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; 2.5 &#176;C) with a humidity of 40%-60%. 
	NOTE: Both male and female mice can be used for this protocol.</li>
	<li>Allow the animal free access to food and water, and acclimatize for at least 1 week before starting the experiment. 
	NOTE: All the animals were group-housed to exclude variables such as isolation stress.</li>
	<li>Assign the mice randomly to the experimental or control group and conduct blind experiments using animal numbers as codes.</li>
	<li>On the day of burn induction, anesthetize the mouse by intraperitoneal (i.p.) injection of 300 &#181;L of alfaxalone at a dose of 100 mg/kg. Wear a surgical gown, gloves, and mask while performing the burn induction.</li>
	<li>After deeply anesthetizing the mouse, disinfect around the right hind paw with 70% ethanol. 
	NOTE: Check the lack of response to pinch stimulation applied to the hind toes or tail to confirm the state of deep anesthesia.</li>
	<li>Apply an ophthalmic ointment to the eyes to prevent corneal drying after induction of anesthesia.</li>
	<li>Immerse the right hind paw of the deeply anesthetized mouse in hot water at 65 &#176;C &#177; 0.5 &#176;C for 3 s. Make a mark on the ankle of each mouse before immersing the hind paw in hot water to maintain consistency in the burned area.</li>
	<li>After induction of burns, bring the mice in a clean home cage and place them on a heating pad until the animals recover from anesthesia. 
	&#8203;NOTE: The analgesic agent, acetaminophen (200 mg/kg), was administered intraperitoneally once daily for 7 days starting from the day of burn injury (Only Burn + Acetaminophen group). The Burn group was treated with saline as vehicle control. The experiment was performed according to the method described in a previous study4.</li>
</ol>
2. Measurement of mechanical allodynia
<ol>
	<li>Bring the mice to the behavioral testing room and let them acclimatize at least for 30 min prior to the test. Wear a surgical gown, gloves, and mask while performing the test.</li>
	<li>Place the mice into a square box (diameter: 13 cm, height: 12 cm) on a metal mesh floor (mesh size: 0.7 cm x 0.7 cm) and let them acclimatize for at least 30 min.</li>
	<li>Assess the mechanical threshold of the hind paw using the ascending stimulus method19,20.</li>
	<li>Gently poke a series of von Frey filaments with 5-8 s intervals to stimulate the hind plantar. Obtain the baseline values on the day before burn induction. 
	NOTE: The 0.16-1.2 g von Frey filaments were used in the test to measure the paw withdrawal threshold in all animals, respectively. The paw withdrawal response test was started with the lowest bending force of von Frey filament (0.16 g in this protocol). If there was no response, then a filament with the next bending force was applied.</li>
	<li>Perform five trials to evaluate mechanical thresholds for each ipsilateral (injured) hind paw. 
	NOTE: The bending force of von Frey filament that produces response more than three times of the five trials in each animal was expressed as paw withdrawal threshold (PWT, g). Mechanical thresholds were measured a day before and at 1, 3, 5, and 7 days after burn injury. Analgesic effect was assessed 1 h after administration of the acetaminophen in the animal.</li>
</ol>
3. Automated gait analysis
<ol>
	<li>Acclimate the mice in the gait analysis system once daily for 10-15 min from 5 days before the burn injury. Wear a surgical gown, gloves, and mask while performing gait analysis.</li>
	<li>On the day of the test, bring the mice to the behavioral test room and acclimatize them for at least 30 min before the test. 
	NOTE: Perform acclimation and gait analysis tests in a dark environment. Set the conditions of the program menu as follows.
	<ol>
		<li>After running the program, click on the Create New Experiment menu to designate the folder to save the data.</li>
		<li>After designation, set the maximum running time to 5 s and maximum allowed speed variations to 50%.</li>
		<li>Select a registered camera and set the walkway length to 30 cm in the Setup tab of the program.</li>
		<li>On the Acquire tab of the program menu, select Open Acquisition.</li>
		<li>Based on the status messages, click on the Snap Background button to acquire a background image of an empty walkway.</li>
	</ol>
	</li>
	<li>Click on the Start Acquisition button, and then place the mouse at the entrance of the left-right traversable walkway. The recording will automatically start following the free movement of the mouse. 
