@article{salzman_merten_panek_fefer_mondino_westermeyer_gruen_olby_mowat_2023, title={Age-associated changes in electroretinography measures in companion dogs}, volume={6}, ISSN={["1573-2622"]}, url={https://doi.org/10.1007/s10633-023-09938-7}, DOI={10.1007/s10633-023-09938-7}, abstractNote={{"Label"=>"PURPOSE", "NlmCategory"=>"OBJECTIVE"} 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. {"Label"=>"METHODS", "NlmCategory"=>"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. {"Label"=>"RESULTS", "NlmCategory"=>"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 {"sup"=>"2"}  flash p < 0.0001) and b-waves (cone flicker p = 0.03, dark-adapted 0.01 cds/m {"sup"=>"2"}  flash p = 0.001). Age was also significantly associated with reduced amplitudes of a-waves (dark-adapted 3 cds/m {"sup"=>"2"}  flash p < 0.0001, 10 cds/m {"sup"=>"2"}  flash p = 0.005) and b-waves (light-adapted 3 cds/m {"sup"=>"2"}  flash p < 0.0001, dark-adapted 0.01 cds/m {"sup"=>"2"}  flash p = 0.0004, 3 cds/m {"sup"=>"2"}  flash p < 0.0001, 10 cds/m {"sup"=>"2"}  flash p = 0.007) and flicker (light-adapted 30 Hz 3 cds/m {"sup"=>"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. {"Label"=>"CONCLUSIONS", "NlmCategory"=>"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.}, journal={DOCUMENTA OPHTHALMOLOGICA}, author={Salzman, Michele M. and Merten, Natascha and Panek, Wojciech K. and Fefer, Gilad and Mondino, Alejandra and Westermeyer, Hans D. and Gruen, Margaret E. and Olby, Natasha J. and Mowat, Freya M.}, year={2023}, month={Jun} }
 @article{lynch_ruterbories_robertson_lunn_mowat_2023, title={Hemostatic profiles in dogs with sudden acquired retinal degeneration syndrome}, volume={4}, ISSN={["1939-1676"]}, url={https://doi.org/10.1111/jvim.16710}, DOI={10.1111/jvim.16710}, abstractNote={Abstract}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={Lynch, Alex M. M. and Ruterbories, Laura K. K. and Robertson, James B. B. and Lunn, Katharine F. F. and Mowat, Freya M. M.}, year={2023}, month={Apr} }
 @article{fefer_panek_khan_singer_westermeyer_mowat_murdoch_case_olby_gruen_2022, title={Use of Cognitive Testing, Questionnaires, and Plasma Biomarkers to Quantify Cognitive Impairment in an Aging Pet Dog Population}, volume={87}, ISSN={["1875-8908"]}, DOI={10.3233/JAD-215562}, abstractNote={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.}, number={3}, journal={JOURNAL OF ALZHEIMERS DISEASE}, author={Fefer, Gilad and Panek, Wojciech K. and Khan, Michael Z. and Singer, Matthew and Westermeyer, Hans D. and Mowat, Freya M. and Murdoch, David M. and Case, Beth and Olby, Natasha J. and Gruen, Margaret E.}, year={2022}, pages={1367–1378} }
 @article{wise_foster_kremers_mowat_2021, title={A modified silent substitution electroretinography protocol to separate photoreceptor subclass function in lightly sedated dogs}, volume={24}, ISSN={["1463-5224"]}, DOI={10.1111/vop.12847}, abstractNote={Abstract}, number={1}, journal={VETERINARY OPHTHALMOLOGY}, author={Wise, E. N. and Foster, M. L. and Kremers, J. and Mowat, F. M.}, year={2021}, month={Jan}, pages={103–107} }
 @article{washington_li_fox_mowat_2021, title={Canine sudden acquired retinal degeneration syndrome: Owner perceptions on the time to vision loss, treatment outcomes, and prognosis for life}, volume={24}, ISSN={["1463-5224"]}, DOI={10.1111/vop.12855}, abstractNote={Abstract}, number={2}, journal={VETERINARY OPHTHALMOLOGY}, author={Washington, Demitrius R. and Li, Zhanhai and Fox, Lani C. and Mowat, Freya M.}, year={2021}, month={Mar}, pages={156–168} }
 @article{panek_murdoch_gruen_mowat_marek_olby_2021, title={Plasma Amyloid Beta Concentrations in Aged and Cognitively Impaired Pet Dogs}, volume={58}, ISSN={["1559-1182"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85091529286&partnerID=MN8TOARS}, DOI={10.1007/s12035-020-02140-9}, abstractNote={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 (r2 = 0.27, p = 0.001; and r2 = 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.}, number={2}, journal={MOLECULAR NEUROBIOLOGY}, author={Panek, Wojciech K. and Murdoch, David M. and Gruen, Margaret E. and Mowat, Freya M. and Marek, Robert D. and Olby, Natasha J.}, year={2021}, month={Feb}, pages={483–489} }
 @article{heyward_reynolds_foster_archibald_stoskopf_mowat_2021, title={Retinal cone photoreceptor distribution in the American black bear (Ursus americanus)}, volume={304}, ISSN={["1932-8494"]}, DOI={10.1002/ar.24472}, abstractNote={Abstract}, number={3}, journal={ANATOMICAL RECORD-ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY}, author={Heyward, Jennifer L. and Reynolds, Benjamin D. and Foster, Melanie L. and Archibald, Kate E. and Stoskopf, Michael K. and Mowat, Freya M.}, year={2021}, month={Mar}, pages={662–672} }
 @article{mowat_avelino_bowyer_parslow_westermeyer_foster_fogle_bizikova_2020, title={Detection of circulating anti-retinal antibodies in dogs with sudden acquired retinal degeneration syndrome using indirect immunofluorescence: A case control study}, volume={193}, ISSN={["1096-0007"]}, DOI={10.1016/j.exer.2020.107989}, abstractNote={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.}, journal={EXPERIMENTAL EYE RESEARCH}, author={Mowat, Freya M. and Avelino, Janelle and Bowyer, Ashley and Parslow, Vanessa and Westermeyer, Hans D. and Foster, Melanie L. and Fogle, Jonathan E. and Bizikova, Petra}, year={2020}, month={Apr} }
 @article{tyrrell_qurollo_mowat_kennedy-stoskopf_2020, title={MOLECULAR PREVALENCE OF SELECTED VECTOR-BORNE ORGANISMS IN CAPTIVE RED WOLVES (CANIS RUFUS)}, volume={51}, ISSN={["1937-2825"]}, DOI={10.1638/2019-0162}, abstractNote={Abstract: 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.}, number={3}, journal={JOURNAL OF ZOO AND WILDLIFE MEDICINE}, author={Tyrrell, Jeffrey D. and Qurollo, Barbara A. and Mowat, Freya M. and Kennedy-Stoskopf, Suzanne}, year={2020}, month={Sep}, pages={663–667} }
 @article{panek_gruen_murdoch_marek_stachel_mowat_saker_olby_2020, title={Plasma Neurofilament Light Chain as a Translational Biomarker of Aging and Neurodegeneration in Dogs}, volume={57}, ISSN={["1559-1182"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85085898712&partnerID=MN8TOARS}, DOI={10.1007/s12035-020-01951-0}, abstractNote={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.}, number={7}, journal={MOLECULAR NEUROBIOLOGY}, author={Panek, Wojciech K. and Gruen, Margaret E. and Murdoch, David M. and Marek, Robert D. and Stachel, Alexandra F. and Mowat, Freya M. and Saker, Korinn E. and Olby, Natasha J.}, year={2020}, month={Jul}, pages={3143–3149} }
 @article{oh_foster_lunn_mowat_2019, title={Circulating neurohormone imbalances in canine sudden acquired retinal degeneration syndrome and canine pituitary-dependent hypercortisolism}, volume={33}, ISSN={["1939-1676"]}, DOI={10.1111/jvim.15646}, abstractNote={Abstract}, number={6}, journal={JOURNAL OF VETERINARY INTERNAL MEDICINE}, author={Oh, Annie and Foster, Melanie L. and Lunn, Katharine F. and Mowat, Freya M.}, year={2019}, month={Nov}, pages={2587–2594} }
