RESUMO
We present a patient-specific finite element model of the human cornea that accounts for the presence of the epithelium. The thin anterior layer that protects the cornea from the external actions has a scant relevance from the mechanical point of view, and it has been neglected in most numerical models of the cornea, which assign to the entire cornea the mechanical properties of the stroma. Yet, modern corneal topographers capture the geometry of the epithelium, which can be naturally included into a patient-specific solid model of the cornea, treated as a multi-layer solid. For numerical applications, the presence of a thin layer on the anterior cornea requires a finer discretization and the definition of two constitutive models (including the corresponding properties) for stroma and epithelium. In this study, we want to assess the relevance of the inclusion of the epithelium in the model of the cornea, by analyzing the effects in terms of uncertainties of the mechanical properties, stress distribution across the thickness, and numerical discretization. We conclude that if the epithelium is modeled as stroma, the material properties should be reduced by 10%. While this choice represents a sufficiently good approximation for the simulation of in vivo mechanical tests, it might result into an under-estimation of the postoperative stress in the simulation of refractive surgery.
Assuntos
Córnea , Epitélio Corneano , Análise de Elementos Finitos , Estresse Mecânico , Humanos , Córnea/fisiologia , Epitélio Corneano/fisiologia , Modelos Biológicos , Fenômenos Biomecânicos , Simulação por Computador , Análise Numérica Assistida por ComputadorRESUMO
The corneal epithelium is one of the first tissue barriers of the eye against the environment. In recent years, many studies provided better knowledge of its healing, its behavior and its essential role in the optical system of the eye. At the crossroads of basic science and clinical medicine, the study of the mechanical stresses applied to the cornea makes it possible to learn the behavior of epithelial cells and better understand ocular surface disease. We describe herein the current knowledge about the adhesion systems of the corneal epithelium and their resistance to mechanical stress. We will also describe the involvement of these mechanisms in corneal healing and their role in epithelial dynamics. Adhesion molecules of the epithelial cells, especially hemidesmosomes, allow the tissue cohesion required to maintain the integrity of the corneal epithelium against the shearing forces of the eyelids as well as external forces. Their regeneration after a corneal injury is mandatory for the restoration of a healthy epithelium. Mechanotransduction plays a significant role in regulating epithelial cell behavior, and the study of the epithelium's response to mechanical forces helps to better understand the evolution of epithelial profiles after refractive surgery. A better understanding of corneal epithelial biomechanics could also help improve future therapies, particularly in the field of tissue engineering.
Assuntos
Lesões da Córnea , Epitélio Corneano , Humanos , Fenômenos Biomecânicos , Mecanotransdução Celular , Córnea/fisiologia , Epitélio Corneano/fisiologia , Cicatrização/fisiologia , Lesões da Córnea/terapiaRESUMO
Recent evidence shows that epithelial stem/progenitor cells in barrier tissues such as the skin, airways and intestines retain a memory of previous injuries, which enables tissues to accelerate barrier restoration after subsequent injuries. The corneal epithelium, the outermost layer of the cornea, is the frontline barrier for the eye and is maintained by epithelial stem/progenitor cells in the limbus. Herein, we provide evidence that inflammatory memory also exists in the cornea. In mice, eyes that had been exposed to corneal epithelial injury exhibited faster re-epithelialization of the cornea and lower levels of inflammatory cytokines following subsequent injury (either the same or a different type of injury) relative to naïve eyes without previous injury. In ocular Sjögren's syndrome patients, corneal punctate epithelial erosions were significantly reduced after experiencing infectious injury compared with before. These results demonstrate that previous exposure of the corneal epithelium to inflammatory stimuli enhances corneal wound healing in response to a secondary assault, a phenomenon which points to the presence of nonspecific inflammatory memory in the cornea.
