Correction to: The kinin B1 and B2 receptors and TNFR1/p55 axis on neuropathic pain in the mouse brachial plexus.

Unfortunately, the Fig. 7 was wrongly downloaded in the original publication.

Spinal blockage of CXCL1 and its receptor CXCR2 inhibits paclitaxel-induced peripheral neuropathy in mice.

Painful peripheral neuropathy is the most dose-limiting side effect of paclitaxel (PTX), a widely used anti-cancer drug to treat solid tumours. The understanding of the mechanisms involved in this side effect is crucial to the development of new therapeutic approaches. CXCL1 chemokine and its receptor CXCR2 have been pointed as promising targets to treat chronic pain. Herein, we sought to evaluate the possible involvement of CXCL1 and CXCR2 in the pathogenesis of PTX-induced neuropathic pain in mice. PTX treatment led to increased levels of CXCL1 in both dorsal root ganglion and spinal cord samples. Systemic treatment with the anti-CXCL1 antibody (10 µg/kg, i.v.) or the selective CXCR2 antagonist (SB225002, 3 mg/kg, i.p.) had minor effect on PTX-induced mechanical hypersensitivity. On the other hand, the intrathecal (i.t.) treatment with anti-CXCL1 (1 ng/site) or SB225002 (10 µg/site) consistently inhibited the nociceptive responses of PTX-treated mice. Similar results were obtained by inhibiting the PI3Kγ enzyme a downstream pathway of CXCL1/CXCR2 signalling with either the selective AS605240 (5 µg/site, i.t.) or the non-selective wortmannin PI3K inhibitor (0.4 µg/site, i.t.). Overall, the data indicates that the up-regulation of CXCL1 is important for the development and maintenance of PTX-induced neuropathic pain in mice. Therefore, the spinal blockage of CXCL1/CXCR2 signalling might be a new innovative therapeutic approach to treat this clinical side effect of PTX.

The kinin B1 and B2 receptors and TNFR1/p55 axis on neuropathic pain in the mouse brachial plexus.

Tumour necrosis factor (TNF) and kinins have been associated with neuropathic pain-like behaviour in numerous animal models. However, the way that they interact to cause neuron sensitisation remains unclear. This study assessed the interaction of kinin receptors and TNF receptor TNFR1/p55 in mechanical hypersensitivity induced by an intraneural (i.n.) injection of rm-TNF into the lower trunk of brachial plexus in mice. The i.n. injection of rm-TNF reduced the mechanical withdrawal threshold of the right forepaw from the 3rd to the 10th day after the injection, indicating that TNF1/p55 displays a critical role in the onset of TNF-elicited neuropathic pain. The connection between TNF1/p55 and kinin B1 and B2 receptors (B1R and B2R) was confirmed using both knockout mice and mRNAs quantification in the injected nerve, DRG and spinal cord. The treatment with the B2R antagonist HOE 140 or with B1R antagonist des-Arg9-Leu8-BK reduced both BK- and DABK-induced hypersensitivity. The experiments using kinin receptor antagonists and CPM inhibitor (thiorphan) suggest that BK does not only activate B2R as an orthosteric agonist, but also seems to be converted into DABK that consequently activates B1R. These results indicate a connection between TNF and the kinin system, suggesting a relevant role for B1R and B2R in the process of sensitisation of the central nervous systems by the cross talk between the receptor and CPM after i.n. injection of rm-TNF.

Kinin Receptors Sensitize TRPV4 Channel and Induce Mechanical Hyperalgesia: Relevance to Paclitaxel-Induced Peripheral Neuropathy in Mice.

