Abstract
purpose. The femtosecond laser has been realized as an advantageous tool for micromachining, and the feasibility of employing it for surgical procedures has been investigated. A prior study demonstrated dose-dependent femtosecond laser photodisruption of peripheral corneal tissue through a gonioscopic lens in enucleated porcine eyes and of the trabecular meshwork (TM) in human corneoscleral rim tissues, with little collateral damage. The present study was undertaken to extend these efforts to ex vivo primate eyes.
methods. Photodisruption of the TM in enucleated baboon eyes and human donor eyes was performed with a gonioscopic lens and a custom femtosecond laser ablation delivery system. Laser ablation was executed with a Titanium:Sapphire laser (800-nm wavelength), focused with a 0.15-NA lens, with the following settings: pulse duration, 45 fs; pulse energy, 60 to 480 μJ; pulse repetition rate, 1 kHz, total exposure time, 0.001 to 0.3 seconds. The laser lesions were evaluated by two-photon microscopy.
results. Laser-induced lesions were consistently observed in the TM of the baboon and human eyes and visualized by two-photon microscopy. These oblique, trough-shaped lesions, which did not penetrate the juxtacanalicular region, had sharp edges and showed no evidence of thermal coagulation. The dimensions of the lesion increased linearly with both pulse energy and exposure time.
conclusions. The present study demonstrates that laser ablation of the TM ab interno in ex vivo primate eyes is feasible by a custom femtosecond laser ablation system with a gonioscopic lens.
There is great need in glaucoma therapy for a simple, effective, and incisionless surgical procedure. Laser trabecular ablation, an example of such a procedure, has been attempted. This technique is intended to create a direct communication between the anterior chamber and Schlemm’s canal with an ab interno approach.
1 Using this procedure, one could, in principle, increase the outflow facility by creating a fistula through the trabecular meshwork (TM), which accounts for approximately 75% of the outflow resistance.
2 The lasers most widely used for trabecular ablation are pulsed Nd:YAG,
3 4 5 6 7 8 9 Er:YAG,
10 11 12 13 14 15 and excimer
16 lasers. Nd:YAG laser trabecular ablation was studied in primates and humans, with the laser pulse delivered through a gonioscopic lens. In monkeys
5 there was an immediate decrease in intraocular pressure (IOP), but the outflow facility returned to baseline levels in 8 days to 3 weeks. Short-term human studies have revealed variable lowering of IOP, with a return to baseline IOP in most patients and gradual filling of the laser holes with scar tissue.
4 These treatments, therefore, were ineffective or not widely accepted.
17 The primary reason for their failure is related to the thermal effects of the lasers used for treatment.
17 18 19 20 21 For pulse durations of several nanoseconds or longer, thermal and mechanical relaxation occur during the laser pulse and compete with material ablation, resulting in collateral damage of tissues and scar formation.
22
More recently, laser trabecular ablation has been performed with the pulsed 2.94 μm Er:YAG laser. The longer wavelength, lying near the peak of the water absorption band, requires the use of an endoprobe, necessitating surgical entry into the anterior chamber to perform the ablation, while visualizing the TM through a gonioscopic lens or endoscope.
12 15 Feltgen et al.
15 reported endoscopic Er:YAG goniopuncture in humans to be a successful adjunct to cataract surgery in lowering IOP by 30% in the treatment group for more than 12 months. The 308-nm XeCl excimer laser has also been used for laser trabecular ablation. The distinct advantage offered by excimer laser trabecular ablation is reduced levels of thermal damage and necrosis. Vogel and Lauritzen
16 demonstrated increased outflow facility in vitro, and they showed modest lowering of IOP (median 7 mm Hg) at 7 months in a study in humans. Huang et al.
23 showed minimal trabecular scarring in a rabbit model of ab interno excimer laser trabecular ablation. The drawbacks of Er:YAG laser and excimer laser trabecular ablation include the need for an incision to introduce the delivery system and difficulty in manipulating the endoprobe in the anterior chamber to visualize the meshwork. The absence of controlled clinical studies may have also contributed to the lack of extensive use of this laser.
The femtosecond Ti:Sapphire laser is a potentially useful tool for laser trabecular ablation for several fundamental reasons. First, the 800-nm wavelength of the Ti:Sapphire laser falls within the absorption window of most tissues. The multiphoton nature of the transition allows the radiation to be focused to a point within the target without damaging the outer layers.
