Comparison of Clinical and Spectral Domain Optical Coherence Tomography Optic Disc Margin Anatomy

Abstract

purpose. To investigate spectral domain optical coherence tomography (SD-OCT)–detected optic disc margin anatomy in the monkey eye by colocalizing disc photographs to SD-OCT scans acquired from the same eyes.

methods. The neural canal opening (NCO) was delineated within 40 digital radial sections generated from SD-OCT volumes acquired from 33 normal monkey eyes (15°, 290 × 768 horizontal grid pattern). Each volume was colocalized to its disc photograph by matching the retinal vessels within each photograph to vessel outlines visible within en face SD-OCT images. Border tissue was delineated where it extended internally to the NCO. A clinician (masked to delineated points) marked the disc margin onto each photograph while viewing the relevant stereophotograph pair. Alignment of the clinician-ascribed disc margin to the NCO and border tissue delineation was assessed. The process was repeated in a single myopic human eye.

results. In 23 eyes, the NCO aligned to the disc margin. In 10 eyes, externally oblique border tissue was detectable in the temporal disc. In these regions of the disc, the termination of border tissue was the disc margin. An exaggerated form of this phenotype was observed in the myopic human eye. In this case, temporal border tissue terminated at the anterior scleral canal opening, which was detected as the disc margin.

conclusions. The termination of Bruch’s membrane, border tissue, and the anterior scleral canal opening may constitute the disc margin within the same eye, depending on the border tissue architecture; this anatomy is consistently visualized by SD-OCT.

An ability to identify, understand, and appreciate the morphologic changes occurring at the optic nerve head (ONH) is vital in the assessment of patients with, or at risk for, glaucoma. The ONH is a complex three-dimensional (3-D) structure, and interpretation of its underlying anatomy can be a challenge for clinicians. The optic disc margin is a fundamental clinical landmark in the assessment of the ONH because it marks the peripheral boundary of neural tissue within the disc. As such the disc margin is an essential component in the assessment of neuroretinal rim loss and of disc size. 

The ring of Elschnig is widely accepted as the structure underpinning the fundoscopic appearance of the optic disc margin in human eyes. ¹ The ring of Elschnig comprises the border tissue of Elschnig (hereby referred to as border tissue), which is collagenous tissue arising from the sclera to join Bruch’s membrane, thereby enclosing the choroid. ² It is this scleral “cuff” or “lip” between the ONH and the choroid that has been thought to account for the white “rim” perceived fundoscopically as the disc margin in the human eye. ^{3 4} The appearance of the disc margin is therefore that of a whitish halo (if present for 360°) or crescent (if present for <360°) at the edge of the disc. However, these landmarks may not be detectable in some regions or may not be detectable at all in certain discs. In this context, the subjective location of the disc margin can be highly variable. 

In a series of recent publications we have proposed three important concepts that are necessary for understanding optic disc margin anatomy within 3-D histomorphometric and spectral domain optical coherence tomography (SD-OCT) reconstructions of the monkey ONH. ^{5 6 7} The first concept is that of a neural canal opening (NCO; Fig. 1A , arrows), which is the anatomic opening in Bruch’s membrane (also referred to as Bruch’s membrane opening [BMO]) through which retinal ganglion cell axons must pass to enter the choroidal and scleral portions of the neural canal. ^{6 7 8} The second concept is that of a neural canal, which is the axonal pathway through the eye wall. ⁵ The neural canal begins at the NCO and extends through a choroidal component, bound on either side by the border tissue (Fig. 1A , arrowheads). Beyond the choroidal component, the neural canal has a scleral component, bound on either side by the internal wall of the sclera. The neural canal terminates where the optic nerve leaves the globe. The third concept is that what the clinician perceives to be the disc margin is not a single anatomic structure but is instead variable; some portions are the NCO and others the border tissue (Fig. 1A , arrowheads) or the anterior scleral canal opening, depending on the 3-D architecture of these structures. ⁷  

The NCO is an important anatomic landmark because it can be identified within SD-OCT images and has been proposed as the basis for a reference plane for SD-OCT imaging. ⁸ Within our 3-D histomorphometric reconstructions, we define the NCO as the termination of the BMO because Bruch’s membrane is within the resolution of our reconstruction technique. ⁶ Within SD-OCT reconstructions we define the NCO as the termination of Bruch’s membrane/retinal pigment epithelium (BM/RPE) complex because it is uncertain whether Bruch’s membrane in isolation can be resolved by SD-OCT. ⁹  

We recently colocalized postmortem 3-D ONH histomorphometric reconstructions to stereophotographs acquired in vivo in 28 normal monkey eyes. ⁷ In that study, we found that the NCO constituted the disc margin throughout its full extent in most monkey eyes. However, in some eyes, border tissue was detectable in disc photographs, and it colocalized to the disc margin in regions in which it demonstrated an externally oblique configuration and in which its junction with the anterior scleral surface was internal to the termination of Bruch’s membrane. 

