June 2012
Volume 53, Issue 7
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Glaucoma  |   June 2012
In Vivo Imaging of Lamina Cribrosa Pores by Adaptive Optics Scanning Laser Ophthalmoscopy
Author Notes
  • From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan. 
  • Corresponding author: Tadamichi Akagi, Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, 54 Shougoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan; akagi@kuhp.kyoto-u.ac.jp
Investigative Ophthalmology & Visual Science June 2012, Vol.53, 4111-4119. doi:10.1167/iovs.11-7536
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      Tadamichi Akagi, Masanori Hangai, Kohei Takayama, Atsushi Nonaka, Sotaro Ooto, Nagahisa Yoshimura; In Vivo Imaging of Lamina Cribrosa Pores by Adaptive Optics Scanning Laser Ophthalmoscopy. Invest. Ophthalmol. Vis. Sci. 2012;53(7):4111-4119. doi: 10.1167/iovs.11-7536.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To visualize and assess the surface-level pores of the lamina cribrosa in patients with glaucoma by using a prototype adaptive optics scanning laser ophthalmoscopy (AOSLO) system.

Methods.: The numbers of laminar pores were compared between color disc photography, scanning laser ophthalmoscopy (SLO) without AO, and AOSLO. The pore area and elongation index were examined for correlation with ocular parameters such as the mean deviation, disc area, cup/disc ratio, disc ovality index, intraocular pressure (IOP), and axial length in the AOSLO images.

Results.: The 40 eyes (20 normal and 20 glaucomatous) of 40 subjects were enrolled. The AOSLO provided laminar pore images of better quality than other imaging methods, and the number of visible pores was significantly greater in the AOSLO images than in the other imaging methods (the color disc photographs [P < 0.001] and SLO without AO images [P < 0.001]) when compared for 26 subjects. When compared for 40 subjects using AOSLO, the pore area was significantly larger in glaucomatous subjects than in normal subjects (P = 0.031), but elongation index was not. The pore area correlated significantly with the axial length (P = 0.008) in normal subjects, with the untreated IOPs (P = 0.002) in the glaucomatous subjects, and with the axial length (P = 0.001) and cup/disc ratio (P = 0.012) in the total subjects. Via multiple regression analysis, significant correlations with pore area were found for axial length in the normal (β = 0.684, P = 0.001) and total subjects (β = 0.496, P < 0.001) and untreated IOP in the glaucomatous (β = 0.506, P = 0.023) and total subjects (β = 0.331, P = 0.014).

Conclusions.: AOSLO is a useful imaging technology for assessing laminar pore morphology. The laminar pore area may be affected by axial length and IOP.

