Rat Burn Model to Study Full-Thickness Cutaneous Thermal Burn and Infection

Summary

A model mimicking the clinical scenario of burn injury and infection is necessary for furthering burn research. The present protocol demonstrates a simple and reproducible rat burn infection model comparable to that in humans. This facilitates the study of burn and infections following burn for developing new topical antibiotic treatments.

Abstract

Burn induction methodologies are inconsistently described in rat models. A uniform burn wound model, which represents the clinical scenario, is necessary to perform reproducible burn research. The present protocol describes a simple and reproducible method to create ~20% total body surface area (TBSA) full-thickness burns in rats. Here, a 22.89 cm² (5.4 cm diameter) copper rod heated at 97 °C in a water bath was applied to the rat skin surface to induce the burn injury. A copper rod with a high thermal conductivity was able to dissipate the heat deeper in the skin tissue to create a full-thickness burn. Histology analysis shows attenuated epidermis with coagulative damage to the full-thickness extent of the dermis and the subcutaneous tissue. Additionally, this model is representative of the clinical situations observed in hospitalized burn patients following burn injury such as immune dysregulation and bacterial infections. The model can recapitulate the systemic bacterial infection by both Gram-positive and Gram-negative bacteria. In conclusion, this paper presents an easy-to-learn and robust rat burn model that mimics the clinical situations, including immune dysregulation and bacterial infections, which is of considerable utility for the development of new topical antibiotic drugs for burn wound and infections.

Introduction

Burn injuries are among the most devastating forms of trauma, with mortality rates reaching 12% even in specialized burn centers^1,2,3. According to recently published reports, ~486,000 burn patients require medical care annually in the United States, with nearly 3,500 deaths^1,2,3,4,5,6. Burn injury imposes a major challenge for patients' immune system and creates a significant open wound, which is slow to heal, leaving them susceptible to cutaneous, pulmonary, and systemic colonization with nosocomial, opportunistic bacteria. Immune dysregulation combined with the bacterial infection is associated with increased morbidity and mortality in burn patients⁷.

An animal burn and infection model is essential for studying the pathogenesis of bacterial infections following skin damage and immune suppression associated with burn trauma. Such models enable the design and evaluation of new methods for treating bacterial infections in burn patients. Rats and humans share similar skin physiological and pathological characteristics that have been previously documented⁸. Additionally, rats are smaller in size, making them easier to handle, more affordable, and easier to procure and maintain than larger animal models.

These characteristics make rats an ideal model animal to study burns and infections⁹. Unfortunately, the technique for burn induction is inconsistent and often minimally described^{10,11,12,13,14}. The present protocol is designed to develop a simple, cost-effective, and reproducible procedure for creating a consistent full-thickness burn injury in a rat model that simulates the clinical scenario and can be used to evaluate immune suppression and bacterial infection.

Protocol

All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of The University of North Carolina and were conducted in accordance with its established guidelines. Male and female Sprague Dawley rats (250-300 g) aged 7-9 weeks old were used for the experiments. All animals were housed in a 12 h:12 h light-dark cycle with free access to food and water ad libitum. Always work with your institutional veterinarian about an analgesic plan prior to study initiation. 

1. Preparing rats for the burn injury

1. Prepare the animals for burn injury 24 h prior to the burn.
2. Anesthetize the rat with 5% isoflurane in 100% oxygen in an induction chamber for 5 min (flow rate: 2 L/min) until breathing has slowed.
3. Once the rat is deeply anesthetized (unresponsive to toe pinch on all limbs), move the rat over to a heating pad in a prone position and reduce the isoflurane to 1.5% in oxygen for maintenance through a nose cone.
4. To prevent corneal drying after anesthesia and during the procedure, apply eye lubricant on the corneas of both eyes using a cotton-tipped applicator.
5. Shave the dorsal area of the rat using an electric clipper (see the Table of Materials) and remove as much hair as possible in a large rectangle from the shoulder blades down to the base of the tail (Figure 2A).
6. Clean the shaved area with a tissue soaked in saline to wipe out the loose hairs. Apply hair removal lotion to the shaved area using a cotton-tipped applicator and leave it on for ~3 min.
   NOTE: Application of the referenced hair removal lotion for more than 3 min will induce red rashes on the skin.
7. Wipe the area with a wet gauze sponge twice to remove the lotion and prevent skin irritation.
8. Turn off the isoflurane, remove the nose cone, and place the rat in the recovery cage.
   NOTE: Put a heating pad in the recovery cage.
9. Transfer the recovered animal to a clean housing cage for the next day's burn procedure (it may take ~10-15 min for the rat to recover from the anesthesia).