From these 3-D histomorphometric reconstructions, we have proposed that there are two principal border tissue orientations (both illustrated in Fig. 2 ) and that border tissue orientation determines what the clinician identifies as the disc margin. The border tissue orientation is most commonly internally oblique, whereby border tissue extends from the sclera into the neural canal to meet Bruch’s membrane (Fig. 2A) . Border tissue is less commonly externally oblique, whereby border tissue extends from the sclera away from the neural canal to meet Bruch’s membrane (Fig. 2B) . Where an overhang of Bruch’s membrane extends beyond the innermost termination of border tissue (whether internally or externally oblique), the termination of the BMO is detected as the disc margin. Where the innermost termination of an externally oblique border tissue is internal to the termination of Bruch’s membrane, the border tissue is visible clinically and comprises the disc margin. ⁷ We now propose that the anatomic basis of the disc margin can also be identified and delineated within SD-OCT volumes. The purpose of this study was to examine whether the NCO and border tissue identified in SD-OCT volumes define the optic disc margin in the normal monkey eye. 

Methods

Animals

The Institutional Animal Care and Use Committee (Legacy Health Care System) approved this study, and all animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All eyes were SD-OCT imaged in vivo as part of other ongoing research studies. Data from 33 normal eyes of 24 rhesus macaque monkeys were included. For each eye, SD-OCT volumes were compared to optic disc stereophotographs. 

Imaging

All imaging was performed while the animal was under pentobarbital or isoflurane anesthesia. The intraocular pressure in the imaged eye was set at 10 mm Hg with a manometer connected to a 27-gauge cannula inserted into the temporal anterior chamber. Pupils were dilated with 1 drop each of 1% tropicamide, 2.5% phenylephrine hydrochloride, and 2% cyclopentolate hydrochloride. Images were acquired through a rigid Plano contact lens placed on the corneal surface, after at least 30 minutes of IOP stabilization. Stereophotographic pairs were acquired with a retinal camera (TRC-WT; Topcon, Paramus, NJ). These images were captured onto 35-mm slide film developed and processed into color slides. SD-OCT imaging of the ONH was performed with a commercially available SD-OCT device (Spectralis; Heidelberg Engineering, Heidelberg, Germany). The technical specifications of the Spectralis device have been covered in detail elsewhere. ^{10 11} Briefly, the Spectralis device makes use of an 870 nm semiluminescent diode light source; its optical resolution is approximately 7 μm in depth and 14 μm transversely. For this study, 290 horizontal B-scans were acquired over a 15° retinal window (768 A-scans per B-scan; each B-scan was acquired nine times and was averaged for speckle noise reduction). In this scan pattern, the Spectralis device software automatically registers all acquired B-scans relative to each other in the z-axis. All image acquisitions were well centered on the ONH, and this scan location was consistent in all images used in this study. 

Delineation of Disc Margin Structures within SD-OCT Volumes

Our delineation method within SD-OCT imaging has been described in detail elsewhere. ⁸ Briefly, a 3-D SD-OCT volume was constructed from each 290 horizontal B-scan set using our custom-built, multiview 3-D visualization and delineation software based on a commercially available system (Visualization Toolkit; Clifton Park, NY). Forty interpolated radial sections (4.5° intervals) were generated from each 3-D SD-OCT volume, centered at the geometric center of origin of each volume. A single experienced operator (NGS) manually delineated all the volumes. No contour smoothing filtering techniques were applied to the interpolated data. 

Figure 1compares a horizontal B-scan with a comparable sagittal serial histologic section taken from the same normal monkey ONH. This eye has not been included in the present study and is used for illustrative purposes only; a detailed account of the comparison between serial histology and SD-OCT B-scans in this eye will be the subject of a future report. Figure 1Billustrates that SD-OCT is capable of identifying both the termination of the RPE/BM complex (NCO, marked with arrows) and the border tissue (marked with arrowheads). The anterior scleral canal opening, which is defined as the internal boundary of the anterior scleral surface, is not detectable in this representative B-scan. For each SD-OCT volume, the operator identified and delineated the NCO (defined as the innermost extent of the posterior surface of the RPE/BM complex) within each interpolated radial SD-OCT section (Fig. 1B , arrows). There are, therefore, two NCO points within each single radial SD-OCT section, one on either side of the neural canal, yielding 80 NCO points per volume. In sections where the innermost (most axial) extent of an externally oblique border tissue was observed to extend further into the neural canal than the NCO point on that side, the innermost termination of the border tissue was also delineated. Within the SD-OCT radial sections, border tissue was defined as the transition from high- to low-intensity signal at the junction of the choroid and the neural canal, visible inferior to the RPE/BM complex (Fig. 1B , arrowheads). Figure 3demonstrates the distinction between internally and externally oblique border tissue within a different SD-OCT B-scan and serial en face (transverse) SD-OCT C-scans relative to their respective features within a disc photograph. 

Once manual delineation was completed, the Cartesian coordinates for each delineated point (both NCO and border tissue) were saved, allowing a 3-D point cloud to be generated. ⁶  

Colocalization of SD-OCT Volumes to the Disc Photographs

For each eye, a stereophotograph pair was selected in which at least one photograph in the pair had sharply defined central retinal vessels. A good stereo effect and a clear focus at the disc margin were desirable secondary considerations, but their absence was not grounds for exclusion. The selected stereophotograph slides were digitized at a resolution of 4800 dpi using a color-calibrated slide scanner (ArtixScan M1; Microtek Laboratory, Inc., Fontana, CA), and the disc image with the most clearly resolved disc margin was selected for the purposes of colocalization. 

