Clinical background
The ocular surface comprises the entire and continuous mucosal outer epithelial lining of the eye lids, conjunctiva, and cornea. This chapter will focus upon ocular surface failure caused by insult to the corneal epithelium and discuss current therapeutic strategies and the underlying pathophysiology.
The cornea on the front surface of the eye is comprised of five layers: the outermost multilayered epithelium, Bowman’s layer (which is acellular), the keratocyte (corneal fibroblast)-populated collagen stroma, and Descemet’s membrane on the inner corneal surface, upon which lies a monolayer of endothelial cells ( Figure 11.1 ). Transparency of the cornea, and therefore vision, is dependent upon the coordinated functionality of all layers.
Integrity of the epithelium is essential for corneal clarity and light refraction. Corneal epithelial cells are constantly lost from the ocular surface during blinking. These desquamated cells are replenished from a population of limbal epithelial stem cells (LESCs) which reside in the basal epithelial layer of the corneoscleral junction, known as the limbus. The specific location of LESCs remains unclear; however, they are likely to reside in or on structures known as the palisades of Vogt at the periphery of the cornea ( Figure 11.2 ). When the LESCs divide asymmetrically they produce daughter transient amplifying cells which migrate, proliferate, and differentiate to maintain the corneal epithelium ( Figure 11.3 ). Hence, LESCs are responsible for homeostatic and posttraumatic regeneration of the corneal epithelium and loss of their function causes ocular surface failure.
Key symptoms and signs
The key symptoms of ocular surface failure include: loss of corneal epithelial transparency, superficial subepithelial corneal neovascularization, epithelial irregularity, history of recurrent epithelial breakdown, stromal inflammation, corneal melting and perforation, loss of limbal palisades of Vogt, and reduction of visual acuity. An example of ocular surface failure caused by a chemical burn injury is shown in Figure 11.4 .
Epidemiology
The diseases and injuries which can cause LESC deficiency can affect either gender.
Chemical/thermal injuries are most prevalent in countries with poor health and safety records, although domestic accidents do occur. Stevens–Johnson syndrome (SJS) has high morbidity and mortality, with an incidence of 1 per million per year. Advanced ocular cicatricial pemphigoid occurs more frequently in females and LESC deficiency associated with this disease can result from inflammatory cytokine damage. Inappropriate overwearing of contact lenses, multiple surgeries, exposure to ultraviolet or ionizing radiation and antimetabolites and extensive microbial infection may also cause LESC deficiency and ocular surface failure. Loss of LESC function due to the inherited eye disease aniridia is associated with vision loss during the early teens.
Diagnostic workup and differential diagnosis
Accurate diagnosis of LESC deficiency is important as patients suffering from these conditions are unlikely to respond well to conventional treatment, including corneal transplantation. LESC deficiency is diagnosed on the basis of the key signs and symptoms described above and by using a technique called impression cytology. This involves placing a filter paper on the front surface of the eye which, when removed, takes with it a sample of the surface layer cells. Immunohistochemical staining and microscopy are then used to detect the profile of cytokeratin expression in the harvested cells. The presence of cytokeratins 3 and 12, identified using monoclonal antibodies, would indicate cells of the correct corneal phenotype. However, cytokeratin 19 positivity together with the presence of mucin-producing goblet cells (following staining with periodic acid – Schiff reagent) is indicative of conjunctivalization of the corneal surface and hence LESC deficiency ( Figure 11.5 ). It is also possible to observe a mixed population of cells which may indicate partial rather than total LESC failure. When fluorescein dye is placed on the eyes of patients with LESC failure, the corneal surface viewed through a slit lamp is often abnormally stained. Areas of the epithelium may be thin with signs of erosion.
Treatment
Previous conservative attempts to correct LESC deficiency have included harvesting healthy autologous limbal tissue from the contralateral eye for transplantion to the diseased eye (keratolimbal autograft). Whilst potentially successful in terms of vision recovery, there is a risk of creating LESC deficiency in the donor eye if too much tissue is harvested. Alternatively, allogeneic tissue from a living related or cadaveric donor may be used in conjunction with long-term systemic immunosuppression ( Box 11.1 ). In 1997, the first description of the successful use of ex vivo expanded autologous LESCs to treat LESC deficiency in chemical burn injury patients was published by Pellegrini et al. For this technique, just a 1–2 mm 2 limbal biopsy is harvested, posing little risk to the donor eye. The isolated epithelial cells are then cultured on a growth-arrested feeder layer of murine 3T3 fibroblasts. Upon formation of a multilayered cell sheet the epithelial cells are released from the culture dish and transferred to a carrier for patient grafting. A number of techniques for ex vivo expansion of autologous and allogeneic limbal epithelial cells have since been developed, including the use of human amniotic membrane, as a surrogate stem cell niche. An example of therapeutic LESC culture methodology and clinical outcome is shown in Figures 11.6 and 11.7 respectively.
