Rat Model Of Inflammatory Pain Research Articles

Signaling Mechanism of Cannabinoid Receptor-2 Activation-Induced β-Endorphin Release.

Activation of cannabinoid receptor-2 (CB2) results in β-endorphin release from keratinocytes, which then acts on primary afferent neurons to inhibit nociception. However, the underlying mechanism is still unknown. The CB2 receptor is generally thought to couple to Gi/o to inhibit cAMP production, which cannot explain the peripheral stimulatory effects of CB2 receptor activation. In this study, we found that in a keratinocyte cell line, the Gβγ subunits from Gi/o, but not Gαs, were involved in CB2 receptor activation-induced β-endorphin release. Inhibition of MAPK kinase, but not PLC, abolished CB2 receptor activation-induced β-endorphin release. Also, CB2 receptor activation significantly increased intracellular Ca(2+). Treatment with BAPTA-AM or thapsigargin blocked CB2 receptor activation-induced β-endorphin release. Using a rat model of inflammatory pain, we showed that the MAPK kinase inhibitor PD98059 abolished the peripheral effect of the CB2 receptor agonist on nociception. We thus present a novel mechanism of CB2 receptor activation-induced β-endorphin release through Gi/o-Gβγ-MAPK-Ca(2+) signaling pathway. Our data also suggest that stimulation of MAPK contributes to the peripheral analgesic effect of CB2 receptor agonists.

Anti-hyperalgesic effects of imidazoline I2 receptor ligands in a rat model of inflammatory pain: interactions with oxycodone.

Emerging preclinical evidence suggests that imidazoline I2 receptor ligands may be effective analgesics. Quantitative analysis of the combined I2 receptor ligands and opioids is needed for the justification of combination therapy. This study systematically examined the anti-hyperalgesic and response rate-suppressing effects of selective I2 receptor ligands (2-BFI and phenyzoline) alone and in combination with oxycodone in rats. Von Frey filament test was used to examine the anti-hyperalgesic effects of drugs in a rat model of complete Freund's adjuvant (CFA)-induced inflammatory pain. Schedule-controlled responding was used to assess the rate-altering effects of study drugs. Duration of actions of individual drugs (2-BFI, phenyzoline, and oxycodone) alone or in combination was studied. Dose-addition analysis was employed to assess the anti-hyperalgesic interactions between drugs. Oxycodone (0.1-3.2 mg/kg, i.p.), 2-BFI (1-17.8 mg/kg, i.p.), and phenyzoline (17.8-56 mg/kg, i.p.) all dose-dependently produced significant antinociceptive effects. When studied as combinations, 2-BFI and oxycodone produced additive interactions while phenyzoline and oxycodone produced supra-additive interactions under all fixed ratios. The same drug combinations did not alter or significantly reduced the operant responding depending on the ratios of the drug combinations. Quantitative analysis of the anti-hyperalgesic effects of I2 receptor ligands strongly supports the therapeutic potential of I2 receptor ligands against inflammatory pain. In addition, the data reveal that phenyzoline is superior to the prototypic I2 receptor ligand 2-BFI for the management of pain and warrants further consideration as a novel analgesic.

Microglial interleukin-1β in the ipsilateral dorsal horn inhibits the development of mirror-image contralateral mechanical allodynia through astrocyte activation in a rat model of inflammatory pain.

Damage on one side of the body can also result in pain on the contralateral unaffected side, called mirror-image pain (MIP). Currently, the mechanisms responsible for the development of MIP are unknown. In this study, we investigated the involvement of spinal microglia and interleukin-1β (IL-1β) in the development of MIP using a peripheral inflammatory pain model. After unilateral carrageenan injection, mechanical allodynia (MA) in both hind paws and the expression levels of spinal Iba-1, IL-1β, and GFAP were evaluated. Ipsilateral MA was induced beginning at 3 hours after carrageenan injection, whereas contralateral MA showed a delayed onset occurring 5 days after injection. A single intrathecal (i.t.) injection of minocycline, a tetracycline derivative that displays selective inhibition of microglial activation, or an interleukin-1 receptor antagonist (IL-1ra) on the day of carrageenan injection caused an early temporary induction of contralateral MA, whereas repeated i.t. treatment with these drugs from days 0 to 3 resulted in a long-lasting contralateral MA, which was evident in its advanced development. We further showed that IL-1β was localized to microglia and that minocycline inhibited the carrageenan-induced increases in spinal Iba-1 and IL-1β expression. Conversely, minocycline or IL-1ra pretreatment increased GFAP expression as compared with that of control rats. However, i.t. pretreatment with fluorocitrate, an astrocyte inhibitor, restored minocycline- or IL-1ra-induced contralateral MA. These results suggest that spinal IL-1β derived from activated microglia temporarily suppresses astrocyte activation, which can ultimately prevent the development of contralateral MA under inflammatory conditions. These findings imply that microglial IL-1β plays an important role in regulating the induction of inflammatory MIP.

