A C T A O P H T H A L M O L O G I C A V O L . 5 5 1 9 7 7

Ttie Tennent Institute of Ophthalmology, (Head: W. S . Foulds),

Ttie University of Glasgow, Glasgow, U . K .

THE APPEARANCE OF THE OUTFLOW APPARATUS OF THE EYE AFTER STAINING WITH RUTHENIUM RED

BY

IAN GRIERSON, WILLIAM R. LEE and SHAHIDA ABRAHAM

The outflow apparatus from adult baboon and rabbit eyes was stained with the inorganic dye ruthenium red. The ruthenium reaction product coated the surface of the trabecular meshwork cells and the canalicular endothelial cells. Deposits also impregnated the various connective tissue elements within the trabeculae and the extracellular spaces of the endo- thelial meshwork. A fine fibrillar network could also be identified with ruthenium red and this was present in the trabecular cores and the extra- cellular spaces of the endothelial meshwork. It was considered that the fibrillar network may represent a matrix of glycosaminoglycans and glyco- proteins. The significance of these materials in relation to aqueous outflow was discussed.

K e y words: transmission electron microscopy - baboon - rabbit - outflow apparatus - ruthenium red - glycoproteins - glycosaminoglycans.

The present ultrastructural investigation was conducted to determine the pattern of ruthenium red staining in the baboon and rabbit outflow apparatus. Ruthe- nium red stains extracellular materials and is considered to have a particular affinity for glycosaminoglycans and glycoproteins (Luft 1964, 1971a,b).

Materials and Methods

The eyes from 5 adult baboons (Papio anubis) weighing between 16.0 and 25.0 kg and 5 adult pigmented dutch rabbits weighing between 1.8 and 2.5 kg provided the material for this study. After enucleation, each eye was bisected,

Received May 13, 1977.

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I a n Grierson, W i l l i a m R. Lee and Sliahida A b r a h a m

the lens excised and segments of limbal tissue were removed. Subsequently, the majority of the tissue was immersed in 2.5 O/O glutaraldehyde buffered with 0.2 M sodium cacodylate which contained ruthenium red. The concentration of ruthenium red in the primary fixative was varied between 300 and 1000 p. p. m. The remaining segments of “fresh” limbal tissue were incubated at 37OC for periods between 30 min and 3 h in either a) buffered testicular hyaluronidase (B.D.H. Laboratories) of various activties between 100 and 2000 IU per ml or b) hyaluronidase inactivated by boiling. Thereafter the incubated tissues were also immersed in buffered glutaraldehyde which contained ruthenium red.

The segments of limbal tissue were trimmed down to blocks with a thickness of approximately 1 mm and then left for 2 to 12 h in the primary fixative solution. Subsequently, the blocks were washed in cacodylate buffer, post-fixed for at least 4 h in 2 O/O buffered osmium tetroxide and then rewashed in caco- dylate buffer. Each solution contained the appropriate concentration of ruthe- nium red.

Fig. 1. Uveal trabecula from the outflow system of the baboon which has been treated with 1000 p .p .m. of ruthenium red. A rich electron-dense deposit of ruthenium red is adherent to the “free” or apical surface (arrow) of the endothelium on the trabecula.

Uranyl acetate section staining (x 10 000).

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The Outflow Apparatus

F i g . 2. Process connections which link adjacent trabeculae in the corneoscleral meshwork of the baboon. The tissue has been treated with 800 p. p. m. of ruthenium red. Deposits of

electron-dense reaction product fill the intercellular spaces. No section staining (x 100 000).

Following fixation, impregnation and washing, the tissue was dehydrated through graded alcohols, cleared with propylene oxide and embedded in Aral- dite. The staining procedure which we adopted was adapted from that outlined by Luft (1964, 1971a,b). Thin sections (600-800 A) were cut on an L.K.B. Ultrotome I11 and viewed in a Phillips 300 or 301 electron microscope either without subsequent section staining or after 10 min exposure to uranyl acetate solution.

Results

The ruthenium red reaction product was identified as an electron-dense homo- genous material within the tissue blocks. However, even with a relatively loose structure like the trabecular meshwork, the effective penetration of the stain was limited to a few microns beyond the cut surfaces of each segment. There- after the distribution of the stain was patchy and deposits of ruthenium red were seldom found a t the centre of a 1 mm thick block. The investigation was therefore restricted to the periphery of the tissue blocks where a constant, regular and reproducable staining reaction was present.

