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PURPOSE: To compare the corneal tissue trauma after the use of an automated preloaded injector and a manual injector and assess scanning electron microscope (SEM) and atomic force microscope (AFM) features of both injector cartridges. SETTING: Ophthalmology Clinic and Laboratory of Stem Cells and Regenerative Medicine University "G. d'Annunzio" of Chieti-Pescara, Chieti, Italy; DESIGN: Prospective randomized clinical study METHODS: Forty eyes of 40 patients for phacoemulsification were divided into two groups: implantation of intraocular lens was performed with AutonoMe automated delivery system (AutonoMe group: 20 eyes) and Monarch III injector system (Monarch group: 20 eyes). In vivo confocal microscopy (IVCM) and anterior segment optical coherence tomography (AS-OCT) were performed before surgery, at 1 h, 1 day and 1 month post-operatively. In addition, SEM and AFM were performed on cartridges of both injector systems after injection of the IOL. RESULTS: A greater increase in central corneal thickness and corneal thickness at the incision site were observed in Monarch group versus AutonoMe group 1 h and 1 day post-operatively (p < 0.05). Endothelial cell count loss was significantly higher in Monarch group compared with AutonoMe group (p < 0.05) at 1 and 24 h. AS-OCT showed less endothelial misalignment at 30 days (p < 0.05), and IVCM showed less tunnel inflammation at all time points (p < 0.05) in AutonoMe group compared with Monarch group; roughness analysis at AFM of the AutonoMe cartridge was significantly lower compared to Monarch D cartridge (p < 0.05). CONCLUSIONS: The AutonoMe injector provided less corneal tissue trauma compared with Monarch III injector. The AutonoMe cartridge showed lower roughness at AFM compared to the Monarch D cartridge.
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Catarata/diagnóstico por imagen , Implantación de Lentes Intraoculares/instrumentación , Lentes Intraoculares , Facoemulsificación/métodos , Anciano , Endotelio Corneal/ultraestructura , Diseño de Equipo , Femenino , Estudios de Seguimiento , Humanos , Masculino , Microscopía Electrónica de Rastreo , Estudios Prospectivos , Tomografía de Coherencia ÓpticaRESUMEN
Neurotrophic keratopathy (NK) is a rare degenerative corneal disorder characterized by instability of epithelial integrity with consequent epithelial defects that can worsen up to persistent epithelial defects with stromal melting and ulceration. The pathogenesis of NK springs from a variable degree of damage to the trigeminal nerve plexus, leading to a reduction or total loss of corneal sensitivity. Mackie classification (1995) distinguishes three stages of NK, based on the severity of clinical presentation. The technological innovations in corneal diagnostic imaging allow clinicians to accurately study the morphometry and morphology of corneal structure with microscopic resolution. In this study, 45 patients affected by NK at different stages underwent in vivo confocal microscopy (IVCM) and anterior segment optical coherence tomography (AS-OCT) with particular attention to analyze subbasal nerve plexus fibers and the stromal structure. At the light of IVCM and AS-OCT observations, we propose a different staging of NK with respect to the Mackie's classification that takes into account the severity of subbasal nerve fibers damage and the extension in depth of stromal ulceration; this classification better defines, at the time of diagnosis, the cellular and structural alterations in the affected corneas, with possible prognostic and therapeutic values in the management of NK.
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Enfermedades de la Córnea/clasificación , Enfermedades de la Córnea/diagnóstico por imagen , Enfermedades de la Córnea/patología , Adulto , Femenino , Humanos , Masculino , Microscopía Confocal , Persona de Mediana Edad , Estudios Retrospectivos , Tomografía de Coherencia ÓpticaRESUMEN
PURPOSE: To investigate, in vivo by means of in vivo confocal microscopy (IVCM) and ex vivo by impression cytology, epithelial cellular damage after excimer laser refractive surgery in patients under different topical lubricant therapies. METHODS: Two hundred eyes of 100 patients, undergone bilateral excimer laser refractive surgery for medium myopic error correction [spherical equivalent refraction from -1.75 to -3.50 dioptres (D) with refractive astigmatism under -0.75 D], have been recruited. All patients received, in addition to standard therapy for refractive surgery, high weight hyaluronic acid 0.2% eyedrops in one randomly selected eye and carboxymethylcellulose 1% eyedrop in the comparator eye (control eye) 4 times daily for 90 days. Follow-up included a baseline visit and further examination 7-, 30- and 90-day intervals [clinical evaluation with Schirmer test and tear break-up time (TBUT), IVCM and impression cytology]. RESULTS: No significant difference in Schirmer test and TBUT was observed during the follow-up period in eyes under different therapies. IVCM showed an improvement of conjunctival and corneal epithelial cells quality in eye in treatment with high weight hyaluronic acid 0.2% when compared to carboxymethylcellulose. Conjunctival impression cytology demonstrated an evident positivity for CD44 in eyes treated with both treatments in all follow-up controls. ICAM1 expression showed an increasing positivity starting at 30 days that became statistically significant after 90 days of high weight hyaluronic acid 0.2% therapy (p = 0.0167). CONCLUSIONS: In vivo and in vitro results showed the effectiveness of high weight hyaluronic acid 0.2% in facilitating cell-cell interaction, migration, cell proliferation, stabilizing epithelial barrier of the ocular surface. Moreover, use of high weight hyaluronic acid in treatment of corneal tissue damage after refractive surgery, in concordance with standard topical corticosteroids and antibiotics therapy, could be effective in promoting corneal epithelial wound healing with consequent good results in clinical outcome and patients' satisfaction.
