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1From the Department of Biomedical Engineering, Tulane University, New Orleans, Louisiana; and the 2LSU Eye Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana.
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Abstract |
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METHODS. Both eyes of four normal (two normal eyes) monkeys and four with early glaucoma (one eye with laser-induced IOP elevation, observed until the onset of CSLT-detected ONH surface change) were enucleated immediately after death and immersion fixed at IOP 0 mm Hg. In an additional four normal monkeys and five with early glaucoma, both eyes were cannulated, and IOP set to 10 mm Hg in one normal eye and either 30 or 45 mm Hg in the other (normal or early-glaucoma) eye. After 15 to 80 minutes of acute IOP elevation, these nine monkeys were perfusion-fixed. Within images of serial sagittal sections of the ONH tissues in all 17 monkeys, anterior lamina cribrosa position, laminar thickness, and scleral canal diameter were measured. For each parameter, differences between the two eyes of each monkey and between treatment groups were assessed by ANOVA.
RESULTS. Within the eyes of the eight monkeys with IOP 0 mm Hg, the lamina cribrosa was posteriorly displaced and thicker and the scleral canal was enlarged at Bruch’s membrane and at the anterior laminar insertion in the early-glaucoma eyes compared with the contralateral normal eyes (plastic deformation). Within the high-IOP normal eyes, the lamina cribrosa was posteriorly displaced compared with that in the low-IOP normal eyes, but there were no significant differences in laminar thickness or scleral canal diameter (normal compliance). Within the high-IOP early-glaucoma eyes, the lamina cribrosa was posteriorly displaced and thicker and the scleral canal enlarged, compared with both low-IOP normal eyes and high-IOP normal eyes (hypercompliant deformation). Differences in laminar position between the high-IOP early-glaucoma eyes and the contralateral low-IOP normal eyes (hypercompliant plus plastic deformation) were more than eight times greater than the differences between the high-IOP normal eyes and the contralateral low-IOP normal eyes (normal compliance).
CONCLUSIONS. Both plastic (permanent) and hypercompliant deformation of the lamina cribrosa and anterior scleral canal wall are present in young adult monkey eyes with early experimental glaucoma. These findings suggest that damage to the ONH connective tissues occurs early in the monkey model of experimental glaucoma.
From a biomechanical standpoint, although the pathophysiology of the axons is likely to be multifactorial and is central to the loss of vision in glaucoma, it is unlikely that axonal damage alone explains the classic posterior deformation and excavation of glaucomatous cupping. It is a central premise of our work that glaucomatous cupping is the predictable result of damage to the ONH connective tissue. In this context, "glaucomatous" describes the predictable appearance the ONH assumes when its load-bearing tissues are damaged (fail mechanically) and deform under the level of IOP-related stress and strain they experience at a given level of IOP. The principal goal of this study was to provide the first histologic evidence of damage to the ONH connective tissues early in experimental glaucoma.
Burgoyne et al.2 have reported the onset of fixed posterior deformation (FPD) and hypercompliance of the ONH surface within 4 to 8 weeks of chronic elevation of IOP in a monkey model of experimental glaucoma and have hypothesized that damage to the connective tissues of the lamina cribrosa and anterior scleral canal wall are responsible for these findings. Although there is a large body of literature that characterizes alterations in connective tissue in moderate and severe human3 4 5 and experimental5 6 7 glaucoma, the histology of damage to the ONH in early glaucoma has not, to our knowledge, been studied.
The purpose of the present study was to test two hypotheses: (1) that plastic (permanent) deformation of the lamina cribrosa and anterior scleral canal wall underlies the onset of confocal scanning laser tomography (CSLT)-detected ONH surface change in early experimental glaucoma and (2) that an increase in the acute, IOP-induced deformation of the lamina cribrosa and anterior scleral canal wall underlies the onset of ONH surface hypercompliance at this same early stage of the neuropathy.
The concepts of compliant, plastic, and hypercompliant deformations of the lamina cribrosa and scleral canal wall are similar to those for other soft tissues. The compliant component of deformation can be thought of as the portion of overall posterior movement caused by an increase in IOP that reverses completely when IOP is lowered. Plastic deformation refers to the residual permanent posterior displacement that remains after an acute IOP elevation is reversed and all recovery is complete. Hypercompliance occurs when a change in material properties causes a tissue to be less rigid and thus more deformable with the same amount of applied pressure.
