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PURPOSE: The aim of this study was to develop and validate a method to generate phenol red thread tests (PRTT) due to the lack of availability of commercial PRTT. METHODS: White cotton thread was dyed with phenol red (pH indicator) for 48 h, dried, cut, and sterilized. To validate its wicking ability, the thread was inserted into solutions of varying pH, flanking the pH of healthy tears, for different time intervals. To assess its diagnostic utility, PRTTs were performed in vivo on wildtype and a murine model of evaporative dry eye, acyl-coA: wax alcohol acyltransferase 2 knockout (Awat2 KO) mice. RESULTS: Two batches of PRTT were produced that had a similar appearance and function to the commercial product. In vitro testing revealed no significant differences in the wicking kinetics at any time point across the pH solutions for batch 1 and only one difference for batch 2 (pH 7.4 vs 7.8 at 5 s, P = 0.029). When comparing both batches, similar wicking kinetics were found with only two significant differences identified (pH 7.6 at 40 s and pH 7.8 at 35 s, P < 0.01). In vivo, our PRTT yielded similar measurements to the commercial PRTT in wildtype and Awat2 KO mice and detected a significant increase in aqueous tear volume in the Awat2 KO mice (commercial: P = 0.015, our PRTT: P = 0.002). CONCLUSION: Our method provides a reproducible diagnostic test that performs similarly to its commercial counterpart in a relevant dry eye model indicating that it can serve as a valid and reliable replacement.
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Although biomechanical changes of the trabecular meshwork (TM) are important to the pathogenesis of glucocorticoids-induced ocular hypertension (GC-OHT), there is a knowledge gap in the underlying molecular mechanisms of the development of it. In this study, we performed intravitreal triamcinolone injection (IVTA) in one eye of 3 rhesus macaques. Following IVTA, we assessed TM stiffness using atomic force microscopy and investigated changes in proteomic and miRNA expression profiles. One of 3 macaques developed GC-OHT with a difference in intraocular pressure of 4.2 mmHg and a stiffer TM with a mean increase in elastic moduli of 0.60 kPa versus the non-injected control eye. In the IVTA-treated eyes, proteins associated with extracellular matrix remodeling, cytoskeletal rearrangement, and mitochondrial oxidoreductation were significantly upregulated. The significantly upregulated miR-29b and downregulated miR-335-5p post-IVTA supported the role of oxidative stress and mitophagy in the GC-mediated biomechanical changes in TM, respectively. The significant upregulation of miR-15/16 cluster post-IVTA may indicate a resultant TM cell apoptosis contributing to the increase in outflow resistance. Despite the small sample size, these results expand our knowledge of GC-mediated responses in the TM and furthermore, may help explain steroid responsiveness in clinical settings.
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Glucocorticoides , Presión Intraocular , Inyecciones Intravítreas , Macaca mulatta , MicroARNs , Proteómica , Malla Trabecular , Animales , MicroARNs/genética , MicroARNs/metabolismo , Malla Trabecular/efectos de los fármacos , Malla Trabecular/metabolismo , Glucocorticoides/farmacología , Glucocorticoides/administración & dosificación , Proteómica/métodos , Presión Intraocular/efectos de los fármacos , Presión Intraocular/fisiología , Hipertensión Ocular/metabolismo , Triamcinolona Acetonida/farmacología , Fenómenos Biomecánicos , Modelos Animales de Enfermedad , Microscopía de Fuerza Atómica , Triamcinolona/farmacología , Triamcinolona/administración & dosificaciónRESUMEN
Understanding and treating human diseases require valid animal models. Leveraging the genetic diversity in rhesus macaque populations across eight primate centers in the United States, we conduct targeted-sequencing on 1845 individuals for 374 genes linked to inherited human retinal and neurodevelopmental diseases. We identify over 47,000 single nucleotide variants, a substantial proportion of which are shared with human populations. By combining rhesus and human allele frequencies with established variant prediction methods, we develop a machine learning-based score that outperforms established methods in predicting missense variant pathogenicity. Remarkably, we find a marked number of loss-of-function variants and putative deleterious variants, which may lead to the development of rhesus disease models. Through phenotyping of macaques carrying a pathogenic OPA1:p.A8S variant, we identify a genetic model of autosomal dominant optic atrophy. Finally, we present a public website housing variant and genotype data from over two thousand rhesus macaques.