2. Inducing the burn injury in rats

1. On the day of the burn, set the water bath temperature to 97 °C and place all four copper rods (420 g each; Figure 1) in the water bath 1 h prior to the burn experiment to let the rods warm up uniformly.
   NOTE: The rods must be immersed in the water. Check the accuracy of the digital temperature display using a thermometer before the experiment.
2. Anesthetize the rat as mentioned in section 1.
3. Once the rat is unresponsive to toe pinch on all limbs, place it on a heating pad in a prone position with 1.5% isoflurane in oxygen for maintenance (Figure 2A).
4. Inject morphine (20 mg/kg body weight) via the intraperitoneal (i.p.) route for pain management⁶.
5. Check the temperature of the water in the water bath. Set up the timer and put on the heat-resistant gloves.
6. Take out one heated copper rod from the water bath and touch it on the dorsum area of the rat for 7 s to induce the burn.
   NOTE: Keep a minimum distance (10-15 cm) between the water bath and the animal to minimize heat loss, and do not put pressure on the rods while inducing the burn (i.e., contact must be maintained by gravity).
7. Apply four burns, using one rod per burn site, one immediately after the other to produce an approximately 20% TBSA full-contact burn (Figure 2B).
8. After the burn, resuscitate the animal by i.p. injection of lactated Ringer's solution (0.1 mL/g body weight).
   NOTE: Use a body temperature-adjusted lactated ringer's solution to resuscitate the rats.
9. Turn off the isoflurane, remove the nose cone, and place the rat on the heat mat for recovery.

3. Preparation of bacterial inoculum and infection

1. Streak the frozen sample of Pseudomonas aeruginosa PAO1 and Staphylococcus aureus ATCC25923 on Muller Hinton Agar (MHA) plates, 2 days prior to the burn experiment.
2. On the next day, select a single colony of grown bacteria from the plate, and using an inoculation loop, slightly scrape it off the plate. Then, place it in the culture tube to inoculate 10 mL of Muller Hinton Broth (MHB) and culture overnight at 37 °C in an incubator shaker.
3. On the day of the burn and infection, centrifuge the culture at 4,000 × g for 5 min. Wash the pellet with normal saline (0.9% NaCl solution).
4. Resuspend the bacterial pellet in saline and dilute up to 0.1 OD_600nm (optical density at 600 nm). Dilute the bacterial inoculum by taking 200 µL of this bacterial suspension and mixing it with 800 µL of saline to get the desired bacterial inoculum of 2 × 10⁷ CFU/mL.
5. Inject 50 µL of P. aeruginosa or S. aureus inoculum prepared in the previous step (infection dose 1 × 10⁶ CFU) into the anesthetized rat 15 min after the burn, using a 29 G needle subcutaneously as close to the burn wound as possible.
6. After infecting the burn wound, place the rat on the heating pad for recovery. Once the animal recovers (~15-20 min), house it in a clean cage.
   NOTE: After the burn injury, house one rat per cage. Use water wet food pellets for easy chewing and place them on the cage floor for easy reach.
7. Fill the water bottles in the cage with morphine-spiked water (0.4 mg/mL) for pain management.
   NOTE: Oral Morphine mirrors the clinical situation with human burn patients. This study utilized oral morphine to keep these experiments comparable to human burn patients after consulting with the veterinary staff on numerous occasions. Drinking and weight logs were maintained throughout the experiment. Use the same drinking system during all the procedures. Other analgesics, such as Buprenorphine, can be administered subcutaneously/intraperitoneally as per institutional animal care guidelines.  
8. Fill out the monitoring checklist and monitor the animals closely for distress or illness for the entire duration of the experiment.

4. Evaluation of the burn injury

1. Evaluate the skin burn injury morphologically in terms of color and margin immediately after the burn injury.
2. Stain the burned skin with hematoxylin and eosin (H&E) to visualize the burn wound structure and epithelial gap¹⁵ (see step 5.6 for sample processing).

5. Postprocessing of rat samples and bacterial enumeration

1. Euthanize the rat at 24, 48, and 72 h post burn with an overdose of anesthesia.
2. Withdraw blood samples from the rats via cardiac puncture and collect them in a mini collect tube.
   1. Analyze complete blood counts from the blood samples to determine the effect of burn induction on the host immune system.
3. Harvest skin, subcutaneous tissue, muscle, lung, and spleen at the time of euthanasia.
   NOTE: Keep one part (~1 cm × 1 cm; weighing ~200-300 mg) of the skin for H&E staining and another part for bacterial enumeration.
4. Collect the tissues in a 10 mL collection tube and place them in normal saline on ice for bacterial enumeration.
5. Normalize the tissue weight with normal saline and homogenize the samples using a tissue homogenizer (see the Table of Materials).
   1. Serially dilute the tissue homogenates in normal saline.
   2. Plate 100 µL of undiluted homogenate and all dilutions of each tissue sample on cetrimide agar plates for samples collected from rats infected with P. aeruginosa.
      NOTE: Use mannitol agar plates for plating samples collected from rats infected with S. aureus.
   3. Incubate the plates at 37 °C in an incubator for 16-18 h.
   4. The next day, count the bacterial colonies on the plates, multiply by the dilution ratio to get the CFU/mL count, and normalize with the tissue's weight to calculate the CFU/g tissue.
   5. Employ data analysis software to plot the bacterial counts in different organs at the different sampling time-points.
6. Perform H&E staining of burned skin to visualize the wound structure and epithelial gap.
   1. Using scissors and toothed forceps, cut a skin patch of 1 cm x 1 cm from the burn area and immerse it in a fixative (10% neutral buffered formalin, NBF) for 48 h at room temperature.
      NOTE: Swirl the container to ensure all tissues are completely immersed in the fixative, with the volume of the fixative 30x the tissue volume.
   2. Dehydrate the skin tissue with 70% (v/v) ethanol for 72 h at room temperature.
   3. Process the dehydrated samples in paraffin blocks to cut the sections and stain with H&E¹⁵.
   4. Digitally image the stained slides in a slide scanner (see the Table of Materials) using a 40x objective.
   5. Analyze the scanned image using software (see Supplementary File 1 for processing of the image for analysis; see the Table of Materials).
   6. Examine all fields of the stained skin section to evaluate the condition of the epidermis, dermis, subcutaneous tissue, and skeletal muscle.

