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CJ Connon, V Siegler, KM Meek, SA Hodson, AJ Quantock; Changes in Proteoglycan Morphology in Embryonic Chick Corneas During Development . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3213.
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Purpose:In the latter stages of development the embryonic chick cornea undergoes significant changes in structure, composition and transparency. In this tissue, as in others, proteoglycan (PG) alterations, collagen matrix reorganisation and corneal transparency are thought to be related. The current study was designed to investigate corneal ultrastructure (collagen and PGs) as a function of development. Methods:Corneas taken from chick embryos at developmental days 13 through 18 were stained for PGs with 0.05% cuprolinic blue at a critical electrolyte concentration of 0.1M MgCl2, and processed for transmission electron microscopy. Quantitative image analysis of PGs, in no less than 10 regions of the mid-stroma at all time-points, was carried out using a digital camera and image analysis software. Sections were unstained for this analysis, but later some sections were stained with 2% uranyl acetate and 2% lead citrate to visualise collagen fibrils. Results:At developmental day 13, corneas contained poorly defined lamellae and numerous small and large PGs, randomly distributed. The identity (i.e. keratan sulphate or chondroitin/dermatan sulphate) and degree of sulphation of these molecules is unknown at present. At day 14 collagen fibrils showed some axial alignment. Interestingly, at this time we occasionally noted a periodic arrangement of small PGs along the axes of the collagen fibrils. By day 16 lamellae were well formed and continued to increase in number and size thereafter. In addition, large PG filaments were less prevalent from day 16 onwards. Between days 16 and 17 there was a significant increase in the number of small PGs identified in the tissue, and this increase persisted into day 18. Conclusion:The size, number and distribution of sulphated PGs in the developing avian cornea made visible by the cationic dye cuprolinic blue changes over time. It is perhaps of interest that the increase in number of small, sulphated PGs between days 16 and 17 of development is concurrent with a significant drop in average collagen fibril spacing.
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