Detecting plastic deformation of the lamina and scleral canal wall at the onset of CSLT-detected ONH surface change is important, because it strongly suggests that a primary insult to the ONH connective tissues occurs early in the pathophysiology of this neuropathy. Detecting acute, IOP-induced hypercompliant deformations of the ONH connective tissues is important, because hypercompliant (as opposed to plastic) deformations are separate evidence of damage to the connective tissue, and the determination of the magnitude of these deformations allows an estimate of the altered material properties of the connective tissue (for future finite element modeling8 ) at this early stage of glaucomatous damage.
Until now, the ability to characterize accurately the position and three-dimensional geometry of the lamina cribrosa within the scleral canal has been limited.9 10 11 In this study, we cut 16 immersion-fixed and 18 perfusion-fixed monkey optic nerve heads from four separate groups of monkeys into serial 4-µm sagittal sections and measured anterior laminar position and thickness and scleral canal diameter in digitized images of every sixth section, based on modifications of a technique first described by Yan et al.,10 to provide a rigorous study of the three-dimensional architecture of the lamina cribrosa and anterior scleral canal wall in the normal and glaucomatous monkey eye at various levels of IOP.
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Materials and Methods |
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In the four normal monkeys in which one eye was perfusion fixed at IOP 10 mm Hg (N10[N] eyes) and the other perfusion fixed at IOP 30 or 45 mm Hg (N30/45 eyes), overall differences between the two groups characterized normal laminar compliance due to acute elevations in IOP. In the five monkeys in which one eye was normal and perfusion fixed at IOP 10 mm Hg (N10[EG] eyes) and the other had early experimental glaucoma and was perfusion fixed at IOP 30 or 45 mm Hg (EG30/45 eyes), overall differences between the groups characterized the combination of plastic posterior deformation (permanent deformation due to early glaucoma) and hypercompliance of the lamina (increased laminar compliance in response to acute IOP elevation, also due to early glaucoma).
To separate plastic posterior deformation from hypercompliance, an experiment-wide analysis was performed of pooled data from the immersion- and perfusion-fixed eyes to characterize the magnitude of differences in ONH connective tissue geometry caused by (1) between-eye physiologic differences and (2) plastic posterior deformation, as well as (3) normal laminar compliance and (4) plastic posterior deformation combined with laminar hypercompliance. Within this combined analysis, subtracting the effect of plastic posterior deformation from the combined effect of plastic posterior deformation and hypercompliance allowed us to isolate the magnitude of the hypercompliance effect alone in these early-glaucoma eyes.
ONH Surface Compliance Testing
In all normal monkeys, both eyes were compliance tested on three separate occasions before death. In the early-glaucoma monkeys, both eyes were compliance tested on three separate occasions, and then one eye was given laser-induced experimental glaucoma. Compliance testing of both eyes continued at 2-week intervals until the onset of CSLT-detected ONH surface change (defined as either plastic posterior deformation of the ONH surface and/or ONH surface hypercompliance in the study eye), at which point each monkey was killed.
We have previously described our CSLT-based ONH surface compliance-testing strategy.12 Briefly, for each individual compliance test, a 27-gauge needle connected to a saline-filled manometer is inserted into the anterior chamber of each eye of a monkey and the IOP adjusted to 10 mm Hg (IOP-10). Six 15° CSLT (TopSS; Laser Diagnostics Technology [LDT], San Diego, CA) images were obtained after 10 (RE) and 20 (LE) minutes (early IOP-10 observation points) and 30 (RE) and 40 (LE) minutes (late IOP-10 observation points). IOP was then elevated to 30 mm Hg (IOP-30), and early and late images were obtained on the same schedule.
LDT TopSS CSLT image registration and alignment have been described in detail.12 ONH surface position within an individual CSLT image was characterized by the parameter, average position of the disc (APD), which was calculated as the average of all the elevation values within the disc margin. Using the 12 IOP-10 images, the parameter, mean position of the discBaseline (MPDBaseline) was calculated as the mean ± 95% confidence interval (CI) for the 12 APD values. Change from MPDBaseline was calculated as the mean ± 95% CI for the differences between the 12 IOP-10 images and the 12 IOP-30 images.
