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1From the Departments of Ophthalmology and 3Orthopedic Surgery, University of California-San Francisco, San Francisco, California; and the 2Department of Mechanical Engineering, University of California-Berkeley, Berkeley, California.
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Abstract |
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METHODS. Scleral discs 18 mm in diameter were dissected from the medial and lateral equatorial regions of 60 cadaveric porcine eyes. Samples were incubated for 24 hours with control solution or methylglyoxal at concentrations of 0.001%, 0.01%, 0.10%, and 1.00%. Nonenzymatic cross-link density in treated and control groups was quantified with the use of papain digest and fluorescence spectrophotometry. Treated scleral discs were mounted in a customized Ussing-type chamber connected to vertical tubing, and specific hydraulic conductivity was determined according to the descent of a column of degassed saline at room temperature. Permeability to diffusion of fluorescein in a static chamber was determined for another set of treated scleral samples.
RESULTS. Methylglyoxal treatment effectively increased nonenzymatic cross-link content, as indicated by the average fluorescence for each group. Specific hydraulic conductivity (m2) was reduced with increasing cross-link density. Similarly, the permeability coefficient for the fluorescein solute consistently decreased with increasing methylglyoxal concentration, indicating diffusion impedance from the treatment.
CONCLUSIONS. Nonenzymatic cross-link density can be significantly increased by treatment with methylglyoxal. Porcine sclera showed a nonlinear reduction in solute permeability and specific hydraulic conductivity with increasing cross-link density. This model indicates that age-related nonenzymatic cross-link accumulation can have a substantial impact on scleral permeability.
In vitro studies show that fluid flow and solute diffusion across the sclera are influenced by scleral thickness, hydration,6 solute size,3 6 and intraocular pressure.7 Specific hydraulic conductivity (also known as intrinsic permeability), a parameter indicating the ease with which a fluid moves through a porous medium, has been reported to decline with age in human scleral samples, a finding that has been attributed to the age-related reduction in scleral hydration.8
Aging is associated with the accumulation of enzymatic and nonenzymatic (glycation) cross-links in collagenous tissues throughout the body.9 10 We have found that the aged human sclera tends to have nonenzymatic cross-link densities nearly three times those of a juvenile porcine model.11 Glutaraldehyde-induced cross-linking has been shown to impair the diffusion of large solutes across rabbit sclera, suggesting that cross-links could be a determinant of permeability, possibly by reducing interfibrillar distances or hydration.6 12 Given the potential of exogenous cross-links to affect fluid transport in the eye, the relationship between cross-links and scleral permeability has not been adequately investigated.
We hypothesized that increasing collagen cross-link density in sclera decreases permeability and solute diffusion. To investigate whether nonenzymatic cross-links are a determinant of permeability, we treated porcine sclera with methylglyoxal (MG), a Maillard pathway intermediate,13 and quantified specific hydraulic conductivity and permeability to fluorescein diffusion.
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Methods |
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Tissue samples were incubated at 37°C in 5% CO2 atmosphere for 24 hours in Dulbecco phosphate-buffered saline (PBS), containing 1% penicillin-streptomycin and protease inhibitor cocktail tablets (10213200; Roche, Mannheim, Germany) before their use in experiments. Selected samples also received 0.001%, 0.01%, 0.10%, or 1.00% MG (Sigma, St. Louis, MO).
Scleral Hydration
Variations in scleral weight after treatment were measured by comparing posttreatment weight to its pretreatment weight. Before incubation in the treatment solutions, scleral samples were blotted dry and weighed, and their thickness was measured. After 24 hours at 37°C, the scleral pieces were removed, blotted, and reweighed, and the percentage difference in water content was measured as the ratio of the difference between posttreatment and pretreatment weight to the pretreatment weight. Thickness was measured after treatment to confirm it was not affected.
Effects of the treatment solutions on scleral hydration were measured in a similar fashion. Tissue samples were blotted dry and weighed before they were incubated in the treatment solutions. After 24 hours, the scleral pieces were removed, blotted, and reweighed. Samples were then dehydrated at 70°C for 24 hours and reweighed. To determine the effects of cross-linking on the hydration of sclera, scleral hydration was calculated as the ratio of the difference between the posttreatment wet weight and the dry weight to the dry weight. Variations in scleral hydration were compared between treatment groups to control (0% MG) sclera to calculate the percentage change in hydration.
