# Three-Dimensional Microarchitecture of Lamina Cribrosa Pores in High and Normal Tension Glaucoma Using Optical Coherence Tomography

Paul Bastelica, MD,* Antoine Labbé, MD, PhD,*†‡

Pascale Hamard, MD, PhD,* Florence Rossant, PhD,§ Hélène Urien, PhD,§

Jérémie Sublime, PhD,§ Hélène Claudel, MD,* Nan Ding, MS,§

Michel Paques, MD, PhD,*‡ and Christophe Baudouin, MD, PhD*†‡

**Précis:** The lamina cribrosa (LC) pores of patients with high-tension glaucoma (HTG) appear to take a more tortuous pathway than the LC pores of patients with non-glaucomatous (NG).

**Objective:** To compare the LC pore microarchitecture in patients with HTG, normal tension glaucoma (NTG), and NG, by reconstructions of the LC made from tomographic images.

**Patients and Methods:** Spectral domain-optical coherence tomography images of 52 eyes (18 NG, 18 HTG, and 16 NTG) of 29 patients were analyzed. Pores were traced using segmentation software. Pore length, tortuosity, and verticality were the 3 quantitative parameters compared among the 3 groups. Correlation analyses were performed to determine the effects of covariates on the 3 quantitative parameters.

**Results:** Pore tortuosity in HTG (1.419 ± 0.093) was significantly higher (*P* = 0.011) than in NG (1.347 ± 0.034) but did not differ from that of NTG eyes (*P* = 0.251). In addition, NTG had significantly shorter pores (*P* = 0.005) than NG. No difference in pore tortuosity or verticality was found between NG and NTG (*P* = 0.587 and *P* = 0.120, respectively). Pore verticality and length in HTG eyes did not significantly differ from that of NG eyes (*P* = 0.049 and *P* = 0.033, respectively) and NTG eyes (*P* = 0.827 and *P* = 0.968, respectively). All of the quantitative parameters measured were not correlated with age but were associated with glaucoma severity (Visual Field Index, mean deviation, retinal nerve fiber layer, and ganglion cell complex), except for pore verticality, which was not correlated with retinal nerve fiber layer.

**Conclusion:** The LC pores of patients with HTG appear to be more tortuous than the pores of patients with NG, and the pores of patients with NTG are shorter than those of patients with NG. Changes in pore parameters appear to be associated with the severity of glaucomatous optic neuropathy.

**Key Words:** optical coherence tomography, high-tension glaucoma, normal tension glaucoma, lamina cribrosa

(J Glaucoma 2024;33:957-963)

Located within the scleral canal, the lamina cribrosa (LC) separates the intraocular portion of the optic nerve from its intraorbital portion. The LC consists of a meshwork whose trabeculae delimit pores which are traversed by the axons of the retinal ganglion cells (RGCs).¹ The work by Quigley et al.²,³ in the early 1980s demonstrated blockage of axonal transport at the level of the LC pores in glaucoma. Because most axonal fibers follow the path of these pores,⁴ it becomes possible to estimate the axon paths by studying the pores and obtaining an indirect assessment of the mechanical stress exerted on them.

Numerous histologic studies have since explored the histomorphological characteristics of the laminar region in both healthy and glaucomatous patients to clarify the role of the LC in the formation of the first axonal lesions in glaucoma.⁵⁻⁸ The glaucomatous LC is subjected to compression/shear forces which distort the axonal fibers within the pores of the LC. These distortions are thought to cause blockage of axonal transport along the intralaminar pathway of the RGC fibers. However, the results of these histologic studies are probably biased by the presence of microarchitectural artifacts induced by optic nerve transection. To overcome these limitations, the resolution of the images and the penetration of the signal obtained with optical coherence tomography (OCT) have made it possible to study the LC in vivo.⁹ Although limited to the analysis of visible laminar tissue not covered by the neuroretinal ring, OCT studies have shown numerous macromorphological changes in the LC in glaucoma.¹⁰⁻¹³ The latest generations of OCT now make it possible to study the microarchitecture by reconstructing the path of the pores of the LC in 3 dimensions.¹⁴

Our team recently developed a semi-automated segmentation software for LC pore detection in vivo, allowing for a three-dimensional (3D) reconstruction of the pore pathway from data acquired on SD-OCT.¹⁵ We herein report the first comparative study performed with this software, analyzing the LC pore paths in patients with high-tension glaucoma (HTG), normal tension glaucoma (NTG), and non-glaucomatous (NG) by 3D LC reconstructions performed semiautomatically.

