The effects of a new class of glaucoma treatment drug netarsudil on cellular connections of Schlemm’s canal endothelium
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Abstract
BACKGROUND: Primary Open Angle Glaucoma (POAG) is a subclass of glaucoma. It is the second most common cause of blindness, within the United States. This disease is characterized by its link to one of its major risk factors, increasing intraocular pressure (IOP). High IOP causes neuronal death of the optic nerve, progressively reducing the visual field until total blindness. IOP elevation is due to an obstruction somewhere along the trabecular outflow pathway, the primary route of drainage for the aqueous humor. One of the most effective treatments to mitigate the progress of POAG involves lowering IOP, by using medicine, laser and surgery. Netarsudil, is one of Rho-Kinase inhibitors, which is a relatively new medication to lower IOP in glaucoma and ocular hypertension. A previous study in Dr. Gong’s lab found that Netarsudil increased outflow facility in human eyes; it induced trabecular meshwork expansion and episcleral vein dilation, which results in an increase in active flow area, consequently increasing outflow facility. However, the effect of Netarsudil on the inner wall endothelial cells has not been carefully studied.
AIM: The purpose of this study was to test the hypothesis that one of mechanisms that Netarsudil increases outflow facility is through reduction of connectivity between the inner wall (IW) endothelial cells of Schlemm’s canal (SC) and their underlying juxtacanalicular cells (JCT) and matrix.
METHODS: Human eyes were perfused with or without Netarsudil for three hours, and perfusion-fixed prior to the onset of this study. Since outflow is not uniform, high- and non-flow regions of each eye were identified and processed for serial block-face scanning electron microscopy (SBF- SEM). SBF-SEM images of these eyes were produced by the Cleveland clinic and returned to the Gong Lab for study. Images of 20 cells from both high- and non-flow regions of the Netarsudi-treated and control groups were analyzed, five cells from each group. Cellular connectivity was counted manually using a software program known as Reconstruct. Raw Data was input into spreadsheets for statistical analysis. Data was then normalized to account for the difference in cellular length. Statistical analyses were performed using Prism.
RESULTS: Netarsudil significantly reduced the total cell-cell (C-C) and cell-matrix (C-M) connections compared to the control. When normalized, a similar reduction is exhibited. When control and treated groups were separated into flow regions, a significant decrease in mean number of total connections was evident in the high-flow region, but not in non-flow region. Once normalized, however, no significant decrease could be noted. When further comparing the effects of Netarsudil on mean C-M connections, between the non- and high-flow regions, no significant findings were evident. When normalized, there were also no significant differences. Analysis of Netarsudil’s effects on C-C connections between treated and control flow regions show no significant difference could be observed for the mean C-C count and normalized means. Cell-cell counts were further specified for 6 different type distributions and compared between high- and non- flow regions of treated and controls. There was a significant decrease in type 2 connections with the Netarsudil administration, while other types (3-7) did not exhibit significant differences in mean connection count in non-flow regions. When normalized, it appears Netarsudil enacted a significant decrease in type 2 connections, and a significant increase in type 5 connections. Type 2 connections were significantly decreased in high-flow regions. Normalization re-affirmed that Netarsudil decreased type 2 connections in high-flow regions.
CONCLUSION: Netarsudil significantly reduced the total cell-cell (C-C) and cell-matrix (C-M) connections compared to the controls, and specifically, Netarsudil significantly decreased type 2 cellular connections. Our data supports our hypothesis. Netarsudil exhibits this relaxing effect on the IW cells of SC and promotes outflow facility by minimization of adherence at the junction of the IW/JCT, resulting in ECM expansion and more uniform flow. The limitation of this study was a small sample size. An increase in sample size would greatly add to the significance and validity of the data and help to reaffirm the findings with more confidence. In addition to what could have been done to improve this study, more work can be done to analyze this effect in the low-flow regions of the TM as well, to develop a more coherent and cohesive understanding of the treatment effects across the entire TM.
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2024