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(Investigative Ophthalmology and Visual Science. 2007;48:4597-4607.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.07-0349
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3-D Histomorphometry of the Normal and Early Glaucomatous Monkey Optic Nerve Head: Lamina Cribrosa and Peripapillary Scleral Position and Thickness

Hongli Yang,1,2 J. Crawford Downs,1,2,3 Christopher Girkin,4 Lisandro Sakata,5 Anthony Bellezza,6 Hilary Thompson,7 and Claude F. Burgoyne1,3

1From the Department of Biomedical Engineering, Tulane University, New Orleans, Louisiana; the 3Devers Eye Institute, Legacy Health System, Portland, Oregon; the 4Department of Ophthalmology, University of Alabama, at Birmingham, Birmingham, Alabama; the 5Singapore Eye Research Institute (SERI), Singapore National Eye Center, Singapore; 6Third Eye Associates, Camden, New Jersey; and the 7School of Public Health, Louisiana State University Health Sciences Center, New Orleans, Louisiana.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
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-µm 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° 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.

We have proposed that intraocular pressure (IOP)–related optic nerve head (ONH) connective tissue damage is a defining feature of glaucomatous cupping.1 We have also demonstrated permanent deformation of the ONH connective tissues within serial histologic sections2 and two-dimensionally delineated, digital, three-dimensional (3-D) reconstructions of monkey ONHs with early experimental glaucoma (EG).3 In both the histologic2 and 3-D reconstruction studies,3 posterior deformation and thickening of the lamina cribrosa were present in the EG monkey ONHs.

The current report is the second in a series of five articles devoted to 3-D histomorphometric quantification of the ONH and peripapillary neural and connective tissues. In the first report,4 we introduced our method for 3-D delineation of 13 ONH and peripapillary scleral landmarks, tested the method’s reproducibility, and used it to describe enlargement and elongation of the neural canal within the EG eyes of three monkeys. In the current report, we concentrate on the position and thickness of the lamina cribrosa, scleral flange, and peripapillary sclera, to characterize the regional pattern of laminar and peripapillary scleral deformation and thickness alterations at the onset of confocal scanning laser tomography (CSLT)–detected ONH surface change in these same three EG eyes.

A series of reports have demonstrated the efficacy of CSLT characterization of ONH surface change in detecting the onset and progression of ONH damage in ocular hypertension.5 6 7 We propose that the onset of CSLT-detected ONH surface change in an ocular hypertensive monkey or human eye is a manifestation of subsurface phenomena that include deformation and thickness changes of the prelaminar neural tissues, lamina cribrosa, and peripapillary sclera. These phenomena are direct manifestations of neural and connective tissue pathophysiology that most likely precede surface change and/or axonal insult.

In the present study, three monkeys were given moderate, laser-induced IOP elevations in one eye and killed at the onset of CSLT-detected ONH surface change2 3 by perfusion fixation, with both eyes set to an IOP of 10 mm Hg by anterior chamber manometer. Both ONHs of each animal were then 3-D reconstructed, and evidence of permanent subsurface, structural change within the EG ONH of each animal was sought within 3-D histomorphometric characterizations of lamina cribrosa, scleral flange, and peripapillary scleral position and thickness.

Our study was designed to detect permanent connective tissue deformation by perfusion fixation with IOP set to 10 mm Hg bilaterally for a 30-minute period of equilibration before death. Detecting permanent deformation of the connective tissues of the lamina cribrosa, scleral flange, and peripapillary sclera in EG is important because it provides evidence that these tissues are damaged early in neuropathy. In addition, because these forms of subsurface structural change may precede clinically identifiable axonal insult, they may one day be detectable by next-generation clinical imaging devices8 9 10 11 12 13 14 15 16 and used to detect the onset of glaucomatous damage before visual field loss.

Three-dimensionally quantifying these deformations is important because it allows us to construct and refine finite element models of IOP-related neural and connective tissue stress and strain within these same ONHs.17 18 19 20 Determination of the structural factors that influence ONH biomechanical behavior should allow us to determine those factors that contribute most to individual susceptibility to glaucoma, and how remodeling of the ONH connective tissues in early glaucoma affects the biomechanical behavior of these tissues.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals
All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Three male cynomolgus monkeys, approximately 8 years of age, were used in the studies (see Table 1 of our previous report3 ).

