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Refractive Surgical Wound Healing Mechanisms Revisited: A Glimpse at the Future of LASEK

James V.Jester, PhD

University of Texas Southwestern Medical Center at Dallas

Dallas, TX

INTRODUCTION

Refractive surgery, from keratomileusis to laser-assisted in situ keratectomy (LASIK), have sequentially offered the promise of permanently correcting refractive visual errors. However, as these procedures have gained initial acceptance and then wider application, invariably problems have been encountered, ranging from regression and reduced visual acuity to progressive corneal instability that ultimately limit the successfulness of these procedures, seemingly leaving unfulfilled the promise of perfectly corrected vision.

Although refractive surgery has developed rapidly over the past 30 years, progress in understanding the basic mechanisms underlying these complications has been limited and at times superficial. For the most part, corneal wound healing has been implicated as a major contributor to the lack of success; however, even with this consensus opinion, the underlying pathophysiology is unknown at the molecular level and even remains controversial at the cellular and tissue level. Furthermore, although experimentation and development of refractive surgery techniques has led to an abundance of clinical experience, this knowledge has yet to be translated into a unified understanding of the basic corneal response to refractive surgery that can help in the development of predictive refractive surgical techniques.

As part of the First International LASEK Congress it is perhaps important to ask the questions as to what differentiates laser-assisted subepithelial keratectomy (LASEK) from its predecessors, photorefractive keratectomy (PRK) and LASIK, and can previous clinical experience combined with basic research on the biology of corneal wound healing provide insights into the success, failure, and future development of this new refractive surgical modality? In addressing these questions, this article reviews our current understanding of the cellular and molecular mechanism underlying the development of corneal haze after PRK, the possible explanations for the absence of haze after LASIK, and assesses the future ability of LASEK and other advanced surface ablation techniques to surgically provide refractive correction without the development of haze.

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blasts. This association has lead to the hypothesis that normally quiescent corneal keratocytes are transparent within the clear cornea, whereas fibroblasts and myofibroblasts markedly scatter light, leading directly to corneal haze.

Figure 2 Three-dimensional reconstructions taken form in vivo confocal microscopy scans of the same rabbit cornea before (A) and after photorefractive keratectomy (PRK) at 1 week (B), 2 weeks, (C) 3 weeks (D), 7 weeks (E), and 17 weeks (F). In the normal cornea, three reflective layers are detected (white arrows), the surface epithelium, basal lamina, and endothelium, respectively. After PRK, a fourth reflective layer appears

separated from the epithelial/stromal interface by approximately 100 µm (black arrows). This fourth reflective layer appears to move anteriorly by 2 weeks (C) and reaches the epithelial/stromal interface at 3 weeks (D), reaching maximum lightscattering. By 17 weeks, lightscattering has decreased, along with an increase in stromal thickness. Bar indicates 100 µm in the x-axis and 70 µm in the z-axis. Taken from Figure 4 in Moller-Pedersen T, Li HF, Petroll WM, Cavanagh HD, Jester JV. Confocal microscopic characterization of wound repair after photo-refractive keratectomy. Invest Ophthalmol Vis Sci 1998a; 39:487–501.

The haze detected by in vivo confocal microscopy is also directly related to specific changes within the corneal keratocytes that were immediately adjacent to the area of injury (Fig. 3). Specifically, when evaluating haze in patients after PRK, increased scattering of light is detected from the enlarged cell bodies of keratocytes at the stromal/epithelial interface (Figs. 3A and 3B). In the rabbit PRK model, early stromal haze is associated with elongation of keratocytes to form spindle-shaped, migratory fibroblasts (Fig. 3C). Importantly, however, after migration of keratocytes/fibroblasts to the stromal surface, there is a dramatic change in keratocyte differentiation to an enlarged cell that markedly scatters light and has broad cellular processes extending and interconnecting to adjacent cells (Fig. 3D). These cells have been identified in other wound models (7) as well as PRK (8) as corneal myofibroblasts, based on detailed immunocytochemical and biochemical characterizations.

While the explanation for the dramatic increase in light-scattering from fibroblasts and myofibroblasts remains unclear, recent studies suggest that keratocytes express abun-

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Figure 3 Human patient (A and B) and rabbit (C and D) cornea after PRK. (A) Slit-lamp photograph of patient’s cornea showing presence of central corneal haze. (B) In vivo confocal microscopy showing broadly thickened and distinct keratocyte cell bodies in the region of haze. (C) Rabbit cornea 7 days after PRK showing migration of spindle-shaped fibroblasts within fourth reflective layer noted in Figure 2B. (D) Same rabbit cornea, 3 weeks after PRK, showing the presence of highly reflective myofibroblasts forming a broad, interconnected meshwork of cells. Bar indicates 100 µm. Taken from Figure 2 of Jester JV, Moller-Pedersen T, Huang J, Sax CM, Petroll WM, Cavanagh HD, Piatigorsky J. The cellular basis of corneal transparency: Evidence for “corneal crystallins.” J Cell Sci 1999b; 112:613–622

dant amounts of a few water-soluble proteins that have been called corneal crystallins (9,10). These proteins at high concentrations are thought to have similar properties as lens crystallin proteins and may contribute to the transparent state of the cornea by destructively interfering with scattered light through short-range physical interactions. Interestingly, water-soluble proteins isolated from rabbit keratocytes obtained from transparent rabbit corneas show high levels of expression of aldehyde dehydrogenase class 1 (ALDH1) and transketolase (TKT). When cells are obtained from hazy corneas, the level of expression of ALDH1 and TKT is markedly reduced or abolished compared to the level of expression within keratocytes from transparent regions immediately adjacent to the hazy corneal regions. Support for a cellular rather than extracellular basis for haze may also be seen in patients who show marked improvement in haze over time, and in patients who show fluctuations in haze that are unlikely to be explained by a simple fibrotic mechanism (11). Together, these findings point to the possibility that corneal haze after refractive surgery represents a defect in the light-scattering properties of keratocytes and is not related to collagen deposition or fibrosis. This possibility is critically important given that transparency may be easily re-established in keratocytes but permanently lost with corneal fibrosis.

Additional support for the cellular basis of haze has also come from basic studies of keratocyte activation and differentiation. Recent developments using defined cell culture systems have been able to establish keratocyte cultures that maintain normal keratocyte differentiation (12–14). Using this serum-free culture system, studies show that transform-ing growth factor-beta (TGF-β) specifically induces activation and myofibroblast differentiation of corneal keratocytes (13,15). Importantly, TGF-β is synthesized by the corneal epithelium and keratocytes and may play a role in epithelialkeratocyte interactions during normal growth and wound repair (16). Importantly, recent studies also show that blocking antibodies to TGF-β when applied topically after stromal injury (17) or PRK (8) remarkably reduces the development of corneal haze. Interestingly, blocking the TGF-β effect on keratocyte differentiation does not block stromal re-growth and regression in the rabbit eye model of PRK, again suggesting that the deposition of new stromal matrix or stromal fibrosis after refractive surgery does not account for corneal haze development.

