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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+and+sensitive+method+for+measuring+thermal+nociception+in+cutaneous+hyperalgesia.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+tactile+allodynia+in+the+rat+paw.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+and+nerve+injury+minimally+affect+mouse+voluntary+behaviors+proposed+as+indicators+of+pain.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-centre+replication+of+suppressed+burrowing+behaviour+as+an+ethologically+relevant+pain+outcome+measure+in+the+rat:+a+prospective+multicentre+study.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unmasking+the+tonic-aversive+state+in+neuropathic+pain.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Conflict+System:+A+Novel+Operant+Method+for+the+Assessment+of+Nociceptive+Behavior.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Pain+Avoidance-Like+Behavior+in+Drug-Dependent+Rats.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sham+surgeries+for+central+and+peripheral+neural+injuries+persistently+enhance+pain-avoidance+behavior+as+revealed+by+an+operant+conflict+test.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+behavioral+test+paradigm+to+measure+the+aversive+quality+of+inflammatory+and+neuropathic+pain+in+rats.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphine+and+gabapentin+decrease+mechanical+hyperalgesia+and+escape/avoidance+behavior+in+a+rat+model+of+neuropathic+pain.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motivational+changes+that+develop+in+a+mouse+model+of+inflammation-induced+depression+are+independent+of+indoleamine+2,3+dioxygenase.">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>Hasco&#235;t, M., Bourin, M., Nic Dhonnchadha, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+light-dark+paradigm:+a+review.">The mouse light-dark paradigm: a review.</a> Progress in Neuropsychopharmacology &amp; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+validation+of+voluntary+gait+and+mechanical+sensitivity+assays+associated+with+inflammatory+and+neuropathic+pain+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+Contribution+of+Myeloid-Lineage+TLR4+to+the+Transition+to+Chronic+Pain:+A+Focus+on+Sex+Differences.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+and+neuropathic+pain+conditions+do+not+primarily+evoke+anxiety-like+behaviours+in+C57BL/6+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathic+pain+drives+anxiety+behavior+in+mice,+results+consistent+with+anxiety+levels+in+diabetic+neuropathy+patients.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burst+and+Tonic+Spinal+Cord+Stimulation+in+the+Mechanical+Conflict-Avoidance+System:+Cognitive-Motivational+Aspects.">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

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...

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Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

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

Research

JoVE Journal

Neuroscience

3.6K Views.  University of Texas MD Anderson Cancer Center. Deze methode kan helpen bij het beantwoorden van vragen over de effectieve of motiverende toestand van dieren onder verschillende pijnomstandigheden. Het is een belangrijke stap in het verbeteren van de vertaling van preklinisch onderzoek. Het belangrijkste voordeel van deze techniek is dat het relatief snel en gemakkelijk uit te voeren is, maar toch complexe motivaties beoordeelt die pijngerelateerd gedrag beïnvloeden.Begin met het construeren van de zijwanden, vloer en plafond van k...

Video: Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

Use of the Operant Orofacial Pain Assessment Device (OPAD) to Measure Changes in Nociceptive Behavior

We present an operant system for the detection of pain in awake, conscious rodents. The Orofacial Pain Assessment Device (OPAD) assesses pain behaviors in a more clinically relevant way by not relying on reflex-based measures of nociception. Food fasted, hairless (or shaved) rodents are placed into a Plexiglas chamber which has two Peltier-based thermodes that can be programmed to any temperature between 7 &deg;C and 60 &deg;C. The rodent is trained to make contact with these in order to access a reward bottle. During a session, a number of behavioral pain outcomes are automatically recorded and saved. These measures include the number of reward bottle activations (licks) and facial contact stimuli (face contacts), but custom measures like the lick/face ratio (total number of licks per session/total number of contacts) can also be created. The stimulus temperature can be set to a single temperature or multiple temperatures within a session. The OPAD is a high-throughput, easy to use operant assay which will lead to better translation of pain research in the future as it includes cortical input instead of relying on spinal reflex-based nociceptive assays.

Use of the Operant Orofacial Pain Assessment Device OPAD to Measure Changes in Nociceptive Behavior

We present a user-friendly, high-throughput operant system for the evaluation of pain behaviors in awake, conscious rodents. The Orofacial Pain Assessment Device (OPAD) can assess pain through a reward/conflict paradigm thus providing a more humane way of testing. This protocol will yield more clinically relevant and translational data from rodents.

We present an operant  system for the detection of pain in awake, conscious rodents. The Orofacial  Pain Assessment Device (OPAD) assesses pain behaviors ...

