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Limbal mesenchymal stromal cells (LMSCs) reside in the limbal niche, supporting corneal integrity and facilitating regeneration. While mesenchymal stem/stromal cells (MSCs) are used in regenerative therapies, there is limited knowledge about LMSC subpopulations and their characteristics. This study characterized human LMSC subpopulations through the flow cytometric assessment of fifteen cell surface markers, including MSC, wound healing, immune regulation, ASC, endothelial, and differentiation markers. Primary LMSCs were established from remnant human corneal transplant specimens and passaged eight times to observe changes during subculture. The results showed the consistent expression of typical MSC markers and distinct subpopulations with the passage-dependent expression of wound healing, immune regulation, and differentiation markers. High CD166 and CD248 expressions indicated a crucial role in ocular surface repair. CD29 expression suggested an immunoregulatory role. Comparable pigment-epithelial-derived factor (PEDF) expression supported anti-inflammatory and anti-angiogenic roles. Sustained CD201 expression indicated maintained differentiation capability, while VEGFR2 expression suggested potential endothelial differentiation. LMSCs showed higher VEGF expression than fibroblasts and endothelial cells, suggesting a potential contribution to ocular surface regeneration through the modulation of angiogenesis and inflammation. These findings highlight the heterogeneity and multipotent potential of LMSC subpopulations during in vitro expansion, informing the development of standardized protocols for regenerative therapies and improving treatments for ocular surface disorders.
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Diferenciação Celular , Imunofenotipagem , Limbo da Córnea , Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Humanos , Limbo da Córnea/citologia , Limbo da Córnea/metabolismo , Células Cultivadas , Proliferação de Células , Biomarcadores/metabolismo , AdultoRESUMO
PURPOSE: To evaluate clinical practice in the diagnosis and treatment of progressive keratoconus with corneal crosslinking (CXL) in four Nordic countries. METHODS: A questionnaire was sent to all centres at which keratoconus patients are evaluated and CXL is performed in Sweden, Denmark, Norway and Iceland. Nineteen of 20 centres participated. RESULTS: CXL is performed approximately 1300 times per year in these four Nordic countries with a population of around 21.7 million (2019). In most cases, progression is evaluated using the Pentacam HR, and the maximum keratometry reading (Kmax ) is considered the most important parameter. The most frequently used treatment protocol in Scandinavia is the 9 mW/cm2 epi-off protocol, using hydroxylpropyl methylcellulose riboflavin (HPMC-riboflavin). The participants deemed the following areas to be in most need of improvement: adaptation of the CXL protocol to individual patients (5/19), the development of effective epi-on treatment protocols (4/19), optimal performance of CXL in thin corneas (4/19), improvement of the definition of progression (2/19), and diagnosis of the need for re-treatment (2/19). CONCLUSIONS: We concluded that the diagnosis of progressive keratoconus and the diagnostic equipment used are similar. Treatment strategies are also similar but are suitably different to provide an interesting basis for the comparison of treatment outcomes. The high degree of participation in this survey indicates the possibility of future scientific collaboration on CXL focusing on the areas deemed to need improvement. It would also be of interest to evaluate the possibility of creating a Nordic CXL Registry. The high number of CXL treatments performed ensures sufficient statistical power to solve many questions. Such a registry could be an important contribution to evidence-based care and would allow for longitudinal evaluation.
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Ceratocone , Fotoquimioterapia , Humanos , Ceratocone/diagnóstico , Ceratocone/tratamento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/uso terapêutico , Acuidade Visual , Reagentes de Ligações Cruzadas/uso terapêutico , Riboflavina/uso terapêutico , Países Escandinavos e Nórdicos/epidemiologia , Topografia da Córnea , Raios UltravioletaRESUMO
The progression of keratoconus is commonly determined by comparing the results of corneal tomographic measurements on different occasions. However, investigations on the repeatability of measurements are commonly performed within the same day, thus not taking the inter-day variation into account. The effect of keratoconus disease severity on the measurement error is also seldom considered. In this post hoc investigation, the parameters A, B and C in the Belin ABCD Progression Display were evaluated in relation to disease severity in intra-day and inter-day measurements. Four consecutive measurements were performed on 61 patients with keratoconus on the same day (intra-day). In another cohort, four consecutive measurements were obtained and then repeated 3 days later in 25 patients with keratoconus and 25 healthy controls (inter-day). The results suggest that the diagnosis of disease progression would benefit from inter-day measurements, and the stratification of the parameters A and C according to disease severity. It is also recommended that tomographic systems such as the Pentacam HR be modified to allow the comparison of both single measurements and the mean of replicate measurements of the parameters used in the assessment of progression of keratoconus.
