[Skip to Content]
Access to paid content on this site is currently suspended due to excessive activity being detected from your IP address 54.159.189.139. Please contact the publisher to request reinstatement.
[Skip to Content Landing]
Download PDF
Figure 1.
Immunoperoxidase staining for matrix metalloproteinase 1 (MMP-1) in the postlaminar region of the human optic nerve head. There was faint immunostaining for MMP-1 in the cytoplasm of a few glial cells or their processes around the axons of the control optic nerve head (A). However, a greater number of glial cells demonstrated immunostaining for MMP-1 in the eyes with primary open-angle glaucoma (B) or normal-pressure glaucoma (C) (nb, nerve bundles; ps, pial septae) (chromagen, diaminobenzidine tetrahydrochloride, nuclear counterstain with Mayer hematoxylin, original magnification ×100).

Immunoperoxidase staining for matrix metalloproteinase 1 (MMP-1) in the postlaminar region of the human optic nerve head. There was faint immunostaining for MMP-1 in the cytoplasm of a few glial cells or their processes around the axons of the control optic nerve head (A). However, a greater number of glial cells demonstrated immunostaining for MMP-1 in the eyes with primary open-angle glaucoma (B) or normal-pressure glaucoma (C) (nb, nerve bundles; ps, pial septae) (chromagen, diaminobenzidine tetrahydrochloride, nuclear counterstain with Mayer hematoxylin, original magnification ×100).

Figure 2.
Immunoperoxidase staining for matrix metalloproteinase 2 (MMP-2) in the postlaminar region of the human optic nerve head. There was a faint immunostaining for MMP-2 in the cytoplasm of a few glial cells or their processes around the axons of the control optic nerve head (A). However, a greater number of glial cells demonstrated immunostaining for MMP-2 in the eyes with primary open-angle glaucoma (B) or normal-pressure glaucoma (C). Notice the intense immunostaining of glial cell processes in areas of cavernous atrophy and around pial blood vessels in the eye with normal-pressure glaucoma (nb, nerve bundles; ps, pial septae; cs, cavernous spaces; and v, vessels) (chromagen, diaminobenzidine tetrahydrochloride, nuclear counterstain with Mayer hematoxylin, original magnification ×100).

Immunoperoxidase staining for matrix metalloproteinase 2 (MMP-2) in the postlaminar region of the human optic nerve head. There was a faint immunostaining for MMP-2 in the cytoplasm of a few glial cells or their processes around the axons of the control optic nerve head (A). However, a greater number of glial cells demonstrated immunostaining for MMP-2 in the eyes with primary open-angle glaucoma (B) or normal-pressure glaucoma (C). Notice the intense immunostaining of glial cell processes in areas of cavernous atrophy and around pial blood vessels in the eye with normal-pressure glaucoma (nb, nerve bundles; ps, pial septae; cs, cavernous spaces; and v, vessels) (chromagen, diaminobenzidine tetrahydrochloride, nuclear counterstain with Mayer hematoxylin, original magnification ×100).

Figure 3.
Immunoperoxidase staining for matrix metalloproteinases (MMPs) in the postlaminar region of the human optic nerve head. A junctional area between preserved (upper right corner) and severely damaged axons (lower left corner) in the optic nerve head of a patient with normal pressure glaucoma was seen. Immunostaining for MMP-2 (A) or MMP-3 (B) was more intense in the cytoplasm of astroglial cells in the areas of preserved axons compared with the areas of severe atrophy (nb, nerve bundles; ps, pial septae; and cs, cavernous spaces) (chromagen, diaminobenzidine tetrahydrochloride, nuclear counterstain with Mayer hematoxylin, original magnification ×100).

Immunoperoxidase staining for matrix metalloproteinases (MMPs) in the postlaminar region of the human optic nerve head. A junctional area between preserved (upper right corner) and severely damaged axons (lower left corner) in the optic nerve head of a patient with normal pressure glaucoma was seen. Immunostaining for MMP-2 (A) or MMP-3 (B) was more intense in the cytoplasm of astroglial cells in the areas of preserved axons compared with the areas of severe atrophy (nb, nerve bundles; ps, pial septae; and cs, cavernous spaces) (chromagen, diaminobenzidine tetrahydrochloride, nuclear counterstain with Mayer hematoxylin, original magnification ×100).

Figure 4.
Immunoperoxidase staining for matrix metalloproteinase 3 (MMP-3) in the postlaminar region of the human optic nerve head. There was faint immunostaining in a few glial cells around the optic nerve axons of control eyes (A) and increased immunostaining in the processes of glial cells around the axons and in the pial septae of the eyes with primary open-angle glaucoma (B) or normal-pressure glaucoma in areas of cavernous atrophy (C) (nb, nerve bundles; ps, pial septae; cs, cavernous spaces; and v, vessel) (chromagen, diaminobenzidine tetrahydrochloride, nuclear counterstain with Mayer hematoxylin, original magnification ×100).

Immunoperoxidase staining for matrix metalloproteinase 3 (MMP-3) in the postlaminar region of the human optic nerve head. There was faint immunostaining in a few glial cells around the optic nerve axons of control eyes (A) and increased immunostaining in the processes of glial cells around the axons and in the pial septae of the eyes with primary open-angle glaucoma (B) or normal-pressure glaucoma in areas of cavernous atrophy (C) (nb, nerve bundles; ps, pial septae; cs, cavernous spaces; and v, vessel) (chromagen, diaminobenzidine tetrahydrochloride, nuclear counterstain with Mayer hematoxylin, original magnification ×100).

Figure 5.
Immunoperoxidase staining for tumor necrosis factor α (TNF-α) in the human optic nerve head. There was faint immunostaining in the processes of a few glial cells around the nerve bundles and blood vessels (v) in the prelaminar region of the control optic nerve head (A). However, the intensity of the immunostaining and the number of stained glial cells were greater in the optic nerve heads from patients with primary open-angle glaucoma (B) or normal-pressure glaucoma (C). There was intense immunostaining of glial cell processes in areas of cavernous atrophy in the eye with normal-pressure glaucoma (gc, glial column; nb, nerve bundles; and cs, cavernous spaces) (chromagen, diaminobenzidine tetrahydrochloride, nuclear counterstain with Mayer hematoxylin, original magnification ×100).

