Name: a simple mechanical procedure to create limbal stem cell deficiency in mouse
Uploaded: 2016-11-17T13:00:00
Description: Watch this Scientific Journal Video about A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse at JoVE.com

The authors thank Ruth Zelkha, MS, for her generous assistance in imaging. This research was supported by Clinical Scientist Development Program Award K12EY021475 to M.E., grant R01 EY024349-01A1 to A.R.D., core grant EY01792 from the National Eye Institute, NIH, and an unrestricted grant from Research to Prevent Blindness. A.R.D. is the recipient of a Career Development Award from Research to Prevent Blindness.

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
<ol>
	<li>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 th...

<ol>
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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 chemicals11, 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 ...

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 inflammation1-6.
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 erosion7, 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 phenotype1, 9, with more restoration of normal corneal epithelium1. Unlike the blunt scraper, the rotating burr removes the epithelial basement membrane as well7, 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 metaplasia11-15. The rotating burr is not associated with these severe complications. Surgically removing the limbal epithelium, alone or with the use of chemicals10, 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.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Rotating burr: AlgerBrush II rust ring remover&#160;</td>
			<td style="width:277px;">Rumex International Co, Clearwater, FL</td>
			<td style="width:144px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 16-141</td>
			<td style="width:351px;">0.5 mm burr. Also available from other companies.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Surgical microscope</td>
			<td style="width:277px;">Wild Heerbrugg, Switzerland</td>
			<td style="width:144px;">&#160;&#160;&#160;&#160;&#160;&#160; Wild M691</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Digital camera</td>
			<td style="width:277px;">Nikon, Thailand</td>
			<td style="width:144px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Nikon FS-2 slit lamp</td>
			<td style="width:277px;">Nikon, Japan</td>
			<td style="width:144px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Polyclonal anti-CK12 antibody</td>
			<td style="width:277px;">Santa Cruz Blotechnology, CA,</td>
			<td style="width:144px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; SC-17101</td>
			<td>1:100 concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#160;Monoclonal anti-CK8 antibody</td>
			<td style="width:277px;">TROMA-I-s, Iowa City, IA</td>
			<td style="width:144px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; AB_531826</td>
			<td>1:50 concentration</td>
		</tr>
	</tbody>
</table>

a simple mechanical procedure to create limbal stem cell deficiency in mouse

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.

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.
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Watch this Scientific Journal Video about A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse at JoVE.com

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

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.

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 ...

The overall goal of this procedure is to create a reproducible, sustainable, and persistent mouse model of limbal stem cell deficiency. This simple technique provides a consistent animal model of limbal stem cell deficiency, without inducing unwanted injury to the eye, which is difficult to achieve with the standard chemical injury models. This model is useful for studying the disease process in long-term at cellular and molecular level.And for testing or comparing the efficacy of novel treatments. After anesthetizing the mouse, pinch the toe to ensure adequate depth of anesthesia. Inspect the eye under a slit lamp to verify the absence of any previous corneal injury.Instill a drop of 1%tetracaine hydrochloride onto the eye, and wait for 60 seconds. Next, gently apply 5%povidone iodine on the eye and the hair around it. After one minute, wipe off the drop or spread it onto the hair around the eye to prevent the hair from interfering with the tip of the tool.Prepare a sterile rotating burr and a pair of sterile bent tweezers, and keep them on a sterile sheet during the procedure. Now, stabilize the eye by grabbing the lids from the nasal side using the bent tweezers, and gently proctozing the eye. Be careful not to apply too much pressure.Under a surgical microscope, shave around the limbal area twice using the sterile rotating burr. Remove the whole corneal epithelium from limbus to limbus with the rotating burr in a circular fashion, and work with the lateral side of its tip. To avoid stroma injury, do not re-brush corneal parts from which the epithelium has been removed.Do not apply any pressure on the eye. Clean the brush tip as required. Apply a drop of sterile PBS on the cornea, and wipe away any detached epithelial sheets with a soft cleaning tissue.Then, shave the remaining epithelium, if any. Pay attention to the mouse tail to ensure adequate depth of anesthesia during the surgery. Apply a drop of Flourescein sodium onto the cornea.Clean the cornea after 30 seconds, and examine it under a microscope with a cobalt blue filter to verify the complete removal of the epithelium. After the procedure, place the animal in its cage on a heating blanket and observe it for 30-45 minutes until complete recovery from anesthesia. In the meantime, apply 1%Erythromycin Ophthalmic ointment to the eye and continue the application of the antibiotic daily for a week.In addition, inject a 0.1mg/kg of Buprenorphine subcutaneously immediately, and 12 hours after the procedure, and continue the injection twice a day for two days. This is the cornea phenotype one month after the procedure. You can see extensive neovascularization which is a feature of limbal stem cell deficiency.A normal cornea expresses Cytokeratin 12 but is devoid of Cytokeratin 8 or goblet cells. In limbal stem cell deficiency, Cytokeratin 8 and goblet cells appear on the cornea, while Cytokeratin 12 is lost. Once mastered, this technique will result in corneal neovascularization that will cover almost the entire mouse corneal surface in one month, and is stable up to three months.It is important to avoid re-brushing the corneal parts that the epithelium has already been removed. This can cause undesired stroma inflammation and opacification. Following development of limbal stem cell deficiency, experimental treatment methods can be tested and compared.Alternatively, treatments can be started shortly after the injury. This video displayed how to effectively remove the limbal and corneal epithelium to induce limbal stem cell deficiency in mouse. With some modifications, it can be performed on other animals as well.

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