Elastosis of the Lamina Cribrosa in Pseudoexfoliation Syndrome with Glaucoma Peter A. Netland, MD, PhD,! Hongqing Ye, MD, 2 Barbara W. Streeten, MD,3 M. Rosario Hernandez, DDS 2 Background: Pseudoexfoliation syndrome is characterized by the presence of glycoprotein fibers in ocular and extraocular tissues, and often is associated with glaucoma. Pseudoexfoliation material may be associated closely with elastic microfibrillar-associated glycoprotein as well as elastin. Methods: Four optic nerve heads of two patients with pseudoexfoliation syndrome and glaucoma were examined using electron microscopy and immunogold detection of elastin. Optic nerve heads from healthy age-matched individuals and patients with primary open-angle glaucoma were used for comparisons. Results: In all eyes with pseudoexfoliation and glaucoma, there was marked and widespread elastosis in the connective tissue of the lamina cribrosa. Elastotic fibers appeared as large and irregular aggregates of electron-dense material labeled with antielastin antibody. Abundant microfibrils were interspersed in the elastotic aggregates, whereas no typical pseudoexfoliation fibers were observed. In contrast, there were less elastotic fibers in the lamina cribrosa from patients with primary open-angle glaucoma compared with pseudoexfoliation glaucoma. Other changes of extracellular matrix were similar to those observed in primary open-angle glaucoma: decreases in collagen fiber density, presence of basement membranes not associated with cell surfaces, and abundant bundles of microfibrils not labeled with elastin antibody. The elastic fibers appeared normal in other locations within the optic nerves of patients with pseudoexfoliation glaucoma, including in the pial septa and blood vessels of the retrolaminar myelinated optic nerve. Conclusion: The authors' findings demonstrate marked and site-specific elastosis in the lamina cribrosa of patients with pseudoexfoliation syndrome with glaucoma, suggesting an abnormal regulation of elastin synthesis and/or degradation in the optic nerve of patients with this disease. Ophthalmology 1995;102:878-886
Originally received: July 25, 1994. Revision accepted: December 31, 1994. I Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston. 2 Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston. 3 Departments of Ophthalmology and Pathology, State University of New York Health Science Center, Syracuse. Presented in part at the AR VO Annual Meeting, Sarasota, May 1994. Supported in part by grants EY06416 and EYO 1602 from the National Eye Institute, Bethesda, Maryland, the Massachusetts Lions Eye Research Fund, and The Glaucoma Foundation, New York, New York. Each author states that she/he has no proprietary interest in any drug or piece of equipment used in this study.
In pseudoexfoliation syndrome, a characteristic fibrillar protein is deposited in the anterior segment of the eye, which can be observed clinically. Immunoelectron microscopic techniques have shown that this material is associated with elastic microfibrillar-associated glycoproteins, including fibrillin, amyloid P, elastin, and tropoelastin. 1-3 All of these macromolecules are associated with the elastic fiber system. In addition to its location in the globe, pseudoexfoliation material has been identified in the conjunctiva and other extrabulbar tissues}-7 The Reprint requests to Peter A. Netland, MD, PhD, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, 243 Charles St, Boston, MA02l14.
Netland et al . Elastosis of the Lamina Cribrosa presence of pseudoexfoliative fibrillopathy in the skin and visceral organs of patients with pseudoexfoliation syndrome suggests that this is a systemic disease rather than a simple ocular disorder. 8- 11 The incidence of pseudoexfoliation varies in different populations, ranging from near 0% to as high as 93%.12 Glaucoma is more common in eyes containing pseudoexfoliation material than in fellow eyes not containing this material. l3 ,14 Pseudoexfoliation glaucoma is thought to be due to blockage of the trabecular meshwork leading to elevated intraocular pressure. 15 .16 Although the optic nerve head is an important site of damage in pseudoexfoliation glaucoma, little is known about the presence of pseudoexfoliation material or the appearance of extracellular matrix components such as elastin in the lamina cribrosa of eyes with pseudoexfoliation syndrome. Changes of the extracellular matrix have been described in eyes with primary open-angle glaucoma. In the cribriform plate, there is loss of collagen fibers, proliferation of basement membranes, and bundles of micro fibrils not associated with collagen or elastic fibers. 17.18 Elastin is a major component of the extracellular matrix of the human lamina cribrosa.17,18 In primary open-angle glaucoma, there is also disorganization and redistribution of the elastic component of the extracellular matrix, with evidence of elastic fiber degeneration that is apparent in early stages of the disease. 19,20 These changes of the elastic component may explain the loss of compliance and resiliency observed in the lamina cribrosa of eyes with primary open-angle glaucoma. 21 The proliferation and degeneration of elastic elements, termed elastosis, is manifested ultrastructurally by increased production of elastic microfibrils and small elastic fibers, atypical aggregation of these elements, development of an abnormal surrounding matrix, and formation of large bizarre elastic fibers. 5 Conjunctival pingueculae and pterygia are examples of severe elastotic degenerations. 22 ,23 To study the elastic component of the lamina cribrosa of patients with pseudoexfoliation and glaucoma, we examined the optic nerves of two patients, using ultrastructural immunocytochemical techniques.
