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Biochemical Basis of Epithelial Dehiscence and Reattachment After LASEK

Eric E.Gabison, MD, Hailton B.Oliveira, MD, Jin-Hong Chang, PhD,

and Dimitri T.Azar, MD

Massachusetts Eye and Ear Infirmary, Schepens Eye Research Institute,

Harvard Medical School

Boston, MA

Several questions have to be answered by refractive surgeons performing laser subepithelial keratomileusis (LASEK) surgery. Is the epithelial sheet viable after alcohol injury? What is the mechanism of epithelial detachment after alcohol application? Can the detached epithelium readhere after stroma injury? The aim of this chapter is to describe the key component of corneal epithelium to the stroma and to provide insight into the role of these molecules in governing the performance of LASEK as well as healing after LASEK.

MOLECULAR BIOLOGY OF THE CORNEAL BASEMENT

MEMBRANE: ULTRASTRUCTURE AND MOLECULAR

COMPOSTION

To understand the principle of LASEK, the molecular basis of the corneal epithelialstromal adhesion complex must be understood. This adhesion complex, coined by Gipson et al. (1,2) and Espana et al. (3), is composed of several linked components including intermediate filaments, hemidesmosomes, anchoring fibrils, and anchoring plaques (Figs. 1 and 2) (1–3). The aim of diluted alcohol application, the first step of the LASEK procedure, is to temporarily dissociate this complex.

Hemidesmosomes—Anchoring Fibrils Adhesion Complex

Intermediate Filament

Keratins are the intermediate filaments that connect the nuclear membrane to the hemidesmosomes of corneal epithelial cell plasma membrane. They are responsible for maintaining corneal epithelium structure.

Several keratins have been detected in the corneal epithelium, each with a differential pattern of expression. Whereas K12 keratin is a cornea-specific intermediate filament distributed in the epithelium and sparing the limbus area, the K14 keratin is ubiquitous

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Figure 1 Schematic illustration of the interaction between the basal epithelial cell and the underlying basement membrane, which consists of the lamina densa made of collagen IV and the lamina lucida made of laminins. Hemidesmosomes are formed by integrin and laminin 5. Collagen VII is distributed in the lamina densa and superficial stroma. (With permission from Espana et al. J Cataract Refract Surg 2003; 29(6):1192–1197.)

and localized to the basal epithelial cells. The K3 keratin is localized to the limbal and peripheral basal and wing cells, and to all the central corneal layers (4,5).

Hemidesmosomes

The hemidesmosomes are connections between basal epithelial cells and the underlying basal lamina. Their structure is divided into outer and inner plaques. The inner plaques are mainly composed of the desmoplakins, connecting the intermediate filament network to the outer plaques. The outer plaques are primary components of α6β4 integrin and the cytoplasmic domain of the 180-kD bullous pemphigoid antigen (BPA type II). This antigen corresponds to the newly discovered type XVII collagen, a membraneintercalated molecule that participates in hemidesmosome formation (6).

Anchoring Filaments

Anchoring filaments connect hemidesmosomes to anchoring fibrils throughout the lamina lucida. Many authors have described the lamina lucida as a transmission electron microscopy (TEM) fixation artifact, and this area may represent a weakness in the epitheliostromal adhesion complex. Anchoring filaments are mainly composed of laminin 5 and of the extracellular domain of the type XVII collagen (BPA type II). The anchoring filaments are connected to the lamina densa.

Figure 2 Representative microphotographs showing the effects of ethanol in cadaver corneas in creating LASEK flaps. (A) Hematoxylin and eosin staining of the lifted epithelial flap. (B) Collagen VII immunofluorescence staining in the area of the lifted flap showing linear staining in the corneal bed but not in the flap. (C) Laminin 5 staining in the area of the lifted flap showing linear

LASEK, PRK, and excimer laser stromal surface ablation 292

staining in the corneal bed and a patchy pattern in the basal cells of the lifted flap. (D) Patchy staining for integrin β4 predominantly localized in the basal area of the lifted flap. (E) Intercellular and pericellular staining in the lifted epithelial flap but not on the stromal bed. (With permission from Espana et al. J Cataract Refract Surg 2003; 29(6): 1192–1197.)

Lamina Densa

The lamina densa is an electron-opaque region composed of type IV collagen, laminins, nidogen-entactin and proteoglycans.

