Excimer Laser Effects on Outflow Facility and Outflow Pathway Morphology

Abstract

purpose. To determine the relative contributions to aqueous outflow resistance of the tissues distal to the inner wall of Schlemm’s canal.

methods. While performing constant pressure perfusion at 10 mm Hg, a 193-nm excimer laser (Questek) was used to precisely remove portions of sclera, unroofing Schlemm’s canal while leaving the inner wall intact. The laser beam was masked to produce a beam 2 mm by 1 mm. The laser output was constant at a fluency of 75 mJ/cm² and 20 Hz. The excimer laser at a frequency of 1 Hz was used as the aiming beam. Photoablation was performed on human cadaver eyes at the limbus at an angle of 0° to 45° from the optical axis. As the excimer photoablations progressed, Schlemm’s canal was visualized by the fluorescence of the Barany’s solution containing fluorescein dye. After perfusion fixation the eyes were immersion-fixed overnight. The facility of outflow before (Co) and after (Ce) the excimer ablation was measured in 7 eyes.

results. The facility of outflow increased in all eyes after the excimer sinusotomy, from a mean of 0.29 ±0.02 before the sinusotomy to 0.37 ± 0.03 μl/min per mm Hg after (P < 0.05). The mean ratio of outflow facility after and before ablation (Ce/Co) was 1.27 ± 0.08 (range, 1.20–1.39), a reduction of outflow resistance of 21.3%. Using the formula of Ellingsen and Grant (1972), percentage of resistance to outflow eliminated = 100[ 1 − αCo/Ce − (1 − α)Co], where α = fraction of the circumference dissected. Assuming that because of circumferential flow approximately 50% of Schlemm’s canal is drained by the single opening made in the outer wall ablation studies, this results in resistance to outflow eliminated of 35%, which is consistent with the calculated eliminated resistance derived from the data of Rosenquist et al., 1989. Light and scanning electron microscopy confirmed the integrity of the inner wall Schlemm’s canal underlying the area of ablation.

conclusions. The results provide direct evidence indicating that approximately one third of resistance to outflow in the human eye lies distal to the inner wall Schlemm’s canal in an enucleated perfused human eye.

Excimer lasers are a source of pulsed high-power ultraviolet radiation. The argon-fluoride (ArF) laser produces 193 nm radiation, which has been used to precisely etch the cornea, sclera, lens, and skin. ^{1 2 3 4} The photoablation excisions produced by the 193-nm laser have sharp borders with minimal thermal or mechanical damage to adjacent tissue. These characteristics have led to the investigation of excimer lasers in corneal refractive surgery and a variety of other applications, including trabeculectomy. ^{2 3 4 5 6}  

Trabeculectomy is the most common surgical procedure for the treatment of chronic open-angle glaucoma. One of the complications of this procedure is failure to maintain a low intraocular pressure, which appears to be related to persistent inflammation leading to closure of the fistula. ³ The ability of the 193-nm excimer laser to make excisions with minimal adjacent tissue damage has been used by Aron–Rosa et al. ³ to produce trabeculectomies in rabbits without signs of inflammation at 5 months. The excimer laser has also been used to create a partial trabeculectomy in human eyes, in which a hole was created in the outer wall Schlemm’s canal while maintaining a competent inner wall. The effect of this lesion on the outflow resistance was examined. ⁴ The precise excisions created by the 193-nm excimer laser can be used to examine fluid dynamics of the aqueous humor such as the outflow resistance. 

In many of the studies examining the aqueous outflow resistance, approximately 75% of the outflow resistance was attributed to the trabecular meshwork and the inner wall Schlemm’s canal. ^{7 8 9 10} However, Rosenquist et al. ¹¹ demonstrated that by using a perfusion pressure of 7 mm Hg this outflow resistance was only 49% compared to 71% at a perfusion pressure of 25 mm Hg. This difference was attributed to the unphysiological high perfusion pressure at 25 mm Hg due to the lack of the normal episcleral venous pressure in the enucleated eyes. This study was performed using internal trabeculotomies. ¹¹  

In the present study, the 193-nm excimer laser was used to remove a portion of the sclera, with Schlemm’s canal intact, and then to penetrate the outer wall Schlemm’s canal. The effect of these lesions on the outflow resistance was studied. The histology of the lesions was examined by light microscopy, transmission electron microscopy (EM), and scanning EM. 

Materials and Methods

Seven pairs of normal human enucleated eyes obtained from the New England Eye Bank and the National Disease Research Interchange (NDRI) were used in this study. Eyes were excluded if there was any history of ocular surgery, trauma, or disease. Enucleation was performed within 6 hours of death, and the eyes were stored in 0.9% saline at 4°C. All eyes were used within 48 hours of enucleation. 