Materials and Methods Four eyes from two patients with a history of pseudoexfoliation with glaucoma were obtained from the Massachusetts Eye and Ear Infirmary and the Glaucoma Research Foundation (San Francisco, CA). Four pairs of normal age-matched human eyes with no history ofpseudoexfoliation or glaucoma were used for controls. Five pairs of eyes with the diagnosis of primary open-angle glaucoma were obtained through the Glaucoma Research Foundation and the New England Eye Bank. The patients with primary open-angle glaucoma were age-matched with the patients with pseudoexfoliation glaucoma and had a similar stage of glaucoma, with significant visual field defects and cup:disc ratios greater than 0.8. The eyes with primary open-angle glaucoma were treated with topical antiglaucoma medications, with intraocular pressures that
were comparable to the patients with pseudoexfoliation glaucoma. The posterior poles were dissected and fixed from 1 to 10 hours after the time of death. The tissues were fixed in either 10% buffered formaldehyde or a mixture of2.5% glutaraldehyde and 2% formaldehyde in either phosphate or cacodylate buffer (pH 7.3) for 4 to 12 hours. 24.25 Tissue preparation and postembedding immunolabeling were performed as previously described. 17 Postembedding immunocytochemical analysis was done using the immunogold technique. We used antiserum against human aorta alpha-elastin (working dilution, 1: 100; Elastin Products, Owenville, MD), which reacts specifically with human alpha-elastin and tropoelastin and less strongly with elastin from other species. Colloidal gold-labeled immunoglobulin G (Jansen Biotech, Olen, Belgium) was diluted 1:12 with 0.05 mol/l TRIS and 1.5% bovine serum albumin, pH 8.3. All specimens were counterstained in uranyl acetate and lead citrate and examined with a Philips 410 electron microscope (Einhoven, The Netherlands). The area of elastin-labeled material was determined using a digital image analysis system (Biological Detection Systems, version 1.3, Pittsburgh, PA). Electron micrographs were captured on video and digitized, and 250,000 pixel areas were analyzed. The percent area occupied by elastin-labeled material was determined after contrast enhancement. The data were obtained from four electron micrographs of each of four eyes from two patients with pseudoexfoliation glaucoma (n = 16); two to three electron micrographs of each of four eyes of four patients with primary open-angle glaucoma (n = 10); and two to three electron micrographs of each of four eyes from four healthy patients (n = 10). The statistical significance of differences between groups was calculated by the Student's t test.
Case Reports Case 1. A 77-year-old white man was followed on the
Glaucoma Service of the Massachusetts Eye and Ear Infirmary with bilateral pseudoexfoliation syndrome and glaucoma. He had a history of mild dementia, and died after aspiration pneumonia developed. Twelve years before his death, a central retinal artery occlusion developed in the left eye, and subsequently he had light perception vision in that eye. Two years before his death, a branch retinal vein occlusion developed in the right eye, which resolved with no effect on the visual acuity. He was intolerant of topical beta-blocker therapy due to hypotension. Before his death, the visual acuity was 20/80 in the right eye and light perception in the left. The intraocular pressure was 15 mmHg in the right eye and 18 mmHg in the left with treatment of topical epinephrine and 2% pilocarpine in both eyes. There was pseudoexfoliation material bilaterally and a cataract in the right eye, consistent with the level of visual acuity. Results of examination of the disc in the right eye showed advanced glaucomatous optic nerve damage (cup:disc ratio, ~0.8). The visual field of the right eye had showed advanced glaucomatous visual field loss. A dense cataract in the left eye limited the view of the fundus. Case 2. A 77-year-old white woman was followed with bilateral pseudoexfoliation and glaucoma. Metastatic cancer de-
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Figure 1. Elastosis in the lamina cribrosa in pseudoexfoliation glaucoma. A, case 2, left eye. Marked elastosis is present in the lamina cribrosa in an eye with pseudoexfoliation syndrome. B, similar area of an agematched normal eye containing normal elastin. NB = nerve bundles; small arrows = cribriform plates; large arrows = elastotic areas (original magnification, X2250).
