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

Abstract

We studied the reproducibility and stability of limbal stem cell deficiency (LSCD) in mice following controlled injuries to the corneal and limbal epithelia. In one method, corneal and limbal epithelia were entirely removed with a 0.5mm metal burr. In the other, limbus to limbus epithelial removal with the burr was followed by thermal injury to the limbus. These two methods were compared with a previously published one. Unwounded corneas were used as control. The corneas were examined monthly for three months by slit lamp with fluorescein staining. Immunofluorescence staining for cytokeratin 12 and 8 on corneal wholemount and cross sections were performed to determine the phenotype of the epithelium. Mechanical shaving of the epithelium, with or without thermal injury, resulted in a reproducible state of LSCD marked by superficial neovascularization, reduce of keratin 12 expression and presence of goblet cells on the cornea. The phenotype was stable in 100% of the eyes up to at least three months. Thermal injury produced a more severe phenotype with more significant stromal opacification. These corneal injury models may be useful for studying the mechanisms leading to limbal stem cell deficiency.

Evaluation of the AlgerBrush II rotating burr as a tool for inducing ocular surface failure in the New Zealand White rabbit

“…Whatever the explanation, since traces of keratin 3 positive cells were sporadically observed in all completely wounded animals (R1 to R3), we must conclude that the level of epithelial cell removal achieved using the Algerbrush II is less than 100%. This conclusion is consistent with prior data produced in mice (Afsharkhamseh et al, 2015).…”

“…During initial selection of a rotating burr, we speculated in a similar manner to prior researchers (Afsharkhamseh et al, 2015), that the undulating crypt-like structure of the limbal stem cell niche may hamper complete removal of more basal progenitor cells. We therefore hypothesized that the finer 1-mm pointed burr tool would penetrate more deeply, thus leading to more efficient removal of the more basal epithelial cells.…”

“…A more limited number of studies have utilized the AlgerBrush II to induce LSCD in mice (Afsharkhamseh et al, 2015;Ksander et al, 2014;Meyer-Blazejewska et al, 2011), but the suitability of this technique for other species including rabbits is unclear. We therefore presently evaluate the suitability of the AlgerBrush II as a tool for inducing LSCD in the New Zealand White rabbit.…”

“…The exceptions are a limited number of studies conducted in mice (Afsharkhamseh et al, 2015;Ksander et al, 2014;Meyer-Blazejewska et al, 2011). In the first of these studies (Meyer-Blazejewska et al, 2011), a 0.5-mm rotating burr was applied to the entire limbal and corneal epithelium.…”

“…Presumably both groups had good reasons for choosing a rotating burr over more conventional methods but a comparison of techniques was not presented. A brief communication (Afsharkhamseh et al, 2015), however, has recently appeared in the literature comparing use of the 0.5-mm rotating burr in mice to a mechanical method based upon scraping with a blunt spatula. The effects of the rotating burr were examined alone and in conjunction with a controlled thermal injury delivered to the limbus using an ophthalmic cautery device.…”