Cross-link Assay
Nonenzymatic glycation-type cross-links formed as the result of the attachment of the carbonyl group of glucose, followed by a ketoamine rearrangement. The cross-links formed because of the Maillard-type reactions that followed then yielded fluorophores characteristic of a reaction of a sugar with a protein. Therefore, fluorescence of a papain digest supernatant in our experiment is an indicator of nonenzymatic cross-link content. There is a linear relationship between fluorescence and increasing cross-link content.14 15 16
Tissue samples were cut from globe tissue adjacent to the medial and lateral scleral discs, weighed, and placed in sealed plastic bags at –80°C for no longer than 2 weeks. Only samples weighing between 10 and 25 mg were used. Before the execution of the assay, the tissue was pulverized to a fine powder to encourage a thorough reaction with the papain. This was accomplished by transferring the tissue to a test tube, submerging that tube in liquid nitrogen for 2 minutes to further cool the tissue into a mechanically brittle state, transferring the tissue to a milling capsule containing a pair of 12-mm-diameter stainless steel balls (also previously submerged in liquid nitrogen for 2 minutes), cyclically displacing the milling capsule and its contents in a milling machine (MM301 ball mill; Retsch Corporation, Haan, Germany) for 3 minutes at 30 Hz, and weighing a portion of the pulverized tissue for use in the assay.
To each pulverized tissue sample was added 1 mL of a 10 mM solution of dithiothreitol in a digestion buffer (0.1 M Na acetate/2.4 mM disodium EDTA). To each tissue sample was then added 20 µL of a 1 mg papain/1 mL digestion buffer solution. The tissue was incubated at 60°C for 12 hours, and then 20 µL of the papain solution was added again. After 12 more hours of incubation, the tubes were centrifuged at 10,000 rpm for 12 minutes. The supernatant was removed and was used to perform fluorescence measurements.
To measure fluorescence, a 100-µL aliquot of each papain supernatant was removed to a black ELISA plate, and fluorescence was measured in the plate reader at an excitation wavelength of 370 nm with emission sensitivity set at 440 nm because these wavelengths give the greatest resolution in detecting nonenzymatic cross-link residuals.
Diffusion Assay
In vitro diffusion studies were conducted using PVC unions (American Valve, Greensboro, NC). The tissue was blotted dry and placed between two stainless steel annular platens having 12.7-mm central apertures. The tissue was mounted without stretching so as to avoid asymmetrical stresses. This assembly was mounted between the two reservoir chambers, and the union housing was threaded together to provide the compression necessary to prevent leakage around the specimen (Fig. 1) . The open ends of the union were sealed (Parafilm; Pechiney Plastic Packaging, Chicago, IL) after both chambers were filled with PBS and were visually inspected for leakage. The apparatus was incubated at 37°C in a 5% CO2 atmosphere for 1 hour to restore normal hydration and temperature.
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Diffusion from the donor chamber to the receiver chamber was characterized by means of a permeability constant P, (cm/s). The apparent permeability coefficient, P, was calculated using the equation
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Fluorescence was measured at room temperature with a fluorescence spectrophotometer (SpectraMax M5; Molecular Devices, Sunnyvale, CA). Excitation and emission wavelengths were 492 and 520 nm, respectively. Standard curves of fluorescence versus concentration were obtained by serial dilution of fluorescein in PBS. Concentrations of samples were determined by linear regression analysis within the linear portion of the standard curve. The values of steady state flux were estimated from the linear portion of the cumulative amount of drug permeated (µg/cm2) versus time (min) profile in each case. All the permeation studies were carried out in duplicate, and the results were expressed as mean ± SD.
Specific Hydraulic Conductivity Determination
In vitro permeability studies were conducted using a modified version of the two-chamber system described (Fig. 1) . Tissue samples were mounted in the reverse fashion, with the uveal side of the sclera facing the donor reservoir and the orbital side facing the receiver reservoir chamber. A reduced-sized washer (6.35-mm diameter central aperture) was used for these experiments. Subsequent tissue mounting steps are similar to those described in the previous section.
The assembled apparatus was connected to identical fluid columns on both ends and filled with degassed PBS containing 1% penicillin-streptomycin and protease inhibitors. The system was visually inspected for fluid leakage and allowed to equilibrate for 1 hour. Periodically, the apparatus was tapped lightly to remove any accumulated air bubbles. The donor column height was set at 25 cm above the receiver column height, and the experiment was begun. This height was chosen because it created a pressure differential within a physiologic range (18 mm Hg). Typical tubing contained 1 mL/14.9 cm. The descent of the water column on the donor side was allowed to proceed for 24 hours. At the end of the trial, the difference in height of the water columns was recorded. The experiment was conducted at room temperature.