 @article{oh_foster_williams_zheng_ru_lunn_mowat_2019, title={Diagnostic utility of clinical and laboratory test parameters for differentiating between sudden acquired retinal degeneration syndrome and pituitary‐dependent hyperadrenocorticism in dogs}, volume={22}, ISSN={1463-5216 1463-5224}, url={http://dx.doi.org/10.1111/vop.12661}, DOI={10.1111/vop.12661}, abstractNote={Abstract}, number={6}, journal={Veterinary Ophthalmology}, publisher={Wiley}, author={Oh, Annie and Foster, Melanie L. and Williams, Jonathan G. and Zheng, Chaowen and Ru, Hongyu and Lunn, Katharine F. and Mowat, Freya M.}, year={2019}, month={Mar}, pages={842–858} }
 @article{mowat_wise_oh_foster_kremers_2019, title={In vivo electroretinographic differentiation of rod, short-wavelength and long/medium-wavelength cone responses in dogs using silent substitution stimuli}, volume={185}, ISSN={["1096-0007"]}, DOI={10.1016/j.exer.2019.05.013}, abstractNote={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/m2) 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/m2). 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/m2). Maximal S-cone driven responses were obtained with low frequency stimuli (4 Hz, maximal range 4–12 Hz) and 32.5–130 cd/m2 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.}, journal={EXPERIMENTAL EYE RESEARCH}, author={Mowat, Freya M. and Wise, Elisabeth and Oh, Annie and Foster, Melanie L. and Kremers, Jan}, year={2019}, month={Aug} }
 @article{mowat_2019, title={Naturally Occurring Inherited Forms of Retinal Degeneration in Vertebrate Animal Species: A Comparative and Evolutionary Perspective}, volume={1185}, ISBN={["978-3-030-27377-4"]}, ISSN={["2214-8019"]}, DOI={10.1007/978-3-030-27378-1_39}, abstractNote={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.}, journal={RETINAL DEGENERATIVE DISEASES: MECHANISMS AND EXPERIMENTAL THERAPY}, author={Mowat, Freya M.}, year={2019}, pages={239–243} }
 @article{westermeyer_salmon_baynes_yeatts_khattab_oh_mowat_2019, title={Safety and efficacy of topically applied 0.5% and 1% pirfenidone in a canine model of subconjunctival fibrosis}, volume={22}, ISSN={["1463-5224"]}, url={https://doi.org/10.1111/vop.12619}, DOI={10.1111/vop.12619}, abstractNote={Abstract}, number={4}, journal={VETERINARY OPHTHALMOLOGY}, author={Westermeyer, Hans D. and Salmon, Beth and Baynes, Ronald and Yeatts, James and Khattab, Ahlam and Oh, Annie and Mowat, Freya}, year={2019}, month={Jul}, pages={502–509} }
 @article{westermeyer_druley_royal_mowat_2019, title={Use of a Versatile, Inexpensive Ophthalmoscopy Teaching Model in Veterinary Medical Student Education Increases Ophthalmoscopy Proficiency}, volume={46}, ISSN={["1943-7218"]}, url={https://doi.org/10.3138/jvme.1117-157r}, DOI={10.3138/jvme.1117-157r}, abstractNote={ 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. }, number={4}, journal={JOURNAL OF VETERINARY MEDICAL EDUCATION}, author={Westermeyer, Hans D. and Druley, Gail E. and Royal, Kenneth D. and Mowat, Freya M.}, year={2019}, pages={518–522} }
 @article{zarfoss_tusler_kass_montgomery_lim_mowat_thomasy_2018, title={Clinical findings and outcomes for dogs with uveodermatologic syndrome}, volume={252}, ISSN={["1943-569X"]}, DOI={10.2460/javma.252.10.1263}, abstractNote={Abstract}, number={10}, journal={JAVMA-JOURNAL OF THE AMERICAN VETERINARY MEDICAL ASSOCIATION}, author={Zarfoss, Mitzi K. and Tusler, Charlotte A. and Kass, Philip H. and Montgomery, Keith and Lim, Christine C. and Mowat, Freya and Thomasy, Sara M.}, year={2018}, month={May}, pages={1263–1271} }
 @article{young_oh_williams_foster_miller_lunn_mowat_2018, title={Clinical therapeutic efficacy of mycophenolate mofetil in the treatment of SARDS in dogs-a prospective open-label pilot study}, volume={21}, ISSN={["1463-5224"]}, DOI={10.1111/vop.12545}, abstractNote={Abstract}, number={6}, journal={VETERINARY OPHTHALMOLOGY}, author={Young, Whitney M. and Oh, Annie and Williams, Jonathan G. and Foster, Melanie L. and Miller, William W. and Lunn, Katharine F. and Mowat, Freya M.}, year={2018}, month={Nov}, pages={565–576} }
 @article{mowat_royal_westermeyer_2018, title={Ophthalmoscopy skills in primary care: a cross-sectional practitioner survey}, volume={182}, ISSN={["2042-7670"]}, url={https://doi.org/10.1136/vr.104569}, DOI={10.1136/vr.104569}, abstractNote={Ophthalmoscopy is challenging to master. Medical professionals regularly practising ophthalmoscopy exhibit greater confidence1–3 and competence.4 Methods practised in veterinary medicine include direct, PanOptic and indirect ophthalmoscopy (Fig 1). Direct ophthalmoscopy is relatively straightforward, but its utility is diminished by a narrow field of view, limiting the ability to visualise the entire fundus during examination, although its use can complement other methods by providing a magnified view of a detected lesion. The PanOptic ophthalmoscope provides a wider field of view, intermediate between direct and indirect ophthalmoscopy. Indirect ophthalmoscopy is arguably the most technically challenging method (requiring optimal orientation of light source, lens and eye), but allows visualisation of a wide fundus area5 facilitating quick identification of lesions during short periods in which an animal’s eye is still. Indirect ophthalmoscopy use by primary care veterinary practitioners is low compared with direct ophthalmoscopy, although no studies have confirmed this speculation. This limited use of indirect ophthalmoscopy could impair detection of ophthalmic diseases with focal or multifocal manifestation that may not be immediately obvious from the small fields of view visible with direct ophthalmoscopy. Since in some cases of systemic illness ophthalmic manifestations may be the only changes present on physical examination, inability to perform comprehensive funduscopic examination may result in delay in diagnosis and poorer patient outcomes. …}, number={15}, journal={VETERINARY RECORD}, publisher={BMJ}, author={Mowat, Freya M. and Royal, Kenneth D. and Westermeyer, Hans D.}, year={2018}, month={Apr}, pages={435-+} }
 @article{oh_loew_foster_davidson_english_gervais_herring_mowat_2018, title={Phenotypic characterization of complete CSNB in the inbred research beagle: how common is CSNB in research and companion dogs?}, volume={137}, ISSN={["1573-2622"]}, DOI={10.1007/s10633-018-9653-y}, abstractNote={{"Label"=>"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. {"Label"=>"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. {"Label"=>"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. {"Label"=>"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.}, number={2}, journal={DOCUMENTA OPHTHALMOLOGICA}, author={Oh, Annie and Loew, Ellis R. and Foster, Melanie L. and Davidson, Michael G. and English, Robert V and Gervais, Kristen J. and Herring, Ian P. and Mowat, Freya M.}, year={2018}, month={Oct}, pages={87–101} }
