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Background: Musculoskeletal pain is a major clinical problem, but researchers lack viable animal models to study this phenomenon. However, formalin injections are widely used to produce tonic and neurogenic nociceptive responses in rodents. Rodents can present a potential animal model for pain. Objectives: To develop a muscle pain model with intramuscular injection of formalin in rats. Materials and Methods: We injected formalin intramuscularly at different concentrations (50 µl; 2%, 4% and 8%) and compared them to its plantar surface subcutaneous injection (50 µl; 2%). Pain responses were scored in the first phase (1-7 minute), interphase (8-14 minute) and the second phases of 2A (15-60 minute) and 2B (61-90 minute). Results: After intramuscular and subcutaneous formalin injection, rats showed nociceptive behaviors for less than 10 minutes, and then nociceptive behaviors decreased significantly or stopped. Subsequently, the second phase showed ongoing peripheral activity. In the first phase subcutaneous injection was more painful and in second phase intramuscular injection with doses of 2% and 4%. Conclusion: We developed a novel muscle pain model based on tonic nociceptive behaviors in rats; following intramuscular formalin injections. This model has great clinical relevance and can facilitate new studies on pain.

The goals of this study were to evaluate the effects of pretreatment by orexin receptor-1 antagonist on the development of morphine tolerance and physical dependence in rat. Animals were rendered dependent on morphine by subcutaneous (SC) injection of morphine sulfate (10 mg/kg) at set intervals of 12 h for 10 days. Just before the morphine administration, the animals received SB-334867, a selective orexin receptor 1 (OXR1) antagonist. To assess morphine tolerance, the antinociceptive responses of morphine were measured using the warm-water tail immersion test before and after its administration. On day 11, naloxone was injected 2 h after morphine administration and the physical dependence evaluated by quantifying/scoring naloxone-precipitated withdrawal signs for 30 min. The effect of chronic SB-334867 on locomotion was carried out by calculating the number of grid crossings as a measure of locomotor activity. Our findings demonstrated that although morphine-tolerance tended to develop in response to repeated injections of morphine, pre-treatment of OXR1 antagonist prevented this effect, causing a delay in the development of morphine-tolerance. Moreover, co-administration of orexin receptor 1 antagonist with morphine significantly decreased the somatic signs of withdrawal including diarrhea, teeth chattering, jumping, and defecation. Administration of SB-334867 alone or in a chronic co-administration with morphine failed to change locomotor activity. These results suggest that the activation of OXR1 might be involved in the development of morphine tolerance and dependence.

In the present study, the effect of orexin-A (ORXA) microinjection into the paragigantocellularis lateralis (LPGI) on nociceptive behaviors, using hot-plate and formalin tests as thermal and chemical models of pain in rat, was examined. Also, we determined whether the pretreatment with SB-334867, a selective OX1-receptor antagonist, would prevent the antinociceptive effect of orexin-A. ORXA (0.1-100 nM/0.5 μL) microinjected into the LPGi nucleus, dose-dependently decreased the formalin induced nociceptive behaviors and also produced a dose-dependent antinociceptive effect in the hot-plate test. Pretreatment with a selective orexin receptor 1 (OX1R) antagonist, SB-334867, also inhibited the effect of ORXA on formalin induced nociceptive behaviors while the SB-334867 (100 μM) alone had no effect on formalin test. These data demonstrated that the ORXA-induced antinociception in formalin test is mainly mediated through the OX1R in LPGi which might play a potential role in processing the pain information associated with descending pain modulation.

Orexin (ORX) plays an important role in pain modulation. ORX receptors have been found in many brain structures and are known to be involved in pain processing. It is well-established that the acute and chronic forms of stress could induce hormonal and neuronal changes that affect both pain threshold and nociceptive behaviors. The role of OX1R receptors in stress-induced analgesia (SIA) has not been fully elucidated. In the present study, using the formalin test, attempts were made to evaluate the effects of acute immobilization restraint stress and swimming stress on pain behavioral responses following OX1R antagonist administration in rats. Animals received OX1R antagonist (SB-334867), vehicle, or naloxone before exposure to acute restraint stress (30min) or swimming stress test (6min, 20±1°C), and immediately submitted to hind paw formalin injection (50μl, 2%). Acute 30-min exposure to restraint stress as well as 6-min exposure to swim stress could significantly reduce the formalin-induced nociceptive behaviors in rats. This antinociceptive effect with either restraint stress or swim stress was fully prevented by OX1R antagonist (SB-334867), while the SB-334867 alone had no effect. However, the opioid receptor antagonist naloxone could not totally reverse the antinociception effect with either form of stress. It is suggested that OX1R might be involved in antinociception behaviors induced by these two forms of stress. These data highlight the significant role of OX1R as a novel target for treatment of stress-related disorders.

Intracerebroventricular injection of orexin-A (hypocretin-1) has been shown to elicit the analgesic responses. However, the locations of central sites that may mediate these effects have not been clearly elucidated. This study was performed using male Sprague Dawley rats to investigate the antinociceptive effects of intra-periaqueductal gray matter (PAG) administration of orexin-A, 5 minutes prior to formalin (50 μL of 2%) injection. Orexin-A had no effect on tail-flick test as thermal and acute model. In the formalin test, intra-PAG injection of orexin-A (10 nM) decreased the formalin-induced nociceptive behaviors in the interphase and phase 2, but not in phase 1, indicating an antinociceptive role of exogenous orexin-A in the PAG. While Orexin-A failed to produce a dose-dependent decrease in formalin-evoked behaviors in phase 1, it may have induced a dose-dependent decrease in formalin-evoked behaviors in early phase 2. Control injections of orexin-A into the sites near the PAG resulted in less or no reduction in pain, indicating that the analgesia observed is probably due to a site of action within the PAG rather than at surrounding neural structures. The antinociceptive effect of orexin-A was compared with intra-PAG administration of morphine (.5 μL of 20 mM, 5 minutes before the formalin injection). Morphine decreased the formalin-induced nociceptive behaviors in all phases. To investigate whether the orexin has a special action on the early part of the second phase, or its delayed effects are related to its pharmacokinetics, the orexin-A was injected into the PAG, 10 minutes before the formalin injection. No difference was observed between 5 and 10 minutes injection of orexin-A prior to formalin injection. The antinociceptive effect of orexin was blocked by intra-PAG injection of SB-334867, a putative type 1 orexin receptor antagonist, suggesting the involvement of orexin receptor type 1 in antinociception produced with intra-PAG injection of orexin-A. The results showed that the orexin-A plays an antinociceptive role in PAG in the interphase and the late phase of formalin test through type 1 orexin receptor dependent mechanism. Perspective Orexin is produced exclusively in the lateral hypothalamus, where it is known to modulate the pain processing through PAG. The antinociceptive effect of orexin in PAG may provide a role for this neurotransmitter in the up-down modulating pain system and further support the development of orexin-1 agonists for pain treatment.