In vivo activation of the SK channel in the spinal cord reduces the NMDA receptor antagonist dose needed to produce antinociception in an inflammatory pain model.

N-methyl-D-aspartate receptor (NMDAR) antagonists have been shown to reduce mechanical hypersensitivity in animal models of inflammatory pain. However, their clinical use is associated with significant dose-limiting side effects. Small-conductance Ca-activated K channels (SK) have been shown to modulate NMDAR activity in the brain. We demonstrate that in vivo activation of SK channels in the spinal cord can alleviate mechanical hypersensitivity in a rat model of inflammatory pain. Intrathecal (i.t.) administration of the SK channel activator, 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309), attenuates complete Freund adjuvant (CFA)-induced mechanical hypersensitivity in a dose-dependent manner. Postsynaptic expression of the SK channel subunit, SK3, and apamin-sensitive SK channel-mediated currents recorded from superficial laminae are significantly reduced in the dorsal horn (DH) after CFA. Complete Freund adjuvant-induced decrease in SK-mediated currents can be reversed in vitro by bath application of NS309. In addition, immunostaining for the SK3 subunit indicates that SK3-containing channels within DH neurons can have both somatic and dendritic localization. Double immunostaining shows coexpression of SK3 and NMDAR subunit, NR1, compatible with functional interaction. Moreover, we demonstrate that i.t. coadministration of NS309 with an NMDAR antagonist reduces the dose of NMDAR antagonist, DL-2-amino-5-phosphonopentanoic acid (DL-AP5), required to produce antinociceptive effects in the CFA model. This reduction could attenuate the unwanted side effects associated with NMDAR antagonists, giving this combination potential clinical implications.

Anti‐hyperalgesic Effects of Imidazoline I2 Receptor Ligands and their Interactions with Opioids in a Rat Model of Inflammatory Pain

Pain affects > 30% of the U.S. population and currently available analgesics are not adequate to meet the clinical needs. Opioids are effective for treating moderate to severe pain; however, their use is limited due to the unwanted effects. We recently found that imidazoline I2 receptor ligands produce antinociceptive effects in rat models of chronic pain and also enhance the antinociceptive effects of morphine. However, little is known of their interaction with other opioids. This study examined the anti‐hyperalgesic effects of selective I2 receptor ligands (2‐BFI and phenyzoline) alone and in combination with oxycodone using the von Frey filament test in rats with complete Freund's adjuvant (CFA)‐induced inflammatory pain. In CFA‐treated rats, 2‐BFI (1‐10 mg/kg, i.p.) and phenyzoline (17.8‐56 mg/kg, i.p.) dose‐dependently produced significant antinociceptive effects. When studied as combinations, 2‐BFI and oxycodone produced additive interactions, while phenyzoline and oxycodone produced synergistic interactions for their effects against mechanical hyperalgesia in CFA‐treated rats. Taken together, these studies suggest that imidazoline I2 receptor ligands alone have anti‐hyperalgesic effects and also enhance the effects of opioids in a rat model of chronic pain. Therefore, drugs acting on the imidazoline I2 receptor may represent a novel class of pharmacotherapy for the management of chronic pain.

Complete Freund's adjuvant-induced reduction of exploratory activity in a novel environment as an objective nociceptive endpoint for sub-acute inflammatory pain model in rats.