Each SD-OCT ONH volume was viewed in the en face orientation, and the z-axis depth was adjusted until the outline of the central retinal vessels was clearly visible. Two en face OCT images were acquired, one showing the vessel outlines and the other showing all the delineated glyphs. The two images were overlaid (Adobe Photoshop CS3; Adobe Systems, Inc., San Jose, CA) to create a perfectly aligned two-layer image file. The disc photographs were then overlaid onto their respective SD-OCT images with the glyphs hidden from view. The same operator (NGS) performed the initial manual colocalizations. Colocalization required the outline of the central retinal vessels and their bifurcations to be matched between the en face OCT image and the photograph. This was achieved by rotating the photograph image layer, moving it in the x and y orientations, and adjusting its magnification in a 1:1 ratio scale. The proximity of alignment between image layers was verified by reducing the opacity of the clinical image so as to view the two overlaid images simultaneously. To not prejudice the colocalization process, the SD-OCT delineation glyphs were hidden throughout. At the completion of colocalization, the coordinates of the SD-OCT delineations were saved, and the glyphs were transferred to the clinical image. The colocalization process is illustrated in Figure 4A 4B 4C . 

Delineation of the Disc Margin

An experienced clinical observer (CFB) viewed each eye’s digitized stereophotograph pair on a computer monitor with a stereoscope (Screen-Vu; PS Mfg., Portland, OR). To ensure that the observer had an optimal view of the disc margin, he also had access to a number of alternative stereophotograph slides that could be viewed using a hand-held stereo viewer (Asahi-Pentax Co., Englewood, CO). For each eye, the observer delineated the disc margin in the colocalized clinical photograph (with the SD-OCT delineations hidden; Fig. 4D ) using a custom-built application designed to capture the x and y coordinates of each selected point. The observer could classify the marks as certain (blue) or uncertain (green), in which case a forced choice was made (Fig. 4E) . There were no constraints on the number of points the observer had to delineate in each photograph. The observer defined the disc margin as the innermost reflective structure that extended beyond the internal termination of pigment at the disc periphery and, in doing so, delineated what clinicians interpret as the Elschnig ring. Where a reflective structure was absent, the observer selected the termination of pigment as the disc margin. The collected coordinates of the marked points were saved, and the points were transferred to the disc photograph image in which the SD-OCT delineation glyphs were also shown. An image of the disc photograph incorporating the colocalized SD-OCT delineations and the disc margin demarcations was then saved for each eye (Fig. 4F) . 

Subjective Assessment of Alignment of SD-OCT Delineations to the Disc Margin

Two observers (NGS and CFB) qualitatively reviewed the disc photographs incorporating the SD-OCT delineations and the disc margin delineations. This was performed to assess how well the colocalized SD-OCT NCO or border tissue delineations aligned to the disc margin. The observers classified good alignment as where there was less than a glyph’s diameter separation between the SD-OCT glyph (either NCO or border tissue) and the nearest adjacent disc margin glyph in all disc sectors. When the two-dimensional SD-OCT image was projected onto the disc photograph in the purpose-built disc margin delineation software, a universal 1:1 pixel ratio was applied. This was performed so as to negate any discrepancy in glyph diameter caused by SD-OCT scaling issues related to differences in monkey axial lengths. The glyphs in this program are 5 pixels in diameter, which means that this is the minimum resolvable separation between adjacent glyphs. Disc sectors were defined according to 0° (vertical), 45°, 90° (horizontal), and 135° axes applied to the clinical photograph, generating superior, inferior, superotemporal, inferotemporal, temporal, nasal, superonasal, and inferonasal disc sectors. Where the SD-OCT NCO delineations aligned to the disc margin delineations, the NCO was defined as the anatomic basis for the disc margin in that region. Where both the NCO and the termination of an externally oblique border tissue had been marked, the landmark most closely aligned to the disc margin delineation was defined as the anatomic basis of the disc margin in that region. In eyes in which good alignment was not observed for 360° of the disc margin, the number of sectors where there was less than one glyph diameter separation between SD-OCT and disc margin glyphs was recorded. To identify causes for discrepancy in these regions of the disc, the relevant stereophotographs and B-scans were systematically reexamined. 

Quantification of Alignment of SD-OCT Delineations to the Disc Margin

In addition to the subjective assessment described, an attempt was made to quantify the degree of alignment between the clinician’s disc margin and the SD-OCT delineations. For the purposes of this analysis, all images were configured into the right eye orientations. The two-dimensional coordinates of all glyphs in x and y spaces were transferred to a numerical computing language (MATLAB; MathWorks, Natick, MA). B-spline curves were fitted between glyphs so that three different continuous curves were generated for each colocalized image. In all images, a continuous disc margin curve and a continuous SD-OCT NCO curve were generated. In those eyes in which externally oblique border tissue had been delineated, a third continuous curve was generated that incorporated the border tissue (where present) and the NCO in the remainder of the disc. The alignment error was assessed along radial spokes generated at 4.5° intervals, from the center of the clinician’s disc margin to the disc margin curve, beginning at the temporal meridian (0°) and proceeding in a counterclockwise fashion. Alignment error at each spoke was expressed as the distance between the disc margin curve and the SD-OCT curve along the spoke, expressed as a percentage of the spoke length from the center of the disc margin to the disc margin curve (Fig. 5) . Percentage alignment error relative to disc margin radius was measured globally and regionally across all eyes (analyses performed using R; R Foundation for Statistical Computing, Vienna, Austria). 

