Antinociceptive Response Research Articles

Evaluating the Antinociceptive Efficacy of Cannabidiol Alone or in Combination with Morphine Using the Formalin Test in Male and Female Mice.

Introduction: Phytocannabinoids have emerged as a potential alternative treatment option for individuals experiencing persistent pain. However, evidence-based research regarding their clinical utility in both males and females remains incomplete. In addition, it is unknown whether combining readily available cannabinoids with opioids has a synergistic or subadditive effect on pain modulation. To begin to fill this knowledge gap, we investigated the antinociceptive effects of the phytocannabinoid, CBD, either alone or in combination with opioids in male and female C57BL/6J mice. Results: Using the formalin test, our results show that CBD (10 mg/kg, i.p.) treatment evoked antinociception in phase I, but not in phase II, of the formalin test in male mice. However, in female mice, CBD showed no significant antinociceptive effect. In addition, a direct sex comparison showed that CBD evoked a significant increase in nociceptive behaviors in female versus male mice during phase I of the formalin test. Furthermore, we show that CBD (10 mg/kg, i.p.) in combination with low-dose morphine (1 mg/kg, i.p.) was ineffective at eliciting a synergistic antinociceptive response in both male and female mice. Lastly, consistent with previous literature, we showed that females treated with a relatively higher dose of morphine (10 mg/kg, i.p.) displayed a significant increase in the variability of nociceptive behaviors compared to morphine-treated male mice. Conclusion: Overall, our results suggest that CBD treatment may have beneficial antinociceptive effects during the acute phase of persistent pain, but these effects are more beneficial to males than females. We provide further pre-clinical support that treatments geared toward reducing nociceptive behaviors differentially affect males and females.

Pilot study on the effects of low intensity focused ultrasound in a swine model of neuropathic pain.

The authors' laboratory has previously demonstrated beneficial effects of noninvasive low intensity focused ultrasound (liFUS), targeted at the dorsal root ganglion (DRG), for reducing allodynia in rodent neuropathic pain models. However, in rats the DRG is 5 mm below the skin when approached laterally, while in humans the DRG is typically 5-8 cm deep. Here, using a modified liFUS probe, the authors demonstrated the feasibility of using external liFUS for modulation of antinociceptive responses in neuropathic swine. Two cohorts of swine underwent a common peroneal nerve injury (CPNI) to induce neuropathic pain. In the first cohort, pigs (14 kg) were iteratively tested to determine treatment parameters. liFUS penetration to the L5 DRG was verified by using a thermocouple to monitor tissue temperature changes and by measuring nerve conduction velocity (NCV) at the corresponding common peroneal nerve (CPN). Pain behaviors were monitored before and after treatment. DRG was evaluated for tissue damage postmortem. Based on data from the first cohort, a treatment algorithm was developed, parameter predictions were verified, and neuropathic pain was significantly modified in a second cohort of larger swine (20 kg). The authors performed a dose-response curve analysis in 14-kg CPNI swine. Specifically, after confirming that the liFUS probe could reach 5 cm in ex vivo tissue experiments, the authors tested liFUS in 14-kg CPNI swine. The mean ± SEM DRG depth was 3.79 ± 0.09 cm in this initial cohort. The parameters were determined and then extrapolated to larger animals (20 kg), and predictions were verified. Tissue temperature elevations at the treatment site did not exceed 2°C, and the expected increases in the CPN NCV were observed. liFUS treatment eliminated pain guarding in all animals for the duration of follow-up (up to 1 month) and improved allodynia for 5 days postprocedure. No evidence of histological damage was seen using Fluoro-Jade and H&E staining. The results demonstrate that a 5-cm depth can be reached with external liFUS and alters pain behavior and allodynia in a large-animal model of neuropathic pain.

Comparison of Thermal and Mechanical Noxious Stimuli for Testing Analgesics in White's Tree Frogs (Litoria caerulea) and Northern Leopard Frogs (Lithobates pipiens).

