Physiologic Intereye Differences in Monkey Optic Nerve Head Architecture and Their Relation to Changes in Early Experimental Glaucoma

Abstract

purpose. To characterize physiologic intereye differences (PIDs) in optic nerve head (ONH) architecture in six normal rhesus monkeys and compare them to intereye differences in three previously reported cynomolgus monkeys with early experimental glaucoma (EEG).

methods. Trephinated ONH and peripapillary sclera from both eyes of six normal monkeys were serial sectioned, 3-D reconstructed, 3-D delineated, and parameterized. For each normal animal and each parameter, PID was calculated (both overall and regionally) by converting all left eye data to the right eye configuration and subtracting the right eye value from that of the left eye. Physiologic intereye percent difference (PIPD) was calculated as the PID divided by the measurement mean of the two eyes. For each EEG monkey, intereye (EEG minus normal) differences and percent differences for each parameter overall and regionally were compared to the PID and PIPD maximums.

results. For all parameters the PID maximums were relatively small overall. Compared to overall PID maximums, overall intereye differences in EEG monkeys were greatest for laminar deformation and thickening, posterior scleral canal enlargement, cupping, and prelaminar neural tissue thickening. Compared with the regional PID maximums, the lamina cribrosa was posteriorly deformed centrally, inferiorly, inferonasally, and superiorly and was thickened centrally. The prelaminar neural tissues were thickened inferiorly, inferonasally, and superiorly.

conclusions. These data provide the first characterization of PID and PIPD maximums for ONH neural and connective tissue parameters in normal monkeys and serve to further clarify the location and character of early ONH change in experimental glaucoma. However, because of the species differences, the findings in EEG should be confirmed in EEG rhesus monkey eyes.

In the monkey model of unilateral experimental glaucoma (EG), chronic intraocular pressure (IOP) elevation is induced in one eye of an animal, and the contralateral eye is maintained as the normal control. ^{1 2 3 4 5 6 7 8 9 10} In our laboratory, animals are killed at clinically defined endpoints of early, moderate, or severe glaucomatous damage on the basis of longitudinal confocal scanning laser tomographic (CSLT) imaging of the study eye, and then in a subset of experiments perfusion fixed with both eyes set to 10 mm Hg by anterior chamber manometer. ^{11 12 13} In these experiments, postmortem histomorphometric ^{11 12 13} analyses are then performed in which the differences between the treated and control eyes are assumed to be due to the pathophysiology of glaucomatous damage. 

We recently introduced our method for three dimensional (3-D) delineation of 13 optic nerve head (ONH) and peripapillary sclera landmarks and used it to quantify enlargement and elongation of the neural canal at the onset of CSLT-detected ONH surface change in three monkeys with early experimental glaucoma (EEG) that occurred after moderate levels of IOP elevation in one eye. ¹¹ In a second report, ¹² we described our method for continuously mapping position and thickness of the lamina cribrosa, scleral flange, and peripapillary sclera and used it to report significant posterior deformation and thickening of the lamina cribrosa accompanied by mild posterior deformation of the scleral flange and peripapillary sclera in the same EEG eyes. In a third report, ¹³ we introduced our concept of prelaminar and laminar cupping, and used four new postmortem 3-D histomorphometric parameters to report that clinical cupping in early glaucoma is primarily due to fixed permanent posterior deformation of the ONH connective tissues and occurs in the setting of prelaminar tissues that are thickened. 

The purpose of the present study was to characterize the upper range of physiologic intereye differences (PIDs) and physiologic intereye percent differences (PIPDs) in ONH neural and connective tissue architecture in six normal monkeys and compare them with intereye differences in the three previously reported monkeys with EEG in one eye, ^{11 12 13} to clarify the most important optic nerve head changes in early experimental glaucoma. Once clarified by region and character and confirmed in a larger number of EEG eyes (manuscripts in preparation), these changes should become important imaging targets in patients with moderate levels of ocular hypertension. 

Materials and Methods

Animals

All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Three male and three female normal rhesus monkeys (age range, 2–10 years) were used. IOP measured under ketamine and xylazine anesthesia ranged from 8 to 14 mm Hg in both eyes of all six monkeys. The three cynomolgus EEG monkeys and their treatment have been extensively described in our four previous reports. ^{11 12 13 14}  

ONH Surface Compliance Testing and Early Glaucoma

We have described our confocal scanning laser tomography (CSLT)-based ONH surface compliance testing strategy (Laser Diagnostic Technologies; LDT, San Diego, CA) and how we use it to detect the onset of EEG. ^{14 15} Briefly, for each compliance test, the animals were initially anesthetized with a combination of 7 mg/kg ketamine and 1 mg/kg xylazine given intramuscularly, IOP was measured (Tono-Pen XL; Bio-Rad, Glendale, CA) three times in each eye, and a contact lens was placed to maintain the corneal surface for CSLT imaging. 

In all normal monkeys, both eyes were compliance tested on three to five separate occasions before euthanatization under pentobarbital anesthesia. In the early glaucoma monkeys, both eyes of each monkey were imaged on three separate occasions while normal, and 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 (the EEG eye), compared to the contralateral eye (the normal eye) using the CSLT-based parameter mean position of the disc. ¹⁴ See Table 1 and Figure 1 in our previous publication regarding the magnitude and duration of IOP elevation experienced by each animal. ¹⁴ Briefly EEG monkeys 2 and 3 were euthanatized 3 weeks and monkey 1 six weeks after CSLT detection of ONH surface change. In EEG monkey 1, detected IOP elevation occurred approximately 1 week before death with the maximum 26 mm Hg (average postlaser IOP 13 mm Hg, IOP not recorded on the day of death). In EEG monkey 2, detected IOP elevation occurred approximately 3 weeks before death, with the IOP maximum of 37 mm Hg recorded on the day of death (average post-laser IOP of 19 mm Hg). No IOP elevation was detected in EEG monkey 3 (average postlaser IOP 13 mm Hg, 18 mm Hg on the day of death). Axon counts in each EEG eye ranged from 16% to 30%, as previously reported. ¹³  

