Orally Administered Paracetamol Does Not Act Locally in the Rat Formalin Test: Evidence for a Supraspinal, Serotonin-dependent Antinociceptive Mechanism

Adult male 200- to 250-g Sprague-Dawley rats (Charles River, France) were housed under standard conditions with food and water ad libitum  for a week before starting the experiments so they could acclimate. This study, including care of the animals involved, was conducted according to the official edict presented by the French Ministry of Agriculture Paris, France, and the recommendations of the Helsinki Declaration. Thus, these experiments were conducted in an authorized laboratory and under the supervision of an authorized researcher.

Following acclimatization for 20 min in the test chamber and drug treatment, the rats received 50 μL of 2.5% formalin injected subcutaneously into the dorsal surface of the hind paw. They were then replaced in the Plexiglas box. Biting and licking time, considered as the best nociceptive parameter, 36was monitored during the two peaks of the typical biphasic nocifensive behavior previously described. 37,38The spontaneous aversive response corresponding to the early phase was assessed during the first 5 min. The second peak of nocifensive behavior was observed from 20 to 40 min after formalin administration.

The rat hind paw was immersed in an electrolyte solution up to the junction between the hairy and nonhairy skin of the ankle, and paw edema was determined electronically as a change in paw volume (in milliliters) using a plethysmometer (Apelex 7150, Massy, France). Volumes were measured before drug treatment, before formalin administration, and at the end of the test. The absolute paw volume was 1.35 ± 0.02 mL.

To observe the maximal antinociceptive activity of paracetamol during both phases of the test, 100 to 400 mg/kg paracetamol or 5 mL/kg NaCl 0.9% was administered orally or 1 mL/kg intravenously 40 min or 5 min before formalin, respectively, according to the kinetics of action previously described. 30 

According to the protocol described by Park et al. , 39the animals received 0.1 to 20 mg/kg paracetamol or 100 μL vehicle subcutaneously into the dorsal surface of their hind paw 30 min before formalin. Indomethacin at a dosage of 1.8 mg/kg was also administered as a positive control. The effect of both drugs was assessed both on nociceptive parameters and on edema volumes. To examine whether a possible diffusion of paracetamol would not minimize its local effect, the drug was also administered 10 min before formalin. Paracetamol at a dosage of 20 mg/kg and indomethacin were also administered subcutaneously in the rat back 30 min before formalin to verify the lack of any systemic action of the maximal intraplantarly injected dose.

Intrathecal injections were performed with the rats under isoflurane anesthesia (4% induction, 2% maintenance) between lumbar vertebrae L5 and L6 (10 μL), as previously described. 40WAY 100,635 at a dosage of 40 μg per rat was administered intrathecally 30 min after 400 mg/kg paracetamol orally (i.e. , 10 min before formalin injection). The effect of 200 μg paracetamol per rat and 50 μg 5-HT per rat administered intrathecally 10 min before formalin was also assessed on the two phases of the test, and the influence of WAY 100,635 that was coadministered with either drug was also investigated.

Intraplantarly administered paracetamol (UPSA, Bristol-Myers-Squibb Group, Rueil-Malmaison, France) was dissolved in dimethyl sulfoxide (Sigma, Saint Quentin Fallavier, France) and then diluted in saline (0.9% NaCl) with a final concentration of 20% dimethyl sulfoxide. For oral and intrathecal routes of administration, paracetamol was dissolved in saline. Indomethacin (Sigma) was dissolved in saline containing 10% ethanol as previously described. 39Formalin (Acros Organics, Noisy-Le-Grand, France), 5-hydroxytryptamin hydrochloride, and WAY 100,635 (N -(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-N -(2-pyridinyl) cyclohexanecarboxamide trihydrochloride) (Sigma) were dissolved in saline.

Drugs and doses were randomized and administered according to the method of blocks to assess the effect of the different treatments at the same lapse of time to avoid any uncontrolled experimental influence. Different animals were used for each experiments (n = 8 per treatment). All the experiments were performed blind in a quiet room by the same experimenter.

The cumulative biting and licking time of each phase of response was generated for every rat. The data (mean ± standard error of mean) expressed in seconds were analyzed by a one-way analysis of variance (ANOVA) followed by a Student-Newman-Keuls test when appropriate. Paw edema measurements were converted into an increase in paw volume (percentage) compared with the preformalin injection values. Analyses were conducted on the original data using ANOVA followed by the Student-Newman-Keuls test. The level of statistical significance was set at 0.05.

In all of the experiments performed, a qualitative assessment of locomotor activity indicated that the observed antinociceptive activity was not the result of motor impairment, because animals displayed a similar behavior regardless of the treatment they received.

