Argon Laser Trabeculoplasty

Studies of Mechanism of Action

E. MICHAEL VAN BUSKIRK, MD, VALERIE POND, MD, ROBERT C. ROSENQUIST, MD, TED S. ACOTT, PhD

Abstract: The effects of argon laser trabeculoplasty (l TP) on outflow facility, Schlemm's canal morphology, trabecular cellularity, and trabecular glycosami­noglycan composition were determined in 33 pairs of eye-bank human eyes. At two levels of intraocular pressure, measured outflow facility did not immediately drop in response to LTP. Schlemm's canal distention was observed only at 40 mmHg intraocular pressure, where the canal normally tends to collapse. Trabecular cell density was reduced by about 40% in the eyes receiving laser treatment. The organ cultured trabecular meshworks in response to L TP altered their incorporation of 355-sulfate, compared to controls, suggesting a change in the synthesis or turnover of the extracellular matrix of the trabeculum after trabeculoplasty.

At least three potential mechanisms were identified in response to laser trabeculoplasty, including some mechanical distortion of the trabeculum at high intraocular pressures. We also hypothesize that laser trabeculoplasty dislodges some trabecular cells and may stimulate the remaining cells to renew more active synthesis and/or turnover of the trabecular extracellular matrix. [Key words: extracellular matrix, laser, trabecular distortion, trabecular hypocellularity, trabeculoplasty.] Ophthalmology 91:1005-1010, 1984

Despite the widespread use of argon laser trabeculo­plasty in medically unresponsive open-angle glaucoma, the mechanism by which it reduces outflow resistance remains unknown. Ignorance about how and why it works inhibits our ability to design better and safer treatment parameters, to predict the response to different kinds of laser energy, and to anticipate how long the laser effect will last and whether or not it may safely be repeated.

One postulated mechanism suggests ·that trabecular photocoagulation produces shortening or "tightening" of the inner trabecular lamellae. 1 This process would

thereby displace the trabeculum internally, possibly pre­venting or delaying collapse of the trabecular lamellae or of Schlemm's canal. Both trabecular and canalicular collapse have been suggested to contribute to the patho­genesis of primary open-angle glaucoma and both have been demonstrated histologically to exist in glaucomatous and normal eyes under appropriate fixation conditions. 2•

3

However, other possibilities also exist including altera­tions in the trabecular cell population and the extracel­lular matrix they synthesize.

From the Department of Ophthalmology, Oregon Health Sciences University, Portland.

Presented at the Eighty-eighth Annual Meeting of the American Academy of Ophthalmology, Chicago, Illinois October 30-November 3, 1983.

Supported in part by NIH Research Grant EY-03279.

Reprint requests to E. Michael Van Buskirk, MD, Department of Oph­thalmology, The Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97201.

MATERIALS AND METHODS

We have performed three different experiments using the trabecular meshworks of 66 human eye-bank eyes, 23 pairs of whole eyes and 10 pairs of anterior segments; the latter had been maintained in organ culture for one week. When not used immediately after enucleation, the whole eyes were stored at 4 °C in a moist chamber and were used within 48 hours postmortem. Immediately before and following argon laser trabeculoplasty, facility

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OPHTHALMOLOGY • SEPTEMBER 1984 • VOLUME 91 • NUMBER 9

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Fig 1. The effects of mean aqueous facility of outflow of laser trabeculoplasty and sham laser trabeculoplasty in excised human eyes. Thirteen pairs were treated at 20 mmHg, and I 0 pairs at 40 mmHg intraocular pressure.

of aqueous outflow was determined by quantitative aqueous perfusion as previously described, using the corneal fitting method of Grant and a 5 mm corneal trephination.4

•5 A radial iridotomy maintained the iris

in a neutral position. The trabeculoplasty, performed through the corneal trephination, consisted of 80 50 ~tm laser bums equally distributed throughout the anterior portion of the trabecular meshwork for 0.1-second du­ration each. The power was titrated between 0. 7 and 0.9 watts to the threshold of bubble formation or "whitening." One member of each pair received the trabeculoplasty, while the fellow underwent a sham procedure, consisting of irrigation of the anterior chamber and examination of the trabecular meshwork through the corneal trephination site at the laser slit lamp.

