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1From the Department of Anatomy II and the 2Medical Department II, University of Erlangen-Nürnberg, Erlangen, Germany; University of Erlangen-Nürnberg; 3Mayo Clinic, Rochester, Minnesota.
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METHODS. Optic nerves obtained at autopsy from 36 eyes with primary open-angle glaucoma (POAG) and 15 with pseudoexfoliation glaucoma (PEXG) were studied quantitatively and qualitatively. Axon counts, fibrosis, capillary number and density, and arteriosclerotic changes were assessed in the postlaminar optic nerve and compared to normal age-matched autopsy eyes. Changes in composition of extracellular matrix components were evaluated by immunohistochemistry and electron microscopy.
RESULTS. Marked differences were found between POAG and PEXG. Axon loss in eyes with POAG but not in PEXG was associated with increasing connective tissue in the septa and surrounding the central retinal vessels, including increased amounts of type IV and VI collagen. The total number of capillaries decreased with the loss of axons in both POAG and PEXG. POAG nerves, however, had a decrease in the density of capillaries, whereas in PEXG the capillary density did not change with axon loss. Arteriosclerotic changes were more common in glaucomatous eyes than in age-matched control eyes.
CONCLUSIONS. The difference in morphology of the optic nerves between POAG and PEXG indicates that in eyes with POAG, elevated IOP cannot be the only pathogenetic factor in glaucomatous optic neuropathy. Additional factors, inducing fibrosis and loss of capillaries, seem to be involved. Such additional factors may also contribute to the clinical finding in POAG that nerves can become damaged without elevation of intraocular pressure.
The cause of the different susceptibilities of optic discs to IOP is not understood. Size and shape of the optic disc, characteristics of the lamina cribrosa, blood supply to the nerve and disc, and inherent axonal characteristics have all been postulated to play a role in susceptibility to damage.1 2 3 Because nearly one half of patients with POAG do not have consistently elevated IOP, this condition has been termed an "optic neuropathy," with IOP being a "risk factor."4 5 The multiple potential damaging factors may represent a spectrum, ranging from elevated IOP on one end to ischemic and other factors on the other end.4 Eyes with POAG may lie in the middle of the spectrum, with optic nerve damage caused by a combination of factors.
Recent histologic studies lend weight to this idea. In eyes with POAG an increase in the amount of elastic tendon sheath material is found in the trabecular meshwork.6 7 This material, also known as sheath-derived (SD)-plaques, is increased in eyes with axon loss in the optic nerve, but does not correlate with IOP.8 This suggests that SD-plaques alone are not responsible for the problem in the meshwork that causes the increase in IOP, but rather accompany the disease process.8 In eyes with PEXG there was a correlation between the PEX material in the meshwork and IOP levels,9 as well as axon loss in the optic nerve.10 We hypothesized from these findings that those factors involved in the pathogenesis of the meshwork changes in POAG leading to the accumulation of SD-plaques may also induce changes in the optic nerve, making the nerve more susceptible to elevated IOP than normal eyes. SD-plaques are not increased in eyes with PEXG.7 10 In addition, PEX material differs profoundly from SD-plaques material in its morphology and distribution within the eye. We therefore assume that factors responsible for PEX and SD-plaque formation are different.
If the factors inducing optic nerve changes in patients with POAG differ from those in patients with secondary glaucomas such as PEXG, or are not present in the secondary glaucomas, the histologic findings in the optic nerve may also differ. To evaluate this hypothesis, we studied the optic nerves from donor eyes with POAG or PEXG.
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POAG Eyes.
Thirty-six eyes from 23 donors (mean age, 75 ± 9 years; range, 58–86) were studied. Clinical details are in Table 1 .
