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

Summary

The following article provides an easy, reproducible technique to effectively create a sustainable mouse model of limbal stem cell deficiency (LSCD). This animal model is useful in testing and comparing the efficacy of treatments for limbal stem cell diseases.

Abstract

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with conjunctiva. Patients suffer from recurrent corneal defects, pain, inflammation, and loss of vision.

Previously, a murine model of LSCD was described and compared to two other models. The goal was to produce a consistent mouse model of LSCD that both mimics the phenotype in humans and lasts long enough to make it possible to study the disease pathophysiology and to evaluate new treatments. Here, the technique is described in more detail.

A motorized tool with a rotating burr has been designed to remove the rust rings from the corneal surface or to smooth the pterygium bed in patients. It is a suitable device to create the desired LSCD model. It is a readily available, easy-to-use tool with a fine tip that makes it appropriate for working on small eyes, as in mice. Its application prevents unnecessary trauma to the eye and it does not result in unwanted injuries, as often is the case with chemical injury models. As opposed to a blunt scraper, it removes the epithelium with the basement membrane. In this protocol, the limbal area was abraded two times, and then the whole corneal epithelium was shaved from limbus to limbus. To avoid stroma injury, care was taken not to brush the corneal surface once the epithelium was already removed.

Introduction

The corneal epithelium is required to maintain the clarity and integrity of the cornea. It is constantly renewed throughout life by the epithelial stem cells residing in the limbus-a narrow zone at the junction of the cornea and the conjunctiva. These self-renewing limbal stem cells play a crucial role in regenerating the corneal epithelium, both normally and after injury. Partial or complete depletion of these stem cells will result in recurrent corneal erosions, pain, corneal scarring and neovascularization, appearance of goblet cells, and if left untreated, corneal blindness. This condition is known as limbal stem cell deficiency (LSCD) and can be idiopathic; hered....

Protocol

All procedures performed on animals comply with the Association for Research in Vision and Ophthalmology statements for animal use in vision research. Fourteen four- to six-month-old male/female C57BL/6J wild type mice were used: ten mice for the injury model and four as control animals (unwounded corneas).

1. Animal Preparation

1. Anesthetize a mouse with an intraperitoneal injection of a 100 mg/kg ketamine and 5 mg/kg xylazine mixture. After the anesthesia has taken effect, pinch the toe to confirm the anesthesia depth; the animal should not react.
2. Inspect the eye under a slit lamp before conducting the experiments to v....

Results

Within a month after performing this technique, 100% of the corneas developed superficial neovascularization (Figure 1A. Photos were taken with a camera connected to a slit lamp under a bright field and cobalt blue filter). In 18/20 (90%) of the eyes, neovascularization involved the whole corneal surface (Figure 1A). In 2/20 (10%), neovascularization was observed in three of four corneal quadrants, but it still reached the corneal center.

Discussion

This article describes a reproducible and relatively simple technique to create a mouse model of LSCD. There are several important aspects of this model that are worth noting. First, unlike models that use chemicals^{11, 12}, the injury primarily involves the surface epithelium (and basement membrane), with minimal damage to the underlying corneal stroma or to other intraocular structures. Thus, there is only limited inflammation and scarring, both of which can complicate the procedure and make it more difficult .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Rotating burr: AlgerBrush II rust ring remover	Rumex International Co, Clearwater, FL	16-141	0.5 mm burr. Also available from other companies.
Surgical microscope	Wild Heerbrugg, Switzerland	Wild M691
Digital camera	Nikon, Thailand
Nikon FS-2 slit lamp	Nikon, Japan
Polyclonal anti-CK12 antibody	Santa Cruz Blotechnology, CA,	SC-17101	1:100 concentration
Monoclonal anti-CK8 antibody	TROMA-I-s, Iowa City, IA	AB_531826	1:50 concentration