Commensal microbiota is fundamental for the development of inflammatory pain

The ability of an individual to sense pain is fundamental for its capacity to adapt to its environment and to avoid damage. The sensation of pain can be enhanced by acute or chronic inflammation. In the present study, we have investigated whether inflammatory pain, as measured by hypernociceptive responses, was modified in the absence of the microbiota. To this end, we evaluated mechanical nociceptive responses induced by a range of inflammatory stimuli in germ-free and conventional mice. Our experiments show that inflammatory hypernociception induced by carrageenan, lipopolysaccharide, TNF-alpha, IL-1 beta, and the chemokine CXCL1 was reduced in germfree mice. In contrast, hypernociception induced by prostaglandins and dopamine was similar in germ-free or conventional mice. Reduction of hypernociception induced by carrageenan was associated with reduced tissue inflammation and could be reversed by reposition of the microbiota or systemic administration of lipopolysaccharide. Significantly, decreased hypernociception in germ-free mice was accompanied by enhanced IL-10 expression upon stimulation and could be reversed by treatment with an anti-IL-10 antibody. Therefore, these results show that contact with commensal microbiota is necessary for mice to develop inflammatory hypernociception. These findings implicate an important role of the interaction between the commensal microbiota and the host in favoring adaptation to environmental stresses, including those that cause pain.

Involvement of LTB4 in zymosan-induced joint nociception in mice: participation of neutrophils and PGE(2)

Leukotriene B-4 (LTB4) mediates different inflammatory events such as neutrophil migration and pain. The present study addressed the mechanisms of LTB4-mediated joint inflammation-induced hypernociception. It was observed that zymosan-induced articular hypernociception and neutrophil migration were reduced dose-dependently by the pretreatment with MK886 (1-9 mg/kg; LT synthesis inhibitor) as well as in 5-lypoxygenase-deficient mice (5LO(-/-)) or by the selective antagonist of the LTB4 receptor (CP105696; 3 mg/kg). Histological analysis showed reduced zymosan-induced articular inflammatory damage in 5LO(-/-) mice. The hypernociceptive role of LTB4 was confirmed further by the demonstration that joint injection of LTB4 induces a dose (8.3, 25, and 75 ng)-dependent articular hypernociception. Furthermore, zymosan induced an increase in joint LTB4 production. Investigating the mechanism underlying LTB4 mediation of zymosan-induced hypernociception, LTB4-induced hypernociception was reduced by indomethacin (5 mg/kg), MK886 (3 mg/kg), celecoxib (10 mg/kg), antineutrophil antibody (100 mu g, two doses), and fucoidan (20 mg/kg) treatments as well as in 5LO(-/-) mice. The production of LTB4 induced by zymosan in the joint was reduced by the pretreatment with fucoidan or antineutrophil antibody as well as the production of PGE(2) induced by LTB4. Therefore, besides reinforcing the role of endogenous LTB4 as an important mediator of inflamed joint hypernociception, these results also suggested that the mechanism of LTB4-induced articular hypernociception depends on prostanoid and neutrophil recruitment. Furthermore, the results also demonstrated clearly that LTB4-induced hypernociception depends on the additional release of endogenous LTs. Concluding, targeting LTB4 synthesis/action might constitute useful therapeutic approaches to inhibit articular inflammatory hypernociception.

Quercetin Reduces Neutrophil Recruitment Induced by CXCL8, LTB(4), and fMLP: Inhibition of Actin Polymerization

Recent in vitro data have suggested that the flavonoid quercetin (1) does not affect the functioning of neutrophils. Therefore, we evaluated in vivo and in vitro whether or not 1 affects neutrophil function, focusing on recruitment. The in vivo treatment with 1 inhibited in a dose-dependent manner the recruitment of neutrophils to the peritoneal cavity of mice induced by known chemotatic factors such as CXCL1, CXCL5, LTB(4), and fMLP. Further-more, 1 also inhibited in a concentration-dependent manner the chemoattraction of human neutrophils induced by CXCL8, LTB(4), and fMLP in a Boyden chamber. In vitro treatment with 1 did not affect human neutrophil surface expression of CXCR1, CXCR2, BLT1, or FLPR1, but rather reduced actin polymerization. These results suggest that 1 inhibits actin polymerization, hence, explaining the inhibition of neutrophil recruitment in vivo and in vitro and highlighting its possible usefulness to diminish excessive neutrophil migration during inflammation.

