A Mouse Model of Limbal Stem Cell Deficiency Induced by Topical Medication With the Preservative Benzalkonium Chloride

Abstract

METHODS. BAC solutions (0%-0.5%) were applied to the mouse ocular surface for 4 weeks. Corneal neovascularization, inflammation, and epithelial status were observed under slit-lamp microscope. The eyeball and ocular surface tissues were collected at 4 and 12 weeks and labeled with a series of antibodies. Limbal structure was evaluated by light and transmission electron microscopy (TEM). Corneal impression cytology was performed at 12 weeks, and specimens were labeled with periodic acid Schiff (PAS) reagents.RESULTS. BAC (0.5%) four times per day for 28 days successfully induced the typical manifestations of LSCD, including corneal neovascularization, severe inflammation in the stroma, and diffuse epithelial defect (P < 0.001). Conjunctival epithelium markers K19 and K13 were positive on the corneal surface. Expression of the putative limbal stem cell markers P63 and ABCG2 was abolished in the limbal epithelium. b-catenin was negative in the basal layer. TEM revealed the irregular basement membrane and the loss of stem cell-specific ultrastructure in the limbal basal epithelium. In the 0.5% BAC group, goblet cells could not be observed on day 28 but emerged after the cessation of BAC, and remained over the cornea after 8 weeks. K13-positive cells were still present over the cornea with the loss of K12.CONCLUSIONS. Topical administration of BAC at high concentration and frequency in mouse induces ocular surface changes resembling those of LSCD in humans, representing a novel model of LSCD.

Corneoscleral Limbus in Glaucoma Patients: In Vivo Confocal Microscopy and Immunocytological Study

“…They proposed that limbal epithelial thickness might be intended as a surrogate of the limbal stem cells status since it appears to directly correlate and represent the stem cell density. 32 To further support this hypothesis, Lin et al 33 reported that the instillation of BAK 0.5% four times per day for 4 weeks in a mouse model induced the typical manifestations of LSCD, including corneal neovascularization, severe inflammation in the stroma, and diffuse epithelial defect. The mechanisms leading to POV alterations in glaucoma were not previously considered and, therefore, are unknown; nevertheless, inflammatory processes occurring within limbal epithelium and diffusing for contiguity in depth finally reaching POV, may be hypothesized.…”

Light aversion and corneal mechanical sensitivity are altered by intrinscally photosensitive retinal ganglion cells in a mouse model of corneal surface damage

“…Benzalkonium chloride (BAC) has been used to cause corneal surface damage to model dry eye disease in mice (Lin et al, 2011;He et al, 2012;Sarkar et al, 2012;Xiao et al, 2012Xiao et al, , 2013Lin et al, 2013;Zhang et al, 2014). The degree of corneal surface damage is proportional to the concentration and duration of BAC.…”

Stability of limbal stem cell deficiency after mechanical and thermal injuries in mice

“…Alkali substances are known to cause severe ocular damage and inflammation with compromising stroma and resulting in corneal ulceration, hyphema, hypopyon, and even corneal perforation (Ma et al, 2006). Other models involve surgical removal of the limbal tissue (Chen and Tseng, 1991), cauterization (Majo et al, 2008) and more recently, using benzalkonium chloride to simulate chronic injury to the ocular surface (Lin et al, 2013). In this study, our goal was to evaluate and validate a reproducible mouse model of LSCD using mechanical and thermal injury in a controlled manner, thereby avoiding extensive damage to the corneal stroma and other anterior chamber structures.…”