Name: chessboard-like burn wound healing model of mice based on digital heating device
Uploaded: 2024-12-27T14:00:00.000+00:00
Duration: 4 min 4 s
Description: Severe burn injuries are among the most traumatic and physically debilitating conditions, impacting nearly every organ system and resulting in ...

This work was supported by the Sichuan Science and Technology Program (23ZYZYTS0120), the West China Hospital of Stomatology Sichuan University grants (RD-03-202011), and the Sichuan Science and Technology Program (2022NSFSC0614). The figures were created with BioRender. com.

All animal procedures in this study were reviewed and approved by the Ethical Committee of the West China School of Stomatology, Sichuan University (WCHSIRB-D-2024-499). Twenty-four eight-week-old C57BL/6 mice (female, body weight 25-30 g) were used for the present study. The details of the reagents and the equipment used are listed in the Table of Materials.
1. Preparing equipment and mouse before burn injury
<ol>
	<li>Acquire the animals and house them in cages for 7 days upon arrival to allow them to acclimatize to the environment.</li>
	<li>Sterilize the working area by spraying and wiping with 70% ethanol (v/v). Cover the operation table with sterile surgical absorbent pads.</li>
	<li>Adjust the temperature of the digital heating device to 97 &#176;C. The heating device consists of three parts (Figure 1). 
	NOTE: Temperature from 50-450 &#176;C can be adjusted through the temperature controller (Figure 1A). The stand base is used to place the tip (Figure 1B), which can be dismantled (Figure 1C) to replace different instrument sizes. Disinfect the tip with iodophor or alcohol.</li>
	<li>Anesthetize the mouse with 5% isoflurane in 100% oxygen in an induction chamber for 3 min (flow rate: 4 L/min) until breathing has slowed. Assess the depth of anesthesia by using a toe pinch test. 
	NOTE: Inhaling isoflurane may lead to drowsiness and harm the cardiovascular system. It is essential to wear protective equipment and handle it within a fume hood in a well-ventilated area.</li>
	<li>Once the mouse is deeply anesthetized, transfer it to a heated pad in a prone position to maintain warmth and prevent hypothermia during recovery from anesthesia. Reduce the isoflurane to 1. 5% oxygen for maintenance via a nose cone.</li>
	<li>Apply lubricating gel to both eyes of the mouse with a cotton swab to prevent corneal drying.</li>
</ol>
2. Inducing full-thickness burn injury
NOTE: The general process of burn induction and analysis is shown in Figure 2.
<ol>
	<li>Shave the skin of the mouse with an electric shaver over an area of approximately 5 cm long and 4 cm wide from the neck to the tail. Work gently to avoid injuring the skin upon shaving (Figure 3C).</li>
	<li>Use a cotton-tipped applicator to apply depilatory cream on the shaved skin area for 3 min (Figure 3D). 
	NOTE: Avoid applying the depilatory cream to other parts of the mouse to prevent burns, which can be hard to detect. The cream caused skin rashes if used for more than 5 min. The precise duration for optimal efficacy varies among products and should be determined by the operator.</li>
	<li>Wipe off the cream using damp gauze until cream and hair are not detected. Disinfect the surgical area with chlorhexidine scrub followed by 70% ethanol (v/v) three times each. Clean and dry the area with sterile gauze.</li>
	<li>Employ a sterilized 1 mL pipette tip with a diameter of 9 mm (Figure 3A) dipped in sterilized ink to pinpoint the desired wounding sites accurately. The murine dorsal skin is characterized by an uneven topography. Defining the wound area apart from the spinal cord of mice is essential to ensure repeatability.</li>
	<li>Cover the skin with a previously trimmed sterile drape, leaving the surgical area free (Figure 3). Put on heat-resistant gloves and check the temperature of the digital heating device. Prior to and after burn induction, sanitize the device&#39;s tip with 70% ethanol (v/v).</li>
	<li>Apply the tip of the heating device to the center of located wound sites (97 &#176;C for 10 s) to create six round wounds with a diameter of 4 mm (Figure 3B). The interval between adjacent burns is 0.5-1 cm. Determine the number of burns according to the experimental design. 