Facilitation of 5-HT(2A/2C)-mediated neurotransmission in the ventromedial hypothalamic nucleus decreases anxiety in the elevated T-maze

Previous evidence has shown that facilitation of GABA/benzodiazepine-mediated neurotransmission in the ventromedial hypothalamus (VMH) inhibits both escape and inhibitory avoidance responses generated in the elevated T-maze test of anxiety (ETM). These defensive behaviors have been associated with panic and generalized anxiety, respectively. Aside from GABA/benzodiazepine receptors, the VMH also contains a significant number of serotonin (5-HT) receptors, including 1A, 2A and 2C subtypes. The purpose of the present study was to investigate the effect of the activation of 5-HT(1A) and 5-HT(2A/2C) receptors in the VMH on defensive behavioral responses in rats submitted to the ETM. For that, male Wistar rats were treated intra-VMH with the 5-HT(1A) agonist 8-OH-DPAT, with the 5-HT(2A/2C) agonist DOI, with the 5-HT(2C) selective agonist MK-212, or with the 5-HT(2A/2C) antagonist ketanserin and 10 min after were submitted to the ETM. Results showed that both DOI and MK-212 significantly decreased avoidance measurements, an anxiolytic-like effect, without altering escape. 8-OH-DPAT and ketanserin were without effect, although the last drug attenuated the effects of DOI. None of the drugs altered locomotor activity in an open field. These results suggest that 5-HT(2A/2C) receptors of the VMH are involved in the regulation of inhibitory avoidance and might be of relevance to the physiopathology of generalized anxiety. (C) 2010 Elsevier B.V. All rights reserved.

Dorsal raphe nucleus regulation of a panic-like defensive behavior evoked by chemical stimulation of the rat dorsal periaqueductal gray matter

Electrical or chemical stimulation of the dorsal periaqueductal gray matter (DPAG) evokes escape, a defensive behavior that has been related to panic attacks. Injection of 5-HT(1A) or 5-HT(2A) receptor agonists into this midbrain area inhibits this response. It has been proposed that the impairment of 5-HT mechanisms controlling escape at the level of the DPAG may underlie the susceptibility to panic attacks that characterizes the panic disorder. In this study we evaluated the effects of the pharmacological manipulation of the dorsal raphe nucleus (DRN), which are the main source of 5-HT input to the DPAG, on the escape response evoked in rats by the intra-DPAG injection of the nitric oxide donor SIN-1. The results showed that DRN administration of the 5-HT(1A) receptor agonist 8-OH-DPAT which inhibits the activity of 5-HT neurons favored the expression of escape induced by SIN-1. Intra-DRN injection of the excitatory amino acid kainic acid or the 5-HT(1A) receptor antagonist WAY-100635 did not change escape expression. However, both compounds fully blocked the escape reaction generated by intra-DPAG injection of the excitatory amino acid D,L-homocysteic acid (DLH). Overall, the results indicate that 5-HT neurons in the DRN exert a bidirectional control upon escape behavior generated by the DPAG. Taking into account the effect of WAY-100635 on DLH-induced escape, they also strengthen the view that DRN 5-HT(1A) autoreceptors are under tonic inhibitory influence by 5-HT. (C) 2010 Elsevier B.V. All rights reserved.

Agglutinin isolated from the red marine alga Hypnea cervicornis J. Agardh reduces inflammatory hypernociception: Involvement of nitric oxide

