Ординатура / Офтальмология / Английские материалы / Ocular Therapeutics Eye on New Discoveries_Yorio, Clark, Wax_2007

clinical outcome of the refractive surgery procedure. In approximately 0.5 to 3% of eyes (Kapadia and Wilson, 2000), however, depending on the laser used for the procedure, the level of refractive error corrected, and other factors, clinically significant haze develops. When severe haze does occur, it is nearly always associated with marked regression of the refractive effect, along with glare, halos, and other visual phenomena that may be visually debilitating to the patient. Such haze nearly always resolves spontaneously, but often requires 1 to 3 years for complete resolution. There are therapeutic options that can be considered to treat patients once severe haze develops – phototherapeutic keratectomy with the excimer laser combined with topical mitomycin C treatment – however, prophylactic treatment to prevent haze formation is the best approach. Unfortunately, however, current approaches, while effective in preventing haze, produce persistent changes in corneal stromal morphology, and the long-term implications of these changes are uncertain and of ongoing concern. This chapter will focus on the pathophysiology, current treatment modalities, and future approaches to treatment of haze associated with excimer laser surface ablation procedures.

II. CLINICAL OBJECTIVES

The clinical objectives of refractive surgeons and scientists involved in investigating and developing treatments for haze, focus on understanding the factors that lead to haze formation in a proportion of human corneas that undergo excimer laser surface ablation, and the development of safe and effective measures to prevent the generation of clinically significant haze.

III. BASIC MECHANISMS

Haze generation is a component of the wound healing response to injury in the cornea. This response has been shown to be qualitatively identical in different species – such as humans, rats, rabbits, mice, and pigs – although individual components of wound healing vary quantitatively between different species. For example, keratocytes apoptosis in response to epithelial injury has been noted in all of these species (Wilson, 2002). Similarly, haze formation is noted in all of these species. However, the level of haze formation in response to similar injury varies substantially between species. Thus, after PRK for 9 diopters of

myopia with the Summit Apex laser (Alcon, Ft Worth, TX), greater than 99% of rabbit corneas develop severe haze compared with only 2 or 3% of human corneas after the identical treatment (Mohan et al., 2003a). Mice are similar to humans with regards to resistance to haze formation and require special measures (marked surface irregularity) to trigger significant haze.

Some of the earliest observations in human corneas that provided clues to the mechanisms underlying haze formation were differences noted between different excimer lasers in the tendency to produce opacity in corneas having similar levels of correction for myopia. Thus, clinically significant haze was much more common after PRK performed with the original Summit Apex and VISX (Santa Clara, CA, now a division of American Medical Optics, Irvine, CA) than it is with excimer lasers that are in current use in patients. Another intriguing observation is that haze is more likely to occur after higher corrections for myopia than lower corrections. Thus, haze is rare following PRK in the human eye (and in the rabbit eye) for less than 6 diopters of myopia, but becomes increasingly more common as the level of attempted correction is increased, even though the low and high correction PRK procedures are otherwise identical. Any hypotheses put forward to explain the mechanism of haze development must account for these clinical observations.

Most manipulations or naturally occurring phenomena that precipitate wound healing responses in the cornea are associated with corneal epithelial injury. Epithelial damage leads to the release of epithelialderived cytokines – such as interleukin-1 (IL-1), tumor necrosis factor alpha (TNFα), transforming growth factor beta (TGFβ), and platelet-derived growth factor (PDGF) – that bind to receptors on keratocytes and alter gene expression and signal transduction and, consequently, keratocytes functions and cellular differentiation (Wilson et al., 1999). Similarly, once cytokines such as IL-1 bind to receptors on keratocytes, these cells upregulate production of other cytokines and chemokines involved in modulating the functions of epithelial cells (i.e. hepatocyte growth factor and keratinocyte growth factor), bone marrow-derived cells (monocyte chemotactic and activating factor and granulocyte chemotactic factor), and, possibly, other cells (Wilson et al., 1999, 2001, 2004). These cytokine mediated cell–cell interactions involved in epithelial–stromal communications and stromal–bone marrow-derived cell communications are fundamental to the corneal wound healing response and a thorough understanding of these cytokinemediated cellular communications has provided critical insights into understanding the development of corneal haze.

