Cold-induced injury to porcine corneal endothelial cells and its mediation by chelatable iron: implications for corneal preservation.
OBJECTIVE
During hypothermic storage of the cornea, corneal endothelial damage restricts storage times. We previously reported a new, iron-dependent mechanism of cold-induced injury to cultured liver cells. In this study, we sought to evaluate whether corneal endothelial cells incur a similar kind of injury.


METHODS
Cultured porcine corneal endothelial cells were exposed to 4 degrees C in either cell culture medium, Krebs-Henseleit buffer, Optisol-GS solution, or McCarey-Kaufman medium for 5 hours to 14 days and then rewarmed under cell culture conditions (3 hours). The cultures were assessed for lethal cell injury (LDH release); cellular, nuclear, and mitochondrial morphologic alterations; lipid peroxidation; and mitochondrial membrane potential.


RESULTS
Corneal endothelial cells sustained substantial injury following cold storage and rewarming in cell culture medium (47% +/- 8% and 64% +/- 20% cell death after 2 and 5 days cold storage, respectively). The injury displayed some apoptotic features, and cells lost mitochondrial membrane potential before cell death occurred. The iron chelators deferoxamine, 1,10-phenanthroline, and 2,2'-dipyridyl and the antioxidant butylated hydroxytoluene completely inhibited this cell injury. Marked iron-dependent cell injury and lipid peroxidation also occurred during and after cold incubation in Krebs-Henseleit buffer and, most importantly, iron-dependent cell injury was also observed after cold incubation in Optisol solution and in McCarey-Kaufman medium.


CONCLUSIONS
Cultured porcine corneal endothelial cells incur a strong iron-dependent injury elicited by hypothermia. This cold-induced injury might provide an explanation for the known corneal endothelial susceptibility to hypothermic preservation injury, which thus might be amenable to therapeutic interventions (ie, by iron chelators).
Rauen
Ursula
U
Institut für Physiologische Chemie, Universitätsklinikum, Essen, Germany. ursula.rauen@uni-essen.de
Kerkweg
Uta
U
Wusteman
Monica C
MC
de Groot
Herbert
H
D015082:2,2'-Dipyridyl / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002084:Butylated Hydroxytoluene / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003080:Cold Temperature / Q000009:adverse effects*; D015925:Cryopreservation; D003470:Culture Media; D003676:Deferoxamine / Q000494:pharmacology; D004728:Endothelium, Corneal / Q000187:drug effects* / Q000293:injuries* / Q000378:metabolism / Q000473:pathology; D007502:Iron Chelating Agents / Q000494:pharmacology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D008954:Models, Biological; D009926:Organ Preservation; D019188:Organ Preservation Solutions; D010618:Phenanthrolines / Q000494:pharmacology; D017694:Rewarming; D013552:Swine; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism
D000975:Antioxidants; D003470:Culture Media; D007502:Iron Chelating Agents; D019188:Organ Preservation Solutions; D010618:Phenanthrolines; D017392:Thiobarbituric Acid Reactive Substances; D002084:Butylated Hydroxytoluene; D015082:2,2'-Dipyridyl; D007770:L-Lactate Dehydrogenase; D003676:Deferoxamine; C025205:1,10-phenanthroline