## PATIENTS AND METHODS

### Population

This single-center retrospective study was conducted between January and July 2022 at Quinze-Vings National Vision Hospital in Paris. The protocol applied during this

Received for publication November 23, 2023; accepted July 15, 2024.

From the *Department of Ophthalmology, Quinze-Vings National Ophthalmology Hospital, IHU FORESIGHT; †Department of Ophthalmology, Ambroise Paré Hospital, IHU FORESIGHT, AP-HP, University of Paris Saclay, Boulogne-Billancourt; ‡IHU FORESIGHT, INSERM-DGOS CIC, Vision Institute, Sorbonne University; and §ISEP Digital Engineering School, Paris, France.

Disclosure: The authors declare no conflict of interest.

Reprints: Paul Bastelica, MD, Hôpital National de la Vision des Quinze-Vings - Service 3 28 rue de Charenton, Paris 75012, France (e-mail: bast.bastelica@gmail.com).

Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.

DOI: 10.1097/IJG.0000000000002494
project was in accordance with the principles of the Declaration of Helsinki. Participants initially underwent a complete ophthalmologic examination by an ophthalmologist, recording maximum untreated intraocular pressure (IOP), Goldman tonometer applanation IOP (measured just before OCT acquisitions), pachymetry (Tonoref III, Nidek), slit lamp examination, gonioscopy, and optic disc examination by indirect ophthalmoscopy. Automated visual field (SITA Standard 24-2, Humphrey visual field analyzer, Carl-Zeiss Meditec) and retinal nerve fiber layer (RNFL) and ganglion cell complex (GCC) OCT (Spectralis, Heidelberg Engineering) were performed. Only cooperative patients over 18 years of age with no history of retinal disease or NG optic neuropathy and with an open iridocorneal angle were included. In addition, we selected only patients with a sufficiently large optic cup to properly visualize the LC. Patients were excluded if the examination could not be performed under optimal conditions or if the quality of the OCT images was insufficient for visual localization of the LC pores. The patients were divided into 3 groups: (1) NG, with IOP < 21 mm Hg, normal RNFL/GCC OCT, and normal visual field, (2) HTG, with characteristic optic nerve head changes, visual field impairment, and/or RNFL/GCC thinning on OCT, and (3) NTG, defined by the presence of optic nerve, OCT and visual field changes similar to HTG but with a maximum untreated IOP < 21 mm Hg. All of these patients underwent brain-orbital magnetic resonance imaging as a systematic examination performed in all patients with NTG to rule out compressive optic neuropathy (performed outside the scope of the study). Because optic cup size has been identified as a risk factor for glaucoma, some “healthy” patients could have a higher risk of glaucoma than the general population and were, therefore, categorized as “non-glaucomatous” according to our diagnostic criteria.¹⁶

## Image Acquisition and Analysis

Image acquisition was acquired at the same time as the RNFL/GCC measurement by 2 trained operators, using an SD-OCT (Spectralis, Heidelberg Engineering) in enhanced depth imaging mode, emitting at a wavelength of 880 nm, with a scanning frequency of 85 kHz. A 5.2×1.8×1.9 mm volume centered on the optic nerve head was scanned, composed of 131 vertical or horizontal en-face images of 496×768 pixels. The axial and lateral sampling density of an en-face image were 4 µm/pixel and 6 µm/pixel, respectively, with a sampling step between two consecutive images of ~11 µ.

