Abstract
Purpose :
ZO-1 binds to Claudins at the apical junctional complex as an intracellular scaffold protein. Thus, it manifests contiguously at the cellular periphery in the endothelium with an intact barrier function. However, stressful stimuli, such as hypothermia, oxidative stress, and allograft rejection, break down the perijunctional actomyosin ring (PAMR) concomitant with the destruction of ZO-1 organization (insets in Fig.). This study aims to quantify such disruptions of ZO-1 through combined immunocytochemistry, confocal microscopy, and automatic image skeletonization.
Methods :
Porcine eyes were subjected to hypothermia (4 °C; 3-7 days) or oxidative stress (100 µM; H2O2 for 60 min). Next, images of PAMR and ZO-1 were registered by immunocytochemistry combined with confocal microscopy at the focal plane of ZO-1. The images were then subjected to automatic segmentation by (U-Net)-based deep learning workflow to delineate ZO-1 distribution at the cellular periphery. Finally, the segmented images were skeletonized using the scikit-image (python) implementation of the Zha84 method, and the corresponding outputs were analyzed for estimating the distribution of branch lengths.
Results :
In untreated cells, ZO-1 was contiguous with tricellular junctions. At moderate hypothermic stress, the linear edges were disrupted along with the loss of tricellular links. The linear edges broke down into smaller pieces and punctate blobs at severe stress levels. The severity of the impact on ZO-1 generally paralled those on PAMR. The U-Net led to reliable segmentation with an F1 score of 82%, IoU of 77%, and an accuracy of 94%. Subsequent skeletonization and determination of branch lengths showed a skewed distribution profile compared to those for untreated cells (Fig.) with the frequency of appearance of small segments increasing significantly with higher stress levels.
Conclusions :
We have developed a workflow to quantify the ZO-1 disruption patterns in response to hypothermia and oxidative stress in the corneal endothelium.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.