	NOTE: The burn is caused mainly by heat conduction; therefore, it is unnecessary to exert pressure on the rod, i.e., keep it in contact with the skin by gravity while burn induction. There were six burns for the experimental design. This is the maximum number of burn wounds that can be built according to the device we used and the area of the mouse dorsum. Multiple replicates of the same experimental group can be achieved on this basis.</li>
</ol>
3. Post-burn care and measurement
<ol>
	<li>Apply ice to the burn wound for 5 min to reduce potential pain for pain management (Figure 3G).</li>
	<li>Turn off the isoflurane, and remove the nose cone after the burn injury. Transfer the mouse to a heating pad until the mouse moves normally (e.g., not crouched or shivering).</li>
	<li>Place each mouse in a recovery cage to prevent them from scratching each other. Soften some food pellets by adding 5-10 drops of drinking water to each pellet and place them on the cage floor to make feeding easier. 
	NOTE: Sterilizing the cages by spraying and wiping with 70% ethanol(vol/vol). The bedding should be replaced regularly to keep the mice warm and to absorb urine.</li>
	<li>Administer buprenorphine (0.1 mg/kg) subcutaneously in a caudal location twice daily for a total of 3 days, ensuring at least 8 h between injections.</li>
	<li>Clean all wounds with sterile gauze moistened with saline after 24 h of burn induction. Blot the skin with gauze and apply a small amount of antibacterial gel (TKH hydrogel) using a syringe to cover the burn wound12,14. Repeat this procedure on days 3, 7, and 10 thereafter.</li>
	<li>Use a breathable, transparent dressing to cover the wounds. The size of the dressing should cover about 1 cm around the wound and should not cover the area without hair removal. 
	NOTE: Change the wound dressing at least once every 2 days. Prolonged use of the dressing without replacement may lead to wound infection with pus formation, resulting in delayed wound healing.</li>
	<li>Throughout the follow-up period, measure the weight every 2-3 days. Generally, mice that lose 15%-20% of their initial body weight should be sacrificed and removed from the experiment.</li>
</ol>
4. Wound collection
<ol>
	<li>At 0, 3, 7, 10, and 14 days, mark the wound with the tip of a sterile 1 mL pipette tip, and record it with a digital camera.</li>
	<li>Euthanize the mice by overdose of anesthesia and cervical dislocation (following institutionally approved protocols). 
	NOTE: Be cautious during the cervical dislocation to avoid excessive pulling, which may damage the dorsum wounds.</li>
	<li>With scissors, isolate the wound site and surrounding skin, including the underlying musculature (1 cm x 1 cm). Prepare for at least three samples for each group.</li>
	<li>Fix the skin specimens in 4% paraformaldehyde (PFA) for 24 h and store at 4 &#176;C. The fixative volume must be 30 times the tissue volume15,16. 
	NOTE: PFA is hazardous. Ensure to work in a well-ventilated area and use appropriate protective equipment.</li>
</ol>
5. Wound healing evaluation
<ol>
	<li>Wash the wound tissue twice with PBS and spread it with filter paper, which should be trimmed to an appropriate size (Figure 2H). Due to the presence of some mucous content within the subcutaneous tissue layer, it adheres to the surface of the filter paper without movement or tissue curling.
	<ol>
		<li>Put the filter paper and sample into a tissue cassette for paraffin dehydration to obtain relatively smooth tissue samples and then embed the tissue15,16. Store samples at 4 &#176;C. 
		NOTE: Stretch the tissue as much as possible and avoid any folding, as these folds will become permanent after fixation.</li>
	</ol>
	</li>
	<li>Cut the paraffin-embedded samples to sections of 5 &#181;m thickness and stain slides via H&#38;E and Masson&#39;s trichrome for evaluating the extent of granulation tissue, angiogenesis formation, and collagen deposition, providing evidence to assess the different stages and the progression of the wound healing17,18.</li>
	<li>Capture images of all samples using a microscope with a 40x objective.</li>
	<li>Use compatible software to analyze the stained skin section, including the epidermis, dermis, subcutaneous tissue, and muscle condition.</li>
</ol>

Consistent Burn Injury Model in Mice for Evaluating Therapeutic Dressings

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The authors declare no conflict of interest.