Hind paw injection of complete Freund's adjuvant (CFA) is a commonly used sub-acute inflammatory pain model in rodents with typical subjective endpoint measurements of paw withdrawal to thermal or mechanical stimuli. Here, we assessed CFA-induced reduction of exploratory activity in a novel environment (CRANE) as an objective nociceptive endpoint in rats. CFA (50%) was subcutaneously injected into the plantar aspect of the hind paw either unilaterally or bilaterally (150μL/paw). Exploratory activity was recorded using an automated locomotor activity system. Bilateral CFA injection reduced exploratory activity 4-48h following injection, compared to sham controls. Unilateral CFA injection produced less reduction of exploratory activity, compared to bilateral injection. Effects of orally dosed standard analgesics on CRANE were examined 48h following bilateral CFA injection. Diclofenac treatment produced dose-related reversal of CRANE at 0.03-1.0mg/kg with a plateau effect observed at higher doses (up to 30mg/kg). Ibuprofen also produced dose-related reversal CRANE at 0.3-3.0mg/kg with a plateau effect at higher doses (up to 60mg/kg). Similarly, celecoxib produced dose-related reversal CRANE at 3-10mg/kg, but not 30mg/kg. Gabapentin (up to 100mg/kg) and duloxetine (up to 30mg/kg) produced no reversal of CRANE. The results presented here demonstrate that CRANE provides an objective assessment of pain behaviours for sub-acute inflammatory pain in rats. The pharmacological profile of standard analgesics supports that CRANE model may potentially be used to identify novel analgesic agents for the treatment of sub-acute inflammatory pain.

AS1069562, the (+)-isomer of indeloxazine, exerts analgesic effects in rat models of nociceptive pain

Objectives:The (+)-isomer of indeloxazine AS1069562 has multiple pharmacological actions, such as serotonin (5-HIT) and norepinephrine (NE) reuptake inhibition and analgesic effects in animal models of neuropathic pain. Here, we investigated the analgesic effects of AS1069562 in rat models of inflammatory and noninflammatory nociceptive pain.Methods:Adjuvant-induced arthritis (AIA) and bradykinin-induced knee joint pain were used as rat models of inflammatory pain. The chronic phase of monoiodoacetate-induced arthritis (MIA) was used as a rat model of noninflammatory pain. Analgesic effects were evaluated by weight-bearing deficit in the AIA and MIA models and by pain response in the bradykinin-induced knee joint pain model.Results:In the AIA model and the bradykinin-induced knee joint pain model, AS1069562 significantly ameliorated the pain-related behavior of weight-bearing deficit and the pain response, respectively. AS1069562 also significantly improved the pain-related behavior of weight-bearing deficit in the chronic phase of the MIA model. Further, following monoiodoacetate injection, repeated administration of AS1069562 or duloxetine significantly improved weight-bearing deficit in the MIA model. Interestingly, the analgesic effect of AS1069562 was sustained for 24 hours after the last administration, although the plasma concentration of AS1069562 was reduced to undetectable levels. In contrast, the analgesic effect of duloxetine did not continue after treatment discontinuation.Discussion:AS1069562 exerts analgesic effects on inflammatory and noninflammatory nociceptive pain in rat models of arthritis pain, and repeated administration of AS1069562 exerts a more persistent analgesic effect on arthritis pain than duloxetine. These findings suggest that AS1069562 has an attractive analgesic profile for the treatment of nociceptive pain.

WITHDRAWN: Intrathecal siRNA against Cyr61 attenuates nociception in a rat model of inflammatory pain

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Micronized/ultramicronized palmitoylethanolamide displays superior oral efficacy compared to nonmicronized palmitoylethanolamide in a rat model of inflammatory pain.