Specific hydraulic conductivity (
) of the tissue samples as derived from Darcy’s law17 was calculated using the equation
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h) of the water column to arrive at a flow volume divided by time, µ is the viscosity (Pa · s) of the diffusion medium under experimental conditions, L (m) is the scleral thickness, A (m2) is the area of the aperture, and P (Pa) is the mean pressure head during the experiment. Results were expressed as mean ± SD.8 18 |
Results |
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Increasing the concentration of the MG treatment was associated with a reduction of the permeability coefficient (Fig. 3) . At the higher concentrations of 0.1% MG (1.94 ± 0.45 x 10–7 cm · s–1) and 1% MG (1.66 ± 0.33 x 10–7 cm · s–1), this decline was significant (P < 0.05) compared with controls (2.72 ± 0.46 x 10–7 cm · s–1). At 0.01% MG (2.42 ± 0.40 x 10–7 cm · s–1), this trend of reducing the permeability coefficient did not reach statistical significance (P = 0.21) compared with controls.
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As with fluorescein permeability, increasing the concentration of the MG treatment was associated with a reduction of specific hydraulic conductivity (Fig. 3) . At the higher concentrations of 0.1% MG (1.22 ± 0.34 x 10–17 m2) and 1% MG (0.97 ± 0.16 x 10–17 m2), this decline was significant (P < 0.05) compared with controls (4.33 ± 2.36 x 10–17 m2). At 0.001% MG (2.67 ± 0.73 x 10–17 m2) and 0.01% MG (2.30 ± 1.04 x 10–17 m2), this trend of reducing the hydraulic conductivity did not reach statistical significance (P = 0.12 and P = 0.07, respectively) compared with controls.
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Discussion |
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According to the analysis of the porous medium model suggested by Bear and Bachmat,19 the determinants of permeability can be shown to depend on three elementary porous medium properties: porosity, average medium conductance, and average tortuosity. Average medium conductance can be thought of as the average size of channels through the porous medium, whereas tortuosity can be considered the complexity of physical pathways through the extracellular matrix. Tortuosity and conductance are primarily determined by collagen fibril architecture. Collagen cross-links serve to alter and constrain fibril architecture. Constraints imposed by cross-links reduce conductance and interstitial space and increase the average level of tortuosity. We speculate that this is the fundamental mechanism behind the changes observed in permeability and diffusion behavior.
The permeability of sclera to passive diffusion of solutes has been evaluated.3 6 7 Generally, the sclera has been found to be permeable to molecules up through 150 kDa. This has been taken as an indication that transscleral delivery of pharmacologic agents is feasible. Based on the results of our study, it seems likely that the density of scleral cross-links could play a role in determining the effectiveness of transscleral drug delivery in a given patient and may have to be taken into account when devising dosing strategies.
Specific hydraulic conductivity of sclera has been measured in human samples by Jackson et al.,8 who found that it declines with age. This was attributed to declining hydration and changes in scleral composition; changes in proteoglycan content were specifically mentioned. This finding has also been proposed by Brown et al.20 and Boubriak et al.6 Although hydration levels and hydrophilicity of the ground substance may very well influence permeability, our data suggest that age-related cross-link accumulation is likely to have played a key role in the results of Jackson et al.8 Although we did not quantify matrix glycosaminoglycans in the present study, our earlier data indicate that natural variability in their levels would be unlikely to explain the permeability data obtained herein.11 To validate this theory, it would be necessary to measure permeability characteristics in human scleral samples and to correlate the findings with cross-link content, water content, proteoglycan content, and perhaps collagen content. It is likely that all the aforementioned factors may influence flow pathways through the sclera. Our results support the idea that cross-link content is a primary factor determining scleral permeability.
We used MG treatment as an in vitro strategy to mimic the accumulation of nonenzymatic cross-links that result from glycation in vivo.21 An added complexity in vivo is the accumulation of enzymatic (lysyl oxidase-dependent) cross-links. This study used pharmacologically induced nonenzymatic cross-links as a proxy for all age-related cross-links because the levels of the two types of cross-links are directly correlated,22 but the lack of enzymatic cross-links remains a limitation of this model.