For burn studies, in vitro models typically focus on the inhibitory effects of local antimicrobial agents or antibiotics on bacteria associated with burns, such as Staphylococcus aureus and Pseudomonas aeruginosa19, as well as the impact of various biomaterials (like elastin, silk, and hydrogel dressings14,20) on post-burn inflammatory cells (such as neutrophils, macrophages) or stem cells (like mesenchymal stem cells). These experiments are usually conducted in artificially controlled environments, and cells cultured in vitro may exhibit growth characteristics and behaviors that differ from those in vivo, which may fail to fully replicate the physiological processes of burn wound healing, such as cell-to-cell interactions and the influence of the extracellular matrix. For these reasons, animal models of burns are essential for elucidating the post-burn pathological mechanisms and evaluating novel therapeutic approaches. The wound healing process in mice and humans undergoes four overlapping but distinct stages: inflammation, proliferation, re-epithelialization, and remodeling21,22. There are several biological advantages to using mice as experimental subjects, including the availability of numerous mouse-specific reagents and the feasibility of creating transgenic models to investigate the molecular signaling pathways involved in the recovery process. This makes mice one of the most commonly utilized animal models for research on burns and wound healing.
The present study established a chessboard-like wound model of full-thickness burn injury and evaluated the efficacy of a hydrogel dressing using this model. The results indicate that this model can effectively monitor the clinical performance of experimental dressings. Within each animal, six burn wounds were symmetrically distributed along the spine. Burn wounds can be created in similar locations but on different mice. Due to the limited skin area of the dorsum and the number of wounds, histological analysis showed that the wounds did not interact with each other, ensuring independent evaluation of each experimental dressing in different locations on the same mouse dorsum, minimizing bias related to the wound healing process. In addition, this model also benefits from ensuring that each mouse serves as its own control, making it possible to compare experimental and control treatments on the dorsum of the same mouse, leading to a significant reduction in the number of animals and experimental cost. Due to the varying impacts of different medications on the overall health of mice, if multiple drugs are to be applied on the skin of the same mouse for the treatment of different wounds and subsequent comparison, it may be necessary to observe and verify whether the application of a single drug or the combined application of different drugs will affect the overall health of the mouse. Additionally, attention should be paid to the stability or adhesion of different drugs on the skin surface to prevent cross-contamination of drugs or wound infection due to the mouse scratching during the healing process. 
Skin sample collection is crucial for subsequent histological analysis. The tissue edge will shrink and fold in the fixative without any treatment16. After paraffin dehydration and section, it will also be curved or even partially overlapped on the slide, resulting in unsightly images under the microscope and potentially affecting further histological analysis, such as the statistical analysis of wound length or thickness. Therefore, using filter paper to flatten the tissue can obtain a smoother section on the slide, which makes the histological image neat and facilitates further statistical analysis. We also tried using paper clips to fix the sample before dehydration, which helped ensure tissue smoothness. However, the application of paper clips may exert pressure on the clamped tissue, leading to tissue squeezing and deformation. Therefore, when using this method, the burn wound part cannot be clamped to avoid affecting observation. 
Numerous factors can influence the healing process, including pain, itching, and bacterial infection23. Effective management of burn pain is crucial for the recovery and reintegration of patients with burn injuries. Inadequate pain control can impede the healing process, leading to enduring physical and psychological challenges, as well as extended hospital stays24. Generally, pain affects feeding behavior in mice. Therefore, each wound was immediately treated with ice after the burn, and injected buprenorphine (0.1 mg/kg) subcutaneously twice a day for 3 days for pain management. Additionally, food pellets were softened and placed on the cage floor to facilitate feeding. In communal housing setups, mice may scratch and lick each other's wounds, potentially delaying wound healing or causing infections. Therefore, individual housing of mice becomes necessary as a preventive measure. Using breathable dressing to cover the dorsum after burn induction can prevent the mice from scratching the wound and reduce the risk of infection25. However, it's essential to select dressings judiciously and cut them to the proper size to allow for normal movement of the mice. Dressings should be promptly replaced if they fall off, with a recommendation to change them at least once every two days. Prolonged use of dressings may lead to inadequate absorption of wound exudates. 
Although the mouse model has its specific advantages, it still cannot fully simulate the wound-healing process in humans. Humans primarily heal through re-epithelialization and granulation. In contrast, mouse skin features a unique panniculus carnosus, a thin layer of skeletal muscle that is only present in the platysma of the neck in humans7, so wound healing occurs primarily through wound contraction. Additionally, mouse skin may possess an enriched pool of progenitor cells, which facilitates rapid skin healing and keratinization26,27. These factors collectively contribute to substantially reduced healing time. In animal models, wound anti-contraction rings have been utilized to mitigate the impact of wound contraction on the healing process and mimic human-like physiological wound healing that heals with granulation tissue and re-epithelization12,17,28. 
The successful establishment of the mouse burn injury model provides a valuable tool for studying the pathophysiology of thermal injuries and evaluating potential therapeutic interventions. The observed histological changes in burned skin tissue align with the characteristic features of thermal injury, highlighting the reliability and validity of the model in recapitulating the clinical manifestations of burns. 
Overall, the mouse burn injury model offers a robust platform for investigating the molecular mechanisms underlying burn-induced tissue damage, inflammation, and wound healing processes. Future studies utilizing this model may contribute to the development of novel therapeutic approaches aimed at improving outcomes for burn patients and reducing the burden of burn-related morbidity and mortality.

