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 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's instructions (i.e., with appropriate PPE in a well-ventilated area).
2. 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.
3. Close off chamber 1 from the rest of the MCA by sliding an opaque acrylic sheet into and out of position.
4. 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.
5. 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.
6. 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.
7. Print with a washable and biocompatible material, such as nylon 12 plastic or similar (recommended).
8. 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.

2. Mouse MCA habituation and testing

1. 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.
2. 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.
3. 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).
4. 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.
   1. 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.
   2. Once recording begins, hold a handheld dry-erase board in the camera's field of view to label the start of the video with identifying information on the animal's testing run (e.g., mouse ID, probe height, date, time point, etc.).
   3. 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.
   4. 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.
   5. 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.
   6. Measure one or more of the several useful outcomes that have been identified (see below; 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.
      1. 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.
   7. 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.
   8. 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.
   9. 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).
   10. Perform a final cleaning with a disinfectant at the end of a testing session.
5. Repeat testing after inducing pain hypersensitivity and/or with drug treatment.
6. Compare each mouse'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.
7. 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.