This work was supported by generous funding from Shriners Children&#39;s Research Grant to S.N.T. Y.B. was supported by Shriners Hospital for Children. We also gratefully acknowledge funding to S.N.T. from the US National Institute of Health (K99/R00 HL1431149; R01HL157803; R01DK134590, R24OD034189), American Heart Association (18CDA34110049), Harvard Medical School Eleanor and Miles Shore Fellowship, Polsky Family Foundation, and the Claflin Distinguished Scholar Award on behalf of the MGH Department of Surgery and/or MGH Executive Committee on Research. Further, we acknowledge the support provided by the Massachusetts General Hospital Executive Committee of Research for awarding the Fund for Medical Discovery (FMD) award to R.J. Finally, support from &#34;Fondation des Gueules Cass&#233;es&#34; (France), Rennes University (France), CHU de Rennes (France) and the French Society of Plastic Surgery to Y.B. is greatly acknowledged. The authors thank Knight Surgery Research Laboratory for their contribution and help with the anesthesia of the animals.

All animal work was performed in accordance with the ARRIVE (Animal Research: Reporting In Vivo Experiments) checklist37 and was compliant with the Massachusetts General Hospital Institutional Animal Care and Use Committee (IACUC) under protocol #2021N000271. Humane care was provided to the animals, following the Guide for the Care and Use of Laboratory Animals38. Five 30 kg female Yucat&#225;n minipigs were used for these experiments. The animals were obtained from a commercial source (see Table of Materials).
1. Pre-operative care and anesthesia
<ol>
	<li>Fast the animals for 12 h before&#160;general anesthesia.</li>
	<li>Initiate anesthesia by an intramuscular injection of 2-4 mg/kg of tiletamine hydrochloride and zolazepam hydrochloride and 1-2 mg/kg of xylazine, followed by inhalation of isoflurane (2-3% in oxygen) (see Table of Materials) for maintaining anesthesia during the procedure.</li>
</ol>
2. Burn wound design and randomization
<ol>
	<li>In order to study both partial and full-thickness burns with negative controls on the same animal (no inter-individual variation), create eight wounds on each pig&#39;s dorsum in the paravertebral area. Perform three full-thickness wounds, three partial-thickness wounds, and two control wounds.</li>
	<li>Number the wounds from 1 to 8, and perform randomization to allocate different burn degrees (partial thickness, full thickness, control) to different anatomical locations on the back. 
	NOTE: Randomization of each wound (depth and location) is performed to improve the significance: Pig dermis is known to vary in terms of thickness and collagen composition39. Therefore, randomization on each animal is used to decrease potential biases.</li>
</ol>
3. Tattoo wound delimitation
NOTE: The first procedure consists of creating circular tattoos on the pig&#39;s dorsum in order to localize and number the randomized wounds (Figure 1). This is performed two days before the initial burn procedure to allow for better acclimatization, but it can be performed on the day of the burn procedure.
<ol>
	<li>After anesthesia (see Step 1)&#160;and orotracheal intubation in the supine position40, place the pig in the prone position, and place soft wedges (bed sheets) under the animal for its comfort and stabilization (under the forelimbs, the umbilical area, and the hindlimbs)41. Place a forced-air warming blanket on the head and neck of the animal to maintain adequate body temperature.</li>
	<li>Place a straight line centrally on the pig&#39;s back to allow the symmetrization of the drawings.</li>