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Carboximetilcelulosa de Sodio/uso terapéutico , Adhesión Celular/efectos de los fármacos , Comunicación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Ácido Hialurónico/uso terapéutico , Gotas Lubricantes para Ojos/uso terapéutico , Miopía/cirugía , Procedimientos Quirúrgicos Refractivos , Adulto , Conjuntiva/citología , Conjuntiva/efectos de los fármacos , Córnea/citología , Córnea/efectos de los fármacos , Epitelio Corneal/citología , Epitelio Corneal/efectos de los fármacos , Femenino , Humanos , Láseres de Excímeros , Masculino , Microscopía Confocal , Procedimientos Quirúrgicos Refractivos/efectos adversosRESUMEN
The aim of this study is to determine modifications in stromal fluorescence intensity after different corneal cross-linking (CXL) procedures and to correlate stromal fluorescence to corneal biomechanical resistance. For confocal microscopy study, 15 human cadaver corneas were examined. Three served as control (group 1), three were just soaked with iontophoresis procedure (group 2), three were treated with standard epi-off technique (group 3), and six underwent iontophoresis imbibition. Three of later six were irradiated for 30 min with 3 mW/cm2 UVA (group 4) and three for 9 min at 10 mW/cm2 UVA (group 5). Confocal microscopy was performed to quantify the fluorescence intensity in the cornea at different stromal depths. For biomechanical study, 30 human cadaver corneas were randomly divided into five groups and treated as previously described. Static stress-strain measurements of the corneas were performed. Iontophoresis imbibition followed by 10mW/cm2 irradiation proved to increase stromal fluorescence into the corneal stroma and significant differences were revealed between group 3 and 5 both at 100 (p = 0.0171) and 250 µm (p = 0.0024), respectively. Biomechanical analysis showed an improvement of corneal resistance in group 5. Iontophoresis imbibition followed by accelerated irradiation increased the stromal fluorescence and is related to an improvement of biomechanical resistance. This approach may represent a new strategy to achieve greater concentrations of riboflavin without removing corneal epithelium and improve clinical results while reducing the side effects of CXL.
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Colágeno/metabolismo , Córnea/patología , Sustancia Propia/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Iontoforesis/métodos , Análisis de Varianza , Fenómenos Biomecánicos , Cadáver , Córnea/efectos de los fármacos , Córnea/fisiopatología , Córnea/efectos de la radiación , Fluorescencia , Humanos , Microscopía Confocal , Fármacos Fotosensibilizantes/farmacología , Rayos UltravioletaRESUMEN
The purpose of this study was to evaluate the incidence of opaque bubble layer (OBL) in femtosecond laser-assisted in situ keratomileusis (LASIK) flaps created with the support of Visumax Carl Zeiss femtosecond laser, planned with different flap diameters (7.90, 8.0, and 8.20 mm) and the same laser energy and power settings. Incidence of intraoperative OBL in flaps of consecutive 108 patients (216 eyes) subjected to bilateral femtosecond-assisted LASIK was considered. Flap creation was performed with the same laser design parameters (spot distance and energy offset) and different presetting diameters of 7.90 mm (72 eyes, group 1), 8 mm (72 eyes, group 2), and 8.20 mm (72 eyes, group 3). The incidence of OBL was considered and its extension was reported measuring involvement of different four corneal flap quadrants in which was theoretically divided the entire flap area; based on these data, OBL presence was classified as none (no evidence of OBL), minimal (minimal presence in not more that one quadrants corneal flap), mild (OBL presence in almost two or three quadrants without tendency to invade central cornea), and moderate (OBL presence in almost three quadrants with tendency to invade central cornea). In group 1, the incidence of OBL was of 23.6 % (17 eyes) with a mild/moderate presence; in group 2, incidence was 20.8 % (15 eyes) with mild presence. Group 3 presented a reduced OBL incidence (4.1 %, 3 eye) with a minimal presence. No statistically significant difference was found between group 1 and 2 (p = 0.8414).We found statistically significant differences between group 1 and group 3 (p = 0.0012) and between groups 2 and 3 (p = 0.0044). A significant reduction and extension of OBL incidence were evident when LASIK flap settings diameter was increased, and flap edge was closer to the contact glass border; this is probably consequent to a more effective gas dispersion outside of corneal flap.
