This study was supported by a grant from the Tri-Service General Hospital; National Defense Medical Center, Taiwan (TSGH-C107-042); Ministry of Science and Technology, Taiwan (MOST 106-2314-B-016 -014); and the National Defense Medical Center (MAB-106-055; MAB-106-010; MAB-107-064).

Procedures involving animal subjects have been approved by the Animal Care Committee at National Defense Medical Center, Taiwan (R.O.C). This study was conducted in the Laboratory Animal Center at the National Defense Medical Center. Swine weighing between 20 and 25 kg has been successfully instrumented using this protocol.
1. Adaptation of the Animals to Human Handling
<ol>
	<li>After arrival in the facility, house the animals solitarily but let them interact with each other.</li>
	<li>Provide the animals ad libitum access to food and water.</li>
	<li>Acclimate swine to human handling and transportation from the animal facility to the experimental laboratory by handling the animal at least once a day for one week.</li>
	<li>Fast the animal for at least 12 hours before surgery to prevent nausea, vomiting, and aspiration of stomach fluids.</li>
</ol>
2. Sedation
<ol>
	<li>Before the burn wound creation, sedate animals via an intravenous injection with Zoletil 50 (25 mg/kg).</li>
</ol>
3. Intubation and Ventilation
<ol>
	<li>Place the animal on a table and/or trolley in sternal&#160;position.</li>
	<li>Open the mouth of the animal with an oral spreader.</li>
	<li>In case of insufficient relaxation of the jaws or presence of swallowing reflexes, which hinder intubation, mask the swine with isoflurane to induce sedation.</li>
	<li>Monitor blood pressure, heart rate, and body temperature by physiological signal monitor during the surgery to prevent potential complications.</li>
</ol>
4. Anesthesia
<ol>
	<li>Induce and maintain anesthesia; preferably anesthetize the animal via an intramuscular injection of tiletamine and zolazepam (25 mg/kg + 25 mg/kg).</li>
	<li>Intubate the animal with the endotracheal tube when muscle relaxation, characterized by loss of jaw tone and palpebral reflexed, was observed.</li>
	<li>Maintain all pigs in an anesthetic state at a vaporizer setting of 0.5&#8211;2.5% (v/v) isoflurane until the end of the surgery.</li>
	<li>Examine the depth of anesthesia by testing pain reflexes with a hind leg toe pinch before surgery. When necessary, add additional anesthesia or wait for a few minutes. Check pain reflexes regularly throughout the surgery.</li>
</ol>
5. Sterilization of the Surgical Site
<ol>
	<li>Shave and clean the skin of the animal over an area of approximately 25 cm width from the vertebral column all the way to the axilla on both sides.</li>
	<li>Scrub the moisturized skin with povidone-iodine scrub (75 mg/mL) for approximately 5 min.</li>
	<li>Remove the povidone-iodine soap from the skin using wet sterile gauzes.</li>
	<li>Sterilize the skin with povidone-iodine lotion (100 mg/mL).</li>
	<li>Cover the animal with sterile surgical drapes to reduce bacterial transfer and subsequent contamination of the surgical site.</li>
</ol>
6. Burn Wound Creation
<ol>
	<li>Use a surgical marking pen to mark the center of six burn wounds symmetrically on the dorsum of the pig. Ensure that the distance between each burn wound is at least greater than the radius of the wound (Figure 1A).</li>
	<li>Fill a modified soldering iron with 50 mL of glycerin and insert an electronic thermometer into it to monitor the temperature. The hot iron possesses a flat area of approximately 9 cm2 (Figure 1B).</li>
	<li>Heat up the iron to 137&#8211;139 &#176;C with a hot plate (Figure 1C).</li>
	<li>Create six uniform burn wounds by placing the iron on the marked area without applying any force for 30 seconds (Figure 1C).</li>
	<li>Wash the burn wounds with 0.9% saline solution (Figure 1D).</li>
	<li>Measure wound dimensions and record the wound by photomicrography (Figure 1E).</li>
</ol>
7. Preparation of Dressings
<ol>
	<li>Cover each wound with the inner contact layer of a four-layer clinical dressing through direct contact. For this layer, use CAPS-containing dressing or alternative materials (Figure 1F).</li>