Determining the clinical efficacy of analgesic drugs in amphibians can be particularly challenging. The current study investigated whether a thermal nociceptive stimulus is useful for the evaluation of analgesic drugs in 2 amphibian species. The objectives of this study were 2-fold: 1) compare 2 models of nociception (thermal and mechanical) using 2 frog species; White's Tree Frogs (Litoria caerulea; WTF) and Northern Leopard Frogs (Lithobates pipiens; NLF) after administration of saline or morphine sulfate; and 2) evaluate antinociceptive efficacy of morphine sulfate at 2 doses in a common amphibian research species, the NLF, using a mechanical stimulus. Neither WTF nor NLF displayed consistent drug-dependent changes in withdrawal responses to a noxious thermal stimulus applied using the Hargreaves apparatus, but NLF exposed to the noxious mechanical stimulus demonstrated a significant dose-dependent antinociceptive response to morphine sulfate. These results indicate that morphine is not antinociceptive in WTF, supporting previously reported results, and demonstrate the importance of using an appropriate experimental antinociceptive test in amphibians. Our data suggest that nociception in amphibian species may be best evaluated by using mechanical nociceptive models, although species differences must also be considered.

Antinociceptive effects of flower extracts and the active fraction from Styrax japonicus

Ethnopharmacological relevanceFlowers from Styrax japonicus sieb. et Zucc. have been used as a Chinese folk medicine to alleviate pain such as toothache and sore throat. Aim of the studyTo testify the analgesic effect of flowers from Styrax japonicus, analyze components of the active fraction, and investigate the mechanism of analgesia. Materials and methodsFlower extracts were obtained by ethanol, petroleum ether and hydrodistillation extraction. Different fractions of ethanol extracts (EE) were isolated by silica gel column chromatography and preparative liquid chromatography. Analgesic effects of EE, petroleum ether extracts (PEE), hydrodistillation extracts (HDE), and fractions of EE were evaluated using hot plate, acetic acid-induced writhing and formalin tests on mice. Components of the active fraction 1 (F1) were determined by the ultrahigh-performance liquid chromatography Q extractive mass spectrometry (UHPLC-QE-MS). Anti-inflammatory and sedative effects involving analgesic mechanisms were evaluated by carrageenan induced hind paw oedema and pentobarbital sodium sleep tests, respectively. In addition, antagonists including naloxone hydrochloride (NXH), flumazenil (FM), SCH23390 (SCH) and WAY100635 (WAY) were used to investigate the possible mechanism of analgesia. Contents of neurotransmitters and relevant metabolites in different brain regions of mice were also quantified by the ultraperformance liquid chromatography with a fluorescence detector (UPLC-FLD). ResultsEE rather than PEE and HDE at medium and high doses (150 mg/kg and 300 mg/kg) significantly prolonged the latency time of the response of mice to the thermal stimulation in the hot plate test. Moreover, EE significantly decreased number of writhes in the acetic acid-induced writhing test, and reduced licking time in both two phases of the formalin test in a dose-dependent manner. The F1 (50 mg/kg) showed effective antinociceptive responses in all mice models. However, fraction 2 (F2) and fraction 3 (F3) at 50 mg/kg performed no analgesic action. Kaempferol-3-O-rutinoside, isorhamnetin-3-O-rutinoside, pinoresinol-4-O-glucoside, forsythin and arctiin were identified from components of the F1. Furthermore, F1 (50 mg/kg) did not significantly affect hind paw oedema of mice induced by carrageenan but significantly shortened sleep latency and increased sleep duration in the pentobarbital sodium sleep test. In addition, the antinociceptive response of F1 was not affected by NXH in two mice models, but significantly blocked by FM and WAY in the hot plate test. In the formalin test, FM avoided the effect of F1 only in the first phase, while the analgesic activity of F1 was totally suppressed by WAY in both two phases. Otherwise, contents of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) increased significantly in hippocampus and striatum of mice in the F1 group. ConclusionEE from flowers of Styrax japonicus, and F1, the active part isolated from EE, showed significant antinociceptive activities. The analgesic effect of F1 appeared to be related to the sedative effect, partially mediated by the GABAergic system, and highly involved in the serotonergic system. This was the first study confirming the analgesic effect of Styrax japonicus flower, which provided a candidate for the development of non-opioid analgesics.

Orexin receptors in the CA1 region of hippocampus modulate the stress-induced antinociceptive responses in an animal model of persistent inflammatory pain

Stress activates multiple neural pathways and neurotransmitters that often suppress pain perception, the phenomenon called stress-induced analgesia (SIA). Orexin neurons from the lateral hypothalamus project to entire brain structures such as the hippocampus. The present study examined this hypothesis that orexinergic receptors in the CA1 region of the hippocampus may play a modulatory role in the development of SIA in formalin test as an animal model of persistent inflammatory pain. One hundred-two adult male Wistar rats were administered with intra-CA1 orexin-1 receptor (OX1r) antagonist, SB334867, at the doses of 3, 10, 30, and 100 nmol or TCS OX2 29 as orexin-2 receptor (OX2r) antagonist at the doses of 1, 3, 10, and 30 nmol. Five min later, rats were exposed to forced swim stress (FSS) for a 6-min period. Then, pain-related behaviors induced by formalin injection were measured at the 5-min blocks during a 60-min period of formalin test. The current study indicated that solely stress exposure elicits antinociception in the early and late phases of the formalin test. The FSS-induced analgesia was prevented by intra-CA1 administration of SB334867 or TCS OX2 29 during either phase of the formalin test. Moreover, the contribution of the OX2r in the mediation of analgesic effect of stress was more prominent than that of the OX1r during both phases of the formalin test. It is suggested that OX1r and OX2r in the CA1 region of the hippocampus are involved in stress-induced analgesia in the animal model of persistent inflammatory pain.

Astaxanthin engages the l-arginine/NO/cGMP/KATP channel signaling pathway toward antinociceptive effects.

One of the main functions of the sensory system in our body is to maintain somatosensory homeostasis. Recent reports have led to a significant advance in our understanding of pain signaling mechanisms; however, the exact mechanisms of pain transmission have remained unclear. There is an urgent need to reveal the precise signaling mediators of pain to provide alternative therapeutic agents with more efficacy and fewer side effects. Accordingly, although the anti-inflammatory, antioxidative and anti-neuropathic effects of astaxanthin (AST) have been previously highlighted, its peripheral antinociceptive mechanisms are not fully understood. In this line, considering the engagement of l-arginine/nitric oxide (NO)/cyclic GMP (cGMP)/potassium channel (KATP) signaling pathway in the antinociceptive responses, the present study evaluated its associated role in the antinociceptive activity of AST. Male mice were intraperitoneally (i.p.) injected with l-arginine (100 mg/kg), SNAP (1 mg/kg), L-NAME (30 mg/kg), sildenafil (5 mg/kg), and glibenclamide (10 mg/kg) alone and prior to the most effective dose of AST. Following AST administration, intraplantarly (i.pl) injection of formalin was done, and pain responses were evaluated in mice during the primary (acute) and secondary (inflammatory) phases of formalin test. The results highlighted that 10 mg/kg i.p. dose of AST showed the greatest antinociceptive effect. Besides, while L-NAME and glibenclamide reduced the antinociceptive effect of AST, it was significantly increased by l-arginine, SNAP and sildenafil during both the primary and secondary phases of formalin test. These data suggest that the antinociceptive activity of AST is passing through the l-arginine/NO/cGMP/KATP pathway.

Sex and dose-dependent antinociceptive effects of the JNK (c-Jun N-terminal kinase) inhibitor SU 3327 are mediated by CB2 receptors in female, and CB1/CB2 receptors in male mice in an inflammatory pain model

Activation of c-Jun N-terminal kinases (JNKs) has been implicated in the development and persistence of inflammatory and neuropathic pain in animal models. Moreover, JNKs have been involved in the maintenance of chronic pain, as well as development of tolerance to antinociceptive agents in the opioid and cannabinoid class of compounds. In this study, we evaluated the antinociceptive effects of the JNK inhibitor SU 3327 (0.3–30 mg/kg) in the formalin pain model with an emphasis on the sex-specific actions of this compound. In wild-type C57BL6J mice, SU 3327 produced strong antinociceptive effects in the formalin pain model which were mediated by CB2 receptors in females, and both CB1 and CB2 receptors in males. SU 3327 at a dose of 10 mg/kg produced antinociception, hypothermia, motor impairment, and hypolocomotion to a similar extent in both males and females. The antinociceptive effects of SU 3327 were more potent in males at lower doses (1 and 3 mg/kg), while females were more sensitive to the hypothermic, and motor-suppression effects at lower (3 mg/kg) doses versus males. Analysis of spinal cords, using qPCR following SU 3327 administration in the formalin test, revealed changes in cannabinoid, tolerance and inflammatory markers in females only, and only in the high (10–30 mg/kg) dose conditions. Indeed, females showed an increase in mRNA levels of cannabinoid (CB2), but a decrease in tolerance (β-arrestin 1) and inflammatory (TNF-α, IL-1β, IL-6)-associated markers. The differences between males and females, in this study, support sex as an important factor in nociception and antinociceptive responses mediated by JNK and the endocannabinoid system.

