3-D histomorphometry of the normal and early glaucomatous monkey optic nerve head: lamina cribrosa and peripapillary scleral position and thickness

Abstract

Purpose: To delineate three-dimensionally the anterior and posterior surfaces of the lamina cribrosa, scleral flange, and peripapillary sclera, to determine the position and thickness of these structures 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 monkeys with early glaucoma (EG; one eye normal, one eye given laser-induced EG) were serially sectioned at 3-mum thickness, with the embedded tissue block's face stained and imaged after each cut. Images were aligned and stacked to create 3-D reconstructions, within which Bruch's membrane opening (BMO) and the anterior and posterior surfaces of the lamina cribrosa and peripapillary sclera were delineated in 40 serial radial (4.5 degrees interval) digital sagittal sections. For each eye, a BMO zero reference plane was fit to the 80 BMO points, which served as the reference from which all position measurements were made. Regional laminar, scleral flange, and peripapillary scleral position and thickness were compared between the normal and EG eyes of each monkey and between treatment groups by analysis of variance.

Results: Laminar thickness varied substantially within the normal eyes and was profoundly thicker within the three EG eyes. Laminar position was permanently posteriorly deformed in all three EG eyes, with substantial differences in the magnitude and extent of deformation among them. Scleral flange and peripapillary scleral thickness varied regionally within each normal ONH with the scleral flange and peripapillary sclera being thinnest nasally. Overall, the scleral flange and peripapillary sclera immediately surrounding the ONH were posteriorly displaced relative to the more peripheral sclera.

Conclusions: Profound fixed posterior deformation and thickening of the lamina are accompanied by mild posterior deformation and thinning of the scleral flange and peripapillary sclera at the onset of confocal scanning laser tomography (CSLT)-detected ONH surface change in young adult monkey eyes with early experimental glaucoma.

Figure 1. 3D delineation of ONH and peripapillary scleral landmark points within colorized, stacked-section, 3D ONH reconstructions

Note that generation of 3D ONH reconstruction from aligned serial section images for each individual ONH are explained in Figures 1 and 2 of our previous publication. A. Sagittal histologic section through a representative normal monkey ONH showing the anatomy, and (B) the associated neural canal landmarks and surface landmarks: Bruch's membrane opening (BMO, red), the anterior scleral canal opening (ASCO, dark blue), the anterior laminar insertion point (ALI, yellow), the posterior laminar insertion point (PLI, green), the posterior scleral canal opening (PSCO, pink), the anterior-most aspect of the subarachnoid space (ASAS, light blue), the anterior laminar/scleral surface (white), the posterior laminar/scleral surface (black) and the neural boundary (light green). C. A total of 40 serial digital, radial, sagittal slices (seen in D, each 7 voxels thick) are served to the delineator at 4.5° intervals. D. A representative digital sagittal slice, showing the marks for 7 landmark surfaces and 6 pairs of landmark points, which are 3D-delineated using linked, simultaneous, co-localization of the sagittal slice (shown) and the transverse section image (E). F. Representative 3D point cloud showing all delineated points for a normal monkey ONH relative to the last serial section image (orbital optic nerve bottom, vitreous above.

Figure 2. ONH anatomy and parameter generation

A) Representative horizontal, digital sagittal section image from the 3D reconstruction of the left eye of Monkey 3 (temporal on left, nasal on right). B) The definition and extent of the major structures quantified in the paper: lamina cribrosa (red) inserts into the scleral flange (green), which transitions into the peripapillary sclera (purple) at the PSCO. The delineated points marking the anterior (white dots) and posterior (black dots) surfaces of the lamina cribrosa and sclera, the neural canal boundary (green dots), and neural canal landmark points are shown. The scleral flange is defined as the peripapillary sclera that extends from the anterior scleral canal opening (ASCO) to the normal from the anterior surface through the posterior scleral canal opening (PSCO normal vector - black arrow). The Bruch's Membrane opening (BMO) zero reference plane is also shown (red line). C) Laminar and scleral position (green arrow) at each delineated anterior laminar surface point (white dot) is defined as the shortest distance from the delineated point to BMO zero reference plane. D) LC and PPS thickness at each delineated anterior surface point is determined by fitting a continuous surface (white line) to all of the delineated anterior surface points (D). E, F) Thickness is defined as the distance along a normal vector to the anterior surface (green arrow in E) from each anterior delineated point to the posterior surface (black line in F). G) Thickness of the scleral flange at each delineated anterior surface point (white dots) is defined as the distance between the neural canal boundary points (green line in G and H), along a vector parallel to the PSCO normal vector.