ONH Surface Compliance Testing and Early Glaucoma
We have previously described our CSLT-based ONH surface compliance testing strategy.3 21 Briefly, both eyes of each monkey were imaged on three separate occasions while they were normal, then lasering of the trabecular meshwork was begun in one eye of each animal to elevate IOP. CSLT imaging was continued at 2-week intervals until the onset of significant permanent posterior deformation of the ONH surface in the lasered eye. See Table 1 and Figure 1 in our previous publication regarding the magnitude and duration of IOP elevation experienced by each animal.3 Monkeys 2 and 3 were killed at 3 weeks and monkey 1 at 6 weeks after CSLT detection of ONH surface change. In monkeys 1 and 2, IOP elevations were moderate, with only one measurement higher than 30 mm Hg. In monkey 3, elevated IOP was not detected.3

Monkey Euthanasia and Perfusion Fixation at Prescribed IOP
With each monkey under deep pentobarbital anesthesia, both eyes were cannulated with a 27-gauge needle, and the IOP was set to 10 mm Hg with the use of an adjustable saline reservoir. After a minimum of 30 minutes, the monkey was perfusion fixed via the descending aorta with 1 L of 4% buffered hypertonic paraformaldehyde solution followed by 6 L of 5% buffered hypertonic glutaraldehyde solution.4 After perfusion fixation, IOP was maintained for 1 hour, after which each eye was enucleated, all extraorbital tissues were removed, and the anterior chamber was removed 2 to 3 mm posterior to the limbus. By gross inspection, perfusion of all six eyes was excellent. The posterior scleral shells with intact ONH, choroid, and retina were placed in 5% glutaraldehyde solution for storage.

Generation of the Aligned Serial Section Images for Each ONH and 3-D ONH Reconstruction
These steps have been described in detail in our previous reports.3 4 Briefly, the ONH and peripapillary sclera were trephinated (6-mm diameter), pierced with alignment sutures, embedded in paraffin, and mounted on a microtome. The block’s surface was stained with a 1:1 (vol/vol) mixture of ponceau S and acid fuchsin and imaged at a resolution of 2.5 x 2.5 µm per pixel. The sections were serially cut at 3.0-µm thickness, and the staining and imaging process was repeated after each cut. Imaging began at the vitreoretinal interface and continued approximately 200 µm into the retrolaminar orbital optic nerve. Serial section images were aligned in the anterior-to-posterior direction and stacked at 3.0-µm intervals into a 3-D reconstruction of the ONH and peripapillary scleral connective tissues, which consist of approximately 1080 x 1520 x 400 voxels, each 2.5 x 2.5 x 3.0 µm in size.

3-D Delineation of ONH and Peripapillary Scleral Landmark Points
Our 3-D delineation technique has been described in detail in our previous report.4 Briefly, using custom software (based on the Visualization Toolkit; VTK, Clifton Park, NY), the 3-D ONH reconstruction is loaded into memory on a remote Linux server. The delineator assigns the approximate center of the neural canal as that reconstruction’s center of rotation, around which 40 7-voxel-thick, digital radial sagittal slices of the digital 3-D reconstruction are serially served at 4.5° intervals to the delineator’s workstation (Fig. 1) .


Figure 1
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  		FIGURE 1. 3-D delineation of ONH and peripapillary scleral landmark points within colorized, stacked-section, 3-D ONH reconstructions. Generation of 3-D ONH reconstruction from aligned serial section images for each ONH are explained in Figures 1 and 2 of our previous publication.2 (A) Sagittal histologic section through a representative normal monkey ONH showing the anatomy, and (B) the associated neural canal landmarks and surface landmarks: 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 (D), each 7 voxels thick, are served to the delineator at 4.5° intervals. (D) 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 image4 (E). (F) Representative 3-D point cloud showing all delineated points for a normal monkey ONH, relative to the prior serial section image (orbital optic nerve bottom, vitreous top).

 
Within each digital section (Fig. 1C) , the delineator marks seven landmark surfaces and six pairs of neural canal landmark points (one point on each side of the canal; Fig. 1D ). In this report, only the anterior and posterior surfaces of the peripapillary sclera and lamina cribrosa, the neural canal wall, and the neural canal landmark points (Fig. 1B) are used.

While marking in the sagittal-section view window (Fig. 1D) , the delineator is simultaneously viewing an adjacent window showing the cursor’s 3-D location within a digital transverse-section image (Fig. 1E) slaved to the sagittal-section view. The delineator also can scroll through the adjacent six 1-voxel-thick sagittal-section images to locate a section in which the landmark can be clearly identified. The 3-D Cartesian coordinates and category for each mark are saved, generating a 3-D point cloud that represents each of the marked structures (Fig. 1F) .

