Clinical background
The safety and predictability of laser in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK) have improved since these procedures were introduced, but the corneal wound-healing response remains a major contributor to variability of results following these procedures. Corneal wound healing entails the complex interactions of different cellular types, including corneal epithelial cells, keratocytes, and, possibly, endothelial cells, in addition to corneal fibroblasts, myofibroblasts, inflammatory cells, lacrimal gland cells, and others. In large part, this communication is mediated by soluble growth factors, cytokines, and chemokines via membrane-bound and soluble receptors.
The unwounded adult cornea is a transparent and avascular structure, providing not only the major refractive surface involved in visual image transmission, but also a protective barrier against external injuries, including microbial infections that are potentially vision-threatening. Activation of these systems during refractive surgery can result in the deposition of opaque fibrotic repair tissue and, possibly, scarring. In order to understand and control these complex interactions better and improve the results and safety of LASIK and PRK, it is important to have a basic understanding of normal and abnormal corneal wound-healing responses. This chapter provides a framework that will allow the clinician not only to understand these interactions, but also at least partially to control them through surgical technique and rational application of medications.
Pathophysiology and pathology
The normal wound-healing response
Corneal stromal fibrils and other matrix components are precisely organized to provide transparency essential to corneal function. However, cellular repair processes during corneal healing can disturb this architecture and lead to visual impairment. The corneal wound-healing response involves a complicated balance of cellular changes, including cell death (apoptosis and necrosis), cell proliferation, cell motility, cell differentiation, expression of cytokines, growth factors, chemokines and their receptors, influx of inflammatory cells, and production of matrix materials ( Box 3.1 ). In large part, communications between corneal cells, nerves, inflammatory cells, bone marrow-derived cells, and other cells are the critical determinants of normal and abnormal corneal wound-healing responses. Although many of these interactions occur simultaneously, for discussion purposes it is convenient to describe the wound-healing response as a pathway, similar to glycolysis or the Kreb’s cycle.
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Epithelial injury
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Stromal cell death (apoptosis and necrosis)
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Influx of inflammatory cells
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Cell proliferation
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Cell motility
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Cell differentiation
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Release of cytokines, growth factors, chemokines, and expression of their receptors
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Production of extracellular matrix materials
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Epithelium healing
Corneal epithelial injury is a common initiator of the corneal wound-healing response to refractive surgical procedures, as well as in trauma and some diseases. Here we will concern ourselves only with surgical injury associated with LASIK and PRK. Corneal epithelial injury triggers the release of a variety of cytokines, such as interleukin-1 (IL-1)-α and -β, transforming growth factor (TGF)-β, tumor necrosis factor (TNF)-α, platelet-derived growth factor (PDGF), and epithelial growth factor (EGF), that regulate keratocyte apoptosis, proliferation, motility, differentiation, and other functions during the minutes to months after surgical insult. In turn, once stimulated by these epithelial-derived soluble factors via membrane-bound receptors, keratocytes not only alter cellular functions, but also produce other soluble modulators that regulate corneal epithelial proliferation and migration (hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF)), attract inflammatory cells (granulocyte chemotactic and stimulating factor (G-CSF), monocyte chemotactic and activating factor (MCAF), neutrophil-activating peptide (ENA-78)), and other corneal changes. Collagenases, metalloproteinases, and other enzymes are activated and released in the stroma during the wound-healing response and function to degrade, remove, and regenerate damaged tissue. The expression of these collagenases and metalloproteinases by keratocytes and corneal fibroblasts is also regulated by IL-1 and fibroblast growth factor-2 derived from the injured corneal epithelial cells.
A recurring theme that must be appreciated to understand corneal wound healing is ongoing communication between epithelial cells and stromal cells mediated by soluble cytokines and chemokines. These interactions occur immediately after injury and continue for weeks, months, or occasionally even years, for example with persistence of haze following PRK.
Many growth factors released during the corneal wound-healing response can be derived from more than one cell type and regulate more than one process. EGF can be used to illustrate this principle. EGF is produced by epithelial cells, keratocytes, corneal fibroblasts, lacrimal cells, and, possibly, other cells. EGF regulates corneal epithelial cell proliferation, motility, and differentiation. EGF also triggers the formation of new hemidemosomes on epithelial cells after injury. EGF also has influence on the proliferation of limbal cells that migrate toward the injury site to seal the wound and to reform a normal stratified epithelial layer. In addition, different growth factors may regulate a single function. For example, EGF, HGF, and KGF all regulate corneal epithelial proliferation. The effect that predominates at a particular point in the wound-healing response likely depends on factors such as receptor expression, cellular localization, cellular differentiation, and the influences of interacting networks of soluble and intracellular factors.
Epithelial injury is typically the initiator of the wound-healing response associated with corneal surgery or injury. For example, epithelial scrape or epithelial ethanol exposure associated with PRK or laser epithelial keratomileusis (LASEK), respectively, epithelial blade penetration associated with Epi-LASEK or LASIK are initiators of corneal wound healing that result in the release of IL-1α, IL-1β, TNF-α, and a host of other modulators that alter the functions of keratocytes, inflammatory cells, and the epithelial cells themselves. Similarly, damage to the epithelium at the edge of the flap in femtosecond LASIK flap formation triggers the wound-healing cascades, although the femtosecond laser has direct stromal necrotic effects that influence the overall wound-healing response of surgery performed with this procedure, as will be covered later.
