Collagen crosslinking impacts stromal wound healing and haze formation in a rabbit phototherapeutic keratectomy model.

Purpose: The purpose of this study was to evaluate the elastic modulus, keratocyte-fibroblast-myocyte transformation, and haze formation of the corneal stroma following combined phototherapeutic keratectomy (PTK) and epithelium-off UV-A/riboflavin corneal collagen crosslinking (CXL) using an in vivo rabbit model. Methods: Rabbits underwent PTK and CXL, PTK only, or CXL 35 days before PTK. Rebound tonometry, Fourier-domain optical coherence tomography, and ultrasound pachymetry were performed on days 7, 14, 21, 42, 70, and 90 post-operatively. Atomic force microscopy, histologic inflammation, and immunohistochemistry for α-smooth muscle actin (α-SMA) were assessed post-mortem. Results: Stromal haze formation following simultaneous PTK and CXL was significantly greater than in corneas that received PTK only and persisted for more than 90 days. No significant difference in stromal haze was noted between groups receiving simultaneous CXL and PTK and those receiving CXL before PTK. Stromal inflammation did not differ between groups at any time point, although the intensity of α-SMA over the number of nuclei was significantly greater at day 21 between groups receiving simultaneous CXL and PTK and those receiving CXL before PTK. The elastic modulus was significantly greater in corneas receiving simultaneous CXL and PTK compared with those receiving PTK alone. Conclusions: We showed that stromal haze formation and stromal stiffness is significantly increased following CXL, regardless of whether it is performed at or before the time of PTK. Further knowledge of the biophysical cues involved in determining corneal wound healing duration and outcomes will be important for understanding scarring following CXL and for the development of improved therapeutic options.

Cell derived matrices from bovine corneal endothelial cells as a model to study cellular dysfunction.

PURPOSE: Fuchs endothelial corneal dystrophy (FECD) is a progressive corneal disease that impacts the structure and stiffness of the Descemet's membrane (DM), the substratum for corneal endothelial cells (CECs). These structural alterations of the DM could contribute to the loss of the CECs resulting in corneal edema and blindness. Oxidative stress and transforming growth factor-ß (TGF-ß) pathways have been implicated in endothelial cell loss and endothelial to mesenchymal transition of CECs in FECD. Ascorbic acid (AA) is found at high concentrations in FECD and its impact on CEC survival has been investigated. However, how TGF-ß and AA effect the composition and rigidity of the CEC's matrix remains unknown. METHODS: In this study, we investigated the effect of AA, TGF-ß1 and TGF-ß3 on the deposition, ultrastructure, stiffness, and composition of the extracellular matrix (ECM) secreted by primary bovine corneal endothelial cells (BCECs). RESULTS: Immunofluorescence and electron microscopy post-decellularization demonstrated a robust deposition and distinct structure of ECM in response to treatments. AFM measurements showed that the modulus of the matrix in BCECs treated with TGF-ß1 and TGF-ß3 was significantly lower than the controls. There was no difference in the stiffness of the matrix between the AA-treated cell and controls. Gene Ontology analysis of the proteomics results revealed that AA modulates the oxidative stress pathway in the matrix while TGF-ß induces the expression of matrix proteins collagen IV, laminin, and lysyl oxidase homolog 1. CONCLUSIONS: Molecular pathways identified in this study demonstrate the differential role of soluble factors in the pathogenesis of FECD.

Identification of putative orthologs of clinically relevant antimicrobial peptides in the equine ocular surface and amniotic membrane.

OBJECTIVES: This 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. PROCEDURES: A 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. RESULTS: Equine 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. CONCLUSIONS: This 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.

Clinical and histopathological features of proliferative corneal lesions in Cyprininae fishes: Implications for treatment and insights into corneal tumors.

Captive fish populations, such as those encompassing aquarium and pet fish, offer significant economic value and are integral to conservation, research, and education. However, these ornamental fish exhibit a reduced ability to protect their ocular surfaces, and our understanding of the ocular diseases that affect them remains limited. Although corneal neoplasms in carp are uncommon, identifying their distinct characteristics is crucial in selecting appropriate therapeutic interventions that aim to preserve vision, prevent the ocular loss, and ultimately ensure the survival of the affected fish. This study provides clinical and histopathological details of various proliferative corneal masses in Cyprininae species, including five koi (Cyprinus carpio) and four goldfish (Carassius auratus). It discusses a spectrum of neoplasms, including soft tissue sarcoma, spindle cell sarcoma, chromatophoroma, and papilloma, in addition to conditions like exuberant granulation tissue and proliferative carp pox. These findings bear significant implications for clinical decision-making and treatment, offering valuable insights into the incidence and characteristics of corneal tumors in captive fish, which could inform further studies in this area.