	<li>Draw two vertical lines using a dermographic pen (see Table of Materials) on both sides of the central line, placed 6.5 cm laterally from it. The two lateral lines will be used to place the center of the circles.</li>
	<li>Draw each pair of 4.5 cm diameter circles symmetrically, keeping a minimum distance of 4 cm between two distinct circles (Figure 1A).</li>
	<li>Once the drawings are done, perform a single-step skin preparation (povidone-iodine 7.5%) to decrease the skin bacterial flora.</li>
	<li>Perform the tattoo procedure42 of eight 4.5 cm circles using a needle-tattoo machine, sterile black ink, and a sterilized 5-point needle (see Table of Materials). The final result is presented in Figure 1B.</li>
	<li>In case of a delayed burn creation procedure, apply triple-antibiotic ointment on the tattoos and cover it with transparent adhesive dressings.</li>
</ol>
4. Burn wound creation and advanced wound dressing
NOTE: Burns will be created by placing the brass block in contact with the skin on the dedicated spot (randomization) for 30 s (invariable). The temperature will determine the burn depth.
<ol>
	<li>On the day of the burn procedure, once the animal is anesthetized and placed in the prone position as discussed in Step 1, place cylindric brass blocks in stainless steel containers filled with aluminum beads (see Table of Materials). Place the containers on a temperature-controlled hot plate. Set one hot plate to 65 &#176;C (for the partial thickness burns), and the second to 93 &#176;C (for the full-thickness burn, Figure 2). 
	NOTE: The container can be kept dry to avoid scalding burns (2-3 h needed to reach the temperature equilibrium) or half-filled with water (30 min to reach the temperature equilibrium). If water is used, the brass blocks need to be thoroughly dried before step 4.5 in order to avoid subsequent burns due to scalding.</li>
	<li>Insert a thermometer into the brass cylinder via its central opening to monitor its core temperature, and confirm the surface temperature with a temperature gun. If needed, increase the hot plate&#39;s temperature to 65 &#176;C or 93&#176; C in the cylinder despite minor heat dissipation. 
	NOTE: Aluminum foil can be used to cover the container and further minimize heat dissipation, reaching the desired temperature faster.</li>
	<li>In parallel, place the animal in the prone position for the burning procedure, similar to the tattoo procedure (step 3.1).</li>
	<li>Once the animal is ready in the prone position, prepare the skin in three steps (povidone-iodine 7.5%, saline solution 0.9%, drying).</li>
	<li>Grasp the brass block with heat-resistant gloves and place it on the dedicated tattoo spot, depending on the randomization. Start the timer as soon as the brass block touches the skin. Be careful not to apply more pressure to the skin than the brass block&#39;s own weight.</li>
	<li>After 30 s, remove the brass block from the skin and put it back in its dedicated heating container. Monitor the temperature until it reaches the target, and repeat the procedure for all the wounds.</li>
	<li>Perform an advanced multi-layer dressing to ensure dressing stability after the animal has recovered.
	<ol>
		<li>Place a petroleum-impregnated gauze on each burn location, including controls, and cover it with a dry non-woven gauze and a large transparent adhesive dressing.</li>
		<li>Spray a tincture of benzoin solution on the surrounding skin for better adherence of the transparent dressings to the skin.</li>
		<li>Wrap the animal in self-adherent adhesive wrap dressing while paying attention not to overtighten it (restriction of pulmonary volumes).</li>