Early Experimental Glaucoma
Early experimental glaucoma was induced in one eye of designated early-glaucoma monkeys by lasering the trabecular meshwork every 2 weeks until an elevation in IOP was observed (Tables 1 2 ; Fig. 1 ). Both eyes of each monkey were then compliance tested every 2 weeks to detect the onset of either plastic posterior deformation of the ONH surface (a statistically significant decrease in MPDBaseline compared with three normal tests) and/or the onset of ONH surface hypercompliance (a statistically significant increase in change from MPDBaseline compared with the three normal tests) in the data from two consecutive compliance tests. However, our definitions of the onset of FPD and/or hypercompliance of the ONH surface were, by necessity, empiric, because the systems for rapid processing, statistical analysis, and graphic presentation of the data for each monkey were not in place at the time of postlaser compliance testing.
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Monkey Death and Fixation
For each immersion-fixed eye, the monkey was placed under ketamine-xylazine anesthesia and killed with an intravenous injection of sodium pentobarbital. Immediately thereafter, both eyes were enucleated and placed in normal saline. For each eye, all extraocular tissues were removed, and the optic nerve was trimmed approximately 1 mm posterior to the globe. The anterior chamber was removed at the limbus; two anterior-to-posterior incisions were made to the equator in the anterior sclera; and the globe, with retina and choroid intact, was gently placed over a fixed polyethylene ball sized to correspond to the internal diameter of the globe, with the optic nerve at the apex of the ball. The optic nerve head and peripapillary sclera were cut from the posterior scleral shell with a 6-mm trephine passed from outside the sclera toward the underlying polypropylene ball. The ONH specimens were immersed in buffered, hypertonic 5% glutaraldehyde solution, and the unfixed posterior scleral shells were processed for other studies.
For each perfusion-fixed eye, the monkey was placed under deep pentobarbital anesthesia, both eyes were cannulated with a 27-gauge needle, and the IOP was set to 10 mm Hg, using an adjustable saline reservoir. Fifteen to 80 minutes before perfusion-fixation, IOP was elevated to either 30 or 45 mm Hg in one eye of the normal monkeys or the glaucomatous eye of the experimental glaucoma monkeys. Each monkey was then perfusion-fixed through the descending aorta with 1 L of 4% buffered hypertonic paraformaldehyde solution followed by 6 L of 5% buffered hypertonic glutaraldehyde solution. After perfusion, IOP was maintained for 1 hour, after which each eye was enucleated, all extra-orbital tissues were removed, and the anterior chamber was removed 2 to 3 mm posterior to the limbus. By gross inspection, perfusion was excellent in all IOP 10 eyes. However, blood was variably present in the retinal arteries and veins, choroidal vasculature, and vortex veins of all the high-IOP eyes, both normal and early glaucoma. The posterior scleral shells with choroid and retina intact were placed in 4% paraformaldehyde solution for long-term storage. The ONH and peripapillary sclera of the normal IOP-10 eye of each monkey was later cut from the posterior scleral shell by passing a 6-mm trephine from the retina side out through the sclera.
Optic Nerve Neural Area Measurements in Perfusion-Fixed Monkey Eyes
Cross sections of the optic nerves from both eyes of all nine perfusion-fixed monkeys were stained with Van Gieson’s stain, oriented using superior and nasal cuts made at the time of tissue processing, and digitally imaged through a 3-charge coupled device (CCD) camera (HV-C20 3; Hitachi, Tokyo, Japan) mounted on a microscope (Optiphot-2; Nikon, Tokyo, Japan). Using a 10x objective lens, pixel resolution was approximately equal to 1.6 µm per pixel. A green filter (Wratten 61; Eastman Kodak, Rochester, NY) was used to enhance contrast between the neural and connective tissues. Images were binarized using image-editing software (Photoshop ver. 5.5; Adobe Systems, Inc., San Jose, CA) to isolate all connective tissue septa. The neural area was calculated in each eye with custom software.
ONH Specimen Preparation and Sectioning
Our method for serial sagittal sectioning is summarized as follows. One edge of each trephined specimen was trimmed for either horizontal or vertical sectioning. In all monkeys, the ONHs from both eyes were sectioned in the same direction to enable direct comparisons of scleral canal diameter and laminar insertion geometry between the two eyes of each monkey. ONH specimens from two of the four normal perfusion-fixed monkeys, three of the five perfusion-fixed early-glaucoma monkeys, and all eight of the monkeys in the immersion-fixation group were vertically sectioned. The remaining ONH specimens were sectioned horizontally. The specimens were dehydrated, infiltrated, and embedded in historesin (Technovit 7100; Kulzer, Wehrheim, Germany). Sagittal sections (4 µm thick) were cut and mounted on glass slides, stained with Van Gieson’s stain, and coverslipped.
