The study was conducted on postmortem tissues, and we cannot exclude the possibility of some necrotic changes. The properties of the sclera, in particular, are important because they are a major determinant of ONH biomechanics.
56,62,70,71 Experiments have suggested that the mechanical properties of the sclera do not change up to 72 hours postmortem.
72 Similarly, the experiments were carried out without a pressurized vascular system, which could have affected the mechanical behavior of the LC.
57 We cannot dismiss the possibility that sectioning the optic nerve to gain access to the LC may have altered LC biomechanics. The results obtained were derived from a two-dimensional (2D) analysis on projected images. Although we were careful to image the LC always in the same direction, some of the more complex effects of the 3D deformations would not be inferred by our analysis and may even result in inaccurate measurements. However, the focal nature of the deformations is inconsistent with whole ONH tilting or rotation. In-plane stretch, compression, or shearing of the retinal ganglion cell axons or of the capillaries supporting the tissues within the canal in the magnitudes we report may be sufficient insult to trigger a detrimental cascade of events. The data acquired through the SHG imaging is 3D, and our technique can be extended to 3D. Hence, analysis in 3D is possible, and is a logical next step. Historically, critical insight on the LC and ONH has been gained from studies using 2D imaging or analysis.
2,9,11,29,30,34,36,65,73,74 We imaged the tissues from the posterior direction, which means that the tissues observed may be different from what is typically observed using OCT, or identified in labeled histology. The horizontal trabecula-like structure makes us confident that we have imaged the LC region, even if incomplete. Laser penetration may have been reduced by effects of the retrolaminar septa.
75 Although our goal was to image the whole LC, imaging the posterior LC is important because experiments
76,77 and modeling
15,52,78 suggest that the posterior LC is an active region, critical for the sensitivity to IOP, remodeling, aging, and progression of glaucoma. For clarity and conciseness, in this work we analyzed the effects of IOP on the LC, although the tissues surrounding the LC, and in particular the sclera, are also critical to LC biomechanics and overall sensitivity to IOP.
14,17,49 In future work we will extend our analysis to neighboring tissues. In this study, we considered only differences between a physiologic and a substantially elevated IOP. Our intention was to find bounds for the displacements and deformations under the assumption that large, but still realistic, increases in IOP would be more likely to result in meaningful insight into the biomechanical effects on the ONH. During the experiments, images were acquired at several intermediate IOP levels. Analysis of these data will allow determining in more detail the complex nonlinear effects of IOP on the ONH. Although we acknowledge that the sample size was small, we point out that the results are consistent across eyes and therefore we believe valuable to the ocular biomechanics community. Because of the sample size, we emphasize direct comparison of the measurements over statistical tests. It is important to point out that the animations (
Supplementary Movies S2–
5) were generated by interpolating between the low and elevated IOP levels. The animations are intended to help distinguish effects of IOP that are difficult to visualize otherwise, such as small deformations or regional differences in response. The paths followed by the structures in intermediate IOP levels may differ. We also acquired images at the intermediate levels, and are currently in the process of analyzing them.