Name: an epithelial abrasion model for studying corneal wound healing
Uploaded: 2021-12-29T14:00:00
Description: Watch this Scientific Journal Video about An Epithelial Abrasion Model for Studying Corneal Wound Healing at JoVE.com

Funding: Supported by: NIH EY018239 (A.R.B., C.W.S., and R.E.R.), P30EY007551 (A.R.B.), and Sigma Xi Grant in Aid of Research (P.K.A.). The content is solely the responsibility of the authors and does not represent the official views of the National Institutes of Health, or Sigma Xi.

All animal protocols were approved by the Institutional Animal Care and Use Committees at the University of Houston and Baylor College of Medicine. The guidelines outlined in the Association for Research in Vision and Ophthalmology (ARVO) statement on the use of animals in vision and ophthalmic research were followed in handling and using the mice.
1. Preparation
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	<li>Preparation of fluorescein solution
	<ol>
		<li>Prepare 1% fluorescein solution by dissolving 10 mg of ...

The purpose of this methods paper was to describe a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud. This murine model has been used to study corneal inflammation and its contribution to wound healing. This type of model can be used to study corneal wound healing mechanisms under normal physiological conditions and in pathologies17,28,29,<sup c...

The cornea is the transparent anterior one-third of the eye. The cornea serves several functions including protecting the inner structures of the eye and forming a structural barrier that protects the eye against infections1. More importantly, the cornea is critical for vision, providing about two-thirds of the refractive power of the eye2,3. Crucial to the role of the cornea in vision is its transparency. However, due to its outward position, the cornea is exposed to a wide variety of injuries on a day-to-day basis that can lead to disruption of its barrier function, loss of transparency, and eventual blindness. Loss of corneal transparency is a leading cause of visual impairment worldwide4,5. Corneal abrasions are a common reason for visits to the emergency room (ER), accounting for half of the eye-related cases presented at the ER6. Over 1 million individuals are estimated to suffer from eye-related injuries annually in the United States7. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of its transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring8,9. Also, the increasing popularity of refractive surgeries places a unique traumatic challenge on the cornea10. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease.
To that end, several animal models of corneal wound healing have been developed11,12,13,14,15. Murine models of corneal wound healing have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Different types of corneal wounds have been employed in studying corneal wound healing, each suitable for investigating different aspects of the wound healing process. The commonest types of wound models used in corneal wound healing studies are the mechanical and chemical wound models. Chemical corneal wounds, mostly involving the creation of alkaline burns on the cornea, are useful for studying corneal ulcers, opacification, and neovascularization13. Mechanical corneal wounds involve debridement (abrasion) wounds and keratectomy wounds14,15,16. An intact or breached corneal epithelial basement membrane defines debridement and keratectomy wounds, respectively. In debridement wounds, the epithelial basement membrane remains intact, while in keratectomy wounds, the basement membrane is breached with penetration mostly into the anterior stroma. Debridement wounds are most useful for studying re-epithelialization, epithelial cell proliferation, immune response, and nerve regeneration following corneal wounding. Keratectomy wounds, on the other hand, are most useful for studying corneal scarring14,15.
Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease17.

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	<tbody>
		<tr>
			<td>Anti-CD31 antibody</td>
			<td>BD Bioscience, Pharmingen</td>
			<td>550274</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD41 antibody</td>
			<td>BD Bioscience, Pharmingen</td>
			<td>553847</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Ly6G antibody</td>
			<td>BD Bioscience, Pharmingen</td>
			<td>551459</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>ThermoFisher scientific</td>
			<td>B14</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Jackson Laboratories</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma Aldrich</td>
			<td>D8417</td>
			<td></td>
		</tr>
		<tr>
			<td>DeltaVision wide-field deconvolution fluorescence microscope</td>
			<td>GE Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic Toploading Balances (Weighing scale)</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>ThermoFisher scientific</td>
			<td>T038181000CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Golf-club spud</td>
			<td>Stephens instruments</td>
			<td>S2-1135</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris curve scissors</td>
			<td>Fisher Scientific</td>
			<td>31212</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson veterinary</td>
			<td>07-893-1389</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Patterson veterinary</td>
			<td>07-890-8598</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospate buffered saline (PBS)</td>
			<td>ThermoFisher scientific</td>
			<td>AM9624</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium fluorescein salt</td>
			<td>Sigma Aldrich</td>
			<td>46970</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical blade (scapel blade)</td>
			<td>Fine Science tools</td>
			<td>10022-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Trephine</td>
			<td>Integra Miltex</td>
			<td>33-31</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX -100</td>
			<td>Fisher Scientific</td>
			<td>50-295-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>Fine Science tools</td>
			<td>11923-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Patterson veterinary</td>
			<td>07-808-1947</td>
			<td></td>
		</tr>
	</tbody>
</table>

