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Drug-loaded nanocarriers (NCs) are new systems that can greatly improve the delivery and targeting of drugs to specific tissues and organs. In our work, a PPAR-γ agonist loaded into polymeric NCs was prepared, stabilized by spray-drying, and tested in vitro, ex vivo, and in vivo (animal models) to provide a safe formulation for optical anti-inflammatory treatments. The NCs were shown to be well tolerated, and no signs of irritancy or alterations of the eye properties were detected by the in vitro HET-CAM test and in vivo Draize test. Furthermore, no signs of cytotoxicity were found in the NC formulations on retinoblastoma cells (Y-79) analyzed using the alamarBlue assay, and the transmittance experiments evidenced good corneal transparency with the formulations tested. The ocular anti-inflammatory study confirmed the significant prevention efficacy using the NCs, and these systems did not affect the corneal tissue structure. Moreover, the animal corneal structure treated with the NCs was analyzed using X-ray diffraction using synchrotron light. Small-angle X-ray scattering (SAXS) analysis did not show a significant difference in corneal collagen interfibrillar spacing after the treatment with freshly prepared NCs or NCs after the drying process compared to the corresponding negative control when inflammation was induced. Considering these results, the PPAR-γ agonist NCs could be a safe and effective alternative for the treatment of inflammatory ocular processes.
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Oftalmopatías , Receptores Activados del Proliferador del Peroxisoma , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Córnea , Oftalmopatías/tratamiento farmacológico , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Pioglitazone-loaded PLGA-PEG nanoparticles (NPs) were stabilized by the spray drying technique as an alternative to the treatment of ocular inflammatory disorders. Pioglitazone-NPs were developed and characterized physiochemically. Interaction studies, biopharmaceutical behavior, ex vivo corneal and scleral permeation, and in vivo bioavailability evaluations were conducted. Fibrillar diameter and interfibrillar corneal spacing of collagen was analyzed by synchrotron X-ray scattering techniques and stability studies at 4 °C and was carried out before and after the spray drying process. NPs showed physicochemical characteristics suitable for ocular administration. The release was sustained up to 46 h after drying; ex vivo corneal and scleral permeation profiles of pioglitazone-NPs before and after drying demonstrated higher retention and permeation through cornea than sclera. These results were correlated with an in vivo bioavailability study. Small-angle X-ray scattering (SAXS) analysis did not show a significant difference in the organization of the corneal collagen after the treatment with pioglitazone-NPs before and after the drying process, regarding the negative control. The stabilization process by Nano Spray Dryer B-90 was shown to be useful in preserving the activity of pioglitazone inside the NPs, maintaining their physicochemical characteristics, in vivo bioavailability, and non-damage to corneal collagen function after SAXS analysis was observed.
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It is well-accepted that hydration water is crucial for the structure, dynamics, and function of proteins. However, the exact role of water for the motions and functions of proteins is still debated. Experiments have shown that protein and water dynamics are strongly coupled but with water motions occurring on a considerably faster time scale (the so-called slaving behavior). On the other hand, water also reduces the conformational entropy of proteins and thereby acts as a plasticizer of them. In this work, we analyze the dynamics (using broadband dielectric spectroscopy) of some specific non-biological water solutions in a broad concentration range to elucidate the role of water in the dynamics of the solutes. Our results demonstrate that at low water concentrations (less than 5 wt. %), the plasticization phenomenon prevails for all the materials analyzed. However, at higher water concentrations, two different scenarios can be observed: the slaving phenomenon or plasticization, depending on the solute analyzed. These results generalize the slaving phenomenon to some, but not all, non-biological solutions and allow us to analyze the key factors for observing the slaving behavior in protein solutions as well as to reshaping the slaving concept.