Burns are considered one of the critical injuries to the skin, caused by heat exposure, electricity, chemical materials, and radiation exposure1,2. It can be classified into four degrees depending on the depth of the injury, ranging from the epidermis to the full thickness of the skin, and even the muscles and bones. Small burns can lead to scar formation and increase the risk of infection. A large area of burn injury not only causes local damage, but also stimulates disorders of the body&#39;s heart, kidneys, and other organs or systems through severe and long-term inflammation and immune responses, leading to serious systemic consequences and high morbidity3. Most burn injury survivors are accompanied by long-lasting physical disabilities, emotional distress, and decreased quality of life4,5. Therefore, it is important to study the pathological process of burns and the mechanisms of regeneration of burned tissue.
Involved in immune responses, tissue regeneration, and systematic homeostasis, in vitro studies could not comprehensively investigate the pathological process of burn wound healing. Thus, over the past two decades, in order to explore potential therapeutic interventions, different burn wound healing animal models were developed to replicate the various features of burn injury6,7. Burn wounds are usually produced on the dorsum surface of pigs, rats, mice, rabbits, and other animals after hair removal. The burn time can last for 3 s to 30 s to form partial to full thickness thermal damage with a range of 5% to 30% total body surface area(TBSA)8. There are currently no standardized models of these methods in burn animal research due to the high variability of the techniques used. Methods used to produce burned surfaces vary among experimental animal models, including gas flame9, burning ethanol bath10, pre-heated single metal plate/bar11,12, boiling or hot water13,14. However, the technique of burn infliction and produced burn depth are often inconsistent and poorly described in previous studies, which is crucial in determining the severity of the burn and method of burn treatment.
This study aims to develop a simple, cost-effective, and user-friendly mouse burn animal model for creating consistent full-thickness burns in simulated clinical scenarios. In this protocol, we used a convenient digital heating device to control the depth of the burn by adjusting the temperature applied to the skin. The tip of this device can be switched to different sizes to induce thermal burns with varying ranges of TBSA. This allows for the creation of a chessboard-like burn wound on the mouse&#39;s back, enabling the comparison of several experimental and control treatments within the same animal. We observed and recorded the wound closure process. Skin samples were harvested for histologic evaluation (Hematoxylin and Eosin (H&#38;E) staining and Masson&#39;s trichrome staining) at different stages of wound healing. This approach reduces the number of animals used in the experiment and, therefore, cuts down on economic costs while being more compatible with animal ethics. This study will provide researchers with essential tools to facilitate the development of novel treatments for burn injury and reveal the pathophysiological mechanisms of burn wound healing.