Multimodal ocular imaging of known and novel corneal stromal disorders in dogs.

BACKGROUND: Imaging 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 PRESENTATION: Lipid 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. CONCLUSIONS: In 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.

Differential effects of Hsp90 inhibition on corneal cells in vitro and in vivo.

The transformation of quiescent keratocytes to activated fibroblasts and myofibroblasts (KFM transformation) largely depends on transforming growth factor beta (TGFß) signaling. Initiation of the TGFß signaling cascade results from binding of TGFß to the labile type I TGFß receptor (TGFßRI), 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 TGFß 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 TGFß1. 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 TGFß1 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.

Correlations between clinical signs and corneal cytology in feline eosinophilic keratoconjunctivitis.

OBJECTIVE: To assess correlations between clinical and cytological features of feline eosinophilic keratoconjunctivitis at the time of cytological diagnosis. ANIMALS STUDIED: Fifteen client-owned, domestic breed cats (18 eyes) examined between 2007 and 2019. PROCEDURES: An 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. RESULTS: Higher 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. CONCLUSIONS: Large 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.

LIQUID NITROGEN CRYOSURGERY FOR CUTANEOUS AND OCULAR NEOPLASMS IN KOI (CYPRINUS CARPIO) AND GOLDFISH (CARASSIUS AURATUS): EIGHT CASES (2018-2019).

Cryosurgery, also known as cryotherapy and cryoablation, is a promising surgical technique that employs highly localized freezing to destroy damaged and diseased tissue, including benign and malignant neoplasms. This procedure has been reported in the treatment of chromatophoromas, fibromas, and peripheral nerve sheath tumors in piscine patients. This study presents eight clinical cases of cryosurgery on cyprinid pet fish for a wide array of neoplastic masses, including chromatophoromas, squamous cell carcinoma, and sarcomas that were diagnosed by histopathology. Surgical excision of external masses, liquid nitrogen cryotherapy, injectable medications (meloxicam and danofloxacin), and topical medical-grade honey were applied to the patients after biopsy sampling. Five out of seven cutaneous cases and two out of three ocular cases had complete resolution without recurrence for at least three months posttreatment. Treatment was unsuccessful for two of the cutaneous cases in which the cutaneous masses were extremely invasive, resulting in severe ulceration and deep invasion into the coelomic cavity. One of the ocular cases involved a corneal mass that did not change in size and had no complications after treatments, suggesting that the treatment might be useful in limiting growth. The effectiveness of cryotherapy appears to correlate with the tumor type, as well as the stage and progression of tumor invasion.

Comparison of automated vs manual analysis of corneal endothelial cell density and morphology in normal and corneal endothelial dystrophy-affected dogs.

OBJECTIVE: To determine the efficacy of automated imaging software of the Nidek ConfoScan 4 confocal biomicroscope at analyzing canine corneal endothelial cell density and morphology in health and disease, by comparing to a manual analysis method. ANIMAL STUDIED: Nineteen eyes of 10 dogs were evaluated and include three Beagles, three Jack Russell Terriers, and four miscellaneous breeds. Twelve clinically normal and seven eyes affected with corneal endothelial dystrophy (CED) were scanned and analyzed. PROCEDURES: Endothelial cell density (ECD), mean and standard deviation (SD) of cell area, percent polymegathism, mean and SD of the number of cell sides, and percent pleomorphism were calculated using automated and manual methods for each scan. RESULTS: The automated analysis showed significantly greater ECD in comparison with the manual frame method due to misidentification of cell domains in CED-affected dogs. No significant differences in ECD were observed between normal and CED-affected dogs in automated analysis, while CED-affected dogs showed significantly lower ECD in manual frame method and planimetry. Using both automated and manual methods, CED-affected dogs showed greater variability of cell area or the number of cell sides than normal dogs. CONCLUSION: The automated imaging software is unable to accurately identify cell borders in CED-affected dogs resulting in inaccurate estimates of ECD. Thus, manual analysis is recommended for use in clinical trials assessing adverse events associated with novel medical treatments and/or surgical procedures.

