Name: Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice (Video) | JoVE Journal
Uploaded: 2022-02-18T14:15:00.000+00:00
Duration: 6 min 57 s
Description: Learn the mechanical conflict-avoidance assay to measure pain sensitivity in mice using a specialized chamber with adjustable probe heights. This method assesses motivational responses and enhances preclinical pain research translation.

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5 minutes

GM is supported by an NDSEG Graduate Fellowship. VLT is supported by NIH NIGMS grant #GM137906 and the Rita Allen Foundation. AJS is supported by Department of Defense grants W81XWH-20-1-0277, W81XWH-21-1-0197, and the Rita Allen Foundation. We are grateful to Dr. Alexxai Kravitz at Washington University School of Medicine for designing and making freely available the 3D printer files for the chamber 2 floor and probe plate.

All experiments involving the use of mice and the procedures followed therein were approved by Institutional Animal Care and Use Committees of MD Anderson Cancer Center and Stanford University, in strict accordance with the National Institutes of Health&#8217;s&#160;Guide for the Care and Use of Laboratory Animals.
1. MCA construction
<ol>
	<li>Construct chamber 1 with the following dimensions: 125 mm x 125 mm x 125 mm (width x depth x height) from opaque white 3 mm thick acrylic used for the sidewalls, floor, ceiling. Use a clear 3 mm thick acrylic for the front-facing wall. Glue all sides together well in advance using dedicated acrylic adhesive. 
	CAUTION: Acrylic adhesive is considered hazardous material (flammable, vapor harmful, may be harmful if swallowed, may irritate skin or eyes). Such adhesives should only be used in accordance with the manufacturer&#39;s instructions (i.e., with appropriate PPE in a well-ventilated area).</li>
	<li>Attach the lid of chamber 1 with a hinge, so that mice can be easily placed into and retrieved from the chamber. Attach self-adhesive light-emitting diode (LED) tape to the inner surface of the lid to provide illumination of ~4800 lux.</li>
	<li>Close off chamber 1 from the rest of the MCA by sliding an opaque acrylic sheet into and out of position.</li>
	<li>Construct the MCA test chamber, chamber 2, as a 270 mm long unlit chamber fabricated from translucent dark red acrylic (3 mm thick) on all sides, with a hinged lid on top. Place a 13 x 31 grid of 2 mm holes on the floor of chamber 2 through which an array of blunt probes with 0.5 mm diameter tips (e.g., blunted map pins) can protrude. 
	NOTE: Blunt pins with a 120-grit sandpaper block or similar. Clean them in warm water with detergent before being disinfected with sporicidal disinfectant.</li>
	<li>Adjust the height of the probes by placing additional acrylic sheets beneath the probe baseplate (Figure 1). Using this approach, configure the device with three settings: 0 mm, 2 mm, and 5 mm probe height.</li>
	<li>As an alternative to blunted map pins or similar materials, use the 3D printer files to print the floor of chamber 2 and the probe plate (see Supplementary File 1: SpikeBed-MCA.stl which refers to the mechanical probes, and Supplementary File 2: MCA_baseplate.stl which forms the floor of chamber 2). 
	NOTE: If 3D printing is not available, glue map pins to an acrylic sheet using the same acrylic adhesive used to construct the walls of the device.</li>
	<li>Print with a washable and biocompatible material, such as nylon 12 plastic or similar (recommended).</li>
	<li>Construct chamber 3 with the following dimensions: 125 mm x 125 mm x 125 mm as an unlit translucent dark red acrylic box (on all sides), placed at the opposite end to chamber 1. Place a hinged lid on the chamber, similar to chambers 1 and 2. This chamber serves as a darkened escape area from the mechanical probes in chamber 2.</li>
</ol>
2. Mouse MCA habituation and testing
<ol>
	<li>As with all experiments involving behavioral outcomes in animals, observe appropriate randomization and blinding throughout to minimize potential bias. 
	NOTE: The representative results were generated by using 8-12 week old male and female C57BL/6J mice (Jackson Laboratories strain number 000664). Mice were socially housed, up to 5 per cage, with access to food and water ad libitum and a 07:00 h to 19:00 h light cycle. MCA took place in the light period, between 09:00 h and 12:00 h.</li>
	<li>One day before baseline testing is scheduled, acclimate mice to the MCA unit for 5 min (minimum) to 15 min (maximum) with their cage mates to facilitate social exploration of the entire device.</li>