Hypnea cervicornis agglutinin (HCA), a lectin isolated from the red marine alga has been previously shown to have an antinociceptive effect. In the present study in rats, mechanisms of action of HCA were addressed regarding mechanical hypernociception induced by carrageenan, ovalbumin (as antigen), and also by prostaglandin E(2) in rats. The lectin administered intravenously inhibited carrageenan- and antigen-induced hypernociception at 1,3, 5 and 7 h. This inhibitory effect was completely prevented when lectin was combined with mucin, demonstrating the role of carbohydrate-binding sites. The inhibition of inflammatory hypernociception by HCA was associated with the prevention of neutrophil recruitment to the plantar tissue of rats but was not associated with the inhibition of the release of pro-hypernociceptive cytokines (TNF-alpha, IL-1 beta and CINC-1). HCA also blocked mechanical hypernociception induced by PGE(2), which was prevented by the administration of nitric oxide synthase inhibitors. These results were corroborated by the increased circulating levels of NO metabolites following HCA treatment. These findings suggest that the anti-hypernociceptive effects of HCA are not associated with the inhibition of pro-inflammatory cytokine production. However, these effects seem to involve the inhibition of neutrophil migration and also the increase in NO production. (C) 2010 Elsevier Inc. All rights reserved.

Cannabidiol injected into the bed nucleus of the stria terminalis modulates baroreflex activity through 5-HT(1A) receptors

Cannabidiol (CBD) is a non-psychotomimetic constituent of the Cannabis sativa plant that inhibits behavioral and cardiovascular responses to aversive situations. facilitating 5-HT(1A)-mediated neurotransmission. Previous results from our group suggest that the bed nucleus of the stria terminalis (BNST) may be involved in CBD`s anti-aversive effects. To investigate whether the cardiovascular effects of the CBD could involve a direct drug effect on the BNST, we evaluated the effects of CBD microinjection into this structure on baroreflex activity. We also verified whether these effects were mediated by the activation of 5-HT(1A) receptors. Bilateral microinjection of CBD (60 nmol/100 nL) into the BNST increased the bradycardiac response to arterial pressure increases. However, no changes were observed in tachycardiac responses evoked by arterial pressure decreases. Pretreatment of the BNST with the selective 5-HT(1A) receptor antagonist WAY100635 (0.37 nmol/100 nL) prevented CBD effects on the baroreflex activity. Moreover, microinjection of the 5-HT(1A) receptor agonist 8-OH-DPAT (4 nmol/100 nL) caused effects that were similar to those observed after the microinjection of CBD, which were also blocked by pretreatment with WAY100635. In conclusion, the present studies show that the microinjection of CBD into the BNST has a facilitatory influence on the baroreflex response to blood pressure increases, acting through the activation of 5-HT(1A) receptors. (C) 2010 Elsevier Ltd. All rights reserved.

IL-17 mediates articular hypernociception in antigen-induced arthritis in mice

IL-17 is an important cytokine in the physiopathology of rheumatoid arthritis (RA). However, its participation in the genesis of nociception during RA remains undetermined. In this study, we evaluated the role of IL-17 in the genesis of articular nociception in a model of antigen (mBSA)-induced arthritis. We found that mBSA challenge in the femur-tibial joint of immunized mice induced a dose-and time-dependent mechanical hypernociception. The local IL-17 concentration within the mBSA-injected joints increased significantly over time. Moreover, co-treatment of mBSA challenged mice with an antibody against IL-17 inhibited hypernociception and neutrophil recruitment. In agreement, intraarticular injection of IL-17 induced hypernociception and neutrophil migration, which were reduced by the pre-treatment with fucoidin, a leukocyte adhesion inhibitor. The hypernociceptive effect of IL-17 was also reduced in TNFR1(-/-) mice and by pre-treatment with infliximab (anti-TNF antibody), a CXCR1/2 antagonist or by an IL-1 receptor antagonist. Consistent with these findings, we found that IL-17 injection into joints increased the production of TNF-alpha, IL-1 beta and CXCL1/KC. Treatment with doxycycline (non-specific MMPs inhibitor), bosentan (ET(A)/ET(B) antagonist), indomethacin (COX inhibitor) or guanethidine (sympathetic blocker) inhibited IL-17-induced hypernociception. IL-17 injection also increased PGE(2) production, MMP-9 activity and COX-2, MMP-9 and PPET-1 mRNA expression in synovial membrane. These results suggest that IL-17 is a novel pro-nociceptive cytokine in mBSA-induced arthritis, whose effect depends on both neutrophil migration and various pro-inflammatory mediators, as TNF-alpha, IL-1 beta, CXCR1/2 chemokines ligands, MMPs, endothelins, prostaglandins and sympathetic amines. Therefore, it is reasonable to propose IL-17 targeting therapies to control this important RA symptom. (C) 2009 International Association for the Study of Pain. Published by Elsevier B. V. All rights reserved.

