Purchase this article with an account.
Sudhir H Ranganath, M. Y. Thanuja, Manjunatha T D, Sangly P Srinivas; Hypothermia breaks down the barrier function of the corneal endothelium. Invest. Ophthalmol. Vis. Sci. 2021;62(8):816.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The donor corneal endothelium (CE) is exposed to hypothermia during cold storage, which is inevitable before transplantation. Here, we have examined the impact of hypothermia on the barrier function of CE with a goal to enhance the success of corneal transplantation.
Primary cultures of porcine CE or freshly isolated porcine corneas were employed for the experiments. Since hypothermia is known to cause microtubule (MT) disassembly, which causes a breakdown of barrier integrity in CE (Srinivas, EER, 2012), we immunostained and imaged MT and ZO-1 with and without prior exposure to epothilone B (a microtubule-stabilizing agent; EpoB). To ensure sustained intracellular levels of the agent, we also formulated EpoB-loaded PLGA nanoparticles (ENPs; coated with poly-L-lysine; zeta potential 25 mV). ENPs were spherical (~ 95 ± 10 nm) with a drug entrapment efficiency of 91% and drug loading of 5% (w/w). The EPNs showed a burst release of EpoB, but the subsequent slow release phase sustained for up to 4 weeks.
An exposure to hypothermia (15 h; 4 °C) led to MT disassembly in cultured CE. The effect was also evident in CE cells associated with ex vivo corneas. Moreover, the impact was similar to that observed in response to TNF-α (20 ng/mL; 24 hrs). Exposure to EpoB (100 nM) or ENPs (0.25 mg/mL) for > 24 h induced MT stabilization with minimal toxicity and importantly opposed the hypothermia-induced MT disassembly. Prior exposure to SB-203580 (a p38 MAPK inhibitor; 20 µM; 1 hr) reduced the hypothermia-induced MT disassembly. The effect of SB-203580 was similar to the inhibition of the response to TNF-α, which activates p38 MAPK (Srinivas, EER, 2012). Concomitant with the impact on MT, hypothermia led to a disruption of the contiguous ZO-1 at the cellular periphery (Fig. 1B; Fig. 1D shows response to TNF-α), indicating a loss of barrier integrity. However, pre-treatment with EpoB (Fig. 1C), ENPs, or SB-203580 opposed the ZO-1 disruption, indicating that MT disassembly underlies the hypothermia-induced breakdown of tight junctions.
Hypothermia induces MT disassembly via activation of p38 MAPK and subsequently breaks down the barrier function of CE. Sustained exposure to MT stabilizers overcomes the hypothermia-induced barrier failure. The ENPs could serve as a modality to obtain sustained protection of CE against hypothermia-induced stress and TNF-α-induced damage to MT in case of allograft rejection.
This is a 2021 ARVO Annual Meeting abstract.
This PDF is available to Subscribers Only