Anchoring Fibrils

The lamina densa is also connected to anchoring fibrils, composed of type VII collagen. This molecule extends from the lamina densa to the anchoring plaques. The globular domains of type VII collagen are present in the lamina densa of the basement membrane and in the anchoring plaque. Therefore, this molecule acts as a “cufflink” between the epithelium and the stroma.

Focal Contacts and Integrins

Focal contacts are inter-hemidesmosomal adhesion molecules essentially composed of integrins and laminins. Like other stratified epithelia, corneal epithelium expresses a combination of integrins, including α2β1, α3β1, α6β1, αvβ1, that mediate attachment to the basement membrane and cell-cell interactions (7–15).

INTERCELLULAR JUNCTIONS IN THE CORNEAL

EPITHELIUM

Intercellular junctions constitute the second ocular surface barrier (after the tear film) protecting the stroma (and therefore the intraocular space) from chemical, bacteriological, or viral threats. The corneal epithelium is composed of four to six layers of stratified squamous nonkeratinized epithelium. Four types of intercellular junctions have been identified in the corneal epithelium: gap junctions, desmosomes, adherens junctions, and tight junctions. The LASEK procedure relies on one premise: intercellular junctions and cell-matrix junctions do not share the same properties. Accordingly, although cell-matrix junctions may be completely released during the first step of the procedure, most intercellular junctions remain functional, allowing the epithelial sheet to remain coherent.

TIGHT JUNCTIONS: ZONULA OCCLUDENS

Tight junctions serve a barrier function in epithelia. Only found in polarized epithelial cells, they are localized to the apical side, forming a continuous band around the cells. Tight junctions include the association of three membrane-bound proteins (the occludins 1 and 2 and the claudins) with intra-cytoplasmic proteins (Z0, 1, 2, and 3). In the normal rat cornea, immunoreactivity of Z0–1, Z0–2, and occludins has been detected in the superficial epithelial cell layers (16–20).

ZONULA ADHERENS

Like the tight junctions, zonula adherens are localized to the apical side of the epithelial and form a continuous band. They are calcium-dependent junctions comprising the association of membrane-bound glycoproteins (the E-cadherins) with cytoplasmic plaques linked to the actin network. In the normal cornea, they are localized to all three epithelial layers (basal, intermediate, superficial). Zonula occludens play a role in the maintenance of the cell shape.

DESMOSOME: MACULA ADHERENS

Desmosomes and adherens junctions belong to the cadherin family of adhesion proteins. Like adherens junctions, they are calcium-dependent and are involved in cell shape maintenance. However, they are not linked to the actin network (they attach to intermediate filaments) and do not form continuous bands around the cells (they are separate junctions). Desmoglein and desmoplakins are the main adhesion proteins of the desmosomes. Unique among stratified epithelia, corneal epithelium expresses only a single pair of desmosomal glycoproteins. Whereas Dsc2 and Dsg2 are expressed in the cornea, expression of Dsc3 and Dsg3 is only present in the limbus and conjunctiva. The association of desemosomes with cell proliferation seen in other epithelia has not been confirmed in corneal epithelial cells during re-epithelialization. Absence of Dsc1 and Dsg3 correlates with lack of keratinization in ocular epithelia (21–26).

GAP JUNCTIONS

Gap junctions are localized to the lateral sides of basal corneal epithelial cells, excluding those of the limbal area. Formed by the association of connexin proteins, GAP junctions mediate intercellular signaling and thereby allow functional synchronization among neighboring cells (27).

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WOUND HEALING PROCESS

Cell-Basement Membrane Junctions

In the absence of a wound, the normal corneal epithelium undergoes a continuous process of regeneration. As basal cells undergo mitosis, daughter cells differentiate in the upper layers and desquamate 7 days later. Therefore, these cells must constantly break and then reform their hemidesmosomes: the newly formed hemidesmosome is a combination of newly synthesized integrins associated with an already-formed anchoring complex (anchoring fibrils and plaques). During the wound-healing process, hemidesmosomes are disassembled as confirmed by the loss of immunoreactivity for the β4 integrin subunit in the leading edge of the migrating corneal epithelium. While the β4 integrin subunit is not present in the corneal epithelial leading edge, the α6 subunit remains and co-localizes with the β1 subunit. After re-epithelialization, the hemidesmosomes completely regenerate within 4 weeks.

