Trabecular meshwork changes in glaucoma

Bernadette E Orsida, BSc, DipImm" Jennifer M Rolland, BSc, PhD* Jerome A Werkmeister, BSc, PhD' Robert H West, FRCS, FRACO+

Glaucoma causes visual impairment in approximately 1% of the population over the age of 40 years. It is characterised by an abnormal increase in intraocular pressure (IOP) associated with temporary or permanent tissue damage. In most cases of glaucoma, the elevated IOP is due to obstruction of the aqueous humour outflow, usu- ally at the angle of the anterior chamber of the eye, i.e. in the region of the trabecular meshwork (TM). Treatment of the angle structures by appli- cation of laser energy (laser trabeculoplasty) is often an effective measure to facilitate aqueous outflow and thus to reduce IOP.' However, the manner in which the pathological changes in glau- coma, and the changes induced by laser treat- ment, alter the aqueous outflow facility is not yet adequately understood.

The TM is a sponge-like tissue composed of connective tissue trabeculae lined by endotheli- um, and normally functions as a one-way valve, a resistor that maintains IOP, a pressure sensor, and a selective filter.' Immunohistochemical studies have shown the connective tissue components to include a variety of extracellular matrix macro- molecules (ECM) - collagens, non-collagenous glycoproteins and glycosaminoglycans. However, there are conflicting reports on the changes in ECM components accompanying glaucoma,S~" presumably due to the use of different detection systems and tissue preparation. Age-associated changes in T M connective tissue is an additional confounder to these studies.

A more complete understanding of the patho- genesis of glaucoma requires additional elucida- tion of the cell types normally producing ECM components in the T M and of the complex net- work of cytokines and growth factors which reg- ulate this process.7 Thus, the objectives of this study were to investigate altered expression of extracellular matrix molecules in the T M of glaucoma patients compared with normal, and to determine whether these changes were associ- ated with alterations in particular cell popula- tions and cytokines. In addition, the effects of laser treatment on reversal of glaucoma-associ- ated changes in the T M were assessed.

Methods

Eye specimens

TM specimens ( n = 19) were surgically removed from glaucoma patients at the Alfred Hospital, Coonara Private Hospital and RoyalVictorian Eye and Ear Hospital. Based on clinical diagnosis, the types of glaucoma were classified as primary open- angle glaucoma (n = 12; six laser-treated), nor- mal-tension glaucoma (n = 2; one laser treated), pseudoexfoliative glaucoma ( n = 2; one laser treated), corticosteroid-induced glaucoma ( n = l), inflammatory (secondary) glaucoma ( T Z = 1) and congenital glaucoma ( p i = 1). Corneoscleral button specimens were obtained through the eye bank at the Royal Victorian Eye and Ear Hospital from normal donors ( n = 7)

* Monask Medizal SL,hool, Alfed Hospital, Melbourne, I'icioria. # CSIRO, Division of Biornolerular Engineering, Afelbourne, I lirtoria. f i l l jred Hospiral anti University of hfdbourne Departments of Ophthalmology, hlzlbourne, T ktoria. Correspondence: Dr RH West, 5 17 St Klda Road, Melbourne 3004, Victoria.

Trabecular meshwork changes in glaucoma 21

within a few hours of donor death. The mean age + SD for the glaucoma patients was 65.5 + 17.5 years, compared with 76.9 + 6.9 years for the nor- mal donors.

Specimen preparation and inz~zzunohistochenzzstry

Tissues were snap-frozen and 5 pm sections placed on to poly-L-lysine-coated glass slides. A representative section from each tissue block was stained with Quick Dip (Histo Labs, Australia) to check for correct orientation and morphology. Sections were fixed either with paraformaldehyde-lysine-periodate (PLP) or, for cytokines, acetone and stained by indirect immunoperoxidase for various ECM compo- nents, cell markers and cytokines.

