Severe Burn Injury in a Swine Model for Clinical Dressing Assessment

Summary

To closely mimic the mode of burn injuries requires the interplay between clinical observation and studies in animal models. In this study, a swine model of severe burn injury was established to assess an experimental dressing in physiological and pathophysiological settings.

Abstract

Wound healing is a dynamic repair process and is the most complex biological process in human life. In response to burn injury, alterations in biological pathways impair the inflammation response, resulting in delayed wound healing. Impaired wound healing frequently occurs in patients with diabetes leading to unfavorable outcomes such as amputation. Hence, dressings having beneficial effect in promoting burn wound repair are needed. However, studies on burn wound treatment are limited due to lack of proper animal models. Our previous study demonstrated wound-healing performance in rat and swine models using a minimally invasive surgical technique. This study aimed to demonstrate a swine model of severe burn injury that eliminates wound contraction and more closely approximates the human processes of re-epithelialization and new tissue formation. This protocol provides a detailed procedure for creating consistent burn wounds and examining the wound-healing performance under the treatment of an experimental dressing in a swine model. Six burn wounds were created symmetrically on the dorsum, which were covered with a clinical dressing composed of four layers: an inner contact layer of experimental materials, an inner intermediate layer of waterproof film, an outer intermediate layer of gauze, and an outer layer of adhesive plaster. Upon the completion of experiments, wound closure, wound area, and Vancouver Scar Scale score were examined. The samples of skin resected from each animal post-sacrifice were histologically prepared and stained using hematoxylin and eosin staining. Antibacterial activity of each dressing in the context of wound healing was also examined. The application of the clinical dressing to the wounds in swine model mimics the biological processes of human wound healing with respect to the processes of epithelialization, cellular proliferation, and angiogenesis. Therefore, this swine model provides an easy-to-learn, cost-effective, and robust method to assess the effect of clinical dressings in severe burn injury.

Introduction

A burn injury initiates the inflammatory process and induces complex pathological effects, which influence numerous body functions immediately after an accident, resulting in negative impact on patients' quality of life. Impaired wound healing causes significant morbidity and mortality among patients with diabetes mellitus^1,2. Most patients with burn injuries experience pain during burn wound debridement, which is known as an excruciating process despite the use of powerful opioid analgesics³.

In contrast to other mammals, swine share several anatomic and physiologic characteristics with humans regarding the process of epithelialization, cellular proliferation, and angiogenesis. This makes swine a potentially better model for certain procedures and studies, and they are often used in subsequent studies that demonstrated promising results in mice. These features have led to the increasing use of swine as a major species in preclinical testing. Recently, a rapid increase in biomedical research with respect to cardiovascular, urinary, integumentary, and digestive systems has been observed^4,5,6. This study aims to demonstrate a swine model of severe burn injury that eliminates wound contraction and provides a closer approximation of the human wound healing processes and the formation of new tissue. Six burn wounds were created symmetrically on the dorsum, three on each side of the spine of the swine. Next,anexperimental dressing was examined in a swine model of severe burn injury, which can be adapted to replicate human wound healing (Figure 1). The wounds were covered with a clinical dressing, which is composed of four layers: an inner contact layer of experimental materials, an inner intermediate layer of waterproof film, an outer intermediate layer of gauze, and an outer layer of adhesive plaster. A waterproof film keeps the wound environment moist while preventing bacterial infection and allowing gases to permeate the dressing. The outer intermediate layer of gauze was applied on the waterproof films and secured by an outer layer of adhesive plaster. At the completion of experiments, wound closure, wound area, and Vancouver Scar Scale (VSS) score were examined. The samples of skin resected from each animal post-sacrifice were histologically prepared and stained using hematoxylin and eosin (HE) staining. Antibacterial activity of each dressing in the context of wound healing was also examined in this model. Our previous study has demonstrated wound-healing performance in rat and swine model using a minimally invasive surgical technique⁷. Since there were six burn wounds on the dorsum within each swine, each experimental dressing was tested and evaluated in all positions to minimize bias related to wound healing process in different spots on the swine dorsum. Therefore, the swine model of severe burn injury established in this study provides a new approach for the evaluation of clinical dressings and facilitates the development of a novel treatment for burn injury. This study provides crucial tools to uncover the pathophysiology of burn wound healing.

Protocol

Procedures involving animal subjects have been approved by the Animal Care Committee at National Defense Medical Center, Taiwan (R.O.C). This study was conducted in the Laboratory Animal Center at the National Defense Medical Center. Swine weighing between 20 and 25 kg has been successfully instrumented using this protocol.

1. Adaptation of the Animals to Human Handling

1. After arrival in the facility, house the animals solitarily but let them interact with each other.
2. Provide the animals ad libitum access to food and water.
3. Acclimate swine to human handling and transportation from the animal facility to the experimental laboratory by handling the animal at least once a day for one week.
4. Fast the animal for at least 12 hours before surgery to prevent nausea, vomiting, and aspiration of stomach fluids.