Clinical Alignment of the 3-D Reconstruction
A high-resolution reconstruction of the central retinal vessels and the neural canal landmark points4 was constructed and three dimensionally overlaid onto a clinical fundus photograph or CSLT image to align the 3-D marks accurately to the anatomic orientation (superior, inferior, nasal and temporal; see Fig. 2 in our previous publication4 ).


Figure 2
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  		FIGURE 2. ONH anatomy and parameter generation. (A) Representative horizontal digital sagittal section image from the 3-D reconstruction of the left eye of monkey 3 (left, temporal; right, nasal). (B) The definition and extent of the major structures quantified: 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 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 the BMO zero reference plane. (D) Lamina cribrosa (LC) and peripapillary sclera (PPS) thicknesses at each delineated anterior surface point are determined by fitting a continuous surface (white line) to all the delineated anterior surface points (D). (E, F) Thickness is defined as the distance along a normal vector to the anterior surface (E, green arrow) from each anterior delineated point to the posterior surface (F, black line). (G) The thickness of the scleral flange at each delineated anterior surface point (white dots) is defined as the distance between the neural canal boundary points (G, H, green line), along a vector parallel to the PSCO normal vector.

 
BMO Zero Reference Plane
For each 3-D ONH reconstruction, a least-squares ellipse was fitted to the 80 marks defining BMO, creating a BMO zero reference plane (Fig. 2B) . All measurements of the anterior laminar and peripapillary scleral positions were made relative to this plane.4

Lamina Cribrosa Position and Thickness
The definition and extent of the lamina cribrosa, scleral flange, and peripapillary sclera as structures are diagrammed and explained in Figure 2 . Independent measures of laminar position and thickness were made at each delineated anterior laminar surface point. At each point, the anterior laminar position was calculated as the distance from the BMO zero reference plane (Fig. 2C) , along a vector normal to that plane. To determine laminar thickness, the anterior and posterior laminar surface points were fitted to continuous anterior and posterior surfaces with a thin-plate B-spline (MatLab; The MathWorks, Natick, MA). The smoothed anterior laminar B-spline surface was used to generate a normal vector at each delineated point (Figs. 2D 2E) . Thickness of the lamina was calculated along each delineated point’s normal vector as the shortest distance to the posterior laminar surface (Fig. 2F) .

Scleral Flange Position and Thickness
The scleral flange (defined in Fig. 2B ) position was determined at each delineated anterior surface point, as described for the lamina. Because of its unique geometry, scleral flange thickness was defined as the distance from the anterior scleral flange surface to the neural canal boundary surface, measured at each delineated anterior surface point along a vector parallel to the posterior scleral canal opening (PSCO) normal vector (Figs. 2G 2H) .

Peripapillary Scleral Position and Thickness
Peripapillary scleral position and thickness were calculated at each delineated anterior scleral surface point, as described for the lamina cribrosa.

Continuous Plots of Laminar, Scleral Flange, and Peripapillary Scleral Position, and Thickness: Intra-animal Difference Map Generation
Continuous plots of position and thickness were generated for each eye by interpolating between the values measured at each delineated anterior surface point (Delaunay-based cubic interpolation in MatLab; The MathWorks). Data for the left eye were converted to right-eye configuration for direct comparison (Figs. 3 4) . Difference maps for each animal were generated by overlaying the BMO centroid for each eye and subtracting the normal data from the EG eye data (Figs. 3 4) .


Figure 3
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  		FIGURE 3. Continuous lamina cribrosa and peripapillary scleral position (in micrometers) maps for both eyes and continuous treatment difference maps for each monkey, colocalized with neural canal landmark points.4 Continuous laminar position maps of the normal and EG eye of each monkey (two leftmost 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 eyes (third and fourth columns; both in right-eye configuration). Continuous treatment difference maps (EG eye data minus normal eye data; EG-N) for laminar, scleral flange, and peripapillary scleral position (fifth and sixth columns) overlaid onto the CSLT image of the EG eye. CSLT images of the EG eye of each monkey (seventh column in the right-eye configuration) for reference.

 

Figure 4
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  		FIGURE 4. Continuous lamina cribrosa and peripapillary scleral thickness (in micrometers) maps for both eyes and continuous treatment difference maps for each monkey colocalized with neural canal landmark points.4 Continuous laminar thickness maps of the normal and EG eye of each monkey (two leftmost 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 (third and fourth columns; both in right-eye configuration). Continuous treatment difference maps (EG eye data minus normal eye data) for laminar, scleral flange, and peripapillary scleral thickness (fifth and sixth columns) overlaid onto the CSLT image of the EG eye. CSLT images of EG eye of each monkey (seventh column in right-eye configuration) for reference.

