Morphology of the Trabecular Meshwork within Monkey (Macaca Speciosa) Eyes after Irradiation with the Free-running Nd:YAG Laser

Morphology of the Trabecular Meshwork within Monkey (Macaca Speciosa) Eyes after Irradiation with the Freerunning Nd:YAG Laser E. VAN DER ZYPEN, MD,* F. FANKHAUSER, MD,t C. ENGLAND, PhD,* S. KWASNIEWSKA, MDt

Abstract: The long-term tissue response of monkey eyes to trabeculoplasty produced at the chamber angle by neodymium:YAG (Nd:YAG) laser (thermal mode) was investigated. Four weeks after irradiation, a monolayer of corneallike endothelial cells extended over the trabeculum in impact zones, with partial occlusion of intertrabecular spaces; this condition persisted at all subsequent stages of observation (viz, up to 13 weeks). Both primary and secondary degeneration of trabecular endothelial cells occurred predominantly at the posterior border of the juxtacanalicular tissue, and continued throughout the observation period. Lysis and degradation of collagen fibrils were also prominent in this region, and collagen-phagocytosing cells could be seen at all stages. Although activated fibroblasts were marked in this zone, regeneration processes were, in general, less marked than those of degeneration. The significance of these findings in relation to the mechanism by which trabeculoplasty works is discussed. [Key words: monkey, morphology, Nd:YAG laser, scanning electron microscopy, trabecular meshwork, trabeculoplasty, transmission electron microscopy.] Ophthalmology 94: 171-179, 1987

Argon laser trabeculoplasty (ALT) was first described by Wise and Witter in 1979,1 and in clinical studies this method has been described to give good results in pressure reduction in most patients. 2- 10 The clinical success of ALT

From the Institute of Anatomy,* University of Berne, and the University Eye Clinic,t Berne, Switzerland. Supported by the Swiss National Science Foundation, grant 3.872-0.83, the Swiss Commission for the Promotion of Scientific Research, and in part by a grant from Alcon-Laboratories. Dedicated to Johannes W. Rohen, MD, Professor of Anatomy at the University of Erlangen, on the occasion of his 65th birthday. Reprint requests to E. van der Zypen, MD, Department of Topographic Anatomy, Institute of Anatomy, University of Berne, Buehlstrasse 26, P.O. Box 139, CH-3000 Berne 9/Switzerland.

in treating open-angle glaucoma is, however, controversial since pressure drops ranging from a satisfactory response of 89%1l down to 4%12 have been reported. Most of the morphologic investigations on the trabecular meshwork after ALT demonstrate a more or less complete closure of the inner intertrabecular spaces in the zone of impact by scar formation. 13- '5 The occlusion of the intertrabecular spaces after ALT is thought to be a reaction to absorbed heat emitted by the continuous wave (cw) argon laser.'6 It is, however, not in the impact zone that the increase in outflow facility occurs. In the monkey eye, it was possible to show that ALT stimulates cell and tissue degeneration deep within the trabecular meshwork, at some distance from the zone of impact, and it is this which causes an improvement in outflow. 17 It had been hoped that the photodisruptive laser might be a useful pressure-reducing tool, and it has been spec171

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Fig 1. Trabecular meshwork of a monkey eye four weeks after irradiation with the free-running Nd:YAG laser (pulse duration, 10 ms; pulse energy, 200 mJ). A monolayer of corneal-like endothelial cells (E) covers the inner trabecular beams near the corneal insertion in the impact zone. Pigmented cells (C) are seen at the surface of the meshwork (SEM; original magnification, X200).

ulated that the explosive effects of this energy modality might tear Schlemm's canal open, thus reducing outflow resistance. This hope was not, however, fulfilled. Instead, when using a Q-switched neodymium:YAG (Nd:YAG) laser for irradiating the trabecular meshwork of the monkey eye, a monolayer of corneal-like endothelial cells was found to extend from the line of Schwalbe over deeper scar tissue eight weeks after treatment. 18 ,19 Correspondingly, the clinical results of photodisruptive trabeculopuncture, performed with an Nd:YAG laser working in the Q-switched mode, were described as "disappointing."20 Later, when using a free-running Nd:YAG laser instead of the argon laser for trabeculoplasty, a reduction of intraocular pressure (lOP) was documented in cases of chronic open-angle glaucoma. 21 ,22 When comparing argon and Nd:YAG laser trabeculoplasty, Brihaye et al 23 found that the final reduction in lOP using both methods is about the same, although the authors believe that complications are less frequent and less severe using the Nd:YAG laser. In the analyses presented here, the long-term effects of the Nd:YAG laser working in the free-running mode upon the trabecular meshwork of the monkey, are described.

