The American Burn Association estimates that 450,000 burn patients are treated in medical institutions every year in the United States. 40000 of them need to be hospitalized, including 30000 in burn centers of specialized hospitals. 1 The most common etiologies requiring burn center admission are fire/flame (43%), followed narrowly by scalds (34%), contact with hot objects (9%), electricity (4%), and chemical agents (3%). 2 Burns often cause extensive skin defects, resulting in loss of skin barrier function and inflammation. 3-8 Therefore, the wound healing time caused by burns and scalds is long, the prognosis is poor and the function recovery is poor.
Generally, the burn is classified according to the depth: Superficial (first-degree) only involves skin epidermis; Superficial partial-thickness (superficial second-degree) involves superficial dermis; Deep partial-thickness (deep second-degree) involves the deeper dermis; Full-thickness (third-degree) includes the whole layer of epidermis and dermis, reaching the subcutaneous tissue, and even muscles and bones are damaged. 9
Deep burns and scalds can cause scar formation, skin contracture, and serious motor dysfunction. People need to study the pathophysiological process and healing mechanism of burns and scalds to explore the healing process of burns and scalds. For burn research, in vitro models are limited in capturing various aspects of burn pathophysiology and complex clinical features of human burn. Therefore, it is necessary to establish a burn animal model to reveal the pathological mechanism after burn and test the new treatment. One of the main limitations of finding practical treatment for burn patients is the lack of an appropriate animal model that can capture all the salient features of burn wounds. However, animal models are still essential to reveal the molecular and cellular characteristics of human burn wounds. 10 Qualified animal models should have the characteristics of simple operation and clear results. In the experiment of burn and scald, researchers used various animal models, including rats, mice, rabbits, pigs, etc. 10-12 Mice are one of the most commonly used animal models to study burn and wound healing. As a research model, due to the feasibility of a variety of mouse specific reagents and transgenic mice, this animal provides great convenience for researchers to study the signal pathway involved in the healing process. 10 In addition, the incidence rate of diseases in mice in the study group was very low due to the significant reduction in healing time 13 and superior in the immune system. 14 In this study, we used boiling water method to make third-degree burn animal models in mice. It has the advantages of simple operation, strong reproducibility, uniform depth and scope of injury. With this method, it is easy to obtain a large number of models with the same conditions, so as to improve the comparability and repeatability of the experimental results and make the results more accurate.
This research protocol was approved by the Dankook University Medical School Research Institutional Animal Care and Use Committee. Eighteen healthy male swiss mice aged 8 weeks were provided by ORIENTBIO Company (Seongnam, Korea) and reared under clean conditions for one week. The preoperative mice weighed 35-36 grams. The experiment was divided into two steps. Step 1 is to clarify the relationship between scald time and scald depth. 12 mice were randomly assigned to three equal groups according to scalding time: (1) scalding time 3s; (2) scalding time 5s; (3) scalding time 8s. Step 2 is to observe the healing process of the third-degree scalded mice. After confirming that 8s scald can cause third degree scald, 12 mice were given 8s scald and randomly divided into 6 groups. On day 2, 4, 6, 8, 10 and 12, two mice were randomly selected for tissue sampling each day.
Narcotics is a combination of tiletamine-zolazepam-xylazine (ZoletilTM 50, Virbac, TX, USA; Rompun ® , Bayer, Leverkusen, Germany), self-made scald mold (15 ml plastic centrifugal tube was used to cut off the tip to form a circular defect with a diameter of 1.13 cm and an area of about 1 cm 2 ), alcohol lamp, flask, flask holder, 10 ml pipette, electric pipetting Electric pusher, stopwatch, hair removal paste, lighter, electric warm pad, etc.
Mice were anesthetized and shaved clean prior to treatment. To make the scald, distilled water was poured into the flask and heated using an alcohol lamp. The temperature of boiling water is determined to be 100°C. One person presses the scald mold on the back of the mouse, avoid using too light force to make water flow out or using too heavy force to damage the mouse skin. Another person uses a pipette to suction 10 ml of boiling water, quickly inject it into the mold, 3 seconds, 5 seconds or 8 seconds later, pour out the boiling water, and spray cold water for cooling (Fig. 1). After scalding, the mice were placed on an electric warm pad to prevent hypothermia and shock. After recovery, the mice were returned in their respective cages and fed normally.