Acute and persistent nociceptive paw sensitisation in mice: The involvement of distinct signalling pathways

Aims: Many fundamental pharmacological studies in pain and inflammation have been performed on rats. However, the pharmacological findings were generally not extended to other species in order to increase their predictive therapeutic value. We studied acute and chronic inflammatory nociceptive sensitisation of mouse hind paws by prostaglandin E(2) (PGE(2)) or dopamine (DA), as previously described in rats. We also investigated the participation of the signalling pathways in acute and persistent sensitisation. Main methods: Mechanical sensitisation (hypernociception) induced by intraplantar administrations of PGE(2) or DA was evaluated with an electronic pressure meter. The signalling pathways were pharmacologically investigated with the pre-administration of adenylyl cyclase (AC), cAMP-dependent protein kinase (PKA), protein kinase C epsilon (PKC epsilon), and the extracellular signal-related kinase (ERK) inhibitors. Key findings: Single or 14 days of successive intraplantar injections of PGE(2) or DA-induced acute and persistent hypernociception (lasting for more than 30 days), respectively. The involvement of AC, PKA or PKC epsilon was observed in the acute hypernociception induced by PGE(2), while PKA or PKC epsilon were continuously activated during the period of persistent hypernociception. The acute hypernociception induced by DA involves activation of ERK, PKC epsilon, AC or PKA, while persistent hypernociception implicated ERK activation, but not PKA, PKC epsilon or AC. Significance: In mice, acute and persistent paw sensitisation involves the different activation of kinases, as previously described for rats. This study opens the possibility of comparing pharmacological approaches in both species to further understand acute and chronic inflammatory sensitisation, and possibly associated genetic manipulations. (C) 2009 Elsevier Inc. All rights reserved.

ACTIVATION OF PERIPHERAL kappa/delta OPIOID RECEPTORS MEDIATES 15-DEOXY-(Delta 12,14)-PROSTAGLANDIN J(2) INDUCED-ANTINOCICEPTION IN RAT TEMPOROMANDIBULAR JOINT

This study assessed the effect of the agonist 15d-PGJ(2) administered into the rat temporomandibular joint (TMJ) on nociceptive behavioral and the anti-inflammatory potential of this prostaglandin on TMJ. It was observed that 15-deoxy-(Delta 12,14)-prostaglandin J(2) (15d-PGJ(2)) significantly reduced formalin-induced nociceptive behavior in a dose dependent manner, however injection of 15d-PGJ(2) into the contralateral TMJ failed to reduce such effects. This antinociceptive effect is dependent on peroxisome proliferator-activated receptors-gamma (PPAR-gamma) since pre-treatment with GW9662 (PPAR-gamma receptor antagonist) blocked the antinociceptive effect of 15d-PGJ(2) in the TMJ. In addition, the antinociceptive effect of 15d-PGJ(2) was also blocked by naloxone suggesting the involvement of peripheral opioids in the process. Confirming this hypothesis pre-treatment with kappa, delta, but not mu receptor antagonists significantly reduced the antinociceptive effect of 15d-PGJ(2) in the TMJ. Similarly to opioid agonists, the 15d-PGJ(2) antinociceptive action depends on the nitric oxide (NO)/guanilate cyclase (cGMP)/ATP-sensitive potassium channel blocker(K(ATP)(+)) channel pathway since it was prevented by the pre-treatment with the inhibitors of nitric oxide synthase (NOS; aminoguanidine), cGMP (ODQ), or the K(ATP)(+) (glibenclamide). In addition, 15d-PGJ(2) (100 ng/TMJ) inhibits 5-HT-induced TMJ hypernociception. Besides, TMJ treated with 15d-PGJ(2) showed lower vascular permeability, assessed by Evan`s Blue extravasation, and also lower neutrophil migration induced by carrageenan administration. Taken together, these results demonstrate that 15d-PGJ(2) has a potential peripheral antinociceptive and anti-inflammatory effect in the TMJ via PPAR-gamma activation. The results also suggest that 15d-PGJ(2) induced-peripheral antinociceptive response in the TMJ is mediated by kappa/delta opioid receptors by the activation of the intracellular L-arginine/NO/cGMP/K(ATP)(+) channel pathway. The pharmacological properties of the peripheral administration of 15d-PGJ(2) highlight the potential use of this PPAR-gamma agonist on TMJ inflammatory pain conditions. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