	<li>Apply a waterproof film onto the clinical dressing to serve as a barrier against bacterial penetration (Figure 1F).</li>
	<li>Cover each wound with a gauze (0.5 cm thick) and fix with paper tape to serve as the mid-layer of the dressing (Figure 1G).</li>
	<li>Secure the gauze with an outer layer of adhesive plaster. Extend this layer to the torso to avoid the displacement of the dressing (Figure 1H-1J).</li>
</ol>
8. Post-burn Care and Measurement
<ol>
	<li>Inject the swine with buprenorphine (0.1 mg/kg, IM) for pain management for every 8-12 h, starting before recovery from anesthesia, for one week to reduce potential pain.</li>
	<li>Allow the pig free access to feed and water.</li>
	<li>Change the clinical dressings every 2 days for the first 10 days and then twice a week for the 6-week study.</li>
	<li>Clean and measure wounds before reapplying clinical dressings. Administer anesthetics during dressing changes.</li>
	<li>Record the wound by photomicrography for comparison of wound healing rate every 2 days for the first 10 days and then twice a week for the 6-week study.</li>
	<li>Calculate the wound re-epithelialization or contraction as the percentage of the original wound size according to a previously described method. The analysis of wound closure was conducted in a double-blinded manner.</li>
	<li>Measure the burn scar using VSS, which consists of four variables: vascularity, height (thickness), pliability, and pigmentation on post-burn days 0, 7, 21, and 42. Each variable has four to six possible scores. The total score ranges from 0 to 14, whereby a score of 0 reflects normal skin.</li>
</ol>
9. Bacterial Growth Experiments of Post-burn Tissues
<ol>
	<li>Swab the wound for antibacterial testing on post-burn days 0, 7, 21, and 42.</li>
	<li>Place the swab into 100 mL of 0.9% sterile saline solution and gently vortex to achieve a homogenous suspension.</li>
	<li>Serially dilute (10&#8722;1&#8211;10&#8722;5) the homogenate and plate 100 &#956;L of each dilution in selective and nonselective media, respectively.</li>
	<li>Incubate all dilutions under aerobic conditions at 37 &#176;C for 24&#8211;72 hours.</li>
	<li>Plate triplicate aliquots of 10 &#956;L each from all dilutions onto blood agar plate supplemented with 5% sheep blood to isolate aerobic Gram-positive organisms.</li>
	<li>Incubate the sample by spreading or pouring the sample uniformly on the surface of an agar plate overnight for determining the number of colony-forming units (CFUs).</li>
	<li>Read the plates after overnight incubation. Invert the sheep blood agar plate and divide the bottom of the dish into four equal quadrants using a marker and small ruler.</li>
	<li>Place the plate onto the stage of a dissection microscope and count the colonies on each plate. By definition, a colony must have a minimum of 300 CFU to be enumerated.</li>
	<li>Count the bacterial colonies in each of the three replicates. Calculate the average value of the three replicates. Determine the CFU per plate by multiplying the average value by the final dilution factor.</li>
</ol>
10. Euthanasia and Tissue Fixation
<ol>
	<li>Intravenously inject an overdose of sodium pentobarbital euthanasia solution (80&#8211;120 mg/kg).</li>
	<li>Perform en bloc excision of burn wound tissue to include the underlying musculature and surrounding unwounded tissue.</li>
	<li>Fix tissues with 10% neutral buffered formalin.
	<ol>
		<li>Mix 10 mL of formaldehyde (37%) in 90 mL of phosphate buffered solution (PBS) and store in 4 &#176;C.</li>
		<li>Transfer tissues to fixative and swirl the container to ensure all tissues are completely immersed in fixative. The volume of fixative must be 30 times the tissue volume.</li>
		<li>Fix tissues overnight at 4 &#730;C.</li>
	</ol>
	</li>
	<li>Dehydrate tissues with ethanol and embed into paraffin blocks. Perform the following steps at 4 &#730;C on a shaker.
	<ol>
		<li>Wash twice with PBS for 30 minutes.</li>
