Effects of different concentrations and volumes of formalin on pain response in rats

Background: Formalin test is commonly used in animal model to assess injury-produced pain response. If the total amount of formaldehyde is fixed, its concentration and volume can be easily adjusted. We evaluated the effect of two sets of three solutions of formalin (one set of same dose of formaldehyde at different concentration and volume, i.e. 2.5%-100 μL, 5%-50 μL, 10%-25 μL, and another set of same volume but at different concentrations, i.e. 2.5%-100 μL, 5%-100 μL, and 10%-100 μL) on the injury-produced pain response in rat. Methods: Male Sprague-Dawley rats weighing 250-300 g were used. Following injection of formalin (n = 8 in each group) or saline (n = 6, control), the flinching frequencies and time spent in licking or biting the injected hindpaw in the early phase 1 (0-5 min after injection) and the late phase 2 (20-60 min after injection) were recorded. Sham-injection rats (n = 5) underwent subcutaneous insertion of the needle, but no substance was injected. Results: Flinching in phase 1 and 2 was more frequent in the 2.5%-100 μL and 5%-50 μL groups than in the control group (P<0.05). Licking (or biting) time in phase 2 in all these three groups was longer than the control group (P<0.05). In the groups of another set of three different solutions (2.5%-100 μL, 5%-100 μL, and 10%-100 μL), flinching in phase 1 and phase 2 was also more frequent than the control group (P<0.05). Regarding lick behavior of another set, it occurred more frequently in 2.5%-100 μL group in phase 1 and in 2.5%-100 μL group as well as 5%-100 μL group in phase 2 than the control group (P<0.05). Conclusions: The 10%-25 μL formalin produces fewer flinching responses than other concentrations. Flinching was a biphasic behavior which was more spontaneous and active than was licking. The volume of formalin was a more important factor than the concentration of formalin in the generation of the active biphasic flinching response in the rat model.