SD-OCT Disc Margin Anatomy in a Myopic Human Eye

To extend our findings to the human ONH and to better demonstrate their clinical implication, the same operator (NGS) delineated an SD-OCT volume acquired from a myopic human eye (optical refraction, −9.00/−1.00 D × 90°; axial length, 29.04 mm). The SD-OCT volume was generated from 97 horizontal B-scans acquired over a 15° by 10° retinal window (768 A-scans per B-scan, each B-scan acquired 25 times and averaged). Human SD-OCT imaging was performed at Dalhousie University (Halifax, Nova Scotia, Canada); the regional Ethics Committee gave its approval, the subject gave his informed consent, and he was treated in accordance with the tenets of the Declaration of Helsinki. 

In addition to the NCO, the full extent of the border tissue was delineated, regardless of whether it was internally or externally oblique or whether it extended beyond the NCO. The anterior scleral canal opening (the innermost extent of the anterior scleral surface) was detectable in this eye and was also delineated within each radial SD-OCT section (blue glyphs). These additional delineations were performed because of the complexity of the anatomy in this eye compared with the normal monkey eyes. After delineation of these landmarks, the en face SD-OCT image was colocalized to the disc photograph, and the clinician (CFB) marked the disc margin, masked to the SD-OCT landmarks, according to the same protocol used in the monkey eyes. 

Results

Assessment of Alignment between SD-OCT and Disc Margin Delineations in Monkey Eyes

In 18 eyes, the delineated NCO was well aligned (within one glyph diameter) to 360° of the disc margin (six examples are illustrated in Fig. 6A ). In five other eyes, NCO was misaligned from the disc margin in a single sector of the disc (examples shown in Figs. 6A 6B 6D ) but was well aligned to the remaining circumference of the disc margin. 

A region of externally oblique border tissue extending internally to the NCO was detectable by SD-OCT in 10 eyes. In all cases, the region of externally oblique border tissue was located in the temporal half of the disc. Within these 10 eyes, where an externally oblique border tissue was delineated, the termination of border tissue rather than the NCO aligned to the disc margin. In eight of these eyes, the NCO aligned well to the remainder of the disc, where border tissue had not been delineated (Fig. 6B) . In one eye, the NCO and the disc margin were misaligned in a single sector. In the remaining eye, misalignment (greater than two glyphs’ diameter separation) between the NCO and the disc margin was encountered in the nasal half of the disc. 

The mean percentage alignment error (distance between the SD-OCT delineations and the disc margin, expressed as a percentage of the center of the disc to the disc margin) was 3.8% ± 1.2%. Figure 7illustrates the regional alignment error for all the eyes in the study. Absolute alignment error has been estimated using the moving average generated from each corresponding 4.5° location and the two locations either side of it (in other words, a moving average based on five consecutive locations). In Figure 7the median (middle lines), 95% (upper lines), and 5% (lower lines) quantiles for alignment error are shown. Median alignment error was <5%, with greatest misalignment of the NCO (solid lines) occurring in the temporal, inferotemporal, and superotemporal regions. The misalignment in these regions was considerably reduced when the disc margin was compared to externally oblique border tissue (dotted lines). 

Sources of Misalignment

In five of the six eyes in which misalignment of the NCO to the disc margin was present in a single disc sector, the disc margin had been marked using green glyphs, indicating that the clinical observer was not certain of the location of the disc margin in that region. These misalignments were all located at the superior or inferior pole of the disc (two examples shown in Figs. 8A 8B ). In the normal monkey eye, the retinal nerve fiber layer can be very thick where it enters the disc at the poles, making it difficult to clearly visualize the disc margin in stereophotographs. It is possible that this may explain the misalignment in these five cases. It should, however, be noted that the degree of misalignment is fairly minimal, amounting to less than two glyphs’ diameter separation. 

In the remaining eye in which misalignment was confined to a single sector, the disc margin in the misaligned sector had been delineated using blue glyphs, indicating that the clinical observer was certain of the disc margin location (Fig. 8D) . Within the radial B-scans at that location, a substantial shadow cast by a blood vessel made precise delineation of the NCO difficult (Fig. 8E) . This problem was not encountered in the remaining SD-OCT ONH volumes examined in this study. 

The reason for the misalignment between the SD-OCT–defined NCO and the nasal disc margin in one eye was less apparent (Fig. 8C) . The degree of misalignment was too large to be explained by the small margin of error inherent in the colocalization process or the potential differences in the size of vessels at the time of SD-OCT imaging relative to their size at the time of stereophotography. 

Assessment of Disc Margin Anatomy in a Myopic Human Eye

The relationships between the photographic disc margin and the SD-OCT NCO, border tissue and anterior scleral canal opening delineations are illustrated in Figures 9A 9B 9C . The 3-D anatomy of this myopic human ONH was ascertained from the point cloud generated from the delineated NCO, border tissue and anterior scleral canal opening (Figs. 9D 9E) . An exaggerated obliquity of the neural canal was clearly visible in the 3-D point cloud. 

As in 10 of the monkey eyes, externally oblique border tissue was present in the temporal half of the disc, whereas internally oblique border tissue was present in the nasal half of the disc. However, the extent and degree of obliquity of the border tissue far exceeded that seen in the normal monkey eyes. 