The images were then processed and analyzed by a physician, blinded to glaucoma status, using dedicated semiautomatic software developed by ISEP (Digital Engineering School of Paris).¹⁵ The first step in image processing was to define the posterior limit of the OCT volume used to segment the pore, which corresponded to the en-face image at which the level of reflectivity no longer allowed tracking of the pores. Pore tracing was performed on the en-face images, where a path was initialized on the highest contrast en-face image, continuing anteriorly and posteriorly to the defined boundary. With a cursor, the examiner specified the center of the pore of interest, and the software indicated, in an automated way, the position of the pore on the upper/lower plane. On each image, the examiner could manually modify the position of the center of the pore. Three indices were then calculated for each pore: length (L), expressed in micrometers, tortuosity (L/L<sub>d</sub>), defined as the ratio of the

<figure><img src="image_1.png" /><figcaption>**FIGURE 1.** Schematic representation of a pore path. L indicates length of the pore path; L<sub>d</sub>, length directly connecting the first and last identified points of the path; L<sub>v</sub>, length separating the projection of the end of the pore path on the vertical axis. Figure 1 can be viewed in color online at www.glaucomajournal.com.</figcaption></figure>

length of the pore path to the length directly connecting the most anterior to the most posterior point of the path, and verticality (Ld/Lv), defined as the length directly connecting the most anterior to the most posterior point of the path related to the length separating the projection of the ends of the pore path on the vertical axis, expressed from 0 to 1, a value of 1 corresponding to a perfectly vertical pathway (Fig. 1).

## Statistical Analysis

A $\chi^2$ test was used to analyze qualitative data. As our analyses were based on a small sample, the distribution of the quantitative parameters analyzed did not follow a normal distribution. We, therefore, used the nonparametric Kruskal-Wallis test to compare the mean of the parameters among the 3 groups studied. When the test was significant at a level set at <0.05, the analysis was completed by a post hoc analysis using a pairwise multiple comparison test to analyze the differences between the groups, with correction of the statistical significance threshold by the Bonferroni method (fixed at 0.0167 for post hoc analyses). Univariate analyses with the Pearson correlation test and multivariate analyses with the ANCOVA test were used to determine whether the measured parameters were correlated with any of the various elements of the clinical and paraclinical examination in the entire study population. The significance level of these correlation analyses was set at a 2-tailed P value of <0.05.

## RESULTS

The study included 31 patients: 11 patients with NG, 12 patients with HTG, and 8 patients with NTG. Nine eyes were excluded due to insufficient image acquisition quality (4 NG and 5 HTG). A total of 52 eyes of 29 patients were analyzed (Fig. 2). The characteristics of the study population are summarized in Table 1. Patients with NG were younger, but the age difference was not statistically significant between groups (P = 0.071). The clinical characteristics of the studied eyes are summarized in Table 2. Visual acuity and pachymetry did not differ among the 3 groups. IOP at the time of acquisition was significantly different among the 3 groups (P = 0.016), and NTG eyes showed a lower IOP than HTG eyes (P = 0.021). Similarly, HTG eyes exhibited a higher maximum pretreatment IOP than NTG (P < 0.0001) and NG (P < 0.0001) eyes. The usual tomographic (RNFL and GCC) and visual field [mean
<figure><img src="image_2.png" /><figcaption>**FIGURE 2.** Flowchart describing the steps for including patients for analysis. HTG indicates high-tension glaucoma; NG, non-glaucomatous; NTG, normal tension glaucoma. Figure 2 can be viewed in color online at www.glaucomajournal.com.</figcaption></figure>

deviation (MD) and Visual Field Index (VFI) parameters for glaucoma diagnosis and follow-up differed between groups. Although these parameters appeared to be more affected in HTG eyes than in NTG eyes, the observed differences were not statistically significant (RNFL: *P* = 0.107; GCC: *P* = 0.827; MD: *P* = 0.717; *P* = 0.922).

