Ординатура / Офтальмология / Английские материалы / Biomaterials and regenerative medicine in ophthalmology_Chirila_2010

proteoglycans that are attached orthogonal to the fibril circumference. While the most predominant collagen in the normal corneal stroma is collagen type I, constituting 71% of its dry weight, a change in the ratio of corneal collagens is found in some disease states. This is also the case after wounding, with an increased amount of collagen type III found to be associated with scars (Newsome et al., 1981).

In the anterior stroma of primates and birds an acellular band approximately 8–10 μm thick can be distinguished immediately subjacent to the epithelial basement membrane (Bergmanson, 2008). This is known as Bowman’s layer

(or the anterior limiting lamina) as seen in Fig. 4.2. The collagen fibrils in

Bowman’s layer are of similar types to the stroma proper but differ in their diameter (narrower) and arrangement (random orientation) with irregular interweaving (Komai and Ushiki, 1991; Jacobsen et al., 1984; Linsenmayer et al., 1998; Wilson and Hong, 2000). Bowman’s layer is generally regarded as separating the epithelium and stroma (Obata and Tsuru, 2007) and being a visible indicator of ongoing stromal–epithelial interactions (Wilson and Hong, 2000). It is also thought to have a role in the mechanical integrity and curvature of the cornea (Müller et al., 2001), although one study reported that the mechanical properties of the cornea were unchanged after its removal (Seiler et al., 1992). The interface of Bowman’s layer with the underlying stromal tissue has been the subject of several studies; which support a role for Bowman’s layer in corneal biomechanics yet question the concept that the collagen lamellae of the stroma extend from limbusto- limbus. An early electron microscopy study of this region showed that some of the fibres do run parallel to the corneal surface while others run at angles into Bowman’s layer (Kayes and Holmberg, 1960). Second harmonic imaging microscopy showed that the anterior lamellae interweave and confirmed that some project into Bowman’s layer with a transverse orientation in normal cornea (Morishige et al., 2007). This differed in keratoconic corneas with weakened corneal biomechanics which have shown less lamellar interweaving and fewer projections (Morishige et al., 2007). High-magnification transmission electron microscopy has confirmed that collagen fibrils link the anterior limiting layer to the anterior stroma (Mathew et al., 2008). A functional biomechanical role for Bowman’s layer and the anterior stroma is supported if one considers the cases where Bowman’s layer has been disrupted leading to a condition affecting vision, such as sub-epithelial fibrosis (corneal dystrophy and bullous keratopathy) or ectasia (keratoconus) (Obata and Tsuru, 2007).

Keratocytes are the cellular elements of the corneal stroma and account for at least 3–5% (Beuerman and Pedroza, 1996) and possibly up to 10% (Müller et al., 1995) of the total stromal mass. Although they are generally regarded as quiescent cells, they are actively involved in the routine maintenance of stromal extracellular matrix and also in its repair after injury. The majority of keratocytes are characterised by flattened triangulate cell bodies with

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clearly defined cell processes (Poole et al., 1993), which appear as long, slender tapering profiles in cross-sections of the corneal stroma (Müller et al., 1995). The relatively sparse distribution of keratocytes in the stroma is thought to retain transparency of the corneal tissue, yet these cells interconnect through the stromal lamellae to form a three-dimensional network allowing for communication at the tips of the cell processes via gap junctions (Poole et al., 2003). A three-dimensional reconstruction of the central corneal stroma showed the keratocytes were arranged in a corkscrew pattern with the highest density in the anterior stroma near Bowman’s layer (Müller et al., 1995). This change in density has since been confirmed by confocal microscopy (Poole et al., 2003; Tervo and Moilanen, 2003; Moilanen et al., 2008). There appear to be different types of keratocytes present in the stroma, with those in the anterior portion possessing twice the number of mitochondria, indicative of a higher metabolic state, which may be associated with oxygen gradients in the stromal tissue and/or the synthesis of different substances (Müller et al., 1995). It is generally accepted that there are few actively proliferating keratocytes in the normal corneal stroma (Zieske, 2004), although data based on stem-cell-specific markers have identified a population of keratocyte progenitor cells near the limbus of the adult cornea (Du et al., 2005).