Assuntos
Lesões da Córnea , Epitélio Corneano , Relesões , Camundongos , Animais , Epitélio Corneano/fisiologia , Córnea , Cicatrização/fisiologia , InflamaçãoRESUMO
The discovery of slow-cycling cells at the corneal periphery three decades ago established the limbus as the putative corneal stem cell niche. Since then, studies have underscored the importance of the limbal stem cells in maintaining the health and function of the ocular surface. Advancements in our understanding of stem cell biology have been successfully translated into stem cell therapies for corneal diseases. Here, we review recent developments in mouse genetics, intravital imaging, and single-cell genomics that have revealed an underappreciated complexity of the limbal stem cells, from their molecular identity, function, and interactions with their niche environment. Continued efforts to elucidate stem cell dynamics of this extraordinary tissue are critical for not only understanding stem cell biology but also for advancing therapeutic innovation and development.
Assuntos
Doenças da Córnea , Epitélio Corneano , Limbo da Córnea , Camundongos , Animais , Epitélio Corneano/fisiologia , Células-Tronco/fisiologia , Nicho de Células-Tronco/genéticaRESUMO
Regulation of innate inflammation is critical for maintaining tissue homeostasis and barrier function, especially in those interfacing the external environments such as the skin and cornea. Expression of pro-inflammatory cytokines by injured tissues has been shown to exacerbate the inflammatory cascade, causing tissue damage. Interleukin 36, a subfamily of the IL-1 superfamily, consists of three pro-inflammatory agonists-IL36α, IL36ß, and IL36γ and an IL36 receptor antagonist (IL36Ra). The current investigation, for the first time, reports that IL36γ is the primary agonist expressed by the corneal epithelium, which is significantly upregulated following corneal injury. The function of IL36γ on non-immune cells, in addition to innate inflammatory cells, in regulating tissue homeostasis has not been well investigated. Using a loss-of-function approach via neutralizing antibody treatment, our data demonstrate that blocking endogenously expressed IL36γ in epithelial cells promotes rapid re-epithelialization in in vitro wound closure assay. Finally, by utilizing a naturally occurring antagonist IL36Ra in a well-established murine model of ocular injury, our study demonstrates that inhibition of IL36γ accelerates epithelial regeneration and suppresses tissue inflammation. Given rapid wound healing is critical for re-establishing normal tissue structure and function, our investigation on the function of IL36γ provides evidence for the development of novel IL36γ-targeting strategies to promote tissue repair.
Assuntos
Córnea/fisiologia , Interleucina-1/metabolismo , Animais , Epitélio Corneano/fisiologia , Inflamação/imunologia , Interleucina-1/imunologia , Camundongos , CicatrizaçãoRESUMO
Mast cells (MCs) regulate wound healing and are influenced by the autonomic nervous system (ANS). However, the underlying mechanisms affecting wound healing outcomes remain elusive. Here, we explored the specific role of the ANS by regulating MC degranulation following corneal epithelium abrasion. A mouse model of corneal abrasion was established by mechanically removing a 2-mm central epithelium. Wound closure, neutrophil infiltration, and transcription of injured corneas were investigated using whole-mount immunostaining, flow cytometry, and RNA-sequencing analysis, respectively. Inhibition of MC degranulation by the MC stabilizers cromolyn sodium and lodoxamide tromethamine increased the infiltration of neutrophils and delayed healing of abraded corneas. Moreover, transcriptomic profiling analysis showed that purified MCs from the limbus expressed adrenergic and cholinergic receptors. Pharmacological manipulation and sympathectomy with 6-hydroxydopamine confirmed that sympathetic nervous system signaling inhibited MC degranulation after corneal abrasion, whereas parasympathetic nervous system signaling enhanced MC degranulation. We conclude that normal degranulation of MCs in the corneal limbus and crosstalk between the ANS and MCs are crucial for the appropriate control of inflammation and the repair progress of wounded corneas. This suggests a potential approach for improving defective corneal wound healing by the administration of clinically available autonomic activity-modulating agents.