Quercetin Reduces Inflammatory Pain: Inhibition of Oxidative Stress and Cytokine Production

Quercetin (1) is known to have both antioxidant and antinociceptive effects. However, the mechanism involved in its antinociceptive effect is not fully elucidated. Cytokines and reactive oxygen species have been implicated in the cascade of events resulting in inflammatory pain. Therefore, we evaluated the antinociceptive mechanism of 1 focusing on the role of cytokines and Oxidative stress. Intraperitoneal and oral treatments with 1 dose-dependently inhibited inflammatory nociception induced by acetic acid and phenyl-p-benzoquinone and also the second phase of formalin- and carrageenin-induced mechanical hypernociception. Compound I also inhibited the hypernociception induced by cytokines (e.g., TNF alpha and CXCL1), but not by inflammatory mediators that directly sensitize the nociceptor such as PGE(2) and dopamine. On the other hand, 1 reduced carrageenin-induced IL-1 beta production as well as carrageenin-induced decrease of reduced glutathione (GSH) levels. These results suggest that I exerts its analgesic effect by inhibiting pro-nociceptive cytokine production and the oxidative imbalance mediation of inflammatory pain.

Fructose-1,6-bisphosphate reduces inflammatory pain-like behaviour in mice: role of adenosine acting on A(1) receptors

Background and purpose: D-Fructose-1,6-bisphosphate (FBP) is an intermediate in the glycolytic pathway, exerting pharmacological actions on inflammation by inhibiting cytokine production or interfering with adenosine production. Here, the possible antinociceptive effect of FBP and its mechanism of action in the carrageenin paw inflammation model in mice were addressed, focusing on the two mechanisms described above. Experimental approach: Mechanical hyperalgesia (decrease in the nociceptive threshold) was evaluated by the electronic pressure-metre test; cytokine levels were measured by elisa and adenosine was determined by high performance liquid chromatography. Key results: Pretreatment of mice with FBP reduced hyperalgesia induced by intraplantar injection of carrageenin (up to 54%), tumour necrosis factor alpha (40%), interleukin-1 beta (46%), CXCL1 (33%), prostaglandin E(2) (41%) or dopamine (55%). However, FBP treatment did not alter carrageenin-induced cytokine (tumour necrosis factor alpha and interleukin-1 beta) or chemokine (CXCL1) production. On the other hand, the antinociceptive effect of FBP was prevented by systemic and intraplantar treatment with an adenosine A(1) receptor antagonist (8-cyclopentyl-1,3-dipropylxanthine), suggesting that the FBP effect is mediated by peripheral adenosine acting on A(1) receptors. Giving FBP to mice increased adenosine levels in plasma, and adenosine treatment of paw inflammation presented a similar antinociceptive mechanism to that of FBP. Conclusions and implications: In addition to anti-inflammatory action, FBP also presents an antinociceptive effect upon inflammatory hyperalgesia. Its mechanism of action seems dependent on adenosine production but not on modulation of hyperalgesic cytokine/chemokine production. In turn, adenosine acts peripherally on its A(1) receptor inhibiting hyperalgesia. FBP may have possible therapeutic applications in reducing inflammatory pain.

