Complete Freund's Adjuvant Model Research Articles

HDAC6-selective inhibitors decrease nerve-injury and inflammation-associated mechanical hypersensitivity in mice.

HDAC6 is a class IIB histone deacetylase expressed at many levels of the nociceptive pathway. This study tested the ability of novel and selective HDAC6 inhibitors to alleviate sensory hypersensitivity behaviors in mouse models of peripheral nerve injury and peripheral inflammation. We utilized the murine spared nerve injury (SNI) model for peripheral nerve injury and the Complete Freund's Adjuvant (CFA) model of peripheral inflammation. We applied the Von Frey assay to monitor mechanical allodynia. Using the SNI model, we demonstrate that daily administration of the brain-penetrant HDAC6 inhibitor, ACY-738, abolishes mechanical allodynia in male and in female mice. Importantly, there is no tolerance to the antiallodynic actions of these compounds as they produce a consistent increase in Von Frey thresholds for several weeks. We observed a similar antiallodynic effect when utilizing the HDAC6 inhibitor, ACY-257, which shows limited brain expression when administered systemically. We also demonstrate that ACY-738 and ACY-257 attenuate mechanical allodynia in the CFA model of peripheral inflammation. Overall, our findings suggest that inhibition of HDAC6 provides a promising therapeutic avenue for the alleviation of mechanical allodynia associated with peripheral nerve injury and peripheral inflammation.

Improved efficacy, tolerance, safety, and abuse liability profile of the combination of CR4056 and morphine over morphine alone in rodent models.

Background and PurposeProlonged use of opioids causes analgesic tolerance and adverse effects including constipation and dependence. Compounds targeting imidazoline I2 receptors are known to potentiate opioid analgesia in rodents. We investigated whether combination with the I2 receptor ligand CR4056 could improve efficacy and safety of morphine and explored the mechanisms of the CR4056–opioid interaction.Experimental ApproachWe used the complete Freund's adjuvant (CFA) model in rats to study the effects of treatments on hyperalgesia, morphine tolerance and microglia activation as measured by immunofluorescence. Opioid‐induced adverse effects were assessed in rodent models of morphine‐induced constipation, sedation (open field, sedation rating scale, and rotarod), physical dependence (naloxone‐induced withdrawal), and abuse (conditioned place preference‐associated reward). Chemiluminescence assays tested CR4056 as allosteric modulator of μ‐opioid receptors.Key ResultsCR4056 (ED50 = 4.88 mg·kg−1) and morphine (ED50 = 2.07 mg·kg−1) synergized in reducing CFA‐induced hyperalgesia (ED50 = 0.52 mg·kg−1; 1:1 combination). Consistently, low doses of CR4056 (1 mg·kg−1) spared one third of the cumulative morphine dose administered during 4 days and prevented/reversed the development of tolerance to morphine anti‐hyperalgesia. These opioid‐sparing effects were associated with decreased activation of microglia, independent of CR4056 interactions on μ‐opioid receptors. Importantly, the low doses of CR4056 and morphine that synergize in analgesia did not induce constipation, sedation, physical dependence, or place preference.Conclusion and ImplicationsWe showed selective synergism between CR4056 and morphine as analgesics. Their combination showed an improved safety and abuse liability profile over morphine alone. CR4056 could be developed as an opioid‐sparing drug in multimodal analgesia.

The Operant Plantar Thermal Assay: A Novel Device for Assessing Thermal Pain Tolerance in Mice.

Pain is a multidimensional experience of sensory-discriminative, cognitive, and affective processes; however, current basic research methods rely heavily on response to threshold stimuli, bypassing the supraspinal processing that ultimately gives rise to the pain experience. We developed the operant plantar thermal assay (OPTA), which utilizes a novel, conflict-based operant task requiring evaluation and active decision-making to obtain reward under thermally aversive conditions to quantify thermal pain tolerance. In baseline measures, male and female mice exhibited similar temperature preferences, however in the OPTA, female mice exhibited greater temperature-dependent tolerance, as defined by choice time spent in an adverse thermal condition to obtain reward. Increasing reward salience (4% vs 10% sucrose solution) led to increased thermal tolerance for males but not females. To determine whether neuropathic and inflammatory pain models alter thermal tolerance, animals with chronic constriction injury (CCI) or complete Freund’s adjuvant (CFA), respectively, were tested in the OPTA. Surprisingly, neuropathic animals exhibited increased thermal tolerance, as shown by greater time spent in the reward zone in an adverse thermal condition, compared with sham animals. There was no effect of inflammation on thermal tolerance. Administration of clonidine in the CCI model led to increased thermal tolerance in both injured and sham animals. In contrast, the non-steroidal anti-inflammatory meloxicam was anti-hyperalgesic in the CFA model, but reduced thermal pain tolerance. These data support the feasibility of using the OPTA to assess thermal pain tolerance to gain new insights into complex pain behaviors and to investigate novel aspects of analgesic efficacy.

