Complete Freund's Adjuvant-induced Inflammation Research Articles

Study of PT1 Peptide Analgesic and Anti-Inflammatory Activity on a Local Inflammation Model in Mice CD-1.

PT1 peptide isolated from the venom of spider Geolycosa sp. is a modulator of P2X3 receptors that play a role in the development of inflammation and the transmission of pain impulses. The anti-inflammatory and analgesic efficacy of the PT1 peptide was studied in a model of complete Freund's adjuvant-induced paw inflammation in CD-1 mice. The analgesic activity of PT1 peptide was maximum after intramuscular injection at a dose of 0.01 mg/kg, which surpassed the analgesic effect of diclofenac at a dose of 1 mg/kg. The anti-inflammatory activity was maximum after intramuscular injection at a dose of 0.0001 mg/kg; a decrease in paw thickness was observed as soon as 2 h after the administration of the PT1 peptide against the background of inflammation development. All tested doses of PT1 peptide showed high anti-inflammatory activity 4 and 24 h after administration. PT1 peptide at a dose of 0.01 mg/kg when injected intramuscularly simultaneously produced high anti-inflammatory and analgesic effects compared to other doses of the peptide. Increasing the dose of PT1 peptide led to a gradual decrease in its analgesic and anti-inflammatory activity; increasing the dose of intramuscular injection to 0.1 and 1 mg/kg is inappropriate.

Transgenic mice overexpressing human ALOX15 under the control of the aP2 promoter are partly protected in the complete Freund's adjuvant-induced paw inflammation model.

Arachidonic acid 15-lipoxygenase (ALOX15) has been implicated in the pathogenesis of inflammatory diseases but since pro- and anti-inflammatory roles have been suggested, the precise function of this enzyme is still a matter of discussion. To contribute to this discussion, we created transgenic mice, which express human ALOX15 under the control of the activating protein 2 promoter (aP2-ALOX15 mice) and compared the sensitivity of these gain-of-function animals in two independent mouse inflammation models with Alox15-deficient mice (loss-of-function animals) and wildtype control animals. Transgenic aP2-ALOX15 mice were tested in comparison with Alox15 knockout mice (Alox15-/-) and corresponding wildtype control animals (C57BL/6J) in the complete Freund's adjuvant induced hind-paw edema model and in the dextran sulfate sodium induced colitis (DSS-colitis) model. In the paw edema model, the degree of paw swelling and the sensitivity of the inflamed hind-paw for mechanic (von Frey test) and thermal (Hargreaves test) stimulation were quantified as clinical readout parameters. In the dextran sodium sulfate induced colitis model the loss of body weight, the colon lengths and the disease activity index were determined. In the hind-paw edema model, systemic inactivation of the endogenous Alox15 gene intensified the inflammatory symptoms, whereas overexpression of human ALOX15 reduced the degree of hind-paw inflammation. These data suggest anti-inflammatory roles for endogenous and transgenic ALOX15 in this particular inflammation model. As mechanistic reason for the protective effect downregulation of the pro-inflammatory ALOX5 pathways was suggested. However, in the dextran sodium sulfate colitis model, in which systemic inactivation of the Alox15 gene protected female mice from DSS-induced colitis, transgenic overexpression of human ALOX15 did hardly impact the intensity of the inflammatory symptoms. The biological role of ALOX15 in the pathogenesis of inflammation is variable and depends on the kind of the animal inflammation model.

Regular Aerobic Exercise Attenuates Pain and Anxiety in Mice by Restoring Serotonin-Modulated Synaptic Plasticity in the Anterior Cingulate Cortex.

