Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

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.