RESUMEN

Refractive error is becoming a significant public health issue. Photorefractive Keratectomy (PRK) is a corneal surface surgical technique that removes the corneal epithelium before stromal photoablation by ultraviolet radiation from the Excimer laser. We designed a retrospective study to characterize corneal remodeling after myopic Photorefractive Keratectomy and assess the accuracy of laser-predicted ablation depth (AD). This study took place in 15-20 National Ophthalmology Hospital, Paris, France. 150 eyes with preoperative manifest spherical equivalent between - 10.00D and - 0.25D and cylinder < 3D, treated with the WaveLight® EX500 laser between 01/2019 and 01/2023, were followed for at least three months. The main outcome measurements were postoperative changes in epithelial (ET) and stromal (ST) thicknesses measured with spectral domain optical coherence tomography and mean simulated keratometry (SimK) assessed with corneal topography. The central ET significantly decreased at M1, increased over the preoperative value from M1 to M6, and stabilized after M6. The increase in central ET after M1 was associated with an increase in mean SimK (r = 0.34). The achieved AD was 7.9 ± 8.0 µm greater than the laser-predicted AD. Stromal over-ablation was significantly and independently associated with myopia > 6D preoperative mean SimK > 44D and transepithelial procedures.

Asunto(s)

Córnea , Láseres de Excímeros , Miopía , Queratectomía Fotorrefractiva , Tomografía de Coherencia Óptica , Humanos , Queratectomía Fotorrefractiva/métodos , Miopía/cirugía , Femenino , Adulto , Masculino , Láseres de Excímeros/uso terapéutico , Estudios Retrospectivos , Córnea/cirugía , Córnea/patología , Córnea/diagnóstico por imagen , Tomografía de Coherencia Óptica/métodos , Adulto Joven , Topografía de la Córnea , Persona de Mediana Edad , Sustancia Propia/cirugía , Epitelio Corneal/cirugía , Epitelio Corneal/patología , Agudeza Visual

RESUMEN

This study investigated the therapeutic effects of exosomes derived from human-induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) on corneal epithelial wound healing. Exosomes were isolated from the culture medium of the hiPSC-derived ROs (Exo-ROs) using ultracentrifugation, and then they were characterized by a nanoparticle tracking analysis and transmission electron microscopy. In a murine model of corneal epithelial wounds, these exosomes were topically applied to evaluate their healing efficacy. The results demonstrated that the exosome-treated eyes showed significantly enhanced wound closures compared with the controls at 24 h post-injury. The 5-ethyl-2'-deoxyuridine assay and quantitative reverse transcription polymerase chain reaction revealed a substantial increase in cell proliferation and a decrease in inflammatory marker contents in the exosome-treated group. The RNA sequencing and exosomal microRNA analysis revealed that the Exo-RO treatment targeted various pathways related to inflammation and cell proliferation, including the PI3K-Akt, TNF, MAPK, and IL-17 signaling pathways. Moreover, the upregulation of genes related to retinoic acid and eicosanoid metabolism may have enhanced corneal epithelial healing in the eyes treated with the Exo-ROs. These findings suggest that hiPSC-derived RO exosomes could be novel therapeutic agents for promoting corneal epithelial wound healing.

Asunto(s)

Proliferación Celular , Epitelio Corneal , Exosomas , Células Madre Pluripotentes Inducidas , Organoides , Cicatrización de Heridas , Exosomas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Organoides/metabolismo , Animales , Epitelio Corneal/metabolismo , Ratones , Retina/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Transducción de Señal

RESUMEN

The corneal epithelium is located on the most anterior surface of the eyeball and protects against external stimuli. The development of the corneal epithelium and the maintenance of corneal homeostasis are essential for the maintenance of visual acuity. It has been discovered recently via the in-depth investigation of ocular surface illnesses that the Wnt/ß-catenin signaling pathway is necessary for the growth and stratification of corneal epithelial cells as well as the control of endothelial cell stability. In addition, the Wnt/ß-catenin signaling pathway is directly linked to the development of common corneal illnesses such as keratoconus, fungal keratitis, and corneal neovascularization. This review mainly summarizes the role of the Wnt/ß-catenin signaling pathway in the development, homeostasis, and pathobiology of cornea, hoping to provide new insights into the study of corneal epithelium and the treatment of related diseases.

Asunto(s)

Epitelio Corneal , Homeostasis , Vía de Señalización Wnt , Epitelio Corneal/metabolismo , Humanos , Homeostasis/fisiología , Vía de Señalización Wnt/fisiología , Animales , beta Catenina/metabolismo , Enfermedades de la Córnea/metabolismo , Enfermedades de la Córnea/patología