Novel selective κ agonists SLL-039 and SLL-1206 produce potent antinociception with fewer sedation and aversion.

SLL-039 (N-cyclopropylmethyl-7α-4'-(N'-benzoyl) amino-phenyl-6,14-endoethano-tetrahydronorthebaine) and SLL-1206 (N-cyclopropylmethyl-7α-3'-(p-methoxybenzyl) amino-phenyl-6,14-endoethano-tetrahydronorthebaine) are two 4,5-epoxymorphinan-based high selective κ receptor agonists that we recently discovered. In the present study we characterized their pharmacological properties in comparison with arylacetamide-based typical κ agonist U50,488H. We showed that both SLL-039 and SLL-1206 produced potent and long-lasting antinociceptive actions in three different rodent models of pain via activation of κ opioid receptor. In hot-plate assay, the antinociceptive potency of SLL-039 and SLL-1206 increased about 11-and 17.3-fold compared to U50,488H and morphine, respectively, with ED50 values of 0.4 mg/kg. Following repeated administration, SLL-1206, SLL-039, and U50,488H all developed analgesic tolerance tested in hot-plate assay. U50,488H and SLL-039 produced antipruritic effects in a dose-dependent manner, whereas SLL-1206 displayed some antipruritic effects only at very low doses. In addition, SLL-1206 was capable of decreasing morphine-induced physical dependence. More importantly, SLL-039 and SLL-1206 at effective analgesic doses did not cause sedation and conditioned place aversion (CPA), whereas U50,488H did. In comparison with SLL-039, SLL-1206 caused similar antinociceptive responses, but fewer sedation and CPA. In conclusion, our results suggest that SLL-039 and SLL-1206 have potential to be developed as novel analgesic agents, and 4,5-expoxymorphinan scaffold is an attractive structure for the development of selective κ agonists with fewer side effects.

Pharmacological activities of Asclepias curassavica L. (Apocynaceae) aerial parts

Ethnopharmacological relevanceAsclepias curassavica L. (Apocynaceae) is a perennial shrub used in the folk treatment of parasitism, pain, and inflammation. Aim of the studyThis work assessed the antiparasitic, anti-inflammatory, antinociceptive, and sedative effects of an ethanol extract from the aerial parts of Asclepias curassavica (ACE). Materials and methodsThe antiparasitic activity against Trichomonas vaginalis was evaluated using the trypan blue exclusion test. The in vitro anti-inflammatory actions of ACE (1–200 μg/ml) were analyzed using LPS-stimulated primary murine macrophages. The in vivo pharmacological activity of ACE (50–200 mg/kg p.o.) was evaluated using animal models of inflammation (TPA-induced ear edema test and carrageenan-induced paw edema test) and nociception (acetic acid-induced writhing test, formalin-induced licking test, and hot plate test). ResultsACE showed poor antiparasitic effects against Trichomonas vaginalis (IC50 = 302 μg/ml). ACE increased the production of IL-10 in both in vitro assays (EC50 = 3.2 pg/ml) and in vivo assays (ED50 = 111 mg/kg). ACE showed good antinociceptive actions (ED50 = 158 mg/kg in phase 1 and ED50 = 83 mg/kg in phase 2) in the formalin test. Pre-treatment with naloxone blocked the antinociceptive response induced by ACE. In addition, ACE did not induce sedative effects or motor coordination deficits in mice. ConclusionFindings showed that the anti-inflammatory activity of ACE is associated with increasing levels of IL-10 in both in vitro and in vivo assays, whereas the antinociceptive effect is associated with the participation of the opioidergic system, without inducing sedation or motor coordination impairment.

Modulatory role of the orexin system in stress-induced analgesia: Involvement of the ventral tegmental area.

