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; hereditary; or acquired due to chemical or thermal injuries, long-term contact lens wear, and chronic inflammation^1-6.

Research on LSCD requires a suitable animal model that not only mimics the disease in humans, but is reproducible and sustainable, with the least amount of injury to other corneal and ocular structures. This model is necessary to assess treatments and to clarify the disease mechanisms at molecular and cellular levels. As described before¹, with the use of a rotating burr, one can easily develop a mouse model of LSCD that features the aforementioned advantages and persists for at least three months. The goal of this study is to present a simple, reproducible, and sustainable mouse model of LSCD.

The rotating burr is a handy tool that evenly removes the epithelium without injuring the underlying stroma¹. It has been used to induce central corneal erosion^{7, 8} in wound healing studies. The hereby-presented technique to create LSCD in a mouse has not been reported before. Previously introduced methods of scraping the epithelium with a blunt spatula result in a less uniform injury-particularly at the limbus-and a more variable phenotype^{1, 9}, with more restoration of normal corneal epithelium¹. 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, n-heptanol, 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^11-15. 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 behind¹⁰.

The below-described technique will result in LSCD with neovascularization and conjunctivalization that mimics the presentation of complete LSCD in patients and lasts for at least three months¹. It is suitable for those who aim to study LSCD or wound healing pathophysiology, immunology, and potential treatments in mice. Possibly, with some modifications, this procedure can be performed in rats or rabbits¹⁰. As the rotating burr efficiently removes the epithelium, it can be utilized in any research pertaining to corneal epithelial abrasion/wounding and in any related treatment or molecular biology studies.

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 verify the absence of any previous corneal injury.
3. Instill a drop of 1% tetracaine hydrochloride onto the eye. After 60 - 90 sec, test the corneal reflex to confirm the absence of sensation. Proparacaine hydrochloride 0.5% can be used instead.
4. When the loss of corneal sensation is certain, gently place a drop of 5% povidone-iodine on the eye and on the hair around the eye. Wipe off the drop after 1 min or spread it on the hair around the eye to prevent the hair from interfering with the tool tip.

2. Abrading the Corneal Epithelium

1. Wear surgical gloves and a gown. Prepare the required tools: a sterile rotating burr and a pair of sterile tweezers. For better control, use bent tweezers. Place the tools on a sterile sheet during the procedure.
2. Using the bent tweezers, stabilize the eye by grabbing the lids from the nasal side and gently proptosing the eye. Avoid using too much pressure.
3. Under a surgical microscope, shave 360° around the limbal area twice using the sterile rotating burr. Go around the limbus with a speed of 5 - 6 sec per round.
   NOTE: The burr has one fixed speed. However, for it to rotate at the established speed, the "end nut" should be completely tightened.
4. Remove the whole corneal epithelium from limbus to limbus with the rotating burr. For better results, move the tool in a circular fashion and hold it somewhat parallel to the corneal surface to work with the lateral side of its tip. Take care not to injure the stroma by not re-brushing the areas where the epithelium has already been removed. Do not apply any pressure on the eye. Clean the brush tip as required.
5. Instill a drop of sterile phosphate-buffered saline on the cornea and wipe away any detached epithelial sheets with a soft cleaning tissue.
6. Shave the remaining epithelium, if any.
7. Pay attention to the mouse tail during the surgery.
   NOTE: If any movement occurs, the anesthesia depth has faded and further injection is required. One-third to two-thirds of the primary injected volume should be sufficient. Do not overdose the animal with the anesthetic medication.
8. Sterilize all the tools before using them on another eye.

3. Confirm Complete Epithelial Removal

1. Apply a drop of 1 mg/ml fluorescein sodium onto the cornea. Clean the cornea after 30 sec and examine it under a microscope with a cobalt blue filter to verify the complete removal of the epithelium.
   NOTE: Corneal areas with epithelial defects are visualized in green.

4. Post-operation Care

1. Put the animal on a heating blanket and keep it in a safe place (e.g., in its cage) while under recovery. Do not keep an unconscious animal in the company of conscious ones.
2. Apply 1% erythromycin ophthalmic ointment. This will also prevent eye dryness. Continue the application the antibiotic daily for a week.
3. Inject 0.1 mg/kg buprenorphine subcutaneously after the procedure and again after 12 hr. Continue the injection twice per day for two days.
4. Observe the animal for 30 - 45 min, until complete recovery from anesthesia.
   NOTE: Mild movement of the respiratory muscles on the ventral surface indicates animal wellness while under recovery.

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

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