Glaucoma is an optic neuropathy initiated at the ONH. Two primary risk factors for glaucoma are higher IOP and older age. In this study, we used a high-resolution ultrasound elastography technique to simultaneously image the ONH and PPS in human donor eyes to quantify IOP-induced 3D mechanical deformation in these tissues and explore associations with age. Compared with optical imaging, ultrasound imaging has a greater penetration depth, which gives us a more comprehensive investigation to include the entire thickness of the ONH and PPS. More important, it allows us to accurately characterize the out-of-plane deformations, including radial compression and shear, which are difficult to obtain using other methods. In this study, we quantified the full strain tensor (six components) at each spatial point within the scanned ONH and PPS volume (approximately 23,000 points per eye) and analyzed the whole and regional ONH and PPS response to IOP increases up to 30 mm Hg. The primary findings are as follows. First, the dominant form of deformation in response to IOP was compressive radial strain εrr in both ONH and PPS, with a much larger magnitude than the in-plane normal strains (εφφ and εθθ) and in-plane shear εθφ. Out-of-plane shear strains (εφr and εθr) were also substantial in both ONH and PPS. Second, most strains were found to be significantly greater in the anterior than the posterior one-half of both the ONH and the PPS. Third, exploratory analyses suggest that radial and volumetric strains in the anterior ONH and PPS increased with age, indicating a greater compression and volume loss in older age during IOP elevation.
Among the six components of the 3D spherical strain tensor, radial strain was the greatest in magnitude, indicating radial compression as the primary form of deformation in both ONH and PPS. This result was consistent with the findings in our previous 2D study that scanned the central cross-section along the nasal–temporal direction of the ONH and PPS.
28 The circumferential and meridional strains in the ONH were very small, suggesting that in the normal eye the PPS is effective in shielding the ONH from in-plane tensile stretch created by an elevated IOP. This is likely accomplished by the strong annulus collagen ring in the PPS immediately adjacent to the ONH, which is present ubiquitously in all studied species including mouse, rabbit, pig, and human. Although the radial compression was smaller in ONH than in PPS, the PPS seemed to be less effective in shielding the ONH from deformations in the out-of-plane direction. Our results showed that the radial compression and the out-of-plane shear strains in the ONH (particularly in the anterior ONH) had a substantial magnitude and increased at higher IOP. It is noted that the strain values in this study are not directly comparable with previous donor eye studies,
12,15,17,37 because these strains were created by a pressure elevation from 15 mm Hg, the average normal pressure in the human eye. Previous donor eye studies reported strains created by an IOP change from a lower baseline (1.5 or 5.0 mm Hg).
12,17
Both our previous 2D study and the present 3D study showed a higher radial strain in the anterior than the posterior ONH. This finding may be explained by the role of LC as it represents a stiffer connective tissue discontinuity in the thickness direction. In our previous investigation of the radial displacement through the thickness of ONH, it was observed that radial displacement decreased from the prelaminar region to the LC and then plateaued in the region posterior to LC.
29,30 Results from the present study showed that out-of-plane shear ε
φr was concentrated in the anterior ONH region. This component of shear describes the outward bending and posterior bowing of the ONH during IOP increases, which may contribute to the biomechanically driven LC remodeling and optic disc cupping, characteristic of clinical glaucoma.
38 These results indicate that the mechanical deformations that contribute to glaucomatous damage in the ONH are likely those in the out-of-plane direction, namely, radial compression and out-of-plane shear. An imaging method that can characterize these deformations in the anterior ONH in vivo accurately may thus provide additional diagnostic information to monitor and predict progression.
Previous studies have shown an age-related stiffening in the collagenous connective tissues of the posterior eye. Albon et al.
11 used laser scanning confocal microscope imaging to determine changes in the volume and strain of ex vivo human LC in response to IOP increase. Higher gradients in the pressure–volume curve were observed in older eyes, suggesting an increased LC stiffness in older age. In addition, the mechanical compliance of LC, as the ability to regain the original shape and size of LC after the removal of applied pressure, decreased with age.
11 As for the sclera, inflation tests of human eyes have observed an age-related stiffening of the pressure–strain response of the scleral surface using different methods for displacement measurements such as electron speckle pattern interferometry
12 and digital image correlation.
17 Inverse computational models were developed that integrated the displacement fields of inflation testing and microstructure of the sclera to examine the elastic properties of the collagen fibers and matrix in the sclera.
39,40 Specifically, the model used by Grytz et al.
41 was based on electron speckle pattern interferometry displacement and constitutive formulation that incorporates scleral collagen fibril crimp and local anisotropic collagen architecture, which reported an increased shear modulus and decreased collagen fibril crimp angle with age. The eye-specific model used by Coudrillier et al.,
42 which was based on digital image correlation-calculated displacement and wide-angle x-ray-measured scleral collagen distribution, showed a greater matrix stiffness and a lower degree of fiber alignment in the sclera of older eyes. Taken together, these findings consistently suggested age-related stiffening in the LC and sclera. However, it remains unclear how age-associated collagenous tissue stiffening may or may not contribute to glaucoma risk.
A major new finding of the present study is that there was a trend of increased radial compression and volume loss with age in the anterior ONH and PPS during IOP increase. This observation was enabled by the 3D ultrasound elastography technique that allows the accurate quantification of displacement and strain fields beyond the tissue surface. The PPS is a highly anisotropic structure with an in-plane tensile modulus orders of magnitude higher than the through-thickness (radial) compressive modulus.