24 Second, the use of subpicosecond pulses allows the radiant energy to be absorbed on a time scale that is much shorter than both the thermal diffusion and shock wave propagation times. This property leads to thermal and stress confinement, thereby reducing the region of material damage to the vicinity of the laser focus.
24 Third, the lower fluence threshold for femtosecond laser ablation reduces the overall thermal and mechanical load on the tissue.
24 Fourth, the femtosecond laser can be applied to the TM without using endoprobes.
Previously, our laboratory showed that photodisruption by a femtosecond laser can be used to create lesions in the human TM in nonfixed corneoscleral rim tissues of donor eyes without damaging the surrounding tissues.
25 In addition, using enucleated porcine eyes and a gonioscopic lens, we demonstrated that femtosecond laser energy can be delivered to the peripheral inner surface of the cornea adjacent to the TM, which is bridged anteriorly by robust pectinate ligaments.
26 The present study was undertaken to extend our previous efforts to ex vivo primate eyes. Using enucleated baboon and human donor eyes, we demonstrate the feasibility of laser trabecular ablation through a gonioscopic lens.
Use of baboon eyes and a human donor eye was in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Declaration of Helsinki on research involving human subjects, respectively. Three baboon eyes were enucleated within 4 hours of death, after the animals were killed for other experiments by other investigators at the Biological Resources Laboratory (BRL) at the University of Illinois at Chicago. A human eye (56 years of age) was obtained within 24 hours of death from the Illinois Eye Bank in Chicago. The eyes were kept at 4°C for several hours before use. Three baboon eyes were examined (1) to assess the feasibility of producing an incisionless trabecular ablation by a femtosecond laser through a gonioscopic lens and (2) to evaluate the relationship between lesion volume in the primate TM and laser parameters. Only the first objective was possible for the human donor eye, because its cornea was insufficiently transparent to achieve perfect focusing of the ablation laser.
The eyes were placed on a gauze pad, with the cornea facing upward, in a Styrofoam orbital model, to approximate the customary positioning of the eye for laser surgery. A gonioscopic lens (Magna View Gonio Laser Lens, OMVGL; Ocular Instruments Inc., Bellevue, WA) was manually placed on the cornea with 2.5% hypromellose ophthalmic demulcent solution (Gonak; Akorn Inc., Buffalo Grove, IL). The TM was observed through the gonioscopic lens with a surgical microscope incorporated in the laser delivery system
(Fig. 1) .
TM photodisruption was conducted in baboon and human eyes using a gonioscopic lens and the femtosecond laser delivery system described in the Laser and Delivery System section. A two-beam focusing technique, developed in our laboratory,
26 was used. The He:Ne guiding beam, which is coaxial with the surgical femtosecond laser beam, was split into two beams at lens L2. When the guide beam was focused onto the target, the two visible beams coalesced. A single spot of the guiding beam indicated precise focusing on the target, as these beams overlapped each other only when the target was at the focal point.
No perfusion system was used during the laser procedure. Air bubbles produced by photodisruption formed in the anterior chamber under the cornea occasionally obstructed the visibility of the TM through the gonioscopic lens. These bubbles were removed with a 1-mL syringe and a 30-gauge needle through a self-sealing paracentesis. The IOP was maintained by the injection of balanced salt solution, also through a self-sealing paracentesis. The pressure was assessed digitally and was found to be approximately 10 to 30 mm Hg. Maintenance of the IOP prevented the eye from either collapsing or being overly compressed. Variable settings of the laser energy (60–480 μJ) and exposure times (0.001–0.3 seconds) were applied to the baboon eyes to investigate the relationship between lesion extent and laser parameters. The laser treatment process was recorded by a video system (DXC-760MD; Sony, Tokyo, Japan) to monitor visible effects of femtosecond laser treatment on the TM and surrounding tissues.
Each lesion in the baboon TM was evaluated using images obtained by two-photon microscopy. The area, perimeter, maximum diameter, and minimum diameter of each lesion produced in the surface tissue of the TM were measured with the Image J software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html). The depth of the lesion was defined as the distance between images of the surface and the bottom of the lesion.
The relationship between lesion extent in the baboon eyes and laser parameters (pulse energy and exposure time) was analyzed statistically in a linear regression model (SPSS, ver. 15.0; SPSS Inc., Chicago, IL). The independent variables were area, perimeter, maximum diameter, minimum diameter, depth, and nominal volume (area multiplied by depth). The pulse energy and exposure time were taken as dependent variables, with the exposure time fixed at 0.1 second for measurements with variable energy, and the laser energy fixed at 800 μJ/pulse for variable exposure time.