		<li>Complete the dressing by adding a final layer of tubular stockinette (size adapted to the animal) (see Table of Materials).&#160;Alternatively, custom fitted swine jackets could&#160;be used&#160;if the animal is unable to keep the dressing clean for the duration of study</li>
		<li>Ensure correct analgesia by placing a transdermal opioid (fentanyl) patch on the neck of the animal after injecting a single dose of buprenorphine (0.05-0.1 mg/kg, IM) and a single dose of carprofen (2-4 mg/kg, IM).</li>
		<li>Following each anesthetization, monitor animals closely up to 2 h after recovery. Use a warm pad if needed, and provide food freely after a full standing position is reached.</li>
	</ol>
	</li>
</ol>
5. Full-thickness burn escharotomy
NOTE: Between 1 and 3 days postoperatively, the animals will receive full-thickness surgical excision of the eschar following the third-degree burns.
<ol>
	<li>After similar preparation of the animal including anesthesia, intubation, and placing the animal in supine position, prepare the skin via the three step skin preparation, perform sterile draping of the back of the animal.</li>
	<li>Perform 4 mm punch biopsies (see Table of Materials) on the wound to confirm the type of injury.</li>
	<li>Perform the escharotomy43,44 by a circular incision of the eschar with a sterile scalpel blade (n&#176;15) until reaching the deep dermis.</li>
	<li>Grasp one side of the eschar firmly with a pair of sterile Adson tissue forceps (with 2/1 teeth) (see Table of Materials) and hitch it upwards to reveal the deep limit of the eschar.</li>
	<li>Continue the escharotomy using the scalpel blade while staying in the same plane within the deep dermis/underlying fascia. A bleeding dermis is considered a sign of tissue viability.
	<ol>
		<li>If an arteriole is cut during the process, perform a minimal cauterization using bipolar coagulation forceps (see Table of Materials). CAUTION: Extensive cauterization of the created wound bed can result in delayed healing and should be strictly avoided.</li>
	</ol>
	</li>
	<li>Put a first set of dry guaze on the wound bed for 5 minutes to enhance local hemostasis before placing the definitive dressing.</li>
	<li>Dress the wounds similarly as previously (step 4.7), with the addition of several dry gauze to fill the cavity. Apply the same layers as previously mentioned: Tincture benzoin spray, adhesive transparent dressing, self-adhesive wrap, tubular stockinette.</li>
	<li>Provide analgesia by changing the opio&#239;d transdermal patch and injecting Caprofen (2-4 mg/kg, IM).</li>
</ol>
6. Follow-up wound dressings
NOTE: Subsequent dressings are performed every 2 to 7 days, depending on the experimental treatment design and the animal&#39;s tolerance. Wound dressings can be stopped after 21 days to allow re-epithelialization in a dry environment and improve the animal&#39;s tolerance. Alternatively, if the treatment group necessitates a moist or wet environment, the dressings can be prolonged until the end of the study. The follow-up period was extended for up to 10 weeks in order to study both the acute and prolonged healing processes.
<ol>
	<li>Anesthetize&#160;the animal for a short period and placed in the supine position with a nose cone (isoflurane 2-5 L/min).</li>
	<li>Remove the previous dressings and clean the wounds using sterile 0.9% saline and sterile gauze.</li>
	<li>Apply the experimental treatment if applicable.</li>
	<li>Similarly, perform subsequent biopsies during this procedure if needed by the study. The animal should then receive pain medication such as carprofen (2-4 mg/kg, IM, 24 h effect).</li>
	<li>Cover all wound locations, including control wounds, with petroleum-impregnated or equivalent gauze, dry, non-woven gauze, and adhesive transparent dressing.</li>
</ol>