		<li>Dehydrate tissues with 70% ethanol for 8 hours, 80% ethanol overnight, 95% ethanol for 8 hours, and then in 100% ethanol overnight.</li>
		<li>Incubate tissues in 100% ethanol for a further 8 hours.</li>
		<li>Incubate tissues in three changes of xylene each for 30 minutes.</li>
		<li>Replace the xylene with freshly melted (52 &#176;C) wax, and incubate at 52 &#176;C in an oven for 1 hour.</li>
		<li>Replace the wax with fresh wax and incubate at 52 &#176;C in an oven for 3 hours, and then replace once more and incubate at 52 &#176;C overnight.</li>
		<li>Incubate tissues with two more changes of wax each for 1 hour, and then embed the tissue and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Cut and stain the paraffin-embedded sections with HE, and visualize via a light microscope with 100&#215; magnification.
	<ol>
		<li>Create paraffin sections using a rotary microtome.</li>
		<li>Dewax sections with three changes of xylene each for 3 minutes.</li>
		<li>Rehydrate tissues with 100%, 95%, 80%, and 70% ethanol each for 3 minutes, and then immerse in distilled water.</li>
		<li>Stain with hematoxylin for 10 minutes, and then rinse in running tap water.</li>
		<li>Differentiate with 0.1% hydrochloric acid ethanol for 5 minutes, and rinse in tap water.</li>
		<li>Stain with 0.5% eosin for 1 minute.</li>
		<li>Dehydrate tissues with 70%, 80%, 95%, and 100% ethanol each for 2 minutes.</li>
		<li>Clear the staining with xylene, and dry in fume hood.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Sen, C. K., 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=Human+skin+wounds:+a+major+and+snowballing+threat+to+public+health+and+the+economy.">Human skin wounds: a major and snowballing threat to public health and the economy.</a> Wound Repair Regeneration. 17, 763-771 (2009).</li><li>Sen, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+essentials:+let+there+be+oxygen.">Wound healing essentials: let there be oxygen.</a> Wound Repair Regeneration. 17, 1-18 (2009).</li><li>Verhaegen, P. D., 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=Differences+in+collagen+architecture+between+keloid,+hypertrophic+scar,+normotrophic+scar,+and+normal+skin:+an+objective+histopathological+analysis.">Differences in collagen architecture between keloid, hypertrophic scar, normotrophic scar, and normal skin: an objective histopathological analysis.</a> Wound Repair Regeneration. 17, 649-656 (2009).</li><li>Swindle, M. M., Adams, 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=.">.</a> Experimental Surgery and Physiology: Induced Animal Models of Human Disease. , (1988).</li><li>Swindle, M. M., Smith, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Swine as Models in Biomedical Research. , (1992).</li><li>Tumbleson, M. E., Schook, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in Swine in Biomedical Research. Vols 1-2. , (1996).</li><li>Wang, C. H., 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=Enhanced+wound-healing+performance+of+a+phyto-polysaccharide-enriched+dressing+-+a+preclinical+small+and+large+animal+study.">Enhanced wound-healing performance of a phyto-polysaccharide-enriched dressing - a preclinical small and large animal study.</a> International Wound Journal. 14, 1359-1369 (2017).</li><li>Rowan, M. P., Cancio, L. C., Elster, E. A., Burmeister, D. M., Rose, L. F., Natesan, S., Chan, R. K., Christy, R. J., Chung, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burn+wound+healing+and+treatment:+review+and+advancements.">Burn wound healing and treatment: review and advancements.</a> Journal of Critical Care. 19, 243 (2015).</li><li>Wong, V. W., Sorkin, M., Glotzbach, J. P., Longaker, M. T., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+approaches+to+create+murine+models+of+human+wound+healing.">Surgical approaches to create murine models of human wound healing.</a> Journal of Biomedicine and Biotechnology. 969618, 8 (2011).</li><li>Gaines, C., Poranki, D., Du, W., Clark, R. A., Van Dyke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+porcine+deep+partial+thickness+burn+model.">Development of a porcine deep partial thickness burn model.