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Sustancia Propia/cirugía , Queratomileusis por Láser In Situ/métodos , Láseres de Excímeros/uso terapéutico , Miopía/cirugía , Refracción Ocular , Colgajos Quirúrgicos , Agudeza Visual , Adulto , Anciano , Paquimetría Corneal , Sustancia Propia/patología , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Miopía/fisiopatología , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
PURPOSE: To correlate a biomicroscopic evaluation, an in vivo confocal microscopy examination, and impression cytologic findings of the corneal center and sclerocorneal limbus after cultured limbal stem cell transplantation and to test the effectiveness of in vivo confocal microscopy as a diagnostic procedure in ocular surface cell therapy reconstructive surgery. METHODS: Six eyes of six patients affected by limbal stem cell deficiency after chemical burns underwent ex vivo expanded limbal stem cell transplantation (two eyes) and ex vivo expanded limbal stem cell transplantation with subsequent penetrating keratoplasty (four eyes) to restore corneal transparency. One year after surgery, all patients underwent a biomicroscopic evaluation, central cornea impression cytology to detect cytokeratin 12 (CK12) positivity, and in vivo confocal microscopy of the central cornea and the sclerocorneal limbus to investigate the epithelial cellular morphology, limbal architecture, and corneal inflammation level. RESULTS: Impression cytology analysis showed CK12 positivity in five of six cases, in concordance with the biomicroscopic evaluation. Confocal microscopy pointed out irregular limbal architecture with the absence of the palisades of Vogt in all cases; the central epithelial morphology presented clear corneal characteristics in three cases and irregular morphology in the remaining three. CONCLUSIONS: After successful ex vivo expanded limbal stem cell transplantation, in the presence of a complete anatomic architecture subversion, documented by support of in vivo confocal microscopy, the sclerocorneal limbus seemed to maintain its primary function. In vivo confocal microscopy confirmed the procedure was a non-invasive, efficacious diagnostic ocular surface procedure in the case of cell therapy reconstructive surgery.
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Limbo de la Córnea/citología , Microscopía Confocal/métodos , Esclerótica/citología , Trasplante de Células Madre , Células Madre/citología , Adulto , Humanos , Inflamación/patología , Persona de Mediana Edad , FenotipoRESUMEN
PURPOSE: The aims of this study are to investigate the expression of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) protein in the normal human cornea and limbus and to analyze modifications of this expression under inflammatory conditions. METHODS: The expression of LGR5 was evaluated in seven limbal epithelial crypts (LECs), collected from healthy cadaver donors, and five inflamed LECs obtained from enucleated eyes. Central corneal buttons were used as controls. LGR5 protein distribution was determined by immunohistochemistry staining analysis. RESULTS: The cytoplasmic expression of LGR5 protein was observed in 100% of healthy LECs. Three out of five inflamed tissues analyzed were completely negative, while in the two remaining cases, we observed a moderate positivity in the basal cells of LECs. No relation was found between the expression of LGR5 and the grade of inflammatory cells. CONCLUSIONS: These findings demonstrate the presence of LGR5-positive cells in human LECs and their decrease in inflamed conditions, which suggests a critical role of this protein during inflammation and its possible use as a marker in normal crypts.
