Non-Peptidergic Nociceptive Neurons Are Essential for Mechanical Inflammatory Hypersensitivity in Mice.

Small nerve fibers that bind the isolectin B4 (IB4+ C-fibers) are a subpopulation of primary afferent neurons that are involved in nociceptive sensory transduction and do not express the neuropeptides substance P and calcitonin-gene related peptide (CGRP). Several studies have attempted to elucidate the functional role of IB4+-nociceptors in different models of pain. However, a functional characterization of the non-peptidergic nociceptors in mediating mechanical inflammatory hypersensitivity in mice is still lacking. To this end, in the present study, the neurotoxin IB4-Saporin (IB4-Sap) was employed to ablate non-peptidergic C-fibers. Firstly, we showed that intrathecal (i.t.) administration of IB4-Sap in mice depleted non-peptidergic C-fibers, since it decreased the expression of purinoceptor 3 (P2X3) and transient receptor potential cation channel subfamily V member 1 (TRPV1) in the dorsal root ganglia (DRGs) as well as IB4 labelling in the spinal cord. Non-peptidergic C-fibers depletion did not alter the mechanical nociceptive threshold, but it inhibited the mechanical inflammatory hypersensitivity induced by glial cell-derived neurotrophic factor (GDNF), but not nerve growth factor (NGF). Depletion of non-peptidergic C-fibers abrogated mechanical inflammatory hypersensitivity induced by carrageenan. Finally, it was found that the inflammatory mediators PGE2 and epinephrine produced a mechanical inflammatory hypersensitivity that was also blocked by depletion of non-peptidergic C-fibers. These data suggest that IB4-positive nociceptive nerve fibers are not involved in normal mechanical nociception but are sensitised by inflammatory stimuli and play a crucial role in mediating mechanical inflammatory hypersensitivity.

Non-peptidergic nociceptive neurons are essential for mechanical inflammatory hypersensitivity in mice

Small nerve fibers that bind the isolectin B4 (IB4+ C-fibers) are a subpopulation of primary afferent neurons that are involved in nociceptive sensory transduction and do not express the neuropeptides substance P and calcitonin-gene related peptide (CGRP). Several studies have attempted to elucidate the functional role of IB4+-nociceptors in different models of pain. However, a functional characterization of the non-peptidergic nociceptors in mediating mechanical inflammatory hypersensitivity in mice is still lacking. To this end, in the present study, the neurotoxin IB4-Saporin (IB4-Sap) was employed to ablate non-peptidergic C-fibers. Firstly, we showed that intrathecal (i.t.) administration of IB4-Sap in mice depleted non-peptidergic C-fibers, since it decreased the expression of purinoceptor 3 (P2X3) and transient receptor potential cation channel subfamily V member 1 (TRPV1) in the dorsal root ganglia (DRGs) as well as IB4 labelling in the spinal cord. Non-peptidergic C-fibers depletion did not alter the mechanical nociceptive threshold, but it inhibited the mechanical inflammatory hypersensitivity induced by glial cell-derived neurotrophic factor (GDNF), but not nerve growth factor (NGF). Depletion of non-peptidergic C-fibers abrogated mechanical inflammatory hypersensitivity induced by carrageenan. Finally, it was found that the inflammatory mediators PGE2 and epinephrine produced a mechanical inflammatory hypersensitivity that was also blocked by depletion of non-peptidergic C-fibers. These data suggest that IB4-positive nociceptive nerve fibers are not involved in normal mechanical nociception but are sensitised by inflammatory stimuli and play a crucial role in mediating mechanical inflammatory hypersensitivity.

Anti-allodynic Effect of Mangiferin in Rats With Chronic Post-ischemia Pain: A Model of Complex Regional Pain Syndrome Type I.

The present study reproduces chronic post-ischemia pain (CPIP), a model of complex regional pain syndrome type I (CRPS-I), in rats to examine the possible transient and long-term anti-allodynic effect of mangiferin (MG); as well as its potential beneficial interactions with some standard analgesic drugs and sympathetic-mediated vasoconstriction and vasodilator agents during the earlier stage of the pathology. A single dose of MG (50 and 100 mg/kg, p.o.) decreased mechanical allodynia 72 h post-ischemia-reperfusion (I/R). MG 100 mg/kg, i.p. (pre- vs. post-drug) increased von Frey thresholds in a yohimbine and naloxone-sensitive manner. Sub-effective doses of morphine, amitriptyline, prazosin, clonidine and a NO donor, SIN-1, in the presence of MG were found to be significantly anti-allodynic. A long-term anti-allodynic effect at 7 and 13 days post-I/R after repeated oral doses of MG (50 and 100 mg/kg) was also observed. Further, MG decreased spinal and muscle interleukin-1ß concentration and restored muscle redox status. These results indicate that MG has a transient and long-term anti-allodynic effect in CPIP rats that appears to be at least partially attributable to the opioid and α2 adrenergic receptors. Additionally, its anti-inflammatory and antioxidant mechanisms could also be implicated in this effect. The association of MG with sub-effective doses of these drugs enhances the anti-allodynic effect; however, an isobolographic analysis should be performed to define a functional interaction between them. These findings suggest the possible clinical use of MG in the treatment of CRPS-I in both early sympathetically maintained pain and long-term sympathetically independent pain.

