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To determine whether hypoxia preconditioning can protect corneal endothelial cells from mechanical stress and perioperative procedures mimicking Descemet stripping automated endothelial keratoplasty (DSAEK).Preconditioning was delivered by 2 hours of 0.5% oxygen incubation in a hypoxia chamber or by exposure to the prolyl hydroxylase inhibitor FG-4592, which prevents hypoxia-inducible factor-1 alpha degradation. Damage to whole corneas was produced by brief sonication. To mimic use with DSAEK, FG-4592-preconditioned and control donor corneas were dissected with a microkeratome, and the posterior donor button was pulled through a transplant insertion device (Busin glide). The area of endothelial damage was determined by trypan blue staining.In all cases, hypoxia preconditioning or incubation with FG-4592 protected corneal endothelial cells from death by mechanical stress. Hypoxia-preconditioned human and rabbit corneas showed 19% and 29% less cell loss, respectively, relative to controls, which were both significant at P < 0.05. FG-4592 preconditioning reduced endothelial cell loss associated with preparation and insertion of DSAEK grafts by 23% relative to the control (P < 0.01).These results support the hypothesis that preconditioning by hypoxia or exposure to FG-4592 improves corneal endothelial cell survival and may also provide protection during surgical trauma.