</a> Burns. 39, 311-319 (2013).</li><li>Gnyawali, S. C., Barki, K. G., Mathew-Steiner, S. S., Dixith, S., Vanzant, D., Kim, J., Dickerson, J. L., Datta, S., Powell, H., Roy, S., Bergdall, V., Sen, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+harmonics+ultrasound+imaging+for+non-invasive+characterization+of+wound+healing+in+a+pre-clinical+swine+model.">High-resolution harmonics ultrasound imaging for non-invasive characterization of wound healing in a pre-clinical swine model.</a> PLoS One. 10 (3), e0122327 (2015).</li><li>Eaglstein, W. H., Mertz, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+methods+for+assessing+epidermal+wound+healing:+the+effects+of+triamcinolone+acetonide+and+polyethelene+film+occlusion.">New methods for assessing epidermal wound healing: the effects of triamcinolone acetonide and polyethelene film occlusion.</a> Journal of Investigative Dermatology. 71, 332-384 (1978).</li><li>Swindle, M. M., Smith, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+anatomy+and+physiology+of+the+pig.">Comparative anatomy and physiology of the pig.</a> Scandinavian Journal of Laboratory Animal Sciences. 25, 1-10 (1998).</li><li>Swindle, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Swine in the Laboratory: Surgery, Anesthesia, Imaging and Experimental Techniques. , (2007).</li><li>Mertz, P. M., 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=IL-1+as+a+potent+inducer+of+wound+re-epithelization.">IL-1 as a potent inducer of wound re-epithelization.</a> Progress in Clinical and Biological Research. 365, 473-480 (1991).</li><li>Eming, S. A., Martin, P., Tomic-Canic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair+and+regeneration:+mechanisms,+signaling,+and+translation.">Wound repair and regeneration: mechanisms, signaling, and translation.</a> Science Translation Medicine. 6, 265-266 (2014).</li><li>Aryza, M. J., Baryza, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Vancouver+Scar+Scale:+an+administration+tool+and+its+interrater+reliability.">The Vancouver Scar Scale: an administration tool and its interrater reliability.</a> The Journal of Burn &amp; Rehabilitation. 16, 535-538 (1995).</li><li>Bystrom, A., Claesson, R., Sundqvist, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibacterial+effect+of+camphorated+paramonochlorophenol,+camphorated+phenol+and+calcium+hydroxide+in+the+treatment+of+infected+root+canals.">The antibacterial effect of camphorated paramonochlorophenol, camphorated phenol and calcium hydroxide in the treatment of infected root canals.</a> Endodontics &amp; Dental Traumatology. 1, 170-175 (1985).</li><li>Reig, A., Tejerina, C., Codina, J., Hidalgo, J., Mirabet, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+new+cicatrization+dressing+in+treating+second-degree+burns+and+donor+sites.">Application of a new cicatrization dressing in treating second-degree burns and donor sites.</a> Annals of Burn and Fire Disasters. 4, 174 (1991).</li><li>Hindy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+between+sodium+carboxymethyl-cellulose+silver,+moist+exposed+burn+ointment,+and+saline-soaked+dressing+for+treatment+of+facial+burns.">Comparative study between sodium carboxymethyl-cellulose silver, moist exposed burn ointment, and saline-soaked dressing for treatment of facial burns.</a> Annals of Burn and Fire Disasters. 22, 131-137 (2009).</li><li>Galiano, R. D., 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=Topical+vascular+endothelial+growth+factor+accelerates+diabetic+wound+healing+through+increased+angiogenesis+and+by+mobilizing+and+recruiting+bone+marrow-derived+cells.">Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells.</a> The American Journal of Pathology. 164, 1935-1947 (2004).</li><li>Thangarajah, H., 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+molecular+basis+for+impaired+hypoxia-induced+VEGF+expression+in+diabetic+tissues.">The molecular basis for impaired hypoxia-induced VEGF expression in diabetic tissues.</a> Proceedings of the National Academy of Sciences of the United States of America. , 13505-13510 (2009).</li></ol>