2. Sedation

1. Before the burn wound creation, sedate animals via an intravenous injection with Zoletil 50 (25 mg/kg).

3. Intubation and Ventilation

1. Place the animal on a table and/or trolley in sternal position.
2. Open the mouth of the animal with an oral spreader.
3. In case of insufficient relaxation of the jaws or presence of swallowing reflexes, which hinder intubation, mask the swine with isoflurane to induce sedation.
4. Monitor blood pressure, heart rate, and body temperature by physiological signal monitor during the surgery to prevent potential complications.

4. Anesthesia

1. Induce and maintain anesthesia; preferably anesthetize the animal via an intramuscular injection of tiletamine and zolazepam (25 mg/kg + 25 mg/kg).
2. Intubate the animal with the endotracheal tube when muscle relaxation, characterized by loss of jaw tone and palpebral reflexed, was observed.
3. Maintain all pigs in an anesthetic state at a vaporizer setting of 0.5–2.5% (v/v) isoflurane until the end of the surgery.
4. Examine the depth of anesthesia by testing pain reflexes with a hind leg toe pinch before surgery. When necessary, add additional anesthesia or wait for a few minutes. Check pain reflexes regularly throughout the surgery.

5. Sterilization of the Surgical Site

1. Shave and clean the skin of the animal over an area of approximately 25 cm width from the vertebral column all the way to the axilla on both sides.
2. Scrub the moisturized skin with povidone-iodine scrub (75 mg/mL) for approximately 5 min.
3. Remove the povidone-iodine soap from the skin using wet sterile gauzes.
4. Sterilize the skin with povidone-iodine lotion (100 mg/mL).
5. Cover the animal with sterile surgical drapes to reduce bacterial transfer and subsequent contamination of the surgical site.

6. Burn Wound Creation

1. Use a surgical marking pen to mark the center of six burn wounds symmetrically on the dorsum of the pig. Ensure that the distance between each burn wound is at least greater than the radius of the wound (Figure 1A).
2. Fill a modified soldering iron with 50 mL of glycerin and insert an electronic thermometer into it to monitor the temperature. The hot iron possesses a flat area of approximately 9 cm² (Figure 1B).
3. Heat up the iron to 137–139 °C with a hot plate (Figure 1C).
4. Create six uniform burn wounds by placing the iron on the marked area without applying any force for 30 seconds (Figure 1C).
5. Wash the burn wounds with 0.9% saline solution (Figure 1D).
6. Measure wound dimensions and record the wound by photomicrography (Figure 1E).

7. Preparation of Dressings

1. Cover each wound with the inner contact layer of a four-layer clinical dressing through direct contact. For this layer, use CAPS-containing dressing or alternative materials (Figure 1F).
2. Apply a waterproof film onto the clinical dressing to serve as a barrier against bacterial penetration (Figure 1F).
3. Cover each wound with a gauze (0.5 cm thick) and fix with paper tape to serve as the mid-layer of the dressing (Figure 1G).
4. Secure the gauze with an outer layer of adhesive plaster. Extend this layer to the torso to avoid the displacement of the dressing (Figure 1H-1J).

8. Post-burn Care and Measurement

1. Inject the swine with buprenorphine (0.1 mg/kg, IM) for pain management for every 8-12 h, starting before recovery from anesthesia, for one week to reduce potential pain.
2. Allow the pig free access to feed and water.
3. Change the clinical dressings every 2 days for the first 10 days and then twice a week for the 6-week study.
4. Clean and measure wounds before reapplying clinical dressings. Administer anesthetics during dressing changes.
5. Record the wound by photomicrography for comparison of wound healing rate every 2 days for the first 10 days and then twice a week for the 6-week study.
6. Calculate the wound re-epithelialization or contraction as the percentage of the original wound size according to a previously described method. The analysis of wound closure was conducted in a double-blinded manner.
7. Measure the burn scar using VSS, which consists of four variables: vascularity, height (thickness), pliability, and pigmentation on post-burn days 0, 7, 21, and 42. Each variable has four to six possible scores. The total score ranges from 0 to 14, whereby a score of 0 reflects normal skin.

9. Bacterial Growth Experiments of Post-burn Tissues

1. Swab the wound for antibacterial testing on post-burn days 0, 7, 21, and 42.
2. Place the swab into 100 mL of 0.9% sterile saline solution and gently vortex to achieve a homogenous suspension.
3. Serially dilute (10⁻¹–10⁻⁵) the homogenate and plate 100 μL of each dilution in selective and nonselective media, respectively.
4. Incubate all dilutions under aerobic conditions at 37 °C for 24–72 hours.
5. Plate triplicate aliquots of 10 μL each from all dilutions onto blood agar plate supplemented with 5% sheep blood to isolate aerobic Gram-positive organisms.
6. Incubate the sample by spreading or pouring the sample uniformly on the surface of an agar plate overnight for determining the number of colony-forming units (CFUs).
7. Read the plates after overnight incubation. Invert the sheep blood agar plate and divide the bottom of the dish into four equal quadrants using a marker and small ruler.
8. Place the plate onto the stage of a dissection microscope and count the colonies on each plate. By definition, a colony must have a minimum of 300 CFU to be enumerated.
9. Count the bacterial colonies in each of the three replicates. Calculate the average value of the three replicates. Determine the CFU per plate by multiplying the average value by the final dilution factor.

