Formalin test: experimental design

The rats were randomly divided into groups of 6–8 animals, according to the following treatment schedule:

1) control group (n=8): saline (30′) + NTG vehicle, 12 ml/kg, i.p.

2) saline + NTG group (n=8): saline (30′) + NTG, 10 mg/kg, i.p.

3) AP 1 + NTG group (n=6): AP extract (30′), 50 mg/kg, i.p. + NTG;

4) AP 2 + NTG group (n=6): AP extract (30′), 25 mg/kg, i.p. + NTG.

In addition, to evaluate the possible interaction of AP extract with NTG vehicle we included another experimental group:

5) AP 1 + NTG vehicle group (n=6): AP extract (30′), 50 mg/kg, i.p. + NTG vehicle

AP or saline was administered 30 minutes (30′) before NTG or NTG vehicle injection. The formalin test was performed 4 hours after NTG or vehicle administration. The investigators responsible for the behavioral testing were blind to treatment group assignment. For the formalin test, a 100 μl volume of 1% formalin (formaldehyde diluted in 0.9% saline) was administered by intraplantar injection. Pain-related behavior was quantified for 1 h by counting spontaneous flinches and shakes of the injected paw: over 60-s periods for the first 5 min (min 1, 2, 3, 4 and 5) and thereafter following 4-min pauses, for 60-s periods up to the hour. Phase I was defined as the period from 1 to 5 min, phase II was defined as the period from 15 to 60 min inclusive. Phase I is considered the result of chemical activation of nociceptors, whereas phase II reflects the inflammatory reaction and central processing (Tjølsen et al., 1992).