ABSTRACTPurposeClinical studies found that regular aerobic exercise has analgesic and antianxiety effects; however, the underlying neural mechanisms remain unclear. Multiple studies have suggested that regular aerobic exercise may exert brain-protective effects by promoting the release of serotonin, which may be a pain modulator. Anterior cingulate cortex (ACC) is a key brain area for pain information processing, receiving dense serotonergic innervation. As a result, we hypothesized that exercise may increase the release of serotonin in the ACC, thus improving pain and anxiety behaviors.MethodsIntegrative methods were used, including behavioral, electrophysiological, pharmacological, biochemical, and genetic approaches, to explore the effects of regular aerobic exercise and the underlying neural mechanisms.ResultsRegular aerobic exercise in the form of voluntary wheel running for 30 min daily for 15 d showed significant effectiveness in relieving pain and concomitant anxiety in complete Freund’s adjuvant–induced chronic inflammation pain models. c-Fos staining and multielectrode array recordings revealed alterations in neuronal activities and synaptic plasticity in the ACC. Moreover, systemic pharmacological treatment with 4-chloro-dl-phenylalanine (PCPA) to deplete endogenous serotonin and local delivery of serotonin to the ACC revealed that exercise-related serotonin release in the ACC bidirectionally modulates pain sensitization and anxiety behaviors by modulating synaptic plasticity in the ACC. Furthermore, we found that 5-HT1A and 5-HT7 receptors mediated the serotonin modulation effects under conditions of regular aerobic exercise through local infusion of a selective antagonist and shRNA in the ACC.ConclusionsOur results reveal that regular aerobic exercise can increase serotonin release and modulate synaptic plasticity in the ACC, ultimately improving pain and concomitant anxiety behaviors through the functions of the 5-HT1A and 5-HT7 receptors.

Role of primary sensory neurone cannabinoid type-1 receptors in pain and the analgesic effects of the peripherally acting agonist CB-13 in mice

BackgroundCannabinoid type-1 receptors (CB1Rs) are expressed in primary sensory neurones, but their role in pain modulation remains unclear. MethodsWe produced Pirt-CB1R conditional knockout (cKO) mice to delete CB1Rs in primary sensory neurones selectively, and used behavioural, pharmacological, and electrophysiological approaches to examine the influence of peripheral CB1R signalling on nociceptive and inflammatory pain. ResultsConditional knockout of Pirt-CB1R did not alter mechanical or heat nociceptive thresholds, complete Freund adjuvant-induced inflammation, or heat hyperalgesia in vivo. The intrinsic membrane properties of small-diameter dorsal root ganglion neurones were also comparable between cKO and wild-type mice. Systemic administration of CB-13, a peripherally restricted CB1/CB2R dual agonist (5 mg kg−1), inhibited nociceptive pain and complete Freund adjuvant-induced inflammatory pain. These effects of CB-13 were diminished in Pirt-CB1R cKO mice. In small-diameter neurones from wild-type mice, CB-13 concentration-dependently inhibited high-voltage activated calcium current (HVA-ICa) and induced a rightward shift of the channel open probability curve. The effects of CB-13 were significantly attenuated by AM6545 (a CB1R antagonist) and Pirt-CB1R cKO. ConclusionCB1R signalling in primary sensory neurones did not inhibit nociceptive or inflammatory pain, or the intrinsic excitability of nociceptive neurones. However, peripheral CB1Rs are important for the analgesic effects of systemically administered CB-13. In addition, HVA-ICa inhibition appears to be a key ionic mechanism for CB-13-induced pain inhibition. Thus, peripherally restricted CB1R agonists could have utility for pain treatment.

Anti-Inflammatory and Antinociceptive Properties of Kielmeyerone A, a Chromenone Isolated from the Roots of Kielmeyera reticulata

Four new chromenones, kielmeyerones A-D (1-4), were obtained from the roots of Kielmeyera reticulata. Their structures were elucidated based on spectroscopic data (NMR and HRESIMS) interpretation. The pharmacological activity of kielmeyerone A (1), the major compound, was evaluated using in vitro and in vivo inflammation and pain models. During in vitro screening, 1, at noncytotoxic concentrations (0.097-1.56 μM), inhibited NO production by J774 macrophages stimulated with LPS and IFN-γ. In the complete Freund's adjuvant-induced inflammation model in mice, 1 (12.5-50 mg/kg) inhibited paw edema, demonstrating an anti-inflammatory effect. Additionally, 1 (12.5-50 mg/kg) induced a dose-dependent antinociceptive effect in the late phase of the formalin test, a profile similar to those of nonsteroidal anti-inflammatory drugs. Mice treated with 1 (100 mg/kg) did not show motor performance alterations using a rota-rod test. Thus, the present study has characterized new chromenones from Kielmeyera reticulata and has provided evidence of the anti-inflammatory and antinociceptive properties of one of these, kielmeyerone A (1).

