Comparative studies between species that do and do not exhibit the washout effect

Abstract

Ocular perfusion studies from all non-human species performed to date consistently demonstrate a perfusion-volume-dependent increase in aqueous outflow facility known as the “washout” effect. However, this “washout” effect does not occur in human eyes. We have recently documented that, in bovine eyes, the washout associated increase in facility correlates with the extent of physical separation between the juxtacanalicular connective tissue (JCT) and the inner wall endothelium lining the aqueous plexus (the bovine equivalent of Schlemm's canal). We hypothesize that if washout truly correlates with inner wall/JCT separation then this separation should not occur in human eyes that do not exhibit the washout effect, even after prolonged perfusion. Eight enucleated human and eight bovine eyes were used in this study. Aqueous humor outflow facility was measured at 15 mmHg for long-duration (3 h) or short-duration (30 min to 1 h) perfusion ( n = 4 for each group). All eyes were perfusion-fixed at 15 mmHg, and examined morphologically with both light and electron microscopy. In bovine eyes, outflow facility increased 81% ( p = 0.049) from 1.06 ± 0.06 μl/min per mmHg (mean ± SEM) at baseline to 1.92 ± 0.30 μl/min per mmHg after 3 h due to washout. The pre-fixation outflow facility in long-duration eyes (1.92 ± 0.30 μl/min per mmHg) was 2-fold greater than pre-fixation facility in short-duration eyes (0.92 ± 0.11 μl/min per mmHg; p = 0.0387). In human eyes, washout was not observed; baseline outflow facility was similar between both groups (0.18 ± 0.02 vs. 0.25 ± 0.08 μl/min per mmHg; p = 0.518); however, pre-fixation outflow facility in long-duration eyes showed a 40% decrease compared to baseline outflow facility in those same eyes ( p = 0.017, paired Student's t-test). In bovine eyes, significant expansion and rarefaction of the JCT and inner wall/JCT separation was much more prevalent in long-duration eyes, and data from all bovine eyes revealed a correlation between the extent of inner wall/JCT separation and the absolute value of outflow facility measured immediately prior to fixation ( p = 0.0024) as well as the washout-induced increase in outflow facility ( p = 0.0006). In human eyes, no significant morphologic differences were observed between long- and short-duration perfusion, with no observed change in inner wall/JCT separation or expansion between the two groups. Morphologic analysis revealed that the previously described “cribriform plexus” of elastic-like fibers was far more extensive in the JCT of human eyes, appearing to form numerous connections to the inner wall endothelium. The cribriform plexus appears to function as a mechanical tether that maintains inner wall/JCT connectivity in human eyes by opposing hydrodynamic forces generated during perfusion, potentially explaining the lack of washout in humans.