	NOTE: If the animal&#39;s gait has been successfully recorded and all footsteps have been detected, it will be marked as Compliant Run with a green icon. If the software does not detect any footsteps, a red icon is displayed, in which case it is recommended to perform the recording again. The authors recommend collecting and analyzing at least five successful compliant runs performed with similar running speeds.</li>
	<li>On the Acquire tab of the program menu, select Classify Runs. 
	NOTE: After selecting the data obtained from the successful compliance run above, go to the video analysis screen where the gait patterns of the mice were recorded.</li>
	<li>Select the run to be analyzed and click on the Auto Classify button.</li>
	<li>After performing automatic classification, remove nose, genital recognition, and the misrecognition of paws to junk data in each run, and then analyze the data. 
	&#8203;NOTE: All statistical parameters are automatically analyzed and saved in the program, and raw data values can be found in the experimenter&#39;s analysis menu. Automatic gait analysis was performed before and at 1, 3, 5, and 7 days after burn injury. The evaluation was performed 30 min after acetaminophen administration in the Burn + Acetaminophen group and 30 min after saline treatment in the Burn group. This experiment was performed according to the method described in the previous studies4,21,22.</li>
</ol>
4. Measurement of depression-like behavior
NOTE: Despair-based behavior, immobility time in the water was measured by the forced swimming test.
<ol>
	<li>Bring the mice to the behavioral test room and acclimatize them for at least 30 min before the test. Wear a surgical gown, gloves, and mask while performing the forced swimming test.</li>
	<li>Put the mouse into a clear plexiglass cylinder (10 cm x 25 cm) containing 15 cm of water (25 &#176;C &#177; 0.5 &#176;C) for 15 min.</li>
	<li>After 24 h, put the mouse into the cylinder of the same conditions and measure the immobility time. 
	NOTE: Immobility time was measured for 5 min of test time, and the time whenever mice stopped climbing or swimming and just floated to keep their head above the water surface was recorded. The forced swimming test was performed on day 7 after the burn injury. The evaluation was performed 1 h after acetaminophen administration in the Burn + Acetaminophen group, and 1 h after saline treatment in the Burn group. The experiment was performed according to the method described in previous studies23,24.</li>
</ol>
5. Measurement of normal motor function
NOTE: The rota-rod test was performed to differentiate the abnormal motor function after burn injury.
<ol>
	<li>Bring the mice to the behavioral test room and acclimatize them for at least 30 min before the test. Wear a surgical gown, gloves, and mask while performing the forced swimming test.</li>
	<li>Place the animals on a rolling cylindrical platform (5.7 cm wide; 3 cm diameter) suspended 16 cm above the bottom of the apparatus.</li>
	<li>Allow each animal to train once a day on a rota-rod for at least 5 days prior to induction of burn injury.</li>
	<li>Perform the rota-rod test every 20 min for 2 h after drug administration. Set the cut-off time to 2 min.</li>
	<li>Measure the duration of time the mouse runs on a rotating rod at the constant speed of 15 revolutions per minute without falling. 
	NOTE: The rota-rod test was performed 7 days after the induction of burn injury. The evaluation was performed immediately after acetaminophen administration in the Burn + Acetaminophen group and after saline treatment in the Burn group. Alfaxalone was used as a positive control for experimentally treated drugs in this test. During the rotarod test, the duration of time the mouse runs on the rotating rod without falling is measured. The experiment was performed according to the method described in previous studies22,25.</li>
</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 such as redness, peeling of the skin, and swelling (Supplementary Figure 1)4.
Mechanical allodynia measurement is a commonly used pain response identification method in animal pain models and was measured using von-Frey filaments in this study. The ascending stimulus method with the von Frey filaments is used to determine the mechanical threshold required to induce an animal&#39;s paw withdrawal response19,20. The experiment started with the filament with the least stimulus. The bending force of the filament responding to a set number (three times in this protocol) was obtained as a paw withdrawal threshold value.