4.1.4Descemet’s membrane and corneal endothelium

The posterior stroma is separated from the underlying endothelium by a thickened basement membrane known as Descemet’s membrane, which is 2–3 μm thick at birth increasing to 7–10 μm in adults (Binder et al., 1991; Joyce, 2003). A single layer of endothelial cells, characteristically 5 μm in height and 15–20 μm in width, binds the anterior chamber (Binder et al., 1991). Both Descemet’s membrane and the endothelium can be seen in Fig. 4.1. The number of corneal endothelial cells decreases with age with approximately 400 000 cells/mm2 of hexagonal shape in young children and 2000 cells/mm2 of less regular shape in adults (Joyce, 2003). The hexagonal morphology is thought to maximise the surface area exposed to neighbouring cells which are linked by specialised cell–cell junctions, including leaky tight junctions, to facilitate the movement of fluid and solutes. This is in keeping with the major function of the corneal endothelium which is to regulate stromal hydration to maintain corneal transparency. The generally accepted ‘pump-leak’ mechanism by which the corneal endothelium maintains corneal hydration was first proposed by David Maurice (Maurice, 1972) and accounts for the leaking of fluids and solutes across the endothelium into the stroma that is balanced by the pumping of solutes and passive fluid transfer back across the endothelium and into the aqueous humour. Injury and disease can upset this balance and this can directly impact on corneal thickness and transparency, diminishing vision. Significant to this, endothelial

cells lack a robust proliferative response and, with the number and density of endothelial cells decreasing with age, it is possible for injury or disease to cause a reduction in density below a critical point causing the corneal stroma to imbibe fluid and become oedematous and opaque (Joyce, 2003;

Bourne and McLaren, 2004). Healing of endothelial wounds occurs by the sliding and enlargement of adjacent cells rather than by mitosis which adds to the changed cell density and shape (Joyce, 2003). The impact of such changes on vision is exemplified in disease states such as Fuch’s endothelial dystrophy, where inherited morphological and functional abnormalities of the endothelium cause a progressive loss of pump function leading to stromal oedema and diminished visual acuity, which can lead to blindness (Wilson and Bourne, 1988; Bourne and McLaren, 2004). Since the endothelium has a limited regenerative capacity, and relies mostly on cell movement and shape changes within the existing cell population to repair wounds, it is particularly vulnerable to insult and injury such as may occur during surgical procedures related to the cornea or lens.

4.1.5Nutrient supply to the cornea

The transparent nature of the cornea is absolutely critical to its optical properties. Transparency is maintained by a highly organised tissue structure and a complete lack of blood vessels in the central cornea. Metabolites (e.g. oxygen and glucose) and waste products (e.g. carbon dioxide and lactate) diffuse in and out of the cornea via the tear film, the abundant blood vessels in the scleral tissue peripheral to the cornea and also from the aqueous humour contained in the anterior chamber. Oxygen in the open eye is supplied by atmospheric oxygen dissolved in the tear film and from the aqueous humour (Friend, 1979). In the closed eye oxygen comes from the blood vessels in the sclera, limbus and the palpebral vessels of the eyelids

(Friend, 1979). Glucose is principally provided by the aqueous humour and is distributed forwards towards the epithelium in the stromal fluid, with smaller contributions diffusing from the tears and limbus (Friend, 1979; McCarey and Schmidt, 1990). Glycogen stores in the epithelium are utilised if there is insufficient free glucose available as a result of anaerobic conditions or trauma (Friend, 1979).

4.1.6Nerve supply to the cornea

Despite its avascular nature, the cornea is well-innervated tissue and has a high density of sensory free nerve endings which make it one of the body’s most sensitive tissues (Rozsa and Beuerman, 1982). The cornea is innervated by the ophthalmic and maxillary branches of the fifth cranial (trigeminal) nerve and sympathetic nerves, which enter the stroma from the periphery

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and fan in a radial pattern parallel to the corneal surface. The majority of these nerve fibre trunks are located in the anterior third of the stroma, where they turn at right angles to penetrate Bowman’s layer and then fan out along the base of the epithelium as epithelial leashes, which are partially interconnected and form a sub-basal nerve plexus (Müller et al., 2003). The diameter of these fibres varies between 1 and 5 μm as measured by confocal microscopy (Stachs et al., 2007). Single nerves or nerve fibres arising from this network protrude between adjacent basal cells of the epithelium (Müller et al., 2003). Equal numbers of nerves penetrate the stroma in all quadrants of the cornea (Müller et al., 2003), although previously it was thought that the majority were located in the nasal and temporal quadrants