Neonatal complete Freund's adjuvant-induced inflammation does not induce or alter hyperalgesic priming or alter adult distributions of C-fibre dorsal horn innervation.

Introduction:Inflammation during the neonatal period can exacerbate pain severity following reinjury in adulthood. This is driven by alterations in the postnatal development of spinal and supraspinal nociceptive circuitry. However, the contribution of alterations in peripheral nociceptor function remains underexplored.Objectives:We examined whether neonatal complete Freund's adjuvant (CFA)-induced inflammation induced or altered adult development of hyperalgesic priming (inflammation-induced plasticity in nonpeptidergic C fibres) or altered postnatal reorganization of calcitonin gene-related peptide (CGRP)-expressing and isolectin B4 (IB4)-binding C fibres in the spinal dorsal horn (DH).Methods:After intraplantar injection of CFA at postnatal day (P) 1, we assessed mechanical thresholds in adult (P60) rats before and after intraplantar carrageenan. One week later, intraplantar PGE2-induced hypersensitivity persisting for 4 hours was deemed indicative of hyperalgesic priming. CGRP expression and IB4 binding were examined in adult rat DH after CFA.Results:P1 CFA did not alter baseline adult mechanical thresholds, nor did it change the extent or duration of carrageenan-induced hypersensitivity. However, this was slower to resolve in female than in male rats. Rats that previously received carrageenan but not saline were primed, but P1 hind paw CFA did not induce or alter hyperalgesic priming responses to PGE2. In addition, CFA on P1 or P10 did not alter intensity or patterns of CGRP or IB4 staining in the adult DH.Conclusion:Complete Freund's adjuvant-induced inflammation during a critical period of vulnerability to injury during early postnatal development does not induce or exacerbate hyperalgesic priming or alter the broad distribution of CGRP-expressing or IB4-binding afferent terminals in the adult dorsal horn.

Uncovering the analgesic effects of a pH-dependent mu-opioid receptor agonist using a model of nonevoked ongoing pain.

Currently, opioids targeting mu-opioid receptors are the most potent drugs for acute and cancer pain. However, opioids produce adverse side effects such as constipation, respiratory depression, or addiction potential. We recently developed (±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide (NFEPP), a compound that does not evoke central or intestinal side effects due to its selective activation of mu-opioid receptors at low pH in peripheral injured tissues. Although we demonstrated that NFEPP effectively abolishes injury-induced pain, hyperalgesia, and allodynia in rodents, the efficacy of NFEPP in nonevoked ongoing pain remains to be established. Here, we examined reward, locomotor activity, and defecation in rats with complete Freund's adjuvant-induced paw inflammation to compare fentanyl's and NFEPP's potentials to induce side effects and to inhibit spontaneous pain. We demonstrate that low, but not higher, doses of NFEPP produce conditioned place preference but not constipation or motor disturbance, in contrast to fentanyl. Using a peripherally restricted antagonist, we provide evidence that NFEPP-induced place preference is mediated by peripheral opioid receptors. Our results indicate that a low dose of NFEPP produces reward by abolishing spontaneous inflammatory pain.

