Dog Retina Research Articles

Differential gene expression between central and peripheral retinal regions in dogs and comparison with humans

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.

Manifestations of systemic disease in the retina and fundus of cats and dogs.

The fundus is unique in that it is the only part of the body that allows for a noninvasive and uninterrupted view of vasculature and nervous tissue. Utilization of this can be a powerful tool in uncovering salient incidental findings which point to underlying systemic diseases, and for monitoring response to therapy. Retinal venules and arterioles allow the clinician to assess changes in vascular color, diameter, outline, and tortuosity. The retina and optic nerve may exhibit changes associated with increased or decreased thickness, inflammatory infiltrates, hemorrhages, and detachments. While some retinal manifestations of systemic disease may be nonspecific, others are pathognomonic, and may be the presenting sign for a systemic illness. The examination of the fundus is an essential part of the comprehensive physical examination. Systemic diseases which may present with retinal abnormalities include a variety of disease classifications, as represented by the DAMNIT-V acronym, for Degenerative/Developmental, Anomalous, Metabolic, Neoplastic, Nutritional, Inflammatory (Infectious/Immune-mediated/ischemic), Toxic, Traumatic and Vascular. This review details systemic illnesses or syndromes that have been reported to manifest in the fundus of companion animals and discusses key aspects in differentiating their underlying cause. Normal variations in retinal anatomy and morphology are also considered.

Ten differentially expressed proteins in the tear film of dogs with diabetic retinopathy

AbstractPurposeA common early lesion of diabetic retinopathy in human patients and a diverse range of animal models is retinal capillary basement membrane thickening occur within 6 months of induction of diabetes. This hallmark pathology of diabetic microvascular disease is used in the evaluation of drug‐mediated modulation of diabetic retinopathy in a range of animal models. Moreover results from published clinical practice have shown that more than 22% of dogs with naturally contracted diabetes mellitus develop ophtalmoscopic signs of retinal hemorrhages or microaneurysms in a relatively short time from onset of the disease. Therefore, we hypothesize that changes in the retina in such dogs results in changes in the protein profile of tear fluid, that can be of the diagnostic or therapeutic value and can contribute to the better understanding the pathophysiology mechanisms underlying that disease. The objective of this study was to identify the protein content of tear film in dogs with naturally contracted diabetes mellitus with and/ without retinopathy, and its comparison with the content of control group. That approach results with mapping out the comprehensive proteome of tear film in diabetic dog and the may contribute to development a noninvasive method to detect potential protein candidates for diabetic retinopathy biomarkers in tear film.MethodsTear film was collected with Schirmer strips from the canine diabetic patients with/ and without retinopathy and sex‐ and age‐matched control group.The study was conducted on 24 dogs (8 dogs in each group).Two‐dimensional electrophoresis was performed followed by MALDI‐TOF mass spectrometry identification of differential proteins. Quantitative analysis of the differentiating electrophoretic spots was done with 2Delta software. Metabolic pathways associated with those proteins were identified with Panther GO software.ResultsAltogether, 5 significantly differentially expressed proteins were identified. Of those, 4 were downregulated, and 1 was upregulated in the tear film of diabetic patients with retinopathy. Pathway analysis revealed that the proteins were associated with multiple pathways involved in biological regulation, cellular and metabolic process and cellular component organization.ConclusionsTear film protein analysis provides a novel way to screen diabetic retinopathy biomarkers in dogs.

Altered transsulfuration pathway enzymes and redox homeostasis in inherited retinal degenerative diseases

