mass in the left caudodorsal lung lobe, and abdominal ultrasound showed multifocal bilateral renal masses. Aspirates of these masses were nondiagnostic. The left globe was removed for palliative reasons, and histopathology showed that fibrosarcoma was infiltrating the iris, choroid, and optic nerve. Despite systemic chemotherapy with doxorubicin, the animal died 4 months after initial presentation. Histopathology confirmed highly angioinvasive metastatic fibrosarcoma also in the right uveal tract, the lungs, and both kidneys., (© 2012 American College of Veterinary Ophthalmologists.)

Molecular oxygen is essential for the development, growth and survival of multicellular organisms. Hypoxic microenvironments and oxygen gradients are generated physiologically during embryogenesis and organogenesis. In the eye, oxygen plays a crucial role in both physiological vascular development and common blinding diseases. The retinal pigment epithelium (RPE) is a monolayer of cells essential for normal ocular development and in the mature retina provides support for overlying photoreceptors and their vascular supply. Hypoxia at the level of the RPE is closely implicated in pathogenesis of age-related macular degeneration. Adaptive tissue responses to hypoxia are orchestrated by sophisticated oxygen sensing mechanisms. In particular, the von Hippel-Lindau tumour suppressor protein (pVhl) controls hypoxia-inducible transcription factor (HIF)-mediated adaptation. However, the role of Vhl/Hif1a in the RPE in the development of the eye and its vasculature is unknown. In this study we explored the function of Vhl and Hif1a in the developing RPE using a tissue-specific conditional-knockout approach. We found that deletion of Vhl in the RPE results in RPE apoptosis, aniridia and microphthalmia. Increased levels of Hif1a, Hif2a, Epo and Vegf are associated with a highly disorganised retinal vasculature, chorioretinal anastomoses and the persistence of embryonic vascular structures into adulthood. Additional inactivation of Hif1a in the RPE rescues the RPE morphology, aniridia, microphthalmia and anterior vasoproliferation, but does not rescue retinal vasoproliferation. These data demonstrate that Vhl-dependent regulation of Hif1a in the RPE is essential for normal RPE and iris development, ocular growth and vascular development in the anterior chamber, whereas Vhl-dependent regulation of other downstream pathways is crucial for normal development and maintenance of the retinal vasculature.

Because retinal ischemia is a common cause of vision loss, we sought to determine the effects of ischemia on neuroretinal function and survival in murine oxygen-induced retinopathy (OIR) and to define the role of endogenous erythropoietin (EPO) in this model. OIR is a reproducible model of ischemia-induced retinal neovascularization; it is used commonly to develop antiangiogenic strategies. We investigated the effects of ischemia in murine OIR on retinal function and neurodegeneration by electroretinography and detailed morphology. OIR was associated with significant neuroretinal dysfunction, with reduced photopic and scotopic ERG responses and reduced b-wave/a-wave ratios consistent with specific inner-retinal dysfunction. OIR resulted in significantly increased apoptosis and atrophy of the inner retina in areas of ischemia. EPO deficiency in heterozygous Epo-Tag transgenic mice was associated with more profound retinal dysfunction after OIR, indicated by a significantly greater suppression of ERG amplitudes, but had no measurable effect on the extent of retinal ischemia, preretinal neovascularization, or neuroretinal degeneration in OIR. Systemic administration of recombinant EPO protected EPO-deficient mice against this additional suppression, but EPO supplementation in wild-type animals with OIR did not rescue neuroretinal dysfunction or degeneration. Murine OIR offers a valuable model of ischemic neuroretinal dysfunction and degeneration in which to investigate adaptive tissue responses and evaluate novel therapeutic approaches. Endogenous EPO can protect neuroretinal function in ischemic retinopathy., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Hypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs) that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators., Methodology/principal Findings: We investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR) in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF) and erythropoietin (Epo) by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO., Conclusions/significance: Both HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation plays a key role in regulating the response of Müller glia to hypoxia.

Purpose: The canine is an important large animal model of human retinal genetic disorders. Studies of ganglion cell distribution in the canine retina have identified a visual streak of high density superior to the optic disc with a temporal area of peak density known as the area centralis. The topography of cone photoreceptors in the canine retina has not been characterized in detail, and in contrast to the macula in humans, the position of the area centralis in dogs is not apparent on clinical funduscopic examination. The purpose of this study was to define the location of the area centralis in the dog and to characterize in detail the topography of rod and cone photoreceptors within the area centralis. This will facilitate the investigation and treatment of retinal disease in the canine., Methods: We used peanut agglutinin, which labels cone matrix sheaths and antibodies against long/medium wavelength (L/M)- and short wavelength (S)-cone opsins, to stain retinal cryosections and flatmounts from beagle dogs. Retinas were imaged using differential interference contrast imaging, fluorescence, and confocal microscopy. Within the area centralis, rod and cone size and density were quantified, and the proportion of cones expressing each cone opsin subtype was calculated. Using a grid pattern of sampling in 9 retinal flatmounts, we investigated the distribution of cones throughout the retina to predict the location of the area centralis., Results: We identified the area centralis as the site of maximal density of rod and cone photoreceptor cells, which have a smaller inner segment cross-sectional area in this region. L/M opsin was expressed by the majority of cones in the retina, both within the area centralis and in the peripheral retina. Using the mean of cone density distribution from 9 retinas, we calculated that the area centralis is likely to be centered at a point 1.5 mm temporal and 0.6 mm superior to the optic disc. For clinical funduscopic examination, this represents 1.2 disc diameters temporal and 0.4 disc diameters superior to the optic disc., Conclusions: We have described the distribution of rods and cone subtypes within the canine retina and calculated a predictable location for the area centralis. These findings will facilitate the characterization and treatment of cone photoreceptor dystrophies in the dog.