Natural chromones as potential anti-inflammatory agents: Pharmacological properties and related mechanisms

Chromones are a group of natural substances with a diversity of biological activities. Herein we assessed the pharmacological potential of three chromones (1, 2 and 3) isolated from Dictyoloma vandellianum as anti-inflammatory agents using in vitro and in vivo approaches. During in vitro screening, the production of NO and cytokines by macrophages stimulated with LPS and IFN-γ was inhibited by all chromones at concentrations (5–20 μM) that did not induce cytotoxicity. Analysis of pharmacokinetic parameters (in vitro half-life and intrinsic clearance) using human liver microsomes revealed that 3 has a superior pharmacokinetic profile, compared to 1 and 2. Treatment with 3 (100 mg/kg, ip) did not affect the mice motor performance, while 1 and 2 induced motor deficit. Taking into account the pharmacokinetic profile and absence of motor impairment, 3 was selected for further pharmacological characterization. Corroborating the data from in vitro screening, treatment of cell cultures with 3 (5–20 μM) reduced TNF-α, IL-6 and IL-1β production by stimulated macrophages. In the complete Freund's adjuvant-induced paw inflammation model in mice, 3 (25 and 50 mg/kg, ip) inhibited mechanical hyperalgesia, edema and cytokine production/release (IL-1β, IL-6 and TNF-α). 3 (5–20 μM) also reduced the transcriptional activity of NF-κB in stimulated macrophages. Furthermore, treatment with RU486, a glucocorticoid receptor (GR) antagonist, partially prevented the inhibitory effect of 3 on macrophages, indicating that this chromone exerts its anti-inflammatory effects in part through the activation of GR. The results presented herein demonstrate the pharmacological potential of natural chromones, highlighting 3 as a possible candidate for the drug discovery process targeting new anti-inflammatory drugs.

MicroRNA-1224 Splicing CircularRNA-Filip1l in an Ago2-Dependent Manner Regulates Chronic Inflammatory Pain via Targeting Ubr5.

Dysfunctions of gene transcription and translation in the nociceptive pathways play the critical role in development and maintenance of chronic pain. Circular RNAs (circRNAs) are emerging as new players in regulation of gene expression, but whether and how circRNAs are involved in chronic pain remain elusive. We showed here that complete Freund's adjuvant-induced chronic inflammation pain significantly increased circRNA-Filip1l (filamin A interacting protein 1-like) expression in spinal neurons of mice. Blockage of this increase attenuated complete Freund's adjuvant-induced nociceptive behaviors, and overexpression of spinal circRNA-Filip1l in naive mice mimicked the nociceptive behaviors as evidenced by decreased thermal and mechanical nociceptive threshold. Furthermore, we found that mature circRNA-Filip1l expression was negatively regulated by miRNA-1224 via binding and splicing of precursor of circRNA-Filip1l (pre-circRNA-Filip1l) in the Argonaute-2 (Ago2)-dependent manner. Increase of spinal circRNA-Filip1l expression resulted from the decrease of miRNA-1224 expression under chronic inflammation pain state. miRNA-1224 knockdown or Ago2 overexpression induced nociceptive behaviors in naive mice, which was prevented by the knockdown of spinal circRNA-Filip1l. Finally, we demonstrated that a ubiquitin protein ligase E3 component n-recognin 5 (Ubr5), validated as a target of circRNA-Filip1l, plays a pivotal role in regulation of nociception by spinal circRNA-Filip1l. These data suggest that miRNA-1224-mediated and Ago2-dependent modulation of spinal circRNA-Filip1l expression regulates nociception via targeting Ubr5, revealing a novel epigenetic mechanism of interaction between miRNA and circRNA in chronic inflammation pain.SIGNIFICANCE STATEMENT circRNAs are emerging as new players in regulation of gene expression. Here, we found that the increase of circRNA-Filip1l mediated by miRNA-1224 in an Ago2-dependent way in the spinal cord is involved in regulation of nociception via targeting Ubr5 Our study reveals a novel epigenetic mechanism of interaction between miRNA and circRNA in chronic inflammation pain.

Anti-inflammatory and Antioxidant Properties of Probiotic Bacterium Lactobacillus mucosae AN1 and Lactobacillus fermentum SNR1 in Wistar Albino Rats.