Exposure to stressful experiences is often accompanied by suppressing pain perception, referred to as stress-induced analgesia. The neuropeptides orexins are essential in regulating the mechanism that responds to stressful and painful stimuli. Meanwhile, the ventral tegmental area (VTA), as a part of descending pain inhibitory system, responds to noxious stimuli. This study aimed to investigate the role of intra-VTA administration of orexin receptor antagonists on stress-induced antinociceptive responses in the animal model of acute pain. Ninety-three adult Wistar rats weighing 230-250g were unilaterally implanted by a cannulae above the VTA. Animals were pretreated with different doses (1, 3, 10 and 30nM/0.3μl) of SB334867 as the orexin-1 receptor antagonist and TCS OX2 29 as the orexin-2 receptor antagonist into the VTA, just 5min before 6min exposure to forced swim stress (FSS). Nociceptive threshold was measured using the tail-flick test as a model of acute pain. The results showed that exposure to FSS could significantly increase analgesic responses. Moreover, intra-VTA administration of SB334768 and TCS OX2 29 blocked the antinociceptive effect of FSS in the tail-flick test. The findings suggest that OX1 and OX2 receptors in the VTA might modulate the antinociceptive behaviours induced by FSS in part. Acute exposure to physical stress suppresses pain-related behaviors in the animal model of acute pain. Blockade of the OX1 and OX2 receptors in the VTA attenuates antinociceptive responses induced by FSS. The contribution of the OX2 receptors in the VTA is more predominant than OX1 receptors in stress-induced analgesia.

Orexin-2 receptor antagonism in the cornu ammonis 1 region of hippocampus prevented the antinociceptive responses induced by chemical stimulation of the lateral hypothalamus in the animal model of persistent pain.

Orexins are excitatory neuropeptides, mainly produced by neurons located in the lateral hypothalamus, which project to many brain areas. The orexinergic system plays a fundamental role in arousal, sleep/wakefulness, feeding, energy homeostasis, motivation, reward, stress and pain modulation. As a prominent part of the limbic system, the hippocampus has been involved in formalin-induced nociception modulation. Moreover, hippocampus regions express both orexin-1 (OX1) and orexin-2 (OX2) receptors. The present study investigated the role of OX2 receptors (OX2R) within the cornu ammonis 1 (CA1) region of the hippocampus in the mediation of lateral hypothalamus-induced antinociception. Fifty-three male Wistar rats were unilaterally implanted with two separate cannulae into the lateral hypothalamus and CA1. Animals were pretreated with intra-CA1 TCS OX2 29 as an OX2R antagonist before intra-lateral hypothalamus administration of carbachol (250 nM) as a muscarinic agonist for chemical stimulation of orexinergic neurons. Formalin test was used as an animal model of persistent pain, following intra-lateral hypothalamus carbachol microinjection. Results showed that the chemical stimulation of the lateral hypothalamus significantly attenuated formalin-evoked nociceptive behaviors during both phases of the formalin test, and administration of TCS OX2 29 into the CA1 blocked these antinociceptive responses in both phases, especially in the late phase. These findings suggest that OX2 receptors in the CA1 partially mediate the lateral hypothalamus-induced antinociceptive responses in persistent inflammatory pain.

A placebo-controlled investigation of the analgesic effects, abuse liability, safety and tolerability of a range of oral cannabidiol doses in healthy humans.

Preclinical studies demonstrate that cannabidiol (CBD) elicits an antinociceptive response in animal models of neuropathic pain; in humans, limited data are available to support such analgesic effects. Few studies have examined CBD's analgesic effects when administered without other compounds, and little is known regarding dose-dependent effects in noncannabis users. This double-blind, placebo-controlled, within-subject outpatient clinical laboratory study sought to determine the analgesic effects, abuse liability, safety and tolerability of acute CBD (0, 200, 400 and 800 mg orally) in healthy noncannabis-using volunteers (n = 17; 8 men, 9 women). Outcomes included experimental pain threshold and pain tolerance using the cold pressor test (CPT), subjective ratings of CPT painfulness and bothersomeness, subjective ratings of abuse liability and mood, and cardiovascular measures, which were assessed at baseline and several time points after drug administration. Data analyses included repeated measures analysis of variance (ANOVA) with planned comparisons. CBD failed to consistently affect pain threshold and tolerance in the CPT relative to placebo. All doses of CBD increased ratings of painfulness compared to placebo (P < .01). Further, CBD had dose-dependent, modest effects on mood and subjective drug effects associated with abuse liability. Oral CBD was safe and well tolerated, producing small decreases in blood pressure (P < .01). CBD did not elicit consistent dose-dependent analgesia and in fact increased pain on some measures. Future studies exploring CBD-induced pain relief should consider using a more extensive pain assessment paradigm in different participant populations.

Synergistic Interaction in the Analgesic-Like Effects of Maqui Berry and Citrus Is Antagonized by Sweeteners.