(Müller et al., 1997). Communication via soluble messengers between the nerve cells, epithelial cells, keratocytes and possibly the endothelial cells is an essential part of normal corneal function and homeostasis (Wilson et al., 2003). One such soluble mediator is Substance P, which is released by the nerve endings and bound by receptors on the epithelial cells promoting cellular activities including proliferation and migration (Garcia-Hirschfeld et al., 1994). It is interesting to note that neural disease states of the cornea, such as neurotrophic keratopathy, are characterised by decreased corneal sensitivity and poor corneal healing and, in these corneas, a breakdown of the epithelium may result in ulceration, infection and degradation (melting) of the underlying stroma (Nishida et al., 2007). Denervation of the cornea results in decreased cell metabolism, increased corneal permeability, with decreased levels of neurotransmitters and decreased cell proliferation, which can lead to an erosion of the epithelium even in the absence of injury as a result of the continuous turnover of corneal epithelial cells.

4.1.7Immune cells in the cornea

The cornea has long been regarded as a site of immune privilege since it was avascular and was thought to lack resident immune cells. Since then, populations of bone marrow-derived leukocytes, which are distinct from stromal keratocytes and include bone marrow-derived cells and macrophages, have been identified in the cornea (Hamrah et al., 2003; Sosnová et al., 2005). Dendritic cells, which are capable of initiating an immune response, including epithelial Langerhans cells and anterior stromal dendritic cells, are not restricted to the peripheral cornea but have been identified in the central portion of healthy corneal tissue, indicating that the entire cornea is able to participate actively in the immune response to foreign antigens and autoantigens (Hamrah and Dana, 2007). While much of this immune activity may remain muted during normal corneal homeostasis, it can be activated by infections or trauma such as that caused by injury or surgery, and epithelial cells and keratocytes respond by chemokine production which

attracts inflammatory cells to the damaged area (Wilson et al., 2003). Indeed, communication between the corneal epithelial cells and stromal keratocytes (and possibly endothelial cells as well) via cell-mediated substances such as cytokines is recognised to be of great importance in the regulation of corneal tissue metabolism (Wilson and Kim, 1998).

4.1.8Corneal renewal and wound healing

Despite its avascular nature, the regenerative capacity of the corneal epithelium is considerable and occurs continuously in normal cornea to maintain the epithelium in good health (Lemp and Mathers, 1991). The corneal epithelial replacement process was originally shown to take 5–7 days to complete in the rat cornea (Hanna and O’Brien, 1960), although more recent cell-labelling studies have shown it to take 2 weeks for complete turnover of the epithelium (Cenedella and Fleschner, 1990). Regeneration is primarily achieved by the migration of the epithelial cells from the periphery of the cornea inwards along the basement membrane and upwards to the superficial layers where they are continuously shed from the corneal surface into the tear film. This was proposed by Thoft and Friend (1983) in their X, Y, Z hypothesis of corneal epithelial cell replacement; where X represented proliferation in the basal cells, Y was the proliferation and migration of the limbal cells, and Z was the loss of epithelial cells from the ocular surface. Equilibrium in regular corneal epithelial cell replacement is maintained if X + Y = Z. The driving force for the centripetal migration of corneal epithelial cells is not fully understood, but is believed to involve desquamation of the central corneal epithelium in a preferential exfoliative process driven at least partly by the shearing force of the upper eyelid (Lavker et al., 1991; Lemp and

Mathers, 1991; Mathers and Lemp, 1992). Davanger and Evensen (1971) first proposed that the regenerative capacity of the corneal epithelium resided in the papillary structure of the limbus at the corneal periphery, which is richly supplied with blood. Corneal epithelial stem cells were later identified in this location using monoclonal antibodies (Schermer et al., 1986; Zieske, 1992) and their slow-cycling nature while in the limbal microenvironment was demonstrated by thymidine labelling (Cotsarelis et al., 1989). Studies in skin (Watt, 1984; Jones and Watt, 1993) and then in cornea lead to the proposal that the pluripotent corneal epithelial stem cells from the limbus gave rise to semi-differentiated, multipotent ‘transient amplifying cells’ that migrated on to the cornea proper to divide and replenish the stratified epithelial layers of the central cornea as non-proliferative ‘terminally differentiated cells’ (Schermer et al., 1986).

