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      A comparison of glycosaminoglycan distributions, keratan sulphate sulphation patterns and collagen fibril architecture from central to peripheral regions of the bovine cornea


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          This study investigated changes in collagen fibril architecture and the sulphation status of keratan sulphate (KS) glycosaminoglycan (GAG) epitopes from central to peripheral corneal regions. Freshly excised adult bovine corneal tissue was examined as a function of radial position from the centre of the cornea outwards. Corneal thickness, tissue hydration, hydroxyproline content, and the total amount of sulphated GAG were all measured. High and low-sulphated epitopes of keratan sulphate were studied by immunohistochemistry and quantified by ELISA. Chondroitin sulphate (CS) and dermatan sulphate (DS) distributions were observed by immunohistochemistry following specific enzyme digestions. Electron microscopy and X-ray fibre diffraction were used to ascertain collagen fibril architecture. The bovine cornea was 1021 ± 5.42 μm thick at its outer periphery, defined as 9–12 mm from the corneal centre, compared to 844 ± 8.10 μm at the centre. The outer periphery of the cornea was marginally, but not significantly, more hydrated than the centre (H = 4.3 vs. H = 3.7), and was more abundant in hydroxyproline (0.12 vs. 0.06 mg/mg dry weight of cornea). DMMB assays indicated no change in the total amount of sulphated GAG across the cornea. Immunohistochemistry revealed the presence of both high- and low-sulphated epitopes of KS, as well as DS, throughout the cornea, and CS only in the peripheral cornea before the limbus. Quantification by ELISA, disclosed that although both high- and low-sulphated KS remained constant throughout stromal depth at different radial positions, high-sulphated epitopes remained constant from the corneal centre to outer-periphery, whereas low-sulphated epitopes increased significantly. Both small angle X-ray diffraction and TEM analysis revealed that collagen fibril diameter remained relatively constant until the outer periphery was reached, after which fibrils became more widely spaced (from small angle x-ray diffraction analysis) and of larger diameter as they approached the sclera. Depth-profiled synchrotron microbeam analyses showed that, at different radial positions from the corneal centre outwards, fibril diameter was greater superficially than in deeper stromal regions. The interfibrillar spacing was also higher at mid-depth in the stroma than it was in anterior and posterior stromal regions. Collagen fibrils in the bovine cornea exhibited a fairly consistent spacing and diameter from the corneal centre to the 12 mm radial position, after which a significant increase was seen. While the constancy of the overall sulphation levels of proteoglycans in the cornea may correlate with the fibrillar architecture, there was no correlation between the latter and the distribution of low-sulphated KS.


          • Proteoglycans (KS, DS, CS) and collagen were correlated with corneal radial position.

          • Total sulfate levels on glycosaminoglycans remained constant across the cornea.

          • KS and DS were ubiquitous; CS was found towards the edge of the cornea onwards.

          • High-sulfated KS remained constant; low-sulfated KS increased peripherally.

          • There was no correlation between fibrillar architecture and sulfation levels of KS.

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          Most cited references57

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          Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue.

          The dimethylmethylene blue assay for sulphated glycosaminoglycans has found wide acceptance as a quick and simple method of measuring the sulphated glycosaminoglycan content of tissues and fluids. The available assay methods have lacked specificity for sulphated glycosaminoglycans in the presence of other polyanions, however, and have not discriminated between the different sulphated glycosaminoglycans. We now describe a modified form of the dimethylmethylene blue assay that has improved specificity for sulphated glycosaminoglycans, and we show that in conjunction with specific polysaccharidases, the dimethylmethylene blue assay can be used to quantitate individual sulphated glycosaminoglycans.
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            Lumican Regulates Collagen Fibril Assembly: Skin Fragility and Corneal Opacity in the Absence of Lumican

            Lumican, a prototypic leucine-rich proteoglycan with keratan sulfate side chains, is a major component of the cornea, dermal, and muscle connective tissues. Mice homozygous for a null mutation in lumican display skin laxity and fragility resembling certain types of Ehlers-Danlos syndrome. In addition, the mutant mice develop bilateral corneal opacification. The underlying connective tissue defect in the homozygous mutants is deregulated growth of collagen fibrils with a significant proportion of abnormally thick collagen fibrils in the skin and cornea as indicated by transmission electron microscopy. A highly organized and regularly spaced collagen fibril matrix typical of the normal cornea is also missing in these mutant mice. This study establishes a crucial role for lumican in the regulation of collagen assembly into fibrils in various connective tissues. Most importantly, these results provide a definitive link between a necessity for lumican in the development of a highly organized collagenous matrix and corneal transparency.
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              Theory of transparency of the eye.

              The present work relates the turbidity of the eye to microscopic spatial fluctuations in its index of refraction. Such fluctuations are indicated in electron microscope photographs. By examining the superposition of phases of waves scattered from each point in the medium, we provide a mathematical demonstration of the Bragg reflection principle which we have recently used in the interpretation of experimental investigations: namely, that the scattering of light is produced only by those fluctuations whose fourier components have a wavelength equal to or larger than one half the wavelength of light in the medium. This consideration is applied first to the scattering of light from collagen fibers in the normal cornea. We demonstrate physically and quantitatively that a limited correlation in the position of near neighbor collagen fibers leads to corneal transparency. Next, the theory is extended to predict the turbidity of swollen, pathologic corneas, wherein the normal distribution of collagen fibers is disturbed by the presence of numerous lakes-regions where collagen is absent. A quantitative expression for the turbidity of the swollen cornea is given in terms of the size and density of such lakes. Finally, the theory is applied to the case of the cataractous lens. We assume that the cataracts are produced by aggregation of the normal lens proteins into an albuminoid fraction and provide a formula for the lens turbidity in terms of the molecular weight and index of refraction of the individual albuminoid macromolecules. We provide a crude estimate of the mean albuminoid molecular weight required for lens opacity.

                Author and article information

                Matrix Biol
                Matrix Biol
                Matrix Biology
                1 September 2014
                September 2014
                : 38
                : 59-68
                [a ]Structural Biophysics Group, School of Optometry and Vision Sciences, Cardiff Centre for Vision Sciences, Cardiff University, Wales, United Kingdom
                [b ]Connective Tissue Biology Laboratories, School of Biosciences, Cardiff University, Wales, United Kingdom
                [c ]Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kawaramachi dori, Kamigyo-Ku, Kyoto, Japan
                [d ]Japan Synchrotron Radiation Research Institute, Spring-8, Sayo, 1-1-1 Kouto, Hyogo, Japan
                Author notes
                [* ]Corresponding author at: Structural Biophysics Research Group, School of Optometry & Vision Sciences, Cardiff Centre for Vision Sciences, College of Biomedical and Life Sciences, Cardiff University, Maindy Road, Cardiff CF24 4HQ, United Kingdom. Tel.: + 44 29 2087 6317; fax: + 44 29 2087 4859. MeekKM@ 123456cf.ac.uk

                Equal contribution.

                © 2014 Published by Elsevier B.V.
                : 21 August 2013
                : 20 June 2014
                : 28 June 2014

                Molecular biology
                cornea,proteoglycans,glycosaminoglycans,collagen structure
                Molecular biology
                cornea, proteoglycans, glycosaminoglycans, collagen structure


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