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Córnea/patologia , Paquimetria Corneana/métodos , Topografia da Córnea/métodos , Ceratocone/patologia , Índice de Gravidade de Doença , Adolescente , Adulto , Estudos de Casos e Controles , Córnea/diagnóstico por imagem , Feminino , Humanos , Ceratocone/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Curva ROC , Reprodutibilidade dos Testes , Tomografia Computadorizada por Raios X/métodos , Adulto JovemRESUMO
PURPOSE: To report a case of iris epithelium detachment following Nd:YAG laser capsulotomy. OBSERVATIONS: We describe the case of an 81 year old woman who developed hyphema and detachment of the iris epithelium following standard Nd:YAG laser capsulotomy. The detachment was readily visualized using anterior segment OCT. The hyphema was managed with topical atropine and steroids. The detachment itself was left untreated. CONCLUSIONS AND IMPORTANCE: No persisting subjective complaints or effect on visual function were noted. To our knowledge, this complication represents a novel observation.
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PURPOSE: To evaluate the interday repeatability in the measurement of parameters used for the detection of progression of keratoconus by prediction limits (PL) for single measurements, and the repeatability coefficient (RC) for the mean of replicate measurements. DESIGN: Prospective reliability analysis for cases and control eyes. METHODS: Twenty-five eyes in 25 subjects with KC and 25 eyes in 25 healthy controls were included. Four consecutive measurements were made, 3 days apart, with a Pentacam HR tomographic instrument (denoted the Pentacam) and a Nidek ARK 560-A auto-keratometer (denoted the keratometer). Main outcome measures were the intra- and interday RC of parameters used in the detection of progression of keratoconus. RESULTS: The most repeatable parameter obtained with the Pentacam was the curvature power of the central flat meridian (K1, 0.44 D [RC], -0.55 to 0.60 diopter [D] [PL]), followed by the central steep meridian (K2, 0.72 D [RC], -0.90 to 0.94 D [PL]). The interday repeatability of K1 and K2 was similar when using the keratometer (K1, 0.32 D [RC], -0.66 to 0.57 D [PL], K2, 0.93 D [RC], -1.36 to 1.08 D [PL]). The interday repeatability of the curvature power of the steepest point (Kmax, 0.84 D [RC], -0.90 to 1.11 D [PL]) would benefit from being stratified: RC = 0.44 D and PL = -0.49 to 0.67 D for Kmax < 49.0 D, and RC = 1.08 D and PL = -1.19 to 1.42 D for Kmax ≥ 49.0 D. CONCLUSIONS: The interday repeatability of measurements, single or replicate, in subjects with keratoconus should be considered when diagnosing progressive disease. K1 exhibited the best intraday repeatability. Kmax benefits from being stratified according to disease severity.
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Córnea/patologia , Ceratocone/diagnóstico , Adulto , Paquimetria Corneana/instrumentação , Topografia da Córnea/instrumentação , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Tomografia/métodosRESUMO
BACKGROUND: Transcriptomic profiling of ex vivo cultured human limbal epithelial stem cells (hLESCs) will foster better understanding of corneal physiology and novel treatment paradigms to limbal stem cell deficiency (LSCD). However, currently such profiling studies are hampered due to difficulties with producing sufficient amounts of intact mRNA for deep RNA sequencing (RNA-seq) from subpopulations sorted on the basis of co-expression of membrane and intracellular antigens by fluorescence-activated cell sorting (FACS). METHODS: To address this problem, we systematically analyzed the critical steps, and found that ethanol fixation together with optimized downstream procedures provided a pipeline that yielded high quality total RNA in amounts to readily support the RNA-seq procedure, while still preserving good discrimination between the individual hLESC immunophenotypes. RESULTS: The average RNA integrity number (RIN) was 7.7 ± 0.4, and the average yield was 4.6 ± 1.7 pg of RNA per cell. The sequencing analysis of the isolated RNA produced high quality data with more than 70% of read pairs mapping uniformly to the reference genome and 80% of bases with a Phred score of at least 30. CONCLUSION: In this study, we developed a reliable FACS-based procedure using ethanol as a fixative that would support accurate isolation of limbal epithelial progenitor subpopulations along with RNA yield and quality sufficient to enable deep transcriptomic profiling.