RESUMEN

Corneal injuries often lead to epithelial damage, apoptosis, and inflammation which impact visual function. Effective epithelial healing is critical for optimal vision and functioning of the cornea. Mesenchymal stem/stromal cells (MSCs)-derived extracellular vesicles (EVs) present promising avenues for cell-free therapy, however, evaluation of their specific roles in corneal epithelial injury requires further investigations with due consideration to the endogenous human corneal epithelial cell-derived EVs (HCEC-EVs). This study aims to isolate and characterize the EVs from a commonly available human corneal epithelial cell line (HCE-2 [50. B1], ATCC) and evaluate their corneal epithelial repair, anti-apoptotic, and immunomodulatory potential in comparison with human bone marrow mesenchymal stem cell-derived EVs (BM-MSC-EVs) in vitro. Both the BM-MSC- and HCEC-EVs exhibited similar morphology with a diameter <150 nm. However, the yield of EVs from HCECs was higher than that of BM-MSCs. Nanoparticle tracking analysis revealed an average EV size of ∼120 nm, while western blotting confirmed the presence of CD63, CD81, and TSG101, whereas Calnexin could not be detected in the BM-MSC- and HCEC-EVs. The corneal epithelial repair was monitored through in vitro wound healing assay, whereas apoptosis was studied through flow cytometry-based Propidium iodide staining in H2O2-treated cells. IL-1ß-stimulated HCECs were treated with BM-MSC- and HCEC-EVs for 24 h and expression of pro- (IL-6 and TNF-α) and anti-inflammatory (IL-10 and TGF-ß) cytokines was evaluated through ELISA. Our results, limited to in vitro investigations, suggest that compared with HCEC-EVs, BM-MSC-EVs showed: i) accelerated corneal epithelial healing, ii) enhanced anti-apoptotic potential, and iii) improved anti-inflammatory properties, in cultured HCECs.

Asunto(s)

Apoptosis , Epitelio Corneal , Vesículas Extracelulares , Inmunomodulación , Células Madre Mesenquimatosas , Cicatrización de Heridas , Humanos , Vesículas Extracelulares/metabolismo , Epitelio Corneal/metabolismo , Apoptosis/fisiología , Células Madre Mesenquimatosas/metabolismo , Inmunomodulación/fisiología , Cicatrización de Heridas/fisiología , Western Blotting , Células Cultivadas , Citocinas/metabolismo , Citometría de Flujo

RESUMEN

PURPOSE: Benzalkonium chloride (BAC) is commonly used as a preservative in ophthalmic medications, despite its potential to induce chemical injury. Extensive research has demonstrated that BAC can lead to adverse effects, including injuries to the ocular surface. Our study aimed to elucidate the underlying mechanism of necroptosis induced by BAC. METHODS: Human corneal epithelial (HCE) cells and mouse corneas were subjected to chemical injury, and the necrostatin-1 (Nec1) group was compared to the dimethylsulfoxide (DMSO) group. The extent of damage to HCE cells was assessed using CCK-8 and flow cytometry. Hematoxylin and eosin staining, as well as fluorescein sodium staining, were used to detect and characterize corneal injury. The activation of inflammatory cytokines and necroptosis-related proteins and genes was evaluated using Western blotting, immunofluorescence staining, and quantitative RT‒PCR. RESULTS: In our study, the induction of necroptosis by a hypertonic solution was not observed. However, necroptosis was observed in HCE cells exposed to NaOH and BAC, which activated the receptor-interacting protein kinase 1 (RIPK1) - receptor-interacting protein kinase 3 (RIPK3) - mixed lineage kinase domain-like protein (MLKL) signaling pathway. In mouse corneal tissues, BAC could induce necroptosis and inflammation. The administration of Nec1 mitigated the inflammatory response and ocular surface damage caused by BAC-induced necroptosis in our experimental models. Furthermore, our in vivo experiments revealed that the severity of necroptosis was greater in the 3-day group than in the 7-day group. CONCLUSIONS: Necroptosis plays a role in the pathological development of ocular surface injury caused by exposure to BAC. Furthermore, our study demonstrated that the administration of Nec1 could mitigate the pathological effects of necroptosis induced by BAC in clinical settings.

Asunto(s)

Compuestos de Benzalconio , Epitelio Corneal , Imidazoles , Indoles , Necroptosis , Proteínas Quinasas , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Necroptosis/efectos de los fármacos , Animales , Ratones , Epitelio Corneal/efectos de los fármacos , Epitelio Corneal/patología , Epitelio Corneal/metabolismo , Indoles/farmacología , Compuestos de Benzalconio/toxicidad , Compuestos de Benzalconio/farmacología , Imidazoles/farmacología , Proteínas Quinasas/metabolismo , Humanos , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Western Blotting , Células Cultivadas , Citometría de Flujo , Transducción de Señal/efectos de los fármacos , Quemaduras Oculares/inducido químicamente , Quemaduras Oculares/patología , Masculino , Quemaduras Químicas/patología , Quemaduras Químicas/metabolismo , Quemaduras Químicas/tratamiento farmacológico , Conservadores Farmacéuticos/toxicidad