Differential Regulation of the Glucocorticoid Receptor in a Rat Model of Inflammatory Pain.

Anti-inflammatory corticosteroids are a common treatment for different conditions involving chronic pain and inflammation. Clinically used steroids target the glucocorticoid receptor (GR) for its anti-inflammatory effects. We previously reported that GR in sensory neurons may play central roles in some pain models and that GR immunoreactivity signal in dorsal root ganglia (DRG) decreased after local inflammation of the DRG (a model of low back pain). In the current study, we aimed to determine if similar changes in GR signal also exist in a skin inflammation model, the complete Freund's adjuvant (CFA) model (a model of peripheral inflammatory pain), in which the terminals of the sensory neurons rather than the somata are inflamed. A low dose of CFA was injected into the hind paw to establish the peripheral inflammation model in Sprague-Dawley rats of both sexes, as confirmed by measurements of behavior and paw swelling. Immunohistochemical and western blotting techniques were used to determine the expression pattern of the GR in the inflamed hind paw and the DRGs. Plasma corticosterone levels were measured with radioimmunoassay. The immunohistochemical staining revealed that GR is widely expressed in the normal DRG and skin tissues. Paw injection with CFA caused upregulation of the GR in the skin tissue on postinjection day 1, mostly detected in the dermis area. However, paw inflammation significantly reduced the GR signal in the L5 DRG 1 day after the injection. The GR downregulation was still evident 14 days after CFA inflammation. On day 1, western blotting confirmed this downregulation and showed that it could also be observed in the contralateral L5 DRG, as well as in the L2 DRG (a level which does not innervate the paw). Plasma corticosterone levels were elevated in both sexes on day 14 after CFA compared to day 1, suggesting autologous downregulation of the GR by corticosterone may have contributed to the downregulation observed on day 14 but not day 1. There are distinctive patterns of GR activation under different pain conditions, depending on the anatomical location. The observed downregulation of the GR in sensory neurons may have a significant impact on the use of steroids as treatment in these conditions and on the regulatory effects of endogenous glucocorticoids.

Anti-hyperalgesic properties of ethanolic crude extract from the peels of Citrus reticulata (Rutaceae).

The therapeutic effects from Citrus reticulata on painful inflammatory ailments are associated to its flavonoids constituent and phytochemical studies with Citrus genus affirm that the peels have important amounts of it. These bioactive compounds have been a considerable therapeutic source and evaluate potential application of the peel extract is significant. This research aims to investigate the influence of ethanolic crude extract from the peels of Citrus reticulata and its possible mechanism of action in different animal models of pain. The extract reduced hyperalgesia in the second phase of formalin test (vehicle: 501.5 ± 40.0 s; C. reticulata extract 300 mg/kg: 161.8 ± 41.1 s), in the carrageenan model (vehicle at 4th h: 82.5 ± 9.6 %; C. reticulata extract 300 mg/kg at 4th h: 47.5 ± 6.5 %) and in Complete Freund's Adjuvant model (vehicle: 501.5 ± 40.0 s; C. reticulata extract 300 mg/kg: 161.8 ± 41.1 s). The possible contribution of opioidergic and adenosinergic systems in the anti-hyperalgesic effect of C. reticulata extract was observed after treatment, with non-selective antagonists for both systems, which produced reversal effects. In conclusion, these properties of C. reticulata extract suggest a potential therapeutic benefit in treating painful conditions.

P.565 Characterisation of pain-, anxiety- and cognition-related behaviour in the complete Freund's adjuvant model of chronic inflammatory pain in Wistar-Kyoto rats

P.565 Characterisation of pain-, anxiety- and cognition-related behaviour in the complete Freund's adjuvant model of chronic inflammatory pain in Wistar-Kyoto rats

Inhibition of chemokine CX3CL1 in spinal cord mediates the electroacupuncture-induced suppression of inflammatory pain.

