We have previously delineated BMO as the NCO and used it as a reference plane within quantitative 3-D histomorphometric reconstructions of the optic nerve head from both eyes of three NHPs with early experimental glaucoma in one eye.25-27
The purposes of the present report were to examine our ability to detect and delineate manually the NCO within 3-D histomorphometric reconstructions of a larger group of normal NHP optic nerve heads and, second, to perform the first rigorous assessment of NCO detection within interpolated SD-OCT ONH volumes, in a separate group of normal eyes. An additional purpose of this study was to characterize the structure of the NCO in both 3-D histomorphometric reconstructions and in interpolated SD-OCT optic nerve head volumes, with particular reference to planarity.
The principal findings of the study are summarized as follows: First, NCO points can be delineated within all digital, radial sagittal section images from interpolated SD-OCT volumes. Second, NCO plane error appears to be of a similar magnitude in both histomorphometric reconstructions and SD-OCT volumes of normal NHP optic nerve heads. The NCO plane error is similarly small relative to the size of the NCO fitted ellipse. Third, the NCO point clouds of both histomorphometric reconstructions and SD-OCT volumes indicate that the NCO appeared to be a biologically continuous structure.
This is the first report of 3-D visualization and delineation of SD-OCT interpolated optic nerve head volumes. As such, this study establishes the NCO to be clearly discernible and biologically continuous within single SD-OCT volumes of 33 normal NHP eyes. The depth resolution of the 870-nm Spectralis imaging system within the 40 serial radial interpolated SD-OCT sections has been shown to be consistently capable of imaging the NCO, as well as the RPE/BM complex, in all 33 eyes. These observations preliminarily support the concept of applying SD-OCT technology to image the optic nerve head in a volumetric and quantifiable fashion by using the NCO as a reference plane.
The fact that NCO plane error was low in both histomorphometric and SD-OCT data sets is important for several reasons. The histomorphometric data in a much larger number of eyes strengthen our use of an NCO reference plane for quantification within our histomorphometric 3-D reconstructions.25-27
Likewise, the SD-OCT data provides support for an identical application of the NCO within clinically obtained 3-D SD-OCT volumes. Indirectly, one may also extrapolate that, as their magnitude of planarity is similar, in vivo SD-OCT imaging may be capable of capturing the 3-D architecture of the NCO in a clinically meaningful manner that is similar to its postfixed, postembedded, poststained, and postreconstructed architecture. This latter statement is made with the important caveat that the present study does not directly compare in vivo SD-OCT imaging to post mortem 3-D histomorphometry in the same eyes. However, in making this point, one should also note that even if the same optic nerve head had been SD-OCT imaged, then 3-D histomorphometrically reconstructed post mortem, the measurements obtained would still not be directly comparable. Although 3-D histomorphometry is capable of measuring the dimension of structures after they have been processed (in summary, after perfusion-fixation, embedding, staining, sectioning, alignment, 3-D visualization, and delineation), these measurements are influenced by tissue shrinkage and measurement error. Likewise, the accuracy of in vivo measurements obtained by SD-OCT is affected by the image acquisition and alignment software, individual eye magnification correction algorithms, as well as the 3-D visualization and 3-D delineation methods. Given their respective sources of measurement error, the comparison of the SD-OCT data to the 3-D histomorphometric data in this report and its application to in vivo human eyes must remain qualitative and preliminary in nature.
Although a correction for SD-OCT magnification error has been included to compensate for the optical system of the monkey eye, it is at present imprecise. Several methods have been published to correct for eye-camera53,54
magnification, all of which make assumptions regarding the optics of the eye. In our method, the error is based on axial length; methods using this technique have been shown to result in smaller errors and to be more accurate than those using ametropia and keratometry.57
The magnification error only affects x
- and y
-axis coordinates, not those within the z-axis. The SD-OCT ellipse dimensions, which are entirely based on x
coordinates, are likely to be greatly influenced by any measurement error. One should therefore regard the SD-OCT NCO dimensions (namely, major ellipse axis, minor ellipse axis, and ellipse area) as being a best available estimate only and not infer importance from the observed similarity with histomorphometrically derived ellipse dimensions.
Eccentricity, however, which is the ratio of the major to minor ellipse axes, is independent of magnification error in either imaging system. Eccentricity is very similar between histomorphometric reconstructions and SD-OCT volumes (median values of 1.38 and 1.43, respectively), suggesting that the scale of measurements is likely to be similar, regardless of differences in magnification error. Among SD-OCT NCO metrics, plane error is likely to be the least influenced by magnification as a significant component of the normal distance between observed NCO points and the fitted plane will be in the z-axis. Plane error appears to be low relative to ellipse magnitude in both imaging systems, suggesting that NCO is a relatively planar structure in both 3-D histomorphometric reconstructions and in SD-OCT volumes.
As noted in the Methods section, the resolution of our 3-D histomorphometric technique improved from a 3.0- to a 1.5-μ
m voxel dimension within the group of histomorphometric reconstructions in this report. However, neither the ability to detect the NCO in the 3-D histomorphometric reconstructions nor its plane error (once detected) appeared to be influenced by this improvement. The increase in 3-D resolution achieved by the newer protocol theoretically achieves a greater “biofidelity” to smaller structures such as individual laminar beams and the extension of unpigmented BM beyond the end of the RPE.25
Our data suggest a relationship between the magnitude of plane error and advancing age in the histomorphometric but not the SD-OCT data sets. However, the maximum age of the NHPs imaged by SD-OCT was only 19 years, whereas there were two NHPs more than 30 years old in the histomorphometric data set. The relationship between age and NCO plane error should be studied in large data sets as volumetric SD-OCT imaging of the human optic nerve head begins to be reported.