Gait analysis of rodents during free walking is used to study Parkinson&#39;s disease or limb movements and position changes in sensory-motor impairment models, including spinal cord injury and stroke27,28. The gait analysis system automatically analyzes various gait parameters, including paw intensity, paw print, stance phase, etc. Alterations of parameters that the gait analysis system can analyze can be used as pain-related indicators in the gait analysis of pain animal models. Therefore, gait analysis may be used as an experimental method to non-invasively quantify spontaneous pain in animal models4,21,22. Based on previous findings that gait parameters were decreased on the pain-induced side in animal models of pain21,22, this presented protocol quantified each gait parameter as the ratio of the burn-induced ipsilateral and contralateral side. In this protocol, paw print area and single stance data were converted into the rate of changes between ipsilateral (injured) and contralateral (non-injured) hind paws. The value of 50% means that the paw print size and the time to reach the floor are the same in both the ipsilateral and contralateral, while the value less than 50% indicates that these parameters are decreased in the burn-induced ipsilateral hind paw. The percentage changes between the ipsilateral and contralateral hind paws were used for obtaining all the data (i.e., normal mice showed ~50%, which means that the ipsilateral: contralateral ratio was 50:50). In normal animals, the parameters related to each hind limb appear the same on both sides when walking freely. However, the analysis of this protocol is focused on the fact that the parameters on the ipsilateral side decrease after pain induction. In addition, there is individual variation in each animal; accurate data may not be obtained when the raw data is analyzed as is. Therefore, each gait parameter value was converted into a ratio to obtain more accurate results during analysis. The present study has shown that the print area size and the single stance time of the ipsilateral hind paw were reduced after burn injury, and this reduction was restored by repeated intraperitoneal acetaminophen administration. These changes coincided with a similar pattern of time-course changes in pain behaviors after burn injury and drug administration.
Although controversial, the forced swim test is the most commonly used method to study the behavior of depressed rodents. The animals attempt to escape from the container full of water but eventually do not move, inducing despair29. However, it is argued that immobility is difficult to evaluate as a measure of depression because this test is associated with endurance as well as feelings of despair. To support the results of the forced swimming experiment, other methods of evaluating depression, such as the tail suspension test, novelty-suppressed feeding test, and sucrose consumption test, may be considered30,31. In the present study, immobilization time was increased after burn injury, and this increase was restored by acetaminophen administration.
The protocols of this study were designed to establish a model of acute pain accompanying depression-like behavior after burn injury. The depression-like behavior in this study may be the secondary effects of physical impairment and changes in thermal sensitivity after burn injury15,32,33. The results could suggest that mice with induced acute pain after burn injury exhibited depression-like behavior. It has been shown to improve pain response and consequent depression-like behaviors by experimentally treated drugs.
The rota-rod test is a performance test based on a rotational load that is generally forcibly applied to rodents with athletic activity. The test measures parameters such as running time and endurance. Some of the test features include, among others, the effects of an experimental drug or the balance of subjects in the neuropathic pain model, grip strength, and motor coordination assessment22,25,34. As shown in the results of this study, there was no change in the rota-rod running time&#160;following burn injury or acetaminophen treatment compared with that of the control group.
This study demonstrates the development of an animal model for studying burn injury-induced pain and depression-like behavior in mice. In this regard, this study has shown that scalding burn injuries induced mechanical allodynia, alterations of gait parameters, and depression-like behavior such as immobility time. This model is suitable for research on the various aspects and outcomes of burn pain and its treatment and is expected to bring important information to this research field.

