Confocal imaging of the keratocyte network in porcine cornea using the fixable vital dye 5-chloromethylfluorescein diacetate

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Abstract
This study reports on the combined use of an aldehyde fixable, cell viability fluoroprobe, 5-chloromethylfluorescein diacetate (CMFDA), confocal laser scanning microscopy and digital image reconstruction, to produce high resolution images of corneal keratocyte preparations in situ. The central region of freshly enucleated porcine corneae were removed and stained overnight at 4 degrees C with CMFDA. The tissue was washed, fixed, and frozen for cryosectioning in either a horizontal or antero-posterior orientation. Sections from anterior, central and posterior stroma were examined with a confocal microscope, and the digital images rendered as three-dimensional stereo reconstructions. Fluorescent CMFDA which completely permeated the cell bodies and extremes of the finest ramifying cell processes of all keratocytes provided exceptional high resolution images of the three morphologically distinct cell subpopulations at different levels of the stroma, and enabled improved characterisation of each cell type. Anteriorly was a thin, dense, non-lamellar network of keratocytes subjacent Bowman's membrane. In the central stroma, keratocytes were arranged in layers, the cell bodies had a flattened pyramidal or stellate shape, and the fine cell processes formed extensive distal ramifications. Immediately anterior to Descemet's membrane a small subpopulation of keratocytes with large cell bodies and short branched processes was identified. Extensive and diverse cell-to-cell contacts were orientated in all stromal planes, including ramping cell bridges between keratocyte lamellae in the central stroma. The use of the cell viability dye CMFDA is feasible and valuable for enhancing the visibility of entire keratocyte population in the intact cornea. Diverse multi-directional cell processes and intercellular contacts throughout the keratocyte network suggest a strong capacity for direct communication and cohesion in the maintenance and repair of the stromal matrix. Keratocytes closely related to the epithelium and endothelium have unique morphologies which may relate to specialised functions of these interface cells.