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Delayed Development of Limbal Stem Cell Deficiency Following Chemical Injury—Pathogenesis and Therapeutic Strategies

US Ophthalmic Review, 2013;6(2):101–4 DOI: http://doi.org/10.17925/USOR.2013.06.02.101

Abstract:

Limbal epithelial stem cell deficiency (LSCD) occurs as a result of damage to the limbal epithelial stem cells (ESC) population. It may derive from direct destructive loss of the ESC (common chemical burn), and/or from dysfunction of the SC niche, leading to delayed death of the cells. This review focuses on delayed-onset LSCD, induced by antineoplastic chemicals, such as mitomycin C, 5-fluorouracil, and mustards, in terms of pathogenesis and management. These agents are used in ocular surface chemotherapy, in ocular surgery procedures, and as warfare agents, and target proliferating cells as slow-cycling cells, such as the ESC, are relatively resistant. Although the mechanism of the delayed loss of ESC is not entirely clear, we have shown, in the rabbit model, pathologic alterations in the limbal stroma, following the application of sulfur mustard, suggesting that dysfunction of the niche triggers the death of the SC later on. The absence of direct cytotoxic effects of these agents on the ESC, indicates a therapeutic window for prevention of the delayed LSCD.
Keywords: Ocular burns, chemical burns, cornea, epithelial stem cells, limbal stem cell deficiency, mustard, mitomycin, 5-fluorouracil
Disclosure: The authors have no conflicts of interest to declare.
Received: July 17, 2013 Accepted: August 12, 2013
Correspondence: Tamar Kadar, Department of Pharmacology, Israel Institute for Biological Research, Ness Ziona, 74100, Israel. E: tamark@iibr.gov.il

Homeostasis of corneal epithelium is essential for the maintenance of healthy ocular surface as well as for corneal transparency and accurate vision. Continuous renewal of corneal epithelium is provided by a population of adult stem/progenitor cells residing in the limbus, the transitional zone between the vascular conjunctiva, and the avascular transparent cornea.1–7 At the limbus, the corneal epithelial stem cells (ESC) reside within the basal layer of the epithelium. Although no single specific marker is available to identify stem cells (SC), a series of markers are used to characterize them. These include the expression of ABCG2, p63, or Notch-1, the absence of differentiation markers, such as CK3 and connexin-43, as well as morphologic criteria, such as small cell size (6–7 um) or high nucleus to cytoplasm ratio.8–14 The limbus differs from the central cornea in the organization of the epithelium and in the composition of the basement membrane and stroma. The distinctive characteristics of the two tissues are thought to play a role in the regulation of their respective populations of epithelial cells.15,16

Limbal ESC require a special environment to retain their SC properties. The environment is provided by the SC niche in which signaling from adjacent cells, as well as properties of the basal membrane, are believed to play a role in the maintenance of their ‘stemness.’7,12,14,17–19 The cells in the niche have been suggested to regulate the preservation, proliferation, and differentiation of the ESC by producing specific matrix components and secreting growth factors and signaling molecules in a tightly regulated spatial and temporal pattern.20,21 Consistently, the extracellular matrix composition of the limbus differs significantly from that of cornea and conjunctiva and specific cell surface receptors and adhesion molecules appear to mediate limbal ESC anchorage to their niche.

When limbal SC are depleted below a certain threshold, clinical signs of limbal epithelial stem cell deficiency (LSCD) appear, causing gradual vision loss.

LSCD occurs as a result of disease or damage to the limbal ESC population. Deficiency can arise from injuries, including chemical or thermal burns, and through diseases, such as Stevens Johnson syndrome and aniridia.22 It could be focal or diffuse depending on the extent of limbal involvement with underlying disease process. Due to the damage in the limbus, the barrier between the vascular conjunctiva and the avascular cornea is impaired and conjunctival epithelial cells migrate toward the corneal surface, accompanied by ingrowth of blood vessels. The clinical signs of LSCD, resulted from conjunctivalization of the cornea, include persistent epithelial defects, corneal vascularization, and chronic stromal inflammation leading to functional impairment and visual loss. Diagnosis of LSCD is based on the symptomatic hallmarks and is supported by identification of conjunctival goblet cells in the cornea, using impression cytology. Recently, the diagnosis of LSCD was supported by in vivo confocal microscopy.19,23 Typical characteristics of LSCD are shown in Figure 1.

LSCD may derive from destructive loss of limbal SC, and/or from dysfunction of the microenvironment of LSC, the SC niche, leading to insufficient support and death of the ESC.5,24,25 Chemical and thermal burns are the most common cause of a direct destruction of limbal SC. In contrast to the immediate loss of SC following a primary destruction, a gradual loss of the SC population with time characterizes the second category. The appearance of the LSCD symptoms in this case is delayed and takes months to years after the initial insult. Neurotrophic keratopathy and chronic limbitis are examples of delayed onset LSCD, deriving from poor nutritional supply of neuronal trophic factors, essential for the maintenance of the epithelium,26 and secretion of undesirable cytokines in the limbus in chronic limbitis.1,2,24

The present article focuses on delayed-onset LSCD induced by chemical agents. This less-familiar type of LSCD will be discussed in terms ofpathogenesis and management.

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Keywords: Ocular burns, chemical burns, cornea, epithelial stem cells, limbal stem cell deficiency, mustard, mitomycin, 5-fluorouracil