ECM antibodies used included monoclonal antibodies against human collagen type I , x 111," V1 and fibrillin-like protein prepared by one of us (JAW), human collagen type IV (Dako, Denmark), laminin, fibronectin (Silenus, Australia), lteratan sulphate, elastin (Sigma Chemicals, USA), and polyclonal goat antibod- ies against human collagen type V (Southern Biotechnology Associates, USA). Cell marker antibodies included monoclonal antibodies a g a in s t C D 4 5 (pa n -1 e u c o c y t e m a r k e r ; Department of Medicine, Monash University, Australia) and CD68 (macrophage and neu- trophil cytoplasmic granules; Dako, Denmark), and polyclonal rabbit antibodies against human von Willebrands factor (endothelial cells; Dako, Denmark). Cytokines were detected using mon- oclonal antibodies against human interferon- gamma (IFN-y; Genzyme, USA) and human basic fibroblast growth factor (bFGF; Upstate Biotechnology Inc., USA), and polyclonal rabbit antibodies against human tumour necrosis fac- tor-alpha (TNF-a; Southern Biotechnology Associates, USA). Antibodies were used at pre- determined optimal dilutions and times, with appropriate blocking and diluent solutions for each system. Antibody binding to the sections was visualised using appropriate secondary anti- bodies labelled with biotin, followed by either streptavidin horse radish peroxidase (Dako, Denmark) or Vectastain ABC reagent (Vector Labs, USA) and DAB as substrate (giving a brown reaction product). For detection of colla- gen type V, before staining, sections were pre- treated with 0.1 M acetic acid for 30 minutes at room temperature in order to swell the type V collagen molecules (normally masked by type I

collagen) and thus allow antibody access to epitopes.'" After staining, sections were counterstained with Harris' haematoxylin and coverslipped. Negative controls included appro- priate immunoglobulin isotype and diluent controls. A standard slide of human slun stained with collagen type VI antibody was included in each run to ensure consistency of staining.

Staining assessment

Staining was assessed independently by two individuals with a light microscope. The intensi- ty of immunoperoxidase staining was graded arbitrarily from negative, -, to strongly positive, +++. The statistical significance of dif- ferences between staining for each clinical group was determined by x' analysis.

Results Processing of the very small specimens of TM excised from glaucomatous eyes presented a con- siderable technical challenge. Careful attention to tissue orientation had to be paid, and optimisation of fixation and staining procedures required extensive preliminary testing. The use of frozen sections with mild fixation was necessary to retain antigenicity of all the tissue markers being studied, with consequent poorer tissue morphology than would have been obtained with formalin-fixed, paraffin-embedded specimens. This resulted in fragmentation of the T M in many cases and thus a difficulty in quantitation of staining intensity using automated image analysis of a standard area. Thus, a semi-quantitative assessment was made visually, with the average of two indepen- dent readings being determined. Despite these limitations, some statistically significant differ- ences in the expression of ECM components were found when normal TM was compared with glau- comatous TM.

The T M beams from normal donor eyes stained strongly for collagen types 111, V and VI, with weaker staining for collagen types I and IV. There was also weak staining for fibrillin-like pro- tein, fibronectin, laminin and keratan sulphate. No staining for elastin was observed in the T M for normal or glaucomatous specimens. When glauco- matous T M was compared with normal TM, increased keratan sulphate staining was noted in the glaucoma specimens (x' = 5.99; P < 0.05) (Table 1). Furthermore, T M from laser-treated glaucomatous eyes showed decreased keratan sul-

22 Australian and New Zealand Journal of Ophthalmology Supplement 1996; 24(2)

Table 1. Keratan sulphate staining of trabecular beams. The number of specimens with a particular staining intensity for each clinical group is shown. -~ _ _ ~ - ~

Staining Intensity - .~ ~ ~ ~

(+I + + (+I ++ ++(+ +++ Clinical group 1 - -~ ~ ~~ - -~ Normal 1 6 Glaucoma.

POAG 3 4 2 1

other types not laser

treated . .

2 3 1 2

1 4

*Chi squared analysis: intensity ++ and above compared with less than ++. 'All glaucoma or POAG TM compared with normal TM. *Laser treated ghucoma compared with not laser treated glaucoma.

phate staining compared with those not laser treated (x' = 4.23; P < 0.05). While sample sizes were too small for comparison of different types of glaucoma, these general trends in keratan sulphate staining were seen for all types studied. With respect to other ECM components, a significant decrease in collagen 111 staining was observed in POAG specimens compared with normals (x' = 3.92; P < 0.05), and there was a trend for increased fibrillin-like protein and fibronectin staining.

