Name: mongolian gerbils as an animal model of wound healing
Uploaded: 2023-01-06T12:30:00
Description: Watch this Scientific Journal Video about Mongolian Gerbils as an Animal Model of Wound Healing at JoVE.com

We would like to thank engineer Rodrigo de la Fuente for his invaluable help and technical support. We also thank Mar&#237;a Eugenia Corbela for the narration and Priscilla Hazr&#250;n for the edition of the figures. Hugo Luj&#225;n allowed us to use the facilities at the Center for Research and Development in Immunology and Infectious Diseases (CIDIE).

All research procedures were approved by the &#34;Institutional Commission for the Care and Use of Laboratory Animals&#34; of Universidad Cat&#243;lica de C&#243;rdoba and followed the National Research Council Guide for the care and use of laboratory animals. These procedures were also approved by the authorities of the &#34;Facultad de Ciencias de la Salud&#34; of Universidad Cat&#243;lica de C&#243;rdoba and &#34;Instituto de la Visi&#243;n Cerro&#34;.
1. Gerbil handling and anesthesi...

<ol>
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R., Stapleton, W. M., Sinha, S., Netto, M. V., Wilson, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+generating+corneal+haze+in+anterior+stroma+of+the+mouse+eye+with+the+excimer+laser.">A novel method for generating corneal haze in anterior stroma of the mouse eye with the excimer laser.</a> Experimental Eye Research. 86 (2), 235-240 (2008).</li><li>Shah, D., Aakalu, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+corneal+epithelial+wound+modeling.">Murine corneal epithelial wound modeling.</a> Methods in Molecular Biology. 2193, 175-181 (2021).</li><li>Zorio, D. A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+sequencing+and+initial+annotation+of+the+Mongolian+gerbil+(Meriones+unguiculatus)+genome.">De novo sequencing and initial annotation of the Mongolian gerbil (Meriones unguiculatus) genome.</a> Genomics. 111 (3), 441-449 (2019).</li><li>Kalha, S., Kuony, A., Michon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+epithelial+abrasion+with+ocular+burr+as+a+model+for+cornea+wound.">Corneal epithelial abrasion with ocular burr as a model for cornea wound.</a> Journal of Visualized Experiments. 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The physiology of corneal wound healing is a balance between tissue regeneration and the maintenance of homeostasis. Excessive wound healing may lead to fibrosis and scarring, which ultimately may result in the loss of organ function. With the rapid evolution of corneal surgical procedures, the importance of understanding corneal wound healing and the physiological and pathological events involved cannot be overestimated11. 
Multiple research works claim that gerbils ha...

Some of the most important aspects of corneal wound healing, which are key concerns for anterior segment surgery, are the integrity of the epithelial architecture, the maintenance of the corneal stroma transparency, and, finally, the outcome in terms of the refractive properties of the cornea1.
The cornea is the outermost clear tissue at the front of the eyeball and is, therefore, susceptible to trauma, infections, and burns; the impaired healing of these wounds can compromise visual health2.
At present, several animal models are available to study corneal healing, and some of them are better than others, depending on the species and the type of mechanism to be studied1.&#160;There are a few records of previous investigations on the retina of gerbils2. However, so far, there is no published literature on the scarring processes in the cornea of these rodents.
Here, we present Meriones unguiculatus (Mongolian gerbil) as an animal model of wound healing in the cornea. Procedures to elicit corneal healing after photorefractive keratectomy are described, which allow us to study the different types of corneal scarring processes, understand wound healing in terms of the dynamic phases of living tissue, and, finally, plan appropriate future treatments3. Phototherapeutic keratectomy is a highly reproducible technique with the possibility of precisely controlling parameters such as the depth and diameter of the corneal injury4. Moreover, this technique does not require procedures with surgical instruments or chemical solutions (e.g., saline solution, formalin, alcohol, etc.) that may add variables that are specific to the instruments or to the operator performing the procedure5.
Three 6-month-old male gerbils of similar sizes and weights (approximately 90 g) were used for the experiment presented in this article. The procedures were only performed in the right eyes.One gerbil (referred to as gerbil 1 or control) did not undergo phototherapeutic keratectomy and was enucleated to evaluate all the normal ocular structures. Phototherapeutic keratectomy involves the controlled delivery of excimer laser-generated ultraviolet light to the cornea and was developed in order to perform refractive surgery6. It has been used in other rodents, such as mice7. The other two gerbils were subjected to phototherapeutic keratectomy. One of them was enucleated at 24 h (referred to as gerbil 2) and the other one at 96 h after surgery (referred to as gerbil 3).
To perform this experiment, a gerbil selected at random was filmed for each condition to be studied, but this experiment was previously performed with 16 gerbils in total for each condition. For editing reasons, it was decided to use a randomly selected gerbil for each condition (three gerbils in total) as an example.
The main objective of this research is to explore the best animal model available. However, it is important to note that not all species have eye characteristics similar to those of the human eye8. This article describes the methodology used to study the cornea of Meriones unguiculatus and the procedure performed to generate the corneal injury, which allow us to study the healing process.

