The authors thank the Division of Comparative Medicine at the University of North Carolina for the provision and care of animals. We thank Lauren Ralph and Mia Evangelista in the Pathology Services Core for expert technical assistance with Histopathology/Digital Pathology, including tissue sectioning and imaging. This research was supported by a research grant from the Department of Defense (Award number W81XWH-20-1-0500, GR and TV).

All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of The University of North Carolina and were conducted in accordance with its established guidelines. Male and female Sprague Dawley rats (250-300 g) aged 7-9 weeks old were used for the experiments. All animals were housed in a 12 h:12 h light-dark cycle with free access to food and water ad libitum. Always work with your institutional veterinarian about an analgesic plan prior to study initiation.&#160;
<p class="jove_t...

<ol>
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F., 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=National+burn+repository+2007+report:+a+synopsis+of+the+2007+call+for+data.">National burn repository 2007 report: a synopsis of the 2007 call for data.</a> Journal of Burn Care &amp; Research. 29 (6), 862-870 (2008).</li><li>Kruger, E., Kowal, S., Bilir, S. P., Han, E., Foster, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+patient+characteristics+and+number+of+procedures+as+well+as+length+of+stay+for+patients+surviving+severe+burn+injuries:+analysis+of+the+American+Burn+Association+National+Burn+Repository.">Relationship between patient characteristics and number of procedures as well as length of stay for patients surviving severe burn injuries: analysis of the American Burn Association National Burn Repository.</a> Journal of Burn Care &amp; Research. 41 (5), 1037-1044 (2020).</li><li>American Burn Association. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burn+incidence+and+treatment+in+the+United+States:+2016.+Burn+Incidence+Fact+Sheet.">Burn incidence and treatment in the United States: 2016. Burn Incidence Fact Sheet.</a> Chicago: American Burn Association. , (2016).</li><li>Willis, M. L., 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=Plasma+extracellular+vesicles+released+after+severe+burn+injury+modulate+macrophage+phenotype+and+function.">Plasma extracellular vesicles released after severe burn injury modulate macrophage phenotype and function.</a> Journal of Leukocyte Biology. 111 (1), 33-49 (2022).</li><li>Kartchner, L. B., 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=One-hit+wonder:+late+after+burn+injury,+granulocytes+can+clear+one+bacterial+infection+but+cannot+control+a+subsequent+infection.">One-hit wonder: late after burn injury, granulocytes can clear one bacterial infection but cannot control a subsequent infection.</a> Burns. 45 (3), 627-640 (2019).</li><li>Abdullahi, A., Amini-Nik, S., Jeschke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+in+burn+research.">Animal models in burn research.</a> Cellular and Molecular Life Sciences. 71 (17), 3241-3255 (2014).</li><li>Cai, E. 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(119), e55281 (2017).</li><li>Naldaiz-Gastesi, N., Bahri, O. A., Lopez de Munain, A., McCullagh, K. J., Izeta, 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+panniculus+carnosus+muscle:+an+evolutionary+enigma+at+the+intersection+of+distinct+research+fields.">The panniculus carnosus muscle: an evolutionary enigma at the intersection of distinct research fields.</a> Journal of Anatomy. 233 (3), 275-288 (2018).</li><li>Weber, B., 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=Modeling+trauma+in+rats:+similarities+to+humans+and+potential+pitfalls+to+consider.">Modeling trauma in rats: similarities to humans and potential pitfalls to consider.</a> Journal of Translational Medicine. 17 (1), 1-19 (2019).</li><li>Nguyen, J. Q. 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=Spatial+frequency+domain+imaging+of+burn+wounds+in+a+preclinical+model+of+graded+burn+severity.">Spatial frequency domain imaging of burn wounds in a preclinical model of graded burn severity.</a> Journal of Biomedical Optics. 18 (6), 066010 (2013).</li><li>Sobral, C., Gragnani, A., Morgan, J., Ferreira, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+proliferation+of+Pseudomonas+aeruginosa+by+KGF+in+an+experimental+burn+model+using+human+cultured+keratinocytes.">Inhibition of proliferation of Pseudomonas aeruginosa by KGF in an experimental burn model using human cultured keratinocytes.</a> Burns. 33 (5), 613-620 (2007).</li><li>Olivera, F., Bevilacqua, L., Anaruma, C., Boldrini Sde, C., Liberti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+changes+in+distant+muscle+fibers+following+thermal+injury+i+n+Wistar+rats.">Morphological changes in distant muscle fibers following thermal injury i n Wistar rats.</a> Acta Cirurgica Brasileira. 25, 525-528 (2010).</li><li>Davies, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Physiological Responses to Burning Injury. , (1982).</li><li>Neely, C. J., 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=Flagellin+treatment+prevents+increased+susceptibility+to+systemic+bacterial+infection+after+injury+by+inhibiting+anti-inflammatory+IL-10++IL-12-neutrophil+polarization.">Flagellin treatment prevents increased susceptibility to systemic bacterial infection after injury by inhibiting anti-inflammatory IL-10+ IL-12-neutrophil polarization.</a> PloS One. 9 (1), e85623 (2014).</li><li>Dunn, J. 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Several burn models have been presented to study the pathophysiology of burn injury8,12,16,17. In the present study, we employed a rat model to develop a simple and reproducible protocol to induce a full-thickness burn followed by bacterial infection to simulate an infected burn trauma in patients. The choice of the rat as the animal model to mimic human conditions is based on a balance of cost...

