A comparison of optic nerve head morphology viewed by spectral domain optical coherence tomography and by serial histology

Abstract

Purpose: To compare serial optic nerve head (ONH) histology with interpolated B-scans generated from a three-dimensional (3-D) spectral domain (SD)-OCT ONH volume acquired in vivo from the same normal monkey eye.

Methods: A 15 degrees ONH SD-OCT volume was acquired in a normal monkey eye, with IOP manometrically controlled at 10 mm Hg. After perfusion fixation at 10 mm Hg, the ONH was trephined, the specimen embedded in a paraffin block, and serial sagittal sections cut at 4-mum intervals. The location of each histologic section was identified within the optic disc photograph by matching the position of the retinal vessels and of Bruch's membrane opening. By altering the angles of rotation and incidence, interpolated B-scans matching the location of the histologic sections were generated with custom software. Structures identified in the histologic sections were compared with signals identified in the matched B-scans.

Results: Close matches between histologic sections and interpolated B-scans were identified throughout the extent of the ONH. SD-OCT identified the neural canal opening as the termination of the Bruch's membrane-retinal pigment complex and border tissue as the innermost termination of the choroidal signal. The anterior lamina cribrosa and its continuity with the prelaminar glial columns were also detected by SD-OCT.

Conclusions: Volumetric SD-OCT imaging of the ONH generates interpolated B-scans that accurately match serial histologic sections. SD-OCT captures the anterior laminar surface, which is likely to be a key structure in the detection of early ONH damage in ocular hypertension and glaucoma.

Figures

Figure 1.

Trephining the ONH in the orientation of the acquired B-scan. (A) The SD-OCT IR image including the 15° acquisition box, viewed at 50% opacity, was aligned to an image of the ONH specimen. (B) The coordinates of the acquisition box were burned onto the image. (C) Cuts were made in the specimen, in an attempt to get the closest approximation to the orientation of the acquired B-scans. The cut made in the superior part of the specimen (red arrows) most closely matched the acquired B-scan orientation. The specimen was embedded with the superior cut surface facing downward.

Figure 2.

Method of orienting histologic sections to the disc photograph. The topographic locations of histologic sections 145, 241, and 313 are shown. The orientation of each section (red lines in the disc photograph) is judged by assessing the relative spacing of the retinal vessels, marked by circular glyphs in the disc photograph and by vertical lines in the histologic sections. The temporal vessel outlined by a red glyph is absent in section 144, but appears in the next section (145). The superior opening in Bruch's membrane (superior disc margin) was near section 145 (described in detail in Fig. 5) and the inferior opening in Bruch's membrane was near section 335 (not shown in this figure, but described in detail in Fig. 6) Hematoxylin and eosin; magnification, ×10.

Figure 3.

Method of identifying interpolated B-scans from the SD-OCT volumetric reconstruction. (A) A representative acquired B-scan is shown within the 3-D volume (enclosed within a red cube) generated from a 290 × 768 horizontal raster scan. The angle of rotation of the B-scan was 90° and the angle of incidence was 0°. (B) The interpolated B-scan shown was generated in a 20° clockwise rotation (angle of rotation, 70°); the angle of incidence was unchanged. (C) The interpolated B-scan shown was generated after a −20° change in the angle of incidence. (D) The interpolated B-scan shown was generated with an angle of rotation of 70° and an angle of incidence of −20°.

Figure 4.

Method of identifying the B-scan angle of rotation. (A) The topographic orientation of serial hematoxylin and eosin–stained sections is depicted as a series of red lines in the disc photograph. There was a minimum separation of 16 μm between sections. Sections that could not be oriented because of poorly preserved retina account for the variable width of gaps between the lines. (B) The image shown (A, viewed at 50% opacity) was colocalized to the en face SD-OCT image (C-scan). Green lines: 0° and 90° orientations of the actual acquired horizontal raster B-scans. (C) The angle of rotation of the histologic sections assessed relative to that of the acquired B-scans varied within three regions: 73.5° in histologic sections from region 1, 87° in sections from region 2, and 89° in sections from region 3.

Figure 5.