Ocular phenotypic consequences of a single copy deletion of the Yap1 gene (Yap1 +/-) in mice.

Purpose: To identify the effects of a single copy deletion of Yap1 (Yap1 +/-) in the mouse eye, the ocular phenotypic consequences of Yap1 +/- were determined in detail. Methods: Complete ophthalmic examinations, as well as corneal esthesiometry, the phenol red thread test, intraocular pressure, and Fourier-domain optical coherence tomography were performed on Yap1 +/- and age-matched wild-type (WT) mice between eyelid opening (2 weeks after birth) and adulthood (2 months and 1 year after birth). Following euthanasia, enucleated eyes were characterized histologically. Results: Microphthalmia with small palpebral fissures, corneal fibrosis, and reduced corneal sensation were common findings in the Yap1 +/- mice. Generalized corneal fibrosis precluded clinical examination of the posterior structures. Histologically, thinning and keratinization of the corneal epithelium were observed in the Yap1 +/- mice in comparison with the WT mice. Distorted collagen fiber arrangement and hypercellularity of keratocytes were observed in the stroma. Descemet's membrane was extremely thin and lacked an endothelial layer in the Yap1 +/- mice. The iris was adherent to the posterior cornea along most of its surface creating a distorted contour. Most of the Yap1 +/- eyes were microphakic with swollen fibers and bladder cells. The retinas of the Yap1 +/- mice were normal at 2 weeks and 2 months of age, but the presence of retinal abnormalities, including retinoschisis and detachment, was markedly increased in the Yap1 +/- mice at 1 year of age. Conclusions: The results show that the heterozygous deletion of the Yap1 gene in mice leads to complex ocular abnormalities, including microphthalmia, corneal fibrosis, anterior segment dysgenesis, and cataract.

YAP and TAZ are distinct effectors of corneal myofibroblast transformation.

PURPOSE: Transforming growth factor ß1 (TGFß1) is elevated in wounds after injury and promotes the transdifferentiation of quiescent cells in the stroma (keratocytes, to activated fibroblasts and subsequently myofibroblasts-KFM transformation). Coactivators of transcription, YAP (Yes-associated protein) and TAZ (Transcriptional coactivator with PDZ-binding motif), are mechanotransducers that intersect with the TGFß pathway via interactions with Smad proteins. Here, we examined the distinct role of YAP and TAZ on TGFß1 induced myofibroblast transformation of primary human corneal fibroblasts (HCFs). METHODS: A knockdown approach was used to silence YAP and TAZ individually in HCFs. Forty-eight hours post siRNA transfection, cells were cultured in the presence or absence of 2 ng/ml TGFß1 for 24h. The cells were subjected to nuclear and cytoplasmic fractionation. The expression of α-smooth muscle actin (αSMA), Smad 2, 3 and 4, CTGF and phospho-Smad2, 3, and 4 were assessed by qPCR and Western blotting. RESULTS: TGFß1 stimulation resulted in the decreased phosphorylation of YAP in the cytosol, and increased levels of phosphorylated TAZ and Smad2/3/4 in the nucleus. Knockdown of TAZ resulted in elevated YAP expression but not vice versa. Additionally, knockdown of TAZ but not YAP resulted in upregulation of αSMA expression in the presence and absence of TGFß1. In the presence of TGFß1 YAP knockdown increased Smad2/3/4 expression and Smad4 phosphorylation, while TAZ knockdown had no effect on Smad2/3/4 expression and phosphorylation. YAP knockdown inhibited CTGF expression while TAZ knockdown resulted in its increased expression. Finally, simultaneous knockdown of YAP and TAZ resulted in cell death. CONCLUSION: Our findings suggest that YAP and TAZ function as distinct modulators of TGFß1 induced myofibroblast transformation and have different roles in signalling. Specifically, TAZ limits YAP's ability to mediate KFM transformation via Smad proteins. The data also suggest that while having distinct effects, YAP and TAZ have redundant or combinatorial functions critical to cell survival. These results suggest that a loss of TAZ may help drive corneal haze and fibrosis and that the balance between YAP/TAZ is essential in controlling myofibroblast differentiation.

Biomechanical changes to Descemet's membrane precede endothelial cell loss in an early-onset murine model of Fuchs endothelial corneal dystrophy.

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.

Clinical findings and normative ocular data for free-living Anna's (Calypte anna) and Black-chinned (Archilochus alexandri) Hummingbirds.