 @article{mowat_gervais_occelli_annear_querubin_bainbridge_smith_ali_petersen-jones_2017, title={Early-Onset Progressive Degeneration of the Area Centralis in RPE65-Deficient Dogs}, volume={58}, ISSN={["1552-5783"]}, DOI={10.1167/iovs.17-21930}, abstractNote={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.}, number={7}, journal={INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE}, author={Mowat, Freya M. and Gervais, Kristen J. and Occelli, Laurence M. and Annear, Matthew J. and Querubin, Janice and Bainbridge, James W. and Smith, Alexander J. and Ali, Robin R. and Petersen-Jones, Simon M.}, year={2017}, month={Jun}, pages={3268–3277} }
 @article{mowat_occelli_bartoe_gervais_bruewer_querubin_dinculescu_boye_hauswirth_petersen-jones_2017, title={Gene Therapy in a Large Animal Model of PDE6A-Retinitis Pigmentosa}, volume={11}, ISSN={["1662-453X"]}, DOI={10.3389/fnins.2017.00342}, abstractNote={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.}, journal={FRONTIERS IN NEUROSCIENCE}, author={Mowat, Freya M. and Occelli, Laurence M. and Bartoe, Joshua T. and Gervais, Kristen J. and Bruewer, Ashlee R. and Querubin, Janice and Dinculescu, Astra and Boye, Sanford L. and Hauswirth, William W. and Petersen-Jones, Simon M.}, year={2017}, month={Jun} }
 @article{lange_mowat_sayed_mehad_duluc_piper_luhmann_nandi_kelly_smith_et al._2016, title={Dimethylarginine dimethylaminohydrolase-2 deficiency promotes vascular regeneration and attenuates pathological angiogenesis}, volume={147}, ISSN={["1096-0007"]}, DOI={10.1016/j.exer.2016.05.007}, abstractNote={Ischemia-induced angiogenesis is critical for tissue repair, but aberrant neovascularization in the retina causes severe sight impairment. Nitric oxide (NO) has been implicated in neovascular eye disease because of its pro-angiogenic properties in the retina. Nitric oxide production is inhibited endogenously by asymmetric dimethylarginines (ADMA and L-NMMA) which are metabolized by dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2. The aim of this study was to determine the roles of DDAH1, DDAH2, ADMA and L-NMMA in retinal ischemia-induced angiogenesis. First, DDAH1, DDAH2, ADMA and L-NMMA levels were determined in adult C57BL/6J mice. The results obtained revealed that DDAH1 was twofold increased in the retina compared to the brain and the choroid. DDAH2 expression was approximately 150 fold greater in retinal and 70 fold greater in choroidal tissue compared to brain tissue suggesting an important tissue-specific role for DDAH2 in the retina and choroid. ADMA and L-NMMA levels were similar in the retina and choroid under physiological conditions. Next, characterization of DDAH1(+/-) and DDAH2(-/-) deficient mice by in vivo fluorescein angiography, immunohistochemistry and electroretinography revealed normal neurovascular function compared with wildtype control mice. Finally, DDAH1(+/-) and DDAH2(-/-) deficient mice were studied in the oxygen-induced retinopathy (OIR) model, a model used to emulate retinal ischemia and neovascularization, and VEGF and ADMA levels were quantified by ELISA and liquid chromatography tandem mass spectrometry. In the OIR model, DDAH1(+/-) exhibited a similar phenotype compared to wildtype controls. DDAH2 deficiency, in contrast, resulted in elevated retinal ADMA which was associated with attenuated aberrant angiogenesis and improved vascular regeneration in a VEGF independent manner. Taken together this study suggests, that in retinal ischemia, DDAH2 deficiency elevates ADMA, promotes vascular regeneration and protects against aberrant angiogenesis. Therapeutic inhibition of DDAH2 may therefore offer a potential therapeutic strategy to protect sight by promoting retinal vascular regeneration and preventing pathological angiogenesis.}, journal={EXPERIMENTAL EYE RESEARCH}, author={Lange, Clemens and Mowat, Freya and Sayed, Haroon and Mehad, Manjit and Duluc, Lucie and Piper, Sophie and Luhmann, Ulrich and Nandi, Manasi and Kelly, Peter and Smith, Alexander and et al.}, year={2016}, month={Jun}, pages={148–155} }
 @article{mowat_hash_mzyk_harned_nagar_mcgahan_2016, title={Evaluation of the proliferative capacity of canine retinal pigment epithelial cells harvested from different regions of the fundus}, volume={57}, number={12}, journal={Investigative Ophthalmology and Visual Science}, author={Mowat, F. M. and Hash, J. and Mzyk, P. and Harned, J. and Nagar, S. and McGahan, M. C.}, year={2016} }
 @article{bainbridge_mehat_sundaram_robbie_barker_ripamonti_georgiadis_mowat_beattie_gardner_et al._2015, title={Long-Term Effect of Gene Therapy on Leber’s Congenital Amaurosis}, volume={372}, ISSN={0028-4793 1533-4406}, url={http://dx.doi.org/10.1056/NEJMOA1414221}, DOI={10.1056/NEJMOA1414221}, abstractNote={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.).}, number={20}, journal={New England Journal of Medicine}, publisher={Massachusetts Medical Society}, author={Bainbridge, James W.B. and Mehat, Manjit S. and Sundaram, Venki and Robbie, Scott J. and Barker, Susie E. and Ripamonti, Caterina and Georgiadis, Anastasios and Mowat, Freya M. and Beattie, Stuart G. and Gardner, Peter J. and et al.}, year={2015}, month={May}, pages={1887–1897} }
 @article{narfström_jalomäki_mowat_samardzija_chadieu_bergstrom_bragadottir_grimm_2014, title={Assessment of a novel pigmentary chorioretinopathy in the Chinese Crested dog}, volume={2}, number={2}, journal={JSciMed Ophthalmology}, author={Narfström, K. and Jalomäki, S. and Mowat, F.M. and Samardzija, M. and Chadieu, G. and Bergstrom, T. and Bragadottir, R. and Grimm, C.}, year={2014}, pages={1018} }
 @article{minella_mowat_willett_sledge_bartoe_bennett_petersen-jones_2014, title={Differential targeting of feline photoreceptors by recombinant adeno-associated viral vectors: implications for preclinical gene therapy trials}, volume={21}, ISSN={0969-7128 1476-5462}, url={http://dx.doi.org/10.1038/gt.2014.65}, DOI={10.1038/gt.2014.65}, abstractNote={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.}, number={10}, journal={Gene Therapy}, publisher={Springer Nature}, author={Minella, A L and Mowat, F M and Willett, K L and Sledge, D and Bartoe, J T and Bennett, J and Petersen-Jones, S M}, year={2014}, month={Jul}, pages={913–920} }
