Role of Oxidative Stress in the Disruption of the Endothelial Apical Junctional Complex During Corneal Cold Storage

To characterize the impact of corneal cold storage (CS) on the endothelial apical junctional complex (AJC). Porcine corneas were held in CS (4°C; 1-7 days) with Cornisol™ preservation medium supplemented with epothilone B (EpoB; microtubule stabilizer; 100 nM), SB-203580 (p38 mitogen-activated protein [MAP] kinase inhibitor; 20 μM), or antioxidants (quercetin, 100 μM; vitamin E, 1 mM; deferoxamine, an iron chelator, 10 mM). After CS termination, the damage to endothelial AJC was characterized by imaging perijunctional actomyosin ring (PAMR) and zonula occludens (ZO-1). The effects of EpoB and SB-203580 were characterized by imaging microtubules. The loss in the barrier function was assessed in cultured cells grown on biotin-coated gelatin by permeability to fluorescein isothiocyanate (FITC)-avidin. The accumulation of reactive oxygen species (ROS), altered mitochondrial membrane potential (MMP), lipid peroxidation, and lactate dehydrogenase (LDH) release were also determined in response to CS. CS led to the loss of microtubules, destruction of PAMR, and breakdown of ZO-1 in the endothelium. The severity of damage increased when CS was prolonged. Although rewarming of the tissue increased the damage, the effect was marginal. CS also induced accumulation of ROS, alteration in MMP, lipid peroxidation, enhanced LDH release, and increased permeability to FITC-avidin. These changes were opposed by EpoB, SB-203580, and antioxidants. Corneal CS destroys AJC of the endothelium, leading to loss of its barrier function. The effects were surmounted by microtubule stabilization, p38 MAP kinase inhibition, and antioxidants. Thus, there is potential for reformulation of the preservation medium to maintain the health of the donor corneal endothelium before transplantation.