Assuntos
Lesões da Córnea , Epitélio Corneano , Animais , Sistema Nervoso Autônomo , Degranulação Celular , Epitélio Corneano/fisiologia , Inflamação , Mastócitos , Camundongos , Camundongos Endogâmicos C57BL , Cicatrização/fisiologiaRESUMO
The purpose of the study was to establish a simple ex vivo corneal re-epithelization model and study the labial mucosal epithelium grafting as a potential approach for ocular surface reconstruction. Four human donor corneal buttons were overstored in a corneal cold storage solution at 4 °C for 32-52 days. Four labial oral mucosa strips were dissected from four patients during fornix reconstruction after they signed informed consent. The substantia propria was trimmed off, and the resulting graft was sutured near the corneal limbus with running sutures (thus forming the tissue construct). Constructs were cultured under the standard conditions with the anterior corneal side outwards. After 3 weeks of culture, constructs were removed, washed, and fixed. Sections were stained with hematoxylin and eosin (HE), anti-keratins 4, 13, 19, and p63. Nuclei were counterstained with Hoechst. After the cultivation, all constructs were integral with the attached graft and non-loosened sutures. The native cells were absent in all donor corneas. Histological evaluation demonstrated that the labial mucosal grafts were attached to the Bowman's membrane (BM), and its cellular outgrowths were found to be transit from the graft to the BM over the anterior surface in all constructs. Cells expressed mucosal epithelial keratins 4, 13, and 19, and several were p63-positive in nuclei. In the study, a simple ex vivo corneal re-epithelization model was successfully established. The model was potent in studying the labial mucosal epithelium grafting as an option for autologous ocular surface reconstruction in patients with bilateral limbal stem cell deficiency.
Assuntos
Células Epiteliais/transplante , Epitélio Corneano/fisiologia , Limbo da Córnea/cirurgia , Mucosa Bucal/citologia , Reepitelização/fisiologia , Adulto , Idoso , Células Cultivadas , Doenças da Córnea/fisiopatologia , Doenças da Córnea/cirurgia , Humanos , Queratinas/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Transplante de Células-Tronco , Células-Tronco/patologia , Técnicas de SuturaRESUMO
Purpose: This work explores the abnormal expression of long noncoding RNAs (lncRNAs), microRNAs (miRNAs) and messenger RNAs (mRNAs) in diabetic corneal epithelial cells (CECs) and constructs an associated competitive endogenous RNA (ceRNA) network. Moreover, we revealed that Rik may exert advantageous effects on diabetic corneal epithelial wound closure by sponging miR-181a-5p. Methods: We obtained the profiles of differentially expressed lncRNAs (DELs) of CECs of type 1 diabetic versus control corneas by microarray and summarized the differentially expressed miRNAs (DEmiRs) and differentially expressed genes (DEGs) data by published literature. Subsequently, the ceRNA network was constructed using bioinformatics analyses. The levels of lncRNA ENSMUST00000153610/3632454L22Rik (Rik) and miR-181a-5p were verified. The localization of Rik was identified with fluorescence in situ hybridization (FISH), and dual-luciferase assays proved the targeted relationship between Rik and miR-181a-5p. Furthermore, we validated the functional impact of Rik in vitro. Results: Overall, 111 upregulated and 117 downregulated DELs were detected in diabetic versus control CECs. The level of Rik located in both the cytoplasm and the nucleus was clearly downregulated, whereas miR-181a-5p was upregulated in vitro and in vivo in the diabetic group versus the control group. Rik can act as a ceRNA to bind to miR-181a-5p, thus promoting diabetic corneal epithelial wound healing in vitro. Conclusions: This work investigated the expression profile of DELs and constructed ceRNA networks of diabetic CECs for the first time. Furthermore, we revealed that Rik may positively impact diabetic corneal epithelial wound healing by sponging miR-181a-5p, providing a novel potential therapeutic target of diabetic keratopathy (DK).