The authors have nothing to disclose.

The present study established a swine model of severe burn injury and examined the model using a CAPS-containing dressing. Our results suggest that this swine model can be used for monitoring the clinical performance of experimental dressings, including antibacterial property. Wound healing rate, wound closure, and antibacterial activity were also analyzed using VSS, H&#38;E staining, and antibacterial test. The use of animal burn models has been developed as a valuable tool to review the pathophysiology of burn injury. ...

A burn injury initiates the inflammatory process and induces complex pathological effects, which influence numerous body functions immediately after an accident, resulting in negative impact on patients&#39; quality of life. Impaired wound healing causes significant morbidity and mortality among patients with diabetes mellitus1,2. Most patients with burn injuries experience pain during burn wound debridement, which is known as an excruciating process despite the use of powerful opioid analgesics3.
In contrast to other mammals, swine share several anatomic and physiologic characteristics with humans regarding the process of epithelialization, cellular proliferation, and angiogenesis. This makes swine a potentially better model for certain procedures and studies, and they are often used in subsequent studies that demonstrated promising results in mice. These features have led to the increasing use of swine as a major species in preclinical testing. Recently, a rapid increase in biomedical research with respect to cardiovascular, urinary, integumentary, and digestive systems has been observed4,5,6. This study aims to demonstrate a swine model of severe burn injury that eliminates wound contraction and provides a closer approximation of the human wound healing processes and the formation of new tissue. Six burn wounds were created symmetrically on the dorsum, three on each side of the spine of the swine. Next,anexperimental dressing was examined in a swine model of severe burn injury, which can be adapted to replicate human wound healing (Figure 1). The wounds were covered with a clinical dressing, which is 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. A waterproof film keeps the wound environment moist while preventing bacterial infection and allowing gases to permeate the dressing. The outer intermediate layer of gauze was applied on the waterproof films and secured by an outer layer of adhesive plaster. At the completion of experiments, wound closure, wound area, and Vancouver Scar Scale (VSS) score were examined. The samples of skin resected from each animal post-sacrifice were histologically prepared and stained using hematoxylin and eosin (HE) staining. Antibacterial activity of each dressing in the context of wound healing was also examined in this model. Our previous study has demonstrated wound-healing performance in rat and swine model using a minimally invasive surgical technique7. Since there were six burn wounds on the dorsum within each swine, each experimental dressing was tested and evaluated in all positions to minimize bias related to wound healing process in different spots on the swine dorsum. Therefore, the swine model of severe burn injury established in this study provides a new approach for the evaluation of clinical dressings and facilitates the development of a novel treatment for burn injury. This study provides crucial tools to uncover the pathophysiology of burn wound healing.

<table><tbody><tr><td>&lt;strong&gt;Sedation:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ketamine</td><td>Merial</td><td>2528 ESP</td><td>10 mL vial</td></tr><tr><td>Azaperone</td><td>China chemical &amp;amp; pharmaceutical</td><td>47W406</td><td>100 mL vial</td></tr><tr><td>Atropine&amp;nbsp;</td><td>Oriental chemical works</td><td>IN120802</td><td>1 mL vial</td></tr><tr><td>&lt;strong&gt;Anesthetic:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tiletamine+Zolazepam</td><td>Virbac</td><td>BC91</td><td>5 mL vial</td></tr><tr><td>Isoflurane</td><td>Baxter</td><td>N002A225</td><td>100 mL vial</td></tr><tr><td>&lt;strong&gt;Surgery:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Hair clippers</td><td>Moser</td><td>-</td><td>-</td></tr><tr><td>Povidone iodine scrub solution</td><td>Ever star</td><td>HA161202</td><td>4 L barrel</td></tr><tr><td>Modified iron</td><td>-</td><td>-</td><td>-</td></tr><tr><td>0.9% saline solution</td><td>CHI SHENG</td><td>KC130</td><td>500 mL vial</td></tr><tr><td>Gauze</td><td>China Surgical Dressings Center</td><td>MO15900080</td><td>10 x10 cm</td></tr><tr><td>CAPS</td><td>CoreLeader Biotech Co., Ltd, Taipei, Taiwan</td><td>-</td><td>-</td></tr><tr><td>Paper tape</td><td>3M</td><td>NDC-8333-1530-01</td><td>2.5 cm x 9.1m</td></tr><tr><td>Waterproof film</td><td>3M</td><td>NDC-8333-1600-40</td><td>10 cm x 10 m</td></tr><tr><td>Adhesive plaster</td><td>Young chemical</td><td>BH1426015</td><td>10 cm x 10 m</td></tr><tr><td>&lt;strong&gt;Dissection:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pair of standard sharp/blunt straight scissors (14 cm)</td><td>Shinetec instruments</td><td>ST-S114</td><td>-</td></tr><tr><td>Halstead-Mosquito (12.5 cm)</td><td>Shinetec instruments</td><td>ST-H012</td><td>-</td></tr><tr><td>Handle(# 4)</td><td>Shinetec instruments</td><td>ST-H004</td><td>-</td></tr><tr><td>Surgical Blade(#21)</td><td>Shinetec instruments</td><td>ST-B021</td><td>-</td></tr><tr><td>&lt;strong&gt;Post-Fixation &amp;amp; Storage:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>50 ml Plastic centrifuge tube&amp;nbsp;</td><td>Falcon</td><td>352070</td><td>-</td></tr><tr><td>10% neutral buffered formalin</td><td>Leica</td><td>3800604EG</td><td>-</td></tr><tr><td>&lt;strong&gt;Bacterial Growth Experiments&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Blood agar plate (BAP) (TSA with 5% sheep blood)&amp;nbsp;</td><td>CMP</td><td>-</td><td>90 mm Mono</td></tr></tbody></table>