Pharmacological Beta-Adrenergic Receptor Activation Attenuates Neutrophil Recruitment by a Mechanism Dependent on Nicotinic Receptor and the Spleen.

The aim of this study was to identify the effect of beta-adrenergic receptor activation on neutrophil migration in experimental peritonitis elucidating the neuroimmune components involved such as nicotinic receptors and the spleen. Mice pre-treated with mecamylamine (nicotinic antagonist) and propranolol (beta-adrenergic antagonist) or splenectomized animals were treated with isoproterenol (beta-adrenergic agonist) prior to intraperitoneal injection of carrageenan. After 4 h, the infiltrating neutrophils and the local cytokine/chemokine levels were evaluated in the peritoneal lavage. The effect of isoproterenol on neutrophil chemotaxis was investigated in a Boyden chamber. Isoproterenol inhibited neutrophil trafficking, reducing the cytokine/chemokine release and neutrophil chemotaxis. Surprisingly, the isoproterenol effect on neutrophil migration was totally reverted by splenectomy and mecamylamine pre-treatment. In contrast, the inhibitory effect of nicotine on neutrophil migration was abrogated only by splenectomy but not by propranolol pre-treatment. Collectively, our data show that beta-adrenergic receptor activation regulates the acute neutrophil recruitment via splenic nicotinic receptor.

Medial plantar nerve ligation as a novel model of neuropathic pain in mice: pharmacological and molecular characterization.

Peripheral neuropathic pain is a consequence of an injury/disease of the peripheral nerves. The mechanisms involved in its pathophysiology are not entirely understood. To better understand the mechanisms involved in the development of peripheral nerve injury-induced neuropathic pain, more experimental models are required. Here, we developed a novel peripheral neuropathic pain model in mice by using a minimally invasive surgery and medial plantar nerve ligation (MPNL). After MPNL, mechanical allodynia was established, and mice quickly recovered from the surgery without any significant motor impairment. MPNL causes an increased expression of ATF-3 in the sensory neurons. At 14 days after surgery, gabapentin was capable of reversing the mechanical allodynia, whereas anti-inflammatory drugs and opioids were ineffective. MPNL-induced neuropathic pain was mediated by glial cells activation and the production of TNF-α and IL-6 in the spinal cord. These results indicate MPNL as a reasonable animal model for the study of peripheral neuropathic pain, presenting analgesic pharmacological predictivity to clinically used drugs. The results also showed molecular phenotypic changes similar to other peripheral neuropathic pain models, with the advantage of a lack of motor impairment. These features indicate that MPNL might be more appropriate for the study of neuropathic pain than classical models.

Joint production of IL-22 participates in the initial phase of antigen-induced arthritis through IL-1ß production.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by neutrophil articular infiltration, joint pain and the progressive destruction of cartilage and bone. IL-22 is a key effector molecule that plays a critical role in autoimmune diseases. However, the function of IL-22 in the pathogenesis of RA remains controversial. In this study, we investigated the role of IL-22 in the early phase of antigen-induced arthritis (AIA) in mice. METHODS: AIA was induced in C57BL/6, IL-22(-/-), ASC(-/-) and IL-1R1(-/-) immunized mice challenged intra-articularly with methylated bovine serum albumin (mBSA). Expression of IL-22 in synovial membranes was determined by RT-PCR. Articular hypernociception was evaluated using an electronic von Frey. Neutrophil recruitment and histopathological analyses were assessed in inflamed knee joint. Joint levels of inflammatory mediators and mBSA-specific IgG concentration in the serum were measured by ELISA. RESULTS: The IL-22 mRNA expression and protein levels in synovial tissue were increased during the onset of AIA. In addition, pharmacological inhibition (anti-IL-22 antibody) and genetic deficiency (IL-22(-/-) mice) reduced articular pain and neutrophil migration in arthritic mice. Consistent with these findings, recombinant IL-22 joint administration promoted articular inflammation per se in WT mice, restoring joint nociception and neutrophil infiltration in IL-22(-/-) mice. Moreover, IL-22-deficient mice showed reduced synovitis (inflammatory cell influx) and lower joint IL-1ß levels, whereas the production of IL-17, MCP-1/CCL2, and KC/CXCL1 and the humoral immune response were similar, compared with WT mice. Corroborating these results, the exogenous administration of IL-22 into the joints induced IL-1ß production in WT mice and reestablished IL-1ß production in IL-22(-/-) mice challenged with mBSA. Additionally, IL-1R1(-/-) mice showed attenuated inflammatory features induced by mBSA or IL-22 challenge. Articular nociception and neutrophil migration induced by IL-22 were also reduced in ASC(-/-) mice. CONCLUSIONS: These results suggest that IL-22 plays a pro-inflammatory/pathogenic role in the onset of AIA through an ASC-dependent stimulation of IL-1ß production.