Section Image Acquisition and Marking and Measurement Parameter Generation
For each ONH, the anterior scleral canal opening was defined as coincident with the opening in Bruch’s membrane. By this definition, the first and last histologic sections in which Bruch’s membrane was intact established the boundaries of the anterior scleral canal opening.
The generation of a composite image of every sixth serial sagittal section and the measurements made within each image are outlined in Figure 2 . Briefly, a digital color image of every sixth section was acquired under a microscope (Optiphot-2; Nikon) fitted with a 3-CCD color camera (HV-C20 3; Hitachi), along with a companion image of a slide-mounted micrometer scale to allow calibration of the exact pixel size. All images were generated at a resolution of approximately 2 µm per pixel. Because multiple images were needed to achieve the required resolution, individual overlapping images were spliced together with an image-processing program (Photoshop, ver. 5.5; Adobe Systems) to form a single composite image (Fig. 2) .
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All section images for a single ONH were marked by one of two operators (CJR or AJB), both of whom were masked to the treatment group of the image being marked. All section images for an ONH were available to the operator, and frequent study of sections before and after the section being marked was often necessary for best placement of the marks. After all section images for an ONH were initially marked, a single operator (AJB) reviewed each section image to ensure overall consistency of the marks.
Overall and Regional Anterior Laminar Position and Laminar Thickness
Within each section image, anterior laminar position and laminar thickness were measured at nine points, which, taken across all measured section images, created a grid of measurement points that covered the entire scleral canal opening (Fig. 3) . For all 34 ONH specimens, the grid of measurement points was superimposed onto a CSLT image of the ONH surface obtained before death. Each point was then assigned to one of five regions: central (centered on the central vascular tree), superior, inferior, nasal, and temporal (Figs. 3F 3G) . For each specimen, mean values for anterior laminar position and laminar thickness were calculated overall (for all points combined) and for each of the five regions.
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Measurement Parameter Reproducibility for Both Eyes of One Monkey
The 67 section images of the normal low-IOP eye and the 71 section images of the normal high-IOP eye of monkey 10 (Table 2) were marked on three separate occasions at least 2 weeks apart by the same operator (AJB). An analysis of variance (ANOVA) was performed to assess the effects of eye (treatment) and measurement day (operator reproducibility) on each parameter.
Statistical Analysis
A nested ANOVA was used to assess the effects of region (central, superior, inferior, nasal, and temporal) and treatment group—normal monkeys: normal eyes N0-RE, N0-LE, N10[N], and N30/45; and early- glaucoma monkeys: normal eyes N0 and N10[EG] and early-glaucoma eyes EG0 and EG30/45—on the parameters anterior laminar position and laminar thickness. A separate ANOVA evaluated the effects of region and treatment between the two eyes of each monkey.
Another ANOVA was used to assess the effects of treatment group on the parameter scleral canal diameter. In this analysis, the same treatment groups were again analyzed. An additional ANOVA was run to examine the effect of treatment on these parameters between the two eyes of each monkey.
From these analyses, we characterized the magnitude of differences between the following treatment groups: normal left and right IOP 0 mm Hg eyes (to show the range of physiologic variability), normal IOP 0 mm Hg and early-glaucoma IOP 0 mm Hg eyes (to show the range of glaucoma-induced plastic deformation), normal IOP 10 mm Hg and normal IOP 30/45 mm Hg eyes (to show the range of normal laminar compliance), and normal IOP 10 mm Hg and early-glaucoma IOP 30/45 mm Hg eyes (to show the range of plastic posterior deformation combined with laminar hypercompliance). To minimize the effect of intermonkey variability, comparisons between paired treatment groups were confined to individual sets of monkeys.
Comparisons between eyes, treatments, regions, and treatment-by-region combinations were made using protected t-tests between least-squares means in which the P values were adjusted for the number of comparisons made. P values reported are derived from these tests.13 In addition to the hypothesis tests performed with the ANOVAs, magnitudes of all measured parameters were assessed by computation of 95% CIs. Magnitudes of differences between two treatment groups were assessed by Tukey’s honestly significant difference method of differences between means (
= 0.05).
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Results |
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Experimental Glaucoma
In all nine early-glaucoma monkeys, IOP elevations were achieved within 5 to 28 weeks of lasering. In monkeys 5, 6, 7, 13, 14, and 15, maximum detected IOP was 30 mm Hg or less. In monkey 8, the maximum detected IOP reached 44 mm Hg; in monkey 16, 47 mm Hg; and in monkey 17, 33 mm Hg. In all nine monkeys, the duration of detected IOP elevation from onset to death ranged from 2 to 7 weeks (Tables 1 2) .