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Modelos Animales de Enfermedad , Variación Genética , Macaca mulatta , Animales , Macaca mulatta/genética , Humanos , Frecuencia de los Genes , Atrofia Óptica Autosómica Dominante/genética , Polimorfismo de Nucleótido Simple , Fenotipo , Aprendizaje Automático , Genotipo , Mutación MissenseRESUMEN
This study evaluated the tolerability and efficacy of the topical rho-kinase inhibitor netarsudil for canine primary corneal endothelial degeneration (PCED). Twenty-six eyes of 21 client-owned dogs with PCED were enrolled in a prospective, randomized, vehicle control clinical trial and received topical netarsudil 0.02% (Rhopressa®) or vehicle control twice daily (BID) for the first 4 months. Then, all patients received netarsudil for the next 4 or 8 months. Complete ophthalmic examination, ultrasonic pachymetry, Fourier-domain optical coherence tomography, and in vivo confocal microscopy were performed at baseline and 1, 2, 4, 6, 8 and 12 months. Effect of netarsudil on central corneal thickness (CCT), percentage of cornea with edema, and endothelial cell density (ECD) were evaluated by repeated measures ANOVA. Kaplan-Meier curves and log-rank test were used to compare corneal edema and clinical progression of eyes in netarsudil versus vehicle control groups. All dogs developed conjunctival hyperemia in at least one eye while receiving netarsudil. Unilateral transient reticulated intraepithelial bullae and stromal hemorrhage were observed respectively in 2 dogs in the netarsudil group. Two dogs showed persistently decreased tear production while receiving netarsudil, requiring topical immunomodulatory treatment. No significant differences in CCT, ECD, corneal edema or clinical progression were observed between netarsudil or vehicle treated eyes. When comparing efficacy of topical netarsudil BID and topical ripasudil 0.4% administered four times daily from our previous study, dogs receiving ripasudil had significantly less progression than those receiving netarsudil.
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Benzoatos , Distrofias Hereditarias de la Córnea , Edema Corneal , Isoquinolinas , Sulfonamidas , beta-Alanina , Animales , Perros , beta-Alanina/análogos & derivados , Edema Corneal/tratamiento farmacológico , Progresión de la Enfermedad , Soluciones Oftálmicas/uso terapéutico , Estudios ProspectivosRESUMEN
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.
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Distrofia Endotelial de Fuchs , Queratocono , Animales , Mecanotransducción Celular , Células Endoteliales , Córnea/fisiologíaRESUMEN
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.
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Queratectomía Fotorrefractiva , Animales , Conejos , Queratectomía Fotorrefractiva/métodos , Córnea/patología , Cicatrización de Heridas , Colágeno , Sustancia Propia/patología , Riboflavina , Inflamación/patología , Reactivos de Enlaces Cruzados/farmacología , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Rayos UltravioletaRESUMEN
Purpose: Choroidal vascular changes occur with normal aging and age-related macular degeneration (AMD). Here, we evaluate choroidal thickness and vascularity in aged rhesus macaques to better understand the choroid's role in this nonhuman primate model of AMD. Methods: We analyzed optical coherence tomography (OCT) images of 244 eyes from 122 rhesus macaques (aged 4-32 years) to measure choroidal thickness (CT) and choroidal vascularity index (CVI). Drusen number, size, and volume were measured by semiautomated annotation and segmentation of OCT images. We performed regression analyses to determine any association of CT or CVI with age, sex, and axial length and to determine if the presence and volume of soft drusen impacted these choroidal parameters. Results: In rhesus macaques, subfoveal CT decreased with age at 3.2 µm/y (R2 = 0.481, P < 0.001), while CVI decreased at 0.66% per year (R2 = 0.257, P < 0.001). Eyes with soft drusen exhibited thicker choroid (179.9 ± 17.5 µm vs. 162.0 ± 27.9 µm, P < 0.001) and higher CVI (0.612 ± 0.051 vs. 0.577 ± 0.093, P = 0.005) than age-matched control animals. Neither CT or CVI appeared to be associated with drusen number, size, or volume in this cohort. However, some drusen in macaques were associated with underlying choroidal vessel enlargement resembling pachydrusen in human patients with AMD. Conclusions: Changes in the choroidal vasculature in rhesus macaques resemble choroidal changes in human aging, but eyes with drusen exhibit choroidal thickening, increased vascularity, and phenotypic characteristics of pachydrusen observed in some patients with AMD.