Monkey Euthanatization and Perfusion Fixation at Prescribed IOP

Under deep pentobarbital anesthesia, both eyes of each monkey were cannulated with a 27-gauge needle and the IOP was set to 10 mm Hg with 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. ¹⁴ After perfusion fixation, IOP was maintained for 1 hour, each eye was enucleated, all extraorbital tissues were trimmed, and the anterior chamber was removed 2 to 3 mm posterior to the limbus. By gross inspection, perfusion was excellent for all 12 eyes. The posterior scleral shell 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. ^{11 14} For this study, all 12 eyes were reconstructed in an enhanced protocol that increases axial and transverse image resolution. Briefly, the ONH and peripapillary sclera were trephinated (6 mm diameter), embedded in paraffin, and mounted on a microtome with the ONH facing out. The fresh block surface was stained with a 1:1 (vol/vol) mixture of ponceau S and acid fuchsin stains, imaged at a transverse resolution of 1.5 × 1.5 μm per pixel and serial sectioned at 1.5-μm increments. Imaging of the stained embedded tissue block surface started at the vitreoretinal interface and ended 200 μm into the retrolaminar orbital optic nerve. The position of the tissue block and camera were recorded by laser displacement sensors (Keyence Corp., Woodcliff Lake, NJ) at each serial section. All acquired images were then stacked and aligned using the laser position data into a digital 3-D reconstruction consisting of approximately 3400 (width) × 3200 (height) × 500 (depth) voxels (depth range, 342–665), each 1.5 × 1.5 × 1.5 μm in size. 

3-D Delineation of ONH and Peripapillary Scleral Landmark Points

Our 3-D delineation technique has been described in detail in previous reports. ^{11 12 13} Briefly, using custom software (based on the Visualization Toolkit [VTK], Clifton Park, NY), the 3-D ONH reconstruction was loaded and the delineator assigned the approximate center of the neural canal as the center of rotation, around which 40, 7-voxel-thick, digital radial sagittal slices of the digital 3-D reconstruction were serially served at 4.5° intervals to the delineator’s workstation (Fig. 1A) . 

Within each digital sagittal section, the delineator marked seven landmark surfaces—lamina cribrosa, sclera, neural boundary, Bruch’s membrane, internal limiting membrane, central retinal vessels, and subarachnoid space—and six pairs of neural canal landmark points: Bruch’s membrane opening (BMO), the anterior scleral canal opening (ASCO), the anterior laminar insertion (ALI), the posterior laminar insertion (PLI), the posterior scleral canal opening (PSCO), and the anteriormost aspect of the subarachnoid space (ASAS) (Fig. 1B) . 

While marking in the sagittal section view window, the delineator simultaneously viewed a slaved window showing the cursor’s 3-D location within a digital transverse section image (Fig. 1C) . The 3-D Cartesian coordinates and category number for each mark were saved, generating a 3-D point cloud that represented each of the marked structures (Fig. 1D) . 

Clinical Alignment of the 3-D Reconstruction

For each ONH, a high-resolution reconstruction of the central retinal vessels was performed and three dimensionally overlaid onto a predeath clinical photograph using the best match of the ONH and retinal vessels. Once preliminarily aligned (using the vessels only) the vessels and BMO points were covisualized to assess the relationship of the clinically visible optic disc margin to the delineated BMO points. A final 3-D adjustment was then performed to best match BMO to the disc margin while maintaining best vessel alignment. 

BMO Zero Reference Plane

For each 3-D ONH reconstruction, a least-squares ellipse was fit to the 80 marks defining BMO, creating a BMO zero reference plane. ¹¹ The centroid of the BMO ellipse established the center point for all measurements. All quantification of neural canal offset, depth, anterior laminar/scleral position, post-BMO cup volume, and post-BMO total prelaminar volume were made relative to this plane. 

Parameterization

The definitions and calculation methods of these parameters have been described in detail in our previous reports. ^{11 12 13} In this report, we have included overall and regional quantification of neural canal offset and depth (Fig. 1E) , lamina cribrosa position (Fig. 1F)and thickness (Fig. 1G) , scleral flange thickness (Fig. 1H) , peripapillary scleral position and thickness, prelaminar tissue thickness (Fig. 1K)and volume (Fig. 1J) , post-BMO cup volume (Fig. 1L) , and post-BMO total prelaminar volume (Fig. 1I) . Scleral position and thickness were calculated in a similar way as lamina cribrosa position and thickness. 

Parameter Regionalization and Difference Map Generation

Neural canal landmark offset and depth data were pooled for the eight clinical regions demonstrated in Figures 2A and 2B , respectively. The S, N, I, and T (superior, nasal, inferior, temporal) regions contain all marks within 60° of the ONH centered about the S–I and N–T clinical axes, and the SN, IN, IT, and ST regions contain all marks in 30° radial sections of the ONH centered about the SN–IT and IN–ST axes, as shown in Figure 2 . 

Lamina cribrosa position and thickness, prelaminar tissue thickness data were assigned to 17 subregions, as shown in Figure 2D . Scleral position data were pooled into 16 subregions as shown in Figure 2E . Scleral thickness data were pooled into the same 24 subregions, but also include flange thickness (the innermost 8 subregions) as shown in Figure 2F . 

Post-BMO cup volume, post-BMO total prelaminar volume, and prelaminar tissue volume were divided into 24, 15° radial regions by using the BMO centroid as the center and BMO normal sections as cutting planes as shown in Figure 2C . 

