Recla2 project protocol

Late phase formalin pain response in a Diversity Outbred (DO) mouse population   (2019)

Recla JM, Bubier JA, Gatti DM, Ryan JL, Robledo RF, Churchill GA, Chesler EJ, Bult CJ
With: Long KH, Garrett AM, Glidden N, Maser RS, Zhang A-W, Burgess RW, Young EE

Project protocol - Contents

Workflow and sampling

Data collected
Mice randomly assigned to testing groups, such that an equal number of males and females are tested each day -
2 Mice acclimated to test room for 1h
- -
3 Formalin injected into left hind paw Syringes and needles -
4 Mice placed into observation chamber Observation chamber, video recording equipment -
5 Video observations scored manually by a single trained investigator Video recordings Formalin score (licking or biting)

Equipment and supplies

  • Syringes (0.3 cc micro-syringe, Hamilton) and needles (30 gauge)
  • Observation chambers
    • Four quadrants (4w x 4l x 5h")
    • Flat glass surface to allow clear video recording
  • Video recording equipment (Noldus Observer 2.1, Noldus Information Technology, Wageningen, The Netherlands) with one camera per observation chamber mounted below the glass surface

Reagents and solutions

  • Formalin
  • Disinfectant

Procedure: Formalin response

  1. Mice are transported to the testing room and left undisturbed to habituate for 1h under ambient light.
  2. Twenty microliters of 2.5% formalin solution are injected subcutaneously into the plantar surface of the left hind paw. (Administration of 2.5% formalin is sufficient to produce the desired biphasic response while increasing test sensitivity and reducing animal suffering compared to the most commonly used 5% formalin solution.)
  3. Injected mice are placed individually into observation chambers equipped with stationary video recording cameras. Mice are recorded for 60 min.
  4. Video observations are grouped into 60 sec bins and scored manually by a single trained observer. A bin is assigned a score of 1 if the mouse is observed licking or biting the injected paw within the first 5 sec of each min, or 0 otherwise. Scores are summed across time points for the acute phase (0-10 min) and chronic response phase (10-60 min). (Chronic response phase data was submitted by the investigator with possible scores of 0-50.)

Data collected by investigator

  • Formalin chronic response phase score
  • Formalin chronic response phase score, square root