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1From the LSU Eye Center and the 3Department of Orthopaedic Surgery, Louisiana State University Health Sciences Center, New Orleans, Louisiana; and the 2Department of Biomedical Engineering, Tulane University, New Orleans, Louisiana.
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Abstract |
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METHODS. Short-term (3–9 weeks), moderate (
44 mm Hg) intraocular pressure (IOP) elevation was induced in one eye of each of eight male monkeys by lasering the trabecular meshwork. This procedure generated early experimental glaucoma, defined as the onset of CSLT-detected optic nerve head (ONH) surface change, in the treated eye. Scleral tensile specimens from the superior and inferior quadrants of the eight early-glaucoma eyes were subjected to uniaxial stress relaxation and tensile tests to failure and the results compared with similar data obtained in a previous study of 12 normal (nonglaucomatous) eyes. Linear viscoelastic theory was used to characterize viscoelastic material property parameters for each specimen. Differences in each parameter due to quadrant and treatment were assessed by analysis of variance (ANOVA).
RESULTS. Peripapillary sclera from the early-glaucoma eyes exhibited an equilibrium modulus (7.46 ± 1.58 MPa) that was significantly greater than that measured in normal eyes (4.94 ± 1.22 MPa; mean ± 95% confidence interval, P < 0.01, ANOVA). Quadrant differences were not significant for the viscoelastic parameters within each treatment group.
CONCLUSIONS. The long-term viscoelastic material properties of monkey peripapillary sclera are altered by exposure to moderate, short-term, chronic IOP elevations and these alterations are present at the onset of CSLT-detected glaucomatous damage to the ONH. Damage to and/or remodeling of the extracellular matrix of these tissues may underlie these changes in scleral material properties.
To characterize the material properties of peripapillary sclera, we constructed a controlled-environment testing apparatus capable of performing uniaxial biomechanical testing of soft tissues in tension. The overall response of a tissue to a load is the combination of instantaneous (elastic) and time-dependent (viscous) responses, which are governed by its viscoelastic material properties. All soft tissues are viscoelastic, and the characterization of the instantaneous and time-dependent aspects of these material properties is important in understanding a tissue’s behavior as a load-bearing structure and its response to short- and long-term changes in the applied load. Accurate characterization of a tissue’s material properties can lead to a broader understanding of the mechanisms that underlie tissue damage, as well as determine the resultant altered load-bearing behavior of a remodeled or healed tissue.
In a previous report we performed quadrant-based uniaxial tensile testing of peripapillary sclera from normal rabbit and normal monkey eyes3 and found no differences in the viscoelastic material properties of the peripapillary sclera by quadrant (superior, inferior, nasal, and temporal to the ONH) in either species. In addition, when normal rabbit and monkey sclera were compared with one another, peripapillary sclera from monkey eyes was stiffer and showed slower stress relaxation than sclera from rabbit eyes.
The present study tests the hypothesis that changes in the viscoelastic material properties of monkey peripapillary sclera are present at the earliest detectable stage of glaucomatous damage to the ONH, defined as the onset of confocal scanning laser tomography (CSLT)–detected ONH surface change.
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Materials and Methods |
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Specimen Preparation and General Testing Methodology
Our methodology, which was described in detail in our previous report,3 is briefly presented here. Each animal was anesthetized with an intramuscular injection of ketamine/xylazine and killed with an intravenous injection of pentobarbital. Immediately after death, each eye was enucleated and cleaned of all extraorbital tissues. The ONH was removed from the surrounding sclera with a 6-mm-diameter trephine for use in another study, with care taken to include as little of the peripapillary sclera in the testing quadrant as possible (typically, 1 mm). A dumbbell-shaped cutting die was used to produce a precise tensile specimen of peripapillary sclera with a gauge length of 8 mm, a gauge width of 3 mm, and grip-end widths of 7 mm. Before testing, the scleral specimens were stored in isotonic (pH 7.4) phosphate-buffered saline (PBS) for a minimum of 30 minutes at zero load to allow for equilibration and rehydration of the tissues.