In this eye, the temporal NCO coincided with the termination of retinal pigment (Fig. 9B) . The border tissue delineations in the temporal disc extended downward and internal to the NCO, where they corresponded to a region of atrophy evident in the photograph. In this region, large underlying choroidal vessels were visible. The temporal disc margin corresponded to the anterior scleral canal opening located at the innermost termination of the externally oblique border tissue. In the disc photograph, the anterior scleral canal opening was visible as a distinct white crescent internal to the variably pigmented border tissue. In this eye, this landmark represents a true scleral lip or scleral crescent, as has been classically described. ^{1 3 12}  

In the nasal half of the disc, the internally oblique border tissue can be seen to sweep external to the NCO, terminating at the anterior scleral canal opening. Within these sectors the NCO corresponded to the disc margin. 

Discussion

The principal findings of this study may be summarized as follows. Border tissue and NCO anatomy were well visualized by volumetric SD-OCT imaging in monkey eyes. The anatomic underpinnings of the disc margin were regionally variable, depending on border tissue obliquity as detected by SD-OCT. These findings could be used to explain the more complicated anatomy of a single myopic human eye. 

The findings of this study are consistent with the results of our previous assessment of disc margin anatomy using 3-D histomorphometric reconstructions. ^{6 7} Chiefly, the NCO (equivalent to the BMO in the monkey eye) corresponded to the disc margin in most monkey eyes. Where an externally oblique border tissue extended into the neural canal internal to the NCO, the disc margin was composed of the innermost termination of the border tissue and the anterior scleral canal opening (where visible). This orientation was observed in approximately one third of the monkey eyes examined in this study and was invariably located in the temporal half of the disc. An extreme form of this phenotype was also seen in the myopic human eye. 

Our findings suggest that disc margin anatomy is consistently detected within SD-OCT volumes. SD-OCT volumes, therefore, have the potential to inform the clinician of the true location of the disc margin when defining the disc margin by fundoscopy or within a disc photograph is difficult. This has important implications in the management of glaucoma patients because it will enable precise identification of the peripheral boundary of neural tissue in the disc and will allow accurate placement of the contour line in optic disc images used in the quantitative assessment of glaucomatous progression. 

The SD-OCT misaligned to the clinician’s ascribed disc margin in more than one sector of the disc in only one monkey eye. In this case, externally oblique border tissue colocalized to the temporal disc margin, whereas the SD-OCT–defined NCO was located at a considerable distance internal to the disc margin nasally. Given the overwhelming consistency regarding the relationship between the NCO, border tissue, and the disc margin in the remaining SD-OCT volumes and in 3-D histomorphometric volumes, ⁷ it is unlikely that the disc margin is the ring of Elschnig nasally. The most valid interpretation in this single exception is that a wide extension of the unpigmented Bruch’s membrane, beyond the termination of retinal pigment epithelium, was detectable by SD-OCT but was not visible within the stereophotograph. As such, the nasal disc margin might have been marked too externally in that disc photograph. Unfortunately, the animal concerned was killed on schedule for a separate study before the completion of this study, and we were unable to confirm the exact location of the nasal disc margin by stereoscopic ophthalmoscopy. 

In our previous examination of the disc margin by 3-D histomorphometry, we proposed that the reason for a tendency toward an internally oblique neural canal orientation nasally and an externally oblique orientation temporally was likely an anatomic adaptation to facilitate the passage of optic nerve axons to reach the chiasm, a midline structure. ⁷ This explanation is supported by the results obtained from delineating a myopic human eye. In this case, the optic nerve axons may have to exit the eye in a more extreme oblique direction toward the midline because of the large size of the myopic globe. 

When examining the 3-D point cloud generated from the human SD-OCT ONH volume, it is apparent that there are a number of potential apertures through which the axons traverse as they pass through the neural canal and then exit the eye through the scleral canal. The first aperture is the NCO; this represents the point at which retinal nerve fiber axons change direction to enter the ONH. The NCO is equivalent to the termination of Bruch’s membrane in our histomorphometric volumes. Within SD-OCT volumes, the innermost termination of the presumed RPE/BM signal was delineated as the NCO. In most cases, the NCO colocalized to a point on the disc photograph beyond the apparent termination of the visible retinal pigment. We interpret this to mean that portions of Bruch’s membrane may be detectable by SD-OCT, beyond the termination of the retinal pigment epithelium. 

Our study used SD-OCT imaging to confirm our previous histomorphometric report that the disc margin is not a single anatomic structure. ⁷ These data suggest that in regions of the disc where the border tissue is externally oblique, the border tissue or anterior scleral canal opening will represent the disc margin. Where Bruch’s membrane extends beyond the termination of border tissue (whether internally or externally oblique), the NCO represents the disc margin. 

Although both the NCO and the disc margin are clinically visible, we believe it is important to recognize them as independent landmarks. First, we predict that the NCO and not the disc margin will become the basis for an SD-OCT reference plane for ONH and peripapillary retinal nerve fiber layer quantification. Furthermore, we believe that clinical colocalization of SD-OCT–detected disc margin anatomy will inform the clinician’s interpretation of the disc in eyes with tilted, highly myopic, or glaucomatous ONH. 

It should be noted that it is possible for an experienced clinician to differentiate between the different anatomic components of a disc margin by judging the stereoscopic depth of the disc margin compared with the retinal pigment. Where the NCO comprises the disc margin, the termination of Bruch’s membrane will be located at the same depth as the retinal pigment. Where the disc margin is at a deeper level (more posterior) than the retinal pigment, it will comprise externally oblique border tissue or the scleral canal opening. 

