The AOSLO provided high-resolution, high-contrast images of the anterior surface of the lamina cribrosa in normal human and macaque eyes. The values of human laminar pore parameters measured in vivo in this study are comparable to in vivo and ex vivo data from previous reports. Mean laminar pore elongation measured using AOSLO imaging in our three normal human eyes (2.00 ± 0.75) was similar to that measured in vivo by Fontana et al.26
in 10 normal eyes with a confocal SLO (1.81 ± 0.1). In addition, the range of human pore areas measured in vivo in this study (154–6637 μm2
) is comparable to that found ex vivo by Quigley and Addicks21
(range in pore diameter from 10–100 μm, or in pore area from ~79–7850 μm2
assuming circular pores) and by Dandona et al.36
in nine normal histologic samples sectioned midway between the anterior and posterior laminar surfaces (range of 410 ± 35 to 5201 ± 621 μm2
for the 10th to 90th percentiles). However, the range of pore areas that we measured in vivo was less than that reported ex vivo by Ogden et al.,37
) and our mean pore area (1713 ± 1414 μm2
) was also less than that measured ex vivo by Jonas et al. 38
(4000 ± 1000 μm2
) at the anterior laminar surface in 35 normal eyes.
Many factors could account for the discrepancies in the range and mean values of pore areas between our in vivo measures in human eyes and the latter two ex vivo reports. Several postmortem studies support the idea that human laminar pores tend to be largest in the superior and inferior poles of the ONH and increase in size toward the periphery of the nerve.21,37
As mentioned earlier, it was difficult to image laminar pores in vivo at the superior and inferior poles in most eyes as the overlying vasculature (e.g., the central retinal artery and vein) typically obstructs the visualization of these pores. In addition, the neuroretinal rim can cast shadows on to the anterior laminar surface, prohibiting the visualization of the most peripheral laminar pores in these circumstances. Therefore, it is possible that we did not image the largest laminar pores in vivo in all our human eyes and that our measures of laminar pore area underestimate some of those found ex vivo.
Another possible confounding factor is that the en face AOSLO images represent a two-dimensional projection of a potentially curved three-dimensional anterior laminar surface. Strouthidis et al.39
recently demonstrated the ability to subjectively identify a contour representing the anterior laminar surface in a primate eye from a cross-sectional image of the ONH acquired using spectral domain OCT. The position of the anterior laminar surface was not measured in our study eyes. Consequently, our AOSLO pore parameters may not represent the true anatomic shape of laminar pores in eyes with nonplanar anterior laminar surfaces. Although it is unknown the extent to which a three-dimensional transformation will alter our measured pore values, we do expect to see larger differences in eyes with more steeply curved anterior laminar surfaces. Therefore, our measured values could represent a lower bound for the true anatomic sizes imaged in vivo. Knowledge of absolute pore geometry may be important for biomechanical modeling of the anterior laminar surface. However, it may not be necessary for detecting changes in laminar pores during early experimental glaucoma in which one may be interested in detecting a relative change in pore structure in relation to the occurrence of changes in other clinical parameters conventionally used to diagnose and assess glaucoma.
Laminar pore parameters measured in vivo in our two normal nonhuman primate eyes can also be compared with the same values measured by Vilupuru et al.27
in four living macaque control eyes with a different AOSLO. Whereas both studies yielded comparable values of mean pore elongation in normal, control eyes (1.77 in the present study vs. ~1.6 in Vilupuru et al.), mean pore area and NND were larger in Vilupuru et al. (~1950 μm2
and ~47 μm, respectively) than in the present study (973 μm2
and 37 μm, respectively). The reasons for these discrepancies in pore area and NND are not clear. We do not know whether the resolution and confocality (e.g., pinhole diameter) of the AOSLO used in this study differed from that used by Vilupuru et al., potentially allowing us to better visualize and define the boundaries of smaller pores. Another possibility is that a different number of pores were analyzed in each study. In addition, it is possible that these differences simply represent intersubject variability in laminar pore geometry given the small number of macaques imaged in this study (n
= 2) and by Vilupuru et al. (n
= 4). Attempts are currently underway to image anterior laminar pore parameters in a greater number of eyes to better define this issue in the normal macaque.
A main goal of this work was to examine the variability in our laminar pore measurements over time. Because we wanted to assess the repeatability with which we could quantify the same laminar pores at each imaging time point, we used a repeated-measures ANOVA to assess the statistical variability in our measurements. A limitation of using a repeated-measures ANOVA is that one can only include pores that were quantified in all imaging sessions. For example, improvements in our imaging and quantification techniques typically enabled us to examine a greater extent of the laminar surface in a given eye over time, thereby allowing us (in general) to quantify a greater number of pores at the end of the experiment. (d represents an example in which we were able to image a greater extent of the laminar surface due to improved techniques.) However, even though an increased number of pores could often be visualized at later time points, these additional pores were not included in the repeated-measures ANOVA for a given eye if they were not imaged at all earlier time points. In addition, although it was possible to measure and track the same laminar pores at multiple time points, we excluded pores from the ANOVA even if they were quantified in all but one session. As a result, the number of pores that were quantified and used to assess repeatability across all imaging sessions was less than the total number of pores used to generate the global statistics on mean pore area, elongation, and NND in a given eye.
This study's method for imaging and quantifying anterior laminar pores with an AOSLO is repeatable in normal eyes. No statistically significant differences were found in pore geometry (ANOVA, P > 0.05) when comparing laminar pore parameters measured in the same eyes imaged over multiple time points. Intrasession variability was also small (), demonstrating a consistent means for identifying pores. Mean pore area tended to have the largest measured intersession variability (, mean standard deviation of 8.3% in humans and 6.1% in macaques). This variability was likely due to small intensity differences between the AOSLO reflectance images acquired across imaging sessions (potentially caused by slight differences in illumination levels, retinal reflectivity, or photomultiplier detector gain). For example, making only a half-pixel error in the identification of the entire boundary of the pores analyzed in this study would result in a 5.1% and 4.5% difference in mean pore area (on average) in humans and macaques, respectively. These values are not vastly dissimilar from our measured variabilities in mean pore area. Also, despite demonstrating good repeatability, the subjective methods used for demarcating and quantifying laminar pores can be very time-consuming and could be challenging to apply to the analysis of several eyes over short periods. We are currently developing algorithms to make this pore analysis method more objective and to decrease the required processing time through the use of semiautomated image processing techniques.
Even though it is possible to acquire excellent en face images of the anterior laminar surface in living eyes, AOSLO imaging is limited in its ability to visualize the entire laminar structure in three dimensions. Therefore, it may not be possible to use this technique to examine changes throughout the entire extent of the lamina in glaucoma. Nevertheless, it is still possible to visualize laminar pores over a finite range in depth behind the anterior laminar surface () despite the AOSLO's limited axial resolution.40
In addition, current models propose that changes in laminar thickness and position occur throughout the entire thickness of the lamina in early experimental glaucoma.41
Therefore, changes in laminar pores should also occur at all depths, including the anterior surface which is visible using AOSLO imaging.
In conclusion, we have established a repeatable method for relocating the same laminar pores and quantifying their geometries in vivo in normal human and nonhuman primate eyes. The methods presented in this article could be extended to assess longitudinal changes in laminar pore structure in glaucomatous neuropathy. These measurements would contribute to a broader and more detailed understanding of the biomechanical properties of the normal and glaucomatous lamina while also enabling the correlation of laminar pore changes with functional and structural changes conventionally used to diagnose glaucoma.