Overall, these finding suggest that corneal haze develops not from corneal fibrosis but from the differentiation of normal stromal keratocytes to an altered phenotype, the myofibroblast, that apparently scatters light to a much greater extent than the normal keratocyte (Fig. 1). The light-scattering by these cells may in part be explained by their failure to express abundant quantities of keratocyte crystallins that act as stealth-like proteins making normal keratocytes transparent. This change in the differentiated state of the keratocyte can be induced in culture by TGF-β and the development of haze clinically blocked by treatment with antibodies that neutralize this important cytokine. Because the normal cornea expresses TGF-β, interactions between the epithelium and keratocytes after injury most likely regulate and control the effects of TGF-β on keratocyte differentiation and development of haze. Understanding these cellular and molecular interactions may ultimately provide us with the tools to control corneal haze after refractive surgery using advanced surface ablation techniques.

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ABSENCE OF HAZE AFTER LASIK

The explanation for why LASIK surgery normally heals without the development of corneal haze is not clearly known. Several hypotheses have been set forth, with the most popular related to the fact that LASIK surgery does not induce the same degree of keratocyte apoptosis at the interface of the microkeratome flap as that observed in the stroma after PRK (5,18). Alternative possibilities include the fact that the corneal epithelium is not injured or Bowman’s/epithelial basement membrane complex remains intact.

Keratocyte apoptosis or programmed cell death after simple epithelial abrasion was first demonstrated by Wilson et al. in 1996 (4). Continued work has suggested that epithelial-derived cytokines, perhaps including interleukin-1α (4) and fas-fas ligand (19), are released during epithelial injury, inducing programmed cell death of the underlying keratocytes. Differing degrees of keratocyte apoptosis have been noted after different refractive surgical procedures with scrape injury and PRK after scrape injury producing significant levels of apoptosis. Other refractive surgical approaches such as LASIK, which does not damage the epithelium except at the edge of the flap, and transepithelial photoablation of the epithelium, which presumably vaporizes any epithelial cytokines, produce little or no apoptosis compared to normal corneas. Based on these findings, it has been hypothesized that the differing degrees of apoptosis may explain the differences between haze and regression after these procedures (5,18).

Unfortunately, detailed studies of haze after manual scrape compared to laser epithelial debridement in patients (20) and laboratory animals (21) have failed to detect any relationship between apoptosis and haze. No difference in the amount of haze was observed in a prospective, paired-eye clinical study of PRK patients for whom one eye received manual debridement while the opposite eye received laser-scraping (20). Additionally, when comparing manual scrape injury to simple laser scrape injury in a rabbit model, haze was noted only to occur in the laser-scraped eye (21), which is the opposite result predicted by the apoptotic hypothesis. Importantly, manual and laserscrape procedures were noted to produce keratocyte cell death, presumably by different mechanisms, i.e., apoptosis and necrosis, respectively. Why, then, was only laser-scrape removal associated with the development of corneal haze?

A closer look at the regions where haze developed showed that activation and differentiation of keratocytes to myofibroblasts occurred only in regions where the epithelial basement membrane had been photoablated and removed (Fig. 4). In immediately adjacent regions, where the basement membrane remained intact after photoablation, keratocytes remained quiescent and did not produce haze. Interestingly, the only parameter that correlated with haze that was identified in this study was the amount of stromal tissue that was photoablated. Overall, this study convincingly shows that apoptosis does not cause corneal haze. Although keratocyte death may be important in the development of haze, the mechanisms by which keratocytes die is not a critical factor, and eliminating apoptosis is not likely to have a major impact on reducing corneal haze after refractive surgery. Rather, the critical factor appears to be the Bowman’s/epithelial basement membrane complex, which may act as a barrier to epithelial-keratocyte interactions, or it regulates the expression of epithelial cytokines that are important in myofibroblast differentiation.

While the definitive experiments with LASIK are still required to answer the question as to why LASIK does not develop haze, the aforementioned experiments suggest that

Figure 4 In vivo morphology of the photoablation edge after excimer laser transepithelial photoablation. (A) Sharply defined epithelial basal lamina at the photoablation edge (arrows) by 1 week after surgery. (B) Immediately below the edge shown in (A), keratocytes outside the photoablation (left side of arrows) appeared quiescent, whereas keratocytes inside the photoablation (right side of arrows) appeared activated with increased nuclear reflectivity. (C) By 3 weeks after ablation, quiescent keratocytes were found immediately outside the photoablation (left side of arrows), whereas stellate wound-healing fibroblasts were found immediately inside the photoablation (right side of arrows). (D) The edge (arrows) appears more irregular by 6 weeks,

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with cavity-like structures inside the photoablation (right side of arrows). Bar indicates 100 µm. Taken from Figure 3 of Moller-Pedersen T, Cavanagh HD, Petroll WM, Jester JV. Stromal wound healing explains refractive instability and haze development after photorefractive kerate ctomy. A 1-year confocal microscopic study. Ophthalmology 2000; 107:1235–1245.

Figure 5 Proposed mechanism for the development of corneal haze after PRK. Removal of the Bowman’s/epithelial basement membrane complex allows interactions between the regenerating corneal epithelium and quiescent corneal keratocytes. These interactions modulate the expression and effects of TGF-β, leading to myofibroblast differentiation and haze progression.

preserving the Bowman’s epithelial basement membrane complex is critical. Damage to the epithelium and the induction of epithelial migration and proliferation would seem to be less important, given that simple scrape injury, which causes both keratocyte apoptosis and epithelial regeneration, does not produce significant haze compared to PRK. In support of this hypothesis is the clinical finding that haze in LASIK is limited to the edge of the microkeratome flap, a region where Bowman’s membrane has been transected, allowing direct interaction between the epithelium and underlying keratocytes. Haze may also occur where epithelium is deposited at the flap interface, again pointing to the importance of direct epithelial-keratocyte interactions in controlling the differentiation of keratocytes to a myofibroblast phenotype and the production of haze (Fig. 5).