Superficial wounding to the epithelium activates a rapid wound-healing response to quickly restore epithelial barrier function and normal vision. Epithelial cells around the wound periphery disassemble their hemidesmosomes

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and rearrange themselves, flattening and migrating inwards as a tissue front, which contracts in a ‘purse-string’ fashion to cover the defect (Buschke, 1949; Crosson et al., 1986; Gipson, 1989; Beuerman and Thompson, 1992;

Dua et al., 1994). Once the wound is covered, the cells proliferate and stratify to restore normal epithelial thickness and hemidesmosomes reform in a wound-healing process that takes only days to complete (Hanna, 1966;

Gipson, 1989; Dua et al., 1994). During this wound-healing phase, the undifferentiated epithelial stem cells located in the limbus and slightly differentiated ‘transient amplifying cells’ in the basal cell layer are stimulated to proliferate, providing additional cells to complete the restratification process

(Dua and Azuara-Blanco, 2000). A direct linkage between differentiation status and proliferative capacity of corneal epithelial cells has been questioned in a study that showed that the daughter cells arising from cell division in the basal layer did not all differentiate synchronously to become wing cells, but rather, some remained in the basal layer with potential to undergo additional rounds of cell division (Beebe and Masters, 1996). Consistent with this are recent findings based on a human organotypic model where donut epithelial wounds were made to the central cornea, with dimensions 7 mm outer diameter and 3 mm inner diameter, and where the limbus was left intact or ablated. Data from the ablated limbus group showed that epithelial cells in the central corneal epithelium had the capacity to undergo sufficient cell division and migration to heal the epithelial wounds in the initial 12 hours post-surgery without recruiting cells from the limbus (Chang et al., 2008).

A substantially longer healing process of weeks to months is involved if the wound has penetrated both the epithelium and the stromal tissue (Stock et al., 1992; Jester et al., 1999). Wound repair in the corneal stroma is undertaken by the stromal keratocytes in a complex process modulated by soluble signalling molecules such as cytokines and growth factors such as platelet-derived growth factor (PDGF), keratocyte growth factor (KGF) and transforming growth factor-beta (TGF-β) that are produced by the injured epithelial cells above. It is not clear whether soluble signalling molecules derived from the epithelium penetrate the full thickness of the stroma or involve the interconnected keratocyte network in transmitting messages (Wilson et al., 2003). The response of the keratocytes to wounding is rapid and causes the keratocytes in the wounded stroma beneath to enter into programmed cell death known as ‘apoptosis’ (Wilson et al., 2003). Keratocytes in the stroma adjacent to the wound are activated to proliferate within hours of wounding and they transform into a fibroblastic phenotype and migrate into the affected area to repair damage (Fini, 1999; Jester et al., 1999; Wilson et al., 2003; West-Mays and Dwivedi, 2006). Repair fibroblasts may develop into a contractile phenotype known as myofibroblast during the wound- healing process and this is strongly mediated by the presence of TGF-β released from epithelial cells (Mohan et al., 2003). Activated fibroblasts

and myofibroblasts synthesise and assemble new extracellular matrix, which has different components and properties to the normal uninjured stromal tissue (Funderburgh et al., 2003; Guo et al., 2007). Notable among these is hyaluronan, which is absent in the stroma of normal cornea but present in abundance in wounded corneas and those with chronic pathology and is regarded as a fibrotic matrix component (Guo et al., 2007).

Stromal–epithelial interactions are recognised to be a core part of the wound-healing processes that take place in response to corneal stromal injury (Melles et al., 1995). Significant to these interactions are the integrity of the epithelium and the exposure of keratocytes to epithelial-derived factors during wounding which determine whether corneal repair will be regenerative or fibrotic in nature (West-Mays and Dwivedi, 2006). Myofibroblasts and other cell types, such as bone marrow-derived cells, may be present in the stroma during the repair process depending on the nature and severity of the wound and their presence at the repair site causes haze (Dupps and Wilson, 2006).