The neural canal extends posteriorly from the NCO to the anterior scleral canal opening, with neural tissue separated from the choroid by the border tissue. The scleral canal further extends from the anterior scleral canal opening to the posterior scleral canal, with the nerve passing through the full thickness of the sclera. ⁶ The anterior scleral surface and the anterior scleral canal opening were not discernible in the monkey eyes examined in this study, but they were detected in the myopic human eye. We were unable to detect the posterior scleral surface and posterior scleral canal opening with 870-nm SD-OCT. However, this may be possible using a 1050-nm light source, which has been reported to achieve increased axial depth penetration. ^{13 14 15 16} An ability to fully image the scleral canal (from anterior to posterior scleral canal openings) may be useful in the detection of glaucoma progression because scleral canal expansion has been identified as a feature of chronic IOP elevation in monkey eyes using 3-D histomorphometric reconstructions. ^{6 17}  

Manassakorn et al. ¹⁸ compared the disc margins delineated in the disc photographs of 17 normal human eyes with the disc margins identified in scans acquired using a novel high-speed, ultrahigh-resolution OCT device. In their study, the termination of the retinal pigment epithelium at either side of the ONH was manually delineated within the 180 consecutive horizontal raster frames within each scan volume. The x-axis coordinates of these delineations were used to transfer these locations to the en face OCT image generated from the completed volume. The en face OCT image was colocalized to the disc photograph without viewing any of the delineations, as in our study. In the 17 eyes studied by Manassakorn et al., ¹⁸ the disc area identified by the cross-sectional OCT images was significantly larger than that identified with the disc photographs, though the geometric center of the disc margin was similar using both techniques. The authors suggested that the cross-sectional OCT disc margins might have larger dimensions than the photograph-defined disc margin because of difficulties delineating the termination of retinal pigment epithelium under large vessel shadows and in areas of peripapillary atrophy. 

Peripapillary atrophy was not a feature of any of the monkey eyes examined in our study, and we encountered difficulty in identifying the NCO because of vessel shadowing in only a single region of one disc. In our study, the detection of NCO despite the presence of retinal vessel shadowing may be attributed to the density of the B-scans acquired, which exceeds what has been attempted to date in human subjects. It is also possible that Bruch’s membrane underlying the retinal vessels in the monkey eye is more reflective than in the human, rendering the signal detectable despite the presence of vessel shadowing. 

Our delineation of the disc margin within B-scans also differed in that we delineated not only the NCO but also regions where the border tissue extended further into the canal than the termination of the RPE/BM complex. It is possible that in some of the eyes examined by Manassakorn et al., ¹⁸ a portion of the disc margin circumference was composed of externally oblique border tissue. If only the termination of the retinal pigment epithelium was marked in these regions, which would have been outside of the termination of the externally oblique border tissue, it would have resulted in an erroneously large disc area compared with the photographic disc area. 

Subsequently, Kotera et al. ¹⁹ compared the photographic disc margin with the SD-OCT defined disc margin in 18 glaucomatous eyes. In that study, a high concordance between the photographic disc margin and the SD-OCT disc margin was found. Unlike the previous study, the observers delineated either the termination of the retinal pigment epithelium or the termination of the so-called highly reflective curved line that connected to the straight signal identified as the retinal pigment epithelium. We believe that this highly reflective curved line is the signal we have identified as border tissue in our study. 

Kotera et al. ¹⁹ also highlighted circumstances in which the termination of the retinal pigment epithelium identified by SD-OCT coincided with the termination of visible retinal pigment at the edge of peripapillary atrophy. We observed the same finding in the myopic human eye examined in this study, with the temporal NCO colocalizing to the edge of the peripapillary atrophy. The appearance of this disc is in keeping with the concept of simple misalignment of the sclera, choroidal and retinal pigment epithelium layers caused by the oblique exit of the nerve in an axially myopic eye. ³ We have identified this curved high-intensity signal as border tissue, as opposed to choroid or sclera, because large choroidal vessels were visible posterior to this signal. These choroidal vessels colocalized to the correct location in the clinical photograph, highlighted by a green arrow in Figures 9A and 9C , suggesting that the signal was derived from tissue anterior to the choroid and the sclera, most likely the compacted connective tissue constituting the border tissue. 

A number of limitations to our methodology should be considered. In particular, our colocalizations were subject to a degree of inherent variability, in part because the caliber of the central retinal vessels is likely to differ between the time of SD-OCT volume acquisition and the time of stereophotography. In addition, the width of the vessels imaged at the surface of the nerve (as in a photograph) might have differed from the width of vessels at the depth of the en face C-scan used for colocalization. These differences may result in a discrepancy between the magnification of the SD-OCT image and the disc photograph. Our colocalization technique is also 2-D and so does not take into account the presence of optic nerve head tilt. This was not a problem for the normal monkey eyes, in which tilt was not a feature. It could, however, lead to misalignment in human eyes with more pronounced tilt, although this was not observed in the single eye examined in this study. It may be necessary to further develop our colocalization technique, perhaps using 3-D viewing software, if this is found to be a problem in other human eyes. Finally, both colocalization and disc margin delineation were limited by the quality of the acquired stereophotographs and SD-OCT volumes. 

In conclusion, border tissue and NCO anatomy are well visualized by volumetric SD-OCT imaging. The anatomic underpinnings of the disc margin vary regionally and depend on border tissue obliquity detected by SD-OCT. These observations held true in the single myopic human eye examined in this study and served to enhance our understanding of its ONH architecture. These findings pave the way for a more detailed exploration of SD-OCT–defined disc margin anatomy in normal and myopic human eyes. 

