Comparison of giant vacuole type and size in the inner wall endothelium of Schlemm’s canal in human eyes perfused at 7 and 15 mmHg using serial block-face scanning electron microscopy

Abstract

Purpose : This study investigated differences in the types and size of giant vacuoles (GVs) at 7 mmHg (physiological pressure in enucleated eyes) and an elevated pressure of 15 mmHg in the inner wall endothelium of Schlemm’s canal (SC), using serial block-face scanning electron microscopy (SBF-SEM) and subsequent three-dimensional (3D) reconstruction of GVs.

Methods : Four normal human eyes from four donors were perfused, two at 7 mmHg and two at 15 mmHg, with fluorescent tracers to label the segmental outflow pattern followed by perfusion-fixation. Three radial wedges of tissue including SC from high-, low-, and non-flow areas of each eye based on tracer distribution was processed for SBF-SEM. A similar number of images was analyzed at 7 mmHg (9586) and 15 mmHg (9802). GVs were counted and typed (Type I: no basal opening or I-pore, Type II: basal opening, no I-pore, Type III: I-pore, no basal opening, and Type IV: basal opening and I-pore). A subset of GVs was randomly selected for 3D reconstruction to measure GV volume. For the reconstructed type II GVs, thickness of cellular lining around GVs was measured.

Results : There was a greater number of GVs at 15 mmHg (3302) compared to 7 mmHg (1312). Type IV GVs were more abundant in the high-flow than non-flow areas at both pressures (P<0.01). GVs with I-pores were significantly larger than GVs without I-pores in all flow-type areas at both pressures (P<0.01). For GV volume across all flow-type areas, GVs with I-pores were not significantly different between pressures. However, GVs without I-pores were significantly larger in volume at elevated pressure (P<0.01). Type II GVs had thinner cellular lining at 15 mmHg (P<0.01).

Conclusions : SBF-SEM and 3D reconstruction allowed for accurate identification of GV types and size. Comparing both pressures, the volume of GVs with I-pores were similar, while the volume of GVs without I-pores were larger at elevated pressures. This may indicate a threshold size of GVs for pore formation. GVs with I-pores were significantly larger than GVs without I-pores in all flow-type areas at both pressures suggesting that larger size of GVs is a contributing factor for GV-associated I-pores. More Type IV GVs observed in the high-flow areas at both pressures suggest increasing this type of GV may increase the total high-flow area.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.