Following the post-treatment facility measurements, 14 eye pairs were fixed by intracameral infusion at the perfusion pressure in cacodylate buffered glutaraldehyde. They were post-fixed in osmium tetroxide, dehydrated in a graded series of alcohols and embedded in Epon® for light microscopy. Each block was sectioned until a laser bum was identified. Sectioning was continued through the block of trabeculum until the next bum was reached, providing sections from the edge of one bum to that of its immediate neighbor. The sections from the trabecular zone midway between adjacent burns were then placed on slides encoded with random numbers to permit masked quantitative measurements. The cross-sectional area of Schlemm's canal was mea­sured using a calibrated eyepiece reticule grid. Trabecular cell density was estimated by counting the number of trabecular cell nuclei observed in the entire thickness of

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the trabecular meshwork overlying the lumen of Schlemm 's canal.

Ten additional eye pair comeo-scleral explants, re­ceived from the eye bank up to 72 hours postmortem, were stabilized in organ culture · for one week using our standard culture conditions. 6 The explants were carefully prepared by excising the anterior segment 3 mm posterior to the limbus, stripping the uveal tissue away from the scleral spur. This left an intact comeoscleral anterior segment that included trabecular meshwork. Other stud­ies from our laboratory have shown that under these conditions in organ culture the trabecular cells incor­porate and synthesize glycosaminoglycans (GAG) simi­larly to normal trabecular cells for at least 18 days. 6 The culture-stabilized anterior segments were then placed in a glass and stainless steel container filled with culture media. The trabecular meshwork was treated with argon laser through the glass walled chamber using the same parameters as used for the eye bank eyes. The meshworks were returned to the organ culture and incubated with 250 ~tCi 35S-sulfate for one to ten days with media and label changed every two days. The trabecular glycosami­noglycans (GAGs) were extracted, partially purified and subjected to electrophoretic analysis on cellulose acetate.6

Briefly, lipids were extracted with 10 volumes chloroform to methanol (2: 1 ); the proteins were digested with papain and precipitated with 10% cold trichloroacetic acid (TCA); the free-labeled, TCA, and small molecules were removed with Bio-gel® P-6DG columns and the GAGs were collected by precipitation with 75% ethanol, 5% sodium acetate. The pellet was dissolved in water and 0.25 ~tL aliquots were electrophoresed for 2 hours at 4 rnA on cellulose acetate strips in a Microzone® apparatus (Beckman) with 3.0 M cadmium acetate buffer at pH 4.1. The strips were stained with alcian blue, sliced and counted in Atomlite® scintillation solution (New England Nuclear). The results were determined in counts per minute above background and were averaged from two separate experiments.

RESULTS

Data results are given as mean± standard error. Student t-tests for paired data were used to determine statistical difference.

There was no significant change in facility of outflow after laser trabeculoplasty at 20 mmHg or at 40 mmHg (Fig 1 ). Of the 13 eyes perfused at 20 mmHg, baseline facility was 0.19 ± 0.04 ~tL/min/mmHg before and 0.20 ± 0.05 ~tL/min/mmHg after trabeculoplasty, a difference not statistically significant. By the same token, at 40 mmHg baseline, facility was 0.16 ± 0.03 ~tL/min/mmHg and increased insignificantly to 0.17 ± 0.03 ~tL/min/ mmHg following trabeculoplasty. After sham trabecu­loplasty, facility also changed by an insignificant amount at both levels of perfusion pressure (Fig 1 ).

Of the six eye pairs fixed at 20 mmHg, there was no significant difference in Schlemm's canal cross-sectional

VAN BUSKIRK, et al • ARGON LASER TRABECULOPLASTY

area between the treated (2114 ± 130 ~m2) and sham­treated (1919 ± 387) eyes (Fig 2). The cross-sectional area approximately 2,000 ~m2, is comparable to that previously observed in other eyes fixed at the same pressure (20 mmHg). 3 At 40 mmHg, however, Schlemm's canal did show evidence of distention after laser trabec­uloplasty. At that pressure, the mean canalicular cross­sectional area in the untreated eyes was 875 ± 129 ~m2, again comparable to eyes from quantitative morpho­metric analytic experiments fixed under the same con­ditions.3 However, the canal cross section in the lasered eyes at that pressure had distended to about twice the area of the sham-treated eyes, 1921 ± 234 ~m2 • The difference was significant (P < 0.01).

Trabecular cell density measured midway between adjacent burns was 39% lower in the treated (31.9 ± 4.5 cells) than the untreated eyes (52.2 ± 5.8 cells), a statis­tically significant difference (P < 0.05) (Fig. 3). Because the counts were taken midway between adjacent burns and the slide identification numbers were masked, no direct evidence of the trabecular burn could be identified in most of the treated eyes. Because counts were taken from all layers of the trabeculum, a propensity for cell loss in a specific trabecular layer was not identified.