 @article{bruewer_mowat_bartoe_boye_hauswirth_petersen-jones_2013, title={Evaluation of Lateral Spread of Transgene Expression following Subretinal AAV–Mediated Gene Delivery in Dogs}, volume={8}, ISSN={1932-6203}, url={http://dx.doi.org/10.1371/journal.pone.0060218}, DOI={10.1371/journal.pone.0060218}, abstractNote={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.}, number={4}, journal={PLoS ONE}, publisher={Public Library of Science (PLoS)}, author={Bruewer, Ashlee R. and Mowat, Freya M. and Bartoe, Joshua T. and Boye, Sanford L. and Hauswirth, William W. and Petersen-Jones, Simon M.}, editor={Li, TiansenEditor}, year={2013}, month={Apr}, pages={e60218} }
 @article{annear_mowat_bartoe_querubin_azam_basche_curran_smith_bainbridge_ali_et al._2013, title={Successful Gene Therapy in Older Rpe65-Deficient Dogs Following Subretinal Injection of an Adeno-Associated Vector Expressing RPE65}, volume={24}, ISSN={1043-0342 1557-7422}, url={http://dx.doi.org/10.1089/hum.2013.146}, DOI={10.1089/hum.2013.146}, abstractNote={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.}, number={10}, journal={Human Gene Therapy}, publisher={Mary Ann Liebert Inc}, author={Annear, Matthew J. and Mowat, Freya M. and Bartoe, Joshua T. and Querubin, Janice and Azam, Selina A. and Basche, Mark and Curran, Paul G. and Smith, Alexander J. and Bainbridge, James W.B. and Ali, Robin R. and et al.}, year={2013}, month={Oct}, pages={883–893} }
 @article{mowat_gornik_dinculescu_boye_hauswirth_petersen-jones_bartoe_2013, title={Tyrosine capsid-mutant AAV vectors for gene delivery to the canine retina from a subretinal or intravitreal approach}, volume={21}, ISSN={0969-7128 1476-5462}, url={http://dx.doi.org/10.1038/gt.2013.64}, DOI={10.1038/gt.2013.64}, abstractNote={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.}, number={1}, journal={Gene Therapy}, publisher={Springer Nature}, author={Mowat, F M and Gornik, K R and Dinculescu, A and Boye, S L and Hauswirth, W W and Petersen-Jones, S M and Bartoe, J T}, year={2013}, month={Nov}, pages={96–105} }
 @article{mowat_gonzalez_luhmann_lange_duran_smith_maxwell_ali_bainbridge_2012, title={Endogenous Erythropoietin Protects Neuroretinal Function in Ischemic Retinopathy}, volume={180}, ISSN={0002-9440}, url={http://dx.doi.org/10.1016/j.ajpath.2011.12.033}, DOI={10.1016/j.ajpath.2011.12.033}, abstractNote={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. 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. Retinal ischemia is a common feature of major causes of vision loss including diabetes,1Bek T. Inner retinal ischaemia: current understanding and needs for further investigations.Acta Ophthalmol. 2009; 87: 362-367Crossref PubMed Scopus (60) Google Scholar retinal vascular occlusion,2Wittström E. Ponjavic V. Lövestam-Adrian M. Larsson J. Andréasson S. Electrophysiological evaluation and visual outcome in patients with central retinal vein occlusion, primary open-angle glaucoma and neovascular glaucoma.Acta Ophthalmol. 2010; 88: 86-90Crossref PubMed Scopus (9) Google Scholar, 3Rehak J. Rehak M. Branch retinal vein occlusion: pathogenesis, visual prognosis, and treatment modalities.Curr Eye Res. 2008; 33: 111-131Crossref PubMed Scopus (337) Google Scholar and retinopathy of prematurity.4Sapieha P. Joyal J.S. Rivera J.C. Kermorvant-Duchemin E. Sennlaub F. Hardy P. Lachapelle P. Chemtob S. Retinopathy of prematurity: understanding ischemic retinal vasculopathies at an extreme of life.J Clin Invest. 2010; 120: 3022-3032Crossref PubMed Scopus (191) Google Scholar The retina has a uniquely high metabolic demand for oxygen that is normally met by a highly efficient vascular supply. Insufficiency of the retinal circulation causes neuroretinal dysfunction and degeneration. Focal retinal ischemia results in selective damage to specific subpopulations of retinal neurons and can result in cellular death by apoptosis or necrosis.5Rosenbaum D.M. Rosenbaum P.S. Gupta A. Michaelson M.D. Hall D.H. Kessler J.A. Retinal ischemia leads to apoptosis which is ameliorated by aurintricarboxylic acid.Vision Res. 1997; 37: 3445-3451Crossref PubMed Scopus (67) Google Scholar Dysfunction and degeneration of the inner retina in ischemic retinopathies leads directly to vision loss and is associated with characteristic changes in electroretinogram (ERG) responses.6Fulton A.B. Hansen R.M. Petersen R.A. Vanderveen D.K. The rod photoreceptors in retinopathy of prematurity: an electroretinographic study.Arch Ophthalmol. 2001; 119: 499-505Crossref PubMed Scopus (91) Google Scholar, 7Fulton A.B. Hansen R.M. Electroretinogram responses and refractive errors in patients with a history of retinopathy prematurity.Doc Ophthalmol. 1995; 91: 87-100Crossref PubMed Scopus (34) Google Scholar, 8Akula J.D. Mocko J.A. Moskowitz A. Hansen R.M. Fulton A.B. The oscillatory potentials of the dark-adapted electroretinogram in retinopathy of prematurity.Invest Ophthalmol Vis Sci. 2007; 48: 5788-5797Crossref PubMed Scopus (57) Google Scholar Endogenous adaptive responses can help protect against ischemic injury. In the eye, however, the associated angiogenic response is typically aberrant, causing severe vision loss through edema, hemorrhage, and fibrosis. Aberrant ischemia-induced angiogenesis exacerbates hypoxic neuroretinal injury and is the target of novel therapies, including inhibitors of vascular endothelial growth factor (VEGF). However, treatments with nonspecific angiostatic agents fail to take into account appropriate beneficial endogenous adaptive responses to hypoxia and so risk compromising critical neuroprotective mechanisms. Therapeutic strategies can be made safer and more effective by considering how to protect and preserve these responses. Ischemia-induced retinal dysfunction and degeneration are features of rodent models of retinal vascular occlusion9Osborne N.N. Casson R.J. Wood J.P. Chidlow G. Graham M. Melena J. Retinal ischemia: mechanisms of damage and potential therapeutic strategies.Prog Retin Eye Res. 2004; 23: 91-147Crossref PubMed Scopus (861) Google Scholar, 10Dijk F. Bergen A.A. Kamphuis W. GAP-43 expression is upregulated in retinal ganglion cells after ischemia/reperfusion-induced damage.Exp Eye Res. 2007; 84: 858-867Crossref PubMed Scopus (50) Google Scholar and diabetes.11Aizu Y. Oyanagi K. Hu J. Nakagawa H. Degeneration of retinal neuronal processes and pigment epithelium in the early stage of the streptozotocin-diabetic rats.Neuropathology. 2002; 22: 161-170Crossref PubMed Scopus (128) Google Scholar, 12Gastinger M.J. Singh R.S. Barber A.J. Loss of cholinergic and dopaminergic amacrine cells in streptozotocin-diabetic rat and Ins2Akita-diabetic mouse retinas.Invest Ophthalmol Vis Sci. 2006; 47: 3143-3150Crossref PubMed Scopus (198) Google Scholar However, the angiogenic response in these models is unpredictable. Oxygen-induced retinopathy (OIR) in the mouse is a reliable model of ischemia-induced retinal neovascularization13Smith L.E. Wesolowski E. McLellan A. Kostyk S.K. D'Amato R. Sullivan R. D'Amore P.A. Oxygen-induced retinopathy in the mouse.Invest Ophthalmol Vis Sci. 1994; 35: 101-111PubMed Google Scholar that has been used extensively in the investigation and preclinical development of novel antiangiogenic treatments such as inhibitors of VEGF14Ozaki H. Seo M.S. Ozaki K. Yamada H. Yamada E. Okamoto N. Hofmann F. Wood J.M. Campochiaro P.A. Blockade of vascular endothelial cell growth factor receptor signaling is sufficient to completely prevent retinal neovascularization.Am J Pathol. 