The immunohistochemical analysis of cellular auto-degradative functions in epithelial cells of the anterior capsule of lens in patients with pseudo-exfoliation syndrome: a preliminary report

Abstract

Purpose : Pseudo-exfoliation syndrome (PXF) is a systemic disease in which fibrillary proteinaceous material is deposited within the tissues such as the eye, which is thought to arise from dysfunctional matrix turnover and intracellular auto-degradation pathways. In this case-control study setting, we aim to evaluate whether defects in mitophagy specifically have a role in PXF etiology and prognosis. Methods : The anterior lens capsules obtained in microincisional cataract surgery were collected both from patients with PXF (n=6) and age-matched healthy controls (n=10). The samples were embedded in paraffin blocks and sliced into 0.4 μm slides for analysis. The ABC® IHC detection kit was used to stain the samples for PTEN-induced kinase 1 (PINK1), Parkin and LC3B. Stained images were captured with 20x magnification under confocal microscopy. The expression intensity was measured using color deconvolution plug-in and integrated density value was calculated on Fiji® (U. S. National Institutes of Health, Bethesda, MD). The Mann-Whitney U test was used for statistical analysis and the results were shown on Graphpad Prism (Graphpad, San Diego, CA). Results : The staining intensity of PINK1 and PARKIN were higher in the epithelia of anterior lens capsule in patients with PXF compared to the control samples, which was only statistically significant for PINK1. (p=0.0002, p=0.999 respectively) LC3B intensity was also found to be increased in PXF patients with statistical significance (p=0.0006). (Images 1 and 2). Conclusions : This increase in the markers of mitophagy, which is the targeted elimination of dysfunctional mitochondria, may be an important indicator of increased oxidative stress in PXF. It can therefore signal an extensive mitochondrial impairment and higher generation of reactive oxygen species, potentially overburdening the cellular clearance and enhancing faulty protein release. Certainly, further in vitro studies with a larger sample number are needed to compare the actual stress responses dynamically in PXF.