BackgroundThe fatty acid amide palmitoylethanolamide (PEA) has been studied extensively for its anti-inflammatory and neuroprotective actions. The lipidic nature and large particle size of PEA in the native state may limit its solubility and bioavailability when given orally, however. Micronized formulations of a drug enhance its rate of dissolution and reduce variability of absorption when orally administered. The present study was thus designed to evaluate the oral anti-inflammatory efficacy of micronized/ultramicronized versus nonmicronized PEA formulations.MethodsMicronized/ultramicronized PEA was produced by the air-jet milling technique, and the various PEA preparations were subjected to physicochemical characterization to determine particle size distribution and purity. Each PEA formulation was then assessed for its anti-inflammatory effects when given orally in the carrageenan-induced rat paw model of inflammation, a well-established paradigm of edema formation and thermal hyperalgesia.ResultsIntraplantar injection of carrageenan into the right hind paw led to a marked accumulation of infiltrating inflammatory cells and increased myeloperoxidase activity. Both parameters were significantly decreased by orally given micronized PEA (PEA-m; 10 mg/kg) or ultramicronized PEA (PEA-um; 10 mg/kg), but not nonmicronized PeaPure (10 mg/kg). Further, carrageenan-induced paw edema and thermal hyperalgesia were markedly and significantly reduced by oral treatment with micronized PEA-m and ultramicronized PEA-um at each time point compared to nonmicronized PeaPure. However, when given by the intraperitoneal route, all PEA formulations proved effective.ConclusionsThese findings illustrate the superior anti-inflammatory action exerted by orally administered, micronized PEA-m and ultramicronized PEA-um, versus that of nonmicronized PeaPure, in the rat paw carrageenan model of inflammatory pain.

Electroacupuncture attenuates mechanical allodynia by suppressing the spinal JNK1/2 pathway in a rat model of inflammatory pain.

Electroacupuncture (EA) has a substantial analgesic effect on inflammatory pain induced by complete Freund's adjuvant (CFA). The activation of the c-Jun N-terminal kinase 1/2 (JNK1/2) signal transduction pathway in the spinal cord is associated with inflammatory pain. However, the relationship between EA's analgesic effect and the JNK1/2 signal transduction pathway in the inflammatory pain remain unclear. In the present study, we used the established rat model of CFA-induced inflammatory pain to investigate the role of the spinal JNK1/2 pathway in EA-mediated analgesia. We observed a decrease in paw withdrawal thresholds and an increase in paw edema at 1 and 3 days after injecting CFA into the right hindpaw. CFA, 3 days after injection, upregulated expression of phospho-c-Jun N-terminal kinase1/2 (p-JNK1/2) protein and its downstream targets, the transcriptional regulators p-c-Jun and activator protein-1 (AP-1), as well as cyclooxygenase-2 (COX-2) and the transient receptor potential vanilloid 1 (TRPV1). EA significantly alleviated CFA-induced inflammatory pain. In addition, EA reduced p-JNK1/2 protein levels and COX-2 mRNA expressions, a degree of down-regulated p-c-Jun protein level and AP-1 DNA binding activity in the spinal dorsal horn of CFA-administered animals, but it had no effect on TRPV1 mRNA expression. Furthermore, EA and the JNK inhibitor SP600125 synergistically inhibited CFA-induced hyperalgesia and suppressed the COX-2 mRNA expression in the spinal dorsal horn. Our findings indicate that EA alleviates inflammatory pain behavior, at least in part, by reducing COX-2 expression in the spinal cord via the JNK1/2 signaling pathway. Inactivation of the spinal JNK1/2 signal transduction pathway maybe the potential mechanism of EA's antinociception in the inflammatory pain model.

A Novel Compound Targeting Kv7.2/3 Channels Relieves Inflammatory and Neuropathic Pain

Encoded by Kv7.2/Kv7.3 subunit assembly, M-channels generate sub-threshold voltage-gated K+ currents that regulate neuronal excitability. Inour quest to target neuronal M-channels, we previously designed noveldiphenylamine carboxylates that are powerful Kv7.2 channel openers. These drugs stabilize the Kv7.2 channel open state by interacting with its voltage sensing domain (VSD). Using the same structural framework, we designed a novel molecule, NH34, to test its activity on neuronal excitability and probe its potency in vivo on inflammatory and neuropathic pain. In transfected CHO cells, NH34 significantly increased Kv7.2 currents, by producing a hyperpolarizing voltage shift of the activation curve (EC50 = 17.5 µM). Interestingly, two S4 mutants, R198A and R207W significantly decreased the opener potency of the drug, suggesting that NH34, like NH29, may interact with the VSD of Kv7.2. NH34 preferentially acts as an opener of Kv7.2 and Kv7.3 channels. NH34 was ineffective on Kv7.4 and Kv7.5 but produced a significant inhibition of TRPV1 cation channels. In CA1 pyramidal neurons from rat hippocampal slices, NH34 reduced the spike discharge frequency evoked by injection of depolarizing current. NH34 significantly depressed the slope of field epsp and the fiber volley amplitude recorded extracellularly in the CA1 pyramidal region following stimulation of the Schaeffer collaterals. NH34 reduced the frequency of evoked spike discharge in dorsal root ganglion neurons. In vivo, NH34 (3-30 mg/kg p.o) significantly relieved the hyperalgesia evoked by mechanical pressure of the paw in a rat model of inflammatory pain. Similarly, NH34 (3-30 mg/kg p.o) was very efficient in alleviating the hyperalgesia evoked by mechanical pressure or cold allodynia in a Seltzer rat model of neuropathic pain. In all, the data suggest that NH34 is a very promising drug template for treating inflammatory and neuropathic pain.