Yucatan Minipig Burn Wound Model for Analysis of Multi-Depth Burns

The authors have no conflict of interest to declare.

Wound healing following burn injuries is a long process that can take up to several months, with various treatment options and considerations for patient care2,13. In order to study it, a reliable and reproducible model is needed. Several animal models have been described, mainly including rodents29,45,46 and swine29,4...

Burns are a major public health problem and affect more than 480,000 patients in the US each year, according to the National Burn Repository1,2. This leads to more than 50,000 yearly hospitalizations for non-fatal complex cases requiring in-depth care2. Moreover, burns are a fundamental cause of military mortality and morbidity and are responsible for 10% to 30% of military casualties3,4. The management of burns has remained nearly unchanged for a long time, despite its immense and diverse impacts on patients, ranging from physical to psychological and emotional5.
Initial diagnosis and evaluation of burn injuries lead to a baseline classification according to the type of burns (first, second, and third) or the depth of the affected tissue (superficial, partial thickness, and deep burns)6,7,8. Partial-thickness burns (first and second-degree) involve the epidermis and different depths of the dermis (superficial or deep dermis, i.e., superficial and deep second-degree burns)9. In particular, damage to the appendages in the deep dermis excludes the possibility of re-epithelialization from the adnexal epithelium10. By definition, full-thickness burns reach the subcutaneous fat, fascia and/or the underlying muscle (third-degree burns), and sometimes the bone (also referred to as fourth-degree burns)11,12.
Following hospitalization, burn patients receive special care involving a strategy consisting of a delicate balance between tissue debridement and preservation. The damaged and/or secondarily infected soft tissue needs to be progressively removed until healthy tissue is exposed, allowing for the use of specific dressings and skin grafts to improve the healing process13,14,15,16. Yet, caution is required during surgery to avoid unintentional removal of healing tissue and reduce complications for optimal recovery. Biologically, burns exhibit a central necrotic area encircled by a &#39;shadow&#39; or &#39;stasis&#39; zone, indicating potentially reversible ischemia. This area can either deteriorate, resulting in an extended necrosis zone, or heal by reversing the apoptotic process17,18. This varying severity of burns presents challenges for surgeons to assess accurately, complicating the balance between conservative treatments and surgical excision19. To date, no efficient tool is available to help characterize this &#34;shadow zone&#34; preceding burn conversion. Developing such tools is crucial for optimizing this delicate balance.
Several treatments have been tested to help decrease secondary burn conversion. Yet, no specific therapy is currently available in the clinic18. Other examples of advances in burn treatments include the development of modern wound dressings and nanomaterials20,21, tissue-engineered skin22,23, and novel epidermal culture approaches24,25. Also, modern reconstructive surgery and fasciocutaneous flaps have improved the management of long-term after-effects, particularly burn contractures following pathologic healing of fold areas26,27. These advancements give promising prospects for burn patients, improving their treatment strategies and quality of life, but recent results show that the functional impact still remains substantial, both in the physical and psychological spheres28. Taken together, the demand for innovative advancements in both burn diagnosis and burn treatment is substantial.
Overall, many approaches are aiming at improving the diagnosis, management, and treatment of complex burn cases, and researchers need a reproducible and relevant model to test these new approaches. Due to its biological complexity, involving several organs and systemic reactions, no in vitro model proved itself relevant to study the burn wound process29. Rodent models have shown major discrepancies with humans due to major differences in biology, skin architecture, elasticity, and lack of adherence to the underlying structures29. In contrast, the swine model has proven to be relevant due to the structural similarity of swine skin to human skin30,31,32. It presents with a similar vascularization, elastic fiber composition, and renewal timing. Moreover, the hair follicle and apocrine annexes allow for islanded re-epithelialization, as can be observed in clinical superficial burns33,34. More specifically, Yucatan minipig models provide interesting features, making them relevant to studying pigmented skin35 and long-term outcomes with minimal physical changes36.