RESUMEN

Dysregulation of calcium homeostasis can precipitate a cascade of pathological events that lead to tissue damage and cell death. Dynasore is a small molecule that inhibits endocytosis by targeting classic dynamins. In a previous study, we showed that dynasore can protect human corneal epithelial cells from damage due to tert-butyl hydroperoxide (tBHP) exposure by restoring cellular calcium (Ca2+) homeostasis. Here we report results of a follow-up study aimed at identifying the source of the damaging Ca2+. Store-operated Ca2+ entry (SOCE) is a cellular mechanism to restore intracellular calcium stores from the extracellular milieu. We found that dynasore effectively blocks SOCE in cells treated with thapsigargin (TG), a small molecule that inhibits pumping of Ca2+ into the endoplasmic reticulum (ER). Unlike dynasore however, SOCE inhibitor YM-58483 did not interfere with the cytosolic Ca2+ overload caused by tBHP exposure. We also found that dynasore effectively blocks Ca2+ release from internal sources. The inefficacy of inhibitors of ER Ca2+ channels suggested that this compartment was not the source of the Ca2+ surge caused by tBHP exposure. However, using a Ca2+-measuring organelle-entrapped protein indicator (CEPIA) reporter targeted to mitochondria, we found that dynasore can block mitochondrial Ca2+ release due to tBHP exposure. Our results suggest that dynasore exerts multiple effects on cellular Ca2+ homeostasis, with inhibition of mitochondrial Ca2+ release playing a key role in protection of corneal epithelial cells against oxidative stress due to tBHP exposure.

Asunto(s)

Calcio , Epitelio Corneal , Hidrazonas , Mitocondrias , Humanos , Epitelio Corneal/metabolismo , Epitelio Corneal/efectos de los fármacos , Calcio/metabolismo , Mitocondrias/metabolismo , Hidrazonas/farmacología , Retículo Endoplásmico/metabolismo , Tapsigargina/farmacología , Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Células Cultivadas , terc-Butilhidroperóxido/farmacología , Homeostasis/fisiología

RESUMEN

This research focused on how upregulation of S100A9 contributed to the pathogenesis of the dry eye disease (DED) and whether S100A9 served as a promising therapeutic target in DED. Public single-cell RNA sequencing (scRNA-seq) data of a lacrimal gland excision (LGE) murine DED model was analyzed. LGE model was established and expression of protein was measured through immunofluorescence and Western blot. DED-related signs were evaluated through tear secretion and fluorescent staining. TUNEL was performed to detect the level of cell death. Briefly, S100A9 was recognized as a highly variable gene in the DED group. LGE model was successfully established, and S100A9 showed a time-dependent increase in the corneal epithelia. Autophagic blockage was predicted by the scRNA-seq data in DED, and further verified by decrease of LC3B-II/LC3B-I and increase of SQSTM1 and p-mTOR/mTOR, while S100A9 inhibitor paquinimod (PAQ) reversed the changes. PAQ also downregulated TLR4, and inhibition of TLR4 also alleviated autophagic blockage in DED. Finally, signs of DED, chronic corneal inflammation and cell death got a remission after either inhibition of S100A9 or TLR4. In general, we deduced a S100A9-TLR4-Autophagic blockage pathway in the pathogenesis of DED.

Asunto(s)

Autofagia , Western Blotting , Calgranulina B , Modelos Animales de Enfermedad , Síndromes de Ojo Seco , Ratones Endogámicos C57BL , Receptor Toll-Like 4 , Animales , Síndromes de Ojo Seco/metabolismo , Síndromes de Ojo Seco/patología , Autofagia/fisiología , Ratones , Calgranulina B/metabolismo , Calgranulina B/genética , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/genética , Aparato Lagrimal/metabolismo , Aparato Lagrimal/patología , Lágrimas/metabolismo , Epitelio Corneal/metabolismo , Epitelio Corneal/patología , Epitelio Corneal/efectos de los fármacos , Etiquetado Corte-Fin in Situ , Femenino , Regulación de la Expresión Génica

RESUMEN

The cornea is the outermost layer of the eye and plays an essential role in our visual system. Limbal epithelial stem cells (LESCs), which are localized to a highly regulated limbal niche, are the master conductors of corneal epithelial regeneration. Damage to LESCs and their niche may result in limbal stem cell deficiency (LSCD), a disease confused ophthalmologists so many years and can lead to corneal conjunctivalization, neovascularization, and even blindness. How to restore the LESCs function is the hot topic for ocular scientists and clinicians around the world. This review introduced LESCs and the niche microenvironment, outlined various techniques for isolating and culturing LESCs used in LSCD research, presented common diseases that cause LSCD, and provided a comprehensive overview of both the diagnosis and multiple treatments for LSCD from basic research to clinical therapies, especially the emerging cell therapies based on various stem cell sources. In addition, we also innovatively concluded the latest strategies in recent years, including exogenous drugs, tissue engineering, nanotechnology, exosome and gene therapy, as well as the ongoing clinical trials for treating LSCD in recent five years. Finally, we highlighted challenges from bench to bedside in LSCD and discussed cutting-edge areas in LSCD therapeutic research. We hope that this review could pave the way for future research and translation on treating LSCD, a crucial step in the field of ocular health.