Introduction
The lamina cribrosa is the site for entry and exit of the retinal blood vessels and optic nerve fibers; the axons of the retinal ganglion cells form bundles to pass through the laminar pores. 1 On the basis of histopathologic findings, the lamina cribrosa has been implicated as the origin of optic nerve damage in glaucoma. 2 Previous histopathologic studies of cadaver or enucleated human eyes have shown morphologic changes in the lamina cribrosa at different stages of glaucoma. 38 To assess the laminar abnormalities associated with glaucoma clinically, several in vivo morphologic studies of the laminar pores in patients with glaucoma have been performed with color disc photography 6,9 or scanning laser ophthalmoscopy (SLO). 5,1013 The results of these studies have included decreased pore area, 9 increased pore area, 5 pore compression, 68 and less-circular pore shape 5 in glaucomatous eyes compared with normal eyes. Precise morphometry of the pore dimensions by in vivo imaging requires clear visualization of all of the laminar pores, including those that are small and deformed. However, such visualization is limited by the poor lateral resolution of the conventional optical fundus imaging methods. Therefore, improved visualization of the laminar pores is required for improved clinical assessment of the laminar abnormalities associated with glaucoma. 
The poor lateral resolution of fundus imaging with optical instruments, such as color fundus photography and SLO, is due primarily to aberrations in the ocular optics. Adaptive optics (AO) systems consisting of a wavefront sensor to measure ocular aberrations, and a deformable mirror or a spatial light modulator to compensate for the measured aberrations, can be used to improve the resolution of fundus imaging of living eyes. 1418 The application of AO to imaging equipment, such as flood-illuminated ophthalmoscopes, scanning laser ophthalmoscopes, and fundus cameras, has allowed researchers to identify abnormalities in fundus microstructures, including individual cone photoreceptors in patients with color blindness, foveal hypoplasia, optic nerve drusen or other optic neuropathies, macular microholes, inherited retinal degeneration, and central serous chorioretinopathy. 1929 For example, Vilupuru et al. 30 have used AOSLO to examine the laminar pores in monkey eyes and obtained high-quality images of these structures. AO technology also has allowed clear visualization of human laminar pores. 31,32 Recently, Ivers et al. 33 have imaged the laminar pores in normal monkey and human eyes and showed that AOSLO was potentially useful for measuring laminar pore dimensions. However, no studies to date have used AOSLO to assess the lamina cribrosa in patients with glaucoma. Therefore, we performed this pilot study by using a prototype AOSLO system to visualize and assess the surface-level pores of the lamina cribrosa in patients with glaucoma. 
Methods
Subjects
Patients with open-angle glaucoma (primary open-angle glaucoma and normal-tension glaucoma) and healthy controls were recruited from the glaucoma service at Kyoto University Hospital. Normal-tension glaucoma was defined as untreated peak intraocular pressure (IOP) ≤21 mm Hg on three repeated measurements obtained at different times on separate visits before the start of antiglaucoma medication. Subjects underwent a comprehensive ophthalmic examination, including measurement of uncorrected and best-corrected visual acuity (BCVA) with the 5-m Landolt chart, slit-lamp examination, IOP measurement with a Goldmann applanation tonometer, gonioscopy, dilated stereoscopic examination of the optic nerve head, stereo disc photography (3-Dx simultaneous stereo disc camera; Nidek, Gamagori, Japan), red-free fundus photography (Heidelberg Retina Angiograph II [HRA II]; Heidelberg Engineering, Heidelberg, Germany), and standard automated perimetry (SAP) through the Humphrey Visual Field Analyzer with the 24-2 Swedish Interactive Threshold Algorithm (HFA + 24-2 SITA; Carl Zeiss Meditec, Inc., Dublin, CA). 
The inclusion criteria for the patients with glaucoma were glaucomatous appearance of the optic nerve disc (diffuse or localized rim thinning) and corresponding abnormalities observed during reliable visual field (VF) testing (considered reliable on the basis of ≤20% fixation loss, ≤15% false positives, and ≤33% false negatives). An abnormal VF was defined as one having an abnormal range during glaucoma hemifield testing or pattern standard deviation of <5% of the normal reference value. The inclusion criteria for the healthy controls were IOP of ≤21 mm Hg, normal-appearing optic disc, no retinal nerve fiber layer defects, and reliable and normal SAP results. The exclusion criteria were BCVA worse than 20/20 in the Snellen equivalent, evidence of concomitant ocular pathologies other than glaucoma affecting the optic nerve or VF, or any other systemic disease that could affect the eye or VF results, such as a cerebrovascular event or blood disorders. 
All investigations adhered to the tenets of the Declaration of Helsinki. The Institutional Review Board and Ethics Committee of Kyoto University Graduate School of Medicine approved the study design. All enrolled subjects gave their written informed consent to participation in the study after receiving an explanation of its nature and possible consequences. 
AOSLO System
A prototype AOSLO system was used to acquire images of the laminar surface as previously described. 28,29 In brief, the system consisted of three primary optical subsystems: a high-resolution confocal SLO imaging subsystem incorporating Badal optics and a wavefront corrector, the wavefront sensing subsystem, and a wide-field imaging subsystem based on the confocal line-scanning SLO principle. 
The SLO subsystem used an 840-nm superluminescent diode (SLD) with 50 nm full width at half-maximum as the light source and a liquid-crystal spatial-light modulator fabricated with liquid-crystal-on-silicon technology as the wavefront corrector. Horizontal raster scans were created by a resonant scanner (SC-30; Electro-Optical Products Corp., Fresh Meadows, NY) and vertical scans by a galvanometer (6230H; Cambridge Technology, Inc., Lexington, MA). We used 2.0 Airy disc confocal pinhole. We also used a head-and-chin rest system that is inclined to stabilize the patients' heads for AO imaging. The high-resolution images were acquired at 10 frames per second. The sinusoidal distortion of the acquired image was corrected offline. The field of view of each AOSLO image after the distortion correction was 1.1° × 1.5° along the horizontal and vertical axes. Each image was stored in Audio Video Interleave (.avi) format on the computer's hard drive. The light source for wavefront sensing was a 780-nm laser diode. The powers into the eye at the subject's pupil were 220 μW for the high-resolution confocal SLO imaging and 70 μW for the wavefront sensing. Although different wavelengths were used for the high-resolution confocal SLO imaging and wavefront sensing, adverse effects were unlikely because (1) the color aberration of the human eye between 780 nm and 840 nm was almost negligible, 34,35 (2) the astigmatisms and higher-order aberrations were corrected by the wavefront corrector with custom control software based on the wavefront error detected by the wavefront sensing subsystem, and (3) the remaining defocus component was adjusted with the Badal optics independently of the detected wavefront error. 
To create wide-field images, we used a 910-nm SLD as the light source and a charge-coupled device–based line scan camera for the linear confocal detection. The field of view was 28° in the horizontal direction and 24° in the vertical direction; the imaging area of the wavefront-corrected high-resolution confocal SLO is indicated in real time by a rectangular frame on the wide-field images. The area of interest for the high-resolution images can be located arbitrarily by moving the rectangular frame on the screen. The rectangular frame on the wide-field images for each high-resolution imaging was recorded. 
AOSLO-Based Imaging
The AOSLO system scanned the optic nerve head by shifting its focus from the surface through to the bottom, and the first images showing clear laminar pore patterns were recorded. In this study, the laminar pores were considered to be at the inner surface of the lamina cribrosa. The scan was repeated at this depth to cover the entire cup area. We built montage images from many individual images captured as single frames from the AOSLO videos. We excluded the AOSLO images in which no laminar pores were visible because of thick neuroretinal rim or retinal vessels from the montage images. The degree to which each montage corresponded to the area of interest was verified by comparing the AOSLO image to the color disc photograph of that eye. In detail, we superimposed multiple neighboring frames on the color disc fundus photograph of Adobe Photoshop (Adobe Systems, San Jose, CA) by referring to the rectangular frame recorded on the wide-field image. 
Measurement of Laminar Pore Dimensions
The mean superior and inferior laminar pore dimensions (laminar pore area and laminar pore elongation index) in each eye were calculated by averaging the laminar pore dimensions measured from the laminar pores within two sectors of the laminar surface in the AOSLO images. We determined the line connecting the centroid of the disc margin (clinically defined by the border tissue of Elschnig) and the foveal center as a reference line in the same color fundus photograph. The centroid of the optic disc was determined by using ImageJ software (National Institutes of Health, Bethesda, MD). The two measurement sectors were chosen by using a modified Garway-Heath regionalization, as shown in Figure 1. Briefly, we located the measurement sectors at 0° to 90° relative to the circumferentially superior and inferior directions in order to include both superior and inferior measurement areas. The centroid of the optic disc was determined by using ImageJ software, and the center of the fovea was marked by hand on the fundus photograph. The laminar pores that extended across the borders of the measurement areas were excluded from evaluation. Each examiner manually determined the margins of the laminar pores by drawing a polygon line along the boundary between the poorly reflective laminar pores and highly reflective lamina plate, using Adobe Photoshop. The area and ovality index (minor axis length/major axis length) were calculated with ImageJ software. The reciprocal of the ovality index (major axis length/minor axis length, termed pore elongation index) was used to represent the lateral pore elongation on the surface of the lamina cribrosa. The pore area was corrected by using Littmann's formula for each axial length. 36 The mean pore area and elongation index were calculated from all of the pores within the two regions (superior and inferior). 
Figure 1. 
 