Dual effects of Rho-kinase inhibitors on a rat model of inflammatory pain.

Rho-kinases (ROCKs), a family of small GTP-dependent enzymes, are involved in a range of pain models, and their inhibition typically leads to antinociceptive effects. To study the effects of inhibiting ROCKs using two known inhibitors, Y27632 and HA1077 (fasudil), administered locally, on nociception and paw edema in rats. A range of doses of Y27632 or HA1077 (2.5 μg to 1000 μg) were injected locally into rat paws alone or in combination with carrageenan, a known proinflammatory stimulus. Nociceptive responses to mechanical stimuli and increased paw volume, reflecting edema formation, were measured at 2 h and 3 h, using a Randall-Selitto apparatus and a hydroplethysmometer, respectively. Animals treated with either ROCK inhibitor showed biphasic nociceptive effects, with lower doses being associated with pronociceptive, and higher doses with antinociceptive responses. In contrast, a monophasic dose-dependent increase in edema was observed in the same animals. Local injection of 8-bromo-cyclic (c)GMP, an activator of the nitric oxide⁄cGMP⁄protein kinase G pathway, also produced biphasic effects on nociceptive responses in rat paws; however, low doses were antinociceptive and high doses were pronociceptive. Local administration of cytochalasin B, an inhibitor of actin polymerization and a downstream mediator of ROCK activity, reversed the antinociceptive effect of Y27632. The results of the present study suggest that ROCKs participate in the local mechanisms associated with nociception⁄antinociception and inflammation, with a possible involvement of the nitric oxide⁄cGMP⁄protein kinase G pathway. Also, drug effects following local administration may differ markedly from the effects following systemic administration. Finally, separate treatment of pain and edema may be needed to maximize clinical benefit in inflammatory pain.

Synthetic self-assembling clostridial chimera for modulation of sensory functions.

Clostridial neurotoxins reversibly block neuronal communication for weeks and months. While these proteolytic neurotoxins hold great promise for clinical applications and the investigation of brain function, their paralytic activity at neuromuscular junctions is a stumbling block. To redirect the clostridial activity to neuronal populations other than motor neurons, we used a new self-assembling method to combine the botulinum type A protease with the tetanus binding domain, which natively targets central neurons. The two parts were produced separately and then assembled in a site-specific way using a newly introduced ‘protein stapling’ technology. Atomic force microscopy imaging revealed dumbbell shaped particles which measure ∼23 nm. The stapled chimera inhibited mechanical hypersensitivity in a rat model of inflammatory pain without causing either flaccid or spastic paralysis. Moreover, the synthetic clostridial molecule was able to block neuronal activity in a defined area of visual cortex. Overall, we provide the first evidence that the protein stapling technology allows assembly of distinct proteins yielding new biomedical properties.