A total of 337 pores were traced from the 52 acquisitions (Fig. 3). The number of traced pores per eye did not differ between the groups of eyes studied (*P* = 0.289). The 3 quantitative parameters measured from the pore tracings were significantly different among the 3 groups, and the results of pairwise comparisons are summarized in Figure 4. Pore tortuosity in HTG (1.419 ± 0.093) was significantly higher (*P* = 0.011) than in NG (1.347 ± 0.034) but did not differ from that of NTG eyes (*P* = 0.251). In addition, NTG had significantly shorter pores (*P* = 0.005) than NG. No difference in pore tortuosity or verticality was found between NG and NTG (*P* = 0.587 and *P* = 0.120, respectively). Pore verticality and length in HTG eyes did not significantly differ from that of NG eyes (*P* = 0.049 and *P* = 0.033, respectively) and NTG eyes (*P* = 0.827 and *P* = 0.968, respectively).

The results of the Pearson correlation analysis performed on the entire sample of patients are shown in Table 3. All of the quantitative parameters measured were correlated with glaucoma severity as measured by VFI, MD, RNFL, and GCC, except for pore verticality, which was not correlated with RNFL. In addition, the pore parameters measured showed no correlation with age or IOP at the time of the examination. In contrast, pore tortuosity was positively correlated with maximum IOP, and pore length was positively correlated with CCT. On multivariate analysis with pore tortuosity defined as the dependent variable, GCC was the dominant explanatory variable. In this model, GCC (*P* = 0.007), MD (*P* = 0.03), and VFI (*P* = 0.025) accounted for 43.1% of the variance (*R*² = 0.438; *P* < 0.0001). With pore verticality considered as the dependent variable, the model incorporating the variables correlated in the univariate analysis (MD, VFI, and GCC) provided no useful information, explaining only 10% of the variance in verticality (*R*² = 0.098; *P* = 0.173). The model, including the variables correlated with pore length (MD, VFI, GCC, RNFL, and CCT), explained 30% of the variance in pore length (*R*² = 0.301; *P* = 0.005). The dominant explanatory variable in this model was CCT (*P* = 0.032).

<table><caption>**TABLE 1.** Demographic Characteristics of the Population</caption><thead><tr><th>Qualitative data</th><th>NG<br>(n = 10);<br>n (%)</th><th>HTG<br>(n = 11);<br>n (%)</th><th>NTG<br>(n = 8);<br>n (%)</th><th>P</th></tr></thead><tbody><tr><td>Sex</td><td></td><td></td><td></td><td>0.531</td></tr><tr><td>F</td><td>5 (50)</td><td>6 (54.5)</td><td>6 (75)</td><td>—</td></tr><tr><td>M</td><td>5 (50)</td><td>5 (45.5)</td><td>2 (25)</td><td>—</td></tr><tr><td>Family history of<br>glaucoma</td><td>1 (10)</td><td>4 (36.4)</td><td>2 (25)</td><td>0.369</td></tr><tr><td>Age (y)</td><td>Mean (SD)</td><td>Mean (SD)</td><td>Mean (SD)</td><td>0.071</td></tr></tbody></table>

HTG indicates high-tension glaucoma; NG, non-glaucomatous; NTG, normal tension glaucoma.