Results

The protocol presented here is highly reproducible and resulted in a third-degree, full-thickness burn injury in rats. The burn wound appears waxy white after burn induction (Figure 2B). The color of the burn injury changed from white to brown over the course of 72 h post burn (Figure 2B-E).

Histological analysis confirmed a full-thickness burn (depth >2.61 mm at 24 h post burn;

Discussion

Several burn models have been presented to study the pathophysiology of burn injury^8,12,16,17. In the present study, we employed a rat model to develop a simple and reproducible protocol to induce a full-thickness burn followed by bacterial infection to simulate an infected burn trauma in patients. The choice of the rat as the animal model to mimic human conditions is based on a balance of cost...

Materials

Name	Company	Catalog Number	Comments
1 mL syringe	BD, USA	309597	Used to inject the analgesic
1.7 mL Microtube	Olympus, USA	24-282	Used to carry morphine
10% NBF	VWR, USA	16004-115	Used to fix the skin piece for staining
30 mL syringe	BD, USA	302832	Used to inject the lactate ringer solution
70% ethyl alcohol	Fischer Scientific, USA	BP28184
Aperio AT2 Digital Pathology  Slide Scanner with ImageScope software	Aperio, Technologies Inc., Vista, CA, USA	n/a	Scanning of H & E slides and analysis
Cetrimide agar plates	BD, USA	285420	Selective media plates for Pseudomonas aeruginosa growth
Copper rods	n/a	n/a	Used to induce the burn injury
Cotton tipped applicators	OMEGA Surgical supply, USA	4225-IMC	Used to apply eye ointment
Electric shaver	Oster, USA	Golden A5	Used to remove the dorsal side hairs
Eye lube	Dechra, UK	n/a	The eye wetting agent to provide long lasting comfort and avoid eye dryness
Fluff filled underpads	Medline, USA	MSC281225	Used in the burn procedure
Forcep	F.S.T.	11027-12	Used to hold the skin piece
Gauze sponges	Oasis, USA	PK412	Used to clean the applied nair cream from the dorsal side
Heat-resistant gloves	n/a	n/a	Used to hold the heated copper rods
Hematology Analyzer	IDEXX laboratories, USA	ProCyte Dx
Induction chamber	Kent Scientific, USA	vetFlo-0730	Used to anesthesize the animals
Insulin syringe	BD, USA	329461
Isoflurane	Pivetal, USA	NDC46066-755-04	Used to anesthesized rats to induce a loss of consciousness
Isoflurane vaporiser	n/a	n/a
Lactated ringer's solution	icumedical, USA	NDC0990-7953-09	Used to resuscitate the rats
L-shaped spreader	Fischer Scientific, USA	14-665-230
Mannitol Agar	BD, USA	211407	Selective media plates for Staphylococcus aureus growth
Minicollect tubes (K2EDTA)	greiner bio-one, USA	450480	Used to collect the blood
Morphine	Mallinckrodt, UK	NDC0406-8003-30	This analgesia was used to induce the inability to feel burn injury pain
Muller Hinton Broth	BD, USA	275730
Muller Hinton II Agar	BD, USA	211438
Nair hair removal lotion	Nair, USA	n/a	Used to remove the residual hairs on dorsal side
Needle 23 G	BD, USA	305193	Used to inject the lactate ringer solution
Normal saline	n/a	n/a
Spectrophotometer	ThermoScientific, USA	Genesys 30
Sprague-Dawley rats, male and female	Charles River Labs	n/a	7-9 weeks old for burn induction
Surgical Scissor	F.S.T.	14501-14	Used to cut the desired skin piece
Tissue collection tubes	Globe Scientific	220101236
Tissue Homogenizer	Kinematica, Inc, USA	POLYTRON PT2100	Used to homogenize the tissue samples
Water bath	Fischer Scientific, USA	n/a	Used to induce the burn injury
Weighted heating pad	Comfytemp, USA	n/a	Used during the procedure to keep rat's body warm