HAZE AFTER LASEK AND THE FUTURE OF ADVANCED

STROMAL ABLATION

While experimental studies of LASEK and the development of haze remain to be conducted, there is no sound biological reason that is currently known to support the contention that LASEK as currently performed will provide improved results over PRK in regard to development of haze. While LASEK may limit keratocyte apoptosis by limiting the release of pro-apoptotic signalling peptides, the reduction of apoptosis using transepithelial laserscraping techniques has not reduced the amount of haze in standard PRK. Furthermore, LASEK ultimately results in the photoablation of Bowman’s/epithelial basement membrane complex, thus allowing direct interactions between the epithelium and keratocyte that lead to myofibroblast differentiation and haze. Although it is possible that maintaining an intact epithelial sheet might limit the release of the epithelial cytokines affecting this process, it is more likely that ultimate control involves important interactions between the epithelium and Bowman’s/basement membrane complex. Because LASEK techniques thus far developed do not clearly maintain this important structural complex, it is not likely that simply maintaining the epithelium intact without its associated attachment structures will affect the long-term development of haze.

Nevertheless, LASEK may prove to be the first step in the development of more advanced surface ablation techniques that will in the future achieve the promise of stable and effective visual correction without the complication of haze. This point of view is based on the fact that the LASEK procedure recognizes that the key to surgically providing complication-free visual correction is the maintenance of normal corneal epithelial differentiation. In this regard, modifications of LASEK already have focused on the maintenance of the Bowman’s/epithelial basement membrane complex as recently discussed at this conference by Pallikaris (22). Whether this approach, removing both epithelium and underlying basement membrane, will be achieved remains to be shown; however, other approaches may also be successful. These might include the control of the epithelial cell TGF-β expression directly by controlling gene transcription through antisense gene therapy or blocking TGF-β synthesis, the major cytokine responsible for myofibroblast differentiation and haze. Alternatively, replacement of critical basement membrane components that control epithelial gene expression through novel coating strategies may limit or block epithelial-keratocyte interactions and lead to the rapid

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recovery of normal epithelial differentiation similar to scrape injury without photoablation. Finally, epithelial/basement membrane grafting techniques may be used to entirely replace the Bowman’s/epithelial basement membrane complex as proposed by Tseng et al. (23). Overall, each of these approaches appears promising and technically feasible in the near future, suggesting that in the next 10 years, refractive surgery may obtain the “Holy Grail” of perfect refractive correction without visual loss using an advanced surface ablation approach.

REFERENCES

1.Petroll WM, Jester JV, Cavanagh HD. In vivo confocal imaging: General principles and applications. Scanning; 1994; 16:131–149.

2.Jester JV, Petroll WM, Cavanagh HD. Measurement of tissue thickness using confocal microscopy Conn PM, Ed. Confocal Microscopy. San Diego. CA: Academic Press, 1999a:230– 245.

3.Moller-Pedersen T, Li HF, Petroll WM, Cavanagh HD, Jester JV. Confocal microscopic characterization of wound repair after photorefractive keratectomy. Invest Ophthalmol Vis Sci; 1998a; 39:487–501.

4.Wilson SE, He YG, Weng J, Li Q, McDowall AW, Vital M, Chwang EL. Epithelial injury induces keratocyte apoptosis: hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization and wound healing. Exp Eye Res; 1996a; 62:325–327.

5.Wilson SE. Everett Kinsey Lecture. Keratocyte apoptosis in refractive surgery. CLAO J; 1998; 24:181–185.

6.Moller-Pedersen T, Vogel M, Li HF, Petroll WM, Cavanagh HD, Jester JV. Quantification of stromal thinning, epithelial thickness, and corneal haze after photorefractive keratectomy using in vivo confocal microscopy. Ophthalmol; 1997; 104:360–368.

7.Jester JV, Petroll WM, Barry PA, Cavanagh HD. Expression of alpha-smooth muscle (alphaSM) actin during corneal stromal wound healing. Invest Ophthalmol Vis Sci; 1995; 36: 809– 819.

8.Moller-Pedersen T, Cavanagh HD, Petroll WM, Jester JV. Neutralizing antibody to TGFb modulates stromal fibrosis but not regression of photoablative effect following PRK. Curr Eye Res 1; 998b; 17:736–747.

9.Piatigorsky J. Gene sharing in lens and cornea. Facts and implications. Prog Retinal Eye Res; 1998; 17:145–174.

10.Jester JV, Moller-Pedersen T, Huang J, Sax CM, Petroll WM, Cavanagh HD, Piatigorsky J. The cellular basis of corneal transparency: Evidence for “corneal crystallins.” J Cell Sci; 1999b; 112:613–622.

11.Moller-Pedersen T, Cavanagh HD, Petroll WM, Jester JV. Stromal wound healing explains refractive instability and haze development after photorefractive keratectomy. A 1-year confocal microscopic study. Ophthalmology; 2000; 107:1235–1245.

12.Kirschner SE, Ciaccia A, Ubels JL. The effect of retinoic acid on thymidine incorporation and morphology of corneal stromal fibroblasts. Curr Eye Res; 1990; 9:1121–1125.

13.Jester JV, Barry PA, Cavanagh HD, Petroll WM. Induction of a-smooth muscle actin (a-SM) expression and myofibroblast transformation in cultured keratocytes. Cornea; 1996; 15: 505– 516.

14.Beales MP, Funderburgh JL, Jester JV, Hassell JR. Proteoglycan synthesis by bovine keratocytes and corneal fibroblasts: Maintenance of the keratocyte phenotype in culture. Invest Ophthalmol Vis Sci; 1999; 40:1658–1663.

15.Jester JV, Huang J, Barry-Lane PA, Kao WW, Petroll WM, Cavanagh HD. TGFb-mediated myofibroblast differentiation and a-smooth muscle actin expression in corneal fibroblasts requires actin re-organization and focal adhesion assembly. Invest Ophthalmol Vis Sci 1; 999b; 40:1959–1967.

16.Wilson SE, Schultz GS, Chegini N, Weng J, He Y-G. Epidermal growth factor, transforming growth factor alpha, transforming growth factor beta, acidic fibroblast growth factor, basic fibroblast growth factor, and interleukin-1 proteins in the cornea. Exp Eye Res; 1994; 59: 63– 72.

17.Jester JV, Barry-Lane PA, Petroll WM, Olsen DR, Cavanagh HD. Inhibition of corneal fibrosis by topical application of blocking antibodies to TGF beta in the rabbit. Cornea; 1997; 16: 177– 187.

18.Helena MC, Baerveldt F, Kim WJ, Wilson SE. Keratocyte apoptosis after corneal surgery. Invest Ophthalmol Vis Sci; 1998; 39:276–283.