Asunto(s)

Epitelio Corneal , Limbo de la Córnea , Regeneración , Células Madre , Humanos , Limbo de la Córnea/citología , Limbo de la Córnea/patología , Células Madre/citología , Epitelio Corneal/citología , Epitelio Corneal/patología , Animales , Medicina de Precisión , Células Epiteliales

RESUMEN

Purpose: Abnormalities in aquaporins are implicated in the pathological progression of dry eye syndrome. Retinoic acid (RA) regulates cellular proliferation, differentiation, and apoptosis in the cornea, thereby being associated with dry eye disease (DED). The objective of this study is to explore the underlying mechanisms responsible for RA metabolic abnormalities in corneas lacking aquaporin 5 (AQP5). Methods: Dry eye (DE) models were induced via subcutaneous scopolamine hydrobromide. Aqp5 knockout (Aqp5-/-) mice and DE mice were utilized to assess corneal epithelial alterations. Tear secretion, goblet cell counts, and corneal punctate defects were evaluated. The impact of Aqp5 on RA-related enzymes and receptors was investigated using pharmacological RA or SR (A JunB inhibitor), a transcription factor JunB inhibitor, treatment in mouse corneal epithelial cells (CECs), or human corneal epithelial cells (HCECs). The HCECs and NaCl-treated HCECs underwent quantitative real-time PCR (qRT-PCR), immunofluorescent, Western blot, and TUNEL assays. The regulation of transcription factor JunB on Aldh1a1 was explored via ChIP-PCR. Results: Aqp5 and Aldh1a1 were reduced in both CECs of DE mice and NaCl-induced HCECs. Aqp5-/- mice exhibited DE phenotype and reduced Aldh1a1. RA treatment reduced apoptosis, promoted proliferation, and improved the DE phenotype in Aqp5-/- mice. JunB enrichment in the Aldh1a1 promoter was identified by ChIP-PCR. SR significantly increased Aldh1a1 expression, Ki67, and ΔNp63-positive cells, and decreased TUNEL-positive cells in CECs and HCECs. Conclusions: Our findings demonstrated the downregulation of Aqp5 expression and aberrant RA metabolism in DE conditions. Knockout of Aqp5 resulted in reduced production of RA through activation of JunB, subsequently leading to the manifestation of DE symptoms.

Asunto(s)

Apoptosis , Acuaporina 5 , Modelos Animales de Enfermedad , Síndromes de Ojo Seco , Ratones Noqueados , Tretinoina , Animales , Acuaporina 5/genética , Acuaporina 5/biosíntesis , Acuaporina 5/metabolismo , Síndromes de Ojo Seco/metabolismo , Síndromes de Ojo Seco/patología , Síndromes de Ojo Seco/genética , Ratones , Tretinoina/farmacología , Epitelio Corneal/metabolismo , Epitelio Corneal/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Ratones Endogámicos C57BL , Western Blotting , Humanos , Células Cultivadas , Lágrimas/metabolismo , Etiquetado Corte-Fin in Situ , Regulación de la Expresión Génica , Proliferación Celular

RESUMEN

Dry eye is a prevalent ophthalmic disease. Ocular surface inflammation in the hyperosmolar environment of the tear film is critical in dry eye progression. Quercetin has strong anti-inflammatory effects; however, its exact mechanism of action in dry eye is not fully understood. Therefore, this study investigated whether quercetin could inhibit the damage sustained to human corneal epithelial cells (HCECs) in a hyperosmolar environment through its anti-inflammatory effects. HCECs were cultured in a complete medium and were divided into four groups: normal, model, quercetin, and inhibitor. The proliferation of HCECs was detected by Ki67 staining; the expression levels of PTEN, p-PI3K and p-AKT were detected by Western blotting and immunofluorescence staining; the relative mRNA expression levels of PTEN, PI3K, AKT, IL-6 and TNF-ɑ were detected by quantitative real-time PCR; the relative expression levels of IL-6 and TNF-α were detected by enzyme-linked immunosorbent assay. In this study, the proliferation of HCECs in the model group was found to be significantly inhibited compared with that in the normal group; however, quercetin was effective in improving the proliferation of HCECs, decreasing the relative expression of p-PI3K, p-AKT, IL-6, TNF-ɑ as well as increasing PTEN. In conclusion, this study demonstrated that quercetin could promote the proliferation of HCECs and reduce the expression of inflammatory factors by inhibiting the PTEN/PI3K/AKT pathway in the hyperosmolarity-induced HCECs model.

Asunto(s)

Epitelio Corneal , Inflamación , Fosfohidrolasa PTEN , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Quercetina , Transducción de Señal , Humanos , Quercetina/farmacología , Fosfohidrolasa PTEN/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Inflamación/patología , Inflamación/metabolismo , Epitelio Corneal/efectos de los fármacos , Epitelio Corneal/metabolismo , Epitelio Corneal/patología , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Proliferación Celular/efectos de los fármacos