Although the data are somewhat variable, CSLT-detected plastic posterior deformation of the ONH surface was present in all nine early-glaucoma eyes at the time of death. In the case of at least three monkeys (monkeys 5, 16, and 17), the onset of plastic posterior deformation of the ONH surface preceded the onset of detected IOP elevation by at least 4 weeks. In the case of monkey 13, death occurred after a single postlaser imaging session in which both plastic posterior deformation and hypercompliance of the ONH surface were apparent. By inspection, the nine early-glaucoma monkeys were killed such that their glaucomatous eyes demonstrated a stage of experimental glaucoma characteristic of the period 3 to 8 weeks after the onset of CSLT-detected plastic posterior deformation for monkeys 5 to 8 and 13 to 15, and perhaps as long as 13 to 15 weeks for monkeys 16 and 17. Although the hypercompliance data are somewhat variable, at least five (monkeys 5, 8, 14, 15, and 17) and perhaps a sixth (monkey 13) of the nine early-glaucoma monkeys demonstrated ONH surface hypercompliance at the time of death. Postlaser IOP, MPDBaseline, and change from MPDBaseline data in the study eye and the contralateral normal eye are shown for four representative early-glaucoma monkeys in Figure 1 .
By inspection, between-eye differences in optic nerve neural area in the five perfusion-fixed early-glaucoma monkeys did not exceed the between-eye differences in optic nerve neural area in the four normal perfusion-fixed monkeys (Table 2) .
Reproducibility of Individual Eye Measurements for the Eyes of Monkey 10
The mean value of each measurement parameter for the three separate markings of the two eyes of monkey 10 are reported in Table 3 . The effect of measurement day was significant for both anterior laminar position (P = 0.0002) and laminar thickness (P < 0.0001). However, even in the face of the variability due to measurement day, statistically significant differences in anterior laminar position (P < 0.0001) were detected.
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Early-Glaucoma Monkeys.
In the early-glaucoma monkeys, marked plastic posterior displacement of the anterior lamina was seen in the immersion-fixed, IOP-0 early-glaucoma eyes, compared with the contralateral normal IOP-0 eyes (EG0, -255 ± 4 µm versus N0, 185 ± 4 µm) and the immersion-fixed IOP-0 eyes from the normal monkeys (EG0, -255 ± 4 µm versus N0-RE, 182 ± 4 µm and N0-LE, 173 ± 4 µm; P < 0.0001 for all comparisons). Deformation of the lamina cribrosa was greatest in the central region (mean central anterior laminar position: EG0, 378 ± 10 µm; N0, 267 ± 10 µm; N0-RE, 267 ± 12 µm; N0-LE, 251 ± 11 µm) and least in the superior region (mean superior anterior laminar position: EG0, 209 ± 8 µm; N0, 152 ± 8 µm; N0-RE, 150 ± 8 µm; N0-LE, 142 ± 8 µm).
In the perfusion-fixed early-glaucoma monkeys, the anterior lamina was profoundly displaced in the five IOP-30/45 early-glaucoma eyes compared with the contralateral IOP-10 normal eyes (EG30/45, 250 ± 3 µm versus N10[EG], 112 ± 3 µm; P < 0.0001). As in the immersion-fixed IOP-0 eyes, the greatest posterior displacement (approximately 200 µm) was seen in the central region. Displacements in the four peripheral regions were smaller and similar to one another in amount.
Thickness of the Anterior Lamina Cribrosa by Treatment Group
Lamina cribrosa thickness data are reported in Table 5 .
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Early-Glaucoma Monkeys.
Again, similar to laminar position, laminar thickness was significantly greater in both groups of early-glaucoma eyes, compared with the contralateral normal eyes (immersion fixation: EG0, 306 ± 2 µm versus N0, 251 ± 2 µm; perfusion-fixation: EG30/45, 241 ± 2 µm versus N10[EG], 182 ± 2 µm; P < 0.0001). However, the difference in thickness between the normal and early-glaucoma eyes immersion-fixed at IOP-0 (N0 versus EG0) was statistically indistinguishable from the difference in thickness between the normal eyes perfusion fixed at 10 mm Hg and the early-glaucoma eyes perfusion-fixed at 30/45 mm Hg (N10[EG] versus EG30/45).
Taken together, these data suggest that although an increase in laminar thickness was present at this early stage of experimental glaucoma, acute IOP elevations lasting 15 to 80 minutes had no detectable effect on laminar thickness in either the normal or early-glaucoma eyes.