 Supported by National Institutes of Health Grant R01-EY11610, Legacy Good Samaritan Foundation, Heidelberg Engineering, and Sears Medical Trust. NGS is funded by an unrestricted educational grant from Heidelberg Engineering and by a Royal College of Ophthalmologists/Pfizer Fellowship.

 Submitted for publication February 18, 2009; revised March 13, 2009; accepted July 13, 2009.

 Disclosure: N.G. Strouthidis, Heidelberg Engineering (F); H. Yang, None; J.F. Reynaud, None; J.L. Grimm, None; S.K. Gardiner, None; B. Fortune, None; C.F. Burgoyne, Heidelberg Engineering (F)

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Neural canal architecture demonstrating the NCO and the border tissue within a histologic section (A) and a colocalized SD-OCT B-scan (B). A representative serial histologic section (hematoxylin and eosin stain, ×2.5 magnification) through the optic nerve head of a normal monkey eye (not part of this report), perfusion fixed at an IOP of 10 mm Hg (A) compared with an equivalent B-scan acquired in vivo from the same optic nerve head at an IOP of 10 mm Hg (B). A rrows: position of the NCO, which in the histologic section (A, black arrows) is at the termination of Bruch’s membrane on either side of the neural canal and in the B-scan (B, white arrows) is at the termination of the retinal pigment epithelium/Bruch’s membrane signal on either side of the neural canal. A rrowheads: position of border tissue, which in the histologic section (A, black arrowheads) is an extension of the scleral connective tissue, enclosing the choroid to meet Bruch’s membrane; in the B-scan (B, white arrowheads) the border tissue is identified as the junction of the choroidal signal with the neural canal. The neural canal extends from the NCO to the point where the optic nerve exits the globe, passing through a choroidal component (bound by the border tissue), followed by a scleral component.

Figure 2.

 

Two principal border tissue configurations, their relationship to a pigmented or unpigmented extension of Bruch’s membrane, and the resultant clinical disc margin anatomy. (A) Internally oblique. The diagram shows the clinical optic disc appearance (top) and a cross-section of the optic nerve head (b ottom). Labeling is as follows: 1, sclera; 2, choriocapillaris; 3, retinal pigment epithelium with Bruch’s membrane; 4, border tissue; 5, neural canal boundary; 6, pigment on the surface of Bruch’s membrane; 7, Bruch’s membrane. L eft i nset: pigmented Bruch’s membrane corresponds to the halo of pigment on the left side of the disc margin. R ight i nset: region of unpigmented Bruch’s membrane is shown; this corresponds to a white crescent internal to the pigment halo at the disc margin, which corresponds to a portion of pigmented Bruch’s membrane. (B) Externally oblique. Labeling is according to the schematic in (A). L eft i nset: Bruch’s membrane is pigmented to its end and does not extend beyond the termination of the border tissue. This Bruch’s membrane extension corresponds to an external crescent of pigment at the disc margin that is internal to the termination of the retinal pigment epithelium. The portion of the border tissue that is internal to the end of Bruch’s membrane (BMO) may be clinically recognizable as an inner reflective (if there is no pigment on the border tissue surface) or a pigmented crescent (if there is pigment on the border tissue surface) that is posterior to the plane of the retinal pigment epithelium. An inner pigmented halo (light gray , stippled) is shown on both sides of the disc diagram. R ight i nset: unpigmented Bruch’s membrane extends internally to the border tissue termination, corresponding to a reflective crescent internal to the pigment crescent. Again, pigmented border tissue (light gray , stippled) extends internally to the reflective crescent. In l eft and right insets the border tissue/scleral junction is depicted without a true scleral lip, which, when present and visible, appears internal and deep to the other structures. Figure previously published in Strouthidis NG, Yang H, Downs JC, Burgoyne CF. Comparison of clinical and three-dimensional histomorphometric optic disc margin anatomy. Invest Ophthalmol Vis Sci. 2009;50:2165–2174. © ARVO.

Figure 3.

 

Disc photograph (A) and SD-OCT (B, C) appearance of internally oblique and externally oblique border tissue in the normal monkey eye. Externally oblique: (A) A rrow 1 marks a thin pale crescent located at the same stereoscopic depth as the retinal pigment. Internal to this is a pigment crescent deep to the retinal pigment; the end of this pigment is the temporal disc margin (arrow 2). In a colocalized horizontal B-scan (B, location of B-scan shown by a white line in A), an externally oblique border tissue configuration is seen temporally. A rrow 1: location of the NCO temporally. A rrow 2: position of the border tissue, which can be seen to extend internally to the NCO. In the disc photograph (A), the pale crescent labeled 1 is a stripe of unpigmented Bruch’s membrane; external to this is pigmented Bruch’s membrane continuous with the retinal pigment epithelium. The pigment crescent internal to 1 is caused by pigment on the surface of an externally oblique border tissue. (C1, C2) Location of these structures (at the center of the red cross-hairs) within the en face OCT images. Note that the externally oblique border tissue (C2) is visible as a high-intensity signal, internal to and at a greater depth than, the NCO (C1). Internally oblique: (A) A rrow 3 highlights the disc margin location, which is at the termination of a crescent of mottled pigment, visible at the same depth as the retinal pigment. Within the B-scan (B), internally oblique border tissue can be clearly seen at the nasal side of the neural canal. A rrow 3: termination of the retinal pigment epithelium/Bruch’s membrane (NCO). The internally oblique border tissue can be seen to meet the retinal pigment epithelium/Bruch’s membrane signal external to the NCO (arrow 4) and to extend away from the neural canal. The pigment crescent at the nasal disc margin represents a region of pigmented Bruch’s membrane. The region of Bruch’s membrane overhang, beyond the point where border tissue meets Bruch’s membrane, is visible as a dark crescent in the en face OCT image (C4) external to the NCO (C3).