We have demonstrated a correlation of increasing MG concentration in the treatment solution with increasing cross-link content, measured by fluorescence quantification. Fluorescence levels are believed to provide a direct indication of nonenzymatic cross-link density.15 The ability to achieve specific cross-link density levels in tissue samples in vitro based on titration of the cross-linking agent, in this case MG, could prove useful in future experiments in which this technique may be used to create models of diseased or aged tissue, with severity correlating with cross-link density levels. Of note, in this porcine model, all the eyes came from a relatively homogeneous population of young pigs. We have previously shown that scleral samples from this porcine population have relatively low cross-link content, as would be expected in a young, nondiabetic population,11 and therefore have the capacity to acquire additional cross-links through exogenous treatment, making this a suitable model system. Because we found it difficult to obtain aged porcine sclera to compare with young porcine sclera, we chose to use this MG treatment model for age-related cross-links. Nevertheless, the lack of a direct comparison of the cross-link content and permeability in young and old porcine sclera is a limitation of this study.
Underlying the use of this exogenous cross-linking treatment as a model for age-related cross-link accumulation is the assumption that the cross-link density achieved with our MG treatment regimes is clinically relevant. If the MG treatment were saturating the tissue with cross-links beyond the levels that occur in vivo, the usefulness of this model’s permeability data would be in question. Our earlier work confirms that our 1% MG treatment achieves scleral cross-link content within the same order of magnitude as occurs in aged human sclera.11 In fact, the density of nonenzymatic cross-linking achieved with MG treatment in this study likely underestimates the amount of cross-links in an aged human eye. The average cross-link content in the strongest treatment group, 1% MG, was 61.4 ± 12.3 fL/mg, whereas we previously determined that aged (66–88 years) human sclera has an average content of 88.6 ± 23.4 fL/mg.15 On this basis, it seems probable that the degree to which aged human scleral permeability is reduced in vivo because of cross-links is likely to be greater than the amount identified in the model system used herein.
This study’s findings have important implications for our understanding of solute permeation into the eye (drug delivery) and fluid egress and homeostasis in the eye, key considerations in intraocular pressure regulation and glaucoma. Our data suggest that drug penetration into the eye would be expected to decline with age because of cross-link accumulation in tissues. In fact, permeation of molecules has been shown to be impaired in aged rabbit cornea, likely through this mechanism.23 In diabetes, in which high levels of cross-links and advanced glycation end products accumulate in tissues, permeability might be expected to be impaired as well, perhaps more severely than by aging alone; whether the reduction in permeability is clinically relevant remains to be determined, though this seems likely given the magnitude of the reduction identified in this study. In vivo pharmacokinetics studies are not generally performed in aged or diabetic patients, but this should be considered if a therapy is intended for use in this population and accurate permeation data are needed. Finally, studies are under way to evaluate exogenous scleral cross-linking as a new therapeutic modality for progressive myopia.24 Our findings suggest that scleral permeability could be impaired as a consequence of this treatment.
In summary, this study has identified nonenzymatic cross-linking as a determinant of scleral permeability and specific hydraulic conductivity using an in vitro model. Exogenous cross-linking was shown to impair solute diffusion and fluid flow through sclera. Further studies are needed to quantify the effects of age- and disease-related cross-link accumulation on these properties in human sclera, but, taken together with our earlier work elucidating the cross-link content of human scleral samples, these findings suggest that exogenous cross-linking can serve as a useful model system for screening for the effects of aging on tissue permeability. Additionally, modulation of cross-link content in vivo may offer an opportunity to affect permeability and thereby influence transscleral drug delivery and uveoscleral fluid outflow.
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Footnotes |
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Submitted for publication May 15, 2008; revised July 18, 2008; accepted October 7, 2008.
Disclosure: J.M. Stewart, None; D.S. Schultz, None; O.-T. Lee, None; M.L. Trinidad, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Jay M. Stewart, Department of Ophthalmology, University of California-San Francisco, K301, 10 Koret Way, San Francisco, CA 94143-0730; stewartj{at}vision.ucsf.edu.
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This article has been cited by other articles:
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  J. M. Stewart, D. S. Schultz, O.-T. Lee, and M. L. Trinidad Collagen Cross-Links Reduce Corneal Permeability Invest. Ophthalmol. Vis. Sci., April 1, 2009; 50(4): 1606 - 1612. [Abstract] [Full Text] [PDF]
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