Diameter of the Anterior Scleral Canal by Treatment Group
Anterior scleral canal diameter data are reported in Table 6 .
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Early-Glaucoma Monkeys.
Again, as with laminar position and thickness, scleral canal diameter was significantly increased in both the immersion-fixed and perfusion-fixed early-glaucoma eyes at both Bruch’s membrane and the anterior laminar insertion, compared with the contralateral normal eyes (P < 0.0001). In the immersion-fixed IOP-0 early-glaucoma eyes (vertically sectioned), average increases in diameter were 80 µm at Bruch’s membrane and 85 µm at the anterior laminar insertion. In the immersion-fixed IOP-30/45 early-glaucoma eyes, the respective increases were 143 µm at Bruch’s membrane and 196 µm at the anterior laminar insertion in the vertically sectioned eyes and 138 µm at Bruch’s membrane and 60 µm at the anterior laminar insertion in the horizontally sectioned eyes.
Experiment-Wide Magnitude of the Four Principal Treatment Effects
The 95% CIs for the following experiment-wide treatment effects are reported for each parameter in Table 7 : (1) intramonkey physiologic between-eye difference; (2) early glaucomatous plastic deformation; (3) normal compliance; (4) early glaucomatous plastic deformation plus early glaucomatous hypercompliance; and (5) early glaucomatous hypercompliance alone.
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Plastic posterior deformation of the lamina cribrosa (62–77 µm) and increases in laminar thickness (50–60 µm) were present in the immersion-fixed early-glaucoma eyes. In addition, a plastic expansion of the anterior scleral canal was present at both Bruch’s membrane (33–129 µm) and the anterior laminar insertion (31–139 µm) within the central vertical sections of these eyes.
Isolated hypercompliant effects are reported in the last column of Table 7 . In the perfusion-fixed monkeys, hypercompliant laminar deformation was present in the early-glaucoma eyes subjected to acute IOP elevation. After the effects of plastic deformation were subtracted, the amount of deformation of the anterior lamina occurring in response to acute IOP elevation in the EG30/45 eyes compared with that in the contralateral normal eyes (55–83 µm) was still more than four times that seen in the normal (N30/45) eyes compared with their contralateral eyes (10–23 µm). No hypercompliant effects on laminar thickness or anterior scleral canal diameter were detected when the plastic components of these parameters were removed.
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Discussion |
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The principal findings of this report are as follows. First, within the four normal perfusion-fixed monkeys, statistically significant deformations of the lamina cribrosa, but not the anterior scleral canal wall, followed acute IOP elevations from 10 to 30 or 45 mm Hg, but their magnitude did not exceed physiologic differences. Second, plastic posterior deformation of the lamina cribrosa, an increase in laminar thickness, and an enlargement of the anterior scleral canal opening were present within histologic sections of monkey eyes that were obtained 3 to 8 weeks after the onset of CSLT-detected ONH surface change in early experimental glaucoma. Third, hypercompliant deformations of the lamina cribrosa, but not the anterior scleral canal wall, were present at this same early stage of glaucomatous damage.
In this study, the magnitude of acute IOP-induced laminar deformation within the N30/45 eyes compared with the N10[N] contralateral normal eyes did not exceed the magnitude of physiologic between-eye differences. This finding suggests that the lamina cribrosa and scleral canal in normal young adult monkey eyes do not profoundly deform under acute, short-term IOP elevations and are thus able to withstand moderate, short-term fluctuations in IOP with only minimal changes in architecture.
Other studies have attempted to quantify laminar deformation after acute elevations in IOP. Levy and Crapps9 reported a 12-µm average posterior movement of the central lamina with acute IOP elevations from 10 to 25 mm Hg. Yan et al.10 found that increasing IOP from 5 to 50 mm Hg for 24 hours produced an average posterior movement of the central lamina of 79 µm. It may be that an IOP increase from 10 mm Hg to either 30 or 45 mm Hg for less than 1 hour, as performed in the present study, is adequately resisted by the connective tissues of the ONH. Laminar and scleral canal wall deformations are likely to be viscoelastic, meaning the tissues reach their full deformation (and recovery) only after a given period.15 16 Therefore, the lamina and scleral canal wall may deform if the period of IOP elevation is extended to hours or days. This may be one explanation for the larger laminar deflections measured by Yan et al.10 after an elevation of IOP to 50 mm Hg for 24 hours, compared with the deformations reported by us in the current study and others.9
Our study is the first to report that plastic deformations of the lamina cribrosa and the anterior scleral canal wall are present at the onset of CSLT-detected ONH surface change in early experimental glaucoma. Although others have measured laminar position in both normal and glaucomatous eyes,3 9 10 our study is important because differences between four early-glaucoma eyes and the contralateral normal eyes in a set of four early-glaucoma monkeys exceeded physiologic between-eye differences, as characterized in the two eyes of a separate group of four normal monkeys.
