Name: mechanical conflict-avoidance assay to measure pain behavior in mice
Uploaded: 2022-02-18T14:15:00
Description: Watch this Scientific Journal Video about Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice at JoVE.com

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...

<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>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#39;spike bed&#39; and &#39;chamber 2 floor&#39;</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 &#38; 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...

Watch this Scientific Journal Video about Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice at JoVE.com

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 ...

This method can help answer questions about the effective or motivational state of animals under different pain conditions. It is an important step in improving the translation of preclinical research. The main advantage of this technique is that it's relatively quick and easy to perform, yet still assesses complex motivations that influence pain-related behavior.Begin by constructing the sidewalls, floor, and ceiling of chamber one using an opaque white three millimeter thick acrylic. For the front facing wall, use a clear three millimeter thick acrylic. Attach the lid of chamber one with a hinge so that mice can be easily placed into and retrieved from the chamber.Then attach self-adhesive light emitting diode tape to the inner surface of the lid to provide illumination of around 4, 800 lux. Slide an opaque acrylic sheet into and out of position to close chamber one from the rest of the MCA device. Next, construct a 270 millimeter long unlit chamber two, the MCA test chamber, using translucent dark red acrylic on all sides with a hinged lid on top.Then place a 13 x 31 grid having two millimeter holes on the floor of the chamber through which an array of blunt probes with 0.5 millimeter diameter tips can protrude. Adjust the height of the probes by placing additional acrylic sheets beneath the probe baseplate. Using this approach, configure the device with zero, two, and five millimeter probe heights.As an alternative to blunted map pins or similar materials, use the 3D printer files to print the floor of chamber two and the probe plate with a washable and biocompatible material such as nylon 12 plastic. Finally, construct a dark chamber three using translucent dark red acrylic on all sides with a hinged lid similar to chambers one and two. Place it at the opposite end of chamber one to serve as a darkened escape area from the mechanical probes in chamber two.One day before scheduling the baseline testing, acclimate the mice to the MCA unit for 5 to 15 minutes with their cage mates to facilitate social exploration of the entire device. Ensure that the LEDs in chamber one are switched off, the barrier between chambers one and two is kept open and the probes are not protruding through the floor of chamber two. Next, set up a video camera capable of recording 1080 pixel 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. Once recording begins, hold a handheld dry erase board in the camera's field view to label the start of the video with identifying information on the animal's testing run. For the first run, set the probe height to zero and transfer the mouse to be tested from its home cage to chamber one with the barrier door in place.Start a timer that's visible in the recorded footage. After 10 seconds, switch on the chamber one LEDs. After the mouse has been in the lit chamber for 20 seconds, withdraw the barrier between chambers one and two.Observe the animal for two minutes and measure latencies or dwell times with a stopwatch while the test is ongoing. Record the latency to the first entry to chamber two, latency to crossing more than halfway across chamber two, the total dwell time in chamber two, the latency to reach chamber three, and the total dwell time in each chamber within two minutes and convert them into proportions. Once testing is complete, return the mouse to its home cage.Clean the MCA chambers with 70%ethanol and allow it to dry completely. After running all mice in the cohort with the probe height set to zero, insert a three millimeter sheet of acrylic beneath the mechanical probe baseplate and repeat the run with a probe height of two millimeters. After running all mice with the probe height set to two millimeters, insert another three millimeter sheet of acrylic beneath the probe baseplate and repeat the run with a probe height of five millimeters.Perform a final cleaning with a disinfectant at the end of the testing session. In the complete Freund's adjuvant-induced inflammatory pain model, a saline injection does not change escape latency compared to baseline. Mice injected with the adjuvant showed a significant increase in escape latency four days post-injection, but only when the probe height was raised to five millimeters.This increased latency was not seen in mice that received carprofen 90 minutes before the beginning of testing. The spared nerve injury model of neuropathic pain significantly increased the latency to escape versus baseline when probe height was set to five millimeters. This increase was prevented by administration of the opioid analgesic buprenorphine 90 minutes before testing.Increased escape latency was also observed in mice that did not undergo a baseline round of MCA testing before nerve injury. The increased escape latency in spared nerve injury mice at five millimeters was prevented by gabapentin administered 90 minutes before testing. In the fracture or casting model, the latency to escape from chamber one increased proportionally to the probe height before injury.Post-injury, the latency remained unchanged at zero millimeters, but significantly increased at the two millimeter and five millimeter probe height for males and the five millimeter probe height for females when compared to baseline. The most important thing to remember when attempting this procedure is to make sure that all mice are habituated to the MCA device, and to maintain proper randomization and blinding throughout the study. This method can be used alongside other measures of pain sensitivity including von Frey or conditional place preference.

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