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.

Burn duration of 30 seconds by hot iron resulted in wounds that were circular with a well-defined margin and uniformly pale with a rim of erythema (Figure 1D). Within each animal, there were six burn wounds on the dorsum. The arrangement of burn wounds was depicted in Figure 1K. Burn wounds were completely covered with CAPS-containing dressing and used to evaluate the depth of scar formation on post-burn days 0, 7, 21, and 42 and...

Watch this Scientific Journal Video about Severe Burn Injury in a Swine Model for Clinical Dressing Assessment at JoVE.com

Severe Burn Injury in a Swine Model for Clinical Dressing Assessment

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 ...

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Research

JoVE Journal

Medicine

9.6K Views.  National Defense Medical Center. 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.

Video: Severe Burn Injury in a Swine Model for Clinical Dressing Assessment

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, ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

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 ...

Clinical Assessment of Spatiotemporal Gait Parameters in Patients and Older Adults

Spatial and temporal characteristics of human walking are frequently evaluated to identify possible gait impairments, mainly in orthopedic and neurological patients1-4, but also in healthy older adults5,6. The quantitative gait analysis described in this protocol is performed with a recently-introduced photoelectric system (see&#160;Materials table) which has the potential to be used in the clinic because it is portable, easy to set up (no subject preparation is required before a test), and does not require maintenance and sensor calibration. The photoelectric system consists of series of high-density floor-based photoelectric cells with light-emitting and light-receiving diodes that are placed parallel to each other to create a corridor, and are oriented perpendicular to the line of progression7. The system simply detects interruptions in light signal, for instance due to the presence of feet within the recording area. Temporal gait parameters and 1D spatial coordinates of consecutive steps are subsequently calculated to provide common gait parameters such as step length, single limb support and walking velocity8, whose validity against a criterion instrument has recently been demonstrated7,9. The measurement procedures are very straightforward; a single patient can be tested in less than 5 min&#160;and a comprehensive report can be generated in less than 1&#160;min.

This protocol is used to evaluate spatial and temporal gait variables of neurological/orthopedic patients and older persons by means of a recently-introduced floor-based photocell system.

Spatial and temporal characteristics of human walking are frequently evaluated to identify possible gait impairments, mainly in orthopedic and ...

A Repetitive Concussive Head Injury Model in Mice

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of knowledge on the injury and its effects. To develop a better understanding of concussions, animals are often used because they provide a controlled, rigorous, and efficient model. Studies have adapted traditional animal models to perform mTBI to stimulate mild injury severity by changing the injury parameters. These models have been used because they can produce morphologically similar brain injuries to the clinical condition and provide a spectrum of injury severities. However, they are limited in their ability to present the identical features of injuries in patients. Using a traditional impact system, a repetitive concussive injury (rCHI) model can induce mild to moderate human-like concussion. The injury degree can be determined by measuring the period of loss of consciousness (LOC) with a sign of a transient termination of breathing. The rCHI model is beneficial to use for its accuracy and simplicity in determining mTBI effects and potential treatments.

Concussion presents the most common type of traumatic brain injury. Therefore, a repetitive concussive animal model, which replicates the important features of an injury in patients, may provide a means to study concussion in a rigorous, controlled, and efficient manner.

