This research was supported by the Chungnam National University and the National Research Foundation of Korea (NRF) grant funded by the government of Korea (NRF-2019R1A6A3A01093963 and NRF-2021R1F1A1062509).

All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Chungnam National University in South Korea, and then conducted based on the ethical guidelines of the International Association for the Study of Pain18.
1. Induction of scalding burn injury on the hind paw
<ol>
	<li>House the male ICR mice weighing 20-25 g in a light and temperature-controlled room (12/12 h light-dark cycle, 22.5 &#176;C &#177;...

<ol>
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The authors have nothing to disclose.

The scalding burn is a kind of thermal burn that is caused by heated liquids. It has been suggested that first- or second-degree burns occur in most cases, but long-term contact with heat sources can cause third-degree burns26. In the present study, burn injury was induced by exposing the right hind paw of mice into hot water at 65 &#176;C for 3 s4,26. Tissue damage was detected in the burn-injured paw, which shows common symptoms of burns...

Tissue damage, such as burn and trauma, is generally associated with the co-occurrence of acute pain. Burn injuries and trauma-related symptoms are an estimated 1,80,000 deaths every year are caused by burns-the vast majority occur in low- and middle-income countries from different types of burns1. According to a worldwide report, burns are common in children and account for about 40%-60% of hospitalized patients2,3. These specific injuries are even more serious as they can occur in everyday life, such as boiling or bathing water4,5. Although acute pain can be resolved spontaneously after recovery from tissue damage in most cases, it may be possible to become chronic due to abnormal changes in the nervous system6,7.
Recently, it has been suggested that acute pain can induce a depressed mood, and chronic pain can cause anxiety and depression8,9,10,11. The coexistence of pain and depression makes it more difficult to treat the patient. Depression also tends to increase pain sensitivity, which is likely to induce more intense depression and pain12. Complications of pain and depression are shown in animal models of peripheral inflammation13,14,15,16. The detailed mechanisms underlying pain-induced depression are not well known until now17. Thus, it is necessary to develop more effective treatments for burns to alleviate the side effects and symptoms.
Thus, the present study was designed to develop an animal model to study burn injury-induced acute pain and depression-like behavior in mice. For this, burn injury-related abnormal tactile sensitivity, altered gait pattern, and depression-like behavior were measured. In addition, this study attempts to validate the model using NSAIDs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL syringe</td>
			<td>BD</td>
			<td>307809</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL tube</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tube</td>
			<td>SPL</td>
			<td>50050</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetaminophen BioXtra, &#8805;99.0%</td>
			<td>Sigma-Aldrich</td>
			<td>A7085-100G</td>
			<td>Positive control (The analgesic agent, acetaminophen (200 mg/kg) was administered intraperitoneally once-daily for 7 days starting from the day of after burn injury (Only Burn + Acetaminophen group. (von-Frey test, gait analysis, and forced swimming test: Used for drug-dependent behavioral testing after burn injury), (Rota-rod test: It was used to investigate the motor and functional impairments of the drug in animals after burn injury).</td>
		</tr>
		<tr>
			<td>Alfaxan multidose (Alfaxalone)</td>
			<td>JUROX Pty.Limited</td>
			<td></td>
			<td>In this experiment, this material used for animal anesthesia, and was used as a positive control for experimentally treated drugs in the rota-rod test.</td>
		</tr>
		<tr>
			<td>CatWalk automated gait analysis system</td>
			<td>Noldus</td>
			<td>CatWalk XT</td>
			<td>Gait analysis in freely walking rodents is used to study the changes in limb movement and positioning in models with sensory-motor dysfunction</td>
		</tr>
		<tr>
			<td>OPTISHIELD (Cyclosporin ophthalmic ointment)</td>
			<td>Ashish Life Science</td>
			<td></td>
			<td>In this experiment, this material was used for an ointment to prevent corneal drying after induction of anesthesia.</td>
		</tr>
		<tr>
			<td>Plexiglass cylinder</td>
			<td>SCITECH KOREA</td>
			<td>custom made products</td>
			<td>Used in forced swimming test</td>
		</tr>
		<tr>
			<td>Rota-rod system</td>
			<td>SCITECH KOREA</td>
			<td>Accelerating rota rod</td>
			<td>Used in the measurement of Normal Motor Function</td>
		</tr>
		<tr>
			<td>von Frey filaments</td>
			<td>North Coast Medical</td>
			<td>NC12775</td>
			<td>Used in the measurement of Mechanical Allodynia</td>
		</tr>
		<tr>
			<td>Waterbath</td>
			<td>CHANGSHIN SCIENCE</td>
			<td>C-WBE</td>
			<td>Used in the burn injury induction</td>
		</tr>
	</tbody>
</table>

burn injury-induced pain and depression-like behavior in mice

Scalding water is the most common cause of burn injury in both elderly and young populations. It is one of the major clinical challenges because of the high mortality and sequelae in low- and middle-income countries. Burns frequently induce intense spontaneous pain and persistent allodynia as well as life-threatening problem. More importantly, excessive pain is often accompanied by depression, which may significantly decrease the quality of life. This article shows how to develop an animal model for the study of burn-induced pain and depression-like behavior. After anesthesia, burn injury was induced by dipping one hind paw of the mouse into hot water (65 &#176;C &plusmn; 0.5 &#176;C) for 3 s. The von Frey test and automated gait analysis were performed every 2 days after burn injury. In addition, depression-like behavior was examined using the forced swimming test, and the rota-rod test was performed to differentiate the abnormal motor function after burn injury. The main purpose of this study is to describe the development of an animal model for the study of burn injury-induced pain and depression-like behavior in mice.

In order to minimize animal suffering and reduce the number of animals used per the Three Rs (Replacement, Reduction, and Refinement) guidelines, this study was designed with the minimum number of animals for the collection of significant data established through preliminary experiment. In this study, behavioral experiments were independently conducted twice as follows. The gait analysis, mechanical allodynia, and depression-like behavior tests were conducted with Control (n = 5), Burn (n = 7; vehicle control; saline), a...

Watch this Scientific Journal Video about Burn Injury-Induced Pain and Depression-Like Behavior in Mice at JoVE.com

Burn Injury-Induced Pain and Depression-Like Behavior in Mice

A transient scald injury (65 &#176;C &plusmn; 0.5 &#176;C, 3 s) of one hind paw decreases the threshold (g) to von Frey filament stimulation of the ipsilateral side and alters gait pattern. Besides, burn injury induces depression-like behavior in the forced swimming test.

Scalding water is the most common cause of burn injury in both elderly and young populations. It is one of the major clinical challenges because of the ...

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.

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JoVE Journal

Neuroscience

2.9K Görüntüleme.  Chungnam National University. Bu protokol, yanık ağrısının ve tedavisinin çeşitli yönleri ve sonuçları üzerine araştırmalar için uygundur ve bu araştırma alanına önemli bilgiler getirmesi beklenmektedir. Bu protokol, sıcak suya bağlı bir yanık hayvan modeli oluşturur ve ilgili ağrıyı, yürüme bozukluklarını ve depresyon benzeri davranışsal tepkileri ölçmek için bir yöntem önerir. Prosedürü gösteren, Dr. Kim'in laboratuvarında yüksek lisans öğrencisi olan Jaehyuk Kim olacak.