The purpose of this article is to describe a reliable multi-degree burn model in Yucat&#225;n pigs, enabling the study of several second and third-degree burns on the same subject. This provides a relevant and reproducible model for studying diagnostic and therapeutic innovations for the management of burns. Further, this model features different burn types and severity, a long-term follow-up allowing the study of burn contracture and pathologic healing, and pigmented skin differential behavior, which is known to have specific characteristics.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adson tissue forceps</td>
			<td>Jarit</td>
			<td>130-234</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum beads</td>
			<td>Lab Armor</td>
			<td>42370-002</td>
			<td>Lab Armor Beads&#160;</td>
		</tr>
		<tr>
			<td>Buprenorphine hydrochloride</td>
			<td>Ranbaxy Pharmaceuticals</td>
			<td>NDC:12469-0757-01</td>
			<td>Buprenex Injectable</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>Pfizer</td>
			<td>NADA 141-199</td>
			<td>Rymadyl 50mg/ml injectable&#160;</td>
		</tr>
		<tr>
			<td>Cylindric brass block</td>
			<td>Hand-made</td>
			<td>N/A</td>
			<td>Engineering drawing included in the manuscript</td>
		</tr>
		<tr>
			<td>Dermographic pen</td>
			<td>McKesson</td>
			<td>Surgical Skin Marker Sterile</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable #15 surgical scalpels</td>
			<td>Medline</td>
			<td>MDS15315</td>
			<td>Scalpel blades</td>
		</tr>
		<tr>
			<td>Fentanyl patch</td>
			<td>Mylan</td>
			<td>NDC:60505-7082</td>
			<td>Fentanyl Transdermal System</td>
		</tr>
		<tr>
			<td>Isoflurane&#160;</td>
			<td>Piramal</td>
			<td>NDC:66794-013-25</td>
			<td>Isoflurane, USP</td>
		</tr>
		<tr>
			<td>McPherson Bipolar coagulation forceps</td>
			<td>Bovie</td>
			<td>A842</td>
			<td>Reusable, autoclavable</td>
		</tr>
		<tr>
			<td>Miltex assorted biopsy punches (3,4 and 5 mm)</td>
			<td>Integra</td>
			<td>33-38</td>
			<td>Biopsy punches- size to adapt to the study</td>
		</tr>
		<tr>
			<td>Non woven gauze</td>
			<td>Starryshine</td>
			<td>GZNW22</td>
			<td>2 x 2&#34; non woven 4 ply medical gauze pads</td>
		</tr>
		<tr>
			<td>Povidone-Iodine</td>
			<td>Betadine</td>
			<td>NDC:0034-9200-88</td>
			<td>Surgical scrub 7.5%&#160;</td>
		</tr>
		<tr>
			<td>Sterile isotonic sodium chloride solution 0.9%</td>
			<td>Aqualite System</td>
			<td>RL-2095</td>
			<td>Sterile saline solution</td>
		</tr>
		<tr>
			<td>Tattoo ink</td>
			<td>Spaulding &#38; Rogers</td>
			<td>Black - 2 oz - #9053</td>
			<td></td>
		</tr>
		<tr>
			<td>Tattoo marker</td>
			<td>Spaulding &#38; Rogers</td>
			<td>Special Electric Tattoo Marker</td>
			<td></td>
		</tr>
		<tr>
			<td>Tattoo needle</td>
			<td>Spaulding &#38; Rogers</td>
			<td>1310251</td>
			<td>Tattoo 5 point needle</td>
		</tr>
		<tr>
			<td>Tegaderm Transparent Film Dressing</td>
			<td>3M</td>
			<td>1.628</td>
			<td>Large transparent adhesive dressing</td>
		</tr>
		<tr>
			<td>Temperature-controlled hot plate</td>
			<td>Cole-Parmer</td>
			<td>03407-11</td>
			<td>StableTemp hot plate stirrer</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td>American Scientific</td>
			<td>U14295</td>
			<td>Tube mercury thermometerr</td>
		</tr>
		<tr>
			<td>Tiletamine and zolazepam hydrochloride</td>
			<td>Zoetis</td>
			<td>NDC:54771-9050</td>
			<td>Telazol</td>
		</tr>
		<tr>
			<td>Tincture of Benzoin Spray</td>
			<td>Smith&#38;Nephew</td>
			<td>407000</td>
			<td>Adhesive layer spray</td>
		</tr>
		<tr>
			<td>Triple Antibiotic ointment</td>
			<td>Fougera</td>
			<td>NDC 0168-0012-31</td>
			<td>Triple antibiotic ointment</td>
		</tr>
		<tr>
			<td>Tubular stockinette</td>
			<td>Medline</td>
			<td>NONNET02</td>
			<td>Curad Medline Latex Free Elastic Nets</td>
		</tr>
		<tr>
			<td>Warming blanket</td>
			<td>3M</td>
			<td>Bair Hugger 750 warming unit</td>
			<td></td>
		</tr>
		<tr>
			<td>Xeroform Occlusive Gauze Strip</td>
			<td>Covidien</td>
			<td>8884433301</td>
			<td>Xeroform petrolatum wound dressings</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Vetone</td>
			<td>NDC:13985-704-10</td>
			<td>AnaSed LA</td>
		</tr>
		<tr>
			<td>Yucat&#224;n minipigs (female, 30 kg)</td>
			<td>Sinclair Bio Resources</td>
			<td>N/A</td>
			<td>Full pigmentation&#160;</td>
		</tr>
	</tbody>
</table>

a swine burn model for investigating the healing process in multiple depth burn wounds

Burn wound healing is a complex and long process. Despite extensive experience, plastic surgeons and specialized teams in burn centers still face significant challenges. Among these challenges, the extent of the burned soft tissue can evolve in the early phase, creating a delicate balance between conservative treatments and necrosing tissue removal. Thermal burns are the most common type, and burn depth varies depending on multiple parameters, such as temperature and exposure time. Burn depth also varies in time, and the secondary aggravation of the &#34;shadow zone&#34; remains a poorly understood phenomenon. In response to these challenges, several innovative treatments have been studied, and more are in the early development phase. Nanoparticles in modern wound dressings and artificial skin are examples of these modern therapies still under evaluation. Taken together, both burn diagnosis and burn treatments need substantial advancements, and research teams need a reliable and relevant model to test new tools and therapies. Among animal models, swine are the most relevant because of their strong similarities in skin structure with humans. More specifically, Yucatan minipigs show interesting features such as melanin pigmentation and slow growth, allowing for studying high phototypes and long-term healing. This article aims to describe a reliable and reproducible protocol to study multi-depth burn wounds in Yucatan minipigs, enabling long-term follow-up and providing a relevant model for diagnosis and therapeutic studies.

Figure 2A,B display the results of multiple burns on the dorsum of a Yucat&#224;n minipig. Wounds (I) and (VII) are control wounds (37 &#176;C). Second-degree wounds (II; III and VIII) present with intense redness and blisters. In contrast, third-degree wounds (IV; V; and VI) are pale and indurated to palpation. It is to be noted that wound VIII looks intermediate between second and third degree: for the purpose of an ongoing study, we increased the contact time to 45 s at 6...

Read this Scientific Article Protocol about Author Spotlight: A Multi-Depth Porcine Model for Comprehensive Study of Burn Injuries and Healing Processes at JoVE.com

Author Spotlight: A Multi-Depth Porcine Model for Comprehensive Study of Burn Injuries and Healing Processes

This protocol describes a reproducible multi-depth burn wound model in a Yucatan minipigs.

A Swine Burn Model for Investigating the Healing Process in Multiple Depth Burn Wounds

Burn wound healing is a complex and long process. Despite extensive experience, plastic surgeons and specialized teams in burn centers still face ...

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850 Views.  Massachusetts General Hospital, Harvard Medical School. This protocol describes a reproducible multi-depth burn wound model in a Yucatan minipigs. 

Video: Author Spotlight: A Multi-Depth Porcine Model for Comprehensive Study of Burn Injuries and Healing Processes

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

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. ...

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine

To date, many pharmacological agents used to treat or prevent arrhythmias in open-heart cases create undesired systemic side effects. For example, antiarrhythmic drugs administered intravenously can produce drops in systemic pressure in the already compromised cardiac patient. While performing open-heart procedures, surgeons will often either create a small port or form a pericardial cradle to create suitable fields for operation. This access yields opportunities for target pharmacological delivery (antiarrhythmic or ischemic preconditioning agents) directly to the myocardial tissue without undesired side effects.
We have developed a swine model for testing pharmacological agents for target delivery within the pericardial fluid. While fully anesthetized, each animal was instrumented with a Swan-Ganz catheter as well as left and right ventricle pressure catheters, and pacing leads were placed in the right atrial appendage and the right ventricle. A medial sternotomy was then performed and a pericardial access cradle was created; a plunge pacing lead was placed in the left atrial appendage and a bipolar pacing lead was placed in the left ventricle. Utilizing a programmer and a cardiac mapping system, the refractory period of the atrioventricular node (AVN), atria and ventricles was determined. In addition, atrial fibrillation (AF) induction was produced utilizing a Grass stimulator and time in AF was observed. These measurements were performed prior to treatment, as well as 30 min&#160;and 60 min&#160;after pericardial treatment. Additional time points were added for selected studies. The heart was then cardiopleged and reanimated in a four chamber working mode. Pressure measurements and function were recorded for 1 hr after reanimation. This treatment strategy model allowed us to observe the effects of pharmacological agents that may decrease the incidence of cardiac arrhythmias and/or ischemic damage, during and after open-heart surgery.

We have developed a swine model for the target delivery of pharmacological agents within the pericardial space/fluid. Using this approach, the relative benefits of administered agents on induced atrial fibrillation, relative refractory periods and/or ischemic protection can be investigated.

To date, many pharmacological agents used to treat or prevent arrhythmias in open-heart cases create undesired systemic side effects. For example, ...

Direct Mouse Trauma/Burn Model of Heterotopic Ossification

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical procedures. The majority of animal models used to study HO rely on the application of exogenous substances, such as bone morphogenetic protein (BMP), exogenous cell constructs, or genetic mutations in BMP signaling. While these models are useful they do not accurately reproduce the inflammatory states that cause the majority of cases of HO. Here we describe a burn/tenotomy model in mice that reliably produces focused HO. This protocol involves creating a 30% total body surface area partial thickness contact burn on the dorsal skin as well as division of the Achilles tendon at its midpoint. Relying solely on traumatic injury to induce HO at a predictable location allows for time-course study of endochondral heterotopic bone formation from intrinsic physiologic processes and environment only. This method could prove instrumental in understanding the inflammatory and osteogenic pathways involved in trauma-induced HO. Furthermore, because HO develops in a predictable location and time-course in this model, it allows for research to improve early imaging strategies and treatment modalities to prevent HO formation.

