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Andrew J. Quantock, Philip N. Lewis, Robert D. Young; Preservation Of Native Structure Of Cells And Developing Collagen Lamellae In Hydrated Embryonic Cornea By High Pressure Cryofixation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1090.
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© ARVO (1962-2015); The Authors (2016-present)
The mechanisms by which keratocytes in the embryonic cornea deposit regularly spaced collagen fibrils into an ordered array of superimposed lamellae are not fully understood. Here we investigate native tissue ultrastructure of the developing chick cornea to understand how cells in the highly-hydrated embryonic connective tissue matrix orientate collagen fibrils for cornea-specific function.
Corneas from developing chicks at embryonic day 14 - when loose collagen bundles begin condensing into lamellae - were trimmed to 1 mm2 x full thickness (approximately 200µm) and rapidly cryofixed in a Leica EMPACT2 high pressure freezer. Frozen specimens were freeze substituted at -80°C in 2% Osmium/Acetone for 24hrs, and embedded in Araldite resin at 20°C. 100nm sections cut on a Leica UC6 ultramicrotome were examined in a Jeol 1010 TEM, and images recorded with a Gatan Orius SC1000 CCD camera.
Ultrastructural preservation was relatively good in cells and extracellular matrix, however, freezing quality varied markedly with location within single specimens. Peripheral and outer sites showed better structural integrity, with gross distortion of structure at the cornea’s centre. In regions of better freezing, keratocyte organelles, cell membranes and surrounding collagenous matrix all showed acceptable structural preservation. This enabled identification of putative cell membrane channels, first recognised in developing tendon, and termed fibripositors, considered crucial for extracellular deposition of orientated collagen fibrils.
We have achieved satisfactory cell and matrix preservation by high pressure freezing of embryonic cornea, although full thickness freezing through out the whole volume of the cornea was not attained. Intracellular structures, potentially crucial for orientated collagen secretion, were identified for the first time in cornea.
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