PurposeChemokine CX3CL1 and its receptor CX3CR1 in the lumbar spinal cord play crucial roles in pain processing. Electroacupuncture (EA) is recognized as an alternative therapy in pain treatment due to its efficacy and safety. However, the analgesic mechanism of EA remains unclear. The aim of this study was to investigate whether EA suppressed complete Freund’s adjuvant (CFA)-induced pain via modulating CX3CL1-CX3CR1 pathway.Materials and methodsInflammatory pain was induced by intraplantar injection of CFA to the left hind paw of Sprague-Dawley rats. EA with 2 Hz for 30 mins was given to bilateral Zusanli acupoints (ST36) on the first and third day after CFA injection. Mechanical allodynia and thermal hyperalgesia were tested with von Frey tests and Hargreaves tests, respectively. The expressions of CX3CL1, CX3CR1 and p38 mitogen-activated protein kinase (MAPK) were quantified with Western blots. The release of IL-1β, IL-6 and TNF-α were evaluated with ELISA. Recombinant CX3CL1 or control IgG were then injected through intrathecal catheters in the EA-treated CFA model rats. The behavioral tests, p38 MAPK activation and cytokine release were then evaluated.ResultsEA significantly inhibited inflammatory pain induced by CFA for 3 days. Meanwhile, EA downregulated the expression of CX3CL1 but not CX3CR1 in the lumbar spinal cord of the CFA rats. Besides, activation of p38 MAPK and the release of pain-related cytokines (IL-1β, IL-6 and TNF-α) were inhibited by EA. Intrathecal injection of CX3CL1 largely reversed the analgesic effect of EA treatment and re-activated p38 MAPK signaling, and resulted in pro-inflammatory cytokines increase in acupuncture-treated rats.ConclusionOur findings indicate that EA alleviates inflammatory pain via modulating CX3CL1 signaling in lumbar spinal cord, revealing a potential mechanism of anti-nociception of EA in inflammatory pain.

Antinociceptive activity of Copaifera officinalis Jacq. L oil and kaurenoic acid in mice.

Copaifera officinalis L. possesses traditional uses as an analgesic, anti-inflammatory, and antiseptic. However, until now the antinociceptive effect and the mechanism of action were not described for Copaifera officinalis L. oil and no compound present in this oil was identified to be responsible for its biological effects. The goal of this study was to identify the presence of kaurenoic acid in Copaifera officinalis oil and investigate its antinociceptive effect, mechanism of action, and possible adverse effects in mice. The quantification of kaurenoic acid in Copaifera officinalis oil was done by HPLC-DAD technique. Male and female albino Swiss mice (25-35g) were used to test the antinociceptive effect of Copaifera officinalis (10mg/kg, intragastric) or kaurenoic acid (1mg/kg) in the tail-flick test, intraplantar injection of capsaicin, allyl isothiocyanate (AITC) or complete Freund's adjuvant (CFA). Copaifera officinalis oil and kaurenoic acid caused the antinociceptive effect in the tail-flick test in a dose-dependent manner, and their effect was reversed by naloxone (an opioid antagonist). Copaifera officinalis oil or kaurenoic acid reduced the nociception caused by capsaicin or AITC and produced an anti-allodynic effect in the CFA model (after acute or repeated administration for 7days). Possible adverse effects were also observed, and non-detectable adverse effect was observed for the intragastric administration of Copaiba officinalis oil or kaurenoic acid and in the same way, the treatments were neither genotoxic nor mutagenic at the doses tested. Thus, Copaiba officinalis oil, and kaurenoic acid possess antinociceptive action without adverse effects.

(407) Preclinical Evaluation of Human Somatostatin Receptor 4 (hSSTR4) Agonist CNTX-0290 for Mixed Pain Conditions