OBJECTIVE: To estimate the prevalence of ocular disease and obtain normative ocular data for free-living hummingbirds. ANIMALS STUDIED: Two hundred and sixty-three free-living, adult Hummingbirds from coastal and inland central California were studied, including Anna's (Calypte anna, n = 186) and Black-chinned (Archilochus alexandri; n = 77) hummingbirds. PROCEDURES: Slit lamp biomicroscopy and indirect ophthalmoscopy were performed on all individuals. Rebound tonometry, measurement of horizontal palpebral fissure length, and streak retinoscopy were performed on select individuals. Five conscious Anna's Hummingbirds underwent ocular imaging including fundus photography, digital slit lamp photography, and anterior segment and retinal optical coherence tomography. RESULTS: The prevalence of ocular disease in this population was 2.28%. Ocular imaging revealed a thin cornea, shallow anterior chamber, large lens, and a single central, deep convexiclivate fovea. Mean ± SD intraocular pressure was 11.21 ± 2.23 mm Hg. Mean ± SD eyelid length was 2.59 ± 0.19 mm. All eyes were emmetropic or mildly hyperopic with a mean (range) ± SD refractive error of +0.32 (-0.25 to +1) ± 0.33 diopters. CONCLUSIONS: Consistent with previous reports, these data suggest that hummingbirds have visual characteristics found in predatory and prey species, as well as a low prevalence of spontaneous ocular disease. This work provides a set of reference values and clinical findings that can be used in the future research on hummingbird vision and ocular disease. It also provides representative diagnostic images of normal birds and demonstrates that advanced ocular imaging can be performed on manually restrained hummingbirds without pharmacologic dilation.

Effects of 5% sodium chloride ophthalmic ointment on thickness and morphology of the normal canine cornea.

OBJECTIVE: To determine the effect of 5% sodium chloride ophthalmic ointment (5% NaCl) on thickness and morphology of the normal canine cornea using ultrasonic pachymetry (USP), in vivo confocal microscopy (IVCM), and Fourier-domain optical coherence tomography (FD-OCT). METHODS: Five healthy laboratory Beagles received ophthalmic examinations including USP, IVCM, and FD-OCT prior to and at fixed intervals following treatment. The right and left eyes were treated with 5% NaCl and artificial tears ophthalmic ointment (AT), respectively, every 2 hours for 4 treatments/d (days 2-9), and then hourly for 7 treatments/d (day 10). Treatment groups were statistically compared using mixed-effects linear regression. RESULTS: Treatment with 5% NaCl resulted in a 12 µm decrease in corneal thickness from baseline (P < .001), while there was no significant difference in corneal thickness between values obtained at baseline and following treatment with AT (P = .82). Epithelial cell density significantly increased from baseline (530 ± 52 cells/mm2 ) to 577 ± 43 and 567 ± 15 cells/mm2 with 5% NaCl and AT, respectively (P = .003 and .005, respectively). However, keratocyte cell density in the anterior and posterior stroma and endothelial cell density did not significantly differ following treatment with 5% NaCl or AT ointment (P > .05). CONCLUSIONS: Short-term topical treatment with 5% NaCl decreased corneal thickness in normal dogs with no observable changes in corneal morphology or signs of ocular toxicity.

Acremonium and trichosporon fungal keratoconjunctivitis in a Leopard Gecko (Eublepharis macularius).

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.

A novel herpesvirus associated with chronic superficial keratitis and proliferative conjunctivitis in a great horned owl (Bubo virginianus).

An adult great-horned owl (Bubo virginianus; GHOW) presented with a history of recurrent corneal ulceration of the right eye (OD). Findings included ulcerative superficial keratitis, proliferative conjunctivitis, and iris pigmentary changes. The ulcer was initially nonresponsive to medical therapy, but showed rapid and appropriate healing following diamond burr debridement. Proliferative conjunctivitis markedly improved following topical antiviral therapy with cidofovir 1%, interferon alpha 2B ophthalmic solutions, and oral l-lysine. Histopathologic evaluation of a conjunctival biopsy revealed epithelial features suspicious for viral cytopathic changes and intranuclear structures suspicious for viral inclusions, suggestive of a possible viral-induced papillomatous conjunctivitis. A novel alphaherpesvirus, referred to as Strigid Herpesvirus 1 (StrHV1), was identified using PCR and gene sequencing. This case represents a new clinical manifestation of a previously unreported herpesvirus in the GHOW. Identification of the herpes virus was critical to administration of appropriate therapy and resolution of the conjunctivitis, and corneal epithelial debridement promoted resolution of the chronic corneal epithelial defect.