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Epitélio Corneano/fisiologia , Regulação da Expressão Gênica/fisiologia , MicroRNAs/genética , Proteínas Serina-Treonina Quinases/genética , RNA Longo não Codificante/genética , Cicatrização/fisiologia , Animais , Glicemia/metabolismo , Linhagem Celular , Diabetes Mellitus Tipo 1/fisiopatologia , Redes Reguladoras de Genes , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise Serial de Proteínas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Transfecção , Regulação para CimaRESUMO
BACKGROUND: Age-related changes affecting the ocular surface cause vision loss in the elderly. Cisd2 deficiency drives premature aging in mice as well as resulting in various ocular surface abnormalities. Here we investigate the role of CISD2 in corneal health and disease. METHODS: We studied the molecular mechanism underlying the ocular phenotypes brought about by Cisd2 deficiency using both Cisd2 knockout (KO) mice and a human corneal epithelial cell (HCEC) cell line carrying a CRISPR-mediated CISD2KO background. We also develop a potential therapeutic strategy that targets the Ca2+ signaling pathway, which has been found to be dysregulated in the corneal epithelium of subjects with ocular surface disease in order to extend the mechanistic findings into a translational application. FINDINGS: Firstly, in patients with corneal epithelial disease, CISD2 is down-regulated in their corneal epithelial cells. Secondly, using mouse cornea, Cisd2 deficiency causes a cycle of chronic injury and persistent repair resulting in exhaustion of the limbal progenitor cells. Thirdly, in human corneal epithelial cells, CISD2 deficiency disrupts intracellular Ca2+ homeostasis, impairing mitochondrial function, thereby retarding corneal repair. Fourthly, cyclosporine A and EDTA facilitate corneal epithelial wound healing in Cisd2 knockout mice. Finally, cyclosporine A treatment restores corneal epithelial erosion in patients with dry eye disease, which affects the ocular surface. INTERPRETATION: These findings reveal that Cisd2 plays an essential role in the cornea and that Ca2+ signaling pathways are potential targets for developing therapeutics of corneal epithelial diseases. FUNDING: This study was supported by the Ministry of Science and Technology (MOST) and Chang Gung Medical Research Foundation, Taiwan.
Assuntos
Epitélio Corneano/fisiologia , Proteínas de Membrana/genética , Regeneração , Animais , Biomarcadores , Cálcio/metabolismo , Linhagem Celular , Biologia Computacional/métodos , Ciclosporina/farmacologia , Células Epiteliais/metabolismo , Epitélio Corneano/citologia , Feminino , Perfilação da Expressão Gênica , Homeostase , Humanos , Leucócitos/imunologia , Leucócitos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Imagem Molecular , Oxigênio/metabolismo , Regeneração/efeitos dos fármacos , Regeneração/genética , Cicatrização/efeitos dos fármacosRESUMO
In the human cornea, regeneration of the epithelium is regulated by the stem cell reservoir of the limbus, which is the marginal region of the cornea representing the anatomical and functional border between the corneal and conjunctival epithelium. In support of this concept, extensive limbal damage, e.g., by chemical or thermal injury, inflammation, or surgery, may induce limbal stem cell deficiency (LSCD) leading to vascularization and opacification of the cornea and eventually vision loss. These acquired forms of limbal stem cell deficiency may occur uni- or bilaterally, which is important for the choice of treatment. Moreover, a variety of inherited diseases, such as congenital aniridia or dyskeratosis congenita, are characterized by LSCD typically occurring bilaterally. Several techniques of autologous and allogenic stem cell transplantation have been established. The limbus can be restored by transplantation of whole limbal grafts, small limbal biopsies or by ex vivo-expanded limbal cells. In this review, the physiology of the corneal epithelium, the pathophysiology of LSCD, and the therapeutic options will be presented.