CNTX-0290 is an oral, nonopioid hSSTR4 agonist under investigation for analgesia. Activity was assessed in acute, nociceptive, and neuropathic rat pain models. In the Complete Freund's Adjuvant (CFA) model, maximum efficacy of CNTX-0290 0.03–3 mg/kg vs vehicle was compared with indomethacin 30 mg/kg and celecoxib 10 mg/kg; minimal effective dose (MED) was determined. In the monosodium iodoacetate (MIA) osteoarthritis model, weight-bearing deficit (WBD) was assessed following CNTX-0290 0.01–30 mg/kg administration. Paw withdrawal threshold (PWT) was determined for CNTX-0290 0.03–0.3 mg/kg vs vehicle in the partial nerve ligation (PNL) model (control: lamotrigine 30 mg/kg) and at 3–30 mg/kg in the streptozocin diabetes model (control: duloxetine 30 mg/kg). In vitro potency and selectivity were evaluated. General/safety pharmacology studies were conducted in rats and cynomologus monkeys. Effects on growth hormone (GH) release and glucose tolerance were compared with octreotide. CNTX-0290 demonstrated potent, selective hSSTR4 agonism (EC50=2.6 nM; Ki=41 nM; hSSTR 1, 2, 3, and 5 Ki >10,000 nM). CNTX-0290 showed dose-dependent antinociceptive effects in the CFA model. Maximum efficacy was achieved at 3 mg/kg; MED was 0.1 mg/kg (minimal effective concentration [MEC]: 32 nM). In the MIA model, CNTX-0290 1–30 mg/kg significantly reversed WBD vs vehicle, and was comparable to morphine at 3 and 30 mg/kg. Efficacy up to 24 hours was observed in both models. In the PNL model, CNTX-0290 achieved maximum efficacy (comparable to lamotrigine) at 0.3 mg/kg; MED: 0.1 mg/kg (MEC: 25 nM). CNTX-0290 10 and 30 mg/kg significantly increased PWT vs vehicle in the streptozocin model, comparable to duloxetine. GH release and glucose tolerance were not significantly altered, and no dose-limiting CNS AEs were observed. CNTX-0290, a potent, selective, full hSSTR4 agonist, showed analgesic efficacy in models of nociceptive and neuropathic pain, demonstrating potential for treating mixed pain conditions. (Funding: Centrexion Therapeutics Corp.)

(258) Reversal of Mechanical Hyperalgesia by a Dual-Acting, Peripherally-Restricted Kappa/Delta Opioid Agonist (CA1001) in a Rat Model of Inflammatory Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory pain condition. In RA and other inflammatory states, normally sequestersed peripheral delta-opioid receptors may become active, allowing delta-opioid agonists to participate in the pain pathway directly and through allosteric modulation of peripheral kappa-opioid receptors. This study evaluated the efficacy of a single intraperitoneal injection of CA1001 (a novel dual-acting, peripherally restricted kappa/delta opioid agonist) on hyperalgesic nociceptive behaviors in the CFA (Complete Freund's Adjuvant) Model of Inflammatory Arthritis Pain in Rats. Following IACUC approval, inflammatory arthritis pain was induced with injection of 50 μL CFA into the tibio-tarsal joint. Mechanical hyperalgesia was assessed via joint compression thresholds (JCTs). 50 animals were randomly assigned to 5 groups (Power: 80%). Ipsilateral and contralateral joint compression thresholds (JCTs) were assessed prior to CFA injection, pre-dosing on Day 0, and 1, 2, and 4 hours post-dosing. Animals were administered a single dose of vehicle, CA1001 (1, 5, or 10 mg/kg IP), or celecoxib (30 mg/kg PO: active control; internal validity) on day 0 (14 days after CFA). All behavioral evaluations were performed by a blinded observer. Mechanical hyperalgesia was measured using a digital Randall-Selitto device. CA1001 (10 mg/kg) significantly increased paw compression thresholds compared to vehicle at all time points, (p

D2-like receptor agonist synergizes the μ-opioid agonist spinal antinociception in nociceptive, inflammatory and neuropathic models of pain in the rat

Opioids are potent analgesic drugs, but their use has been limited due to their side effects. Antinociceptive effects of D2-like receptor agonists such as quinpirole have been shown at the spinal cord level; however, their efficacy is not as high as that of opioids. Dopaminergic agonists are long-prescribed and well-tolerated drugs that have been useful to treat clinically and experimentally painful conditions. Because current pain treatments are not completely effective, the aim of this work was to determine if a D2-like receptor agonist improves the antinociceptive effects of a μ-opioid receptor agonist.Drugs were intrathecally administered in adult rats; mechanonociceptive and thermonociceptive tests were carried out. Intraplantar injection of complete Freund's adjuvant (CFA) and sciatic loose ligation (SLL) were used for inflammatory and neuropathic models of pain, respectively.In intact animals, D-Ala2, N-MePhe4, Gly-ol-enkephalin (DAMGO; a µ-opioid receptor agonist) increased the paw withdrawal latencies (PWL) in thermal and mechanical nociceptive tests in a dose-dependent manner. Quinpirole (D2-like receptor agonist) increased PWL only in mechanonociception. In the presence of quinpirole (1 nmol), the ED50 of the mechanical antinociceptive effect of DAMGO was significantly decreased (8-fold). Coadministration of 1 nmol quinpirole and 30 pmol DAMGO completely reversed hyperalgesia in the CFA model, whereas 100 pmol DAMGO plus 1 nmol quinpirole reversed the allodynia in the SLL model.This work offers evidence about a synergistic antinociceptive effect between opioidergic and dopaminergic drugs. This combination may relieve painful conditions resistant to conventional treatments, and it may reduce the adverse effects of chronic opioid administration.

