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PurposeCorneal dysmorphologies (CDs) are typically classified as either regressive degenerative corneal dystrophies (CDtrs) or defective growth and differentiation-driven corneal dysplasias (CDyps). Both eye disorders have multifactorial etiologies. While previous work has elucidated many aspects of CDs, such as presenting symptoms, epidemiology, and pathophysiology, the genetic mechanisms remain incompletely understood. The purpose of this study was to analyze phenotype data from 8,707 knockout mouse lines to identify new genes associated with the development of CDs in humans.Methods8,707 knockout mouse lines phenotyped by the International Mouse Phenotyping Consortium were queried for genes associated with statistically significant (P

The cornea, as a dynamic and responsive tissue, constantly interacts with mechanical forces in order to maintain its structural integrity, barrier function, transparency and refractive power. Cells within the cornea sense and respond to various mechanical forces that fundamentally regulate their morphology and fate in development, homeostasis and pathophysiology. Corneal cells also dynamically regulate their extracellular matrix (ECM) with ensuing cell-ECM crosstalk as the matrix serves as a dynamic signaling reservoir providing biophysical and biochemical cues to corneal cells. Here we provide an overview of mechanotransduction signaling pathways then delve into the recent advances in corneal mechanobiology, focusing on the interplay between mechanical forces and responses of the corneal epithelial, stromal, and endothelial cells. We also identify species-specific differences in corneal biomechanics and mechanotransduction to facilitate identification of optimal animal models to study corneal wound healing, disease, and novel therapeutic interventions. Finally, we identify key knowledge gaps and therapeutic opportunities in corneal mechanobiology that are pressing for the research community to address especially pertinent within the domains of limbal stem cell deficiency, keratoconus and Fuchs' endothelial corneal dystrophy. By furthering our understanding corneal mechanobiology, we can contextualize discoveries regarding corneal diseases as well as innovative treatments for them.

Sibling female and male Chihuahuas were evaluated for a 9-month history of tachypnea that failed to respond to fenbendazole, doxycycline, amoxicillin-clavulanate, and prednisone. Physical examination identified tachypnea, hyperpnea, and harsh bronchovesicular lung sounds. Fundic examination disclosed diffuse chorioretinitis, manifested as multifocal chorioretinal granulomas in the female dog and occasional chorioretinal scars in the male dog. Thoracic radiographs indicated moderate to severe interstitial to broncho-interstitial infiltrates in both dogs. Serum and urine antigen and antibody testing in the female dog failed to identify infectious agents, but cytologic assessment of hepatic lymph node, liver, and splenic aspirates identified Pneumocystis trophozoites. Infection was confirmed in both dogs by 28S rRNA PCR sequencing from multiple tissue samples. The female dog responded well to trimethoprim-sulfamethoxazole, but the male dog was euthanized because of liver failure, presumably related to antimicrobial treatment.

PurposeWe sought to define the role of Wwtr1 in murine ocular structure and function and determine the role of mechanotransduction in Fuchs' endothelial corneal dystrophy (FECD), with emphasis on interactions between corneal endothelial cells (CEnCs) and Descemet's membrane (DM).MethodsA Wwtr1 deficient mouse colony was established, and advanced ocular imaging, atomic force microscope (AFM), and histology/immunofluorescence were performed. Corneal endothelial wound healing was assessed using cryoinjury and phototherapeutic keratectomy in Wwtr1 deficient mice. Expression of WWTR1/TAZ was determined in the corneal endothelium from normal and FECD-affected patients; WWTR1 was screened for coding sequence variants in this FECD cohort.ResultsMice deficient in Wwtr1 had reduced CEnC density, abnormal CEnC morphology, softer DM, and thinner corneas versus wildtype controls by 2 months of age. Additionally, CEnCs had altered expression and localization of Na/K-ATPase and ZO-1. Further, Wwtr1 deficient mice had impaired CEnC wound healing. The WWTR1 transcript was highly expressed in healthy human CEnCs comparable to other genes implicated in FECD pathogenesis. Although WWTR1 mRNA expression was comparable between healthy and FECD-affected patients, WWTR1/TAZ protein concentrations were higher and localized to the nucleus surrounding guttae. No genetic associations were found in WWTR1 and FECD in a patient cohort compared to controls.ConclusionsThere are common phenotypic abnormalities seen between Wwtr1 deficient and FECD-affected patients, suggesting that Wwtr1 deficient mice could function as a murine model of late-onset FECD. Despite the lack of a genetic association between FECD and WWTR1, aberrant WWTR1/TAZ protein subcellular localization and degradation may play critical roles in the pathogenesis of FECD.

ObjectivesThis study aimed to define the antimicrobial peptide (AMP) expression pattern of the equine ocular surface and amniotic membrane using a targeted qPCR approach and 3'Tag-sequencing. It will serve as a reference for future studies of ocular surface innate immunity and amniotic membrane therapies.ProceduresA targeted qPCR approach was used to investigate the presence of orthologs for three of the most highly expressed beta-defensins (DEFB1, DEFB4B, and DEFB103A) of the human ocular surface and amniotic membrane in equine corneal epithelium, conjunctiva, and amniotic membrane. 3'Tag-sequencing was performed on RNA from one sample of corneal epithelium, conjunctiva, and amniotic membrane to further characterize their AMP expression.ResultsEquine corneal epithelium, conjunctiva, and amniotic membrane expressed DEFB1, DEFB4B, and DEFB103A. DEFB103A was expressed at the highest amounts in corneal epithelium, while DEFB4B was most highly expressed in conjunctiva and amniotic membrane. 3'Tag-sequencing from all three tissues confirmed these findings and identified expression of five additional beta-defensins, 11 alpha-defensins and two cathelicidins, with the alpha-defensins showing higher normalized read counts than the beta-defensins.ConclusionsThis study identified AMP expression in the equine cornea and conjunctiva, suggesting that they play a key role in the protection of the equine eye, similar to the human ocular surface. We also determined that equine amniotic membrane expresses a substantial number of AMPs suggesting it could potentiate an antimicrobial effect as a corneal graft material. Future studies will focus on defining the antimicrobial activity of these AMPs and determining their role in microbial keratitis.

