The formalin test for nociception, which is predominantly used with rats and mice, involves moderate, continuous pain generated by injured tissue. In this way it differs from most traditional tests of nociception which rely upon brief stimuli of threshold intensity. In this article we describe the main features of the formalin test, including the characteristics of the stimulus and how changes in nociceptive behaviour may be measured and interpreted. The response to formalin shows an early and a late phase. The early phase seems to be caused predominantly by C-fibre activation due to the peripheral stimulus, while the late phase appears to be dependent on the combination of an inflammatory reaction in the peripheral tissue and functional changes in the dorsal horn of the spinal cord. These functional changes seem to be initiated by the C-fibre barrage during the early phase. In mice, the behavioural response in the late phase depends on the ambient temperature. We argue that the peripheral tissue temperature as well as other factors influencing the peripheral inflammation may affect the response, possibly confounding the results obtained with the test. Furthermore, we discuss the methods of recording the response and the value of observing more than one aspect of behaviour. Scoring of several behavioural variables provides a means of assessing motor or sensorimotor function as possible causes for changes in behaviour. In conclusion, the formalin test is a valuable addition to the battery of methods available to study nociception.

The effect of different formalin concentrations on the nociceptive response in the formalin test was examined in mice. Subcutaneous formalin injection induces 2 distinct periods of high licking activity: an early phase lasting the first 5 min, and a late phase lasting 20-30 min after the injection. Formalin concentrations of 0.02-0.2% induced only the early phase, while concentrations of 1% or more induced both the early phase and the late phase. The ability of the test to show the antinociceptive effect of morphine and acetylsalicylic acid was similar for high and low formalin concentrations. For both these analgesics, a lower dose was needed to induce antinociception in the late phase than in the early phase using the same formalin concentration. Indomethacin had no effect in the early phase. In the late phase indomethacin induced antinociception when 1% formalin was used, while no significant effect was observed using 5% formalin. Clear histological changes in the paw were demonstrated after formalin concentrations that induced both phases. Lower formalin concentrations induced only very small changes. Using a low formalin concentration (0.2%), repeated testing using the same paw could be performed at intervals of 1 week without any significant change in the response. It was concluded that the formalin concentration should be kept as low as possible to minimize the suffering of the animal. Formalin concentrations of 0.05-0.2% are recommended for studying the early phase. Formalin concentrations of 1% or higher have to be used when studying the nociceptive response in the late phase.

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In conscious rats, we investigated the change of nociceptive paw withdrawal reflexes elicited by mechanical and heat stimuli during intramuscular (i.m.) 5.8% hypertonic (HT) saline elicited muscle nociception. i.m. injection of HT saline caused rapid onset, long lasting (around 7 days), bilateral mechanical hyperalgesia, while it induced bilateral, slower onset (1 day after the HT saline injection), long-term (about 1-2 weeks) heat hypoalgesia. Ipsilateral topical pre-treatment of the sciatic nerve with 1% capsaicin significantly prevented the occurrence of both the bilateral mechanical hyperalgesia and the contralateral heat hypoalgesia. Intrathecal administration of either 6-hydroxydopamine hydrobromide (6-OHDA) or 5,7-dihydroxytryptamine (5,7-DHT), and intraperitoneal injection of naloxone all markedly attenuated the HT saline induced bilateral heat hypoalgesia, but not the mechanical hyperalgesia. Combined with experiments with lesioning of the rostroventral medulla with kainic acid, the present data indicate that unilateral i.m. injection of HT saline elicits time-dependent bilateral long-term mechanical hyperalgesia and heat hypoalgesia, which were modulated by descending facilitatory and inhibitory controls, respectively. We hypothesize that supraspinal structures may function to discriminate between afferent noxious inputs mediated by Aδ-and C-fibres, either facilitating Aδ-fibre mediated responses or inhibiting C-fibre mediated activities. However, this discriminative function is physiologically silent or inactive, and can be triggered by stimulation of peripheral C-fibre afferents. Importantly, in contrast to the rapid onset of descending facilitation, the late occurrence of descending inhibition suggests a requirement of continuous C-fibre input and temporal summation. Thus, a reduction of C-fibre input using exogenous analgesic agents, i.e. opioids, may counteract the endogenous descending inhibition.