Oxidative Stress-Induced Mitochondrial Dysfunction In Corneal Endothelium

Corneal transplantation restores visual function but long-term survival rates for corneal grafts are low.  A new surrogate measure to quantify the health of corneal grafts, by measuring the function of endothelial cells instead of  cell density, may be beneficial. In this study, we create a model of oxidative stress to determine if it will disrupt mitochondrial function and intercellular tight junction Zonula Occludens-1 (ZO-1) through an analysis of cell and tissue immunofluorescent staining.  A non-invasive assay (Acridan Lumigen) is evaluated for potential use in an eye bank setting, to distinguish between suitable and unsuitable corneal graft tissues based on reactive oxygen species production in media.

Laboratory. Whole porcine corneal segments and three different cell types were used:primary porcine corneal endothelial cells (PCEC), human corneal endothelial cell line (HCEC-12 cells; ACC 636, DSMZ)  and human trabecular meshwork cells (HTMC) at the Department of Eye and Vision science, University of Liverpool.

The corneal segments and cultured HTMC, PCEC and HCEC-12 cells were subjected to various hydrogen peroxide (H₂O₂) concentrations for 30 mins at 37°C. These were evaluated using mitochondrial density (MitoTracker Orange CM-H2TMRos, ThermoFisher) and live/ dead cell viability assays (Live/Dead viability kit (Thermo Fisher)). Cells were imaged using an epifluorescence inverted microscope, Zeiss Axiolmager M1 (Zeiss). Mitochondrial function was assessed through ROS measurement using fluorogenic probes (MitoSOX™ Red, ThermoFisher) and fluorescence microscopy.  HCEC-12 corneal endothelial cell medium was tested with the Acridan Lumigen PS-3 Assay after cell exposure to varying concentrations of H₂O_2.

The percentage of live cells reduced with increased H₂O₂. Corneal segments were more resilient in retaining up to 22% of live cell population at 100μM H₂O₂. Oxidative stress significantly decreased mitochondrial density in the corneal endothelium culture and ex vivo (p <0.001). PCEC cultures were more sensitive to stress as most cells lost fluorescence at 80μM whereas corneal segments retained MitoTracker fluorescence even at 100μM. Cell membrane continuity and structural integrity of Na+/K+ ATPase was disrupted in high concentrations of H₂O₂. On the contrary, tight junction ZO-1 retained its structure even with H₂O₂ above 5 mM. The Acridan Lumigen assay was unable to distinguish between HCEC-12 treated with various concentrations of H₂O₂.

Impairment of cell viability, mitochondrial density and function were observed in our model of oxidative stress in corneal segments and HTMC, PCEC and HCEC-12 cells. The whole cornea was more resilient to oxidative stress than cultured cells. Intercellular junctions were interrupted but the hexagonality was still retained under oxidative stress. This supports our hypothesis that assessment of endothelial cell count and hexagonality alone are not sufficient measures to predict donor graft health. The Acridan Lumigen assay proved not to be a suitable surrogate marker for these changes, however, an increased understanding of evaluating donor corneal mitochondrial parameters may help to determine the health and survival of corneal graft tissue.