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J. H. Mathew, J. D. Goosey, A. R. Burns, J. P. G. Bergmanson; Immunohistochemistry and Ultrastructure of Anterior Stromal Cells in Keratoconus. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6230.
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The presence of increased number of stromal cells in keratoconus (Kc) was investigated immunohistochemically and ultrastructurally.
Twelve surgically removed Kc corneal buttons (8 LKP, 4 LKP) and six control human cadaver corneas were immediately preserved and processed for transmission electron microscopy using an established corneal protocol, ensuring minimal tissue shrinkage and distortion. Two further corneas (1 Kc, 1 normal) were fixed in 2% paraformaldehyde and prepared for viewing on the DeltaVision Deconvolution Core Microscope. The tissue was exposed to antibodies directed against CD34, CD45, and CD68.
Keratocytes were recognized, by TEM, on their electron dense cytoplasm, long and thin cytoplasmic processes and nuclear heterochromatin pattern. Cells not fitting these characteristics, and not associated with scar formation, were seen at the interface of the ALL and stroma. These cells, lacking granules but containing vesicles, appeared to be involved in the removal of ALL and anterior stromal lamellae. They lined up closely to resident keratocytes leaving a separation of less than 100nm between the two opposing plasmalemmas over long stretches. Preliminary immunofluorescent study to identify these cells using anti-CD34 indicated that keratocytes, a normal resident of the stroma, were present in both central and less affected peripheral portions of the Kc cornea. However, cell shape was altered in the central cone, where it had adopted a more slender outline from which more processes extended. Labeling for CD68, a macrophage marker, was absent in the Kc cornea. In addition, white blood cells (CD45 labeled) were abundantly present in the cone region of the Kc epithelium, but not in the stroma.
TEM revealed that the Kc cornea contained, in the anterior stroma, cells other than keratocytes, which were assumed to be involved in the removal of ALL and anterior lamellae. These cells were closely aligned to keratocytes, which suggested that some intercellular interaction may be taking place between these two cells. This cellular activity may play a key role in lamellar fragmentation and corneal ectasia associated with keratoconus.
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