Factors affecting functional giant vacuole formation in Schlemm’s canal endothelial cells

Abstract

Purpose : This study investigated factors affecting giant vacuoles (GV) with pores (i.e. functional GV) of the inner wall (IW) endothelium of Schlemm’s canal (SC) using three-dimensional (3D) reconstruction of serial block-face scanning electron microscopy (SBF-SEM) images.

Methods : Two normal human donor eyes were perfused at 15 mmHg with fluorescent tracers to label the outflow pattern followed by perfusion-fixation. A small radial wedge of trabecular meshwork including SC from each flow-type area (high-, low-, and non-flow) based on the distribution of tracers from each eye (total: 6 wedges) was processed for SBF-SEM. GV and pores were labeled throughout each block (~1600 images per block). The percentage of GV with pores, density of GV/IW area, and number of pores/GV were determined. Additionally, 180 GV (90 with pores and 90 without) were randomly-selected and 3D reconstructed. GV volume and shape were analyzed and compared. Statistical analysis was performed using R.

Results : Overall, 15.5% of GV were functional. A higher percentage of functional GV were observed in high-flow areas (21.4%), compared to low- (16.7%) and non-flow (8.5%) areas. Density of functional GV of total GV per unit IW area was highest in high-flow area (4,358 of 26,520 GV/mm²), then low-flow (1,976 of 14,581 GV/mm²), and lowest in non-flow (1,845 of 24,966 GV/mm²). Overall, 83.1% of functional GV had a single pore; 16.9% had more than one pore (range: 2-6). Mean GV volume was not different among flow-type areas (P > 0.05). Mean volume of GV with pores (N = 90) was significantly larger than GV without pores (N = 90) for all GV 3D reconstructed (P < 0.01). Volume of GV with more than one pore was larger than GV with a single pore, but this difference was not significant (P = 0.07). 90% of functional GV were round in shape (81/90). Percentage of GV with pores was similar between round (49.7%) and collapsed GV (52.9%).

Conclusions : SBF-SEM and 3D reconstruction provided an accurate method to investigate GV density, volume, shape, and percentage of GV with pores. GV volume appears to be a factor contributing to the formation of functional GV and GV with multiple pores, whereas GV shape does not. More functional GV were observed in high-flow areas, suggesting that increasing the number of functional GV could be a possible therapeutic strategy to increase aqueous outflow for glaucoma treatment.

This is a 2020 ARVO Annual Meeting abstract.