19.Wilson SE, Li Q, Weng J, Barry-Lane PA, Jester JV, Liang Q, Wordinger RJ. The Fas-Fas ligand system and other modulators of apoptosis in the cornea. Invest Ophthalmol Vis Sci; 1996b; 37:1582–1592.

20.Lee YG, Chen WY, Petroll WM, Cavanagh HD, Jester JV. Corneal haze after photorefractive keratectomy using different epithelial removal techniques: mechanical debridement versus laser scrape. Ophthalmology; 2001; 108:112–120.

21.Moller-Pedersen T, Cavanagh HD, Petroll WM, Jester JV. Corneal haze development after PRK is regulated by volume of stromal tissue removal. Cornea; 1998c; 17:627–639.

22.Pallikaras I. SEL, Sub Epithelial LASIK. 1st International LASEK Conference. In: Krueger RWYRR, Ed. Houston. Texas, 2002.

23.Tseng SCG. Role of basement membrane in corneal wound healing. 1st International LASEK Conference. In: Krueger RWYRR, Ed. Houston. Texas, 2002.

26

Mitomycin C and Haze: Natural

Progression

Mujtaba A.Qazi, MD, and Jay S.Pepose, MD, PhD

Pepose Vision Institute

Chesterfield, MO; Washington University School of Medicine

St. Louis, MO

Irwin Y.Cua, MD, Saira A.Choudhri, MD, and M.Azim Mirza, MD

Pepose Vision Institute

Chesterfield, MO

INTRODUCTION

Laser subepithelial keratomileusis (LASEK) offers a means of improving the safety profile of refractive procedures while potentially providing a smoother stromal surface for ablation than photorefractive keratectomy (PRK) (1–3). For these reasons, LASEK has carved out a niche in the repertoire of the refractive surgeon for patients with steeper, flatter, and thinner corneas than average and in whom anatomical considerations, such as deep-set eyes and narrow orbits, may preclude microkeratome use (4). In fact, some surgeons have selected LASEK as their vision correction technique of choice, with excellent outcomes and minimal adverse events (5). Furthermore, the advent of customized interventions draws attention to refractive procedures such as LASEK that avoid the biomechanical shifts induced by lamellar flap formation (6,7).

The term corneal haze has been used since 1988 (8) to describe alterations in corneal transparency caused by the reflection or scattering of light after refractive surgery (Fig. 1). The relationship between corneal haze and regression has been well-documented in PRK literature; as a result of aggressive wound healing, stromal thickness and subsequent corneal refractive power increase, resulting in a myopic shift (Fig. 2) (9–13). As with PRK, corneal haze and myopic regression are among the most significant short-term and long-term complications associated with LASEK (Fig. 3) and decrease the predictability of refractive results (3,14).

There has been a great deal of discussion recently as to whether PRK and LASEK are in fact different surgeries. Although some investigators (1–3,15–17) find support for one technique over the other, it appears that the postoperative discomfort and healing time are similar for each and are certainly greater than that of laser in situ keratomileusis (LASIK). However, preservation of an epithelial flap and its basement membrane components (3,18) over an ablated stromal bed may modify the risk of corneal haze and regres-

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Figure 1 Dense, central, reticular corneal haze after photorefractive keratectomy. (Courtesy of J.S.Pepose).

sion. This chapter reviews the natural history, pathophysiology, and treatment of haze formation after LASEK. Because of its recent introduction (1) in 1998, there are relatively few experimental, histological, in vitro, in vivo, and clinical descriptions of the changes induced by LASEK. For this reason, we review findings seen in PRK, given the similarity of these two techniques, and highlight how an epithelial flap may modulate wound healing after excimer photoablation.

Visual Consequences of Haze

Subepithelial fibrosis, or haze, has been described after myopic epikeratoplasties (19) and radial keratotomy (Fig. 4). Most researchers agree that some degree of corneal haze is seen after all cases of PRK, with an onset of 2 days to 2 months, a peak intensity between 1 and 6 months, and resolution by 3 to 24 months postoperatively (Fig. 5) (8–12,20–22) This variability is related to differences in species studied, targeted refractions, lasers used, postoperative regimens, and techniques for assessing haze. A number of investigators (12,23) have appreciated the importance of a clinical grading system to describe the quality or pattern of haze. The most common method uses a scale from zero to four based on slit-lamp findings (Fig. 6). The slit-lamp appearance of corneal haze, being a composite of reflected and back-scattered light, is highly dependent, however, on

the viewing angle of the observer and does not necessarily correlate with effect on visual function (24). It is only the forward-scattered light that degrades the retinal image. More objective techniques (25,26) have also been developed, including analysis of digital images to detect light-scatter and corneal opacification. In fact, assessment with scatterometers has shown closer correlation with logMAR acuity than the clinical evaluation of haze (27). Another diagnostic modality is confocal microscopy, a noninvasive technique for obtaining high-resolution microscopic images of the cornea in vivo. Digital image analysis of continuous, high-speed confocal z-scans (confocal microscopy through focusing [CMTF]; Fig. 7) has been used to observe details of ultrastructural changes in multiple layers of the cornea after excimer refractive surgery in animals (28) and humans (24,29,30).

While mild haze has been routinely reported in the early postoperative period after PRK and generally resolves without visual sequelae after several months, a dense, lateronset, reticular haze has been noted to reduce visual acuity in 1% to 3% of cases overall, but in up to 10% to 15% of eyes with myopia greater than −10 diopters (D) (31,32). Difficulties with night vision and diminished contrast sensitivity appear to correlate with the extent of corneal haze after PRK (24). Visual compromise is rare in patients with attempted correction less than 5 D and increases in frequency with

Figure 5 Timeline of haze formation after excimer photorefractive keratectomy. Grading of early and late phases of corneal haze, including lateonset corneal haze (LOCH), which is generally associated with higher haze gradings (21). “Reprinted from the Journal of Cataract and Refractive Surgery, © 2001, with permission from the ASCRS & ESCRS.”

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Figure 6 Slit-lamp grading of corneal haze is based upon the pattern of translucence (reticular versus confluent) and degree of obscuration of iris details. “Courtesy of J.S. Pepose.”

larger (Fig. 8) and multiple treatments (11,22,32,33). Patients with atopy or autoimmune conditions are also at higher risk for corneal haze after excimer photoablation (34).