The constant pressure perfusion technique described by Grant was used with Barany’s mock aqueous humor. ^{12 13 14} A 4-mm corneal trephine excision was used to place Grant fittings. To maintain the normal anterior angle during perfusion one radial incision was made in the iris. The perfusion pressure determinations were performed at 37°C and a pressure of 10 mm Hg. Barany’s solution containing 0.02% fluorescein dye was used to help visualize Schlemm’s canal. 

The enucleated eyes were perfused for 45 minutes to establish a baseline facility of outflow. After the excimer ablations, which required approximately 15 minutes, the postablation facility of outflow was determined for 60 minutes. The facility of outflow was calculated for the last 5 minutes of every 15-minute period. 

A Questek argon fluoride excimer laser (193 nm) was used for photoablation. The laser beam was masked to produce a beam 2 mm by 1 mm. The laser output was constant at a fluency of 75 mJ/cm² and 20 Hz. Before the ablation the conjunctiva was dissected from the limbus. The excimer laser at a frequency of 1 Hz was used as the aiming beam. Photoablation was performed at the limbus at an angle of 0° to 45° from the optical axis. 

As the excimer photoablations progressed, Schlemm’s canal was visualized by the fluorescence of the Barany’s solution containing fluorescein dye. In some of the outflow studies, visualization of Schlemm’s canal without ablation of the outer wall was used as the end point of the ablation. In other outflow studies, the excimer laser was used to penetrate the outer wall Schlemm’s canal, with the leakage of aqueous as a marker for completion. 

After completion of the outflow resistance determinations, a fixative solution (2% formaldehyde, 2.5% glutaraldehyde in Sorensen’s buffer, pH 7.35) was perfused at the same pressure for 60 minutes. After perfusion–fixation the eyes were immersion–fixed overnight. The anterior segments of the eyes were dissected into quadrants. Meridional wedges (2 mm) were prepared and washed in Sorensen’s buffer. One half of the tissue samples were prepared for light microscopy and transmission EM by infiltration and embedding in Epon/Araldite. The other samples were prepared for scanning EM by standard laboratory procedures. One-micrometer-thick sections stained with toluidine blue were examined by light microscopy. Ultrathin sections stained with uranyl acetate and lead citrate were examined by transmission EM. 

Results

The progression of the excimer excisions was followed using an operating microscope. The photoablations produced smooth excisions that progressively increased in depth. Each pulse of the 193-nm radiation resulted in a slight fluorescence of the limbal tissue; however, as the excisions progressed, Schlemm’s canal was clearly visualized by the strong fluorescence of the aqueous (which contained fluorescein dye). In the excimer sinusotomy experiments, the photoablations continued until the outer wall Schlemm’s canal was ablated, as indicated by the release of the fluorescing aqueous. 

The ability of the excimer laser to specifically ablate the outer wall Schlemm’s canal (Fig. 1) explains why it was used to examine the sites of resistance to aqueous outflow. The facility of outflow was determined for seven enucleated eyes in which the outer wall Schlemm’s canal was ablated. The facility of outflow before (Co) and after (Ce) the excimer ablation is illustrated in Table 1 . In all seven samples, the facility of outflow increased after the excimer sinusotomy. The change in facility of outflow from a mean of 0.29 μl/min per mm Hg before the sinusotomy to 0.37 μl/min per mm Hg after the excimer ablation was statistically significant (P < 0.05). The ratio of the outflow after the ablation to the outflow before the ablation (Ce/Co) ranged from 1.20 to 1.39 for the seven samples, with a mean of 1.27 (which is statistically different when compared to a ratio of 1). Using the equation 100 (1 − Co/Ce), the percentage of the outflow resistance eliminated was calculated to be 21.3%. 

The resistance to aqueous outflow was examined further by using the excimer laser to ablate tissue until Schlemm’s canal was visualized by fluorescence, without ablating the outer wall of the canal. The facility of outflow was determined for seven enucleated eyes, in which the outer wall Schlemm’s canal was preserved. The facility of outflow before (Co) and after (Ce) the excimer ablations is shown in Table 2 . In all seven samples, the facility of outflow showed only minor changes. Six samples had a slight increase in the facility of outflow after the excimer ablation; however, in one sample the facility of outflow showed a minor decrease. In this experiment, there was not a statistically significant difference between the mean facility of outflow before and after the ablation (0.30 versus 0.31). The ratio (Ce/Co) ranged from 0.95 to 1.08, with a mean of 1.04 (which was not statistically different from a ratio of 1). The change in facility of outflow corresponded to a decrease of 3.8% of the resistance. The mean facility of outflow in the experiment preserving the outer wall Schlemm’s canal is illustrated in Table 2 . 