A schematic diagram of the modified Garway-Heath regionalization in a right eye. The two measurement sectors are shown in red (superior) and blue (inferior). The line connecting the centroid of the disc margin (green circle) and the fovea (green cross) was designated as the reference line.
Figure 1. 
 
A schematic diagram of the modified Garway-Heath regionalization in a right eye. The two measurement sectors are shown in red (superior) and blue (inferior). The line connecting the centroid of the disc margin (green circle) and the fovea (green cross) was designated as the reference line.
Comparison of Numbers of Visible Laminar Pores
For the comparison between AOSLO, SLO without AO, and color disc photography, the numbers of all visible pores in the entire optic disc of the eyes in which the laminar pores on the color fundus photographs were sufficiently clear for counting were counted. For comparisons between the normal and glaucomatous groups, the numbers of visible pores in the measurement sectors on the AOSLO images (described in Fig. 1) in all the 40 eyes (20 normal eyes and 20 glaucomatous eyes) were counted with AOSLO. 
Measurement of Ocular Parameters
A Heidelberg Retina Tomograph II (HRT II; Heidelberg Engineering) was used to measure the disc area and linear cup/disc ratio, and to obtain in-focus SLO images without AO. HRT II was also used to obtain best-focused laminar pore images as clinically available SLO images without AO. HRT II provides serial en face images by shifting the focus from the surface of the optic disc to the deep layers of the lamina cribrosa, from which we selected the depth that best showed the laminar pores. The axial length was measured with the IOLMaster (Carl Zeiss Meditec). We used the mean sensitivity values in the threshold map within the superior or inferior hemisphere as parameters to represent the degree of VF loss within each hemisphere. Mean sensitivity values were calculated by dividing the sum of the sensitivity values of all test points in the superior or inferior hemisphere by the number of test points. The threshold map “<0” was regarded as zero in the Humphrey Visual Field Analyzer.  
Interobserver, Intraobserver, and Intervisit Reproducibilities
To evaluate the interobserver reproducibility of our measuring method, the montage AOSLO images of all 40 subjects were evaluated independently by two examiners blinded to any information other than the laminar pore images, and intraclass correlation coefficients [ICCs (2,1)] were calculated. 
To evaluate the intraobserver reproducibility and variability in measuring the same pores, 30 pores of the same images from two normal subjects were evaluated three times by the same examiner to calculate ICC (1,1) and coefficient of variation (CV) of the areas and elongation indices. 
To evaluate the intervisit variability, the data of the seven normal subjects who agreed to a second visit were compared between the two different days (the two visits were 35–77 days apart). The ICCs (1,2) of the mean areas and elongation indices of the pore measurement regions were calculated. To calculate the ICCs (1,2) of the areas and elongation indices of the same pores obtained on the two different days, the same 30 pores obtained on each day.  
Statistical Analysis
All statistical evaluations were performed by using commercially available software (SPSS version 18; SPSS, Inc., Chicago, IL). A P value of <0.05 was considered statistically significant; the data were presented as the mean ±1 SD. We analyzed the relationships between the pore dimensions (mean pore area and elongation index) and the ocular parameters (axial length, mean deviation [MD], disc area, and linear cup/disc ratio) by using Spearman's correlation coefficients (ρ) and unpaired t-tests. Analysis of variance (ANOVA) and Tukey's post hoc test were used to compare the numbers of laminar pores counted by three different imaging methods. Stepwise forward multivariate linear regression analyses were performed to evaluate the effects of age, disc area, axial length, IOP, and disc ovality index on the pore area and elongation index, and multiple logistic regression analysis was applied to correlate these factors, with presence of glaucoma.  
Results
Clinical Characteristics
Forty eyes (20 glaucomatous and 20 normal) were examined in this study (Table 1). All of the subjects were of Japanese ethnicity. Among all of the cases of glaucoma, eight were primary open-angle glaucoma and 12 normal-tension glaucoma. There were no significant differences in sex (Pearson's χ2 = 1.616, P = 0.204) or age (healthy controls, 51.3 ± 11.2 years [range, 33–81 years]; patients with glaucoma, 57.9 ± 14.6 years [range, 27–78 years]; unpaired t-test: P = 0.114) between the healthy controls (men: 13, women: 7) and the patients with glaucoma (men: 9, women: 11). The untreated maximum IOPs and the IOPs at the time of AOSLO of the patients with glaucoma and the untreated IOPs of the normal subjects are shown in Table 1. The MD value and cup/disc ratio differed significantly between the groups, whereas no differences were observed for age, disc area, axial length, refractive errors, disc ovality index, disc rotation angle, and IOP (Table 1). 
Table 1. 
 
Subject Characteristics and Ocular Measurements
Table 1. 
 
