Three-dimensional histomorphometry of the normal and early glaucomatous monkey optic nerve head: neural canal and subarachnoid space architecture

Abstract

Purpose: To delineate three dimensionally the neural canal landmarks-Bruch's membrane opening (BMO), anterior sclera canal opening (ASCO), anterior laminar insertion (ALI), posterior laminar insertion (PLI), and posterior scleral canal opening (PSCO)-and the anterior-most aspect of the subarachnoid space (ASAS), within digital three-dimensional (3-D) reconstructions of the monkey optic nerve head (ONH).

Methods: The trephinated ONH and peripapillary sclera from both eyes of three early glaucoma (EG) monkeys (one eye normal, one eye with laser-induced EG) were serial sectioned at 3-microm thickness, with the embedded tissue block face stained and imaged after each cut. The images were aligned and stacked in a 3-D volume, within which the BMO, ASCO, ALI, PLI, PSCO, and ASAS were delineated in 40 digital, radial, and sagittal sections. An ellipse was fitted to the 80 BMO points to establish a BMO zero reference plane, on which all other points were projected. The distance from each projected point to the BMO centroid (offset) and BMO zero reference plane (depth) were calculated and compared regionally between normal and EG eyes, both overall and within each monkey, by analysis of variance.

Results: BMO was the clinically visible optic disc margin in all six eyes. The neural canal architecture was highly variable in the three normal eyes. Radial expansion of the neural canal was greatest posteriorly in the EG eyes. Axial elongation of the canal was less pronounced overall but was regionally present within all three EG eyes. ASAS was regionally radially expanded and anteriorly displaced within two of the three EG eyes.

Conclusions: Profound deformation of the neural canal and ASAS architecture are present in young adult monkey eyes at the onset of ONH surface change in early experimental glaucoma.

Figure 1

3-D delineation of ONH and peripapillary scleral landmark points within colorized, stacked-section, 3-D ONH reconstructions. (A) Digital image of a representative central horizontal histologic section from a normal monkey eye perfusion fixed at an IOP of 10 mm Hg with the nasal side of the canal magnified in (B). (C) Histologic landmarks within (B) are labeled as follows: a, BMO; b, insertion of the border tissues of Elschnig into Bruch's membrane; c, border tissues of Elschnig; d, ASCO; e, border tissues of Jacoby, which in this region are heavily pigmented; f, PLI; g, PSCO; h, ASAS; i, BM flange, extending centrally beyond the border tissues and in this case without pigmentation and not covered by RPE; j, RPE sitting on BM. (D) Principle tissues colored as follows: yellow, BM; dark blue, border tissues of Elschnig; green, sclera; red hatched, branches of posterior ciliary arteries; light blue, subarachnoid space. (E) Neural canal landmark points analyzed in the study. (F–J) 3-D delineation methodology. Note that generation of 3-D ONH reconstructions from aligned serial section images are explained in Figures 1 and 2 of our previous publication. (F) A total of 40 serial digital, radial, and sagittal slices (each 7 voxels thick) are served to the delineator at 4.5° intervals. (G) A representative digital sagittal slice, showing the marks for seven landmark surfaces and six pairs of landmark points, which are 3-D-delineated using linked, simultaneous, colocalization of the sagittal slice (shown), and the transverse section image (H). (I) Representative 3-D point cloud showing all delineated points for a normal monkey ONH relative to the last section image of the reconstruction. (J) The subset of the 3-D point cloud showing the neural canal landmarks reported in this study. (K) The 80 delineated BMO points are fit to an ellipse (L) that defines the BMO zero reference plane and the BMO centroid. All other neural canal landmark points are projected onto the BMO zero reference plane (M) shown for the PSCO, only, then the distance from the BMO centroid (offset, blue lines) and distance from the BMO zero reference plane (depth, green lines) are noted for each marked point (N).

Figure 2

Clinical overlay of neural canal landmark points in both eyes of each monkey with overlay maps and regionalization for each monkey. Neural canal landmark points projected onto the BMO zero reference plane for the normal (left) and EG eye (middle) of each monkey (both in right eye configuration). To the right, offset overlay maps for each monkey (solid lines: EG eye data; dotted lines: normal eye data) in right eye configuration with coincident BMO centroids. Beneath and to the left of each offset overlay map, the depth data (in micrometers) for the central vertical section (left) and central horizontal section (bottom) are plotted in scale for reference. S, superior; SN, superonasal; N, nasal; IN, inferonasal; I, inferior; IT, inferotemporal; T, temporal; ST, superotemporal.

Figure 3

Regional treatment effects in offset and depth (both in micrometers) by monkey. Values are the magnitude of change in the EG eye relative to its contralateral normal control (positive is radial expansion and negative is radial contraction for offset; positive is axial elongation and negative is axial contraction or anteriorization for depth). Concentric rings represent the different neural canal landmarks from internal to external: BMO, ASCO, ALI, PLI, PSCO, ASAS, respectively. The regions that achieve statistical significance (P < 0.05, ANOVA) are colored by landmark (bottom right).

Figure 4

Bruch's membrane opening is the clinically visible optic disc margin within clinical photographs of monkey 3. (A) Clinical photograph of the normal right eye of monkey 3. White boxed area is in (G) and (J). (B) 3-D reconstruction of the central retinal vessels and BMO (red) and ASCO points (blue) for this eye are overlaid onto the clinical photograph to achieve the best qualitative alignment of the vessel branch points. Note the alignment of BMO points with the optic disc margin in the clinical photograph. Green line: approximate location of the digital sagittal section shown in (C). (C) Digital sagittal section in which the operator has delineated BMO on the nasal side of the canal (red dot) by positioning the cursor in this image, while simultaneously noting its position (red dot within green cross-hairs) in the slaved transverse section image shown in (D). White boxes in (C) and (D) are shown in magnified view in (E) and (F), along with an estimate of their location in the clinical photograph shown in (G). Labeling in (H), (I), and (J) is as follows: a, BM extending over and beyond the border tissues of Elschnig, covered by variably mottled pigment; b, probable start of viable RPE with a greater density of pigment; c, BM points that are not clearly seen in this image but are present when the brightness and contrast are adjusted (part of the normal delineation sequence); d, BMO point (red), which is marked by the delineator by colocalizing the cursor in image (E) with the green cross hairs of the cursor in the slaved transverse view (F); e, the junction of the border tissues of Elschnig with BM; f, the border tissues of Elschnig; g, ASCO; h, heavy pigmentation within the choroid. White dotted lines in (I) and (J): the border of more densely mottled pigment on BM that is visible within the clinical photographs and serial transverse section images of the 3-D reconstruction. Taken together, the images demonstrate that in this region of this eye, lightly pigmented BM extend beyond the border tissues of Elschnig and is the clinically visible optic disc margin.

Figure 5

Enlargement and obliqueness of the neural canal in representative digital, sagittal, and nasotemporal sections of the normal eye of each monkey. BMO (red), ASCO (yellow), ALI (dark blue), PLI (green), PSCO (purple), and ASAS (blue) landmarks delineated within the central N–T sagittal section from the normal eye of each monkey, showing the obliqueness (angle between the BMO zero reference plane normal vector and the line connecting the BMO centroid [top black dot] and PSCO centroid [bottom black dot]), and anterior-to-posterior enlargement (distance between BMO marks relative to PSCO marks) of the neural canal. Neural canal enlargement and obliqueness measures for the normal eye of each monkey are given in Table 7.