Assuntos
Córnea/patologia , Córnea/fisiologia , Epitélio Corneano/patologia , Epitélio Corneano/fisiologia , Animais , Doenças da Córnea/patologia , Células Epiteliais/patologia , Células Epiteliais/fisiologia , Humanos , Transplante de Células-Tronco/métodos , Células-Tronco/patologia , Células-Tronco/fisiologiaRESUMO
A properly functioning cornea is critical to clear vision and healthy eyes. As the most anterior portion of the eye, it plays an essential role in refracting light onto the retina and as an anatomical barrier to the environment. Proper vision requires that all layers be properly formed and fully intact. In this article, we discuss the role of the epidermal growth factor receptor (EGFR) in maintaining and restoring the outermost layer of the cornea, the epithelium. It has been known for some time that the addition of epidermal growth factor (EGF) promotes the restoration of the corneal epithelium and patients using EGFR inhibitors as anti-cancer therapies are at increased risk of corneal erosions. However, the use of EGF in the clinic has been limited by downregulation of the receptor. More recent advances in EGFR signaling and trafficking in corneal epithelial cells have provided new insights in how to overcome receptor desensitization. We examine new strategies for overcoming the limitations of high ligand and receptor expression that alter trafficking of the ligand:receptor complex to sustain receptor signaling.
Assuntos
Doenças da Córnea/fisiopatologia , Epitélio Corneano/fisiologia , Receptores ErbB/metabolismo , Transdução de Sinais , Animais , HumanosRESUMO
Corneal transplantation is an effective treatment for corneal blindness. However, it brings risk factors for the occurrence of bacterial keratitis, which can affect the repair effect and even lead to transplantation failure. The difficulty in re-epithelialization is also a main problem faced by corneal transplantation. Herein, a collagen-GelMA composite membrane containing lysozyme (CGL) was developed as an antibacterial corneal implant to fill stromal defect and support re-epithelialization. Characterizations of physicochemical properties and in vitro biocompatibility revealed that the composite membranes have proper water content, light transmittance and mechanical strength as well as good biocompatibility. Particularly, the cell adhesion force and adhesion-related genes expression were evaluated and exhibited an improvement after the addition of GelMA. Furthermore, the formed CGL membrane could continuously release lysozyme and exhibited a bactericidal rate of 96% and 64% after 2 h and 72 h, respectively. The results demonstrated that this CGL membrane has promising application in corneal repair.
Assuntos
Antibacterianos/química , Colágeno/química , Transplante de Córnea/métodos , Membranas Artificiais , Muramidase/química , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Adesão Celular , Células Cultivadas , Transplante de Córnea/instrumentação , Reagentes de Ligações Cruzadas/química , Liberação Controlada de Fármacos , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/fisiologia , Muramidase/administração & dosagem , Muramidase/farmacologia , Coelhos , Staphylococcus aureus/efeitos dos fármacosRESUMO
Platelet extravasation during inflammation is under-appreciated. In wild-type (WT) mice, a central corneal epithelial abrasion initiates neutrophil (PMN) and platelet extravasation from peripheral limbal venules. The same injury in mice expressing low levels of the ß2-integrin, CD18 (CD18hypo mice) shows reduced platelet extravasation with PMN extravasation apparently unaffected. To better define the role of CD18 on platelet extravasation, we focused on two relevant cell types expressing CD18: PMNs and mast cells. Following corneal abrasion in WT mice, we observed not only extravasated PMNs and platelets but also extravasated erythrocytes (RBCs). Ultrastructural observations of engorged limbal venules showed platelets and RBCs passing through endothelial pores. In contrast, injured CD18hypo mice showed significantly less venule engorgement and markedly reduced platelet and RBC extravasation; mast cell degranulation was also reduced compared to WT mice. Corneal abrasion in mast cell-deficient (KitW-sh/W-sh) mice showed less venule engorgement, delayed PMN extravasation, reduced platelet and RBC extravasation and delayed wound healing compared to WT mice. Finally, antibody-induced depletion of circulating PMNs prior to corneal abrasion reduced mast cell degranulation, venule engorgement, and extravasation of PMNs, platelets, and RBCs. In summary, in the injured cornea, platelet and RBC extravasation depends on CD18, PMNs, and mast cell degranulation.
