Relationships between increased aqueous outflow facility during washout with the changes in hydrodynamic pattern and morphology in bovine aqueous outflow pathways

Previous studies suggest that the structural correlate for the increased outflow facility (C) during washout in the bovine eye is separation between the inner wall (IW) and underlying juxtacanalicular connective tissue (JCT). However, how these structural changes affect hydrodynamic patterns of outflow during washout has not been studied. We hypothesize that an increase in the outflow facility during washout is associated with an increase in the effective filtration area (EFA) of aqueous outflow, which is regulated by a loss of the connectivity between the IW and JCT. To test this hypothesis, the relationship between C and the hydrodynamic patterns of outflow as well as the morphological changes in JCT and IW during the washout were investigated. Ten bovine eyes were perfused at 15 mmHg with Dulbecco's PBS + 5.5 mM glucose (DPBS) for 30 min to establish stable baseline C. After measuring baseline C, five eyes (short-duration group) were perfused with 0.5 mL DPBS containing 0.002% microspheres (0.5 μm) to trace the hydrodynamic pattern of outflow. Five other eyes (long-duration group) were perfused for 3 h to elicit a significant washout effect followed by subsequent perfusion of the same volume (0.5 mL) of microspheres to map out the outflow pattern after washout. All eyes were then perfusion-fixed. Anterior segments were sectioned and prepared for confocal and light microscopy. Total length (TL) and filtration length (FL) of the IW were measured in ≥15 images/eye to calculate percent effective filtration length (PEFL = FL/TL) while TL and length exhibiting JCT/IW separation (SL) were measured in ≥13 images/eye to calculate percent separation length (PSL = SL/TL). In long-duration eyes, C increased 170.5 ± 21.3% (mean ± SEM, 1.55 ± 0.24 vs 4.13 ± 0.55 μl/min/mmHg, p = 0.001) above baseline. Pre-fixation C (4.13 ± 0.55 μl/min/mmHg) in long-duration was 1.6-fold greater than that (2.14 ± 0.61 μl/min/mmHg; p = 0.042) in short-duration. A more uniform tracer labeling was observed in the JCT/IW of long-duration eyes compared to short-duration. PEFL was 2.3-fold larger (52.82 ± 6.06 vs. 22.2 ± 6.0%; p = 0.007) and PSL was 2.6-fold larger (54.2 ± 6.0 vs. 20.5 ± 1.3%; p = 0.004) in long-duration eyes compared to short-duration. Data from all eyes revealed a positive correlation between PEFL and PSL (p = 0.02). Both PEFL and PSL demonstrated significant positive correlations with the relative increase in C due to washout (p ≤ 0.05). An additional experiment was performed in which unequal volumes of tracer (0.5 and 1.0 mL) were perfused in paired eyes for both short- and long-duration (N = 2 for each condition) to examine the affect on PEFL. No significant change in PEFL was found in eyes perfused with 0.5 and 1.0 mL within the same group. These data support our hypothesis that separations between the IW and JCT result in an increase in the EFA that then influences C. Altogether, these data suggest that outflow hydrodynamics and the tissue structure work together to regulate outflow resistance.