C57BL/6 substrain differences in inflammatory and neuropathic nociception and genetic mapping of a major quantitative trait locus underlying acute thermal nociception.

Sensitivity to different pain modalities has a genetic basis that remains largely unknown. Employing closely related inbred mouse substrains can facilitate gene mapping of nociceptive behaviors in preclinical pain models. We previously reported enhanced sensitivity to acute thermal nociception in C57BL/6J (B6J) versus C57BL/6N (B6N) substrains. Here, we expanded on nociceptive phenotypes and observed an increase in formalin-induced inflammatory nociceptive behaviors and paw diameter in B6J versus B6N mice (Charles River Laboratories). No strain differences were observed in mechanical or thermal hypersensitivity or in edema following the Complete Freund’s Adjuvant model of inflammatory pain, indicating specificity in the inflammatory nociceptive stimulus. In the chronic constrictive nerve injury, a model of neuropathic pain, no strain differences were observed in baseline mechanical threshold or in mechanical hypersensitivity up to one month post-chronic constrictive nerve injury. We replicated the enhanced thermal nociception in the 52.5°C hot plate test in B6J versus B6N mice from The Jackson Laboratory. Using a B6J × B6N-F2 cross (N = 164), we mapped a major quantitative trait locus underlying hot plate sensitivity to chromosome 7 that peaked at 26 Mb (log of the odds [LOD] = 3.81, p < 0.01; 8.74 Mb-36.50 Mb) that was more pronounced in males. Genes containing expression quantitative trait loci associated with the peak nociceptive marker that are implicated in pain and inflammation include Ryr1, Cyp2a5, Pou2f2, Clip3, Sirt2, Actn4, and Ltbp4 (false discovery rate < 0.05). Future studies involving positional cloning and gene editing will determine the quantitative trait gene(s) and potential pleiotropy of this locus across pain modalities.

Memantine selectively prevented the induction of dynamic allodynia by blocking Kir2.1 channel and inhibiting the activation of microglia in spinal dorsal horn of mice in spared nerve injury model.

BackgroundMemantine is one of the important clinical medications in treating moderate to severe Alzheimer disease. The effect of memantine on preventing or treating punctate allodynia has been thoroughly studied but not on the induction of dynamic allodynia. The aim of this study is to investigate whether memantine could prevent the induction of dynamic allodynia and its underlying spinal mechanisms.Results(1) In in vivo spared nerve injury pain model, pretreatment with memantine at a lower dose (10 nmol, intrathecal; memantine-10) selectively prevented the induction of dynamic allodynia but not the punctate allodynia. (2) Pretreatment with either MK801-10 (MK801-10 nmol, intrathecal) or higher dose of memantine (30 nmol, intrathecal; memantine-30) prevented the induction of both dynamic and punctate allodynia. (3) Memantine-10 showed significant effect on the inhibition of the spared nerve injury-induced overactivation of microglia in spinal dorsal horn. (4) In contrast, in complete freund′s adjuvant (CFA) model, memantine-10 neither affected the CFA injection-induced activation of microglia in spinal dorsal horn nor the induction of dynamic allodynia. (5) Immunohistological studies showed Kir2.1 channel distributed widely and co-localized with microglia in the spinal dorsal horn of mice. (6) Pretreatment with either minocycline, a microglia inhibitor, or ML133, a Kir2.1 inhibitor, both selectively prevented the overactivation of microglia in spinal dorsal horn and the induction of dynamic allodynia following spared nerve injury.ConclusionThe selective inhibitory effect on the induction of dynamic allodynia in spared nerve injury model by low dose of the memantine (memantine-10) was tightly correlated with the blockade of microglia Kir2.1 channel to suppress the microglia activation.

In vivo and in vitro Approach to Anti-arthritic and Anti-inflammatory Effect of Crocetin by Alteration of Nuclear Factor-E2-Related Factor 2/hem Oxygenase (HO)-1 and NF-κB Expression.

