The argon fluoride excimer laser is able to ablate corneal tissue with submicron accuracy and with minimal damage to adjacent tissue.1 Many variables that could affect reepithelialization and healing of the cornea have been studied in animals and in humans, including edge profile, wound depth, and size of the ablated zone.2,3
The ablated area can be shaped using masks or lenses.4 Based on this principle, Del Pero and colleagues used progressively smaller circular masks to perform myopic ablations in monkeys and humans.5 McDonald and colleagues6 used a manuallycontrolled, constricting iris diaphragm to excise anterior stromal tissue in rabbits to change the refractive power of the cornea, and they reported that multiple small steps without sharp edges resulted in better healing than did fewer steps.
Microscopic studies have revealed an electron dense pseudomembrane on the anterior surface of corneas ablated with the excimer laser.7-9 We previously compared the ultrastructural surface features of corneas that had been ablated using an iris aperture diameter that progressively increased with those ablated with a progressively smaller iris diameter using the same laser; we found the pseudomembrane that formed after corneal ablation to have fewer discontinuities when an expanding aperture was used.10 To evaluate the effects on the corneal healing of contracting versus expanding iris apertures, we performed myopic cornea ablations in rabbits using these two strategies.
MATERIALS AND METHODS
The animals used in this study were maintained in animal-care facilities fully accredited by the American Association of Laboratory Animal Science, and all studies were in accordance with the ARVO resolution on the Use of Animals in Research. Eight New Zealand pigmented rabbits (16 eyes) were used in these studies. All procedures were performed on anesthetized rabbits (ketamine hydrochloride 40 mg/kg and xylazine 7 mg/kg). Rabbits were examined preoperatively with the slit-lamp microscope and keratometer (American Optical, Buffalo, NY), and all had normal, clear corneas.
Figure 1: Size of the corneal epithelial defect of rabbits following photorefractive keratectomy. Rabbits were treated using an expanding diaphragm (group 1) or a contracting diaphragm (group 2). The values represent the means of each group.
Laser and Delivery System
The laser used was the model Twenty-Twenty argon-fluoride excimer laser (VISX, Ine, Sunnyvale, Calif). The delivery system of this laser includes an iris diaphragm that is continuously variable in diameter, thereby allowing ablation of a series of circular areas of superficial corneal tissue. To perform an ablation for myopia, in which the tissue centrally is removed to a greater depth than is peripherally located tissue, the diaphragm can be continuously expanded or contracted in up to 240 steps. Eyes were randomized so that each rabbit had one eye treated with an expanding diaphragm and the other with a contracting diaphragm. The eyes operated on with an expanding aperture were referred to as group 1, and eyes operated on with a contracting aperture were referred to as group 2. A vacuum fixation ring, equipped with jets that blow nitrogen gas across the corneal surface, was applied to each globe. To remove most of the corneal epithelium, all eyes were submitted to a phototherapeutic keratectomy of 6 mm diameter, 45 pm depth. The laser fluence was 180 mJ/cmp 2, pulse rate was 5 Hz, with a 0.3-micron average depth of ablation per pulse. Any remaining epithelium was then easily removed using cellulose sponges (Weck-Cel Surgical Spears, Edward Week & Co, Research Triangle Park, NC). The denuded area was then submitted to a myopic ablation (photorefractive keratectomy ) of 5.00 diopters with a treatment zone diameter of 5 mm, using a total of 149 pulses.
After surgery, combination tobramycin (0.3%) and dexamethasone (0.1%) ointment were applied daily to each eye, until reepithelialization was complete at about 1 week. No topical corticosteroids were used subsequently in the study. Rabbits were examined with a biomicroscope each day until the cornea had completely reepithelialized, and then weekly for 4 weeks after surgery. Documentary photographs, with and without topical fluorescein, were obtained at each examination. At the last examination, keratometric readings were performed. Corneal clarity was graded as previously described:11 grade 0, no visible opacity; grade 0.5, trace corneal haze seen only by indirect tangential illumination; grade 1, minimal corneal haze seen with difficulty by direct illumination; grade 2, easily visible mild haze; grade 3, dense opacity with partial obscuration of iris details; and grade 4, completely opaque corneas. Two observers, without knowledge of the treatment group, examined the rabbits and graded the corneal opacity. The size of the epithelial defect was measured using an Image Digitizer System (Video Image Analysis System, Millbrae, Calif). The vertical diameter of each cornea was used as a scale in each photograph analyzed. The photographs obtained at each examination were scanned and the surface area of the epithelial defect measured in mmp 2.