Retinal degenerative diseases result from apoptotic photoreceptor cell death. As endogenously produced gaseous molecules such as hydrogen sulfide (H2S) and nitric oxide (NO) play a key role in apoptosis, we compared the expression levels of genes and proteins involved in the production of these molecules in the retina of normal dogs and three canine models (rcd1, crd2, and xlpra2) of human inherited retinal degeneration (IRD). Using qRT-PCR, Western blot, and immunohistochemistry (IHC), we showed that mRNA and protein levels of cystathionine β–synthase (CBS), an enzyme that produces H2S in neurons, are increased in retinal degeneration, but those of cystathionine γ-lyase (CSE), an enzyme involved in the production of glutathione (GSH), an antioxidant, are not. Such findings suggest that increased levels of H2S that are not counterbalanced by increased antioxidant potential may contribute to disease in affected retinas. We also studied the expression of neuronal and inducible nitric oxide synthase (nNOS and iNOS), the enzymes responsible for NO production. Western blot and IHC results revealed increased levels of nNOS and iNOS, resulting in increased NO levels in mutant retinas. Finally, photoreceptors are rich in polyunsaturated fatty acids (PUFAs) that can make these cells vulnerable to oxidative damage through reactive oxygen species (ROS). Our results showed increased levels of acrolein and hydroxynonenal (4HNE), two main toxic products of PUFAs, surrounding the membranes of photoreceptors in affected canines. Increased levels of these toxic products, together with increased NO and ROS, likely render these cells susceptible to an intrinsic apoptotic pathway involving mitochondrial membranes. To assess this possibility, we measured the levels of BCL2, an anti-apoptotic protein in the mitochondrial membrane. Western blot results showed decreased levels of BCL2 protein in affected retinas. Overall, the results of this study identify alterations in the expression of enzymes directly involved in maintaining the normal redox status of the retina during retinal degeneration, thereby supporting future studies to investigate the role of H2S and NO in retinal degeneration and apoptosis.

Biodistribution of adeno-associated virus type 2 carrying multi-characteristic opsin in dogs following intravitreal injection.

Gene therapy of retinal diseases using recombinant adeno‐associated virus (rAAV) vector‐based delivery has shown clinical success, and clinical trials based on rAAV‐based optogenetic therapies are currently in progress. Recently, we have developed multi‐characteristic opsin (MCO), which has been shown to effectively re‐photosensitize photoreceptor‐degenerated retina in mice leading to vision restoration at ambient light environment. Here, we report the biodistribution of the rAAV2 carried MCO (vMCO‐I) in live samples and post‐mortem organs following intraocular delivery in wild‐type dogs. Immunohistochemistry showed that the intravitreal injection of vMCO‐I resulted in gene transduction in the inner nuclear layer (INL) but did not induce detectable inflammatory or immune reaction in the dog retina. Vector DNA analysis of live body wastes and body fluids such as saliva and nasal secretions using quantitative polymerase chain reaction (qPCR) showed no correlative increase of vector copy in nasal secretions or saliva, minimal increase of vector copy in urine in the low‐dose group 13 weeks after injection and in the faeces of the high‐dose group at 3–13 weeks after injection suggesting clearance of the virus vector via urine and faeces. Further analysis of vector DNA extracted from faeces using PCR showed no transgene after 3 weeks post‐injection. Intravitreal injection of vMCO‐I resulted in few sporadic off‐target presences of the vector in the mesenteric lymph node, liver, spleen and testis. This study showed that intravitreal rAAV2‐based delivery of MCO‐I for retinal gene therapy is safe.

Short prolactin isoforms are expressed in photoreceptors of canine retinas undergoing retinal degeneration

Prolactin (PRL) hormone functions as a pleiotropic cytokine with a protective role in the retina. We recently identified by transcriptome profiling that PRL is one of the most highly upregulated mRNAs in the retinas of mutant rcd1 (PDE6B) and xlpra2 (RPGR) dogs at advanced stages of photoreceptor disease. In the present study, we have identified the expression of a short PRL isoform that lacks exon 1 in canine retinas and analyzed the time-course of expression and localization of this isoform in the retinas of these two models. Using laser capture microdissection to isolate RNA from each of the retinal cellular layers, we found by qPCR that this short PRL isoform is expressed in photoreceptors of degenerating retinas. We confirmed by in situ hybridization that its expression is localized to the outer nuclear layer and begins shortly after the onset of disease at the time of peak photoreceptor cell death in both models. PRL protein was also detected only in mutant dog retinas. Our results call for further investigations into the role of this novel PRL isoform in retinal degeneration.

Candidate Genetic Modifiers for RPGR Retinal Degeneration.