The timeline of haze formation after LASEK is similar to that in PRK, Claringbold (5) notes trace haze in 13% of 222 eyes 3 months after LASEK (mean myopia −4.89, 6.0- mm ablation zone, VISX Star S2), with resolution of all cases by 12 months. Another series (14) (n=58, mean myopia −7.80, Alcon Autonomous) reports 8% of eyes with visually significant haze after LASEK. A retrospective review of 62 eyes (mean myopia −7.96, VISX Star S3) at our center, with at least 3 months of follow-up after LASEK, demonstrates a haze rate of 47%, with almost all receiving only the lowest grading. Three eyes (4.8%) had a haze grading of 2 or more, of which one eye required surgical intervention (see later) because of loss of best-corrected visual acuity (BCVA). The preoperative spherical equivalence of this eye was −8.38 D. The higher haze rate in our series appears to be related to greater attempted correction, and supports Yee, who identified an ablation depth of more than 100 µm and an ablation depth-to-corneal thickness ratio more than 0.18 as independent risk factors for haze formation after LASEK (35). In a prospective study of 27 patients with low to moderate myopia who were randomized to LASEK in one eye and PRK in the fellow eye, Lee et al. (17) found

lower clinical haze ratings in the LASEK eyes at 1 month (p=0.02). Comparable gradings were described between eyes by 3 months of follow-up (p=0.22).

Figure 7 Anterior stromal wound healing morphologic characteristics (A-D) and corresponding CMTF profiles (A’-D’) obtained from the same patient before and after PRK. Images obtained within the anterior 50 µm of the stroma. (A) Preoperative normal quiescent keratocyte nuclei (arrows). (B) One month after PRK, activated keratocytes with increased reflectivity of both nuelei (arrows) and cell bodies. (C) Six months after PRK, increased density of activated

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keratocytes (arrows). Bar indicates 110 µm. (D) Twelve months after PRK, return to moderately reflective, quiescent keratocyte nuclei (arrows). (A’) preoperative CMTF peaks corresponding to epithelium (a), subepithelial nerve plexus (b), anterior layer of keratocytes (c), and endothelium (d). (B’, C’) One and 6 months after PRK, the peak corresponding to subepithelial haze (c’) dominates the CMTF profile. (D’) By 12 months, the degree of reflectivity of the anterior stromal keratocyte layer (c’) has declined. Clinically on the slit-lamp, the patient had grade 1 subepithelial haze at 1 month and grade 0 haze at 12 months (29). “Reprinted from Ophthalmology, © 2000, with permission from the American Academy of Ophthalmology.”

Location and Components of Haze

In vivo investigations of the components responsible for corneal haze after PRK reveal a nontranslucent layer at the epithelial-stromal junction (Fig. 9) that develops 1 week postoperatively, peaks between 1 and 3 months, and declines gradually thereafter. This sequence is characteristic of the onset and duration of mild, early haze after both PRK and LASEK. Histological studies in primates confirm that the subepithelial haze is restricted to the ablation zone itself (23), which becomes comprised of proliferating keratocytes and atypical extracellular matrix (ECM) components, including glycosaminoglycans (GAGs), such as heparan and keratan sulfate, newly synthesized collagen (types III, IV, V, and VII), fibronectin, laminin, tenascin, and hyaluronic acid (Fig. 10) (36,37). The new collagen fibrils are placed in a nonorthogonal arrangement with consequent alteration of corneal transparency. There is evidence to suggest that deeper ablation causes greater accumulation of these materials.

Figure 8 Correlation of CMTF-haze with attempted correction. In 15 patients with moderate light scattering, a cumulative measure of CMTF haze development over a 12-month followup period correlated significantly (r=0.68, p<0.01) with the photoablation depth [preoperative stromal thickness (STpreop)-stromal thickness at 1 month (ST1m)], demonstrating that higher PRK corrections generally are associated with more corneal opacification (29). “Reprinted from Ophthalmology, © 2000, with permission from the American Academy of Ophthalmology.”

Overall, the temporal patterns and levels of messenger RNAs (mRNA) for ECM proteins measured after PRK reveal a series of sequential phases that combine to heal the wound. In a study of rat corneas after PRK (36), mRNA levels for fibronectin increased from undetectable preoperatively to more than 600 copies per cell within 1 week after PRK, suggesting that this factor is important in initiating wound healing; fibronectin is known to promote migration of corneal epithelial cells. Meanwhile, levels of other ECM proteins such as collagen IV do not peak until after 3 months.

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Antibodies against laminin-labeled basal epithelium in ablated and adjacent nonablated regions. Weak staining of the basement membrane was also noted (arrow). (D) Antibodies against fibronectin label both basal epithelium and anterior stroma of the ablated region. (E) Lysozyme expression is shown in basal epithelium and anterior stroma of the ablated area. (F–H) Antibodies against collagen type III, IV, and VII label subepithelial haze. Arrows indicate positive findings (37). “Reprinted from the Journal of Refractive Surgery, © 2001, with permission from Slack, Inc.”

Animal studies further reveal that within 24 hours after excimer laser treatment, the anterior stroma beneath the treatment zone is transiently hypocellular, with few stromal keratocytes (22). Soon thereafter, in mammals and primates, activated keratocytes migrate into the treated zone and begin to synthesize new collagen and ECM. Stromal fibroblasts within haze tissue contain vimentin and smooth muscle actin, cytoskeletal components characteristic of myofibroblasts (37). Vimentin is also manufactured by sliding corneal epithelial cells during wound healing. Myofibroblasts may contain greater quantities of cytosolic, water-soluble proteins such as crystallins (29), which appear to affect the optical properties of keratocytes, suggesting an intracellular contribution to corneal haze. Myofibroblasts are characterized by greater light reflectivity during CMTF and play a significant role in corneal light back-scatter. Restoration of keratocyte densities and reflectivity to preoperative levels, as myofibroblast activity subsides, is temporally associated with the disappearance of haze.

In 1968, Dohlman et al. (38) described the disappearance of superficial keratocytes after corneal epithelial injury. Wilson et al. (39) later reported that this was mediated by apoptosis, programmed cell death in which the cell is dismantled and eliminated with negligible release of intracellular components and minimal damage to surrounding tissues. Many investigators believe that the transient hypocellularity seen as an early event in PRK models is caused by keratocyte apoptosis (Fig. 11) (22). Support for this hypothesis stems from the finding that many regulators of apoptosis, including interleukin-1 (IL-1), Fas ligand, and tumor necrosis factor alpha (TNF-α), are constitutively produced and stored by the epithelium, and then instantly released upon injury to bind to keratocyte receptors. Increased levels of TNF-α have been identified in human tear fluid collected within 2 days of PRK. Injection of IL–1α into mouse central stroma has been shown to trigger keratocyte apoptosis (39). Furthermore, topical

application of factors that inhibit apoptosis can prevent the loss of superficial keratocytes in rabbit corneas after mechanical epithelial debridement (40). Other cytokines implicated in regulation of epithelial and stromal healing after excimer laser keratectomy include epidermal growth factor, keratinocyte growth factor, hepatocyte growth factor, fibroblast growth factor, vascular endothelial growth factor, and transforming growth factor beta (TGF-β) (22,39,41–43). Potential sources for these agents include the main and accessory tear glands, corneal epithelial and stromal cells, conjunctival cells and vessels, and inflammatory cells.