Assuntos
Plaquetas/fisiologia , Antígenos CD18/fisiologia , Degranulação Celular , Córnea/irrigação sanguínea , Eritrócitos/fisiologia , Hiperemia/fisiopatologia , Mastócitos/fisiologia , Neutrófilos/fisiologia , Migração Transendotelial e Transepitelial/fisiologia , Vasculite/imunologia , Vênulas/metabolismo , Animais , Antígenos CD18/deficiência , Movimento Celular , Quimiotaxia de Leucócito , Lesões da Córnea/metabolismo , Lesões da Córnea/patologia , Epitélio Corneano/fisiologia , Feminino , Hiperemia/sangue , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microcirculação , Microscopia Eletrônica , Modelos Animais , Fagocitose , Regeneração/fisiologia , Vasculite/sangue , Vênulas/patologia , Cicatrização/fisiologiaRESUMO
The cornea, while appearing to be simple tissue, is actually an extremely complex structure. In order for it to retain its biomechanical and optical properties, perfect organization of its cells is essential. Proper regeneration is especially important after injuries and in the course of various diseases. Eph receptors and ephrin are mainly responsible for the proper organization of tissues as well as cell migration and communication. In this review, we present the current state of knowledge on the role of Eph and ephrins in corneal physiology and diseases, in particular, we focused on the functions of the epithelium and endothelium. Since the role of Eph and ephrins in the angiogenesis process has been well established, we also analyzed their influence on conditions with corneal neovascularization.
Assuntos
Córnea/fisiologia , Doenças da Córnea/etiologia , Efrinas/fisiologia , Receptores da Família Eph/fisiologia , Animais , Doenças da Córnea/tratamento farmacológico , Neovascularização da Córnea/etiologia , Endotélio Corneano/patologia , Endotélio Corneano/fisiologia , Epitélio Corneano/patologia , Epitélio Corneano/fisiologia , Humanos , Terapia de Alvo MolecularRESUMO
Homeostasis in adult tissues relies on the replication dynamics of stem cells, their progenitors and the spatial balance between them. This spatial and kinetic coordination is crucial to the successful maintenance of tissue size and its replenishment with new cells. However, our understanding of the role of cellular replicative lifespan and spatial correlation between cells in shaping tissue integrity is still lacking. We developed a mathematical model for the stochastic spatial dynamics that underlie the rejuvenation of corneal epithelium. Our model takes into account different spatial correlations between cell replication and cell removal. We derive the tradeoffs between replicative lifespan, spatial correlation length, and tissue rejuvenation dynamics. We determine the conditions that allow homeostasis and are consistent with biological timescales, pattern formation, and mutants phenotypes. Our results can be extended to any cellular system in which spatial homeostasis is maintained through cell replication.
Assuntos
Epitélio Corneano/fisiologia , Homeostase , Modelos Biológicos , Regeneração , Processos EstocásticosRESUMO
Adult stem cell identity, plasticity, and homeostasis are precisely orchestrated by lineage-restricted epigenetic and transcriptional regulatory networks. Here, by integrating super-enhancer and chromatin accessibility landscapes, we delineate core transcription regulatory circuitries (CRCs) of limbal stem/progenitor cells (LSCs) and find that RUNX1 and SMAD3 are required for maintenance of corneal epithelial identity and homeostasis. RUNX1 or SMAD3 depletion inhibits PAX6 and induces LSCs to differentiate into epidermal-like epithelial cells. RUNX1, PAX6, and SMAD3 (RPS) interact with each other and synergistically establish a CRC to govern the lineage-specific cis-regulatory atlas. Moreover, RUNX1 shapes LSC chromatin architecture via modulating H3K27ac deposition. Disturbance of RPS cooperation results in cell identity switching and dysfunction of the corneal epithelium, which is strongly linked to various human corneal diseases. Our work highlights CRC TF cooperativity for establishment of stem cell identity and lineage commitment, and provides comprehensive regulatory principles for human stratified epithelial homeostasis and pathogenesis.