Immunoperoxidase staining for tumor necrosis factor α (TNF-α) in the human optic nerve head. There was faint immunostaining in the processes of a few glial cells around the nerve bundles and blood vessels (v) in the prelaminar region of the control optic nerve head (A). However, the intensity of the immunostaining and the number of stained glial cells were greater in the optic nerve heads from patients with primary open-angle glaucoma (B) or normal-pressure glaucoma (C). There was intense immunostaining of glial cell processes in areas of cavernous atrophy in the eye with normal-pressure glaucoma (gc, glial column; nb, nerve bundles; and cs, cavernous spaces) (chromagen, diaminobenzidine tetrahydrochloride, nuclear counterstain with Mayer hematoxylin, original magnification ×100).

Figure 6.
Immunoperoxidase staining for tumor necrosis factor α (TNF-α) receptor 1 in the human optic nerve head. Faint immunostaining of the prelaminar region of the optic nerve head was noted for TNF-α receptor 1 in the control optic nerve head (A). Immunostaining was mostly perivascular (v). The intensity of the immunostaining and the number of stained glial cells were greater in optic nerve heads from patients with primary open angle glaucoma (B) or normal pressure glaucoma (C). Nerve bundles in the prelaminar region also exhibited some immunostaining (gc, glial column; nb, nerve bundles) (chromagen, diaminobenzidine tetrahydrochloride, nuclear counterstain with Mayer hematoxylin, original magnification ×100).

Immunoperoxidase staining for tumor necrosis factor α (TNF-α) receptor 1 in the human optic nerve head. Faint immunostaining of the prelaminar region of the optic nerve head was noted for TNF-α receptor 1 in the control optic nerve head (A). Immunostaining was mostly perivascular (v). The intensity of the immunostaining and the number of stained glial cells were greater in optic nerve heads from patients with primary open angle glaucoma (B) or normal pressure glaucoma (C). Nerve bundles in the prelaminar region also exhibited some immunostaining (gc, glial column; nb, nerve bundles) (chromagen, diaminobenzidine tetrahydrochloride, nuclear counterstain with Mayer hematoxylin, original magnification ×100).