The potent antioxidant probiotic strains Lactobacillus mucosae AN1 and Lactobacillus fermentum SNR1 were assessed for anti-inflammatory properties in carrageenan (acute) and complete Freund’s adjuvant-induced inflammation (chronic) models in the present study. The two probiotic strains were administered orally along with feed to the Wistar albino male rats as whole cell as well as microencapsulated form. The following experiments were performed to evaluate the anti-inflammatory properties of probiotic strains and the results were observed that the encapsulated and unencapsulated probiotic strains have exhibited statistically significant decrease in paw thickness. Percentage of inhibition in paw thickness of microencapsulated probiotic bacteria (Group VIII), unencapsulated strains (Group IX) were revealed 85 ± 13% and 77 ± 25%, respectively. In Hematoxylin and Eosin staining, results were revealed that the probiotic strains were exhibited anti-inflammatory effects on inflammation-induced paw tissues. qRT-PCR studies revealed upregulation of anti-inflammatory cytokine genes and down-regulation pro-inflammatory cytokine genes in probiotic-treated rat paw tissues. Further, the expression of anti-inflammatory and pro-inflammatory cytokines were examined using immunohistochemistry and ELISA methods. The probiotic administered rat paw tissue in different groups have exhibited the low level of lipid peroxides formation and higher anti-oxidant activities when compared to the control and inflammation control tissues.

Intraplantar injection of sialidase reduces mechanical allodynia during inflammatory pain

Sialic acids are highly charged glycoresidues that are attached to glycoproteins or glycosphingolipids, and they are associated with various biological functions. Gangliosides, sialic acid-containing glycosphingolipids, are abundant in neural tissues and play important roles in the nervous system. Previous studies revealed that peripheral gangliosides are involved in nociceptive behavior and hyperalgesia. These observations prompted us to determine whether the sialic acid-cleaving enzyme sialidase affects pain signaling. Intraplantar injection of sialidase reduced mechanical allodynia during complete Freund's adjuvant-induced inflammation. We also found that ganglioside induces mechanical allodynia in naïve mice. These results suggest that sialyl conjugates in subcutaneous tissues modify allodynia.

Skin-nerve preparation to assay the function of opioid receptors in peripheral endings of sensory neurons.

This chapter describes the methodology of the in vitro skin-saphenous nerve preparation and its application to test for the modulatory effects of opioids on the function of cutaneous sensory neurons in experimental models of pain. We detail the skin-nerve setup requirements and the technique to record action potentials from single sensory fibers. We address how to test for inhibitory effects of opioid receptor activation on mechanical and thermal sensitivity of nociceptors and mechanoreceptors in the complete Freund's adjuvant-induced inflammation and the chronic constriction injury model of neuropathic pain.

LPA5 receptor plays a role in pain sensitivity, emotional exploration and reversal learning

Lysophosphatidic acid (LPA) is a bioactive lipid acting on the nervous system through at least 6 different G protein-coupled receptors. In this study, we examined mice lacking the LPA5 receptor using an extensive battery of behavioral tests. LPA5-deficient mice showed decreased pain sensitivity in tail withdrawal, faster recovery in one inflammatory pain procedure (complete Freund's adjuvant-induced inflammation) and attenuated responses under specific neuropathic pain conditions. Notably, deletion of LPA5 also induced nocturnal hyperactivity and reduced anxiety in the mutant mice. Several exploratory tasks revealed signs of reduced anxiety in LPA5 knockout mice including increased visits to the arena center and reduced thigmotaxis in the open field, and more open arm entries in the elevated plus maze. Finally, LPA5 knockout mice also displayed marked reduction in social exploration, although several other tests indicated that these mice were able to respond normally to environmental stimuli. While learning and memory performance was not impaired in LPA5-deficient mice, we found differences, e.g., targeted swim strategy and reversal learning, as well as scheduled appetitive conditioning that might indicate differential motivational behavior. These results imply that LPA5 might be involved in both nociception and mechanisms of pain hypersensitivity, as well as in anxiety-related and motivational behaviors. These observations further support the proposed involvement of LPA signaling in psychopathology.

