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 anesthesia
NOTE: All animals were specific pathogen-free(SFP) male Mongolian gerbils and were kept in the Center for Research and Development in Immunology and Infectious Diseases (CIDIE) facilities (C&#243;rdoba, Argentina). They were obtained from Universidad de La Plata (Buenos Aires, Argentina).
<ol>
	<li>House the gerbils socially in polysulfone cages bedded with corncob bedding. Provide food and filtered tap water in water bottles ad libitum. Ensure the room temperature range is 18 &#176;C to 24 &#176;C and a 12:12 h light-dark cycle is used.</li>
	<li>Weigh each gerbil separately, and identify each one to avoid confusion. Make a mark with an indelible ink marker on the base of the gerbil&#39;s tail. Use tweezers to hold the gerbil&#39;s ear and make a mark using the indelible marker on the rodent&#39;s ear. If a laboratory and a large biotherium are available, assign a unique cage for each rodent with the corresponding identification.</li>
	<li>Disinfect the laminar flow hood with 70% ethanol solution. Place all the surgical and disposable instruments including needles, syringes, and racks in the work area inside the hood. Place a disposable surgical foam in the hood as well.</li>
	<li>Use a small open plastic container to keep the rodent on the precision balance to facilitate measurement.</li>
	<li>Use three 6-month-old male Mongolian gerbils of similar sizes and weights (~80 g) for this experiment. One at a time, place each cage with the rodent inside the laminar flow hood. Open the cages, identify each of the gerbils, and weigh them on the scale. 
	NOTE: Gerbils are harmless but delicate animals. Wear disposable gloves when handling the gerbils.</li>
	<li>Grasp the gerbil with the non-dominant hand to hold it firmly by the tail. Use the dominant hand, with the thumb and index finger behind the ears, to hold the animal with the ventral region facing upward. Use the little finger to hold the tail.</li>
	<li>Fill a syringe with a 30 G needle with 1 mL of ketamine and xylazine. Administer the anesthesia intraperitoneally into the rodent (50-100 mg/kg ketamine and 2 mg/kg xylazine)9 using the dominant hand. The duration of the effect will be approximately 20-50 min (variations may occur).</li>
	<li>To ensure the gerbil is fully anesthetized, check with a toe pinch, tail pinch, and corneal reflection, etc., prior to making incisions in the cornea. 
	&#8203;NOTE: Perform all the procedures in the right eye only.</li>
</ol>
2. Optical coherence tomography (OCT) of the cornea
<ol>
	<li>Place sterile surgical drapes to protect the equipment from secretions or animal hair.</li>
	<li>Ensure one of the operators holds the animal while another operator takes the images. The operator should rest his hands on the equipment while holding the gerbil so that the gerbil&#39;s eye is as stable and still as possible to be studied. Rest the hand holding the gerbil on the chin rest..</li>
	<li>Start the software that controls the OCT, and press Take Image and then Save Desired Image. Perform multiple sagittal and coronal slices of the cornea. Image the eye under the OCT, and make multiple slices to view the anterior segment of the rodent cornea. 
	NOTE: If the obtained image is not sharp and the eye has moved slightly, repeat the procedure several times to obtain enough images.</li>
	<li>Using the OCT software, perform pachymetric measurements of the central and peripheral regions. In the main screen of the software, press Take Image, and then press the Save Desired Image button.</li>
	<li>Perform measurements on the normal or control eye and immediately after phototherapeutic keratectomy on the other rodent eyes.</li>
</ol>
3. Excimer laser phototherapeutic keratectomy (PTK)
<ol>
	<li>Place sterile surgical drapes on the excimer laser device to protect the equipment from secretions or animal hair.</li>
	<li>Instill a drop of topical proparacaine hydrochloride (0.5%) in the eye to be treated 5 min before the surgical procedure.</li>
	<li>Use the non-dominant hand to hold the gerbil firmly. With the dominant hand, open the animal&#39;s eyelids so that the images can be captured properly. To be able to focus and obtain a sharp image, ensure that the hands of the person holding the gerbil rest on the head of the equipment. Place the hands holding the animal where a patient would place their neck.</li>
	<li>Perform PTK ablation on the right eye. Use the following parameters: an ablation between 60 &#181;m and 62 &#181;m thick, an optical zone of 3 mm, a duration of 4 s, and a total of 1,867 pulses. 
	NOTE: PTK is performed only on gerbil 2 and gerbil 3. In this step, the second operator prepares and activates the laser to ablate the corneal tissue.</li>
	<li>Immediately after the procedure, take photographs and perform OCT analysis to record and document surface changes in the treated eyes.</li>
	<li>Once the procedure is completed, place the rodent back in the cage, monitor the vital signs (heart rate: 360 beats per minute; rectal temperature: 37-38.5 &#176;C; respiratory rate: 90 breaths per minute), and let the animal recover from the anesthesia.</li>
</ol>
4. Awakening of the gerbils after corneal PTK
<ol>
	<li>Administer buprenorphine (0.1mg/kg to 0.05mg/kg) and atipamezole (0.1-1 mg/kg) via intraperitoneal injections.</li>
	<li>Place each gerbil in its respective home cage, and monitor the vital signs for normal awakening (the normal body temperature is 37-39 &#176;C).</li>
	<li>Apply an erythromycin ointment to keep the surface clean and prevent infection. Perform this procedure twice per day.</li>
	<li>Administer buprenorphine (every 6-12 h) subcutaneously (0.01-0.05 mL) for analgesia and eye ointment on two consecutive days after PTK.</li>
</ol>
5. Euthanasia method
<ol>
	<li>Perform euthanasia in the home cage whenever possible.</li>
	<li>Introduce compressed carbon dioxide (CO2) gas into the home cage. A filling rate of 30%-70% of the chamber volume per minute with CO2 added to the existing air in the home cage is adequate to achieve a mixture that meets the objective (for a 10 L volume chamber, use a flow rate of 3-7 L/min). Use cervical dislocation (as a secondary method of euthanasia) to ensure the death of the rodent.</li>
	<li>At 24 h and 96 h after surgery for gerbil 2 and gerbil 3, respectively, remove the animal from the home cage to perform the enucleation of the eyeball (both the normal eyeball and the one undergoing surgery) to observe corneal healing.</li>
	<li>Place the animal on the operating table, and check for the absence of a heartbeat for approximately 1 min.</li>
</ol>
6. Eye surgery
<ol>
	<li>Remove the upper and lower eyelids to gain access to the eyeball. Use surgical forceps and scissors to remove the eyelids. The size of the working area is so small and delicate that removing the eyelids allows the eyeball to be enucleated without damaging it.</li>
	<li>To enucleate the eyeball, make an incision in the external canthus, and guide the scissors in a posterior direction. Repeat this procedure from the internal canthus by separating the eyeball from the orbit.</li>
	<li>Section the optic nerve at the back of the eyeball. It should be clarified that the posterior orbital plexus usually generates slight bleeding when performing this technique, which makes the work difficult.</li>
	<li>Introduce the eyeball into a microcentrifuge tube with sterile saline solution for 30 s to 1 min to wash out any residual blood.</li>
	<li>Place the eyeball in a microcentrifuge tube containing 10% formaldehyde for subsequent anatomo-pathological analysis as described below. Take multiple images and photographs.</li>
</ol>
7. Anatomo-pathological analysis
<ol>
	<li>Embed the whole eye in 10% buffered formalin for 6-24 h.</li>
	<li>Cut the tissue using a microtome. Ensure that the cut tissue has a thickness of 3 mm.</li>
	<li>Soak the tissue in 96% alcohol for 30-90 min, and repeat this procedure twice.</li>
	<li>Place the tissue in isopropyl alcohol for 30-90 min, and repeat this procedure twice.</li>
	<li>Put the tissue in xylene or xylene substitute for 1-3 h.</li>
	<li>Embed the tissue in liquid kerosene for 1 h minimum.</li>
	<li>Use a block to place the tissue and embed it in liquid paraffin. Allow it to solidify (place it in a cold place), and cut it.</li>
	<li>Prepare the microtome for sectioning according to the manufacturer&#39;s instructions.</li>
	<li>Subsequently, use stains such as hematoxylin and eosin.</li>
	<li>Obtain pictures with the camera added to the microscope.</li>
</ol>