Figure 2: Slit-lamp microscope photograph showing mild anterior stromal haze 4 weeks after photorefractive keratectomy.
A paired Student i-test was used to compare differences between groups. Ap-value of ≤ .05 was considered significant.
By slit-lamp microscopic examination, all rabbit corneas appeared well healed within 2 weeks. The time (mean ± SD) required for complete epithelial healing (no area of staining with fluorescein) in group 1 was 7.8 ± 5.6 days and in group 2 was 6.5 ± 4.2 days (p = .4). The rate of epithelial healing, as measured with image analysis of serial slit-lamp microscope photographs, was essentially identical for the two groups (Fig 1).
Beginning at 1 week after photorefractive keratectomy, reticular superficial stromal haze was present in all eyes (Fig 2). The intensity of the haze remained fairly constant during the 1st 3 weeks, but a slight decrease in the haze was detected in both groups during the last week of the experiment (Table). During the course of this experiment, no statistically significant difference in haze was found between the two groups.
Keratometric analysis indicated that corneal flattening was obtained in both groups.
Group 1 had an average flattening of 3.60 ± 1.50 D and group 2 had an average corneal flattening of 4.1 ± 1.5 D. The difference between the two groups was not statistically significant (p = .69).
Photorefractive keratectomy is a recently developed technique for altering anterior corneal curvature. In contrast with radial keratotomy, in which incisions are created to a depth of 90% or greater of the corneal thickness, photorefractive keratectomy involves ablation of superficial corneal stroma.6 When performing myopic ablations using the excimer laser, a greater amount of tissue is removed centrally than peripherally. Regional variability in the amount of tissue removed is possible with the use of a computer-controlled iris diaphragm or the use of rotating disks that shape the laser beam.4
Central Corneal Haze Determined by Slit-Lamp Microscopy in Rabbit Corneas
To date, several reports have been published regarding the use of the iris diaphragm or masks to perform myopic ablations. 3,5-7 Techniques employing contracting or expanding apertures have been used, with little consideration of how these different apertures might affect corneal reepithelialization and healing. Recently, we demonstrated that the pseudomembrane that forms after laser ablation had fewer discontinuities when an expanding iris diaphragm was used.10 According to a report by Marshall and colleagues,12 the sealing promoted by the pseudomembrane contributes to the healing of the ablated zone. Other functions of the pseudomembrane, such as prevention of corneal swelling13 or promotion of more organized corneal healing, have been proposed.14 Trokel14 stated that the pseudomembrane appears to maintain the integrity of sectioned cells and serves as a template for reepithelialization during corneal healing.14 We hypothesized that a pseudomembrane with fewer discontinuities might promote faster reepithelialization or possibly limit anterior stromal keratocyte response, and, thereby, alleviate the consequent haze. In the pigmented rabbits used in this study, however, no differences in epithelial healing rates, amount of anterior stromal haze, or amount of corneal flattening were demonstrable between eyes treated with contracting versus expanding iris apertures.
The small sample size used in this experiment required that relatively large differences in reepithelialization rates or stromal haze would have to be present to allow statistical significance. Also, the use of a subjective assessment of haze may make it more difficult to appreciate small differences in haze. Also, the length of follow up in this study was relatively short, but extended beyond the time of maximum haze (3 weeks) in the rabbits. Because of possible differences in healing between human and rabbit corneas and the lack of Bowman's layer in the rabbit cornea, however, these results are not necessarily applicable to the human cornea. A definitive trial designed to demonstrate, in humans, a difference between expanding and contracting iris diaphragm would require a very large sample size, based on our observations in a relative small pilot study in animals. Sample-size calculations, based upon observations in these rabbits, suggested that two groups of at least 137 eyes would be required to have a 90% power of detecting differences at an alpha level of 0.05.
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13. Holland SP, Ng W, Marshall J. Corneal swelling following excimer laser photorefractive keratectomy. Lasers and Light in Ophthalmology. In press.
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Central Corneal Haze Determined by Slit-Lamp Microscopy in Rabbit Corneas