Although physiologically pain has a protective function, in many diseases, it is one of the most prominent symptoms. Today, new trends are focused on finding more natural alternatives to conventional treatments to alleviate it. Thereby, the purpose of this investigation was to obtain preclinical data of the antinociceptive properties of a lyophilized obtained from a newly designed maqui–citrus beverage alone and added with different sweeteners. To achieve this objective, maqui berry and citrus pharmacological activity were studied separately, as well as the interaction of both ingredients. In addition, due to the controversy generated regarding the intake of sugars, related to different metabolic diseases, the influence of different sweeteners (stevia, sucralose, or sucrose) was studied to determine their possible influence on the bioactive compounds of this product. For the attainment of our goals, a pharmacological evaluation, using the 1% formalin test, a nociceptive pain model in mice, was performed by using a sub-efficacious dosage of Maqui (25 mg/kg, i.p.) alone and combined with citrus, and then compared with the effects obtained in the presence of the different sweeteners. As a result, the antinociceptive response of the maqui was synergized in the presence of citrus in the neurogenic and inflammatory phases of the formalin test. However, this response was partially or totally reduced in the presence of the sweeteners. Our study gives preclinical evidence that a combination of maqui and citrus might exert beneficial actions to relieve pain, whereas the presence of sweeteners could reduce or avoid it.

Loss of SUR1 subtype KATP channels alters antinociception and locomotor activity after opioid administration

Opioid signaling can occur through several downstream mediators and influence analgesia as well as reward mechanisms in the nervous system. KATP channels are downstream targets of the μ opioid receptor and contribute to morphine-induced antinociception. The aim of the present work was to assess the role of SUR1-subtype KATP channels in antinociception and hyperlocomotion of synthetic and semi-synthetic opioids. Adult male and female mice wild-type (WT) and SUR1 deficient (KO) mice were assessed for mechanical and thermal antinociception after administration of either buprenorphine, fentanyl, or DAMGO. Potassium flux was assessed in the dorsal root ganglia and superficial dorsal horn cells in WT and KO mice. Hyperlocomotion was also assessed in WT and KO animals after buprenorphine, fentanyl, or DAMGO administration. SUR1 KO mice had attenuated mechanical antinociception after systemic administration of buprenorphine, fentanyl, and DAMGO. Potassium flux was also attenuated in the dorsal root ganglia and spinal cord dorsal horn cells after acute administration of buprenorphine and fentanyl. Hyperlocomotion after administration of morphine and buprenorphine was potentiated in SUR1 KO mice, but was not seen after administration of fentanyl or DAMGO. These results suggest SUR1-subtype KATP channels mediate the antinociceptive response of several classes of opioids (alkaloid and synthetic/semi-synthetic), but may not contribute to the “drug-seeking” behaviors of all classes of opioids.

Antinociceptive Activity of Compounds from the Aqueous Extract of Melampodium divaricatum.

A decoction prepared from the aerial parts of Melampodium divaricatum showed antinociceptive and antihyperalgesic responses when tested in the formalin model in mice. From the CH2 Cl2 fraction of the decoction, two non-previously reported secondary metabolites, 3-O-β-D-glucopyranosyl-16α-hydroxy-ent-kaurane (1) and melampodiamide (2) [(2'R*,4'Z)-2'-hydroxy-N-[(2S*,3S*,4R*)-1,3,4-trihydroxyoctadec-2-yl]tetracos-4-enamide] were separated and characterized by spectroscopic, spectrometric, and computational techniques. The flavonoids isoquercitrin and hyperoside, which possessed noted antinociceptive properties, were obtained from the active AcOEt fraction of the decoction. The chemical composition of the essential oil of the plant was also analyzed by gas chromatography-mass spectrometry. The major constituents were (E)-caryophyllene, germacrene D, β-elemene, δ-elemene, γ-patchoulene, and 7-epi-α-selinene. Headspace solid-phase microextraction analysis detected (E)-caryophyllene as the main volatile compound of the plant.

Pharmacological evaluation of the gabapentin salicylaldehyde derivative, gabapentsal, against tonic and phasic pain models, inflammation, and pyrexia.