Paracetamol given orally (300, 400 mg/kg) reduced significantly the two phases of spontaneous aversive behavior monitored as biting and licking time of the rat paw injected with formalin (300 mg/kg: 36.7 ± 10.1% and 49.3 ± 14% of inhibition in phases 1 and 2, respectively, P < 0.05; 400 mg/kg: 36.9 ± 4.6% and 61.5 ± 5.2% of inhibition in phases 1 and 2, respectively, P < 0.05, fig. 1A). The second phase, measured from 20 to 40 min after induction of the noxious stimulus, was more sensitive to paracetamol, as the drug was significantly effective at 200 mg/kg (33.6 ± 9.9% of inhibition, P < 0.05), whereas only higher doses reduced both phases. The same profile was observed when the drug was administered intravenously (fig. 1B). Paracetamol reduced biting/licking in the second phase from 100 mg/kg (42.6 ± 13.1% of inhibition, P < 0.05). A dose of 200 mg/kg significantly reduced the late phase by 60.9 ± 14.3% (P < 0.05), and a dose of 300 mg/kg was necessary to decrease both phases of nociception (47.6 ± 17.6 and 72.5 ± 6.3% of inhibition in phases 1 and 2, respectively, P < 0.05).

It was first observed that the 20% dimethyl sulfoxide–containing vehicle used did not affect formalin responses (data not shown). Paracetamol administered intraplantarly 30 min before formalin produced a statistically significant reduction of nocifensive behavior in phase 1 at 10 and 20 mg/kg (38.2 ± 10.0% and 33.3 ± 7.9% of inhibition, respectively, P < 0.05, fig. 2A). The dose of 20 mg/kg was the maximum tested because of the poor solubility of the drug and the limited volume injection allowed in the rat paw. However, the second phase (20–40 min) remained unaffected by paracetamol. In contrast, 1.8 mg/kg of indomethacin, a dose similar to that used by Park et al. , 39significantly reduced the late phase only by 37.8 ± 11.9% without attenuating the first one. This difference of profile of action between the two drugs was also seen with the assessment of the antiedematous effect, because only indomethacin decreased significantly the development of paw edema at the end of the second phase (25.2 ± 6.2% of reduction in paw volume, P < 0.05, fig. 2B). Paracetamol at a dosage of 20 mg/kg injected intraplantarly 10 min before formalin did not affect the late phase, and its antinociceptive activity in the first phase was comparable to that observed when the injection was performed 30 min before formalin (data not shown).

Paracetamol at a dosage of 400 mg/kg given orally was significantly effective in inhibiting the two phases of aversive behavior induced by formalin (36.6 ± 9.6% and 39.6 ± 9.9% inhibition in phases 1 and phase 2, respectively, P < 0.05;fig. 3). WAY 100,635 at a dosage of 40 μg per rat administered intrathecally 10 min before formalin had no intrinsic action on the duration of the behavioral response. However, it significantly and totally prevented the antinociception elicited by paracetamol during both phases. Scores of the group treated by paracetamol plus WAY 100,635 were statistically different from those of the paracetamol/saline-treated animals but not from the saline/saline- or saline/WAY 100,635-treated groups.

Reversal by WAY 100,635 of the antinociceptive activity of intrathecally injected paracetamol was then studied. An intrathecal administration of 200 μg per rat of paracetamol did not significantly affect the early phase of aversive behavior, whereas the second phase was significantly inhibited by 32.0 ± 7.1% (P < 0.05, fig. 4). This effect was not reversed when paracetamol was coinjected with 40 μg per rat of WAY 100,635 because the score was not significantly different from the paracetamol/saline group (22.7 ± 7.3% of inhibition in biting/licking time, P < 0.05). In the same blind experiment, 50 μg 5-HT per rat was administered intrathecally as a positive control. In accordance with previous results, 415-HT inhibited both phases with 43.5 ± 13.7% and 33.5 ± 11% of inhibition in phase 1 and phase 2, respectively. Its efficacy in the second phase was similar to that produced by paracetamol. When coadministered with WAY 100,635, 5-HT lost its antinociceptive property in both phases, the reversal being more marked during the late phase.

The primary aim of our study was to investigate the site(s) of action of paracetamol involved in its antinociceptive effect using the formalin model of tonic pain in rats. Several data obtained in this work suggest a limited, if any, local antinociceptive effect of paracetamol in the vicinity of the injury: (1) Intraplantar injection of paracetamol, but not indomethacin, failed to induce any antiinflammatory effect and did not modify scores of the late phase, whereas high doses were needed to reduce biting/licking behavior in the first phase that could result from a direct activation of C-fiber primary afferents. 34(2) As seen in another work, 29paracetamol administered either intravenously or orally primarily reduced behavioral scores in phase 2, which is thought to reflect both ongoing activity of nociceptors and central sensitization. 34(3) The effect of systemically administered paracetamol in both phases was totally blocked by an intrathecal administration of WAY 100,635, a 5-HT^1Areceptor antagonist. Few data report any peripheral activity after local administration of paracetamol. The results obtained by Abbott and Hellemans 42using the same test were not conclusive, whereas Ferreira et al.  21found a locally mediated antinociception in the rat paw pressure test at a dose of 50 μg per rat.