Each scleral tensile specimen was clamped into custom-built, soft-tissue grips and mounted in a testing environment chamber. The custom-built chamber was designed to maintain the specimen in a fog of 37°C PBS for the duration of each testing sequence. The chamber was mounted on a servo-hydraulic test frame (Bionix 858; MTS, Inc., Eden Prairie, MN), fitted with an integrated computer-based test controller and digital data acquisition system (Teststar II; MTS, Inc.). A biomedical extensometer (model 632.32F; MTS, Inc.) and a 250-N load cell (in the 10% range, 0–25 N; model 661.11A; MTS, Inc.) were used to measure strain and load, respectively.
Uniaxial Testing in Tension
Peripapillary scleral specimens were tested as follows, using a protocol identical with that reported previously.3 The specimen was fitted in the testing chamber and warmed to 37°C in a fog of PBS at zero load for approximately 15 minutes. After an 0.08-N tensile preload was applied and the extensometer was mounted, the specimen was initially preconditioned with 10 cycles of 0 to 150 µm of grip-to-grip displacement at a rate of 150 µm/s in displacement control, followed by a 360-second recovery period at zero displacement. The specimen was then subjected to a second preconditioning phase consisting of 10 cycles of 50- to 350-µm displacement at 175 µm/s and rested again for 360 seconds. The second phase of preconditioning generated strain amplitudes of approximately 1% in the central 5-mm gauge section, and was performed to provide a cyclic loading regimen that could be used for validation of future computational models of scleral tensile specimens. The specimen was then subjected to a stress relaxation test consisting of a tensile ramp in strain rate control at a strain rate of 1% per second to 1% strain, with the ramp-ending displacement held for 1000 seconds. After a third 360-second recovery period at zero displacement, each specimen was subjected to a tensile ramp to failure (or a maximum of 20% strain) at a strain rate of 1%/s. Data for tensile load and gauge strain were recorded at 0.01-second intervals.
Estimation of Elastic and Viscoelastic Parameters for Each Specimen
Four principal viscoelastic parameters were derived from the stress relaxation test of each scleral tensile specimen. The instantaneous modulus, E0+, is a measure of the instantaneous stiffness of the tissue. The short-term and long-term time constants, 
S and L, describe the time-dependent stress relaxation response of the sclera and are measures of how quickly the tissue relaxes to an equilibrium stress after a rapid deformation (Fig. 1) . The short- and long-term time constants are measures of the shortest and longest time-scale components, respectively, of the tissue’s stress relaxation response. The equilibrium modulus, E
, is a measure of the tissue’s stiffness after it has fully relaxed after a rapid deformation (Fig. 1) and is a measure of the long-term stiffness of the tissue. The full derivation of these parameters, which is described in our previous report,3 is briefly described in the next section.
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A, exhibits a stress relaxation response, 
, that can be expressed as
 | (1) |
e (A) is the linear elastic stress response defined at equilibrium (time, t = ) and G(t) is the time-dependent, reduced-relaxation function that satisfies G() = 1.
We used the following functional form to define a spectral reduced-relaxation function
 | (2) |
1 = S and n = L are the short- and long-term relaxation time constants, respectively. Stress was normalized with respect to (t = ) and we used Hooke’s law as it applies to the fully relaxed state: () = E(), where E is Young’s modulus at equilibrium (equilibrium modulus). For given short- and long-term relaxation time constants, S and L, n spectral relaxation time constants were uniquely determined7 by the formula
 | (3) |
S and L on the log time scale. We calculated the instantaneous modulus of the tissue, E0+ = (0)/0, by evaluating equation 1 at time, t = 0+.