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Histologic Analysis
Because eyes were received from various eye banks around the United States, they had been fixed in a variety of fixatives, from paraformaldehyde-glutaraldehyde mixtures to 10% formalin. Portions from the postbulbar region of the optic nerve, 1 to 2 mm behind the globe, were embedded in Epon for electron microscopy, or in paraffin for immunohistochemistry. In this region, the central retinal artery and vein are still located in the center of the nerve. Semithin cross sections were stained with toluidine blue and fuchsin. Quantitative analysis of sections was performed similar to our previous reports,8 10 based on the method of Quigley et al.11 The area of the entire nerve, excluding the meninges, was measured. The area of the nerve fiber bundles and the area of connective tissue between the bundles and surrounding the central retinal vessels were measured separately. For determination of the number of axons, the cross section of the optic nerve was divided into eight sectors. In each sector, axons in five sample areas of 1000 µm2 were counted. These sample areas extended from the central to the peripheral nerve at equal distances from each other. The mean of all measurements was multiplied with the nerve fiber area, to yield the total axon counts.
The postbulbar retrolaminar portion of the optic nerve was chosen for study because of the regularity of the tissue and ease of making quantitative measurements when compared with the bowing and cupping of the laminar region. This retrolaminar portion of the optic nerve receives most of its blood supply from the pial arterioles, which penetrate the connective tissue septa and branch into capillaries; only a few capillaries originate at the central retinal artery (CRA).12 Evaluation of both the capillaries and arterioles was performed. Within the connective tissue septa, both the total number and the density of capillaries were determined. The total number of capillaries was determined by dividing the cross section of the nerve into quadrants. In the middle of each quadrant, three regions with an area of 0.155 mm2 each were analyzed at 40x magnification. These regions were spaced at equal distances from the central to the peripheral portions of the nerve. The mean of these 12 regions was multiplied with the entire cross-sectional area of the nerve, to produce the capillary number per nerve. The potential tortuosity of capillaries did not allow the determination of their absolute number, and so a simple count of the number of visible capillary lumens (or capillary profiles) present in a cross-section was made (Fig. 1) . The measurements were performed by two of the authors (JG, AK) independently, and the average of the data was taken. The density of capillaries was defined as the total number of capillaries per square millimeter of total nerve area.
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Immunohistochemistry
Depending on the fixation protocols, in only nine eyes with POAG (mean age, 76 ± 12.8 years; range, 58–92) and five eyes with PEXG (mean age, 83 ± 7.8 years; range, 83–93) was immunostaining possible. These results were compared to five control eyes (mean age, 82 ± 6.7 years; range, 74–90).
Immunohistochemistry was performed with affinity-purified rabbit and mouse polyclonal antibodies, according to the manufacturers’ instructions. Immunoreactivity was visualized on paraffin-embedded sections. Ten- to 20-µm cross sections through the postbulbar optic nerve were placed on poly-L-lysine-coated slides and initially incubated with dry milk solution (Blotto; Santa Cruz Biotechnology, Heidelberg, Germany) at room temperature for 30 minutes, to reduce nonspecific background staining. Incubation with the primary antibody (Table 3) was performed in a moist chamber for 12 to 36 hours at room temperature. The sections were rinsed in Tris-buffered saline (TBS) three times for 10 minutes each and then incubated for 1 hour with the appropriate secondary antibodies (Table 3) . After they were rinsed in PBS, the sections were mounted in Kaiser’s glycerin jelly (Merck KGaA; Darmstadt, Germany). The sections were viewed either with a fluorescence microscope (Aristoplan; Ernst Leitz, Wetzlar, Germany) or with a confocal laser-scanning microscope (LSM 5 Pascal; Carl Zeiss, Jena, Germany).
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< 0.05 was considered significant. |
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The number of capillaries within the connective tissue septa was 450 ± 78 (range, 277–564). The number of capillaries per square millimeter—the capillary density—was 61.1 ± 5.9. There was no correlation between number of capillaries or capillary density and age or axon counts. The number of pial arterioles in the pial connective tissue surrounding the optic nerve ranged from 9 to 24 (mean, 13.7 ± 3.4).
