A biological membrane-based novel excisional wound-splinting model in mice (With video)

Rodents have robust wound healing mechanism compared to other animal species. The major mechanisms of wound healing differ between rodents and humans. In humans, wound healing primarily depends on re-epithelialization and granulation tissue (GT) formation, whereas wound contraction is more important during rodent wound closure. In this study, we described a novel excisional wound-splinting model in mice with a new biological membrane to imitate wound healing in humans. In this model, wound contraction can be effectually prevented, and the extent of re-epithelialization and the amount of granulation tissue can be determined easily. Furthermore, the harvested tissues can be analyzed with different methods according to the research aim. In conclusion, we have developed a biological membrane-based, novel, excisional wound-splinting model in mice that has unique advantages for wound healing research compared with the conventional animal model.


Introduction
Wound healing is a dynamic and coordinated process, and there are various animal models used to study wound healing. Incontestably, rodents are used for a wide range of research applications due to low cost and ease of breeding and genetic modifications. However, the major mechanisms of wound healing differ between the humans and rodents. In humans, the skin closely connects to the subcutaneous tissues. Thus, wound healing depends on re-epithelialization and granulation tissue

A B S T R A C T
Rodents have robust wound healing mechanism compared to other animal species. The major mechanisms of wound healing diff er between rodents and humans. In humans, wound healing primarily depends on re-epithelialization and granulation tissue (GT) formation, whereas wound contraction is more important during rodent wound closure. In this study, we described a novel excisional wound-splinting model in mice with a new biological membrane to imitate wound healing in humans. In this model, wound contraction can be eff ectually prevented, and the extent of re-epithelialization and the amount of granulation tissue can be determined easily. Furthermore, the harvested tissues can be analyzed with diff erent methods according to the research aim. In conclusion, we have developed a biological membrane-based, novel, excisional wound-splinting model in mice that has unique advantages for wound healing research compared with the conventional animal model.

Animal preparation
Depilate the mice the day before surgery. Anesthetize the mice with an intraperitoneal injection of 1% sodium pentobarbital (10 μl/g).
Remove the fur from the dorsal skin (from neck to tail) with an electric clipper. Apply hair removal cream (Veet, 20070747) to the back for 3-5 min (not exceeding 6 min to avoid damaging the skin).
Clean the skin with the sterile cotton balls. Then, wipe again with sterile cotton balls soaked in warm water.
Keep the mice individually to avoid scratching and chewing.
Place the mice under a warm lamp until they are completely recovered.

Skin wound procedure (With video)
Anesthetize the mice with an intraperitoneal injection of 1% sodium pentobarbital (10 μl/g). Lift the dorsal skin slightly along the posterior midline. Perform 2 symmetrical 3 mm diameter, full-thickness, cutaneous biopsy punch wounds at point A and B. The wound sizes should be chosen according to your research aims and the strain of mice. Drugs, bioactive factors and cells can be applied to the wound bed or injected into the tissue to determine their effects on wound healing.
Glue the biological membrane (NPWT-1, Negative pressure wound therapy kit, China) with the adhesive dressings immediately onto the surface of the wound before contraction, and single-house the mice. For long-term wound observation, you should place simple, surgical sutures around the margin of the membrane to guarantee a higher success rate.
Place the mice under a warm lamp until they are completely recovered.
Place the contrast (the same diameter as the wound) beside the wound, and take photographs perpendicular to the individual wounds with a digital camera.
The photographing time points should be decided according to your research aims and should be discussed in the experimental design.
Place the mice under a warm lamp until they are completely recovered.
Draw a circle along the 2 mm surrounding wound edge, and shear the entire wound tissue sample carefully along the circle. The wound tissue should be used according to your research purposes and should be discussed in the experimental design.

Wound analysis
Wound size measurement Measure the pixels of the actual wound and the control with Photoshop.
Calculat e the percent wound closure as follows: (pixels of actual wound/pixels of control)/(pixels of original wound/ pixels of control) × 100.

Histological analysis
The wound tissue should be harvested and divided across the center into two equal pieces. Carefully place the wound tissue on a piece of filter paper to avoid crispation. Fix the wound tissue in 4% paraformaldehyde at 4°C overnight.
The paraformaldehyde-fixed tissues can be embedded in paraffin, sectioned at 6 μm, and stained with hematoxylin and eosin (HE).
Examine the HE sections of the wound tissue with a light microscope. Then photograph the wound.
Wound re-epithelialization begins at the wound edges and moves toward the center. Take low-magnification images of the wound center and measure the extent of epithelialization and the gap between the epithelialized areas using Image-Pro Plus.
Measure the amount of GT at different time points. The HE sections are photographed under a light micro-scope, and the granulation thickness is calculated using Image-Pro Plus.
Specific markers can also be detected using immumohistochemical staining with specific antibodies according to your research purpose. Three to five animals are required in each time point.