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Proteínas/química , Agua/química , Dextranos/química , Movimiento (Física) , Oligopéptidos/química , Povidona/químicaRESUMEN
Nitric oxide (NO)-releasing nonsteroidal anti-inflammatory drugs (NSAIDs) have been developed to overcome the gastrointestinal and cardiovascular toxicity of NSAIDs, by chemically associating a NO-releasing moiety with commercial NSAIDs. Since increasing evidence supports that NSAIDs toxicity is related to their topical actions in membrane lipids, this work aims to evaluate the impact of adding a NO-releasing moiety to parent NSAIDs regarding their effect on lipid bilayers. Thus, the interactions of NO-indomethacin and indomethacin (parent drug) with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers were described herein at pH 3.0 and 7.4. Diverse experimental techniques were combined to characterize the partitioning and location of drugs in DMPC bilayers, and to analyze their effect on the lipid phase transition and the bilayer structure and dynamics. The partitioning of NO-indomethacin into DMPC bilayers was similar to that of charged indomethacin and smaller than that of neutral indomethacin. Both drugs were found to insert the DMPC bilayer and the membrane location of indomethacin was pH-dependent. NO-indomethacin and indomethacin induced a decrease of the main phase transition temperature of DMPC. The effect of these drugs on the bilayer structure and dynamics was dependent on diverse factors, namely drug ionization state, drug:lipid molar ratio, temperature and lipid phase. It is noteworthy that NO-indomethacin induced more pronounced alterations in the biophysical properties of DMPC bilayers than indomethacin, considering equivalent membrane concentrations. Such modifications may have in vivo implications, particularly in the gastric mucosa, where NO-NSAIDs-induced changes in the protective properties of phospholipid layers may contribute to the occurrence of adverse effects.
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Mucosa Gástrica/efectos de los fármacos , Indometacina/farmacología , Membrana Dobles de Lípidos/química , Óxido Nítrico/farmacología , Fosfolípidos/química , Humanos , Concentración de Iones de Hidrógeno , Indometacina/administración & dosificación , Indometacina/química , Estructura Molecular , Óxido Nítrico/administración & dosificación , Óxido Nítrico/química , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Gastrointestinal (GI) toxicity is a major drawback of the chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs). The NSAIDs topical actions on the protective phospholipid layers of the GI mucosa seem to be a central toxicity mechanism of these pharmaceuticals. This work describes the interactions of acemetacin, a commercialized NSAID, with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers at pH 3.0, 5.0, and 7.4. This pH range was chosen to mimic the pH gradient found in the gastric mucosa, and to ultimately gain insights into the mechanisms underlying the acemetacin-induced gastric toxicity. Various experimental techniques were combined to characterize the partitioning of acemetacin in DMPC bilayers, and its effects on the phase transition behavior, as well as the structure and dynamics of DMPC bilayers. The acemetacin-DMPC interactions were clearly pH-dependent. The neutral (protonated) form of acemetacin had more affinity for the DMPC bilayer than the negatively charged form. Due to the higher affinity of neutral acemetacin, the drug effects on the phase transition and the structure and dynamics of the DMPC bilayer were more pronounced at lower pH values. In general, acemetacin decreased the temperature and the cooperativity of the lipid phase transition and induced changes in the packing and dynamics of the DMPC bilayer. These results support the hypothesis that acemetacin-induced gastric toxicity may be related to its effects on the protective phospholipid layers of the mucosal barrier.
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Antiinflamatorios no Esteroideos/química , Indometacina/análogos & derivados , Fosfatidilcolinas/química , Concentración de Iones de Hidrógeno , Indometacina/química , Cinética , Membrana Dobles de Lípidos/química , Estructura Molecular , Transición de Fase , Electricidad Estática , TemperaturaRESUMEN
The primary aim of this study was to quantify the relationship between corneal structure and hydration in humans and pigs. X-ray scattering data were collected from human and porcine corneas equilibrated with polyethylene glycol (PEG) to varying levels of hydration, to obtain measurements of collagen fibril diameter, interfibrillar spacing (IFS) and intermolecular spacing. Both species showed a strong positive linear correlation between hydration and IFS2 and a nonlinear, bi-phasic relationship between hydration and fibril diameter, whereby fibril diameter increased up to approximately physiological hydration, H = 3.0, with little change thereafter. Above H = 3.0, porcine corneas exhibited a larger fibril diameter than human corneas (p < 0.001). Intermolecular spacing also varied with hydration in a bi-phasic manner but reached a maximum value at a lower hydration (H = 1.5) than fibril diameter. Human corneas displayed a higher intermolecular spacing than porcine corneas at all hydrations (p < 0.0001). Human and porcine corneas required a similar PEG concentration to reach physiological hydration, suggesting that the total fixed charge that gives rise to the swelling pressure is the same. The difference in their structural responses to hydration can be explained by variations in molecular cross-linking and intra/interfibrillar water partitioning.