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Degeneración Macular , Drusas Retinianas , Humanos , Animales , Macaca mulatta , Estudios Retrospectivos , Retina , Coroides/irrigación sanguínea , Envejecimiento , Tomografía de Coherencia Óptica/métodosRESUMEN
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.
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Purpose: There is a pressing need to investigate the impact of type II diabetes mellitus on the posterior cornea in donor tissues given its increasing prevalence and potential impact on endothelial keratoplasty surgical outcomes. Methods: Immortalized human cultured corneal endothelial cells (CECs; HCEC-B4G12) were grown in hyperglycemic media for 2 weeks. Extracellular matrix (ECM) adhesive glycoprotein expression and advanced glycation end products (AGEs) in cultured cells and corneoscleral donor tissues, as well as the elastic modulus for the Descemet membrane (DMs) and CECs of diabetic and nondiabetic donor corneas, were measured. Results: In CEC cultures, increasing hyperglycemia resulted in increased transforming growth factor beta-induced (TGFBI) protein expression and colocalization with AGEs in the ECM. In donor corneas, the thicknesses of the DM and the interfacial matrix (IFM) between the DM and stroma both increased from 8.42 ± 1.35 µm and 0.504 ± 0.13 µm in normal corneas, respectively, to 11.13 ± 2.91 µm (DM) and 0.681 ± 0.24 µm (IFM) in non-advanced diabetes (P = 0.013 and P = 0.075, respectively) and 11.31 ± 1.76 µm (DM) and 0.744 ± 0.18 µm (IFM) in advanced diabetes (AD; P = 0.0002 and P = 0.003, respectively). Immunofluorescence in AD tissues versus controls showed increased AGEs (P < 0.001) and markedly increased labeling intensity for adhesive glycoproteins, including TGFBI, that colocalized with AGEs. The elastic modulus significantly increased between AD and control tissues for the DMs (P < 0.0001) and CECs (P < 0.0001). Conclusions: Diabetes and hyperglycemia alter human CEC ECM structure and composition, likely contributing to previously documented complications of endothelial keratoplasty using diabetic donor tissue, including tearing during graft preparation and reduced graft survival. AGE accumulation in the DM and IFM may be a useful biomarker for determining diabetic impact on posterior corneal tissue.
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Queratoplastia Endotelial de la Lámina Limitante Posterior , Diabetes Mellitus Tipo 2 , Hiperglucemia , Humanos , Lámina Limitante Posterior/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Células Endoteliales , Queratoplastia Endotelial de la Lámina Limitante Posterior/métodos , Córnea , Matriz Extracelular , Hiperglucemia/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Donantes de Tejidos , Endotelio Corneal/metabolismoRESUMEN
Purpose: We 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). Methods: A 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. Results: Mice 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. Conclusions: There 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.
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Células Endoteliales , Distrofia Endotelial de Fuchs , Humanos , Ratones , Animales , Células Endoteliales/metabolismo , Mecanotransducción Celular , Distrofia Endotelial de Fuchs/patología , Endotelio Corneal/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
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.