Figure 11 Location of apoptotic keratocytes 1 week after phototherapeutic keratectomy in a rabbit cornea. (A) Thymidine-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay detects apoptotic keratocytes in the subepithelial stroma. (B) Propidium iodide staining of nucleic acid shows the position of all epithelial and stromal cells (70). “Reprinted from the Journal of Cataract and Refractive Surgery, © 2001, with permission from the ASCRS & ESCRS.”

TGF-β is a major player in fibrosis and scar formation in many tissues, including skin, lung, and liver (36). TGF-β treatment markedly increases the levels of mRNAs for fibronectin and type I collagen in cultures of mouse 3T3 fibroblasts. A TGF-β response element is found in the promoter region of the α1 (1) collagen gene. TGF-β may modulate some of its effects via connective tissue growth factor (CTGF), which has been shown to mediate increases in matrix synthesis. TGF-β has also been reported to suppress production of matrix metalloproteinases (MMP) and increase production of tissue inhibitors of metalloproteinases (TEIMP), having the overall effect of preventing scar tissue remodeling.

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All three TGF-β isoforms are expressed in regenerating epithelial cells of rats after PRK (36). The rate of release of TGF-β1 into tears increases approximately 18-fold within 2 days after PRK. Furthermore, 4 weeks after PRK, the expression of TGF-β isomers is upregulated in keratocytes that proliferate in the subepithelial fibrous layer (44). The increase in expression of key components of the TGF-β system coincides with an increase in synthesis of ECM proteins deposited subepithelially after PRK (Fig. 12), supporting the hypothesis that TGF-β is a key growth factor promoting corneal stromal haze formation and suggests that limiting the TGF-β system may reduce corneal scarring after excimer laser ablation. Systemic administration of TGF-β neutralizing antibody during the first 10 days after PRK in rats reduces stromal cell density and immunostaining of laminin and fibronectin in the subepithelial stroma (36). Topical treatment of rabbit corneas with neutralizing antibodies to TGF-β for the first 3 days after PRK mitigates subsequent haze formation as quantified by light reflectivity measurements. Further investigations may demonstrate a role for topical anti-TGF elements in the prophylaxis and treatment of corneal haze formation in humans after excimer refractive surgery.

Modification of corneal stromal structure can also occur via activity of oxygen free radicals, which may form directly from ultraviolet (UV) irradiation during laser treatment or postoperatively from ambient UV exposure and after infiltration of leukocytes (45,46). Polymorphonuclear cells (PMN) first appear at the ablated margin within 6 hours of excimer application, with cytokines such as IL-1 serving as chemotactic mediators. They release oxygen free radicals, which cause tissue damage by reacting with lipid components of the cell membranes, nucleic acids and sulfur-containing enzymes. Lipid peroxidation has been identified in superficial corneal stroma after laser photoablation (47).

Another facet of wound healing after excimer laser treatment of the cornea is the role of matrix metalloproteinases (MMP), a group of proteolytic enzymes responsible for remodeling the extracellular matrix. They require zinc and calcium for catalytic activity. Among the MMPs operating in the cornea are collagenases, gelatinases, and stromelysin. The transcription of MMP genes is regulated by several agents. Steroids, TGF-β, IL-1 receptor antagonist (IL-1ra), and chelating agents such as cysteine, EDTA, and tetracycline inhibit MMP synthesis (48). The major collagenase of the cornea is MMP-1, which is secreted by keratocytes and leukocytes. It cleaves the helical structure of the collagen (types I, II, and III) fibrils, thereby making them susceptible to the action of gelatinases such as MMP-2 and MMP-9. Although the latter two MMPs have the same activity, they have different predominant sites of production: MMP-2 by keratocytes and MMP-9 by epithelium. In the normal cornea, MMP-2 is present as an inactive proenzyme that is locally activated to metabolize damaged stromal collagen molecules. The active form of MMP-2 is detected within the first week after stromal injury and may remain elevated for at least 9 months. MMP-9 is not expressed in the normal cornea, but its activity increases immediately after injury, then falls rapidly as re-epithelialization occurs, until it is absent at 2 weeks. This, together with its epithelial site of production, suggests that it may be involved in the degradation and reassembly of the epithelial basement membrane. Additional investigations may unveil modes of intervention to promote or inhibit, appropriately, MMP activity to prevent tissue damage from these agents or to use them to reverse haze formation.

Figure 12 Levels of mRNA for the TGF-βs, TGF-β receptor (TGF-β II), and ECM proteins in pooled rat corneas before and after PRK. Competition-based quantitative reverse transcription polymerase chain reaction was performed on pooled rat corneas before (day 0) and at 1.5 days, 6 days, 21 days, 42 days, and 91 days after PRK (36). “Reprinted from Investigative Opthalmology & Visual Science, © 2000, with permission from the Association for Research in Vision and Ophthalmology.”

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Summary of Mechanisms of Stromal Haze Formation

As we have seen, a key event after excimer photoablation, regardless of technique used, is epithelial injury, which triggers release of cytokines by the lacrimal gland and epithelium. Epithelial-keratocyte interactions initiate epithelial regeneration and keratocyte apoptosis. This is accompanied by inflammatory cell infiltration, which further mediates corneal injury directly through free radical formation and indirectly through the release of additional cytokines. The cytokine milieu, particularly TGF-β, promotes transformation of keratocytes at the borders of the ablation zone into myofibroblasts, which migrate into the subepithelial space. These highly reflective cells and the atypical matrix elements that they synthesize combine to reduce light transmission. Once the epithelial defect is healed, there is a shift in the cytokines expressed by mature epithelial cells, with disappearance of myofibroblasts and a return to quiescent keratocytes with normal morphology. Metalloproteinases then assist in remodeling stromal tissue, restoring a more orthogonal arrangement of collagen fibrils. Any imbalance, particularly prolonged delay in epithelial healing or sloughing of the epithelial sheet, in this complex process of wound healing may shift the equilibrium to formation of subepithelial haze, and also permits a number of entry points for pharmacological and surgical intervention (Fig 13. ).