Behavior

Investigating Pain-Related Avoidance Behavior using a Robotic Arm-Reaching Paradigm

Avoidance behavior is a key contributor to the transition from acute pain to chronic pain disability. Yet, there has been a lack of ecologically valid paradigms to experimentally investigate pain-related avoidance. To fill this gap, we developed a paradigm (the robotic arm-reaching paradigm) to investigate the mechanisms underlying the development of pain-related avoidance behavior. Existing avoidance paradigms (mostly in the context of anxiety research) have often operationalized avoidance as an experimenter-instructed, low-cost response, superimposed on stimuli associated with threat during a Pavlovian fear conditioning procedure. In contrast, the current method offers increased ecological validity in terms of instrumental learning (acquisition) of avoidance, and by adding a cost to the avoidance response. In the paradigm, participants perform arm-reaching movements from a starting point to a target using a robotic arm, and freely choose between three different movement trajectories to do so. The movement trajectories differ in probability of being paired with a painful electrocutaneous stimulus, and in required effort in terms of deviation and resistance. Specifically, the painful stimulus can be (partly) avoided at the cost of performing movements requiring increased effort. Avoidance behavior is operationalized as the maximal deviation from the shortest trajectory on each trial. In addition to explaining how the new paradigm can help understand the acquisition of avoidance, we describe adaptations of the robotic arm-reaching paradigm for (1) examining the spread of avoidance to other stimuli (generalization), (2) modeling clinical treatment in the lab (extinction of avoidance using response prevention), as well as (3) modeling relapse, and return of avoidance following extinction (spontaneous recovery). Given the increased ecological validity, and numerous possibilities for extensions and/or adaptations, the robotic arm-reaching paradigm offers a promising tool to facilitate the investigation of avoidance behavior and to further our understanding of its underlying processes.

Avoidance is central to chronic pain disability, yet adequate paradigms for examining pain-related avoidance are lacking. Therefore, we developed a paradigm that allows investigating how pain-related avoidance behavior is learned (acquisition), spreads to other stimuli (generalization), can be mitigated (extinction), and how it may subsequently re-emerge (spontaneous recovery).

Avoidance behavior is a key contributor to the transition from acute pain to chronic pain disability. Yet, there has been a lack of ecologically valid ...

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols. 
We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.

We describe a protocol to examine the development of opioid-induced hyperalgesia and tolerance in mice. Based on the measurement of thermal and mechanical nociceptive responses of na&#239;ve and morphine-treated animals, it allows to quantify the increase in pain sensitivity (hyperalgesia) and decrease in analgesia (tolerance) associated with chronic opiate administration.

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular ...

Back Mechanical Sensitivity Assessment in the Rat for Mechanistic Investigation of Chronic Back Pain

Low back pain is the leading cause of disability worldwide, with dramatic personal, economic, and social consequences. To develop novel therapeutics, animal models are needed to examine the mechanisms and effectiveness of novel therapies from a translational perspective. Several rodent models of back pain are used in current investigations. Surprisingly, however, no standardized behavioral test was validated to assess mechanical sensitivity in back pain models. This is critical to confirm that animals with presumed back pain present local hypersensitivity to nociceptive stimuli, and to monitor sensitivity during interventions designed to relieve back pain. The objective of this study is to lay down a simple and accessible test to assess mechanical sensitivity in the back of rats. A test cage was fabricated specifically for this method; length x width x height: 50 x 20 x 7 cm, having a stainless-steel mesh on the top. This test cage allows the application of mechanical stimuli to the back. To perform the test, the back of the animal is shaved in the region of interest, and the test area is marked to repeat the test on different days, as needed. The mechanical threshold is determined with Von Frey filaments applied to the paraspinal muscles, utilizing the up-down method described previously. The positive responses include (1) muscle twitching, (2) arching (back extension), (3) rotation of the neck (4) scratching or licking the back, and (5) escaping. This behavioral test (Back Mechanical Sensitivity (BMS) test) is useful for mechanistic research with rodent models of back pain for the development of therapeutic interventions for the prevention and management of back pain.

To develop novel therapeutic interventions for the prevention and management of back pain, animal models are required to examine the mechanisms and effectiveness of these therapies from a translational perspective. The present protocol describes the BMS test, a standardized method to assess back mechanical sensitivity in the rat.

Low back pain is the leading cause of disability worldwide, with dramatic personal, economic, and social consequences. To develop novel therapeutics, ...