PurposeTo define genetic variants associated with variable severity of X-linked progressive retinal atrophy 1 (XLPRA1) caused by a five-nucleotide deletion in canine RPGR exon ORF15.MethodsA genome-wide association study (GWAS) was performed in XLPRA1 phenotype informative pedigree. Whole genome sequencing (WGS) was used for mutational analysis of genes within the candidate genomic region. Retinas of normal and mutant dogs were used for gene expression, gene structure, and RNA duplex analyses.ResultsGWAS followed by haplotype phasing identified an approximately 4.6 Mb candidate genomic interval on CFA31 containing seven protein-coding genes expressed in retina (ROBO1, ROBO2, RBM11, NRIP1, HSPA13, SAMSN1, and USP25). Furthermore, we identified and characterized two novel lncRNAs, ROBO1-AS and ROBO2-AS, that display overlapping gene organization with axon guidance pathway genes ROBO1 and ROBO2, respectively, producing sense-antisense gene pairs. Notably, ROBO1-AS and ROBO2-AS act in cis to form lncRNA/mRNA duplexes with ROBO1 and ROBO2, respectively, suggesting important roles for these lncRNAs in the ROBO regulatory network. A subsequent WGS identified candidate genes within the genomic region on CFA31 that might be implicated in modifying severity of XLPRA1. This approach led to discovery of genetic variants in ROBO1, ROBO1-AS, ROBO2-AS, and USP25 that are strongly associated with the XLPRA1 moderate phenotype.ConclusionsThe study provides new insights into the genetic basis of phenotypic variation in severity of RPGRorf15-associated retinal degeneration. Our findings suggest an important role for ROBO pathways in disease progression further expanding on our previously reported changes of ROBO1 expression in XLPRA1 retinas.

Retinal Diseases of Senior Dogs

Abstract Aging consists of a physiological decline of an organism’s functional activity. During the aging process, the structural and functional changes of the retina can be observed. In most cases, progressive vision loss occurs due to the age related changes of the anterior segment. Retinal diseases, characteristic for senior dogs are: retinal detachment, hypertensive chorioretinopathy, sudden acquired retinal degeneration syndrome (SARDS), progressive retinal atrophy (PRA), glaucoma, retinopathy, cystoid degeneration and neoplasms. The examination of the retina in senior dogs is based on: ophthalmoscopic examination, electroretinography, spectral-domain optical coherence tomography (AD-OCT) and if necessary, histopathological examinations. Comprehensive knowledge regarding the senior dog’s health, significantly increases their quality of life.

Diagnostic Imaging of Retina in Dogs

Oküler yapıların önemli bir bölümü doğrudan da dolaylı yoldan muayene edilebilir. Kapsamlı bir oftalmik muayene yapılarak göz hastalıklarının birçoğuna hızlı ve hatasız teşhis koymak mümkün olabilmektedir. Retina, optik sinir vasıtası ile direk beynin ön bölümüne bağlanır. Görme bozukluklarının değerlendirilmesi için retinayı etkileyen hastalıkların önceden belirlenmesi sağaltıma erken başlama ve prognoz açısından oldukça önemlidir. Gelişmiş görüntüleme teknolojileri sayesinde, hastaların patolojilerinin gösterilmesinde, sunulmasında ve paylaşılmasında büyük yol alınmıştır. Bu “derleme” köpeklerde retinanın tanısal görüntülenmesi ile ilgili bilgileri toplayarak hastalıkların tespitini kolaylaştırmak amacıyla sunulmuştur.

Critical Decrease in the Level of Axon Guidance Receptor ROBO1 in Rod Synaptic Terminals Is Followed by Axon Retraction.