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Ion Grierson, Wi l l i am R. Lee and Shahida Abraham

An electron-dense coating of ruthenium red covered the surface of the trabe- cular meshwork cells and canalicular endothelial cells in both the baboon and the rabbit. As well as forming a deposit on “free” cell surfaces (Fig. I ) , ruthe- nium red penetrated intercellular spaces fairly readily (Fig. 2). The thickness

F i g . 3. Meshwork cells from the baboon treated with 400 p. p. m. of ruthenium red. a) At this concentration, the surface layer on the meshwork cells is thin and discontinuous but there is selective staining for b) micropinosomes and c) the viable space (arrow) within the gap junctions which modify the intercellular clefts. Uranyl acetate section staining

((a) x 120 000, (b) x 150 000, and (c) x 220 000).

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The Outflow Apparatus

Fig. 4. Part of a baboon corneoscleral trabecula stained with ruthenium red (1000 p. p. m.). The basement material (BM), elastic-like substances (E) and the collagen (arrow) are electron dense. In longitudinal sections, the periodicity in the collagen and the clumps of elastic-like material is emphasised. The insert shows collagen fibrils from a rabbit trabecula stained with ruthenium red (800 p. p. m.) and cut in cross section. Each fibril is surrounded with a coating of electron-dense reaction product. No subsequent section

staining (x 54 000 insert x 100 000).

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Ian Grierson, Will iam R. Lee and Shahida Abraham

and intensity of the electron-dense surface coat varied with the concentration of ruthenium in the fixative solutions. At 300 and 400 p. p. m. the ruthenium deposit was patchy (Figs. 3 a and b), whereas with stronger solutions of up to 800 p. p. m. a regular and continuous deposit was observed (Fig. 2). At the highest concentration (1000 p. p. m.) the electron-dense material formed a dif- fuse layer on the surfaces of the cells (Figs. 1 and 4).

F i g . 5. Shows some of the extracellular constituents of a trabecular core in a rabbit which are stained with ruthenium red (800 p. p. m.). Collagen fibrils can be seen both in cross section (small arrows) and in longitudinal sections (large arrows). A network of fibrillar

material connects the various collagen fibrils. Uranyl acetate section staining (x 45 000).

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The Outflow Afifiaratus

Fig. 6. The network of electron dense material in the extracellular spaces of the endothelial

meshwork after ruthenium red treatment (400 p. p. m.). Baboon tissue, no section staining ( x 100 000).

With low concentrations of ruthenium red, there was preferential staining for the interspaces of the gap junctions and maculae adhaerentes which were sometimes found between adjoining baboon and rabbit meshwork cells (Fig. 3 c). In addition, deposits were found selectively on the limiting membranes of micro- pinosomes where these vesicles opened onto the surface of meshwork and canal endothelial cells (Fig. 3 b).

Ruthenium red increased the electron density of the connective tissue elements in the trabeculae (Fig. 4). The basement layer had an increased electron density and, generally, had a more granular appearance than in conventionally stained material. When the elastic-like material and the collagen of the trabecular core was cut longitudinally, ruthenium red emphasised the banding pattern in both materials. In cross section, the elastic-like substance and each collagen fibril was seen to be surrounded by an electron-dense deposit of ruthenium red (Fig. 4). The various connective tissue elements in the trabeculae were sometimes seen to be connected by a web or network of fine filamentous material (Fig. 5 ) .

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Ian Griersim, Will iam R . Lee and Shaliida Abraham

The extracellular spaces of the endothelial meshwork had a particular affinity for ruthenium and this fine-grain deposit demarcated an even more delicate network of interconnecting filaments than that present within the trabeculae (Fig. 6). The network extended between neighbouring cells and enmeshed the aggregates of collagen, curly collagen and elastic like material. T o see the net- work to its best effect, the concentration of ruthenium red in the fixative solu- tion had to be low otherwise the fine filaments were obscured by electron dense stain deposits.

Ruthenium formed a surface coat on the endothelial cells which surrounded Schlemm’s canal. The layer was thicker on the luminal than the trabecular aspect of the endothelium. The material also covered the limiting membrane of giant vacuoles and penetrated the lateral cell borders to the level of the intercellular junctions.

The chemical nature of the ruthenium positive material which was present in the outflow apparatus was not determined. After incubating blocks of fresh tissue in active hyaluronidase, the staining reaction was less pronounced but, in addition, incubation in boiled hyaluronidase also produced an effective re- duction of the subsequent staining reaction. I t would appear that either a) the pretreatments had adverse effect on the staining mechanism or b) the hyaluroni- dase molecule in its deactivated form had a “masking” effect.