Dry eye disease is a complex ophthalmic disorder that consists of two main subtypes, aqueous deficient dry eye (ADDE) and evaporative dry eye disease (EDED). Due to the complex underlying physiology, human dry eye disease can be difficult to model in laboratory animal species. Thus, the identification and characterization of a spontaneous large animal model of dry eye disease is desirable. Dogs have been described as an ideal spontaneous model of ADDE due to the similar pathophysiology between dogs and humans. Recently, EDED and meibomian gland dysfunction (MGD) have been increasingly recognized and reported in dogs. These reports on EDED and MGD in dogs have identified similarities in pathophysiology, clinical presentations, and diagnostic parameters to humans with the comparable disorders. Additionally, the tests that are used to diagnose EDED and MGD in humans are more easily applicable to dogs than to laboratory species due to the comparable globe sizes between dogs and humans. The reported response of dogs to EDED and MGD therapies are similar to humans, suggesting that they would be a valuable preclinical model for the development of additional therapeutics. Further research and clinical awareness of EDED and MGD in dogs would increase their ability to be utilized as a preclinical model, improving the positive predictive value of therapeutics for EDED and MGD in both humans and dogs.

Graphene-based nanomaterials (GBNs) are widely used due to their chemical and physical properties for multiple commercial and environmental applications. From an occupational health perspective, there is concern regarding the effects of inhalation on the respiratory system, and many studies have been conducted to study inhalation impacts on lung. Similar to the respiratory system, the eyes may also be exposed to GBNs and thus impacted. In this study, immortalized human corneal epithelial (hTCEpi) cells and rabbit corneal fibroblasts (RCFs) were used to investigate the toxicity of eight types of GBN: graphene oxide (GO; 400 nm), GO (1 μm), partially reduced graphene oxide (PRGO; 400 nm), reduced graphene oxide (RGO; 400 nm), RGO (2 μm), graphene (110 nm), graphene (140 nm), and graphene (1 μm). We next examined the effects of these GBNs on hTCEpi cell migration. We also determined whether the expression of α-smooth muscle actin (αSMA), a myofibroblast marker, is altered by the GBNs using RCFs. We found that RGO (400 nm) and RGO (2 μm) were highly toxic to hTCEPi cells and RCFs meanwhile, PRGO (400 nm) was toxic only to hTCEpi cells. In addition, PRGO (400 nm), RGO (400 nm), and RGO (2 μm) inhibited hTCEpi cell migration and significantly increased αSMA mRNA expression. Further study in vivo is required to determine if RGO nanomaterials delay corneal epithelial healing and induce scar formation.

INTRODUCTION: Equine recurrent uveitis (ERU), an immune mediated disease characterized by repeated episodes of intra-ocular inflammation, affects 25% of horses in the USA and is the most common cause of glaucoma, cataracts, and blindness. Mesenchymal stromal cells (MSCs) have immunomodulatory properties, which are upregulated by preconditioning with toll-like receptor agonists. The objective was to evaluate safety and migration of TLR-3 agonist polyinosinic, polycytidylic acid (pIC)-activated MSCs injected subconjunctivally in healthy horses prior to clinical application in horses with ERU. We hypothesized that activated allogeneic MSCs injected subconjunctivally would not induce ocular or systemic inflammation and would remain in the conjunctiva for >14 days. METHODS: Bulbar subconjunctiva of two horses was injected with 10 × 106 pIC-activated (10 μg/mL, 2 h) GFP-labeled MSCs from one donor three times at two-week intervals. Vehicle (saline) control was injected in the contralateral conjunctiva. Horses received physical and ophthalmic exams [slit lamp biomicroscopy, rebound tonometry, fundic examination, and semiquantitative preclinical ocular toxicology scoring (SPOTS)] every 1-3 days. Systemic inflammation was assessed via CBC, fibrinogen, and serum amyloid A (SAA). Horses were euthanized 14 days following final injection. Full necropsy and histopathology were performed to examine ocular tissues and 36 systemic organs for MSC presence via IVIS Spectrum. Anti-GFP immunohistochemistry was performed on ocular tissues. RESULTS: No change in physical examinations was noted. Bloodwork revealed fibrinogen 100-300 mg/dL (ref 100-400) and SAA 0-25 μg/mL (ref 0-20). Ocular effects of the subjconjucntival injection were similar between MSC and control eyes on SPOTS grading system, with conjunctival hypermia, chemosis and ocular discharge noted bilaterally, which improved without intervention within 14 days. All other ocular parameters were unaffected throughout the study. Necropsy and histopathology revealed no evidence of systemic inflammation. Ocular histopathology was similar between MSC and control eyes. Fluorescent imaging analysis did not locate MSCs. Immunohistochemistry did not identify intact MSCs in the conjunctiva, but GFP-labeled cellular components were present in conjunctival phagocytic cells. DISCUSSION: Allogeneic pIC-activated conjunctival MSC injections were well tolerated. GFP-labeled tracking identified MSC components phagocytosed by immune cells subconjunctivally. This preliminary safety and tracking information is critical towards advancing immune conditioned cellular therapies to clinical trials in horses.