Kinin B1 (B1R) and B2 receptors (B2R) and the transient receptor potential vanilloid 4 (TRPV4) channel are known to play a critical role in the peripheral neuropathy induced by paclitaxel (PTX) in rodents. However, the downstream pathways activated by kinin receptors as well as the sensitizers of the TRPV4 channel involved in this process remain unknown. Herein, we investigated whether kinins sensitize TRPV4 channels in order to maintain PTX-induced peripheral neuropathy in mice. The mechanical hyperalgesia induced by bradykinin (BK, a B2R agonist) or des-Arg9-BK (DABK, a B1R agonist) was inhibited by the selective TRPV4 antagonist HC-067047. Additionally, BK was able to sensitize TRPV4, thus contributing to mechanical hyperalgesia. This response was dependent on phospholipase C/protein kinase C (PKC) activation. The selective kinin B1R (des-Arg9-[Leu8]-bradykinin) and B2R (HOE 140) antagonists reduced the mechanical hyperalgesia induced by PTX, with efficacies and time response profiles similar to those observed for the TRPV4 antagonist (HC-067047). Additionally, both kinin receptor antagonists inhibited the overt nociception induced by hypotonic solution in PTX-injected animals. The same animals presented lower PKCε levels in skin and dorsal root ganglion samples. The selective PKCε inhibitor (εV1-2) reduced the hypotonicity-induced overt nociception in PTX-treated mice with the same magnitude observed for the kinin receptor antagonists. These findings suggest that B1R or B2R agonists sensitize TRPV4 channels to induce mechanical hyperalgesia in mice. This mechanism of interaction may contribute to PTX-induced peripheral neuropathy through the activation of PKCε. We suggest these targets represent new opportunities for the development of effective analgesics to treat chronic pain.

The transient receptor potential ankyrin-1 mediates mechanical hyperalgesia induced by the activation of B1 receptor in mice.

The kinin receptor B1 and the transient receptor potential ankyrin 1 (TRPA1) work as initiators and gatekeepers of nociception and inflammation. This study reports that the nociceptive transmission induced by activation of B1 receptor is dependent on TRPA1 ion channel. The mechanical hyperalgesia was induced by intrathecal (i.t.) injection of B1 agonist des-Arginine9-bradykinin (DABK) or TRPA1 agonist cinnamaldehyde and was evaluated by the withdrawal response after von Frey Hair application in the hind paw. After behavioral experiments, lumbar spinal cord and dorsal root ganglia (DRG) were harvested to assess protein expression and mRNA by immunohistochemistry and real time-PCR, respectively. The pharmacological antagonism (HC030031) or the down-regulation of TRPA1 greatly inhibited the mechanical hyperalgesia induced by DABK. Intrathecal injection of DABK up regulated the ionized calcium binding adaptor molecule (Iba-1) in lumbar spinal cord (L5-L6); TRPA1 protein and mRNA in lumbar spinal cord; and B1 receptor mRNA in both lumbar spinal cord and DRG. The knockdown of TRPA1 prevented microglia activation induced by DABK. Furthermore, the mechanical hyperalgesia induced by either DABK or by cinnamaldehyde was significantly reduced by inhibition of cyclooxygenase (COX), protein kinase C (PKC) or phospholipase C (PLC). In summary, this study revealed that TRPA1 positively modulates the mechanical hyperalgesia induced by B1 receptor activation in the spinal cord and that the classical GPCR downstream molecules PLC, diacylglycerol (DAG), 3,4,5-inositide phosphate (IP3) and PKC are involved in the nociceptive transmission triggered by these two receptors.

Mechanisms Underlying the Scratching Behavior Induced by the Activation of Proteinase-Activated Receptor-4 in Mice.

A role for proteinase-activated receptor-4 (PAR-4) was recently suggested in itch sensation. Here, we investigated the mechanisms underlying the pruriceptive actions of the selective PAR-4 agonist AYPGKF-NH2 (AYP) in mice. Dorsal intradermal (i.d.) administration of AYP elicited intense scratching behavior in mice, which was prevented by the selective PAR-4 antagonist (pepducin P4pal-10). PAR-4 was found to be coexpressed in 32% of tryptase-positive skin mast cells, and AYP caused a 2-fold increase in mast cell degranulation. However, neither the treatment with cromolyn nor the deficiency of mast cells (WBB6F1-Kit(W/Wv) mice) was able to affect AYP-induced itch. PAR-4 was also found on gastrin-releasing peptide (GRP)-positive neurons (pruriceptive fibers), and AYP-induced itch was reduced by the selective GRP receptor antagonist RC-3095. In addition, AYP evoked calcium influx in ∼1.5% of cultured DRG neurons also sensitive to TRPV1 (capsaicin) and/or TRPA1 (AITC) agonists. Importantly, AYP-induced itch was reduced by treatment with either the selective TRPV1 (SB366791), TRPA1 (HC-030031), or NK1 (FK888) receptor antagonists. However, genetic loss of TRPV1, but not of TRPA1, diminished AYP-induced calcium influx in DRG neurons and the scratching behavior in mice. These findings provide evidence that PAR-4 activation by AYP causes pruriceptive itch in mice via a TRPV1/TRPA1-dependent mechanism.