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><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, &amp;ge;99.0%</td><td>Sigma-Aldrich</td><td>A7085-100G</td><td>This analgesic agent is used as a positive control.</td></tr><tr><td>Alfaxan multidose (Alfaxalone)</td><td>JUROX Pty.Limited</td><td></td><td>In this experiment, this material was 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>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), and Burn + Acetaminophen (n = 7) groups. In the rota-rod test, Control (n = 3), Burn (n = 4; vehicle control; saline), Burn + Acetaminophen (n = 4), Positive control (n = 4; Alfaxalone) groups were designed. Alfaxalone is a type of neuroactive steroid and anesthetic that is currently used in veterinary medicine as an injectable general anesthetic inducer. In this study, alfaxalone was used in animal anesthesia for burn induction and used as a positive control drug for motor impairments in the rota-rod test.
Data were expressed as mean &#177; S.E.M. In addition, experimental data obtained at different times were analyzed independently. Pain behavioral responses were calculated as the area under the curve (AUC). Two-way repeated measures ANOVA was performed to determine differences in the data from mechanical allodynia test, gait analysis, and rota-rod test over time. Dunnett&#39;s test was used for Post-hoc analysis to determine the P-value among the experimental groups. P-values less than 0.05 were considered significant. GraphPad Prism 6.0 software was used to analyze this statistical validity. All statistical analysis procedures were performed blindly in relation to the experimental conditions. A figure illustrating the burn injury-induced tissue damage is shown in Supplementary Figure 1.
Time-course changes in the paw withdrawal threshold (PWT, g) after burn injury are shown in Figure 1. The PWT (g) of burn injury-induced mice was decreased 1 day after burn-induction and sustained for 7 days compared with that of the control group. Acetaminophen administration (200 mg/kg, i.p., once daily for 7 days starting from the day of burn-induction) significantly reduced the burn-induced decrease in PWT (Figure 1A, ** p &#60; 0.01 versus Burn group). In addition, the AUC analysis (for 7 days) showed that acetaminophen administration significantly decreased the burn injury-induced mechanical allodynia (Figure 1B, *** p &#60; 0.001 versus Control group, ** p &#60; 0.01 versus Burn group).
Changes in the hind paw print area after burn injury over time are shown in Figure 2. Burn injury significantly reduced the ipsilateral hind paw print area from the day after induction and persisted for 7 days. The hind paw print area was significantly improved by administering acetaminophen (200 mg/kg, i.p., once daily for 7 days starting from the day of burn-induction) compared with the vehicle-treated group (Figure 2A,B, * p &#60; 0.05 and ** p &#60; 0.01 versus Burn group).
Time course changes in a single stance after burn injury are shown in Figure 3. Burn injury reduced the single stance (%) of the ipsilateral hind paw 1 day after burn-induction, and this reduction was maintained for 7 days. The hind paw single stance was improved by acetaminophen administration (200 mg/kg, i.p., once daily for 7 days starting from the day of burn-induction) compared with the vehicle-treated group (Figure 3A,B, * p &#60; 0.05 versus Burn group).
Changes in the immobility time obtained from the forced swimming test are shown in Figure 4. Immobility time&#160;of the burn injury-induced mice were increased 7 days after burn induction as compared with that of the control group. In burn injury-induced mice, acetaminophen administration (200 mg/kg, i.p., once daily for 7 days starting from the day of burn induction) significantly reduced the burn injury-induced increase in immobility time (** p &#60; 0.01 and *** p &#60; 0.001 versus Burn group).
The normal motor function was assessed based on the changes in running time on the rota-rod, as shown in Figure 5. Running time&#160;of the burn injury-induced mice did not change at 7 days after burn induction compared with that of the control group. By contrast, the running time&#160;of alfaxalone-treated mice (positive control) were significantly decreased during about 60 min. This result indicates that a burn injury used in this study does not cause motor impairment (*** p &#60; 0.001 versus Burn group).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62817/62817fig01.jpg" /> 
Figure 1: Mechanical allodynia assessed by von Frey test in burn injury-induced mice. (A) The paw withdrawal threshold (PWT, g) in the ipsilateral hind paw of mice was decreased 1 day after burn injury and sustained for 7 days as compared with the Control group. Acetaminophen administration (200 mg/kg, i.p., once daily for 7 days starting from the day of burn induction) significantly reduced the burn injury-induced mechanical allodynia. (B) The PWT was analyzed as the area under the curve (AUC). Arrows indicate the day of drug administration. *** p &#60; 0.001 versus Control group, ** p &#60; 0.01 versus Burn group. Two-way repeated measures ANOVA was performed to determine overall effects in the time-course of the von Frey test. Post-hoc analysis was performed using Dunnett&#39;s test in order to determine the P-value. <a href="https://www.jove.com/files/ftp_upload/62817/62817fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62817/62817fig02.jpg" /> 