Assuntos
Células-Tronco Adultas/metabolismo , Plasticidade Celular/genética , Doenças da Córnea/patologia , Epitélio Corneano/fisiologia , Redes Reguladoras de Genes/fisiologia , Adolescente , Adulto , Idoso , Linhagem da Célula/genética , Células Cultivadas , Criança , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Doenças da Córnea/genética , Epitélio Corneano/citologia , Feminino , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Limbo da Córnea/citologia , Masculino , Pessoa de Meia-Idade , Fator de Transcrição PAX6/metabolismo , Cultura Primária de Células , RNA-Seq , Proteína Smad3/genética , Proteína Smad3/metabolismoRESUMO
OBJECTIVE: We sought to compare the efficacy and safety of conjunctival autograft (CAG), amniotic membrane transplantation (AMT) with postoperative interferon alfa-2b (IFN alfa-2b), and modified conjunctival autograft plus amniotic membrane transplantation (mCAG plus AMT) with postoperative IFN alfa-2b for primary pterygium. DESIGN: Randomized controlled clinical trial. METHODS: Eyes with nasal and primary pterygia were randomized in a 1:1:1 ratio to receive CAG, AMT with IFN alfa-2b, or mCAG plus AMT with IFN alfa-2b. Subjects were followed up for 12 months. Primary outcomes included recurrence rate and complications. Secondary outcomes included corneal epithelium status, ocular surface symptom score, and visual acuity change. RESULTS: Eighty-five subjects (30 in the CAG group, 25 in the AMT group, and 30 in the CAG+AMT group) completed the 12-month follow-up. No complication or grade 4 recurrence was found. There was no significant difference among the 3 groups in recurrence grade, corneal epithelium status, and visual acuity change. Compared with mCAG+AMT, CAG has a negative effect (ß = -0.62, P = .001), and AMT has a negative effect (ß = -2.02, P < .001) on postoperative symptom scores. Compared with AMT, CAG has a positive effect (ß = 1.28, P < .001) on postoperative symptom scores. CONCLUSIONS: All 3 strategies had good safety and clinical efficacy in the study. Compared with conjunctival autograft, the 2 surgeries using no autograft or limited autograft was less traumatic and gave more flexibility for future ocular surface condition changes.
Assuntos
Âmnio/transplante , Antineoplásicos/administração & dosagem , Túnica Conjuntiva/cirurgia , Interferon alfa-2/administração & dosagem , Pterígio/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Epitélio Corneano/fisiologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Pterígio/fisiopatologia , Transplante Autólogo , Resultado do TratamentoRESUMO
A murine model of corneal epithelial wounding can be performed using simple injury and imaging methods. Here, we describe the creation of a central corneal epithelial defect using mechanical debridement under ophthalmic microscopic visualization. Subsequent monitoring with vital dye application and slit-lamp bio microscopy (slit-lamp) is described in detail.