For a linear increase in strain at a finite rate, , beginning at time 0 and ending at time t0, the stress at time, t, according to equations 1 , 2 and 3 is
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 | (5) |
Equations 4 and 5 were fit simultaneously to the two stages of the experimental stress relaxation data6 (the tensile ramp and the relaxation phase), by using the Levenberg-Marquardt nonlinear, least-squares algorithm, which yields unique estimates7 of c, S, L, and E for a given value of n(Fig. 1) .
For a sufficiently large n, the parameters c, S, and L in the spectral relaxation function defined by equations 2 and 3 are equivalent to Fung’s well-known reduced-relaxation function,3 5 7 and c, S, and L can be uniquely defined independent of n.7 Chi-squared (
2, a measure of goodness of fit) was minimized to determine the minimum value of n necessary to guarantee the uniqueness of these parameters.3 The minimum n required for the parameters to converge to their unique, terminal values varied from 3 to 7, depending on the specimen. No significant change in the parameters resulted from increasing the number of spectral terms beyond that necessary to minimize 2.
Statistical Analysis
Viscoelastic Parameters.
An ANOVA was used to determine the effects of quadrant and treatment on the short- and long-term time constants, and the instantaneous and equilibrium moduli. Confidence intervals were calculated using the SE, which employs the pooled variance of all values in the ANOVA for each parameter.
Tensile Ramp to Failure Testing.
A second ANOVA was used to assess the effects of quadrant and treatment on the stress values obtained at the integer percentage values of strain (1%, 2%, ... , 10%) for the tensile ramp-to-failure tests.
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Results |
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The mean stress relaxation responses of peripapillary sclera (superior and inferior quadrants pooled) from both normal and early-glaucoma monkey eyes are shown in Figure 2 . Note the high degree of stress relaxation over the relatively short duration of the test and the higher equilibrium stress evident in the early-glaucoma group, which is a manifestation of the significantly higher equilibrium modulus in the early-glaucoma eyes.
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Discussion |
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The principal findings of the current report are as follows. First, peripapillary sclera (superior and inferior quadrants only) of early-glaucoma eyes exhibited an equilibrium modulus that was significantly greater than that measured in normal eyes (P < 0.01, ANOVA). Second, we detected no differences between peripapillary sclera from early-glaucoma and normal eyes in the instantaneous modulus and the short- and long-term time constants (P > 0.05, ANOVA). This finding suggests that the long-term elastic properties of monkey peripapillary sclera are altered by exposure to moderate (23–44 mm Hg), short-term (3–9 weeks), chronic IOP elevations and that these alterations are present at the onset of CSLT-detected glaucomatous damage4 to the ONH. Third, we detected no differences in the viscoelastic material properties of peripapillary sclera from the superior and inferior quadrants of early-glaucoma eyes. Finally, stress–strain curves from a tensile test to failure at a 1% per second strain rate were virtually identical in sclera from both normal and early-glaucoma eyes.
For the material properties of peripapillary sclera to be altered at this early stage of glaucomatous damage, the extracellular matrix (ECM) of the tissue must be damaged and/or remodeled. Cellular mechanotransduction and ECM remodeling have been reported in other load-bearing collagenous tissues, in which fibroblasts secrete factors that elicit changes in the surrounding ECM in response to an increase in strain.8 It is likely that such strain-induced changes in protein and genetic factor expression act to increase the resistance of the ECM to further strain, thereby shielding the tissues and cells from future damage. Changes in the ECM of the lamina cribrosa in glaucomatous human and monkey eyes with more advanced glaucomatous damage have been reported by other investigators.9 10 11 12 Quigley et al.13 reported a decrease in collagen fibril density within the lamina cribrosa and peripapillary sclera in human eyes with moderate or severe glaucomatous damage. Although their study did not detect this effect in monkeys, our results indicate that alterations in the ECM of peripapillary sclera occur early in response to chronic IOP elevation and that these alterations are sufficiently large to be detected using mechanical testing.