Crocetin (apo carotenoid dicarboxylic acid) is a common constituent of saffron. Its importance is well documented in Chinese medicine. Some studies have reported the inhibitory effect on inflammation in rats. The aim of the current experimental investigation to scrutinize the anti-inflammatory effect of Crocetin using the lipo polysaccharide (LPS) induced mouse macrophages (RAW 264.7) in vitro and complete Freund’s adjuvant-induced arthritis model and to explore in vivo possible mechanism of action. RAW 264.7 macrophages were used for estimation of the effect of crocetin on the cyclooxygenase (COX-2), nitric oxide (NO)production, anti-inflammatory and along with pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10). Single intraperitoneal injection of complete freund’s adjuvant (CFA) was used to induce arthritis. The rats were divided into different group and received the oral administration of crocetin in a dose-dependent manner with indomethacin till 28 days. The paw edema and body weight was estimated at regular interval of time. The biochemical parameters, hematological and pro-inflammatory cytokines such as tumor necrosis factor receptor 1 (TNF-R1), IL-6, and IL-1β, Vascular endothelial growth factor (VEGF); heme oxygenase-1/nuclear factor erythroid 2–related factor 2 (HO-1/Nrf-2) expression were estimated at end of the experimental study. Crocetin inhibited the COX-2 catalyzed prostaglandin (PGE2) and inducible nitric oxide synthase catalyzed NO production on RAW 264.7. The paw edema and body weight was significantly (P < 0.001) modulated by the Crocetin in a dose-dependent manner. Crocetin treatment increased the level of red blood cells (RBC), hemoglobin (Hb) and decreased level of white blood cells (WBC), erythrocyte sedimentation rate (ESR), alkaline phosphatase (ALP), serum glutamic pyruvic transaminase (SGPT), and serum glutamic-oxaloacetic transaminase (SGOT) parameters, with reduction of TNF-α, IL-6, and IL-1β.The protective effect of crocetin was substantiated with a reduction in expression of IL-6, IL-1β, VEGF, and TNF-R1, respectively. Crocetin also increased the HO-1/Nrf-2 and decreased the nuclear factor kappa-B (NF-κB) mRNA, protein expression. On the basis of the result, we can conclude that the reduction of HO-1/Nrf-2 expression, as well as inflammatory mediators, may be involved in the protective effect of Crocetin in the CFA model.

Cannabinoid CB2 Agonist AM1710 Differentially Suppresses Distinct Pathological Pain States and Attenuates Morphine Tolerance and Withdrawal.

AM1710 (3-(1,1-dimethyl-heptyl)-1-hydroxy-9-methoxy-benzo(c) chromen-6-one), a cannabilactone cannabinoid receptor 2 (CB2) agonist, suppresses chemotherapy-induced neuropathic pain in rodents without producing tolerance or unwanted side effects associated with CB1 receptors; however, the signaling profile of AM1710 remains incompletely characterized. It is not known whether AM1710 behaves as a broad-spectrum analgesic and/or suppresses the development of opioid tolerance and physical dependence. In vitro, AM1710 inhibited forskolin-stimulated cAMP production and produced enduring activation of extracellular signal-regulated kinases 1/2 phosphorylation in human embryonic kidney (HEK) cells stably expressing mCB2. Only modest species differences in the signaling profile of AM1710 were observed between HEK cells stably expressing mCB2 and hCB2. In vivo, AM1710 produced a sustained inhibition of paclitaxel-induced allodynia in mice. In paclitaxel-treated mice, a history of AM1710 treatment (5 mg/kg per day × 12 day, i.p.) delayed the development of antinociceptive tolerance to morphine and attenuated morphine-induced physical dependence. AM1710 (10 mg/kg, i.p.) did not precipitate CB1 receptor-mediated withdrawal in mice rendered tolerant to Δ9-tetrahydrocannabinol, suggesting that AM1710 is not a functional CB1 antagonist in vivo. Furthermore, AM1710 (1, 3, 10 mg/kg, i.p.) did not suppress established mechanical allodynia induced by complete Freund's adjuvant (CFA) or by partial sciatic nerve ligation (PSNL). Similarly, prophylactic and chronic dosing with AM1710 (10 mg/kg, i.p.) did not produce antiallodynic efficacy in the CFA model. By contrast, gabapentin suppressed allodynia in both CFA and PSNL models. Our results indicate that AM1710 is not a broad-spectrum analgesic agent in mice and suggest the need to identify signaling pathways underlying CB2 therapeutic efficacy to identify appropriate indications for clinical translation.

