In this study, we characterized scleral biomechanical behavior in both eyes of eight adult monkeys in which one eye had been exposed to chronic, laser-induced IOP elevations of modest to substantial magnitude and duration. The following are the principal findings of this report.
When considered as a group of 16 monkey eyes without regard for experimental condition (normal versus EG) the eyes in this report behaved similarly to the eyes from bilaterally normal monkeys from a previous report,8
as follows. First, the monkey posterior sclera is thicker in the peripapillary and temporal regions. Second, the monkey posterior sclera is a nonlinear, anisotropic, and inhomogeneous soft tissue. Third, concentrations of tangent modulus, structural stiffness, and maximum principal stress and strain were evident adjacent to the scleral canal in each eye. Fourth, maximum principal stress magnitude was substantially higher than IOP. Fifth, maximum principal strain magnitude was low. A detailed discussion of these results can be found in our previous report.8
Age-Related Changes in Scleral Biomechanics
Scleral biomechanical properties (i.e., tangent modulus and structural stiffness; ) in the eight normal adult eyes (mean age, 17.3 ± 5.1 years) from this study fell between those from young (1.5 ± 0.7 years) and old (22.9 ± 5.3 years) monkeys from our previous aging study.8
These findings separately confirm our initial report of age-related stiffening of the posterior sclera by establishing that adult eyes are stiffer than young ones but less stiff than old eyes. This result is likely to be related to increasing scleral cross-links with age28,52
; details on this topic can be found in our previous report.8
We have reported a decrease in scleral thickness in old compared with young normal monkey eyes. In that study, we found that adult monkey sclera was not significantly thinner than young monkey sclera, but was significantly thicker than sclera from old monkey eyes. This finding is important because it suggests that the rate of scleral thinning, if present, may accelerate with age. Whether scleral thinning with age could accelerate the progression of glaucoma in elderly patients remains unclear. In monkeys, we observed a reduction in peripapillary scleral thickness of approximately 8.4% from adult to older eyes on average. In comparison, we observed an increase in tangent modulus of 37.4% (at 30 mm Hg). If both tangent modulus and thickness contribute similarly to the overall stiffness of the scleral shell (i.e., structural stiffness), then age-related changes in peripapillary scleral thickness will have a lesser impact on IOP-induced deformation than those of the tangent modulus. The relationship between glaucomatous susceptibility and scleral stiffness is likely to be complex, individual specific, and involve many interacting factors.
Changes in Scleral Biomechanics after Exposure to Chronic IOP Elevation
The major findings were as follows: First, significant changes in tangent modulus and structural stiffness were observed in some monkeys between the normal eyes and those exposed to chronic IOP elevation. Second, changes in FE-based scleral anisotropy measures were not observed between normal and glaucomatous monkey eyes. Finally, although all glaucomatous eyes exhibited thinner peripapillary sclera than their contralateral normal eyes (), these differences did not achieve statistical significance.
We observed significant changes in scleral tangent modulus () and structural stiffness () between the normal and glaucomatous eyes of monkeys 1, 4, 5, 6, and 7. The glaucomatous eye in monkey 1 exhibited a smaller tangent modulus at 10 mm Hg () and structural stiffness at 10 and 30 mm Hg () than those of its contralateral normal. Burgoyne et al.53
have reported hypercompliance of the ONH surface within 4 to 8 weeks of chronic IOP elevation in monkeys and suggested that weakening of the load-bearing structures of the eye (lamina cribrosa and peripapillary sclera) through connective tissue damage might be responsible for this phenomenon at the earliest stage of glaucomatous optic neuropathy. Moreover, a computational modeling study by Downs et al.54
reported weakening of the lamina cribrosa structure at the earliest stage of glaucomatous optic neuropathy. It is interesting to note that weakening of the sclera was only present in the glaucomatous eye of monkey 1, the eye that received the smallest cumulative IOP insult (432 mm Hg × days) and had the lowest maximum measured IOP (24 mm Hg). It should also be noted that the decrease in tangent modulus in the glaucomatous eye of monkey 1 was only significant at low IOP (10 mm Hg).