Burn injuries are among the most devastating&#160;forms of trauma, with mortality rates reaching 12% even in specialized burn centers1,2,3. According to recently published reports, ~486,000 burn patients require medical care annually in the United States, with nearly 3,500 deaths1,2,3,4,5,6. Burn injury imposes a major challenge for patients&#39; immune system and creates a significant open wound, which is slow to heal, leaving them susceptible to cutaneous, pulmonary, and systemic colonization with nosocomial, opportunistic bacteria. Immune dysregulation combined with the bacterial infection is associated with increased morbidity and mortality in burn patients7.
An animal burn and infection model is essential for studying the pathogenesis of bacterial infections following skin damage and immune suppression associated with burn trauma. Such models enable the design and evaluation of new methods for treating bacterial infections in burn patients. Rats and humans share similar skin physiological and pathological characteristics that have been previously documented8. Additionally, rats are smaller in size, making them easier to handle, more affordable, and easier to procure and maintain than larger animal models.
These characteristics make rats an ideal model animal to study burns and infections9. Unfortunately, the technique for burn induction is inconsistent and often minimally described10,11,12,13,14. The present protocol is designed to develop a simple, cost-effective, and reproducible procedure for creating a consistent full-thickness burn injury in a rat model that simulates the clinical scenario and can be used to evaluate immune suppression and bacterial infection.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringe</td>
			<td>BD, USA</td>
			<td>309597</td>
			<td>Used to inject the analgesic</td>
		</tr>
		<tr>
			<td>1.7 mL Microtube</td>
			<td>Olympus, USA</td>
			<td>24-282</td>
			<td>Used to carry morphine</td>
		</tr>
		<tr>
			<td>10% NBF</td>
			<td>VWR, USA</td>
			<td>16004-115</td>
			<td>Used to fix the skin piece for staining</td>
		</tr>
		<tr>
			<td>30 mL syringe</td>
			<td>BD, USA</td>
			<td>302832</td>
			<td>Used to inject the lactate ringer solution</td>
		</tr>
		<tr>
			<td>70% ethyl alcohol</td>
			<td>Fischer Scientific, USA</td>
			<td>BP28184</td>
			<td></td>
		</tr>
		<tr>
			<td>Aperio AT2 Digital Pathology&#160; Slide Scanner with ImageScope software</td>
			<td>Aperio, Technologies Inc., Vista, CA, USA</td>
			<td>n/a</td>
			<td>Scanning of H &#38; E slides and analysis</td>
		</tr>
		<tr>
			<td>Cetrimide agar plates</td>
			<td>BD, USA</td>
			<td>285420</td>
			<td>Selective media plates for Pseudomonas aeruginosa growth</td>
		</tr>
		<tr>
			<td>Copper rods</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>Used to induce the burn injury</td>
		</tr>
		<tr>
			<td>Cotton tipped applicators</td>
			<td>OMEGA Surgical supply, USA</td>
			<td>4225-IMC</td>
			<td>Used to apply eye ointment</td>
		</tr>
		<tr>
			<td>Electric shaver</td>
			<td>Oster, USA</td>
			<td>Golden A5</td>
			<td>Used to remove the dorsal side hairs</td>
		</tr>
		<tr>
			<td>Eye lube</td>
			<td>Dechra, UK</td>
			<td>n/a</td>
			<td>The eye wetting agent to provide long lasting comfort and avoid eye dryness</td>
		</tr>
		<tr>
			<td>Fluff filled underpads</td>
			<td>Medline, USA</td>