PID (PIPD) Range and Maximum in the Six Normal Monkeys

Overall and regional values were calculated for each parameter in each eye. For each animal, PID was calculated for each parameter (both overall and regionally) by converting all left eye data to right eye configuration, subtracting the right eye value from the left eye value and taking the absolute value. PIPD was calculated for each parameter as the PID divided by the absolute mean of the measurements of the two eyes. For all six animals, the measurement mean (both overall and regionally) for each parameter was calculated as the mean value of all six right eyes. The PID range was the range of PID values and the PID maximum was the largest PID (the upper range) among the six animals. The PIPD range was the range of PIPD values and the PIPD maximum was the largest PIPD (the upper range) among the six animals. 

EEG Animal Intereye Differences and Comparison to the PID and PIPD Maximum

EEG minus normal eye differences and percent differences (the intereye difference divided by the normal eye value) for each parameter overall and regionally were calculated for each previously reported EEG monkey and compared to the PID/PIPD maximums established by the six normal animals. Parameter change for each EEG animal required the intereye difference and percent difference (overall or regionally) to exceed the PID and PIPD maximum for that parameter. 

Results

Descriptive data for the six normal rhesus monkeys and the histomorphometric optic disc size for each eye are reported in Table 1 . Descriptive data for the three previously reported cynomolgus EEG monkeys is contained in our previous reports. ¹⁴  

Optic Disc Size

BMO vertical length ranged from 1355 to 1662 μm and horizontal length ranged from 959 to 1193 μm. The PID maximum for BMO vertical length was 40 μm and the PID maximum for BMO horizontal length was 25 μm. Disc area ranged from 1.034 to 1.539 mm², whereas the PID maximum was 0.036 mm². 

Qualitative Inspection of Quantitative 3-D Neural Canal Size and Shape Data by Monkey

The size and shape of the clinical disc as defined by BMO and the neural canal landmark points of both eyes of each monkey were overlaid and presented in right eye configuration in Supplementary Figure S1. For all six animals, the two eyes demonstrated remarkably similar neural canal size, shape, enlargement (the degree to which the PSCO is bigger than the BMO) and obliqueness (the degree to which the PSCO is off center relative to BMO). However, there were clear differences in the size and shape of the anteriormost aspect of the subarachnoid space in three of the monkeys, especially in the inferior and inferior–nasal regions. 

Qualitatively, BMO in the two eyes of each monkey was almost identical (Supplementary Fig. S1) and in each eye BMO closely colocalized to the clinically visible optic disc margin when the retinal and ONH vessels were used to maximize the colocalization (Supplementary Figs. S2 and S3). 

Overall Measurement Mean and Range of Each Parameter

The measurement mean and range (for six right eyes of six animals) for each parameter are listed in Table 2 . 

Overall PID and PIPD Maximum for Each Parameter

For all six animals considered together, the PID and PIPD maximum for each parameter are listed in Table 2 . The PID maximum for neural canal offset and depth parameters were as little as 9 μm for BMO offset to 27 μm for PSCO depth. The PID maximum for prelaminar, laminar, and scleral position and thickness data were also small, ranging from 5 μm for scleral flange thickness to 21 μm for peripapillary scleral position. The PID maximum for all volumetric parameters varied from 0.0056 mm³ for post-BMO cup volume to 0.0440 mm³ for post-BMO total prelaminar volume. 

Regional Measurement Means

The measurement mean (for six right eyes of six animals) and the PID and PIPD maximum (for six animals) are reported for all regional parameters in Figures 3to 9 . 

Regional Neural Canal Landmark Offset and Depth Measurement Means and PID Maximum

The regional PID maximum for the neural canal landmark offset varied from 12 μm in the inferior regions of BMO to 109 μm in the inferonasal region of the ASAS (Fig. 3) . The regional PID maximum for neural canal landmark depth varied from 7 μm in the superior region of BMO to 65 μm in the nasal region of the ASAS. Neural canal landmark offset and depth PID maximums were lowest in the inner canal landmarks (BMO and ASCO) and highest in the outer canal landmarks (the PSCO and ASAS). 

Regional Lamina Cribrosa Position and Thickness Measurement Means and PID Maximums

The PID maximum varied from 7 to 42 μm for laminar position and 9 to 36 μm for laminar thickness (Fig. 4) . In general, regional PID maximums for laminar position were greatest centrally and inferonasally, whereas laminar thickness PID maximums were greatest in the peripheral regions. 

Regional Post-BMO Total Prelaminar Volume Measurement Means and PID Maximums

The regional PID maximums for post-BMO total prelaminar volume (a volumetric parameter that incorporates features of, and is related to, the laminar position and neural canal offset parameters outlined earlier) ranged from 0.0012 to 0.0038 mm³ and was greatest superiorly and inferonasally (Fig. 5) . 

Regional Peripapillary Scleral Position and Thickness Measurement Means and PID Maximums

The regional PID maximums for peripapillary scleral position ranged from 17 to 64 μm and were greatest peripherally, superiorly, inferiorly, and nasally (Fig. 6) . The PID maximums for scleral flange and peripapillary scleral thickness ranged from 16 to 57 μm and were greatest in the inferior nasal periphery. 

Regional Prelaminar Tissue Thickness and Volume Measurement Means and PID Maximums

The regional PID maximums for prelaminar tissue thickness ranged from 23 to 62 μm and were greatest inferiorly and superotemporally (Figs. 7 8) . The PID maximums for prelaminar tissue volume varied from 0.0011 mm³ to 0.0050 mm³ and were greatest superonasally and inferiorly. 

Regional Post-BMO Cup Volume Measurement Means and PID Maximums

Regional PID maximums for post-BMO cup volume varied from 0.0001 to 0.0013 mm³ and were greatest superonasally and nasally (Fig. 9) . 

Comparison of Overall PID and PIPD Maximums to Overall Intereye Differences in the Three Previously Reported EEG Animals

Data to make these comparisons are reported in Table 3 . Whereas overall laminar thickness differences exceeded the PID maximum in all three EEG monkeys, their percent differences exceeded the PIPD maximum in only monkeys 1 and 3 (equaling but not exceeding the PIPD maximum in monkey 2). EEG to normal eye differences exceeded the overall PID and PIPD maximums in all three EEG monkeys for parameters related to laminar deformation, posterior scleral canal enlargement, cupping, and prelaminar neural tissue thickening. 