Glaucomatous Eyes
In both POAG and PEXG eyes, loss of axons was accompanied by a decrease in cross-sectional area of the optic nerve and an increase in the relative proportion of the amount of connective tissue. When the absolute amount of connective tissue was quantitated, however, PEXG eyes had only a minimal increase in the thickness of the septa, and the proportional increase in connective tissue was due to the decreased size of the axon bundles (see quantitation below). In contrast, one half of the POAG eyes with significant axon loss had not only a proportional increase in connective tissue but also an actual increase in the amount of connective tissue.
In both POAG and PEXG the total number of capillaries was decreased when compared to age-matched normal eyes (quantitation described later). The density of capillaries (total number of capillaries per square millimeter of total nerve area), however, differed between the diseases. In PEXG the density of capillaries remained constant. In contrast, the density was less in POAG. As in the normal eyes, in both PEXG and POAG, capillaries were seen only within the connective tissue septa, usually in the middle. In POAG with increased thickness of the septa, the "diffusion distance" between the capillary wall and nerve fiber bundles was therefore enlarged.
Arteriosclerosis was more common and more advanced in both the CRA and the pial arterioles in POAG eyes than in age-matched control eyes.
Quantitative Findings
Connective Tissue.
In POAG eyes, a decrease in overall nerve cross-sectional area was found with axon loss (r = 0.7144, P < 0.001; Fig. 3b ). As axons decreased, an increase in the total amount of connective tissue was found in 16 of 36 eyes, resulting in an overall 26% increase in the mean amount (1.96 ± 0.71 mm2, n = 36 POAG eyes, compared with 1.55 ± 0.27 mm2 in age-matched normal eyes; P = 0.04). This increase occurred in both the connective tissue in the region surrounding the central retinal vessels and also in the septa. The amounts ranged as high as 3.83 mm2, compared with the highest value of 2.04 mm2 in normal eyes and 2.52 mm2 in PEXG (Fig. 3c) . The proportion of the nerve composition changed with increasing axon loss: the connective tissue in the septa comprised a proportionately larger fraction of the nerve (r = –0.7608, P < 0.001; Fig. 3d ).Comparison of the entire group of POAG eyes (including all stages of axon loss) with age-matched normal eyes showed a 65% increase in thickness of the connective tissue septa (33.30% ± 11.9% vs. 20.2% ± 1.5% of total nerve cross-sectional area; P = 0.0002; Tables 5, 6).
The connective tissue surrounding the central retinal vessels also increased with axonal loss (r = 0.4117, P < 0.05; Fig. 4a ). Overall this increase was 17% when comparing all POAG eyes with age-matched normal eyes (0.106 ± 0.019 mm2 vs. 0.090 ± 0.014 mm2; P = 0.02; Tables 5 6 ).
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0.001; Fig. 3f ) with increasing axon loss. Comparison of all POAG eyes (all stages of axon loss combined) with age-matched normal eyes showed a 30% decrease in the number of capillary profiles in the septa (304 ± 205 vs. 435 ± 80; P = 0.01). The corresponding decrease in capillary density was 23% (48.2 ± 27.1 vs. 62.4 ± 5.7 capillaries/mm2; P = 0.01; Tables 5 6 ). In keeping with this finding, nerves with more connective tissue septa had fewer capillaries (r = –0.36; P < 0.05) and a reduced capillary density (r = –0.33; P < 0.05; Fig. 3g ). Fewer arterioles in the pia were present in POAG when compared with age-matched normal control eyes (25% decrease: 10.2 ± 2.4 vs. 13.6 ± 3.6; P < 0.0001; Tables 5 6 ). The decrease in number of pial arterioles correlated with the decrease in density of capillaries in the septa (r = 0.35; P < 0.05).
In POAG eyes there were more arteriosclerotic changes than in the age-matched normal eyes for both the CRA and pial vessels. The arteriosclerosis score for the CRA was 1.9 ± 0.6 vs. 1.6 ± 0.5 in age-matched control eyes (P = 0.03; scores range from 1 to 3: see the Methods section). The mean arteriolosclerosis score for the pial arterioles was 1.9 ± 0.6 vs. 1.3 ± 0.5 (P = 0.001; Tables 5 6 ). In the CRA, 5 of the POAG eyes had advanced arteriosclerosis (grade 3), and 20 eyes had mild to moderate changes (grade 2). Similarly, in the pial vessels five eyes showed advanced arteriolosclerosis (grade 3), but these were different eyes from different donors than those with advanced arteriosclerosis of the CRA. In the pial vessels, 21 eyes had mild to moderate changes (grade 2). Only four eyes had no evidence of arteriolosclerosis in any of the vessels.