	<li>Throughout the process, ensure that the LEDs in chamber 1 are switched off, the barrier between chambers 1 and 2 is left open, and the probes are set to a height of zero (i.e., not protruding through the floor of chamber 2).</li>
	<li>Perform a baseline test of mice (optional) if the study incorporates negative control animals (i.e., sham surgery, or vehicle injection controls). If desired, use a baseline test to exclude any uninjured outliers that never cross into chamber 2, though this has not proven necessary. If used, report all criteria for exclusion and the number of mice excluded.
	<ol>
		<li>Before beginning testing, set up a video camera capable of recording 1080p footage on a tripod with a side-facing view of the entire MCA device. Adjust the field of view such that the MCA fills the recorded image.</li>
		<li>Once recording begins, hold a handheld dry-erase board in the camera&#39;s field of view to label the start of the video with identifying information on the animal&#39;s testing run (e.g., mouse ID, probe height, date, time point, etc.).</li>
		<li>For the first run, set the probe height to zero. Transfer the mouse to be tested from its home cage to chamber 1 with the barrier door in place. Start a timer that is visible in the recorded footage. 
		NOTE: The timer ensures that the intervals between the different parts of the test are consistent between runs.</li>
		<li>After 10 s, switch on the chamber 1 LEDs. After the mouse has been in the lit chamber for 20 s, withdraw the barrier between chambers 1 and 2.</li>
		<li>Observe the animal for 2 min. Measure latencies and/or dwell times with a stopwatch while the test is ongoing. Alternatively, the video footage can be analyzed once testing is complete. 
		NOTE: For reasons of throughput and avoiding prolonged exposure to aversive stimuli, the cutoff was set at 2 min.</li>
		<li>Measure one or more of the several useful outcomes that have been identified (see below;&#160;Figure 1). Recommended to analyzie all 5 outcome measures when beginning testing, in order to ascertain which aspects of behavior differ in a given experimental setup.
		<ol>
			<li>Option I: Record the latency to the first entry to chamber 2. Option II: Record the latency to crossing more than halfway across chamber 2. Option III: Record the total dwell time in chamber 2. Option IV: Record the latency to reach chamber 3 (escape). Option V: Similar to option II, record the total dwell time in each chamber within 2 min and convert them into proportions. 
			NOTE: Since every experiment is unique and may be influenced by biological factors and behavioral changes unique to the disease model, investigators can experiment with these and other measures in their own hands.</li>
		</ol>
		</li>
		<li>Once testing is complete, return the mouse to its home cage, clean the MCA chambers with 70% ethanol, and allow it to dry completely. 
		NOTE: Fecal boli can usually be cleaned from the chamber relatively easily with paper towels prior to ethanol/disinfectant. If more thorough cleaning becomes necessary, chambers 2 and 3 can be disassembled and immersed in warm, soapy water.</li>
		<li>After running all mice in a cohort with the probe height set to zero, insert a 3 mm sheet of acrylic beneath the mechanical probe baseplate and repeat steps 2.4.2 to 2.4.7 with a probe height of 2 mm.</li>
		<li>After running all mice with the probe height set to 2 mm, insert a second 3 mm sheet of acrylic beneath the probe base plate and repeat steps 2.4.2 to 2.4.7 with a probe height of 5 mm. 
		NOTE: A group of 8 mice can be tested in approximately 2 h using this approach. Use smaller group sizes if more precise post-drug timing is required (e.g., for a drug time course experiment).</li>
		<li>Perform a final cleaning with a disinfectant at the end of a testing session.</li>
	</ol>
	</li>
	<li>Repeat testing after inducing pain hypersensitivity and/or with drug treatment.</li>
	<li>Compare each mouse&#39;s performance at baseline with their performance after the pain is induced. Assess the impact of a pharmacological intervention by comparing vehicle-treated animals with drug-treated animals at the same timepoint.</li>
	<li>Perform non-parametric statistical analysis (e.g., the Mann Whitney U Test) if animals reach the 2 min cutoff without satisfying the desired outcome measure, resulting in non-continuous data.</li>
</ol>