<ol>
	<li>Kuo, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+wound+healing.">Corneal wound healing.</a> Current Opinion in Ophthalmology. 15 (4), 311-315 (2004).</li><li>Agrawal, V. B., Tsai, R. J. <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+wound+healing.">Corneal epithelial wound healing.</a> Indian Journal of Ophthalmology. 51 (1), 5-15 (2003).</li><li>Lu, L., Reinach, P. S., Kao, W. W. <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+wound+healing.">Corneal epithelial wound healing.</a> Experimental Biology and Medicine. 226 (7), 653-664 (2001).</li><li>Rathi, V. M., Vyas, S. P., Sangwan, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototherapeutic+keratectomy.">Phototherapeutic keratectomy.</a> Indian Journal of Ophthalmology. 60 (1), 5-14 (2012).</li><li>Baumeister, M., B&#252;hren, J., Ohrloff, C., Kohnen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+re-epithelialization+following+phototherapeutic+keratectomy+for+recurrent+corneal+erosion+as+in+vivo+model+of+epithelial+wound+healing.">Corneal re-epithelialization following phototherapeutic keratectomy for recurrent corneal erosion as in vivo model of epithelial wound healing.</a> Ophthalmologica. 223 (6), 414-418 (2009).</li><li>Fagerholm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototherapeutic+keratectomy:+12+years+of+experience.">Phototherapeutic keratectomy: 12 years of experience.</a> Acta Ophthalmologica Scandinavica. 81 (1), 19-32 (2003).</li><li>Mohan, R. 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. (137), e58071 (2018).</li><li>Yang, S., 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=The+electroretinogram+of+Mongolian+gerbil+(Meriones+unguiculatus.):+Comparison+to+mouse.">The electroretinogram of Mongolian gerbil (Meriones unguiculatus.): Comparison to mouse.</a> Neuroscience Letters. 589, 7-12 (2015).</li><li>Baker, A. G., Emerson, V. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grating+acuity+of+the+Mongolian+gerbil+(Meriones+unguiculatus).">Grating acuity of the Mongolian gerbil (Meriones unguiculatus).</a> Behavioural Brain Research. 8 (2), 195-209 (1983).</li><li>Govardovskii, V. I., R&#246;hlich, P., Sz&#233;l, A., Khokhlova, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cones+in+the+retina+of+the+Mongolian+gerbil,+Meriones+unguiculatus:+An+immunocytochemical+and+electrophysiological+study.">Cones in the retina of the Mongolian gerbil, Meriones unguiculatus: An immunocytochemical and electrophysiological study.</a> Vision Research. 32 (1), 19-27 (1992).</li><li>Zanandr&#233;a, L. I., Oliveira, G. M., Abreu, A. S., Pereira, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+lesions+in+gerbils+(Meriones+unguiculatus)+infected+with+low+larval+burden+of+Toxocara+canis:+Observations+using+indirect+binocular+ophthalmoscopy.">Ocular lesions in gerbils (Meriones unguiculatus) infected with low larval burden of Toxocara canis: Observations using indirect binocular ophthalmoscopy.</a> Revista da Sociedade Brasileira de Medicina Tropical. 41 (6), 570-574 (2008).</li><li>Cheng, S., 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=Enhancement+of+de+novo+sequencing,+assembly+and+annotation+of+the+Mongolian+gerbil+genome+with+transcriptome+sequencing+and+assembly+from+several+different+tissues.">Enhancement of de novo sequencing, assembly and annotation of the Mongolian gerbil genome with transcriptome sequencing and assembly from several different tissues.</a> BMC Genomics. 20 (1), 903 (2019).</li><li>Henriksson, J. 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The authors have nothing to disclose.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 ...

