Name: mongolian gerbils as an animal model of wound healing
Uploaded: 2023-01-06T12:30:00.000+00:00
Duration: 7 min 54 s
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 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>

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	<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><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&amp;nbsp;</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&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</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

Research

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Medicine

2.1K 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. ...

Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, proliferation and remodeling. Murine wounds have a histologically well-defined and easily recognizable wound bed over which these different phases of the healing process are measurable. Within the field, it is common to use an arbitrarily defined &#8220;middle&#8221; of the wound for histological analyses. However, wounds are a three-dimensional entity and often not histologically symmetrical, supporting the need for a well-defined and robust method of quantification to detect morphometric defects with a small effect size. In this protocol, we describe the procedure for creating bilateral, full-thickness excisional wounds in mice as well as a detailed instruction on how to measure morphometric parameters using an image processing program on select serial sections. The two-dimension measurements of wound length, epidermal length, epidermal area, and wound area are used in combination with the known distance between sections to extrapolate the three-dimension epidermal area covering the wound, overall wound area, epidermal volume and wound volume. Although this detailed histological analysis is more time and resource consuming than conventional analyses, its rigor increases the likelihood of detecting novel phenotypes in an inherently complex wound healing process.

This protocol describes how to generate bilateral, full-thickness excisional wounds in mice and how to subsequently monitor, harvest, and prepare the wounds for morphometric analysis. Included is an in-depth description of how to use serial histological sections to define, precisely quantify and detect morphometric defects.

The murine excisional wound model has been used extensively to study each of the sequentially overlapping phases of wound healing: inflammation, ...

Developmental Biology

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

Immunology and Infection

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine models provide valuable mechanistic insight into these processes; however, no single model fully addresses all aspects of the wound healing response. Instead, it is ideal to use multiple models to address the different aspects of wound healing. Here, two different methods that address diverse aspects of the wound healing response are described. In the first model, polyvinyl alcohol sponges are subcutaneously implanted along the mouse dorsum. Following sponge retrieval, cells can be isolated by mechanical disruption, and fluids can be extracted by centrifugation, thus allowing for a detailed characterization of cellular and cytokine responses in the acute wound environment. A limitation of this model is the inability to assess the rate of wound closure. For this, a tail skin excision model is utilized. In this model, a 10 mm x 3 mm rectangular piece of tail skin is excised along the dorsal surface, near the base of the tail. This model can be easily photographed for planimetric analysis to determine healing rates and can be excised for histological analysis. Both described methods can be utilized in genetically altered mouse strains, or in conjunction with models of comorbid conditions, such as diabetes, aging, or secondary infection, in order to elucidate wound healing mechanisms.

Here, two murine wound healing models are described, one designed to assess cellular and cytokine wound healing responses and the other to quantify the rate of wound closure. These methods can be used with complex disease models such as diabetes to determine mechanisms of various aspects of poor wound healing.

Wound healing is a complex process that requires the orderly progression of inflammation, granulation tissue formation, fibrosis, and resolution. Murine ...

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

Digital Planimetry for Assessing Wound Closure Kinetics in a Mouse Model

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, thereby reducing the risk of complications and alleviating the economic burden on healthcare systems. Although numerous experimental studies have investigated wound healing, most rely on qualitative observations or quantitative direct measurements. The objective of this study was to standardize an indirect wound measurement method using digital planimetry, incorporating digital scaling and segmentation. This approach addresses the lack of detailed, step-by-step methodologies for accurate wound assessment. A photodocumentation booth was designed and constructed, and computer-assisted digital planimetry tools were employed to minimize variability in measurements of the wound area, perimeter, and the distance from the wound center to its edges. A circular traumatic wound (5 mm in diameter) was created on the dorsal midline at the shoulder blade level of male CD1 mice (n = 4, 10 weeks old, 30-35 g). Wound evolution was photodocumented for 14 days using the custom-designed photo booth, which controlled lighting conditions, focal distance, and subject positioning. Scaling and wound measurements were performed using segmentation in ImageJ software, and statistical analysis was conducted using statistical analysis software. The kinetics of wound closure showed a slight increase in wound size and perimeter between day 0 and day 2, followed by a gradual decrease until complete closure by day 14. The photodocumentation booth and computer-assisted digital planimetry enabled quantitative measurements with minimal variability. In conclusion, these tools provide a reliable and reproducible method for evaluating wound closure kinetics in pre-clinical models.

Wounds represent a global health challenge. This study developed a standardized photo booth utilizing digital planimetry to minimize wound measurement variability. Monitoring wounds in mice over 14 days revealed an initial increase in wound area and perimeter, followed by gradual closure. This methodology may aid in evaluating wound closure kinetics in pre-clinical models.

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, ...

Bioengineering

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.

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 ...

A Protocol for Constructing a Rat Wound Model of Type 1 Diabetes

A single high dose of streptozotocin injection followed by full-thickness skin excision on the dorsum of rats is a common method for constructing animal models of type 1 diabetic wounds. However, improper manipulation can lead to model instability and high mortality in rats. Unfortunately, there are few existing guidelines on type 1 diabetic wound modeling, and they lack detail and do not present specific reference strategies. Therefore, this protocol details the complete procedure for constructing a type 1 diabetic wound model and analyzes the progression and angiogenic characteristics of the diabetic wounds. Type 1 diabetic wound modeling involves the following steps: preparation of the streptozotocin injection, induction of type 1 diabetes mellitus, and construction of the wound model. The wound area was measured on day 7 and day 14 after wounding, and the skin tissues of the rats were extracted for histopathological and immunofluorescence analysis. The results revealed that type 1 diabetes mellitus induced by 55 mg/kg streptozotocin was associated with lower mortality and a high success rate. The blood glucose levels were relatively stable after 5 weeks of induction. The diabetic wound healing rate was significantly lower than that of normal wounds on day 7 and day 14 (p &#60; 0.05), but both could reach more than 90% on day 14. Compared with the normal group, the epidermal layer closure of diabetic wounds on day 14 was incomplete and had delayed re-epithelialization and significantly lower angiogenesis (p &#60; 0.01). The type 1 diabetic wound model constructed based on this protocol has the characteristics of chronic wound healing, including poor closure, delayed re-epithelialization, and decreased angiogenesis compared to normal rat wounds.

The streptozotocin-induced diabetic wound model in male SD rats is currently the most widely used model for studying wound healing in type I diabetes mellitus. This protocol describes the methods used to construct this model. It also presents and addresses potential challenges and examines the progression and angiogenic characteristics of diabetic wounds.

A single high dose of streptozotocin injection followed by full-thickness skin excision on the dorsum of rats is a common method for constructing animal ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

Mongolian Gerbils as an Animal Model of Wound Healing

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A Simplified Technique for Producing an Ischemic Wound Model

Murine Model of Wound Healing

Murine Excisional Wound Healing Model and Histological Morphometric Wound Analysis

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

Assessment of Acute Wound Healing using the Dorsal Subcutaneous Polyvinyl Alcohol Sponge Implantation and Excisional Tail Skin Wound Models.

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Digital Planimetry for Assessing Wound Closure Kinetics in a Mouse Model

An Epithelial Abrasion Model for Studying Corneal Wound Healing

A Protocol for Constructing a Rat Wound Model of Type 1 Diabetes

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft