Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

Summary

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.

Abstract

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.

Introduction

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' 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....

Protocol

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’s Guide for the Care and Use of Laboratory Animals.

1. MCA construction

1. 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.......

Discussion

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.

Materials

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