pathological dilemmas in the outflow system in primary open-angle glaucoma

ACTA OPHTHALMOLOGICA SCANDJNAVICA 1997 -

Pathological dilemmas in the outflow system in primary open-angle glaucoma Ian Grierson’, Ashraf Swalem2, Helen Davies’, Penny Hogg’, Mark Batterbury’ and Peter Watson3

Department of Ophthalmology’, University of Liverpool, U.K., Department of Ophthalmology*, University of Mansoura, Egypt, Department of Ophthalmology3, Addenbrooke’s Hospital, Cambridge, U.K.

he outflow system in glaucoma, T particularly primary open-angle glaucoma (POAG), has been the sub- ject of intensive research over the years. However, despite the efforts of laboratory technicians, ocular pathologists and clinical ophthalmologists, a thorough understanding of the drainage pathobiology associated with POAG remains elusive. We still do not know precisely how compromise of the outflow system in chronic disease leads to elevated intraocular pressure COP).

able material. Although trabeculectomy specimens are abundant they are small and may be unrepresentative of a patchy pathology. In addition, specimens are frequently traumatised by surgical removal and an anterior approach may remove only a limited amount of relevant material or none at all; indeed, it has long been known that removal of meshwork tissue is not a re- quirement for surgical success (Taylor 1976). Moreover, trabeculectomy is generally performed following the failure of maximal medical therapy when the disease is in an advanced stage and thus specimens tell us little The outflow system

The conventional outflow route, via the trabecular meshwork and Schlemm’s canal (Fig. l), is thought to account for about 90% of the drainage of aqueous humour from the normal human eye (Bill & Phillips 1971). In POAG the intrinsic resistance of the trabecular meshwork is elevated, leading to raised IOP, pathology in the retinal nerve fibre layer and cupping of the optic nerve head. However, the initiat- ing change in the outflow system (i.e. that which precipitates the downward spiral leading to elevated resistance) remains shrouded in controversy.

One problem is that investigations have been hampered by a lack of suit-

about the early precipitating factors. Post-mortem glaucomatous eyes have rarely been available for examination but with the development of glaucoma tous eye donor schemes in various countries, particularly the US, this is no longer the case. However, the absence of suitable material is only one of the factors that has limited our progress towards a meaningful understanding of the disease process in the outflow system.

Another problem, perhaps even more important, is how researchers have focused on the dilemma of ident- ifying the key event in outflow disease.

Key words: trabecular meshwork - Schlemm’s canal - juxtacanalicular connective tissue.

Fig. 1. Light micrograph of a semithin section of the human aqueous humour outflow system, showing trabecular meshwork 0, scleral spur (S) and Schlemm’s canal (arrows) ( X 350).

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Fig. 2. Part of the inner wall of Schlemm’s canal showing the juxtacanalicular connective tissue (A), canal endothelium (B) and the canal (C). The JCT comprises a network of cells, between which are a coarse matrix (of extracellular materials including collagen and plaque material) and a fine matrix (predominantly glycosaminoglycans). The vacuolar pore system (single arrow) is the main route by which aqueous humour passes from the JCT to the canal.

Because the early lesion is subtle it has been argued that it must arise in that part of the outflow system that offers most resistance to flow in order to have any effect on IOP, as a subtle change at a site with low resistance would have a negligible impact on IOP. Consequent- ly, anatomists, pathologists, physiolog- ists, biochemists, immunochemists and mathematicians expended considerable efforts to identify the site where aqueous flow is most impeded.

It has long been thought that the bulk of resistance to outflow occurs on the meshwork side of Schlemm’s canal whereas very little resistance is associated with the collector channels (through which aqueous humour flows from Schlemm’s canal to the episcleral venous system and the subconjunctival vessels). Using a micropuncture tech- nique to measure pressure drop in various regions of the outflow system, Maepea & Bill (1989) calculated a

Fig. 3. Transmission electron micrograph of part of the juxtacanalicular connective tissue which is rich in plaque material (indicated by arrows); the trabeculectomy specimen was taken from a patient with primary open-angle glaucoma who was on maximal medical therapy prior to surgery (x 10,000).

figure of 90% for the precanalicular (i.e. meshwork) contribution to resistance and 10% for the postcanalicular (collector channels, etc.) contribution. However, on the precanalicular side, neither the uveal portion of the trabecular meshwork (next to the chamber angle) nor the corneoscleral meshwork (which constitutes the bulk of the trabecular tissue) are high resistance zones (Inomata et al. 1972; Maepea & Bill 1989, 1992). The wall on the trabecular side of Schlemm’s canal consists of a lining epithelium on top of a loose connective tissue called the juxtacanalicular connective tissue (JCT). The endothelium is leaky and con- tributes little to outflow resistance (Grierson et al. 1979; Eriksson & Svedbergh 1980) (although this has been questioned) (Ethier et al. 1995), so by default the JCT is considered the area of high resistance. Aqueous humour must pass through narrow channels formed by the extracellular spaces between JCT cells. However, the main factor which determines resistance is not the size and shape of JCT cells but the presence of extracellular matrix materials, particularly glycosaminoglycans (GAGS), that retard aqueous humour flow (Fig. 2) (Kamm et al. 1983; Seiler & Wollensak 1985).