Discussion

Luft (1964, 197la,b) proposed that ruthenium red binds to the polysaccharide moieties of glycosaminoglycans and glycoproteins but the evidence presented by this author was somewhat circumstantial and the precise nature of the reac- tion remains obscure. In the present study ruthenium red was used to stain the outflow apparatus in both the baboon and the rabbit. Although enzymatic treatment was found to be unhelpful, the similarity of the staining pattern prcduced in the outflow apparatus with ruthenium compared to that produced by other cationic staining systems (Segawa 1970; Armaly & Wang 1975; Grier- son & Lee 1975) makes Luft’s proposals seem reasonable.

Ruthenium red has the particular advantage a) of fine resolution and accurate location and b) its staining reaction takes place at physiological p H levels. The major disadvantage of the material is its extremely poor penetration into tissue blocks. Because effective staining is limited to only a few microns of surface tissue, comparative investigations using this dye are fraught with difficulty.

Recently, Segawa (1975) has shown that an extremely intense ruthenium staining reaction can be produced in the endothelial meshwork of patients suf- fering from primary open angle glaucoma. The staining reaction in these patients

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T h e Outflow Apparatus

appeared to be more extensive than that found in either the baboon or the rabbits of the present study. Segawa found that the ruthenium positive material was f a r more sensitive to chondroitinase ABC than to streptomycetes hyaluroni- dase, therefore the material was thought to be sulphated polysaccharides rather than hyaluronic acid. I t must be borne in mind that Segawa used trabeculectomy specimens as the main source of his tissue. Cellular damage due to surgical manipulation is a hazard association with trabeculectomy specimens (Lee & Grierson 1974) and the possibility exists that some of the increased staining could be due to extruded cytoplasmic polysaccharides rather than extracellular glycosaminoglycans (Luft 197 la) .

Probably the most significant findings of the present investigation is the demonstration of a delicate fibrillar network i n the trabecular and endothelial meshwork. I t is considered that this may be a framework of glyccsaminoglycans or glycoproteins. Such a framework would be of particular importance in the extracellular spaces of the endothelial meshwork which are the narrowest and most tortuous portion of the outflow pathway. T h e fine polysaccharide mesh would help to bind cellular and extracellular elements together, have a selective filtration effect on the passage of solute molecules and, because the polymers a re strongly hydrophilic they will bind water into the tissues (Ogston 1970). Thus the distribution and density of the mucopolysaccharides will influence both the general conductance of aqueous humour through the system and the direction of fluid movement to the canal endothelium.

Acknowledgments

The investigation was supported by grants 359 and 442 from the Scottish Hospitals Endowments Research Trust. This assistance is gratefully acknowledged. We wish to thank Miss 0. M. Rankin for her secretarial assistance.

References

Armaly M. F. & Wang Y. (1975) Demonstration of acid mucopolysaccharides in the

Grierson I. & Lee W. R. (1975) Acid mucopolysaccharides in the outflow apparatus.

Lee W. R. & Grierson I. (1974) Relationships between intraocular pressure and the

Luft J. H. (1964) Electron microscopy of cell extraneous coats as revealed by ruthenium

Luft J. H. (1971a) Ruthenium red and violet. I Chemistry, purification, methods of use

trabecular meshwork of the rhesus monkey. Invest . Ophthal. 14, 507-516.

Exp . E y e Res. 21, 417-431.

morphology of the outflow apparatus. Trans. ophthal. SOC. U . K . 94, 430-449.

red staining. J. Cel l Biol. 23, 54a.

for electron microscopy and mechanism of action. Anat. Rec. 171, 347-368.

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Ian Grierson, Will iam R. Lee and Shaliida Abraham

Luft J. H. (1971b) Ruthenium red and violet. I1 Fine structural localisation in animal tissues. A n d . Rec. 171, 369-416.

Ogston A. G. (1970) The biological functions of the glycosaminoglycans. In: Chemistry and Molecular Biology of the Intercelliilar Matrix. (Balazs E. A,, Ed.). Vol. 3, p. 1231. Academic Press, London.

Segawa K. (1970) Localisation of acid mucopolysaccharides in the human trabecular meshwork. J . clin. Oplithal. (Jut.) 24, 363-367.

Segawa K. (1975) Ultrastructural changes of the trabecular tissues in primary open angle glaucoma. Jab. J . Ophtlial. 19, 317-338.

Antlzor’s address: Ian Grierson, The Tennent Institute of Ophthalmology, University of Glasgow, Church Street, Glasgow, G11. GNT, U. K.

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