Subject Characteristics and Ocular Measurements
Parameter Glaucomatous Group (n = 20) Normal Group (n = 20) P
Age, y 57.9 ± 14.6 (27–78) 51.3 ± 11.2 (33–81) 0.114
MD, dB −10.42 ± 7.79 (−1.19 to −29.41) −0.17 ± 1.05 (1.63 to −3.66) <0.001*
Axial length, mm 25.35 ± 1.88 (21.64–27.90) 24.79 ± 1.62 (21.85–27.79) 0.319
Refraction, diopters −4.10 ± 3.77 (−11.75 to 1.25) −2.71 ± 2.67 (−8.75 to 1.75) 0.188
IOP when sampling, mm Hg (untreated IOP, mm Hg) 15.7 ± 2.5 (12–20) (21.4 ± 6.9 [14-46]) 14.4 ± 1.7 (12–18) 0.053
Disc area, mm2 2.28 ± 0.63 (1.09–3.91) 2.30 ± 0.34 (1.66–3.01) 0.884
Disc ovality index 0.85 ± 0.06 (0.72–0.98) 0.89 ± 0.08 (0.76–1.04) 0.116
Disc rotation angle, ° 7.50 ± 4.32 (2–15) 5.55 ± 3.10 (0–12) 0.110
Vertical cup/disc ratio 0.74 ± 0.12 (0.47–0.94) 0.52 ± 0.12 (0.34–0.81) <0.001*
Pore area, μm2 3103.0 ± 857.0 (1893.3–4775.0) 2507.7 ± 825.5 (1211.7–5079.8) 0.031*
Pore area (superior), μm2 3489.5 ± 1190.2 (1594.9–6431.3) 2927.8 ± 1225.9 (1318.1–5017.3) 0.012*
Pore area (inferior), μm2 2927.8 ± 1225.9 (1174.0–6370.4) 2459.6 ± 903.1 (1109.7–5156.0) 0.177
Pore elongation index 2.38 ± 0.79 (1.67–5.24) 2.13 ± 0.47 (1.59–3.23) 0.231
Pore elongation index (superior) 2.21 ± 0.94 2.00 ± 0.46 0.365
Pore elongation index (inferior) 2.48 ± 0.76 2.27 ± 0.54 0.307
Laminar Pore Imaging by AOSLO, SLO without AO, and Color Disc Photography
The laminar pores were clearly visible as hyporeflective spots on the AOSLO images. Each montage of the AOSLO images could be successfully fitted to the area of interest on the color disc photograph. As a result, the locations of the laminar dots on the color disc photographs corresponded to the hyporeflective spots on the AOSLO images in all the eyes examined. The borderlines of the laminar pores were better visualized in the AOSLO images than in the color disc photographs or SLO without AO images. In particular, AOSLO clearly revealed the small, thin, and irregularly shaped pores that were not clearly visible by color disc photography or SLO without AO (Fig. 2). 
Figure 2. 
 
The comparison of (1) color disc photographs, (2) HRA SLO images, (3) HRT SLO images when best focused on the laminar pores at the superficial level of the lamina cribrosa, and (4) AOSLO montage overlaid on the same HRA SLO images. (A) The left eye of a 50-year-old man with normal VF. (B) The right eye of a 39-year-old woman with normal-tension glaucoma. Resolution and contrast are improved following AO correction. Green arrowheads show that the borders of the pores are not clear in SLO without AO (2, 3) but are relatively clear in AOSLO (4).
Figure 2. 
 
The comparison of (1) color disc photographs, (2) HRA SLO images, (3) HRT SLO images when best focused on the laminar pores at the superficial level of the lamina cribrosa, and (4) AOSLO montage overlaid on the same HRA SLO images. (A) The left eye of a 50-year-old man with normal VF. (B) The right eye of a 39-year-old woman with normal-tension glaucoma. Resolution and contrast are improved following AO correction. Green arrowheads show that the borders of the pores are not clear in SLO without AO (2, 3) but are relatively clear in AOSLO (4).
We compared the numbers of laminar pores clearly visible in the entire optic disc of the 26 eyes (14 normal eyes and 12 glaucomatous), for which the color disc photographs revealed laminar pores with sufficient clarity for counting in each of the AOSLO, color disc photography, and SLO without AO images. SLO without AO and AOSLO were able to visualize laminar pores clearly in all the 26 eyes, for which the color disc photographs displayed laminar pores with sufficient clarity for counting. We found that the ICCs (2,1) for the numbers of laminar pores counted by color disc photography, SLO without AO, and AOSLO were 0.815, 0.833, and 0.855, respectively. The number of clearly visible laminar pores differed significantly among the groups (ANOVA, P < 0.001). The pore number of the AOSLO images (47.1 ± 14.8/eye [range, 24–80/eye]) was larger than that of the color disc photographs (18.2 ± 14.8/eye [range, 0–50/eye]) or SLO without AO images (25.6 ± 12.8/eye [range, 4–55/eye]); statistically significant differences were found between the AOSLO and color disc photography images (P < 0.001) and between the AOSLO and SLO without AO images (P < 0.001), but not between the color disc photographs and SLO without AO images (P = 0.138). The mean numbers of laminar pores detected with AOSLO in the entire optic discs of 14 glaucomatous and 12 normal eyes were 51.0 ± 11.0/eye and 42.6 ± 17.8/eye, respectively. There was no statistical difference between these two groups (unpaired t-test: P = 0.172). The total numbers of pore in the measurement regions (the sectors described in Fig. 1) of the 20 glaucomatous and 20 normal eyes were 516 (268 superior and 248 inferior) and 471 (241 superior and 230 inferior), respectively. 
Interobserver, Intraobserver, and Intervisit Reproducibility
The superior and inferior laminar pore dimensions in each eye were manually measured within two sectors of the laminar surface (Fig. 3). The interobserver reproducibility of the measurements of the mean pore area [ICC (2,1) = 0.867] and elongation index [mean pore elongation index, ICC (2,1) = 0.931] was excellent as was the intraobserver reproducibility [pore area, ICC (1,1) = 0.968; pore elongation index, ICC (1,1) = 0.958] when the same examiner measured the same 30 randomly selected pores at three different times. The CVs for pore area and elongation index ranged from 0.9% to 16.4% (mean, 7.6%) and from 0.1% to 14.4% (mean, 7.0%), respectively. When the results for the seven normal subjects who agreed to a second visit were reanalyzed between the two different days (ranging from 35 to 77 days apart) to evaluate intervisit variability, the ICCs (1,2) for pore area and elongation index were 0.734 and 0.880, respectively. The mean pore area and elongation index were, respectively, 3432.0 ± 894.2 and 2.33 ± 0.61 on day 1 and 2985.3 ± 1185.4 and 2.56 ± 0.87 on day 2 within the measurement area. The ICCs (1,2) of the areas and elongation indices of the same pores obtained on the two different days were 0.915 and 0.900, respectively, when the same examiner measured the same 30 pores obtained on the different days. 
Figure 3. 
 