Figure 2: Paw print area obtained from automated gait analysis in burn injury-induced mice. (A) Representative images of both the ipsilateral and contralateral hind paws of mice were captured by the gait analysis software. The contact size of the paw is reduced after burn injury compared to that of the Control group. This reduction was partially recovered by administering acetaminophen (200 mg/kg, i.p., once daily for 7 days starting from the day of burn induction). White rectangles indicate the hind paws analyzed by the software, (B) a graph shows the time course changes in the paw print area (%). Data are calculated as the percent of changes in the print area between the ipsilateral (right) and contralateral (left) hind paws (e.g., the value of 50% indicates the same paw print areas in the right and left hind paws). Arrows indicate the day of drug administration. * p &#60; 0.05 and ** p &#60; 0.01 versus Burn group. Two-way repeated measures ANOVA was performed to determine overall effects in the time-course of the print area on gait analysis. Post-hoc analysis was performed using Dunnett&#39;s test in order to determine the P-value. <a href="https://www.jove.com/files/ftp_upload/62817/62817fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62817/62817fig03.jpg" /> 
Figure 3: Single stance obtained from automated gait analysis in burn injury-induced mice. (A) Representative images of a single stance were captured by the gait analysis software. Different colors indicated the stance of each paw: blue, right front paw; pink, right hind paw; yellow, left front paw; green, left hind paw. The single stance of the ipsilateral hind paw was shortened after burn injury. This change was partially recovered by administering acetaminophen (200 mg/kg, i.p., once daily for 7 days starting from the day of burn induction). (B) A graph shows time-course changes in the single stance (%). Data are summarized as a line graph after calculating the percent of changes in single stance between the ipsilateral (right) and contralateral (left) hind paws (e.g., the value of 50% indicates the same single stance in the right and left hind paws). Arrows indicate the day of drug administration. * p &#60; 0.05 versus Burn group. Two-way repeated measures ANOVA was performed to determine overall effects in the time-course of single stance on gait analysis. Post-hoc analysis was performed using Dunnett&#39;s test in order to determine the P-value. <a href="https://www.jove.com/files/ftp_upload/62817/62817fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62817/62817fig04.jpg" /> 
Figure 4: The immobility time of the forced swimming test in burn injury-induced mice. Immobility time&#160;of the burn injury-induced mice were increased 7 days after burn induction as compared with that of the Control group. In burn injury-induced mice, acetaminophen administration (200 mg/kg, i.p., once daily for 7 days starting from the day of burn induction) significantly alleviated burn injury-induced enhanced immobility time. *** p &#60; 0.001 versus Control group, ** p &#60; 0.01 versus Burn group. One-way repeated measures ANOVA was performed to determine overall effects in the time-course of the forced swimming test. Post-hoc analysis was performed using Dunnett&#39;s test in order to determine the P-value. <a href="https://www.jove.com/files/ftp_upload/62817/62817fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62817/62817fig05.jpg" /> 
Figure 5: The normal motor function assessment based on the changes in running time of the rota-rod test in burn injury-induced mice. There was no change in running time&#160;of the burn injury-induced mice at 7 days after burn induction as compared with those of Control and acetaminophen-treated burn injury groups. However, the running time of alfaxalone-treated mice (Positive control) was significantly decreased to ~60 s. This result indicates that a burn injury used in this study does not cause motor impairment. ***&#160;p &#60; 0.001 versus Burn group. Two-way repeated measures ANOVA was performed to determine overall effects in the time-course of the rota-rod test. Post-hoc analysis was performed using Dunnett&#39;s test in order to determine the P-value. <a href="https://www.jove.com/files/ftp_upload/62817/62817fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Changes in tissue damage over time after induction of burns. After the scalding burn induction, significant tissue damage was observed, which increased gradually over time. In this study, Acetaminophen, used as a positive control drug, has shown a protective effect on tissue damages. <a href="https://www.jove.com/files/ftp_upload/62817/Supplementary Figure 1 (Revision).pdf" target="_blank">Please click here to download this File.</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 ...