			<td>MSC281225</td>
			<td>Used in the burn procedure</td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>F.S.T.</td>
			<td>11027-12</td>
			<td>Used to hold the skin piece</td>
		</tr>
		<tr>
			<td>Gauze sponges</td>
			<td>Oasis, USA</td>
			<td>PK412</td>
			<td>Used to clean the applied nair cream from the dorsal side&#160;</td>
		</tr>
		<tr>
			<td>Heat-resistant gloves</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>Used to hold the heated copper rods</td>
		</tr>
		<tr>
			<td>Hematology Analyzer</td>
			<td>IDEXX laboratories, USA</td>
			<td>ProCyte Dx</td>
			<td></td>
		</tr>
		<tr>
			<td>Induction chamber</td>
			<td>Kent Scientific, USA</td>
			<td>vetFlo-0730</td>
			<td>Used to anesthesize the animals</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>BD, USA</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Pivetal, USA</td>
			<td>NDC46066-755-04</td>
			<td>Used to anesthesized rats to induce a loss of consciousness</td>
		</tr>
		<tr>
			<td>Isoflurane vaporiser</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Lactated ringer&#39;s solution</td>
			<td>icumedical, USA</td>
			<td>NDC0990-7953-09</td>
			<td>Used to resuscitate the rats</td>
		</tr>
		<tr>
			<td>L-shaped spreader</td>
			<td>Fischer Scientific, USA</td>
			<td>14-665-230</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol Agar</td>
			<td>BD, USA</td>
			<td>211407</td>
			<td>Selective media plates for Staphylococcus aureus growth</td>
		</tr>
		<tr>
			<td>Minicollect tubes (K2EDTA)</td>
			<td>greiner bio-one, USA</td>
			<td>450480</td>
			<td>Used to collect the blood</td>
		</tr>
		<tr>
			<td>Morphine</td>
			<td>Mallinckrodt, UK</td>
			<td>NDC0406-8003-30</td>
			<td>This analgesia was used to induce the inability to feel burn injury pain</td>
		</tr>
		<tr>
			<td>Muller Hinton Broth</td>
			<td>BD, USA</td>
			<td>275730</td>
			<td></td>
		</tr>
		<tr>
			<td>Muller Hinton II Agar</td>
			<td>BD, USA</td>
			<td>211438</td>
			<td></td>
		</tr>
		<tr>
			<td>Nair hair removal lotion</td>
			<td>Nair, USA</td>
			<td>n/a</td>
			<td>Used to remove the residual hairs on dorsal side</td>
		</tr>
		<tr>
			<td>Needle 23 G</td>
			<td>BD, USA</td>
			<td>305193</td>
			<td>Used to inject the lactate ringer solution</td>
		</tr>
		<tr>
			<td>Normal saline</td>
			<td>n/a</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>ThermoScientific, USA</td>
			<td>Genesys 30</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague-Dawley rats, male and female</td>
			<td>Charles River Labs</td>
			<td>n/a</td>
			<td>7-9 weeks old for burn induction</td>
		</tr>
		<tr>
			<td>Surgical Scissor</td>
			<td>F.S.T.</td>
			<td>14501-14</td>
			<td>Used to cut the desired skin piece</td>
		</tr>
		<tr>
			<td>Tissue collection tubes</td>
			<td>Globe Scientific</td>
			<td>220101236</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Homogenizer</td>
			<td>Kinematica, Inc, USA</td>
			<td>POLYTRON PT2100</td>
			<td>Used to homogenize the tissue samples</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Fischer Scientific, USA</td>
			<td>n/a</td>
			<td>Used to induce the burn injury</td>
		</tr>
		<tr>
			<td>Weighted heating pad</td>
			<td>Comfytemp, USA</td>
			<td>n/a</td>
			<td>Used during the procedure to keep rat&#39;s body warm</td>
		</tr>
	</tbody>
</table>