Comparison of Regional PID and PIPD Maximums to Regional Intereye Differences in the Three Previously Reported EEG Animals

Data to make these comparisons are reported in Figure 10 . Compared to the regional PID maximums, whereas regional intereye differences in each of the three EEG monkeys were substantial for parameters related to laminar thickening and posterior scleral canal enlargement when considered individually (data not shown), ^{11 12 13} only inferior, inferonasal, and superior intereye differences for laminar deformation, cupping, and prelaminar tissue thickening and central laminar deformation and thickening were present in all three EEG monkeys, considered together. 

Discussion

The purpose of the present study was to characterize PIDs in ONH neural and connective tissue architecture in six normal monkeys and compare them to intereye differences in three previously reported monkeys with early experimental glaucoma in one eye. ^{11 12 13} The principal findings of this report are as follows. First, for all parameters considered, the PID maximums in these six normal animals were relatively small. EEG compared with normal eye differences in all three previously reported EEG monkeys consistently and substantially exceeded these overall PID and PIPD maximums for parameters related to laminar deformation, posterior scleral canal enlargement, cupping, and prelaminar neural tissue thickening. Second, regional PID and PIPD maximums were greater than the overall PID and PIPD maximums for most parameters. Only parameters related to laminar deformation and thickening in the central region and laminar deformation and prelaminar neural tissue thickening in the inferior, inferonasal, and superior regions exceeded these maximums in all three EEG animals. 

That our overall PID and PIPD maximum data focuses attention on the parameters related to laminar deformation, posterior scleral canal enlargement, cupping, and prelaminar neural tissue thickening in EEG is important for two reasons. First, it establishes that at least for these three EEG monkeys relative to the six bilaterally normal monkeys, the model and our methods were sensitive enough to detect similar forms of early damage in all three EEG eyes. Second, it suggests that these processes may not just be the most detectable in our model/method system, but rather that they may be the most consistent early processes in experimental glaucomatous damage to the monkey ONH that occurs at moderate levels of IOP elevation. 

Our regional PID maximums create a more conservative benchmark for the detection of early glaucomatous damage and further clarify that in these three EEG animals the earliest changes were laminar deformation and thickening in the central region and laminar deformation and prelaminar neural tissue thickening in the inferior, inferonasal, and superior regions. Although the human and monkey optic nerve heads may fundamentally differ in their clinical behavior, we propose that these changes, (now clarified by region and character), suggest important new imaging targets for patients with moderate levels of ocular hypertension. A study assessing these findings in a larger group of EEG monkeys is nearing completion and will be the subject of a future report. 

Apart from our principal findings, several additional points bear discussing. First, while we report both the PID and PIPD maximums for each overall and regional parameter, we emphasize the PID maximum without extensively discussing it as a percentage difference (PIPD). We believe that the PID value alone is most relevant to the monkey experimental glaucoma model for cross-sectional detection of change in one eye compared with its contralateral normal control. Whereas PID estimates show the variability inherent in our method, PID data are not variability data alone but rather are estimates of the biological difference between the two normal eyes of an animal. Because our method achieves 1.5-μm voxel resolution, its accuracy is adequate to detect that the intereye differences among bilaterally normal eyes are different from the intereye differences of monkeys with experimental glaucoma in one eye. As such, we propose that requiring that the intereye difference in a given parameter exceed the upper range of its PID is a valid benchmark for change detection in the early glaucomatous eye of an animal, whether it is 0.1% or 50% of the measurement value in the contralateral normal eye. 

However, we have included PIPD data (and used it as a criteria for change that is likely to be important), because in so doing, the data we report may be more applicable to similar measurements made with a clinical instrument in which the exact scale of the measurement cannot be certain. In the case of laminar position as measured by clinical Spectral Domain Optical Coherence Tomography (SD-OCT) in both eyes of an animal, the PIPD maximum for overall or regional laminar position conservatively estimates the percentage difference (between the treated and contralateral normal eye of a study animal) required to exceed the highest percent difference that occurred among these six pairs of bilaterally normal eyes. 

Although we report measurement means and their range for each parameter, we do not extensively discuss their significance, as these data will soon be supplemented by the report of measurement means, 95% CI, and upper and lower range for each parameter in 41 normal eyes of 41 monkeys, ranging from 2 to 31 years of age, all perfusion fixed at 10 mm Hg IOP. These data will form the most comprehensive characterization of normal monkey ONH neural and connective tissue architecture to date and provide the source dimensions for parameterized finite element models, which will characterize the most important determinants of IOP-related stress and strain at all levels of IOP. ^{16 17 18 19 20}  

Our report should be considered in the context of the following limitations. First, the true range of physiologic intereye variation would be best characterized in hundreds of pairs of normal eyes and include groups designed to assess the effects of species, age, and asymmetries in ocular dimensions and refractive errors. Although we did not preselect these animals, that we happened to study six rhesus monkeys in which the eyes of each animal were very similar does not mean that more marked intereye differences are not present in some or even most normal rhesus monkeys. We therefore view our report as preliminary and hope to gradually add to these data through additional postmortem 3-D histomorphometric reconstruction of dedicated bilaterally normal animals as well as clinical SD-OCT ONH reconstructions ²¹ of bilaterally normal monkeys before their inclusion in our longitudinal studies of experimental glaucoma. 

Because of circumstances beyond our control, we were forced to establish PID and PIPD maximums in rhesus monkeys that may not directly apply to the three cynomolgus EEG monkeys in our previous reports. ^{11 12 13} Although there may be differences in measurement means between the species, we believe that important species differences in PID and PIPD maximum are unlikely. By gross comparison, the right eye measurement ranges of all parameters for the six rhesus monkeys (Table 2)are very similar to the normal eye ranges of the three cynomolgus EEG monkeys (Table 3) . However, without having studied a large population of animals, the species difference between our normal and EEG groups add to the preliminary nature of our findings. 

The measurement means and PID values reported herein are not the actual physiologic dimensions of the living monkey before death, because of uncharacterized tissue shrinkage effects (from both fixation and embedding) associated with our 3-D histomorphometric technique. However, since both eyes of each animal were treated identically (i.e., at the same time and in the same dehydration solutions), comparisons between the two eyes of each monkey should still be valid. It is within this context that the PIPD maximum we report may have the greatest relevance to any future study in which a consistent but uncertain shrinkage or measurement artifact is present. 

It is possible that lowering IOP to 10 mm Hg 30 minutes before perfusion fixation induced the prelaminar tissue thickening seen in all three EEG eyes. However, we think this is unlikely for the following reasons. First, IOP was lowered to 10 mm Hg slowly (over 1–2 minutes), and second, in those eyes in which it was measured, IOP was not that high on the day of death: 37 mm Hg in the EEG eye of monkey 2, 18 mm Hg in the EEG eye of monkey 3, and not measured in the EEG eye of monkey 1 (but the maximum IOP ever detected in that monkey was 26 mm Hg and IOP was 22 mm Hg 4 days before death). Although it is possible that the change from 37 to 10 mm Hg caused the prelaminar thickening in EEG monkey 2, we doubt that the IOP change in EEG monkeys 1 and 3 accounts for this finding. It should also be noted that when we discussed these same data in our previous publication ¹³ we erroneously reported which animal had no IOP data on the day of death (monkey 3 instead of monkey 1) and the highest IOP recorded (32 mm Hg instead of 37 mm Hg). However, our interpretation of these data remains unchanged from that report. ¹³ We predict that longitudinal SD-OCT imaging of prelaminar tissue volume and thickness (once clinically available), will confirm this finding in hypertensive human and monkey eyes. 

Finally, previous studies have demonstrated that IOPs in ketamine-anesthetized normal monkeys (mean ± SD, 14.9 ± 2.1 mm Hg) ²² are similar to those in normal humans (mean ± SD, 15.5 ± 2.6 mm Hg). ²³ However, our IOP measurements in these normal animals were made after administration of a combination of intramuscular ketamine and xylazine, and the IOP in our animals (range, 8–14 mm Hg) are overall slightly lower than the mean IOP in monkeys. ²² There are two possible reasons for the lower IOP measurements: (1) the effects of ketamine can be time dependent ^{24 25 26} ; thus, IOP may have become more variable and lower than actual pressures in both the normal and glaucomatous monkey eye in our experimental settings; and (2) xylazine may have an additional pressure-lowering effect, which has been reported to significantly reduce IOP in rabbits, cats, and monkeys. ²⁷  

In conclusion, we have rigorously characterized the range of PID and PIPD maximums in ONH neural and connective tissue architecture in six normal rhesus monkeys. The PID and PIPD maximums of this report serve to clarify our previous studies in early experimental glaucoma and will now be used as reference values for a series of articles on intereye differences in ONH architecture in bilaterally normal monkeys as well as those with early, moderate, and severe EG, perfusion fixed with each eye at either identical or different levels of IOP. 

 Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2008.

 Supported in part by National Eye Institute Grant R01EY011610 (CFB); a grant from the American Health Assistance Foundation, Rockville, Maryland (CFB); a grant from The Whitaker Foundation, Arlington, Virginia (CFB); a Career Development Award (CFB); The Legacy Good Samaritan Foundation, Portland, Oregon; and the Sears Trust for Biomedical Research, Mexico, and Missouri.

 Submitted for publication June 20, 2008; revised August 17, 2008; accepted November 17, 2008.

 Disclosure: H. Yang, None; J.C. Downs, 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.

 

Figure 1.

 

3-D delineation within colorized, stacked-section, 3-D ONH reconstructions and parameters definitions. (A) A total of 40 serial digital radial sagittal slices, each 7 voxels thick, were served to the delineator at 4.5° intervals. (B) A representative digital sagittal slice showing all 13 marks, which were 3-D delineated by linked, simultaneous colocalization of the sagittal slice (shown) and the transverse section image through the delineated point (C). (D) Representative 3-D point cloud showing all delineated points for a normal monkey ONH, relative to the posterior serial section image (top, vitreous; bottom, orbital optic nerve). (E) BMO zero reference plane (red line) colocalized with the neural canal landmarks BMO (red), ASCO (dark blue), ALI (dark yellow, hidden behind the ASCO in dark blue), PLI (green), and PSCO, (pink), ASAS (light blue); the distance from the BMO centroid (offset) and distance from the BMO zero reference plane (depth) are noted for ASAS. (F) The laminar position (green arrow) is defined as the shortest distance from the delineated anterior laminar surface point (white dot) to the BMO zero reference plane. (G) Lamina cribrosa thickness at each delineated anterior surface point is determined by fitting a continuous surface (white line) to all anterior surface points and then measuring the distance along a normal vector of the anterior surface (green arrow) from each anterior delineated point to the posterior surface. (H) 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 (green line), along a vector parallel to the PSCO normal vector (blue arrow). (I) Post-BMO total prelaminar volume (light green), a measure of the laminar or connective tissue component of cupping, is the volume beneath the BMO zero reference plane (cyan), above the lamina cribrosa and within the neural canal wall; (J) prelaminar tissue volume (purple) is the volume above the lamina, inside the neural canal, and below the internal limiting membrane (ILM) within the cylinder defined by the BMO projection; and (F) prelaminar tissue thickness (purple with black arrows) is the distance along a normal vector from each delineated anterior laminar surface point (white dots) to the ILM surface (pink line). (J) Post-BMO cup volume (pink: a measure of the clinical cup) is the volume (of the clinical cup) beneath BMO zero reference plane but above the ILM.

Figure 2.

