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A. J. Quantock, B. P. Palka, R. D. Young, K. M. Meek, H. Tanioka, S. Kinoshita, N. Yagi; Synchrotron Microbeam Analysis of the Developing Chick Corneal Stroma and Depth-Profiled Changes in Collagen Fibril Arrangement. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3927. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
As it is becoming transparent in the week before hatching the developing chick cornea undergoes a structural reorganisation of its collagen fibrillar array. Previous synchrotron X-ray diffraction studies of chick embryogenesis have characterised the corneal matrix compaction over this developmental period, with the collagen interfibrillar spacing measured as an average throughout the whole thickness of the stroma (Siegler and Quantock, Exp Eye Res 2002;74:427-431). The current study was conducted to investigate changes in collagen interfibillar spacing at different intrastromal depths as chick cornea develops.
Chick corneas at embryonic days E12 to E18 and post-hatch were collected and fixed in 4% paraformaldehyde to help preserve matrix structure. After 24 hrs they were transferred to transport buffer containing 0.5% paraformaldehyde before data collection on the high flux beamline, 40XU, at the SPring-8 synchrotron facility. From all specimens, small-angle X-ray fibre diffraction patterns were obtained with sub-second exposure times using a 25µm X-ray beam. From this data the collagen interfibrillar spacing was calculated. One cornea of a pair was examined front-on with the beam moved across the whole cornea in 50µm steps. A thin strip was dissected through the centre of the other cornea, approximately 400µm thick, and this was orientated edgeways so that the side of the cornea was exposed, and X-ray patterns were obtained in 25µm steps across the whole depth of the cornea as described previously (Quantock et al., J Appl Crystal 2007;40:335-340).
As has been found in the corneas of humans and mice, the collagen interfibrillar spacing was higher at the edge of the chick cornea than at the centre before and after hatch. Moreover, in the very centre of pre-E15 corneas, a region of even more closely packed collagen was seen. In line with our previous synchrotron studies, collagen fibril spacing reduced between E12 and E18, and by E18 the spacing was constant across the stromal depth. Collagen spacing from E15 to E17, however, tended to be lower in posterior stromal regions.
Depth-profiled interfibrillar collagen spacing measurements obtained using synchrotron microbeam technology indicated that matrix compaction from E15 onwards proceeds initially in the deepest stromal layers, spreading forward with time.
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