Assessment of Nerve Injury-Induced Mechanical Hypersensitivity in Rats Using an Orofacial Operant Pain Assay

Pain has sensory and affective components. Unlike traditional, reflex-based pain assays, operant pain assays can produce more clinically relevant results by addressing the cognitive and motivational aspects of pain in rodents. This paper presents a protocol for assessing mechanical hypersensitivity following chronic constriction injury of the infraorbital nerves (CCI-ION) in rats using an orofacial operant pain system. Before CCI-ION surgery, rats were trained in an orofacial pain assessment device (OPAD) to drink sweetened condensed milk while making facial contact with the metal spiked bars and lick-tube.
In this assay, rats can choose between receiving milk as a positive reinforcer or escaping an aversive mechanical stimulus that is produced by a vertical row of small pyramid-shaped spikes on each side of the reward access hole. Following 2 weeks of training in the OPAD and before the CCI-ION surgery, baseline mechanical sensitivity data were recorded for 5 days for each rat during a 10 min testing session. During a session, the operant system automatically records the number of reward bottle activations (licks) and facial contacts, contact duration, and latency to the first lick, among other measures.
Following baseline measurements, rats underwent either CCI-ION or sham surgery. In this protocol, mechanical hypersensitivity was quantified by measuring the number of licks, latency to the first lick, the number of contacts, and the ratio of licks to facial contacts (L/F). The data showed that CCI-ION resulted in a significant decrease in the number of licks and the L/F ratio and an increase in the latency to the first lick, indicating mechanical hypersensitivity. These data support the use of operant-based pain assays to assess mechanical pain sensitivity in preclinical pain research.

This protocol describes the assessment of mechanical hypersensitivity in a rat model of neuropathic orofacial pain using an operant-based orofacial pain assessment device.

Pain has sensory and affective components. Unlike traditional, reflex-based pain assays, operant pain assays can produce more clinically relevant results ...

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 ...

Development of Recombinant Proteins to Treat Chronic Pain

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates for treating debilitating pain conditions due to their capacity to regulate aberrant neuro-immune interactions. However, physiologically they work in a network of various cytokines, and therefore their therapeutic effect may not be optimal when used as stand-alone drugs. To overcome this limitation, we developed a fusion protein of the anti-inflammatory cytokines IL4 and IL10. Here, we describe the methods for production and quality control of IL4-10 recombinant fusion protein and we test the effectiveness of the IL4-10 fusion protein to resolve pain in a mouse model of persistent inflammatory pain.

In this paper we provide details for the methods of production and quality control of an IL4-10 recombinant fusion protein. We also show how to test the effectiveness of this protein to resolve pain in a mouse model of inflammatory pain.

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates ...

Medicine

Cheek Injection Model for Simultaneous Measurement of Pain and Itch-related Behaviors

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts are typically counted to evaluate itch induced by pruritogens. However, previous reports have shown that algesic substances also induce scratching behaviors in a mouse neck injection model, which is the most common test used for scratching behaviors. This finding makes it difficult to study itch in mice. &#160;In contrast, capsaicin, a common algogen, reduced scratching behaviors in some neck injection experiments. Therefore, the effect of pain on scratching behaviors remains unclear. It is thus necessary to develop a method to concurrently investigate itch and pain sensation using behavioral tests. Here, a cheek injection model is introduced which can be used to simultaneously measure pain- and itch-related behaviors. In this model, pruritogens induce scratching behaviors while algesic substances induce wiping behaviors. Using this model, lysophosphatidic acid (LPA), an itch mediator found in cholestatic patients with itch, is shown to exclusively induce itch but not pain. However, in mouse models, LPA has been reported to be both a pruritogen and an algogen. Investigation into the effects of LPA in a mouse cheek injection model showed that LPA only induced scratching, but not wiping behaviors. This indicates that LPA acts as a pruritogen similarly in mice and humans, and demonstrates the utility of a cheek injection model for itch research.

Typically, the mouse neck injection model is used for evaluate pruritogen-induced scratch behaviors. However, the model provides information only on itch, not pain. Here, a cheek injection model is introduced in mice which can be used to simultaneously measure pain and itch-related behaviors.

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts ...

Spontaneous and Evoked Measures of Pain in Murine Models of Monoarticular Knee Pain

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are necessary and useful for studying the mechanisms of pain and for evaluating efficacy of arthritis therapies. Measuring pain in animal models of arthritis is challenging. We have developed methods for measuring evoked and spontaneous pain in three models of murine arthritis. We quantitate the evoked pain responses of mice subjected to firm palpation of a painful knee. We also evaluate spontaneous pain by the proportion of weight and the amount of time placed on each of their 4 limbs after induction of arthritis pain in one knee. Joint pain in these mouse models produces a significant increase in evoked pain responses and an alteration in weight bearing. Since mice are quadrupeds, they offload the painful limb to the contralateral limb, to the forelimbs, or some combination. These methods are simple, require minimal equipment, and are reproducible and sensitive for detecting pain. They are useful for studying both disease-modifying arthritis treatments and analgesics in mice.

We have developed an evoked measure of arthritis pain and coupled it with a standardized method for measuring spontaneous pain in different murine models of chemically induced arthritis. These measures are sensitive and reproducible for different types of joint pain.

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are ...

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

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