Abstract
Purpose :
Posterior capsule opacification following cataract surgery resulting from surgical induced wound-healing remains a common problem. Glutathione (GSH) is associated with cell survival and maintenance. In the present study, we assessed the impact of glutathione depletion by Menadione on lens cell survival, maintenance and growth.
Methods :
The human lens epithelial cell line FHL124 and the in vitro human capsular bag model were used as experimental models. Cell viability was assessed using the Cell-TitreGlo (CTG) Luminescent assay. ROS levels were determined using a DCFDA cellular ROS assay. Protein levels were assessed by Western blotting to detect ER stress and autophagy markers. To visualise mitochondrial networks, cells were treated with Mitotracker Red CMXROS prior to fixation in 4% formaldehyde and viewed using confocal microscopy.
Capsular bags were prepared from human donor eyes obtained with ethical approval by following the key stages of cataract surgery. A ciliary body/zonule capsular bag complex was transferred to a 6 well plate and maintained in 6ml EMEM. Match-paired experiments (N=3 for each comparison) were performed (treatment v control) and cultured for at least 15 days.
Results :
In capsular bag cultures, 10µM Menadione slowed cell cover of the central posterior capsule, while 30µM lead to total cell loss relative to their non-treated controls. This concentration response was reflected in FHL124 cell viability. Menadione treatment (30µM) of FHL124 cells was also associated with elevated ROS levels, mitochondrial network disruption, ER stress and increased autophagy. Treatment with NAC, a reactive oxygen species scavenger, or GSH significantly prevented menadione-induced cell death protected mitochondria network integrity, prevented endoplasmic reticulum (ER) stress and autophagy.
Conclusions :
Menadione depletion of GSH increases oxidative stress in lens epithelial cells, which in turn triggers disruption of critical cell processes leading to cell death. Menadione is therefore an interesting candidate to explore further to improve the management of PCO following cataract surgery.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.