Figure 3. Continuous lamina cribrosa and peripapillary scleral position (μm) maps for both eyes and continuous treatment difference maps for each monkey co-localized with neural canal landmark points

Continuous laminar position maps of the Normal and EG eye of each monkey (2 left columns; both in right eye configuration) overlaid with a subset of neural canal landmark points for reference. Continuous scleral flange and peripapillary scleral position maps of Normal and EG eye (3^rd and 4^th columns; both in right eye configuration). Continuous treatment difference maps (EG eye data – Normal eye data) for laminar, scleral flange, and peripapillary scleral position (5^th and 6^th columns) overlaid onto the CSLT image of the EG eye. CSLT images of EG eye of each monkey (far right column in right eye configuration) for reference.

Figure 4. Continuous lamina cribrosa and peripapillary scleral thickness (μm) maps for both eyes and continuous treatment difference maps for each monkey co-localized with neural canal landmark points

Continuous laminar thickness maps of the Normal and EG eye of each monkey (2 left columns; both in right eye configuration) overlaid with a subset of neural canal landmark points for reference. Continuous scleral flange and peripapillary scleral thickness maps of Normal and EG eye (3^rd and 4^th columns; both in right eye configuration). Continuous treatment difference maps (EG eye data – Normal eye data) for laminar, scleral flange, and peripapillary scleral thickness (5^th and 6^th columns) overlaid onto the CSLT image of the EG eye. CSLT images of EG eye of each monkey (far right column in right eye configuration) for reference.

Figure 5. Regionalization method and overall regional position and thickness treatment differences (EG-N) within the lamina cribrosa, scleral flange, and peripapillary sclera

Within the lamina, position and thickness data are pooled into 17 regions according to the three radial regions (central; MP, middle periphery; P, periphery) and eight quadrants (S, superior; SN, supero-nasal; N, nasal; IN, infero-nasal; I, inferior; IT, infero-temporal; T, temporal; ST, supero-temporal) (top left). Scleral flange and peripapillary sclera data, together, are pooled into 8 quadrants (top right). Overall laminar position treatment difference (middle left) and pooled peripapillary scleral position treatment difference (middle right). Regional thickness difference in the lamina cribrosa (bottom left), scleral flange (bottom right inner ring), and peripapillary sclera (bottom right outer ring). Values shown (in μm) are the magnitude of change in the EG eye relative to its contralateral Normal control (EG-N). The colored regions achieved statistically significant difference between the normal and EG eyes (P<0.05, ANOVA). Color intensity represents magnitude as illustrated in the color bar. For position data, red indicates posterior deformation and green indicates anterior deformation in the EG eyes relative to their contralateral Normal controls. For thickness data, yellow to red indicates an increase in thickness and green to blue indicates a decrease in thickness in the EG eyes relative to their contralateral Normal controls.

Figure 6. Regional treatment differences in position (μm) and thickness (μm) of the lamina cribrosa, scleral flange, and peripapillary sclera by monkey (Delineator 1 (JCD) data)

The colored regions indicate statistically significant differences between Normal and EG eyes (P<0.05, ANOVA). Color intensity represents the magnitude of difference as illustrated in the color bars. For the position data, red indicates posterior deformation and green indicates anterior deformation in the EG eyes compared to their contralateral Normal controls. For the thickness data, yellow to red indicates an increase in thickness and green to blue indicates a decrease in thickness in the EG eyes compared to their contralateral Normal controls.

Figure 7. Neural canal landmarks and scleral flange architecture within the nasal region of the Normal eye of each monkey

Neural canal landmarks within a digital sagittal section of the nasal ONH for each Normal eye (left). Border Tissues of Elschnig (purple) and scleral flange (green) (right); note the relationship between regional neural canal obliqueness and scleral flange obliqueness in each eye. In general, a more oblique neural canal results in a more oblique scleral flange. While regional neural canal and scleral flange obliqueness are related, they may have separate clinical implications. The clinical importance of these anatomic features remains to be determined.

Figure 8. Schematic diagram of the macro-scale connective tissue architecture in the Normal and EG monkey eye

Normal lamina cribrosa, scleral flange and peripapillary position and thickness are schematically represented in the upper illustration. In EG (middle), the lamina cribrosa is thickened and posteriorly deformed, greatest centrally, with focal progression to the periphery (not shown). The scleral flange and peripapillary sclera are together displaced posteriorly, with most of the displacement in the sclera occurring closest to the neural canal. The posterior aspect of the neural canal is diffusely expanded in EG, while the anterior entrance to the neural canal is only focally expanded (not shown). Diagrams of Normal (black) and EG (grey) ONH architecture are overlaid in the bottom illustration.