DAMGO in the central amygdala alleviates the affective dimension of pain in a rat model of inflammatory hyperalgesia

Pain has sensory-discriminative and emotional-affective dimensions. Recent studies show that the affective component can be assessed with a conditioned place avoidance (CPA) test. We hypothesized that systemic morphine before a post-conditioning test would more potently attenuate the affective aspect compared to the sensory component and that [d-Ala2-N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO), a μ-selective opioid receptor agonist, injected into the central nucleus of the amygdala (CeA) would reduce established CPA. A rat model of inflammatory pain, produced by a complete Freund adjuvant (CFA) injection into the hind paw, was combined with a CPA test. Three experiments were performed on adult male Sprague–Dawley rats. Systemic morphine (0.5 or 1.0mg/kg) in Experiment 1, intrathecal (i.t.) morphine (2.5μg/rat) in Experiment 2, and intra-CeA DAMGO (7.7–15.4ng/0.4μl) in Experiment 3 were given to CFA-injected rats (n=6–8/group) prior to a post-conditioning test. Saline-injected rats were used as control. Time spent in a pain-paired compartment was recorded twice, before conditioning and after a post-conditioning test. Paw withdrawal latency (PWL) to a noxious thermal stimulus was measured before experiment at day-1 and after the post-conditioning test; hyperalgesia was defined as a decrease in PWL. The data showed that CFA-injected rats had significantly negative CPA compared to those of saline-injected rats (P<0.05). Low-dosage systemic morphine significantly (P<0.05) reduced CFA-induced CPA but had no effect on PWL. I.t. morphine did not inhibit the display of CPA but significantly increased PWL, suppressing hyperalgesia (P<0.05). Intra-CeA DAMGO significantly inhibited the display of CPA compared to saline (P<0.05) but had no effect on PWL. The data demonstrate that morphine attenuates the affective component more powerfully than it does the sensory and suggests that the sensory and the emotional-affective dimensions are underpinned by different mechanisms.

The K + channel GIRK2 is both necessary and sufficient for peripheral opioid‐mediated analgesia

The use of opioid agonists acting outside the central nervous system (CNS) is a promising therapeutic strategy for pain control that avoids deleterious central side effects such as apnea and addiction. In human clinical trials and rat models of inflammatory pain, peripherally restricted opioids have repeatedly shown powerful analgesic effects; in some mouse models however, their actions remain unclear. Here, we investigated opioid receptor coupling to K+ channels as a mechanism to explain such discrepancies. We found that GIRK channels, major effectors for opioid signalling in the CNS, are absent from mouse peripheral sensory neurons but present in human and rat. In vivo transgenic expression of GIRK channels in mouse nociceptors established peripheral opioid signalling and local analgesia. We further identified a regulatory element in the rat GIRK2 gene that accounts for differential expression in rodents. Thus, GIRK channels are indispensable for peripheral opioid analgesia, and their absence in mice has profound consequences for GPCR signalling in peripheral sensory neurons.GIRK channels are indispensable for peripheral opioid analgesia. The absence of GIRK channels from mouse dorsal root ganglion neurons questions the predictive validity of mice as a model organism for investigating peripheral GPCRmediated analgesia.

Peripheral antinociceptive efficacy and potency of a novel opioid compound 14-O-MeM6SU in comparison to known peptide and non-peptide opioid agonists in a rat model of inflammatory pain

This study compared the peripheral analgesic effects of a novel opioid agonist 14-O-methylmorphine-6-O-sulfate (14-O-MeM6SU), to that of non-peptide (morphine, fentanyl) and peptide opioid agonists (Met-enkephalin; met-ENK and β-endorphin; β-END) in a model of localized inflammatory pain evoked by intraplantar (i.pl.) Freund's complete adjuvant (FCA). Nociceptive responses to local opioid agonists were measured by pressure paw-withdrawal procedures. In addition, the antinociceptive efficacy and potency of these test compounds in vivo was compared to that in vitro using the rat vas deferens (RVD) bioassay. Intraplantar 14-O-MeM6SU (0.32–2.53nmol/rat), morphine (14.95–112.15nmol/rat), fentanyl (0.19–2.36nmol/rat), met-ENK (0.10–10nmol/rat) and β-END (0.77–5.00nmol/rat) dose dependently increased paw pressure thresholds exclusively in inflamed hindpaws. At higher doses analgesic effects were also seen in noninflamed paws for 14-O-MeM6SU, morphine and fentanyl but not for met-ENK or β-END. The maximal possible local analgesic effect (%) measured in inflamed paws was 50.6±2.7, 18.23±1.78, 37.44±2.17, 36.00±1.43, and 40.69±0.91 for 14-O-MeM6SU, morphine, fentanyl, met-ENK and β-END, respectively. Interestingly, i.pl. administered opioid peptides met-ENK and β-END displayed a peripheral analgesic ceiling effect. This local antinociception was antagonized by co-administered opioid antagonist naloxone-methiodide (NAL-M). Similar to the analgesic testing, the RVD showed the following efficacy order of the test compounds: 14-O-MeM6SU>β-END>fentanyl>met-ENK⪢morphine. Taken together, 14-O-MeM6SU was more potent than morphine, fentanyl and met-ENK and β-END and displayed superiority in the maximum antinociceptive effects. The superiority of local antinociceptive effects of 14-O-MeM6SU might be due to both pharmacodynamic and pharmacokinetic factors.