<table><tbody><tr><td>1 mL pipette tip</td><td>KIRGEN,USA</td><td>KG1333</td><td>Used to locate burn wound sites</td></tr><tr><td>3 M Tegaderm film</td><td>3M,USA</td><td>1624WCN 6 cm x 7 cm</td><td>For the wound cover after burn induction</td></tr><tr><td>4% paraformaldehyde(PFA)</td><td>Biosharp,China</td><td>BL539A</td><td>Used to fix the skin samples</td></tr><tr><td>Buprenorphine</td><td>Sigma-Aldrich,USA</td><td>PHR8955-50MG</td><td>For the pain management of the mice</td></tr><tr><td>C57BL/6 mice</td><td>Chengdu Dashuo experimental animal company,China</td><td>none</td><td>For the establishment of burn model</td></tr><tr><td>Depilatory cream</td><td>Veet,China</td><td>&amp;mdash;</td><td>For the dorsum hair removal of the mice</td></tr><tr><td>Digital Heating Device</td><td>Shenzhen Kapper Technology Company,China</td><td>No.936D</td><td>For the burn induction of the mice</td></tr><tr><td>Electric shaver</td><td>AUX,China</td><td>AUX-A5</td><td>For the dorsum hair removal of the mice</td></tr><tr><td>Filter paper</td><td>&amp;mdash;</td><td>&amp;mdash;</td><td>Used to unfold of the skin samples</td></tr><tr><td>GraphPad software</td><td>&amp;mdash;</td><td>GraphPad prism 9.5.0</td><td>For the analysis of burn wound area</td></tr><tr><td>Heat-resistant gloves</td><td>&amp;mdash;</td><td>&amp;mdash;</td><td>Used to hold the digital heating device tip</td></tr><tr><td>Hematoxylin and Eosin Stain kit</td><td>Solarbio,China</td><td>G1120</td><td>For the histological analysis of the slides</td></tr><tr><td>ImageJ software</td><td>&amp;mdash;</td><td>ImageJ 1.54f</td><td>For the analysis of burn wound area</td></tr><tr><td>Isoflurane</td><td>RWD,China</td><td>R510-22-10</td><td>For the anesthesia of the mice</td></tr><tr><td>Masson&#039;s Trichrome Stain Kit</td><td>Solarbio,China</td><td>G1340</td><td>For the histological analysis of the slides</td></tr><tr><td>Microscope</td><td>Olympus,Japan&amp;nbsp;</td><td>VS200 ASW</td><td>Used to scan the H&amp;amp;E and Masson stained slides</td></tr><tr><td>Tissue cassette</td><td>CITOTEST LABWARE MANUFACTURING Co., LTD,China</td><td>31050102W</td><td>For tissue paraffin dehydration and paraffin embedding</td></tr></tbody></table>

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.

