Abstract
Purpose :
The limbus, which intersects cornea and conjunctiva, is purported to harbor stem cells (SCs) that replenish the corneal epithelium throughout life. Damage to this site or depletion of its SCs can have dire consequences for eye health and vision. SC markers can help identify this site, but none are definitive discriminators of its boundaries. Here, we investigated whether morphological, phenotypic, and structural features associated with epithelial and neuronal cells and vascular tracks can identify the limbus and its boundaries with the cornea and conjunctiva.
Methods :
Globes with intact conjunctiva were procured from 8-10-week-old C57BL/6 male/female mice (n=25) and dissected into superior-nasal, superior-temporal, inferior-nasal and inferior-temporal quadrants. Tissues were fixed and either sectioned or prepared as flat-mounts for histology, transmission electron microscopy and immunofluorescence with cell-type specific antibodies. BrdU was pulse-chased to identify label-retaining cells, and limbal width computed with ImageJ.
Results :
Despite revealing differences in cell morphology between regions of the ocular surface, limbal boundaries could not be accurately defined by histology. Moreover, putative limbal SCs markers (ABCB5, GPHA2, K14, K15, Lrig1) also labelled conjunctival epithelia, and thus could not delineate this region. However, immunostaining with corneal K12 and conjunctival K8 revealed an epithelial band lacking immunoreactivity which we propose represents the limbal expanse. This zone was widest in the superior-nasal and narrowest in the inferior-temporal (190.39±15.46µm vs 18.64±8.38µm; p<0.0001) quadrant. Other determinants of limbal over corneal and conjunctival precincts included differences in epithelial nuclei dimensions (56.77±1.50µm2 vs 40.53±0.83µm2; p<0.0001), presence of slow-cycling BrdU+ cells, vascular diameter (4.28±0.15µm vs 5.08±0.20µm, p<0.0001) and intraepithelial corneal basal nerve directionality, length and density (2.92±0.44% vs 0.94±0.21%, p<0.05).
Conclusions :
Herein, we recorded morphological, phenotypical and structural features attributed to cell-types within the ocular surface that define the limbal boundaries. Mapping the limbal expanse is imperative for pinpointing SC location, improving their extraction to increase the potency of therapeutics, and gauging efficacy of treatments in restoring cellular composition, tissue architecture and sight.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.