Table 1. 
Clinical Data of Postmortem Glaucomatous Eyes*
Clinical Data of Postmortem Glaucomatous Eyes*
Table 2. 
Semiquantitative Evaluation of the Intensity of Immunostaining in Optic Nerve Head*
Semiquantitative Evaluation of the Intensity of Immunostaining in Optic Nerve Head*
1.
Wax  MBTezel  GEdward  DP Clinical and histopathological findings of a patient with normal-pressure glaucoma. Arch Ophthalmol. 1998;116993- 1001Article
2.
Morrison  JCDorman-Pease  MEDunkelberger  GRQuigley  HA Optic nerve head extracellular matrix in primary optic atrophy and experimental glaucoma. Arch Ophthalmol. 1990;1081020- 1024Article
3.
Hernandez  MRAndrzejewska  WMNeufeld  AH Changes in the extracellular matrix of the human optic nerve head in primary open-angle glaucoma. Am J Ophthalmol. 1990;109180- 188
4.
Quigley  HADorman-Pease  MEBrown  AE Quantitative study of collagen and elastin of the optic nerve head and sclera in human and experimental monkey glaucoma. Curr Eye Res. 1991;10877- 888Article
5.
Hernandez  MR Ultrastructural immunocytochemical analysis of elastin in the human lamina cribrosa: changes in elastic fibers in primary open-angle glaucoma. Invest Ophthalmol Vis Sci. 1992;332891- 2903
6.
Varela  HJHernandez  MR Astrocyte responses in human optic nerve head with primary open-angle glaucoma. J Glaucoma. 1997;6303- 313Article
7.
Minckler  DSSpaeth  GL Optic nerve damage in glaucoma. Surv Ophthalmol. 1981;26128- 148Article
8.
Quigley  HAHohman  RMAddicks  EMMassof  RWGreen  WR Morphologic changes in the lamina cribrosa correlated with neural loss in open-angle glaucoma. Am J Ophthalmol. 1983;95673- 691
9.
Okada  YGonoji  YNakanishi  INagase  HHayakawa  T Immunohistochemical demonstration of collagenases and tissue inhibitor of metalloproteinases (TIMP) in synovial lining cells of rheumatoid synovium. Virchows Arch B Cell Pathol Incl Mol Pathol. 1990;59305- 312Article
10.
Woessner  JF  Jr Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J. 1991;52145- 2154
11.
Backstrom  JRMiller  CATokes  ZA Characterization of neural proteinases from Alzheimer-affected and control brain specimens: identification of calcium-dependent metalloproteinases from the hippocampus. J Neurochem. 1992;58983- 992Article
12.
Giraudon  PBuart  SBernard  AThomasset  NBelin  MF Extracellular matrix-remodeling metalloproteinases and infection of the central nervous system with retrovirus human T-lymphotropic virus type I (HTLV-I). Prog Neurobiol. 1996;49169- 184Article
13.
Rosenberg  GANavratil  MBarone  FFeuerstein  G Proteolytic cascade enzymes increase in focal cerebral ischemia in rat. J Cereb Blood Flow Metab. 1996;16360- 366Article
14.
Ridet  JLMalhotra  SKPrivat  AGage  FH Reactive astrocytes: cellular and molecular cues to biological function. Trends Neurosci. 1997;20570- 577Article
15.
Gottschall  PEYu  X Cytokines regulate gelatinase A and B (matrix metalloproteinase 2 and 9) activity in cultured rat astrocytes. J Neurochem. 1995;641513- 1520Article
16.
Gottschall  PEDeb  S Regulation of matrix metalloproteinase expression in astrocytes, microglia and neurons. Neuroimmunomodulation. 1996;369- 75Article
17.
Migita  KEguchi  KKawabe  Y  et al.  TNF-α–mediated expression of membrane-type matrix metalloproteinase in rheumatoid synovial fibroblasts. Immunology. 1996;89553- 557Article
18.
Chandler  SMiller  KMClements  JM  et al.  Matrix metalloproteinases, tumor necrosis factor and multiple sclerosis: an overview. J Neuroimmunol. 1997;72155- 161Article
19.
Apodaca  GRutka  JTBouhana  K  et al.  Expression of metalloproteinases and metalloproteinase inhibitors by fetal astrocytes and glioma cells. Cancer Res. 1990;502322- 2329
20.
Eddleston  MMucke  L Molecular profile of reactive astrocytes: implications for their role in neurologic disease. Neuroscience. 1993;5415- 36Article
21.
Romanic  AMMadri  JA Extracellular matrix-degrading proteinases in the nervous system. Brain Pathol. 1994;4145- 156Article
22.
Nakagawa  TKubota  TKabuto  M  et al.  Production of matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 by human brain tumors. J Neurosurg. 1994;8169- 77Article
23.
Maeda  ASobel  RA Matrix metalloproteinases in the normal human central nervous system, microglial nodules, and multiple sclerosis lesions. J Neuropathol Exp Neurol. 1996;55300- 309Article
24.
Johnson  ECDeppmeier  LMHVarner  ACMorrison  JC Matrix metalloproteinases and TIMP-1 in the primate optic nerve head and retina.  Presented at: the Association for Research in Vision and Ophthalmology Annual Meeting May 5, 1993 Sarasota, Fla.
25.
Emi  KSawaguchi  SYue  BHara  HFukuchi  TIwata  K Increased levels of matrix metalloproteinase in the optic nerve head of monkey eyes with laser-induced glaucoma.  Presented at: the Association for Research in Vision and Ophthalmology Annual Meeting May 6, 1993 Sarasota, Fla.
26.
Sawaguchi  SFukuchi  THanyu  J  et al.  Matrix metalloproteinase were over-expressed in the optic nerve heads of experimental primate glaucoma.  Presented at: the Association for Research in Vision and Ophthalmology Annual Meeting May 12, 1998 Fort Lauderdale, Fla.
27.
Raff  MC Glial cell diversification in the rat optic nerve. Science. 1989;2431450- 1455Article
28.
Radany  EHBrenner  MBesnard  FBigornia  VBishop  JMDeschepper  CF Directed establishment of rat brain cell lines with the phenotypic characteristics of type 1 astrocytes. Proc Natl Acad Sci U S A. 1992;896467- 6471Article
29.
Ye  HHernandez  MR Heterogeneity of astrocytes in human optic nerve head. J Comp Neurol. 1995;362441- 452Article
30.
Hernandez  MRPena  JD The optic nerve head changes in glaucomatous optic neuropathy. Arch Ophthalmol. 1997;115389- 395Article
31.
Nordstrom  LALochner  JYeung  WCiment  G The metalloproteinase stromelysin-1 (transin) mediates PC12 cell growth cone invasiveness through basal laminae. Mol Cell Neurosci. 1995;656- 68Article
32.
Schwartz  MCohen  AStein-Izsak  CBelkin  M Dichotomy of the glial cell response to axonal injury and regeneration. FASEB J. 1989;32371- 2378
33.
Muir  D Metalloproteinase-dependent neurite outgrowth within a synthetic extracellular matrix is induced by nerve growth factor. Exp Cell Res. 1994;210243- 252Article
34.
Hernandez  MRIgoe  FNeufeld  AH Extracellular matrix of the human optic nerve head. Am J Ophthalmol. 1986;102139- 148Article
35.
Rosenberg  GAKornfeld  MEstrada  EKelley  ROLiotta  LAStetler-Stevenson  WG TIMP-2 reduces proteolytic opening of blood-brain barrier by type IV collagenase. Brain Res. 1992;576203- 207Article
36.
Liu  TClark  RKMcDonnell  PC  et al.  Tumor necrosis factor-α expression in ischemic neurons. Stroke. 1994;251481- 1488Article
37.
Barone  FCArvin  BWhite  RF  et al.  Tumor necrosis factor-α: a mediator of focal ischemic brain injury. Stroke. 1997;281233- 1244Article
38.
Hsu  HXiong  JGoeddel  DV The TNF receptor 1-associated protein TRADD signals cell death and NF-kappa B activation. Cell. 1995;81495- 504Article
39.
Lin  XKashima  YKhan  MHeller  KBGu  XSadun  AA An immunohistochemical study of TNF-α in optic nerves from AIDS patients. Curr Eye Res. 1997;161064- 1068Article
40.
Romero  LITatro  JBField  JAReichlin  S Roles of IL-1 and TNF-alpha in the endotoxin-induced activation of nitric oxide synthase in cultured rat brain cells. Am J Physiol. 1996;270R326- R332
41.
Goureau  OAmiot  FDautry  FCourtois  Y Control of nitric oxide production by endogenous TNF-alpha in mouse retinal pigmented epithelial and Muller glial cells. Biochem Biophys Res Commun. 1997;240132- 135Article
42.
Heneka  MTLoschmann  PAGleichmann  M  et al.  Induction of nitric oxide synthase and nitric oxide–mediated apoptosis in neuronal PC12 cells after stimulation with tumor necrosis factor alpha/lipopolysaccharide. J Neurochem. 1998;7188- 94Article
43.
Neufeld  AH Nitric oxide: a potential mediator of retinal ganglion cell damage in glaucoma. Surv Ophthalmol. 1999;43(suppl 1)S129- S135Article
44.
Neufeld  AHSawada  ABecker  B Inhibition of nitric-oxide synthase 2 by aminoguanidine provides neuroprotection of retinal ganglion cells in a rat model of chronic glaucoma. Proc Natl Acad Sci U S A. 1999;969944- 9948Article
Laboratory Sciences
May 2000

Matrix Metalloproteinases and Tumor Necrosis Factor α in Glaucomatous Optic Nerve Head

Author Affiliations

From the Departments of Ophthalmology and Visual Sciences, University of Illinois at Chicago (Drs Yan and Edward) and Washington University School of Medicine, St Louis, Mo (Drs Tezel and Wax). The authors have no proprietary interest in any of the materials used in this study.