The representative images were obtained by (1) color disc photography, (2) AOSLO with color disc photography, (3) AOSLO with framed squares (measurement areas). Panel (4) shows magnified images (left) and the same images with the pores marked in red (right) within the measurement areas. Panel (5) shows HFA + 24-2 SITA of the (A) left eye of a 46-year-old woman with normal VF (MD = 0.26 dB), (B) left eye of a 62-year-old man with primary open-angle glaucoma (SAP showed more severe VF defects in the inferior hemifield, MD = −15.34 dB), and (C) right eye of a 70-year-old woman with primary open-angle glaucoma (SAP showed unilateral VF defects in the superior hemifield, MD = −13.82 dB). Scale bar = 500 μm.
Figure 3. 
 
The representative images were obtained by (1) color disc photography, (2) AOSLO with color disc photography, (3) AOSLO with framed squares (measurement areas). Panel (4) shows magnified images (left) and the same images with the pores marked in red (right) within the measurement areas. Panel (5) shows HFA + 24-2 SITA of the (A) left eye of a 46-year-old woman with normal VF (MD = 0.26 dB), (B) left eye of a 62-year-old man with primary open-angle glaucoma (SAP showed more severe VF defects in the inferior hemifield, MD = −15.34 dB), and (C) right eye of a 70-year-old woman with primary open-angle glaucoma (SAP showed unilateral VF defects in the superior hemifield, MD = −13.82 dB). Scale bar = 500 μm.
Comparison of Pore Analysis between Normal and Glaucomatous Subjects
The mean numbers of the pores within the measurement areas of AOSLO images were comparable (total, P = 0.366; superior, P = 0.325; inferior, P = 0.513) between the 20 glaucomatous (total, 25.8 ± 6.0 [range, 15–40]; superior, 13.4 ± 3.3 [range, 7–20] ; inferior, 12.4 ± 3.9 [range, 5–20]) and 20 normal (total, 23.6 ± 9.2 [range, 8–40]; superior, 12.1 ± 5.1 [range, 4–22]; inferior, 11.5 ± 4.7 [range, 4–21]) subjects. The mean pore area (unpaired t-test: P = 0.031), but not the mean pore elongation (unpaired t-test: P = 0.231), differed significantly between the groups (Table 1). When we performed multiple logistic regression analysis to correlate the several factors, such as age, disc area, axial length, IOP, disc ovality index, pore area, and pore elongation index, with presence of glaucoma, glaucoma was significantly associated with a larger mean pore area (P = 0.014), older age (P = 0.012), higher IOP (P = 0.014), and a smaller disc ovality index (P = 0.021), but not the mean pore elongation (P = 0.092), disc area (P = 0.789), or axial length (P = 0.418). 
Correlations of the Pore Dimensions with the Ocular Parameters in Normal and Glaucomatous Groups
We examined the pore dimensions and ocular parameters in each group as shown in Table 2. The mean pore area correlated significantly with the axial length in normal subjects, with the untreated maximum IOPs in the glaucomatous subjects, and with the axial length and cup/disc ratio in the total subjects (Fig. 4). The pore elongation index did not correlate with any ocular parameter. 
Table 2. 
 
Univariate Analysis of the Associations between Pore Dimensions and Ocular Parameters
Table 2. 
 
Univariate Analysis of the Associations between Pore Dimensions and Ocular Parameters
Subject Pore Area Pore Elongation Index
Normal Glaucomatous Total Normal Glaucomatous Total
ρ P ρ P ρ P ρ P ρ P ρ P
Age, y −0.279 0.234 −0.124 0.603 −0.036 0.827 −0.352 0.128 −0.294 0.208 −0.237 0.141
MD value, dB 0.010 0.967 0.003 0.990 −0.272 0.089 −0.068 0.774 0.367 0.112 −0.090 0.582
Axial length, mm 0.576 0.008* 0.424 0.062 0.519 0.001* 0.253 0.283 0.039 0.870 0.181 0.264
Disc area, mm2 −0.451 0.056 0.280 0.232 −0.022 0.892 −0.394 0.086 −0.015 0.950 −0.202 0.212
Disc ovality index −0.002 0.995 0.414 0.070 0.094 0.563 −0.176 0.458 0.044 0.853 −0.140 0.390
Cup/disc ratio −0.040 0.867 0.093 0.698 0.395 0.012* −0.315 0.176 −0.103 0.665 −0.008 0.959
IOP, mm Hg −0.582 0.007 0.192 0.417 0.064 0.693 −0.312 0.181 0.204 0.389 0.027 0.870
Untreated maximum IOPs, mm Hg 0.645 0.002* 0.308 0.053 −0.298 0.201 −0.039 0.810
Figure 4. 
 
The scatter diagram shows the linear regression (y = 348.972x – 6144.034) of the laminar pore area against the axial length in the normal subjects (P < 0.01, r 2 = 0.468).
Figure 4. 
 