<ol><li>Hargreaves, K., Dubner, R., Brown, F., Flores, C., Joris, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+and+sensitive+method+for+measuring+thermal+nociception+in+cutaneous+hyperalgesia%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia.</a> Pain. 32 (1), 77-88 (1988).</li>
<li>Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M., Yaksh, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+assessment+of+tactile+allodynia+in+the+rat+paw%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">Quantitative assessment of tactile allodynia in the rat paw.</a> Journal of Neuroscience Methods. 53 (1), 55-63 (1994).</li>
<li>Sheahan, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inflammation+and+nerve+injury+minimally+affect+mouse+voluntary+behaviors+proposed+as+indicators+of+pain%5BTitle%5D)+AND+%22Neurobiology+of+Pain%22%5BJournal%5D)">Inflammation and nerve injury minimally affect mouse voluntary behaviors proposed as indicators of pain.</a> Neurobiology of Pain. 2, 1-12 (2017).</li>
<li>Wodarski, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cross-centre+replication+of+suppressed+burrowing+behaviour+as+an+ethologically+relevant+pain+outcome+measure+in+the+rat%3A+a+prospective+multicentre+study%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">Cross-centre replication of suppressed burrowing behaviour as an ethologically relevant pain outcome measure in the rat: a prospective multicentre study.</a> Pain. 157 (10), 2350-2365 (2016).</li>
<li>King, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unmasking+the+tonic-aversive+state+in+neuropathic+pain%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">Unmasking the tonic-aversive state in neuropathic pain.</a> Nature Neuroscience. 12 (11), 1364-1366 (2009).</li>
<li>Harte, S. E., Meyers, J. B., Donahue, R. R., Taylor, B. K., Morrow, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanical+Conflict+System%3A+A+Novel+Operant+Method+for+the+Assessment+of+Nociceptive+Behavior%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Mechanical Conflict System: A Novel Operant Method for the Assessment of Nociceptive Behavior.</a> PLoS One. 11 (2), 0150164(2016).</li>
<li>Pahng, A. R., Edwards, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measuring+Pain+Avoidance-Like+Behavior+in+Drug-Dependent+Rats%5BTitle%5D)+AND+%22Current+Protocols+in+Neuroscience%22%5BJournal%5D)">Measuring Pain Avoidance-Like Behavior in Drug-Dependent Rats.</a> Current Protocols in Neuroscience. 85 (1), 53(2018).</li>
<li>Odem, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sham+surgeries+for+central+and+peripheral+neural+injuries+persistently+enhance+pain-avoidance+behavior+as+revealed+by+an+operant+conflict+test%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">Sham surgeries for central and peripheral neural injuries persistently enhance pain-avoidance behavior as revealed by an operant conflict test.</a> Pain. 160 (11), 2440-2455 (2019).</li>
<li>LaBuda, C. J., Fuchs, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+behavioral+test+paradigm+to+measure+the+aversive+quality+of+inflammatory+and+neuropathic+pain+in+rats%5BTitle%5D)+AND+%22Experimental+Neurology%22%5BJournal%5D)">A behavioral test paradigm to measure the aversive quality of inflammatory and neuropathic pain in rats.</a> Experimental Neurology. 163 (2), 490-494 (2000).</li>
