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Cell Death during Corneal Storage at 4°C

From the Departments of ¹ Ophthalmology, ² Biostatistics, and ³ Gastroenterology and Hepatology, Mayo Clinic, and Mayo Foundation, Rochester, Minnesota.

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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