Arch Ophthalmol. 2000;118(5):666-673. doi:10.1001/archopht.118.5.666
Abstract

Objective  To study expression and location of matrix metalloproteinases (MMPs) and tumor necrosis factor α (TNF-α) in glaucomatous optic nerve heads, which are known to be secreted in response to a variety of neuronal injury.

Methods  Four postmortem eyes from patients with primary open-angle glaucoma, 7 eyes from patients with normal-pressure glaucoma, and 4 eyes from age-matched normal donors were studied by immunohistochemistry. The sections of the optic nerve heads were examined after immunostaining with antibodies to MMPs (MMP-1, MMP-2, and MMP-3), TNF-α, or TNF-α receptor 1.

Results  The intensity of the immunostaining and the number of stained cells for MMPs, TNF-α, or TNF-α receptor 1 were greater in the glaucomatous optic nerve heads, particularly in eyes with normal-pressure glaucoma compared with age-matched controls. Positive immunostaining was observed in all regions of the glaucomatous optic nerve heads, but most prominently in the postlaminar region. Immunostaining was observed mainly in glial cells and their processes around the axons and blood vessels and in pial septae.

Conclusion  There is increased immunostaining for MMPs, TNF-α and TNF-α receptor 1 in the glaucomatous optic nerve head, which suggests increased expression of these proteins in glaucoma and thereby implies a role in the tissue remodeling and degenerative changes seen in glaucomatous optic nerve heads.

Clinical Relevance  The MMPs and TNF-α may be components of astroglial activation that occurs in glaucomatous optic nerve heads. The biological alterations in the expression of these proteins may play a role in the progression of glaucomatous optic neuropathy.

HISTOPATHOLOGIC studies of the glaucomatous optic nerve head in primary open-angle glaucoma (POAG) reveal astroglial activation and tissue remodeling, which accompanies neuronal damage. As a part of tissue remodeling, backward bowing and disorganization of the laminar cribriform plates are common characteristics of glaucomatous eyes with either normal or high intraocular pressure.1 These histologic changes are accompanied by the up-regulation of extracellular matrix components, including collagen and proteoglycan, and adhesion molecules by optic nerve head astrocytes in glaucomatous eyes.26 The astroglial activation seen in glaucomatous optic nerve heads likely represents an attempt to limit the extent of the injury and promote the tissue repair process. However, despite the astroglial activation, there is limited deposition of extracellular matrix in glaucomatous optic nerve atrophy, which does not retain characteristics of scar tissue formation.7,8 This suggests that there are diverse cellular responses to the initial event or subsequent tissue injury, which preferentially results in tissue degradation.

Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade components of extracellular matrix. Increased secretion of MMPs by activated glial cells have been implicated in various extracellular matrix remodeling events that occur during normal development and in a number of pathologic processes, including atherosclerosis, arthritis, tumor growth, and metastasis.913 In addition, reactive astrocytes after neuronal injury produce various neurotrophic factors and cytokines, including tumor necrosis factor α (TNF-α),14 which play a critical role in the regulation of the synthesis of MMPs.1517 Furthermore, the release of TNF-α from its membrane-bound precursor is a MMPs-dependent process.18

Recently, we reported the histopathologic features of a pair of eyes with normal-pressure glaucoma (NPG) that demonstrated Schnabel optic atrophy.1 In addition to the eyes presented in the report, our histopathologic examinations in an additional 5 postmortem eyes from patients with NPG revealed Schnabel cavernous atrophy. Like eyes with POAG, the eyes with NPG exhibited bowing of the lamina cribrosa, areas of glial activation, and axonal atrophy. However, the remarkable histopathologic distinctions between the eyes with NPG or POAG were the large cavernous spaces in the optic nerve head, extensive loss of pial septae, and the absence of a fibroglial response. These findings suggested that one possible mechanism for the cavernous degeneration might be an exaggerated astroglial response to degrade extracellular matrix by protelytic enzymes such as the MMPs.

In this immunohistochemical study, antibodies against MMPs (MMP-1, MMP-2, and MMP-3), TNF-α, and TNF-α receptor 1 were used to label optic nerve head sections from postmortem eyes with POAG or NPG and from age-matched normal donors. Our observations suggest that there is increased expression of MMPs, TNF-α, and TNF-α receptor 1 in the glaucomatous optic nerve head, particularly with NPG, which likely contributes to the tissue remodeling seen in glaucomatous optic neuropathy.

PATIENTS AND METHODS
PATIENTS

Four postmortem human eyes with a diagnosis of POAG and 7 human eyes with a diagnosis of NPG were obtained. The age of patients ranged from 68 to 84 years. Table 1 outlines the clinical findings that were available from glaucomatous eyes. Four human donor eyes with no history of eye disease were used as age-matched controls (age range, 61-81 years). The death-to-fixation time for the specimens ranged between 6 and 9 hours.

IMMUNOHISTOCHEMISTRY

After enucleation, all eyes were fixed in 10% neutral buffered formalin for 24 hours, dehydrated in graded alcohol, and embedded in paraffin. Since some of the specimens contained only the optic nerve head and small portions of the peripapillary retina, retinal distribution of immunostaining was not studied. After deparaffinization, 5-µm-thick longitudinal sections of optic nerve heads were incubated with monoclonal antibodies against MMP-1, MMP-2, or MMP-3 (2.5 µg/mL) (Oncogene Science, Cambridge, Mass) or polyclonal antibodies against TNF-α or TNF-α receptor 1 (2 µg/mL) (R &D Systems, Minneapolis, Minn) overnight at 4°C, after endogenous peroxidase was blocked with 2% hydrogen peroxide in methanol and followed by several washes in phosphate-buffered saline solution. The 3 anti-MMP antibodies recognized both latent as well as active forms of MMPs. Prior to incubation with primary antibodies, the sections were incubated with either mouse skin extract (during MMP staining) or 20% nonimmune donkey serum (during TNF-α and TNF-α receptor 1 staining) for 20 minutes to block background staining. Biotinylated secondary antibody (anti-mouse or anti-goat IgG) (Dako Corp, Carpinteria, Calif) was applied to the sections for 30 minutes at room temperature. The slides were then incubated with horseradish peroxidase–labeled streptavidin solution (Dako Corp) for 30 minutes, and the reaction was visualized by incubation in a solution of 0.02% 3,3′-diaminobenzidine tetrahydrochloride and 0.006% hydrogen peroxide in 0.05M Tris-HCl (pH, 7.6). The slides were lightly counterstained with Mayer hematoxylin. Sections incubated with mouse serum or phosphate-buffered saline solution in place of the primary antibody served as negative controls. Sections from biopsy specimens of infiltrating ductal breast carcinoma served as a positive control for all antibodies used in this study.

