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

Abstract

The New Zealand White rabbit has been widely used as a model of limbal stem cell deficiency (LSCD). Current techniques for experimental induction of LSCD utilize caustic chemicals, or organic solvents applied in conjunction with a surgical limbectomy. While generally successful in depleting epithelial progenitors, the depth and severity of injury is difficult to control using chemical-based methods. Moreover, the anterior chamber can be easily perforated while surgically excising the corneal limbus. In the interest of creating a safer and more defined LSCD model, we have therefore evaluated a mechanical debridement technique based upon use of the AlgerBrush II rotating burr. An initial comparison of debridement techniques was conducted in situ using 24 eyes in freshly acquired New Zealand White rabbit cadavers. Techniques for comparison (4 eyes each) included: (1) non-wounded control, (2) surgical limbectomy followed by treatment with 100% (v/v) n-heptanol to remove the corneal epithelium (1-2 minutes), (3) treatment of both limbus and cornea with n-heptanol alone, (4) treatment of both limbus and cornea with 20% (v/v) ethanol (2-3 minutes), (5) a 2.5-mm rounded burr applied to both the limbus and cornea, and (6) a 1-mm pointed burr applied to the limbus, followed by the 2.5-mm rounded burr applied to the cornea. All corneas were excised and processed for histology immediately following debridement. A panel of four assessors subsequently scored the degree of epithelial debridement within the cornea and limbus using masked slides. The 2.5-mm burr most consistently removed the corneal and limbal epithelia. Islands of limbal epithelial cells were occasionally retained following surgical limbectomy/heptanol treatment, or use of the 1-mm burr. Limbal epithelial cells were consistently retained following treatment with either ethanol or n-heptanol alone, with ethanol being the least effective treatment overall. The 2.5-mm burr method was subsequently evaluated in the right eye of 3 live rabbits by weekly clinical assessments (photography and slit lamp examination) for up to 5 weeks, followed by histological analyses (hematoxylin & M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT3 eosin stain, periodic acid-Schiff stain and immunohistochemistry for keratin 3 and 13). All 3 eyes that had been completely debrided using the 2.5-mm burr displayed symptoms of ocular surface failure as defined by retention of a prominent epithelial defect (~40% of corneal surface at 5 weeks), corneal neovascularization (2 to 3 quadrants), reduced corneal transparency and conjunctivalization of the corneal surface (demonstrated by the presence of goblet cells and/or staining for keratin 13). In conclusion, our findings indicate that the AlgerBrush II rotating burr is an effective method for the establishment of ocular surface failure in New Zealand White rabbits. In particular, we recommend use of the 2.5-mm rotating burr for improved efficiency of epithelial debridement and safety compared to surgical limbectomy.

Epithelial Cell Migration and Proliferation Patterns During Initial Wound Closure in Normal Mice and an Experimental Model of Limbal Stem Cell Deficiency

“…4 A), as previously shown. 30 – 34 The successful removal of LSCs using our surgical procedure for debriding the limbal rim was confirmed immediately after wounding (results not shown) and also 10 hours after injury ( Figs. 4 B and C).…”

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

“…. Unlike the blunt scraper, the rotating burr removes the epithelial basement membrane as well 7,8,10 . Other reported methods to sever stem cells involve the use of chemicals, such as sodium hydroxide, nheptanol, and benzalkonium chloride, which not only may induce unwanted injury to underlying eye structures, but also may lead to significant inflammation and subsequent corneal opacification or epithelial squamous metaplasia [11][12][13][14][15] .…”

“…The rotating burr is not associated with these severe complications. Surgically removing the limbal epithelium, alone or with the use of chemicals 10,11,16 , is more difficult to perform and is not the best option in animals with small eyes and a thin limbal epithelia (like mice). In addition, surprisingly, limbectomy may still leave some of the limbal epithelium behind 10 .…”

The Impact of Limbal Mesenchymal Stromal Cells on Healing of Acute Ocular Surface Wounds Is Improved by Pre-cultivation and Implantation in the Presence of Limbal Epithelial Cells

“…Female New Zealand White rabbits (2.5–3.0 kg) were sourced and cared for as previously described in detail 8 . Likewise, anesthesia, post-operative care, and clinical assessments (including general photography, slit lamp examination, and quantification of re-epithelialization) were conducted as described previously 8 . Notably, post-operative pain management consisted of alternating doses of meloxicam (morning; 0.05 mg/kg) and buprenorphine (afternoon; 50 µg/kg) for up to 72 h following surgery.…”

“…We have therefore presently investigated the effects of allogeneic RLMSC when applied alone or in conjunction with human limbal epithelial (HLE) cells cultivated on human amniotic membrane (HAM). As in our previous study 8 , epithelial tissue is removed from across the full width of the cornea including the limbus. A mechanical method of epithelial debridement is used in order to create a more defined wound than that caused by caustic chemicals.…”