## DISCUSSION

With recent OCT devices, it is now possible to analyze in vivo the path of the pores within the LC to better understand the mechanical stress exerted by LC trabeculae on RGC axons in glaucoma. Wang et al¹⁴ first studied the LC pore pathway in vivo. Using 3D reconstructions of tomographic acquisitions made from the optic nerves of healthy patients, glaucoma suspects, and glaucomatous patients, the pores were traced using customized software. The pores were more tortuous and shorter in patients with glaucoma. The authors also assessed the trajectory of the pores in relation to the center of the optic nerve head. The authors found no difference in the trajectory of the pores within the laminar tissue among the 3 groups of patients
<table><caption>TABLE 2. Clinical Characteristics of the Eyes and Pore Analysis</caption><thead><tr><th rowspan="2"></th><th colspan="3">Mean (SD)</th><th rowspan="2">P</th></tr><tr><th>NG (n = 18)</th><th>HTG (n = 18)</th><th>NTG (n = 16)</th></tr></thead><tbody><tr><td colspan="5"><strong>Quantitative clinical data</strong></td></tr><tr><td>Log VA</td><td>0.006 (0.02)</td><td>0.078 (0.24)</td><td>0</td><td>0.374</td></tr><tr><td>CCT (μm)</td><td>525.6 (31.6)</td><td>525.2 (47.4)</td><td>520.3 (19.2)</td><td>0.854</td></tr><tr><td>IOP</td><td>14.8 (3.5)</td><td>14.7 (3.2)</td><td>12.1 (1.9)</td><td>0.016</td></tr><tr><td>mIOP</td><td>16.5 (3.1)</td><td>26.6 (5.4)</td><td>15.7 (2.0)</td><td>&lt;0.0001</td></tr><tr><td>No. glaucoma medications</td><td>0</td><td>2.4 (1.2)</td><td>1.5 (1.0)</td><td>&lt;0.0001</td></tr><tr><td>Cup/disc ratio</td><td>0.717 (0.086)</td><td>0.783 (0.129)</td><td>0.794 (0.085)</td><td>0.046</td></tr><tr><td>MD 24-2 (dB)</td><td>-0.07 (0.08)</td><td>-7.19 (8.43)</td><td>-4.38 (4.40)</td><td>&lt;0.0001</td></tr><tr><td>VFI (%)</td><td>99.7 (0.6)</td><td>79.2 (27.1)</td><td>86.7 (14.1)</td><td>0.001</td></tr><tr><td>Mean RNFL thickness (μm)</td><td>96.0 (7.6)</td><td>65.0 (12.1)</td><td>74.4 (12.1)</td><td>&lt;0.0001</td></tr><tr><td>Mean GCC thickness (μm)</td><td>81.3 (3.1)</td><td>63.1 (10.4)</td><td>64.9 (7.6)</td><td>&lt;0.0001</td></tr><tr><td colspan="5"><strong>Pore analysis</strong></td></tr><tr><td>Pore paths (n)</td><td>6.33 (1.46)</td><td>7.1 (1.78)</td><td>6.31 (1.49)</td><td>0.289</td></tr><tr><td>Pore tortuosity</td><td>1.347 (0.034)</td><td>1.419 (0.093)</td><td>1.373 (0.067)</td><td>0.018</td></tr><tr><td>Pore length (μm)</td><td>139.9 (23.6)</td><td>113.7 (29.2)</td><td>110.2 (21.9)</td><td>0.004</td></tr><tr><td>Pore vertically</td><td>0.967 (0.031)</td><td>0.932 (0.047)</td><td>0.946 (0.036)</td><td>0.037</td></tr></tbody></table>

CCT indicates central corneal thickness; GCC, ganglion cell complex; HTG, high-tension glaucoma; IOP, intraocular pressure; MD, mean deviation; mIOP, maximum intraocular pressure; NG, non-glaucomatous; NTG, normal tension glaucoma; RNFL, retinal nerve fiber layer; VA, visual acuity; VFI, visual field index.

<figure><img src="image_3.png" /><figcaption>FIGURE 3. LC pore paths are identified by colored lines on the 3D reconstruction (left) by colored squares on the en-face OCT (center). The pore tracing can be visualized within the boundaries of the LC (right). A, Patient with NG. B, Patient with HTG. 3D indicates three-dimensional; HTG, high-tension glaucoma; LC, lamina cribrosa; NG, non-glaucomatous; OCT, optical coherence tomography. Figure 3 can be viewed in color online at www.glaucomajournal.com.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**FIGURE 4.** Box plots comparing (pairwise multiple comparisons) quantitative data measured on OCT in the NG, NTG, and HTG groups. Results were significant when *P* values were less than the Bonferroni corrected significance level calculated at 0.0167. HTG indicates high-tension glaucoma; NG, non-glaucomatous; NTG, normal tension glaucoma; OCT, optical coherence tomography. Figure 4 can be viewed in color online at www.glaucomajournal.com.</figcaption></figure>

studied. In our study, we looked at a new parameter, verticality, which not take into account the direction of the pores, but only their position relative to the orientation of the laminar tissue. Our results are consistent with those of the study by Wang and colleagues. The LC pores were

indeed more tortuous and the orientation of the pores did not differ in HTG eyes compared with NG eyes. The pores thus appeared distorted within the laminar tissue. These results are also consistent with previous experimental studies which have suggested that these changes are the