Gabapentinoids are effective drugs in most animal models of pain and inflammation with variable effects in humans. The current study evaluated the pharmacological activity of gabapentin (GBP) and its salicylaldehyde derivative (gabapentsal; [2-(1-(((2-hydroxybenzylidene) amino) methyl) cyclohexyl) acetic acid]; GPS) in well-established mouse models of nociceptive pain, inflammatory edema, and pyrexia at doses of 25-100 mg/kg. GPS allayed tonic visceral pain as reflected by acetic acid-induced nociception and it also diminished thermally induced nociception as a mimic of phasic thermal pain. Antagonism of GPS-induced antinociceptive activities by naloxone (NLX, 1.0 mg/kg, subcutaneously, s.c), beta-funaltrexamine (β-FNT, 5.0 mg/kg, s.c), naltrindole (NT, 1.0 mg/kg, s.c), and nor-binaltorphimine (NOR-BNI, 5.0 mg/kg, s.c), and pentylenetetrazole (PTZ-15 mg/kg, intraperitoneally, i.p) implicated an involvement of both opioidergic and GABAergic mechanisms. Tail immersion test was conducted in order to delineate the mechanistic insights of antinociceptive response. Inflammatory edema induced by carrageenan, histamine, or serotonin was also effectively reversed by GPS in a fashion analogous to aspirin (150 mg/kg, i.p), chlorpheniramine (1.0 mg/kg, i.p), and mianserin (1.0 mg/kg, i.p), respectively. Additionally, yeast-induced pyrexia was decreased by GPS in a comparable manner to acetaminophen (50 mg/kg, i.p). These observations suggest that GPS possesses ameliorative properties in tonic, phasic, and tail immersion tests of nociception via opioidergic and GABAergic mechanisms, curbs inflammatory edema, and is antipyretic in nature.

Sex-specific mechanisms of tolerance for the cannabinoid agonists CP55,940 and delta-9-tetrahydrocannabinol (Δ9-THC).

Tolerance to cannabinoids could limit their therapeutic potential. Male mice expressing a desensitization-resistant form (S426A/S430A) of the type-1 cannabinoid receptor (CB1R) show delayed tolerance to delta-9-tetrahydrocannabinol (∆9-THC) but not CP55,940. With more women than men using medical cannabis for pain relief, it is essential to understand sex differences in cannabinoid antinociception, hypothermia, and resultant tolerance. Our objective was to determine whether female mice rely on the same molecular mechanisms for tolerance to the antinociceptive and/or hypothermic effects of cannabinoids that we have previously reported in males. We determined whether the S426A/S430A mutation differentially disrupts antinociceptive and/or hypothermic tolerance to CP55,940 and/or Δ9-THC in male and female S426A/S430A mutant and wild-type littermates. The S426A/S430A mutation conferred an enhanced antinociceptive response for ∆9-THC and CP55,940 in both male and female mice. While the S426A/S430A mutation conferred partial resistance to ∆9-THC tolerance in male mice, disruption of CB1R desensitization had no effect on tolerance to ∆9-THC in female mice. The mutation did not alter tolerance to the hypothermic effects of ∆9-THC or CP55,940 in either sex. Interestingly, female mice were markedly less sensitive to the antinociceptive effects of 30mg/kg ∆9-THC and 0.3mg/kg CP55,940 compared with male mice. Our results suggest that disruption of the GRK/βarrestin2 pathway of desensitization alters tolerance to Δ9-THC but not CP55,940 in male but not female mice. As tolerance to Δ9-THC appears to develop differently in males and females, sex should be considered when assessing the therapeutic potential and dependence liability of cannabinoids.

Spinal A3 adenosine receptor activation acutely restores morphine antinociception in opioid tolerant male rats.

Opioids are potent analgesics, but their pain-relieving effects diminish with repeated use. The reduction in analgesic potency is a hallmark of opioid analgesic tolerance, which hampers opioid pain therapy. In the central nervous system, opioid analgesia is critically modulated by adenosine, a purine nucleoside implicated in the beneficial and detrimental actions of opioid medications. Here, we focus on the A3 adenosine receptor (A3 AR) in opioid analgesic tolerance. Intrathecal administration of the A3 AR agonist MRS5698 with daily systemic morphine in male rats attenuated the reduction in morphine antinociception over 7days. In rats with established morphine tolerance, intrathecal MRS5698 partially restored the antinociceptive effects of morphine. However, when MRS5698 was discontinued, these animals displayed a reduced antinociceptive response to morphine. Our results suggest that MRS5698 acutely and transiently potentiates morphine antinociception in tolerant rats. By contrast, in morphine-naïve rats MRS5698 treatment did not impact thermal nociceptive threshold or affect antinociceptive response to a single injection of morphine. Furthermore, we found that morphine-induced adenosine release in cerebrospinal fluid was blunted in tolerant animals, but total spinal A3 AR expression was not affected. Collectively, our findings indicate that spinal A3 AR activation acutely potentiates morphine antinociception in the opioid tolerant state.