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Sustancia Propia/fisiología , Agua/metabolismo , Animales , Bovinos , Humanos , Polietilenglicoles , Ovinos , Especificidad de la Especie , PorcinosRESUMEN
Macromolecular crystallography (MX) and small-angle X-ray scattering (SAXS) studies on proteins at synchrotron light sources are commonly limited by the structural damage produced by the intense X-ray beam. Several effects, such as aggregation in protein solutions and global and site-specific damage in crystals, reduce the data quality or even introduce artefacts that can result in a biologically misguiding structure. One strategy to reduce these negative effects is the inclusion of an additive in the buffer solution to act as a free radical scavenger. Here the properties of uridine as a scavenger for both SAXS and MX experiments on lysozyme at room temperature are examined. In MX experiments, upon addition of uridine at 1â M, the critical dose D1/2 is increased by a factor of â¼1.7, a value similar to that obtained in the presence of the most commonly used scavengers such as ascorbate and sodium nitrate. Other figures of merit to assess radiation damage show a similar trend. In SAXS experiments, the scavenging effect of 40â mM uridine is similar to that of 5% v/v glycerol, and greater than 2â mM DTT and 1â mM ascorbic acid. In all cases, the protective effect of uridine is proportional to its concentration.
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Dispersión del Ángulo Pequeño , Sincrotrones , Uridina/química , Proteínas/química , Difracción de Rayos XRESUMEN
AIM: In this work the effect of infrared (IR) radiation, at temperatures between 25 and 30°C, on the formation of free radicals (FRs) in the skin is studied. Additionally, the influence of IR radiation at high temperatures in the degradation of skin collagen is evaluated. In both experiments the protective effect against IR radiation of phospholipid nanostructures (bicosomes) incorporating ß-carotene (Bcb) is also evaluated. METHODS: The formation of FRs in skin under IR exposure was measured near physiological temperatures (25-30°C) using 5,5-dimethyl-1-pyrroline-N-oxide spin trap and electron paramagnetic resonance (EPR) spectroscopy. The study of the collagen structure was performed by small-angle X-ray scattering using synchrotron radiation. RESULTS: EPR results showed an increase in the hydroxyl radical in the irradiated skin compared to the native skin. The skin collagen was degraded by IR exposure at high temperatures of approximately 65°C. The treatment with Bcb reduced the formation of FRs and kept the structure of collagen. CONCLUSIONS: The formation of FRs by IR radiation does not depend on the increase of skin temperature. The decrease of FRs and the preservation of collagen fibers in the skin treated with Bcb indicate the potential of this lipid system to protect skin under IR exposure.
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Rayos Infrarrojos/efectos adversos , Nanoestructuras/administración & dosificación , Fosfolípidos/administración & dosificación , Piel/efectos de los fármacos , Piel/efectos de la radiación , beta Caroteno/administración & dosificación , Colágeno/efectos de los fármacos , Colágeno/metabolismo , Colágeno/efectos de la radiación , Espectroscopía de Resonancia por Spin del Electrón/métodos , Radicales Libres/antagonistas & inhibidores , Radicales Libres/metabolismo , Humanos , Nanoestructuras/química , Fosfolípidos/química , Piel/metabolismoRESUMEN
The J aggregates of 4-sulfonatophenyl meso-substituted porphyrins are non-covalent polymers obtained by self-assembly that form nanoparticles of different morphologies. In the case of high aspect-ratio nanoparticles (bilayered ribbons and monolayered nanotubes), shear hydrodynamic forces may modify their shape and size, as observed by peak force microscopy, transmission electron microscopy of frozen solutions, small-angle X-ray scattering measurements in a disk-plate rotational cell, and cone-plate rotational viscometry. These nanoparticles either show elastic or plastic behaviour: there is plasticity in the ribbons obtained upon nanotube collapse on solid/air interfaces and in viscous concentrated nanotube solutions, whereas elasticity occurs in the case of dilute nanotube solutions. Sonication and strong shear hydrodynamic forces lead to the breaking of the monolayered nanotubes into small particles, which then associate into large colloidal particles.