Using cellulose acetate electrophoresis to examine the incorporation profile of radioactive sulfate into the extracellular matrix of lasered and control trabeculae, the expected incorporation pattern observed in our standard organ cultured eyes was maintained in the control eyes.6 In the lasered eyes, however, a new

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Fig 2. The effects of laser and sham-laser trabeculoplasty on mean Schlemm's canal cross-sectional area at two levels of intraocular pressure.

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Fig 3. The effects on trabecular cell density of laser trabeculoplasty (L TP) and sham trabeculoplasty in excised human eyes. (Mean nuclear counts in pre-canalicular meshwork ± standard error of the mean.)

distribution of label was observed with more rapid electrophoretic migration of the predominant glycosami­noglycan complex. By the second day, this new peak dominated and was maintained through the fourth and sixth day (Fig 4). By the tenth day, two explants appeared to have reverted to the normal pattern while the others continued to exhibit the new peak. This may indicate a progressive reversion to the normal incorpo­ration pattern with time, suggesting a reparative process:

DISCUSSION

Collapse of the trabecular lamellae upon each other and into the Schlemm's canal has been suggested as a contributory mechanism in some cases of primary open angle glaucoma.2 Laser trabeculoplasty reduces outflow resistance by an unknown mechanism, but reversal or at least prevention of canalicular collapse by mechanically tensing or "tightening" the trabecular meshwork has been proposed.' Our previous experiments in nonglau­comatous eyebank eyes, using a lens depression apparatus to mechanically "tighten" the trabecular meshwork, demonstrated that the "non-tightened" meshwork does collapse into the lumen of the canal as the trans­trabecular pressure exceeds 20 mmHg. This canalicular collapse was nearly complete at 40 mmHg.3

The present experiments with laser trabeculoplasty were likewise performed in ostensibly normal, nonglau­comatous, eyes. However, since we had previously dem­onstrated canalicular collapse and its reversal, accom-

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OPHTHALMOLOGY • SEPTEMBER 1984 • VOLUME 91 • NUMBER 9

10

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Cll SJ.ICI IUIIII Fig 4. Cellulose acetate electrophoretic migration of glycosaminoglycans (GAGs) extracted from organ cultured trabecular meshworks 4 days after laser or sham (control) trabeculoplasty. The more distal migration of the predominant GAG complex in the laser-treated eyes indicates greater sulfate incorporation or a change in the relative synthesis of the individual GAGs.

panied by a facility increase, with trabecular tension in similar eyes, we felt the experimental model was capable of demonstrating increased trabecular tension, if it oc­curred.3·5·7 However, in contrast to the lens-depressed eyes, but like in the clinical setting, trabeculoplasty did not produce an immediate outflow facility increase at the two levels of intraocular pressure studied. This does not necessarily indicate that no trabecular tension was induced. The apparent lack of effect could merely reflect two mutually counteracting factors of trabecular tension tending to increase outflow while released tissue debris could block it downstream.

Perhaps the strongest evidence for mechanical trabec­ular distortion was the canalicular distention induced by trabeculoplasty at 40 mmHg. Canalicular distention was not detectable at the lower pressure levels of 20 mmHg but our previous experiments have suggested that the canal is only beginning to collapse at that pressure. These data seem to suggest that immediate trabecular distortion, as would result from heat-induced collagen shrinkage, comes into play mainly at high levels of intraocular pressure where the previous studies showed Schlemm's canal to collapse.3 More progressive trabecular distortion could possibly occur at lower pressures with the passage of time ( eg.) as the result of fibroblastic scarring), but the model does not permit examination of that factor.

1008

The trabecular meshworks that had received laser treatment were relatively acellular compared to control eyes. We can only speculate about the possible clinical consequences of this laser-induced loss of trabecular cells. In the immediate postoperative period, clogging of downstream outflow pathways by denuded cellular debris could well account for the immediate post-trabeculo­plasty pressure rise often observed clinically. However; long-term reduced cell density could foretell a deleterious effect, especially if the relative acellularity of the aged and glaucomatous trabeculum as demonstrated by Al­varado and others proves to be an important pathogenetic factor. 8•

9 What we do not know is whether this laser­induced hypocellularity persists with time or whether the laser trabeculoplasty, by expunging diseased cells, permits the regeneration of new, more active cells. Such cellular stimulation with accelerated mitosis occurs in trabecular and corneal cells in culture, but has not been investigated in the intact living eye. I0-!3