2000; 156: 697-707Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 15Pechan P. Rubin H. Lukason M. Ardinger J. DuFresne E. Hauswirth W.W. Wadsworth S.C. Scaria A. Novel anti-VEGF chimeric molecules delivered by AAV vectors for inhibition of retinal neovascularization.Gene Ther. 2009; 16: 10-16Crossref PubMed Scopus (99) Google Scholar, 16Konopatskaya O. Churchill A.J. Harper S.J. Bates D.O. Gardiner T.A. VEGF165b, an endogenous C-terminal splice variant of VEGF, inhibits retinal neovascularization in mice.Mol Vis. 2006; 12: 626-632PubMed Google Scholar and erythropoietin (EPO).17Watanabe D. Suzuma K. Matsui S. Kurimoto M. Kiryu J. Kita M. Suzuma I. Ohashi H. Ojima T. Murakami T. Kobayashi T. Masuda S. Nagao M. Yoshimura N. Takagi H. Erythropoietin as a retinal angiogenic factor in proliferative diabetic retinopathy.N Engl J Med. 2005; 353: 782-792Crossref PubMed Scopus (429) Google Scholar, 18Chen J. Connor K.M. Aderman C.M. Willett K.L. Aspegren O.P. Smith L.E. Suppression of retinal neovascularization by erythropoietin siRNA in a mouse model of proliferative retinopathy.Invest Ophthalmol Vis Sci. 2009; 50: 1329-1335Crossref PubMed Scopus (151) Google Scholar, 19Xiong S.Q. Xia X.B. Xu H.Z. Jiang J. 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Favazza T.L. Vyhovsky T.C. Fulton A.B. The neurovascular relation in oxygen-induced retinopathy.Mol Vis. 2008; 14: 2499-2508PubMed Google Scholar, 22Reynaud X. Hansen R.M. Fulton A.B. Effect of prior oxygen exposure on the electroretinographic responses of infant rats.Invest Ophthalmol Vis Sci. 1995; 36: 2071-2079PubMed Google Scholar The availability of transgenic strains, however, makes the mouse an attractive species to investigate disease mechanisms and to develop therapeutic strategies addressing ischemia-induced retinal neurodegeneration and neovascularization. EPO is a widely distributed oxygen-regulated hormone and paracrine cytokine that acts on multiple pathways, including erythropoiesis, angiogenesis, and neuroprotection. Expression of EPO is predominantly in the kidney and liver postnatally,23Eckardt K.U. Ratcliffe P.J. Tan C.C. Bauer C. Kurtz A. Age-dependent expression of the erythropoietin gene in rat liver and kidneys.J Clin Invest. 1992; 89: 753-760Crossref PubMed Scopus (104) Google Scholar but local expression is also evident in neuronal tissues, including the brain24Yu X. Shacka J.J. Eells J.B. Suarez-Quian C. Przygodzki R.M. Beleslin-Cokic B. Lin C.S. Nikodem V.M. Hempstead B. Flanders K.C. Costantini F. Noguchi C.T. Erythropoietin receptor signalling is required for normal brain development.Development. 2002; 129: 505-516PubMed Google Scholar, 25Chavez J.C. Baranova O. Lin J. Pichiule P. The transcriptional activator hypoxia inducible factor 2 (HIF-2/EPAS-1) regulates the oxygen-dependent expression of erythropoietin in cortical astrocytes.J Neurosci. 2006; 26: 9471-9481Crossref PubMed Scopus (201) Google Scholar and retina.26Grimm C. Wenzel A. Groszer M. Mayser H. Seeliger M. Samardzija M. Bauer C. Gassmann M. Reme C.E. HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration.Nat Med. 2002; 8: 718-724Crossref PubMed Scopus (475) Google Scholar, 27Morita M. Ohneda O. Yamashita T. Takahashi S. Suzuki N. Nakajima O. Kawauchi S. Ema M. Shibahara S. Udono T. Tomita K. Tamai M. Sogawa K. Yamamoto M. Fujii-Kuriyama Y. HLF/HIF-2alpha is a key factor in retinopathy of prematurity in association with erythropoietin.EMBO J. 2003; 22: 1134-1146Crossref PubMed Scopus (212) Google Scholar Specific disease processes may influence the location of EPO receptors in the retina.28Shah S.S. Tsang S.H. Mahajan V.B. Erythropoetin receptor expression in the human diabetic retina.BMC Res Notes. 2009; 2: 234Crossref PubMed Scopus (24) Google Scholar Because EPO is proangiogenic and is expressed in the retina, it presents a potential target for antiangiogenic therapy in retinal neovascular disorders. Vitreous EPO levels are elevated in patients with ischemic retinal diseases, including retinal vascular occlusion,29Stahl A. Buchwald A. Martin G. Junker B. Chen J. Hansen L.L. Agostini H.T. Smith L.E. Feltgen N. Vitreal levels of erythropoietin are increased in patients with retinal vein occlusion and correlate with vitreal VEGF and the extent of macular edema.Retina. 2010; 30: 1524-1529Crossref PubMed Scopus (35) Google Scholar diabetic retinopathy,17Watanabe D. Suzuma K. Matsui S. Kurimoto M. Kiryu J. Kita M. Suzuma I. Ohashi H. Ojima T. Murakami T. Kobayashi T. Masuda S. Nagao M. Yoshimura N. Takagi H. Erythropoietin as a retinal angiogenic factor in proliferative diabetic retinopathy.N Engl J Med. 2005; 353: 782-792Crossref PubMed Scopus (429) Google Scholar, 30Inomata Y. Hirata A. Takahashi E. Kawaji T. Fukushima M. Tanihara H. Elevated erythropoietin in vitreous with ischemic retinal diseases.Neuroreport. 2004; 15: 877-879Crossref PubMed Scopus (54) Google Scholar, 31Katsura Y. Okano T. Matsuno K. Osako M. Kure M. Watanabe T. Iwaki Y. Noritake M. Kosano H. Nishigori H. Matsuoka T. Erythropoietin is highly elevated in vitreous fluid of patients with proliferative diabetic retinopathy.Diabetes Care. 2005; 28: 2252-2254Crossref PubMed Scopus (82) Google Scholar and retinopathy of prematurity.32Sato T. Kusaka S. Shimojo H. Fujikado T. Vitreous levels of erythropoietin and vascular endothelial growth factor in eyes with retinopathy of prematurity.Ophthalmology. 2009; 116: 1599-1603Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar In the hypoxic phase of murine OIR, retinal EPO expression is increased, and supplementation with high doses of EPO can be proangiogenic in this context.33Chen J. Connor K.M. Aderman C.M. Smith L.E. Erythropoietin deficiency decreases vascular stability in mice.J Clin Invest. 2008; 118: 526-533PubMed Google Scholar Systemic EPO supplementation also has been shown to be an independent risk factor for the development of retinopathy of prematurity in premature babies.34Suk K.K. Dunbar J.A. Liu A. Daher N.S. Leng C.K. Leng J.K. Lim P. Weller S. Fayard E. Human recombinant erythropoietin and the incidence of retinopathy of prematurity: a multiple regression model.J AAPOS. 