Investigation of giant vacuoles in the inner wall endothelium of Schlemm's canal at physiologic pressure and comparison of the types and size of giant vacuoles in human eyes perfused at 7- and 15-mm Hg using serial block-face scanning electron microscopy
OA Version
Citation
Abstract
Glaucoma is a disease characterized by elevated intra-ocular pressure and chronic, irreversible loss of vision. The precise mechanism by which outflow resistance is generated remains elusive. We investigated a unique structure called the giant vacuole (GV) in the inner wall endothelium of Schlemm’s canal (SC) that contribute to regulating outflow resistance. This study aimed to: 1) investigate the types and size of GVs in eyes perfused at 7 mmHg (physiological pressure in enucleated eyes) in the inner wall endothelium of SC using serial block-face scanning electron microscopy (SBF-SEM) and subsequent three-dimensional (3D) reconstruction of GVs; 2) to compare differences in the types and sizes of GVs in eyes perfused at 7 mmHg with published results in eyes perfused at 15 mm Hg (Swain et al, 2021). Two normal human donor eyes were perfused at 7 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 were processed for SBF-SEM. GVs were counted and typed (Type I: no intracellular pore (I-pore), no basal opening, Type II: no I-pore, present basal opening, Type III: no basal opening, present I-pore, and Type IV: present I-pore and basal opening). 3D reconstruction was performed on a GV subset. A subset of GVs was randomly selected for 3D reconstruction to measure GV volume. GVs in eyes perfused at 7 mmHg were compared with a previous GV study in eyes perfused at 15 mmHg. A similar number of images were analyzed at 7 mmHg (9586 images) and 15 mmHg (9802 images). Statistical analyses were performed using R statistical computing package.
There was a greater number of GVs at 15 mmHg (3302 GVs) compared to 7 mmHg (1312 GVs). 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). GVs with I-pores were similar in volume at both pressures in all GVs and in high- and non-flow areas. However, GVs without I-pores were significantly larger in volume at elevated pressure (P≤0.01). 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-pore formation. More Type IV GVs observed in the high-flow areas at both pressures suggest that Type VI GV formation may play a role in regulating segmental outflow and increasing this type of GV may increase the total high-flow area.
Description
License
Attribution 4.0 International