title: A comparison of glycosaminoglycan distributions, keratan sulphate sulphation patterns and collagen fibril architecture from central to peripheral regions of the bovine cornea.
abstract: 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.
authors.affiliation: Structural Biophysics Group, School of Optometry and Vision Sciences, Cardiff Centre for Vision Sciences, Cardiff University, Wales, United Kingdom.
authors.forename: Leona T Y
authors.initials: LT
authors.lastname: Ho
authors.affiliation: Connective Tissue Biology Laboratories, School of Biosciences, Cardiff University, Wales, United Kingdom.
authors.forename: Anthony M
authors.initials: AM
authors.lastname: Harris
authors.affiliation: Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kawaramachi dori, Kamigyo-Ku, Kyoto, Japan.
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Tanioka
authors.affiliation: Japan Synchrotron Radiation Research Institute, Spring-8, Sayo, 1-1-1 Kouto, Hyogo, Japan.
authors.forename: Naoto
authors.initials: N
authors.lastname: Yagi
authors.affiliation: Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kawaramachi dori, Kamigyo-Ku, Kyoto, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Kinoshita
authors.affiliation: Connective Tissue Biology Laboratories, School of Biosciences, Cardiff University, Wales, United Kingdom.
authors.forename: Bruce
authors.initials: B
authors.lastname: Caterson
authors.affiliation: Structural Biophysics Group, School of Optometry and Vision Sciences, Cardiff Centre for Vision Sciences, Cardiff University, Wales, United Kingdom.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Quantock
authors.affiliation: Structural Biophysics Group, School of Optometry and Vision Sciences, Cardiff Centre for Vision Sciences, Cardiff University, Wales, United Kingdom.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Young
authors.affiliation: Structural Biophysics Group, School of Optometry and Vision Sciences, Cardiff Centre for Vision Sciences, Cardiff University, Wales, United Kingdom. Electronic address: MeekKM@cf.ac.uk.
authors.forename: Keith M
authors.initials: KM
authors.lastname: Meek
mesh_terms: D000818:Animals; D002417:Cattle; D003315:Cornea / Q000033:anatomy & histology* / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D024022:Fibrillar Collagens / Q000648:ultrastructure*; D006025:Glycosaminoglycans / Q000378:metabolism*; D007150:Immunohistochemistry; D007632:Keratan Sulfate / Q000378:metabolism*; D008751:Methylene Blue / Q000031:analogs & derivatives; D046529:Microscopy, Electron, Transmission; D014961:X-Ray Diffraction
chemical_list: D024022:Fibrillar Collagens; D006025:Glycosaminoglycans; C435946:dimethylmethylene blue; D007632:Keratan Sulfate; D008751:Methylene Blue
keywords: Collagen structure; Cornea; Glycosaminoglycans; Proteoglycans