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	<tbody>
		<tr>
			<td>Anesthesia</td>
			<td>Tododrogas</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td>Tododrogas</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Excimer Laser</td>
			<td>Technolas</td>
			<td>2022445</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein</td>
			<td>Poen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Ofcor</td>
			<td>3339</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Tododrogas</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td>Tododrogas</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optical coherence tomography</td>
			<td>Optovue</td>
			<td>659007</td>
			<td></td>
		</tr>
		<tr>
			<td>Proparacaine</td>
			<td>Poen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scisors</td>
			<td>Ofcor</td>
			<td>3336</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile drapes</td>
			<td>Soporte hospitalario</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gauzes</td>
			<td>Soporte hospitalario</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes and needles</td>
			<td>Tododrogas</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

mongolian gerbils as an animal model of wound healing

Corneal wound healing studies have been conducted for a long time and have helped to reduce suffering and develop treatments that contribute to improving patients' eye health. Historically, corneal healing has been studied in rodents such as mice and rats, but these models might not completely mimic human disorders. However, information on other rodents such as Mongolian gerbils (Meriones unguiculatus) is scant in corneal research.
Here, we describe a technique to develop a novel animal model for studying corneal healing after photorefractive keratectomy. Due to the limited literature available on the cornea of M. unguiculatus, we also describe a histological analysis of the normal cornea. These research techniques can also be employed in the study of eye diseases because of the similarity between the corneas of Mongolian gerbils and humans in terms of genetics, anatomy, and physiology.

In the present study, the entire corneal structure was thoroughly analyzed using histological techniques and complementary studies of the anterior segment, such as optical coherence tomography. The image analysis using optical coherence tomography of the anterior segment structures shows a normal epithelium and stroma (Figure 1), with central and peripheral corneal thicknesses of 160 &#181;m and 106 &#181;m &#177; 2 &#181;m, respectively. Other publications have also shown that the cornea of...

Watch this Scientific Journal Video about Mongolian Gerbils as an Animal Model of Wound Healing at JoVE.com

Mongolian Gerbils as an Animal Model of Wound Healing

This article describes a new animal model developed to study the anatomy and histology of the cornea and its healing processes. This new animal model uses the Mongolian gerbil, which has a cornea with many similarities to the human cornea.

Corneal wound healing studies have been conducted for a long time and have helped to reduce suffering and develop treatments that contribute to improving ...