RESUMEN

Purpose: The purpose of this study was to investigate the ocular morphological characteristics of Col4a3-/- mice as a model of Alport syndrome (AS) and the potential pathogenesis. Methods: The expression of collagen IV at 8, 12, and 21 weeks of age was evaluated by immunohistochemistry in wild-type (WT) and Col4a3-/- mice. Hematoxylin and eosin (H&E) staining and thickness measurements were performed to assess the thickness of anterior lens capsule and retina. Ultrastructure analysis of corneal epithelial basement membrane, anterior lens capsule, internal limiting membrane (ILM), and retinal pigment epithelium (RPE) basement membrane was performed using transmission electron microscopy. Finally, Müller cell activation was evaluated by glial fibrillary acidic protein (GFAP) expression. Results: Collagen IV was downregulated in the corneal epithelial basement membrane and ILM of Col4a3-/- mice. The hemidesmosomes of Col4a3-/- mice corneal epithelium became flat and less electron-dense than those of the WT group. Compared with those of the WT mice, the anterior lens capsules of Col4a3-/- mice were thinner. Abnormal structure was detected at the ILM Col4a3-/- mice, and the basal folds of the RPE basement membrane in Col4a3-/- mice were thicker and shorter. The retinas of Col4a3-/- mice were thinner than those of WT mice, especially within 1000 µm away from the optic nerve. GFAP expression enhanced in each age group of Col4a3-/- mice. Conclusions: Our results suggested that Col4a3-/- mice exhibit ocular anomalies similar to patients with AS. Additionally, Müller cells may be involved in AS retinal anomalies. Translational Relevance: This animal model could provide an opportunity to understand the underlying mechanisms of AS ocular disorders and to investigate potential new treatments.

Asunto(s)

Membrana Basal , Colágeno Tipo IV , Modelos Animales de Enfermedad , Ratones Noqueados , Nefritis Hereditaria , Animales , Nefritis Hereditaria/patología , Nefritis Hereditaria/genética , Nefritis Hereditaria/metabolismo , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Colágeno Tipo IV/deficiencia , Ratones , Membrana Basal/metabolismo , Membrana Basal/patología , Membrana Basal/ultraestructura , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/ultraestructura , Microscopía Electrónica de Transmisión , Ratones Endogámicos C57BL , Cápsula del Cristalino/metabolismo , Cápsula del Cristalino/patología , Cápsula del Cristalino/ultraestructura , Epitelio Corneal/patología , Epitelio Corneal/ultraestructura , Epitelio Corneal/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteína Ácida Fibrilar de la Glía/genética , Retina/patología , Retina/metabolismo , Retina/ultraestructura , Autoantígenos/genética , Autoantígenos/metabolismo , Células Ependimogliales/patología , Células Ependimogliales/metabolismo , Células Ependimogliales/ultraestructura , Inmunohistoquímica , Masculino

RESUMEN

Microplastics (MPs) are ubiquitously dispersed in the environment, and undergoing the process of oxidation that alters their physical and chemical properties. Eyes, which directly interface with the external milieu, inevitably encounter MPs. Nonetheless, the ophthalmic toxicity of MPs towards organisms remains unclear. In this study, primary mouse corneal epithelial cells (MCECs), C57BL/6 mice, and CX3CrlGFP/+ mice were utilized to evaluate the toxicity and differences between oxidized low-density polyethylene MPs (modified-MPs) and low-density polyethylene MPs (virgin-MPs) on eyes. The results manifested that virgin-MPs and modified-MPs could be endocytosed by primary MCECs, resulting in a range of cellular damage. Furthermore, they could diminish tear secretion, increase intraocular pressure, and could be internalized into cornea and retina in mice, instigating a series of detrimental reactions. Importantly, modified-MPs exhibited heightened toxicity towards mouse eyes, seemingly due to oxidation enhances the interaction between virgin-MPs/modified-MPs and tissues/cells, and leading to the release of toxic substances increased. In conclusion, our discoveries demonstrate that oxidation exacerbates the harm of virgin-MPs to eyes, and are of great significance for evaluating the risk of MPs to ocular health.

Asunto(s)

Ratones Endogámicos C57BL , Microplásticos , Oxidación-Reducción , Polietileno , Animales , Microplásticos/toxicidad , Ratones , Polietileno/toxicidad , Ojo/efectos de los fármacos , Córnea/efectos de los fármacos , Córnea/metabolismo , Epitelio Corneal/efectos de los fármacos , Epitelio Corneal/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo

RESUMEN

BACKGROUND: To compare the repeatability and reproducibility of corneal and corneal epithelial thickness mapping using anterior segment optical coherence tomography (AS-OCT) according to tear film break-up time (TBUT). METHODS: The included eyes were divided into three subgroups according to TBUT (group 1: TBUT ≤ 5 s, group 2: 5 s < TBUT ≤ 10 s, and group 3: TBUT > 10 s). All eyes were imaged separately thrice by two operators to obtain the thickness maps (TMs) of the cornea and corneal epithelium based on spatial zones encompassing a 9-mm-diameter area. Each TM consisted of 25 areas. Intraoperator (repeatability) and interoperator (reproducibility) standard deviations (Sws), coefficients of variation (CoVs), and intraclass correlation coefficients (ICCs) among the tests were calculated and compared in all the areas. RESULTS: Altogether, 132 eyes of 67 subjects were included (50, 47, and 35 eyes in groups 1, 2, and 3; respectively). The ICCs of corneal epithelial thickness and corneal thickness were > 0.75 in most of the areas. Pairwise comparisons showed that AS-OCT exhibited lower repeatability in group 1 than in groups 2 and 3 (P < 0.05). However groups 2 and 3 showed similar results. Sws and CoVs of corneal epithelial thickness exhibited no significant interoperator differences. While no significant differences were observed in corneal thickness in most of the areas. CONCLUSIONS: TBUT significantly influences the repeatability of corneal and corneal epithelial thickness measurements. Poor tear film stability requires careful evaluation of corneal epithelial thickness.