BackgroundImaging features obtained with Fourier-domain optical coherence tomography (FD-OCT) and in vivo confocal microscopy (IVCM) for corneal stromal disorders have been sparsely reported in dogs. This case report is a compilation of imaging features for three cases of different stromal disorders of the canine cornea which have not yet been reported elsewhere.Case presentationLipid deposition in case 1 appeared as needle-shaped hyperreflective lines along the collagen lamellae, which correlated histologically with lipid clefts. In case 2, glycosaminoglycan accumulation by mucopolysaccharidosis type 1 caused diffuse stromal hyperreflectivity and depletion of keratocytes on IVCM and was associated with secondary corneal degeneration presumed to be calcium deposition. In case 3, posterior corneal stromal opacities in the absence of ocular inflammation were identified. Hyperreflective particles were scattered in the middle and posterior corneal stroma on FD-OCT. With IVCM, hyperreflective deposits were identified within keratocytes and the number of enlarged keratocytes containing hyperreflective deposits increased towards the posterior stroma. The bilateral, non-inflammatory nature and unique appearance with IVCM is most consistent with a posterior stromal dystrophy reminiscent of pre-Descemet corneal dystrophy described in humans.ConclusionsIn vivo multimodal corneal imaging facilitated instantaneous microstructural analysis and may be valuable in the differential diagnosis of corneal stromal disorders in veterinary clinical practice. The non-specific nature of imaging findings occurs in some conditions such as mucopolysaccharidosis, thus in vivo corneal imaging should be complemented with other gold standard methods of definitive diagnosis.

PurposeTo define the normal range of central corneal thickness (CCT) and corneal endothelial cell density (ECD) in rhesus macaques (Macaca mulatta) and the effects of age, body weight, sex, and intraocular pressure (IOP) on these parameters.MethodsOphthalmic examinations were performed on 144 rhesus macaques without anterior segment pathology. The CCT was measured via ultrasound pachymetry (USP) and specular microscopy, and the ECD was semiautomatically and manually counted using specular microscopy. Rebound tonometry was used to measure IOP. Linear regression and mixed-effects linear regression models were used to evaluate the effects of age, body weight, sex, and IOP on CCT and ECD.ResultsWe included 98 females and 46 males with an age range of 0.2 to 29.4 years. The mean CCT by USP and specular microscopy were 483 ± 39 and 463 ± 33 µm, respectively, and were statistically different (P < 0.001). The ECDs were 2717 ± 423 and 2747 ± 438 cells/mm2 by semiautomated and manual analysis, respectively. Corneal endothelial degeneration was identified in one aged rhesus macaque.ConclusionsThe mean USP and specular microscopy CCT values differed significantly, whereas the semiautomatic and manual ECD did not. The CCT was associated with the IOP and sex, whereas the ECD was associated with body weight and age (P < 0.05). As in humans, corneal disease in rhesus macaques is uncommon.Translational relevanceEstablishing reference values is fundamental to use rhesus macaques as a model for corneal disease or to identify toxicity in studies of ocular drugs or devices.

PurposeThe purpose of this study was to evaluate the tolerability and efficacy of topical rho-kinase inhibitor ripasudil in the treatment of primary corneal endothelial degeneration (PCED) in dogs.MethodsTwenty-one eyes of 12 client-owned, PCED-affected dogs received topical ripasudil 4 times daily. Ophthalmic examination, ultrasonic pachymetry (USP), Fourier-domain optical coherence tomography (FD-OCT), and in vivo confocal microscopy were performed at baseline and 1, 3, 6, and 12 months. Effects of treatment on corneal thickness, corneal edema extent, and endothelial cell density (ECD) were evaluated by repeated-measures ANOVA or Friedman test. Individual eyes were classified as improved, progressed, or stable at 12 months using clinical response criteria. Kaplan-Meier curves and log-rank test were used to compare ripasudil-treated eyes to age-, breed/size-, and disease stage-matched historical controls.ResultsDuring treatment, 12 dogs developed conjunctival hyperemia, 4 demonstrated reticular bullous epithelial edema, and 2 developed corneal stromal hemorrhage. No adverse event necessitated permanent cessation of ripasudil. Central corneal thickness measured by USP significantly progressed from baseline to 12 months. Corneal thickness by FD-OCT, ECD, and edema extent did not differ over time. Considered individually, 5 eyes improved, 8 remained stable, and 8 progressed. The log-rank test found less edema progression in ripasudil-treated eyes compared to historical controls.ConclusionsRipasudil was well-tolerated in PCED-affected dogs. Response to therapy varied; 62% of eyes showed improved or stable disease whereas 38% progressed. Ripasudil-treated eyes progressed more slowly than historical controls.Translational relevanceTopical ripasudil offered a therapeutic benefit in a subset of patients using a canine model of endothelial degeneration, which may guide future trials in humans.

ObjectiveTo describe the clinical findings, multimodal corneal imaging features and treatment in canine patients diagnosed with endotheliitis.Animals studiedFour canine patients met inclusion criteria for bilateral corneal disease with endothelial inflammation and secondary corneal edema that responded to topical anti-inflammatory treatment.MethodsThe patients selected underwent a complete ophthalmic examination with emphasis on the cornea including ultrasound pachymetry (USP), Fourier-domain optical coherence tomography (FD-OCT), in vivo confocal microscopy (IVCM), and digital slit lamp photography.ResultsAll patients in this study demonstrated thickened corneas due to edema with USP and FD-OCT. With IVCM, mild to severe polymegathism and pleomorphism of corneal endothelial cells, reduced endothelial cell density, hyperreflective keratic precipitates (KPs), and extracellular debris as well as hyporeflective pseudoguttata were observed. With FD-OCT, hyperreflective KPs were commonly observed on the inferior cornea. Clinical examination and advanced imaging results were consistent with a diagnosis of endotheliitis. All patients initially responded to topical anti-inflammatory treatment and required continued therapy; two patients also received topical netarsudil, a rho-associated coiled-coil kinase inhibitor.ConclusionEndotheliitis should be considered for canine patients with bilateral edema that is most severe in the inferior cornea. Careful inspection of Descemet's membrane-endothelial complex should be performed for KPs or inflammatory debris. Chronic administration of topical anti-inflammatories may be necessary to prevent flare-ups of endotheliitis.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.