<li>LaBuda, C. J., Fuchs, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphine+and+gabapentin+decrease+mechanical+hyperalgesia+and+escape%2Favoidance+behavior+in+a+rat+model+of+neuropathic+pain%5BTitle%5D)+AND+%22Neuroscience+Letters%22%5BJournal%5D)">Morphine and gabapentin decrease mechanical hyperalgesia and escape/avoidance behavior in a rat model of neuropathic pain.</a> Neuroscience Letters. 290 (2), 137-140 (2000).</li>
<li>Vichaya, E. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Motivational+changes+that+develop+in+a+mouse+model+of+inflammation-induced+depression+are+independent+of+indoleamine+2%2C3+dioxygenase%5BTitle%5D)+AND+%22Neuropsychopharmacology%22%5BJournal%5D)">Motivational changes that develop in a mouse model of inflammation-induced depression are independent of indoleamine 2,3 dioxygenase.</a> Neuropsychopharmacology. 44 (2), 364-371 (2019).</li>
<li>Hascoët, M., Bourin, M., Nic Dhonnchadha, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mouse+light-dark+paradigm%3A+a+review%5BTitle%5D)+AND+%22Progress+in+Neuropsychopharmacology+%26+Biological+Psychiatry%22%5BJournal%5D)">The mouse light-dark paradigm: a review.</a> Progress in Neuropsychopharmacology & Biological Psychiatry. 25 (1), 141-166 (2001).</li>
<li>Shepherd, A. J., Mohapatra, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pharmacological+validation+of+voluntary+gait+and+mechanical+sensitivity+assays+associated+with+inflammatory+and+neuropathic+pain+in+mice%5BTitle%5D)+AND+%22Neuropharmacology%22%5BJournal%5D)">Pharmacological validation of voluntary gait and mechanical sensitivity assays associated with inflammatory and neuropathic pain in mice.</a> Neuropharmacology. 130, 18-29 (2018).</li>
<li>Huck, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temporal+Contribution+of+Myeloid-Lineage+TLR4+to+the+Transition+to+Chronic+Pain%3A+A+Focus+on+Sex+Differences%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Temporal Contribution of Myeloid-Lineage TLR4 to the Transition to Chronic Pain: A Focus on Sex Differences.</a> Journal of Neuroscience. 41 (19), 4349-4365 (2021).</li>
<li>Pitzer, C., La Porta, C., Treede, R. D., Tappe-Theodor, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inflammatory+and+neuropathic+pain+conditions+do+not+primarily+evoke+anxiety-like+behaviours+in+C57BL%2F6+mice%5BTitle%5D)+AND+%22European+Journal+of+Pain%22%5BJournal%5D)">Inflammatory and neuropathic pain conditions do not primarily evoke anxiety-like behaviours in C57BL/6 mice.</a> European Journal of Pain. 23 (2), 285-306 (2019).</li>
<li>Sieberg, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuropathic+pain+drives+anxiety+behavior+in+mice%2C+results+consistent+with+anxiety+levels+in+diabetic+neuropathy+patients%5BTitle%5D)+AND+%22Pain+Reports%22%5BJournal%5D)">Neuropathic pain drives anxiety behavior in mice, results consistent with anxiety levels in diabetic neuropathy patients.</a> Pain Reports. 3 (3), 651(2018).</li>
<li>Meuwissen, K. P. V., van Beek, M., Joosten, E. A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Burst+and+Tonic+Spinal+Cord+Stimulation+in+the+Mechanical+Conflict-Avoidance+System%3A+Cognitive-Motivational+Aspects%5BTitle%5D)+AND+%22Neuromodulation%22%5BJournal%5D)">Burst and Tonic Spinal Cord Stimulation in the Mechanical Conflict-Avoidance System: Cognitive-Motivational Aspects.</a> Neuromodulation. 23 (5), 605-612 (2020).</li>
</ol>