Anterior stromal keratocyte apoptosis is the first observable event following epithelial injury (Figure 7.2a) and can be noted

to begin immediately following the insult using transmission electron microscopy, although analysis with the TUNEL assay peaks between 2 and 4 hours (Wilson, 2002). Ongoing cell apoptosis, and subsequently cell necrosis, in the anterior stroma can be detected for approximately 1 week following epithelial injury. After the initial wave of keratocyte apoptosis, the cells likely continuing to undergo programmed cell death and necrosis in the anterior stroma include corneal fibroblasts derived from proliferation of residual keratocytes in the peripheral and posterior stroma, as well as bone marrow-derived cells, including monocytes, granulocytes, and lymphocytes, that migrate into the cornea in response to chemokines released by the injured epithelium and upregulated in keratocytes or their progeny by cytokines like IL-1 released from injured epithelium (Wilson et al., 2004).

Beginning approximately 12 hours after injury, residual keratocytes in the peripheral and posterior stroma begin to undergo mitosis – detected using immunocytochemistry for Ki67 (Figure 7.2b) (Wilson, 2002; Zieske et al., 2001). Resulting progeny corneal fibroblasts migrate into the anterior stroma where apoptosis occurred. There is evidence that this migration cannot occur posterior to anterior through stromal lamellae, but only along lamellae from peripheral to central (Mohan et al., 2003b). A few days after PRK injury, the anterior stroma may become hypercellular – having been repopulated with a combination of corneal fibroblasts, bone marrow-derived monocytes, and, possibly, other cells (Wilson, 2002). The anterior stroma cellular mixture at this point during wound healing has not been well characterized, except that corneal fibroblasts and bone marrowderived cells have been identified (Mohan et al., 2003b; Wilson et al., 2004).

If the PRK injury is sufficient, depending on the insult and the species – for example, 9 diopter PRK in rabbits – myofibroblasts begin to be detected in the most superficial

anterior corneal stroma immediately beneath the basement membrane of the corneal epithelium from 2 to 3 weeks after surgery (Figure 7.2c). These cells are detected by immunocytochemistry for alpha smooth muscle actin, a marker for myofibroblasts, and at the ultrastructural level are noted to have prominent rough endoplasmic reticulum, abundant matrix, and peripherally located smooth-muscle type myofilaments. The myofibroblasts produce large amounts of matrix materials, including collagens, glycosaminoglycans, and other extracellular components that, lacking the normal regular structure of matrix components in the normal corneal stroma, contribute to a lack of anterior stromal transparency after PRK. In addition, myofibroblasts themselves are opaque compared with keratocytes due to down-regulation of corneal crystallins (Jester et al., 1999a,c).

Several intriguing questions regarding myofibroblast generation and fate after corneal injury, which are important to the development of therapeutic agents to pharmacologically limit haze formation, remain either totally or partially unanswered. These include:

1.What are the progenitor cells for corneal myofibroblasts – keratocytes and corneal fibroblasts or bone marrowderived cells?

2.What is the status of the progenitor cells during the interval between the injury and the time myofibroblasts are first detected in the anterior stroma after PRK – approximately 2 weeks after surgery?

3.What are the factors that determine whether or not a particular cornea generates myofibroblasts and haze following PRK?

4.How do myofibroblasts and haze disappear over time when corneal transparency is reestablished?

It has long been a dogma among scientists interested in the cornea that myofibroblasts are derived from keratocytes or their

progeny corneal fibroblasts generated in the stroma after corneal injury. Supporting this view, multiple in vitro studies have demonstrated that cultured corneal fibroblasts are capable of differentiating into myofibroblasts under the influence of transforming growth factor beta (TGFβ), and possibly other cytokines like PDGF (Masur et al., 1996; Jester et al., 1999b). Recent studies in the skin (Mori et al., 2005), lung (Epperly et al., 2003), heart valves (Deb et al., 2005), gut (Brittan et al., 2005), and liver (Forbes et al., 2004), however, have demonstrated that myofibroblasts may also develop from bone marrow-derived cells. A connection between corneal haze and inflammatory cells in some patients has long been suspected on the basis of clinical responsiveness of haze to corticosteroid treatment. Thus, in most patients who have PRK, corneal haze is completely unresponsive to treatment with topical corticosteroids – even intensive every hour drops for weeks. In an occasional patient, however, the haze proves to be highly responsive – disappearing completely within 1 week of instituting topical corticosteroid treatment. The difference between these groups of patients has long puzzled clinicians. Could it be that in some patients steroid-unresponsive, keratocyte-derived myofibroblasts predominate, while in others steroid-responsive myofibroblasts that differentiated from bone marrow-derived cells are in the majority? We are currently investigating myofibroblast generation with chimeric mice in which bone marrowderived cells express fluorescent green protein (Wilson et al., 2004). Preliminary results suggest that at least some myofibroblasts are generated from bone marrow-derived cells (Mohan, R.R., Netto, M.V., Perez, V., Wilson, S.E., unpublished data, 2006), but further experiments are in progress to confirm this finding.

