Refractive surgery has progressed rapidly since the development of the 193 nm excimer laser, which ablates tissue without coagulation or deformity of the surrounding tissue. Among the various refractive surgery procedures, photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK) are the most frequently performed to correct myopia and astigmatism.
Photorefractive keratectomy makes changes in the corneal curvature by removing part of Bowman's layer and anterior corneal stromal tissue. In contrast, LASIK does not remove the corneal epithelium, Bowman's layer, or anterior stromal tissue but does remove deep stroma. Photorefractive keratectomy with a 6.0 mm ablation zone was the first refractive surgical procedure to receive premarket approval from the United States Food and Drug Administration. However, myopic regression and corneal haze frequently occur, especially in highly myopic patients in whom large amounts of corneal tissue must be removed. These are the major complications of this method.1,2 There is no remarkable myopic regression or corneal haze observed clinically in LASIK, even in patients with high myopia.3 However, there are few experimental studies of the differences in the wound-healing process between the 2 methods. In this study, we evaluated the differences; our analysis included quantitative measurement of regenerated stromal tissue and ultramicroscopic changes in the surgical sites.
Materials and Methods
Twenty-four rabbits (24 eyes) were divided into 2 groups: PRK (n = 12 eyes) and LASIK (n = 12 eyes). In both groups, 1 mL/kg of ketamine hydrochloride 5% was injected intramuscularly and a topical anesthetic agent was applied to the eyes preoperatively.
The VISX Star 193 nm excimer laser was used. In the PRK group, a 4.0 mm diameter and 15.0 μm deep phototherapeutic keratectomy was performed first to ablate the thick epithelium of the rabbit cornea. A 4.0 mm diameter and 20.0 μm deep stromal PRK was then performed in the same site.
In the LASIK group, an 8.0 mm diameter and 150.0 μm deep hinged corneal flap was made with a microkeratome (MicroTech Co.), and a 4.0 mm diameter and 20.0 μm deep ablation was performed in the exposed anterior corneal stroma.
After surgery, dichlorotriazinyl aminofluorescein (DTAF) 0.5% dye (Molecular Probes Inc.), diluted with 0.2 M sodium bicarbonate buffer, was instilled in all treated eyes. The DTAF was washed out with normal saline after 60 seconds.
Regenerated Corneal Stromal Thickness During Wound Healing.
Dichlorotriazinyl is a fluorescent dye and a derivative of thiocyanate and triazinyl chloride. It forms covalent bonds with amino groups in tissue protein. It is stable in body tissues for more than a year and does not interfere with the wound-healing process. Instilled in an eye having photorefractive surgery, it can be used to distinguish newly regenerated collagen, which will not be stained, from preformed collagen.4–6
The rabbits were killed 1, 4, 8, and 12 days after surgery, and the globes were fixed with neutral formalin 10%, embedded in paraffin after dehydration, and cut into 6 μm slices. Fluorescence microscopy (Axiophot Photomicroscope, Zeiss) was performed and histology photographs with a ×200 magnification were obtained. The thickness of the regenerated stromal tissue was calculated using an image-analysis system (Kontron) at 4 spots, 2 in the middle of the surgical site and 2 in the periphery. Each site was checked 4 times in spots at least 50.0 μm apart. The mean value of the 16 calculations represented the regenerated corneal stromal thickness. In both groups, regenerated collagen fibers were also examined after Masson trichrome staining.
Number of Keratocytes in Regenerated Stroma.
In both groups, some of the globe slices were stained with hematoxylin–eosin at 1, 4, 8, and 12 weeks. Microphotographs were then obtained with a light microscope (Axioskip, Zeiss). The number of regenerated keratocytes per 10 000 μm2 of the surgical site were calculated. Calculations were performed at 4 spots, 2 in the middle of the surgical site and 2 in the periphery, and provided the mean number of keratocytes.
Ultramicroscopic Changes in Stromal Tissue.
Four weeks after surgery, half of each globe tissue was prefixed with paraformaldehyde 2%–glutaraldehyde 2.5% (pH 7.2) for 4 hours, postfixed with osmium tetroxide 1% for 2 hours, and then embedded in Epon 812 after dehydration. After ultrathin (80 μm) tissue slices were obtained and double-stained with uranyl acetate and lead citrate, transmission electron microscopy (TEM) (1200 ES, JEOL) was performed. Some fixed tissues were dehydrated with 50% alcohol, substituted with isoamyl acetate, dried with a critical point dryer (E3000, Polaron), and mounted. After surface treatment with gold by ion sputter, scanning electron microscopy (SEM) (JSM-5410LV, JEOL) was performed.