Corneal opacification due to fibrosis is a leading cause of blindness worldwide. Fibrosis occurs from many causes including trauma, photorefractive surgery, microbial keratitis (infection of the cornea), and chemical burns, yet there is a paucity of therapeutics to prevent or treat corneal fibrosis. This study aimed to determine if andrographolide, a labdane diterpenoid found in Andrographis paniculate, has anti-fibrotic properties. Furthermore, we evaluated if andrographolide could prevent the differentiation of fibroblasts to myofibroblasts in vitro, given that the transforming growth factor beta-1(TGF-β1) stimulated persistence of myofibroblasts in the cornea is a primary component of fibrosis. We demonstrated that andrographolide inhibited the upregulation of alpha smooth muscle actin (αSMA) mRNA and protein in rabbit corneal fibroblasts (RCFs), thus, demonstrating a reduction in the transdifferentiation of myofibroblasts. Immunofluorescent staining of TGF-β1-stimulated RCFs confirmed a dose-dependent decrease in αSMA expression when treated with andrographolide. Additionally, andrographolide was well tolerated in vivo and had no impact on corneal epithelialization in a rat debridement model. These data support future studies investigating the use of andrographolide as an anti-fibrotic in corneal wound healing.

Acute primary angle closure glaucoma is a potentially blinding ophthalmic emergency requiring prompt treatment to lower the elevated intraocular pressure in humans and dogs. The PACG in most of canine breeds is epidemiologically similar to humans with older and female patients overrepresented with the condition. The American Cocker Spaniel (ACS) is among the most common breeds observed with PACG development in dogs. This study initially sought to identify genetic risk factors to explain the high prevalence of PACG in ACSs by using a case-control breed-matched genome-wide association study. However, the GWAS failed to identify candidate loci associated with PACG in this breed. This study then assessed intrinsic ocular morphologic traits that may relate to PACG susceptibility in this breed. Normal ACSs without glaucoma have a crowded anterior ocular segment and narrow iridocorneal angle and ciliary cleft, which is consistent with anatomical risk factors identified in humans. The ACSs showed unique features consisting of posterior bowing of iris and longer iridolenticular contact, which mirrors reverse pupillary block and pigment dispersion syndrome in humans. The ACS could hold potential to serve as an animal model of naturally occurring PACG in humans.

Microbial keratitis is a common cause of ocular pain and visual impairment worldwide. The ocular surface has a relatively paucicellular microbial community, mostly found in the conjunctiva, while the cornea would be considered relatively sterile. However, in patients with microbial keratitis, the cornea can be infected with multiple pathogens including Staphylococcus aureus, Pseudomonas aeruginosa, and Fusarium sp. Treatment with topical antimicrobials serves as the standard of care for microbial keratitis, however, due to high rates of pathogen resistance to current antimicrobial medications, alternative therapeutic strategies must be developed. Multiple studies have characterized the expression and activity of antimicrobial peptides (AMPs), endogenous peptides with key antimicrobial and wound healing properties, on the ocular surface. Recent studies and clinical trials provide promise for the use of AMPs as therapeutic agents. This article reviews the repertoire of AMPs expressed at the ocular surface, how expression of these AMPs can be modulated, and the potential for harnessing the AMPs as potential therapeutics for patients with microbial keratitis.

The ocular surface, comprised of the transparent cornea, conjunctiva, and protective tear film, forms a protective barrier defending deeper structures of the eye from particulate matter and mechanical trauma. This barrier is routinely exposed to a multitude of naturally occurring and engineered nanomaterials (ENM). Metallic ENMs are particularly ubiquitous in commercial products with a high risk of ocular exposure, such as cosmetics and sunscreens. Additionally, there are several therapeutic uses for metallic ENMs owing to their attractive magnetic, antimicrobial, and functionalization properties. The increasing commercial and therapeutic applications of metallic ENMs come with a high risk of ocular exposure with poorly understood consequences to the health of the eye. While the toxicity of metallic ENMs exposure has been rigorously studied in other tissues and organs, further studies are necessary to understand the potential for adverse effects and inform product usage for individuals whose ocular health may be compromised by injury, disease, or surgical intervention. This review provides an update of current literature on the ocular toxicity of metallic ENMs in vitro and in vivo, as well as the risks and benefits of therapeutic applications of metallic ENMs in ophthalmology.

Purpose: Instability of the tear film leads to evaporative dry eye disease (EDED), but the Harderian gland in some terrestrial vertebrates may produce novel lipids that stabilize the tear film and protect against dry eye. Here, the nonpolar lipids in the Harderian gland and tears of the rabbit but absent in human tears were identified and tested in preclinical studies to determine whether they could treat severe EDED. Methods: Lipids were identified primarily by atmospheric pressure chemical ionization mass spectrometry (MS) and fragmentation MS/MS. An identified lipid was synthesized and formulated as an emulsion and as a cyclodextrin (CD) clathrate. Following doses with test agents and controls, tear film breakup time (TBUT), tear production, corneal fluorescein staining, macrophage infiltration, and goblet cell survival were measured using standard tests at 0, 2 and 4 weeks in an animal model of EDED. Results: The lipid emulsion increased TBUT (P

ObjectiveTo assess correlations between clinical and cytological features of feline eosinophilic keratoconjunctivitis at the time of cytological diagnosis.Animals studiedFifteen client-owned, domestic breed cats (18 eyes) examined between 2007 and 2019.ProceduresAn electronic search and medical record review of cats diagnosed with feline eosinophilic keratitis or keratoconjunctivitis (FEK) based on clinical examination findings and eosinophils detected on corneal cytology were conducted. Clinical severity was graded using a modified version of a previously validated semiquantitative preclinical ocular toxicology scoring (SPOTS) system. Clinical grades were assigned following review of clinical images and medical record descriptions, and cytological grades were assigned following review of archived corneal cytology slides. Correlations were analyzed for significance using Spearman's rank correlation coefficient.ResultsHigher total corneal scores correlated with higher total conjunctival scores, but not with total fluorescein scores. Small lymphocyte scores correlated negatively with scores for collagen degeneration or mineralization. Globule leukocytes, a unique cell type not previously described in ocular cytology, were identified in 4 of 18 cytological samples. Higher globule leukocyte scores were correlated with higher scores for mast cells or plasma cells. Specimens with lower eosinophil scores had higher globule leukocyte scores.ConclusionsLarge variability was detected in the cytological characteristics and clinical features of FEK-affected cats. This is the first report of globule leukocytes being identified in ocular cytology from any species. The role of globule leukocytes in the etiopathogenesis and progression of FEK remains unknown and warrants further investigation.