As the wound-healing response continues, stromal cells – such as keratocytes, fibroblasts, myofibroblasts and inflammatory cells – die by necrosis (Mohan et al., 2003). Damaged cells release pro-inflammatory chemokines which attract great numbers of bone-marrow-derived cells to clean up degenerative cells by engulfing debris into their cytoplasm in a process of phagocytosis (Dupps and Wilson, 2006). A study of myofibroblasts in tissues other than cornea (Dupps and Wilson, 2006) has suggested that myofibroblasts may originate in the bone marrow. These cells tend to be present in the stroma near the epithelium or sites of epithelial ingrowth into the stroma, implying that cytokines produced by epithelial cells are linked to their presence.

Myofibroblasts are found where abnormalities of the stromal surface or regenerated basement membrane occur, as seen following refractive surgical procedures, such as surface laser ablation (Netto et al., 2006). Adult human corneal stromal wounds heal slowly and incompletely and may result in abnormalities such as scar tissue and reduplicated basement membrane (Melles et al., 1995; Dawson et al., 2008). These findings are consistent with the idea that the structural integrity of the epithelial basement membrane is significant in minimising the fibrotic response of the keratocytes and any subsequent scarring and loss of corneal clarity (West-Mays and Dwivedi,

2006).

4.2Using the cornea to correct refractive error

4.2.1Refractive error

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into images. The cornea is responsible for 70% of the refractive power of the eye. Vision is blurred if light does not bend or refract correctly and focus directly on the retina. Blurred vision is a refractive error, which is the most common disorder of the eye. Myopia (short-sightedness) occurs when the eyeball is too long in relation to the refractive power of the eye and light rays focus in front of the retina making distant objects appear blurry.

Myopia is the leading cause of vision loss in the Asia–Pacific region and affects approximately one-third of people in the world. Hyperopia (longsightedness) occurs when the image is focused on a point beyond the retina because the eyeball is too short in relation to the refractive power of the eye. Approximately one-quarter of the world’s population is hyperopic. Many people with myopia or hyperopia also have some astigmatism which is caused by the shape of the cornea. A normal cornea is spherically shaped and astigmatism occurs if the curvature of the cornea is irregular/unequal causing light rays to have more than one focal point, which results in blur and distortion to both distant and near objects. Presbyopia is an age-related condition where the flexibility of the natural lens is gradually reduced and is accompanied by a reduction in the ability of the muscles to change the shape of the lens to focus light on the retina when observing near objects (accommodation). The global increase in life expectancy translates to an increased number of people over the age of 45 years with presbyopia.

4.2.2Alternative approaches in using the cornea to correct refractive error

There is a need for more permanent and convenient solutions for the correction of refractive error as an alternative to spectacles or contact lenses (rigid gaspermeable, soft and orthokeratology lenses). Currently, there are alternative technologies available to treat all types of refractive errors and this section deals with those that involve the cornea. Technologies that address this demand can be categorised broadly into two groups: those that remove corneal tissue to correct refractive error (subtractive) and those technologies that add to corneal tissue to achieve a correction (additive). Subtractive solutions, such as the laser-based procedures, are non-reversible in the sense that tissue is permanently removed. The excimer laser is used to flatten the cornea in the treatment of myopia, to steepen the cornea to correct hyperopia, and to reshape the corneal topography in the treatment of astigmatism. Depending on the type of laser used, the corneal epithelium may need removal or lifting and replacement to expose the stroma for the laser ablation and this has given rise to the development of a range of associated surgical techniques. Newer laser technologies such as the femtosecond laser are able to pass through the tissue to the target area with apparently minimal damage and therefore leave the epithelium intact. Additive solutions embrace a range of

intracorneal devices made from either biological or synthetic materials or a combination of both, which are technically removable and are therefore reversible procedures. There is some cross-over between these groups, since all devices require some form of surgical procedure to implant them. Frequently, surgical techniques that have been developed to enable laser procedures are used for implanting intracorneal devices. Sections 4.3 and 4.4 examine different approaches taken in the correction of refractive errors involving the cornea. Optical and biological outcomes of the technologies that have been developed are considered along with the knowledge gained from those endeavours.