Previous studies show that the photorefractive keratectomy or transepithelial PRK, is one of the best animal model for the study of corneal healing, and also, the changes associated to histopathological debridement of the epithelial cornea. Working with Mongolian gerbils as animal model has several advantages over other rodents. First, the cornea has the same histologic layers as the human cornea.Second, the eye is anatomically larger than the eye of other rodents, allowing for easier manipulation. All this research was carried out in the facilities of the Center for Research and Development in Immunology and Infectious Diseases of the Universidad Catolica De Cordoba. The first step was to disinfect the working surface of the laminar flow cabinet.For this purpose, 70%alcohol was used. Surgical instruments to be used. Disposable materials such as needles and syringes, and drugs to anesthetize the rodent, were placed in the work area.A disposable surgical field should also be placed to contain secretions and rodent hair if necessary. Three six-month old male Mongolian gerbils of similar size and weight, approximately 90 grams were used in the experiment. The procedures to be performed later were performed on the right eyes only.The cage with the rodent is taken and placed inside the laminar flow cabinet. We proceed to open the box, identify each of the gerbils, and weigh them on the scale. They're harmless but delicate animals.Disposable gloves should be worn at all times. For it correct handling, the operator should use his unskilled hand to hold it firmly by the tail, and with the skilled hand, gently press down with a thumb and index finger behind the ears to hold the animal and with the little finger, hold the tail. Based on the weight of the animal, the amount of anesthesia needed for each of the gerbils is calculated.A one milliliter syringe should be filled with ketamine, 50 over 100 milligram per kilogram, and xylazine, two milligram per kilogram. Prior to performing the optical coherence tomography, sterile surgical drapes were placed to protect the equipment from secretions or animal hair. While one of the operators hold the animal, another operator must take the captures.The eye was photographed microscopically and multiple slices were made of the interior segment of the rodent. The entire cornea, the anterior chamber, the iris, the iridocorneal angle and lens, which protrudes into the anterior chamber can be observed. In addition, central and peripheral corneal thickness measurements were taken showing a central thickness of 160 micrometers decreasing to approximately 110 micrometers in the periphery.Prior to perform excimer laser PTK, sterile surgical drapes were placed to protect the equipment from secretions or animal hair. Between 60 and 62 micrometers of corneal thickness were ablated at the diameter of three millimeters. The cornea was photographed to evaluate immediate morphologic changes.In this photograph, it can be seen, the eye eyeball of the gerbil right eye. A, irregular surface of the normal eye eyeball is observed. B, photograph taken five minutes after PTK was performed showing that the corneal surface presents irregularities.See evidence of the corneal ulcer stained with fluorescein. On the OCT, the right arrow mark the area where detritus has accumulated in the center and the left arrow mark the edge of the corneal ulcer. The method of sacrifice used in this experiment was through the use of a carbon dioxide chamber.They were sacrificed at different time intervals and enucleation of the normal eyeball and post-surgical eyeballs were performed at 24 and 96 hours post-surgery to observe corneal healing. Each enucleated eyeball was placed in formaldehyde and processed by a pathologist. Multiple sections, measurements and photographs were taken.Histologically, the corneal thickness consists with that obtained by OCT, and there is a progressive thinning towards the periphery. This is because the layers of epithelium and stromal fibers decrease in the periphery. The Mongolian gerbil cornea has the same layers as the human, stratified anterior epithelium with four to six layers of cells representing 28%of the total thickness of the cornea.Bowman's layer, stroma representing 66%of the total thickness. This cements membrane and endothelium. At 24 hours postsurgery, the corneal ulcer can be seen where the arrow is marked.Also observed multiple patches of epithelial achantholysis and isolated dyskeratocis, acute subepithelial inflammatory infiltrate, edema at the stromal level. The changes observed in the operated gerbil at 96 hours after PTK are, presence of increased edema, disaggregation of stromal fibers and cells, and it should be noted that the interior epithelium was already completely regenerated and did not present inflammatory infiltrate. The present study show the positive result of the current animal model in gerbils and confirming it as a new research option in corneal wound healing.To date, there are no publications that analyze, in detail, the anatomy and histology of the Mongolian gerbil cornea. Therefore, we hope that this animal model will be the basis for future scientific investigations.

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