Asunto(s)

Córnea , Lágrimas , Tomografía de Coherencia Óptica , Humanos , Tomografía de Coherencia Óptica/métodos , Femenino , Reproducibilidad de los Resultados , Masculino , Lágrimas/fisiología , Córnea/diagnóstico por imagen , Adulto , Persona de Mediana Edad , Epitelio Corneal/diagnóstico por imagen , Adulto Joven , Paquimetría Corneal/métodos , Anciano

RESUMEN

Given that the corneal epithelium is situated on the outermost part of the eye, its functions can be influenced by external temperatures and chemical substances. This study aimed to elucidate the expression profile of chemosensory receptors in corneal epithelial cells and analyze their role in eye function regulation. A comprehensive analysis of 425 chemosensory receptors in human corneal epithelial cells-transformed (HCE-T) revealed the functional expression of TRPV4. The activation of TRPV4 in HCE-T cells significantly increased the expression of membrane-associated mucins MUC1, MUC4, and MUC16, which are crucial for stabilizing tear films, with efficacy comparable to the active components of dry eye medications. The present study suggests that TRPV4, which is activated by body temperature, regulates mucin expression and proposes it as a novel target for dry eye treatment.

Asunto(s)

Epitelio Corneal , Mucina-1 , Mucina 4 , Mucinas , Canales Catiónicos TRPV , Humanos , Antígeno Ca-125/metabolismo , Antígeno Ca-125/genética , Células Epiteliales/metabolismo , Células Epiteliales/citología , Epitelio Corneal/metabolismo , Epitelio Corneal/citología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Mucina-1/metabolismo , Mucina-1/genética , Mucina 4/metabolismo , Mucina 4/genética , Mucinas/metabolismo , Mucinas/biosíntesis , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/genética

RESUMEN

BACKGROUND: To compare the epithelial thickness map of ptotic eyes of blepharoptosis patients with contralateral non- ptotic eyes. METHODS: Unilateral blepharoptosis patients were enrolled consecutively. Patients were underwent full ophthalmologic examination and their demographic data such as age and gender and specific ptosis findings e.g. the cause and duration, MRD-1, and levator palpebralis superioris function were registered. Anterior segment imaging for epithelial thickness measurements was done using the Avanti RTVue-XR platform. The corneal epithelial thickness maps of ptotic and non-ptotic eyes were compared. RESULTS: 44 patients with unilateral blepharoptosis were included in the study. 27 (61.4%) of them were female and 17 (38.6%) cases were male. The mean of the patients' ages was 24.40 ± 15.16 years. Ptotic eyes had significantly thinner superior (p = 0.000), superior-temporal (p = 0.000) and superior-nasal (p = 0.005) sectors of the cornea and slightly thicker corneal epithelium (CE) in the inferior-nasal sector. The correlation of difference of superior-inferior CE was evaluated with different parameters including patient's age (p = 0.457), type of blepharoptosis (p = 0.786), duration of blepharoptosis (p = 0.477) and MRD1 (p = 0.248), but no correlation was found. CONCLUSIONS: This study revealed that lid position in blepharoptosis may have effects on the corneal epithelial thickness map. Because of the lower position of upper eyelid, a thinning effect on superior corneal sectors may happen.

Asunto(s)

Blefaroptosis , Epitelio Corneal , Humanos , Blefaroptosis/diagnóstico , Femenino , Masculino , Adulto , Epitelio Corneal/patología , Epitelio Corneal/diagnóstico por imagen , Adulto Joven , Adolescente , Persona de Mediana Edad , Niño , Preescolar , Anciano

RESUMEN

BACKGROUND: Dysfunction or deficiency of corneal epithelium results in vision impairment or blindness in severe cases. The rapid and effective regeneration of corneal epithelial cells relies on the limbal stem cells (LSCs). However, the molecular and functional responses of LSCs and their niche cells to injury remain elusive. METHODS: Single-cell RNA sequencing was performed on corneal tissues from normal mice and corneal epithelium defect models. Bioinformatics analysis was performed to confirm the distinct characteristics and cell fates of LSCs. Knockdown of Creb5 and OSM treatment experiment were performed to determine their roles of in corneal epithelial wound healing. RESULTS: Our data defined the molecular signatures of LSCs and reconstructed the pseudotime trajectory of corneal epithelial cells. Gene network analyses characterized transcriptional landmarks that potentially regulate LSC dynamics, and identified a transcription factor Creb5, that was expressed in LSCs and significantly upregulated after injury. Loss-of-function experiments revealed that silencing Creb5 delayed the corneal epithelial healing and LSC mobilization. Through cell-cell communication analysis, we identified 609 candidate regeneration-associated ligand-receptor interaction pairs between LSCs and distinct niche cells, and discovered a unique subset of Arg1+ macrophages infiltrated after injury, which were present as the source of Oncostatin M (OSM), an IL-6 family cytokine, that were demonstrated to effectively accelerate the corneal epithelial wound healing. CONCLUSIONS: This research provides a valuable single-cell resource and reference for the discovery of mechanisms and potential clinical interventions aimed at ocular surface reconstruction.