A selective CCR2 antagonist showed monocytes migration-independent analgesia in rat models of inflammatory pain

Purpose: The aim of this study is to demonstrate whether the Chemokine C-C motif (CCR2) receptor is involved in inflammatory pain and which mechanisms could describe its physiological contribution in pain pathways. Methods: Compound A was tested for in vitro properties in native cells (rat whole blood assay) and recombinant cells lines (CHO cells expressing rat CCR2). Binding assay was performed in rat CCR2 receptor membrane (RMB) while functional assays were rat whole blood monocytes shape change and calcium mobilization assay in CHO cell line. Selectivity over CCR5 was investigated by binding assay on CHO cells expressing rat CCR5. To assess the in vivo profile of compound A, adult (200-275 g) male Wistar-Han (Charles River, Germany) rats undergone intraplantar injection of complete Freund's adjuvant (CFA, 25ug/ul by Sigma Aldrich) or intraarticular injection with 1 mg of monosodium iodoacetate (MIA, 50 ul by Sigma Aldrich) for induction of paw inflammatory pain or osteoarthritis (OA), respectively. Analgesic properties of compound A were assessed by using paw pressure test (Randall Selitto test, UgoBasile, Italy) at 24 hrs post CFA injection and by Incapacitance tester (manufactured by Boehringer-Ingelheim) the weight bearing (WB) deficit was assessed at 3 days post MIA injection. In addition anti-edema properties were investigated in the CFA model measuring changes in the paw volume as assessed by pletysmography (UgoBasile, Italy). Tissues were collected from injured paws or joints, for further histological examination. All experimental procedures were approved by the ethics committee and the Regierungspräsidium Tübingen (VVH 06-007). Results: In rat compound A is a selective CCR2 antagonist with Ki in rat RMB of 3.6 nM, with no binding properties to the closest homologous CCR5 (Ki=2920 nM). In addition, it reversed monocytes shape changes (IC50 4.3 nM) and inhibited Calcium mobilization in CHO cells (IC50=1.4 nM) proving to be a full antagonist devoid of any agonistic activity. In the CFA model, Compound A showed a dose dependent anti-hyperalgesic effect after acute treatment. The highest dose of 10 mg/kg, orally (PO) achieved full effect comparable to celecoxib (30 mg/kg, PO) while the minimal effective dose was measured at 1 mg/kg, PO. Moreover, unlike celecoxib repeated doses of compound A did not prevent edema formation in the CFA model. In the MIA model compound A, like celecoxib, showed no effect in the WB deficit when dosed acutely, however it significantly reversed pain-like behaviour when given by subchronic treatment. Furthermore histological findings revealed that compound A has no effect in reducing monocytes migration in the CFA paw or MIA joint. Conclusions: Overall our data support a CCR2 antagonist to have the potential to provide analgesia in nociceptive pain condition. In addition preclinical data suggest that analgesic properties of CCR2 antagonists in pain inflammatory models can not be attributed to blockage of monocytes migration.

