Corneal Wound Healing after Excimer Laser Ablation Effects of Nitrogen Gas Blower Mauro Campos, MD, Kevin Cuevas, BA, Jenny Garbus, BS, Martha Lee, PhD, Peter]. McDonnell, MD
Purpose: To examine the effects of blowing nitrogen gas over the cornea during photorefractive keratectomy. Methods: Excimer laser ablations for myopia were performed on rabbit corneas with or without the blowing of nitrogen across the surface of the cornea. All eyes underwent a 5-diopter myopic ablation; in 8 eyes, a ring was used to blow nitrogen gas across the cornea, and, in 8 eyes, the same ring was used, but no nitrogen gas was blown. Results: Epithelial healing occurred more rapidly in the eyes that were not treated with the gas (3.8 ± 1.3 days) than in the gas-treated group (6.1 ± 0.8 days; P = 0.0025). Corneal haze was greater in the group treated with gas. Results of histologic examination showed the ablated area to have a smoother surface when nitrogen was not blown across the cornea surface. Conclusion: Superficial corneal deturgescence produced by the nitrogen gas appears to result in a rougher surface immediately postoperatively with undesirable effects on surface healing, but further studies will be necessary to determine the applicability of these results to humans. Ophthalmology 1992;99:893-897
Excimer laser photorefractive keratectomy involves ablation of the anterior surface of the central cornea to change its radius of curvature. 1 After ablation, epithelial healing and superficial stromal keratocyte migration and new collagen formation are processes that can influence the success of the procedure. 2 Postoperative corneal clarity can range from completely clear corneas to slight haze or even dense opacity that obscures anterior chamber details. 3
Originally received: December 5, 1991. Revision accepted: February 24, 1992. From the Doheny Eye Institute and the Department of0phtha1mology, University of Southern California School of Medicine, Los Angeles. Supported by a grant from the Autry Foundation, Los Angeles, California. The authors have no financial interest in the subject matter or materials discussed in the article. Reprint requests to Peter J. McDonnell, MD, Doheny Eye Institute, 1450 San Pablo St, Los Angeles, CA 90033.
Many variables that could affect re-epithelialization and stromal healing of the cornea have been studied, including edge profile, wound depth and diameter, and the role of pharmacologic modulators of wound healing. 2- 5 Much of the effort toward improving photorefractive keratectomy has been directed at laser delivery systems and at tissue interactions, with less attention directed toward coupling systems. 6 Studies by Puliafito and colleagues7 demonstrated that tissue is ejected from the cornea during the ablation process. This material, which accumulates over the area that is being ablated, could absorb some of the laser energy from subsequent pulses, 8 possibly reducing the amount of tissue ablated with each pulse. Preliminary studies with cadaver eyes showed that this vaporized debris led to uneven ablations, which were avoided with the use of a nitrogen air flow designed to remove the particles during surgery. 9 Conversely, blowing nitrogen over the cornea might dehydrate and thin the cornea, possibly invalidating previous calculations. 6
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To our knowledge, this study is the first attempt to evaluate the effects of a vacuum fixation ring that blows nitrogen gas across the cornea surface during photorefractive keratectomy.
Materials and Methods Animals Nine New Zealand pigmented rabbits (18 eyes) were used in these studies. All procedures were performed on anesthetized rabbits (ketamine hydrochloride, 40 mg/kg, and xylazine, 7 mgjkg). All rabbits were examined preoperatively with the biomicroscope, keratometer (American Optical, Buffalo, NY), and ultrasonic pachometer (Storz Instruments, St. Louis, MO). The animals used in this study were maintained in animal care facilities fully accredited by the American Association of Laboratory Animal Science, and all animal studies were in accordance with the ARVO Resolution on the Use of Animals in Research.