Effect of the Croton rhamnifolioides Essential Oil and the Inclusion Complex (OEFC/β-CD) in Antinociceptive Animal Models

This study aims to evaluate the antinociceptive effect of the C. rhamnifolioides leaf essential oil (OEFC) and the β-cyclodextrin inclusion complex (COEFC) and investigate the pain signaling pathways involved in the antinociceptive response. The effects of the OEFC and COEFC on the central nervous system (CNS) were determined by open field and rota-rod assays, and the antinociceptive effect was evaluated via the acetic acid-induced abdominal contortions, formalin, and hot plate models. Swiss (Mus musculus) male mice (20–30 g) were used in both trials. The OEFC (200 mg/kg/v.o-orally) and COEFC (83.5 mg/kg/v.o.) did not present alterations in the CNS. The OEFC (25, 50, 100, and 200 mg/kg/vo.) and COEFC (8.35, 41.75, and 83.5 mg/kg/v.o.) demonstrated antinociceptive effects in the abdominal contortions, formalin, and hot plate tests. The OEFC (25 mg/kg/v.o.) and COEFC (8.35 mg/kg/v.o.) doses showed that the antinociceptive effect involves the activation of the opioid, cholinergic, and vanilloid systems, as well as the L-arginine/NO and α-2 adrenergic receptor pathways. The antinociceptive potential the OEFC and COEFC demonstrate possible alternatives for the therapy of pain. However, the COEFC presented more significant effects at lower doses than the isolated OEFC, where this action may be justified by the properties and advantages of the complexation.

CGRP monoclonal antibody prevents the loss of diffuse noxious inhibitory controls (DNIC) in a mouse model of post-traumatic headache.

Determine the role of calcitonin-gene related peptide in promoting post-traumatic headache and dysregulation of central pain modulation induced by mild traumatic brain injury in mice. Mild traumatic brain injury was induced in lightly anesthetized male C57BL/6J mice by a weight drop onto a closed and unfixed skull, which allowed free head rotation after the impact. We first determined possible alterations in the diffuse noxious inhibitory controls, a measure of net descending pain inhibition called conditioned pain modulation in humans at day 2 following mild traumatic brain injury. Diffuse noxious inhibitory control was assessed as the latency to a thermally induced tail-flick that served as the test stimulus in the presence of right forepaw capsaicin injection that provided the conditioning stimulus. Post-traumatic headache-like behaviors were assessed by the development of cutaneous allodynia in the periorbital and hindpaw regions after mild traumatic brain injury. We then determined if intraperitoneal fremanezumab, an anti-calcitonin-gene related peptide monoclonal antibody or vehicle administered 2 h after sham or mild traumatic brain injury induction could alter cutaneous allodynia or diffuse noxious inhibitory control responses on day 2 post mild traumatic brain injury. In naïve and sham mice, capsaicin injection into the forepaw elevated the latency to tail-flick, reflecting the antinociceptive diffuse noxious inhibitory control response. Periorbital and hindpaw cutaneous allodynia, as well as a loss of diffuse noxious inhibitory control, was observed in mice 2 days after mild traumatic brain injury. Systemic treatment with fremanezumab blocked mild traumatic brain injury-induced cutaneous allodynia and prevented the loss of diffuse noxious inhibitory controls in mice subjected to a mild traumatic brain injury. Sequestration of calcitonin-gene related peptide in the initial stages following mild traumatic brain injury blocked the acute allodynia that may reflect mild traumatic brain injury-related post-traumatic headache and, additionally, prevented the loss of net descending inhibition within central pain modulation pathways. As loss of conditioned pain modulation has been linked to multiple persistent pain conditions, dysregulation of descending modulatory pathways may contribute to the persistence of post-traumatic headache. Additionally, evaluation of the conditioned pain modulation/diffuse noxious inhibitory controls response may serve as a biomarker of vulnerability for chronic/persistent pain. These findings suggest that early anti-calcitonin-gene related peptide intervention has the potential to be effective both for the treatment of mild traumatic brain injury-induced post-traumatic headache, as well as inhibiting mechanisms that may promote post-traumatic headache persistence.