rat burn model to study full-thickness cutaneous thermal burn and infection

Burn induction methodologies are inconsistently described in rat models. A uniform burn wound model, which represents the clinical scenario, is necessary to perform reproducible burn research. The present protocol describes a simple and reproducible method to create ~20% total body surface area (TBSA) full-thickness burns in rats. Here, a 22.89 cm2 (5.4 cm diameter) copper rod heated at 97&#160;&#176;C in a water bath was applied to the rat skin surface to induce the burn injury. A copper rod with a high thermal conductivity was able to dissipate the heat deeper in the skin tissue to create a full-thickness burn. Histology analysis shows attenuated epidermis with coagulative damage to the full-thickness extent of the dermis and the subcutaneous tissue. Additionally, this model is representative of the clinical situations observed in hospitalized burn patients following burn injury such as immune dysregulation and bacterial infections. The model can recapitulate the systemic bacterial infection by both Gram-positive and Gram-negative bacteria. In conclusion, this paper presents an easy-to-learn and robust rat burn model that mimics the clinical situations, including immune dysregulation and bacterial infections, which is of considerable utility for the development of new topical antibiotic drugs for burn wound and infections.

The protocol presented here is highly reproducible and resulted in a third-degree, full-thickness burn injury in rats. The burn wound appears waxy white after burn induction (Figure 2B). The color of the burn injury changed from white to brown over the course of 72 h post burn (Figure 2B-E).
Histological analysis confirmed a full-thickness burn (depth &#62;2.61 mm at 24 h post burn; <strong class="xfi...

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Rat Burn Model to Study Full-Thickness Cutaneous Thermal Burn and Infection

A model mimicking the clinical scenario of burn injury and infection is necessary for furthering burn research. The present protocol demonstrates a simple and reproducible rat burn infection model comparable to that in humans. This facilitates the study of burn and infections following burn for developing new topical antibiotic treatments.

Burn induction methodologies are inconsistently described in rat models. A uniform burn wound model, which represents the clinical scenario, is necessary ...