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Queratitis/metabolismo , Limbo de la Córnea/citología , Limbo de la Córnea/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Nicho de Células Madre/fisiología , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Estudios de Casos y Controles , Endoftalmitis/metabolismo , Endoftalmitis/patología , Humanos , Inmunohistoquímica , Queratitis/patología , Limbo de la Córnea/patología , Persona de Mediana EdadRESUMEN
PURPOSE: To evaluate cellular inflammation and apoptosis induced in the central portion of capsulorhexes/capsulotomies during cataract surgery, comparing a conventional manual technique and a femtosecond laser-assisted procedure at different energy settings using two laser systems. METHODS: Fifty-six capsulorhexes/capsulotomies were divided into four groups: the manual group (14 capsulorhexes) performed with the manual technique; the 7.0-µJ group (14 capsulotomies) (LensAR laser system; Lensar, Inc., Orlando, FL); the 10-µJ group (14 capsulotomies) (LenSx laser system; Alcon Laboratories, Inc., Fort Worth, TX); and the 13.0-µJ group (14 capsulotomies) (LenSx laser system). All samples were stained for cellular apoptosis analysis (TUNEL assay) and cellular induced inflammation (NF-κB). RESULTS: One-way analysis of variance indicated a statistically significant difference in the percentage of NF-κB and TUNEL positive cells between the four groups, (F [3.52] = 14.717, P < .001) and (F [3.52] = 139.561, P < .001), respectively. Post-hoc analysis indicated a statistically significant difference in the percentage of NF-κB positive cells between the 13.0-µJ group and the manual, 7.0-µJ, and 10-µJ groups (P < .001, = .037, and < .001, respectively). Post-hoc analysis of differences in TUNEL positive cells indicated a significant difference between the 7.0-µJ and 10-µJ groups (P <.017) and between the 13.0-µJ group and the manual, 7.0-µJ, and 10-µJ groups (P < .001, < .001, and < .001, respectively). CONCLUSION: The results show a higher percentage of NF-κB and TUNEL positive cells in the 13.0-µJ group compared to the 7.0-µJ, 10-µJ, and manual groups. Therefore, inflammatory response and cell death increased at increasing energies. An effective capsulotomy in femtosecond laser-assisted cataract surgery with minimal detrimental apoptotic and inflammatory effects is possible if the laser system is set to use the minimum energy level.
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Apoptosis , Capsulorrexis/métodos , Células Epiteliales/patología , Cápsula del Cristalino/patología , Capsulotomía Posterior/métodos , Uveítis Anterior/etiología , Adulto , Anciano , Anciano de 80 o más Años , Células Epiteliales/metabolismo , Femenino , Humanos , Etiquetado Corte-Fin in Situ , Cápsula del Cristalino/metabolismo , Implantación de Lentes Intraoculares , Masculino , Persona de Mediana Edad , FN-kappa B/metabolismo , Facoemulsificación , Método Simple Ciego , Uveítis Anterior/diagnóstico , Uveítis Anterior/metabolismoRESUMEN
The aim of this study is to investigate in vivo and ex vivo ocular surface alterations induced by dry eye disease and modification after osmoprotective therapy. Forty-eight eyes of 24 patients suffering from dry eye have been recruited. All patients received Optive (compatible solutes) eye drops in one randomly selected eye and Hylogel (sodium hyaluronate 0,2%) in the other. Follow-up included a baseline visit and further examination 30-, 60-, and 90-day intervals (which comprises clinical evaluation, in vivo confocal microscopy-IVCM-of the ocular surface, and conjunctival impression cytology). No significant difference in Schirmer I Test, TBUT, and vital staining results was observed during the follow-up period in both groups. IVCM showed in all patients an improvement of ocular surface epithelial morphology and signs of inflammation (oedema and keratocyte activation). However, these modifications were more evident in patients treated with Optive therapy. A significant reduction of the expression of MMP9 and IL6 in Optive group was observed during the follow-up period in comparison to Hylogel treatment. Our results show that in dry eye disease therapy based on osmoprotective eye drops determines a reduction of inflammatory activation of ocular surface, with consequent improvement of the quality of corneal and conjunctival epithelium.
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Síndromes de Ojo Seco/tratamiento farmacológico , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Soluciones Oftálmicas , Adulto , Síndromes de Ojo Seco/inmunología , Femenino , Humanos , Inflamación/tratamiento farmacológico , Masculino , Microscopía Confocal , Persona de Mediana Edad , Adulto JovenRESUMEN
PURPOSE: To analyze, using in vivo laser scanning confocal microscopy (LSCM), the conjunctival features in glaucomatous patients receiving prostaglandin analogues (PGA). METHODS: Eighty eyes of 80 consecutive glaucomatous patients naive for therapy were enrolled; 30 eyes of 30 healthy subjects served as a control. Patients were randomized to: preservative-free (PF) and preserved latanoprost (groups 1 and 2, respectively), PF and preserved timolol (groups 3 and 4), and controls to vehicle of latanoprost or physiological buffered saline solution (groups 5 and 6). All subjects underwent LSCM of bulbar conjunctiva at baseline and 3 months after initiating therapy. The main outcomes were: mean density (MMD: cysts/mm(2)) and mean area (MMA: cysts/mm(2)) of epithelial microcysts. The relations between MMA and MMD with intraocular pressure (IOP), age, and mean defect (MD), were analyzed. RESULTS: At baseline, microcysts were found in all subjects. At month three, MMD did not change in all groups (p > 0.05). MMA significantly increased only in group 1 from 2,158.81 ± 524.09 to 3,877.77 ± 867.31, and in group 2 from 2,019.71 ± 541.03 to 5,560.39 ± 1,176.14, with values significantly higher in group 2 (p < 0.001). Significant relations were not found between MMD and MMA with IOP, MD, and age (p > 0.05). CONCLUSIONS: PGA increased MMA in therapy-naive glaucomatous patients, indicating a possible enhancement of the trans-conjunctival aqueous humor outflow. Therefore, conjunctiva seems an additional target tissue to evaluate the hydrodynamic pathways in glaucoma and modifications induced by medical therapy.
