The effect of netarsudil on the size and types of giant vacuoles in the inner wall endothelium of Schlemm’s canal of perfusion-fixed human eyes using serial block-face scanning electron microscopy
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Citation
Abstract
BACKGROUND: Primary open-angle glaucoma (POAG) is predominantly caused by impaired aqueous humor (AH) drainage via the trabecular meshwork (TM) into the Schlemm’s canal (SC). On a cellular level, one route on how the AH enters the lumen of SC is to pass giant vacuoles (GVs) with a basal opening and luminal intracellular pores (I-pores) in inner wall (IW) endothelial cells of SC. There are four types of GVs. The stage of a GV with a basal opening is called Type II GVs and when both the basal opening and luminal I-pore(s) are present is called a Type IV GV.
Netarsudil, a newly approved topical Rho-kinase (ROCK)/norepinephrine (NET) inhibitor medication for glaucoma, has been shown to increase outflow facility via multiple mechanisms in human eyes. While netarsudil has been shown to increase GVs size, its impact on the density and size of Type IV GVs in IW cells of SC remains unexplored.
AIM: To determine whether the density and size (span) of Type IV GVs increase in both high- and non-flow areas of netarsudil-treated human eye-bank eyes when compared to untreated controls perfusion-fixed at 15 mmHg using serial block-face scanning electron microscopy (SBF-SEM).
METHODS: Two pairs of human donor eyes were perfused with green, fluorescent tracers to label the AH outflow pattern prior to netarsudil treatment. Then, one eye of each pair was perfused with netarsudil, while the contralateral eye was perfused with vehicle solution for three hours. All eyes were then perfused with red fluorescent tracers to label the outflow pattern after treatment, followed by perfusion-fixation at 15 mmHg. Global imaging was performed for all eyes to visualize high- and non-flow areas in the TM and ESV. Eight tissue wedges (one high-flow and one non-flow areas from each eye) including the IW of SC and TM were processed and imaged using SBF-SEM (total: 16,378 images). This study analyzed a total of 1,950 GVs, the percentage of GVs in each of the four types (Type I, II, III, IV), their size (median spans), and densities (per IW nucleus and surface area) between high- and non-flow areas of netarsudil-treated and control eyes. RStudio package was used for statistical analysis.
RESULTS: The size of total GVs, GVs with I-pores, and Type II and Type IV GVs increased significantly in netarsudil-treated eyes compared to controls (each P ≤ 0.01). The percentage of Type IV GVs was significantly greater in netarsudil-treated than control eyes in high-flow areas (χ 2 = 7.04, P ≤ 0.01). There was a significantly higher percentage of GVs with I-pores in high-flow areas compared to non-flow areas in both netarsudil-treated (χ2 =43.43, P ≤ 0.01) and control eyes (χ2 =3.84, P ≤ 0.05) . Overall density of GVs showed a trend of greater total GVs, GVs with I-pores, specifically Type IV GVs, in netarsudil-treated compared to control eyes. The same trend of greater total GVs, GVs with I-pores, specifically Type IV GVs, was also observed in high-flow than non-flow areas in both netarsudil-treated and control eyes. Since n=2 for each group, statistical analysis was not performed for the density of GVs.
CONCLUSION: Netarsudil treatment increased the size of Types II and IV GVs, and the percentage of Type IV GVs significantly when compared to control. In addition, there was significantly a higher percentage of Type IV GVs in high-flow areas than in non-flow areas in both netarsudil-treated and control eyes. Taken together, the results of this study suggest that increased size of Type II GVs may contribute to higher percentage of Type IV GVs found in netarsudil-treated eyes. One of the mechanism by which netarsudil increases outflow facility and lowers IOP may act through increasing percentage of Type IV GVs.