In 1957, Roach and Burton55
studied the roles of elastin and collagen in arteries. They found that elastin alone was responsible for resisting pressure-induced arterial deformations at low pressure and that collagen alone was responsible for resisting pressure-induced arterial deformations at high pressure. These results were recently confirmed by Fonck et al.56
Based on these studies, it could be that only damage to elastin occurred in the glaucomatous eye of monkey 1, and this represents the earliest stage of scleral damage and/or remodeling in glaucomatous progression.
We observed a significant increase in scleral tangent modulus at IOPs of 30 and 45 mm Hg in both the peripapillary and peripheral regions in monkeys 4, 5, and 6, and a significant increase in scleral tangent modulus at 45 mm Hg in the peripapillary region in monkey 7 (). The glaucomatous eyes in monkeys 4, 5, and 6 also exhibited larger structural stiffness in the peripapillary sclera at IOPs of 30 and 45 mm Hg (). These findings suggest that at higher levels of cumulative IOP insult, the posterior sclera stiffens through extracellular matrix remodeling. This finding is consistent with that in our previous uniaxial study of scleral biomechanics in glaucomatous monkey eyes11
and may be a protective mechanism that limits scleral canal expansion and related deformation of the ONH at elevated IOPs. Zeimer and Ogura57
reported that human ONHs became more rigid with severe glaucoma, a result that is consistent with this hypothesis. Roach and Burton55
also demonstrated that collagen alone was responsible for limiting arterial deformations at high pressure, which suggests that the observed increase in tangent modulus in glaucomatous eyes at high IOPs may be due primarily to collagen remodeling.
Even though tangent modulus changes occurred in five monkeys (1, 4, 5, 6, and 7), significant changes in maximum principal strains (monkeys 1 and 4) and maximum principal stress (monkeys 4 and 6, in the peripapillary sclera only) were each observed in only two monkeys. This result suggests that overall changes in scleral thickness (generally lower in the glaucoma eyes) and tangent modulus (generally higher in the glaucoma eyes) at this stage of damage interact to maintain the stress and strain ranges typical of sclera in normal eyes.
In this study, we observed no significant changes in either scleral tangent modulus () or structural stiffness () between the normal and glaucomatous eyes in three monkeys (2, 3, and 8). Since monkey 8 received the highest cumulative IOP insult (7714 mm Hg × days), we would have expected to observe the largest changes in the glaucomatous eye of this monkey. Of note, at IOPs of 30 and 45 mm Hg, the normal eyes of monkeys 3 and 8 had the two highest mean tangent moduli and structural stiffnesses in the peripapillary sclera. Assuming that before IOP insult, the glaucomatous eyes in monkeys 3 and 8 were as stiff as their contralateral normals, these results suggest that stiffer scleral shells may be less prone to biomechanical changes when exposed to chronic IOP elevation, presumably because they are more resistant to IOP-induced strain.
Several studies have reported that mechanical strain applied to scleral fibroblasts can trigger the release of MMPs and TIMPs,58–61
which can then lead to remodeling of the scleral extracellular matrix. IOP-induced scleral shell expansion increases scleral strain in all eyes, but in eyes with the stiffest sclera, these increases may be too small to elicit a fibroblast response. Note that mean strain levels at 45 mm Hg in monkeys 3 and 8 were 0.30% and 0.32% respectively, which were smaller than all the reported values in the aforementioned in vitro studies on scleral fibroblast response to strain (a minimum of 0.45%58
In the case of the third monkey, which demonstrated no change in tangent modulus or structural stiffness (monkey 2), the normal eye had the most scleral thickness (), and lowest tangent modulus () among all normal eyes. Its mean structural stiffness was therefore comparable with the mean structural stiffness of all normal eyes. These data suggest that mean structural stiffness may not always predict which eyes will undergo IOP-induced scleral remodeling and that scleral thickness may also be an important contributor in predicting which eyes will be less sensitive to biomechanical change.5,46
In this study, we did not observe significant differences in predicted scleral anisotropy (preferred fiber orientation and fiber concentration factor) between normal and glaucomatous monkey eyes. The preservation of anisotropy was recently reported for the remodeled monkey lamina cribrosa in EG,15
suggesting that remodeling of the peripapillary and posterior sclera must be accompanied by changes in elastin/collagen content and/or fiber diameter, but not fiber reorientation. This is consistent with the work of Quigley et al.,62
who reported glaucomatous changes in collagen and elastin content and collagen fiber diameter in the peripapillary sclera in human and monkey eyes.