Asunto(s)

Plasticidad de la Célula , Células Madre Limbares , Limbo de la Córnea , Cicatrización de Heridas , Animales , Ratones , Epitelio Corneal/metabolismo , Epitelio Corneal/patología , Epitelio Corneal/lesiones , Células Madre Limbares/citología , Células Madre Limbares/metabolismo , Limbo de la Córnea/metabolismo , Limbo de la Córnea/citología , Limbo de la Córnea/patología , Ratones Endogámicos C57BL , Nicho de Células Madre , Cicatrización de Heridas/genética

RESUMEN

Currently, in vitro cultured corneal epithelial transplantation is effective in treating limbal stem cell dysfunction (LSCD). Selecting carriers is crucial for constructing the corneal epithelium through tissue engineering. In this study, the traditional amniotic membrane (AM) was modified, and mesenchymal stem cells (MSCs) were inoculated into the ultra-thin amniotic membrane (UAM) stroma to construct a novel UAM-MSC tissue-engineered corneal epithelial carrier, that could effectively simulate the limbal stem cells (LSCs) microenvironment. The structure of different carriers cultured tissue-engineered corneal epithelium and the managed rabbit LSCD model corneas were observed through hematoxylin-eosin staining. Cell phenotypes were evaluated through fluorescence staining, Western blotting, and RT-qPCR. Additionally, cell junction genes and expression markers related to anti-neovascularization were evaluated using RT-qPCR. Corneal epithelium cell junctions were observed via an electron microscope. The tissue-engineered corneal epithelium culture medium was analyzed through mass spectrometry. Tissue-engineered corneal epithelial cells expanded by LSCs on UAM-MSCs had good transparency. Simultaneously, progenitor cell (K14, PNCA, p63) and corneal epithelial (PAX6) gene expression in tissue-engineered corneal epithelium constructed using UAM-MSCs was higher than that in corneal epithelial cells amplified by UAM and de-epithelialized amniotic membrane. Electron microscopy revealed that corneal epithelial cells grafted with UAM-MSCs were closely connected. In conclusion, the UAM-MSCs vector we constructed could better simulate the limbal microenvironment; the cultured tissue-engineered corneal epithelium had better transparency, anti-neovascularization properties, closer intercellular connections, and closer resemblance to the natural corneal epithelial tissue phenotype.

Asunto(s)

Amnios , Epitelio Corneal , Células Madre Mesenquimatosas , Ingeniería de Tejidos , Amnios/citología , Ingeniería de Tejidos/métodos , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Epitelio Corneal/citología , Epitelio Corneal/metabolismo , Animales , Conejos , Humanos , Células Cultivadas , Limbo de la Córnea/citología , Limbo de la Córnea/metabolismo , Diferenciación Celular

RESUMEN

Tissue-specific delivery of oligonucleotide therapeutics beyond the liver remains a key challenge in nucleic acid drug development. To address this issue, exploiting exosomes as a novel carrier has emerged as a promising approach for efficient nucleic acid drug delivery. However, current exosome-based delivery systems still face multiple hurdles in their clinical applications. Herein, this work presents a strategy for constructing a hybrid exosome vehicle (HEV) through a DNA zipper-mediated membrane fusion approach for tissue-specific siRNA delivery. As a proof-of-concept, this work successfully fuses a liposome encapsulating anti-NFKBIZ siRNAs with corneal epithelium cell (CEC)-derived exosomes to form a HEV construct for the treatment of dry eye disease (DED). With homing characteristics inherited from exosomes, the siRNA-bearing HEV can target its parent cells and efficiently deliver the siRNA payloads to the cornea. Subsequently, the NFKBIZ gene silencing significantly reduces pro-inflammatory cytokine secretions from the ocular surface, reshapes its inflammatory microenvironment, and ultimately achieves an excellent therapeutic outcome in a DED mouse model. As a versatile platform, this hybrid exosome with targeting capability and designed therapeutic siRNAs may hold great potential in various disease treatments.

Asunto(s)

Exosomas , Liposomas , Fusión de Membrana , ARN Interferente Pequeño , Exosomas/metabolismo , Exosomas/química , ARN Interferente Pequeño/metabolismo , Animales , Ratones , Liposomas/química , Síndromes de Ojo Seco/terapia , Humanos , Epitelio Corneal/metabolismo , Epitelio Corneal/patología , Silenciador del Gen , Córnea/metabolismo