Modulation of human corneal stromal cell differentiation by hepatocyte growth factor and substratum compliance.

Corneal wound healing is a complex process that consists of cellular integration of multiple soluble biochemical cues and cellular responses to biophysical attributes associated with the matrix of the wound space. Upon corneal stromal wounding, the transformation of corneal fibroblasts to myofibroblasts is promoted by transforming growth factor-ß (TGFß). This process is critical for wound healing; however, excessive persistence of myofibroblasts in the wound space has been associated with corneal fibrosis resulting in severe vision loss. The objective of this study was to determine the effect of hepatocyte growth factor (HGF), which can modulate TGFß signaling, on corneal myofibroblast transformation by analyzing the expression of α-smooth muscle actin (αSMA) as a marker of myofibroblast phenotype particularly as it relates to biomechanical cues. Human corneal fibroblasts were cultured on tissue culture plastic (>1 GPa) or hydrogel substrates mimicking human normal or wounded corneal stiffness (25 and 75 kPa) in media containing TGFß1 ±â€¯HGF. The expression of αSMA was analyzed by quantitative PCR, Western blot and immunocytochemistry. Cellular stiffness, which is correlated with cellular phenotype, was measured by atomic force microscopy (AFM). In primary human corneal fibroblasts, the mRNA expression of αSMA showed a clear dose response to TGFß1. The expression was significantly suppressed when cells were incubated with 20 ng/ml HGF in the presence of 2 ng/ml of TGFß1. The protein expression of αSMA induced by 5 ng/ml TGFß1 was also decreased by 20 ng/ml of HGF. Cells cultured on hydrogels mimicking human normal (25 kPa) and fibrotic (75 kPa) cornea also showed an inhibitory effect of HGF on αSMA expression in the presence or absence of TGFß1. Cellular stiffness was decreased by HGF in the presence of TGFß1 as measured by AFM. In this study, we have demonstrated that HGF can suppress the myofibroblast phenotype promoted by TGFß1 in human corneal stromal cells. These data suggest that HGF holds the potential as a therapeutic agent to improve wound healing outcomes by minimizing corneal fibrosis.

The role of hepatocyte growth factor in corneal wound healing.

Hepatocyte growth factor (HGF) is a glycoprotein produced by mesenchymal cells and operates as a key molecule for tissue generation and renewal. During corneal injury, HGF is primarily secreted by stromal fibroblasts and promotes epithelial wound healing in a paracrine manner. While this mesenchymal-epithelial interaction is well characterized in various organs and the cornea, the role of HGF in corneal stromal and endothelial wound healing is understudied. In addition, HGF has been shown to play an anti-fibrotic role by inhibiting myofibroblast generation and subsequent production of a disorganized extracellular matrix and tissue fibrosis. Therefore, HGF represents a potential therapeutic tool in numerous organs in which myofibroblasts are responsible for tissue scarring. Corneal fibrosis can be a devastating sequela of injury and can result in corneal opacification and retrocorneal membrane formation leading to severe vision loss. In this article, we concisely review the available literature regarding the role of HGF in corneal wound healing. We highlight the influence of HGF on cellular behaviors in each corneal layer. Additionally, we suggest the possibility that HGF may represent a therapeutic tool for interrupting dysregulated corneal repair processes to improve patient outcomes.

Latrunculin B and substratum stiffness regulate corneal fibroblast to myofibroblast transformation.