Although IOP-related stress remains constant across eyes with identical peripapillary geometry (thickness and curvature), strain (local deformation) within sclera that is identically loaded depends on the sclera’s material properties. Once the material properties of the peripapillary sclera are altered, the eye deforms differently under the same level of IOP-related stress. IOP-induced deformation within the peripapillary sclera should be an important contributing factor to the overall biomechanical response of the ONH. Peripapillary scleral deformation may be directly transferred to the peripheral laminar insertions through the scleral canal wall. Large scleral deformations may secondarily diminish the volume flow of blood through the contained branches of the posterior ciliary arteries.14 In addition, in those conditions, such as myopia, in which either the sclera is thinned15 16 or its ECM altered,17 scleral deformation after a similar change in IOP is likely to be increased.
We recently reported that posterior sclera of young adult monkeys thins in response to exposure to chronic IOP elevations,18 and this may also hold true for peripapillary sclera. If sclera from early-glaucoma eyes is thinner than sclera from normal eyes, then the scleral stress would be higher in the early-glaucoma eyes at a given IOP. With respect to the testing reported herein, this phenomenon would result in an increase in the instantaneous and equilibrium moduli we report in the early-glaucoma eyes, thereby accentuating the difference in the equilibrium modulus.
In physical terms, the increased equilibrium modulus that we report indicates that peripapillary sclera from early-glaucoma eyes is stiffer than that from normal eyes for IOP increases that last longer than 2 minutes (Fig. 2) . A stiffer scleral shell stretches less at a given elevated IOP and is thus less able to reduce IOP over time through globe expansion. Hence, after an IOP elevation of fixed magnitude lasting longer than 2 minutes (such as diurnal IOP fluctuations), IOP remains higher in an early-glaucoma eye than in a normal eye (Fig. 2) . However, this phenomenon would not be present during transient IOP elevations (blink, squint, or eye rub; Figs. 2 3 ), the response to which is largely dependent on the instantaneous modulus and short- and long-term time constants, parameters that exhibited no detectable difference by treatment. The virtually identical stress–strain curves plotted in Figure 3 confirm that the instantaneous elastic properties of sclera are not detectably altered in response to chronic IOP elevation.
Zeimer19 has suggested that a mismatch of the material properties of the lamina cribrosa and peripapillary sclera is an important contributor to the pathophysiology of glaucomatous damage to the tissues of the ONH. We have reported that hypercompliance of the ONH surface4 20 21 and underlying connective tissues4 is present at the same stage of early glaucomatous damage reported in the current study. If the ONH connective tissues have become more compliant and the peripapillary sclera less compliant in an eye with early glaucoma, IOP elevations (such as diurnal IOP fluctuations) may expose the glaucomatous ONH to higher shear stresses at the insertion of the lamina cribrosa into the sclera.
Our study is limited by the following considerations. First, due to the size of the harvested specimens and the nature of tensile testing, we could generate only one peripapillary scleral tensile specimen and one measurement of each of the material property parameters per eye. Thus, due to the lack of repeated measures, we were not able to directly compare the material properties of the peripapillary sclera from the normal and early-glaucoma eyes within individual monkeys.
Second, only eight early-glaucoma eyes were generated in this study, and so we were limited to testing specimens from the superior and inferior quadrants to maintain an adequate sample size of n = 4 per quadrant. Peripapillary scleral material properties of specimens from the nasal and temporal quadrants may be altered differently as a result of exposure to IOP elevations, compared with the superior and inferior quadrant data reported here. Future studies are necessary to characterize the peripapillary sclera fully at this early stage of connective tissue damage.
