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Each nerve cross section was divided into 4 quadrants (S, T, I, and N indicate superior, temporal, inferior, and nasal quadrants, respectively) by 2 meridional lines. Each quadrant was divided into peripheral and central sectors. Two white frames in the superior quadrant indicate the position where 6 counting frames were placed in the peripheral and central sectors. The insert on the right shows unbiased counting of circles representing optic nerve fibers. Only the profiles completely inside the frame and those intersecting the lower or right-hand border of the frame were counted. The area of each counting frame was 100 µm2.

Each nerve cross section was divided into 4 quadrants (S, T, I, and N indicate superior, temporal, inferior, and nasal quadrants, respectively) by 2 meridional lines. Each quadrant was divided into peripheral and central sectors. Two white frames in the superior quadrant indicate the position where 6 counting frames were placed in the peripheral and central sectors. The insert on the right shows unbiased counting of circles representing optic nerve fibers. Only the profiles completely inside the frame and those intersecting the lower or right-hand border of the frame were counted. The area of each counting frame was 100 µm2.

Table 1. 
Global Optic Disc Topographic Parameters*
Global Optic Disc Topographic Parameters*
Table 2. 
Estimated Total Number of Nerve Fibers for Control Left and Glaucomatous Right Optic Nerves
Estimated Total Number of Nerve Fibers for Control Left and Glaucomatous Right Optic Nerves
Table 3. 
Correlation of Optic Nerve Fiber Number and Optic Disc Topography Parameters in Glaucomatous Eyes
Correlation of Optic Nerve Fiber Number and Optic Disc Topography Parameters in Glaucomatous Eyes
Table 4. 
Correlation of the Differences Between Optic Nerve Fiber Number and Optic Disc Topographic Parameters Between Left Control and Right Glaucomatous Eyes
Correlation of the Differences Between Optic Nerve Fiber Number and Optic Disc Topographic Parameters Between Left Control and Right Glaucomatous Eyes
1.
Weinreb  RNDreher  AWBille  J Quantitative assessment of the optic nerve head with the laser tomographic scanner. Int Ophthalmol. 1989;1325- 29Article
2.
Dreher  AWTso  PCWeinreb  RN Reproducibility of topographic measurements of the normal and glaucomatous optic nerve head with the laser tomographic scanner. Am J Ophthalmol. 1991;111221- 229
3.
Dreher  AWWeinreb  RN Accuracy of topographic measurements in a model eye with the laser tomographic scanner. Invest Ophthalmol Vis Sci. 1991;322992- 2996
4.
Mikelberg  FSWijsman  KSchulzer  M Reproducibility of topographic parameters obtained with the Heidelberg Retina Tomograph. J Glaucoma. 1993;2101- 103Article
5.
Lusky  MBosem  MEWeinreb  RN Reproducibility of optic nerve head topography measurements in eyes with undilated pupils. J Glaucoma. 1993;2104- 109Article
6.
Weinreb  RNLusky  MBartsch  D-UMorsman  D Effect of repetitive imaging on topographic measurements of the optic nerve head. Arch Ophthalmol. 1993;111636- 638Article
7.
Rohrschneider  KBurk  ROKruse  FEVolcker  HE Reproducibility of the optic nerve head topography with a new laser tomographic scanning device. Ophthalmology. 1994;1011044- 1049Article
8.
Gaasterland  DKupfer  C Experimental glaucoma in the rhesus monkey. Invest Ophthalmol Vis Sci. 1974;13455- 457
9.
Weibel  ER Stereological Methods: Practical Methods for Biological Morphometry. Vol 1. New York, NY Academic Press Inc1980;
10.
Gundersen  HJG Notes on the estimation of the numerical density of arbitrary profiles: the edge effect. J Microsc. 1977;111219- 223Article
11.
Sanchez  RMDunkelberger  GRQuigley  HA The number and diameter distribution of axons in the monkey optic nerve. Invest Ophthalmol Vis Sci. 1986;271342- 1350
12.
Mikelberg  FSDrance  SMSchulzer  MYidegiligne  HWeis  M The normal human optic nerve: axon count and axon diameter distribution. Ophthalmology. 1989;961325- 1328Article
13.
Jonas  JBMüller-Bergh  JASchlotzer-Schrehardt  UMNaumann  GOH Histomorphometry of the human optic nerve. Invest Ophthalmol Vis Sci. 1990;31736- 744
14.
Uchida  HBrigatti  LCaprioli  J Detection of structural damage from glaucoma with confocal laser image analysis. Invest Ophthalmol Vis Sci. 1996;372393- 2401
15.
Coleman  ALQuigley  HAVitale  SDunkelberger  G Displacement of the optic nerve head by acute changes in intraocular pressure in monkey eyes. Ophthalmology. 1991;9835- 40Article
16.
Burgoyne  CFQuigley  HAThompson  HWVitale  SVarma  R Early changes in optic disc compliance and surface position in experimental glaucoma. Ophthalmology. 1996;1021800- 1818Article
Laboratory Sciences
April 1998

