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W tym Artykule

  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

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.

Streszczenie

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 °C ± 0.5 °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.

Wprowadzenie

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.

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Protokół

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

  1. House the male ICR mice weighing 20-25 g in a light and temperature-controlled room (12/12 h light-dark cycle, 22.5 °C ± 2.5 °C) with a humidity of 40%-60%.
    NOTE: Both male and female mice can be used for this protocol.
  2. 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.
  3. Assign the mice randomly to the experimental or control group and conduct blind experiments using animal numbers as codes.
  4. On the day of burn induction, anesthetize the mouse by intraperitoneal (i.p.) injection of 300 µL of alfaxalone at a dose of 100 mg/kg. Wear a surgical gown, gloves, and mask while performing the burn induction.
  5. 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.
  6. Apply an ophthalmic ointment to the eyes to prevent corneal drying after induction of anesthesia.
  7. Immerse the right hind paw of the deeply anesthetized mouse in hot water at 65 °C ± 0.5 °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.
  8. 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.
    ​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.

2. Measurement of mechanical allodynia

  1. 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.
  2. 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.
  3. Assess the mechanical threshold of the hind paw using the ascending stimulus method19,20.
  4. 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.
  5. 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.

3. Automated gait analysis

  1. 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.
  2. 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.
    1. After running the program, click on the Create New Experiment menu to designate the folder to save the data.
    2. After designation, set the maximum running time to 5 s and maximum allowed speed variations to 50%.
    3. Select a registered camera and set the walkway length to 30 cm in the Setup tab of the program.
    4. On the Acquire tab of the program menu, select Open Acquisition.
    5. Based on the status messages, click on the Snap Background button to acquire a background image of an empty walkway.
  3. 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'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.
  4. 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.
  5. Select the run to be analyzed and click on the Auto Classify button.
  6. After performing automatic classification, remove nose, genital recognition, and the misrecognition of paws to junk data in each run, and then analyze the data.
    ​NOTE: All statistical parameters are automatically analyzed and saved in the program, and raw data values can be found in the experimenter'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.

4. Measurement of depression-like behavior

NOTE: Despair-based behavior, immobility time in the water was measured by the forced swimming test.

  1. 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.
  2. Put the mouse into a clear plexiglass cylinder (10 cm x 25 cm) containing 15 cm of water (25 °C ± 0.5 °C) for 15 min.
  3. 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.

5. Measurement of normal motor function

NOTE: The rota-rod test was performed to differentiate the abnormal motor function after burn injury.

  1. 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.
  2. Place the animals on a rolling cylindrical platform (5.7 cm wide; 3 cm diameter) suspended 16 cm above the bottom of the apparatus.
  3. Allow each animal to train once a day on a rota-rod for at least 5 days prior to induction of burn injury.
  4. Perform the rota-rod test every 20 min for 2 h after drug administration. Set the cut-off time to 2 min.
  5. 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.

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Wyniki

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...

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Dyskusje

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 °C for 3 s4,26. Tissue damage was detected in the burn-injured paw, which shows common symptoms of burns...

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Ujawnienia

The authors have nothing to disclose.

Podziękowania

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).

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Materiały

NameCompanyCatalog NumberComments
1 mL syringeBD307809
1.5 mL tubeAxygenMCT-150-C
50 mL tubeSPL50050
Acetaminophen BioXtra, ≥99.0%Sigma-AldrichA7085-100GThis analgesic agent is used as a positive control.
Alfaxan multidose (Alfaxalone)JUROX Pty.LimitedIn 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.
CatWalk automated gait analysis systemNoldusCatWalk XTGait analysis in freely walking rodents is used to study the changes in limb movement and positioning in models with sensory-motor dysfunction
OPTISHIELD (Cyclosporin ophthalmic ointment)Ashish Life ScienceThis material was used for an ointment to prevent corneal drying after induction of anesthesia.
Plexiglass cylinderSCITECH KOREAcustom made productsUsed in forced swimming test
Rota-rod systemSCITECH KOREAAccelerating rota rodUsed in the measurement of Normal Motor Function
von Frey filamentsNorth Coast MedicalNC12775Used in the measurement of Mechanical Allodynia
WaterbathCHANGSHIN SCIENCEC-WBEUsed in the burn injury induction

Odniesienia

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Burn InjuryPain AssessmentDepression like BehaviorMouse ModelMechanical ThresholdGait AnalysisAnesthetization ProtocolExperimental DesignBehavioral TestingICR MiceTreatment OutcomesSurgical Protocol

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