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The role of Decorin in organising the extracellular matrix was examined in normal human corneas and in corneas from patients with Congenital Stromal Corneal Dystrophy (CSCD). In CSCD, corneal clouding occurs due to a truncating mutation (c.967delT) in the decorin (DCN) gene. Normal human Decorin protein and the truncated one were reconstructed in silico using homology modelling techniques to explore structural changes in the diseased protein. Corneal CSCD specimens were also examined using 3-D electron tomography and Small Angle X-ray diffraction (SAXS), to image the collagen-proteoglycan arrangement and to quantify fibrillar diameters, respectively. Homology modelling showed that truncated Decorin had a different spatial geometry to the normal one, with the truncation removing a major part of the site that interacts with collagen, compromising its ability to bind effectively. Electron tomography showed regions of abnormal stroma, where collagen fibrils came together to form thicker fibrillar structures, showing that Decorin plays a key role in the maintenance of the order in the normal corneal extracellular matrix. Average diameter of individual fibrils throughout the thickness of the cornea however remained normal.
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Colágeno/metabolismo , Distrofias Hereditarias de la Córnea/metabolismo , Decorina/metabolismo , Condroitinasas y Condroitín Liasas/metabolismo , Córnea/patología , Distrofias Hereditarias de la Córnea/patología , Decorina/química , Humanos , Imagenología Tridimensional , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Multimerización de Proteína , Dispersión del Ángulo Pequeño , Homología Estructural de Proteína , Tomografía , Difracción de Rayos XRESUMEN
The cornea is the primary refractive lens in the eye and transmits >90% of incident visible light. It has been suggested that the development of postoperative corneal haze could be due to an increase in light scattering from activated corneal stromal cells. Quiescent keratocytes are thought to produce crystallins that match the refractive index of their cytoplasm to the surrounding extracellular material, reducing the amount of light scattering. To test this, we measured the refractive index (RI) of bovine corneal stromal cells, using quantitative phase imaging of live cells in vitro, together with confocal microscopy. The RI of quiescent keratocytes (RI = 1.381 ± 0.004) matched the surrounding matrix, thus supporting the hypothesis that keratocyte cytoplasm does not scatter light in the normal cornea. We also observed that the RI drops after keratocyte activation (RI = 1.365 ± 0.003), leading to a mismatch with the surrounding intercellular matrix. Theoretical scattering models showed that this mismatch would reduce light transmission in the cornea. We conclude that corneal transparency depends on the matching of refractive indices between quiescent keratocytes and the surrounding tissue, and that after surgery or wounding, the resulting RI mismatch between the activated cells and their surrounds significantly contributes to light scattering.
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Sustancia Propia/fisiología , Sustancia Propia/efectos de la radiación , Imagen Óptica/métodos , Dispersión de Radiación , Animales , Bovinos , Tamaño de la Célula , Células Cultivadas , Queratocitos de la Córnea/fisiología , Queratocitos de la Córnea/efectos de la radiación , Sustancia Propia/citología , Citoplasma/fisiología , Citoplasma/efectos de la radiación , Microscopía Confocal , Modelos Biológicos , Células del Estroma/fisiologíaRESUMEN
[This corrects the article on p. e68166 in vol. 8.].
RESUMEN
PURPOSE: To quantify long-term changes in stromal collagen ultrastructure following penetrating keratoplasty (PK), and evaluate their possible implications for corneal biomechanics. METHODS: A pair of 16 mm post-mortem corneo-scleral buttons was obtained from a patient receiving bilateral penetrating keratoplasty 12 (left)/28 (right) years previously. Small-angle x-ray scattering quantified collagen fibril spacing, diameter and spatial order at 0.5 mm or 0.25 mm intervals along linear scans across the graft margin. Corresponding control data was collected from two corneo-scleral buttons with no history of refractive surgery. Wide-angle x-ray scattering quantified collagen fibril orientation at 0.25 mm (horizontal)×0.25 mm (vertical) intervals across both PK specimens. Quantification of orientation changes in the graft margin were verified by equivalent analysis of data from a 13 year post-operative right PK specimen obtained from a second patient in a previous study, and comparison made with new and published data from normal corneas. RESULTS: Marked changes to normal fibril alignment, in favour of tangentially oriented collagen, were observed around the entire graft margin in all PK specimens. The total number of meridional fibrils in the wound margin was observed to decrease by up to 40%, with the number of tangentially oriented fibrils increasing by up to 46%. As a result, in some locations the number of fibrils aligned parallel to the wound outnumbered those spanning it by up to five times. Localised increases in fibril spacing and diameter, with an accompanying reduction in matrix order, were also evident. CONCLUSIONS: Abnormal collagen fibril size and spatial order within the PK graft margin are indicative of incomplete stromal wound remodelling and the long term persistence of fibrotic scar tissue. Lasting changes in collagen fibril orientation in and around PK wounds may alter corneal biomechanics and compromise the integrity of the graft-host interface in the long term.