ObjectiveTo compare intraocular pressures (IOPs) estimated by rebound and applanation tonometry for dogs with lens instability.Animals66 dogs.ProceduresMedical records of dogs examined between September 2012 and July 2018 were reviewed for diagnoses of anterior (ALL) or posterior (PLL) lens luxation or lens subluxation.ResultsEstimates of IOP obtained with rebound and applanation tonometry significantly differed from each other for all types of lens instability considered collectively (mean ± SE difference between tonometric readings, 8.1 ± 1.3 mm Hg) and specific types of lens instability considered individually (mean ± SE difference between tonometric readings: ALL, 12.8 ± 2.5 mm Hg; PLL, 5.9 ± 1.7 mm Hg; subluxation, 2.8 ± 0.8 mm Hg). Median (range) differences between rebound and applanation tonometer readings for dogs with ALL was 5 mm Hg (-9 to 76 mm Hg), with PLL was 3 mm Hg (-1 to 19 mm Hg), and with lens subluxation was 3 mm Hg (-9 to 18 mm Hg). In eyes with ALL, rebound tonometer readings exceeded applanation tonometer readings on 44 of 60 (73%) occasions.Conclusions and clinical relevanceRebound tonometry yielded higher estimates of IOP than did applanation tonometry in eyes with ALL and with all types of lens luxation considered collectively. Estimates of IOP in eyes with lens instability should ideally be obtained with both rebound and applanation tonometers. Veterinarians with only one type of tonometer should interpret results for dogs with lens instability concurrent with physical examination findings.

PurposeCorneal keratocyte-fibroblast-myofibroblast (KFM) transformation plays a critical role in corneal stromal wound healing. However, the impact of engineered nanomaterials (ENMs), found in an increasing number of commercial products, on this process is poorly studied. This study investigates the effects of metal oxide ENMs on KFM transformation in vitro and in vivo.MethodsCell viability of rabbit corneal fibroblasts (RCFs) was tested following treatment with 11 metal oxide ENMs at concentrations of 0.5 to 250 µg/ml for 24 hours. Messenger RNA (mRNA) and protein expression of αSMA, a marker of myofibroblast transformation, were measured using RCFs after exposure to 11 metal oxide ENMs at a concentration that did not affect cell viability, in media containing either 0 or 10 ng/ml of TGF-β1. Additionally, the effect of topical Fe2O3 nanoparticles (NPs) (50 ng/ml) on corneal stromal wound healing following phototherapeutic keratectomy (PTK) was determined.ResultsV2O5, Fe2O3, CuO, and ZnO ENMs were found to significantly reduce cell viability as compared to vehicle control and the other seven metal oxide ENMs tested. V2O5 nanoflakes significantly reduced mRNA and protein αSMA concentrations in the presence of TGF-β1. Fe2O3 NPs significantly increased αSMA mRNA expression in the presence of TGF-β1 but did not alter αSMA protein expression. Topically applied Fe2O3 NPs in an in vivo rabbit corneal stromal wound healing model did not delay healing.ConclusionsFe2O3 NPs promote corneal myofibroblast induction in vitro but do not impair corneal stromal wound healing in vivo.Translational relevanceThese experimental results can apply to human nanomedical research.

Progressive corneal endothelial disease eventually leads to corneal edema and vision loss due to the limited regenerative capacity of the corneal endothelium in vivo and is a major indication for corneal transplantation. Despite the relatively high success rate of corneal transplantation, there remains a pressing global clinical need to identify improved therapeutic strategies to address this debilitating condition. To evaluate the safety and efficacy of novel therapeutics, there is a growing demand for pre-clinical animal models of corneal endothelial dysfunction. In this review, experimentally induced, spontaneously occurring and genetically modified animal models of corneal endothelial dysfunction are described to assist researchers in making informed decisions regarding the selection of the most appropriate animal models to meet their research goals.

BackgroundCeroid lipofuscinosis type 8 belongs to a heterogenous group of vision and life-threatening neurodegenerative diseases, neuronal ceroid lipofuscinosis (NCL). Effective therapy is limited to a single drug for treatment of ceroid lipofuscinosis type 2, necessitating animal disease models to facilitate further therapeutic development. Murine models are advantageous for therapeutic development due to easy genetic manipulation and rapid breeding, however appropriate genetic models need to be identified and characterized before being used for therapy testing. To date, murine models of ocular disease associated with ceroid lipofuscinosis type 8 have only been characterized in motor neuron degeneration mice.MethodsCln8-/- mice were produced by CRISPR/Cas9 genome editing through the International Mouse Phenotyping Consortium. Ophthalmic examination, optical coherence tomography, electroretinography, and ocular histology was performed on Cln8-/- mice and controls at 16 weeks of age. Quantification of all retinal layers, retinal pigmented epithelium, and the choriocapillaris was performed using images acquired with ocular coherence tomography and planimetry of histologic sections. Necropsy was performed to investigate concurrent systemic abnormalities. Clinical correlation with human patients with CLN8-associated retinopathy is provided.ResultsRetinal degeneration characterized by retinal pigment epithelium mottling, scattered drusen, and retinal vascular attenuation was noted in all Cln8-/- mice. Loss of inner and outer photoreceptor segment demarcation was noted on optical coherence tomography, with significant thinning of the whole retina (P=1e-9), outer nuclear layer (P=1e-9), and combined photoreceptor segments (P=1e-9). A global reduction in scotopic and photopic electroretinographic waveforms was noted in all Cln8-/- mice. Slight thickening of the inner plexiform layer (P=0.02) and inner nuclear layer (P=0.004), with significant thinning of the whole retina (P=0.03), outer nuclear layer (P=0.01), and outer photoreceptor segments (P=0.001) was appreciated on histologic sections. Scattered lipid vacuoles were noted in splenic red pulp of all Cln8-/- mice, though no gross systemic abnormalities were detected on necropsy. Retinal findings are consistent with those seen in patients with ceroid lipofuscinosis type 8.ConclusionsThis study provides detailed clinical characterization of retinopathy in adult Cln8-/- mice. Findings suggest that Cln8-/- mice may provide a useful murine model for development of novel therapeutics needed for treating ocular disease in patients with ceroid lipofuscinosis type 8.