4.3Subtractive approaches to correct refractive error: refractive surgery

4.3.1Incisional refractive surgery techniques

The demand for solutions to the correction of refractive error that offer convenience and cosmesis has driven the development of a range of refractive surgical techniques that have been well reviewed in the literature (Kaufman, 1989; Waring, 1992; Aquavella, 1994; Tervo and Moilanen, 2003; Sakimoto et al., 2006). Initially, ophthalmic surgeons addressed this need with incisional refractive procedures such as radial keratotomy (RK), where the cornea was flattened by a series of radial incisions to treat myopia (Sato et al., 1953). Regression in many patients following this type of surgery cast doubt on its predictability and this problem was attributed to contraction of the wound bed during healing. Studies of incisional gape wounds in animals, such as used in RK, showed healing involved the rapid migration of the corneal epithelium to cover the wounded surface and fill the wound site with an epithelial plug (over the first few days) that was gradually replaced by stromal fibroblasts which produced new extracellular matrix material (by 2 weeks), which eventually contracted (by 4 weeks) (Garana et al., 1992). The wound-healing process was biphasic and strongly linked to the presence of epithelium in the wound bed (pre-contractile phase), with contraction occurring once this was replaced by fibroblastic tissue (contractile phase) (Jester et al., 1992). Further animal studies identified that the tension responsible for the contraction was generated by actin stress fibres formed in myofibroblasts interacting with fibronectin in the local extracellular matrix (Garana et al., 1992). Animal data showing the initiation of wound contraction by myofibroblasts and re- steepening of the cornea observed 2 weeks post-operatively were found to correlate temporally with the progressive hyperopic shift observed in 30% of patients following incisional refractive surgery (Waring et al., 1994; Jester et al., 1999).

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4.3.2Ablative refractive surgery techniques

The transition from incisional to ablative refractive surgery occurred with the advent of excimer laser technology in the 1980s. Photorefractive keratectomy (PRK) used the excimer laser in a surface ablation technique to change the curvature of the central cornea after debridement of the epithelium, which healed afterwards (Trokel et al., 1983; McDonald et al., 1990). While clinically this procedure has successfully corrected refractive errors, it has been associated with complications including post-surgical pain, regression of the refractive effect and post-surgical sub-epithelial haze (Azar et al., 1998; Azar et al., 2001). Histological analyses of corneas have shown reduced keratocyte densities, undulations in Bowman’s layer, incomplete stromal wound healing and scar formation months to years after PRK (Hanna et al., 1990; SundarRaj et al., 1990; Linna and Tervo, 1997). Studies in animals and humans have linked the regression and haze issues to the presence of myofibroblasts in the healing wound bed and the deposition of new collagen above the photoablated stromal surface (Møller-Pedersen et al., 1998; Møller-Pedersen et al., 2000). In recent years, problems relating to cutting a flap and wound-healing issues with laser in situ keratomileusis (LASIK) procedures have revived the use of PRK. Alternative methods of epithelial removal in the PRK procedure have been investigated to address the issues of post-operative pain and re-epithelialisation of the wound bed following surface ablation. The aim of the new approaches was to make a superficial corneal flap minimising damage to the underlying stromal tissue. Instruments for blunt dissection are used to cleave the epithelium through the basement membrane zone creating a flap of epithelial tissue, which is repositioned after the stromal surface has been ablated. The techniques vary in the method of removal of the epithelial sheet (Pallikaris et al., 2003).

In laser-assisted sub-epithelial keratomileusis (LASEK), diluted alcohol is used to loosen the epithelial sheet which is freed with a spatula (Azar et al., 2001). Brief periods (25–30 seconds) of exposure to diluted alcohol

(15–20%) allow the creation of a reproducible flap with a smooth cleavage plane through the basement membrane zone (Browning et al., 2003; Espana et al., 2003), with low levels of cell death found in epithelium and underlying stromal keratocytes (Lee et al., 2002). In vitro studies on the effect of various alcohol dilutions on cultured epithelial monolayers resulted in increasing cell death in a doseand time-dependent manner (Chen et al., 2002). Nerve damage is also caused by the LASEK procedure and persists for some time after the procedure. A confocal microscopy study of 35 patients reported that the sub-basal nerves were not recovered 6 months after the LASEK procedure although corneal sensitivity, evaluated using aesthesiometry, was restored after 3 months (Darwish et al., 2007). Overall, LASEK may be useful for patients unable to have LASIK but they would have to accept