This protocol is suitable for research on the various aspects and the outcomes of burn pain and its treatment, and is expected to bring important information to this research field. This protocol establishes a hot water-induced burn animal model and proposes a method to measure related pain, gait disturbances, and depression-like behavioral responses. Demonstrating the procedure will be Jaehyuk Kim, a master student in Dr.Kim's laboratory.
To begin, house the male ICR mice weighing 20 to 25 grams in a light and temperature-controlled room. Allow the mice to have free access to food and water and acclimatize for at least one week before the start of the experiment. After one week, assign the mice randomly to the experimental or control group and use animal numbers as codes for conducting blind experiments.
Before performing the burn induction, wear a surgical gown, gloves, and mask. Next, after deeply anesthetizing the mouse, disinfect around the right hind paw with 70%ethanol and apply an ophthalmic ointment to the eyes to prevent corneal drying, then make a mark on the ankle of each mouse and immerse the right hind paw in hot water at 65 degrees Celsius for three seconds. After the burn injury, place the mouse on a heating pad in a clean home cage until it recovers from anesthesia.
Bring the mice to the behavioral testing room and let them acclimatize for at least 30 minutes before the test. While the mice get acclimatized, put on a surgical gown, gloves, and mask. Next, place the mice into a square box on a metal mesh floor and let them acclimatize for another 30 minutes.
Then to assess the mechanical threshold of the hind paw using the ascending stimulus method, gently poke the paw with a series of Von Frey filaments in five to eight-second intervals to stimulate the hind plantar. Perform five trials to evaluate mechanical thresholds for each ipsilateral hind paw. For gait analysis, acclimatize the mice in the gait analysis system daily for 10 to 15 minutes beginning five days before the burn injury.
On the test day, after wearing surgical gown, gloves, and mask, bring the mice to the behavioral test room and let them acclimatize for 30 minutes before the test, then start the gait analysis program and click on the create new experiment menu to designate the folder to save the data. After designation, set the maximum running time to five seconds and maximum allowed speed variations to 50%Next, in the setup tab of the program, select a registered camera and set the walkway length to 30 centimeters. Then on the acquired tab of the program menu, select open acquisition and based on the status messages, click on the snap background button to acquire a background image of an empty walkway.
Next, click on the start acquisition button and place the mouse at the entrance of the left/right transversable walkway. The recording will start automatically following the free movement of the mouse. After a successful recording, select classify runs from the acquired tab of the program menu, then select the run to be analyzed and click on the auto-classify button.
After the automatic classification is complete, remove nose, genital recognition, and misrecognition of paws to junk data in each run, then analyze the data. After a 30-minute acclimatization period in the behavioral test room, place the mouse into a clear Plexiglas cylinder containing 15 centimeters of water for 15 minutes. The next day, place the mouse into the cylinder again under the same conditions and measure the immobility time, that is when the mouse stops climbing or swimming and just floats with its head above the water surface.
Next, to measure normal motor function, place the animal on a rolling cylindrical platform suspended 16 centimeters above the bottom of the apparatus. Before inducing burn injury, allow the mice to train once a day on the rotarod for at least five days. After drug administration, perform a rotarod test every 20 minutes for two hours with a cutoff time of two minutes.
Measure the duration of time the mouse runs on a rotating rod at the constant speed of 15 revolutions per minute without falling. The paw withdrawal threshold of burn injury-induced mice decreased after day one and was sustained for seven days compared to the control group. Acetaminophen administration significantly reduced the burn-induced decrease in the paw withdrawal threshold.
Additionally, the area under the curve analysis showed that acetaminophen administration significantly decreased the burn injury-induced mechanical allodynia. The hind paw print area also significantly reduced after the induction of burn injury and persisted for seven days. Acetaminophen administration significantly improved the hind paw print area compared to the vehicle treatment.
Further, burn injury reduced the single stance of the ipsilateral hind paw after day one and this reduction was maintained for seven days. The hind paw single stance was improved by acetaminophen administration. In the forced swimming test, the immobility times of the burn injury-induced mice were increased seven days after injury compared to those of the control group.
Acetaminophen administration significantly reduced the burn injury-induced increase in immobility time. In the rotarod test, running times of the burn injury-induced mice were similar to those of the control group seven days after burn induction. In contrast, the running times of alphaxolone-treated mice were significantly decreased by about 60 minutes, indicating that this burn injury method does not cause motor impairment.
The extent of the burn applied to each animal must be kept constant to obtain accurate results. The various behavioral experimental analysis methods proposed in this study can also be applied to other animal models related to pain, gait disturbance, and depression-like behavior. The proposed analysis methods can quantify various symptoms after induction of burn injury, which will be helpful for the development of burn-related research and treatment methods in the future.