Orofacial operant behaviors and electrophysiological properties of trigeminal ganglion neurons following masseter muscle inflammation in rats

Orofacial muscle pain is a significant clinical problem because it affects eating, speaking, and other orofacial functions in patients. However, mechanisms underlying orofacial muscle pain are not fully understood. In the present study we induced orofacial muscle pain by injecting Complete Freund’s Adjuvant (CFA) into masseter muscle of rats and assessed pain by the orofacial operant test. In comparison with the control group, CFA-injected animals (CFA group) showed decreases in operant behaviors, suggesting the presence of orofacial pain. Trigeminal ganglion (TG) neurons innervating masseter muscles were retrograde-labeled with DiI and their electrophysiological properties studied using patch-clamp recordings. About 20% of DiI-labeled TG neurons showed spontaneous action potentials (APs) in the CFA group but none in the control group. AP rheobase levels were significantly lower in DiI-labeled TG neurons of the CFA group than in the control group. Membrane input resistance of DiI-labeled TG neurons was significantly higher in the CFA group than in the control group. Several other membrane parameters were also different between DiI-labeled TG neurons of the CFA and control groups. Voltage-dependent currents were examined and the most significant changes following CFA were background K+ currents, which showed significantly smaller in DiI-labeled TG neurons of CFA group compared to the control group. Collectively, orofacial muscle pain in CFA model is accompanied with changes of electrophysiological properties and background K+ currents in TG neurons that innervate masseter muscles.

The analgesic efficacy of morphine varies with rat strain and experimental pain model: implications for target validation efforts in pain drug discovery

BackgroundTranslating efficacy of analgesic drugs from animal models to humans remains challenging. Reasons are multifaceted, but lack of sufficiently rigorous preclinical study design criteria and phenotypically relevant models may be partly responsible. To begin to address this fundamental issue, we assessed the analgesic efficacy of morphine in three inbred rat strains (selected based on stress reactivity and affective/pain phenotypes), and outbred Sprague Dawley (SD) rats supplied from two vendors.MethodsSensitivity to morphine (0.3–6.0 mg/kg, s.c.) was evaluated in the hot plate test of acute thermal nociception, the Complete Freund's Adjuvant (CFA) model of inflammatory‐induced mechanical hyperalgesia, and in a locomotor motility assay in male rats from the following strains; Lewis (LEW), Fischer (F344), Wistar Kyoto (WKY), and SD's from Envigo and Charles River.ResultsF344 and SD rats were similarly sensitive to morphine in hot plate and CFA‐induced inflammatory hyperalgesia (Minimum Effective Dose (MED) = 3.0 mg/kg). WKY rats developed a less robust mechanical hypersensitivity after CFA injection, and were less sensitive to morphine in both pain tests (MED = 6.0 mg/kg). LEW rats were completely insensitive to morphine in the hot plate test, in contrast to the reversal of CFA‐induced hyperalgesia (MED = 3.0 mg/kg). All strains exhibited a dose‐dependent reduction in locomotor activity at 3.0–6.0 mg/kg.ConclusionSensory phenotyping in response to acute thermal and inflammatory‐induced pain, and sensitivity to morphine in various inbred and outbred rat strains indicates that different pathophysiological mechanisms are engaged after injury. This could have profound implications for translating preclinical drug discovery efforts into pain patients.SignificanceThe choice of rat strain used in preclinical pain research can profoundly affect the outcome of experiments in relation to (a) nociceptive threshold responses, and (b) efficacy to analgesic treatment, in assays of acute and tonic inflammatory nociceptive pain.

The central amygdala to periaqueductal gray pathway comprises intrinsically distinct neurons differentially affected in a model of inflammatory pain.

The central nucleus of the amygdala (CeA) encompasses the main output pathways of the amygdala, a temporal lobe structure essential in affective and cognitive dimensions of pain. A major population of neurons in the CeA send projections to the periaqueductal gray (PAG), a key midbrain structure that mediates coping strategies in response to threat or stress. CeA-PAG neurons are topographically organized based on their targeted subregion within the PAG. PAG-projecting neurons in the central medial (CeM) and central lateral (CeL) regions of CeA are intrinsically distinct. CeL-PAG neurons are a homogeneous population of intrinsically distinct neurons while CeM-PAG neurons are intrinsically heterogeneous. Membrane properties of distinct CeM-PAG subtypes are altered in the complete Freund's adjuvant model of inflammatory pain. A major population of neurons in the central nucleus of amygdala (CeA) send projections to the periaqueductal gray (PAG), a key midbrain structure that mediates coping strategies in response to threat or stress. While the CeA-PAG pathway has proved to be a component of descending anti-nociceptive circuitry, the functional organization of CeA-PAG neurons remains unclear. We identified CeA-PAG neurons in C57BL/6 mice of both sexes using intracranial injection of a fluorescent retrograde tracer into the PAG. In acute brain slices, we investigated the topographical and intrinsic characteristics of retrogradely labelled CeA-PAG neurons using epifluorescence and whole-cell electrophysiology. We also measured changes to CeA-PAG neurons in the complete Freund's adjuvant (CFA) model of inflammatory pain. Neurons in the central lateral (CeL) and central medial (CeM) amygdala project primarily to different regions of the PAG. CeL-PAG neurons consist of a relatively homogeneous population of intrinsically distinct neurons while CeM-PAG neurons are intrinsically heterogeneous. Membrane properties of distinct CeM-PAG subtypes are altered 1day after induction of the CFA inflammatory pain model. Collectively, our results provide insight into pain-induced changes to a specific population of CeA neurons that probably play a key role in the integration of noxious input with endogenous analgesia and behavioural coping response.