Whether haze-associated corneal myofibroblasts are derived from corneal fibroblasts, bone marrow-derived cells, or both, there remains an interesting dilemma

concerning the progenitor cells. The myofibroblasts expressing alpha smooth muscle actin are first detected approximately 2 weeks after PRK and continue to increase in numbers to a peak at 1 to 3 months after surgical injury. What is the nature of the progenitor cells during the interval between PRK and the time the myofibroblasts are detected? There is no definitive answer to this question. It is our hypothesis that progenitor cells that do not express alpha smooth muscle actin are present early on in the anterior stroma, and under the influence of TGFβ derived from the epithelium these cells transition, possibly via intermediate cell types also not expressing alpha smooth muscle actin, to myofibroblasts. Further work is in progress to study myofibroblast development.

What are the factors that determine whether or not a particular cornea develops large numbers of myofibroblasts and associated haze after PRK? Stated another way, why is it that in both humans and rabbits a cornea that undergoes PRK for 4.5 diopters of myopia is much less likely to develop severe haze than a cornea that undergoes PRK for 9.0 diopters of myopia? Moller-Pedersen and coworkers (1998) reported that corneal haze development after PRK is regulated by volume of stromal tissue removal. This is clearly an association, but does not prove a cause- and-effect relationship. For many years clinicians have been intrigued about the concept that surface smoothness versus roughness following PRK was associated with transparency versus opacity, respectively – as was most cogently presented by Vinciguerra and coworkers (1998). We explored the relationship between surface irregularity and stromal haze in a recent study (Netto et al., 2006a). This study, in which the excimer laser and a fine mesh screen were utilized to precisely regulate surface irregularity, demonstrated that there is a direct relationship between surface irregularity at the end of a PRK procedure and myofibroblast density or anterior

stromal opacity developing two to four weeks later. Thus, in the rabbit model, a4.5 diopter PRK for myopia – that is associated with very little haze – generated dense haze if the last 50% of the PRK were performed over a fine mesh screen to produce surface irregularity. Conversely, if the irregular 4.5 diopter PRK surface were smoothed with excimer laser phototherapeutic keratectomy with a smoothing agent (1% methylcellulose or a solution of hyaluronic acid), no haze was generated. Most importantly, this study demonstrated defects in the regenerating epithelial basement membrane could be detected overlying anterior stromal myofibroblasts, likely allowing penetration of epitheliumderived TGFβ and other cytokines into the stroma. Stramer and coworkers (2003) have also reported studies demonstrating the key role of the epithelial basement membrane in modulating haze formation. Thus, it seems likely, based on our studies and work of Fini and coworkers, that defects in the epithelial basement membrane structure and function after PRK allow key cytokines (such as TGFβ) required for the generation of myofibroblasts to penetrate into the anterior stroma. Surface irregularity, or any other factors including possible genetic influences that promote basement membrane incompetence, would likely be associated with greater myofibroblast and haze generation.

Once myofibroblasts and haze are generated in the cornea, how do they spontaneously disappear over time? Spontaneous return of transparency occurs in corneas with severe haze, even though years may be required for complete resolution. Typically, multiple clear areas called lacunae appear within the confluent haze beginning approximately 1 year after surgery.Overtimetheselacunaeslowlycoalesce until the entire cornea is clear once again. Since both the myofibroblasts themselves and the extracellular matrix materials they produce are the source of haze, both of these components must be removed or

altered for a return of transparency. Maltsev and coworkers (2001) have demonstrated transdifferentiation of myofibroblasts to corneal fibroblasts in vitro. However, our careful long-term analysis of corneal tissues following PRK for 9 diopters of myopia have detected late apoptosis in myofibroblasts associated with haze (Netto et al., 2006a). We believe this mechanism likely provides a better explanation for the slow disappearance of these cells over a year or more following the generation of haze.

What is the cellular control that triggers myofibroblast apoptosis? Although no studies have been undertaken to explore late myofibroblast apoptosis, it seems likely that repair of the epithelial basement membrane structural and functional defects (discussed earlier) results in diminished levels of cytokines needed to maintain myofibroblast viability. Once myofibroblasts and ongoing production of collagen and other matrix materials are eliminated by apoptosis, keratocytes repopulate the area. A primary function of keratocytes is to reabsorb wound healing associated matrix components and replace them with specific collagens, glycosaminoglycans, and other stromal components associated with transparency in the normal cornea.