Fluorescence activated cell sorting (FACS) analysis
The harvested wound tissue can be incubated in 0.5% trypsin in 37° C for 30 min to digest single-cell suspensions.
Add an equal volume of phosphate-buffered saline (PBS) containing 3% fetal bovine serum (FBS) to the digest and strain through a 70 μm cell strainer.
Centrifuge the digest at 1000 rpm for 10 min at room temperature and resuspend the cells in 500 μl PBS with 3% FBS.
Count the cells with a cell counter and calculate the total cell number per wound.
Measure the percentage of green fluorescence protein (GFP)-positive cells or marked cells by FACS analysis in the single cell suspension. Figure 1 demonstrates the steps of the biological membrane-based, novel, excisional and wound-splinting model [ Figure 1]. This model minimizes wound closure caused by skin contraction compared to the conventional model. In the conventional model, wound closure will reach 30% in 48 h to the wound contraction. In this novel model, wound closure remained above 80% [ Figure 2]. The extent of re-epithelialization should be measured using Image-Pro Plus at different time points [ Figure 3]. The percentage of GFP-positive cells can be measured with FACS on day 3 and day 5 [ Figure 4].

Discussion
The major mechanisms of wound healing are different between rodents and humans due to differential skin orga-nizational structure. Rodent models are not ideal because the major mechanism of wound healing in rodents is wound contraction, which is significantly different from that in humans. [2] In our model, we used a biological membrane, which is an adhesive dressing and is commonly used for continuous negative pressure therapy for the deep wounds. The biological membrane can glue the skin stably for 10 days. With the biological membrane, skin contraction can be efficiently prevented. The wound will heal by re-epithelialization and GT formation, which reasonably reflects human process. Additionally, this model is easy to perform and low cost. Drugs, bioactive factors and cells can be applied to the wound bed or injected into the tissue directly to determine their effects on wound healing and provide new treatment. Furthermore, with this model, all the genetic modifications and disease models in mice can be used to study wound healing. Meanwhile, the tissue harvested in this model can be used with various methods according the research aims (for example, with FACS analysis of single-cell suspensions or western blot analysis). Some new animal wound models are also being used to prevent wound contraction in other studies. The rabbit ear dermal ulcer model is widely used as a splinted model. The rabbit ear skin is attached to the underlying cartilage, which restricts wound contraction and forces wound healing to occur via new tissue formation. [3] This model is used to investigate mechanisms of hypertrophic scaring and wound healing. [4] However, the disadvantage of this model is the high cost of breeding, and rabbits are hard to genetically modify. The cranial and tail excision models in rodents are other examples of anchored skin. [5,6] However, the underlying soft connective tissue is lacking, and these models require complex surgery. The excisional woundsplinting model described in the present study is a novel wound healing model in mice using wound-splinting, which efficiently prevents skin contraction and forces the wound to heal through granulation and re-epithelialization. Galiano et al. found that the rate of re-epithelialization in splinted wounds is similar to unsplinted wounds, and the amount of GT deposition is increased compared to unsplinted wounds. [7] Stem cells can be implanted into the wound bed to study cutaneous repair or regeneration using the mouse excisional, wound-splinting model. [8] In this model, special frames prevent wound contraction. For example, silicone rubber, metal rings and polydimethylsiloxane devices, require microfabrication techniques, and some laboratories cannot afford these techniques. These devices also require complex surgeries to fix the rings in the skin, which may aggravate the wound. [9,10] Chung et al. attached the Tegaderm HP dressing over the wound in diabetic and non-diabetic mice and found that the Tegaderm HP dressing retarded contraction in a large proportion of diabetic mice and, to a lesser extent, in non-diabetic mice. [11] Here we describe in detail every operational step. For example, we confirmed the wound point according to the different lines drawn on the back, which is very important for the stability of this model and is not referenced in other articles. In our previous study, we found that the animal's activities influence the position of the wound on the back. When the wound is on the top or middle of the back, the wound will be greatly influenced. In our study, the wound point placement is more constant and stable. Additionally, we propose more applications for this model. For example, drugs, bioactive factors and cells can be applied to the wound bed or injected into the tissue to determine their effects on wound healing, and the biological membrane could prevent the loss of drugs or cells.
In this study, we describe a new, excisional, wound-splinting model in mice to simulate human wound healing, which has some advantages compared to other animal models. We hope that this model can be used in more studies.