Relationship of Optic Disc Topography to Optic Nerve Fiber Number in Glaucoma

Author Affiliations

From the Glaucoma Center (Drs Yücel, Gupta, de Souza Lima, Zangwill, and Weinreb) and the Department of Pathology (Drs Kalichman and Mizisin), University of California, San Diego; the Department of Ophthalmology, University of Toronto, Ontario (Dr Gupta); and the Department of Biological Sciences, Allergan Inc, Irvine, Calif (Dr Hare). Dr Yücel is now with the Department of Ophthalmology, University of Toronto. The authors have no proprietary interest in the Heidelberg Retina Tomograph.

Arch Ophthalmol. 1998;116(4):493-497. doi:10.1001/archopht.116.4.493
Abstract

Objective  To assess the relationship between in vivo measurements of optic disc topography and histomorphometric measurements of optic nerve fiber number in glaucoma.

Methods  Both eyes of 10 monkeys (Macaca fascicularis) with laser-induced glaucoma in the right eye were studied. Optic disc topography was measured in vivo with a confocal scanning laser ophthalmoscope. Histomorphometry was performed on optic nerve cross sections using bright-field microscopy with camera lucida. Nerve fiber density was estimated by unbiased random sampling. Nerve fiber number was estimated for each sector by multiplying nerve fiber density with neuroglial area. Nerve fiber count was compared with each of 13 global optic disc topographic parameters.

Results  For neuroretinal measurements in the glaucomatous eyes, rim area, retinal nerve fiber layer (RNFL) cross-sectional area, rim volume, and RNFL thickness correlated significantly with optic nerve fiber number. Differences in nerve fiber count between control and glaucomatous optic nerves showed the strongest correlation with differences in mean height contour; this was followed by RNFL cross-sectional area, RNFL thickness, rim volume, and differences in rim area. For cup measurements in the glaucomatous eyes, cup volume below reference, cup area, mean cup depth, the ratio of cup area to disc area, and cup shape correlated significantly with nerve fiber number. Differences in nerve fiber number between control and glaucomatous optic nerves showed the strongest correlation with differences in cup shape; this was followed by mean cup depth, cup volume below reference, the ratio of cup area to disc area, cup area, and differences in cup volume below surface. No association was found between optic nerve fiber number and optic disc area in glaucomatous eyes.

Conclusions  In experimental glaucoma, most optic disc topography measures correlated significantly with optic nerve fiber number. The results of this histomorphometric study support the use of confocal scanning laser ophthalmoscopy to evaluate optic nerve damage in glaucoma.

CONFOCAL scanning laser provides rapid and reproducible in vivo measurements of optic disc topography.17 However, the relationship between optic disc topography and optic nerve fiber loss in glaucoma has not been established. An understanding of this relationship is important to guide the clinician in the use of the best topographic parameters to diagnose glaucoma and detect progressive change. The current study was undertaken to compare measurements of optic disc topography with the number of surviving optic nerve fibers in experimental glaucoma.

METHODS

Experimental glaucoma was induced in the right eyes of 10 monkeys (Macaca fascicularis), according to the protocol described by Gaasterland and Kupfer.8

INTRAOCULAR PRESSURE MEASUREMENTS

Intraocular pressure (IOP) measurements were performed with a pneumatonometer (Digilab, Norwell, Mass) on monkeys that had received light sedation (intramuscular injection of ketamine, 5 mg/kg) and local anesthesia (proparacaine hydrochloride). Compared with the control left eye (mean±SD) (19.75±1.92 mm Hg), the IOP was significantly increased in the glaucomatous right eye (39±14.24 mm Hg; P=.002) prior to optic disc topography measurements.