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Colágeno/química , Córnea/química , Córnea/citología , Anciano , Córnea/cirugía , Sustancia Propia/química , Sustancia Propia/citología , Humanos , Queratoplastia Penetrante , Persona de Mediana Edad , Donantes de Tejidos , Difracción de Rayos XRESUMEN
PURPOSE: To examine the effect of riboflavin/UVA corneal crosslinking on stromal ultrastructure and hydrodynamic behaviour. METHODS: One hundred and seventeen enucleated ungulate eyes (112 pig and 5 sheep) and 3 pairs of rabbit eyes, with corneal epithelium removed, were divided into four treatment groups: Group 1 (28 pig, 2 sheep and 3 rabbits) were untreated; Group 2 (24 pig) were exposed to UVA light (3.04 mW/cm(2)) for 30 minutes and Group 3 (29 pig) and Group 4 (31 pig, 3 sheep and 3 rabbits) had riboflavin eye drops applied to the corneal surface every 5 minutes for 35 minutes. Five minutes after the initial riboflavin instillation, the corneas in Group 4 experienced a 30 minute exposure to UVA light (3.04 mW/cm(2)). X-ray scattering was used to obtain measurements of collagen interfibrillar spacing, spatial order, fibril diameter, D-periodicity and intermolecular spacing throughout the whole tissue thickness and as a function of tissue depth in the treated and untreated corneas. The effect of each treatment on the hydrodynamic behaviour of the cornea (its ability to swell in saline solution) and its resistance to enzymatic digestion were assessed using in vitro laboratory techniques. RESULTS: Corneal thickness decreased significantly following riboflavin application (p<0.01) and also to a lesser extent after UVA exposure (p<0.05). With the exception of the spatial order factor, which was higher in Group 4 than Group 1 (p<0.01), all other measured collagen parameters were unaltered by cross-linking, even within the most anterior 300 microns of the cornea. The cross-linking treatment had no effect on the hydrodynamic behaviour of the cornea but did cause a significant increase in its resistance to enzymatic digestion. CONCLUSIONS: It seems likely that cross-links formed during riboflavin/UVA therapy occur predominantly at the collagen fibril surface and in the protein network surrounding the collagen.
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Colágeno/química , Sustancia Propia/efectos de los fármacos , Sustancia Propia/efectos de la radiación , Hidrodinámica , Riboflavina/farmacología , Rayos Ultravioleta , Animales , Colágeno/metabolismo , Sustancia Propia/metabolismo , Sustancia Propia/ultraestructura , Enzimas/metabolismo , Técnicas In Vitro , Conejos , Ovinos , Porcinos , Difracción de Rayos XRESUMEN
PURPOSE: The mouse has become an important wound healing model with which to study corneal fibrosis, a frequent complication of refractive surgery. The aim of the current study was to quantify changes in stromal ultrastructure and light scatter that characterize fibrosis in mouse corneal debridement wounds. METHODS: Epithelial debridement wounds, with and without removal of basement membrane, were produced in C57BL/6 mice. Corneal opacity was measured using optical coherence tomography, and collagen diameter and matrix order were quantified by x-ray scattering. Electron microscopy was used to visualize proteoglycans. Quantitative PCR (Q-PCR) measured mRNA transcript levels for several quiescent and fibrotic markers. RESULTS: Epithelial debridement without basement membrane disruption produced a significant increase in matrix disorder at 8 weeks, but minimal corneal opacity. In contrast, basement membrane penetration led to increases in light scatter, matrix disorder, and collagen diameter, accompanied by the appearance of abnormally large proteoglycans in the subepithelial stroma. This group also demonstrated upregulation of several quiescent and fibrotic markers 2 to 4 weeks after wounding. CONCLUSIONS: Fibrotic corneal wound healing in mice involves extensive changes to collagen and proteoglycan ultrastructure, consistent with deposition of opaque scar tissue. Epithelial basement membrane penetration is a deciding factor determining the degree of ultrastructural changes and resulting opacity.