The JCT in POAG As the JCT is considered the most important site of resistance in the normal eye (Inomata et al. 1972; Grierson et al. 1979; Eriksson & Svedbergh 1980; Kamm et al. 1983; Seiler & Wollensak 1985; Maepea & Bill 1989, 1992) it has been intensively investigated in order to identify the earliest changes ‘n POAG. In medically controlled ’OAG, marked alterations to the JCT ire surprisingly difficult to find (Tripa- hi 1977). However, quantitative inves- igations have shown that the elastic ICT fibre network (often called plaque naterial (Lutjen-Drecoll et al. 1986)) hickens with ageing and that the

changes are even more pronounced in POAG (Alvarado et al. 1986; Lutjen- Drecoll et al. 1986); it is the surround- ing sheath, rather than the electron- dense core, which significantly in- creases in ageing and disease (Fig. 3) (Lutjen-Drecoll et al. 1986). It appears

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that enlargement of plaque material is part of the natural history of POAG rather than secondary to the pro- tracted use of topical antiglaucoma medication. There was no significant difference in the JCT plaque material in trabeculectomy specimens taken from patients where surgery was the first form of treatment compared with those where surgery followed maximal medical therapy; however, in both types of specimens a significant in- crease in plaque material was evident in comparison with age-matched normal tissue

However, no clear relationship between IOP on the day of surgery and plaque accumulation has been found in the small number of specimens from patients who underwent early surgery (Fig. 4). As plaque material abounds in advanced POAG and also in the underperfused meshwork (Rohen et al. 1993) it may be that plaque accumulation does not cause IOP elevation but is merely a consequence of localised poor tissue perfusion. Furthermore, when computer models are used to determine the effect of alterations to the elastic network and other coarse framework materials on outflow resistance in the glaucomatous JCT, their influence is - at best - marginal (Ethier et al. 1986).

Coarse framework materials such as elastic tissue and collagen have little influence on bulk flow. In contrast, fine materials such as GAGs and glycopro- teins are thought to have a much greater influence on flow as they trap large amounts of water in their rnolecu- lar domains. Unfortunately these sub- stances are notoriously labile and are difficult to retain and stain within the tissue (Carreras et al. 1992). Some in- vestigators have located GAGs in the normal and ageing JCT but few at- tempts have been made to study them in POAG. Segawa (1979) stained GAGs in trabeculectomy specimens using ruthenium red and provided quantitative evidence (using electron microscopy) that GAGs were particularly abundant in POAG.

In a similar study, transmission electron microscopic montages were made of the central one third of the drainage wall of Schlemm’s canal from 25 POAG specimens and 10 controls (Swalem 1991). GAGs were visualised

b

: * 0

0 . * * * *

0

IS M 25 30 35 40 45 M

Fig. 4. Scattergram of plaque material in the juxtacanalicular connective tissue against intraocular pressure at the time of surgery from 29 patients with primary open-angle glaucoma treated by primary surgery (trabeculectomy). There was an upward trend but no significant correlation.

IOP (mmHg)

using ruthenium red staining (Fig. 5 ) and quantified using a 100-mesh rec- tangular overlay similar to that de- scribed by Lutjen-Drecoll et al. (1986) and Rohen et al. (1993). The overlay procedure was adopted in preference

Fig. 5. Transmission electron micrograph of the ruthenium red-stained fine material of the extracellular matrix in the juxtacanalicular connective tissue. A glycosaminog- lycan network can be seen beneath the endothelium (E) of Schlemm’s canal (x 60,000).

to other techniques in order that the results could be more easily compared with previously published work. The extracellular material was divided into two components, coarse (mostly plaque material and collagen) and fine

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Table 1. Analyses of extracellular materials in the juxtacanalicular connective tissue of patients with primary open-angle glaucoma and healthy controls. The coarse material, consist- ing of plaque and collagen fibres, was increased (* p < 0.05. Student’s t-test), whereas fine material (predominantly ruthenium red-stained glycosaminoglycans), was not.

