Involvement of bradykinin, cytokines, sympathetic amines and prostaglandins in formalin-induced orofacial nociception in rats

Abstract

1. This study characterises some of the mechanisms and mediators involved in the orofacial nociception triggered by injection of formalin into the upper lip of the rat, by assessing the influence of various treatments on behavioural nociceptive responses (duration of facial rubbing) elicited either by a low subthreshold (i.e. non-nociceptive; 0.63%) or a higher concentration of the algogen (2.5%). 2. The kininase II inhibitor captopril (5 mg kg(-1), s.c.) and prostaglandin(PG) E(2) (100 ng lip(-1)) potentiated both phases of the response to 0.63% formalin, whereas tumour necrosis factor (TNF alpha; 5 pg lip(-1)), interleukin(IL)-1 beta (0.5 pg lip(-1)), IL-6 (2 ng lip(-1)) and IL-8 (200 pg lip(-1)), or the indirectly acting sympathomimetic drug tyramine (200 microg lip(-1)), each augmented only the second phase of nociception. 3. Conversely, both phases of nociception induced by 2.5% formalin were inhibited by the bradykinin (BK) B(2) receptor antagonist HOE140 (5 microg lip(-1)) or the selective beta(1)-adrenoceptor antagonist atenolol (100 microg lip(-1)). However, the BK B(1) receptor antagonist des-Arg(9)-Leu(8)-BK (1 and 2 microg lip(-1)), antibody and/or antiserum against each of the cytokines, the adrenergic neurone blocker guanethidine (30 mg kg(-1) day(-1), s.c., for 3 days) and the cyclooxygenase(COX)-2 inhibitor celecoxib (50 and 200 microg lip(-1), s.c.; or 1 and 3 mg kg(-1), i.p.) reduced only the second phase of the response. The nonselective COX inhibitor indomethacin and the 5-lipoxygenase activating protein inhibitor MK886 did not change formalin-induced nociception. 4. Our results indicate that BK, TNF-alpha, IL-1 beta, IL-6, IL-8, sympathetic amines and PGs (but not leukotrienes) contribute significantly to formalin-induced orofacial nociception in the rat and the response seems to be more susceptible to inhibition by B(2) receptor antagonist and selective COX-2 inhibitor than by B(1) receptor antagonist or nonselective COX inhibitor.

Figure 1

Nociceptive responses induced by formalin administration into the upper lip of rats, evaluated as the time the animals spent rubbing the injected site, in seconds. (a) Time course of rubbing responses displayed throughout consecutive 3-min bins during the first 30 min after injection of formalin (50 μl at 0.63, 1.25, 2.5, 5.0%) or saline. (b, c) Total rubbing time induced by formalin (open bars) and saline (S, closed bars) during the first 3 min (0–3 min; phase I) and between 12 and 30 min (phase II) following injection, respectively. Each value represents the mean±s.e.m. recorded from 10 animals. Asterisks denote P<0.05 relative to the corresponding saline-injected control value (ANOVA followed by Bonferroni's test).

Figure 2

Influence of captopril and BK B₁ and B₂ receptor antagonist treatment on nociceptive behaviour induced by formalin injection into the upper lip of rats. Upper and lower panels show total rubbing time induced by formalin during the first 3 min (0–3 min; phase I) and between 12 and 30 min (phase II) following injection, respectively. Rats received (a) the kininase II inhibitor captopril (CAP; 5 mg kg⁻¹, s.c., 2 h before), (b) the B₁ receptor antagonist Des-Arg⁹-Leu⁸-BK (DALBK; 0.5, 1.0 or 2.0 μg lip⁻¹, 30 min before) or (c) the B₂ receptor antagonist HOE 140 (1.25, 2.5 or 5.0 μg lip⁻¹, 30 min before) or vehicle alone (saline, S; closed bars). Note that all animals received 50 μl of formalin, either at 0.63% in (a) or at 2.5% in (b) and (c). Each value represents the mean±s.e.m. recorded from six animals. Asterisks denote P<0.05 relative to the corresponding saline-injected control value (ANOVA followed by Bonferroni's test).

Figure 3

Influence of prior local treatment with various cytokines on nociceptive behaviour induced by formalin injection into the upper lip of rats. Upper and lower panels show total rubbing time induced by 50 μl of 0.63% formalin during the first 3 min (0–3 min; phase I) and between 12 and 30 min (phase II) following its injection, respectively. Rats received (a) TNFα, (b) IL-1β, (c) IL-6 or (d) IL-8 or vehicle alone (saline, S; closed bars), 2 h before formalin. Note that all animals received formalin injection. Each value represents the mean±s.e.m. recorded from six animals. Asterisks denote P<0.05 relative to the corresponding saline-injected control value (ANOVA followed by Bonferroni's test).

Figure 4

Influence of prior local treatment with antibody/antiserum against various cytokines on nociceptive behaviour induced by formalin injection into the upper lip of rats. (a, b) Total rubbing time induced by 50 μl of 2.5% formalin during the first 3 min (0–3 min; phase I) and between 12 and 30 min (phase II) following its injection, respectively. Rats received either antibody against TNFα, or antisera against IL-1β, IL-6 or IL-8 (each diluted 1 : 5 in saline), and control animals were given the corresponding vehicle (nonimmune serum, NIS or saline, S; closed bars), 1 h beforehand. Note that all animals received formalin injection. Each value represents the mean±s.e.m. recorded from six animals. Asterisks denote P<0.05 relative to the corresponding NIS- or saline-injected control value (ANOVA followed by Bonferroni's test).

Figure 5

Influence of sympathetic activation or blockade on nociceptive behaviour induced by formalin injection into the upper lip of rats. Upper and lower panels show total rubbing time induced by formalin during the first 3 min (0–3 min; phase I) and between 12 and 30 min (phase II) following its injection, respectively. Rats received either (a) the noradrenaline releasing drug tyramine (Tyr, 200 μg lip⁻¹, 2 h beforehand), (b) daily injections of the sympathetic postganglionic neurone-blocking agent guanethidine (Gua, 30 mg kg⁻¹ day⁻¹, s.c.) for 3 days or (c) the β1-adrenoceptor antagonist atenolol (6.25, 25 and 100 μg lip⁻¹, 30 min before), or vehicle alone (saline, S; closed bars). Note that all animals received a 50 μl injection of formalin, either at 0.63% in (a) or at 2.5% in (b) and (c), and also that a group of Tyr-treated rats in (b) was treated first with Gua. Each value represents the mean±s.e.m. recorded from six to 10 animals. Asterisks denote P<0.05 relative to the corresponding saline-injected control value (ANOVA followed by Bonferroni's test).

Figure 6

Influence of PGE₂ and selective COX-2 inhibition with celecoxib on nociceptive behaviour induced by formalin injection into the upper lip of rats. Upper and lower panels show total rubbing time induced by formalin during the first 3 min (0–3 min; phase I) and between 12 and 30 min (phase II) following injection, respectively. Rats received either (a) PGE₂ (100 ng lip⁻¹, 3 h before), (b) systemic celecoxib (0.3, 1 or 3 mg kg⁻¹, i.p., 30 min before) or (c) local celecoxib (12.5, 50 or 200 μg kg⁻¹, 30 min before), or the corresponding vehicle (Veh; closed bars). Note that all animals received a 50 μl injection of formalin, either at 0.63% in (a) or at 2.5% in (b) and (c). Each value represents the mean±s.e.m. recorded from six animals. Asterisks denote P<0.05 relative to the corresponding saline-injected control value (ANOVA followed by Bonferroni's test).