The zebrafish (Danio rerio) has been proposed as a low-cost and simple alternative to the use of higher vertebrates in laboratory research on novel compounds with antinociceptive potential. In this study, we tested adult zebrafish (Danio rerio) as an alternative behavioral model of formalin-induced nociception. We evaluated the nociceptive effect of 0.1% formalin (3 or 5 μL; intramuscularly [i.m.]), applied into the tail or lips, on locomotor activity, using as parameter the number of times the fish crossed the lines between the quadrants of a glass Petri dish during the neurogenic stage (0-5 min) and the inflammatory stage (15-30 min). The behavioral model was validated by testing the antinociceptive effect of morphine and indomethacin (standard analgesic drugs used in the formalin test of rodents). We also tested whether the effect of morphine could be modulated by naloxone, an opioid antagonist. The effect of morphine and indomethacin on zebrafish locomotor behavior was evaluated with the open field test. The white/black test was used to rule out the anxiolytic effect of 0.1% formalin injected into the tail on adult zebrafish. Formalin (0.1%; 3 and 5 μL injected into the tail) increased significantly the nociceptive behavior of the adult zebrafish in both stages (p < 0.001 vs. control). Morphine and indomethacin (both 0.2 mg/mL; 20 μL; intraperitoneally [i.p.]) significantly inhibited nociception induced with formalin (5 μL injected i.m. into the tail) in both stages (p < 0.001). Naloxone blocked the antinociceptive effect of morphine. No influence on locomotion was observed. Locally administered formalin (injected into the tail) induced nociception, but not anxiety. The results suggest that the adult zebrafish behavioral model is a feasible alternative to more conventional laboratory models used in research on novel compounds with antinociceptive potential.
Neem fruit (Azadirachta indica A. Juss.) are popularly used to treat infections, diarrhea, fever, bronchitis, skin diseases, infected burns and hypertension. Although the antinociceptive and anti-inflammatory potential of A. indica has already been investigated in experimental models of pain and inflammation in mice, the current research is the first to report the evaluation of the capacity of A. indica fruit ethanolic extract (EtFrNeem) in acute pain attenuation using the adult zebrafish (Danio rerio) as an alternative model to the use in rodents. EtFrNeem was submitted to antioxidant action, preliminary chemical prospecting, FT-IR and determination of phenol and flavonoid content tests. Subsequently, EtFrNeem was tested for acute nociception and abdominal inflammation, locomotor activity, and acute toxicity in adult zebrafish. Possible neuromodulation mechanisms were also evaluated. EtFrNeem showed low antioxidant activity, but was shown to be rich in flavonoids. EtFrNeem showed no anti-inflammatory action, did not alter the locomotor system, and it was not toxic. However, EtFrNeem significantly reduced the nociceptive behavior induced by formalin, glutamate and acidic saline, when compared to the control group. These effects of EtFrNeem were significantly similar to those of morphine, used as a positive control. The antinociceptive effect of EtFrNeem was inhibited by naloxone, ketamine and amiloride. EtFrNeem has the pharmacological potential for acute pain treatment and this effect is modulated by the opioid system, NMDA receptors and ASICs channels. These results lead us to studies of isolation and characterization of EtFrNeem bioactive principles, using adult zebrafish as an experimental model.
Terpenes have a wide range of pharmacological properties, including antinociceptive action. The anti-inflammatory and antinociceptive effects of eucalyptol are well established. The purpose of this study was to evaluate the antinociceptive effect of eucalyptol on acute and neuropathic orofacial pain in rodent models. Acute orofacial and corneal nociception was induced with formalin, capsaicin, glutamate and hypertonic saline in mice. In another series, animals were pretreated with capsazepine or ruthenium red to evaluate the involvement of TRPV1 receptors in the effect of eucalyptol. In a separate experiment, perinasal tissue levels of IL-1β, TNF-α and IFN-γ were measured. Rats were pretreated with eucalyptol before induction of temporomandibular joint pain with formalin or mustard oil. In another experiment, rats were submitted to infraorbital nerve transection (IONX) to induce chronic pain, followed by induction of mechanical hypersensitivity using Von Frey hairs. Locomotor performance was evaluated with the open-field test, and molecular docking was conducted on the TRPV1 channel. Pretreatment with eucalyptol significantly reduced formalin-induced nociceptive behaviors in all mouse strains, but response was more homogenous in the Swiss strain. Eucalyptol produced antinociceptive effects in all tests. The effect was sensitive to capsazepine but not to ruthenium red. Moreover, eucalyptol significantly reduced IFN-γ levels. Matching the results of the experiment in vivo, the docking study indicated an interaction between eucalyptol and TRPV1. No locomotor activity changes were observed. Our study shows that eucalyptol may be a clinically relevant aid in the treatment of orofacial pain, possibly by acting as a TRPV1 channel antagonist.
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