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 microl/min per mmHg (mean+/-SEM) at baseline to 1.92 +/- 0.30 microl/min per mmHg after 3 h due to washout. The pre-fixation outflow facility in long-duration eyes (1.92 +/- 0.30 microl/min per mmHg) was 2-fold greater than pre-fixation facility in short-duration eyes (0.92 +/- 0.11 microl/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 microl/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.

Figure 1. Morphologic Assessment of Separation between the Inner Wall/JCT in the Bovine Eye

The degree of inner wall/JCT separation observed in each section was assessed based on a scale of 0 (no separation), + (less than approximately 1/3 of total length of the inner wall), ++ (more than approximately 1/3 but less than approximately 2/3 of the total length of inner wall), and +++ (more than approximately 2/3 of the total length of the inner wall). The length of the black lines corresponded to the estimated length of inner wall/JCT separation in each case.

Figure 2. Human and Bovine Outflow Facility

Outflow facility in long-duration bovine eyes increases (81%; p=0.049) vs. short-duration. Human long-duration eyes show no significant change in outflow facility (p>0.3) vs. short-duration.

Figure 3

A. Light Micrograph of Bovine Aqueous Plexus after Short-Duration Perfusion. No distention or separation between the JCT and inner wall (IW) of the bovine aqueous plexus (AP) is observed. The JCT/inner wall region appears organized and intact. B. Light Micrograph of Bovine Aqueous Plexus after Long-Duration Perfusion. A clear separation (Double headed arrow) can be seen between the JCT and the inner wall (IW) of the aqueous plexus (AP). The inner wall distends into the lumen of the AP while the underlying tissue appears disorganized.

Figure 4. Correlation of Absolute Value of Pre-Fixation Facility and JCT/Inner Wall Separation Scores

The absolute value of outflow facility measured prior to fixation correlated with the extent of inner wall/JCT separation in all bovine eyes (p=0.0024). Closed circles = long-duration, open circles = short-duration.

Figure 5

A Transmission Electron Micrograph of Bovine Aqueous Plexus after Long-Duration Perfusion Significant separation between the basal lamina (BL) of the inner wall (IW) of the bovine aqueous plexus (AP) and underlying extracellular matrix of the JCT region is observed (double headed arrow). Scale bar: 5μm B Transmission Electron Micrograph of Bovine Aqueous Plexus after Short-Duration Perfusion No distention of the JCT or separation of the IW lining of the aqueous plexus (AP) was observed. Giant vacuoles (GV) were seen along the inner wall. Scale bar: 5μm

Figure 6

A Light Micrograph of Human JCT/Inner Wall Region after Short-Duration Perfusion. No distention or separation between the JCT and inner wall (IW) of Schlemm’s canal (SC) is observed. The JCT/inner wall region appears organized and intact. Scale bar: 5μm B Light Micrograph of Human JCT/Inner Wall Region after Long-Duration Perfusion. No distention or separation between the JCT and inner wall (IW) of Schlemm’s canal (SC) is observed. The JCT/inner wall region appears organized and intact. Scale bar: 5μm

Figure 7

A Transmission Electron Micrograph of Human Schlemm’s Canal after Short-Duration Perfusion B Transmission Electron Micrograph of Human Schelmm’s Canal after Long-Duration Perfusion. No separation is observed between the inner wall (IW) of Schlemm’s canal (SC) and the underlying JCT in both short and long-duration perfusion (A and B, respectively). Giant vacuoles (GV) are seen along the IW of SC with a similar distribution in both short and long-duration perfusion. Compared to bovine eyes (Figure 5A & B) there is more complex array of elastic fibers (EL) in the JCT region (single headed arrows), especially in the area immediately underneath the inner wall (IW). Scale bars: 3μm