Impaired Nociception and Peripheral Opioid Antinociception in Mice Lacking Both Kinin B1 and B2 Receptors

Kinins (e.g., bradykinin) acting through the constitutively expressed B2 and the injury-induced B1 receptors are involved in pain and hyperalgesia, as previously shown by use of receptor-selective antagonists and single-receptor knockout models. Because the overall contribution of kinins to painful processes remains unclear, the aim of this study was to analyze pain-related behaviors of mice unable to respond to kinins because of a lack of both B1 and B2 receptors. In knockout mice lacking both B1 and B2 receptors and in wild-type mice (n = 8-21 per group) the authors assessed nociceptive thresholds to mechanical and heat stimuli (von Frey and Hargreaves tests, respectively) in healthy animals and after induction of inflammatory and neuropathic pain, acid-induced visceral nociception, and modulation of nociceptive responses by peripherally administered opioid agonists. In knockout mice lacking both B1 and B2 receptors baseline nociceptive responses to heat were unaltered, nocifensive responses to bradykinin were abolished, acute acetic acid-induced visceral nociception was reduced by approximately 70% (mean difference: 19.5 writhes/30 min) and heat hypersensitivity in carrageenan-induced paw inflammation was decreased 48 h after injection (mean difference 2.88 s), hypersensitivities in chronic complete Freund's adjuvant-induced paw inflammation or after chronic constriction injury of the sciatic nerve were unchanged, and peripheral μ- and δ-opioid-induced analgesia after chronic constriction injury was reduced by 30-35% (mean differences: μ-agonist: 0.495 g, δ-agonist: 0.555 g). These data suggest that kinins are important for nociception associated with acute short-lasting inflammation but are less essential in chronic stages of pain. The results also highlight a new protective function of kinins via interactions with the opioid system.

Achillea santolina reduces serum interlukin-6 level and hyperalgesia during complete Freund's adjuvant-induced inflammation in male Wistar rats

Immune system is involved in the etiology and pathophysiologic mechanisms of inflammation. Medicinal plants are an important source of substances which are claimed to induce non-specific immunomodulatory effects. In view of this and on account of the interleukin (IL)-6's role in inflammation and pain induction, this study investigated the effects of Achillea santolina extracts on inflammation which was induced by complete Freund's adjuvant (CFA) in male Wistar rats. Both methanolic and defatted extracts prepared from aerial parts of the plant were examined. Inflammatory symptoms such as hyperalgesia and paw edema in CFA-injected rats' paw were measured by radiant heat and plethysmometer during different stages of study respectively. Serum IL-6 level was checked by rat standard enzyme-linked immunosorbent assay specific kit. The results indicated dose-related effects of methanolic extract on paw edema, hyperalgesia and serum IL-6 level reduction in rats. Methanolic extract of A. santolina exhibited significant antihyperalgesic and anti-inflammatory effects during pretreatment and short-term treatment at dose of 200 mg/kg and there was no significant difference between 200 and 400 mg/kg doses of this extract. Defatted extract did not show significant effect on CFA-induced inflammation during different stages of treatment (P>0.05). Short-term treatment with methanolic extract at dose of 200 mg/kg was more effective than indomethacin in edema, hyperalgesia and serum IL-6 level reduction (P<0.01, P<0.01 and P<0.05 respectively). These results suggest that methanolic extract of A. santolina possesses potent anti-inflammatory and immunomodulatory activities during pretreatment and short-term administration.