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AIM OF DATABASE: To monitor the development of diabetic eye disease in Denmark and to evaluate the accessibility and effectiveness of diabetic eye screening programs with focus on interregional variations. TARGET POPULATION: The target population includes all patients diagnosed with diabetes. Denmark (5.5 million inhabitants) has ~320,000 diabetes patients with an annual increase of 27,000 newly diagnosed patients. The Danish Registry of Diabetic Retinopathy (DiaBase) collects data on all diabetes patients aged ≥18 years who attend screening for diabetic eye disease in hospital eye departments and in private ophthalmological practice. In 2014-2015, DiaBase included data collected from 77,968 diabetes patients. MAIN VARIABLES: The main variables provide data for calculation of performance indicators to monitor the quality of diabetic eye screening and development of diabetic retinopathy. Data with respect to age, sex, best corrected visual acuity, screening frequency, grading of diabetic retinopathy and maculopathy at each visit, progression/regression of diabetic eye disease, and prevalence of blindness were obtained. Data analysis from DiaBase's latest annual report (2014-2015) indicates that the prevalence of no diabetic retinopathy, nonproliferative diabetic retinopathy, and proliferative diabetic retinopathy is 78%, 18%, and 4%, respectively. The percentage of patients without diabetic maculopathy is 97%. The proportion of patients with regression of diabetic retinopathy (20%) is greater than the proportion of patients with progression of diabetic retinopathy (10%). CONCLUSION: The collection of data from diabetic eye screening is still expanding in Denmark. Analysis of the data collected during the period 2014-2015 reveals an overall decrease of diabetic retinopathy compared to the previous year, although the number of patients newly diagnosed with diabetes has been increasing in Denmark. DiaBase is a useful tool to observe the quality of screening, prevalence, and progression/regression of diabetic eye disease.
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Fuchs endothelial corneal dystrophy (FECD), is the most common corneal endothelial dystrophy, and contributes up to 50% of all corneal transplantations performed in developed countries. FECD develops in Descemet's membrane (DM) and possibly alters the mechanical properties and internal structures in this basal lamina. In this work, the morphology and mechanical properties of FECD-DMs are studied by transmission electron microscopy (TEM) and quantitative dynamic atomic force microscopy (QD-AFM) at nano scale. Pathological wide-space collagens that are typical of FECD display different mechanical properties in that they are softer than the remaining tissue both for dehydrated- and fully hydrated samples. Additionally, the hydration level has major influence on the mechanical properties. These findings could help to further understand the structural changes in FECD, and possibly be useful for further characterization of the disease, the diagnosis and assessment or even pathologic analysis.
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Córnea/fisiopatologia , Lâmina Limitante Posterior/fisiopatologia , Distrofia Endotelial de Fuchs/fisiopatologia , Fenômenos Mecânicos , Algoritmos , Córnea/patologia , Córnea/ultraestrutura , Lâmina Limitante Posterior/ultraestrutura , Módulo de Elasticidade , Humanos , Microscopia de Força Atômica , Microscopia Eletrônica de TransmissãoRESUMO
The purpose of this article was to review the existing evidence on emergency treatment of chemical eye burns. Clinical studies show that patients receiving prompt eye irrigation after chemical burns had a significantly better clinical outcome. This is further collaborated in animal studies where prompt irrigation with diphoterine or borate buffer significantly lowered pH in the eye after alkali burns. Two of three studies showed that tap water significantly lowered pH as well, but only if it was administered within 60 seconds after exposure. Saline, however, did not cause any significant decrease in pH at all.
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Queimaduras Químicas/terapia , Queimaduras Oculares/induzido quimicamente , Álcalis/efeitos adversos , Queimaduras Químicas/patologia , Tratamento de Emergência/métodos , Queimaduras Oculares/epidemiologia , Queimaduras Oculares/patologia , Queimaduras Oculares/terapia , Humanos , Soluções Oftálmicas/administração & dosagem , Soluções Oftálmicas/uso terapêutico , Cloreto de Sódio/administração & dosagem , Cloreto de Sódio/uso terapêutico , Irrigação Terapêutica/métodos , Fatores de Tempo , Água/administração & dosagemRESUMO
The structures and mechanical properties of human tissues are significantly influenced by water. The functionality of the human cornea can be linked to the hydrated collagen fibers. By applying quantitative dynamic atomic force microscopy to investigate morphological and mechanical property variations of corneal stroma under different hydration levels, we found that the collagen fibers in the stromal tissue show the specific periodicities and the stiffness of giga-Pa magnitude at 40% humidity. However, under increasing hydration, the collagen fibers clearly show nanoparticle structures along the fibers with the stiffness in mega-Pa magnitude. By increasing the hydration time, the stroma regains the fiber structure but with larger diameter. The age-dependency in stiffness was further investigated. The interplay of structures and nanomechanical mapping may be applied for the future diagnosis and assessment or even pathologic analysis.