If a limited local antinociceptive effect of paracetamol can be observed only after high doses administered intraplantarly, a central serotonin-mediated action is demonstrated after oral administration. This observation is in agreement with previous results from our group 30,32or from other authors. 28,29,31However, the present data give information on the central site(s) of action of paracetamol and the nature of the 5-HT receptors involved. Concerning the central site(s) of action, the results obtained on the early phase suggest a supraspinal action of paracetamol. Orally, but not intrathecally, administered paracetamol reduced scores in phase 1. This effect was inhibited by WAY 100,635 (intrathecal), a 5-HT^1Areceptor antagonist, which also inhibited the antinociception induced by intrathecally injected 5-HT in the early phase. Thus, paracetamol injected intrathecally does not seem to be able to activate the 5-HT system. The spinal serotonergic effect observed after oral administration of paracetamol could be the result of an activation of a supraspinal component able to activate this system. This is in agreement with previous results showing that the action of paracetamol was reduced in the early phase of the formalin test after lesion of the bulbospinal 5-HT neurons using 5,6-DHT 28and with the lack of binding of paracetamol on the 5-HT reuptake sites. 30Activation of the 5-HT descending pathways could be correlated with the diminution of cortical high-affinity 5-HT^2Areceptors evoked by paracetamol, 29because a close relationship between this decrease and the analgesic activity of paracetamol has been reported. 31Moreover, some studies proposed that the stimulation of supraspinal 5-HT²receptors could produce a decrease in the activity of bulbospinal pathways. 43,44 

The data observed in phase 2 tend to confirm a supraspinally mediated activation of the serotonergic bulbospinal pathways. The antinociceptive effect of orally administered paracetamol was inhibited by an intrathecal injection of WAY 100,635, similar to the effect of intrathecally injected 5-HT. On the other hand, the antinociceptive effect of high doses of intrathecally injected paracetamol produced an antinociception comparable to that of 5-HT in the second phase only as previously described. 45Besides, this action was not reversed by the 5-HT^1Areceptor antagonist, suggesting a WAY 100,635-insensitive spinal activity. Thus, the serotonergic effect observed after an oral administration of paracetamol might not involve a spinal but instead primarily a supraspinal site of action, which could promote the activation of bulbospinal pathways. However, a direct spinal effect of paracetamol, WAY 100,635-insensitive, cannot be ruled out for high doses, at least in the late phase. Indeed, it has been observed that paracetamol still exerts a part of its antinociceptive effect in the late phase following the inactivation of the 5-HT system. 28,29 

Finally, this work also informs on the nature of the 5-HT receptors involved in the effect of paracetamol and shows, in the rat formalin test, the involvement of the spinal 5-HT^1Areceptors, which are also involved in the antinociceptive effect of 5-HT. 46However, we must keep in mind that we do not control rostral spreading of WAY 100,625 into the brain. These results disagree with those obtained with paracetamol and 5-HT in the rat paw pressure test by Courade et al.  32and Bardin et al.  47Indeed, their antinociceptive actions in this mechanical pain test were inhibited by several 5-HT receptor antagonists, but not by WAY 100,635. This indicates that various 5-HT receptors are involved according to the nature of the noxious stimulus and, consequently, to the nature of the afferent fibers involved. This is in line with the comments recently reported by Millan 48about the pharmacologic complexity of the serotonergic system in controlling nociceptive pathways.

In conclusion, our data seem to indicate that paracetamol, long thought to act by a mechanism similar to the nonsteroidal antiinflammatory drugs, exerts an antinociceptive action in both phases of the rat formalin test that is not mediated by a local antinociceptive action contrary to indomethacin. Its antinociceptive effect observed during the early phase might result from a modulation of the activity of supraspinal neuronal areas leading to the activation of the serotonergic inhibitory descending pathways and the involvement of spinal 5-HT^1Aserotonergic receptors. Reduction of the nocifensive behavior in phase 2 might also largely involve the bulbospinal serotonergic system and spinal 5-HT^1Areceptors when the drug is given systemically, but a direct modulation of other spinal pro- or antinociceptive systems might also be implicated in this phase. Finally, a potential interaction between supraspinal and spinal sites of action could also occur as proposed by Raffa et al.  49