Both overall and within each of the four early-glaucoma eyes considered individually (data not shown), laminar deformation was greatest in the central region (surrounding the central vascular tree) and was relatively similar, but less, in the four peripheral regions. This finding was also described by Levy and Crapps,9 who reported maximum displacements centrally and minimum displacements near the canal wall.
Although it is likely that the central vasculature provides substantial support to the lamina and may prevent even further posterior laminar displacement, these data suggest that the vascular stalk is not rigid but moves in tandem with the lamina. Thus, at this early stage of damage, shear stresses within the peripheral laminar insertion sites, which may bow back with the peripapillary sclera12 but are still relatively immobile, exceed shear stresses at the insertions of the lamina into the central vessels, which deform posteriorly.
Laminar thickness in both the immersion-fixed and perfusion-fixed early-glaucoma eyes was significantly increased compared with their respective contralateral normal eyes. It is interesting to note that Quigley et al.3 reported compression of the lamina cribrosa without detectable posterior deformation in scanning electron micrographs of trypsin-digested human cadaver eyes with an early (pre-Goldmann visual field loss) stage of glaucomatous damage. Thus, these investigators observed neither the posterior deformation nor the thickening of the lamina seen in the young adult early-glaucoma monkey eyes in the current report.
The increase in laminar thickness in the monkey eyes in the present study is probably the result of axonal swelling that is secondary to axonal transport blockage within the laminar trabeculae. Several studies have shown that both retrograde and orthograde axonal transport are compromised by increases in IOP of less than 1 week’s duration.17 18 19 Even minimal elevation of IOP in monkey eyes has been shown to produce blockage of retrograde axoplasmic flow at the lamina within 24 hours of IOP elevation.20
Several points regarding the presence or absence of laminar deformation in early glaucomatous human versus monkey eyes should be mentioned. First, it may be that laminar deformation was, in fact, present in the earlier study of glaucomatous human cadaver eyes,3 but was hidden within uncharacterized physiologic differences between eyes, given that comparison with contralateral normal eyes was not possible. Second, the pattern of connective tissue damage in the elevated-IOP form of the neuropathy may be fundamentally different in the adult human eye, compared with the more compliant young adult monkey eye.
The scleral canal diameters of both the immersion-fixed and perfusion-fixed early-glaucoma eyes were significantly larger than those of the contralateral normal eyes, both at Bruch’s membrane and at the anterior laminar insertion. This finding in these nine eyes suggests that the connective tissues of the anterior scleral canal wall are permanently deformed at this early stage of glaucomatous damage. Although several researchers have reported that glaucomatous eyes measured clinically21 22 23 and after death3 24 do not have larger canal diameters, those studies could not compare early-glaucoma eyes with contralateral normal eyes.
Robin et al.25 reported enlargement of the scleral canal in the eyes of children with glaucoma. It may be that the elasticity of the scleral canal connective tissues determines whether the scleral canal enlarges during glaucomatous damage. If this is true, the scleral canal enlargement in young adult monkeys may occur because their connective tissues are more compliant (i.e., less rigid) than those of older human eyes.
Our study is the first to report that hypercompliant deformations of the lamina cribrosa, but not the anterior scleral canal wall, are present at this early stage of glaucomatous damage. Zeimer and Ogura26 have reported that human eyes with severe glaucoma are rigid, when compared with normal human eyes. That there is a hypercompliant phase of damage to the connective tissue in young adult monkey eyes may be a product of the age of the tissues and should be confirmed in old monkey eyes at this same stage of damage.