10. Euthanasia and Tissue Fixation

1. Intravenously inject an overdose of sodium pentobarbital euthanasia solution (80–120 mg/kg).
2. Perform en bloc excision of burn wound tissue to include the underlying musculature and surrounding unwounded tissue.
3. Fix tissues with 10% neutral buffered formalin.
   1. Mix 10 mL of formaldehyde (37%) in 90 mL of phosphate buffered solution (PBS) and store in 4 °C.
   2. Transfer tissues to fixative and swirl the container to ensure all tissues are completely immersed in fixative. The volume of fixative must be 30 times the tissue volume.
   3. Fix tissues overnight at 4 ˚C.
4. Dehydrate tissues with ethanol and embed into paraffin blocks. Perform the following steps at 4 ˚C on a shaker.
   1. Wash twice with PBS for 30 minutes.
   2. Dehydrate tissues with 70% ethanol for 8 hours, 80% ethanol overnight, 95% ethanol for 8 hours, and then in 100% ethanol overnight.
   3. Incubate tissues in 100% ethanol for a further 8 hours.
   4. Incubate tissues in three changes of xylene each for 30 minutes.
   5. Replace the xylene with freshly melted (52 °C) wax, and incubate at 52 °C in an oven for 1 hour.
   6. Replace the wax with fresh wax and incubate at 52 °C in an oven for 3 hours, and then replace once more and incubate at 52 °C overnight.
   7. Incubate tissues with two more changes of wax each for 1 hour, and then embed the tissue and store at 4 °C.
5. Cut and stain the paraffin-embedded sections with HE, and visualize via a light microscope with 100× magnification.
   1. Create paraffin sections using a rotary microtome.
   2. Dewax sections with three changes of xylene each for 3 minutes.
   3. Rehydrate tissues with 100%, 95%, 80%, and 70% ethanol each for 3 minutes, and then immerse in distilled water.
   4. Stain with hematoxylin for 10 minutes, and then rinse in running tap water.
   5. Differentiate with 0.1% hydrochloric acid ethanol for 5 minutes, and rinse in tap water.
   6. Stain with 0.5% eosin for 1 minute.
   7. Dehydrate tissues with 70%, 80%, 95%, and 100% ethanol each for 2 minutes.
   8. Clear the staining with xylene, and dry in fume hood.

Results

Burn duration of 30 seconds by hot iron resulted in wounds that were circular with a well-defined margin and uniformly pale with a rim of erythema (Figure 1D). Within each animal, there were six burn wounds on the dorsum. The arrangement of burn wounds was depicted in Figure 1K. Burn wounds were completely covered with CAPS-containing dressing and used to evaluate the depth of scar formation on post-burn days 0, 7, 21, and 42 and...

Discussion

The present study established a swine model of severe burn injury and examined the model using a CAPS-containing dressing. Our results suggest that this swine model can be used for monitoring the clinical performance of experimental dressings, including antibacterial property. Wound healing rate, wound closure, and antibacterial activity were also analyzed using VSS, H&E staining, and antibacterial test. The use of animal burn models has been developed as a valuable tool to review the pathophysiology of burn injury. ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sedation:
Ketamine	Merial	2528 ESP	10 mL vial
Azaperone	China chemical & pharmaceutical	47W406	100 mL vial
Atropine	Oriental chemical works	IN120802	1 mL vial
Anesthetic:
Tiletamine+Zolazepam	Virbac	BC91	5 mL vial
Isoflurane	Baxter	N002A225	100 mL vial
Surgery:
Hair clippers	Moser	-	-
Povidone iodine scrub solution	Ever star	HA161202	4 L barrel
Modified iron	-	-	-
0.9% saline solution	CHI SHENG	KC130	500 mL vial
Gauze	China Surgical Dressings Center	MO15900080	10 x10 cm
CAPS	CoreLeader Biotech Co., Ltd, Taipei, Taiwan	-	-
Paper tape	3M	NDC-8333-1530-01	2.5 cm x 9.1m
Waterproof film	3M	NDC-8333-1600-40	10 cm x 10 m
Adhesive plaster	Young chemical	BH1426015	10 cm x 10 m
Dissection:
Pair of standard sharp/blunt straight scissors (14 cm)	Shinetec instruments	ST-S114	-
Halstead-Mosquito (12.5 cm)	Shinetec instruments	ST-H012	-
Handle(# 4)	Shinetec instruments	ST-H004	-
Surgical Blade(#21)	Shinetec instruments	ST-B021	-
Post-Fixation & Storage:
50 ml Plastic centrifuge tube	Falcon	352070	-
10% neutral buffered formalin	Leica	3800604EG	-
Bacterial Growth Experiments
Blood agar plate (BAP) (TSA with 5% sheep blood)	CMP	-	90 mm Mono