An Achilles tenotomy and burn injury model of heterotopic ossification allows for the reliable study of trauma induced ectopic bone formation without the application of exogenous factors.

Heterotopic ossification (HO) is the formation of bone outside of the skeleton which forms following major trauma, burn injuries, and orthopaedic surgical ...

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

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 ...

A Model of Free Tissue Transfer: The Rat Epigastric Free Flap

Free tissue transfer has been increasingly used in clinical practice since the 1970s, allowing reconstruction of complex and otherwise untreatable defects resulting from tumor extirpation, trauma, infections, malformations or burns. Free flaps are particularly useful for reconstructing highly complex anatomical regions, like those of the head and neck, the hand, the foot and the perineum. Moreover, basic and translational research in the area of free tissue transfer is of great clinical potential. Notwithstanding, surgical trainees and researchers are frequently deterred from using microsurgical models of tissue transfer, due to lack of information regarding the technical aspects involved in the operative procedures. The aim of this paper is to present the steps required to transfer a fasciocutaneous epigastric free flap to the neck in the rat.
This flap is based on the superficial epigastric artery and vein, which originates from and drain into the femoral artery and vein, respectively. On average the caliber of the superficial epigastric vein is 0.6 to 0.8 mm, contrasting with the 0.3 to 0.5 mm of the superficial epigastric artery. Histologically, the flap is a composite block of tissues, containing skin (epidermis and dermis), a layer of fat tissue (panniculus adiposus), a layer of striated muscle (panniculus carnosus), and a layer of loose areolar tissue.
Succinctly, the epigastric flap is raised on its pedicle vessels that are then anastomosed to the external jugular vein and to the carotid artery on the ventral surface of the rat's neck. According to our experience, this model guarantees the complete survival of approximately 70 to 80% of epigastric flaps transferred to the neck region. The flap can be evaluated whenever needed by visual inspection. Hence, the authors believe this is a good experimental model for microsurgical research and training.

This paper describes the steps required to raise a fasciocutaneous epigastric free flap and transfer it to the neck in the rat.

Free tissue transfer has been increasingly used in clinical practice since the 1970s, allowing reconstruction of complex and otherwise untreatable defects ...

Severe Burn Injury in a Swine Model for Clinical Dressing Assessment

Wound healing is a dynamic repair process and is the most complex biological process in human life. In response to burn injury, alterations in biological pathways impair the inflammation response, resulting in delayed wound healing. Impaired wound healing frequently occurs in patients with diabetes leading to unfavorable outcomes such as amputation. Hence, dressings having beneficial effect in promoting burn wound repair are needed. However, studies on burn wound treatment are limited due to lack of proper animal models. Our previous study demonstrated wound-healing performance in rat and swine models using a minimally invasive surgical technique. This study aimed to demonstrate a swine model of severe burn injury that eliminates wound contraction and more closely approximates the human processes of re-epithelialization and new tissue formation. This protocol provides a detailed procedure for creating consistent burn wounds and examining the wound-healing performance under the treatment of an experimental dressing in a swine model. Six burn wounds were created symmetrically on the dorsum, which were covered with a clinical dressing composed of four layers: an inner contact layer of experimental materials, an inner intermediate layer of waterproof film, an outer intermediate layer of gauze, and an outer layer of adhesive plaster. Upon the completion of experiments, wound closure, wound area, and Vancouver Scar Scale score were examined. The samples of skin resected from each animal post-sacrifice were histologically prepared and stained using hematoxylin and eosin staining. Antibacterial activity of each dressing in the context of wound healing was also examined. The application of the clinical dressing to the wounds in swine model mimics the biological processes of human wound healing with respect to the processes of epithelialization, cellular proliferation, and angiogenesis. Therefore, this swine model provides an easy-to-learn, cost-effective, and robust method to assess the effect of clinical dressings in severe burn injury.

To closely mimic the mode of burn injuries requires the interplay between clinical observation and studies in animal models. In this study, a swine model of severe burn injury was established to assess an experimental dressing in physiological and pathophysiological settings.

Wound healing is a dynamic repair process and is the most complex biological process in human life. In response to burn injury, alterations in biological ...

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 ...