Laser and Delivery System The laser used was the model Twenty-Twenty argon-fluoride excimer laser (VISX, Inc, Sunnyvale, CA). The delivery system of the laser includes an iris diaphragm that is continuously variable in diameter, and that ablates a series of circular areas of superficial corneal tissue. To perform an ablation for myopia, in which tissue is removed to a greater depth centrally than peripherally, the diaphragm can be continuously expanded or contracted in up to 240 steps. An expanding diaphragm was used in these experiments. A vacuum fixation ring equipped with jets to blow nitrogen gas over the cornea surface and an aspirator vacuum to remove the floating debris was used. Eyes were randomized so that each eye of each rabbit underwent a different surgical technique. Nine of the eyes (group 1) were operated on using the fixation ring with the nitrogen gas blowing across the cornea surface (5liters per minute). The other 9 eyes (group 2) were operated on using the fixation ring but no nitrogen gas. To remove most of the epithelium, all eyes first underwent a phototherapeutic keratectomy of 6 mm in diameter and 45 p,m depth. The laser fluence was 160 mJ/ cm2, pulse rate was 5Hz, and average depth of ablation per pulse was 0.3 p,. A cellulose sponge (Weck-Cel Surgical Spears, Edward Week and Co, Research Triangle Park, NC) was gently applied to all corneas to remove any remaining epithelium. The denuded area was then submitted to a myopic ablation (photorefractive keratectomy) of 5 diopters (D) with a treatment zone diameter of 5 mm, using a total of 149 pulses. After surgery, tobramycin (0.3%) and prednisolone ( 1%) ointments were applied daily to each eye. Eight rabbits were examined with a biomicroscope daily until the
corneas had completely re-epithelialized and then weekly for 4 weeks after surgery. One rabbit was killed just after surgery. Documentary photographs, with and without topical fluorescein, were obtained at each examination. Postoperative keratometry and pachometry were performed weekly by the same observer who was masked as to treatment group. At each examination, three readings were obtained and the average of the measurements was considered for each time point. Corneas were considered completely healed when no detectable epithelial defect stained with fluorescein. Corneal clarity was graded as previously described. 4 Two masked observers examined the rabbits without knowledge of the surgery protocol, and average grades were obtained. Criteria used to grade the density of corneal opacity were: grade 0, totally clear corneas; grade 0.5, trace or faint corneal haze seen only by indirect tangential illumination; grade 1, minimal haze seen by direct illumination; grade 2, a mild corneal opacity seen easily by direct focal slit lamp illumination; grade 3, a dense corneal opacity that partially obscured iris details; and grade 4, completely opaque cornea. One rabbit was killed, and the eyes were enucleated for ultrastructural studies immediately after surgery. The corneas were excised at the limbus and were fixed in halfstrength Karnovsky fixative (2% paraformaldehyde, 2.5% glutaraldehyde, and 0.1 M sodium calcodylate buffer). After 48 hours of fixation, the corneas were divided in 2, washed in 0.1 M sodium cacodylate buffer, postfixed in 2% sodium tetroxide in 0.1 M osmium cacodylate buffer for 2 hours, washed in 0.1 M sodium cacodylate buffer, stained in 1% uranyl acetate in 0.05 M sodium acetate buffer. They were dehydrated in a graded series of alcohol and then in pure polypropylene oxide, and embedded under vacuum in epoxy plastic resin. Sections for light microscopy were cut with a diamond knife ultramicrotome and stained with toluidine blue. Photographs were obtained to document the findings. Variables of the two surgical groups over time were compared by analysis of variance for repeated measures. Group and time were considered as two within-subject effects. Pairwise comparisons were made by paired t test with adjusted standard errors. All tests were two-tailed and the significance level was 0.05. Data are reported as mean ± standard error.
Table 1. Central Corneal Haze Evaluated by Biomicroscopy in Rabbit Corneas Postoperative Interval (Mean± Standard Error)
Type of Treatment
With nitrogen Without nitrogen
1.1 ± 0.2. 0.6 ± 0.1
1.3 ± 0.2 0.9 ± 0.1
1.1 ± 0.1 0.8 ± 0.1
Campos et al · Nitrogen Blower Effects
Figure 1. Slit-lamp photograph of both corneas of 1 rabbit 2 weeks after photorefractive keratectomy. A, cornea operated on with the nitrogen blower shows more intense haze than does B, the cornea operated on without gas augmentation.
Results By slit-lamp examination, all corneas appeared healed within 1 week. The time required for complete epithelial healing (no area of staining with fluorescein) in group 1 was 6.1 ± 0.8 days and in group 2 was 3.8 ± 1.4 days (P = 0.0025). All eyes developed some degree of corneal haze after surgery. The intensity of haze observed in both groups is shown in Table 1. Although no statistically significant differences were detected during the four weeks of the experiment (P = 0.1 0), at each time point the average intensity of haze was always greater in the group operated on with the nitrogen blower (Fig 1).
Results of keratometric analysis showed a significant degree of corneal flattening in all eyes. One month after surgery, the amount of corneal flattening obtained in group 1 was 4.8 ± 1.3 D, while group 2 had a total flattening of 4.1 ± 2.2 D (Fig 2). The difference between groups was not statistically significant at 4 weeks after surgery (P = 0.27). Results of pachometry immediately after surgery showed that the corneas from group 1 eyes were significantly thinner than the corneas from group 2 eyes. Average corneal thickness after surgery was 0.29 ± 0.01 mm in group 1 and 0.31 ± 0.02 mm in group 2 (P = 0.0 15). Two weeks after surgery, the average corneal thickness in group 1 (0.41 ± 0.01 mm) exceeded that of group 2 (0.40 ± 0.02 mm) (P = 0.03). At the last examination (week 4), the pachometry readings were not different between the two groups (Fig 3). Histopathologic examination of the cornea showed that the surface ablated with the use of the gas blower was more irregular than the ablated surface of the corneas operated on without the blowing of nitrogen across the
Time after surgery
Figure 2. The mean (± standard error) corneal flattening obtained in eight rabbits submitted to photorefractive keratectomy with (group 1) or without (group 2) the nitrogen blower. No significant differences were found during the 4 weeks of the experiment.
Figure 3. Pachometry measurements (mean ± standard error) of eight rabbits submitted to photorefractive keratectomy with (group 1) or without (group 2) the nitrogen blower.