CONFOCAL LASER SCANNING TOMOGRAPHY

Optic disc topographic measurements were acquired and analyzed using a confocal scanning laser ophthalmoscope (Heidelberg Retina Tomograph [HRT], Heidelberg Engineering GmbH, Heidelberg, Germany), which employs a diode laser (wavelength, 670 nm) to scan a surface in the x, y, and z directions. Image acquisition and processing with the HRT have been described in detail elsewhere.6 Briefly, an image series is obtained from 32 transverse optical section images taken at consecutive height planes with a scan depth of 1.5 to 2.5 mm. An effort was made to maintain a constant distance between the objective lens of the HRT and the eye. Each image series was then analyzed to generate a topographic map containing 256 × 256 picture elements. Optic nerve topography was measured in the anesthesized (ketamine and xylazine) animals 10 months after laser treatment. Three HRT images of 15° field of view were taken for each eye. Image quality was monitored by the software and the operator. The mean topography image was calculated using HRT software version 2.01 (Heidelberg Retina Tomograph [HRT], Heidelberg Engineering GmbH). To estimate the reproducibility of the HRT measurements in subjects receiving general anesthesia, the mean SD of 3 images was averaged for all picture elements by the software. Disc margins were outlined while viewing stereophotographs obtained 10 months after inducing glaucoma, prior to HRT measurements. The keratometric readings were used to correct for magnification error. Software-determined parameters were retinal nerve fiber layer (RNFL) thickness, RNFL cross-sectional area, rim volume, rim area, mean height contour, cup volume below reference, cup volume below surface, cup shape, mean cup depth, maximum cup depth, cup area, optic disc area, and the ratio of cup area to disc area (cup-disc area ratio). The RNFL thickness, RNFL cross-sectional area, rim volume, rim area, cup area, and cup volume below reference are measured relative to the standard reference plane. The position of the standard reference plane is 50 µm posterior to the mean height of the optic disc margin contour line in a temporal segment between 350° and 356°. The RNFL thickness and RNFL cross-sectional area are indirect estimates of the retinal nerve fiber layer thickness and cross-sectional area along the disc margin.

HISTOLOGY

Animals were killed by intracardiac perfusion with fixative, 4% paraformaldehyde, and 0.1% glutaraldehyde in 0.1-mol/L phosphate buffer (pH, 7.4) while receiving deep general anesthesia (intramuscular injection of ketamine and xylazine) 14 months after laser treatment. After enucleation, optic nerves were fixed by immersion in 2.5% glutaraldehyde in 0.1-mol/L phosphate buffer (pH, 7.4) for at least 48 hours. Following 3 washes with 0.1-mol/L phosphate buffer (pH, 7.4), optic nerves were placed in a solution of 2% osmium tetroxide in phosphate buffer for 1 hour and then washed again with 0.1-mol/L phosphate buffer. Optic nerve cross sections of 1-mm thickness were taken 2 mm posterior to the sclera and marked with 1 razor-blade slit in the superior quadrant and 2 slits in the nasal quadrant to indicate the orientation. Cross sections were placed in 2% osmium tetroxide for 2 hours, dehydrated in alcohol, and embedded in a resin mixture. Semithin, 0.5-µm-thick cross sections were cut with a microtome, mounted on glass slides, and stained for myelin with p-phenylenediamine.

MORPHOMETRY

Morphometry (Figure 1) was performed using bright-field microscopy with camera lucida. At low power, optic nerve cross sections were divided into 4 quadrants (superior, inferior, temporal, and nasal) of approximately equal area. Each quadrant was further divided into peripheral and central sectors according to the paler staining observed in the peripheral zone that contains less myelin. For each sector on the optic nerve cross sections, neuroglial area, interfascicular septal area, central retinal artery and surrounding connective tissue area, and total area were estimated by point counting.9 Each point corresponded to an area of 0.0196 mm2. The neuroglial area included myelinated nerve fibers and glial cells.