 

Parameter regionalization. Neural canal offset (A) and depth (B) data for each neural canal landmark were pooled for eight anatomic regions: S, SN, N, IN, I, IT, T, and ST. The S, N, I, and T regions contained all marks within 60° sections of the ONH centered about the S–I and N–T clinical axes, and the SN, IN, IT, and ST regions contained all marks in 30° radial sections of the ONH centered about the SN–IT and IN–ST axes. Concentric rings represent the different neural canal landmarks from its internal entrance BMO to its external exit PSCO, as shown in the superior region of (A) and (B). Neural canal depth measurements start with the ASCO rather than the BMO. (C) From the center of the BMO, 12 radial sections perpendicular to the BMO zero reference plane divide the volumetric parameters into 24, 15° radial regions. Regional volumes are projected onto the BMO zero reference plane, color coded by region, and overlaid onto a standard ellipse. (D) Within the lamina, position and thickness and prelaminar tissue thickness data were pooled into 17 regions according to the three radial regions (central; MP, middle periphery; P, periphery) and eight quadrants, as in (A) and (B). (E) Peripapillary sclera position data were pooled into 16 regions according to two radial regions (MP, the inner boundary starting from the ASCO ellipse [bold black line] to an ellipse 1.62 times the size of the ASCO ellipse; P, inner boundary starting from the outer boundary of the MP regions to an ellipse 2.34 times the size of the ASCO ellipse). (F) Peripapillary scleral thickness data were pooled into 24 regions according to three radial regions (F, flange thickness, covered area from ASCO to PSCO; MP, inner boundary starting from PSCO to an ellipse 1.62 times the size of the ASCO ellipse; P, periphery regions are the same as peripapillary scleral position periphery regions).

Table 1.

 

Animal and Eye Data

No.	ID	Weight (kg)	Age (y)	Sex	Eye	IOP, † (mm Hg)	Number of serial Section Images	Histomorphometric Optic Disc Size
																Optic Disc Dimension (μm)*							Optic Disc Area (mm2)
																Vertical, ‡	Vertical PID, § (PIPD %), ∥	Horizontal, ¶	Horizontal PID (PIPD %)	Area, #	Area PID (PIPD %)
1	99R1753	3	4	Male	Right	11	342	1636	26 (1.6)	1193	14 (1.2)	1.532	0.007 (0.4)
					Left	11	434	1662		1179		1.539
2	97R0793	5	6	Male	Right	14	491	1444	3 (0.2)	1060	17 (1.7)	1.201	0.023 (1.9)
					Left	14	456	1447		1077		1.224
3	23540	5.3	9	Female	Right	13	524	1520	10 (0.7)	1072	16 (1.4)	1.280	0.027 (2.1)
					Left	10	665	1530		1088		1.307
4	23533	3.68	8	Female	Right	8	607	1355	18 (1.3)	984	25 (2.6)	1.048	0.014 (1.3)
					Left	8	528	1373		959		1.034
5	23510	4.86	10	Female	Right	12	561	1439	40 (2.8)	1044	18 (1.7)	1.180	0.012 (1.0)
					Left	11	558	1479		1026		1.192
6	25341	5.4	2	Male	Right	13	484	1420	14 (1.0)	1076	21 (1.9)	1.199	0.036 (3.0)
					Left	12	478	1434		1097		1.235

* Optic disc dimension as determined by the clinically visible optic disc margin which is BMO in these monkey eyes (See Supplementary Figs. S2, S3).

† Mean IOP of all n = 3 to 5 baseline measurements under ketamine/xylazine anesthesia.

‡ Vertical length as determined by the major length of the BMO ellipse.

§ Physiologic intereye difference (PID) is the absolute difference between right and left eye measures.

∥ Physiologic intereye percent difference (PIPD) is the PID divided by the mean measurement magnitude of the two eyes.

¶ Horizontal length as determined by the minor length of the BMO ellipse.

# Disc area is determined by the area of the BMO ellipse.

Table 2.

 

Overall Measurements Means, PIDs, and PIPDs for all Parameters within Six Bilaterally Normal Rhesus Monkeys

	Measurement Mean*	Measurement Range	PID Range, †	PIPD Range (%), ‡
Optic Disk Size
Vertical length (μm)	1469	1355–1636	3–40	0.2–2.8
Horizontal length (μm)	1071	984–1193	14–25	1.2–2.6
Disk area (mm2)	1.240	1.048–1.532	0.007–0.036	0.4–3.0
Neural Canal Architecture
BMO offset (μm)	623	573–694	1–9	0.1–1.5
ASCO offset (μm)	716	652–778	5–16	0.7–2.1
ALI offset (μm)	720	650–784	5–16	0.7–2.2
PLI offset (μm)	808	753–848	2–18	0.3–2.1
PSCO offset (μm)	840	789–893	1–22	0.1–2.7
ASAS offset (μm)	936	892–992	2–18	0.2–2.1
ASCO depth (μm)	31	19–49	1–15	2.8–100.9
ALI depth (μm)	36	21–50	1–20	2.8–89.6
PLI depth (μm)	121	90–181	4–20	3.3–16.8
PSCO depth (μm)	143	93–196	4–27	3.1–17.1
ASAS depth (μm)	162	97–234	1–25	0.5–19.4
ONH Connective Tissue
Lamina cribrosa position (μm)	−84	−103–−68	6–16	6.1–21.1
Peripapillary scleral position, § (μm)	−11	−39–32	0–21	0–400
Lamina cribrosa thickness (μm)	104	81–136	1–16	0.7–18
Peripapillary scleral thickness, § (μm)	160	137–195	0–11	0–7.1
Sclera flange thickness (μm)	66	54–101	3–5	4.6–8.8
Mid-peripapillary scleral position, ∥ (μm)	−16	−38–14	2–18	5.4–180
Mid-peripapillary scleral thickness , ∥ (μm)	147	121–179	1–14	0.8–9.2
ONH Prelaminar Neural Tissue and Cupping
Prelaminar tissue thickness, § (μm)	160	95–193	1–15	0.5–8.9
Post-BMO cup volume, # (mm3)	0.0081	0.0030–0.0159	0.0018–0.0056	13.1–174.4
Post-BMO total prelaminar volume (mm3)	0.1358	0.1084–0.1870	0.0052–0.0440	3.7–28.4
Prelaminar tissue volume, § (mm3)	0.2255	0.1682–0.2543	0.0098–0.0334	3.8–18.1

*  Mean value of the six right eyes.