RESUMEN

PURPOSE: To evaluate curvature changes in different regions and their correlation with corneal epithelial remodeling in myopic patients undergoing femtosecond laser-assisted in situ keratomileusis (FS-LASIK) and transepithelial refractive keratectomy (Trans-PRK) after surgery. METHODS: One hundred and sixty-three patients (163 right eyes) undergoing Trans-PRK and LASIK were evaluated for up to 6 months using anterior segment optical coherence tomography (OCT) to measure the epithelial thickness and corneal topography to measure corneal curvature in different areas (2 mm, 5 mm, and 7 mm). We calculated the curvature ΔK (ΔK = preoperative - postoperative), ΔK5-2 (ΔK5-2 = K5mm - K2mm), ΔK7-5 (ΔK7-5 = K7mm - K5mm), and the epithelial thickness ΔET5-2 (ΔET5-2 = ET5mm - ET2mm) and ΔET7-5 (ΔET7-5= ET7mm - ET5mm). RESULTS: Corneal curvature flattened in each region of the two groups (all p < 0.001) and gradually steepened during the follow-up period. The Trans-PRK group flattened more significantly within 2 mm and 5 mm, while the FS-LASIK group at 7 mm. Both groups of ΔK decreased over time. Both groups of ΔK5-2 and ΔK7-5 gradually decreased during the follow-up period (P5-2=0.025 and P7-5 < 0.001 for Trans-PRK, P5-2 = 0.011 and P7-5 < 0.001 for FS-LASIK). The corneal epithelium of the two groups gradually thickened during the follow-up period, with Trans-PRK significantly thickening in the central and peripheral regions and FS-LASIK in the central and paracentral regions. There is a significant correlation between the ΔK5-2 and ΔET5-2, ΔK7-5 and ΔET7-5 (all r > 0.37, p < 0.001). CONCLUSIONS: All groups showed significant curvature flattening after surgery and gradually steepening during the follow-up period. The corneal epithelium thickness in both groups of 17 regions became thicker,. In contrast, Trans-PRK group showed more significant thickening to the periphery and the central 5 mm area of the FS-LASIK. This study indicates a significant positive correlation between differences in epithelial thickening in different regions and differences in curvature changes in the corresponding areas PRK, FS-LASIK, curvature, corneal epithelial thickness.

Asunto(s)

Córnea , Topografía de la Córnea , Epitelio Corneal , Queratomileusis por Láser In Situ , Láseres de Excímeros , Miopía , Queratectomía Fotorrefractiva , Tomografía de Coherencia Óptica , Agudeza Visual , Humanos , Epitelio Corneal/patología , Epitelio Corneal/diagnóstico por imagen , Masculino , Femenino , Adulto , Tomografía de Coherencia Óptica/métodos , Queratomileusis por Láser In Situ/métodos , Queratectomía Fotorrefractiva/métodos , Miopía/cirugía , Miopía/fisiopatología , Láseres de Excímeros/uso terapéutico , Agudeza Visual/fisiología , Adulto Joven , Córnea/patología , Córnea/diagnóstico por imagen , Refracción Ocular/fisiología , Estudios de Seguimiento , Persona de Mediana Edad , Estudios Prospectivos

RESUMEN

BACKGROUND AND PURPOSE: The adenosine A2A receptor (A2AR) is involved in various physiological and pathological processes in the eye; however, the role of the A2AR signalling in corneal epithelial wound healing is not known. Here, the expression, therapeutic effects and signalling mechanism of A2AR in corneal epithelial wound healing were investigated using the A2AR agonist CGS21680. EXPERIMENTAL APPROACH: A2AR localization and expression during wound healing in the murine cornea were determined by immunofluorescence staining, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting. The effect of CGS21680 on corneal epithelial wound healing in the lesioned corneal and cultured human corneal epithelial cells (hCECs) by modulating cellular proliferation and migration was critically evaluated. The role of Hippo-YAP signalling in mediating the CGS21680 effect on wound healing by pharmacological inhibition of YAP signalling was explored. KEY RESULTS: A2AR expression was up-regulated after corneal epithelial injury. Topical administration of CGS21680 dose-dependently promoted corneal epithelial wound healing in the injured corneal epithelium by promoting cellular proliferation. Furthermore, CGS21680 accelerated the cellular proliferation and migration of hCECs in vitro. A2AR activation promoted early up-regulation and later down-regulation of YAP signalling molecules, and pharmacological inhibition of YAP signalling reverted CGS21680-mediated wound healing effect in vivo and in vitro. CONCLUSION AND IMPLICATIONS: A2AR activation promotes wound healing by enhancing cellular proliferation and migration through the YAP signalling pathway. A2ARs play an important role in the maintenance of corneal epithelium integrity and may represent a novel therapeutic target for facilitating corneal epithelial wound healing.

Asunto(s)

Adenosina , Epitelio Corneal , Fenetilaminas , Receptor de Adenosina A2A , Transducción de Señal , Cicatrización de Heridas , Proteínas Señalizadoras YAP , Cicatrización de Heridas/efectos de los fármacos , Animales , Fenetilaminas/farmacología , Transducción de Señal/efectos de los fármacos , Adenosina/análogos & derivados , Adenosina/farmacología , Receptor de Adenosina A2A/metabolismo , Epitelio Corneal/efectos de los fármacos , Epitelio Corneal/metabolismo , Humanos , Ratones , Agonistas del Receptor de Adenosina A2/farmacología , Movimiento Celular/efectos de los fármacos , Masculino , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ratones Endogámicos C57BL , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Lesiones de la Cornea/tratamiento farmacológico , Lesiones de la Cornea/metabolismo , Lesiones de la Cornea/patología