At high power magnification, nerve fiber density was estimated for the peripheral and central sectors of each quadrant using an oil immersion ×100 objective. The final magnification was ×1562. The sampling of the fields for each sector was determined in an unbiased and systematic fashion on a line from the borders of the quadrant at the half radius of the optic nerve cross section (for the central sector) and at 120 th of the radius (for the peripheral sector) on a drawing of optic nerve boundary independent of its content. Unbiased counting frames were positioned at the center of a visual field in each sector. Six frames were selected to avoid interfascicular septae. As described by Gundersen,10 sampling bias was minimized by counting only those profiles completely inside the frame and those intersecting the lower or right-hand border of the frame. The area of each counting frame was 100 µm2. Nerve fiber density for each sector was calculated by dividing the number of nerve fibers in the 6 counting frames by 600 µm2. The total number of nerve fibers per sector was calculated by multiplying nerve fiber density by neuroglial area. The total number of nerve fibers per quadrant was calculated by adding the peripheral sector nerve fiber number to the central sector nerve fiber number. Total nerve fiber number per optic nerve was estimated by adding nerve fiber numbers of all 4 quadrants.

STATISTICAL ANALYSIS

To compare means of the right and left optic nerve fiber counts, a Wilcoxon signed rank test was used. Nonparametric Spearman rank correlation was used to compare number of optic nerve fibers with each of 13 global HRT parameters.

All studies were performed following the guidelines of the Association for Research in Vision and Ophthalmology Resolution on the use of animals in research.

RESULTS

Compared with the control left eye, 11 of 13 global optic disc topographic parameters were statistically different in the glaucomatous right eye (Table 1). Only the differences in the optic disc area and rim area did not reach a level of statistical significance. Differences between control and glaucomatous eyes were significant for RNFL thickness, RNFL cross-sectional area, rim volume, and mean height contour, as were differences in cup area, mean cup depth, cup volume below reference, cup volume below surface, maximum cup depth, cup shape, and differences in the ratio of cup area to disc area.

Morphometric estimates of the number of optic nerve fibers are shown in Table 2. The estimated mean (±SD) number of fibers per optic nerve in 10 control left optic nerves was 1073205 (±198254), and the estimated number of fibers per nerve ranged from 796250 to 1431519. In contrast, the estimated mean (±SD) number of fibers per glaucomatous nerve was 519610 (±470273), and the estimated number of fibers ranged from 0 to 1184203. The mean of the number of nerve fibers was significantly different in glaucomatous right optic nerves compared with control left optic nerves (P=.002). The mean (±SD) of the difference in number of optic nerve fibers between control and glaucomatous optic nerves was 553595 (±426731). Optic nerve fiber number in the glaucomatous optic nerves correlated with intraocular pressure prior to death (ρ= −0.88).

The correlation coefficients between the number of optic nerve fibers in the glaucomatous optic nerves and the 13 optic disc topography parameters are shown in Table 3. For neuroretinal rim measurements, optic nerve fiber number showed the strongest correlation with rim area (ρ=0.82); this was followed by RNFL (ρ=0.75), rim volume (ρ=0.72), and RNFL thickness (ρ=0.71). For cup measurements, optic nerve fiber number showed the strongest correlation with cup volume below reference (ρ=−0.87); this was followed by cup area (ρ=−0.82), mean cup depth (ρ=−0.81), ratio of cup area to disc area (ρ=0.81), and cup shape (ρ=−0.65). The number of optic nerve fibers in glaucomatous eyes showed weaker associations with maximum cup depth (ρ=−0.61), cup volume below surface (ρ=−0.53), mean height contour (ρ=−0.49), or optic disc area (ρ=0.42).