Validating Rat Model of Empathy for Pain: Effects of Pain Expressions in Social Partners.

Pain can be socially transferred between familiar rats due to empathic responses. To validate rat model of empathy for pain, effects of pain expressions in a cagemate demonstrator (CD) in pain on empathic pain responses in a naïve cagemate observer (CO) after 30 min priming dyadic social interactions (PDSI) were evaluated. The CD rats were prepared with four pain models: bee venom (BV), formalin, complete Freund's adjuvant (CFA), and spared nerve injury (SNI). Both BV and formalin tests are characterized by displayable and eye-identifiable spontaneous pain-related behaviors (SPRB) immediately after treatment, while CFA and SNI models are characterized by delayed occurrence of evoked pain hypersensitivity but with less eye-identifiable SPRB. After 30 min PDSI with a CD immediately after BV and formalin, respectively, the empathic mechanical pain hypersensitivity (EMPH) could be identified at both hind paws in CO rats. The BV—or formalin-induced EMPH in CO rats lasted for 4–5 h until full recovery. However, EMPH failed to develop in CO after socially interacting with a CD immediately after CFA, or 2 h after BV when SPRB completely disappeared. The CO's EMPH was partially relieved when socially interacting with an analgecized CD whose SPRB had been significantly suppressed. Moreover, repeated exposures to a CD in pain could enhance EMPH in CO. Finally, social transfer of pain hypersensitivity was also identified in CO who was being co-housed in pairs with a conspecific treated with CFA or SNI. The results suggest that development of EMPH in CO rats would be determined not only by extent of familiarity but also by visually identifiable pain expressions in the social partners during short period of PDSI. However, the visually unidentifiable pain can also be transferred to naïve cagemate when being co-housed in pairs with a distressed conspecific. In summary, the vicariously social contagion of pain between familiar rats is dependent upon not only expressions of pain in social partners but also the time that dyads spent in social communications. The rat model of empathy for pain is a highly stable, reproducible and valid model for studying the neural mechanisms of empathy in lower animals.

Anti-inflammatory and anti-arthritic effects of methanol extract of the stem bark of Boswellia dalzielii Hutch (Burseraceae) in rats.

Boswellia dalzielii is a tall tree (more than 13 m high) that produces aromatic white flowers. This plant is commonly used in indigenous medicine across Africa against diarrhea, malaria, vomiting, inflammation and arthritis. The present study focuses on the anti-inflammatory and anti-arthritis potential of methanol extract of Boswellia dalzielii (BDME). Anti-inflammatory activity was evaluated in inflammatory models induced by carrageenan, arachidonic acid, histamine, serotonin, prostaglandin and bradykinin. Anti-arthritis activity was measured using complete Freund's adjuvant model. Intracellular and extracellular ROS production and proliferation of T-cells were evaluated using chemiluminescence and liquid scintillation counter techniques, respectively. TNF-α and IL-1β production were assessed using ELISA and MTT assay performed for cytotoxicity. BDME revealed a significant anti-inflammatory effect by preventing the development of edema caused by carrageenan, arachidonic acid, histamine, serotonin, prostaglandin and bradykinin. For anti-arthritic properties of BDME, the results showed a significant reduction of the joint diameter and a decrease in pain in the treated animals. The extract also showed a noticeable systemic effect, maintaining the values of the evaluated parameters close to normal in treated rats with an inhibition of joint destruction as shown in histopathological analysis. Furthermore, BDME exhibited significant inhibition of extracellular and intracellular ROS production. Still, the extract displayed significant inhibitory activity on T-cell proliferation as well as a reduced production of TNF-α and IL-1β. Boswellia dalzielii could be considered as a promising tract in the prevention and/or management of inflammatory diseases.