Involvement of nuclear factor kappa B in the maintenance of persistent inflammatory hypernociception.

The pathophysiology of chronic inflammatory pain remains poorly understood. In this context, we developed an experimental model in which successive daily injection of prostaglandin E2 (PGE2) for 14days into rat hind paws produces a persistent state of hypernociception (i.e. decrease in mechanical nociceptive threshold). This state persists for more than 30days after discontinuing PGE2 injection. In the present study, we investigated the participation of nuclear factor kappa B (NF-κB), in the maintenance of this process. Mechanical hypernociception was evaluated using the electronic von Frey test. Activation of NF-κB signaling was measured through the determination of NF-κB p65 subunit translocation to the nucleus of dorsal root ganglion neurons (DRG) by immunofluorescence and western blotting. Herein, we detected an increase in NF-κB p65 subunit translocation to the nucleus of DRG neurons along with persistent inflammatory hypernociception compared with controls. Intrathecal treatment with either dexamethasone or PDTC (NF-κB activation inhibitor) after ending of the induction phase of the persistent inflammatory hypernociception, curtailed the hypernociception period as well as reducing NF-κB p65 subunit translocation. Treatment with antisense oligonucleotides against the NF-κB p65 subunit for 5 consecutive days also reduced persistent inflammatory hypernociception. Inhibition of PKA and PKCε reduced persistent inflammatory hypernociception, which was associated with inhibition of NF-κB p65 subunit translocation. Together these results suggest that peripheral activation of NF-κB by PKA and PKC in primary sensory neurons plays an important role in maintaining persistent inflammatory pain.

Interleukin-10 limits intense acute swimming-induced muscle mechanical hyperalgesia in mice.

NEW FINDINGS: What is the central question of this study? This study investigated the role of the endogenous anti-inflammatory cytokine interleukin-10 in intense acute swimming-induced muscle mechanical hyperalgesia in mice. What is the main finding and its importance? Endogenous interleukin-10 has a key role in limiting exercise-induced muscle pain in a model presenting similarities to delayed-onset muscle soreness in mice. Interleukin-10 reduced muscle pain by diminishing leucocyte recruitment, hyperalgesic cytokine production, oxidative stress and myocyte damage. Interleukin-10 (IL-10) is an antihyperalgesic cytokine. In this study, IL-10-deficient (IL-10(-/-) ) mice were used to investigate the role of endogenous IL-10 in intense acute swimming-induced muscle mechanical hyperalgesia, which presents similarities with delayed-onset muscle soreness. An intense acute swimming session of 1 or 2 h induced significant muscle mechanical hyperalgesia in a time-dependent manner in wild-type mice compared with the sham group 24 h after the session, which was further increased in IL-10(-/-) mice (P Ë‚ 0.05). Intraperitoneal treatment of wild-type mice with IL-10 (1-10 ng) reduced muscle mechanical hyperalgesia in a dose-dependent manner and reversed the enhanced muscle hyperalgesia in IL-10(-/-) mice (P Ë‚ 0.05). The 2 h swimming session induced increases in tumour necrosis factor-α, interleukin-1ß and IL-10 production in the soleus muscle. However, tumour necrosis factor-α and interleukin-1ß production in the soleus muscle were even higher in IL-10(-/-) mice between 2 and 6 h after the stimulus (P Ë‚ 0.05). There was no statistical difference in the levels of the antihyperalgesic cytokines interleukin-4, interleukin-5, interleukin-13 and transforming growth factor-ß between wild-type and IL-10(-/-) mice (P Ëƒ 0.05). Interleukin-10 deficiency also resulted in increased myeloperoxidase activity, greater depletion of reduced glutathione levels, increased superoxide anion production and the maintenance of high plasma concentrations of creatine kinase (until 24 h after the swimming session) in soleus muscle (P Ë‚ 0.05). These results demonstrate that endogenous IL-10 controls intense acute swimming-induced muscle mechanical hyperalgesia by limiting oxidative stress and cytokine production.

