Evaluation of antinociceptive effect of Citral in two chronic nociception experimental models: partial sciatic nerve ligation (PSNL) and chronic post ischemia-reperfusion pain (CPIP) in mice.

CM Nishijima1, J Stramosk0,2, L Martins-Mazzardo0,2, DF Martins0,2, LRM Rocha1, ARS Santos0,2, CA Hiruma-Lima1. 1UNESP, Physiology, Brazil, 2UFSC, Physiological sciences, Brazil

This study aimed to evaluate the antinociceptive effects of Citral in two chronic nociception models and its mechanisms of action. Male Swiss mice (n=5-9) were anesthetized with volatile anesthetic isoflurane (1% to 2% in O2) and PSNL was performed by tying the distal one third to one half of the dorsal portion of the sciatic nerve, according to the procedure described by Malmberg and Basbaum (1998). The CPIP was induction by elastic ring placed around the mice’s left hindlimb just proximal to the ankle joint, producing a tight-fit (ischemia) and were left on the limb for 3 h (Coderre et al. 2004). Both experimental models used group of mice (30 ± 2 g) divided in operated, sham or naive groups. The operated groups of mice were treated by oral route with Citral (25; 100; 300 mg/kg) or vehicle (Tween 1% 10 ml/kg) 7 days after surgery. Groups of mice were also evaluated 2 days after ischemia-reperfusion (early phase) in the CPIP´s model. The hypernociception responses were recorded before (0) and after 1, 2, 3, 4 hours of treatment with vehicle or Citral to evaluate the time course effect. To investigate the effect of Citral (100 mg/kg – the dose that present greatest antinociceptive effect) at the long-term treatment on hypernociceptive mechanisms, this compound was administered orally during 9 days. The withdrawal response frequency to 10 applications of 0.4 g Von Frey filaments was considered as nociceptive index value. In order to evaluate the mechanisms of action from Citral, groups of mice received orally Citral or vehicle 1 hour before intrathecal injection of 5 µl of glutamatergic agonists receptors (NMDA or trans-ACPD), TNF-α or substance P. The amount of time the animal spent biting the caudal region was taken as evidence of nociception action. Rota-rod assay was also performed to exclude muscle relaxant effect of Citral. The statistical difference between groups was analyzed by 1-way ANOVA followed by Dunnet´s test or 2-way ANOVA followed by Bonferroni´s test. P values less than 0.05 were considered to indicate significance and the results were expressed as mean ± S.E.M. In the time course, Citral has shown the most antinociceptive activity in the dose of 100 mg/kg, 2 hours after oral administration in the model of PSNL (p<0.01; control: 9,0 ± 0,5; citral 100mg/kg: 4,5 ± 0,7) and it was also effective in the long-term treatment. In the model of CPIP, group of animals treated with citral has not exhibited antinociceptive activity in the early phase (2 days after induction). However Citral (100 mg/kg, p.o) presents significant antinociceptive activity during 3hs (p< 0.01) 7 days after ischemia-reperfusion procedure. The highest nociceptive inhibition was obtained 1 hour after administration of Citral in the time course evaluation (control: 7,8 ± 0,4; citral 100 mg/kg: 3,2 ± 1). Moreover, the activity was maintained during 9 days over prolonged treatment. Mice treated with Citral (100 mg/kg) presents strongly inhibited behaviour bitting induced by intrathecal injection of trans-ACPD (control: 260,6 ± 20,9 ; citral: 59,7± 10,8), NMDA (control: 163 ± 15,5; citral: 74,9 ± 14,8), substance P (control: 101 ± 9,5 ; citral:58,1 ± 6,5), TNF-α (control: 233,1 ± 28,9 ; citral: 65 ± 18,8) in relation to control group. These results shown that prolonged systemic treatment with Citral is effective in preventing persistent mechanical allodynia caused by PNSL and CPIP in mice. The antinociceptive mechanism of action of Citral involves glutamatergic receptors, substance P and cytokine TNF-α pathways.