<table><caption>**TABLE 3.** Matrix of Pearson Correlation Coefficients of Pore Tortuosity, Length, and Verticality in the Study Population as a Function of Clinical, Tomographic, and Visual Field Data</caption><thead><tr><th>Variable</th><th>Tortuosity</th><th>Length</th><th>Verticality</th><th>MD</th><th>VFI</th><th>RNFL</th><th>GCC</th><th>Cup/disc</th><th>IOP</th><th>mIOP</th><th>ECC</th><th>Age</th></tr></thead><tbody><tr><td>Tortuosity</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>r</td><td>1</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>P</td><td>0</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Length</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>r</td><td>-0.230</td><td>1</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>P</td><td>0.100</td><td>0</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Verticality</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>r</td><td>-0.340</td><td>0.557</td><td>1</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>P</td><td>0.014</td><td>&lt;0.0001</td><td>0</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>MD</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>r</td><td>-0.445</td><td>0.297</td><td>0.276</td><td>1</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>P</td><td>0.001</td><td>0.033</td><td>0.048</td><td>0</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>VFI</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>r</td><td>-0.399</td><td>0.317</td><td>0.281</td><td>0.986</td><td>1</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>P</td><td>0.003</td><td>0.022</td><td>0.044</td><td>&lt;0.0001</td><td>0</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>RNFL</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>r</td><td>-0.455</td><td>0.386</td><td>0.198</td><td>0.629</td><td>0.574</td><td>1</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>P</td><td>0.001</td><td>0.005</td><td>0.159</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>0</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>GCC</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>r</td><td>-0.560</td><td>0.418</td><td>0.290</td><td>0.685</td><td>0.681</td><td>0.773</td><td>1</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>P</td><td>&lt;0.0001</td><td>0.002</td><td>0.037</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>0</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Cup/disc</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>r</td><td>0.220</td><td>-0.233</td><td>-0.139</td><td>-0.485</td><td>-0.456</td><td>-0.474</td><td>-0.474</td><td>1</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>P</td><td>0.116</td><td>0.096</td><td>0.326</td><td>0</td><td>0.001</td><td>0.002</td><td>0</td><td>0</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>IOP</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>r</td><td>0.017</td><td>0.045</td><td>-0.042</td><td>0.087</td><td>0.107</td><td>0.129</td><td>0.129</td><td>-0.235</td><td>1</td><td>—</td><td>—</td><td>—</td></tr><tr><td>P</td><td>0.903</td><td>0.749</td><td>0.766</td><td>0.538</td><td>0.452</td><td>0.675</td><td>0.361</td><td>0.094</td><td>0</td><td>—</td><td>—</td><td>—</td></tr><tr><td>mIOP</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>r</td><td>0.422</td><td>-0.200</td><td>-0.237</td><td>-0.531</td><td>-0.480</td><td>-0.371</td><td>-0.371</td><td>0.023</td><td>0.023</td><td>1</td><td>—</td><td>—</td></tr><tr><td>P</td><td>0.002</td><td>0.156</td><td>0.091</td><td>&lt;0.0001</td><td>0</td><td>&lt;0.0001</td><td>0.007</td><td>0.871</td><td>0.018</td><td>0</td><td>—</td><td>—</td></tr><tr><td>CCT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>r</td><td>-0.154</td><td>0.326</td><td>0.210</td><td>0.183</td><td>0.188</td><td>0.098</td><td>0.098</td><td>-0.234</td><td>0.438</td><td>0.183</td><td>1</td><td>—</td></tr><tr><td>P</td><td>0.276</td><td>0.018</td><td>0.135</td><td>0.193</td><td>0.182</td><td>0.433</td><td>0.487</td><td>0.095</td><td>0.001</td><td>0.194</td><td>0</td><td>—</td></tr><tr><td>Age</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>r</td><td>0.067</td><td>0.042</td><td>-0.151</td><td>-0.005</td><td>0.016</td><td>0.235</td><td>0.235</td><td>-0.019</td><td>0.293</td><td>0.311</td><td>0.016</td><td>1</td></tr><tr><td>P</td><td>0.638</td><td>0.766</td><td>0.286</td><td>0.971</td><td>0.910</td><td>0.352</td><td>0.094</td><td>0.894</td><td>0.035</td><td>0.025</td><td>0.908</td><td>0</td></tr></tbody></table>

Bold values are statically significant 0.05.