ObjectivesThe aim of this retrospective case-control study was to report the efficacy of subcutaneous triamcinolone as part of a regimen for feline eosinophilic keratoconjunctivitis (FEK).MethodsRecords and clinical photographs were reviewed and lesions semiquantitatively graded for cats with cytologically confirmed FEK. Clinical data were compared between a study population of nine cats (11 eyes) treated with, and a reference population of seven cats (eight eyes) treated without, a median of 0.11 mg/kg (range 0.10-0.20 mg/kg) of triamcinolone acetonide subcutaneously.ResultsBreed, sex, age and prevalence of corneal ulceration at presentation; corneal disease severity before and at the initiation of immunomodulation; and duration of antiviral treatment before immunomodulation did not differ significantly between populations (P ⩾0.059). Corneal plaques resolved in five cats each from the study and reference populations (P = 0.366). Median (range) time from immunomodulation to corneal plaque resolution did not significantly differ (P = 0.246) between the study (median 14 days; range 8-38 days) and reference (median 28 days, range 14-46 days) populations. No adverse reactions were attributed to triamcinolone administration, and all corneal ulcers in the study population re-epithelialized within 14 days (range 8-38 days) following triamcinolone injection. Time to corneal ulcer re-epithelialization following triamcinolone injection varied minimally in those receiving antivirals prior to (8 or 30 days until re-epithelialization), simultaneously with (38 days) or after (14 or 24 days) triamcinolone.Conclusions and relevanceIn otherwise healthy cats with FEK, subcutaneous administration of triamcinolone appears to be well tolerated and as efficacious as conventional topical immunomodulatory therapies. It may be especially useful in ulcerated eyes where topical immunomodulation is contraindicated.

PurposeTo retrospectively evaluate the clinical data, diagnostic tests, treatments, and outcomes for dogs with corneal endothelial dystrophy (CED) and determine risk factors for CED when compared with a canine reference population.MethodsMedical records of 99 dogs (1991-2014) diagnosed with CED at the University of California Davis Veterinary Medical Teaching Hospital were reviewed and compared with 458,680 dogs comprising the general hospital population during the study period. Retrieved data included signalment, examination findings, diagnoses, treatments, and outcomes associated with CED. The exact Pearson χ2 test or exact Kruskal-Wallis test was used to compare parameters between the groups. Progression of corneal edema was assessed using 3 independent Kaplan-Meier curves, identifying clinically significant changes in corneal opacity.ResultsBoston terriers, German wirehaired pointers, and Dachshunds were overrepresented in the CED-affected group, whereas Labradors were underrepresented. Dogs older than 11 years were overrepresented in the CED-affected group, whereas intact dogs were underrepresented. Surgical intervention was performed (n = 11) based on the severity of disease and secondary complications from CED. Median time to progression of corneal edema was 1) 368 days when an at-risk eye initially without edema developed edema at a subsequent visit, 2) 701 days when there was progression from mild to marked corneal edema, and 3) 340 days when there was progression from focal to diffuse corneal edema.ConclusionsMany CED-affected dogs progress over months to years without surgical intervention, making dogs with CED a useful model for studying genetic predispositions and development of novel therapeutics for Fuchs endothelial corneal dystrophy.

The transformation of quiescent keratocytes to activated fibroblasts and myofibroblasts (KFM transformation) largely depends on transforming growth factor beta (TGFβ) signaling. Initiation of the TGFβ signaling cascade results from binding of TGFβ to the labile type I TGFβ receptor (TGFβRI), which is stabilized by the 90 kDa heat shock protein (Hsp90). Since myofibroblast persistence within the corneal stroma can result in stromal haze and corneal fibrosis in patients undergoing keratorefractive therapy, modulation of TGFβ signaling through Hsp90 inhibition would represent a novel approach to prevent myofibroblast persistence. In vitro, rabbit corneal fibroblasts (RCFs) or stratified immortalized human corneal epithelial cells (hTCEpi) were treated with a Hsp90 inhibitor (17AAG) in the presence/absence of TGFβ1. RCFs were cultured either on tissue culture plastic, anisotropically patterned substrates, and hydrogels of varying stiffness. Cellular responses to both cytoactive and variable substrates were assessed by morphologic changes to the cells, and alterations in expression patterns of key keratocyte and myofibroblast proteins using PCR, Western blotting and immunocytochemistry. Transepithelial electrical resistance (TEER) measurements were performed to establish epithelial barrier integrity. In vivo, the corneas of New Zealand White rabbits were wounded by phototherapeutic keratectomy (PTK) and treated with 17AAG (3× or 6× daily) either immediately or 7 days after wounding for 28 days. Rabbits underwent clinical ophthalmic examinations, SPOTS scoring and advanced imaging on days 0, 1, 3, 7, 10, 14, 21 and 28. On day 28, rabbits were euthanized and histopathology/immunohistochemistry was performed. In vitro data demonstrated that 17AAG inhibited KFM transformation with the de-differentiation of spindle shaped myofibroblasts to dendritic keratocyte-like cells accompanied by significant upregulation of corneal crystallins and suppression of myofibroblast markers regardless of TGFβ1 treatment. RCFs cultured on soft hydrogels or patterned substrates exhibited elevated expression of α-smooth muscle actin (αSMA) in the presence of 17AAG. Treatment of hTCEpi cells disrupted zonula occludens 1 (ZO-1) adherens junction formation. In vivo, there were no differences detected in nearly all clinical parameters assessed between treatment groups. However, rabbits treated with 17AAG developed greater stromal haze formation compared with controls, irrespective of frequency of administration. Lastly, there was increased αSMA positive myofibroblasts in the stroma of 17AAG treated animals when compared with controls. Hsp90 inhibition promoted reversion of the myofibroblast to keratocyte phenotype, although this only occurred on rigid substrates. By contrast, in vivo Hsp90 inhibition was detrimental to corneal wound healing likely due to impairment in corneal epithelial closure and barrier function restoration. Collectively, our data demonstrated a strong interplay in vitro between biophysical cues and soluble signaling molecules in determining corneal stromal cell phenotype.