Three to 5 sections from each optic nerve head were examined by immunohistochemistry for each protein including MMPs, TNF-α, and TNF-α receptor 1. To obtain comprehensive semiquantitative evaluation of the immunostaining, the intensity of immunostaining for MMPs and TNF-α and its receptor in the prelaminar, laminar, and postlaminar regions of the optic nerve head was graded using an arbitrary score in which each region was graded from − to +++. A semiquantitative score (indicated in parentheses) was then calculated for each optic nerve head (−, absent [0]; ±, ranging from absent to weak [0.5]; +, weak staining [1]; ++, moderate staining [2]; +++, strong staining [3]). The grading of the immunostaining was performed in a masked fashion by an observer who was skilled in grading immunohistochemical staining but was not familiar with the pathologic changes in the optic nerve head. The observer graded the intensity of immunostaining in optic nerve head regions (prelaminar, laminar, and postlaminar) that were pointed out by one of the authors (X.Y.). Both the scored results and the photographs of representative sections from each group are presented.

RESULTS

The normal eyes exhibited glial columns and nerve bundles in the prelaminar region when observed by light microscopy. In the lamina cribrosa, there were glial cells lining the collagenous laminar beams; in the postlaminar region, the glial cells were mainly distributed along the pial septae and were also scattered among the axonal bundles.

The glaucomatous eyes either with POAG or NPG demonstrated backward bowing of the lamina cribrosa and axonal atrophy. The degree of the laminar bowing was comparable in the eyes with POAG or NPG. The degree of axonal atrophy was mild to moderate in the eyes with POAG and was especially noted in the postlaminar region. In the eyes with NPG, the axonal atrophy was moderate in most eyes and characterized with focal loss in the areas of cavernous degeneration. In 1 eye with NPG, severe axonal loss was noted through the optic disc cup, with axonal preservation in more peripheral areas. The postlaminar region of the optic nerve head in the eyes with POAG demonstrated mild disorganization of the pial septae without tissue destruction. These changes were uniformly consistent in all eyes with POAG. In contrast, the eyes from the patients with NPG exhibited varying stages of Schnabel cavernous degeneration, which was evident mainly at the lamina cribrosa and the postlaminar optic nerve. Axonal atrophy accompanied multifocal destruction of the cribriform laminar plates or pial septae within the cavernous degeneration areas seen in the eyes with NPG. In some eyes, the areas of degeneration coalesced to form large cavernous spaces. In the areas of preserved axons, the arrangement of laminar plates and pial septae remained intact and the distribution of glial cells remained unchanged.

Examinations of the optic nerve heads using immunohistochemistry revealed that the intensity of the immunostaining and the number of stained cells for MMPs, TNF-α, or TNF-α receptor 1 were greater in the glaucomatous optic nerve heads, particularly with NPG, when compared with age-matched controls. The immunolabeling seemed to be mainly in cells that resembled astrocytes by morphology. The semiquantitative scores of the immunostaining in samples examined are presented in Table 2. Since Table 2 reflects the changes in the intensity of immunostaining but not the number of stained cells, photographs are also presented to optimally reflect changes that occur in glaucomatous optic nerve heads. Immunostaining patterns of optic nerve heads for MMPs, TNF-α, and TNF-α receptor 1 are also given below.

MMP-1

In normal eyes, faint immunostaining for MMP-1 was observed in the cytoplasm of a few glial cells, located mostly in the laminar and postlaminar regions of the optic nerve head. Faint staining was also noted around the axons or in the pial septae.

Although the intensity of immunostaining for MMP-1 was similar in all regions of the optic nerve head of glaucomatous eyes and in control eyes, the number of positively stained glial cells was greater in glaucomatous eyes, either with NPG or POAG. In addition, in glaucomatous eyes, immunostaining was positive around the axons in the postlaminar region (Figure 1).

MMP-2

In normal eyes, the prelaminar, laminar, and postlaminar regions of the optic nerve head exhibited faint immunostaining for MMP-2 in a few glial cells and around the axons.

However, both the intensity of the immunostaining and the number of stained glial cells were moderately increased in the prelaminar region of the optic nerve head, as well as along the laminar beams, in the glaucomatous eyes with either NPG or POAG. In the postlaminar region, positive immunostaining was seen around the axons and around the pial blood vessels in the glaucomatous eyes, which was more prominent in the eyes with NPG compared with those with POAG. The MMP-2 labeling in the eyes with NPG was also noted along the degenerating laminar plates and pial septae lining the cavernous spaces as well as within the astrocytes in these structures (Figure 2). In the postlaminar region of some eyes with NPG, areas of axonal preservation were seen adjacent to areas of severe axonal atrophy. In the areas of preserved axons, intracytoplasmic MMP immunolabeling was more intense than seen in the glial cells located in the areas of severe atrophy (Figure 3, A).

MMP-3

In the normal eyes, faint immunostaining for MMP-3 was observed in rare glial cells and around the axons in the all regions of the optic nerve head.

In the glaucomatous eyes, immunostaining of glial cells and their processes around the axons for MMP-3 demonstrated an increase in the all regions of the optic nerve head compared with controls. The increase in the labeling intensity was particularly evident in the glial cells and along the axons and pial septae in the eyes with NPG (Figure 4). Perivascular anti–MMP-3 immunostaining was also noted along the pial septae. In the eyes with NPG, glial cells located in the areas of preserved axons demonstrated more intensive immunostaining compared with the cells located in the areas of cavernous atrophy (Figure 3, B).

TNF-α

In control eyes, there was faint immunostaining for TNF-α and TNF-α receptor 1 in the processes of a few glial cells and around the nerve bundles and blood vessels of the optic nerve head.