The third question concerns alterations in the bio­chemical activity of trabecular cells induced by laser radiation. We are only now beginning to understand the complex extracellular matrix of glycoproteins and pro­teoglycans which are an integral part of the trabecu­lum.6·10 Mucinous glycosaminoglycans (GAGs) are major components of the proteoglycans which line the trabec­ular cell surfaces and the inter-trabecular spaces, as

VAN BUSKIRK, et al • ARGON LASER TRABECULOPLASTY

observed by Zimmerman over two decades ago. 14 These compounds serve, in connective tissues, to sequester water, to provide mechanical protection and to regulate fluid flow; they are likely to serve similar functions in trabecular meshwork. Although the results of the bio­chemical studies reported here are preliminary, we do have evidence that laser trabeculoplasty affects the pro­duction of the extracellular matrix, as indicated by the observed alterations of radioactive sulfate incorporation into lasered trabecular meshworks in culture when com­pared to controls. This is not to imply that the extant trabecular glycosaminoglycans actually were changed by the laser radiation, but rather that the trabecular cells themselves had changed their incorporation into sulfated macromolecules. This process, if allowed to persist, would produce a different composition of trabecular extracellular matrix. Since we have not fully uncovered the normal array of trabecular extracellular glycosami­noglycans, the precise clinical implications ofthese laser­induced effects are unknown. We may logically speculate, however, that these changes could permit the washing downstream of outflow-obstructing material, the rejuvi­nation of diseased basement membranes, or the produc­tion of new protective and conductive trabecular cell coats.

The studies reported suggest that at least three mech­anisms, mechanical, cellular, and biochemical, take place within the trabecular meshwork in response to argon laser trabeculoplasty. It appears that there is some mechanical effect to displace the trabeculum internally but it seems most prominent at high levels of intraocular pressure. In addition, trabecular cell loss and alteration of the extracellular matrix simultaneously occur. Our working hypothesis is that trabeculoplasty, in removing some trabecular cells, may stimulate the division, mi­gration and renewed synthesis and turnover of the extracellular matrix by those trabecular cells left behind.

ACKNOWLEDGMENTS

The authors thank the Oregon Eye Bank and the Oregon Lion's Eye Bank for providing the donor eyes and Mary Westcott-Hodson for technical assistance with the experiments.

REFERENCES

1. Wise JB, Witter SL. Argon laser therapy for open-angle glaucoma; a pilot study. Arch Ophthalmol 1979; 97:319-22.

2. Nesterov AP, Batmanov YE. Study on morphology and function of the drainage area of the eye of man. Acta Ophthalmol 1972; 50:337-50.

3. Van Buskirk EM. Anatomic correlates of changing aqueous outflow facility in excised human eyes. Invest Ophthalmol Vis Sci 1982; 22:625-32.

4. Grant WM. Further studies on facility of flow through the trabecular meshwork. Arch Ophthalmol 1958; 60:523-33.

5. Van Buskirk EM, Grant WM. Lens depression and aqueous outflow in enucleated primate eyes. Am J Ophthalmol 1973; 76:632-40.

6. Acott TS, Westcott M, Van Buskirk EM. Explant organ culture of human trabecular meshwork: growth conditions, morphology and synthesis of glycosaminoglycans. Invest Ophthalmol Vis Sci 1984, submitted.

7. Van Buskirk EM. Changes in the facility of aqueous outflow induced by lens depression and intraocular pressure in excised human eyes. Am J Ophthalmol 1976; 82:736-40.

8. Alvarado J, Murphy C, Polansky J, Juster R. Age-related changes in trabecular meshwork cellularity. Invest Ophthalmol Vis Sci 1981; 21:714-27.

9. Alvarado J, Murphy C, Juster R. Trabecular meshwork cellularity in primary open-angle glaucoma of nonglaucomatous normals. Oph­thalmology 1984; 91 :564-79.

10. Rohen JW, Schachtschabel DO, LUtjen-Drecoll E, et al. Morphological and biochemical studies of cell and tissue cultures of human trabecular meshwork. In: Krieglstein GK, Leydhecker W, eds. Glau­coma Update II; Glaucoma Society of the International Congress of Ophthalmology, Carmel, California, October 22-27, 1982. Berlin: Springer-Verlag, 1983; 39-43.

11. Grierson I, Marshall J, Robins E. Human trabecular meshwork in primary culture: a morphological and autoradiographic study. Exp Eye Res 1983; 37:349-65.

