Mice randomly assigned to testing groups, such that an equal number of males and females are tested each day
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2
Mice acclimated to test room for 1h
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3
Formalin injected into left hind paw
Syringes and needles
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4
Mice placed into observation chamber
Observation chamber, video recording equipment
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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
Mice are transported to the testing room and left undisturbed to habituate for 1h under ambient light.
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.)
Injected mice are placed individually into observation chambers equipped with stationary video recording cameras. Mice are recorded for 60 min.
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.)