The authors have no relevant conflicts of interest to disclose.

As with all behavioral tests, proper handling, randomization, and blinding to the treatment of animals is essential throughout. Given the multifactorial inputs into complex behaviors and decision-making, it is imperative that animals are handled, habituated, and tested as consistently as possible while minimizing distress. Care should also be taken to reproduce the timing of mouse placement in chamber 1, switching on the LED lights, and removing the barrier, since differences here could influence subsequent behavior. </p...

Pain is a complex experience with sensory and affective components. A reduction in the threshold of pain perception and hypersensitivity to thermal and/or mechanical stimuli are key features of this experience, which stimulus-evoked pain behavior tests can capture (like Hargreaves&#39; test of heat sensitivity and the von Frey test of mechanical sensitivity)1,2. Although such tests give robust and reproducible results, they are limited by their reliance on reflexive withdrawal from a perceived noxious stimulus. This has called into question an ongoing reliance of pain research on these tests alone. To that end, pain researchers have for several years been exploring alternative/complementary behavioral tests for use in rodent pain models in an effort to capture more of the affective and/or motivational components of pain. These un-evoked, voluntary, or non-reflexive measures (e.g., wheel running, burrowing activity, conditioned place preference3,4,5) are being implemented in an attempt to improve the translatability of preclinical pain research.
The mechanical conflict avoidance (MCA) assay was originally described by Harte et al. in 20166, is used predominantly in rats7,8, and represents a modification of an earlier approach - the place escape-avoidance paradigm. In this approach, a noxious stimulus of the hind paw is performed in an otherwise desirable (dark) chamber to drive purposeful behavior of the animal to escape/avoid such stimulation9,10. Instead of relying on manual noxious stimulation of the hind paw by an observer, the MCA assay forces mice to negotiate a potentially noxious stimulus to escape an aversive environment and reach the dark chamber. The conflict/avoidance that gives the assay its name arises from these two competing motivations: escape brightly-lit areas and avoid noxious stimulation of the paws. The MCA assay also shares features with conditioned place-preference testing, where the pairing of pain relief with environmental cues drives changes in behavior that reflect a preference for the pain-relieving/rewarding context11.
Fundamentally speaking, all these assays share a similar approach: using a shift in an animal&#39;s preference for one aversive environment over another as an indicator of their affective/motivational state. The MCA assay is a 3 chamber paradigm consisting of a brightly lit chamber followed by a dark middle chamber with adjustable height probes and a dark third chamber without any aversive stimuli. An uninjured mouse is typically motivated to escape to a darkened chamber, given the innate aversion of rodents to bright light12. In this example, the natural motivation to escape a brightly-lit environment overcomes the disinclination to encounter hind paw stimulation (the adjustable height probes), which occurs exclusively in the darkened environment. In contrast, a mouse experiencing pain (due to inflammation or neuropathy, for example) may opt to spend more time in the brightly-lit environment, since there is motivation to avoid the unpleasant tactile experience of the mechanical probes in the setting of ongoing tactile hypersensitivity.
This article describes a modified version of the MCA assay. We have adapted the original method (which was performed in rats6) for use in mice. We have also reduced the number of probe heights tested from six to three (0, 2, and 5 mm above floor height) in order to streamline data acquisition. This approach has been tested across multiple pain models, and validated with known analgesics, indicating that pain hypersensitivity and/or the associated affective and motivational changes are driving these changes in behavior. This approach is relatively quick to conduct and adaptable when compared to other non-reflexive measures, which can take many days of habituation and training1,2. In concert with other measures of pain, MCA can generate valuable insights into the affective and motivational aspects of pain.