MATERIALS AND METHODS Five monkeys (Macaca speciosa) were used in this investigation, and irradiations were made under KetalarValium-Rompun anesthesia. One eye of each animal was irradiated with an Nd:YAG laser (Microruptor II) working in the free-running (thermal) mode (i.e., 1 producing a photocoagulative effect). Delivered to the chamber angle were lO-ms pulses, with energies varying betwe~n 200 and 800 mJ. The focus spot diameter (in air) was 50 ~m. No visible effects occurred at pulse energies lower than 200 mJ, but by using energies above this value impact sites were recognizable as "whitish" patches, which sometimes 172

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appeared as depressions within the tissue. With increasing pulse energy, the intensity of the impacts increased, and gas bubbles were visible. Via a goniolens, 20 to 25 impacts were distributed over the anterior and posterior meshwork around the whole circumference in each eye. During the irradiation procedure, blood was occasionally seen within the nonperforated Schlemm's canal. This observation indicates that no disruption of the inner wall ofSchlemm's canal occurred. Monkeys were killed with an overdose of anesthetic 2 hours and 4, 7, and 13 weeks after irradiation. They were perfused via the aorta initially with Ringer's solution containing heparin (25,000 units-United States Pharmacopoeia/I) and procaine (5 gil), and then with 2.5% glutaraldehyde in 0.1 mol phosphate buffer (pH 7.4; final osmolarity, 5lO mosm). After enucleation of the eyes, the irradiated areas were removed and immersed in glutaraldehyde solution at 4°C for either four hours (for transmission electron microscopy [TEM]) or 24 hours (for scanning electron microscopy [SEM]). Tissue specimens destined for TEM were postfixed in osmium tetroxide (1 %) in 0.2 mol cacodylate buffer. Blocks were stained with uranyl acetate during dehydration, and subsequently embedded in Epon. Thin sections were cut, and after contrasting with lead citrate (according to Reynolds24 ) and uranyl acetate they were examined in a Philips 300 electron microscope (Philips, The Netherlands). For examination by SEM, pieces of the irradiated trabecular meshwork were dried using the critical point method and thereafter sputtered with gold vapor up to a thickness of 300 A. Five impact zones from each eye were used for SEM and 15 to 18 for TEM. Hence, 20 to 25 impacts per eye were examined at each stage of repair (i.e., about 90 total).

RESULTS MORPHOLOGIC FINDINGS

Two hours after irradiation. Because we were interested in long-term morphologic responses, our attention was directed mainly to these effects with less emphasis being placed upon the acute reactions. In SEM, the acute effects are characterized by a widening of the inner intertrabecular spaces caused by a disruption of the cellular bridges connecting trabecular beams. Within the connective tissue cores of large trabecular beams, most of the collagen fibrils are dissolved into subfiQrillar structures with concomitant loss of their periodicity. These findings correspond to a heat effect between 60° and 70°C as comparative studies on the connective tissue of the iris have shown. 25 The heat response extends to the trabeculum cribriforme (juxtacanalicular meshwork). Four weeks after irradiation. Stimulated cells, with morphologic features similar to those of the corneal endothelium and continuous with them, migrate from the line of Schwalbe over the inner iridocorneal trabeculae, where they build a monolayer of hexagonal cells occluding the intertrabecular spaces (Fig 1). Descemet's membrane

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Fig 2. Chamber angle of a monkey eye 13 weeks after irradiation with the free-running Nd:YAG laser (pulse duration, 10 ms; pulse energy, 800 mJ). The trabecular meshwork is traversed by pigmented phagocytes (C). Near the region where trabecular meshwork and cornea meet, a monolayer of cuboidal cells is built against the anterior chamber (AC). Descemet's membrane (D) is indicated. The hyperplasia of cells observed within the operculum is illustrated in the insert as a higher magnification view of the boxed region (toluidine blue; original magnification, X40) (insert, original magnification, X50).