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Grafito , Nanoestructuras , Animales , Humanos , Conejos , Grafito/toxicidad , Córnea , Cicatrización de HeridasRESUMEN
Purpose: Foveoschisis involves the pathologic splitting of retinal layers at the fovea, which may occur congenitally in X-linked retinoschisis (XLRS) or as an acquired complication of myopia. XLRS is attributed to functional loss of the retinal adhesion protein retinoschisin 1 (RS1), but the pathophysiology of myopic foveoschisis is unclear due to the lack of animal models. Here, we characterized a novel nonhuman primate model of myopic foveoschisis through clinical examination and multimodal imaging followed by morphologic, cellular, and transcriptional profiling of retinal tissues and genetic analysis. Methods: We identified a rhesus macaque with behavioral and anatomic features of myopic foveoschisis, and monitored disease progression over 14 months by fundus photography, fluorescein angiography, and optical coherence tomography (OCT). After necropsy, we evaluated anatomic and cellular changes by immunohistochemistry and transcriptomic changes using single-nuclei RNA-sequencing (snRNA-seq). Finally, we performed Sanger and whole exome sequencing with focus on the RS1 gene. Results: Affected eyes demonstrated posterior hyaloid traction and progressive splitting of the outer plexiform layer on OCT. Immunohistochemistry showed increased GFAP expression in Müller glia and loss of ramified Iba-1+ microglia, suggesting macro- and microglial activation with minimal photoreceptor alterations. SnRNA-seq revealed gene expression changes predominantly in cones and retinal ganglion cells involving chromatin modification, suggestive of cellular stress at the fovea. No defects in the RS1 gene or its expression were detected. Conclusions: This nonhuman primate model of foveoschisis reveals insights into how acquired myopic traction leads to phenotypically similar morphologic and cellular changes as congenital XLRS without alterations in RS1.
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Miopía Degenerativa , Retinosquisis , Animales , Macaca mulatta , Retina , Fóvea Central , Tomografía de Coherencia ÓpticaRESUMEN
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.
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Antiinfecciosos , alfa-Defensinas , beta-Defensinas , Humanos , Animales , Caballos , beta-Defensinas/genética , beta-Defensinas/metabolismo , alfa-Defensinas/metabolismo , Amnios/metabolismo , Córnea/metabolismo , Conjuntiva/metabolismoRESUMEN
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.
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Distrofia Endotelial de Fuchs , Factor de Crecimiento Transformador beta1 , Animales , Bovinos , Factor de Crecimiento Transformador beta1/metabolismo , Células Endoteliales/metabolismo , Factor de Crecimiento Transformador beta3/metabolismo , Distrofia Endotelial de Fuchs/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Endotelio Corneal/metabolismoRESUMEN
Purpose: Non-human primates (NHPs) are useful models for human retinal disease. Chromatic pupillometry has been proposed as a noninvasive method of identifying inherited retinal diseases (IRDs) in humans; however, standard protocols employ time-consuming dark adaptation. We utilized shortened and standard dark-adaptation protocols to compare pupillary light reflex characteristics following chromatic stimulation in rhesus macaques with achromatopsia to wild-type (WT) controls with normal retinal function. Methods: Nine rhesus macaques homozygous for the p.R656Q mutation (PDE6C HOMs) and nine WT controls were evaluated using chromatic pupillometry following 1-minute versus standard 20-minute dark adaptations. The following outcomes were measured and compared between groups: pupil constriction latency, peak constriction, pupil constriction time, and constriction velocity. Results: Pupil constriction latency was significantly longer in PDE6C HOMs with red-light (P = 0.0002) and blue-light (P = 0.04) stimulation versus WT controls. Peak constriction was significantly less in PDE6C HOMs with all light stimulation compared to WT controls (P < 0.0001). Pupil constriction time was significantly shorter in PDE6C HOMs versus WT controls with red-light (P = 0.04) and white-light (P = 0.003) stimulation. Pupil constriction velocity was significantly slower in PDE6C HOMs versus WT controls with red-light (P < 0.0001), blue-light (P < 0.0001), and white-light (P = 0.0002) stimulation. Dark adaptation time only significantly affected peak (P = 0.008) and time of pupil constriction (P = 0.02) following blue-light stimulation. Conclusions: Chromatic pupillometry following 1- and 20-minute dark adaptation is an effective tool for screening NHPs for achromatopsia. Translational Relevance: Rapid identification of NHPs with IRDs will provide animal research models to advance research and treatment of achromatopia in humans.