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Integrity and functionality of the cornea are essential for vision
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The cornea is comprised of five layers: epithelium, Bowman’s layer, stroma, Descemet’s membrane, and endothelium
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Epithelium is regenerated by stem cells in the limbus at the edge of the cornea
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Limbal epithelial stem cell (LESC) deficiency can result from a variety of inherited or acquired conditions causing blinding ocular surface failure
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LESC deficiency is diagnosed by epithelial cell cytokeratin profile and clinical appearance
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Treatment includes transplantation of cultured autologous or allogeneic LESCs
Prognosis and complications
With no standard protocol for assessing clinical outcome, prognosis following cultured LESC therapy can be difficult to predict since patients are of mixed etiological background and have usually undergone a variety of surgical procedures. Nonetheless, the majority of reports agree that LESC cultures may be a useful addition to the management protocols for LESC deficiency since improved visual acuity has been reported in approximately 70% of cases (extensively reviewed by Shortt et al. ). Complications associated with cultured LESC therapy have included graft loss due to recurrence of residual infection and corneal neovascularization.
Etiology
Primary causes of LESC deficiency
Genetic risk factors
A variety of primary disorders can lead to a deficiency of LESCs as a result of inadequate stem cell support by the stromal microenvironment. These include heritable genetic disorders such as aniridia which is caused by a mutation in the eye development gene Pax6 . Multiple endocrine deficiencies can also lead to keratitis and LESC failure.
Secondary causes of LESC deficiency
Environmental risk factors
LESC deficiency occurs more commonly as a result of acquired factors which destroy the stem cells, such as chemical or thermal injury. In heavily industrialized areas it is common for manual workers to sustain chemical or thermal burn injuries to the eyes, resulting in partial or complete physical destruction of the limbal palisades of Vogt, causing LESC deficiency and ocular surface failure, particularly where health and safety practices are not optimal or are not strictly adhered to. LESC deficiency has occurred in some patients as the result of soft contact lens wear, the effects of which have included ulceration, stromal scarring, neovascularization and decreased visual acuity ( Box 11.2 ). Occasionally surgical or medical intervention may cause temporary or permanent loss of LESC function. Examples include the use of antimetabolite drugs, cryotherapy, and radiation therapy. Multiple ocular surgery procedures can also increase the risk, as can extensive microbial infection. The onset of inflammatory disorders and autoimmune diseases, including SJS and advanced cicatricial pemphigoid, have also been linked with LESC failure. If the neighbouring conjunctival cells are also depleted, the cornea surface becomes heavily keratinized.
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Genetic risk factors such as Pax6 gene haploinsufficiency cause limbal epithelial stem cell (LESC) failure
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More commonly, LESC deficiency occurs due to physical insult such as chemical burn
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LESC deficiency can also occur following inappropriate contact lens wear, ocular surgery, infection, and inflammation
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The cornea undergoes “conjunctivalization” with neovascularization, ulceration, and/or scarring
Usually, when LESC deficiency occurs, the neighboring conjunctival epithelial cells and blood vessels migrate over the corneal surface. This conjunctivalization process causes persistent epithelial breakdown and superficial vascularization of the cornea. Patients experience impaired vision and chronic discomfort. LESC deficiency may be partial or total depending upon the insult.
Chemical/thermal injury
LESCs reside in a specialized niche environment at the palisades of Vogt which regulates self-renewal and cell fate decisions. Properties of this niche have recently been described and include specific tissue architecture, the presence of a vascular and neural network, and close proximity of stromal cells. Physical destruction of this niche can occur with chemical/thermal injury with subsequent ocular surface failure. However, transplantation of cultured autologous LESCs can restore a normal corneal epithelial phenotype ( Box 11.3 ). Allogeneic cells can also produce a similar clinical outcome, which is very interesting since the transplanted cells do not appear to survive on the cornea for longer than 28 weeks, as determined by polymerase chain reaction genotyping of sampled cells. It is possible that the cultured transplanted cells create a permissive environment for any remaining host LESCs to resume function or that bone marrow stem cells are recruited. It is also not clear whether surviving transplanted cells are able to regenerate the niche to any extent.