is often found to extend over the inner trabecular meshwork in anterior regions near the cornea (Fig 2). Cells of the operculum were also stimulated, and a hyperplastic cell mass is observed in this region (Fig 2, insert). The operculum, which is built up in some species of monkey but is absent from human eyes, consists of a group of cells lying near the anterior insertion of the trabecular sheets. The expansion of corneal-like endothelial cells appears to be more extensive if the center of the impact zone is located more anteriorly near the operculum. Giant pigmented macrophages (like the clump cells of the iris) are found to traverse the trabecular meshwork within irradiated zones (Fig 2) and to phagocytose cell and tissue debris, as demonstrated by electron microscopy. Pigmented macrophages may be found at all subsequent stages of observation (i.e., up to 13 weeks after irradiation). They may break through the continuous superficial cell layer and lie isolated or in groups on the corneal-like endothelium that covers the trabecular meshwork (Fig 1). These clump cells have a spherical form, are not surrounded by a basement lamina, and exhibit an abundance of microplicae on their surfaces. In the juxtacanalicular tissue, fibroblasts, characterized by a dilated rough endoplasmic reticulum containing a homogeneous granular mass, are found (Fig 3). At the posterior border of Schlemm's canal, many microfibrillar structures, having a diameter of 10 nm, are observed lying between collagen fibrils exhibiting a normal diameter of 60 nm. In contrast to the normal situation, the cross diameters of collagen fibrils within trabecular cores range from 60 to 700 nm (Fig 4). Degenerating cells are found predominantly in the posterior border of the juxtacanalicular tissue, near the scleral spur. The degeneration process is characterized by a lysis

of organelles and ground substance within the cytoplasm. This process occurs alongside a swelling of the pericaryon and a retraction of cell processes (Fig 5). The occurrence of this latter phenomenon is suggested by the appearance of basement laminae, which maintain their original position over a long time (Fig 5). In various places, collagen fibrils seem to be reduced in number due to phagocytosis

Fig 3. Juxtacanalicular tissue of a monkey chamber angle four weeks after irradiation with the free-running Nd:YAG laser (pulse duration, 10 ms; pulse energy, 200 mJ). Part ofa fibroblast is illustrated. Activity in collagen synthesis is indicated by a well-developed dilated rough endoplasmic reticulum containing a fine granular amorphous mass (TEM; original magnification, X22,OOO).

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Fig 4. Trabecular beam within a monkey chamber angle four weeks after irradiation with the free-running Nd: YAG laser (pulse duration, 10 ms; pulse energy, 200 mJ). Collagen fibrils exhibit considerable variation in cross diameter indicative of different stages of polymerization. Elastic-like fibers (e) and the trabecular endothelium (Te) are indicated (TEM; original magnification, X 17,(00).

by collagen-phagocytosing cells. This process is, however, better demonstrated at later stages (Fig 11). Seven weeks after irradiation. The superficial layer of corneal-like endothelial cells covering the inner iridocor-

neal trabecular meshwork persists. These cells often build cellular plates, which spread over trabecular beams (Fig 6). Clump cells may often be seen passing through the intertrabecular spaces (Fig 6). lit...

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Fig 5. Degeneration oftrabecular endothelial cells within the chamber angle of a monkey eye four weeks after irradiation with the free-running Nd:YAG laser (pulse duration, 10 ms; pulse energy, 500 mJ). During the lytic degeneration process, cells retract their processes (Pr); basement laminae (BI) do not, however, participate in this process (TEM; original magnification, x6000).