In this protocol, chessboard-like burn wounds were created with a burn duration of 10 s at 97 &#176;C by the digital heating device (Figure 1). The device&#39;s tip is made of pure copper, which is known for its excellent heat conduction and fast heating capabilities. The grip held by the experimenter is made of polycarbonate material, which provides heat resistance and non-flammability. Compared to the pre-heated single metal plate/bar or boiling or hot water methods, this device demonstrates superior safety performance and reduces anesthesia time in mice, consequently shortening the overall experiment duration.
The general process of burn induction and analysis is shown in Figure 2. Each animal had six burn wounds on the dorsum, arranged as depicted in Figure 2E. Wound size in these animals was evaluated, and skin samples were harvested for histological analysis, including H&#38;E and Masson trichrome staining on post-burn days 1, 3, 7, 10, and 14, respectively.
Each burn wound was circular in shape with equal area as well as uniform depth after burn induction. The edge color of the burn injury shifted from waxy white to red, accompanied by a hyperemic zone due to the immediate inflammatory response to the injury. Additionally, the size of the burn wound increased slightly over the 72 h following the burn (Figure 4A).
Microscopic analysis of H&#38;E and Masson trichrome-stained sections of the wounds (Figure 4B) revealed complete destruction of the epidermal layer and damage to the full thickness of the dermis, affecting both subcutaneous fat and muscle. Histological examination indicated that the wound healing process was in the inflammatory stage on day 3, characterized by vasodilation and a substantial infiltration of inflammatory cells. A notable reduction in wound area was observed on post-burn day 7 compared to day 3, as seen in microscopic images of the samples. Granulation tissue and cardiovascular formed, and collagen fibers are deposited densely on day 14, which means wound healing enters the remodeling stage. The wounds recovered without obvious infection throughout the healing process.
Additionally, the wound-healing abilities were evaluated by quantifying wound-healing data and analyzing wound tracings (Figure 5). Wound recovery was observed to be enhanced in wounds treated with TKH peptide hydrogel12,14, evidenced by significantly smaller wound areas on days 7, 10, and 14.
The interval between adjacent wounds (Figure 6), either transverse or longitudinal, confirmed no difference compared to non-burn skin. The epidermis, dermis, and subcutaneous tissues were intact, and there was no lymphocyte infiltration and no vasodilation. The healing process of the two wounds does not interfere with each other. Therefore, different drugs or dressings can be applied to wound treatment on the basis of the model to evaluate the effect in vivo.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67375/67375fig01.jpg" /> 
Figure 1: Digital heating device for burn induction. The digital heating device used for inducing burn injuries. (a) The temperature controller allows temperature settings ranging from 50-450 &#176;C. (b) The stand base is used to hold the burning tip. (c) The tip of the device can be switched to different sizes. <a href="https://www.jove.com/files/ftp_upload/67375/67375fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67375/67375fig02.jpg" /> 
Figure 2: Schematic of the chessboard-like wound model. A step-by-step outline for reproducing the chessboard-like wound model described in the article. (A) An 8-week-old mouse is used as the model for wound-healing experiments. (B) The hair is removed from the designated wounding area. (C) The six wounding sites are marked using a 1 mL pipette tip. (D) A digital heating device is used to create six burn wounds in the center of each marked site (E). (F) A close-up image of the dorsum after burn induction. (G) Skin samples (about 1 cm &#215; 1 cm) are harvested on days 3, 7, 10, and 14. (H) The skin samples are flattened using filt paper and transferred into embedding cassettes. (I) Paraffin dehydration is performed, and (J) the samples are sectioned to 5 &#181;m thickness. (K) The sections are stained using hematoxylin and eosin (H&#38;E), and Masson&#39;s trichrome. <a href="https://www.jove.com/files/ftp_upload/67375/67375fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67375/67375fig03.jpg" /> 
Figure 3: Full-thickness burn injury induction process. (A) The diameter of a 1 mL pipette tip and (B) the tip of the digital heating device are 9 mm and 4 mm, respectively. (C) The dorsal area of the mouse is shaved with an electric shaver. (D) Depilatory cream is applied for 3 min. (E) The shaved area is cleaned and dried, and the wounding points are marked with a sterilized 1 mL pipette tip. (F) The digital heating device is applied at 97 &#176;C for 10 s to create six burn wounds. (G) Ice is applied to the burn wounds for pain management. (H) The entire back of the mouse shows the six full-thickness burn wounds. <a href="https://www.jove.com/files/ftp_upload/67375/67375fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67375/67375fig04.jpg" /> 
Figure 4: Representative images of burn wounds and histological analysis. (A) Images of burn wounds on days 3, 7, 10, and 14, showing the progression of healing. (B) Histological images stained with H&#38;E and Masson&#39;s trichrome demonstrate wound healing and skin regeneration. Scale bars = 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/67375/67375fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67375/67375fig05.jpg" /> 
Figure 5: Wound size variation over time. (A) The variation in wound size in control and TKH hydrogel-treated animals over time, analyzed using ImageJ software. (B) Corresponding wound trace data is presented. <a href="https://www.jove.com/files/ftp_upload/67375/67375fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67375/67375fig06.jpg" /> 
Figure 6: Adjacent burn wounds and histological analysis. (A) Representative images of adjacent burn wounds on day 1. (B) Adjacent wound samples harvested for histological analysis on day 3 (circled in red). (C) Filter paper is used to flatten the tissue samples before paraffin dehydration. (D) H&#38;E and Masson&#39;s trichrome analysis of adjacent wounds. Scale bars: 1 mm and 200 &#181;m (magnified). <a href="https://www.jove.com/files/ftp_upload/67375/67375fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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 ...

chessboard-like-burn-wound-healing-model-mice-based-on-digital

Nanyang Technological University

Research

JoVE Journal

Biology

376 Views.  Sichuan University. 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.

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

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Medicine

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, repairing broken cell membrane before the cellular contents leak out. The giant unicellular organism Stentor coeruleus, in which cells can be more than one millimeter in size, have been a classical model organism for studying wound healing in single cells. Stentor cells can be cut in half without loss of viability, and can even be cut and grafted together. But this high tolerance to cutting raises the question of why the cytoplasm does not simply flow out from the size of the cut. Here we present a method for cutting Stentor cells while simultaneously imaging the movement of cytoplasm in the vicinity of the cut at high spatial and temporal resolution. The key to our method is to use a &#34;double decker&#34; microscope configuration in which the surgery is performed under a dissecting microscope focused on a chamber that is simultaneously viewed from below at high resolution using an inverted microscope with a high NA lens. This setup allows a high level of control over the surgical procedure while still permitting high resolution tracking of cytoplasm.