In PEXG, the number of capillaries decreased with axon loss, but in contrast to the POAG eyes, the capillary density did not change with axon loss (Figs. 3e 3f) . No correlation was found between the number of capillaries or capillary density and the area of septa (Fig. 3g) . The number of pial arterioles was comparable to that in the age-matched control (Tables 5, 7).
In eyes with PEXG, the arteriosclerotic changes were somewhat less prominent than in eyes with POAG, but the differences were not statistically significant (Tables 5 7) . Only 1 of 15 PEXG eyes had advanced arteriolosclerosis in the pial vessels (grade 3), whereas seven had mild to moderate changes (grade 2). In the CRA the arteriosclerotic changes were somewhat more pronounced: 2 of 15 eyes had advanced changes (grade 3) and 6 had mild to moderate changes. Almost one half of the donors with PEXG had normal-appearing vessels within and surrounding the nerve.
Immunohistochemistry and Electron Microscopy
Fixation of the material gave reliable results in POAG eyes only for type IV and VI collagen staining and in PEXG eyes only for type VI collagen. Staining for type I, II, and III collagen was not possible.
In normal eyes, a thin layer of type IV collagen–immunoreactive (IR) material surrounded the blood vessels and the nerve fiber bundles (Fig. 4a) . This finding was the same in all areas of the nerve and in all five eyes investigated. In contrast, in all POAG eyes, the collagen IV layers were markedly thickened at the rim of the nerve fiber bundles, and stained fibers extended into the periphery of the nerve bundles, forming an irregular or spike-like pattern (Fig. 4b) . The pattern was similar in both the central and peripheral regions of the nerves. Ultrastructurally, the basement membranes surrounding the nerve fiber bundles were thin in the normal eyes, even in the advanced age groups investigated (Fig. 4c) . In most eyes with PEXG the morphology of the basement membranes resembled that in the normal control eyes. In contrast, in eyes with POAG, there was a thickening of the basement membrane and protrusions of this thickened basement-membrane–like material extended into the nerve fiber bundles (Fig. 4d) .
In control eyes, a thin layer of type VI collagen was located between the collagen IV layer around both the blood vessels and nerve fiber bundles and the adjacent connective tissue of the septa. In addition, thin strands of collagen VI were also present within the connective tissue septa (Fig. 5a) . PEXG nerves showed a pattern and thickness of type VI collagen staining similar to that in the control eyes. In contrast, in POAG eyes collagen VI layers appeared thickened around the nerve fiber bundles, and within the septa (Fig. 5b) .
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In normal and PEXG, only a thin sheath of fine fibrillar material surrounded the elastic fibers in the septa, regardless of age or amount of axon loss (Fig. 5c) . In contrast, in POAG eyes with increased thickness of the septa, an increased amount of this fine fibrillar material was present surrounding the elastic fibers and also adjacent to basement membranes (Fig. 5d) .
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Discussion |
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This increase in connective tissue in POAG eyes seems to be more than a simple reaction to axon loss, especially when compared with PEXG eyes with similar amounts of axon loss. A significant correlation between axon loss and age was even found in normal eyes, but without concomitant increase in connective tissue. It is possible that a growth factor or other stimulus caused this increase in connective tissue. Increased levels of TGF-ß2 have been reported in the optic nerves of eyes with POAG.14 TGF-ß2 can induce increased expression of ECM components in monolayer cultures of human optic nerve astrocytes.15 TGF-ß2 levels are increased in the aqueous humor and vitreous of 50% patients with POAG.16 17 In anterior chamber perfusion experiments, treatment with TGF-ß2 leads to an increase in extracellular material under the inner wall of Schlemm’s canal and a simultaneous decrease in outflow facility.18 It is therefore tempting to speculate that TGF-ß2 is one possible common factor inducing trabecular meshwork and optic nerve changes in eyes with POAG.