veloped, and the patient died of pneumonia. She was intolerant of topical betaxolol due to bronchoconstriction. The patient had been treated with argon laser trabeculoplasty in the left eye. One month before her death, visual acuity was 20/80 in the right eye and 20/50 in the left. The intraocular pressure was 20 mmHg in the right eye and 25 mmHg in the left with treatment of 0.1 % dipivefrin and methazolamide tablets (50 mg 2 times daily). There was bilateral pseudoexfoliation material, and the opacity of the lens was consistent with the level of visual acuity. Results of examination of the discs showed glaucomatous optic nerve damage, worse on the left (cup:disc ratio 0.8 and 0.9 in the right and left eyes, respectively). Automated perimetry showed general
depression bilaterally, with superior and inferior arcuate defects in the left eye.
Results In all eyes with pseudoexfoliation and glaucoma, there was marked elastosis of the connective tissue of the lamina cribrosa (Fig 1). Elastotic fibers appeared as large irregular aggregates of electron-dense material labeled with antialpha-elastin antibody (Fig 2). At higher magnification,
Netland et al . Elastosis of the Lamina Crib rosa
Figure 2. Higher power magnification shows dense elastotic material labeled with elastin antibody in the lamina cribrosa of an eye with pseudoexfoliation glaucoma (A, case 1, left eye), which contrasts with the normal morphology of elastic fibers in the cribriform plates of a normal eye (B) (original magnification, X16,640).
Figure 3. Elastotic material in the lamina cribrosa in pseudoexfoliation glaucoma. High power magnification shows large clumps of elastin-labeled material and microfibrils (m£) in the lamina cribrosa of an eye with pseudoexfoliation glaucoma (case 1, right eye). Notice the sparse collagen fibers (Co) and basement membrane not associated with cell surfaces (asterisk). Arrow indicates the basement membrane of a lamina cribrosa cell (LCC) (original magnification, X26,OOO). Inset, morphology of normal elastic fibers surrounded by· microfibrils, in collagen (Co) of pial septa from the same patient (original magnification, X26,OOO).
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the normal elastic fiber morphology was absent in these anti-elastin-labeled aggregates (Fig 3). Clumps of microfibrils were interspersed in the elastotic aggregates, and there were apparent basement membranes not associated with cell surfaces. As in primary open-angle glaucoma, the collagen matrix appeared loose and there were fewer collagen fibers. We did not observe any pseudoexfoliation fibers in the lamina cribrosa from patients with pseudoexfoliation glaucoma. As shown in Figure 4, the elastosis of the lamina cribrosa was more severe in pseudoexfoliation glaucoma than in primary open-angle glaucoma. Although all eyes with pseudoexfoliation and primary open-angle glaucoma had advanced clinical disease, the eyes with pseudoexfoliation had more widespread elastosis and appeared to have increased amounts of elastotic material. The right and left eyes of the two patients with pseudoexfoliation glaucoma were similar in the degree of elastosis, although there was mild asymmetry of intraocular pressure noted on some clinical visits before death. The electron micrographs were digitized, and the percent of total area due to elastin-labeled material was quantitated using an image analysis system (Biological Detection Systems). The elastin-labeled material in normal eyes was normal-appearing elastic fibers with a characteristic thick, cylindric, tubular morphology of the
Table 1. Percent Total Area Containing Elastin-labeled Material
Pseudoexfoliation glaucoma Primary open-angle glaucoma Control
Mean ± SD
3S.9 ± 9.0
8.1 ± 2.0 2.8 ± 0.7
• Compared with control and primary open-angle glaucoma groups.
