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

Summary

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.

Abstract

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 °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&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.

Introduction

Burns are considered one of the critical injuries to the skin, caused by heat exposure, electricity, chemical materials, and radiation exposure^1,2. 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'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 life^4,5. 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 injury^6,7. 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/bar^11,12, boiling or hot water^13,14. 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'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&E) staining and Masson'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.

Protocol

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

1. Acquire the animals and house them in cages for 7 days upon arrival to allow them to acclimatize to the environment.
2. Sterilize the working area by spraying and wiping with 70% ethanol (v/v). Cover the operation table with sterile surgical absorbent pads.
3. Adjust the temperature of the digital heating device to 97 °C. The heating device consists of three parts (Figure 1).
   NOTE: Temperature from 50-450 °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.
4. 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.
5. 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.
6. Apply lubricating gel to both eyes of the mouse with a cotton swab to prevent corneal drying.

2. Inducing full-thickness burn injury

NOTE: The general process of burn induction and analysis is shown in Figure 2.

1. 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).
2. 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.
3. 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.
4. 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.
5. 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's tip with 70% ethanol (v/v).
6. Apply the tip of the heating device to the center of located wound sites (97 °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.

3. Post-burn care and measurement

1. Apply ice to the burn wound for 5 min to reduce potential pain for pain management (Figure 3G).
2. 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).
3. 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.
4. 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.
5. 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 wound^12,14. Repeat this procedure on days 3, 7, and 10 thereafter.
6. 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.
7. 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.

4. Wound collection

1. 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.
2. 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.
3. 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.
4. Fix the skin specimens in 4% paraformaldehyde (PFA) for 24 h and store at 4 °C. The fixative volume must be 30 times the tissue volume^15,16.
   NOTE: PFA is hazardous. Ensure to work in a well-ventilated area and use appropriate protective equipment.

5. Wound healing evaluation

1. 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.
   1. Put the filter paper and sample into a tissue cassette for paraffin dehydration to obtain relatively smooth tissue samples and then embed the tissue^15,16. Store samples at 4 °C.
      NOTE: Stretch the tissue as much as possible and avoid any folding, as these folds will become permanent after fixation.
2. Cut the paraffin-embedded samples to sections of 5 µm thickness and stain slides via H&E and Masson'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 healing^17,18.
3. Capture images of all samples using a microscope with a 40x objective.
4. Use compatible software to analyze the stained skin section, including the epidermis, dermis, subcutaneous tissue, and muscle condition.

Results

In this protocol, chessboard-like burn wounds were created with a burn duration of 10 s at 97 °C by the digital heating device (Figure 1). The device'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&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&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 hydrogel^12,14, 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.

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 °C. (b) The stand base is used to hold the burning tip. (c) The tip of the device can be switched to different sizes. Please click here to view a larger version of this figure.

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 × 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 µm thickness. (K) The sections are stained using hematoxylin and eosin (H&E), and Masson's trichrome. Please click here to view a larger version of this figure.

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 °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. Please click here to view a larger version of this figure.

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&E and Masson's trichrome demonstrate wound healing and skin regeneration. Scale bars = 200 µm. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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&E and Masson's trichrome analysis of adjacent wounds. Scale bars: 1 mm and 200 µm (magnified). Please click here to view a larger version of this figure.

Discussion

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 dressings^14,20) 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 remodeling^21,22. 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 keratinization^26,27. 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-epithelization^12,17,28.

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.

Materials

List of materials used in this article

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