In the present study, we demonstrated that resistance decreased as volume perfused through the AC increased during outflow facility determination by two-level constant pressure perfusion in both rhesus and cynomolgus monkeys. We also found an increase in the rate of change in resistance as a function of volume perfused that was significant for cynomolgus monkeys, but not for rhesus monkeys. As in previous studies10,17
we found an overall decrease in baseline outflow resistance (increase in outflow facility) with volume perfused in both rhesus and cynomolgus monkeys that was not correlated with age in either species. However, after correcting for total volume perfused, we did find a significant age-related decrease in the rate of change in resistance in rhesus monkeys that was not present in cynomolgus monkeys. This indicates that age may play a role in the rate of resistance change during AC perfusion. That there was not a significant difference in the rate of change in resistance with age in cynomolgus monkey eyes may be explained by the fact that we were able to obtain data only from young and middle-aged animals, unlike the rhesus monkey group that included more middle-aged and elderly animals. We also found a nearly significant decrease in total volume perfused with age in rhesus monkeys and a similar, although not significant, trend in cynomolgus monkeys.
The exact mechanism(s) for washout are not entirely clear. Changes in resistance could be the result of mechanical disruption or removal of extracellular material from the outflow pathways.5,29,30
Outflow resistance in the trabecular meshwork (TM) outflow pathways has been shown to increase with age in humans and rhesus monkeys.11,12,14
Buildup of extracellular material within the interstices of the TM has been implicated in the age-related increase in outflow resistance seen in humans.31,32
Similarly, increases in sheath-derived plaques and fibrillar material in the juxtacanalicular tissue region and a decrease in overall cellularity of the TM have been found with increasing age in rhesus monkeys.23
Loss of trabecular cells with age could result in a reduction in matrix metalloproteinase (MMP) activity in the TM. This may result in a reduced capacity of the TM to break down extracellular material.23
In fact, passage-number–related reduction in MMP activity has been found in porcine trabecular cell cultures exposed to 15–50 mm Hg of pressure for 72 hours (Ehrich D, et al. IOVS
2001;42:ARVO Abstract 748) and in human TM cells in vitro (Williams GC, et al. IOVS
2001;42:ARVO Abstract 764). This increase in extracellular material and loss of cellularity seen in older animals might affect the rate of change in outflow resistance during AC perfusion. Removal of extracellular material from the TM during AC perfusion may need to reach a “critical mass” in these older monkeys (a certain amount of time may need to elapse and/or a sufficient amount of fluid may need to pass through the outflow tissues) before the rate of change in resistance is similar to that seen in young and middle-aged monkeys. In the present study, we measured outflow resistance for only 30 to 45 minutes, whereas outflow resistance washout typically occurs for the first 1 to 2 hours during AC perfusion.1
Therefore, it may be useful to determine whether the rate of change in resistance is similar in groups of young, middle-aged, and older monkeys when perfused for a longer period.
Previous studies have indicated that decreased resistance may be due to a disruption in the connectivity between the inner wall endothelium of Schlemm's canal and the juxtacanalicular tissue6,8,33,34
) that may lead to an increase in the effective filtration area of aqueous outflow.35
Scott et al.8
found that a greater separation of the inner wall and juxtacanalicular tissue was significantly correlated with a larger absolute value of outflow facility in enucleated bovine eyes. In fact, studies have shown that enucleated human eyes do not exhibit the washout effect36
; moreover, human eyes, evaluated by light and electron microscopy after undergoing long-duration perfusion (~3 hours), were found to have no apparent separation between the inner wall and juxtacanalicular tissue,8
supporting the theory that decreased resistance may be caused by this separation of outflow tissues rather than actual “washout” of extracellular material.
Our findings support previous reports indicating that outflow resistance decrease during perfusion is largely, and often primarily, dependent on volume rather than on age.9,11,37
) However, in populations with old/elderly animals, such as our rhesus group, we found that age does play a significant role in the rate of change of resistance, and may be an even more important factor than total perfusion volume when examining the rate of resistance washout. In our study, eyes were perfused for the same length of time, so that any change in outflow resistance over the course of the perfusion would have been due to some factor other than time, such as volume perfused or age. The number of previous experimental procedures an animal has undergone could theoretically influence the rate of change in resistance, thus contributing to the change we observed in the older rhesus monkeys. However, this seems highly unlikely since all animals in the rhesus monkey group had only one perfusion and no monkey in either group had more than four previous perfusions. Since the older rhesus did not undergo any more ocular experimental manipulations than did the younger rhesus, the number of procedures cannot explain the decrease in rate of change in resistance seen with age. Many of the cynomolgus monkeys underwent more than one perfusion, but no difference in rate of change in resistance was observed among monkeys in this group, irrespective of whether they underwent one perfusion or more than one perfusion.
The present study suggests that the factors that lead to changes in outflow resistance, such as removal of extracellular material and/or the separation of the inner wall and juxtacanalicular tissue, may occur at a slower rate with increasing age. This may be partly explained by the fact that the age-related increase in overall outflow resistance allows less volume to pass through the outflow pathways, which leads to a slower rate of resistance washout.