The scatter diagram shows the linear regression (y = 348.972x – 6144.034) of the laminar pore area against the axial length in the normal subjects (P < 0.01, r 2 = 0.468).
By multiple regression analysis, significant correlations with pore area were found for axial length in normal (β = 0.684, P = 0.001) and total subjects (β = 0.496, P < 0.001) and untreated IOP in glaucoma (β = 0.506, P = 0.023) and total subjects (β = 0.331, P = 0.014). Pore elongation index did not correlate with any ocular parameter. 
Discussion
Previous histologic studies have revealed that the surface and pores of the lamina cribrosa become deformed as glaucoma advances. 37,38 In addition, previous in vivo reports have shown that the laminar pores are more elongated in glaucomatous eyes than in normal eyes. 5,6,11,30 Although the “laminar dot sign,” the representation of the visible laminar pores by color disc photography, has been reported to predict glaucomatous visual field loss, 39 this feature is also seen in normal eyes, particularly in those with large cupping. 40 In order to understand the changes in the lamina cribrosa of patients with glaucoma, it is important to determine which morphologic characteristics are actually specific to glaucomatous eyes. Therefore, an improved method for imaging the laminar pores (relative to conventional color disc photography) is required. In this study, we found that AOSLO enables better visualization of the laminar pores than do color photography and SLO without AO. 
Previous researchers have used color disc photography, 6,9 SLO, 5,1013 and spectral-domain optical coherence tomography (SD-OCT) 41 for noninvasive assessment of lamina cribrosa deformations. In particular, SD-OCT enables the visualization of pores throughout the depth of the lamina cribrosa. However, the lateral resolution of optical fundus imaging without AO is limited to approximately 5–20 μm, primarily because of aberrations in the ocular optics 28 ; in contrast, AOSLO has high lateral resolution of approximately 2 μm. Although AOSLO may not delineate some laminar pores, it appears to be a promising technology for assessing these structures, especially as the number of laminar pores detected is significantly higher by AOSLO than by color photography or SLO without AO. 
The intervisit pore area measurements appeared to be somewhat variable. As we obtained clear lamina cribrosa images by shifting the focus to scan from the surface to the bottom of the optic nerve head, it is possible that the focusing differed slightly between imaging sessions on different visits. 
We found that the mean laminar pore area correlated positively with the axial length in normal subjects. In this study, the pore area measurements were corrected by using Littmann's formula for each axial length; this is the first report on the relationship between the laminar pore dimensions and the axial length. In most of the previous in vivo studies, pore area measurements have not been corrected for axial length. 5,11,40 Because fundus dimensions are affected by magnification errors caused by the axial length and refraction error, our result indicates that magnification errors caused by the axial length may have interfered with the laminar pore area measurements in previous studies using optical imaging technologies. The reason for the association between laminar pore area and axial length is currently uncertain; elongation of the axial length may possibly lead to lateral extension of the lamina cribrosa. 42,43  
Using SLO, Fontana et al. 5 have demonstrated that larger pore elongation index correlates with greater severity of VF defect (worse MD). However, we did not find a correlation between the mean pore elongation index and the MD value. Although the reason for this disagreement is uncertain, it may be attributable to the different methods of measurement, subjects, and sample sizes (smaller in our study), and/or to corrections for the axial length (in our study). 
In this study, we found larger pore areas in the glaucomatous eyes than in the healthy eyes, consistent with the results of Vilupuru et al. 30 and Fontana et al., 5 who have reported that the pore areas are larger in monkey eyes with experimental glaucoma and in patient eyes with glaucoma than in normal control eyes, as imaged by AOSLO and SLO, respectively. In contrast, Tezel et al., 9 using color disc photography, have demonstrated that the mean pore area, but not the total pore area, significantly decreases during the mean follow-up time of 3.9 years. The discrepancies between these studies may be attributable to the different imaging modalities, measurement of pore area, and study designs (longitudinal versus cross-sectional). It is possible that the lamina pore areas depicted by color fundus photographs and AOSLO differ because of the remodeling of laminar pores, such as astroglial migration and extracellular matrix deposition within the laminar pores, which occurs following glaucomatous nerve fiber loss. Lamina pores are usually visualized as low-reflective areas because retinal nerve fibers passing along the pores almost in parallel to the imaging light are hyporeflective, whereas the lamina plate is visualized as a highly reflective region. However, the replacement of retinal nerve fibers during extracellular matrix deposition during remodeling would alter (probably increase) the reflectivity, likely leading to unclear borders of the laminar pores. AOSLO may allow for clearer depiction of the borders of the remodeled lamina pores because of the high signal/noise ratio and high lateral resolution. Further longitudinal investigation using AOSLO would be helpful. 
Changes in the laminar pore dimensions may be attributable to various factors such as tissue remodeling and mechanical deformation of the lamina cribrosa. Some changes may depend on age, axial length, and optic nerve head deformations, such as disc tilting and rotation, while others may be glaucoma-specific. The laminar pores comprise mainly collagen fibers when observed after trypsin digestion and dehydration, but in vivo, these structures could contain not only collagen fibers but also blood vessels, extracellular matrix other than collagen fibers, and/or astroglia, some of which are lost after trypsin digestion. 1 Histopathologic studies of the glaucomatous optic nerve head have demonstrated persistent glial activation accompanied by upregulated synthesis of extracellular matrix components 4448 and migration of astrocytes from the laminar cribriform plates into the laminar pores, 49 where they then occupy the spaces that appear within the pores during glaucoma progression. In addition to these cellular events associated with tissue remodeling in the glaucomatous optic nerve head, the laminar pore dimensions would be affected by compression, posterior bowing of the lamina cribrosa, or optic nerve head deformations, particularly optic disc tilting and rotation following axial elongation. Such optic nerve head deformations may affect the laminar pore dimensions, although the relationship between optic nerve head deformation and laminar pore deformation remains unknown. The contributions of these effects would vary with the severity of the loss of retinal ganglion cell axons and the integrity of the laminar plate. These multiple factors could induce variable alterations in the visible pore dimensions, such as enlargement or contraction and elongation, likely forming a complicated visualization of laminar pores. This complexity may be responsible for the inconsistency of laminar pore dimensions on AOSLO with the severity of the VF defects. However, regardless of the presumed complicated factors involved in laminar pore deformation, in the multiple logistic regression analysis, glaucoma was significantly associated with a larger mean pore area, as well as older age, higher IOP, and a smaller disc ovality index. 
In our univariate and multivariate correlation analyses, a larger laminar pore area was significantly associated with longer axial length in normal and total subjects and untreated IOP in the glaucomatous subjects, whereas pore elongation index was not associated with any factors. It is uncertain why the correlation of the larger laminar pore with axial length observed in the normal subjects was lost in the glaucomatous subjects. As we discussed earlier, the remodeling of laminar pores, such as extracellular matrix deposition within the laminar pores, may cause underestimation of the laminar pore area in glaucomatous eyes. It is interesting that although the laminar pore area was not associated with the severity of the VF defects, it was significantly associated with untreated IOP. This means that larger lamina pores may be at least in part caused by higher IOP but do not necessarily damage the retinal nerve fibers running along the pore. The changes in the laminar pore area after axial length elongation and glaucomatous damage remain to be investigated in longitudinal studies. 
This study has several limitations. First, it is a pilot study with a small number of subjects, which may not have provided sufficient statistical power to obtain statistically significant results in correlation analyses. Second, AOSLO cannot be used to visualize the laminar pores beneath the neuroretinal rim and blood vessels, hindering assessment of the entire laminar region. As a consequence, this study was limited to evaluation of the areas in which AOSLO can visualize laminar pores. Because the rim width and disc (lamina cribrosa) and cup sizes vary among subjects, the regions that could be selected for measuring pore dimensions in our study were not necessarily identical among subjects. Laminar pores, which are located within the measurement area (described in Fig. 1) but partially obscured by vessels, can cause bias in our analyses. Third, laminar pores are three-dimensional structures with collagenous extensions of the sclera, 50 and the morphologic characteristics of the lamina cribrosa vary at different depths. 51 Because AOSLO can only be used to visualize the superficial laminar structures, it cannot show possible full-depth deformations of the laminar pores, such as tilting and narrowing. Fourth, it is difficult to completely divide the optic nerve head into superior and inferior halves, particularly in eyes with torted optic discs. For this purpose, we used the connecting line between the centroid of the disc margin and the fovea as the reference line and excluded any pores that contacted the border lines. Although it is uncertain whether this is the best method for performing the regional analysis, it is at least useful for decreasing the confounding effects of torted optic discs. Recently it has been shown that the Bruch's membrane opening detected by SD-OCT does not always coincide with the clinically defined disc margin. 5254 Disc margin has been histologically defined by the scleral ring of Elschnig so far, 55 and clinical disc margin has been determined from this understanding by using disc photographs/SLO images. Because the recent study has shown that the clinical disc margin is highly variable within individual eyes and between different eyes, and is constructed by complex structures including Bruch's membrane and border tissue, Bruch's membrane opening may be desirable to determine the centroid of the disc margin more consistently within each eye and among eyes. Thus, we need to keep in mind that our results were based on the conventional definition of the disc margin. The method of determining the disc margin might need to be considered for future regional analysis of the optic disc. Furthermore, although AOSLO improves the visibility of the boundaries of laminar pores, they are still not as sharp as are cone photoreceptors visualized by AOSLO. The interobserver and intraobserver reproducibilities of our measurement method were excellent, but we hope to be able to further improve the image quality. 
Despite these limitations, AOSLO is a useful imaging technology for assessing laminar pore morphology. A larger-scale study is required to confirm the role of laminar pore deformation in glaucomatous optic neuropathy. 
Acknowledgments
We thank Akiko Hirata and Mayumi Yoshida for counting and measuring the laminar pores, and Sachiko Yoshida for collecting the AOSLO images. 
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Footnotes
 Supported in part by a Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Young Scientists (22890097) and the New Energy and Industrial Technology Development Organization (NEDO; P05002).
Footnotes
 Disclosure: T. Akagi, None; M. Hangai, None; K. Takayama, None; A. Nonaka, None; S. Ooto, None; N. Yoshimura, None
Figure 1. 
 