The role of keratinocyte-derived chemokine (KC) on hyperalgesia caused by peripheral nerve injury in mice.

Chemokines are associated with both inflammatory and immune responses and play an important role in the pathophysiological process associated with neuropathic pain following peripheral nerve injury. Here, we investigated the involvement of peripheral keratinocyte-derived chemokine (KC) in the pathogenesis of neuropathic pain induced by the partial ligation of the sciatic nerve (PLSN) in mice. PLSN increased KC levels and its mRNA in both the sciatic nerve and spinal cord when compared with sham-operated mice. In addition, PLSN-induced mechanical and thermal hyperalgesia was prevented by systemic (i.v.) treatment with anti-KC antibody either at the time of surgery or on the 4th day after surgery. Also, intrathecal (i.t.) injection of anti-KC antibody prevented mechanical hyperalgesia induced by PLSN when administered at the time of surgery or on the 4th day after surgery. Importantly, the intraneural (i.n.) injection of KC in the mouse sciatic nerve elicited long-lasting mechanical hyperalgesia, which was prevented by the selective CXCR2 antagonist SB225002. The established mechanical hyperalgesia induced by KC was expressively reduced by the treatment with gabapentin, a drug widely used to treat chronic pain in humans. Intraneural KC injection also caused neutrophil migration into the mouse sciatic nerve and the depletion of neutrophils, by pre-treating animals with vinblastine, significantly reduced KC-induced mechanical hyperalgesia. Similar results were obtained for the pre-treatment with indomethacin, a non-selective COX inhibitor. We also demonstrated an increased level of cytokines (IL-1ß, IL-6, and MCP-1, but not TNF-α) after i.n. injection of KC in the mouse sciatic nerve. Together, these findings suggest a role for KC in the development of neuropathic pain in mice by attracting neutrophils to the injured site and increasing the production of proinflammatory mediators. Therefore, strategies to inhibit the action or the release of this chemokine could constitute a therapeutic tool for the management of neuropathic pain in humans.

The role of kinin B1 and B2 receptors in the persistent pain induced by experimental autoimmune encephalomyelitis (EAE) in mice: evidence for the involvement of astrocytes.

Multiple sclerosis (MS) is a progressive, demyelinating inflammatory disease of the human central nervous system (CNS). While the primary symptoms of MS affect motor function, it is now recognized that chronic pain is a relevant symptom that affects both animals and MS patients. There is evidence that glial cells, such as astrocytes, play an important role in the development and maintenance of chronic pain. Kinins, notably bradykinin (BK) acting through B1 (B1R) and B2 (B2R) receptors, play a central role in pain and inflammatory processes. However, it remains unclear whether kinin receptors are involved in neuropathic pain in MS. Here we investigated by genetic and pharmacological approaches the role of kinin receptors in neuropathic pain behaviors induced in the experimental autoimmune encephalomyelitis (EAE) mouse model. Our results showed that gene deletion or antagonism of kinin receptors, especially B1R, significantly inhibited both tactile and thermal hypersensitivity in EAE animals. By contrast, animals with EAE and treated with a B1R selective agonist displayed a significant increase in tactile hypersensitivity. We also observed a marked increase in B1R mRNA and protein level in the mouse spinal cord 14days after EAE immunization. Blockade of B1R significantly suppressed the levels of mRNAs for IL-17, IFN-γ, IL-6, CXCL-1/KC, COX-2 and NOS2, as well as glial activation in the spinal cord. Of note, the selective B1 antagonist DALBK consistently prevented IFN-induced up-regulation of TNF-α and IL-6 release in astrocyte culture. Finally, both B1R and B2R antagonists significantly inhibited COX-2 and NOS2 expression in primary astrocyte culture. The B1R was co-localized with immunomarker of astrocytes in the spinal cord of EAE-treated animals. The above data constitute convincing experimental evidence indicating that both kinin receptors, especially the B1 subtype, exert a critical role in the establishment of persistent hypersensitivity observed in the EAE model, an action that seems to involve a central inflammatory process, possibly acting on astrocytes. Thus, B1 selective antagonists or drugs that reduce kinin release may have the potential to treat neuropathic pain in patients suffering from MS.