In glaucomatous optic nerve heads, both the intensity of immunostaining and the number of stained cells for TNF-α or TNF-α receptor 1 were increased in all regions of the glaucomatous optic nerve head compared with controls. Immunostaining was positive in glial cells around the axons and vessels in the prelaminar and laminar regions of the optic nerve head in the glaucomatous eyes. In the postlaminar region, the glial cells distributed along the pial septae and scattered among the nerve bundles exhibited immunostaining. Although immunostaining for TNF-α was mostly associated with glial cells, an increased immunostaining for TNF-α receptor 1 was also observed in the nerve bundles, which was prominent in the prelaminar region of the glaucomatous optic nerve heads (Figure 5 and Figure 6).

COMMENT

The integrity and turnover of the extracellular matrix are influenced by many factors, including MMPs. The MMPs are a family of proteolytic enzymes secreted by glial cells, and are capable of degrading almost all components of the extracellular matrix. The MMPs have been divided into the following 3 broad families based on their domain structure and substrate specificity. (1) Interstitial collagenase (MMP-1) and neutrophil collagenase (MMP-8) belong to the collagenase family; their major substrates are fibrillar collagen types I, II, and III. (2) The enzymes MMP-2 and MMP-9 are members of the gelatinase family; their substrates include types IV and V collagen, and fibronectin, proteoglycans, and gelatin. (3) Members of the stromelysin family include MMP-3 (stromelysin, transin) and MMP-7 (matrilysin); they act on a wide range of substrates, including proteoglycans, laminin, fibronectin, gelatin, and procollagen precursor peptides.15,1923

Although they are implicated in several diseases of the central nervous system,1113 little is known about the role of MMPs in either normal or glaucomatous human optic nerves. The localization of MMP-3 and MMP-2 and tissue inhibitor of metalloproteinases (TIMP-1) have been shown to be present in the normal primate optic nerve head and retina.24 In addition, increased gelatinase activity has been found in glaucomatous monkey eyes.25,26 Our observation of the mild MMP immunolabeling of the glial cells in normal optic nerve heads and increased immunolabeling of MMPs in glaucomatous eyes is consistent with these limited studies.

Our observations revealed that the intensity of immunostaining for MMPs, TNF-α, and TNF-α receptor 1 was greater in glaucomatous optic nerve heads compared with controls. In addition, differential immunostaining patterns for these proteins were noted in the prelaminar, laminar, and postlaminar regions of the optic nerve head. Some of these differential patterns included the most prominent labeling of MMPs in the postlaminar region and the most prominent labeling of TNF-α and TNF-α receptor 1 in the prelaminar region of the glaucomatous optic nerve heads. One possible explanation of these findings may be based on the recently described regional and functional heterogeneity of glial cells in the optic nerve head. For example, the size and the density of type 1B astrocytes in the prelaminar and laminar regions, and the type 1A astrocytes in the postlaminar region, are greater in glaucomatous eyes than in normal tissue.2729

Increased immunostaining of MMPs was noted in the cytoplasm of astroglial cells and their processes as well as in the extracellular matrix of optic nerve head in the eyes with POAG or NPG. The distribution of increased immunostaining for MMPs in the different regions of optic nerve head was comparable in the eyes with POAG or NPG. However, the intensity of immunostaining for MMPs, especially for MMP-2, was greater in the eyes with NPG compared with the eyes with POAG. In the eyes with NPG, immunostaining along the pial septae was moderately increased in the region of cavernous degeneration.

Cells secrete MMPs in an inactive form and the proenzyme can be activated in the extracellular space by various molecules. The antibodies used to recognize MMPs in this study identify both MMP precursors and the proteolytically processed active forms. Therefore, immunohistochemistry cannot distinguish the functional state in which the MMPs are present within the tissue. The abundance of immunoreactivity in the astrocytes suggests the presence of a large pool of intracellular MMPs that might function, under normal conditions, at relatively low levels in the extracellular space. Such pools could possibly be rapidly activated to act on substrates in the extracellular matrix under pathologic conditions.23

The generalized increase in the expression of MMPs in the glaucomatous optic nerve head may have various consequences. Since MMPs are responsible for the degradation of the extracellular matrix components, their increased expression in the glaucomatous optic nerve head may represent a physiological response to counteract the increased extracellular matrix deposition that occurs in glaucomatous optic nerve head.30 This may explain the absence of glial scar tissue in glaucomatous optic nerves despite astroglial activation. It is tempting to speculate that tissue degeneration resulting from increased MMP activity may in part account for the excavated appearance of optic disc cupping that accompanies glaucomatous optic neuropathy, regardless of other factors such as intraocular pressure.

Matrix metalloproteinases have been proposed to play a role in axonal growth by preventing scar tissue formation in vivo,31,32 which is thought to be a barrier to trophic substances necessary for neuronal regeneration.33 Therefore, our observation of prominent immunostaining for MMPs in the areas of preserved axons may signify that activated glial cells increase secretion of MMPs for the dual purposes of preventing scar tissue formation while simultaneously promoting neuronal growth.

The pial septae of the normal optic nerve contains collagen types III and IV and fibronectin mainly around the blood vessels.34 These are the major substrates of MMP-2 and MMP-3. The increased immunostaining of MMP-2 and MMP-3 in the astrocytes and along the pial septae in the glaucomatous optic nerve head suggests that these MMPs may play a role in the disruption of pial septa seen in the areas of cavernous degeneration.

In addition, we observed increased expression of MMP-2 in the astrocytic processes enveloping blood vessels in the glaucomatous optic nerve head, particularly in the eyes with NPG. Since MMP-2 causes a thinning of the basal lamina and an increase in the capillary permeability,35 it seems possible that increased expression of MMPs in the perivascular area may influence the blood-brain barrier in this area.

Another finding we observed was increased immunostaining of TNF-α and TNF-α receptor 1 in the glaucomatous optic nerve heads either with POAG or NPG. Tumor necrosis factor α is a potent immunomediator and proinflammatory cytokine that is rapidly up-regulated in the brain after injury.36,37 It is also known as an inducer of apoptotic cell death via TNF-α receptor 1 occupancy.38 Tumor necrosis factor α has been implicated in the pathogenesis of several diseases of the nervous system, such as multiple sclerosis and autoimmune encephalomyelitis; it has also been thought to account for axonal degeneration and glial changes observed in the optic nerves of patients with acquired immunodeficiency syndrome.39 Although our studies demonstrated that the TNF-α immunostaining was mostly positive in the glial cells of the optic nerve head, TNF-α receptor 1 immunostaining was more prominently positive in nerve bundles located in the prelaminar section of the optic nerve head, which was increased in the glaucomatous eyes. This observation suggests that neuronal tissue is an important target for the effects of TNF-α. Our findings that the expression of TNF-α and MMPs are both increased in the glaucomatous optic nerve head is not surprising, since it is well known that there are interactions between TNF-α and MMPs for the regulation of their secretion and function.1418 Therefore, increased expression of TNF-α in the glaucomatous optic nerve head suggests that this cytokine may play a role in tissue remodeling as a part of the astroglial activation process and/or may participate in tissue injury.

In addition to its potential to directly activate the cell death cascade in retinal ganglion cells and to facilitate remodeling of the optic nerve head in glaucoma, TNF-α may also contribute to the pathogenesis of glaucomatous neuropathy, as it is a potent stimulator of nitric oxide synthesis.4042 Recent evidence suggests that up-regulation of nitric oxide synthase occurs in human and experimental glaucomatous eyes.43 Furthermore, pharmacological inhibition of nitric oxide synthase–2 was shown to decrease ganglion cell death in an experimental animal model of glaucoma.44 Therefore, blockade, amelioration, or attenuation of retinal or optic nerve head TNF-α may have therapeutic potential in treating patients with glaucoma. Such compounds could effectively inhibit, reduce, or prevent nitric oxide synthase–related ganglion cell death, which may be an important causal factor in glaucoma.

CONCLUSIONS

Increased expression of MMPs, TNF-α, and TNF-α receptor 1 may be collective components of the astroglial activation process that occurs in the glaucomatous optic nerve head. They may serve to prevent scar tissue formation and thus facilitate neuronal viability and repair. However, their increased expression may also have a role in the degenerative process of glaucomatous optic neuropathy as a result of facilitating formation of cavernous spaces, cupping, and the progression of neuronal damage.

Back to top
Article Information

Accepted for publication December 8, 1999.

This study was supported in part by the Otsuka Research Fellowship from the American Glaucoma Society, San Francisco, Calif, and gifts from the Laura K. Binder Fund and Pondill Glaucoma Research Fund, Chicago, Ill (Dr Edward), institutional core grants EY001792 (Dr Edward) and EY012314 (Dr Wax) from the National Eye Institute, Bethesda, Md, a grant from the Glaucoma Foundation, New York, NY (Dr Tezel), and an unrestricted grant to Washington University School of Medicine, Department of Ophthalmology and Visual Sciences, St Louis, Mo, from Research to Prevent Blindness Inc, New York, NY.

We thank David Brocato, ASCP, and Belinda McMahan for preparing the histopathologic sections.

Corresponding author: Deepak P. Edward, MD, Department of Ophthalmology and Visual Sciences, University of Illinois, 1855 W Taylor St, Room 217, Chicago, IL 60612 (e-mail: deepedwa@uic.edu).