CCT indicates central corneal thickness; GCC, ganglion cell complex thickness; IOP, intraocular pressure; MD, mean deviation; mIOP, maximum intraocular pressure; RNFL, retinal nerve fiber layer; VFI, visual field index.
consequence of a compressive rearrangement of the successive sheets of the LC and are the cause of axon shear in glaucoma.⁶

We also explored the microarchitectural characteristics of the LC in patients with NTG. NTG is a specific entity defined by the presence of glaucomatous optic neuropathy with measured IOP < 21 mm Hg in the absence of any other cause of optic neuropathy.¹⁷ In our study, the laminar pores of patients with NTG presented a particular profile; they were neither more tortuous nor more vertical compared with the pores of patients with NG or with those of patients with HTG. However, the pores of patients with NTG were significantly shorter than those of patients with NG, whereas the difference between the length of NTG pores and HTG pores was not significant. The pores of patients with NTG appeared to have an intermediate microarchitectural profile between that of patients with NG and the pores of patients with HTG, which were more tortuous. Moreover, in this study, the greater the maximum IOP, the more tortuous the pores were, at IOP levels at the time of examination uncorrelated with the microarchitectural parameters. In these patients with NTG, the axons may, therefore, be less subject to the shear/compression stresses exerted by the LC trabeculae in response to IOPs that are consistently below 21 mm Hg. These results are consistent with the hypotheses formulated regarding the pathogenesis of NTG, according to which other factors independent of the mechanical stresses exerted by the IOP might be involved, in particular vascular factors.¹⁸,¹⁹

Other factors appear to be contributory to changes to the microarchitecture of laminar tissue. Wang et al¹⁴ found no link between tortuosity and the severity of visual field damage measured by MD and VFI. However, studies have demonstrated the presence of numerous changes (increased curvature of the anterior surface and thinning of the LC, intratissular defects) which appear in the early stages of glaucoma and worsen with disease severity.⁶,¹³,²⁰ These results are consistent with our observations. The pores of the LC tended to be shorter, more tortuous and less vertical in relation to disease severity. Tortuosity, verticality and length were indeed correlated with visual field and tomographic parameters of glaucoma follow-up, except for verticality, which was not correlated with RNFL. The microarchitectural changes found in our study thus seem to accompany the progression of glaucomatous optic neuropathy. Furthermore, in the study by Wang et al,¹⁴ the tortuosity of the pores of patients with early-stage glaucoma was significantly different from that of healthy patients. We did not perform subgroup analyses because the number of patients was too small. However, on multivariate analysis, the most informative parameter to explain pore tortuosity in our study was the GCC. Since damage to the GCC occurs early in the natural history of glaucoma,²¹ this result supports the hypothesis that microarchitectural damage to the LC may occur early or even precede the first axonal lesions. However, it remains difficult to establish the precise chronological sequence between the appearance of these microarchitectural alterations and the onset of the first axonal lesions in the RGCs. Longitudinal studies are still needed to determine whether the distortion of LC pores predispose to axonal lesions, but it would seem that the use of these new parameters could be of interest for the early diagnosis and monitoring of glaucomatous optic neuropathies.