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Cells at the operculum are associated in an epitheliallike manner, with intercellular spaces being closed by junctional complexes (Fig 7). In some places, morphologic findings indicate that these cells may participate in the new formation of trabecular beams. Near the posterior border ofSchlemm's canal, lytic degeneration of cells has further increased. In various places, folded remnants of basement lamina material indicate the former extension of trabecular cells before degeneration or retraction (Fig 8). The quantity of collagenous material within trabecular cores is reduced. Curly (longspacing) collagen, normally having a periodicity of about 100 nm in the monkey's trabecular meshwork, disintegrates during the degeneration process. The dark crossbanding distance is increased fivefold or more, and the light bridges between dark bands exhibit tree-like ramifications (Fig 9). The trabecular core normally contains groups of microfibrils embedded in a fine granular mass. In ultrastructure, they have morphologic features similar to elastic fibers. However, these fibers are not stainable with elastic-staining techniques (in the light microscope). They are therefore generally referred to as elastic-like fibers. These fibers are more resistant to decay than collagenous material (Fig 9). Thirteen weeks after irradiation. Pigmented clump cells are still observed at this stage. The degeneration process of the trabecular endothelial cells and collagen fibrils persists. Both processes appear to be localized in scattered zones up to 0.5 mm in diameter, concentrated within the juxtacanalicular tissue (Fig 10). In those regions where there is almost complete degeneration of collagenous tissue and trabecular cells, the former organization of the network is approximately indicated by basement lamina remnants (Fig 10). At the borders of these degeneration zones, cells characterized by long processes, and containing fragments of collagen fibrils enclosed in small cisternae are observed (Fig 11). Some of the ingested collagen fibrils show a periodic banding of 620 A, whereas others have lost their cross banding completely (Fig 11).

Fig 6. Inner surface of the trabecular meshwork within the chamber angle of a monkey eye seven weeks after irradiation with the free-running Nd:YAG laser (pulse duration, 10 ms; pulse energy, 800 mJ). Newly formed cellular plates spreading over trabecular beams are illustrated. Spherical pigmented phagocytes (C) migrate through the intertrabecular spaces to the inner surface ofthe meshwork (SEM; original magnification, X200).

DISCUSSION Both the cw argon laser and the Nd: Y AG laser working in the free-running mode induce thermal damage within the trabecular meshwork. Unlike the impact of argon laser light (wavelength, 488-514 nm), which reaches only the first two to three layers of the trabecular meshwork,26 Nd: Y AG laser light (wavelength, 1064 nm) in the free-running mode produces a cone-like heat effect, the apex of which reaches the juxtacanalicular trabeculum cribriforme26 (Fig 12). Irradiation by both lasers induces the formation of a superficial compact scar towards which corneal-like endothelial cells contribute. The results of the current investigation indicate that stimulated opercular cells also participate in scar formation at the insertion of the iridocorneal trabeculae. The nature of the tissue reaction produced using argon (cw) and Nd:YAG (free-running

Fig 7. Trabecular meshwork near the line of Schwalbe in a monkey eye seven weeks after irradiation with the free-running Nd:YAG laser (pulse duration, 10 ms; pulse energy, 400 mJ). A hyperplasia of cells within the operculum is seen. A close relationship between these cells and trabecular endothelial cells exists. AC = anterior chamber (TEM; original magnification, X3200).

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mode) light is similar; in each case, there is a hyperplasia of cells occurring alongside a secondary degeneration process. Degeneration and new formation of collagen also

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Fig 8. Juxtacanalicular meshwork within the monkey chamber angle seven weeks after irradiation with the free-running Nd:YAG laser (pulse duration, 10 ms; pulse energy, 800 mJ). Many of the trabecular cells (Te) have degenerated or retracted their processes. Folded remnants of basement lamina material (BI) indicate the former extent of the cells before degeneration. e = elasticlike fiber (TEM; original magnification, X20,500).

•" occurs in parallel within the same region. In relation to the position of impact, no differences in cell and tissue reaction were documented (i.e., impacts located anteriorly

Fig 9. Trabecular meshwork of a monkey eye seven weeks after irradiation with the freerunning Nd: YAG laser (pulse duration, 10 ms; pulse energy, 200 mJ). Long-spacing (curly) collagen within the trabecular cores degenerates. This process is shown initially by irregularities of the light bands (CCI), which later exhibit tree-like ramifications (CC2). The dark cross bands also become more dispersed with consequent reduction in electron density. e = elasticlike fiber (TEM; original magnification, X 14,000).