Discussion

Several investigators have provided insight into the location of resistance to aqueous outflow; however, the exact characteristics of aqueous outflow remain controversial. In several studies of trabeculotomy in enucleated human eyes, approximately 75% of the outflow resistance was attributed to the trabecular meshwork and the inner wall Schlemm’s canal. ^{7 8 9 10} This observation was confirmed and supported for over 20 years. Then Bill and Svedbergh, ¹⁵ using an in vivo micropuncture technique in monkeys, attributed approximately 90% of the aqueous outflow resistance to the trabecular meshwork and the inner wall Schlemm’s canal. This finding was consistent with a previously published study by Ellingsen and Grant, ¹⁰ which found that 83% to 97% of the outflow resistance was proximal to the outer wall Schlemm’s canal in enucleated monkey eyes. These studies all provide support to the hypothesis that the principal site of resistance to aqueous outflow is proximal to the outer wall Schlemm’s canal; however, Rosenquist et al. ¹¹ demonstrated that the characteristics of resistance to aqueous outflow were sensitive to different perfusion pressures. In their study using trabeculotomy in enucleated human eyes and a perfusion pressure of 7 mm Hg, only 49% of the resistance to outflow was located proximal to the outer wall Schlemm’s canal,compared with 71% of the resistance at a perfusion pressure of 25 mm Hg. The perfusion pressure of 25 mm Hg, which had been used in several previous studies, was determined to be unphysiologically high due to the lack of normal (approximately 10 mm Hg) episcleral venous pressure in enucleated eyes. This is based on the assumption that the anatomic channels through which aqueous humor flows distal to Schlemm’s canal are intact in the enucleated human eye, and that the resistances are the same in these vessels in the absence of blood flow and in the absence of vasoactive control. In support of this assumption, our outflow facilities in this study were similar to what one would measure in the living eye. 

Another approach to characterizing the resistance to aqueous outflow is examining the effect of structures distal to the inner wall Schlemm’s canal. This was examined in a study by Ellingsen and Grant, ¹⁰ in which sinusotomies were performed in enucleated human eyes by dissection. In that study, successful externalization of Schlemm’s canal without damage to the inner wall was difficult, and gross trauma was apparent in a significant number of eyes. Because of the technical difficulties associated with sinusotomies, this approach was not heavily pursued to examine the resistance to aqueous outflow. However, with the advent of the excimer laser and its ability to perform precise excisions, the sinusotomy approach developed a renewed interest. The excimer laser was initially used to examine aqueous outflow by Seiler. In that study, the excimer laser was used to ablate the outer wall Schlemm’s canal, using the outpouring of aqueous as the end point of the ablation. However, the perfusion studies produced data with large standard errors, and the histology showed damage to the inner wall Schlemm’s canal in all samples. 

In this study several changes were made to improve the precision of the ablation and the perfusion studies. The three parameters changed for the ablation were as follows: (1) 0.02% fluorescein was used, (2) the angle of ablation was changed, and 3) there was a lower fluency of 75 mJ/cm². Fluorescein dye allowed visualization of Schlemm’s canal before ablation of the outer wall. This allowed precise monitoring of the photoablations’ gradual progression, from the ablation of limbal tissue, to initial visualization of Schlemm’s canal, to progressively increasing fluorescence and visualization of Schlemm’s canal, and, finally, the ablation of the outer wall Schlemm’s canal. The fluorescein dye also demonstrated the precise location of Schlemm’s canal, which permitted changes in the angle of ablation to maximize the ablation of Schlemm’s canal and minimize ablation of surrounding structures. In addition, a lower fluency of 75 mJ/cm² was used, compared with 160 to 180 mJ/cm² used by Seiler. The lower fluency provided a decreased depth of ablation per pulse and thus improved control of the ablation. All these changes were used to precisely ablate the outer wall Schlemm’s canal, while maintaining the integrity of the inner wall Schlemm’s canal. The one major change in the perfusion techniques was the use of Grant fittings, instead of a 25-gauge needle. In some of the initial experiments, Grant fittings resulted in more consistent and reproducible outflow measurements. 