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Antihipertensivos/uso terapéutico , Humor Acuoso/fisiología , Conjuntiva/metabolismo , Glaucoma/tratamiento farmacológico , Prostaglandinas F Sintéticas/uso terapéutico , Administración Tópica , Recuento de Células , Quistes/metabolismo , Método Doble Ciego , Células Epiteliales/metabolismo , Femenino , Glaucoma/metabolismo , Humanos , Presión Intraocular/efectos de los fármacos , Latanoprost , Masculino , Microscopía Confocal , Persona de Mediana Edad , Soluciones Oftálmicas , Conservadores Farmacéuticos , Estudios Prospectivos , Timolol/uso terapéutico , Tonometría Ocular , Agudeza Visual/fisiologíaRESUMEN
Purpose: The purpose of this study was to explore the optimal shape of customized lenticules for stromal lenticule addition keratoplasty (SLAK) for off-centered ectasia. Methods: Two different methods to create ex vivo models of eccentric-keratoconus were investigated. Twelve human corneas were used to create model 1 by a hyperopic photorefractive keratectomy (PRK), and model 2 by masked phototherapeutic keratectomy (PTK) on the anterior corneal surface, whereas both types received myopic ablation of the posterior surface. Keratoconus models underwent a modified femtosecond laser (FSL) flap-cut to create stromal pockets. Sixteen human corneas underwent FSL dissection to obtain four lenticule types: type I (planar) and type II (negative) lenticules were used without modifications, whereas type III (customized-planar), and type IV (customized-negative) lenticules underwent further masked-PRK to obtain an asymmetric bow-tie shape. Topographic, aberrometric analysis, and anterior segment optical coherence tomography (AS-OCT) were performed in all recipient corneas before and after lenticule implantation. Results: Keratoconus model was successfully reproduced. Tomographic analysis showed a significant inferiorly decentered corneal steepening with coherent stromal thinning. Model 2 reproduced better the curvature of real keratoconus. Lenticules type I implantation induced a homogeneous corneal thickening, type III produced higher thickening in the inferior half of the cornea. Type II determined a maximal peripheral pachymetric increase, with a gradual reduction toward the center, and type IV presented an asymmetric peripheral thickening. Topographic assessment showed a cone apex flattening in all cases, but it was significantly higher in types II and IV. Customized lenticules improved significantly corneal surface regularity regarding types I and II. Conclusions: The approach of customizing lenticules by increasing their asymmetry and tailoring the re-shaping effects, may improve SLAK outcomes in eccentric keratoconus.
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Sustancia Propia , Topografía de la Córnea , Queratocono , Queratectomía Fotorrefractiva , Tomografía de Coherencia Óptica , Humanos , Sustancia Propia/cirugía , Sustancia Propia/trasplante , Queratocono/cirugía , Queratocono/fisiopatología , Queratectomía Fotorrefractiva/métodos , Láseres de Excímeros/uso terapéutico , Femenino , Masculino , Adulto , Colgajos Quirúrgicos , Aberrometría , Refracción Ocular/fisiología , Trasplante de Córnea/métodos , Persona de Mediana EdadRESUMEN
PURPOSE: To study the expression of S100 A and B family proteins in normal human limbus and to analyze modification of the expression in inflammatory conditions. METHODS: The total expression of members of the S100 family and the expression of A4, A8, A9, and B individually were evaluated in nine normal human corneal limbi, collected from cadaver healthy donors, in particular in the limbal epithelial crypts (LECs), and in five inflamed limbi obtained from enucleated eyes. S100 protein distribution was determined with immunohistochemistry staining analysis. RESULTS: Cytoplasmic expression of total S100 proteins was observed in 100% of LECs; in contrast, the inflamed tissues were completely negative, and faint positivity was observed in only one case. Moreover, cytoplasmic expression of S100 A4 and A9 was uniformly found in the entire LECs in all samples analyzed, while S100 A8 positivity was observed in only 44.4% of cases and only in the cells localized in the central area of the LEC. Positivity for S100 B was not observed in all samples analyzed. CONCLUSIONS: As reported in the literature, normal limbal epithelial cells show strong expression of S100 proteins. A novel finding of this study was the expression for the limbal epithelial crypts. In particular, S100 A4 and A9, which are normally involved in regulating a wide range of biologic effects, including cell motility, survival, and differentiation, are the most expressed members in healthy limbal crypts. In inflamed tissues, expression of S100 proteins was dramatically decreased. S100 proteins, and in particular S100 A4 and S100 A9, can be useful as markers of early changes in stem cell niches due to inflammation.
