BACKGROUND: Primary open angle glaucoma (POAG) is the second-leading cause of blindness in the U.S. It is caused by a buildup of aqueous humor resulting from abnormally increased resistance to outflow in the trabecular drainage pathway, which leads to an increase in intraocular pressure (IOP) in the eye. Elevated IOP is the primary risk factor for development and progression of POAG. Higher IOP compresses and damages the optic nerve, causing visual field loss, and ultimately leading to blindness. Reducing the IOP slows the progression of glaucoma. Netarsudil, sold under the brand name “Rhopressa,” is a rho-kinase/NE transporter inhibitor that lowers elevated IOP in patients with open-angle glaucoma or ocular hypertension. A previous study showed that netarsudil uses a number of mechanisms to increase the outflow facility of aqueous humor in human eyes (Gong & Swain 2020). However, netarsudil’s effects on the endothelial cells of Schlemm’s canal (SC) have yet to be investigated.
AIM: The goal of this study was to determine the effects of netarsudil-treatment on cell dimensions, volumes, and GVs of the inner wall endothelial cells of SC in comparison to those of non-treated control eyes using serial block-face scanning electron microscopy (SBF-SEM) and 3D reconstruction.
METHODS: The two pairs of eyes were perfused with green fluorescent tracer to determine the correct outflow pattern (either high-flow or non-flow) before administering either 0.3 µM netarsudil or vehicle solution at constant pressure of 15 mmHg for 3 hours. Afterwards, the eyes were perfused with red fluorescent tracer to determine the outflow pattern post-treatment followed by perfusion fixation. Global imaging was performed for the eyes to visualize these high- and non-flow areas in the trabecular meshwork (TM) and ESV’s. A SBF-SEM was used to image the inner wall cells of SC in high- and non-flow areas of each eye. This study analyzed a total of 9,257 images and 3D reconstructed 16 inner wall endothelial cells of SC (4 cells in each group) to compare the cell dimensions, cell volume, nuclear volume, giant vacuole (GV) volume, and multi-cell GV (mcGV) volume, as well as GV types between cells from high- or non-flow regions of netarsudil-treated and un-treated control eyes.
RESULTS: The mean cell length appears to be greater in the non-flow regions of netarsudil-treated eyes compared to that in the control group. The mean cell width in both nuclear and non-nuclear regions did not differ much among the control and treatment groups in both high-flow and non-flow regions. The mean cell thickness in both nuclear and non-nuclear regions appeared to be greater in high-flow regions of the treated eyes than that of control eyes. Netarsudil treatment increased the mean cellular volume and the mean nuclear volume in areas of high-flow. The number of GVs associated with endothelial cells from both high- and non-flow regions increased after netarsudil treatment. When treated with netarsudil, cells from high-flow regions appear to have increased mean mcGV volume. Type II GVs appear to be the most common type of GV, and no type III GVs were observed. The treated cells had a greater percentage of type I GVs, while the control cells had a greater percentage of type II GVs. There was no apparent difference between the treatment and control groups in the percentage of type IV GVs.
CONCLUSIONS: SBF-SEM and 3D reconstruction provided a reliable method to evaluate cellular changes after netarsudil treatment. Preliminary data shows that, when treated with netarsudil, inner wall endothelial cells of SC appear to have increased cellular thickness, nuclear, cell and GV volumes (including mcGVs). Previous studies indicated that size of GVs plays a role in pore formation. A large number of cells will need to be studied to determine whether increased GV size leads to increased pore or type IV GV formations, contributing to netarsudil-induced increase in aqueous outflow.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/45530 |
| Date | 27 January 2023 |
| Creators | Arquiza, Roviel |
| Contributors | Gong, Haiyan, Trinkaus Randall, Vickery E. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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