mongolian-gerbils-as-an-animal-model-of-wound-healing

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2.0K Views.  Catholic University of Córdoba. 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.

Video: Mongolian Gerbils as an Animal Model of Wound Healing

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

Targeting Gray Rami Communicantes in Selective Chemical Lumbar Sympathectomy

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the lower extremities, hyperhidrosis, etc. It is commonly practiced to position the puncture needle tip in front of the anterior fascia of the psoas major muscle and inject the inactivating agent around the sympathetic trunk, which is defined as conventional CLS. Although relatively rare, ureteropelvic damage is the most frequently reported complication of conventional CLS and can cause serious harm to patients. We found that injecting the inactivating agent behind the anterior fascia, which only targets gray rami communicantes, helped achieve therapeutic efficacy in&#160;vasodilation, sweat reduction, and pain relief comparable to conventional CLS, and serious complications were largely reduced. We define this procedure as selective CLS. Here, we present a protocol of selective CLS. The precise needle tract and accurate evaluation of the spreading of the contrast agent are critical to ensure that the drug is injected behind the anterior fascia of the psoas major muscle. The needle tip is at approximately one-third the dividing line of the vertebral body in the lateral view of a lumbar X-ray. The contrast is mainly confined around the needle tip and spreads outward and downward along the psoas muscle fibers. In this way, the anterior fascia provides a natural barrier for the ureteropelvic area, and the psoas major muscle provides a natural barrier for the lumbar nerve root. There are several highlights of this article, including 1) a detailed description of the selective CLS procedures, 2) an explanation of the anatomical basis for the implementation of selective CLS, and 3) an explanation of the differences between selective and conventional CLS.

We present a protocol for selective chemical lumbar sympathectomy (CLS), which inactivates only gray rami communicantes and not the sympathetic trunk. Selective CLS can help achieve therapeutic efficacy in vasodilation, sweat reduction, and pain relief that are comparable to conventional CLS, and serious complications, particularly ureteropelvic damages, can be reduced.

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the ...