 
Parameter Regionalization
To perform statistical inference testing, we pooled position and thickness data at each delineated point on the anterior surface into laminar, scleral flange, and peripapillary scleral structures, as shown in Figure 5 , top. Within the lamina cribrosa, position and thickness data at each delineated point were assigned to one of 17 subregions, as depicted in Figure 5 , top left. Scleral flange and peripapillary scleral position data were pooled together because they were identically obtained and assigned to one of eight sub-regions as depicted in Figure 5 , top right. Scleral flange and peripapillary scleral data thickness data were considered separately, because their thicknesses were measured differently (described earlier) but were assigned to the same eight subregions as depicted in Figure 5 .


Figure 5
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  		FIGURE 5. Regionalization method and overall regional position and thickness treatment differences (EG minus normal; EG-N) within the lamina cribrosa, scleral flange, and peripapillary sclera. Within the lamina, position and thickness data were pooled into 17 regions according to the three radial regions (central; MP, middle periphery; P, periphery) and eight quadrants (S, superior; SN, superonasal; N, nasal; IN, inferonasal; I, inferior; IT, inferotemporal; T, temporal; ST, superotemporal; top left). Scleral flange and peripapillary sclera data were pooled into eight 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). Data (in micrometers) are the magnitude of change in the EG eye relative to its contralateral normal control (EG-N). The colored regions showed a 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 control eyes. 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 control eyes.

 
Interdelineator Variability and Intradelineator Reproducibility
Interdelineator variability was assessed in the data for all five delineators, in which both eyes of all three monkeys were delineated. Intradelineator reproducibility was assessed in the data for two of the five delineators, in which both eyes of monkey 3 were delineated two additional times at least 2 weeks apart. The effect of delineator (overall and within each monkey) and delineation day were assessed within separate factorial analyses of variance (ANOVA).

Statistical Analyses
Factorial ANOVAs were used to assess the effects of delineator, region, and treatment group (normal or EG) on the parameters lamina cribrosa position, lamina cribrosa thickness, scleral flange and peripapillary scleral position, scleral flange thickness, and peripapillary scleral thickness, both by treatment group and between the two eyes of each monkey.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Continuous Position Maps
Continuous maps of laminar, scleral flange, and peripapillary scleral position relative to the BMO zero reference plane are presented for each eye and for the difference between the EG and normal eye of each monkey in Figure 3 . It should be noted that in this description, and throughout this report, we use the histologic terms "anterior" and "posterior" to mean "inward" and "outward" movement of these structures relative to the vitreous cavity, as detected by clinical examination.

Continuous maps of the differences in laminar, scleral flange, and peripapillary scleral position between the EG and normal eyes of each monkey are presented in Figure 3 . The laminar position was very similar in the three normal monkey eyes, but the lamina was profoundly posteriorly displaced in all three EG eyes. The lamina was most posteriorly displaced centrally in all three EG eyes, with substantial extension of the posterior displacement into the inferonasal region in monkeys 1 and 3. Scleral flange and peripapillary scleral position relative to the BMO zero reference plane in the three EG eyes was anterior compared with its position in the contralateral normal eyes, which is the likely result of overall posterior deformation of the scleral flange and peripapillary sclera in all three EG eyes. In monkeys 1 and 2, there was additional posterior displacement of the nasal scleral flange and peripapillary sclera, which suggests an additional component of tilt (nasal down, temporal up) in these two eyes.

Continuous Thickness Maps
Continuous maps of laminar, scleral flange, and peripapillary scleral thickness are presented in Figure 4 for both the normal and EG eye of each monkey. Laminar thickness varied substantially among the three normal eyes, being thinnest inferiorly and superiorly in monkeys 1 and 3 and diffusely thicker in monkey 2. The lamina was substantially thicker in all three EG eyes. The scleral flange and peripapillary sclera were thinnest immediately adjacent to the neural canal, particularly nasally, in both eyes of all three monkeys.

Continuous maps of the differences in laminar and scleral flange and peripapillary scleral thickness between the EG and normal eye of each monkey are shown in Figure 4 . Whereas the lamina was focally thinner within the superior and inferior regions of monkey 2 and nasal and temporal regions of monkey 3, the lamina is diffusely thicker within most regions of all three EG eyes. Both the scleral flange and peripapillary sclera are mildly thinner within the EG eyes of all three monkeys, with each EG eye demonstrating variable focal nasal and inferonasal thickening.