2008; 12: 233-238Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar Recent studies have advocated the use of EPO inhibitors such as EPO-targeting short interfering RNA (siRNA)18Chen J. Connor K.M. Aderman C.M. Willett K.L. Aspegren O.P. Smith L.E. Suppression of retinal neovascularization by erythropoietin siRNA in a mouse model of proliferative retinopathy.Invest Ophthalmol Vis Sci. 2009; 50: 1329-1335Crossref PubMed Scopus (151) Google Scholar or antibodies17Watanabe D. Suzuma K. Matsui S. Kurimoto M. Kiryu J. Kita M. Suzuma I. Ohashi H. Ojima T. Murakami T. Kobayashi T. Masuda S. Nagao M. Yoshimura N. Takagi H. Erythropoietin as a retinal angiogenic factor in proliferative diabetic retinopathy.N Engl J Med. 2005; 353: 782-792Crossref PubMed Scopus (429) Google Scholar in the hypoxic phase of OIR, to inhibit neovascularization. There is emerging evidence that proangiogenic factors such as VEGF and EPO have important roles in neuroprotection in the retina35Saint-Geniez M. Maharaj A.S. Walshe T.E. Tucker B.A. Sekiyama E. Kurihara T. Darland D.C. Young M.J. D'Amore P.A. Endogenous VEGF is required for visual function: evidence for a survival role on muller cells and photoreceptors.PLoS One. 2008; 3: e3554Crossref PubMed Scopus (541) Google Scholar, 36Nishijima K. Ng Y.S. Zhong L. Bradley J. Schubert W. Jo N. Akita J. Samuelsson S.J. Robinson G.S. Adamis A.P. Shima D.T. Vascular endothelial growth factor-A is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury.Am J Pathol. 2007; 171: 53-67Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar and in the central nervous system.37Brines M.L. Ghezzi P. Keenan S. Agnello D. de Lanerolle N.C. Cerami C. Itri L.M. Cerami A. Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury.Proc Natl Acad Sci USA. 2000; 97: 10526-10531Crossref PubMed Scopus (1295) Google Scholar Supplemental EPO can protect the adult retina from ischemic damage38Junk A.K. Mammis A. Savitz S.I. Singh M. Roth S. Malhotra S. Rosenbaum P.S. Cerami A. Brines M. Rosenbaum D.M. Erythropoietin administration protects retinal neurons from acute ischemia-reperfusion injury.Proc Natl Acad Sci USA. 2002; 99: 10659-10664Crossref PubMed Scopus (412) Google Scholar and from light-induced retinal degeneration.26Grimm C. Wenzel A. Groszer M. Mayser H. Seeliger M. Samardzija M. Bauer C. Gassmann M. Reme C.E. HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration.Nat Med. 2002; 8: 718-724Crossref PubMed Scopus (475) Google Scholar, 39Grimm C. Wenzel A. Stanescu D. Samardzija M. Hotop S. Groszer M. Naash M. Gassmann M. Remé C. Constitutive overexpression of human erythropoietin protects the mouse retina against induced but not inherited retinal degeneration.J Neurosci. 2004; 24: 5651-5658Crossref PubMed Scopus (114) Google Scholar Many retinal neuronal cell types express receptors for EPO (EPO-R),40Grimm C. Hermann D.M. Bogdanova A. Hotop S. Kilic U. Wenzel A. Kilic E. Gassmann M. Neuroprotection by hypoxic preconditioning: HIF-1 and erythropoietin protect from retinal degeneration.Semin Cell Dev Biol. 2005; 16: 531-538Crossref PubMed Scopus (83) Google Scholar, 41Weishaupt J.H. Rohde G. Pölking E. Siren A.L. Ehrenreich H. Bähr M. Effect of erythropoietin axotomy-induced apoptosis in rat retinal ganglion cells.Invest Ophthalmol Vis Sci. 2004; 45: 1514-1522Crossref PubMed Scopus (191) Google Scholar and EPO-R expression is up-regulated after retinal ischemia.38Junk A.K. Mammis A. Savitz S.I. Singh M. Roth S. Malhotra S. Rosenbaum P.S. Cerami A. Brines M. Rosenbaum D.M. Erythropoietin administration protects retinal neurons from acute ischemia-reperfusion injury.Proc Natl Acad Sci USA. 2002; 99: 10659-10664Crossref PubMed Scopus (412) Google Scholar EPO may also have an important role in neurodevelopment in the eye and the brain.24Yu X. Shacka J.J. Eells J.B. Suarez-Quian C. Przygodzki R.M. Beleslin-Cokic B. Lin C.S. Nikodem V.M. Hempstead B. Flanders K.C. Costantini F. Noguchi C.T. Erythropoietin receptor signalling is required for normal brain development.Development. 2002; 129: 505-516PubMed Google Scholar Neurodevelopmental abnormalities in premature infants with systemic EPO deficiency42Bierer R. Peceny M.C. Hartenberger C.H. Ohls R.K. Erythropoietin concentrations and neurodevelopmental outcome in preterm infants.Pediatrics. 2006; 118: e635-e640Crossref PubMed Scopus (128) Google Scholar can be ameliorated by EPO supplementation.43Brown M.S. Eichorst D. Lala-Black B. Gonzalez R. Higher cumulative doses of erythropoietin and developmental outcomes in preterm infants.Pediatrics. 2009; 124: e681-e687Crossref PubMed Scopus (69) Google Scholar To date, however, similar studies examining the effects of EPO on retinal neurodevelopment have not been performed. The purpose of the present study was to determine the effect of ischemia on neuroretinal function and survival in murine OIR and to investigate the role of EPO in this model. We investigated the effect of ischemia in murine OIR on inner retinal function and neurodegeneration by electroretinography and detailed morphology, both in the short and long term. Using this model, we found that endogenous EPO can protect neuroretinal function. The Epo-Tag transgenic mouse is described elsewhere.44Maxwell P.H. Osmond M.K. Pugh C.W. Heryet A. Nicholls L.G. Tan C.C. Doe B.G. Ferguson D.J. Johnson M.H. Ratcliffe P.J. Identification of the renal erythropoietin-producing cells using transgenic mice.Kidney Int. 1993; 44: 1149-1162Crossref PubMed Scopus (349) Google Scholar Briefly, Epo-Tag mice have a 2.7-kb insertion of the viral simian virus 40 T (SV40T) antigen coding sequence into the 5′ untranslated region of the Epo gene.44Maxwell P.H. Osmond M.K. Pugh C.W. Heryet A. Nicholls L.G. Tan C.C. Doe B.G. Ferguson D.J. Johnson M.H. Ratcliffe P.J. Identification of the renal erythropoietin-producing cells using transgenic mice.Kidney Int. 1993; 44: 1149-1162Crossref PubMed Scopus (349) Google Scholar This insertion leads to relative EPO deficiency, although homozygous mice do have a low level of EPO production from transcripts running through the viral polyA signal into the EPO coding sequence. Homozygous Epo-Tag−/− mice are profoundly anemic, whereas heterozygous Epo-Tag+/− adult mice are only mildly anemic.45Raja K.B. Maxwell P.H. Ratcliffe P.J. Salisbury J.R. Simpson R.J. Peters T.J. Iron metabolism in transgenic mice with hypoplastic anaemia due to incomplete deficiency of erythropoietin.Br J Haematol. 