Normals POAG

No. 10 25 Age (years) 59.0f 13.2 69.0f 15.7 Area of JCT coarse material 95.5 f 42.5* Area of JCT fine material 53.0 f 34.4

43.1 f 30.0 60.5 f 32.5

(ground materials and GAG net- works). Variation was pronounced but, as predicted from the study by Lutjen- Decoll et al. (1986), coarse matrix materials were significantly increased (p < 0.05, Student’s t-test) in POAG trabeculectomy specimens. In contrast, the fine material was slightly reduced (no significant difference) compared to controls (Table 1). These results were unexpected, but as these materials are notoriously difficult to preserve in situ the findings must be treated with cau- tion. In addition, trabeculectomy specimens are extremely limited samples of tissues that are subjected to considerable trauma and disruption dur- ing surgical removal, making interpre- tation even more difficult. To date, the case for excessive fine matrix material and accumulation of GAGS in the JCT as the primary pathological event in POAG remains unproven.

In a study of age-related changes in the JCT extracellular matrix, McMe- namin and coworkers (McMenamin et al. 1986) found that the coarse plaque material increased with age whereas the fine material (or ground substance) decreased. One cannot make a close comparison between this and the study by Swalem et al. because McMe- namin’s ‘ground substance’ comprised only part of Swalem’s ruthenium red- stained ‘fine component’. Nonetheless, it may be that the changes in the extracellular matrix in POAG are, in the initial stages, only an exaggeration of what is seen in the ageing process.

Clearly, considerable effort has been focused on the drainage wall of Schlemm’s canal with limited return. This philosophy of focusing on the re- gion of high resistance, to the exclusion of all other parts of the outflow system, has meant that key changes in other areas may have been overlooked or ig- nored.

Schlemm’s canal in POAG Little emphasis has been placed on the importance of Schlemm’s canal and its endothelial monolayer in the early pathology of POAG. Because of its trans- cellular pore system (Grierson et al. 1979; Eriksson & Svedbergh 1980), the presence of numerous giant va- cuoles (Inomata et al. 1972; Johnstone & Grant 1973; Grierson & Lee 1974; Grierson et al. 1979; Eriksson & Sved- bergh 1980) and the leaky nature of the cell-to-cell junctions (Raviola & Ravi- ola 1981), it has been calculated that the endothelium on the trabecular side of the canal has only a small resistance to outflow. Although it has recently been shown that in POAG the porosity of the endothelium is decreased such that the hydraulic conductivity is mar- kedly reduced (Allingham et al. 1992), the intrinsic conductivity of the endo-

thelium is so high that even pronounced alterations could not account for the increased resistance which characterises POAG (Allingham et al. 1992).

It has been reported that acute in- creases in IOP (above the normal range) produce narrowing of Schlemm’s canal in the human eye in vitro and the rhesus monkey eye in vivo (Fig. 6) (Grierson & Lee 1974). This raises the question of whether com- plete or partial closure of the canal could be central to pathological IOP elevation. Early morphological studies of POAG noted that Schlemm’s canal was altered (Nesterov et al. 1974), narrowed (Lee 1995) or even obliterated (Ashton 1960). In contrast, others (Johnson & Kamm 1983; Grierson 1987) dismissed changes in Schlemm’s canal and associated collector channels as occumng late in the pathological process and having little to do with the primary events.

However, rejecting change in Schlemm’s canal as an important factor in POAG may have been prema- ture. In a study of the effects of age and high pressure fixation on Schlemm’s canal, Ainsworth & Lee (1990) showed a decrease in the length of the inner wall of Schlemm’s canal with increasing age. In addition, in an examination of 100 trabeculectomy speci-

Fig. 6. Light micrograph of a semithin section of part of the trabecular meshwork (M) and Schlemm’s canal (arrows) from a rhesus monkey exposed to an intraocular pressure of 50 mmHg for one hour before intracameral perfusion fixation at that pressure. Schlemm’s canal is narrowed as a result of distension of the damaged meshwork ( X 400).

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mens Lee (1995) found that 50% had a narrowed Schlemm’s canal. Because trabeculectomy specimens are re- moved and fixed in the absence of a pressure head they are not ideal for evaluating canalicular narrowing. However, Allingham et al. (1996) examined POAG donor eyes under conditions in which Schlemm’s canal could be evaluated by measuring outflow facility in the enucleated eyes before fixation and then fixing the eyes at 15 mmHg. In POAG eyes, the canal cross sectional area, canal perimeter and inner wall length were significantly reduced compared to control eyes. When the POAG and control eyes were pooled, a correlation was found between outflow facility and canalicular area. From this well-controlled ex- periment the authors concluded that reductions in the dimensions of Schlemm’s canal could account for around 50% of the resistance observed in POAG. These studies indicate that gross changes in Schlemm’s canal may be a more important factor in the dis- eased outflow system than previously suspected.

Meshwork cells in POAG It was not until the 1980s that Alva- rado and coworkers (Alvarado et al. 1981, 1984) conclusively demon- strated that cells are lost from all regions of the trabecular meshwork with age. Parallel investigations demon- strated that the human meshwork cell population is around 750,000 at 20 years but decreases to 400,000 by 80 years, with a loss rate of around 6,000 cells per year (Grierson et al. 1982; Grierson & Howes 1987). Age-related loss of meshwork cells has also been reported in monkeys (Rohen & Lut- jen-Drecoll 1993) and dogs (Renwick 1992).