Intercellular Junctions

Each layer of the intact corneal epithelium expresses a different combination of intercellular junctions. Gap junctions (connexin 43 and 50) are present in the basal cell layer, desmosomes (desmoglein 1 and 2) in the wing cell layer, adherens junctions (E- cadherin) in all cell layers, and tight junctions (occluding) in the superficial cell layers of the epithelium. Although migrating epithelial cells lack connexins 43 and 50 during wound-healing processes, these components are present in the transition zone between migrating and nonmigrating cells. Tight junctions represent the main intercellular junction of migrating epithelial cells that do not express gap junctions, E-cadherins, or desmosomes.

Alcohol Treatment

Local application of 20% ethanol for 30 seconds on the corneal surface has been proven to be a safe technique to remove the corneal epithelium preceding photorefractive keratectomy (PRK). However, little is known concerning the mechanism of alcohol action. Accordingly, alcohol-assisted removal of corneal epithelium has been primarily tested “empirically” and established by the majority of corneal surgeons performing LASEK (28,29).

Vitality of corneal epithelial cells after the exposure to 20% alcohol has been recently assessed. In this study, vital epithelial cells were seen after up to 45 seconds of exposure to 20% alcohol, whereas longer exposition times (up to 120 seconds) have been shown to be lethal for these cells.

Because TEM is the “gold standard” to study epithelial cell adhesion molecules, we used TEM to evaluate epithelial sheets removed after 20% alcohol exposure. Results from this investigation revealed that the site of cleavage may be the anchoring fibrils and that most of the basement membrane is preserved during this process. In light of these

findings, alcohol mode of action may be compared with that of dithiothreitol (DTT) (30,31). This molecule is an alcohol and a reducing agent, and it has been shown to disrupt basement membrane adhesion complexes at the level of the anchoring fibrils. Therefore, the DTT has been used to cleave adhesion molecules of several mucosa to study type VII collagen distribution in adhesion complex (Figure 1). Other techniques of corneal sheet removal include incubating with EDTA or dispase II. The action of EDTA and dispase II appears different from that of EtOH, because EDTA, acting as a calcium chelator, disrupts intracellular junctions and dispase II disconnects epithelial cells at the hemidesmosome level (2,32–36).

REASSEMBLY OF CORNEAL EPITHELIAL ADHESION

STRUCTURES AFTER LASEK

Gipson et al have studied hemidesmosome formation in vitro and in vivo. Sheets of epithelium removed using dispase II and then placed on corneal stroma were able to reform their hemidesmosomes. They subsequently described treatment of corneal wounds with corneal epithelium transplantation in vivo. Sheets of corneal epithelium removed using the same technique were placed on abraded (basement membrane intact) or keratectomized corneas and protected with soft contact lenses. After 24 hours, the transplanted epithelium was adherent to both abraded and keratectomized corneas. Hemidesmosomes formation between basal cells of donor epithelium and denuded host membrane were detected. Interestingly, transplants of corneal epithelial sheets to abraded corneas were most successfully maintained if the host basement membrane was present. This pioneering in vivo experiment has been followed by ex vivo experiments studying epithelial-basement membrane interaction after recombination of epithelial sheets with basement membrane denuded corneal stromas. Results from these investigations revealed the viability of epithelial sheets transplanted on corneal stromas (in rats), that 6 hours were necessary for hemidesmosome reformation in epithelial basement membrane recombination, and that hemidesmosomal reformation requires both healthy epithelium and stroma (37–40).

Viability of Epithelial Cells in LASEK

Several studies have evaluated the viability of epithelial cells after LASEK. Chen et al. determined the effect of dilute alcohol on human corneal epithelial cellular morphology and viability (41). A 20-second time exposure of cultured immortalized human cells to various concentrations of EtOH-H2O showed significant reduction of viable cells when EtOH-H2O concentration exceeded 25% (v/v) (P=0.005). Increasing the duration of 20% EtOH-H2O beyond 30 seconds resulted in a significant reduction in viable cells. TdTmediated dUTP nick-end labeling (TUNEL) assay for apoptosis of cultured human corneal epithelial cells exposed to 20% EtOH-H2O for 20 and 40 seconds showed maximal labeling at 24 hours (58.05%±33.10%) and 8 hours (94.12%±1.21%), respectively.