<table><tbody><tr><td>32.8ft 3000K-6000K Tunable White LED Strip Lights, Dimmable Super Bright LED Tape Lights with 600 SMD 2835 LEDs</td><td>Lepro</td><td>SKU: 410087-DWW-US</td><td>For lighting chamber 1. https://www.lepro.com/32ft-dimmable-tunable-white-led-strip-lights.html</td></tr><tr><td>3D printed &#039;spike bed&#039; and &#039;chamber 2 floor&#039;</td><td>Shapeways</td><td>N/A</td><td>Optional, for mechanical probes as an alternative to blunted map pins.</td></tr><tr><td>70% ethanol</td><td>Various</td><td>N/A</td><td>To clean MCA between mice.</td></tr><tr><td>Acryl-Hinge 2</td><td>TAP Plastics</td><td>N/A</td><td>for attaching chamber lids to rear walls. https://www.tapplastics.com/product/plastics/handles_hinges_latches/acryl_hinge_2/122</td></tr><tr><td>Chemcast Cast Acrylic Sheet, Clear</td><td>TAP Plastics</td><td>N/A</td><td>3mm thick. For front wall of chamber 1. https://www.tapplastics.com/product/plastics/cut_to_size_plastic/acrylic_sheets_cast_clear/510</td></tr><tr><td>Chemcast Cast Transparent Colored Acrylic, Transparent Dark Red - 50%</td><td>TAP Plastics</td><td>N/A</td><td>3mm thick. 50% light transmission. For walls and lids of chambers 2 and 3. https://www.tapplastics.com/product/plastics/cut_to_size_plastic/acrylic_sheets_transparent_colors/519</td></tr><tr><td>Chemcast Translucent &amp;amp; Opaque Colored Cast Acrylic, Sign Opaque White - 0.1%</td><td>TAP Plastics</td><td>N/A</td><td>3mm thick. For side walls and lid of chamber 1. https://www.tapplastics.com/product/plastics/cut_to_size_plastic/acrylic_sheets_color/341</td></tr><tr><td>Disinfectant (e.g. Quatricide)</td><td>Pharmacal Research Laboratories, Inc.</td><td>65020F</td><td>To disinfect MCA at the end of a testing session.</td></tr><tr><td>Dry-erase markers and board</td><td>Various</td><td>N/A</td><td>To add experimental info to the beginning of video footage.</td></tr><tr><td>Map pins</td><td>Various</td><td>N/A</td><td>Optional, for mechanical probes. Use sandpaper to blunt sharp points before use. Can be used in place of 3D-printed parts.</td></tr><tr><td>Paper towels</td><td>Various</td><td>N/A</td><td>To clean/disinfect MCA.</td></tr><tr><td>SCIGRIP Weld-On #3 Acrylic Cement</td><td>TAP Plastics</td><td>N/A</td><td>For assembling acrylic sheets into chambers and affixing hinges. https://www.tapplastics.com/product/repair_products/plastic_adhesives/weld_on_3_cement/131</td></tr><tr><td>Stopwatch</td><td>Various</td><td>N/A</td><td>To record escape latencies/dwell times in real-time or from recorded video.</td></tr><tr><td>Timer</td><td>Various</td><td>N/A</td><td>To ensure LED turn-on, barrier removal and test completion are timed consistently.</td></tr><tr><td>Video camera</td><td>Various</td><td>HDRCX405 Handycam Camcorder</td><td>To record mouse behavior in the MCA device. Can be substituted with any consumer-grade video camera capable of 1080p resolution.</td></tr><tr><td>Tripod</td><td>Famall</td><td>N/A</td><td>Any tripod that can hold the camera at bench height for recording MCA footage is acceptable.</td></tr></tbody></table>

mechanical conflict-avoidance assay to measure pain behavior in mice

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 MCA assay has been used successfully with several mechanistically distinct mouse pain models. Figure 2 shows data where the outcome measure of choice was crossing the mid-point of chamber 2 (Figure 2A). The data obtained by using the halfway point versus escape into chamber 3 are very similar, ~40 s for halfway versus ~45 s for chamber 3 escape in the spared nerve injury (SNI) model of neuropathic pain with 5 mm probe height13.
<p...

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 ...

mechanical-conflict-avoidance-assay-to-measure-pain-behavior-in-mice

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

The protocol describes how to measure knee hyperalgesia in mice. We show examples in mouse models of acute knee pain as well as in models of osteoarthritis (OA).

Assessment of Knee Hyperalgesia in Mice using Pressure Application Measurement

Learn the mechanical conflict-avoidance assay to measure pain sensitivity in mice using a specialized chamber with adjustable probe heights. This method assesses motivational responses and enhances preclinical pain research translation.

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

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