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Cell death during corneal storage at 4 degrees C
A Komuro1, D O Hodge, G J Gores
1Department of Ophthalmology, Mayo Clinic, and Mayo Foundation, Rochester, Minnesota 55905, USA.
Purpose:
To evaluate cell death in human donor corneas stored at 4 degrees C, to determine whether terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labeling (TUNEL) discriminates between apoptosis and necrosis in corneas stored at 4 degrees C.
Methods:
Ten human corneas were stored in Optisol (Chiron Ophthalmics, Irvine, CA) at 4 degrees 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 degrees 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 degrees C may prolong acceptable storage times.
Insights
Cell death, including apoptosis and necrosis, occurs in human corneas during cold storage. The TUNEL assay can identify apoptosis but not necrosis, potentially aiding in extending corneal storage viability.
Area of Science:
- Ophthalmology
- Cell Biology
- Tissue Engineering
Background:
- Human donor corneas are stored at 4 degrees C for transplantation.
- Understanding cell death mechanisms during storage is crucial for optimizing graft survival.
Purpose of the Study:
- To evaluate cell death in human donor corneas stored at 4 degrees C.
- To determine if the terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labeling (TUNEL) assay can differentiate between apoptosis and necrosis in stored corneas.
Main Methods:
- Human corneas were stored at 4 degrees C for up to 21 days.
- Corneal sections were analyzed using transmission electron microscopy (TEM) and the TUNEL assay.
- Keratocytes, epithelial, and endothelial cells were graded as normal, apoptotic, or necrotic.
Main Results:
- TEM revealed apoptosis in 23% and necrosis in 12% of keratocytes.
- The TUNEL assay identified apoptosis in 11% of keratocytes, correlating with TEM findings for apoptosis.
- Apoptosis was detected in 13% of epithelial cells and 8% of endothelial cells by TUNEL assay.
Conclusions:
- Both apoptosis and necrosis occur in corneal cells during cold storage, with apoptosis being more prevalent.
- The TUNEL assay is effective in identifying apoptosis but not necrosis in corneal tissue.
- Inhibiting apoptosis may extend the viable storage time of donor corneas.