Gabler et al. evaluated the vitality of epithelial cells after various exposure times to 20% ethanol and epithelial flap preparation in laser-assisted subepithelial keratectomy

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(LASEK) (42). Human cadaver eyes were exposed to 20% ethanol for 15, 30, 45, 60, and 120 seconds, respectively. After a 15-and 30-second exposure, most epithelial cells were vital. This changed substantially after 45 seconds, when viable and dead cells were approximately equal in number. Longer exposure times (60 seconds and 120 seconds) showed predominantly dead epithelial cells.

Lee et al. evaluated the morphologic changes in the white leghorn chick cornea caused by 20% alcohol after LASEK surgery (43). Exposure of the corneal epithelium to 20% alcohol for 30 seconds or longer allowed reproducible separation of epithelial flaps from stroma in white leghorn chick eyes. Transmission electron microscopy immediately after alcohol treatment showed that exposure to 20% alcohol for 30 seconds or less had minimal adverse effects on the corneal epithelium structure. TUNEL-positive cells in the central superficial stroma around the epithelial flap margin and in the epithelial flap itself were identified (particularly in the basal epithelial layer). Transmission electron microscopy showed similar evidence of apoptosis in the epithelium and anterior stroma.

Importance of Basement Membrane Integrity

It is not known whether the separation of the basement membrane from Bowman’s layer and its replacement after laser offer LASEK an advantage over PRK. The layer at which epithelium and stroma separates depends on the particular LASEK surgical technique. In Azar’s and Chen’s studies, variable morphologic patterns of separation were seen in the basement membrane zone by electron microscopy. Various basement complex configurations were observed beneath the epithelial basal cells including unilamellar basement membrane, irregular basement membrane with intact hemidesmosomes, and basement membrane containing dense bundles of anchoring fibrils. Similarly, Browning et al. have shown that a small amount of basement membrane was left attached to the LASEK flap; thus, this remaining basement membrane was of variable thickness (44). The residual basement membrane also had an undulating appearance, coinciding with the position of hemidesmosomes.

The action of alcohol may in part be caused by disruption of the binding of hemidesmosomes to basement membrane. During corneal wound healing, the disruption of the basement membrane has been shown to induce inflammatory cytokine production. Stramer et al. have demonstrated that penetrating incision or ablation injury to the corneal stroma stimulates a typical fibrotic repair response involving hypercellularity, expression of smooth muscle actin, and deposition of a disorganized extracellular matrix (45). The fibrotic response in vascularized tissues is controlled by bioactive substances, including PDGF and TGF-β, which are released from platelets at the wound site. In the cornea, TGF-β2 may be produced by epithelial cells activated by injury. Thus, debridement of corneal epithelium from basement membrane causes stromal cell apoptosis but no obvious hypercellularity or deposition of matrix. Nakamura et al. have shown that the fibrotic response is stimulated in PRK; thus, less fibrotic response was found in LASIK and, thus, TGF-β2 induced by injured corneal epithelium may play a role in the fibrotic phenotype.

Whether these inflammatory cytokines are produced during LASEK is not yet known. There may be a difference in the release of TGF-β2 between surviving epithelium vs. killed epithelium after LASEK procedures and the barrier function offered by the

presence of intact basement membrane in the prevention of scar formation. Preliminary results, using rabbit and chicken models, show that corneal scarring may be greater in PRK than in LASEK. Because the experimental evidence is limited, further experiments are needed to determine whether LASEK induces less scarring than PRK in human subjects.

CONCLUSION

Several lines of evidence suggest the feasibility of LASEK as a good alternative procedure to PRK in reducing scar formation. Our theory based on current data is that alcohol weakens the adhesions between epithelium and stroma, and the mechanical separation between the two layers during surgery depends on the plane of the mechanical cut. In general, one can assume that with current technologies and techniques, the separation of corneal epithelial layer from stromal layer is within the basement membrane. The preservation of an intact, viable layer in conjunction with intact basement membrane may explain the potential reduction in stromal complications after LASEK as compared to PRK. We hypothesize that the intact basement membrane may reduce the alteration of fibrotic pheno-type. Despite the potential for LASEK, important questions remain. Will LASEK patients have a greater rate of recurrent erosion syndromes given the effect of EtOH on hemidesosomes? Will diabetic patients be particularly prone to recurrent erosions after LASEK? Ultimately, will the benefits of LASEK justify the greater complexity of the procedure?

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