Assuntos
Lesões da Córnea/patologia , Modelos Animais de Doenças , Animais , Lesões da Córnea/etiologia , Desbridamento/instrumentação , Desbridamento/métodos , Epitélio Corneano/patologia , Epitélio Corneano/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Procedimentos Cirúrgicos Oftalmológicos/instrumentação , Procedimentos Cirúrgicos Oftalmológicos/métodos , CicatrizaçãoRESUMO
The purpose of this study was to investigate the expression and localization of transforming growth factor (TGF) ß1 and TGFß2 in rabbit corneas that healed with and without stromal fibrosis, and to further study defective perlecan incorporation in the epithelial basement membrane (EBM) in corneas with scarring fibrosis. A total of 120 female rabbits had no surgery, -4.5D PRK, or -9D PRK. Immunohistochemistry (IHC) was performed at time points from unwounded to eight weeks after surgery, with four corneas at each time point in each group. Multiplex IHC was performed for TGFß1 or TGFß2, with Image-J quantitation, and keratocan, vimentin, alpha-smooth muscle actin (SMA), perlecan, laminin-alpha 5, nidogen-1 or CD11b. Corneas at the four-week peak for myofibroblast and fibrosis development were evaluated using Imaris 3D analysis. Delayed regeneration of both an apical epithelial growth factor barrier and EBM barrier function, including defective EBM perlecan incorporation, was greater in high injury -9D PRK corneas compared to -4.5D PRK corneas without fibrosis. Defective apical epithelial growth factor barrier and EBM allowed epithelial and tear TGFß1 and tear TGFß2 to enter the corneal stroma to drive myofibroblast generation in the anterior stroma from vimentin-positive corneal fibroblasts, and likely fibrocytes. Vimentin-positive cells and unidentified vimentin-negative, CD11b-negative cells also produce TGFß1 and/or TGFß2 in the stroma in some corneas. TGFß1 and TGFß2 were at higher levels in the anterior stroma in the weeks preceding myofibroblast development in the -9D group. All -9D corneas (beginning two to three weeks after surgery), and four -4.5D PRK corneas developed significant SMA + myofibroblasts and stromal fibrosis. Both the apical epithelial growth factor barrier and/or EBM barrier functions tended to regenerate weeks earlier in -4.5D PRK corneas without fibrosis, compared to -4.5D or -9D PRK corneas with fibrosis. SMA-positive myofibroblasts were markedly reduced in most corneas by eight weeks after surgery. The apical epithelial growth factor barrier and EBM barrier limit TGFß1 and TGFß2 entry into the corneal stroma to modulate corneal fibroblast and myofibroblast development associated with scarring stromal fibrosis. Delayed regeneration of these barriers in corneas with more severe injuries promotes myofibroblast development, prolongs myofibroblast viability and triggers stromal scarring fibrosis.
Assuntos
Membrana Basal/fisiologia , Córnea/metabolismo , Substância Própria/patologia , Epitélio Corneano/fisiologia , Regeneração/fisiologia , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Animais , Opacidade da Córnea/metabolismo , Opacidade da Córnea/patologia , Substância Própria/metabolismo , Feminino , Fibrose/metabolismo , Fibrose/patologia , Proteínas de Membrana/metabolismo , Microscopia Confocal , CoelhosRESUMO
Keratoconus (KC) is a corneal dystrophy characterized by progressive ectasia that leads to severe visual impairment and remains one of the leading indications for corneal transplantation. The etiology is believed to be multifactorial and alterations have been documented in the biomechanical, biochemical and ultrastructural characteristics of the cornea. While the exact site of disease origin is still debated, changes in the corneal epithelium are believed to occur even before the disease is clinically manifested. In this study we investigate the possible role of ß-catenin as mechanotransducer in KC corneal epithelium. The sheets of corneal epithelium removed from keratoconic eyes when they underwent collagen crosslinking as a therapeutic procedure were used for this study. The healthy corneal epithelium of patients undergoing Laser Refractive Surgery for the correction of their refractive error, served as controls. Immunoblotting and tissue immunofluorescence studies were performed on KC epithelium to analyse the expression and localization of ß-catenin, E-cadherin, ZO1, α-catenin, Cyclin D1, α-actinin, RhoA, and Rac123. Co-immunoprecipitation of ß-catenin followed by mass spectrometry of KC epithelium was performed to identify its interacting partners. This was further validated by using epithelial tissues grown on scaffolds of different stiffness. Histology data reported breaks in the Bowman's layer in KC patients. We hypothesize that these breaks expose the epithelium to the keratoconic corneal stroma, which, is known to have a decreased elastic modulus and that ß-catenin acts as a mechanotransducer that induces structural changes such as loss of polarity (Syntaxin3) and barrier function (ZO1) through membrane delocalization. The results of our study strongly suggest that ß-catenin could be a putative mechanotransducer in KC epithelium, thus supporting our hypothesis.