Compared with control group.
amorphous component, whereas nearly all of the labeled material in eyes with primary open-angle glaucoma and pseudoexfoliation glaucoma was abnormal-appearing elastotic material. The percentage of the total area containing elastin-labeled material in the lamina cribrosa of eyes with pseudoexfoliation glaucoma was 4.4-fold and 12.8-fold higher than in primary open-angle glaucoma and normal eyes, respectively (Table I). Both primary open-angle glaucoma and pseudoexfoliation glaucoma
Figure 4. Elastin in pseudoexfoliation glaucoma and open-angle glaucoma. A, case 2, right eye. In pseudoexfoliation glaucoma, there is severe elastosis of the lamina cribrosa. B, in an age-matched patient with open-angle glaucoma, there is less elastosis compared with the lamina cribrosa in pseudoexfoliation (original magnification, X6000).
Netland et al . Elastosis of the Lamina Cribrosa
Figure 5. The elastic fibers in the pial septa (A) and the sclera (B) of an eye with pseudoexfoliation glaucoma appear normal (case 1, right eye). Notice the normal cylindrical morphology of the elastic fibers, which are cut in cross-section in SA and longitudinally in 5B. The small septal elastic fibers are surrounded by a fine microfibrillar framework and thin collagen fibrils (original magnification: A, x31,200; B, X26,OOO).
eyes had a significantly greater mean area of elastin-labeled material when compared with the controls (P < 0.05 and P < 0.001, respectively). There was a significantly greater mean area of elastin-labeled material in eyes with pseudoexfoliation glaucoma compared with that in eyes with primary open-angle glaucoma (P < 0.001). In eyes with pseudoexfoliation glaucoma, the elastic fiber morphology in other parts of the eye examined was normal (Fig 5). Elastic fibers in the sclera had a normalappearing thick cylindric shape with a central electronlucent area labeled for alpha-elastin. A delicate framework of microfibrils not labeled for elastin was observed around some of the elastic fibers. Elastic fibers in locations other than the lamina cribrosa in the optic nerve, including the pial septa and blood vessels, were also normal. These elastic fibers were surrounded by dense bundles of normal collagen. When compared with normal eyes without pseudoexfoliation or glaucoma, the elastic fibers in areas other than the lamina cribrosa in eyes with pseudoexfoliation were indistinguishable from normal elastic fibers. The specificity of the immunolabeling was tested by replacing the antiserum with nonimmune rabbit serum or by incubation in buffer without antiserum. In these controls, gold particles were not observed, indicating the specificity ofimmunolabeling (Fig 6). Figure 6 also shows
anti-alpha-elastin labeling closely associated with glial cells in the lamina cribrosa, suggesting production of elastotic material by these cells.
Discussion In human eyes, elastin is a major component of the conjunctival stroma, blood vessels, Bruch membrane, and the lamina cribrosa.1 7,26 Elastin also has been identified in human trabecular meshwork and the eyelid. 27 - 29 Normal elastic fibers are composed of two morphologically distinct components: the central amorphous fiber with an electron-lucent core containing elastin and the microfibrillar component. 30 The microfibrils primarily are located around the periphery of the amorphous core fiber and, to some extent, interspersed within it. Elastosis, due to proliferation and degeneration of elastic elements, is characterized by increased production of microfibrils and elastic fibers, formation of large irregular aggregates of elastin, and abnormalities of the surrounding matrix. 5 In this study, we used specific antibodies against human alpha-elastin and postembedding immunogold labeling to identify the elastic component of the extracellular matrix in the lamina cribrosa of patients with pseudoexfoliation
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Figure 6. Case 1, left eye. Specificity of labeling with immunogold for elastin. A, elastotic areas label with immunogold for elastin. Arrows indicate material in close association with basement membrane of glial cell (0). B, in sections not pretreated with antielastin antibody, the immunogold labeling is absent (original magnification,
glaucoma. In all eyes with pseudoexfoliation and glaucoma, we found marked and widespread elastosis in the connective tissue of the lamina cribrosa. The elastosis observed in eyes with pseudoexfoliation glaucoma was more severe than that in eyes with primary open-angle glaucoma. Changes of elastin in the optic nerve head in primary open-angle glaucoma and experimental glaucoma have been described. 17,20,31 In advanced disease, there is loss of the normal elastic fiber morphology and