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.0K Views.  Chungnam National University. A transient scald injury (65 °C ± 0.5 °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.

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

Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or knockdown gene expression in a relatively focused region without significant damage to the brain tissue. After recovery, the injected mouse can be tested on various behavioral tasks such as the Open Field Test (OFT) and the Forced Swim Test (FST). The OFT is designed to assess locomotion and the anxious phenotype in mice by measuring the amount of time that a mouse spends in the center of a novel open field. A more anxious mouse will spend significantly less time in the center of the novel field compared to controls. The FST assesses the anti-depressive phenotype by quantifying the amount of time that mice spend immobile when placed into a bucket of water. A mouse with an anti-depressive phenotype will spend significantly less time immobile compared to control animals. The goal of this protocol is to use the stereotactic injection of a lentivirus in conjunction with behavioral tests to assess how genetic factors modulate animal behaviors.

Stereotactic injection of lentiviruses expressing cDNAs or shRNAs can modulate gene expression in specific brain areas of mice. Here, we present a protocol to combine stereotactic injections with behavioral tasks, such as the Open Field Test (OFT) and the Forced Swim Test (FST).

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or ...

Behavior

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal experimentation whenever possible. When there is no alternative to animal experimentation, the total number of laboratory animals used should be the minimum needed to obtain valuable data. Moreover, appropriate refinement measures should be applied to minimize pain, suffering, and distress accompanying the experimental procedure. The categories used to classify the degree of pain, suffering, and distress are non-recovery, mild, moderate, or severe (EU Directive 2010/63). To determine which categories apply in individual cases, it is crucial to use scientifically sound tools.
The well-being-assessment protocol presented here is designed for procedures during which general anesthesia is used. The protocol focuses on home cage activity, the Mouse Grimace Scale, and luxury behaviors such as burrowing and nest building behavior as indicators of well-being. It also uses the free exploratory paradigm for trait anxiety-related behavior. Fecal corticosterone metabolites as indicators of acute stress are measured over the 24-h post-anesthetic period.
The protocol provides scientifically solid information on the well-being of mice following general anesthesia. Due to its simplicity, the protocol can easily be adapted and integrated in a planned study. Although it does not provide a scale to classify distress in categories according to the EU Directive 2010/63, it can help researchers estimate the degree of severity of a procedure using scientifically sound data. It provides a way to improve the assessment of well-being in a scientific, animal-centered manner.

We developed a protocol to assess well-being in mice during procedures using general anesthesia. A series of behavioral parameters indicating levels of well-being as well as glucocorticoid metabolites were analyzed. The protocol can serve as a general aid to estimate the degree of severity in a scientific, animal-centered manner.

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal ...

A Chronic Immobilization Stress Protocol for Inducing Depression-Like Behavior in Mice

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, therapeutic treatments for depression or research on depression is scarce. Thus, in the present paper, we propose a mouse model of depression induced by movement restriction. Chronic mild stress (CMS) is a well-known technique to induce depressive-like behavior. However, it necessitates a complex procedure consisting of a combination of various mild stresses. In contrast, chronic immobilization stress (CIS) is a readily accessible chronic stress model, modified from a restraint model that induces depressive behavior by restricting movement using a restrainer for a certain period. To evaluate the depressive-like behaviors, the sucrose preference test (SPT), the tail suspension test (TST), and the ELISA assay to measure stress marker corticosterone levels are combined in the present experiment. The described protocols illustrate the induction of CIS and evaluation of the changes in behavior and physiological factors for the validation of depression.