SD-OCT NCO delineation was performed using interpolated B-scans generated from 290 × 768 horizontal grid B-scan acquisitions. It is likely that our delineation technique will be improved by using interpolated sections generated from higher density acquisitions (for example, 768 A-scans per each of 768 B-scans) or by using actual radial B-scan acquisitions, which will have a better image quality than the current interpolated scans. Despite this, our ability to delineate NCO points in this study is encouraging, with a manageable detrimental influence from motion artifact and/or shadowing from blood vessels.
The current 290 horizontal B-scan acquisition protocol takes 70 seconds in an anesthetized NHP. It is likely that the same scan will take considerably longer in an awake human subject and longer still for a test of higher B-scan density. Acquiring 40 or 80 radial B-scans will likely be of shorter duration and will have the advantage that one will be able to delineate structures within the acquired B-scan, rather than an interpolated SD-OCT section. A disadvantage to this approach is that interpolated 3-D volumes cannot, at present, be generated from radial B-scan acquisition patterns. As a consequence, true 3-D delineation of the NCO points (in which the delineator can cross-reference demarcated points between the location in the transverse and sagittal view before actual delineation), which powerfully aided this study, are not possible using radial B-scan acquisitions in isolation.
NCO visualization with volumetric (or serial B-scan) SD-OCT images may be enhanced with clinical application of a higher wavelength source. In an in vivo study of high-resolution OCT imaging of the human retina, a 1050-nm wavelength source was found to achieve greater depth resolution than an 870-nm source (equivalent to that used in the Spectralis device).58
Visualization beyond the RPE and into the choroidal vasculature was achieved, and this depth resolution was maintained, even in the presence of significant cataract. It will therefore be necessary in the future to investigate the effect of a 1050 nm light source on the ability to delineate NCO and deep optic nerve head structures in the monkey eye.
Although our results show that SD-OCT is capable of reliably discerning the NCO in the monkey eye, this may not necessarily be consistent in the human eye. The reason for potential discrepancy is due to differences in the amount of pigment at the level of the RPE/BM. In normal monkey eyes, RPE and choroidal atrophy is uncommon, whereas it is relatively common in humans, particularly in the aged eye. It will not be apparent whether the NCO is detectable in this context until our methodology can be applied to human eyes.
The location of the reference plane—effectively the landmark from which all structural quantification is measured—is critical in longitudinal imaging of the optic nerve head. The topographical height of the reference plane, determined by tomography (Heidelberg Retina Tomograph [HRT]; Heidelberg Engineering) has been shown to be the major contributor to parameter variability. The default reference plane used in the HRT operational software is the standard reference plane, which is located 50 μ
m posterior to the temporal disc margin. The choice of the temporal disc margin was based on the assumption that its height would be stable until the latest stages of glaucoma, when papillomacular bundle thinning would be expected to take place.32
However, OCT evidence suggests that retinal nerve fiber layer thinning occurs at a much earlier time frame in the disease process.59
Given this caveat, any measurements generated using a reference plane anchored to the retinal surface height at the disc margin are likely to fluctuate longitudinally as the topographical surface shifts posteriorly as glaucoma progresses.
Studies have shown that HRT reference planes anchored to the retinal surface height at a reference ring located in the image periphery generate less variable stereometric parameters, particularly rim area, and this most likely reflects the more stable topographical surface height at locations distal to the optic disc.35,37,38,46
However, the peripheral surface-anchored reference planes have the shortcoming that they generate erroneous measurements when applied to atypical disc morphology, particularly those with gross tilt or advanced parapapillary atrophy. Indeed, defining the reference plane relative to the topographic surface is always likely to be problematic as the height of the internal limiting membrane alters as the nerve fiber layer thins in progressing glaucoma.
A reference plane source-structure deep within the optic nerve head structure, such as the NCO, is a more logical option, if it is reasonable to assume that this structure will be more stable than the internal limiting membrane through the onset and progression of glaucomatous damage. We propose that, relative to the optic nerve head neural and connective tissues (primary sites of glaucomatous damage and known to be damaged early in the neuropathy),18-23
the position of the NCO will remain relatively stable throughout the course of the neuropathy. If this is true, alterations in the anterior laminar surface and prelaminar neural tissue internal limiting membrane should be more sensitively detected relative to the NCO during periods in which retinal surface–based reference planes are themselves being altered by the neuropathy.
However, the position of the NCO and the same optic nerve head target tissues may change relative to the peripapillary sclera due to glaucomatous (outward) bowing of the peripapillary sclera.26
Since we believe this bowing, when it is present, is part of the neuropathy, it may be advisable to use the NCO as a source-structure for a secondary (peripheral) reference plane that is located at a fixed distance from the NCO on the BM/RPE complex. The advantage of such a peripheral zero-reference plane would be the detection of both glaucomatous scleral deformation and optic nerve head neural and connective tissue alterations at all stages of the neuropathy. The relative stability of NCO and peripheral (NCO-anchored) and internal limiting membrane–based reference planes within longitudinal SD-OCT images of NHPs with early, moderate, and severe experimental glaucoma is currently under study in our laboratories.
Our study is the first in which deep optic nerve head structures were visualized and delineated within SD-OCT 3-D volumes acquired in vivo. The NCO has been shown to be continuous and reasonably planar, which supports its adoption as the SD-OCT reference plane source-structure within the NHP and human optic nerve head.