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Robert D Young, Carlo Knupp, Elena Koudouna, Philip N Lewis, Keith M Meek, Andrew J Quantock; Studies of collagen organisation and early stages of cell infiltration in the primary stroma of developing chick cornea. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1909. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Primary stroma, the earliest rudiment of developing corneal matrix in the chick embryo, has been implicated in organisation of cells and lamellar structure in the mature tissue, and yet it is absent in the immature corneas of many species, including man. We examined early stages in chick corneal development to characterise the 3-dimensional structure of the primary stroma to determine if inwardly migrating neural crest cells exhibit alignment in relation to its components.
Eyes from embryonic chicks at 3 to 7 days of development were fixed in buffered aldehyde solution. Dissected corneas, or whole eyes, were embedded in epoxy resin after immersion in osmium tetroxide with potassium ferricyanide, tannic acid, osmium tetroxide and uranyl acetate, each for 1 h, then dehydration in ethanol. Toluidine blue-stained tissue sections were cut for orientation and the polished block surface imaged with a backscatter electron detector in a Zeiss Sigma VP FEG scanning electron microscope. Sequences of up to 1000 serial images were taken, each alternating with removal of a 100nm surface slice by an in-chamber Gatan 3View2 microtome. 3-dimensional reconstructions of tissue architecture were made by manual or automated segmentation using Amira 5.6 or ImageJ software.
The primary stroma forms a layer of loosely distributed collagen fibrils (mean diameter at E4, 25.5nm, S.D. 3.5, n=750), increasing in thickness from 15 to 100µm between E3 and E7. Fibril distribution was not homogeneous, but showed a central condensation, approximately 5µm thick, which undulated regularly with respect to the epithelial basal lamina between more-sparsely dispersed fibrils distally and proximally. Immediately below the basal lamina, fibrils showed orthogonal arrangement, but some zones almost free of fibrils, and others with fibrils orientated at large angles to the basal lamina were also present. Preliminary observations seem to indicate that cells migrating into the primary stroma at E5-E6 exhibit no preferential alignment with respect to the collagen fibrils.
Absence of any clear alignment between presumptive keratocytes entering the primary stroma and collagen fibrils of this rudimentary tissue would challenge the long-held view that it serves a role in guidance and organisation of cells and thus lamellae of the mature stroma.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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