Activation of the alpha-7 nicotinic acetylcholine receptor (α7 nAchR) reverses referred mechanical hyperalgesia induced by colonic inflammation in mice.

In the current study, we investigated the effect of the activation of the alpha-7 nicotinic acetylcholine receptor (α7 nAchR) on dextran sulphate sodium (DSS)-induced colitis and referred mechanical hyperalgesia in mice. Colitis was induced in CD1 male mice through the intake of 4% DSS in tap water for 7 days. Control mice received unadulterated water. Referred mechanical hyperalgesia was evaluated for 7 days after the beginning of 4% DSS intake. Referred mechanical hyperalgesia started within 1 day after beginning DSS drinking, peaked at 3 days and persisted for 7 days. This time course profile perfectly matched with the appearance of signs of colitis. Both acute and chronic oral treatments with nicotine (0.1-1.0 mg/kg, p.o.) were effective in inhibiting the established referred mechanical hyperalgesia. The antinociceptive effect of nicotine was completely abrogated by cotreatment with the selective α7 nAchR antagonist methyllycaconitine (MLA) (1.0 mg/kg). Consistent with these results, i.p. treatment with the selective α7 nAchR agonist PNU 282987 (0.1-1.0 mg/kg) reduced referred mechanical hyperalgesia at all periods of evaluation. Despite their antinociceptive effects, nicotinic agonists did not affect DSS-induced colonic damage or inflammation. Taken together, the data generated in the present study show the potential relevance of using α7 nAchR agonists to treat referred pain and discomfort associated with inflammatory bowel diseases.

Plant derived alkaloid (-)-cassine induces anti-inflammatory and anti-hyperalgesics effects in both acute and chronic inflammatory and neuropathic pain models.

Natural products have been revealed as relevant sources of therapeutic agents including those for the management of pain states. In this study, the anti-nociceptive and anti-inflammatory effects of (-)-cassine, isolated from Senna spectabilis were evaluated using pharmacological, behavioural and biochemical approaches. Oral treatment with (-)-cassine (3-30 mg/kg) reduced carrageenan-induced mechanical and thermal nociception associated with the suppression of myeloperoxidase activity in the mouse paw. Moreover, (-)-cassine (1-10 µg/site) prevented mechanical hyperalgesia induced by carrageenan when given through the intraplantar (i.pl.), spinal and intracerebroventricular routes. Additionally, oral treatment with (-)-cassine (3-60 mg/kg) prevented the mechanical hyperalgesia elicited by intraplantar injection of prostaglandin E(2), complete Freund's adjuvant, interleukin-1ß, interleukin-6 and keratinocyte-derived chemokine. Furthermore, (-)-cassine inhibited the mechanical nociceptive response induced by ligation of the sciatic nerve and also significantly reduced the levels of cytokines/chemokines in paw tissue following i.pl. injection of carrageenan. In addition, the anti-nociceptive and anti-inflammatory actions of (-)-cassine were associated with its ability to interact with both TRPV1 and TRPA1 receptors and by inhibiting the upregulation of cyclooxigenase-2 as well as inhibiting the phosphorylation of MAPK/ERK and the transcription factor NF-κB. It is important to highlight that oral treatment with (-)-cassine did not produce any effects related to temperature, locomotor activity or catalepsy. Altogether, the present data demonstrate that (-)-cassine has systemic, spinal and supraspinal anti-nociceptive properties when assessed in inflammatory and neuropathic pain models. These effects are associated with its ability to block several signalling pathways associated with inflammatory and nociceptive responses. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

The role of kinin receptors in preventing neuroinflammation and its clinical severity during experimental autoimmune encephalomyelitis in mice.