Overall Position and Thickness of the Lamina Cribrosa, Scleral Flange, and Peripapillary Sclera by Treatment and Delineator
Table 1 reports the normal eye data and overall treatment difference of all five parameters for all five delineators. Overall, the lamina was more posterior (–90 to –106 µm) and thicker (30 to 62 µm) and the scleral flange and peripapillary sclera was more anterior (16 to 44 µm) in the EG eyes relative to BMO within the data for all five delineators (P < 0.05, ANOVA). Changes in scleral flange and peripapillary scleral thickness were of smaller magnitude and inconsistent direction and significance.


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  		TABLE 1. Position and Thickness of Lamina Cribrosa, Scleral Flange, and Peripapillary Sclera by Delineator

 
Overall Position and Thickness of the Lamina Cribrosa, Scleral Flange, and Peripapillary Sclera within Each Monkey
Table 2 reports the normal eye data and overall treatment difference of all five parameters within each monkey for delineator 1. Overall, the laminar position was significantly more posterior in all three EG eyes (–79 to –118 µm). The scleral flange and peripapillary sclera were significantly more anterior in two of the three EG eyes, with monkey 2 demonstrating no significant overall difference. It should be noted that because the BMO zero reference plane is tethered to the scleral flange and moves in concert with it, the relative anterior position of the peripheral peripapillary sclera in the EG eyes (Fig. 3) is the likely result of an overall posterior deformation of the ONH, scleral flange, and immediate peripapillary sclera in these eyes. Overall laminar thickness was significantly increased (23–61 µm) in the EG eyes of all 3 monkeys. Scleral flange thickness was not significantly different by treatment within any monkey, and peripapillary scleral thickness decreased only within the EG eye of monkey 2. These differences were similar within the data of all five delineators (data not shown).


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  		TABLE 2. Position and Thickness of Lamina Cribrosa, Scleral Flange, and Peripapillary Sclera by Monkey

 
Overall Regional Position and Thickness of the Lamina Cribrosa and Peripapillary Sclera by Treatment
The differences in regional position and thickness between the pooled normal and EG eyes are presented for delineator 1 in Figure 5 . Overall, most regions of the lamina cribrosa were significantly more posterior (–36 to –163 µm) in the EG eyes, with the highest deformations in the central and inferior midperipheral regions (P < 0.05, ANOVA). Overall, the scleral flange and peripapillary sclera were significantly more anterior (15–60 µm) in the superior, superotemporal, temporal, and inferotemporal subregions, and significantly more posterior in the superonasal and nasal subregions (–7 to –20 µm) in the EG eyes (P < 0.05, ANOVA). Again, the relative anterior position of the peripheral peripapillary sclera in the EG eyes is the likely result of an overall posterior deformation of the ONH, scleral flange, and immediate peripapillary sclera in these eyes.

The lamina was significantly thicker (21–61 µm) in all but 2 of 17 subregions in the EG eyes (P < 0.05, ANOVA), with the greatest thickness increases present in the inferior and inferonasal quadrants. In contrast, the scleral flange demonstrated no significant differences in subregional thickness, and the peripapillary sclera demonstrated significant thinning (up to 44 µm) in 3 of 16 regions in the EG eyes (P < 0.05, ANOVA). The reported treatment effects were similar within the data from all five delineators (data not shown).

Regional Position and Thickness of the Lamina Cribrosa and Peripapillary Sclera by Treatment within Each Monkey
Regional position and thickness differences between the normal and EG eye of each monkey are presented for delineator 1 in Figure 6 . The lamina was significantly more posterior within all three EG eyes (–118 to –199 µm centrally), with the largest posterior deformations in the central, inferior, and inferonasal subregions (P < 0.05, ANOVA). The scleral flange and peripapillary sclera again measured more anterior relative to the BMO reference plane within the temporal regions of each EG eye.


Figure 6
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  		FIGURE 6. Regional treatment differences in position (micrometers) and thickness (micrometers) 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 with their contralateral normal control eyes. 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 with their contralateral normal control eyes.