This article provides a simplified and standardized protocol for induction of depressive-like behavior in chronically immobilized mice by using a restrainer. In addition, behavior and physiological techniques to verify induction of depression are explained.

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, ...

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the &#34;cuff model&#34; of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around the main branch of the sciatic nerve. It induces a long-lasting mechanical allodynia, i.e., a nociceptive response to a normally non-nociceptive stimulus that can be evaluated by using von Frey filaments. Besides the detailed surgery and testing procedures, the interest of this model for the study of neuropathic pain mechanism, for the study of neuropathic pain sensory and anxiodepressive aspects, and for the study of neuropathic pain treatments are also discussed.

Neuropathic pain is a consequence of a lesion or disease affecting the somatosensory system. The &#8220;cuff model&#8221; of neuropathic pain in mice consists of the implantation of a polyethylene cuff around the main branch of the sciatic nerve. Mechanical allodynia is tested using von Frey filaments.

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in ...

Medicine

Animal Models of Depression - Chronic Despair Model (CDM)

Major depressive disorder is one of the most prevalent forms of mental illnesses and causes tremendous individual suffering and socioeconomic burden. Despite its importance, current pharmacological treatment is limited, and novel treatment options are urgently needed. One key factor in the search for potential new drugs is evaluating their anti-depressive potency in appropriate animal models. The classical Porsolt forced swim test was used for this purpose for decades to induce and assess a depressive-like state. It consists of two short periods of forced swimming: the first to induce a depressed state and the second on the following day to evaluate the antidepressant effect of the agent given in between the two swim sessions. This model might be suitable as a screening tool for potential antidepressive agents but ignores the delayed onset of action of many antidepressants. The CDM was recently established and represented a modification of the classical test with notable differences. Mice are forced to swim for 5 consecutive days, following the idea that in humans, depression is induced by chronic rather than by acute stress. In a resting period of several days (1-3 weeks), animals develop sustained behavioral despair. The standard read-out method is the measurement of immobility time in an additional delayed swim session, but several alternative methods are proposed to get a broader view of the mood status of the animal. Multiple analysis tools can be used targeting behavioral, molecular, and electrophysiological changes. The depressed phenotype is stable for at least 4 weeks, providing a time window for rapid but also subchronic antidepressant treatment strategies. Furthermore, alterations in the development of a depressive-like state can be addressed using this approach. CDM, therefore, represents a useful tool to better understand depression and to develop novel treatment interventions.

Animal Models of Depression - Chronic Despair Model CDM

The chronic despair mouse model (CDM) of depression consists of repetitive forced swim sessions and another delayed swim phase as a read-out. It represents a suitable model for induction of a chronic depressive-like state stable for at least 4 weeks, amendable to evaluate subchronic and acute treatment interventions.

Major depressive disorder is one of the most prevalent forms of mental illnesses and causes tremendous individual suffering and socioeconomic burden. ...

Urokinase-type Plasminogen Activator-induced Mouse Back Pain Model

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid induction of a persisting back pain model in mice are provided here using an injection of urokinase-type plasminogen activator (urokinase), a serine protease present in humans and other animals. The methodology for induction of persisting lower back pain in mice involves a simple injection of urokinase along the ligamentous insertion region of the lumbar spine. The urokinase inflammatory agent activates plasminogen to plasmin. Typically, the model can be induced within 10 min and hypersensitivity persists for at least 8 weeks.
Hypersensitivity, gait disturbance, and other standard anxiety- and depression-like measures can be tested in the persisting model.&#160;Back pain is the most prevalent type of pain. To improve awareness of back pain, the International Association for the Study of Pain (IASP) named 2021 the &#34;Global Year about Back Pain&#34; and 2022 the &#34;Global Year for Translating Pain Knowledge to Practice.&#34; One limitation of the therapeutic advancement of pain therapeutics is the lack of suitable models for testing persistent and chronic pain. The&#160;features of this model&#160;are suitable for testing potential therapeutics aimed at the reduction of back pain and its ancillary characteristics, contributing to IASP&#39;s naming 2022 as the Global Year for Translating Pain Knowledge to Practice.