Anxiolytic-like effects induced by blockade of transient receptor potential vanilloid type 1 (TRPV1) channels in the medial prefrontal cortex of rats

The endocannabinoid anandamide, in addition to activating cannabinoid type 1 receptors (CB1), may act as an agonist at transient receptor potential vanilloid type 1 (TRPV1) channels. In the periaqueductal gray, CB1 activation inhibits, whereas TRPV1 increases, anxiety-like behavior. In the medial prefrontal cortex (mPFC), another brain region related to defensive responses, CB1 activation induces anxiolytic-like effects. However, a possible involvement of TRPV1 is still unclear. In the present study, we tested the hypothesis that TRPV1 channel contributes to the modulation of anxiety-like behavior in the mPFC. Male Wistar rats (n = 5-7 per group) received microinjections of the TRPV1 antagonist capsazepine (1-60 nmol) in the ventral portion of the mPFC and were exposed to the elevated plus maze (EPM) or to the Vogel conflict test. Capsazepine increased exploration of open arms in the EPM as well as the number of punished licks in the Vogel conflict test, suggesting anxiolytic-like effects. No changes in the number of entries into the enclosed arms were observed in the EPM, indicating that there were no changes in motor activity. Moreover, capsazepine did not interfere with water consumption or nociceptive threshold, discarding potential confounding factors for the Vogel conflict test. These data suggest that TRPV1 in the ventral mPFC tonically inhibits anxiety-like behavior. TRPV1 could facilitate defensive responses opposing, therefore, the anxiolytic-like effects reported after local activation of CB1 receptors.

Differential regulation of TRP channels in a rat model of neuropathic pain

Neuropathic pain is a chronic disease resulting from dysfunction of the nervous system often due to peripheral nerve injury. Hypersensitivity to sensory Stimuli (mechanical, thermal or chemical) is a common source of pain in patients and ion channels involved in detecting these Stimuli are possible candidates for inducing and/or maintaining the pain. Transient receptor potential (TRP) channels expressed on nociceptors respond to different sensory stimuli and a few of them have been studied previously in the models of neuropathic pain. Using real-time PCR for quantification of all known TRP channels we identified several TRP channels, which have not been associated with nociception OF neuropathic pain before, to be expressed in the DRG and to be differentially regulated after spared nerve injury (SNI). Of all TRP channel members, TRPML3 showed the most dramatic change in animals exhibiting neuropathic pain behaviour compared to control animals. fit situ hybridisation showed a widespread increase of expression ill neurons of small, medium and large cell sizes, indicating expression ill multiple subtypes. Co-localisation of TRPML3 with CGRP, NF200 and IB4 staining confirmed a broad Subtype distribution. Expression studies during development showed that TRPML3 is all embryonic channel that is induced upon nerve injury in three different nerve injury models investigated. Thus. the current results link for the first time a re-expression of TRPML3 with the development of neuropathic pain conditions. In addition, decreased mRNA levels after SNI were seen for TRPM6, TRPM8, TRPV1, TRPA1, TRPC3, TRPC4 and TRPC5. (C) 2009 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

GABAergic mediation of stress-induced secretion of corticosterone and oxytocin, but not prolactin, by the hypothalamic paraventricular nucleus

The hypothalamus-pituitary-adrenal axis (HPA) participates in mediating the response to stressful stimuli. Within the HPA, neurons in the medial parvocellular region of paraventricular nucleus (PVN) of the hypothalamus integrate excitatory and inhibitory signals triggering secretion of corticotropin-releasing hormone (CRH), the main secretagogue of adrenocorticotropic hormone (ACTH). Stressful situations alter CRH secretion as well as other hormones, including prolactin and oxytocin. Most inputs to the PVN are of local origin, half of which are GABAergic neurons, and both GABA-A and GABA-B receptors are present in the PVN. The objective of the present study was to investigate the role of GABA-A and GABA-B receptors in the PVN`s control of stress-induced corticosterone, oxytocin and prolactin secretion. Rats Were microinjected with saline or different doses (0.5, 5 and 50 pmol) of GABA-A (bicuculine) or GABA-B (phaclofen) antagonists in the PVN. Ten minutes later, they were subjected to a stressor (ether inhalation) and blood samples were collected 30 min before and 10, 30, 60, 90 and 120 min after the stressful stimulus to measure hormone levels by radioimmunoassay. Our results indicate that GABA acts in the PVN to inhibit stress-induced corticosterone secretion via both its receptor subtypes, especially GABA-B. In contrast, GABA in the PVN stimulates oxytocin secretion through GABA-B receptors and does not alter prolactin secretion. (C) 2008 Elsevier Inc. All rights reserved.