This burn injury model is able to recapitulate the clinical situations observed in hospitalized burn patients following burn injuries such as immune dysregulation and bacterial infections. This method of inducing burn in rats is easy to learn and can be used for the development and evaluation of new tropical drugs for burn wounds and infections. This model can be used to evaluate wound healing as well as the progression of bacterial infection at the burn site.This burn induction method can also be used in mouse and other animal models for burn induction and to study the bacterial infection of burn wounds. Begin preparing the anesthetized rat for the burn injury by moving it over a heating pad in a prone position. Using a cotton-tipped applicator, apply eye lubricant on both eyes to prevent corneal drying.Shave the rat's dorsal area with an electric clipper in a large rectangle from the shoulder blades down to the base of the tail. Wipe out the loose hairs from the shaved area using the tissue soaked in saline. Apply the hair removal lotion to the shaved area using a cotton-tipped applicator.After three minutes, wipe the area with a wet gauze sponge twice to remove the lotion and prevent skin irritation. Turn off the isoflurane and remove the nose cone before placing the rat in the recovery cage. On the day of the burn injury experiment, set the water bath temperature to 97 degrees Celsius and one hour prior to the experiment, place 420 grams of four copper rods in the water bath.Once the rat is unresponsive to toe pinch on all the limbs, place him on the heating pad in a prone position and inject 20 milligrams per kilogram of body weight of morphine via the intraperitoneal, or IP root, for pain management. Next, check the water bath temperature. Set the timer and put on the heat-resistant gloves.Take one heated copper rod from the water bath and touch it on the dorsum area for seven seconds. Immediately apply the next three burns one after the other using one rod per burn site and produce an approximately 20%total body surface area full-contact burn. After the burn, resuscitate the animal by injecting 0.1 milliliter per gram body weight of lactated Ringer's solution through the IP route.Once done, turn off the isoflurane. Remove the nose cone and place the rat on the heat mat. On the burn and infection day, centrifuge the Pseudomonas aeruginosa or Staphylococcus aureus culture separately at 4, 000 times g for 5 minutes.Wash the pellet with normal saline before diluting the pellet using saline to 0.1 optical density at 600 nanometer. Dilute 200 microliters of the bacterial suspension with 800 microliters of saline and get the desired bacterial inoculum of 2 by 10 to the 7th colony-forming units or CFU per milliliter. 15 minutes after the burn, subcutaneously inject 50 microliters of diluted inoculum of the bacteria near the burn area using a 29 gauge needle.Place the rat on the heating pad for recovery. After 15 to 20 minutes, transfer the recovered animal to a clean cage. Immediately after the burn injury, evaluate the skin injury morphologically in terms of color and margin.After euthanization, analyze complete blood counts to determine the effect of burn induction on the host immune system. Collect the tissue of the euthanized rat in a 10-milliliter collection tube. Homogenize the samples using a tissue homogenizer and serially dilute the tissue homogenates in normal saline.Plate 100 microliters of undiluted homogenate and all dilutions of each tissue sample collected from P.Aeruginosa-infected rats on cetrimide agar plates. After incubating the plates for 16 to 18 hours at 37 degrees Celsius in an incubator, count the bacterial colonies on the plates. Multiply them by the dilution ratio to get the CFU per milliliter count and normalize it with the tissue's weight to calculate the CFU program tissue.This highly reproducible protocol resulted in a third-degree full-thickness burn injury in rats. The color of the burn injury changed from white to brown in 24 hours to 72 hours post-burn. In histological analysis, the skin samples from burned animals showed injury across all layers at 24, 48, and 72 hours post-burn injury, whereas the non-burn skin showed a clear distinction of epidermis, dermis, and subcutaneous tissue layers.Destruction of the epidermal layer and damage to the dermis'full thickness with subcutaneous fat and skeletal muscle were observed in burned skin samples. In 24-hour post-burn, a full thickness burn was more than 2.61 millimeters in depth. In bacterial clearance evaluation, bacterial recovery was observed in all burn injury rats.During P.aeruginosa PAO1 infection, the number of bacteria recovered 24 hours post-infection from the skin of burned rat was less than the initial inoculum of 1 by 10 to the 6th CFU. However, it increased 48 and 72 hours post-infection. In S.aureus, ATC25923 infection, 2 log 10 increase was observed at all time points in the skin compared to the initial inoculum, suggesting that the establishment of S.aureus infection resulted due to the active replication in the tissues.The subcutaneous tissue and muscles showed a higher bacterial load than the lung and spleen. The data showed that burned rats developed a systemic infection 24 hours following P.aeruginosa inoculation and 48 hours following S.aureus inoculation. Completely remove hair on the rat dorsum and keep the temperature and burn induction time as mentioned in the protocol.The leftover hair and different temperature of the copper rods can affect the quality of the burn. The rat burn model can be explored to evaluate novel topical therapeutics for treatment of burn wound infections and for evaluating different wound dressings.

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Research

JoVE Journal

Immunology and Infection

4.1K 조회수.  University of North Carolina at Chapel Hill. 이 화상 손상 모델은 면역 조절 장애 및 세균 감염과 같은 화상 부상 후 입원한 화상 환자에서 관찰된 임상 상황을 요약할 수 있습니다. 쥐에서 화상을 유도하는이 방법은 배우기 쉽고 화상 상처 및 감염에 대한 새로운 열대 약물의 개발 및 평가에 사용할 수 있습니다. 이 모델은 화상 부위에서 상처 치유와 세균 감염의 진행을 평가하는 데 사용할 수 있습니다.이 화상 유...