Comparative studies between species that do and do not exhibit the washout effect
Introduction
The “washout effect” is a phenomenon in which perfusion of an eye at physiological pressure results in a volume-dependent increase in the measured facility of aqueous humor outflow. Washout was originally believed to result from a “washing out” of extracellular glycosaminoglyans (Bárány and Scotchbrook, 1954, Bárány and Woodin, 1955, Bárány, 1962, Bárány, 1964) from the outflow pathway, but biochemical studies have failed to detect an appreciable change in either sulfated proteoglycans (Johnson et al., 1993) or hyaluronic acid (Knepper et al., 1984) from the outflow pathway following prolonged perfusion. While the rate of facility increase during washout can be slowed by perfusion with either serum (Kee et al., 1996, Johnson et al., 1993) or serum proteins (Epstein et al., 1978, Sit et al., 1997) or by perfusion with either pooled homologous aqueous humor (Bárány and Woodin, 1955, Gaasterland et al., 1978) or mock aqueous humor with similar biochemical constituents (Erickson and Kaufman, 1981, Gaasterland et al., 1979), the washout effect cannot entirely be eliminated (Bárány and Scotchbrook, 1954, Van Buskirk and Brett, 1978).
Possibly the most intriguing aspect of the washout effect, however, is that it does not occur in the human eye (Erickson-Lamy et al., 1990). This suggests that there is some unique aspect of outflow anatomy or physiology that distinguishes human eyes from most other species, including from non-human primate eyes that exhibit washout during perfusion both in vivo (Bárány, 1962, Bárány, 1964, Erickson and Kaufman, 1981, Gaasterland et al., 1978, Gaasterland et al., 1979, Kaufman et al., 1988) and in vitro (Epstein et al., 1982, Hashimoto and Epstein, 1980, Peterson and Joscon, 1974), despite their anatomical similarity to humans. A thorough understanding of the mechanism of washout, and the reason for its absence in humans would likely provide important insight into the fundamental mechanisms that generate outflow resistance. Such understanding might also permit us to artificially induce a washout-like response in human eyes as a means of reducing intraocular pressure in glaucoma.
Two recent studies (Overby et al., 2002, Sabanay et al., 2004) have documented that washout is a reversible process in both bovine and monkey eyes. A structural correlate to the facility increase during washout in bovine eyes appeared to be the degree of separation of the JCT from the inner wall of the aqueous plexus—the bovine equivalent to Schlemm's canal (Tripathi, 1971). This separation was proposed to increase outflow facility by disrupting a hydrodynamic interaction between the inner wall and JCT known as “funneling” (Johnson et al., 1992). The funneling theory states that the patterns of outflow through the JCT are confined to those regions nearest the pores in the inner wall, and this flow confinement reduces the filtration area through the JCT, thereby increasing its effective hydrodynamic resistance. Based upon our prior work in bovine eyes, we hypothesized that washout resulted from a disruption in the connectivity between inner wall and JCT that decreased outflow resistance by eliminating the funneling effect (Overby et al., 2002). The washout hypothesis and funneling theory emphasize the role of cellular and extracellular matrix adhesions that maintain the connectivity between the inner wall and JCT in the face of an opposing pressure gradient and thereby influence outflow resistance by controlling the local hydrodynamic patterns of outflow.
In this study, we hypothesize that if the structural correlate for washout is separation between the inner wall and JCT, then these morphological changes should not be found in human eyes subject to prolonged perfusion. Light and electron microscopy were used to compare the baseline morphological differences in the outflow pathway and morphological changes following prolonged perfusion in bovine eyes that exhibit washout and human eyes that do not exhibit washout. Our goal was to determine whether the absence of washout in human eyes relates to morphological differences unique to the human outflow pathway. A corollary of our hypothesis is that the absence of washout in the human eye may result from an enhanced connectivity between the inner wall and JCT, which would have important consequences for the regulation of outflow resistance in this species.
Section snippets
Materials and methods
Enucleated eyes from both bovine and human donors were used in this study. Eight bovine eyes were obtained from a local abattoir (Arena and Sons, Hopkinton, MA) and delivered on ice within 6 h post-mortem. Eyes with any discernible damage or accumulated blood in the angle of the anterior chamber were excluded. Eight human eyes from anonymous donors with no known history of eye disease (ranging from 33 to 89 years of age) were obtained from National Disease Research Interchange (Philadelphia, PA)
Outflow facility in human and bovine eyes
A summary of outflow facility data from all human and bovine eyes is presented in Table 1. The average baseline facility for the short- and long-duration perfused bovine eyes was 0.98 ± 0.06 μL/min per mmHg (mean ± SEM), which was nearly 5-fold larger than the average human baseline facility 0.21 ± 0.04 μL/min per mmHg. In bovine eyes, long-duration perfusion led to washout with an 81% increase (Table 1; Fig. 2) in outflow facility (p = 0.049) from 1.06 ± 0.06 μl/min per mmHg at baseline to 1.92 ± 0.30 μl/min
Discussion
During experimental perfusion of non-human eyes, outflow facility progressively increases due to a phenomenon known as washout. Because washout occurs in many different species (Bárány, 1953, Bárány, 1964, Gaasterland et al., 1978, Kaufman et al., 1988, Melton and Deville, 1960, Pandolfi, 1967, Van Buskirk and Brett, 1978, Yan et al., 1991) including non-human primates (Bárány, 1962, Bárány, 1964, Erickson and Kaufman, 1981, Kaufman et al., 1988) and is observed despite differences in perfusion
Acknowledgements
Supported by NIH grants EY007149 and EY-09699, the Glaucoma Research Foundation, National Glaucoma Research, a program of the American Health Assistance Foundation and The Massachusetts Lions Eye Research Fund, Inc to Boston University. We thank Dr Kristine Erickson for the generous use of her ocular perfusion system. We are grateful for the technical assistance of Rozanne Richman, MS.
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2011, Experimental Eye ResearchCitation Excerpt :This may be due to the shorter time and smaller volume perfused in the Y27 treated group (30 min) than washout group (3 h). Similar to our previous bovine studies[Lu et al., 2008; Overby et al., 2002; Scott et al., 2007], a positive correlation was found between the outflow facility and PSL in monkey eyes after either Y-27 treatment or induction of washout. However, separation in bovine eyes was found between the basal lamina of the inner wall endothelial cells and the extracellular matrix of the JCT (matrix–matrix separation).