Figure 4.

 

Colocalization and disc margin delineation process. (A) Location of the neural canal opening points (NCO, visible as red glyphs) within an en face OCT image. (B) During colocalization, the en face OCT image is used with the NCO glyphs not visible. (C) Clinical photograph, at 50% opacity, laid on the en face OCT image. By adjusting x- and y-axis shifts and z-axis rotation and by scaling at a 1:1 ratio, the central retinal vessels are matched between the photograph and the en face OCT image. (D) The disc photograph, colocalized to the en face OCT image, is then presented to the clinical observer for disc margin delineation. While delineating the disc margin, the observer simultaneously views the representative stereophotograph pair. (E) Completed disc margin delineation, with blue glyphs representing certainty regarding disc margin location and green glyphs representing forced choice. (F) Once disc margin delineation is completed, the colocalized OCT delineated points are revealed so that their alignment to the disc margin can be compared. In this case, the SD-OCT–defined NCO is well aligned to the disc margin because there is less than a glyph’s separation between adjacent glyphs for 360° of the disc margin.

Figure 5.

 

Schematic to illustrate the estimation of alignment error in the superonasal quadrant of a colocalized image (all images in right eye orientation; all four quadrants used in the analysis [not shown in diagram]). The clinician-ascribed disc margin is shown in black, and the SD-OCT delineated curve is shown in red. Radial spokes are generated from the center of the disc margin at 4.5° intervals from the temporal meridian. Alignment error is the distance between the clinician’s disc margin and the SD-OCT curve (red line, 2), expressed as a percentage of the disc radius (black line, 1). Alignment errors are calculated for each radial spoke, enabling an estimation of global and regional alignment error for each eye.

Figure 6.

 

Examples of good alignment between disc margin delineations and SD-OCT delineations. (A) Six discs in which the NCO (red glyphs) aligns to within one glyph’s diameter for 360° of the disc margin (blue glyphs, certain disc margin location; green glyphs, forced-choice disc margin location). (B) Three examples in which the termination of externally oblique border tissue (yellow glyphs) aligns to the temporal disc margin, and the NCO aligns to the remainder of the disc margin.

Figure 7.

 

Regional alignment error for all eyes included in the study. The lines shown are for the median (middle lines), 95% (upper lines), and 5% (lower lines) quantiles of alignment error. This is measured at each 4.5° location as a moving average of that location and of the two locations on either side of it. S olid lines: degree of misalignment from the disc margin of the curves generated by the NCO. D otted lines: degree of misalignment from the disc margin of the curves generated by a combination of the NCO and the border tissue. T, temporal; I, inferior; N, nasal; S, superior.

Figure 8.

 

Examples of misalignment between the disc margin and SD-OCT delineations. (A) NCO is internal to the disc margin, beyond one glyph’s diameter, at the superior pole. (B) NCO is misaligned at the superior pole. In both cases, the remainder of the disc margin is well aligned to the NCO. A possible reason for misalignment in these two cases is the fact that the disc margin may be indistinct in regions of the disc poles where the nerve fiber layer is particularly thick. (C) Left disc where the NCO is misaligned from the nasal disc margin. It is possible in this case that a wide crescent of unpigmented Bruch’s membrane was not visible at the nasal disc margin on the stereophotographs but was picked up by SD-OCT imaging. (D) NCO at the inferior pole is external to the disc margin, which had been marked “with certainty” (blue glyphs) in this region. When examining the B-scan (E) acquired at the location highlighted by the white line in the disc photograph, the superior NCO, which is aligned to the disc margin, is clearly visible (white arrows). The inferior NCO and the retinal pigment epithelium/Bruch’s membrane complex are obscured by a shadow cast by a blood vessel (white arrowheads), causing difficulties in accurate delineation of the NCO.

Figure 9.

 

SD-OCT disc margin anatomy in a myopic human left eye. (A) Disc photograph; a white line highlights the location of the B-scan shown in (C). (B) Alignment of the disc margin (blue and green square glyphs) to the SD-OCT delineations. (B, C) Representative B-scan. (D, E) 3-D point clouds. C ircular red glyphs are the NCO; circular blue glyphs are the anterior scleral canal opening; yellow lines represent the full extent of the border tissue. (B) Anterior scleral canal opening, at the termination of externally oblique border tissue, can be seen to align to the temporal disc margin. This is seen clinically as a true scleral lip at the disc margin. The NCO is aligned to the nasal disc margin. A green arrow in (C) highlights the location of a choroidal vessel within the border tissue signal, which is also shown in its colocalized position within the disc photograph (A). (D, E) 3-D point clouds generated from the delineation of the SD-OCT images.

The authors thank Galen Williams, Erica Dyrud, Wenxia Wang, and Christy Hardin for SD-OCT imaging, and Balwantry Chauhan for supplying the human SD-OCT volume and for his careful and considered comments regarding this manuscript.