The transformation of keratocytes and fibroblasts to myofibroblasts is important to corneal wound healing as well as formation of stromal haze. The purpose of this study was to determine the effect of latrunculin B, an actin cytoskeleton disruptor in conjunction with a fundamental biophysical cue, substrate stiffness, on myofibroblast transformation in vitro and in vivo. Rabbit corneal fibroblasts were cultured on substrates of differing compliance (1.5, 22, and 71 kPa) and tissue culture plastic (TCP; > 1 GPa) in media containing 0 or 10 ng/ml TGFß1 for 72 h. Cells were treated with 0.4 µM Lat-B or DMSO for 30 min every 24 h for 72 h. RNA was collected from cells and expression of alpha-smooth muscle actin (α-SMA), keratocan, and ALDH1A1 determined using qPCR; immunocytochemistry was used to assess α-SMA protein expression. A rabbit phototherapeutic keratectomy (PTK) model was used to assess the impact of 0.1% Lat-B (n = 3) or 25% DMSO (vehicle control, n = 3) on corneal wound healing by assessment of epithelial wound size with fluorescein stain and semi-quantitative stromal haze scoring by an observer masked to treatment group as well as Fourier-domain optical coherence tomography (FD-OCT) at set time points. Statistical analysis was completed using one-way or two-way analysis of variance. Treatment with Lat-B versus DMSO resulted in significantly less αSMA mRNA (P ≤ 0.007) for RCF cells grown on 22 and 71 kPa substrates as well as TCP without or with TGFß1, and significantly decreased α-SMA protein expression in RCFs cultured on the intermediate (22 kPa) stiffness in the absence (P = 0.028) or presence (P = 0.018) of TGFß1. Treatment with Lat-B versus DMSO but did not significantly alter expression of keratocan or ALDH1A1 mRNA in RCFs (P > 0.05) in the absence or presence of TGFß1, but RCFs grown on stiff hydrogels (71 kPa) had significantly more keratocan mRNA expression versus the 22 kPa hydrogel or TCP (P < 0.001) without TGFß1. Administration of topical Lat-B BID was well tolerated by rabbits post-PTK but did not significantly alter epithelial wound closure, stromal haze score, stromal haze thickness as measured by FD-OCT in comparison to DMSO-treated rabbits. When corneal stromal cells are cultured on substrates possessing biologically relevant substratum stiffnesses, Lat-B modulates mRNA and protein expression of α-SMA and thus modulates myofibroblast transformation. At a dose and dose-frequency that reduced IOP in human glaucoma patients, Lat-B treatment did not substantially impact corneal epithelial or stromal wound healing in a rabbit PTK model. While a significant impact on wound healing was observed at the concentration and dose frequency reported here was not found, encouraging in vitro data support further investigations of topically applied Lat-B to determine if this compound can reduce stromal fibrosis.

Intraocular lens power calculation for the equine eye.

BACKGROUND: Phacoemulsification and intraocular lens (IOL) implantation during cataract surgery in horses occur with increasing frequency. To reduce the postoperative refractive error it is necessary to determine the proper IOL power. In the present study retinoscopy, keratometry and ultrasonographic biometry were performed on 98 healthy equine eyes from 49 horses. The refractive state, corneal curvature (keratometry) and the axial location of all optical interfaces (biometry) were measured. The influences of breed, height at the withers, gender and age on values obtained and the comparison between the left and right eye were evaluated statistically. Corresponding IOL power were calculated by use of Binkhorst and Retzlaff theoretical formulas. RESULTS: Mean ± SD refractive state of the horses was + 0.32 ± 0.66 D. Averaged corneal curvature for Haflinger, Friesian, Pony, Shetland pony and Warmblood were 21.30 ± 0.56 D, 20.02 ± 0.60 D, 22.61 ± 1.76 D, 23.77 ± 0.94 D and 20.76 ± 0.88 D, respectively. The estimated postoperative anterior chamber depth (C) was calculated by the formula C = anterior chamber depth (ACD)/0.73. This formula was determined by a different research group. C and axial length of the globe averaged for Haflinger 9.30 ± 0.54 mm and 39.43 ± 1.26 mm, for Friesian 10.12 ± 0.33 mm and 42.23 ± 1.00 mm, for Pony 8.68 ± 0.78 mm and 38.85 ± 3.13 mm, for Shetland pony 8.71 ± 0.81 mm and 37.21 ± 1.50 mm and for Warmblood 9.39 ± 0.51 mm and 40.65 ± 1.30 mm. IOL power was calculated with the Binkhorst and Retzlaff theoretical formulas. Calculated IOL power for the several breeds ranged from 18.03 D to 19.55 D. The mean value across all horses was 18.73 D determined with Binkhorst formula and 18.54 D determined with Retzlaff formula. CONCLUSIONS: Mean result of this study is: an 18.5 D IOL seemed to be the most appropriate to achieve emmetropia after IOL implantation in horses. Cataract surgery without IOL implantation results in hyperopic and visual compromised horses. Retinoscopy, keratometry and ultrasonographic biometry should be performed on every affected horse and postoperative visual outcome should be determined.