PurposeTo define remodeling of photoreceptor synaptic terminals and second-order retinal neurons in canine X-linked progressive retinal atrophy 1 caused by a five-nucleotide deletion in the RPGR exon ORF15.MethodsRetinas of normal and mutant dogs were used for gene expression, Western blot, and immunohistochemistry. Cell-specific markers were used to examine disease-dependent retinal remodeling.ResultsIn mutant retinas, a number of rod axon terminals retract into the outer nuclear layer. This neuritic atrophy preceded significant loss of rods and was evident early in disease. Rod bipolar and horizontal cell processes were found to extend into the outer nuclear layer, where they seemed to form contacts with the spherules of rod photoreceptors. No ectopic rewiring was observed. Because cytoskeletal reorganization was previously shown to underlie photoreceptor axon retraction, we examined normal and mutant retinas for expression of axon guidance receptors ROBO1 and ROBO2, which are known to regulate actin cytoskeleton dynamics. We found that the overall expression of both ROBO1 and ROBO2 is retained at the same level in premature and fully developed normal retinas. However, analysis of predisease and early disease retinas identified markedly decreased levels of ROBO1 in rod spherules compared with controls. In contrast, no differences in ROBO1 signals were noted in cone pedicles in normal and mutant retinas, where ROBO1 levels remained similarly low.ConclusionsDepletion of ROBO1 in rod synaptic terminals correlates with the remodeling of axonal and dendritic processes in the outer retina of dogs with X-linked progressive retinal atrophy 1 and may play a role in the retraction of rod axons.

The effects of physical luminance on colour discrimination in dogs: A cautionary tale

Due to the composition of the cone photoreceptors in the retina of dogs, it has been proposed they might demonstrate human-like red/green colour blindness. However, some assessments have shown that dogs may still be able to distinguish between red and green. This suggests that dogs may be differentiating between the two colours on the basis of their brightness. To further explore this issue, the present investigation tested dogs’ abilities to differentiate between two colours at different physical luminance levels. Seven dogs were trained on a simultaneous size discrimination task in which two circles were presented on a monitor. The colours of the circles were then varied such that dogs would choose one stimulus if they could discriminate between two colours and a different stimulus if they could not. Four experiments were conducted. The first presented dogs with yellow, blue, green, and red stimuli at their maximum red, green, and blue (RGB) values. All seven dogs were equally proficient at discriminating between red and green, yellow and blue, and black and white stimuli as no difference in performance was observed (p = 0.52). In Experiments 2–4, the four colours were presented with equal physical luminance across three different intensities. All seven dogs successfully discriminated between all four colours when the colours were isoluminant at 34.6 cd/m2 as no differences in performance was observed (p = 0.10). When presented with isoluminant stimuli at 41.9 cd/m2, a difference was observed between conditions, with the dogs failing to detect red stimuli presented on a green background compared to the achromatic controls (p = 0.03). When presented with stimuli at 49.3 cd/m2, a difference was again observed between conditions, but this time the dogs were unable to detect blue stimuli presented on a yellow background compared to the achromatic controls (p < 0.01). These findings demonstrate that luminance levels of stimuli can affect colour discrimination and what dogs are able to see on computer screens. These results have important implications in how cognition is studied in dogs through the use of presenting stimuli on monitors.

Long-term expression of melanopsin and channelrhodopsin causes no gross alterations in the dystrophic dog retina.

Several preclinical studies have investigated the potential of algal channelrhodopsin and human melanopsin as optogenetic tools for vision restoration. In the present study, we assessed the potentially deleterious effects of long-term expression of these optogenes on the diseased retina in a large animal model of retinal degeneration, the RPE65-deficient Briard dog model of Leber congenital amaurosis. Intravitreal injection of adeno-associated virus vectors expressing channelrhodopsin and melanopsin had no effect on retinal thickness over a 16-month period post injection. Our data support the safety of the optogenetic approach for the treatment of blindness.

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

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. 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. 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. 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.

Localization of Rod Bipolar Cells in the Mammalian Retina Using an Antibody Against the α1c L-type Ca(2+) Channel.

Bipolar cells transmit stimuli via graded changes in membrane potential and neurotransmitter release is modulated by Ca2+ influx through L-type Ca2+ channels. The purpose of this study was to determine whether the α1c subunit of L-type voltage-gated Ca2+ channel (α1c L-type Ca2+ channel) colocalizes with protein kinase C alpha (PKC-α), which labels rod bipolar cells. Retinal whole mounts and vertical sections from mouse, hamster, rabbit, and dog were immunolabeled with antibodies against PKC-α and α1c L-type Ca2+ channel, using fluorescein isothiocyanate (FITC) and Cy5 as visualizing agents. PKC-α-immunoreactive cells were morphologically identical to rod bipolar cells as previously reported. Their cell bodies were located within the inner nuclear layer, dendritic processes branched into the outer plexiform layer, and axons extended into the inner plexiform layer. Immunostaining showed that α1c L-type Ca2+ channel colocalized with PKC-α in rod bipolar cells. The identical expression of PKC-α and α1c L-type Ca2+ channel indicates that the α1c L-type Ca2+ channel has a specific role in rod bipolar cells, and the antibody against the α1c L-type Ca2+ channel may be a useful marker for studying the distribution of rod bipolar cells in mouse, hamster, rabbit, and dog retinas.