The cornea is the most external layer of the eye and serves two important roles in (1) the refraction of light and (2) protection from the outside environment, both of which are highly dependent on the collagen assembly of the corneal stroma. This study sought to determine the collagen fiber arrangement of the canine corneal stroma and correlate the stromal organization with tissue stiffness in the anterior and posterior cornea. Collagen organization of the canine cornea was visualized through second-harmonic generation (SHG) imaging, and tissue stiffness of the anterior and posterior corneal stroma was determined by atomic force microscopy. Analysis of the canine anterior corneal stroma using SHG imaging documented intertwining of the collagen fibers with a high degree of fiber branching, with a more lamellar and non-branching posterior stroma. The anterior stroma had significantly higher tissue stiffness in both dogs and humans, when compared with the posterior corneal stroma (canine median: 1.3 kPa vs. 0.3 kPa; human median: 14.6 kPa vs. 2.1 kPa, respectively). There was a direct correlation between corneal collagen stromal organization and tissue stiffness in the dog, which was consistent with other mammalian species previously examined and likely reflects the need for maintenance of rigidity and corneal curvature.

Ocular exposure to metal oxide engineered nanomaterials (ENMs) is common as exemplified by zinc oxide (ZnO), a major constituent of sunscreens and cosmetics. The ocular surface that includes the transparent cornea and its protective tear film are common sites of exposure for metal ENMs. Despite the frequency of exposure of the ocular surface, there is a knowledge gap regarding the effects of metal oxide ENMs on the cornea in health and disease. Therefore, we studied the effects of metal oxide ENMs on the cornea in the presence or absence of injury. Cell viability of immortalized human corneal epithelial (hTCEpi) cells was assessed following treatment with 11 metal oxide ENMs with a concentration ranging from 0.5 to 250 μg/mL for 24 hours. An epithelial wound healing assay with a monolayer of hTCEpi cells was then performed using 11 metal oxide ENMs at select concentrations based on data from the viability assays. Subsequently, based on the in vitro results, in vivo testing of precorneal tear film (PTF) quantity and stability as well as a corneal epithelial wound healing were tested in the presence or absence ZnO or vanadium pentoxide (V2O5) at a concentration of 50 μg/mL. We found that WO3, ZnO, V2O5 and CuO ENMs significantly reduced hTCEpi cell viability in comparison to vehicle control or the other metal oxide ENMs tested. Furthermore, ZnO and V2O5 ENMs also significantly decreased hTCEpi cell migration. Although ZnO and V2O5 did not alter PTF parameters of rabbits in vivo, corneal epithelial wound healing was significantly delayed by topical ZnO while V2O5 did not alter wound healing. Finally, hyperspectral images confirmed penetration of ZnO and V2O5 through all corneal layers and into the iris stroma. Considering the marked epithelial toxicity and corneal penetration of ZnO, further investigations on the impact of this ENM on the eye are warranted.

The cornea is the most external layer of the eye and serves two important roles in (1) the refraction of light and (2) protection from the outside environment, both of which are highly dependent on the collagen assembly of the corneal stroma. This study sought to determine the collagen fiber arrangement of the canine corneal stroma and correlate the stromal organization with tissue stiffness in the anterior and posterior cornea. Collagen organization of the canine cornea was visualized through second-harmonic generation (SHG) imaging, and tissue stiffness of the anterior and posterior corneal stroma was determined by atomic force microscopy. Analysis of the canine anterior corneal stroma using SHG imaging documented intertwining of the collagen fibers with a high degree of fiber branching, with a more lamellar and non-branching posterior stroma. The anterior stroma had significantly higher tissue stiffness in both dogs and humans, when compared with the posterior corneal stroma (canine median: 1.3 kPa vs. 0.3 kPa; human median: 14.6 kPa vs. 2.1 kPa, respectively). There was a direct correlation between corneal collagen stromal organization and tissue stiffness in the dog, which was consistent with other mammalian species previously examined and likely reflects the need for maintenance of rigidity and corneal curvature.

PurposeTo perform a comprehensive clinical, diagnostic, and imaging characterization of the ocular surface in West Highland White Terriers (WHWTs) diagnosed with aqueous deficient dry eye (ADDE) disease.MethodsSix ADDE-affected and 13 ADDE-unaffected WHWT dogs were enrolled and underwent clinical assessment and disease scoring, tear osmolarity, phenol red thread test, Schirmer tear test, tear film breakup time, fluorescein staining, Rose bengal and lissamine green vital dye staining, meibometry, corneal esthesiometry, ultrasound pachymetry, optical coherence tomography, in vivo confocal microscopy, and conjunctival biopsy. Subjective assessment of their condition was provided by owner-reported surveys.ResultsADDE-affected WHWT dogs had higher median clinical disease (conjunctiva: 5.75 vs. 0.00; cornea: 14.00 vs. 5.00; total: 17.50 vs. 5.00), vital staining (Rose bengal: 2.25 vs. 1.50; lissamine green: 2.00 vs. 1.00), and histologic disease (conjunctiva: 2 vs. 0) scores when compared with the controls. In addition, ADDE-affected WHWTs had significantly lower phenol red thread test (5.0 vs. 17.5, mm/15 s), Schirmer tear test (3 vs. 20, mm/min), tear film breakup time (3.6 vs. 13.9, s) values and higher area under the curve values for meibometry (394 vs. 245, meibometry units [MU]). There were no significant differences in other tear film tests performed. Advanced imaging revealed decreased tear meniscus height (optical coherence tomography) and variable pigment deposition within corneal epithelial cells (in vivo confocal microscopy).ConclusionsThis comprehensive assessment of ADDE-affected WHWTs depicts the ocular surface changes associated with quantitative lacrimal gland dysfunction. Importantly, ADDE-affected WHWTs may prove a valuable naturally occurring ADDE model for investigating underlying pathophysiological mechanisms and the development of novel therapeutics.