Regulation of the β-endorphin precursor proopiomelanocortin in lymphocytes in a rat model of inflammatory pain

Our previous studies have shown that monocytes/macrophages, granulocytes, and lymphocytes release opioid peptides in the presence of different releasing factors at the site of peripheral tissue injury or inflammation and thereby contribute to opioid-mediated reduction of pain. We are interested in the regulation of β-endorphin and its precursor proopiomelanocortin (POMC) at the transcriptional and posttranslational level in lymphocytes, as such cells play a role in chronic inflammation. We hypothesized that (i) POMC gene expression in the lymph node draining inflamed tissue is up-regulated in inflammation, and (ii) that the precursor is cleaved by the prohormone convertases (PC)1 and PC2 similar to neuroendocrine cells in the pituitary gland. We found elevated POMC gene expression and β-endorphin levels in cells of lymph nodes draining inflamed tissue, as assessed by qPCR and radioimmunoassay. Our PCR and immunocytochemistry data show that PC1 mRNA and protein expression, respectively, are induced after induction of inflammation. PC2 required no stimulation and was detectable using PCR and immunocytochemistry, both, in cells from nai¨ve and activated lymph nodes. Our findings indicate that β-endorphin synthesis in lymphocytes may depend on the presence of PC1/3, while PC2 alone does not seem to be sufficient. Functional experiments are necessary to verify the specific roles of these enzymes for POMC processing in cells of the immune system.

Changes in expression of Nogo-A protein in dorsal root ganglion and spinal dorsal horn in a rat model of inflammatory pain

Objective To evaluate the changes in the expression of Nogo-A protein in the dorsal root ganglion (DRG) and spinal dorsal horn in a rat model of inflammatory pain.Methods One hundred and twenty male Sprague-Dawley rats,weighing 250-300 g,were randomly divided into 2 groups (n =60 each):control group (group C) and formalin group (group F).The inflammatory pain was induced by injection of 3％ formalin 100 μl into the plantar surface of left hindpaw in group F.The equal volume of normal saline was given instead of formalin in group C.Mechanical withdrawal threshold to yon Frey filament stimulation was measured at 1 h and 1,2,3 and 7 days after injection.Twelve rats in each group were chosen at each time point and sacrificed.The L5 DRG and L4,5 segment of spinal cord on the operated side were removed for determination of Nogo-A protein expression by immunofluorescence and Western blot.Remlts Compared with group C,the mechanical withdrawal threshold was significantly decreased at 1 h,1,2,3 and 7 days after injection,and the expression of Nogo-A protein in the DRG and L4,5 segment of spinal cord was up-regulated at 1 h and 2,3 and 7 days after injection in group F (P ＜0.05).Conclusion Up-regulation of Nogo-A protein in the DRG and spinal dorsal horn may play an important role in the development of formalin-induced inflammatory pain in rats. Key words: Infammation; Pain; Spinal cord; Ganglia, spinal; Nogo-A protein

Intervention of Electroacupuncture on Spinal p38 MAPK/ATF-2/VR-1 Pathway in Treating Inflammatory Pain Induced by CFA in Rats

BackgroundPrevious studies have demonstrated that p38 MAPK signal transduction pathway plays an important role in the development and maintenance of inflammatory pain. Electroacupuncture (EA) can suppress the inflammatory pain. However, the relationship between EA effect and p38 MAPK signal transduction pathway in inflammatory pain remains poorly understood. It is our hypothesis that p38 MAPK/ATF-2/VR-1 and/or p38 MAPK/ATF-2/COX-2 signal transduction pathway should be activated by inflammatory pain in CFA-injected model. Meanwhile, EA may inhibit the activation of p38 MAPK signal transduction pathway. The present study aims to investigate that anti-inflammatory and analgesic effect of EA and its intervention on the p38 MAPK signal transduction pathway in a rat model of inflammatory pain.ResultsEA had a pronounced anti-inflammatory and analgesic effect on CFA-induced chronic inflammatory pain in rats. EA could quickly raise CFA-rat’s paw withdrawal thresholds (PWTs) and maintain good and long analgesic effect, while it subdued the ankle swelling of CFA rats only at postinjection day 14. EA could down-regulate the protein expressions of p-p38 MAPK and p-ATF-2, reduced the numbers of p-p38 MAPK-IR cells and p-ATF-2-IR cells in spinal dorsal horn in CFA rats, inhibited the expressions of both protein and mRNA of VR-1, but had no effect on the COX-2 mRNA expression.ConclusionsThe present study indicates that inhibiting the activation of spinal p38 MAPK/ATF-2/VR-1 pathway may be one of the main mechanisms via central signal transduction pathway in the process of anti-inflammatory pain by EA in CFA rats.