Scald area were photographed at 4, 6, 8, 10, 12 and 14 days to observe wound contraction. Lesions were photographed with a digital camera at a standard distance of 15 cm. Adobe Photoshop CC (Adobe Systems, Mountain View, CA, USA) was used to measure the size of the wound (Unit: pixel area). The region of interest (ROI) was carefully selected using an elliptical marquee tool drawn around the wound area. WRI was determined from the selected ROI and calculated by the following equation: WRI (%) = (initial area – area on the day of euthanasia) ¡À initial area ×100. 15 The water intake of mice was observed and the weight of mice was measured by electronic scale.
On the 4th, 6th, 8th, 10th, 12th day after scald, the scald wounds were cut off after anesthesia. After flattening and fixing, the wounds were immersed in 10% formalin solution. Paraffin sections were prepared by conventional method, and the thickness of the sections was 7 μm. After hematoxylin-eosin (H&E) staining, Olympus microscope (DP74; Olympus, Tokyo, Japan) was used to observe and photograph.
After scalding mice with boiling water, there were regular circle scald wounds on the back of experimental mice without blisters and exudation. The mice were observed to be in poor mental and dietary condition.
In order to find out the relationship between the time and the depth of scald, we made 3 seconds, 5 seconds and 8 seconds scald wounds respectively, and made histological examination after scald (Fig. 2).
Forty-eight hours after scald, tissue in the scald area was necrosed as observed in (Fig. 2), and blood crust have not formed yet, there were obvious regular round scald wounds on the back of experimental mice, without blisters and exudation (Fig. 2A). The epithelial tissue structure is incomplete and the epithelial cells are atrophic and necrotic. Meanwhile, the structures of skin appendages and glands was atrophic. Hair follicles in the dermis were destroyed. The morphology of dermis was abnormal, indicating necrosis. Scald leads to the degeneration of collagen in the dermis and changes the cord like collagen fiber into hyaline necrosis tissue. There was no neovascularization in dermis. After 3 seconds of scalding, the wound depth reaches to the superficial dermis, which can be determined as superficial second-degree scald. After 5 seconds of scalding, the depth of the wound reaches deep dermis, which can be determined as deep second-degree scald. After 8 seconds of scalding, the depth includes the whole skin layer and reaches the subcutaneous fat layer, which can be determined as third-degree scald (Fig. 2B). 9 Since the experiment usually requires a long healing time to determine the effect of treatment, we choose the third-degree burn as the main study object. The mice were in poor mental state and poor diet.
Fig. 3 shows the changes of wounds in mice after scalding. On day 4, blood crust appeared on the wound surface and the wound partly contracted.
From day 4 to day 6, the wound contracted most in this period (
In normal skin tissue (Fig. 4A), the epidermis and dermis are intact. Collagen fibers in dermis were distributed regularly and the hair follicles and skin organs are plump. There are normal vessels in the dermis.
On day 2 after scald (Fig. 4B), the necrosis of tissue in the burn area was formed, and no blood crust was found. Epithelial tissue structure is incomplete and epithelial cells atrophy and necrosis. The collagen cells in the dermis lost normal fibrous structure and exhibits hyaloid or caseous necrosis. Skin appendages and lands are atrophic in morphology and abnormal in structure. Hair follicles were destroyed. Compared with normal skin (279.7 ± 33.9), the number of nucleated cells in dermis decreased significantly (164.5 ± 14.8,
On day 4 after scald (Fig. 4C), there was still necrosis in the epidermis and dermis. The epidermis was filled with collagen fibers without epithelial tissue formation. The dermis is filled with a large number of collagen fibers arranged in parallel, and exhibits hyaloid or caseous necrosis. Skin appendages were disappeared with no angiogenesis. Compared with day 2 (164.5 ± 14.8), the number of nucleated cells in dermis was further reduced (63.5 ± 12.2 ,
On day 6 after scald (Fig. 4D), blood crust formation was observed in the scalded part, but no epidermal formation was observed. A large number of inflammatory cells and necrotic cells were found at the junction of the crust and dermis. At this time, no skin appendages and glands were found in the dermis, but there was angiogenesis formed.