 
In general, the lamina was significantly thicker in all three EG eyes than in their contralateral normal control eyes, although the pattern and magnitude of glaucomatous thickening was different in each monkey (Fig. 6 ; P < 0.05, ANOVA). Lamina cribrosa thickness increased significantly in monkey 1, with the greatest changes occurring superiorly and inferiorly. Lamina cribrosa thickness increased nasally and temporally in the EG eye monkey 2 and thickened more diffusely in the EG eye of monkey 3, although the treatment effect was greatest inferiorly. There were no statistically significant regions of laminar thinning or differences in scleral flange thickness in any of the EG eyes. Statistically significant thinning of the peripheral peripapillary sclera was present in the EG eye of monkey 2, but was present only within a single region of monkeys 1 and 3.

Interdelineator Variability
All five thickness and position parameters are presented for all five delineators in Table 1 . Although the effect of delineator was significant for position and thickness by treatment (P < 0.001, ANOVA), all five delineators agreed in the direction and magnitude of glaucomatous change in all parameters except the thickness of the peripapillary sclera (Table 1) . The effect of delineator was significant (P < 0.001) for both position and thickness within all three monkeys considered individually; however, their effect compared to treatment was small for position and modest for thickness.

Intradelineator Repeatability
All five thickness and position parameters are presented for two delineators marking both eyes of monkey 3 on three different days in Table 3 . The effect of delineation day was statistically significant for all parameters (P < 0.05, ANOVA) except the thickness of the peripapillary scleral flange (P = 0.31). However, the magnitude and direction of intereye differences between the two eyes of monkey 3 are very similar within and between the three sessions of each delineator (Table 3) .


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  		TABLE 3. Position and Thickness of Lamina Cribrosa, Scleral Flange, and Peripapillary Sclera of Delineators 3 and 4 by Day

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Accurately characterizing ONH and peripapillary scleral connective tissue architecture has been the goal of a large and important body of literature, which was extensively reviewed in two previous reports.2 4 8 In a previous report, we introduced our method for high-resolution, digital, and 3-D reconstruction of the ONH tissues and used it to reconstruct the normal and EG ONHs of three monkeys with early experimental glaucoma in one eye.3 In the first article of this series,4 we introduced a new method for 3-D delineation and 3-D quantification of ONH and peripapillary scleral neural and connective tissue architecture and used the 3-D-delineated point clouds to characterize neural canal architecture changes quantitatively in early experimental glaucoma within the same reconstructions.

In the present study, we used a subset of the 3-D-delineated point clouds to generate continuous maps of lamina cribrosa, scleral flange, and peripapillary scleral position and thickness within both the normal and EG eye of each animal so as to characterize the principal inter- and intra-animal differences between the normal and EG eyes.

Our method of delineation of laminar and peripapillary scleral surfaces and the subsequent measurements of thickness and position of those structures exhibit low inter- and intradelineator variability (Tables 1 3) . Generally, the absolute position and thickness as well as the magnitude of the EG treatment effects, both overall and within each monkey, were similar among delineators. For all parameters, two delineators consistently measured similar magnitudes and detected similar treatment effects within delineation sessions performed in both eyes of one monkey on three different days. However, both interdelineator variability and intradelineator reproducibility were better for position than for thickness. Two factors may contribute to this difference. First, the biological graduation from the lamina to the retrolaminar septa can be difficult to discern, which makes the posterior laminar surface more difficult to delineate than the anterior surface. Second, treatment differences in position were larger than thickness, and therefore easier to detect.

The principal findings of this report are as follows. First, lamina cribrosa thickness varied substantially within the three normal eyes and was profoundly thicker in the EG eye of all three monkeys. Second, the lamina cribrosa was permanently posteriorly displaced in all three EG eyes. Although the lamina cribrosa’s position relative to the BMO zero reference plane was similar in the three normal eyes, the magnitude and area of the posterior laminar deformation were very different between the EG eyes. Third, scleral flange architecture and peripapillary scleral thickness varied regionally within each normal ONH, with the flange being thinnest nasally due to the oblique path of the nerve though the sclera (Fig. 7) . Interanimal differences in these anatomic features were substantial within normal eyes and were not profoundly changed in EG. Fourth, the scleral flange and immediate peripapillary sclera was posteriorly deformed overall and in two of the three EG eyes.


Figure 7
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  		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. Although 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. LC, lamina cribrosa.

 
Our findings suggest that permanent posterior deformation and thickening of the lamina cribrosa, and posterior deformation of the scleral flange and immediate peripapillary sclera are present at the earliest detectable stage of glaucomatous damage in the young adult monkey eye. Since each animal had IOP lowered to 10 mm Hg in both eyes for at least 30 minutes before perfusion fixation, and in a previous study, only minimal levels of anterior ONH surface movement were detected even 45 minutes after IOP lowering in glaucomatous eyes,22 these changes probably represent permanent, irreversible ONH and peripapillary scleral deformation.