Apoptosis and necrosis in initiation, modulation, and termination of wound healing ( Box 3.2 )
The first stromal change that is noted following epithelial injury is apoptosis of the underlying keratocyte cells ( Figure 3.1 ). Apoptosis, or programmed cell death, is a gentle, regulated form of cell death that occurs with the release of only limited intracellular components such as lysosomal enzymes that would potentially damage surrounding tissue. Keratocytes undergoing apoptosis are found to have chromatin condensation, DNA fragmentation, cell shrinkage, and formation of membrane-bound vesicles called apoptotic bodies that contain intracellular contents. The localization of the apoptosis response is related to the type of injury, and in large part determines the localization of the subsequent wound-healing events. For example, in PRK, LASEK, and Epi-LASEK, keratocyte apoptosis occurs in the anterior stroma beneath the site of epithelial injury ( Figure 3.1A ). In contrast, keratocyte apoptosis associated with microkeratome LASIK occurs at the site of blade penetration at the edge of the flap and along the lamellar cut in the central stroma ( Figure 3.1B ).
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Apoptosis of the underlying keratocyte cells
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Modulation by eliminating excess inflammatory, fibroblast, and other cells
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Elimination of myofibroblasts
The apoptosis process is likely regulated by soluble cytokines such as IL-1 and TNF-α released from injured epithelial cells and the Fas-Fas ligand system expressed in keratocytes. Apoptosis is an extremely rare event in unwounded normal cornea. Once an injury to the epithelium occurs, however, keratocytes undergoing apoptosis can be detected within moments. This early wave of relatively pure apoptosis makes a transition into a later phase in which both apoptosis and necrosis occur in many stromal cells, including keratocytes, corneal fibroblasts, and invading inflammatory cells. Although all of these cells are typically labeled with the terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, careful analysis with transmission electron microscopy demonstrates that cellular necrosis, a more random death associated with release of intracellular enzymes and other components, also makes a major contribution. It is unknown whether necrosis that occurs during corneal wound healing is a regulated event or merely a result of cells being killed by inflammation or other contributors to healing. A much later low-level phase of apoptosis occurring in myofibroblasts is also noted in corneas that develop haze.
Precise regulation of the apoptosis processes that occur during corneal wound healing implies an important function besides a merely reactionary response to the injury. Studies have suggested that the earliest apoptosis response is likely a defense mechanism designed to limit the extension of viral pathogens, such as herpes simplex and adenovirus, into the stroma and eye after initial infection of the corneal epithelium. The second phase of stromal apoptosis extending from hours to a week after injury likely functions to modulate the corneal wound-healing response by eliminating excess inflammatory, fibroblast, and other cells. The latest phase of stromal apoptosis that occurs in corneas with haze serves to rid the stroma of myofibroblasts that are no longer needed.
Mitosis and migration of stromal cells
Mitosis and migration of stromal cells are noted approximately 8–12 hours after the initial corneal injury. Initially, most cells undergoing mitosis appear to be keratocytes, but corneal fibroblasts and other cells may make subsequent contributions to this response. This cellular mitosis response provides corneal fibroblasts and other cells that participate in corneal wound healing and replenish the stroma. Once again, localization of the stromal mitosis response is related to the type of injury. Thus, in PRK stromal mitosis tends to occur in the anterior stroma, as well as in the peripheral and posterior stroma outside the zone of apoptosis ( Figure 3.2 ). In LASIK, stromal mitosis occurs at the periphery of the flap where the epithelium was injured, and anterior and posterior to the lamellar cut.
Mitosis and migration of stromal cells are regulated by cytokines released from the epithelium and its basement membrane. For example, PDGF is produced by corneal epithelium and bound to basement membrane due to heparin-binding properties of the cytokine. It is released from the epithelial basement membrane after injury and stimulates mitosis of corneal fibroblasts. It is also highly chemotactic to corneal fibroblasts, tending to attract them to the source of the cytokine. Thus, in PRK, for example, PDGF released from the injured epithelium and basement membrane stimulates surviving keratocytes in the peripheral and posterior stroma to undergo mitosis and the daughter cells are attracted to the ongoing PDGF release and repopulate the anterior stroma. Other cytokines such as TGF-β also likely contribute to this keratocyte/corneal fibroblast mitosis and migration.
Corneal fibroblasts derived from keratocytes produce collagen, glycosaminoglycans, collagenases, gelatinases, and metalloproteinases used to restore corneal stromal integrity and function. These cells also produce cytokines such as EGF, HGF, and KGF that direct mitosis, migration, and differentiation of the overlying healing epithelium. After total epithelialization, the fibronectin clot disappears and the nonkeratinized stratified epithelium is re-established.
Inflammatory cell influx ( Box 3.3 )
Beginning approximately 8–12 hours after the initial epithelial injury, and lasting for several days, a wave of inflammatory cells migrates into the cornea ( Figure 3.3 ) from the limbal blood vessels and tear film. These cells function to clear cellular and other debris from the injury and to respond to pathogens that could be associated with injuries such as viral or bacterial infections.
