Name: rat burn model to study full-thickness cutaneous thermal burn and infection
Uploaded: 2022-08-23T14:40:00
Description: Watch this Scientific Journal Video about Rat Burn Model to Study Full-Thickness Cutaneous Thermal Burn and Infection at JoVE.com

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;
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<ol>
	<li>Peck, M., Molnar, J., Swart, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+plan+for+burn+prevention+and+care.">A global plan for burn prevention and care.</a> Bulletin of the World Health Organization. 87, 802-803 (2009).</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:+2011+fact+sheet.">Burn incidence and treatment in the United States: 2011 fact sheet.</a> Chicago: American Burn Association. , (2011).</li><li>Miller, S. 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. Z., 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=Creation+of+consistent+burn+wounds:+a+rat+model.">Creation of consistent burn wounds: a rat model.</a> Archives of Plastic Surgery. 41 (4), 317 (2014).</li><li>Pessolato, A. G. T., dos Santos Martins, D., Ambr&#243;sio, C. E., Man&#231;anares, C. A. F., de Carvalho, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propolis+and+amnion+reepithelialise+second-degree+burns+in+rats.">Propolis and amnion reepithelialise second-degree burns in rats.</a> Burns. 37 (7), 1192-1201 (2011).</li><li>Gurung, S., &#352;kalko-Basnet, N. <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+properties+of+Carica+papaya+latex:+in+vivo+evaluation+in+mice+burn+model.">Wound healing properties of Carica papaya latex: in vivo evaluation in mice burn model.</a> Journal of Ethnopharmacology. 121 (2), 338-341 (2009).</li><li>Eloy, R., Cornillac, A. <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+of+burns+in+rats+treated+with+a+new+amino+acid+copolymer+membrane.">Wound healing of burns in rats treated with a new amino acid copolymer membrane.</a> Burns. 18 (5), 405-411 (1992).</li><li>Upadhyay, N., 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=Safety+and+healing+efficacy+of+Sea+buckthorn+(Hippophae+rhamnoides+L.)+seed+oil+on+burn+wounds+in+rats.">Safety and healing efficacy of Sea buckthorn (Hippophae rhamnoides L.) seed oil on burn wounds in rats.</a> Food and Chemical Toxicology. 47 (6), 1146-1153 (2009).</li><li>El-Kased, R. F., Amer, R. I., Attia, D., Elmazar, 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=Honey-based+hydrogel:+In+vitro+and+comparative+In+vivo+evaluation+for+burn+wound+healing.">Honey-based hydrogel: In vitro and comparative In vivo evaluation for burn wound healing.</a> Scientific Reports. 7 (1), 1-11 (2017).</li><li>Fan, G. -. Y., 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=Severe+burn+injury+in+a+swine+model+for+clinical+dressing+assessment.">Severe burn injury in a swine model for clinical dressing assessment.</a> Journal of Visualized Experiments. (141), e57942 (2018).</li><li>Davenport, L., Dobson, G., Letson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+for+standardising+and+treating+thermal+injury+in+the+rat.">A new model for standardising and treating thermal injury in the rat.</a> MethodsX. 6, 2021-2027 (2019).</li><li>Kaufman, T., Lusthaus, S., Sagher, U., Wexler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+partial+skin+thickness+burns:+a+reproducible+animal+model+to+study+burn+wound+healing.">Deep partial skin thickness burns: a reproducible animal model to study burn wound healing.</a> Burns. 16 (1), 13-16 (1990).</li><li>Casal, 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=Blood+supply+to+the+integument+of+the+abdomen+of+the+rat:+a+surgical+perspective.">Blood supply to the integument of the abdomen of the rat: a surgical perspective.</a> Plastic and Reconstructive Surgery Global Open. 5 (9), (2017).</li><li>Casal, 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=A+model+of+free+tissue+transfer:+the+rat+epigastric+free+flap.">A model of free tissue transfer: the rat epigastric free flap.</a> Journal of Visualized Experiments. (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. 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=Direct+detection+of+blood+nitric+oxide+reveals+a+burn-dependent+decrease+of+nitric+oxide+in+response+to+Pseudomonas+aeruginosa+infection.">Direct detection of blood nitric oxide reveals a burn-dependent decrease of nitric oxide in response to Pseudomonas aeruginosa infection.</a> Burns. 42 (7), 1522-1527 (2016).</li><li>Gouma, E., 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=A+simple+procedure+for+estimation+of+total+body+surface+area+and+determination+of+a+new+value+of+Meeh's+constant+in+rats.">A simple procedure for estimation of total body surface area and determination of a new value of Meeh's constant in rats.</a> Laboratory Animals. 46 (1), 40-45 (2012).</li><li>Dawson, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+surface-area/body-weight+relationship+in+mice.">The surface-area/body-weight relationship in mice.</a> Australian Journal of Biological Sciences. 20 (3), 687-690 (1967).</li><li>Moins-Teisserenc, 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=Severe+altered+immune+status+after+burn+injury+is+associated+with+bacterial+infection+and+septic+shock.">Severe altered immune status after burn injury is associated with bacterial infection and septic shock.</a> Frontiers in Immunology. 12, 529 (2021).</li><li>Robins, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosuppression+of+the+burned+patient.">Immunosuppression of the burned patient.</a> Critical Care Nursing Clinics. 1 (4), 767-774 (1989).</li></ol>

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...

Watch this Scientific Journal Video about Rat Burn Model to Study Full-Thickness Cutaneous Thermal Burn and Infection at JoVE.com

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

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging. Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux.

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected ...

Mouse Footpad Inoculation Model to Study Viral-Induced Neuroinflammatory Responses

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus infection in mice. As alphaherpesviruses are main invaders of the peripheral nervous system (PNS), this model is suitable to characterize the kinetics of viral replication, its spread from the PNS to CNS, and associated neuroinflammatory responses. The footpad inoculation model allows virus particles to spread from a primary infection site in the footpad epidermis to sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads via the sciatic nerve to the dorsal root ganglia (DRG) and ultimately through the spinal cord to the brain. Here, a mouse footpad is inoculated with pseudorabies virus (PRV), an alphaherpesvirus closely related to herpes simplex virus (HSV) and varicella-zoster virus (VZV). This model demonstrates that PRV infection induces severe inflammation, characterized by neutrophil infiltration in the footpad and DRG. High concentrations of inflammatory cytokines are subsequently detected in homogenized tissues by ELISA. In addition, a strong correlation is observed between PRV gene and protein expression (via qPCR and IF staining) in DRG and the production of pro-inflammatory cytokines. Therefore, the footpad inoculation model provides a better understanding of the processes underlying alphaherpesvirus-induced neuropathies and may lead to the development of innovative therapeutic strategies. In addition, the model can guide research on peripheral neuropathies, such as multiple sclerosis and associated viral-induced damage to the PNS. Ultimately, it can serve as a cost-effective in vivo tool for drug development.

The footpad inoculation model is a valuable tool for characterizing viral-induced neuroinflammatory responses in vivo. In particular, it provides a clear assessment of viral kinetics and associated immunopathological processes initiated in the peripheral nervous system.

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus ...