Name: rat burn model to study full-thickness cutaneous thermal burn and infection
Uploaded: 2022-08-23T14:40:00.000+00:00
Duration: 8 min 40 s
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;
1. Preparing rats for the burn injury
<ol>
	<li>Prepare the animals for burn injury 24 h prior to the burn.</li>
	<li>Anesthetize the rat with 5% isoflurane in 100% oxygen in an induction chamber for 5 min (flow rate: 2 L/min) until breathing has slowed.</li>
	<li>Once the rat is deeply anesthetized (unresponsive to toe pinch on all limbs), move the rat over to a heating pad in a prone position and reduce the isoflurane to 1.5% in oxygen for maintenance through a nose cone.</li>
	<li>To prevent corneal drying after anesthesia and during the procedure, apply eye lubricant on the corneas of both eyes using a cotton-tipped applicator.</li>
	<li>Shave the dorsal area of the rat using an electric clipper (see the Table of Materials) and remove as much hair as possible in a large rectangle from the shoulder blades down to the base of the tail (Figure 2A).</li>
	<li>Clean the shaved area with a tissue soaked in saline to wipe out the loose hairs. Apply hair removal lotion to the shaved area using a cotton-tipped applicator and leave it on for ~3 min. 
	NOTE: Application of the referenced hair removal lotion for more than 3 min will induce red rashes on the skin.</li>
	<li>Wipe the area with a wet gauze sponge twice to remove the lotion and prevent skin irritation.</li>
	<li>Turn off the isoflurane, remove the nose cone, and place the rat in the recovery cage. 
	NOTE: Put a heating pad in the recovery cage.</li>
	<li>Transfer the recovered animal to a clean housing cage for the next day&#39;s burn procedure (it may take ~10-15 min for the rat to recover from the anesthesia).</li>
</ol>
2. Inducing the burn injury in rats
<ol>
	<li>On the day of the burn, set the water bath temperature to 97 &#176;C and place all four copper rods (420 g each; Figure 1) in the water bath 1 h prior to the burn experiment to let the rods warm up uniformly. 
	NOTE: The rods must be immersed in the water. Check the accuracy of the digital temperature display using a thermometer before the experiment.</li>
	<li>Anesthetize the rat as mentioned in section 1.</li>
	<li>Once the rat is unresponsive to toe pinch on all limbs, place it on a heating pad in a prone position with 1.5% isoflurane in oxygen for maintenance (Figure 2A).</li>
	<li>Inject morphine (20 mg/kg body weight) via the intraperitoneal (i.p.) route for pain management6.</li>
	<li>Check the temperature of the water in the water bath. Set up the timer and put on the heat-resistant gloves.</li>
	<li>Take out one heated copper rod from the water bath and touch it on the dorsum area of the rat for 7 s to induce the burn. 
	NOTE: Keep a minimum distance (10-15 cm) between the water bath and the animal to minimize heat loss, and do not put pressure on the rods while inducing the burn (i.e.,&#160;contact must be maintained by gravity).</li>
	<li>Apply four burns, using one rod per burn site, one immediately after the other to produce an approximately 20% TBSA full-contact burn (Figure 2B).</li>
	<li>After the burn, resuscitate the animal by i.p. injection of lactated Ringer&#39;s solution (0.1 mL/g body weight). 
	NOTE: Use a body temperature-adjusted lactated ringer&#39;s solution to resuscitate the rats.</li>
	<li>Turn off the isoflurane, remove the nose cone, and place the rat on the heat mat for recovery.</li>
</ol>
3. Preparation of bacterial inoculum and infection
<ol>
	<li>Streak the frozen sample of Pseudomonas aeruginosa PAO1 and Staphylococcus aureus ATCC25923 on Muller Hinton Agar (MHA) plates, 2 days prior to the burn experiment.</li>
	<li>On the next day, select a single colony of grown bacteria from the plate, and using an inoculation loop, slightly scrape it off the plate. Then, place it in the culture tube to inoculate 10 mL of Muller Hinton Broth (MHB) and culture overnight at 37 &#176;C in an incubator shaker.</li>
	<li>On the day of the burn and infection, centrifuge the culture at 4,000 &#215; g for 5 min. Wash the pellet with normal saline (0.9% NaCl solution).</li>
	<li>Resuspend the bacterial pellet in saline and dilute up to 0.1 OD600nm (optical density at 600 nm). Dilute the bacterial inoculum by taking 200 &#181;L of this bacterial suspension and mixing it with 800 &#181;L of saline to get the desired bacterial inoculum of 2 &#215; 107 CFU/mL.</li>
	<li>Inject 50 &#181;L of P. aeruginosa or S. aureus inoculum prepared in the previous step (infection dose 1 &#215; 106 CFU) into the anesthetized rat 15 min after the burn, using a 29 G needle subcutaneously as close to the burn wound as possible.</li>
	<li>After infecting the burn wound, place the rat on the heating pad for recovery. Once the animal recovers (~15-20 min), house it in a clean cage. 
	NOTE: After the burn injury, house one rat per cage. Use water wet food pellets for easy chewing and place them on the cage floor for easy reach.</li>
	<li>Fill the water bottles in the cage with morphine-spiked water (0.4 mg/mL) for pain management. 
	NOTE: Oral Morphine mirrors the clinical situation with human burn patients. This study utilized oral morphine to keep these experiments comparable to human burn patients after consulting with the veterinary staff on numerous occasions. Drinking and weight logs were maintained throughout the experiment. Use the same drinking system during all the procedures. Other analgesics, such as Buprenorphine, can be administered subcutaneously/intraperitoneally as per institutional animal care guidelines.&#160;&#160;</li>
	<li>Fill out the monitoring checklist and monitor the animals closely for distress or illness for the entire duration of the experiment.</li>
</ol>
4. Evaluation of the burn injury
<ol>
	<li>Evaluate the skin burn injury morphologically in terms of color and margin immediately after the burn injury.</li>
	<li>Stain the burned skin with hematoxylin and eosin (H&#38;E) to visualize the burn wound structure and epithelial gap15 (see step 5.6 for sample processing).</li>
</ol>
5. Postprocessing of rat samples and bacterial enumeration
<ol>
	<li>Euthanize the rat at 24, 48, and 72 h post burn with an overdose of anesthesia.</li>
	<li>Withdraw blood samples from the rats via cardiac puncture and collect them in a mini collect tube.
	<ol>
		<li>Analyze complete blood counts from the blood samples to determine the effect of burn induction on the host immune system.</li>
	</ol>
	</li>
	<li>Harvest skin, subcutaneous tissue, muscle, lung, and spleen at the time of euthanasia. 
	NOTE: Keep one part (~1 cm &#215; 1 cm; weighing ~200-300 mg) of the skin for H&#38;E staining and another part for bacterial enumeration.</li>
	<li>Collect the tissues in a 10 mL collection tube and place them in normal saline on ice for bacterial enumeration.</li>
	<li>Normalize the tissue weight with normal saline and homogenize the samples using a tissue homogenizer (see the Table of Materials).
	<ol>
		<li>Serially dilute the tissue homogenates in normal saline.</li>
		<li>Plate 100 &#181;L of undiluted homogenate and all dilutions of each tissue sample on cetrimide agar plates for samples collected from rats infected with P. aeruginosa. 
		NOTE: Use mannitol agar plates for plating samples collected from rats infected with S. aureus.</li>
		<li>Incubate the plates at 37 &#176;C in an incubator for 16-18 h.</li>
		<li>The next day, count the bacterial colonies on the plates, multiply by the dilution ratio to get the CFU/mL count, and normalize with the tissue&#39;s weight to calculate the CFU/g tissue.</li>
		<li>Employ data analysis software to plot the bacterial counts in different organs at the different sampling time-points.</li>
	</ol>
	</li>
	<li>Perform H&#38;E staining of burned skin to visualize the wound structure and epithelial gap.
	<ol>
		<li>Using scissors and toothed forceps, cut a skin patch of 1 cm x 1 cm from the burn area and immerse it in a fixative (10% neutral buffered formalin, NBF) for 48 h at room temperature. 
		NOTE: Swirl the container to ensure all tissues are completely immersed in the fixative, with the volume of the fixative 30x the tissue volume.</li>
		<li>Dehydrate the skin tissue with 70% (v/v) ethanol for 72 h at room temperature.</li>
		<li>Process the dehydrated samples in paraffin blocks to cut the sections and stain with H&#38;E15.</li>
		<li>Digitally image the stained slides in a slide scanner (see the Table of Materials) using a 40x objective.</li>
		<li>Analyze the scanned image using software (see Supplementary File 1 for processing of the image for analysis; see the Table of Materials).</li>
		<li>Examine all fields of the stained skin section to evaluate the condition of the epidermis, dermis, subcutaneous tissue, and skeletal muscle.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to disclose.