†  Physiologic intereye difference (PID) is the absolute difference between right and left eye measures.

‡  Physiologic intereye percent difference (PIPD) is the PID divided by the mean measurement magnitude of the two eyes.

§  Data for only five animal (animal 2 - animal 6) are available for this measurement.

∥  Overall measurements for the mid-periphery only (this is the same region as our previous three EEG monkeys ^{11 12 13} ).

#  Data for only four animals (2, 3, 5, 6) are available for this measurement.

Figure 3.

 

Regional neural canal landmark offset and depth measurement means, PID maximums, and PIPD (%) maximums. Concentric rings represent the different neural canal landmarks from its internal entrance (BMO) to its external exit (PSCO) as depicted in Figure 2(BMO, ASCO, ALI, PLI, PSCO, and ASAS, respectively). Depth measurements start with the ASCO rather than BMO, because the BMO points form the BMO zero reference plane for these measurements. Within each region, the measurement mean is the mean of the six right eye measurements in each animal and is gray-scale mapped in micrometers. The PID maximum is the upper range of the PID within each region for the six PID values from the six animals and is gray-scale mapped in micrometers. PIPD maximum is the upper range of the PIPD within each region (expressed as a percentage) for the six PID values from the six animals and is nested next to the PIPD value inside the bracket. All data are plotted in right eye configuration. See Figure 2for a detailed description of regionalization.

Figure 4.

 

Regional lamina cribrosa position and thickness measurement means, PID maximums, and PIPD (%) maximums. Measurement means are the mean of the six right eyes of each animal and are negative for laminar position (top left) because the position of the lamina cribrosa surface is below the BMO zero reference plane. Within each region, the measurement mean is the mean of the six right eye measurements in each animal and is gray-scale mapped in micrometers. PID and PIPD maximums and the plotting of the data are as described in Figure 3 . The laminar position and thickness data are pooled into 17 concentric regions as described in Figure 2 .

Figure 5.

 

Regional post-BMO total prelaminar volume (in cubic millimeters) measurement means, PID maximums, and PIPD (%) maximums. Post–BMO total prelaminar volume (Fig. 1I)is a volumetric parameter that attempts to capture both the posterior deformation of the lamina and the expansion of the neural canal that are central to a “glaucomatous” or “laminar” form of cupping (see Fig. 1for an explanation of these relationships). It incorporates features of (and is related to) the laminar position and neural canal offset parameters outlined in Figures 3 and 4 . Within each region, the measurement mean is the mean of the six right eyes of each animal and is gray-scale mapped in cubic micrometers on the left. The PID maximum as described in Figure 3 . The PIPD maximum is as described in Figure 3and is gray-scale mapped on the right. The 15° radial region maps (Fig. 2)are presented in right eye configuration.

Figure 6.

 

Regional peripapillary scleral position and thickness measurement means, PID maximums, and PIPD (%) maximums. Measurement means are the mean of the six right eyes of each animal and are negative or positive for peripapillary scleral position (top left) because its position is above or below the BMO zero reference plane depending on the region. PID and PIPD maximums are as described in Figure 3 . Peripapillary scleral position data are pooled into 16 concentric regions and scleral flange and peripapillary thickness data are pooled into 24 concentric regions, as described in Figure 2 . All data are plotted in right eye configuration.

Figure 7.

 

Regional prelaminar tissue thickness measurement means, PID maximums, and PIPD (%) maximums. Within each region, the measurement mean is the mean of the six right eye measurements for each animal and is gray-scale mapped in micrometers. PID and PIPD maximums are as described in Figure 3 .

Figure 8.

 

Regional prelaminar tissue volume (in cubic millimeters) measurement means, PID maximums, and PIPD (%) maximums. Within each region, the measurement mean is the mean of the six right eyes of each animal and is gray-scale mapped in cubic micrometers on the left. PID is as described in Figure 3and is gray-scale mapped in cubic micrometers in the middle. PIPD is as described in Figure 3and is gray-scale mapped on the right. The 15° radial region maps (Fig. 2)are presented in right eye configuration.

Figure 9.

 

Regional post-BMO cup volume (in cubic millimeters) measurement means, PID maximums, and PIPD (%) maximums. Post-BMO cup volume (Fig. 1L)is a measure of the clinical cup defined as the volume of space beneath the BMO zero reference plane but above the internal limiting membrane (ILM). Within each region, the measurement mean is the mean of the six right eyes of each animal and is gray-scale mapped in cubic micrometers on the left. PID is as described in Figure 3and is gray-scale mapped in cubic micrometers in the middle. PIPD is as described in Figure 3and is gray-scale mapped on the right. The 15° radial region maps (Fig. 2)are presented in right eye configuration.

Figure 11.

Figure 12.

Table 3.

 

Normal Eye Range and Treatment Eye Differences for Three Previously Reported Cynomolgus EEG monkeys ^{11 12 13} Compared with the PID (PIPD) Maximum

	Normal Eye Range of EEG Monkeys	EEG Monkey 1 ΔEEG (ΔEEG/N%)*	EEG Monkey 2 ΔEEG (ΔEEG/N%)	EEG Monkey 3 ΔEEG (ΔEEG/N%)	PID (PIPD%) Maximum, †
Optic Disk Size
Vertical length, ‡ (μm)	1337–1459	108 (8.1)	1 (0.1)	3 (0.2)	40 (2.8)
Horizontal length, ‡ (μm)	1017–1055	47 (4.6)	2 (0.2)	68 (6.5)	25 (2.6)
Disk area, ‡ (mm2)	1.068–1.209	0.140 (13.1)	0.001 (0.1)	0.081 (6.7)	0.036 (3)
Neural Canal Architecture
BMO offset (μm)	579–615	39, § (6.7)	2 (0.3)	21 (3.4)	9 (1.5)
ASCO offset (μm)	648–724	71, § (9.8)	11 (1.7)	1 (0.1)	16 (2.1)
ALI offset (μm)	649–736	70, § (9.6)	11 (1.7)	4 (0.5)	16 (2.2)
PLI offset (μm)	721–904	99, § (11.0)	39, § (5.4)	54, § (6.5)	18 (2.1)
PSCO offset (μm)	743–940	85, § (9.0)	19, § (2.6)	30, § (3.5)	22 (2.7)
ASAS offset (μm)	831–1038	98, § (9.4)	39, § (4.7)	74, § (7.5)	18 (2.1)
ASCO depth (μm)	22–53	7 (31.8)	4 (13.8)	2 (3.8)	15 (100.9)
ALI depth (μm)	22–64	7 (31.8)	18, § (47.4)	14, § (21.9)	20 (89.6)
PLI depth (μm)	86–166	−5 (5.8)	39, § (23.5)	14, § (9.7)	20 (16.8)
PSCO depth (μm)	104–181	−9 (8.7)	25, § (13.8)	7 (4.6)	27 (17.1)
ASAS depth (μm)	125–185	−25, § (20.0)	9 (4.9)	−36, § (24.7)	25 (19.4)
ONH Connective Tissue
Lamina cribrosa position (μm)	−111–−105	−118, § (115.7)	−97, § (87.4)	−79, § (75.2)	16 (21.1)
Lamina cribrosa thickness (μm)	83–128	61, § (59.2)	23, § (18.0)	26, § (31.3)	16 (18.0)
Sclera flange thickness (μm)	60–76	7 (10.1)	1 (1.3)	8 (13.3)	5 (8.8)
Mid-peripapillary sclera position (μm)	−14–45	21, § (46.7)	3 (21.4)	35, § (94.6)	18 (180)
Mid-peripapillary sclera thickness (μm)	113–136	−4 (2.94)	−32, § (25.4)	1 (0.9)	14 (9.2)
ONH Prelaminar Neural Tissue and Cupping
Prelaminar tissue thickness (μm)	95–110	82, § (74.5)	47, § (47)	58, § (61.1)	15 (8.9)
Post-BMO cup volume, ‡ (mm3)	0.010–0.054	0.127 (235.2)	0.058 (446.2)	0.090 (900)	0.0056 (174.4)
Post-BMO total prelaminar volume, ‡ (mm3)	0.159–0.231	0.308 (133.3)	0.122 (76.7)	0.191 (119.4)	0.0440 (28.4)
Prelaminar tissue volume, ‡ (mm3)	0.232–0.289	0.167 (62.3)	0.025 (10.8)	0.002 (0.692)	0.0334 (18.1)

*  The treatment difference (ΔEEG) using overall EEG eye data minus normal eye data in three EEG monkeys and also expressed as percent treatment difference (difference magnitude divided by the normal eye measurement magnitude). Neural canal architecture, ONH connective tissue, and prelaminar neural tissue. and cupping parameters data od the 3 EEG monkeys were adapted from previous reports. ^{11 12 13}

†  PID/PIPD maximum is the upper limit of the PID/PIPD range reported in Table 2for each parameter.

‡  ANOVA not applicable to these measurements in the 3 EEG monkeys.

§  EEG eye value was significant different (P < 0.05 ANOVA) from the contralateral normal eye.

  Both treatment difference magnitude and percent treatment difference exceeded the PID/PIPD maximums in the region.

  Only treatment difference magnitude exceeded the PID maximum in the region.

Figure 10.

 

Regional intereye differences within 3 previously reported EEG monkeys that exceeded the PID and PIPD maximums in six bilaterally normal monkeys. For each EEG animal, intereye (EEG minus normal) difference and percentage difference (intereye difference divided by the normal eye measurement value) for each parameter was calculated within the regions and compared to the regional PID and PIPD maximums reported in Figures 3to 9 . Dark gray: regions in which the regional intereye differences (magnitude) in all three EEG monkeys exceeded the regional PID and PIPD maximums.

Supplementary Materials

Supplementary Figure S1 - (JPG) Neural canal landmark offset data points for both eyes of each animal overlaid in right eye configuration. Neural canal landmarks points projected onto the BMO zero reference plane for OS and OD eyes (solid lines: OS eye data; dotted lines: OD eye data) in right eye configuration with coincident BMO centroids (black dot). Overall the neural canal structures were very similar in all the six animals except for obvious differences in the ASAS for monkey 1, 2 and 3. To view BMO points co-localized to clinical color photos of each eye, please see Supplementary Figure S2 and Supplementary Figure S3. 

Supplementary Figure S2 - (JPG) 

Clinical co-localization of the 3D histomorphometric reconstruction to pre-sacrifice clinical color photos for Monkeys 1, 2 and 3 demonstrated close co-localization of the 3-D histomorphometrically delineated BMO points (in dark red) to the clinically visible optic disc margin in most eyes, when retinal and ONH vessel (in pink) co-localization were maximized. 

Supplementary Figure S3 - (JPG) 

Clinical co-localization of the 3D histomorphometric reconstruction to pre-sacrifice clinical color photos for Monkeys 4, 5 and 6 demonstrate findings similar to Supplementary Figure S2, above. ONH vessels are in pink or gray. 

The authors thank Jonathon Grimm, Juan Reynaud, and Michael Roberts for assistance with software for volumetric and thickness quantification; Galen Williams for help with delineation; Wenxia Wang for help with animal testing; Hillary Thompson and Stuart Gardiner for statistical consultation; and Joanne Couchman for assistance with manuscript preparation and figures.