The chemokine Bv8/prokineticin 2 is up-regulated in inflammatory granulocytes and modulates inflammatory pain

Neutrophil migration into injured tissues is invariably accompanied by pain. Bv8/prokineticin 2 (PK2), a chemokine characterized by a unique structural motif comprising five disulfide bonds, is highly expressed in inflamed tissues associated to infiltrating cells. Here, we demonstrate the fundamental role of granulocyte-derived PK2 (GrPK2) in initiating inflammatory pain and driving peripheral sensitization. In animal models of complete Freund's adjuvant-induced paw inflammation the development and duration of pain temporally correlated with the expression levels of PK2 in the inflamed sites. Such an increase in PK2 mRNA depends mainly on a marked up-regulation of PK2 gene transcription in granulocytes. A substantially lower up-regulation was also detected in macrophages. From a pool of peritoneal granulocytes, elicited in rats by oyster glycogen, we purified the GrPK2 protein, which displayed high affinity for the prokineticin receptors (PKRs) and, when injected into the rat paw, induced hypersensitivity to noxious stimuli as the amphibian prokineticin Bv8 did. Mice lacking PKR1 or PKR2 developed significantly less inflammation-induced hyperalgesia in comparison with WT mice, confirming the involvement of both PKRs in inflammatory pain. The inflammation-induced up-regulation of PK2 was significantly less in pkr1 null mice than in WT and pkr2 null mice, demonstrating a role of PKR1 in setting PK2 levels during inflammation. Pretreatment with a nonpeptide PKR antagonist, which preferentially binds PKR1, dose-dependently reduced and eventually abolished both prokineticin-induced hypernociception and inflammatory hyperalgesia. Inhibiting PK2 formation or antagonizing PKRs may represent another therapeutic approach for controlling inflammatory pain.

Mycobacteria Attenuate Nociceptive Responses by Formyl Peptide Receptor Triggered Opioid Peptide Release from Neutrophils

In inflammation, pain is regulated by a balance of pro- and analgesic mediators. Analgesic mediators include opioid peptides which are secreted by neutrophils at the site of inflammation, leading to activation of opioid receptors on peripheral sensory neurons. In humans, local opioids and opioid peptides significantly downregulate postoperative as well as arthritic pain. In rats, inflammatory pain is induced by intraplantar injection of heat inactivated Mycobacterium butyricum, a component of complete Freund's adjuvant. We hypothesized that mycobacterially derived formyl peptide receptor (FPR) and/or toll like receptor (TLR) agonists could activate neutrophils, leading to opioid peptide release and inhibition of inflammatory pain. In complete Freund's adjuvant-induced inflammation, thermal and mechanical nociceptive thresholds of the paw were quantified (Hargreaves and Randall-Selitto methods, respectively). Withdrawal time to heat was decreased following systemic neutrophil depletion as well as local injection of opioid receptor antagonists or anti-opioid peptide (i.e. Met-enkephalin, β-endorphin) antibodies indicating an increase in pain. In vitro, opioid peptide release from human and rat neutrophils was measured by radioimmunoassay. Met-enkephalin release was triggered by Mycobacterium butyricum and formyl peptides but not by TLR-2 or TLR-4 agonists. Mycobacterium butyricum induced a rise in intracellular calcium as determined by FURA loading and calcium imaging. Opioid peptide release was blocked by intracellular calcium chelation as well as phosphoinositol-3-kinase inhibition. The FPR antagonists Boc-FLFLF and cyclosporine H reduced opioid peptide release in vitro and increased inflammatory pain in vivo while TLR 2/4 did not appear to be involved. In summary, mycobacteria activate FPR on neutrophils, resulting in tonic secretion of opioid peptides from neutrophils and in a decrease in inflammatory pain. Future therapeutic strategies may aim at selective FPR agonists to boost endogenous analgesia.