BACKGROUND: Multiple sclerosis (MS) is a demyelinating and neuroinflammatory disease of the human central nervous system (CNS). The expression of kinins is increased in MS patients, but the underlying mechanisms by which the kinin receptor regulates MS development have not been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Experimental autoimmune encephalomyelitis (EAE) was induced in female C57BL/6 mice by immunization with MOG(35-55) peptide emulsified in complete Freund's adjuvant and injected with pertussis toxin on day 0 and day 2. Here, we report that blockade of the B(1)R in the induction phase of EAE markedly suppressed its progression by interfering with the onset of the immune response. Furthermore, B(1)R antagonist suppressed the production/expression of antigen-specific T(H)1 and T(H)17 cytokines and transcription factors, both in the periphery and in the CNS. In the chronic phase of EAE, the blockade of B(1)R consistently impaired the clinical progression of EAE. Conversely, administration of the B(1)R agonist in the acute phase of EAE suppressed disease progression and inhibited the increase in permeability of the blood-brain barrier (BBB) and any further CNS inflammation. Of note, blockade of the B(2)R only showed a moderate impact on all of the studied parameters of EAE progression. CONCLUSIONS/SIGNIFICANCE: Our results strongly suggest that kinin receptors, mainly the B(1)R subtype, play a dual role in EAE progression depending on the phase of treatment through the lymphocytes and glial cell-dependent pathways.

Anti-nociceptive effect of kinin B1 and B2 receptor antagonists on peripheral neuropathy induced by paclitaxel in mice.

BACKGROUND AND PURPOSE: In the current study, we investigated the role of both kinin B1 and B2 receptors in peripheral neuropathy induced by the chronic treatment of mice with paclitaxel a widely used chemotherapeutic agent. EXPERIMENTAL APPROACH: Chemotherapy-evoked hyperalgesia was induced by i.p. injections of paclitaxel (2 mg·kg⁻¹) over 5 consecutive days. Mechanical and thermal hyperalgesia were evaluated between 7 and 21 days after the first paclitaxel treatment. KEY RESULTS: Treatment with paclitaxel increased both mechanical and thermal hyperalgesia in mice (C57BL/6 and CD1 strains). Kinin receptor deficient mice (B1, or B2 receptor knock-out and B1B2 receptor, double knock-out) presented a significant reduction in paclitaxel-induced hypernociceptive responses in comparison to wild-type animals. Treatment of CD1 mice with kinin receptor antagonists (DALBK for B1 or Hoe 140 for B2 receptors) significantly inhibited both mechanical and thermal hyperalgesia when tested at 7 and 14 days after the first paclitaxel injection. DALBK and Hoe 140 were also effective against paclitaxel-induced peripheral neuropathy when given intrathecally or i.c.v. A marked increase in B1 receptor mRNA was observed in the mouse thalamus, parietal and pre-frontal cortex from 7 days after the first paclitaxel treatment. CONCLUSIONS AND IMPLICATIONS: Kinins acting on both B1 and B2 receptors, expressed in spinal and supra-spinal sites, played a crucial role in controlling the hypernociceptive state caused by chronic treatment with paclitaxel.

Mechanisms involved in IL-6-induced muscular mechanical hyperalgesia in mice.

Interleukin-6 (IL-6) is an inflammatory cytokine known to modulate muscle pain. However, the mechanisms underlying this effect still remain unclear. Here we show that the injection of IL-6 into mice gastrocnemius muscle evoked a time- and dose-dependent mechanical hyperalgesia. This effect is in part dependent on the presence of gp130 expression in inflammatory cells in the gastrocnemius muscle as well as in DRG neurons. We also demonstrated an increased inflammatory cell recruitment and cytokines levels, namely TNF-α, IL-1ß and KC. TNFR1(-/-) mice or mice pre-treated with the selective CXCR2 antagonist, SB225002, with the anti-macrophage, anti-TNF-α or anti-KC antibodies or with IL-1 receptor antagonist (IL-1RA) showed decreased IL-6-mediated mechanical hyperalgesia. Furthermore, systemic pre-treatment with the classically used drugs indomethacin, celecoxib, guanetidine, morphine, thalidomide or dexamethasone, also prevented IL-6-induced muscle pain. Likewise, local pre-treatment with inhibitors of phospholipase A2 (PACOCF3), phospholipase C (U73122), protein kinase C (GF109203X), protein kinase A (KT-5720) or with phosphatidylinositol 3-kinase (AS605204) also consistently diminished IL-6-induced muscle hyperalgesia. The intramuscular injection of the selective inhibitors of p38 MAPK (SB203580), ERK (PD98059) or JNK (SP60015) also prevented IL-6-mediated muscular pain. Simultaneous flow cytometry measurements revealed that ERK, p38 MAPK and JNK were phosphorylated as early as 5 min after IL-6 injection. These findings provided new evidence indicating that IL-6 exerts a relevant role in the development and maintenance of muscular hyperalgesia. The IL-6-mediated muscular pain response involves resident cell activation, polymorphonuclear cell infiltration, cytokine production, prostanoids and sympathomimetic amines release and the activation of intracellular pathways, especially MAPKs.