Significant correlations also were found for differences in optic disc topography parameters between control and glaucomatous eyes and differences in nerve fiber numbers (Table 4). For neuroretinal rim measurements, differences in nerve fiber number between control and glaucomatous optic nerves showed the strongest correlation with differences in mean height contour (ρ=−0.88); this was followed by differences in RNFL thickness (ρ=0.84), RNFL cross-sectional area (ρ=0.84), rim volume (ρ=0.81), and differences in rim area (ρ=0.72). For cup measurements, differences in nerve fiber count between control and glaucomatous optic nerves showed the strongest correlation with differences in cup shape (ρ=−0.83); this was followed by differences in mean cup depth (ρ=−0.76), cup volume below reference (ρ=−0.75), ratio of cup area to disc area (ρ=−0.75), and differences in cup volume below surface (ρ=−0.71). No association was found between differences in the nerve fiber count between control and glaucomatous optic nerves and differences in optic disc area (ρ<0.01).

COMMENT

In the glaucomatous eyes, most of the optic disc topographic parameters correlated significantly with optic nerve fiber number. Optic nerve fiber number in glaucomatous optic nerves showed the strongest correlation with cup volume below reference; this was followed by rim area, cup area, mean cup depth, and the ratio of cup area to disc area. Nerve fiber number difference between the control and glaucomatous optic nerves showed the strongest correlation with the differences in mean height contour; this was followed by difference in RNFL cross-sectional area, RNFL thickness, and difference in cup shape. For most of the optic disc topographic parameters, differences between the control and glaucomatous eyes showed stronger correlation with nerve fiber number differences compared with measurements in glaucomatous eyes alone.

In this study, the mean (±SD) of the estimated nerve fiber number of the 10 control monkey optic nerves was 1073205±198254, similar to that observed in an earlier study of perfusion-fixed monkey optic nerves (1169227±227957).11 The coefficient of variation for optic nerve fiber was 0.185, similar to those calculated in previous studies: 0.195, 0.24, and 0.169.1113

In the current study, the difference in nerve fiber number between control and glaucomatous optic nerves was used to estimate nerve fiber loss. The analysis assumed that differences between optic nerves in an individual monkey were smaller than differences among the various animals. This method of analysis is supported by a previous report in which the difference between optic nerve fiber numbers in left and right normal human optic nerves ranged from 16% to 25%.11 In contrast, the nerve fiber counts of control optic nerves in the current study showed large differences among animals. For example, in the present study, the difference in nerve fiber number between the control optic nerves of monkey 6 and monkey 7 was 41%. These data suggest that the variation between left and right optic nerve fiber counts in the normal monkey may be less than the variation observed among animals.

The RNFL thickness, RNFL cross-sectional area, and rim volume measurements are estimates of the retinal nerve fiber layer thickness, cross-sectional area along the disc margin, and neuroretinal rim volume, respectively. These parameters correlated significantly with optic nerve fiber count in the glaucomatous optic nerves. In addition, differences in each of these 3 parameters correlated strongly with nerve fiber loss. Hence, these topographic parameters, which are based on a standard reference plane, may be particularly useful for the evaluation of nerve fiber loss in glaucoma.

Mean height contour differences between control and glaucomatous eyes showed the strongest correlation with nerve fiber loss. Therefore, this neuroretinal rim parameter, which measures the mean of the absolute retinal height along the contour line, independent of a reference plane, might be useful in monitoring optic disc changes related to optic nerve loss. On the other hand, mean height contour correlated weakly with optic nerve fiber numbers in glaucomatous eyes. This may be explained by the fact that, compared with all other parameters, mean height contour showed the greatest variation among animals.

In our study, cup measurements—including cup volume, the ratio of cup area to disc area, and cup shape— were significantly associated with nerve fiber number. In addition, differences in cup measurements between control and glaucomatous eyes correlated strongly with nerve fiber loss. Cup shape has been shown to discriminate between normal persons and those with early glaucomatous damage.14

Optic disc topography also may be dependent on variation in IOP in the monkey.15 We cannot exclude that pressure-related changes, which are explained by mechanical deformation not related to optic nerve fiber loss, could be responsible for some of the HRT-parameter differences found between control and glaucomatous eyes. Pressure-related topographic changes have been described for chronic IOP elevation for as long as 4 months.16 In the current study, optic nerve fiber damage also was caused by chronic elevation of intraocular pressure, and optic nerve fiber loss correlated highly (ρ=−0.88) with the level of IOP. This may explain why the HRT parameters that correlated with optic nerve fiber loss also correlated with IOP of glaucomatous optic nerves. Future experimental histomorphometric studies designed to detect optic nerve head topographic changes due only to optic nerve fiber loss, independent of IOP changes, may be useful in determining the best HRT parameters and the most meaningful analysis strategies for detecting or following the progression of glaucoma.