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Colágeno/ultraestructura , Córnea/cirugía , Desbridamiento/efectos adversos , Dispersión de Radiación , Animales , Membrana Basal/patología , Membrana Basal/cirugía , Membrana Basal/ultraestructura , Biomarcadores/análisis , Córnea/patología , Córnea/ultraestructura , Lesiones de la Cornea , Opacidad de la Córnea/patología , Sustancia Propia/fisiopatología , Sustancia Propia/ultraestructura , Epitelio Corneal/lesiones , Epitelio Corneal/metabolismo , Epitelio Corneal/patología , Epitelio Corneal/ultraestructura , Fibrosis , Luz , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , ARN/análisis , ARN/biosíntesis , ARN Mitocondrial , Tomografía de Coherencia Óptica , Cicatrización de Heridas/fisiologíaRESUMEN
PURPOSE: In the visually debilitating condition of climatic droplet keratopathy, corneal transparency is progressively lost. Although the precise cause of the disease and the mechanism by which it progresses are not known, a lifetime exposure to high solar radiation and a vitamin C-deficient diet may be involved in its development. This study examines the effect of dietary ascorbate levels and ultraviolet (UV)-B exposure on corneal stromal structure. METHODS: Eight guinea pigs were divided into four treatment groups (A, B, C, and D). For 15 weeks, Groups A and C were fed an ascorbate-rich diet (2 mg/100 g bodyweight/day), while Groups B and D received an ascorbate-deficient diet (0.07 mg/100 g bodyweight/day). For the last 12 weeks of the study, Groups C and D also experienced chronic UVB exposure (0.12 J/cm² for 40 min/day). Following euthanasia, the corneas were enucleated and their stromal ultrastructure examined using X-ray scattering and electron microscopy. RESULTS: UVB exposure resulted in an increased corneal thickness (p<0.001), but this was not accompanied by a widespread expansion of the collagen fibrillar array, and in the case of ascorbate-deficient animals, stromal thickening was associated with the compaction of collagen fibrils (p<0.01). Neither UVB exposure nor ascorbic acid deficiency caused any change in the average diameter or D-periodicity of the stromal collagen fibrils. CONCLUSIONS: UVB-induced changes in the corneal ultrastructure were most pronounced in animals fed an ascorbic acid-deficient diet. This suggests that ascorbic acid may play a vital role in protecting the corneal stroma from the harmful effects of UVB.
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Deficiencia de Ácido Ascórbico/patología , Córnea/efectos de la radiación , Córnea/ultraestructura , Rayos Ultravioleta , Animales , Peso Corporal , Córnea/patología , Sustancia Propia/patología , Sustancia Propia/efectos de la radiación , Sustancia Propia/ultraestructura , Cobayas , Masculino , Microscopía , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
PURPOSE: To determine the effect of Ultraviolet-A collagen cross-linking with hypo-osmolar and iso-osmolar riboflavin solutions on stromal collagen ultrastructure in normal and keratoconus ex vivo human corneas. METHODS: Using small-angle X-ray scattering, measurements of collagen D-periodicity, fibril diameter and interfibrillar spacing were made at 1 mm intervals across six normal post-mortem corneas (two above physiological hydration (swollen) and four below (unswollen)) and two post-transplant keratoconus corneal buttons (one swollen; one unswollen), before and after hypo-osmolar cross-linking. The same parameters were measured in three other unswollen normal corneas before and after iso-osmolar cross-linking and in three pairs of swollen normal corneas, in which only the left was cross-linked (with iso-osmolar riboflavin). RESULTS: Hypo-osmolar cross-linking resulted in an increase in corneal hydration in all corneas. In the keratoconus corneas and unswollen normal corneas, this was accompanied by an increase in collagen interfibrillar spacing (p<0.001); an increase in fibril diameter was also seen in two out of four unswollen normal corneas and one unswollen keratoconus cornea (p<0.001). Iso-osmolar cross-linking resulted in a decrease in tissue hydration in the swollen normal corneas only. Although there was no consistent treatment-induced change in hydration in the unswollen normal samples, iso-osmolar cross-linking of these corneas did result in a compaction of collagen fibrils and a reduced fibril diameter (p<0.001); these changes were not seen in the swollen normal corneas. Collagen D-periodicity was not affected by either treatment. CONCLUSION: The observed structural changes following Ultraviolet-A cross-linking with hypo-osmolar or iso-osmolar riboflavin solutions are more likely a consequence of treatment-induced changes in tissue hydration rather than cross-linking.