Antibodies to Nerve Growth Factor Reverse Established Tactile Allodynia in Rodent Models of Neuropathic Pain without Tolerance

A considerable body of evidence implicates endogenous nerve growth factor (NGF) in conditions in which pain is a prominent feature, including neuropathic pain. However, previous studies of NGF antagonism in animal models of neuropathic pain have examined only the prevention of hyperalgesia and allodynia after injury, whereas the more relevant issue is whether treatment can provide relief of established pain, particularly without tolerance. In the current work, we studied the effects of potent, neutralizing anti-NGF antibodies on the reversal of tactile allodynia and thermal hyperalgesia in established models of neuropathic and inflammatory pain in rats and mice. In the complete Freund's adjuvant-induced hind-paw inflammation, spinal nerve ligation and streptozotocin-induced neuropathic pain models, a single intraperitoneal injection of a polyclonal anti-NGF antibody reversed established tactile allodynia from approximately day 3 to day 7 after treatment. Effects on thermal hyperalgesia were variable with a significant effect observed only in the spinal nerve ligation model. In the mouse chronic constriction injury (CCI) model, a mouse monoclonal anti-NGF antibody reversed tactile allodynia when administered 2 weeks after surgery. Repeated administration of this antibody to CCI mice for 3 weeks produced a sustained reversal (days 4 to 21) of tactile allodynia that returned 5 days after the end of dosing. In conclusion, NGF seems to play a critical role in models of established neuropathic and inflammatory pain in both rats and mice, with no development of tolerance to antagonism. Antagonists of NGF, such as fully human monoclonal anti-NGF antibodies, may have therapeutic utility in analogous human pain conditions.

Trigeminal P2X 3 receptor expression differs from dorsal root ganglion and is modulated by deep tissue inflammation

The distribution and modulation of the P2X 3 receptor was studied in trigeminal ganglion neurons to provide insight into the role of ATP in craniofacial sensory mechanisms. Binding to the d-galactose specific lectin IB4 was found in 73% of P2X 3-positive neurons while only 16% of IB4 neurons expressed P2X 3. Neurons expressing P2X 3 alone were significantly larger than IB4-or IB4/P2X 3-positive neurons. Investigation of target-specificity revealed that 22% of trigeminal ganglion muscle afferent neurons were positive for P2X 3 versus 16% of cutaneous afferent neurons. Muscle P2X 3 afferents were significantly smaller than the overall muscle afferent population while P2X 3 cutaneous afferent neurons were not. Presumptive heteromeric (P2X 2/3) muscle afferent neurons were also identified and comprised 77% of the P2X 3 muscle afferent population. Muscle afferent neurons co-expressed P2X 3 with either calcitonin gene-related peptide (15%) or substance P (4%). The number of P2X 3-positive muscle afferent neurons significantly increased one and four days following complete Freund's adjuvant-induced masseter muscle inflammation, but significantly decreased after 12 days. These results indicate that within trigeminal ganglia: (1) the P2X 3 receptor is expressed in both small and medium-sized neurons; (2) the P2X 3 receptor is not exclusively expressed in IB4 neurons; (3) P2X 3 is co-expressed with neuropeptides; (4) differences in the proportion of cutaneous versus muscle P2X 3 afferents are not apparent. Trigeminal P2X 3 neurons therefore differ markedly from dorsal root ganglion P2X 3 afferents. This study also shows that deep tissue inflammation modulates expression of the P2X 3 receptor and thus may warrant exploration as a target for therapeutic intervention.

Inflammation alters sodium currents and excitability of temporomandibular joint afferents

Inflammation-induced changes in voltage-gated sodium currents ( I Na) in primary afferent neurons may contribute to hyperexcitability and pain. The present study was designed to test the hypothesis that persistent inflammation of the temporomandibular joint (TMJ) increases I Na in TMJ afferents. Acutely dissociated retrogradely labeled TMJ afferents were studied using whole-cell patch clamp techniques three days following Complete Freund's Adjuvant-induced inflammation of the TMJ. Inflammation was associated with a decrease in tetrodotoxin (TTX)-sensitive Na + conductance and no significant change in slowly inactivating TTX-resistant Na + conductance. However, inflammation increased the excitability of TMJ afferents. These results suggest that changes in ion channels other than those underlying TTX-sensitive and the slowly inactivating TTX-resistant Na + conductance are likely to account for the inflammation-induced increase in the excitability of TMJ afferents.