Comparison of a histologic section located near the superior Bruch's membrane opening (disc margin) with matched interpolated B-scans. (A) Section 145, with its topographical location shown as a black line in the disc photograph (inset, also shown in Fig. 2). Note the presence of a small vessel in the nasal (left) part of the section (red arrow) and an oblique vessel in the temporal (right) part of the section (green asterisk). There was an opening in the choroid and sclera, but the RPE and Bruch's membrane were intact over the optic nerve. (B) An interpolated section generated from the same topographic location, at an angle of rotation of 73.5°, but with an angle of incidence of 0° (perpendicular to the surface). Neither of the vessels highlighted in (A) was detected, and the choroidal signal appeared to be continuous. (C) An interpolated B-scan generated from the same location as (B), but with an angle of incidence of −18° (angled toward the nerve). The two vessels became visible in the correct location (labeled with a red arrow and a green asterisk). An opening in the choroid was visible, the RPE/BM complex remained intact, but no signal from the deeper optic nerve and lamina cribrosa was detected. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PR, photoreceptor layer; RPE/BM, retinal pigment epithelium/Bruch's membrane complex. Hematoxylin and eosin; magnification, ×10.

Figure 6.

Comparison of a histologic section located near the inferior Bruch's membrane opening (disc margin) with its matched interpolated B-scan. (A) Section 335 is shown with its topographical location depicted (black line) in the clinical photograph (inset). (B) The matched interpolated B-scan was acquired at an angle of rotation of 89°, but the angle of incidence was kept at 0°. The retinal pigment epithelium/Bruch's membrane complex was intact, but the opening in the choroid was present, as in the histologic section. The prelaminar neural tissue and the intact sclera were most likely not detected because of shadowing from the central retinal vessels. Hematoxylin and eosin; magnification, ×10.

Figure 7.

Identification of the neural canal opening. Right: Magnification of highlighted regions (boxes, left). Differences in anterior surface tilt between the B-scan and the histologic sections can be explained by the presence of retinal elevation/detachment and of a rip in the retina on the left side of the sections (both features cropped out of A and C). The retinal layers in (A) and (C) are as identified in Figure 5. (A) Section 257 (hematoxylin and eosin). The termination of Bruch's membrane (the neural canal opening) is highlighted with a white arrow in the magnified view. At this location, the retinal layers taper to a point (black arrows) at the photoreceptor layer, internal to the NCO. (B) Matched interpolated B-scan. The same relationship between the retinal layers (black arrows) and the termination of the retinal pigment epithelium/Bruch's membrane complex (white arrow), as was detected in (A), are seen in the magnified view. (C) Section 260 (Ponceau S). With this stain, the distinction between the termination of the retinal pigment epithelium and an extension of unpigmented Bruch's membrane (blue arrows) was clearly visible. Magnification: (A, C) ×10.

Figure 8.

Detection of border tissue of Elschnig. (A) Section 258, with border tissue highlighted with white arrows. This histologic section was located immediately adjacent to the section shown in Figure 7. The border tissue of Elschnig was seen as a connective tissue strut (stained blue) connecting the anterior sclera to Bruch's membrane and enclosing the choroid. A more complete view of this section is shown in Figure 9. Masson trichrome; magnification, ×10. (B, white arrows) The border tissue signal in the matched interpolated B-scan. SD-OCT appeared to accurately capture the orientation of the border tissue with an internally oblique configuration seen nasally (left) and an externally oblique configuration seen temporally (right).

Figure 9.

Detection of the lamina cribrosa. (A) Section 257. Prelaminar glial columns appear to correspond to vertical striations in the matched interpolated B-scan (bottom; marked with red arrows). Hematoxylin and eosin. (B) Section 258. The anterior lamina surface delineated with white glyphs. The peripheral insertion of the lamina was not clearly visible in the interpolated B-scan, although the approximate level of insertion could be ascertained by following the contour of the anterior laminar signal to the periphery of the neural canal (white arrows). Masson trichrome. (C) Section 256. Posterior lamina surface delineated with black glyphs in the histologic section. The posterior surface was not detectable in the matched interpolated B-scan, as the signal faded rather than coming to a discrete termination (region highlighted by a white bracket) Luxol fast blue stain. Magnification, ×10.