The limitations of the testing methodology have been described in detail3 and are recounted briefly here. First, we did not measure thickness within each specimen, but instead assigned a thickness of 400 µm to all our specimens based on our previous characterization of monkey peripapillary scleral thickness.22 The specimens were taken beginning approximately 1 mm from the scleral canal, which is outside the region of greatest variability in peripapillary scleral thickness.22 We were unable to measure scleral thickness with ultrasound or other nondestructive techniques, as it is not possible to distinguish the load-bearing sclera from the adjacent episclera in fresh tissues. Assigning a fixed scleral thickness for the calculation of stress is a major assumption and is a likely source of error in the reported results, although previous work has shown no statistically significant differences in the thickness of sclera from the superior and inferior quadrants in the peripapillary region.22 That the inferior and superior quadrants exhibit similar material properties suggests that there is little difference in scleral thickness between quadrants within treatment groups. Also, the nearly identical short-term stress–strain response of sclera from normal and early-glaucoma eyes (Fig. 3) and the lack of significant treatment effect in the instantaneous modulus and time constants (Table 2) suggest that scleral thickness did not vary significantly between treatment groups in this study. It is possible that sclera from the glaucomatous eyes is thinner and stiffer than sclera from normal eyes, resulting in the similar instantaneous responses we report, but that would only accentuate the reported difference in equilibrium modulus.
Second, the preload necessary to assure consistent mounting of the extensometer arms (0.08 N) generated scleral stresses that are relatively high compared with normal levels of IOP. Using this type of contact extensometer was necessary to employ strain-rate–controlled testing. A uniaxial tensile preload of 0.08 N is equivalent to an IOP of approximately 40 mm Hg.23 This is higher than the IOPs of 8 to 18 mm Hg we measured in the normal eyes of resting, anesthetized monkeys (Table 1) .
Third, uniaxial tensile testing was used to ascertain the isotropic (uniform) properties of an anisotropic (nonuniform) tissue. Sclera is likely to be stiffer in the direction of the predominant collagen fibril orientation, which depends on location in the globe. Our assumption of isotropic properties is based on previous work on fibril orientation in the peripapillary region, where the fibrils follow a circular path ringing the scleral canal.13 15 24 25 26 Uniaxial testing of tensile specimens from the peripapillary region should provide a valid first estimate of the material properties of sclera in the axial direction of the specimen, which coincides with the direction of predominant fiber orientation. The moduli of sclera in any direction not parallel to that of the predominant fibril orientation are likely to be lower than that reported here.
Finally, it should be noted that the early-glaucoma specimens were predominantly from cynomolgus monkeys and the normal specimens were predominantly from rhesus monkeys (Table 1) . This was inadvertent and may be a source of error if there are species-specific differences in peripapillary scleral material properties. We performed an ANOVA to compare the viscoelastic properties of inferior scleral tensile specimens 11 to 14 (rhesus) to that of specimens 19 and 20 (cynomolgus; Table 1 ), and found no significant differences in any of the parameters by species (data not shown).
The uniaxial viscoelastic material properties reported in Table 2 are being incorporated into finite element models of the posterior scleral shell and ONH of the normal and early-glaucoma monkey to study the role of IOP in the development and progression of glaucoma (Bellezza AJ, et al. IOVS 2003;44:ARVO E-Abstract 1094; Downs JC, et al. IOVS 2002;43:ARVO E-Abstract 4042). Future improvements in testing will allow us to generate biaxially determined anisotropic material properties of the sclera and incorporate these refined properties into our models.
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Footnotes |
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Supported in part by Grants R01EY011610 (CFB) and P30EY002377 (departmental core grant) from the National Eye Institute; a grant from The Whitaker Foundation, Arlington, Virginia (CFB); and a Career Development Award (CFB) and an unrestricted departmental grant (LSU Eye Center) from Research to Prevent Blindness, Inc., New York, New York.
Submitted for publication February 5, 2004; revised July 19, 2004; accepted August 16, 2004.
Disclosure: J.C. Downs, None; J-K.F. Suh, None; K.A. Thomas, None; A.J. Bellezza, None; R.T. Hart, None; C.F. Burgoyne, 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: J. Crawford Downs, LSU Eye Center, 2020 Gravier Street, Suite B, New Orleans, LA 70112; jdowns{at}lsuhsc.edu.
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