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><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&amp;nbsp; Slide Scanner with ImageScope software</td><td>Aperio, Technologies Inc., Vista, CA, USA</td><td>n/a</td><td>Scanning of H &amp;amp; E slides and analysis</td></tr><tr><td>Cetrimide agar plates</td><td>BD, USA</td><td>285420</td><td>Selective media plates for &lt;em&gt;Pseudomonas aeruginosa&lt;/em&gt; 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&amp;nbsp;</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&#039;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 &lt;em&gt;Staphylococcus aureus &lt;/em&gt;growth</td></tr><tr><td>Minicollect tubes (K&lt;sub&gt;2&lt;/sub&gt;EDTA)</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&#039;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 &gt;2.61 mm at 24 h post burn; <strong class="xfig...

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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 ...

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Research

JoVE Journal

Immunology and Infection

4.5K Views.  University of North Carolina at Chapel Hill. Dit brandwondenmodel is in staat om de klinische situaties samen te vatten die worden waargenomen bij gehospitaliseerde brandwondenpatiënten na brandwonden zoals immuunontregeling en bacteriële infecties. Deze methode voor het induceren van brandwonden bij ratten is gemakkelijk te leren en kan worden gebruikt voor de ontwikkeling en evaluatie van nieuwe tropische geneesmiddelen voor brandwonden en infecties. Dit model kan worden gebruikt om wondgenezing en de progressie van bacteriële infe...

Video: Rat Burn Model to Study Full-Thickness Cutaneous Thermal Burn 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 ...

Severe Burn Injury in a Swine Model for Clinical Dressing Assessment

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

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

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

Medicine

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 ...

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

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

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

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

Biology

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.

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

Consistent Burn Injury Model in Mice for Evaluating Therapeutic Dressings

Chessboard-like Burn Wound Healing Model of Mice Based on Digital Heating Device

Severe burn injuries are among the most traumatic and physically debilitating conditions, impacting nearly every organ system and resulting in considerable morbidity and mortality. Given their complexity and the involvement of multiple organs, various animal models have been created to replicate different facets of burn injury. Methods used to produce burned surfaces vary among experimental animal models. This study describes a simple, cost-effective, and user-friendly mouse burn model for creating consistent full-thickness burns using a digital heating device. The tip of this device was applied to the dorsum of mice for 10 s at 97 &#176;C to establish a chessboard-like burn and examine wound healing under the treatment of an experimental dressing. Skin samples were collected for histological analysis, including Hematoxylin and Eosin (H&amp;E) staining and Masson's staining. Wound healing was assessed through analysis of the wound area and microscopic examination of inflammatory infiltration, re-epithelialization, and granulation tissue formation. The mouse burn injury model can serve as a fundamental tool in studying the pathophysiology of thermal injuries and evaluating therapeutic interventions.

A streamlined protocol is presented for establishing a burn wound healing model in mice using a digital heating device. The chessboard-like experimental sites created on the skin facilitate further functional analysis for the wound healing assay.

Severe burn injuries are among the most traumatic and physically debilitating conditions, impacting nearly every organ system and resulting in ...

Burn Injuries

Burn injuries occur when the skin and underlying tissues are damaged due to exposure to heat, electricity, chemicals, radiation, or friction. They can vary in severity, from minor superficial burns to severe deep burns that can be life-threatening.
The damage results in the death of skin cells, which can lead to a massive loss of fluid. Dehydration, electrolyte imbalance, and renal and circulatory failure follow, which can be fatal. Burn patients are treated with intravenous fluids to offset dehydration, as well as intravenous nutrients that enable the body to repair tissues and replace lost proteins. Another serious threat to the lives of burn patients is infection. Burned skin is extremely susceptible to bacteria and other pathogens, due to the loss of protection by intact layers of skin.
In addition to the depth of the burn, burns are also classified by the total body surface area (TBSA) affected. The &#34;Rule of Nines&#34; is commonly used to estimate the TBSA involved in adults, where each major body part is assigned a percentage (e.g., the head is 9%, each arm is 9%, the front chest and abdomen are 18%, etc.). For children, a modified rule is used to account for the differences in body proportions.
Burns are also classified by the degree of their severity:
A first-degree burn is a superficial burn that affects only the epidermis. Although the skin may be painful and swollen, these burns typically heal on their own within a few days. Mild sunburn fits into the category of a first-degree burn.
A second-degree burn goes deeper and affects both the epidermis and a portion of the dermis. These burns result in swelling and painful blistering of the skin. It is important to keep the burn site clean and sterile to prevent infection. If this is done, the burn will heal within several weeks.
A third-degree burn fully extends into the epidermis and dermis, destroying the tissue and affecting the nerve endings and sensory function. These are serious burns that may appear white, red, or black; they require medical attention and will heal slowly without it. Oddly, third-degree burns are usually not as painful because the nerve endings themselves are damaged. Full-thickness burns cannot be repaired by the body, because the local tissues used for repair are damaged. They require excision (debridement), or amputation in severe cases, followed by grafting of the skin from an unaffected part of the body, or from skin grown in tissue culture for grafting purposes.
Treatment for burn injuries depends on the severity and extent of the burns. It may involve cleaning and debriding the wound, applying topical medications or dressings, administering pain medications, and providing supportive care such as intravenous fluids and nutritional support. Severe burns often require specialized care in burn centers or hospitals with expertise in burn management.
This text is adapted from <a href="https://openstax.org/books/anatomy-and-physiology/pages/5-4-diseases-disorders-and-injuries-of-the-integumentary-system">Openstax, Anatomy and Physiology 2e, Section 5.4: Diseases, Disorders, and Injuries of the Integumentary System.</a>

Burn injuries occur when the skin and underlying tissues are damaged due to exposure to heat, electricity, chemicals, radiation, or friction. They can ...

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

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