To investigate the underlying mechanisms for these ECM changes, the cell types and their cytokine products in the T M were also investigat- ed. Only a few specimens showed appreciable numbers of CD45 positive or CD68 positive cells, and there was no apparent relationship between these cell numbers and clinical condition. Furthermore, the endothelial cells lining the tra- becular beams proved difficult to characterise. Although endothelial cells lining Schlemms canal and corneal vessels stained with von Willebrands factor, T M endothelial cells did not. The most striking staining of these cells was with bFGF anti- body. Strong staining for bFGF was seen on the beam edges of all normalTM specimens, with sig- nificantly reduced staining of glaucomatous T M (x' = 5.80; P < 0.05; Table 2). Other cytokines tested for their relevance to ECM production and breakdown included IFN-y and TNF-a. However, little staining for these cytokines was observed; a few specimens showed some rounded cells stain- ing for IFN-y in the TM region, and some cases had staining of TNF-a on T M edges, but no dif- ferences between normal and glaucomatous eyes were seen.

1 2

1

2

- ~~

Significance of difference*

-

x' = 6 . 4 7 , P < 0.05+

Discussion

We have demonstrated changes in expression of ECM components in glaucomatous TM com- pared with normal TM. That these changes were not due to the normal ageing process was indi- cated by the use of age-matched normal controls. As reported b y others,'," we found strong stain- ing for collagen types II1,V and VI in the normal TM with weaker staining for collagen types I and IV. Other non-collagenous components (fibrillin- like protein, laminin and fibronectin) and a gly- cosaminoglycan (keratan sulphate) were also detected in normal TM, but elastin was not detected. Ultrastructural analysis by others revealed elastin in normal and glaucomatous TM," suggesting that further studies using dif- ferent antibodies and tissue processing should be pursued.

The most notable change observed in ECM components in glaucoma was increased keratan sulphate. It is likely that altered sulphated proteo- glycan content in the region of the T M contributes to increased resistance of the outflow system in glaucoma, but the parallel observation of decreased collagen I11 indicates that the mecha- nisms for regulation of ECM deposition are com- plex. One such regulatory factor is suggested by our finding of decreased bFGF in glaucomatous TM. This cytokine appeared to be localised to the endothelial cells lining the trabecular spaces, but to determine whether it had been synthesised by these cells or was bound to ECM components at these sites, further analysis such as by in-situ hybridisa- tion for bFGF mRNA expression is required. Of further interest is the effect of laser treatment on

Trabecular meshwork changes in glaucoma 23

Table 2. of specimens with a particular staining intensity for each clinical group is shown.

Basic fibroblast growth factor staining of trabecular beam edges. The number

Clinical group

- _ _ _ _ _ _ _ _ _ _ _ _ _ _ - _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - - - ~ _ - _ _ _ _ _ _ _ _ _ - _ _ _ _ _ _ - _ _ _ Normal 3 3 1 Glaucoma

POAG 2 3 3 1 1 x = 4 96, P < 0 05i

other 1 1 1 all 3 4 4 1 1 x = 5 80,

P < 0 05' Glaucoma laser 2 1 2

treated not laser 1 3 2 1 1

treated

*Chi squared analysis intensity ++ and above compared with less than ++ iAll glaucoma or POAG T M compared with normal T M

_ _ _ _ - _ - - _ _ _ _ _ _ _ _ _ _ _ _ _ _ - - - - _ - _ _ _ _ _ _ _ _ _ _ _ _ ~ - _ - - _ _ _ _ _ _ _ _ _ - _ _ _ - - ~ _ _ _ _ _

these changes. Although specimens from laser- treated eyes in this study necessarily comprised those where additional surgery was required to improve clinical condition, an indication of reversal of the glaucoma-associated changes in ECM was seen, especially for keratan sulphate.

The precise role of different cell types and cytokines in the regulation of T M composition and thus function awaits further study. In agree- ment with others," we found few leukocytes in the tissues studied, suggesting that resident endothelial cells and fibroblasts may be the key cell types involved. Elucidation of these events together with development of cytokine-targeted therapy will provide new prospects for non-sur- gical treatment for glaucoma.

Acknowledgements Supported by the Alfred Hospital Foundation and the Glaucoma Investigation and Research Unit, Royal Victorian Eye and Ear Hospital, Victoria.

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Australian and New Zealand Journal of Ophthalmology Supplement 1996; 24(2)