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Defectos de la Visión Cromática , Adaptación a la Oscuridad , Modelos Animales de Enfermedad , Macaca mulatta , Reflejo Pupilar , Animales , Reflejo Pupilar/fisiología , Adaptación a la Oscuridad/fisiología , Defectos de la Visión Cromática/genética , Defectos de la Visión Cromática/fisiopatología , Defectos de la Visión Cromática/diagnóstico , Pupila/fisiología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Masculino , Estimulación Luminosa , FemeninoRESUMEN
Purpose: Uveal coloboma is a congenital eye malformation caused by failure of the optic fissure to close in early human development. Despite significant progress in identifying genes whose regulation is important for executing this closure, mutations are detected in a minority of cases using known gene panels, implying additional genetic complexity. We have previously shown knockdown of znf503 (the ortholog of mouse Zfp503) in zebrafish causes coloboma. Here we characterize Zfp503 knockout (KO) mice and evaluate transcriptomic profiling of mutant versus wild-type (WT) retinal pigment epithelium (RPE)/choroid. Methods: Zfp503 KO mice were generated by gene targeting using homologous recombination. Embryos were characterized grossly and histologically. Patterns and level of developmentally relevant proteins/genes were examined with immunostaining/in situ hybridization. The transcriptomic profile of E11.5 KO RPE/choroid was compared to that of WT. Results: Zfp503 is dynamically expressed in developing mouse eyes, and loss of its expression results in uveal coloboma. KO embryos exhibit altered mRNA levels and expression patterns of several key transcription factors involved in eye development, including Otx2, Mitf, Pax6, Pax2, Vax1, and Vax2, resulting in a failure to maintain the presumptive RPE, as evidenced by reduced melanin pigmentation and its differentiation into a neural retina-like lineage. Comparison of RNA sequencing data from WT and KO E11.5 embryos demonstrated reduced expression of melanin-related genes and significant overlap with genes known to be dynamically regulated at the optic fissure. Conclusions: These results demonstrate a critical role of Zfp503 in maintaining RPE fate and optic fissure closure.
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Coloboma , Neuropéptidos , Animales , Humanos , Ratones , Coloboma/genética , Coloboma/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Melaninas/metabolismo , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Neuropéptidos/genética , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Pez Cebra/genéticaRESUMEN
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.
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Glaucoma de Ángulo Cerrado , Glaucoma de Ángulo Abierto , Perros , Humanos , Animales , Femenino , Glaucoma de Ángulo Cerrado/genética , Glaucoma de Ángulo Cerrado/veterinaria , Glaucoma de Ángulo Cerrado/complicaciones , Estudio de Asociación del Genoma Completo , Fitomejoramiento , Iris , Glaucoma de Ángulo Abierto/complicaciones , Enfermedad Aguda , Presión IntraocularRESUMEN
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-ß1) 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-ß1-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.
Asunto(s)
Diterpenos , Factor de Crecimiento Transformador beta1 , Conejos , Ratas , Animales , Factor de Crecimiento Transformador beta1/metabolismo , Miofibroblastos/metabolismo , Actinas/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Diterpenos/farmacología , Córnea/metabolismo , Fibrosis , ARN Mensajero/genéticaRESUMEN
OBJECTIVE: To identify genetic associations with primary glaucoma (PG) in American Cocker Spaniels using a genome-wide association study (GWAS). ANIMALS: A nationwide ambidirectional case-control cohort study was performed in American Cocker Spaniels that had an ophthalmic examination performed by a veterinarian. Ninety-four dogs with PG (cases) and 111 dogs without glaucoma (controls) met phenotypic criteria and had a blood sample collected after receiving informed owner consent. PROCEDURES: Genomic DNA was extracted from whole blood samples and genotyped (CanineHD BeadChip, Illumina Inc). A case-control GWAS using a linear mixed model was performed, and 3 significance thresholds were calculated (1) using a Bonferroni correction on all single nucleotide polymorphisms (SNPs) included in the GWAS, (2) using a Bonferroni correction on only the unlinked SNPs from a pruned data set, and (3) using 10,000 random phenotype permutations. RESULTS: Following genotype data quality control, 89 cases and 93 controls were included in the GWAS. We identified an association on canine chromosome (CFA10); however, it did not reach statistical significance. Potential candidate genes within the surrounding linkage disequilibrium interval include coiled-coil domain containing 85A (CCDC85A) and extracellular growth factor containing fibulin extracellular matrix protein 1 (EFEMP1). CLINICAL RELEVANCE: Primary glaucoma in the American Cocker Spaniel is a complex heterogeneous disease that may be influenced by a locus on CFA10. The candidate genes CCDC85A and EFEMP1 within the identified linkage disequilibrium interval have been shown to be involved in human open-angle glaucoma.