Oxidative stress increases in retinas of dogs in acute glaucoma but not in chronic glaucoma.

To test the hypothesis that oxidative stress occurs early in the pathogenesis of glaucoma in dogs. Sections from eight control retinas and 25 retinas from dogs with primary glaucoma. For retinas embedded in paraffin, sections were immunohistochemically stained for malondialdehyde (MDA) and 3-nitrotyrosine (NT). For retinas embedded in plastic, serial 0.5-μm sections were immunogold-stained for total glutathione, taurine, and glutamate. Increased immunostaining for MDA and NT, markers of oxidative stress, occurred in retinal ganglion cells (RGCs) and other neurons in acute glaucoma, but not in chronic glaucoma. In minimally damaged regions, immunostaining for the antioxidant glutathione was decreased in RGCs, neurons of the inner nuclear layer (INL), and Müller cell processes. The loss of glutathione immunostaining in RGCs occurred without a decrease in glutamate immunostaining. Neurons with nuclear damage in the INL had low levels of glutathione, taurine, and glutamate. In severely damaged regions, immunostaining for glutathione was increased in the remaining retinal tissue. Immunohistochemical staining revealed an increase in markers of oxidative stress and loss of glutathione in neurons with minimal damage during acute glaucoma. Oxidative changes were no longer present in chronic glaucomatous retinas, suggesting transient oxidative stress occurs early in glaucoma. The loss of glutathione in minimally damaged regions occurred without a significant redistribution of glutamate, suggesting oxidative stress may occur before glutamate redistribution. Alteration in markers of oxidative stress occurs early in canine glaucoma, suggesting oxidative stress may contribute to subsequent glutamate redistribution and other damaging processes.

Immuno-Histochemical Analysis of Rod and Cone Reaction to RPE65 Deficiency in the Inferior and Superior Canine Retina

Mutations in the RPE65 gene are associated with autosomal recessive early onset severe retinal dystrophy. Morphological and functional studies indicate early and dramatic loss of rod photoreceptors and early loss of S-cone function, while L and M cones remain initially functional. The Swedish Briard dog is a naturally occurring animal model for this disease. Detailed information about rod and cone reaction to RPE65 deficiency in this model with regard to their location within the retina remains limited. The aim of this study was to analyze morphological parameters of cone and rod viability in young adult RPE65 deficient dogs in different parts of the retina in order to shed light on local disparities in this disease. In retinae of affected dogs, sprouting of rod bipolar cell dendrites and horizontal cell processes was dramatically increased in the inferior peripheral part of affected retinae, while central inferior and both superior parts did not display significantly increased sprouting. This observation was correlated with photoreceptor cell layer thickness. Interestingly, while L/M cone opsin expression was uniformly reduced both in the superior and inferior part of the retina, S-cone opsin expression loss was less severe in the inferior part of the retina. In summary, in retinae of young adult RPE65 deficient dogs, the degree of rod bipolar and horizontal cell sprouting as well as of S-cone opsin expression depends on the location. As the human retinal pigment epithelium (RPE) is pigmented similar to the RPE in the inferior part of the canine retina, and the kinetics of photoreceptor degeneration in humans seems to be similar to what has been observed in the inferior peripheral retina in dogs, this area should be studied in future gene therapy experiments in this model.