On day 8 after scald (Fig. 4E), there were new epidermal cells covering the surface of dermis, and the new epithelial layer was thicker (63.84 ± 13.45 μm) than normal skin (31.21 ± 11.3 µm,
On day 10 after scald (Fig. 4F), epithelial layer was thicker (127.96 ± 20.63 μm) than that on day 8 (63.84 ± 13.45 µm,
On day 12 after scald (Fig. 4G), the morphology of collagen in dermis was improved and the number of inflammatory cells in dermis was decreased. The morphology of dermis was not different from that at day 10.
Burn and scald is a serious and complex injury. Despite the limitations of using animal models to simulate human burn and scald, rodents have been accepted as good model carriers to simulate burn and scald. 16 Because of low cost, low incidence rate of disease 13,14 and the existence of multiple transgenic mice, mouse model is very convenient as animal model, and has advantages in the research of burn as well. 10
Techniques that have been used to generate burn surfaces in experimental animal models include direct contact with a heated metal, 11,17,18 burning fuel, 19 electricity, 20 and heated water. 11,21,22 The method of heating metal blocks can effectively control scald area and scald time, but the back surface of mice is non-planar. Different pressure in different areas may affect the depth of scald. In addition, the temperature of the metal block by heating is not easy to control, and the heat dissipation is faster, which may cause larger errors in the experiment. The drawback of this method is the lack of a homogenous uniform burn depth. The method of heating phosphate powder is direct burn, which can simulate the burn process in life. However, high concentration of phosphorus itself has a certain toxicity, which will cause organ damage in the whole body after absorption, 23 and white phosphorus burning point is low and flammable, storage risk is large. Electrical burns usually require higher animals, such as monkeys, to achieve damage similar to that of humans. 20 Among the above models, the boiling water model has been widely used, which is considered as the standard of burn animal model by some experts. Burns caused by hot liquids are the most common cause of burns in children and the elderly. 10 Moreover, scalding is the easiest mechanism of provoking an experimental dermal burn. The possibility of varying water temperature, time of exposure and the burned area makes this method ideal for reproducing almost every kind of thermal aggression. 11 The method of whole-body scald of mouse has a large wound, which is only suitable for simulating large area scald process. In this experiment, we designed a standardized method, using self-made mold and boiling water to create 1 cm 2 scald wounds, and obtained third-degree scald wounds with uniform scald area and depth. The experimental model is easy to operate and low cost. It has the characteristics of constant scald time, constant scald pressure and constant scald area. As a result, it can ensure that every scald wound has the same pathological characteristics and does not cause excessive damage to animals. This is the key factor in making animal models.
In our study, we used the method of boiling water 24 scalding for 3 seconds, 5 seconds, 8 seconds, and the obtained wound was proved to be superficial second-degree, deep second-degree, and third-degree scald by histological analysis. In the actual experiment, the desired depth of scald can be obtained by adjusting the scald time according to the need of the experiment. In our experiments, we found that the process of collagen changes and wound recovery was similar to that of human, but the time of inflammation was different. Inflammatory exudation did not occur in the burned area of mice until day 4, whereas inflammatory exudation occurred immediately after human skin burns. The reasons for this difference are not clear, and need to be further studied and solved.
We thank Beckman Laser Institute Korea, Dankook University, for providing necessary financial assistance and the experimental equipment and animals. We also thank Prof. Abueva for useful comments and language editing which have greatly improved the manuscript.
The authors have no conflict of interest to disclose.
Hua C, Lyu L, Ryu HS, Park SY, Lim NK, Abueva C, Chung PS. Design and evaluation of a scalding animal model by the boiling water method. Med Laser 2020;9:51-57. https://doi.org/10.25289/ML.2020.9.1.51