This study investigated the morphologic differences of the giant vacuoles (GVs) in the inner wall endothelium of Schlemm’s canal (SC) in human eyes perfused at either 30 mmHg or 7 mmHg (physiologic pressure in enucleated eyes) using serial block-face scanning electron microscopy (SBF-SEM) paired with three-dimensional reconstruction software. Two normal human eyes were perfused at 30 mmHg with fluorescent tracers to mark regions of active and inactive flow, followed by perfusion-fixation. Tissue wedges (n = 6) of trabecular meshwork including SC from high-, low-, and non-flow areas of each eye (determined by tracer distribution) were dissected and processed for SBF-SEM. Four types of GVs were identified: Type I GVs which lack both a basal opening and an Ipore; Type II GVs which have a basal opening but lack an I-pore; Type III GVs which have an I-pore but lack a basal opening; and Type IV GVs which possess both a basal opening and an I-pore. Types and spans of GVs were collected from the SBF-SEM images, and volumes of GVs from a random subset were measured using 3D reconstruction. Results were compared with findings from an earlier study conducted with two eyes perfused at 7 mmHg and prepared in the same manner (Soares, 2022). In total, 19,047 SBF-SEM images were analyzed between 7 mmHg (n = 9586) and 30 mmHg (n = 9461) using Reconstruct. Statistical analysis comparing data between the two pressures was performed using R. There were more GVs found at 30 mmHg (n = 1541) when compared with 7 mmHg (n = 1312), and there were more Type IV GVs at 30 mmHg when compared with 7 mmHg. Type IV GVs occurred most frequently in high-flow areas at both pressures. GVs with I-pores were greater in size (both span and volume) than GVs without I-pores in all flow areas at both pressures. Type IV GVs were larger than Type II GVs which were larger than Type I GVs at both pressures. The span of GVs without I-pores was significantly greater at 7 mmHg. However, there was no significant difference between the volumes of GVs with or without I-pores between the two pressures. The result that GVs with I-pores were larger in size than GVs without I-pores at all conditions appears to support the theory that GV size is an important contributing factor to I-pore formation. The differences in span but not volume of GVs without I-pores between two pressures suggest that GVs at high pressure may be more convex in shape and may protrude further into SC, a situation which could contribute to thinning of the cellular membrane of GVs. Finally, the result that more Type IV GVs were found in high-flow areas at both pressures implies that the changing percentage of Type IV GVs likely plays a role in regulating segmental flow. / 2025-02-26T00:00:00Z

PURPOSE: To determine, under flow conditions, whether reduced connections between Schlemm’s canal (SC) inner wall (IW) and juxtacanalicular tissue (JCT) cells play a role in giant vacuole (GV) formation; and whether decreased amount of cell margin overlap between adjacent IW cells promotes paracellular pore formation using serial block-face scanning electron microscopy (SBF-SEM). METHODS: Normal human eyes were immersion-fixed (0 mmHg, N=2) or perfusion-fixed (15 mmHg, N=1). Frontal and radial sections of SC were processed for SBF-SEM. IW and JCT cells, GVs, and pores were 3D-reconstructed. In each IW cell, total number of connections with underlying JCT cells/matrix was determined. Total cell margin length (TCML) and zero-overlap length (ZL) of each IW cell were measured to calculate percent zero-overlap length (PZL=ZL/TCML). All data were compared between the eyes fixed at 0 and 15 mmHg. RESULTS: Total number of IW/JCT connections in individual IWs significantly decreased in the eye fixed at 15 mmHg (33±5, N=5 cells) compared to those fixed at 0 mmHg (189±12, N=4 cells, p<0.001). The summed GV volume in individual cells significantly increased in the eye fixed at 15mmHg (218.03±19.65 μm3) compared to those fixed at 0 mmHg (82.33±27.22 μm3, p=0.0043). PZL increased 26.68% (p=0.001) in the eye fixed at 15mmHg vs. those fixed at 0mmHg, and all paracellular pores were found only in regions where the overlap length was 0 μm. CONCLUSIONS: Cellular connections between IW/JCT and IW/IW cells play a role in GV and pore formation in normal human eyes under flow conditions. Our results provide a baseline for future comparison with primary open angle glaucoma eyes.

Primary open-angle glaucoma (POAG) is one of the leading causes of blindness worldwide. The only modifiable risk factor for POAG is elevated intraocular pressure, resulting from increased aqueous humor production or decreased drainage. Resistance to drainage in the aqueous outflow pathway is believed to reside in the juxtacanalicular connective tissue (JCT) and to be modulated by the inner wall (IW) endothelium of Schlemm’s canal (SC); however, the mechanisms that increase resistance in POAG remain unclear. To cross the IW, aqueous humor passes through I-pores on giant vacuoles (GVs) or B-pores between adjacent endothelial cells. Additionally, outflow around the circumference of the eye is segmental, or non-uniform, and fluorescent tracers can be used to label areas of high-flow and non-flow. The morphological differences in the endothelial cells of SC and their GVs in high- vs. non-flow areas have not been fully elucidated. In this project, we investigated the role of GVs and pores in the IW endothelial cells of SC in regulating segmental outflow in human eyes. We used serial block-face scanning electron microscopy to generate thousands of serial images and visualize these structures in 3D at the ultrastructural level. First, we 3D-reconstructed 45 individual IW cells and their GVs and quantified the number of connections each cell makes with the underlying JCT matrix/cells. We found that cells in high-flow areas made significantly fewer connections to JCT matrix/cells compared to cells in non-flow areas. Secondly, we analyzed 3,302 GVs for I-pores and basal openings and found a significantly greater percentage of GVs with both basal openings and I-pores in high-flow area compared to non-flow area, suggesting this type of GVs form a channel through which aqueous humor passes from JCT to SC. We also found that GVs with I-pores were significantly larger than those without I-pores. Our results suggest that decreasing number of cellular connections and increasing number of GVs with pores may be potential strategies to increase the amount of high-flow area and aqueous outflow for glaucoma treatment. Together, these studies add to our understanding of the role of GVs and pores in regulating segmental flow around the eye.