All results are presented as mean ± standard deviation (SD). In both groups, the number of regenerated keratocytes and the thickness of regenerated stromal tissue were analyzed using the Wilcoxon rank sum test. The level of significance was a P value of 0.05.
In the PRK group, 2 or 3 layers of migrated corneal epithelial cells covered the ablated corneal stroma 1week after surgery. However, at 4, 8, and 12 weeks, epithelial hyperplasia was observed over 10 layers in some areas. Their arrangement was irregular, and some elongated basal cells were also observed (Table 1; Figure 1 A). In the stromal tissue in the wound site, there was no detectable regenerated tissue 1 week after surgery (Figure 2 A). But at 4, 8, and 12 weeks, irregularly arranged regenerated collagen became detectable, and the amount increased with time (Figure 2B). In the untreated corneal area, stroma maintained a well-arranged lamellar structure, and there was no abnormality in the size or shape of the endothelial cells.
In the LASIK group, all eyes had similar features and the pattern of wound healing was relatively regular despite various examination times (Figure 1B). In the microkeratotomy wound site, epithelial hyperplasia and elongation of basal cells were observed, as well as epithelial ingrowth between the corneal flap and underlying stromal bed (Figures 1C, 1D). Despite newly formed, irregularly arranged collagen fibers in the microkeratotomy wound site, the stroma generally maintained its lamellar structure in the central surgical site (Figures 2C, 2D). The number of regenerated keratocytes increased in the wound margin but not in the wound center. This finding was the same in both superficial and deep stromal tissues (Table 1).
In the PRK group, at 4 weeks, newly regenerated stromal tissue, unstained by DTAF, was clearly detectable between the corneal epithelium and the DTAF-stained stromal bed, and its thickness increased with time (Table 2; Figures 3A, 3B).
In the LASIK group, there was no detectable regenerated stromal tissue unstained by DTAF between the DTAF-stained corneal flap and the stromal bed, except in the microkeratotomy wound margin (Table 2; Figures 3C, 3D).
In the PRK group, SEM showed normal corneal epithelial features. However, TEM showed a loss of parallel lamellar structure and deposits of irregularly arranged collagen fibers in the stroma beneath the epithelium. Multiple spaces were also observed because of insufficient stromal maturation (Figures 4A and 5A). The number of keratocytes in the wound area was increased, and their shape was bizarre and irregular. Cytoplasmic organelles, such as rough and smooth endoplasmic reticuli, mitochondria, and ribosomes, had an activated appearance (Figure 5B).
In the LASIK group, SEM showed normal epithelial features (Figure 4B). Transmission electron microscopy showed that the collagen fibers maintained their well-arranged lamellar structure in the corneal flap and stromal bed. A small number of collagen fibers, which seemed to be newly regenerated tissue, was observed between the flap and bed, but the arrangement was relatively regular. Superficial and deep stroma also maintained their lamellar structure (Figure 5C). Keratocytes in the wound site remained stable in size and shape and were arranged parallel to the stromal structure. Cytoplasmic micro-organelles had inactive features (Figure 5D).
The cornea is a unique tissue in the body. It has no vessels, and because of its parallel and lamellated structure, light can pass through it. However, every refractive surgery in the cornea evokes the wound-healing process, which can reduce corneal clarity and influence the outcome of refractive surgery. Many organelles can be involved in corneal cloudiness during wound healing. The first stage of wound healing in the cornea after photorefractive surgery is epithelial migration along the ablated stromal bed. Epithelial proliferation and stromal regeneration follow. Excessive stromal regeneration during wound healing causes corneal cloudiness and myopic regression. Corneal haziness after refractive surgery has been observed in rabbits, monkeys, and humans, and several causes have been proposed. One is the abnormal deposition of type III and type IV collagen in the wound site.7–10 A recent study shows the importance of the corneal epithelium in the occurrence of corneal haziness.11
Corneal epithelial regeneration is followed by stromal regeneration. In rabbits, epithelial cell regeneration is complete 2 days after surgery, and the epithelial basement membrane is complete in 1 week. Any problems during this period can influence stromal regeneration and result in corneal haziness. Gauthier et al.12 found that the thickness of regenerated corneal epithelium increased until 32 months after surgery and that epithelial hyperplasia directly influenced corneal clarity. The fact that the size and shape of the ablated zone influence corneal clarity may indicate that corneal epithelial hyperplasia, growth factors in the tear film, and various cytokines are involved in corneal haziness.
