This burn injury model is able to recapitulate the clinical situations observed in hospitalized burn patients following burn injuries such as immune dysregulation and bacterial infections. This method of inducing burn in rats is easy to learn and can be used for the development and evaluation of new tropical drugs for burn wounds and infections. This model can be used to evaluate wound healing as well as the progression of bacterial infection at the burn site.
This burn induction method can also be used in mouse and other animal models for burn induction and to study the bacterial infection of burn wounds. Begin preparing the anesthetized rat for the burn injury by moving it over a heating pad in a prone position. Using a cotton-tipped applicator, apply eye lubricant on both eyes to prevent corneal drying.
Shave the rat's dorsal area with an electric clipper in a large rectangle from the shoulder blades down to the base of the tail. Wipe out the loose hairs from the shaved area using the tissue soaked in saline. Apply the hair removal lotion to the shaved area using a cotton-tipped applicator.
After three minutes, wipe the area with a wet gauze sponge twice to remove the lotion and prevent skin irritation. Turn off the isoflurane and remove the nose cone before placing the rat in the recovery cage. On the day of the burn injury experiment, set the water bath temperature to 97 degrees Celsius and one hour prior to the experiment, place 420 grams of four copper rods in the water bath.
Once the rat is unresponsive to toe pinch on all the limbs, place him on the heating pad in a prone position and inject 20 milligrams per kilogram of body weight of morphine via the intraperitoneal, or IP root, for pain management. Next, check the water bath temperature. Set the timer and put on the heat-resistant gloves.
Take one heated copper rod from the water bath and touch it on the dorsum area for seven seconds. Immediately apply the next three burns one after the other using one rod per burn site and produce an approximately 20%total body surface area full-contact burn. After the burn, resuscitate the animal by injecting 0.1 milliliter per gram body weight of lactated Ringer's solution through the IP route.
Once done, turn off the isoflurane. Remove the nose cone and place the rat on the heat mat. On the burn and infection day, centrifuge the Pseudomonas aeruginosa or Staphylococcus aureus culture separately at 4, 000 times g for 5 minutes.
Wash the pellet with normal saline before diluting the pellet using saline to 0.1 optical density at 600 nanometer. Dilute 200 microliters of the bacterial suspension with 800 microliters of saline and get the desired bacterial inoculum of 2 by 10 to the 7th colony-forming units or CFU per milliliter. 15 minutes after the burn, subcutaneously inject 50 microliters of diluted inoculum of the bacteria near the burn area using a 29 gauge needle.
Place the rat on the heating pad for recovery. After 15 to 20 minutes, transfer the recovered animal to a clean cage. Immediately after the burn injury, evaluate the skin injury morphologically in terms of color and margin.
After euthanization, analyze complete blood counts to determine the effect of burn induction on the host immune system. Collect the tissue of the euthanized rat in a 10-milliliter collection tube. Homogenize the samples using a tissue homogenizer and serially dilute the tissue homogenates in normal saline.
Plate 100 microliters of undiluted homogenate and all dilutions of each tissue sample collected from P.Aeruginosa-infected rats on cetrimide agar plates. After incubating the plates for 16 to 18 hours at 37 degrees Celsius in an incubator, count the bacterial colonies on the plates. Multiply them by the dilution ratio to get the CFU per milliliter count and normalize it with the tissue's weight to calculate the CFU program tissue.
This highly reproducible protocol resulted in a third-degree full-thickness burn injury in rats. The color of the burn injury changed from white to brown in 24 hours to 72 hours post-burn. In histological analysis, the skin samples from burned animals showed injury across all layers at 24, 48, and 72 hours post-burn injury, whereas the non-burn skin showed a clear distinction of epidermis, dermis, and subcutaneous tissue layers.
Destruction of the epidermal layer and damage to the dermis'full thickness with subcutaneous fat and skeletal muscle were observed in burned skin samples. In 24-hour post-burn, a full thickness burn was more than 2.61 millimeters in depth. In bacterial clearance evaluation, bacterial recovery was observed in all burn injury rats.
During P.aeruginosa PAO1 infection, the number of bacteria recovered 24 hours post-infection from the skin of burned rat was less than the initial inoculum of 1 by 10 to the 6th CFU. However, it increased 48 and 72 hours post-infection. In S.aureus, ATC25923 infection, 2 log 10 increase was observed at all time points in the skin compared to the initial inoculum, suggesting that the establishment of S.aureus infection resulted due to the active replication in the tissues.
The subcutaneous tissue and muscles showed a higher bacterial load than the lung and spleen. The data showed that burned rats developed a systemic infection 24 hours following P.aeruginosa inoculation and 48 hours following S.aureus inoculation. Completely remove hair on the rat dorsum and keep the temperature and burn induction time as mentioned in the protocol.
The leftover hair and different temperature of the copper rods can affect the quality of the burn. The rat burn model can be explored to evaluate novel topical therapeutics for treatment of burn wound infections and for evaluating different wound dressings.