A schematic diagram of the modified Garway-Heath regionalization in a right eye. The two measurement sectors are shown in red (superior) and blue (inferior). The line connecting the centroid of the disc margin (green circle) and the fovea (green cross) was designated as the reference line.
Figure 1. 
 
A schematic diagram of the modified Garway-Heath regionalization in a right eye. The two measurement sectors are shown in red (superior) and blue (inferior). The line connecting the centroid of the disc margin (green circle) and the fovea (green cross) was designated as the reference line.
Figure 2. 
 
The comparison of (1) color disc photographs, (2) HRA SLO images, (3) HRT SLO images when best focused on the laminar pores at the superficial level of the lamina cribrosa, and (4) AOSLO montage overlaid on the same HRA SLO images. (A) The left eye of a 50-year-old man with normal VF. (B) The right eye of a 39-year-old woman with normal-tension glaucoma. Resolution and contrast are improved following AO correction. Green arrowheads show that the borders of the pores are not clear in SLO without AO (2, 3) but are relatively clear in AOSLO (4).
Figure 2. 
 
The comparison of (1) color disc photographs, (2) HRA SLO images, (3) HRT SLO images when best focused on the laminar pores at the superficial level of the lamina cribrosa, and (4) AOSLO montage overlaid on the same HRA SLO images. (A) The left eye of a 50-year-old man with normal VF. (B) The right eye of a 39-year-old woman with normal-tension glaucoma. Resolution and contrast are improved following AO correction. Green arrowheads show that the borders of the pores are not clear in SLO without AO (2, 3) but are relatively clear in AOSLO (4).
Figure 3. 
 