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Calgranulina A/genética , Calgranulina B/genética , Mediadores de Inflamación/metabolismo , Queratitis/metabolismo , Limbo de la Córnea/metabolismo , Limbo de la Córnea/patología , Factores de Crecimiento Nervioso/metabolismo , Proteínas S100/genética , Proteínas S100/metabolismo , Anciano , Anciano de 80 o más Años , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Estudios de Casos y Controles , Citoplasma/metabolismo , Marcadores Genéticos , Humanos , Queratitis/genética , Queratitis/patología , Limbo de la Córnea/citología , Persona de Mediana Edad , Subunidad beta de la Proteína de Unión al Calcio S100 , Proteína de Unión al Calcio S100A4 , Nicho de Células Madre/genéticaRESUMEN
PURPOSE: The corneoscleral limbus is the site of corneal epithelial stem cells (SC). The aim of this study is to evaluate the expression of different SC markers in the normal human limbus and to determine how this is affected by inflammation. METHODS: Corneoscleral specimens from healthy and inflamed donor eyes were examined by immunohistochemistry/immunofluorescence for p63, vimentin, laminin 5, integrin α6, ß1, ß4, ABCG2, desmoglein 3, connexin 43, N-cadherin, and cytokeratins 12 and 15. The distribution and anatomic structure of the limbal crypts and the percentage of SC marker antigens in healthy donors were analyzed. In inflamed tissues, we evaluated the anatomic structure of the limbal epithelial crypt (LEC) and the positivity for SC markers. RESULTS: In normal limbus, the niche structures were distributed differently. The variability of their number correlated with the percentage of p63 positivity. Integrin ß1 staining directly correlated with p63 positivity while the remaining proteins were variably and widely distributed. Double staining for p63 and vimentin did not reveal any co-localization. In inflamed eyes, the basal cells in the crypts were "stretched" and surrounded by inflammatory cells, and only a few SC markers were still present. CONCLUSIONS: Diseases involving the limbus may result in marked changes of expression of SC markers within the LEC and also alter the crypt structure.
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Mediadores de Inflamación/metabolismo , Queratitis/metabolismo , Queratitis/patología , Limbo de la Córnea/metabolismo , Limbo de la Córnea/patología , Nicho de Células Madre , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Estudios de Casos y Controles , Proteínas del Ojo/metabolismo , Humanos , Inflamación/metabolismo , Inflamación/patología , Persona de Mediana EdadRESUMEN
AIM: Ultraviolet (UV) B irradiation induces gene expression that leads to skin cancer. Among the transcription factors induced by UVB radiation exposure, the cyclic AMP response element-binding protein (CREB) is significant. Since several factors downstream of CREB signaling are known to be involved in pterygium pathogenesis, we investigated CREB expression in pterygial and human conjunctival tissues to evaluate if a similar expression pattern is present. Moreover, we analyzed the correlation with CREB expression and other known pterygium markers. METHODS: Primary pterygium samples and normal bulbar conjunctivas surgically removed were analyzed. Formalin-fixed, paraffin-embedded tissues were stained by immunohistochemistry with anti-CREB, anti-vimentin, anti-ki-67, anti-survivin, anti-MMP7, anti-p63, anti-cyclin D1, or anti-p53 antibodies. RESULTS: 94.4% of pterygium samples were positive for CREB with a significant difference compared to the control group (p = 0.002). The staining was localized in the epithelium and absent in the stroma. An increased expression was found for cyclin D1 (p = 0.019), ki-67 (p = 0.005), vimentin (p = 0.003), survivin (p < 0.001), p63 (p = 0.003), and MMP7 (p = 0.002). CREB expression showed a significant correlation with cyclin D1 (ρ = 0.49; p = 0.035), ki-67 (ρ = 0.61; p = 0.007), and p53 (ρ = 0.57; p = 0.013) in pterygium. CONCLUSIONS: These results permit to hypothesize that CREB is involved in pterygium pathogenesis. Since various molecules have been discovered to inhibit CREB, these data could be of interest for pterygium treatment.