Strategies for the prevention and treatment of post-LASEK haze are discussed below. Once again, these are often extensions of our experience with haze after PRK. Research has been directed toward controlling postoperative development of haze with agents such as corticosteroids, nonsteroidal anti-inflammatory agents (NSAIDs), mitomycin C (MMC), idoxuridine, a-interferon, and others (45,48–53).

Steroids

Corticosteroids specifically inhibit phospholipase A2, preventing arachidonic acid production. They down-regulate DNA synthesis in stromal fibroblasts and, therefore, limit subepithelial collagen deposition and corneal haze in animal models (45). While experimental models show a beneficial effect of topical corticosteroid use, clinical trials have, for the most part, shown mixed result (54,55). Vertugno et al. (56), in a randomized, double-masked trial, compared outcomes of a cohort of PRK patients given 0.1% fluoromethalone versus 0.5% ketorolac during the re-epithelialization phase. Both groups received a topical steroid for a number of months after re-epithelialization. Twelve months after PRK, mean refractive error in the NSAID group was −1.1 D, but only −0.65 D in the steroid group (p<0.0001). Haze was significantly reduced in the steroid group (p=0.005), especially for myopic correction greater than −5 D. Interestingly, despite this topical regimen, the authors describe grade 2 haze in 12.5% and 20% of myopes below and above −5 D, respectively.

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Because of the well-known ocular complications, such as prolonged epithelial healing, increased risk for microbial infection, elevated ocular tensions, and cataract formation, associated with topical corticosteroid therapy, some investigators have suggested that their routine use after laser refractive procedure ought to be curtailed (57). Other authors (58) have recommended continued use for deeper PRK ablations. In 1996, Arshinoff (59) reported that 86% of PRK surgeons prescribed steroids immediately after PRK. There are no surveys as such with respect to topical steroid use after LASEK, although most reports in the literature proscribe a regimen similar to what the surgeon used with PRK. We routinely prescribe, after bandage contact lens removal, prednisolone acetate 1% four times daily to all PRK and LASEK patients, with a conservative taper regimen. It may be that earlier implementation of corticosteroids is required to more effectively short circuit the cycle of cytokine activity and myofibroblast activation.

Mitomycin C

Mitomycin-C (MMC) is derived from Streptomyces caespitosus. Its alkylating properties enable it to cross-link DNA between adenine and guanine, thereby inhibiting DNA synthesis. It has been widely used as a systemic chemotherapeutic agent, because rapidly dividing cells are most susceptible to its effects. Over the past years, the use of topical MMC has gained popularity in ophthalmologic surgery. Intraoperative MMC is commonly utilized in glaucoma surgery to prevent scarring of filtering blebs through its ability to inhibit subconjunctival fibroblast proliferation (60). It is also used topically intraoperatively and postoperatively after pterygium surgery (61) to prevent recurrence and has been advocated for the treatment of conjunctival and corneal intraepithelial neoplasia (62).

Application of MMC after excimer laser surgery was first investigated by Talamo et al. (63). Rabbit eyes after PRK were randomized for treatment with topical mitomycin C 0.05%, steroids, and erythromycin or topical steroids and erythromycin or simply erythromycin alone. All treatment regimens started immediately after surgery and were instituted twice daily for 2 weeks. Results of light, fluorescence, and electron microscopy showed reduced subepithelial collagen formation in the group treated with MMC, corticosteroid, and erythromycin. Yamamoto et al. (64) found that mitomycin C 0.001%, 0.01%, and 0.1% could suppress proliferation of keratocytes in vivo, with greater effect at higher concentrations. Several investigators have shown a decrease in keratocyte proliferation in rabbits treated with topical MMC after PRK. In one report (65), immediately after a bilateral −10-D ablation, a 5-minute application of 0.02% MMC was placed on the ablated stromal bed of one eye only. Decreased corneal haze was noted in eyes treated with MMC. Histopathological findings showed a significantly lower keratocyte density in the anterior stroma of the rabbits treated with MMC at 1, 2, and 4 weeks after PRK. However, there was no statistically significant difference in keratocyte density between the PRK alone and PRK+MMC groups by week 12. Lamellar arrangement in the anterior stroma of the PRK+MMC group was more orderly (Fig. 14). Additionally, the corneal epithelium of

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12, the lamellae in the anterior stroma of the PRK alone group have become more regular and smooth. (D) At week 12, the lamellae in the anterior stroma of the PRK+MMC group were arranged normally (65). “Reprinted from the Journal of Refractive Surgery, © 2001, with permission from Slack, Inc.”

the mitomycin C group was thinner than in the control group, although this finding is not seen by other investigators after single-dose application of similar concentrations of MMC (0.02%−0.04%) (51). The ability of a brief exposure of MMC to inhibit fibrosis may be the result of sustained tissue binding and possible modulation of cell migration and extracellular matrix production. It is unclear what role intraoperative MMC application may have in the regulation of keratocyte apoptosis.

While all of these refer to prophylactic measures, Majmudar et al. (66) illustrate the use of MMC as an adjunct to debridement for the treatment of subepithelial scarring after refractive corneal surgery. They describe that a 2-minute intraoperative application of MMC (0.02%) after epithelial and stromal debridement results in recovery of BCVA and successfully prevents recurrence of subepithelial fibrosis. The mean interval between initial surgery and MMC treatment was 31 months (range, 5–60 months), during which time topical steroids were used in each case without significant effect.

We report a 21-year-old patient in our experience in whom gradual onset of significant haze developed within 3 months of bilateral LASEK. Preoperative spherical equivalence was −8.38 D right eye (OD) and −9.00 D left eye (OS), with BCVA of 20/20 each eye (OU). Delayed epithelial healing in the right eye marked the early postoperative course. At 6 months, 3+ haze was noted centrally OD and 2+ haze OS, with loss of four lines of BCVA OD (UCVA 20/20 OS). After manual debridement with adjuvant MMC OD, as described by Majmudar et al. (66), UCVA improved to 20/25 with only 1+ haze after 1 week. Some myopic regression was noted over the next few months along with return of corneal haze despite a regimen of prednisolone acetate 1% four times daily with monthly taper. A repeat debridement/MMC procedure was performed OD within 10 months of the original LASEK surgery. After bandage contact lens removal, the patient had gained two lines of BCVA with 1+ haze visible on biomicroscopy (Fig. 15). This illustrates the applicability of interventions (67) used to successfully treat post-PRK haze to cases after LASEK as well.