Although we did observe a trend toward scleral thinning in the glaucomatous eyes, this difference did not exceed the physiologic intereye differences in the eight normal monkeys. Using traditional histology, we previously reported significant thinning of the posterior10
and peripapillary sclera13,14
in monkey eyes subjected to chronic IOP elevation. Scleral morphologic changes should be assessed in future studies to understand whether scleral thinning due to chronic IOP elevation is a consequence of strain-induced fluid exudation, tissue loss, or a combination of the two.
Overall, our data suggest that a stiffer scleral shell is biomechanically beneficial and is less apt to remodel in response to elevated IOP. In normal eyes, we suggest that the sclera shields the ONH from biomechanical insult by resisting large overall deformation (through uncrimping and stiffening of the collagen fibers: nonlinearity) and large scleral canal expansion (through the presence of a circumferential peripapillary ring of collagen fibers: anisotropy).3,4,8
We hypothesize that the scleral stiffening in response to moderate exposure to chronic IOP elevations shown herein is an effort to maintain the biomechanical homeostasis of the scleral shell and indirectly, the ONH. As such, this response could act to slow glaucomatous progression in eyes with higher IOPs and/or more compliant scleral shells. Surprisingly, the argument that a stiff scleral shell shields the ONH from glaucomatous damage seems to contradict the facts that (1) the sclera stiffens with age.8
and (2) the elderly are more susceptible to glaucoma.63
To reconcile all these observations, it should be noted that collagen fibers become brittle with age28
and therefore it is plausible to suggest that scleral stiffening with age is an unhealthy mechanism as opposed to scleral stiffening because of the remodeling seen in this study. In addition, there are other factors that are likely to contribute to glaucomatous damage, including blood flow and cellular reactivity, and these factors change with age as well. We also caution the reader that stiffness is a broad term, and reporting a single stiffness value for the sclera does not represent its biomechanical response well. Scleral stiffness is a function of IOP (nonlinearity), orientation (anisotropy), and location (heterogeneity). This complexity should be taken into account when evaluating the contribution of scleral biomechanics to glaucoma pathogenesis.
Several limitations should be recognized when viewing this work. Although the general limitations of the method have been discussed at length in our previous reports,3,8,9
we will briefly revisit them and then discuss those inherent to this study.
First, IOP was measured in both eyes of each monkey over time (), but we did not characterize the diurnal and nocturnal IOP fluctuations that are known to exist in monkeys. Diurnal IOP fluctuations have been shown to be large in monkeys, especially in glaucomatous eyes, in which they are highly individual-specific and can exceed 10 mm Hg.64
Therefore, our definition of cumulative IOP insult (change in area under the IOP/time curve) should be regarded with caution and was used here only to provide a rough estimate of the cumulative insult from IOP. An implantable telemetric IOP transducer is currently being developed for monkeys that should allow for continuous measurements of diurnal and nocturnal IOP fluctuations and more accurate characterization of IOP insult (Downs JC, et al. IOVS
2008;49:ARVO E-Abstract 2043).
Second, because the sclera is approximately 90% collagen by weight,65
we assumed that collagen is the primary fibrous element66
of this tissue. We lumped all other tissue constituents into the ground substance matrix, a common practice in soft-tissue FE modeling.4,30,67–71
Because our study suggests that both elastin and collagen play important roles in glaucomatous damage, further experimental and theoretical work is needed to address how these components separately contribute to scleral biomechanics.
Third, we did not characterize scleral biomechanics between 0 and 5 mm Hg in the tested eyes, because the scleral shells required an IOP of approximately 4 mm Hg to sustain their shape. IOPs in the range of 0 to 5 mm Hg are rarely measured in monkey eyes, and so ignoring the initial IOP loading should not compromise the clinical importance of our results. We are developing methods that will allow the initial scleral stress and strain that exist in the tissues at 5 mm Hg to be estimated and included in future studies.
Fourth, the ONH tissues were assumed to be linearly elastic, with a common elastic modulus of 1 MPa assigned to the ONH region for both normal and glaucomatous monkey eyes. Our previous sensitivity study showed that ONH elastic moduli in the range of 0.1 to 5 MPa did not affect the results of our scleral biomechanical property fitting9
; hence, our assumption should not affect the results presented herein.