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Biomarcadores/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas del Ojo/metabolismo , Pterigion/metabolismo , Anciano , Anciano de 80 o más Años , Ciclina D1/metabolismo , Femenino , Humanos , Inmunohistoquímica , Proteínas Inhibidoras de la Apoptosis/metabolismo , Antígeno Ki-67/metabolismo , Masculino , Metaloproteinasa 7 de la Matriz/metabolismo , Proteínas de la Membrana/metabolismo , Pterigion/cirugía , Survivin , Proteína p53 Supresora de Tumor/metabolismo , Vimentina/metabolismoRESUMEN
Refractive surgery is one of the most common elective surgeries performed worldwide. The incidence of dry eye disease (DED) after corneal refractive surgery varies among different studies. Pre-existing untreated DED has been identified as a risk factor for postsurgical dry eye symptoms. On the basis of both evidence and clinical experience, some recommendations for ocular surface and DED management pre- and post-refractive surgery are described. In aqueous deficiency Dry Eye Disease, preservative-free lubricating drops should be preferred, in addition to ointment and gel forms. Topical anti-inflammatory agents (Cyclosporine 0.1%, hydrocortisone phosphate, fluorometholone) should be used for 3-6 months in cases of ocular surface damage. The therapy of evaporative DED includes lifestyle modifications, lid hygiene (either performed by the patient or offered as professional lid hygiene by the physician), use of lubricating eye drops with lipid components, topical and/or systemic antibiotic treatment with anti-inflammatory properties and Intense Pulsed Light (IPL-) Treatment for meibomian gland dysfunction.
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Neurotrophic Keratopathy (NK), classified as an orphan disease (ORPHA137596), is a rare degenerative corneal disease characterized by epithelial instability and decreased corneal sensitivity caused by the damage to the corneal nerves. The administration of human recombinant nerve growth factor (rhNGF) eye drops, as a licensed-in-Europe specific medication for treatment of moderate and severe NK, has added promising perspectives to the management of this disorder by providing a valid alternative to the neurotization surgery. However, few studies have been conducted to the molecular mechanism underlying the response to the treatment. Here, we carried out tears proteomics to highlight the protein expression during pharmacological treatment of NK (Data are available via ProteomeXchange with identifier PXD025408).Our data emphasized a proteome modulation during rhNGF treatment related to an increase in DNA synthesis, an activation of both BDNF signal and IL6 receptor. Furthermore, the amount of neuronal Extracellular Vesicles EVs (CD171+) correlated with the EVs carrying IL6R (CD126+) together associated to the inflammatory EVs (CD45+) in tears. Such scenario determined drug response, confirmed by an in vivo confocal microscopy analysis, showing an increase in length, density and number of nerve fiber branches during treatment. In summary, rhNGF treatment seems to determine an inflammatory micro-environment, mediated by functionalized EVs, defining the drug response by stimulating protein synthesis and fiber regeneration.
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Enfermedades de la Córnea/tratamiento farmacológico , Factor de Crecimiento Nervioso/uso terapéutico , Proteoma , Lágrimas/metabolismo , Administración Tópica , Enfermedades de la Córnea/metabolismo , Vesículas Extracelulares/metabolismo , Femenino , Humanos , Masculino , Microscopía Confocal , Factor de Crecimiento Nervioso/farmacología , Estudios Prospectivos , Enfermedades Raras , Proteínas RecombinantesRESUMEN
Small incision lenticule extraction (SMILE), is a surgical procedure for the myopia correction, during which a corneal stromal lenticule is extracted. Given that we have previously demonstrated how this discarded tissue could be repurposed as a bio-scaffold for stromal engineering, this study aimed to explore its use as an ocular drug delivery system of active molecules, using neurotrophic factor Nerve Growth Factor (NGF). We employed human stromal lenticules directly collected from healthy donors undergoing SMILE. Following a sodium dodecylsulfate (SDS) treatment, decellularized lenticules were incubated with a suspension of polylactic-co-glycolic-acid (PLGA) microparticles (MPs) loaded with recombinant human NGF (rhNGF-MPs). Fluorescent MPs (Fluo-MPs) were used as control. Data demonstrated the feasibility to engineer decellularized lenticules with PLGA-MPs which remain incorporated both on the lenticules surface and in its stromal. Following their production, the in vitro release kinetic showed a sustained release for up to 1 month of rhNGF from MPs loaded to the lenticule. Interestingly, rhNGF was rapidly released in the first 24 h, but it was sustained up to the end of the experiment (1 month), with preservation of rhNGF activity (around 80%). Our results indicated that decellularized human stromal lenticules could represent a biocompatible, non-immunogenic natural scaffold potential useful for ocular drug delivery. Therefore, combining the advantages of tissue engineering and pharmaceutical approaches, this in vitro proof-of-concept study suggests the feasibility to use this scaffold to allow target release of rhNGF in vivo or other pharmaceutically active molecules that have potential to treat ocular diseases.