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Figure 4. A, the ablated surface operated on with the gas blower shows marked irregularity, while B, the surface ablated without the gas blower shows a smooth appearance (toluidine blue; original magnification, Xl60).
surface. The anterior stroma had a "saw tooth" appearance in the corneas operated on with the gas (Fig 4). The effects of the surgery were limited to the anterior portion of the stroma, regardless of the ablative technique used.
Discussion Numerous variables involved in photorefractive surgery of the cornea can affect the outcome. Studies have been performed on the laser systems used for such procedures, as well as on the responses of the cornea to such surgery. A recent study demonstrated that use of mitomycin and steroids could modulate the corneal wound healing process in rabbits. 5 We demonstrated here that blowing nitrogen gas across the cornea during surgery also might affect the response of the cornea to excimer laser injury. Use of nitrogen gas during the ablation induced a significant delay in the re-epithelialization of the cornea, and it may be that corneal dehydration induced by the nitrogen gas accounts for this difference. Results of microscopic examination showed that corneas treated with the blower presented a more irregular surface than did corneas operated on without the gas. It is possible that the smoother surface encourages faster corneal re-epithelialization. Furthermore, the greater corneal haze observed in corneas operated on with the gas might reflect the irregularity of these ablated corneal surfaces. Other studies have shown that the use of masking fluids of moderately viscosity enhanced the smoothness of the ablated surface. 10• 11 In effect, a hydrated surface may generate a more regular ablated area, decreasing irregularities in the corneal surface. Corneal thickness measurements performed immediately after surgery demonstrated that corneas treated with the nitrogen blower were thinner than were those treated without the blower. This might result from dehydration
of the anterior stroma, or, alternatively, removing the debris might have resulted in a greater depth per laser pulse. The finding at 2 weeks after surgery that corneas operated on with the gas were thicker than the corneas operated on without the gas might be related to epithelial hyperplasia or to enhanced formation of new collagen. The clinical significance of the 10 JI-m difference is not proven, however. We should consider also that at 2 weeks after surgery, the degree of haze was maximal in the corneas operated on with the gas, suggesting that the increase in thickness might relate to production of anterior stroma new collagen. Although some authors believe that more effective surgery might be performed by blowing nitrogen gas across the corneal surface, 9 the keratometric findings in the current study showed that a significant degree of corneal flattening was achieved in both groups. No statistically significant difference in the keratometric readings was detected between corneas treated with or without the blowing of nitrogen. Our data suggest that use of a nitrogen gas blower during excimer laser refractive ablation might have negative effects on epithelial healing and may promote greater corneal stromal haze. If these effects are caused by dehydration of the stroma induced by the nitrogen, studies using humidified gases might be more appropriate. The use of gases of lower molecular weight also might influence the result of surgery by altering the speed of the shock wave and material leaving the ablated surface. Further studies are necessary to evaluate these findings in human corneas.
References 1. Marshall J, Trokel S, Rothery S, Krueger RR. Photoablative
reprofiling of the cornea using an excimer laser: photorefractive keratectomy. Lasers Ophthalmol 1986;1:21-48.
Campos et al · Nitrogen Blower Effects 2. Hanna KD, Pouliquen Y, Waring GO III, et al. Corneal stromal wound healing in rabbits after 193-nm excimer laser surface ablation. Arch Ophthalmol 1989; 107:895-901. 3. McDonald MB, Frantz JM, Klyce SD, et al. Central photorefractive keratectomy for myopia. The blind eye study. Arch Ophthalmol 1990; 108:799-808. 4. Fantes FE, Hanna KD, Waring GO III, et al. Wound healing after excimer laser keratomileusis (photorefractive keratectomy) in monkeys. Arch Ophthalmol 1990;108:665-75. 5. Talamo JH, Gollamudi S, Green WR, et al. Modulation of corneal wound healing after excimer laser keratomileusis using topical mitomycin C and steroids. Arch Ophthalmol 1991;109:1141-6. 6. Waring GO III. Development of a system for excimer laser corneal surgery. Trans Am Ophthalmol Soc 1989;87:854983.
7. Puliafito CA, Stern D, Krueger RR, Mandel ER. High-speed photography of excimer laser ablation of the cornea. Arch Ophthalmol1987;105:1255-9. 8. Srinivasan R, Sutcliffe E. Dynamics of the ultraviolet laser ablation of corneal tissue. Am J Ophthalmol1987;103:470-l. 9. Del Pero RA, Gigstad JE, Roberts AD, et al. A refractive and histopathologic study of excimer laser keratectomy in primates. Am J Ophthalmol 1990;109:419-29. 10. Kornmehl EW, Steinert RF, Puliafito CA. A comparative study of masking fluids for excimer laser phototherapeutic keratectomy. Arch Ophthalmol 1991;109:860-3. 11. Fasano AP, Moreira H, McDonnell PJ, Sinbawy A. Excimer laser smoothing of a reproducible model of anterior corneal surface irregularity. Ophthalmology 1991 ;98: 1782-5.