A 6-year-old male leopard gecko (Eublepharis macularius) was presented with a 2-year history of recurrent dysecdysis involving the ocular surface of both eyes. Ophthalmic examination revealed ocular surface desiccation and multifocal superficial ulcerative keratitis with patchy remnants of retained shed. Other abnormalities included stomatitis and mandibular and maxillary osteomyelitis. Topical and systemic antibiotic therapy, oral vitamin A, and improved husbandry conditions resolved the stomatitis and osteomyelitis but did not improve the ocular surface. Corneal cytology collected with a cytobrush revealed branching hyphae and budding yeast consistent with fungal keratitis. Fungal culture grew Acremonium sp. and Trichosporon sp. The addition of topical antifungal therapy improved the ocular surface health, but the patient was euthanized 7 weeks after initial presentation for persistent vomiting and dyspnea. Necropsy was declined. This case describes the first case of fungal keratitis caused by Acremonium sp. and Trichosporon sp. in a reptile.

CASE SERIES SUMMARY:Described are 13 cats diagnosed with deep ulcerative keratitis and successfully managed medically without grafting procedures. Typical treatment involved frequent topical application of serum and antibiotics (usually a fluoroquinolone and a cephalosporin). Seven cats also received systemic antibiotics. Analgesia was achieved using various combinations of topical atropine and systemic buprenorphine, robenacoxib or corticosteroids. Six cats were hospitalized for a median (range) period of 2.5 (1-8) days, typically because of frequent medication administration. Median (range) follow-up time was 41.5 (9-103) days. Median (range) number of recheck examinations was 4 (2-6). Median (range) time to corneal re-epithelialization was 21 (9-103) days. Median (range) topical antibiotic course was 29.5 (16-103) days. Median (range) duration of Elizabethan collar use was 28 (13-73) days. At the time of writing, no further recheck examinations were recommended for 10 cats; median (range) time between initial to final examinations in these cats was 35 (20-103) days. All cats retained the affected globes and were apparently comfortable and visual at the latest recheck examination. RELEVANCE AND NOVEL INFORMATION:These cases reveal that aggressive medical management is highly successful in select cats with deep ulcerative keratitis, and can result in a cosmetically acceptable, apparently comfortable and visual globe. However, therapy is intensive with frequent administration of multiple topical and sometimes systemic medications, and requires multiple veterinary visits over many weeks. Referral to a veterinary ophthalmologist for consideration of surgical stabilization is recommended, as not all cases may be amenable to the medical therapy described here.

CASE SERIES SUMMARY:Described are 13 cats diagnosed with deep ulcerative keratitis and successfully managed medically without grafting procedures. Typical treatment involved frequent topical application of serum and antibiotics (usually a fluoroquinolone and a cephalosporin). Seven cats also received systemic antibiotics. Analgesia was achieved using various combinations of topical atropine and systemic buprenorphine, robenacoxib or corticosteroids. Six cats were hospitalized for a median (range) period of 2.5 (1-8) days, typically because of frequent medication administration. Median (range) follow-up time was 41.5 (9-103) days. Median (range) number of recheck examinations was 4 (2-6). Median (range) time to corneal re-epithelialization was 21 (9-103) days. Median (range) topical antibiotic course was 29.5 (16-103) days. Median (range) duration of Elizabethan collar use was 28 (13-73) days. At the time of writing, no further recheck examinations were recommended for 10 cats; median (range) time between initial to final examinations in these cats was 35 (20-103) days. All cats retained the affected globes and were apparently comfortable and visual at the latest recheck examination. RELEVANCE AND NOVEL INFORMATION:These cases reveal that aggressive medical management is highly successful in select cats with deep ulcerative keratitis, and can result in a cosmetically acceptable, apparently comfortable and visual globe. However, therapy is intensive with frequent administration of multiple topical and sometimes systemic medications, and requires multiple veterinary visits over many weeks. Referral to a veterinary ophthalmologist for consideration of surgical stabilization is recommended, as not all cases may be amenable to the medical therapy described here.

Early-onset Fuchs endothelial corneal dystrophy (FECD) has been associated with nonsynonymous mutations in collagen VIII α2 (COL8A2), a key extracellular matrix (ECM) protein in Descemet's membrane (DM). Two knock-in strains of mice have been generated to each express a mutant COL8A2 protein (Col8a2L450W/L450W and Col8a2Q455K/Q455K) that recapitulate the clinical phenotype of early-onset FECD including endothelial cell loss, cellular polymegathism and pleomorphism, and guttae. Due to abnormalities in ECM protein composition and structure in FECD, the stiffness of DM in Col8a2 knock-in mice and wildtype (WT) controls was measured using atomic force microscopy at 5 and 10 months of age, coinciding with the onset of FECD phenotypic abnormalities. At 5 months, only sporadic guttae were identified via in vivo confocal microscopy (IVCM) in Col8a2Q455K/Q455K mice, otherwise both strains of Col8a2 transgenic mice were indistinguishable from WT controls in terms of endothelial cell density and size. By 10 months of age, Col8a2L450W/L450W and Col8a2Q455K/Q455K mice developed reduced corneal endothelial density, increased endothelial cell area and guttae, with the Col8a2Q455K/Q455K strain exhibiting a more severe phenotype. However, at 5 months of age, prior to the development endothelial cell abnormalities, Col8a2L450W/L450W and Col8a2Q455K/Q455K mice knock-in mice had reduced tissue stiffness of DM that was statistically significant in the Col8a2Q455K/Q455K mice when compared with wildtype controls. These data indicate that alterations in the tissue compliance of DM precede phenotypic changes in endothelial cell count and morphology, and may play a role in onset and progression of FECD.