IV. CURRENT THERAPY

Two forms of therapy are currently utilized to prevent haze after surface ablation PRK, LASEK, or Epi-LASIK. Topical mitomycin C prophylaxis is the most common therapy. Some surgeons, however, use smoothing with phototherapeutic keratectomy with the excimer laser at the end of the surface ablation procedure to retard haze formation.

Over the past few years, it has become commonplace for refractive surgeons to apply topical mitomycin C to the exposed stromal bed at the end of a surface ablation procedure to prevent haze formation. Mitomycin C is a powerful alkylating agent

that forms covalent linkages with deoxyribonucleic acid (DNA), inhibits DNA synthesis and consequently suppresses ribonucleic acid (RNA) and protein synthesis (Galm et al., 2005). As a result of these effects, it has been assumed that mitomycin C (MMC) triggers apoptosis of corneal stromal myofibroblast progenitor cells; although, until recently, little work has been performed to establish the mechanism of action of MMC in vivo or the appropriate dosage or exposure times. Recent studies in our laboratory (Netto et al., 2006b) have demonstrated that, while MMC does trigger some increase in anterior stromal cell apoptosis relative to corneal epithelial scrape alone, the most profound effect of the agent is to inhibit proliferation of all stromal cells throughout the anterior stroma. This study in rabbit corneas also confirmed the recent clinical impressions of some surgeons that the medication is effective when used at 1/10 the concentration routinely used on an empirical basis in clinical practice (0.005% versus 0.05%) at lower exposure times than commonly used (15 seconds versus 1 to 2 minutes). Unfortunately, this study also uncovered a disturbing effect of topical MMC treatment following PRK. The anterior stroma of corneas treated with MMC, even using the lower concentration for a shorter period of time, remains almost completely devoid of cells to a follow-up of 6 months. Longerterm studies are in progress, but if the acellularity continues, there is concern about long-term degenerative changes in the anterior stroma and, possibly, the overlying epithelium that is dependent on stromal cytokines for viability and normal function. Clinicians point to the lack of serious complications noted to date in patients treated with MMC, but no conclusions regarding safety can be made until decades following treatment if these corneal structural changes persist for many years.

The alternative treatment used by some surgeons to prevent haze after surface ablation is the use of phototherapeutic keratectomy with a smoothing agent

(Vinciguerra et al., 1998). This treatment has not gained widespread acceptance because it is technically more complex than MMC treatment, takes a few additional minutes per eye, and some lasers do not have the capacity to perform phototherapeutic keratectomy.

V. FUTURE THERAPY

BOX 7.1

Clearly, more directed treatments that specifically inhibit myofibroblast generation, without affecting surrounding cells or corneal structure, would be a desirable addition to the refractive surgeon’s armamentarium, and efforts should be directed to this end. An obvious target is inhibition of the effects of TGFβ in promoting myofibroblast development. Jester and coworkers (1997) demonstrated that topical application of blocking antibodies to TGFβ was successful in inhibiting stromal haze in the rabbit. This type of treatment would appear to be the most promising for directed therapy. It is not clear how long TGFβ effects on stromal cells would need to be inhibited to prevent myofibroblast generation in the human. If prolonged TGFβ inhibition is necessary, then gene therapy approaches would likely be needed when safe and effective vectors are available (Mohan et al., 2005). Other potential targets for more directed pharmacological prevention of myofibroblast generation will likely be suggested by data from ongoing studies to elucidate the factors regulating myofibroblast development and viability.

VI. SUMMARY

•Corneal haze is noted following all surface ablation procedures – including PRK, LASEK, and Epi-LASIK.

•Myofibroblast generation is a key event in haze formation.

•Surface irregularity and resultant abnormal basement membrane regeneration augments myofibroblast generation and haze formation.

•Mitomycin C blocks haze through profound effects on stromal cell proliferation, but leaves the anterior stroma devoid of cells for prolonged periods after surgery.

VII. ACKNOWLEDGMENTS

Supported in part by US Public Health Service grants EY10056 and EY15638 from the National Eye Institute, National Institutes of Health, Bethesda, MD.

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I. INTRODUCTION

Cataract is a term that describes any opacification of the lens of the eye. Cataracts can be present at birth (congenital), or develop early in life ( juvenile), or in response to ocular trauma. However, the vast majority of cataracts occur in later life. Cataracts are the most common cause of blindness worldwide, accounting for nearly 50% of blindness, and cataract surgery is the most

frequently performed surgical procedure. Despite aggressive attempts to provide adequate surgical treatment, the worldwide increase in longevity has meant that the number of individuals with impaired sight due to cataract is increasing. In developed countries, the cost of cataract surgery is a significant burden on the health care system. In the developing world, cataracts markedly reduce productivity and, in regions where subsistence is marginal, cataract