Abstract
Purpose :
Cold storage of donor corneas is inevitable before endothelial transplantation or preparation of cultured cells for “cell injection therapy”. Here we have examined the effects of hypothermia on endothelial tight junctions (TJs) and its principal regulator, the perijunctional actomyosin ring (PAMR).
Methods :
Ex vivo porcine corneas were exposed to hypothermia (HYPO: 4 °C; 1 to 7 days) in Cornisol™ medium with and without pretreatment with a microtubule-stabilizing agent (epothilone B, EpoB; 100 nM), p38 MAP kinase inhibitor (SB-203580, SB; 20 µM), antioxidants (quercetin; 100 µM, or Vitamin E; 1 mM), or iron chelator (deferoxamine; 10 mM) and examined subsequently with and without rewarming (37 °C for 3 h). The damage to microtubules, PAMR, and ZO-1 (a marker of TJs) in the endothelium was assessed by immunocytochemistry. In addition, we imaged paracellular flux of FITC-avidin across endothelial cells grown on biotinylated gelatin to evaluate the functional integrity of TJs.
Results :
Exposure to HYPO led to disassembly of microtubules, disruption of PAMR (Figs. A,B), and loss of contiguous distribution of ZO-1 at the cellular periphery (Fig. E,F). These responses were slightly augmented by rewarming after HYPO. However, the damage to PAMR and ZO-1 could be abated by pretreatment with EpoB, SB, antioxidants, or the iron chelator (Figs. C,G). Deliberate oxidative stress to endothelium by exposure to H2O2 and t-butyl hydroperoxide (tBHP) also led to widespread damage to the organization of PAMR and ZO-1 (Figs. D,H) in a p38 MAP kinase-dependent manner. Furthermore, photodynamic treatment with riboflavin or tryptophan + UV-A also caused damage to PAMR and ZO-1, which could be abolished by cotreatment with catalase. In cultured cells, both HYPO and oxidative stress caused a significant increase in the flux of FITC-avidin, indicating a loss in barrier integrity.
Conclusions :
Prolonged cold storage of corneas induces destruction of the cytoskeleton in the endothelium via activation of oxidative stress, leading to a breakdown of the barrier function. Thus, the inclusion of microtubule stabilizers, antioxidants, and iron chelators in the storage medium has the potential to overcome the endothelial barrier failure during cold storage of donor corneas.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.