1997; 96: 248-253Crossref PubMed Scopus (10) Google Scholar We chose to evaluate heterozygous Epo-Tag mice in the present study, because the severe anemia in Epo-Tag−/− mice might influence OIR through an effect on oxygen delivery to the eye. Epo-Tag+/− mice were maintained on a C57Bl6J background (Harlan United Kingdom, Bicester, UK) as mixed litters of Epo-Tag+/− and wild-type animals (Epo-Tag+/+). All animals were used with institutional ethical approval and under a United Kingdom Home Office project license and personal license. All procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Genotyping by PCR analysis was performed at postnatal day 7 (p7) and additionally after euthanasia. Ear-clip samples were digested using proteinase K (0.1 mg/mL in 30 mmol/L Tris-HCl pH 8.0 0.5% w/v SDS at 55°C for 16 hours; Sigma-Aldrich, Gillingham, UK), and genomic DNA was purified by ethanol. A commercial master mix containing Taq polymerase [1.1× ReddyMix PCR master mix and 1.5 mmol/L MgCl2 (ABgene; Thermo Scientific, Epsom, UK)] was used according to the manufacturer's instructions. Primers used for genotyping mice were 5′-CGCACACACAGCTTCACCC-3′ (mEPO forward), 5′-CTGTAGGGCCAGATCACC-3′ (mEPO reverse), and 5′-GCCTAGGCCTCCAAAAAAGC-3′ (SV40T reverse). Separate reactions were performed on each ear-clip sample to determine the presence of the wild-type mEPO allele and the SV40T allele. Nursing mothers and their pups were placed in a 75% oxygen supply chamber from p7 to p12, as described previously.13Smith L.E. Wesolowski E. McLellan A. Kostyk S.K. D'Amato R. Sullivan R. D'Amore P.A. Oxygen-induced retinopathy in the mouse.Invest Ophthalmol Vis Sci. 1994; 35: 101-111PubMed Google Scholar A constant low flow of 80% oxygen (with the balance as nitrogen) was provided to a closed acrylic glass chamber. The oxygen level was monitored twice daily, and maintained at 75% ± 3% O2. The mice were exposed to a standard 12-hour light-dark cycle. Samples were collected at various time points after the return to room air. Hematocrit measurement was performed as a terminal procedure, because of the limited blood volume in young mice and the potential for induced anemia from repeated blood sampling. Animals were terminally anesthetized, and venous blood was obtained from the right atrium immediately after death. Blood was collected directly into heparinized microhematocrit capillary tubes (Hawksley; Lancing, UK), to prevent clotting. Samples were spun at 5,400 × g for 5 minutes in a microhematocrit centrifuge (Biodynamics Selectafuge 24; BCL, Lewes, UK), and the percentage packed cell volume (%PCV) was calculated using a premeasured scale. Supplementation with recombinant human erythropoietin (rHuEPO) (Epoetin alfa, 40,000 U/mL; Janssen-Cilag, High Wycombe, UK) was administered systemically by intraperitoneal injection starting at p14 and ending at p25 at a dose rate of 5000 U/kg diluted in PBS, as described previously.26Grimm C. Wenzel A. Groszer M. Mayser H. Seeliger M. Samardzija M. Bauer C. Gassmann M. Reme C.E. HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration.Nat Med. 2002; 8: 718-724Crossref PubMed Scopus (475) Google Scholar, 33Chen J. Connor K.M. Aderman C.M. Smith L.E. Erythropoietin deficiency decreases vascular stability in mice.J Clin Invest. 2008; 118: 526-533PubMed Google Scholar Each animal was weighed daily, and dosing was administered per 0.1 g body weight in a maximum volume of 0.15 mL. Previous studies have shown that systemic supplementation with erythropoietin in murine OIR from p14 to p16 does not affect the vascular phenotype at p17.33Chen J. Connor K.M. Aderman C.M. Smith L.E. Erythropoietin deficiency decreases vascular stability in mice.J Clin Invest. 2008; 118: 526-533PubMed Google Scholar Mice were anesthetized using an intraperitoneal injection of a mixture containing medetomidine hydrochloride (1 mg/mL Domitor; Pfizer Animal Health, Kent, UK), ketamine (100 mg/mL; Fort Dodge Animal Health, Southampton, UK), and sterile water in the ratio 5:3:42. Anesthesia was reversed using an intraperitoneal injection of the reversal agent atipamezole hydrochloride (Antisedan, 5 mg/mL; Pfizer Animal Health) in sterile water (ratio 1:50). Electroretinography (ERG) was performed at p26 and p60. Mice were dark-adapted for 16 hours. Animals were anesthetized for ERG, and pupils were dilated using 1% tropicamide (Minims; Bausch & Lomb, Kingston-on-Thames, UK) applied topically. Water-based ocular lubricant (Viscotears; Novartis Pharmaceuticals United Kingdom, Camberley, UK) was used to improve electrical contact with the electrode and to keep the eyes moist during the procedure. All manipulations before scotopic ERG were performed under dim red-light illumination. ERGs were obtained using an Espion ERG system (Diagnosys, Cambridge, UK). Ganzfeld ERGs were obtained from both eyes simultaneously using contact platinum corneal electrodes on each eye; one reference electrode was placed sublingually, and the ground electrode was placed subdermally in the midline at the base of the tail. Electrical impedance was balanced for each eye before recording (∼10 kΩ). A scotopic flash intensity series was performed, followed by light-adapted photopic flicker measurements. In all measurements, 400 ms of response was recorded. Recordings were filtered from 0 to 1 kHz and were digitized with a sampling frequency of 5 kHz. The first 10 ms before the stimulus onset of each recording was automatically used to set zero for the trace. For scotopic flash examination, a multiple white-light flash intensity series was performed, with light intensity increasing in six steps from 0.001 cd · s/m2 to 5 cd · s/m2. Ten responses (for intensities <1 cd · s/m2) or five responses (for intensities ≥1 cd · s/m2) per intensity were collected and averaged for use in subsequent analysis. Measurements for amplitude and implicit time were taken from the trough of the a-wave to the peak of the b-wave. The photopic a-wave was defined as the maximal negative amplitude after the onset of light exposure. The photopic b-wave was defined as the maximal positive amplitude after the onset of light exposure, compensating visually for the waveforms of the OPs. Statistical comparison was performed at the 1 cd · s/m2 intensity representing a mixed rod-cone response46Lei B. Yao G. Zhang K. Hofeldt K.J. Chang B. Study of rod- and cone-driven oscillatory potentials in mice.Invest Ophthalmol Vis Sci. 2006; 47: 2732-2738Crossref PubMed Scopus (83) Google Scholar and provided the maximal a-wave amplitude. OPs were obtained with a fifth-order Butterworth filter and 65- to 235-Hz bandpass using a custom-made program coded in MATLAB (MathWorks, Natick, MA), as described previously.8Akula J.D. Mocko J.A. Moskowitz A. Hansen R.M. Fulton A.B. The oscillatory potentials of the dark-adapted electroretinogram in retinopathy of prematurity.Invest Ophthalmol Vis Sci. 2007; 48: 5788-5797Crossref PubMed Scopus (57) Google Scholar, 46Lei B. Yao G. Zhang K. Hofeldt K.J. Chang B. Study of rod- and cone-driven oscillatory potentials in mice.Invest Ophthalmol Vis Sci. 2006; 47: 2732-2738Crossref PubMed Scopus (83) Google Scholar OP 1 was not analyzed, because of the potential for a-wave contamination. Analysis of OPs was performed to determine the sum of the OP amplitudes of OP 2 to 5 (SOPA).8Akula J.D. Mocko J.A. Moskowitz A. Hansen R.M. Fulton A.B. The oscillatory potentials of the dark-adapted electroretinogram in retinopathy of prematurity.Invest Ophthalmol Vis Sci. 2007; 48: 5788-5797Crossref PubMed Scopus (57) Google Scholar The OPs were also analyzed in the frequency domain by using a fast Fourier transformation}, number={4}, journal={The American Journal of Pathology}, publisher={Elsevier BV}, author={Mowat, Freya M. and Gonzalez, Francisco and Luhmann, Ulrich F.O. and Lange, Clemens A. and Duran, Yanai and Smith, Alexander J. and Maxwell, Patrick H. and Ali, Robin R. and Bainbridge, James W.B.}, year={2012}, month={Apr}, pages={1726–1739} }