The role of kinin B1 and B2 receptors in the scratching behaviour induced by proteinase-activated receptor-2 agonists in mice.

BACKGROUND AND PURPOSE: Activation of the proteinase-activated receptor-2 (PAR-2) induces scratching behaviour in mice. Here, we have investigated the role of kinin B(1) and B(2) receptors in the pruritogenic response elicited by activators of PAR-2. EXPERIMENTAL APPROACH: Scratching was induced by an intradermal (i.d.) injection of trypsin or the selective PAR-2 activating peptide SLIGRL-NH(2) at the back of the mouse neck. The animals were observed for 40 min and their scratching response was quantified. KEY RESULTS: I.d. injection of trypsin or SLIGRL-NH(2) evoked a scratching behaviour, dependent on PAR-2 activation. Mice genetically deficient in kinin B(1) or B(2) receptors exhibited reduced scratching behaviour after i.d. injection of trypsin or SLIGRL-NH(2). Treatment (i.p.) with the non-peptide B(1) or B(2)receptor antagonists SSR240612 and FR173657, respectively, prevented the scratching behaviour caused by trypsin or SLIGRL-NH(2). Nonetheless, only treatment i.p. with the peptide B(2)receptor antagonist, Hoe 140, but not the B(1)receptor antagonist (DALBK), inhibited the pruritogenic response to trypsin. Hoe 140 was also effective against SLIGRL-NH(2)-induced scratching behaviour when injected by i.d. or intrathecal (i.t.) routes. Also, the response to SLIGRL-NH(2) was inhibited by i.t. (but not by i.d.) treatment with DALBK. Conversely, neither Hoe 140 nor DALBK were able to inhibit SLIGRL-NH(2)-induced scratching behaviour when given intracerebroventricularly (i.c.v.). CONCLUSIONS AND IMPLICATIONS: The present results demonstrated that kinins acting on both B(1) and B(2) receptors played a crucial role in controlling the pruriceptive signalling triggered by PAR-2 activation in mice.

The effects of the selective and non-peptide CXCR2 receptor antagonist SB225002 on acute and long-lasting models of nociception in mice.

This study evaluated the antinociceptive effects of the selective and non-peptide CXCR2 antagonist SB225002 in mouse models of pain. As assessed in different tests of spontaneous nociception, intraperitoneal (i.p.) administration of SB225002 caused consistent and dose-related reduction of acetic acid-induced abdominal constrictions, whereas it did not significantly affect the nociception evoked by formalin, capsaicin, glutamate or phorbol ester acetate (PMA). Systemic treatment with SB225002 strikingly reduced the spontaneous nociception induced by 8-bromo-cAMP (8-Br-cAMP), or mechanical hypernociception induced by prostaglandin E(2) (PGE(2)), epinephrine, or the keratinocyte-derived chemokine (KC). In the carrageenan model, SB225002 markedly reduced mechanical hypernociception when administered by i.p., intrathecal (i.t.) or intracerebroventricular (i.c.v.) routes, or even when co-administered with carrageenan into the mouse paw, indicating peripheral and central sites of action for SB225002. In addition, i.p. treatment with SB225002 significantly attenuated the increase in MPO activity or the elevation of IL-1beta, TNFalpha or KC levels following carrageenan injection. In the persistent models of pain evoked by complete Freund's adjuvant (CFA) or by the partial ligation of the sciatic nerve (PLSN), the repeated administration of SB225002 displayed prominent and long-lasting antinociceptive effects. Notably, SB225002 did not evoke unspecific central effects, as evaluated in the open-field and rota-rod tests, or even in the latency responses for thermal stimuli. Our data confirm the previous notion on the critical role exerted by chemokines in pain, indicating that selective CXCR2 antagonists, such as SB225002, might well represent interesting and innovative alternatives for the management of both acute and chronic pain.