12. Tripathi RC, Tripathi BJ. Human trabecular endothelium, corneal endothelium, keratocytes, and scleral fibroblasts in primary cell culture; a comparative study of growth characteristics, morphology, and phagocytic activity by light and scanning electron microscopy. Exp Eye Res 1982; 35:611-24.

13. Polansky JR, Weinreb AN, Baxter JD, Alvarado J. Human trabecular cells. I. Establishment in tissue culture and growth characteristics. Invest Ophthalmol Vis Sci 1979; 18:1043-9.

14. Zimmerman LE. Demonstration of hyaluronidase-sensitive acid mu­copolysaccharide; in trabecula and iris in routine paraffin sections of adult human eyes. Am J Ophthafmol1957; 44:1-4.

Discussion by

James B. Wise, MD

Using 23 pairs of autopsy eyes, Dr. Van Buskirk has treated one eye of each pair with laser trabeculoplasty. In the treated eyes, he has found an increased resistance to collapse of Schlemm's canal, but no increase in outflow. He also found reduction in trabecular cell count and alteration of glycosami­noglycan metabolism in these eyes. His work has been carefully done, but is subject to the limitation that these autopsy eyes did not have glaucoma. One of the principal objections to the early attempts to use the laser for open-angle glaucoma was

that the laser did not produce increased outflow or decreased pressure when applied to the normal trabecular meshwork of experimental animals. 1

-3 The results reported today confirm

those animal experiments by showing that laser treatment of the normal human meshwork does not produce any increase in outflow, proving that trabecular collapse is not significant in the normal eye. In the glaucomatous eye, however, trabecular collapse is a consistent finding.4 In a laser-treated human eye whose pressure went from 36 mmHg on 4% pilocarpine to 12

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OPHTHALMOLOGY • SEPTEMBER 1984 • VOLUME 91 • NUMBER 9

Fig 1. Scanning electron micrograph of trabecular meshwork of a human eye treated with 25 laser bums in 90° three weeks before enucleation

for ciliary body melanoma. Laser settings were 50 !lm spot, 0.1 second, 1000 mw. The bums involve only a small percentage of the total

trabecular area.

mmHg on no therapy, and stable for two years, trabecular collapse was no longer present. 5 Failure of the laser to further increase outflow in eyes with open meshwork and normal outflow does not predict the result when the laser is applied to eyes with reduced outflow due to trabecular collapse. Dr. Van Buskirk's excellent studies should therefore be extended to human eyes with glaucoma, which will of course be difficult because they are so seldom available.

Another problem with autopsy eyes is the lack of tone in the ciliary muscle. Laser shrinkage of the meshwork may be offset by anterior displacement of the scleral spur. In other studies, Dr. Van Buskirk has shown that outflow of autopsy eyes is increased by lens depression, which causes backward displacement of the scleral spur simulating contraction of the ciliary body.6 Response to lens depression may well be different in laser-treated eyes, and should be studied.

Dr. Van Buskirk also observed a 40% reduction in trabecular cell count between the burns. Part of this apparent reduction could be simply spreading and stretching of the layers due to laser effect, but some of the reduction is probably real and merits further study. However, laser trabeculoplasty treats only a small percentage of the meshwork, as seen in this human eye treated with the laser prior to removal for melanoma (Fig 1 ). Measurement of cell loss anterior and posterior to the

1010

burns is also necessary before concluding that the laser causes severe cell depletion. The changes in metabolism need to be studied in the living eye to see if they persist beyond the immediate post-bum period.

References

1. Wickham MG, Worthen DM, Binder PS. Physiological effects of

laser trabeculotomy in rhesus monkey eyes. Invest Ophthalmol Vis

Sci 1977; 16:624-8. 2. Ticho U. Cadet JC, Mahler J, et al. Argon laser trabeculotomies in

primates: evaluation by histological and perfusion studies. Invest

Ophthalmol Vis Sci 1978; 17:667-74.

3. Gaasterland DE, Kuwabara T. Effects on monkey eyes of argon

laser glaucoma treatment. ARVO Abstracts. Invest Ophthalmol Vis

Sci 1980; 19(Suppl):83-4.

4. Fine BS, Yanoff M, Stone RA. A clinicopathologic study of four

cases of primary open·angle glaucoma compared to normal eyes.

Am J Ophthalmol1981; 91 :88-105.

5. Wise JB. Long·term control of adult open angle glaucoma by argon

laser treatment. Ophthalmology 1981; 88:197-202.

6. Van Buskirk EM. Anatomic correlates of changing aqueous outflow

facility in excised human eyes. Invest Ophthalmol Vis Sci 1982;

22:625-32.