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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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.

Protokół

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.

Wyniki

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 demonstrat...

Dyskusje

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 ...

Materiały

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