Morphological investigation on the effects of netarsudil on the volume of giant vacuoles in Schlemm’s canal endothelial cells of human eyes
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Abstract
Primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide, primarily due to its irreversible nature and its association with elevated intraocular pressure (IOP). The exact mechanisms by which outflow resistance is regulated remains difficult to identify. Aqueous outflow is not uniform, rather segmented into high-, low-, and non-flow areas. Lowering IOP is the only effective treatment for POAG. One newly approved class of drug that is currently used to lower IOP in POAG is netarsudil, a Rho kinase/norepinephrine transporter inhibitor. Previous studies conducted in the Gong lab showed that netarsudil increases outflow facility through trabecular meshwork expansion and episcleral vein dilation, leading to an increase in high-flow areas, consequently increasing outflow facility and lowering IOP. However, the effect of netarsudil on the inner wall endothelial cells of Schlemm’s canal has not been fully explored. Giant vacuoles (GVs) are a unique structure in the inner wall endothelium of Schlemm’s canal that play an important role in regulating aqueous outflow resistance via their I-pore formation. Previous studies have found that the size of GVs with I-pores is significantly larger than that of GVs without I-pores, suggesting that GV’s play a role in I-pore formation. This study aimed to test the hypothesis that netarsudil increases GV volume and percentage of GVs with basal opening and I-pores compared to control eyes. Two pairs of human donor eyes were perfused with netarsudil in one eye and vehicle solution in the other eye of each pair for 3 hours at constant pressure (15 mmHg). Two different colors of fluorescent tracer were perfused into the eye before and after netarsudil treatment to label flow pattern changes prior to perfusion-fixation. Tissue wedges from high-, low-, and non-flow areas, as well as newly-recruited high-flow or low-flow areas (identified by comparing green and red fluorescein tracer global imaging) were processed and imaged (~2000 images per wedge) using serial block face scanning electron microscopy. Three hundred GVs, with I-pores and without I-pores from four different flow regions of each eye, were identified and traced. The volumes of GVs across these groups were compared using RStudio.
Mean GV volume was significantly larger in netarsudil-treated eyes compared to control eyes overall (p < 0.01) and GVs with I-pores were significantly larger than GVs without I-pores (p < 0.05) in both groups. When analyzing different flow type areas, mean GV volume was significantly greater in treated high-flow areas than in control high- and non-flow areas (p < 0.01). Finally, mean GV volume was significantly larger in recruited-flow than control non-flow (p < 0.05). These findings suggest that one mechanism by which netarsudil lowers IOP may be its ability to increase GV size and the percentage of GVs with I-pores. Further study is needed to confirm whether netarsudil increases the number of GVs with I-pores as sample size increases and to determine whether netarsudil affects membrane thinning by measuring the thickness of the cellular membrane of GVs.
Description
2025