References
1.
Wax  MBTezel  GEdward  DP Clinical and histopathological findings of a patient with normal-pressure glaucoma. Arch Ophthalmol. 1998;116993- 1001Article
2.
Morrison  JCDorman-Pease  MEDunkelberger  GRQuigley  HA Optic nerve head extracellular matrix in primary optic atrophy and experimental glaucoma. Arch Ophthalmol. 1990;1081020- 1024Article
3.
Hernandez  MRAndrzejewska  WMNeufeld  AH Changes in the extracellular matrix of the human optic nerve head in primary open-angle glaucoma. Am J Ophthalmol. 1990;109180- 188
4.
Quigley  HADorman-Pease  MEBrown  AE Quantitative study of collagen and elastin of the optic nerve head and sclera in human and experimental monkey glaucoma. Curr Eye Res. 1991;10877- 888Article
5.
Hernandez  MR Ultrastructural immunocytochemical analysis of elastin in the human lamina cribrosa: changes in elastic fibers in primary open-angle glaucoma. Invest Ophthalmol Vis Sci. 1992;332891- 2903
6.
Varela  HJHernandez  MR Astrocyte responses in human optic nerve head with primary open-angle glaucoma. J Glaucoma. 1997;6303- 313Article
7.
Minckler  DSSpaeth  GL Optic nerve damage in glaucoma. Surv Ophthalmol. 1981;26128- 148Article
8.
Quigley  HAHohman  RMAddicks  EMMassof  RWGreen  WR Morphologic changes in the lamina cribrosa correlated with neural loss in open-angle glaucoma. Am J Ophthalmol. 1983;95673- 691
9.
Okada  YGonoji  YNakanishi  INagase  HHayakawa  T Immunohistochemical demonstration of collagenases and tissue inhibitor of metalloproteinases (TIMP) in synovial lining cells of rheumatoid synovium. Virchows Arch B Cell Pathol Incl Mol Pathol. 1990;59305- 312Article
10.
Woessner  JF  Jr Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J. 1991;52145- 2154
11.
Backstrom  JRMiller  CATokes  ZA Characterization of neural proteinases from Alzheimer-affected and control brain specimens: identification of calcium-dependent metalloproteinases from the hippocampus. J Neurochem. 1992;58983- 992Article
12.
Giraudon  PBuart  SBernard  AThomasset  NBelin  MF Extracellular matrix-remodeling metalloproteinases and infection of the central nervous system with retrovirus human T-lymphotropic virus type I (HTLV-I). Prog Neurobiol. 1996;49169- 184Article
13.
Rosenberg  GANavratil  MBarone  FFeuerstein  G Proteolytic cascade enzymes increase in focal cerebral ischemia in rat. J Cereb Blood Flow Metab. 1996;16360- 366Article
14.
Ridet  JLMalhotra  SKPrivat  AGage  FH Reactive astrocytes: cellular and molecular cues to biological function. Trends Neurosci. 1997;20570- 577Article
15.
Gottschall  PEYu  X Cytokines regulate gelatinase A and B (matrix metalloproteinase 2 and 9) activity in cultured rat astrocytes. J Neurochem. 1995;641513- 1520Article
16.
Gottschall  PEDeb  S Regulation of matrix metalloproteinase expression in astrocytes, microglia and neurons. Neuroimmunomodulation. 1996;369- 75Article
17.
Migita  KEguchi  KKawabe  Y  et al.  TNF-α–mediated expression of membrane-type matrix metalloproteinase in rheumatoid synovial fibroblasts. Immunology. 1996;89553- 557Article
18.
Chandler  SMiller  KMClements  JM  et al.  Matrix metalloproteinases, tumor necrosis factor and multiple sclerosis: an overview. J Neuroimmunol. 1997;72155- 161Article
19.
Apodaca  GRutka  JTBouhana  K  et al.  Expression of metalloproteinases and metalloproteinase inhibitors by fetal astrocytes and glioma cells. Cancer Res. 1990;502322- 2329
20.
Eddleston  MMucke  L Molecular profile of reactive astrocytes: implications for their role in neurologic disease. Neuroscience. 1993;5415- 36Article
21.
Romanic  AMMadri  JA Extracellular matrix-degrading proteinases in the nervous system. Brain Pathol. 1994;4145- 156Article
22.
Nakagawa  TKubota  TKabuto  M  et al.  Production of matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 by human brain tumors. J Neurosurg. 1994;8169- 77Article
23.
Maeda  ASobel  RA Matrix metalloproteinases in the normal human central nervous system, microglial nodules, and multiple sclerosis lesions. J Neuropathol Exp Neurol. 1996;55300- 309Article
24.
Johnson  ECDeppmeier  LMHVarner  ACMorrison  JC Matrix metalloproteinases and TIMP-1 in the primate optic nerve head and retina.  Presented at: the Association for Research in Vision and Ophthalmology Annual Meeting May 5, 1993 Sarasota, Fla.
25.
Emi  KSawaguchi  SYue  BHara  HFukuchi  TIwata  K Increased levels of matrix metalloproteinase in the optic nerve head of monkey eyes with laser-induced glaucoma.  Presented at: the Association for Research in Vision and Ophthalmology Annual Meeting May 6, 1993 Sarasota, Fla.
26.
Sawaguchi  SFukuchi  THanyu  J  et al.  Matrix metalloproteinase were over-expressed in the optic nerve heads of experimental primate glaucoma.  Presented at: the Association for Research in Vision and Ophthalmology Annual Meeting May 12, 1998 Fort Lauderdale, Fla.
27.
Raff  MC Glial cell diversification in the rat optic nerve. Science. 1989;2431450- 1455Article
28.
Radany  EHBrenner  MBesnard  FBigornia  VBishop  JMDeschepper  CF Directed establishment of rat brain cell lines with the phenotypic characteristics of type 1 astrocytes. Proc Natl Acad Sci U S A. 1992;896467- 6471Article
29.
Ye  HHernandez  MR Heterogeneity of astrocytes in human optic nerve head. J Comp Neurol. 1995;362441- 452Article
30.
Hernandez  MRPena  JD The optic nerve head changes in glaucomatous optic neuropathy. Arch Ophthalmol. 1997;115389- 395Article
31.
Nordstrom  LALochner  JYeung  WCiment  G The metalloproteinase stromelysin-1 (transin) mediates PC12 cell growth cone invasiveness through basal laminae. Mol Cell Neurosci. 1995;656- 68Article
32.
Schwartz  MCohen  AStein-Izsak  CBelkin  M Dichotomy of the glial cell response to axonal injury and regeneration. FASEB J. 1989;32371- 2378
33.
Muir  D Metalloproteinase-dependent neurite outgrowth within a synthetic extracellular matrix is induced by nerve growth factor. Exp Cell Res. 1994;210243- 252Article
34.
Hernandez  MRIgoe  FNeufeld  AH Extracellular matrix of the human optic nerve head. Am J Ophthalmol. 1986;102139- 148Article
35.
Rosenberg  GAKornfeld  MEstrada  EKelley  ROLiotta  LAStetler-Stevenson  WG TIMP-2 reduces proteolytic opening of blood-brain barrier by type IV collagenase. Brain Res. 1992;576203- 207Article
36.
Liu  TClark  RKMcDonnell  PC  et al.  Tumor necrosis factor-α expression in ischemic neurons. Stroke. 1994;251481- 1488Article
37.
Barone  FCArvin  BWhite  RF  et al.  Tumor necrosis factor-α: a mediator of focal ischemic brain injury. Stroke. 1997;281233- 1244Article
38.
Hsu  HXiong  JGoeddel  DV The TNF receptor 1-associated protein TRADD signals cell death and NF-kappa B activation. Cell. 1995;81495- 504Article
39.
Lin  XKashima  YKhan  MHeller  KBGu  XSadun  AA An immunohistochemical study of TNF-α in optic nerves from AIDS patients. Curr Eye Res. 1997;161064- 1068Article
40.
Romero  LITatro  JBField  JAReichlin  S Roles of IL-1 and TNF-alpha in the endotoxin-induced activation of nitric oxide synthase in cultured rat brain cells. Am J Physiol. 1996;270R326- R332
41.
Goureau  OAmiot  FDautry  FCourtois  Y Control of nitric oxide production by endogenous TNF-alpha in mouse retinal pigmented epithelial and Muller glial cells. Biochem Biophys Res Commun. 1997;240132- 135Article
42.
Heneka  MTLoschmann  PAGleichmann  M  et al.  Induction of nitric oxide synthase and nitric oxide–mediated apoptosis in neuronal PC12 cells after stimulation with tumor necrosis factor alpha/lipopolysaccharide. J Neurochem. 1998;7188- 94Article
43.
Neufeld  AH Nitric oxide: a potential mediator of retinal ganglion cell damage in glaucoma. Surv Ophthalmol. 1999;43(suppl 1)S129- S135Article
44.
Neufeld  AHSawada  ABecker  B Inhibition of nitric-oxide synthase 2 by aminoguanidine provides neuroprotection of retinal ganglion cells in a rat model of chronic glaucoma. Proc Natl Acad Sci U S A. 1999;969944- 9948Article
×