The long-term use of topical MMC may be associated with significant ocular toxicity. Transient side effects, such as hyperemia, pain, and punctate epithelial keratopathy, have been noted with topical application of MMC 0.02% to 0.04%, but resolve with cessation of the drug. More serious complications include corneal perforation, iritis, and secondary glaucoma (68). However, these adverse outcomes are seen, as a rule, after prolonged topical administration in eyes with underlying pathologic conditions. Single intraoperative application of MMC has the advantages of full compliance, minimal side effects, and controlled

Figure 15 A 21-year old with postLASEK haze treated with manual debridement and intraoperative MMC 0.02%. (A) At 6 months after LASEK, 3+ haze is noted centrally. (B) Central cornea 4 days after repeat manual debridement with intraoperative MMC. (Courtesy of J.S.Pepose.)

drug delivery. Although previous studies recognize the potential of MMC for preventing the development of stromal haze and in reducing pre-existing stromal scarring, further studies are needed to define the optimal method of application and dosage before its routine use in patients undergoing excimer laser surgery. Jain et al. (69) have suggested, based on their observations after phototherapeutic keratectomy (PTK) in rabbits, that annular application of MMC may more effectively reduce light-scatter and corneal toxicity. To minimize complications, the lowest possible therapeutic concentration should be applied for the shortest effective period, ensuring minimal corneal contact, particularly when epithelial defects are present. The authors currently prophylactically use 2-minute application of MMC 0.02% to the stromal bed for all cases of LASEK −6 D or higher. We have not noted any adverse intraoperative events or significant delay in epithelial healing postoperatively. We have not made any nomogram adjustments when using intraoperative MMC.

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identified by the propidium iodide (red) fluorescence. Apoptotic nuclei, detected by apop-FITC (green) fluorescence, are readily seen in the subepithelial region of the control cornea (a), but not in the experimental cornea (c) (70). “Reprinted from the Journal of Cataract and Refractive Surgery, © 2001, with permission from the ASCRS & ESCRS.”

the amniotic membrane stromal matrix instead, where they exhibited markers of apoptosis. Histopathological findings in these eyes also demonstrated a lesser degree of subsequent epithelial hyperplasia and keratocyte proliferation.

Amniotic membrane transplantation is an impractical procedure to perform customarily in patients undergoing refractive surgery but may be attempted in patients with severe corneal scarring after PRK or LASEK. In situations in which mechanical debridement of the scar tissue is performed, removal of the overlying epithelium may restart the cascade of haze formation. Application of amniotic membrane after mechanical debridement may be beneficial in preventing recurrent inflammation and scarring. The routine use of amniotic membranes after LASEK, however, may require their incorporation into a contact lens placed intraoperatively, as described by Wang (71).

Superficial Lamellar Keratectomy

In cases in which medical therapy fails and the scarring involves deeper layers of the cornea, penetrating keratoplasty has been the procedure of last resort. Rasheed and Rabino witz (72) instead advise superficial lamellar keratectomy, microkeratome-assisted excision of a corneal cap. If deep enough, this technique results in a smooth optical surface, which would be difficult to achieve via freehand mechanical debridement or dissection. In their case report, a 180-µm footplate was used. The treated patient regained good BCVA. Some authors (73), however, advise that this technique be used only before proceeding to penetrating keratoplasty in patients with severe corneal haze, especially given the encouraging results of manual debridement techniques with MMC, as described.

CONCLUSION

Several studies correlate severity of epithelial trauma to the degree of subsequent anterior stromal hypocellularity (22,46,70), presumably because more cytokines are released and can access stromal receptors to induce keratocyte apoptosis. In vitro studies (41) confirm the role of differentiating epithelium in the recruitment of fibroblasts to a wound. Nakamura et al. (74) have, in fact, demonstrated subepithelial fibrosis after LASIK when

the epithelium is denuded intraoperatively. They identified myofibroblast activity in rabbit corneas after PRK and LASIK with epithelial debridement, but not after debridement alone or LASIK alone, suggesting that epithelial and stromal injury are required for haze formation. This has certain implications in LASEK, in which an epithelial flap is maintained to protect the ablated stromal surface; the preserved epithelium and basement membrane complex (3) may act as a barrier to cytokine penetration and PMN infiltration of the stroma, thereby reducing the extent of keratocyte apoptosis and ensuing myofibroblast activation. Although there is a certain amount of epithelial cell death related to chemical injury in a LASEK flap, it is likely that the overall cytokine load within the ablated zone is reduced because of both relative preservation of epithelial cells and alcohol denaturing of acutely released cytokines. Furthermore, Marshall (75) has suggested that there is a temporal window after stromal ablation during which keratocytes are more susceptible to apoptotic signals. It is possible that modification of epithelial regeneration patterns via formation of a central epithelial flap may shift the timeline for introduction of apoptotic cytokines outside the susceptibility period of stromal fibroblasts.

These considerations may explain the observation by Carones et al. (2) of lower haze rates (p=0.04) in human eyes treated with the excimer laser after de-epithelialization using 20% alcohol versus those de-epithelialized manually. They felt that this was related to a smoother Bowman’s surface and improved corneal regularity after alcohol-assisted debridement. This result is corroborated by Lee et al. (42) in a prospective study comparing LASEK and PRK in the same patient, in which haze scores were significantly lower at 1 month (p=0.005) in LASEK eyes. This was associated with lower tear TGF-β levels and release of TGF-β during the early postoperative period in LASEK eyes. Tseng (76) has suggested that some constituents of the basement membrane and subepithelial region after LASEK are also found in amniotic membranes and thus may inhibit haze formation. Further developments in LASEK flap creation, such as the use of methylcellulose (77) to dissect free an epithelial flap or microkeratome-assisted (78) epithelial flap formation, may offer even more protection by preserving the integrity of epithelial cells and basement membrane components.

The complex wound healing response of the cornea has important implications in refractive surgery. The end result is variable stromal remodeling and epithelial hyperplasia associated with myopic regression and haze. Although the specific cellular events of corneal wound healing after LASEK remain unclear, it is speculated that the epithelial flap protects the bare surface of the stroma and prevents the influx of cytokines and inflammatory cells from the tears, reducing the apoptotic and inflammatory insult to the stroma. This may decrease the initial loss of anterior stromal keratocytes and late subepithelial myofibroblast activity, effectively reducing haze formation. Experimental and in vivo investigations are required to confirm this premise. While several researchers have identified keratocyte apoptosis blockers, further investigations are needed to determine the efficacy of topical agents (40) and vector gene therapy (79) for the management of postsurgical corneal haze. Controlled clinical trails may reveal the benefits of surgical techniques that further preserve epithelial integrity, or of earlier and more uniform use of modulating agents such as corticosteroids, MMC, IL-1 inhibitors, TGF-β inhibitors, or amniotic membrane factors.

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