Changes in Pore Densities of the Inner Wall Endothelium of Schlemm’s Canal with Increasing Pressures Using 3D Serial Block-Face Scanning Electron Microscopy

Abstract

Purpose : Previous studies of pore density in the inner wall endothelium of Schlemm’s canal (SC) used traditional scanning electron microscopy, which was limited to the surface view and may have led to uncertainties for a portion of pore identification. In this study, we investigated changes in pore densities of the inner wall endothelial cells of SC with increasing perfusion pressures in normal human donor eyes using 3D serial block-face scanning electron microscopy (3D SBF-SEM).

Methods : Six normal human eyes were perfusion-fixed at 7, 15, or 30 mmHg (n=2 per pressure). Two or three radial wedges of trabecular meshwork tissues including SC were dissected per eye. Tissues were sectioned serially (0.13 µm) and imaged using SBF-SEM. A total of 17 wedges and 26,953 serial SEM images were screened for intracellular pores (I-pores) and paracellular or border pores (B-pores). Tears or artifacts were excluded. The surface area of the inner wall of SC was calculated. Pore densities were compared between pressures. Statistical analysis was performed by one-way ANOVA using RStudio statistical computing package.

Results : Between pressures, total pore density was higher in 15 mmHg (3713.1±445.9 pores/mm², mean±SEM) than in 7 mmHg (820.0±472.2 pores/mm², p=0.045) and 30 mmHg (1369.7±485.7 pores/mm², p=0.078); no difference was detected between 7 and 30 mmHg (p=0.719). Of all pores, more I-pores (88.7%) than B-pores (11.3%) were observed. Overall, 96.6 % of I-pores were associated with giant vacuoles (GVs); 39.7% of which were located on the apex of GVs and 56.9% were located on the lateral. 14.0% of lateral I-pores positioned near the edge of cells could be mistaken as B-pores by SEM. 19.8% of all pores (I-pores: 18.5%, B-pores: 1.3%) could have been missed by SEM because they were obstructed from surface view.

Conclusions : 3D SBF-SEM improved the accuracy for identifying pores, especially I-pores that could be mistaken as B-pores and pores that could be missed from the surface view by SEM. Our findings suggest that perfusion pressure may be one of the factors that contribute to pore formation and tight junctions between the neighboring inner wall cells of SC may have a higher resistance than cell membrane of GVs to form pores. Further study is warranted to understand the other factors that contribute to pore formation.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.