The LC thickness has been incriminated in the pathogenesis of glaucomatous optic neuropathy, particularly that of NTG.²² A thin LC, by increasing the translaminar

pressure gradient, would partly explain the development and progression of glaucomatous optic neuropathy despite normal measured IOP. In our study, a parameter related to LC thickness, pore length, was correlated with CCT. The lower the CCT, the shorter the pores in the entire population including patients with glaucomatous and NG. Moreover, in the multivariate model in which pore tortuosity was defined as the dependent variable, CCT was the dominant explanatory variable. This result is surprising, but raises the question of the possible links between corneal biomechanics and LC microarchitecture. This hypothesis has been suggested in light of the similarity in histologic composition of the extracellular matrix of the cornea to that of the LC. However, Jonas et al²² found no link between LC thickness and corneal pachymetry.²³ The authors pointed out the potential bias introduced into their histomorphometric study, notably due to the swelling of post-mortem tissue and the induced artifacts in the LC and measured corneal parameters. Although imperfect, in particular due to the attenuation of the OCT signal by the prelaminar tissue, in vivo OCT exploration makes it possible to avoid these postmortem artifacts. In light of our results, a thin cornea could also be associated with a thin LC, and thus constitute an indirect sign of microarchitectural susceptibility to axonal lesions in glaucoma. It would be interesting to combine the measurement of our parameters with that of corneal hysteresis, which gives a more complete picture of the biomechanical properties of the cornea in clinical practice than CCT alone.²⁴ Moreover, several OCT studies have shown that low corneal hysteresis is associated with more pronounced dynamic morphometric changes (posterior displacement and posterior curvature of the anterior surface of the LC).²⁵,²⁶ These complex links are still poorly understood, and further work is obviously needed to better understand the potential association between LC and corneal biomechanics.

This study has limitations. The work was based on the analysis of a small sample of the population of interest. As a result, the statistical analyses lacked power, and further work on larger numbers is still needed. Moreover, because of the overall hyperreflectivity of the images and the presence of intralaminar defects probably related to fibrous remodeling of the LC,⁸ fewer pores could be traced correctly in the 2 groups of patients with glaucoma. We thus decided to limit the evaluation to between 5 to 10 pores for each eye. Another well-known and unavoidable limitation of tomographic imaging of the LC is that the images obtained on OCT allow only partial visualization of the laminar tissue. Areas covered by vessels or the neuroretinal rim cannot be analyzed, because the OCT signal is absorbed by these structures. Consequently, some patients with very small optic discs and small or no cups were excluded, because the images obtained were of poor quality and did not allow pores to be traced. This was obviously a selection bias of the present study inherent to the imaging technology used. In addition, the study was carried out only in patients with a visible LC, whose characteristics are certainly different from those of the general population. However, as this selection process was identical for all 3 groups, it might not have influenced the results. As explained previously, the size of the optic cup has been identified as a risk factor for glaucoma.¹⁶ Thus, our patients with NG, although totally normal regarding glaucoma diagnostic criteria, are more susceptible to future glaucoma than the general population. Another limitation of the present study is that the hyperreflectivity of the LC and prelaminar tissue
tends to attenuate the OCT signal and, therefore, reduce visualization of the deeper layers of the LC. It is likely that the entire pores were not traced. In addition, the analyses focused on pores not covered by the neuroretinal rim. Analysis of these “empty” pores does not necessarily reflect the architecture of the pores still containing RGC axons, which are probably more representative of the impact of LC microarchitecture on axonal lesions. Moreover, it has been shown that the trajectory of the pores differs according to their location in the laminar tissue.²⁷ Peripheral pores are longer and more tortuous than central pores in healthy patients. Thus, the analysis of NG eyes should theoretically have been based on more central and less tortuous pores, artificially increasing the differences between NG and glaucomatous eyes. The importance of this potential bias was reduced by the small difference in LC visibility between the two groups.

## CONCLUSION

This study further characterized the changes in the path of the LC pores in HTG and NTG. The pores of patients with HTG appear to be more tortuous than the LC pores of patients with NG. Changes in LC pore parameters appear to be associated with the severity of glaucomatous optic neuropathy. As it appears correlated to GCC damage, the increase in pore tortuosity could be altered as early as, or even precede, the first axonal lesions and thus constitute a predisposing factor for glaucoma. Further studies are needed to understand the chronological sequence of the changes in these new parameters and the first axonal lesions related to glaucoma and to clarify the role that this new imaging technique could play in the diagnosis and monitoring of patients with glaucoma.

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