Variations in active outflow along the trabecular outflow pathway

Previous tracer studies have shown segmental outflow in the trabecular meshwork (TM) and along the inner wall (IW) of Schlemm's canal (SC). Whether segmental outflow is conserved distal to SC has not yet been investigated. This study aims to investigate whether the segmented pattern of outflow is conserved in distal outflow pathways by using a newly developed global imaging method and to evaluate variations of active outflow in three distinct regions along trabecular outflow pathway. Six normal whole globe human eyes were first perfused at 15 mmHg to establish a stable baseline outflow facility. The anterior chamber was then exchanged (5 mL) and perfused with fluorescent microspheres (0.002% v/v, 200 μL) to label areas of active outflow. All eyes were perfusion fixed and dissected into anterior segments. The TM and scleral surface were en face imaged globally. Effective filtration area (EFA) and fluorescent tracer distribution and intensity were analyzed in global images for both the TM and episcleral veins (EPVs). Anterior segments were further dissected into a minimum of 16 radial wedges, from which frontal sections were cut, stained, and imaged, using confocal microscopy. EFA from all three locations along the trabecular outflow pathway were measured and compared. Additionally, TM thickness, SC height, and total number of collector channels (CC) were analyzed and compared between active and inactive areas of outflow. Statistical analysis was performed using Student's t-tests and Wilcoxon signed-rank test with a required significance of p ≤ 0.05. All three locations showed a segmental outflow pattern. The TM had a significantly higher mean EFA (86.3 ± 3.5%) compared to both the IW (34.7 ± 2.9%; p ≤ 0.01) and EPVs (41.1 ± 3.8%; p ≤ 0.01). No significant difference in mean EFA was found between IW and EPVs. Preferential active outflow was observed in the nasal and inferior quadrants. TM thickness was significantly larger in areas of active outflow (103.3 ± 4.0 μm; p ≤ 0.01) compared to areas of inactive outflow (78.5 ± 6.5 μm), but there was no significant difference in SC height between active and inactive outflow areas. Among all eyes, a total of 80 CCs were counted with 63 associated with active outflow and 17 associated with inactive outflow. A higher number of CCs associated with areas of active outflow were found in the nasal (26 of 63) and inferior (20 of 63) quadrants compared to the temporal (9 of 63) and superior (8 of 63) quadrants. A segmental nature of outflow is conserved along the trabecular outflow pathway with variations in three distinct locations (TM, IW, and EPVs). IW and EPVs showed a similar mean EFA. Preferential active outflow was observed in the nasal and inferior quadrants of the eye, which are associated with more expanded TM and higher number of CCs. Normal outflow patterns and its variations along the outflow pathway reported in this study will provide the basis for future studies of the outflow changes in eyes with glaucoma.