The results of these experiments are consistent with the hypothesis of incomplete circumferential flow in Schlemm’s canal. Ellingsen and Grant described an equation for the resistance to outflow that assumed that there was no resistance to circumferential flow in Schlemm’s canal: percentage of resistance to outflow eliminated = 100[ 1 − αCo/Ce − (1 − α)Co], where α = fraction of the circumference dissected (Table 3) . By placing the facility outflow data from the outer wall ablation experiments into this equation and assuming an α = 0.05, the resistance to outflow eliminated by ablation of the outer wall was 84%. This value is elevated due to the assumption in this equation of no resistance to circumferential flow. Rosenquist et al. ¹¹ found no significant difference in the outflow resistance for trabeculotomy of 4 clock hours compared with 12 clock hours; however, for 1 clock hour the resistance to outflow eliminated was decreased due to the circumferential resistance. The resistance to outflow for 1 clock hour (α = 0.05) was 60% of the resistance at 12 clock hours for a perfusion pressure of 7 mm Hg and 41% for a pressure of 25 mm Hg. Thus, we assume that approximately 50% of the resistance was eliminated for the outer wall ablation studies (α = 0.50), because in these experiments a perfusion pressure of 10 mm Hg was used. This resulted in a resistance to outflow eliminated of 35%, which is consistent with the Rosenquist data. 

In conclusion, these results indicate that 21.3% of the resistance to outflow is eliminated for a 1 clock hour ablation of the tissue from the outer wall Schlemm’s canal and distal. This resistance appears to be located in the outer wall Schlemm’s canal or tissue surrounding it, because there was no significant decrease in resistance by ablating tissue up to the outer wall Schlemm’s canal. In addition, our results are consistent with the presence of circumferential resistance in Schlemm’s canal and the data of Rosenquist et al. ¹¹ in which 49% of the resistance to outflow was determined to be proximal to the outer wall Schlemm’s canal at a perfusion pressure of 7 mm Hg and 71% of the resistance at 25 mm Hg. 

 Reprint requests: Joel S. Schuman, New England Eye Center, Tufts University School of Medicine, 750 Washington Street, Box 450, Boston, MA 02111.

 Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Sarasota, Florida, May 1992.

 Submitted for publication September 24, 1998; revised January 28, 1999; accepted March 2, 1999.

 Proprietary interest category: N.

 

Figure 1.

 

Light micrograph demonstrates ablation just through outer wall Schlemm’s canal. Note sharp margins of ablation, without evidence of thermal damage. Note also giant vacuoles on inner wall Schlemm’s canal, which is patent (magnification, ×10).

Table 1.

 

Ablating Outer Wall of Schlemm’s Canal

	Co	SE	Ce	SE	Ce/Co
Eye 1	0.22	0.00	0.27	0.02	1.22
Eye 2	0.30	0.03	0.36	0.00	1.20
Eye 3	0.21	0.03	0.25	0.02	1.18
Eye 4	0.31	0.00	0.40	0.03	1.26
Eye 5	0.39	0.03	0.51	0.03	1.32
Eye 6	0.32	0.00	0.42	0.05	1.33
Eye 7	0.26	0.01	0.37	0.01	1.39
Mean	0.29	0.01	0.37	0.03	1.27
Mean Ce/Co 1.27± 0.02
Resistance	21.3%

 Each baseline value (Co) represents the mean of three consecutive 15-minute measurement periods; each experimental value (Ce) represents the mean of four consecutive 15-minute measurement periods.

Table 2.

 

Preserving Outer Wall of Schlemm’s Canal

	Co	SE	Ce	SE	Ce/Co
Eye 1	0.27	0.00	0.29	0.02	1.07
Eye 2	0.38	0.02	0.41	0.03	1.08
Eye 3	0.23	0.02	0.24	0.02	1.04
Eye 4	0.35	0.01	0.36	0.02	1.02
Eye 5	0.24	0.02	0.26	0.02	1.05
Eye 6	0.42	0.02	0.43	0.03	1.02
Eye 7	0.22	0.01	0.21	0.00	0.95
Mean	0.30	0.01	0.31	0.01	1.04
Mean Ce/Co 1.04± 0.02
Resistance	3.8%

 Each baseline value (Co) represents the mean of three consecutive 15-minute measurement periods; each experimental value (Ce) represents the mean of four consecutive 15-minute measurement periods.

Table 3.

 

Resistance Changes with Outer Wall Ablation

	α = 0.05	α = 0.50
Eye 1	82%	31%
Eye 2	80%	29%
Eye 3	78%	28%
Eye 4	85%	37%
Eye 5	86%	38%
Eye 6	86%	39%
Eye 7	89%	46%
Mean	84%	35%

 Calculated from our ablations, using the formula of Ellingsen and Grant. ¹⁰