The representative images were obtained by (1) color disc photography, (2) AOSLO with color disc photography, (3) AOSLO with framed squares (measurement areas). Panel (4) shows magnified images (left) and the same images with the pores marked in red (right) within the measurement areas. Panel (5) shows HFA + 24-2 SITA of the (A) left eye of a 46-year-old woman with normal VF (MD = 0.26 dB), (B) left eye of a 62-year-old man with primary open-angle glaucoma (SAP showed more severe VF defects in the inferior hemifield, MD = −15.34 dB), and (C) right eye of a 70-year-old woman with primary open-angle glaucoma (SAP showed unilateral VF defects in the superior hemifield, MD = −13.82 dB). Scale bar = 500 μm.
Figure 3. 
 
The representative images were obtained by (1) color disc photography, (2) AOSLO with color disc photography, (3) AOSLO with framed squares (measurement areas). Panel (4) shows magnified images (left) and the same images with the pores marked in red (right) within the measurement areas. Panel (5) shows HFA + 24-2 SITA of the (A) left eye of a 46-year-old woman with normal VF (MD = 0.26 dB), (B) left eye of a 62-year-old man with primary open-angle glaucoma (SAP showed more severe VF defects in the inferior hemifield, MD = −15.34 dB), and (C) right eye of a 70-year-old woman with primary open-angle glaucoma (SAP showed unilateral VF defects in the superior hemifield, MD = −13.82 dB). Scale bar = 500 μm.
Figure 4. 
 
The scatter diagram shows the linear regression (y = 348.972x – 6144.034) of the laminar pore area against the axial length in the normal subjects (P < 0.01, r 2 = 0.468).
Figure 4. 
 
The scatter diagram shows the linear regression (y = 348.972x – 6144.034) of the laminar pore area against the axial length in the normal subjects (P < 0.01, r 2 = 0.468).
Table 1. 
 
Subject Characteristics and Ocular Measurements
Table 1. 
 
Subject Characteristics and Ocular Measurements
Parameter Glaucomatous Group (n = 20) Normal Group (n = 20) P
Age, y 57.9 ± 14.6 (27–78) 51.3 ± 11.2 (33–81) 0.114
MD, dB −10.42 ± 7.79 (−1.19 to −29.41) −0.17 ± 1.05 (1.63 to −3.66) <0.001*
Axial length, mm 25.35 ± 1.88 (21.64–27.90) 24.79 ± 1.62 (21.85–27.79) 0.319
Refraction, diopters −4.10 ± 3.77 (−11.75 to 1.25) −2.71 ± 2.67 (−8.75 to 1.75) 0.188
IOP when sampling, mm Hg (untreated IOP, mm Hg) 15.7 ± 2.5 (12–20) (21.4 ± 6.9 [14-46]) 14.4 ± 1.7 (12–18) 0.053
Disc area, mm2 2.28 ± 0.63 (1.09–3.91) 2.30 ± 0.34 (1.66–3.01) 0.884
Disc ovality index 0.85 ± 0.06 (0.72–0.98) 0.89 ± 0.08 (0.76–1.04) 0.116
Disc rotation angle, ° 7.50 ± 4.32 (2–15) 5.55 ± 3.10 (0–12) 0.110
Vertical cup/disc ratio 0.74 ± 0.12 (0.47–0.94) 0.52 ± 0.12 (0.34–0.81) <0.001*
Pore area, μm2 3103.0 ± 857.0 (1893.3–4775.0) 2507.7 ± 825.5 (1211.7–5079.8) 0.031*
Pore area (superior), μm2 3489.5 ± 1190.2 (1594.9–6431.3) 2927.8 ± 1225.9 (1318.1–5017.3) 0.012*
Pore area (inferior), μm2 2927.8 ± 1225.9 (1174.0–6370.4) 2459.6 ± 903.1 (1109.7–5156.0) 0.177
Pore elongation index 2.38 ± 0.79 (1.67–5.24) 2.13 ± 0.47 (1.59–3.23) 0.231
Pore elongation index (superior) 2.21 ± 0.94 2.00 ± 0.46 0.365
Pore elongation index (inferior) 2.48 ± 0.76 2.27 ± 0.54 0.307
Table 2. 
 
Univariate Analysis of the Associations between Pore Dimensions and Ocular Parameters
Table 2. 
 
Univariate Analysis of the Associations between Pore Dimensions and Ocular Parameters
Subject Pore Area Pore Elongation Index
Normal Glaucomatous Total Normal Glaucomatous Total
ρ P ρ P ρ P ρ P ρ P ρ P
Age, y −0.279 0.234 −0.124 0.603 −0.036 0.827 −0.352 0.128 −0.294 0.208 −0.237 0.141
MD value, dB 0.010 0.967 0.003 0.990 −0.272 0.089 −0.068 0.774 0.367 0.112 −0.090 0.582
Axial length, mm 0.576 0.008* 0.424 0.062 0.519 0.001* 0.253 0.283 0.039 0.870 0.181 0.264
Disc area, mm2 −0.451 0.056 0.280 0.232 −0.022 0.892 −0.394 0.086 −0.015 0.950 −0.202 0.212
Disc ovality index −0.002 0.995 0.414 0.070 0.094 0.563 −0.176 0.458 0.044 0.853 −0.140 0.390
Cup/disc ratio −0.040 0.867 0.093 0.698 0.395 0.012* −0.315 0.176 −0.103 0.665 −0.008 0.959
IOP, mm Hg −0.582 0.007 0.192 0.417 0.064 0.693 −0.312 0.181 0.204 0.389 0.027 0.870
Untreated maximum IOPs, mm Hg 0.645 0.002* 0.308 0.053 −0.298 0.201 −0.039 0.810
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