aggregate formation with irregular staining and a honeycomb appearance. These changes are similar to the elastosis we observed in pseudoexfoliation glaucoma, but; as indicated by our quantitative image analysis, the elastotic changes are significantly more severe in pseudoexfoliation glaucoma compared with primary open-angle glaucoma (35.9% ± 9.0% and 8.1 % ± 2.0% of total area, respectively; P < 0.001). The similar findings in all eyes with pseudoexfoliation glaucoma indicate that the vascular events
Netland et al . Elastosis of the Lamina Cribrosa described in case 1 (branch vein occlusion in one eye and central retinal artery occlusion in the other) do not account for the marked elastosis that we observed in these patients. In eyes with pseudoexfoliation glaucoma, we also observed decreased collagen content and presence of basement membranes not associated with cell surfaces. These changes are similar to those observed in primary openangle glaucoma. 18 .20,31 ,32 We did not identify any pseudoexfoliative material in the lamina cribrosa, which differs from the close association of pseudoexfoliative material with elastotic fibers in the conjunctiva. 5 In eyes with pseudoexfoliation glaucoma, there were bundles of microfibrils in the lamina cribrosa, some of which stained with immunogold for elastin and had a granular appearance, suggesting increased synthesis of elastin. 33 During elastogenesis, microfibrils containing material that stains positively for elastin appear before formation of the mature elastic fiber in areas adjacent to the elastogenic cells. 34 ,35 Our finding of positively stained microfibrils adjacent to the basement membrane of glial cells suggests that these cells may be the source of the elastotic material in the lamina cribrosa in pseudoexfoliation glaucoma. The elastosis in the lamina cribrosa in pseudoexfoliation glaucoma appeared to be a site-specific abnormality, with normal elastic fibers found in the other locations within the same optic nerves, including the pial septa and blood vessels of the retrolaminar myelinated optic nerve. Elastic fibers were also normal in the sclera of the same eyes with pseudoexfoliation glaucoma. This may represent a pathologic response to cellular injury in the lamina cribrosa, as may be the case with the localized elastosis in pterygium. 22•23 Alternatively, altered gene expression may be responsible for increased synthesis and/or degradation of elastin in pseudoexfoliation. It has been reported that elastin gene expression is upregulated locally in the lamina cribrosa in eyes with primary open-angle glaucoma, leading to abnormal synthesis of elastic fibers. 17 ,36 Perhaps a similar mechanism is responsible for the accumulation of elastotic fibers in the lamina of eyes with pseudoexfoliation glaucoma. Pseudoexfoliation with glaucoma may have a more severe clinical course compared with primary open-angle glaucoma. Patients with pseudoexfoliation glaucoma have higher mean intraocular pressure, more extensive cupping and field defects, and more rapidly progressive glaucoma compared with primary open-angle glaucoma. 37 - 4o Glaucomatous optic nerve damage is more likely to develop in patients with ocular hypertension and pseudoexfoliation than in those without pseudoexfoliation. 13 ,40 Although these differences may be due to higher intraocular pressure in patients with pseudoexfoliation, the abnormalities we observed of elastin in the lamina cribrosa may playa role. Several heritable and acquired diseases are characterized by abnormalities of elastin. 29.30.33 Increased synthesis of elastin and elastin mRNA have been demonstrated in experimental systemic hypertension. 41 In this model, increased synthesis in response to elevated pressure subsides, whereas changes in elastin and extracellular matrix persist
after lowering of blood pressure. It has been suggested that pseudoexfoliation syndrome is a metabolic disorder involving elastogenic cells. II Changes in intraocular pressure in individuals with pseudoexfoliation may induce exaggerated abnormal synthesis and/or degradation of elastin by elastogenic cells in the lamina cribrosa, leading to marked elastosis. These pathologic changes in the elastic component could alter the mechanical properties of the lamina cribrosa significantly. Elastosis of the extracellular matrix in the lamina cribrosa could initiate or increase susceptibility to elevated intraocular pressure, leading to optic nerve damage in patients with pseudoexfoliation. In summary, we found marked elastosis in the connective tissue of the lamina cribrosa in eyes with pseudoexfoliation glaucoma. Other changes ofthe extracellular matrix in the lamina cribrosa were similar to those observed in primary open-angle glaucoma, including decreased collagen fiber density, basement membranes not associated with cell surfaces, and bundles of microfibrils. The elastic fibers appeared normal in the sclera and in other locations within the optic nerves, indicating a sitespecificity of elastotic changes of the lamina cribrosa in pseudoexfoliation glaucoma. These findings may be due to abnormal elastic fiber biosynthesis and/or degradation in the lamina cribrosa in pseudoexfoliation glaucoma.
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