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

The giant ciliate Stentor coeruleus is a classical system for studying regeneration and wound healing in single cells.&#160;By imaging Stentor cells simultaneously at low and high magnification it is possible to measure cytoplasmic flows before, during, and after wounding.

Although wound-healing is often addressed at the level of whole tissues, in many cases individual cells are able to heal wounds within themselves, ...

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.

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 ...

Immunology and Infection

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 ...

Digital Planimetry for Assessing Wound Closure Kinetics in a Mouse Model

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, thereby reducing the risk of complications and alleviating the economic burden on healthcare systems. Although numerous experimental studies have investigated wound healing, most rely on qualitative observations or quantitative direct measurements. The objective of this study was to standardize an indirect wound measurement method using digital planimetry, incorporating digital scaling and segmentation. This approach addresses the lack of detailed, step-by-step methodologies for accurate wound assessment. A photodocumentation booth was designed and constructed, and computer-assisted digital planimetry tools were employed to minimize variability in measurements of the wound area, perimeter, and the distance from the wound center to its edges. A circular traumatic wound (5 mm in diameter) was created on the dorsal midline at the shoulder blade level of male CD1 mice (n = 4, 10 weeks old, 30-35 g). Wound evolution was photodocumented for 14 days using the custom-designed photo booth, which controlled lighting conditions, focal distance, and subject positioning. Scaling and wound measurements were performed using segmentation in ImageJ software, and statistical analysis was conducted using statistical analysis software. The kinetics of wound closure showed a slight increase in wound size and perimeter between day 0 and day 2, followed by a gradual decrease until complete closure by day 14. The photodocumentation booth and computer-assisted digital planimetry enabled quantitative measurements with minimal variability. In conclusion, these tools provide a reliable and reproducible method for evaluating wound closure kinetics in pre-clinical models.

Wounds represent a global health challenge. This study developed a standardized photo booth utilizing digital planimetry to minimize wound measurement variability. Monitoring wounds in mice over 14 days revealed an initial increase in wound area and perimeter, followed by gradual closure. This methodology may aid in evaluating wound closure kinetics in pre-clinical models.

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, ...

Bioengineering

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 ...

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 ...

Optimized Protocol for Mouse Wound Healing Model with Stem Cell-Derived Exosomes

Mouse Wound Models and Preparation of Single-Cell Suspensions 

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving diverse cell populations and the intricate wound microenvironment, complicates the assessment of treatment effects and their interactions using traditional experimental approaches. Single-cell transcriptome sequencing technology has revolutionized the understanding of cellular functions and mechanisms during wound healing. However, the variability in methodologies for creating mouse wound models introduces confounding factors that can impact experimental results. Furthermore, the process of dissociating mouse skin tissues presents significant technical hurdles, highlighting the critical need for high-quality single-cell suspensions for accurate transcriptome sequencing. Therefore, this study aims to optimize the construction of mouse wound models to accurately assess changes in wound closure while eliminating variables that could influence the subsequent sequencing result. Additionally, the preparation of single-cell suspensions was performed using both enzyme preparation protocols and test kit protocols to optimize cost and effectiveness.

Mouse Wound Models and Preparation of Single-Cell Suspensions

This study presents two crucial experimental techniques: the construction of a murine wound model for assessing wound closure and the preparation of single-cell suspensions from murine skin to preserve cellular viability.

The management of acute full-thickness skin injuries presents a considerable challenge in clinical practice. The complexity of wound healing, involving ...

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

Summary

Explore More Videos

Chapters in this video

Murine Model of Wound Healing

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Visualizing Cytoplasmic Flow During Single-cell Wound Healing in Stentor coeruleus

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

Swine Model of Biofilm Infection and Invisible Wounds

Digital Planimetry for Assessing Wound Closure Kinetics in a Mouse Model

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

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

Mouse Wound Models and Preparation of Single-Cell Suspensions