Fifth, within our models the ONH was represented as an elliptical cylinder, with its external boundary located at the outer aspect of the dural sheath insertion (as measured with the 3-D digitizer) and therefore included regions of the immediate peripapillary sclera and scleral flange. As a result of this anatomic convention, we could not estimate biomechanical properties for that region of the sclera that lies immediately under the dural sheath insertion.
Sixth, we divided each reconstructed scleral shell into eight regions to assess regional variations in predicted collagen fiber orientation (θp1–θp8). This division was made because a preliminary study confirmed that a combination of eight regions and 13 model parameters was the minimum requirement to obtain good agreement between the FE-predicted and experimentally measured displacements (), while also ensuring a unique set of model parameters. Future studies will include a more rigorous assessment of regional variability.
Seventh, all experiments were conducted at room temperature (22°C) because the incorporation of a heated saline chamber at 37°C proved incompatible with the speckle interferometry sensor we used. Because all experiments were consistently performed at the same temperature, all hypotheses tested regarding the changes in scleral biomechanics should be valid.
Eighth, collagen fiber alignment was not measured directly, but was predicted using the models' fits to experimental scleral deformations (). Because of the relatively coarse discretization of the regions (discussed above) we could only report an overall approximation of the preferred collagen fiber orientation within each region. Future inclusion of experimental measures of collagen fiber orientation as input parameters, as currently being measured with small-angle light-scattering (Girard MJ, et al. IOVS 2010;51:ARVO E-Abstract 2128), will allow for finer regional discretization for the remaining fitted parameters and improve our characterization of scleral anisotropy.
Finally, seven of the eight monkeys (monkeys 2–8) began this study while the investigators laboratories were still located at the LSU Eye Center in New Orleans, and were unavailable for study for more than 3 months after Hurricane Katrina. Monkeys 2 and 3 and 6 to 8, however, had already undergone initial laser, and so the accuracy of their cumulative IOP exposure calculations is affected by the lack of IOP data between 30 August and 15 December 2005, (, shaded area) and the diminished frequency of testing after their arrival at Devers. In addition, these monkeys (2, 3, and 6 to 8) were examined only sporadically until the laboratory at Devers became fully operational. At the time of the hurricane, monkeys 4 and 5 had not yet been lasered, and so the frequency of IOP measurement and assessment of pre- and postlaser cumulative IOP exposure in these animals was unaffected by the hurricane. Monkey 1 was purchased at Devers and was altogether unaffected by these events.
In summary, new experimental and computational methodologies3,9
were used in this study to characterize scleral biomechanics in normal and glaucomatous monkey eyes. These methods have broad applicability and can be applied to other thin, soft tissues with multidirectional collagen fibers. The long-term goal of this work is to establish the pathophysiologic mechanisms linking connective tissue stress and strain within the peripapillary sclera and lamina cribrosa to retinal ganglion cell axon damage and death. Given the important role of scleral biomechanics in determining the biomechanics of the ONH, new strategies for the clinical visualization and measurement of peripapillary and posterior scleral biomechanics are needed.
Overall, our results suggest that significant changes in the biomechanical behavior of the sclera are associated with exposure to chronic IOP elevations. These changes are complex and individual-specific and are likely to be the result of scleral extracellular matrix remodeling. Our findings suggest that (1) scleras with initially large tangent modulus or thickness are more resistant to IOP-induced biomechanical changes, (2) a decrease in tangent modulus (possibly due to elastin damage) occurs at the earliest stage of glaucoma, (3) an increase in tangent modulus (possibly associated with collagen changes) occurs at moderate stages of glaucoma, and (4) collagen fiber reorientation does not occur on a large scale in glaucomatous eyes. The sclera drives the levels of stress and strain transmitted to the ONH, so changes in scleral biomechanical behavior will inevitably perturb the biomechanics of the ONH.7
Scleral remodeling induced by exposure to chronic IOP elevations may provide a protective mechanism for the contained ONH and act to keep stress and strain levels within the normal range, but future work is necessary to establish the link between scleral/ONH biomechanics and glaucomatous vision loss.72–74