Several of the key age-related changes, such as trabecular thickening, trabecular fusion and thickening of the scleral spur, can be explained in terms of meshwork cell loss (Grierson et al. 1982). As these features are all more pronounced in POAG, the question arises whether depletion of the meshwork cell population is more severe in POAG than in age-matched controls. More importantly, is cell loss an early

Table 2. Area nuclear counts (by light microscopy) from four semithin sections for each specimen. A standard frame placed in the middle of the meshwork, with one side immediately below Schlemm’s canal, was used for each count; the frame covered approximately one fifth of the meshwork. Distinction was made between the juxtacanalicular connective tissue and the trabecular meshwork m. * p < 0.05, Student’s t-test. ‘

Nuclear count

Group No. Age (years) J C T TMW Total

Control 18 66.9f 14.0 20.0f 7.2 78.0f30.0 98.0f34.8 Primary surgery 42 70.9f 9.1 26.8f11.6 52.4f12.4* 72.2f18.0* Maximal medical therapy 27 69.6 f 13.2 22.4 f 8.8 40.4 f 14.8* 62.8 f 14.8*

event or a secondary (late onset) alter- ation?

Alvarado and coworkers found that the cellularity decrease in patients with POAG was outside the range expected for an ageing population. They were convinced that the depressed cellularity was an early event and rep- resented the definitive milestone in the pathology affecting the drainage pathways in POAG (Alvarado et al. 1984). Our initial findings from a small number of specimens seemed to disagree; patients who had had maximal medical therapy before surgery had a significantly lower meshwork cell count than age-matched controls but there was no difference between control patients and POAG patients who had first-line surgery (Grierson et al. 1982). How- ever, a bias in our counting procedure favoured the tissue closest to Schlemm’s canal. In fact, glaucomatous cell loss is not uniform; differen- tial loss is restricted to the trabecular meshwork whereas the JCT loses cells only at the rate associated with normal ageing. Separation of trabecular from JCT cells and increasing the number of specimens allowed demonstration of excessive cell loss from the trabeculae that was not replicated in the JCT (Table 2) (Joseph & Grierson 1994; Grierson & Hogg 1996). Importantly, trabecular cell loss was evident in specimens both from patients who had maximal medical therapy before surgery and those who had surgery as the first method of antiglaucoma treatment (Grierson 1987; Grierson & Hogg 1996). Consequently, excessive cell loss is now considered to be an important event in the pathogenesis of glaucomatous disease in the outflow system.

It is not yet clear whether meshwork

cells are lost at a greater rate in POAG or whether POAG patients have fewer cells than those who do not develop glaucoma. It may be that POAG patients lose meshwork cells at the normal rate but, because they have fewer cells to begin with, at a critical level they decompensate. Current results are conflicting; one group suggests a higher rate of cell loss in POAG (Grier- son & Hogg 1996) whereas the other suggests an initial low number of cells (Alvarado et al. 1984) and more research is needed to resolve this issue. In addition, it is not known why meshwork cells do not replace their losses in vivo. Nor is the mechanism of meshwork cell loss clear (Grierson & Hogg 1996); natural wear and tear through ciliary muscle action, aqueous humour toxicity and chemotaxis of meshwork cells are all possibilities. Furthermore, it is not yet clear whether excessive depletion of the trabecular meshwork cell population is a feature of all forms of glaucoma; a severely reduced cellularity has also been reported in some types of secondary open-angle glaucoma (Joseph & Grierson 1994).

It is important to elucidate the mechanism by which meshwork cell loss leads to increased resistance to aqueous outflow, and whether decreased cellularity in the meshwork and the presumed accumulation of extracellular material in the JCT are in- terrelated.

Con c 1 us i o n A fertile area of future research is un- folding but it should not be forgotten that the change in cellularity remained unrecognised because of a ‘blinkered’ approach to the disease process in the

ACTA OPHTHALMOLOGICA SCANDINAVICA 1997

outflow system. Hundreds of research workers studied extracellular material accumulation; although their findings aided understanding of the trabecular tissue they contributed little to the mechanisms underlying POAG. Al- most 10 years ago Epstein stated that ‘Our fundamental knowledge of the normal and abnormal processes of the trabecular meshwork is too primitive to allow the development of specific trabecular medical or surgical ther- apies,’ (Epstein 1987) and that situ- ation remains largely unchanged today.

Acknowledgements Our research is supported by grants from the Guide Dogs for the Blind, the International Glaucoma Association and the British Council for the Prevention of Blindness.

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pathological dilemmas in the outflow system in primary open-angle glaucoma

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