Methods for simple, rapid induction of a back pain model in mice are provided here using an intraligament injection of urinary plasminogen activator.

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid ...

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using ...

Dual Hind Paw Injection for Enhanced Analgesic Testing

A Modified Inflammatory Pain Model to Study the Analgesic Effect in Mice

The hot plate test is widely used to evaluate analgesic effects on inflammatory pain in mice. A commonly used model of inflammatory pain was induced with an intraplantar injection of carrageenan in one hind paw. However, the findings from our laboratory showed that mice with a single-hind-paw injection of carrageenan lifted their paws to avoid thermal nociception during the hot plate test. Because of this response,&#160;previous injection method cannot accurately reflect the thermal pain threshold. Thus, we investigated a new method to avoid this issue. In the present study, we modified the previous method by injecting carrageenan into both hind paws to establish the model of inflammatory pain. The results demonstrated that both-hind-paw injection with carrageenan was sensitive and a better method to induce inflammatory pain when using the hot plate test than single-hind-paw injection. On the basis of these findings, we designed further experiments in which mice with either both-hind-paw or single-hind-paw injection of carrageenan were treated intragastrically with celecoxib (30 mg/kg). The results of the hot plate test showed that celecoxib augmented the thermal pain threshold in mice with both-hind-paw injection of carrageenan but not in mice with single-hind-paw injection of carrageenan. In summary, we developed a superior method to induce a model of inflammatory pain to evaluate analgesic effect.

Author Spotlight: Enhanced Method for Evaluating Analgesic Effects &#8212; Dual Hind Paw Carrageenan Injection in Mice

Here, we present a modified inflammatory pain model with the both-hind-paw carrageenan injection to evaluate the analgesic effect.

The hot plate test is widely used to evaluate analgesic effects on inflammatory pain in mice. A commonly used model of inflammatory pain was induced with ...

Consistent Burn Injury Model in Mice for Evaluating Therapeutic Dressings

Chessboard-like Burn Wound Healing Model of Mice Based on Digital Heating Device

Severe burn injuries are among the most traumatic and physically debilitating conditions, impacting nearly every organ system and resulting in considerable morbidity and mortality. Given their complexity and the involvement of multiple organs, various animal models have been created to replicate different facets of burn injury. Methods used to produce burned surfaces vary among experimental animal models. This study describes a simple, cost-effective, and user-friendly mouse burn model for creating consistent full-thickness burns using a digital heating device. The tip of this device was applied to the dorsum of mice for 10 s at 97 &#176;C to establish a chessboard-like burn and examine wound healing under the treatment of an experimental dressing. Skin samples were collected for histological analysis, including Hematoxylin and Eosin (H&amp;E) staining and Masson's staining. Wound healing was assessed through analysis of the wound area and microscopic examination of inflammatory infiltration, re-epithelialization, and granulation tissue formation. The mouse burn injury model can serve as a fundamental tool in studying the pathophysiology of thermal injuries and evaluating therapeutic interventions.

A streamlined protocol is presented for establishing a burn wound healing model in mice using a digital heating device. The chessboard-like experimental sites created on the skin facilitate further functional analysis for the wound healing assay.

Severe burn injuries are among the most traumatic and physically debilitating conditions, impacting nearly every organ system and resulting in ...

Biology

Inducing Depression-like Behavior in a Mouse via Immobilization Stress

Source: Son, H., et al., <a href="https://app.jove.com/v/59546">A Chronic Immobilization Stress Protocol for Inducing Depression-Like Behavior in Mice.</a> J. Vis. Exp. (2019).
This video demonstrates the method of inducing chronic stress in a mouse through repeated immobilization, leading to corticosterone release and reduced glutamate receptor activity in neurons, which results in depression-like behavior.