Purpose
Reduced outflow facility in glaucoma is thought to be associated with decreased pore formation on the inner wall endothelium of Schlemm’s Canal (SC). Recent evidence (Overby, PNAS, 2014) indicates that elevated mechanical stiffness of glaucomatous SC cells contributes to reduced pore formation in glaucomatous eyes. Corticosteroids are known to affect cell stiffness and can cause ocular hypertension. We here examine the effect of dexamethasone on SC cell stiffness.
Methods
Normal (n=2) and glaucomatous (n=2) SC cell strains were treated with 0 (Control), 0.01μM, 0.1μM, 1μM of dexamethasone for 7 days. Atomic Force Microscopy (AFM) was then used to characterize cortical and subcortical stiffness using sharp (20 nm) and spherical tips (10 μm), respectively (15-20 cells in each group). Two-tailed t-tests were used for statistical analysis. Samples were then fixed with 4% paraformaldehyde, stained with Alexafluor 568 Phalloidin (F-actin) and Hoechst (nucleus), respectively, and then were imaged using confocal microscopy.
Results
When compared to controls (Figure 1), cell strains showed 1.5-2.1 fold higher cortical stiffness at a dexamethasone concentration of 1 μM (p<0.05), while differences with lower doses were not significant. No difference was found in subcortical stiffness between treatment doses in any cell strain. Imaging showed increased stress fibers with increasing dexamethasone concentration (Figure 2). No change in cortex thickness was seen. No differences were seen between normal and glaucomatous cell stains.
Conclusions
Our results suggest that dexamethasone treatment increases cell stiffness, which may impede pore formation in the inner wall endothelium and thereby increase outflow resistance and potentially cause glaucoma. Increased assembly of actomyosin machinery might be the responsible mechanism for elevated cortical stiffness. However, in spite of increased stress fibers caused by dexamethasone treatment, we saw no change in subcortical stiffness. This suggests that other cytoskeletal elements, possibly intermediate filaments, play a more dominant role in regulation of the subcortical stiffness.