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Aoi Komuro, David O. Hodge, Gregory J. Gores, William M. Bourne; Cell Death during Corneal Storage at 4°C. Invest. Ophthalmol. Vis. Sci. 1999;40(12):2827-2832.
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purpose. To evaluate cell death in human donor corneas stored at 4°C, to
determine whether terminal deoxynucleotidyl transferase–mediated
dUTP-fluorescein nick-end labeling (TUNEL) discriminates between
apoptosis and necrosis in corneas stored at 4°C.
methods. Ten human corneas were stored in Optisol (Chiron Ophthalmics, Irvine,
CA) at 4°C for periods ranging from 0 to 21 days and then fixed for
histologic examination. Central corneal sections from each cornea were
examined by transmission electron microscopy (TEM) and by the TUNEL
assay. Electron micrographs of at least 15 keratocytes each from the
anterior, middle, and posterior stroma were examined by three masked
observers who graded each cell as normal, apoptotic, or necrotic.
Central sections from the same corneas were processed by the TUNEL
assay and evaluated with a laser scanning confocal microscope to
determine the percentage of apoptotic cells.
results. By TEM, apoptosis occurred in 23% of the keratocytes and necrosis in
12%. By TUNEL assay, apoptosis occurred in 11% of the keratocytes,
with the results in individual corneas being similar to the findings by
TEM for apoptosis, rather than for necrosis. By TUNEL assay, apoptosis
occurred in 13% of the epithelial cells and in 8% of the endothelial
cells. The percentage of apoptotic cells and storage time correlated
significantly for the epithelium, but not for the keratocytes or
endothelium in this small sample.
conclusions. Both apoptosis and necrosis occur in cells during corneal storage at
4°C, with apoptosis appearing to predominate. The TUNEL assay
identifies cells undergoing apoptosis, but not necrosis, in corneal
tissue. Inhibition of apoptosis in corneas stored at 4°C may prolong
acceptable storage times.
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