A decrease in the total number of capillaries was found with axon loss in both POAG and PEXG. A difference in capillary density—the number of capillaries per area of optic nerve—was present between POAG and PEXG. In PEXG, the capillary density remained constant despite axon loss. This is similar to changes in monkey eyes with laser-induced ocular hypertension.19 20 Both Quigley et al.19 and our group20 studied monkeys with experimentally increased IOP and found that the percentage of capillary area (capillary density) in the prelaminar portion of the optic nerves was the same as in control nerves. These findings support the idea that PEXG behaves as a secondary glaucoma, with pressure-induced optic nerve damage rather than damage caused by primarily ischemic or other factors.
In contrast to PEXG, the optic nerves of POAG eyes had a decrease in capillary density with axon loss. Thus, the loss of capillary density cannot be caused solely by axon loss. The decrease in capillary density was also correlated with increased thickness of connective tissue in the septa of eyes with POAG but not PEXG. Common factors could be responsible for the increase in connective tissue and the decrease in capillary density in POAG. It is tempting to speculate that these factors also induce the changes in the trabecular meshwork in POAG eyes.
A decrease in the number of pial arteries was found in POAG. We are not certain why. It could exist before the development of POAG and thus make the nerves more susceptible to damage. In keeping with this, atherosclerosis appeared more prominent in POAG eyes than in normal and PEXG eyes. Second, the decrease in pial arterioles could be caused by the same factors responsible for the pathogenesis of the nerve damage, and thus change in parallel with the nerve damage. Finally, the decrease in arterioles could be a secondary reaction to atrophy of the nerve.
In summary, this study suggests that, in addition to IOP, other factors are involved in the degeneration of the optic nerve in POAG eyes. This could contribute to the development of optic neuropathy in eyes without elevated IOP. The elevated levels of TGF-ß2 reported in the aqueous and vitreous of some eyes with POAG and the known effects of TGF-ß2 on the trabecular meshwork and optic nerve astrocytes may play a role in this development.
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Acknowledgements |
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Footnotes |
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Submitted for publication March 8, 2005; revised June 20 and July 29, 2005; accepted September 20, 2005.
Disclosure: J. Gottanka, None; A. Kuhlmann, None; M. Scholz, None; D.H. Johnson, None; E. Lütjen-Drecoll, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Elke Lütjen-Drecoll, Department of Anatomy II, Universitätsstrasse 19, 91054 Erlangen, Germany; anat2.gl{at}anatomie2.med.uni-erlangen.de.
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) and PEXG (
). (
) Mean of control eyes ± SD. (a) Correlation of axon counts and age in optic nerves of control eyes (r = –0.6598; P < 0.001). (b) Correlation of axon counts and cross-sectional area. In both groups the correlation is significant (rPOAG = 0.7144, P < 0.001; rPEXG = 0.4727, P < 0.05). (c) Correlation of axon counts and the total area of connective tissue. Whereas in POAG eyes, the total area increased with axon loss, in PEXG eyes, the area did not increase (rPOAG = 0.1593, not significant; rPEXG = 0.0245, not significant). (d) Correlation of axon counts and proportion of connective tissue septa. In both groups, the correlation is significant (rPOAG = –0.7608, P < 0.001; rPEXG = –0.7217, P < 0.001). (e) Correlation of axon counts and number of capillaries. In both groups, the correlation is significant (rPOAG = 0.6877, P < 0.001; rPEXG = 0.5078, not significant). (f) Correlation of axon counts and capillary density. Whereas in POAG the capillary density decreased significantly with axon loss, in PEXG it remained nearly constant (rPOAG = –0.5780, P < 0.001; rPEXG = –0.1497, not significant). (g) Correlation of the amount of connective tissue and capillary density in optic nerves with POAG (