 @inbook{petersen-jones_annear_bartoe_mowat_barker_smith_bainbridge_ali_2012, series={Advances in Experimental Medicine and Biology}, title={Gene Augmentation Trials Using the Rpe65-Deficient Dog: Contributions Towards Development and Refinement of Human Clinical Trials}, ISBN={9781461406303 9781461406310}, ISSN={0065-2598}, url={http://dx.doi.org/10.1007/978-1-4614-0631-0_24}, DOI={10.1007/978-1-4614-0631-0_24}, abstractNote={Dogs with a spontaneous mutation in Rpe65 have been key in the testing of gene augmentation therapy using viral vectors to introduce a normal copy of the Rpe65 gene. These ground-breaking experiments have led to Phase I/II human clinical trials for treatment of Leber congenital amaurosis type II (LCAII). The Rpe65-deficient dog remains a useful model for studies to refine this treatment approach. A recent question it has been used to answer is whether or not immune response resulting from the gene augmentation treatment of one eye interferes with the success of the same treatment in the second eye. Fortunately, it was shown that treatment of the second eye had a similar success to that of the first eye, clearly demonstrating potential for gene augmentation therapy in the second eye of LCAII patients.}, booktitle={Retinal Degenerative Diseases}, publisher={Springer US}, author={Petersen-Jones, Simon M. and Annear, Matthew J. and Bartoe, Joshua T. and Mowat, Freya M. and Barker, Susie E. and Smith, Alexander J. and Bainbridge, James W. and Ali, Robin R.}, editor={Lavail, M. and Ash, J. and Anderson, R. and Hollyfield, J. and Grimm, C.Editors}, year={2012}, month={Nov}, pages={177–182}, collection={Advances in Experimental Medicine and Biology} }
 @article{mowat_breuwer_bartoe_annear_zhang_smith_bainbridge_petersen-jones_ali_2012, title={RPE65 gene therapy slows cone loss in Rpe65-deficient dogs}, volume={20}, ISSN={0969-7128 1476-5462}, url={http://dx.doi.org/10.1038/gt.2012.63}, DOI={10.1038/gt.2012.63}, abstractNote={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.}, number={5}, journal={Gene Therapy}, publisher={Springer Nature}, author={Mowat, F M and Breuwer, A R and Bartoe, J T and Annear, M J and Zhang, Z and Smith, A J and Bainbridge, J W B and Petersen-Jones, S M and Ali, R R}, year={2012}, month={Sep}, pages={545–555} }
 @article{lange_luhmann_mowat_georgiadis_west_abrahams_sayed_powner_fruttiger_smith_et al._2012, title={Von Hippel-Lindau protein in the RPE is essential for normal ocular growth and vascular development}, volume={139}, ISSN={0950-1991 1477-9129}, url={http://dx.doi.org/10.1242/dev.070813}, DOI={10.1242/dev.070813}, abstractNote={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.}, number={13}, journal={Development}, publisher={The Company of Biologists}, author={Lange, C. A. K. and Luhmann, U. F. O. and Mowat, F. M. and Georgiadis, A. and West, E. L. and Abrahams, S. and Sayed, H. and Powner, M. B. and Fruttiger, M. and Smith, A. J. and et al.}, year={2012}, month={May}, pages={2340–2350} }
 @article{mowat_luhmann_smith_lange_duran_harten_shukla_maxwell_ali_bainbridge_2010, title={HIF-1alpha and HIF-2alpha Are Differentially Activated in Distinct Cell Populations in Retinal Ischaemia}, volume={5}, ISSN={1932-6203}, url={http://dx.doi.org/10.1371/journal.pone.0011103}, DOI={10.1371/journal.pone.0011103}, abstractNote={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.}, number={6}, journal={PLoS ONE}, publisher={Public Library of Science (PLoS)}, author={Mowat, Freya M. and Luhmann, Ulrich F. O. and Smith, Alexander J. and Lange, Clemens and Duran, Yanai and Harten, Sarah and Shukla, Deepa and Maxwell, Patrick H. and Ali, Robin R. and Bainbridge, James W. B.}, editor={Koch, Karl-WilhelmEditor}, year={2010}, month={Jun}, pages={e11103} }
 @article{georgiadis_tschernutter_bainbridge_balaggan_mowat_west_munro_thrasher_matter_balda_et al._2010, title={The Tight Junction Associated Signalling Proteins ZO-1 and ZONAB Regulate Retinal Pigment Epithelium Homeostasis in Mice}, volume={5}, ISSN={1932-6203}, url={http://dx.doi.org/10.1371/journal.pone.0015730}, DOI={10.1371/journal.pone.0015730}, abstractNote={Cell-cell adhesion regulates the development and function of epithelia by providing mechanical support and by guiding cell proliferation and differentiation. The tight junction (TJ) protein zonula occludens (ZO)-1 regulates cell proliferation and gene expression by inhibiting the activity of the Y-box transcription factor ZONAB in cultured epithelial cells. We investigated the role of this TJ-associated signalling pathway in the retinal pigment epithelium (RPE) in vivo by lentivirally-mediated overexpression of ZONAB, and knockdown of its cellular inhibitor ZO-1. Both overexpression of ZONAB or knockdown of ZO-1 resulted in increased RPE proliferation, and induced ultrastructural changes of an epithelial-mesenchymal transition (EMT)-like phenotype. Electron microscopy analysis revealed that transduced RPE monolayers were disorganised with increased pyknosis and monolayer breaks, correlating with increased expression of several EMT markers. Moreover, fluorescein angiography analysis demonstrated that the increased proliferation and EMT-like phenotype induced by overexpression of ZONAB or downregulation of ZO-1 resulted in RPE dysfunction. These findings demonstrate that ZO-1 and ZONAB are critical for differentiation and homeostasis of the RPE monolayer and may be involved in RPE disorders such as proliferative vitroretinopathy and atrophic age-related macular degeneration.}, number={12}, journal={PLoS ONE}, publisher={Public Library of Science (PLoS)}, author={Georgiadis, Anastasios and Tschernutter, Marion and Bainbridge, James W. B. and Balaggan, Kamaljit S. and Mowat, Freya and West, Emma L. and Munro, Peter M. G. and Thrasher, Adrian J. and Matter, Karl and Balda, Maria S. and et al.}, editor={Klymkowsky, MichaelEditor}, year={2010}, month={Dec}, pages={e15730} }
 @article{mowat_petersen-jones_williamson_williams_luthert_ali_bainbridge_2008, title={Topographical characterization of cone photoreceptors and the area centralis of the canine retina}, volume={14}, journal={Molecular Vision}, author={Mowat, F.M. and Petersen-Jones, S.M. and Williamson, H. and Williams, D.L. and Luthert, P.J. and Ali, R.R. and Bainbridge, J.W.}, year={2008}, pages={2518–2527} }