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Colágeno/metabolismo , Sustancia Propia/efectos de los fármacos , Sustancia Propia/efectos de la radiación , Reactivos de Enlaces Cruzados/farmacología , Queratocono/metabolismo , Riboflavina/farmacología , Rayos Ultravioleta , Anciano , Anciano de 80 o más Años , Sustancia Propia/metabolismo , Humanos , Técnicas In Vitro , Persona de Mediana EdadRESUMEN
The collagen microstructure of the peripheral cornea is important in stabilizing corneal curvature and refractive status. However, the manner in which the predominantly orthogonal collagen fibrils of the central cornea integrate with the circumferential limbal collagen is unknown. We used microfocus wide-angle x-ray scattering to quantify the relative proportion and orientation of collagen fibrils over the human corneolimbal interface at intervals of 50 µm. Orthogonal fibrils changed direction 1-1.5 mm before the limbus to integrate with the circumferential limbal fibrils. Outside the central 6 mm, additional preferentially aligned collagen was found to reinforce the cornea and limbus. The manner of integration and degree of reinforcement varied significantly depending on the direction along which the limbus was approached. We also employed small-angle x-ray scattering to measure the average collagen fibril diameter from central cornea to limbus at 0.5 mm intervals. Fibril diameter was constant across the central 6 mm. More peripherally, fibril diameter increased, indicative of a merging of corneal and scleral collagen. The point of increase varied with direction, consistent with a scheme in which the oblique corneal periphery is reinforced by chords of scleral collagen. The results have implications for the cornea's biomechanical response to ocular surgeries involving peripheral incision.
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Colágeno/química , Córnea/metabolismo , Anciano , Anciano de 80 o más Años , Colágenos Fibrilares/química , Humanos , Limbo de la Córnea/metabolismo , Persona de Mediana Edad , Modelos Biológicos , Difracción de Rayos XRESUMEN
PURPOSE: Retinopathy, globe enlarged (RGE) is an inherited genetic disease of chickens with a corneal phenotype characterized by loss of tissue curvature and changes in peripheral collagen fibril alignment. This study aimed to characterize the material behavior of normal and RGE chicken corneas under inflation and compare this with new spatial- and depth-resolved microstructural information to investigate how stromal fibril architecture determines corneal behavior under intraocular pressure (IOP). METHODS: Six RGE chicken corneas and six age-matched normal controls were tested using trephinate inflation and their stress-strain behavior determined as a function of posterior pressure. Second harmonic generation mulitphoton microscopy was used to compare the in-plane appearance and degree of through-plane interlacing of collagen lamellae between normal and mutant corneas. RESULTS: RGE corneas displayed a 30-130% increase in material stiffness [E(tangent)(RGE) = 0.94 ± 0.18 MPa to 3.09 ± 0.66 MPa; E(tangent)(normals) = 0.72 ± 0.13 MPa to 1.34 ± 0.35 MPa] (P ≤ 0.05). The normal in-plane disposition of anterior collagen in the peripheral cornea was altered in RGE but through-plane lamellar interlacing was unaffected. CONCLUSIONS: This article demonstrates changes in corneal material behavior in RGE that are qualitatively consistent with microstructural collagen alterations identified both herein and previously. This study indicates that, in general, changes in stromal fibril orientation may significantly affect corneal material behavior and thereby its response to IOP.