an epithelial abrasion model for studying corneal wound healing

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Figure 3 shows a transmission electron micrograph of a corneal wound created with the blunt golf club spud, demonstrating that the epithelial&#160;basement membrane is indeed intact after injury.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63112/63112fig03.jpg" /> 
Figure 3: Epithelial basement membrane remains intact after corneal abrasion. Transmission electron mi...

Watch this Scientific Journal Video about An Epithelial Abrasion Model for Studying Corneal Wound Healing at JoVE.com

An Epithelial Abrasion Model for Studying Corneal Wound Healing

Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This corneal wound healing model is highly reproducible and is now being used to evaluate compromised corneal wound healing in the context of diseases.

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its ...

This protocol is significant because it is an excellent model for studying corneal inflammation and its contribution to wound healing under normal and pathological conditions. The main advantage of this technique is that it creates a precise and reproducible epithelial wound without breaching the basement membrane. This model of corneal wounding is straightforward and quick to perform.Demonstrating the procedure will be Prince Akowuah, a PhD student from my laboratory. To begin, prepare a 1%fluorescein solution by dissolving 10 milligrams of sodium fluorescein salt in one milliliter of sterile saline or PBS. Next, for anesthesia, prepare 10 milliliters of a ketamine-xylazine cocktail by mixing two milliliters of ketamine, one milliliter of xylazine, and seven milliliters of sterile PBS.After anesthetizing an 8-to 12-week-old C57 black 6 wild-type mouse, evaluate the depth of anesthesia by assessing the pedal reflex after toe pinch. To create the epithelial corneal wound, place the anesthetized mouse under a dissection microscope. Wound the right or left eye only and maintain consistency with the eye being wounded when moving from mouse to mouse.Keep the eye wide open by holding the eyelids with the thumb and index finger. Then, using a two-millimeter-diameter sterile trephine, demarcate the center of the cornea and gently twirl the trephine to make an impression on the corneal epithelium. Do not apply excessive pressure, as this can result in corneal perforation.Next, holding the sterile blunt golf club spud at an approximately 45-degree angle from the cornea's surface, debride the epithelium by careful and continuous scraping within the demarcated area using the spud. Do not apply excessive force and maintain hydration using sterile PBS if necessary. For monitoring wound closure and re-epithelization, pipette 1 to 1.5 microliters of 1%fluorescein solution onto the wounded surface and image the cornea using a digital microscope with a blue light source.Image the wounded cornea at specific times after wounding. Take images within the first minute of fluorescein solution addition to avoid spreading of the solution to the surrounding epithelium, which can lead to an overestimation of the wound size. Next, using an image analysis software, trace the wound area and express the wound area at each time point as a percentage of the original wound area at zero hour.A transmission electron micrograph of the corneal wound demonstrates that the epithelium basement membrane remains intact even after the injury. In 8-to 12-week-old wild-type mice, wound closure is complete 24 hours after wounding. Inflammatory response to wounding is characterized by cell imaging at the limbus.Shown here is the limbal vasculature of an unwounded cornea with extra-vascular neutrophils, and that of a wounded cornea with extravascular platelets and neutrophils 30 hours after abrasion. To successfully reproduce this protocol, the same strain in each range of mouse must be used, and the wound size and depth must be created as described.

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