In conclusion, several optic disc topographic parameters of the neuroretinal rim and cup showed strong correlation with nerve fiber loss in an experimental model of glaucoma. The results of this histomorphometric study support the potential for using the confocal scanning laser ophthalmoscope to evaluate optic nerve damage in glaucoma.

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Article Information

Accepted for publication December 19, 1997.

This study was supported in part by R. S. McLaughlin Foundation, University of Toronto, Ontario (Dr Gupta); grant EY11008 from the National Eye Institute, Bethesda, Md (Dr Zangwill); Joseph Drown Foundation, Los Angeles, Calif, National Eye Institute grant EY11158 (Dr Weinreb); and Foundation for Eye Research Rancho Santa Fe, Calif (Drs Yäcel and de Souza Lima).

Corresponding author: Robert N. Weinreb, MD, Glaucoma Center 0946, University of California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0946.

References
1.
Weinreb  RNDreher  AWBille  J Quantitative assessment of the optic nerve head with the laser tomographic scanner. Int Ophthalmol. 1989;1325- 29Article
2.
Dreher  AWTso  PCWeinreb  RN Reproducibility of topographic measurements of the normal and glaucomatous optic nerve head with the laser tomographic scanner. Am J Ophthalmol. 1991;111221- 229
3.
Dreher  AWWeinreb  RN Accuracy of topographic measurements in a model eye with the laser tomographic scanner. Invest Ophthalmol Vis Sci. 1991;322992- 2996
4.
Mikelberg  FSWijsman  KSchulzer  M Reproducibility of topographic parameters obtained with the Heidelberg Retina Tomograph. J Glaucoma. 1993;2101- 103Article
5.
Lusky  MBosem  MEWeinreb  RN Reproducibility of optic nerve head topography measurements in eyes with undilated pupils. J Glaucoma. 1993;2104- 109Article
6.
Weinreb  RNLusky  MBartsch  D-UMorsman  D Effect of repetitive imaging on topographic measurements of the optic nerve head. Arch Ophthalmol. 1993;111636- 638Article
7.
Rohrschneider  KBurk  ROKruse  FEVolcker  HE Reproducibility of the optic nerve head topography with a new laser tomographic scanning device. Ophthalmology. 1994;1011044- 1049Article
8.
Gaasterland  DKupfer  C Experimental glaucoma in the rhesus monkey. Invest Ophthalmol Vis Sci. 1974;13455- 457
9.
Weibel  ER Stereological Methods: Practical Methods for Biological Morphometry. Vol 1. New York, NY Academic Press Inc1980;
10.
Gundersen  HJG Notes on the estimation of the numerical density of arbitrary profiles: the edge effect. J Microsc. 1977;111219- 223Article
11.
Sanchez  RMDunkelberger  GRQuigley  HA The number and diameter distribution of axons in the monkey optic nerve. Invest Ophthalmol Vis Sci. 1986;271342- 1350
12.
Mikelberg  FSDrance  SMSchulzer  MYidegiligne  HWeis  M The normal human optic nerve: axon count and axon diameter distribution. Ophthalmology. 1989;961325- 1328Article
13.
Jonas  JBMüller-Bergh  JASchlotzer-Schrehardt  UMNaumann  GOH Histomorphometry of the human optic nerve. Invest Ophthalmol Vis Sci. 1990;31736- 744
14.
Uchida  HBrigatti  LCaprioli  J Detection of structural damage from glaucoma with confocal laser image analysis. Invest Ophthalmol Vis Sci. 1996;372393- 2401
15.
Coleman  ALQuigley  HAVitale  SDunkelberger  G Displacement of the optic nerve head by acute changes in intraocular pressure in monkey eyes. Ophthalmology. 1991;9835- 40Article
16.
Burgoyne  CFQuigley  HAThompson  HWVitale  SVarma  R Early changes in optic disc compliance and surface position in experimental glaucoma. Ophthalmology. 1996;1021800- 1818Article
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