The clinical variability of ONH and peripapillary scleral connective tissue thickness within normal eyes holds important implications for connective tissue and axonal susceptibility to glaucomatous damage. We have previously proposed that the central pathophysiology of glaucomatous cupping is primary or secondary damage to the ONH connective tissues, which results1 in the associated permanent deformation reported herein. From an engineering standpoint, ONH connective tissue susceptibility to damage and permanent deformation should be directly related to its structural stiffness, which is a combination of the structure’s geometry (tissue volume and morphology) and material properties (tissue stiffness). If all other components of structural stiffness were equal in two eyes, eyes with thicker lamina and thicker sclera should be more resistant to deformation and mechanical damage. Structurally stiffer eyes should therefore demonstrate a shallower form of glaucomatous cupping if damage and deformation do occur.

However, because the mechanisms of IOP-related axonal insult are likely to be multifactorial,1 23 the relationship between ONH connective tissue thickness and axonal susceptibility are likely to be complex. Because the principle insults to the axons within the lamina cribrosa have yet to be elucidated, the implications of differences in lamina cribrosa thickness are not certain. A thicker lamina should be of benefit in those regions in which axonal transport blockage occurs due to high translaminar tissue pressure gradients,24 as increased laminar thickness serves to expand the length of the axon over which the pressure difference between the vitreous cavity and cerebrospinal fluid (CSF) is dissipated.

Our finding of increased lamina cribrosa thickness within these three EG eyes confirms the histologic findings of our previous report2 in a new group of EG monkey eyes and has important implications. A considerable body of literature reports compression of the lamina cribrosa within moderately and severely damaged glaucomatous eyes.25 26 27 Quigley et al.26 reported compression of the lamina cribrosa in scanning electron micrographs of trypsin-digested human cadaveric eyes with an early (pre-Goldmann visual field loss) stage of glaucomatous damage.

The increase in laminar thickness that we report could be the result of several factors that include axonal swelling that is secondary to axonal transport blockage within the laminar trabeculae, edema of the neural and/or connective tissues, and remodeling and/or synthesis of laminar connective tissue in response to IOP-related damage. Several studies have shown that both retrograde and orthograde axonal transport are compromised by increases in IOP of less than 1 week’s duration, but the degree to which intralaminar axonal swelling and/or edema could contribute to laminar thickening is unknown. We have gained the ability to quantify the microarchitecture of the lamina cribrosa and will report the results in the fourth paper of this series. These data suggest a very substantial increase in the volume of lamina cribrosa connective tissue (total connective tissue voxels) in these same three EG eyes, compared with their normal fellow eyes. Molecular and biochemical characterizations of EG changes within the monkey lamina cribrosa are currently under way in our laboratory.

Early deformation of the lamina cribrosa in all three EG eyes were greatest centrally, with the areas of maximum deformation extending inferiorly and inferonasally in two of the three EG eyes. Laminar deformation was confined to the central region in monkey 2, which had the highest detected IOP exposure and the most robust connective tissue architecture (thickest lamina, least oblique canal, thickest scleral flange, and thickest peripapillary sclera). These results suggest that posterior deformation of the central lamina appears to be the earliest permanent deformation of these tissues in experimental EG.

In this report, we have introduced the concept of the scleral flange as the transition zone between the lamina cribrosa and peripapillary sclera and suggest that it should be treated as a unique component of the ONH connective tissue architecture. The scleral flange contains the penetrating branches of the circle of Zinn-Haller and underlies important clinical landmarks and behavior that are central to peripapillary scleral involvement in the neuropathy. Figure 7 illustrates scleral flange architecture within the normal eyes of all three monkeys and demonstrates the fundamental relationship between neural canal and scleral flange obliqueness at any given BMO clock-hour location.

The peripapillary sclera has been shown to be the site of substantial stress and strain concentrations within initial finite element models of the human18 20 and monkey ONH.17 To date, these models have not been detailed enough to incorporate scleral flange architecture into their estimates. Our results demonstrate that the scleral flange is susceptible to posterior deformation, which may significantly influence laminar deformation and strain. Regions of the flange that are thin or oblique should be most susceptible to posterior deformation.

