This study has several limitations. First, the preconditioning cycles used a much faster loading rate than the actual inflation tests. We used faster preconditioning cycles to reduce the total experimental time and minimize tissue decay. However, the preconditioning can be affected by loading rate, loading limits, resting periods, and age- or species-related difference in tissue structures.
45 Our preliminary tests in pig and donor eyes showed that tissue displacements generally stabilized after several cycles. This observation was consistent with previous reports of minimal preconditioning effects in pig and human donor eye inflation tests.
26–28 However, given that our preconditioning cycles had a much faster rate than the actual testing, future studies are needed to further optimize the preconditioning protocol to ensure a stabilized response during the actual testing. Second, the sample size of human donor eyes was limited (
N = 9 pairs, 18 globes), which provided 80% power to detect correlations at
R = 0.8 and 62% power for
R = 0.7 at a significance level of 0.05. This sample size does not provide sufficient power to detect weaker correlations that may exist between other types of strains or between CCT and strains. Although the observed strong correlation between the cornea and ONH shear strains was confirmed by both Pearson correlation and Spearman correlation analyses indicating the likelihood of such association, future studies in a larger sample size are needed to verify this result. Another limitation with the small sample size is the potential effect of outliers. One of the tested eyes had a large PPS shear strain and also a large PPS tangential strain. If data from this eye are removed from analysis, correlations between PPS and cornea shear strains (see
Fig. 5B) and PPS and cornea tangential strains (see
Fig. 5C) would no longer achieve statistical significance. It is noted that the correlation between ONH and cornea shear strains remains significant after removing data from this eye. Interestingly, the PPS was abnormally thin in this eye (approximately 582 µm), suggesting a potential relationship between thin PPS and large PPS shear/tangential strains in response to IOP. Future studies in larger sample sizes are needed to elucidate these relationships. Third, ex vivo tissue may have experienced a certain level of biomechanical alterations as compared to in vivo tissue. Our laboratory has been developing in vivo ultrasound elastography methods to characterize corneal biomechanics
30,46,47 and is pursuing similar techniques for the posterior eye. When these approaches become available, it will offer tools to measure the cornea and posterior eye mechanical responses in vivo to further our understanding of the biomechanical connection between the cornea and the posterior eye and how that impacts an eye's risk for glaucoma. Fourth, we measured the cornea and the ONH/PPS in paired eyes instead of measuring cornea and ONH/PPS in the same eye. Logistically it is difficult for our current imaging setup and protocols to perform inflation tests of both the cornea and the ONH/PPS in the same globe. Dissecting the cornea and ONH/PPS shells are not ideal because dissection could cause damage to the more delicate tissues, such as the retina and the corneal endothelium whose integrity is important for this study. Tissue clamping also introduces unnatural boundary effects. For these reasons, we opted to perform two separate inflation tests in the paired eyes of the same donor. Although there is generally a high degree of interocular symmetry in biometric (e.g. CCT and axial length) and biomechanical parameters (e.g. IOP and corneal displacements) in healthy subjects,
46–50 the potential variance between the left and right eyes could have obscured some correlations and rendered them undetectable in the present study. A comparison between the left and right eyes ONH/PPS shear strains in six pairs of donor eyes is presented in the
Supplementary Material to provide data on the potential variance. Our laboratory is currently developing ultrasound-based techniques for in vivo biomechanical characterization of both the cornea and the posterior eye to enable future in vivo studies of their correlations in the same eye.