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Introduction: The main role of preservatives in eyedrops is to ensure sterility and microbiological integrity of the drug, and to facilitate the penetration of active compounds into the eye. However, several studies documented significant toxic effects induced by preservatives, especially on the ocular surface. Consequently, most of the ophthalmic medications became progressively available in preservative-free (PF) formulations.Areas covered: We analyzed pre-clinical and clinical studies on PF eyedrops with particular attention to common chronic diseases such as dry eye and glaucoma. We discussed about the pros and cons of using PF eyedrops, in terms of efficacy, safety, and social-economic aspects.Expert opinion: There are still unresolved issues that make hard for PF medications to definitively conquer the drug market. Despite robust pre-clinical evidences of less toxicity, the low number of randomized clinical trials does not permit to state that PF eyedrops have, in clinical practice, a similar efficacy or a higher safety compared to preserved forms. These aspects limit their use to chronic diseases requiring long-term therapies with multiple daily instillations, especially in the presence of concomitant ophthalmic diseases that expose to a risk of ocular surface worsening.
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Antihipertensivos , Glaucoma , Glaucoma/tratamiento farmacológico , Humanos , Soluciones Oftálmicas , Conservadores Farmacéuticos/efectos adversosRESUMEN
Purpose: The purpose of this study was to describe the face mask (FM)-related ocular surface changes using clinical tests, in vivo confocal microscopy (IVCM) and impression cytology (IC), and to investigate the Dry Eye-related Quality of life Score (DEQS). Methods: Sixty-six patients with dry eye disease (DED) and 62 healthy subjects (group 2) using FM were enrolled. Groups were divided into: groups 1A and 2A: < 3 hours of FM wear; groups 1B and 2B: 3 to 6 hours; and groups 1C and 2C: > 6 hours. Patients underwent DEQS questionnaire, break-up time (BUT), Schirmer test I (STI), fluorescein and lissamine staining (FS and LS), IVCM to determine corneal dendritic cell density (DCD) and goblet cell density (GCD), and IC to measure HLA-DR, at baseline and after 3 months. Results: FM use duration before enrollment was 27 ± 2.3 and 30 ± 4.1 (days ± SD) for groups 1 and 2 (P > 0.05). After 3 months, DEQS worsened in groups 1B and 1C, STI in groups 1A to 1C, FS and LS in group 1C (P < 0.05); in controls, BUT and FS worsened only in group 2C (P < 0.05). DCD significantly increased in groups 1A to 1C and HLA-DR in groups 1B and 1C (P < 0.05), whereas GCD did not significantly change. DCD and HLA-DR increased only in group 2C (P < 0.05). DEQS significantly correlated with DCD (P = 0.05, r = 0.698; P < 0.001, r = 0.832) and HLA-DR (P = 0.043, r = -0.687; P < 0.001, r = 0.861) at baseline and 3 months. Conclusions: Use of FM increases ocular surface inflammation and negatively impacts the quality of life in patients with DED. Translational Relevance: The study of the prolonged use of FM effects may be relevant to managing DED.
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COVID-19 , Pandemias , Humanos , Máscaras , Microscopía Confocal , Calidad de Vida , SARS-CoV-2RESUMEN
The purpose of this study was to evaluate corneal epithelium and stromal remodelling with anterior segment optical coherence tomography in patients who have undergone stromal lenticule addition keratoplasty (SLAK) for advanced keratoconus. This was a prospective non-comparative observational study. Fifteen eyes of 15 patients with advanced keratoconus underwent implantation with a cadaveric, donor negative meniscus-shaped intrastromal lenticule, produced with a femtosecond laser, into a stromal pocket dissected in the recipient cornea at a depth of 120 µm. Simulated keratometry, central corneal thickness (CTT), corneal thinnest point (CTP), central epithelial thickness (CET), central and peripheral lenticule thickness, anterior and posterior stromal thickness were measured. Regional central corneal epithelial thickness (CET) and variations in the inner annular area (IAT) and outer annular area (OAT) were also analysed. All parameters were measured preoperatively and 1, 3, and 6 months postoperatively. The average anterior Sim-k decreased from 59.63 ± 7.58 preoperatively to 57.19 ± 6.33 D 6 months postoperatively. CCT, CTP, CET, and OAT increased and IAT decreased significantly after 1 month. All parameters appeared unchanged at 6-months except that of OAT that further increased. Lenticule thickness was stable. In conclusion we observed that SLAK reshapes the cornea by central flattening with stromal thickening and epithelial thickness restoration.