Despite the concussion/ mild traumatic brain injury (mTBI) being the most frequent occurrence of traumatic brain injury, there is still a lack of ...

Semi-quantitative Assessment Using [18F]FDG Tracer in Patients with Severe Brain Injury

Patients with severe traumatic brain injury (sTBI) have difficulty knowing whether they are accurately expressing their thoughts and emotions because of disorders of consciousness, disrupted higher brain function, and verbal disturbances. As a consequence of an insufficient ability to communicate, objective evaluations are needed from family members, medical staff, and caregivers. One such evaluation is the assessment of functioning brain areas. Recently, multimodal brain imaging has been used to explore the function of damaged brain areas. [18F]-fluorodeoxyglucose positron emission tomography-computed tomography ([18F]FDG-PET/CT) is a successful tool for examining brain function. However, the assessment of brain glucose metabolism based on [18F]FDG-PET/CT is not standardized and depends on several varying parameters, as well as the patient&#39;s condition. Here, we describe a series of semiquantitative assessment protocols for a region-of-interest (ROI) image analysis using self-produced [18F]FDG tracers in patients with sTBI. The protocol focuses on screening the participants, preparing the [18F]FDG tracer in the hot lab, scheduling the acquisition of [18F]FDG-PET/CT brain images, and measuring glucose metabolism using the ROI analysis from a targeted brain area.

Semi-quantitative Assessment Using [18F]FDG Tracer in Patients with Severe Brain Injury

[18F]-fluorodeoxyglucose (FDG) positron emission tomography-computed tomography is useful for studying glucose metabolism related to brain function. Here, we present a protocol for an [18F]FDG tracer set-up and semiquantitative assessment of the region-of-interest analysis for targeted brain areas associated with clinical manifestations in patients with severe traumatic brain injury.

Patients with severe traumatic brain injury (sTBI) have difficulty knowing whether they are accurately expressing their thoughts and emotions because of ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Developing a Long-Term Swine Model for Biofilm-Infected Burn Wound Studies

Swine Model of Biofilm Infection and Invisible Wounds

Biofilm infection is a major contributor to wound chronicity. The establishment of clinically relevant experimental wound biofilm infection requires the involvement of the host immune system. Iterative changes in the host and pathogen during the formation of such clinically relevant biofilm can only occur in vivo. The swine wound model is recognized for its advantages as a powerful pre-clinical model. There are several reported approaches for studying wound biofilms. In vitro and ex vivo systems are deficient in terms of the host immune response. Short-term in vivo studies involve acute responses and, thus, do not allow for biofilm maturation, as is known to occur clinically. The first long-term swine wound biofilm study was reported in 2014. The study recognized that biofilm-infected wounds may close as determined by planimetry, but the skin barrier function of the affected site may fail to be restored. Later, this observation was validated clinically. The concept of functional wound closure was thus born. Wounds closed but deficient in skin barrier function may be viewed as invisible wounds. In this work, we seek to report the methodological details necessary to reproduce the long-term swine model of biofilm-infected severe burn injury, which is clinically relevant and has translational value. This protocol provides detailed guidance on establishing an 8 week wound biofilm infection using P. aeruginosa (PA01). Eight full-thickness burn wounds were created symmetrically on the dorsum of domestic white pigs, which were inoculated with (PA01) at day 3 post-burn; subsequently, noninvasive assessments of the wound healing were conducted at different time points using laser speckle imaging (LSI), high-resolution ultrasound (HUSD), and transepidermal water loss (TEWL). The inoculated burn wounds were covered with a four-layer dressing. Biofilms, as established and confirmed structurally by SEM at day 7 post-inoculation, compromised the functional wound closure. Such an adverse outcome is subject to reversal in response to appropriate interventions.

Author Spotlight: Studying Host-Microbe Interactions in Wound Biofilm Formation 

Chronic wounds that are resistant to antibiotics are a major threat to the healthcare system. Biofilm infections are stubborn and hostile and can cause deficient functional wound closure. We report a clinically relevant swine model of biofilm-infected full-thickness chronic wounds. This model is powerful for mechanistic studies as well as for testing interventions.

Biofilm infection is a major contributor to wound chronicity. The establishment of clinically relevant experimental wound biofilm infection requires the ...

Immunology and Infection

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

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.

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.