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Fig 10. Iuxtacanalicular tissue of the monkey trabecular meshwork 13 weeks after irradiation with the Nd:YAG laser (pulse duration, 10 ms; pulse energy, 600 mI). In some places, most trabecular cells have degenerated, and collagen fibrils have dissolved. The former organization of the netwOlk is approximately indicated by the folded basement laminae (Bl) (TEM; original magnification, X8(00).

and posteriorly within the trabecular meshwork produced similar responses). Cell proliferation and new formation of trabecular beams occurs more anteriorly within the trabecular meshwork, near and at the operculum, whereas secondary cell degeneration appears to be more prominent at the posterior end, near Schlemm's canal. The production of collagen, which takes place in all regions, is indicated by the presence of activated fibroblasts. Fibroblast-like cells with clast-like activity ("fibroclasts") were observed to engulf fragments of cross-banded collagen fibrils, which, as shown previously using the acid phosphatase reaction, they subsequently digest.27 - 29 There are indications that trabecular cells can digest extracellular tissue components,3O and the collagen-phagocytozing cells observed in this and other investigations27 - 29 may thus represent transformed trabecular endothelial cells. Irradiation with either argon (cw) or Nd:YAG (freerunning mode) lasers operating at temperatures up to 80 a C, stimulates an increased turnover of collagen. Degeneration appears, however, to eclipse proliferation; and in regions where this occurs concomitantly with loss (without replacement) of trabecular cells (Le., predominantly in the juxtacanalicular cribriforme) a widening of the intertrabecular spaces occurs. These factors may contribute to an improved aqueous outflow facility. Although it is not possible to predict the effects of trabeculoplasty upon human glaucomatous eyes from those produced in normal monkey eyes, the decrease in lOP obtained after trabeculoplasty in glaucomatous human

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Fig 11. I uxtacanalicular tissue of the monkey trabecular meshwork 13 weeks after irradiation with the free-running Nd:YAG laser (pulse duration, 10 ms; pulse energy, 800 mJ). Collagen-phagocytosing cell at the border of the degeneration zone is illustrated. Collagen fibrils lie singly or in small groups within small cisternae of cell processes. Cisternae containing both cross-sectioned (cfd and longitudinally sectioned (cf2 ) collagen fibrils are indicated (TEM; original magnification, X22,OOO).

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Fig 12. Scheme of part of the monkey chamber angle, indicating the differences in penetration depth between argon laser (Ar) and Nd: YAG laser (Nd) light effects during trabeculoplasty. TM, = the inner trabecular meshwork in frontal view; TM2 = the trabecular meshwork in crosssection; SC = Schlemm's canal; and S = sclera.

eyes coincides in time with that at which widening of the intertrabecular spaces by degeneration of cells and degradation of intercellular material was seen. A survey of the literature shows the findings of two groups to be of possible importance in relation to our hypothesis. Tengroth et al 31 ,32 have shown that changes in the composition of collagen within the lamina cribrosa and the trabecular meshwork may be of primary importance in the development of chronic open-angle glaucoma. One could hypothetically assume that the composition of collagen may change again if a new generation of cells is activated, and the turnover of collagenous material accelerated, as the presented findings suggest. Furthermore, van Buskirk et al 33 have demonstrated that laser irradiation may stimulate trabecular cells to furnish compositional changes within the extracellular matrix; such factors may also contribute towards increasing aqueous outflow. Assuming this hypothesis is correct, one may further speculate that the mechanism by which collagen turnover is stimulated is not triggered iftrabeculotomy is produced using a Q-switched laser. This may be the reason for the disappointing results obtained using the Q-switched Nd: Y AG laser (Epstein et al 20 ; van der Zypen and Fankhauser, unpublished data). It would thus appear that thermal rather than mechanical damage is necessary to stimulate the mechanism whereby aqueous outflow is facilitated. From the morphologic viewpoint, there are qualitatively no differences in the tissue responses induced by either the cwargon or the free-running Nd:YAG lasers. However, the greater depth of tissue penetration possible using the Nd:YAG laser (free-running mode) raises the

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possibility of performing trabeculoplasty via the transscleral route, which is not feasible using the argon laser. Whether this approach offers any advantages has yet to be assessed.

ACKNOWLEDGMENTS The authors thank Miss Nina Caratsch, Mrs. Susanne Zimmermann, and Mrs. Franziska Schweizer for their excellent technical assistance, and Mrs. Regina Channi for her secretarial help.

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