43 Age-associated collagen cross-linking may result in stiffening of the in-plane properties, whereas through-thickness stiffness may decrease with age owing to decreased proteoglycan and hydration.
44–46 Our results indicated that the radial compression in the anterior ONH also showed a trend of increasing with age. Although a larger sample size is needed to confirm this result, two implications are worth noting. First, this finding points to a potential biomechanical mechanism underlying the greater glaucoma risk in older age. Despite connective tissue in-plane stiffening, the anterior ONH, which is composed mostly of neuroglial and capillary tissues, may be subject to an increased through-thickness compression during aging. This factor could contribute to optic disc cupping, a key clinical feature of glaucoma, as well as axonal blockage, glial reactivity, and decreased blood flow in this region. Second, because radial strains in the anterior ONH and anterior PPS were correlated strongly, further studies are needed to investigate the interactions between the ONH and PPS radial responses to the IOP. For example, would a decrease in PPS radial strain alleviate ONH radial compression? Previous studies have investigated the effect of PPS stiffening on ONH and LC in-plane deformation
47,48 and found decreased in-plane strains after PPS stiffening. However, the responses in the radial (through-thickness) direction were not examined. In vivo rodent models of experimentally induced glaucoma did not show a protective effect of PPS stiffening against glaucoma damage, despite reduced in-plane strains.
49,50 These results, along with the findings in the present study, indicated that the ONH and PPS radial compression may be potentially a more important driver in IOP-related glaucomatous damage and warrants further study.
We also observed a small volume loss in the anterior ONH and PPS in response to elevated IOP. Most biological tissues are modeled as nearly incompressible with a Poisson's ratio close to 0.5 (essentially no volume change during mechanical deformation). Tissues such as cartilage are known to change volume during deformation; for example, patella cartilage volume decreased by 6% after knee bends.
51 A recent study reported that cells undergo slow deformation at constant volume, whereas fast deformation leads to volume loss.
52 Very few studies have reported the volume ratio or volumetric strains in ONH or PPS. The 3D approach used in this study allowed us to characterize the full strain tensor and calculate these parameters. Interestingly, there was a significantly larger volume loss (i.e., larger volumetric strain magnitude) in older eyes, which was consistent with the larger radial compression discussed elsewhere in this article. Further studies are needed to explore the biomechanical and pathophysiological implications of volume loss in ONH and PPS during IOP elevation.
In addition to deformation characterization, with the advantage of high-frequency 3D ultrasound, morphological characterization was also made possible. Taking into account the exsanguinated choroid layer, the measured PPS thickness was in a similar range as previously reported in our
28 and others
53–55 studies. Similarly, PPS curvature, the BMO radius, and area also agreed with reported ranges.
56,57 We did not find reports of human PSCO radius and area. Our measured values were comparable with the reported intraorbital optic nerve sheath diameter.
58 We also did not observe significant correlations between morphometric parameters and age. In the case of PPS thickness, previous studies reported mixed results. Some reported no association with age,
54,59 whereas others reported a decrease with age.
17
This study has several limitations. First, ex vivo testing is limited to only include IOP without other important forces that are exerted on the living eye, such as cerebrospinal fluid pressure, central retinal artery blood pressure, and tension on the optic nerve. Because IOP is a dominant factor for determining the biomechanical insults in the development and progression of glaucoma, our focus was IOP-induced deformation with an understanding that in vivo deformation would be affected by other factors as well. Second, the exsanguinated choroid is indistinguishable from the PPS in the ultrasound images and, thus, was not separated out from the PPS in the current analysis. In the ex vivo donor eye, the choroid is approximately 200 µm in thickness near the posterior pole.
60 Evaluating the strain maps of the PPS, the anterior 200-µm layer did not show a distinct strain response. As such, the mechanical response of the PPS volume described in this study would be close to that of the PPS if choroid was removed. Third, our current sample size was limited. The sample size of 15 provides 80% power to detect a Pearson correlation of greater than 0.65 at a significance level of 0.05. Thus, future studies with a larger sample size (30 or more) are needed to verify age-associated changes. Our current sample size provided approximately 90% power to detect an effect size of 1 (1 SD difference) between two regions based on paired
t tests. Last, our analysis of the anterior and posterior halves of the ONH and PPS was not based on natural tissue boundaries, such as the LC surface. The LC was not separately segmented out for either morphometric or strain analysis in the present study. We are currently developing 3D segmentation methods and aim to delineate anatomical features such as the LC in future studies to characterize the mechanical deformation in the pre-LC, LC, and post-LC regions.
In conclusion, high-resolution ultrasound elastography was used to measure the 3D mechanical responses of the human ONH and PPS to IOP elevation. We found that radial compression was the dominant form of deformation in the anterior PPS and anterior ONH, and its magnitude increased with age. Although this trend seemed to contrast with age-associated stiffening, increased radial compression may be the predominant IOP-related mechanical insult at the ONH, potentially contributing to age-related glaucoma risk. Future studies in glaucomatous eyes are needed to further understand the connection with glaucoma progression. The unique capability of high-frequency ultrasound elastography to quantify 3D tissue deformation may provide a useful tool in such studies to gain new insights into the age-associated biomechanical susceptibility to glaucoma, particularly in the anterior ONH, where glaucomatous damages initiate.