Altered miRNA expression in canine retinas during normal development and in models of retinal degeneration

BackgroundAlthough more than 246 loci/genes are associated with inherited retinal diseases, the mechanistic events that link genetic mutations to photoreceptor cell death are poorly understood. miRNAs play a relevant role during retinal development and disease. Thus, as a first step in characterizing miRNA involvement during disease expression and progression, we examined miRNAs expression changes in normal retinal development and in four canine models of retinal degenerative disease.ResultsThe initial microarray analysis showed that 50 miRNAs were differentially expressed (DE) early (3 vs. 7 wks) in normal retina development, while only 2 were DE between 7 and 16 wks, when the dog retina is fully mature. miRNA expression profiles were similar between dogs affected with xlpra2, an early-onset retinal disease caused by a microdeletion in RPGRORF15, and normal dogs early in development (3 wks) and at the peak of photoreceptor death (7 wks), when only 2 miRNAs were DE. However, the expression varied much more markedly during the chronic cell death stage at 16 wks (118 up-/55 down-regulated miRNAs). Functional analyses indicated that these DE miRNAs are associated with an increased inflammatory response, as well as cell death/survival. qRT-PCR of selected apoptosis-related miRNAs (“apoptomirs”) confirmed the microarray results in xlpra2, and extended the analysis to the early-onset retinal diseases rcd1 (PDE6B-mutation) and erd (STK38L-mutation), as well as the slowly progressing prcd (PRCD-mutation). The results showed up-regulation of anti-apoptotic (miR-9, -19a, -20, -21, -29b, -146a, -155, -221) and down-regulation of pro-apoptotic (miR-122, -129) apoptomirs in the early-onset diseases and, with few exceptions, also in the prcd-mutants.ConclusionsOur results suggest that apoptomirs might be expressed by diseased retinas in an attempt to counteract the degenerative process. The pattern of expression in diseased retinas mirrored the morphology and cell death kinetics previously described for these diseases. This study suggests that common miRNA regulatory mechanisms may be involved in retinal degeneration processes and provides attractive opportunities for the development of novel miRNA-based therapies to delay the progression of the degenerative process.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-172) contains supplementary material, which is available to authorized users.

Primary primitive neuroectodermal tumors of the retina and ciliary body in dogs

We describe the clinical, histological, and immunohistochemical features of primary intraocular primitive neuroectodermal tumors in eight dogs. Four of eight tumors exhibited histological features similar to human retinoblastomas characterized by Flexner-Wintersteiner rosettes, and fleurettes, and demonstrated variable immunoreactivity for retinal markers opsin, S-antigen (S-Ag) and interphotoreceptor retinoid-binding protein (IRBP). All dogs with tumors displaying histological and immunohistochemical features of retinal differentiation were ≤2years of age. All tumors diagnosed as medulloepitheliomas (n=4) did not display histological and immunohistochemical features of retinal differentiation and were present in dogs 7years or older. Age of onset, in conjunction with immunohistochemistry for opsin, S-Ag, and IRBP, is an important aid in the differentiation of primary, primitive neuroectodermal tumors arising within the canine ciliary body, retina, and optic papilla.

Restoration of Vision in the pde6β-deficient Dog, a Large Animal Model of Rod-cone Dystrophy

Defects in the β subunit of rod cGMP phosphodiesterase 6 (PDE6β) are associated with autosomal recessive retinitis pigmentosa (RP), a childhood blinding disease with early retinal degeneration and vision loss. To date, there is no treatment for this pathology. The aim of this preclinical study was to test recombinant adeno-associated virus (AAV)-mediated gene addition therapy in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6β deficiency that strongly resembles the human pathology. A total of eight rcd1 dogs were injected subretinally with AAV2/5RK.cpde6β (n = 4) or AAV2/8RK.cpde6β (n = 4). In vivo and post-mortem morphological analysis showed a significant preservation of the retinal structure in transduced areas of both AAV2/5RK.cpde6β- and AAV2/8RK.cpde6β-treated retinas. Moreover, substantial rod-derived electroretinography (ERG) signals were recorded as soon as 1 month postinjection (35% of normal eyes) and remained stable for at least 18 months (the duration of the study) in treated eyes. Rod-responses were undetectable in untreated contralateral eyes. Most importantly, dim-light vision was restored in all treated rcd1 dogs. These results demonstrate for the first time that gene therapy effectively restores long-term retinal function and vision in a large animal model of autosomal recessive rod-cone dystrophy, and provide great promise for human treatment.

RPE65 gene therapy slows cone loss in Rpe65-deficient dogs

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.