There appears to be a relationship between corneal haziness and stromal hyaluronic acid presentation. The duration of corneal epithelial regeneration and corneal hydration may also be related. Many investigators have proposed that corneal haziness after refractive surgery is correlated with the amount of ablation.9,13–15 In highly myopic eyes in which it is necessary to remove large amounts of corneal tissue, more corneal haze and a larger amount of regenerated stromal tissue are observed than in eyes with low to moderate myopia.16–20 To prevent corneal haze and myopic regression, steroid eyedrops are now widely used after surgery.21–25 However, their effect is not stable and there are some problems associated with them, such as intraocular pressure elevation.18,26–32 Thus, many refractive surgeons prefer LASIK to PRK in highly myopic eyes. Although Amm et al.33 found less stromal regeneration after LASIK than after PRK in their histopathologic study, few studies of the wound-healing process after LASIK have been done, particularly a quantitative comparison of regenerating stromal tissue after LASIK and PRK.
In rabbit eyes, there is no Bowman's layer, unlike in human eyes. Despite this anatomic difference, we chose rabbits as the experimental animal in this study because the stromal wound healing is similar to that in humans, and many previous studies of wound healing after refractive surgery have been performed in rabbits. This made it easy to compare our results with those of other investigators. We also had a 12 week follow-up because corneal haze and myopic regression generally occur rapidly for 3 months after surgery and then the progression slows.16,17,19,20
We stained the rabbit corneas after surgery with 2 different dyes. One of them, DTAF, attaches to amino groups in tissue protein with a covalent bond so it is stable for more than 1 year in tissues and does not interfere with wound remodeling.5 Furthermore, it is easy to wash unbound dye from the cornea, and DTAF does not evoke an immunologic reaction. Therefore, we could easily differentiate newly regenerated unstained tissue from preformed DTAF stained tissue in the wound site by microscopic examinations. The second dye was Masson trichrome, which enabled us to identify the arrangement of collagen tissue in both groups.
In all eyes that had PRK, notable corneal haze occurred. This did not occur in eyes that had LASIK. Histopathologic examinations showed that both corneal epithelium and stroma were involved in corneal haze after surgery. Corneal epithelial hyperplasia, an increased number of stromal keratocytes, and irregularly arranged collagen fibers beneath the epithelium were observed at the wound site.
In the PRK group, fluorescence microscopy showed no newly regenerated stromal tissue until 1 week after surgery; but after 4 weeks, the thickness of regenerated stroma increased rapidly and irregularly arranged regenerated stromal collagen was also observed with Masson trichrome staining. We think this may represent a clinically detectable corneal haze after refractive surgery. However, in the LASIK group, we did not see any of these findings at the wound site except in the keratotomy wound margin. We also observed more active keratocytes in the PRK group than in the LASIK group, and the arrangement of collagen fibers in the PRK group was more irregular.
We think the more aggressive wound healing after PRK may reflect continuous stromal damage along the anterior portion of the cornea, including the epithelium. In LASIK, the anterior portion of the cornea was preserved, and the amount of irregularly regenerated collagen in stromal tissue was quite small when examined by light, fluorescence, and electron microscopy. Nevertheless, around the keratotomy wound margin, severe distortion of corneal tissue was observed, and this may be the cause of the ring-shaped microkeratotomy wound mark. A dehydrated and contracted corneal flap or the loss of corneal epithelium during the keratotomy procedure might result in stromal exposure after surgery, and this evokes excessive wound healing in a ring-shaped area as in PRK.
Electron microscopy was performed 4 weeks after surgery because former studies have shown that regeneration of stromal tissue and keratocyte activation peak at 4 weeks and then slow.1,8 Our study also showed activated keratocytes at the surgical site beneath the epithelium in the PRK group 4 weeks after surgery. However, this was not seen at the surgical site or beneath the corneal epithelium in the LASIK group. We found that in cases in which the anterior portion of the cornea was preserved, as in the LASIK group, there was little irregularly regenerated stromal tissue. Therefore, in highly myopic patients in whom the removal of a large amount of tissue is inevitable, LASIK may be the better choice for good refractive results. However, further studies are needed to understand why removing the anterior portion of the cornea evokes excessive wound healing while preserving the anterior cornea does not, as well as the mechanisms of interaction between deep and superficial stromal tissue in the wound-healing process. The reasons for the near absence of wound healing after LASIK have to be studied since LASIK is widely used to correct high myopia, despite insufficient animal studies and insufficient consideration of the serious potential complications such as loss of the corneal flap and wound infection.
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