Other researchers have suggested that a change in connective tissue material properties may be associated with IOP-induced increases in stress and strain. Tezel et al.27 reported that proteolytic degradation accompanied ONH astrocyte migration in response to 48 hours of hydrostatic pressure elevation. Other laboratories have reported the presence of proteolytic enzymes in postmortem glaucomatous ONHs.28 29 Finally, changes in the synthesis of extracellular matrix in the glaucomatous ONH have been reported by a large group of investigators.4 6 30 31
Although connective tissue hypercompliance and plastic deformation are almost certainly related, they may be caused by different types of damage to the extracellular matrix. Damage to the elastin and collagen components of the lamina and scleral canal wall may manifest differently. A breakdown of elastin fibrils may lessen the ability of a tissue to contract after an increase in pressure is applied, leading to plastic deformation. Separately, damage to the collagen fibrils, which provide the main source of rigidity in these tissues, may cause the lamina and canal wall to expand more easily in response to increases in IOP, leading to tissue hyperelasticity.
Zeimer16 proposed a model in which the individual material properties of the scleral canal wall and lamina may be less important than a mismatch between the two tissue properties. In this model, a scleral canal that is less rigid than the lamina cribrosa would force the laminar beams to withstand a relatively high load, potentially leading to damage at their insertion into the canal wall. A breakdown of the laminar beams, in turn, would allow for easier expansion of the scleral canal at the level of the laminar insertion.
From a biomechanical engineering standpoint, mechanical failure of the ONH connective tissues should occur as either a primary or secondary event at all levels of IOP, after both IOP-related and non-IOP-related insults. Primary mechanical failure can be defined as occurring when unaltered connective tissues fail as a direct result of the IOP-related stress and strain they experience, whether the level of IOP is normal or elevated. Secondary failure can be defined as occurring when the load-bearing connective tissues have been weakened as a result of some other insult and then fail under the existing level of IOP-related stress and strain.
The laminar deformations we report may have been dampened by chronic and/or acute swelling within the laminar beams. Chronic swelling could have been caused by chronic early glaucomatous IOP elevation, whereas acute swelling could have resulted from ischemia that may have occurred during the 15 to 80 minutes of high IOP before death. At the time of death, the retinal and choroidal vasculature of the high-IOP eyes (both normal and early glaucomatous) were often partially filled with blood. Yet in all eyes, the tissues were well fixed by light microscopy. Poor perfusion of the ocular vessels caused us to decrease the time of acute IOP elevation from 80 minutes to 15 minutes in most of the high-IOP eyes. If fixation was less than ideal in the high-IOP eyes, our data may underestimate the magnitude of acute IOP-induced deformation of the ONH connective tissues in both the normal and early-glaucoma monkey eye.
Because the animals were killed 3 to 8 weeks after the onset of CSLT-detected ONH surface change, we may be characterizing a more advanced, rather than a very early stage of glaucomatous damage. However, differences in optic nerve neural area between the early-glaucoma eyes and the normal eyes of the five perfusion-fixed early-glaucoma monkeys were not significantly larger than differences between the two eyes of the four perfusion-fixed normal monkeys (Table 2) . This suggests that the findings in this report characterize a stage of glaucomatous damage that precedes a detectable change in neural area. However, it is likely that more sensitive measures of axonal damage, such as counting of axons, may reveal substantial axonal involvement at this early stage of connective tissue damage.
Five of the 17 monkeys used in this study were cynomolgus, and 12 were rhesus. Although numerous studies have used a combination of these species, to our knowledge no one has assessed differences in ONH physiology between the two monkey types. All four immersion-fixed early-glaucoma monkeys were cynomolgus. The plastic deformation present within all four early-glaucoma eyes may be partially explained by interspecies differences. However, Figures 4 5 6 7 show no obvious morphological differences between the cynomolgus and rhesus ONHs. Although variations in the relative amounts of elastin and collagen in the connective tissues of the two species may contribute to the differences we describe, we think that this is unlikely.
We propose that progressive mechanical failure of the load-bearing connective tissues of the lamina cribrosa, scleral canal wall, and peripapillary sclera underlies the posterior deformation and excavation of the glaucomatous optic neuropathies. The data in this report provide the first direct evidence that primary damage to the ONH connective tissues occurs early in an elevated-IOP form of the neuropathy.
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Acknowledgements |
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Footnotes |
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Supported in part by Grant R01EY11610 (CFB) and departmental Core Grant P30EY02377 from the National Eye Institute; 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) and an unrestricted departmental grant (LSU Eye Center) from Research to Prevent Blindness; and a graduate student stipend from the Board of Regents, State of Louisiana (AJB).
Submitted for publication December 26, 2001; revised August 20, 2002; accepted August 30, 2002.
Commercial relationships policy: N.
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, LSU Eye Center, 2020 Gravier Street, Suite B, New Orleans, LA 70112; cburgo{at}lsuhsc.edu.
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