RESUMEN
PURPOSE: Limbal stem cell deficiency (LSCD) secondary to ocular surface alkali burn is a blinding condition that features corneal conjunctivalization. Mechanistic insights into its pathophysiology are lacking. Here, we developed a mouse model that recapitulates human disease to comprehensively delineate the clinicopathological features of a conjunctivalized cornea. METHODS: LSCD was induced in the right eyes of 6-8-week-old C57BL/6 male and female mice (n = 151) by topical administration of 0.25N sodium hydroxide on the cornea. Uninjured left eyes served as controls. Clinical, histological, phenotypic, molecular, and immunological assessments were performed at multiple time-points over 6-months. RESULTS: Clinically, alkali burn caused persistent corneal opacity (p = 0.0014), increased punctate staining (p = 0.0002), and reduced epithelial thickness (p = 0.0082) compared to controls. Total LSCD was confirmed in corneal whole mounts by loss of K12 protein (p < 0.0001) and mRNA expression (p = 0.0090). Instead, K8+, K13+, K15+ and MUC5AC+ conjunctival epithelia prevailed. 20 % of injured corneas developed islands of K12+ epithelia, suggesting epithelial transdifferentiation. Squamous metaplasia was detected in 50 % of injured corneas. Goblet cell density peaked early post-injury but decreased over time (p = 0.0047). Intraepithelial corneal basal nerve density remained reduced even at 6-months post-injury (p = 0.0487). CONCLUSIONS: We developed and comprehensively characterized a preclinical mouse model of alkali-induced LSCD. Understanding the pathophysiological processes that transpire on the ocular surface in LSCD is key to discovering, testing, and advancing biological and pharmacological interventions that can be dispensed prior to or in conjunction with stem cell therapy to rehabilitate the cornea and restore vision.
RESUMEN
The narrow intersection between the cornea and conjunctiva, otherwise known as the limbus, is purported to harbor stem cells (SCs) that replenish the ocular surface epithelium throughout life. Damage to this site or depletion of its SCs can have dire consequences for eye health and vision. To date, various SC and keratin proteins have been used to identify the limbus, however, none could definitively mark its boundaries. Herein, we use the mouse as a model system to investigate whether structural and phenotypic features can be used to define the limbus and its boundaries with adjacent tissues. We demonstrate that differentially aligned blood and lymphatic vessels, intraepithelial nerves, and basal epithelial cellular and nuclei dimensions can be used as structural landmarks of the limbus. Identification of these features enabled approximation of the limbal expanse, which varied across distinct ocular surface quadrants, with the superior nasal and inferior temporal limbus being the widest and narrowest, respectively. Moreover, label-retaining SCs were unevenly distributed across the ocular circumference, with increased numbers in the superior temporal and inferior temporal moieties. These findings will heighten our current understanding of the SC niche, be beneficial for accurately predicting SC distribution to improve their isolation and devising efficacious cell therapies, and importantly, aid the ongoing search for novel SC markers.
RESUMEN
Continuous replenishment of the corneal epithelium is pivotal for maintaining optical transparency and achieving optimal visual perception. This dynamic process is driven by limbal epithelial stem cells (LESCs) located at the junction between the cornea and conjunctiva, which is otherwise known as the limbus. In patients afflicted with diabetes, hyperglycemia-induced impairments in corneal epithelial regeneration results in persistent epithelial and other defects on the ocular surface, termed diabetic keratopathy (DK), which progressively diminish vision and quality of life. Reports of delayed corneal wound healing and the reduced expression of putative stem cell markers in diabetic relative to healthy eyes suggest that the pathogenesis of DK may be associated with the abnormal activity of LESCs. However, the precise role of these cells in diabetic corneal disease is poorly understood and yet to be comprehensively explored. Herein, we review existing literature highlighting aberrant LESC activity in diabetes, focusing on factors that influence their form and function, and emerging therapies to correct these defects. The consequences of malfunctioning or depleted LESC stocks in DK and limbal stem cell deficiency (LSCD) are also discussed. These insights could be exploited to identify novel targets for improving the management of ocular surface complications that manifest in patients with diabetes.