Our overall findings suggest the following model for early permanent deformation of the ONH and peripapillary scleral connective tissues in monkey experimental glaucoma (Fig. 8) . First, 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 displaced together posteriorly. The outer aspect of the neural canal is diffusely expanded as a result of the posterior deformation of the peripapillary sclera, whereas the inner entrance to the neural canal is only focally expanded4 (not shown).


Figure 8
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  		FIGURE 8. Schematic diagram of the macroscale connective tissue architecture in the normal and EG monkey eye. Top: normal lamina cribrosa, scleral flange, and peripapillary sclera positions and thicknesses. Middle: in EG eyes, 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 displaced together 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,4 whereas the anterior entrance to the neural canal is only focally expanded (not shown). Bottom: overlays of diagrams of normal (black) and EG (gray) ONH architecture.

 
The pattern of axonal loss in early human glaucoma has been ascribed traditionally to the superior and inferior poles of the ONH.28 29 However, in these three EG eyes, early laminar, scleral flange, and peripapillary scleral deformations and thickness alterations are not consistently localized to the superior and inferior quadrants in the EG eyes (Figs. 5 6) . A definitive study colocalizing alterations in the ONH prelaminar neural tissue and lamina cribrosa with orbital optic nerve axonal loss in these three EG eyes will be the subject of a future report.

The limitations of our method of 3-D reconstruction have been discussed3 4 and include: (1) Anterior-to-posterior resolution is limited to 3 µm by the fact that the current stain penetrates approximately 2.5 µm into the embedded tissue block face; (2) the stain is applied manually to the block face, and so staining variation between section images can be substantial; (3) there are tissue shrinkage effects (both from fixation and embedding) associated with this technique, but because all eyes were treated identically, comparisons between the two eyes of each monkey and within treatment groups should be valid; and (4) we have not yet characterized physiologic, intereye differences for these parameters.

In addition, although all future monkey eye reconstructions will be aligned to clinical photos, clinical alignment of the 3-D reconstructions for these three monkeys were performed on CSLT images, which may not provide as clear an image of the clinical optic disc margin. However, in a subsequent alignment of both eyes of monkey 3 to clinical photographs (the only animal for which they were available)4 no substantial shift in alignment over that achieved with CSLT images was required.

Continuum and microfinite element models of each eye, which are derived from the 3-D landmark point clouds described earlier, are currently under construction. These models will characterize the magnitude and distribution of IOP-related stress and strain within the connective tissues of each 3-D-reconstructed ONH. This characterization will allow us to establish the relationships between IOP-related stress and strain, ONH anatomy, and the permanent EG connective tissue deformations reported herein. These relationships, once elucidated, will provide valuable insight into the biomechanical factors that contribute to individual ONH susceptibility to glaucomatous damage.

Finally, we propose that visualization of the laminar, scleral flange, and peripapillary scleral landmarks described in this report become an important goal of clinical glaucoma imaging. In assessing the susceptibility of an individual ONH to a given level of IOP, the 3-D architecture of these structures may be central to the estimation of ONH susceptibility and the clinical assignment of target IOP. In addition, clinically detectable deformation and thickness changes of these deep structures may precede surface-detected structural and functional change at all stages of the neuropathy, and serve as a marker for the development and progression of the disease.


   Acknowledgements
 
The authors thank Jonathon Grimm, Budd Hirons, Juan Reynaud, and Pris Zhou, without whom the development and implementation of this methodology would not have been possible.


   Footnotes
 
2 Contributed equally to the work and therefore should be considered equivalent authors. Back

Supported in part by National Eye Institute Grants R01EY011610 (CFB), K23EY13959 (CAG), and P30EY002377 (HWT; departmental Core Grant); grants from the American Health Assistance Foundation, Rockville, MD (CFB), The Whitaker Foundation, Arlington, VA (CFB), the Eyesight Foundation of Alabama (CAG), and the Sears Trust, Mexico, MO; and a Career Development Award (CFB) and a Physician-Scientist Award (CAG) from Research to Prevent Blindness, Inc.

Submitted for publication March 22, 2007; revised April 25, 2007; accepted July 16, 2007.

Disclosure: H. Yang, None; J.C. Downs, None; C. Girkin, None; L. Sakata, None; A. Bellezza, None; H. Thompson, None; C.F. Burgoyne, None

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.

Corresponding author: Claude F. Burgoyne, Optic Nerve Head Research Laboratory, Devers Eye Institute, 1225 NE 2nd Avenue, Box 3950, Portland, OR 97208-3950; cfburgoyne{at}deverseye.org.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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