Anti-hypernociceptive effect of mangiferin in persistent and neuropathic pain models in rats.

The present study examines the possible effect of the glucosylxanthone mangiferin (MG) on pain-related behaviors in a tonic acute pain model (formalin test at 5%) and in a chronic constriction injury (CCI) model to clarify the underlying transient and long-term mechanisms. Acute administration of MG (10-100mg/kg, i.p.) reduced licking/biting exclusivity in the tonic phase of formalin test in a naloxone and yohimbine-sensitive manner. This effect was enhanced by a nonselective nitric oxide synthase (NOS) inhibitor (NG-monomethyl-L-arginine) and by a non-competitive N-methyl-D-aspartate (NMDA) antagonist (ketamine), but it was reversed by the NOS substrate (L-arginine). Pre-treatment with intrathecal yohimbine prevented the anti-hypernociceptive effect of systemic MG. Pre-treatment during 4 days before surgical and 3 days after CCI with MG (50mg/kg, i.p.) reduced mechanical hypernociception and decreased the signs of Wallerian degeneration (WD) of the sciatic nerve. MG improved the PC-12 cellular viability exposure to glutamate-mediated neuronal death, also involved in neuropathic pain. The findings of this study suggest that MG shows ability to decrease tonic pain in the formalin test. A transient activity of this xanthone on nociceptive pathways mediated by α2 adrenergic receptors in cooperation with the opioid system could be involved, at least in part, in this effect. Its neuroprotective effect by preventing WD in mononeuropathic rats could be implicated in the mechano-antihypernociceptive long term mechanisms.

Nitroxyl inhibits overt pain-like behavior in mice: role of cGMP/PKG/ATP-sensitive potassium channel signaling pathway.

BACKGROUND: Several lines of evidence have indicated that nitric oxide (NO) plays complex and diverse roles in modulation of pain/analgesia. However, the roles of charged and uncharged congeners of NO are less well understood. In the present study, the antinociceptive effect of the nitroxyl (HNO) donor, Angeli's salt (Na2N2O3; AS) was investigated in models of overt pain-like behavior. Moreover, whether the antinociceptive effect of nitroxyl was dependent on the activation of cGMP (cyclic guanosine monophosphate)/PKG (protein kinase G)/ATP-sensitive potassium channels was addressed. METHODS: The antinociceptive effect of AS was evaluated on phenyl-p-benzoquinone (PBQ)- and acetic acid-induced writhings and via the formalin test. In addition, pharmacological treatments targeting guanylate cyclase (ODQ), PKG (KT5923) and ATP-sensitive potassium channel (glybenclamide) were used. RESULTS: PBQ and acetic acid induced significant writhing responses over 20min. The nociceptive response in these models were significantly reduced in a dose-dependent manner by subcutaneous pre-treatment with AS. Furthermore, AS also inhibited both phases of the formalin test. Subsequently, the inhibitory effect of AS in writhing and flinching responses were prevented by ODQ, KT5823 and glybenclamide, although these inhibitors alone did not alter the writhing score. Furthermore, pretreatment with L-cysteine, an HNO scavenger, confirmed that the antinociceptive effect of AS depends on HNO. CONCLUSION: The present study demonstrates the efficacy of a nitroxyl donor and its analgesic mechanisms in overt pain-like behavior by activating the cGMP/PKG/ATP-sensitive potassium channel (K(+)) signaling pathway.

Targeting interleukin-1ß reduces intense acute swimming-induced muscle mechanical hyperalgesia in mice.

OBJECTIVES: The role of interleukin (IL)-1ß in intense acute swimming-induced muscle mechanical hyperalgesia was investigated in mice. METHODS: Untrained mice were submitted to one session of intense acute swimming for 120 min or were submitted to sham conditions (30 s exposure to water), and muscle mechanical hyperalgesia (before and 6-48 h after swimming session), IL-1ß production (skeletal muscle and spinal cord), myeloperoxidase activity, reduced glutathione (GSH) levels (skeletal muscle and spinal cord), and cortisol, glucose, lactate and creatine kinase (CK) levels (plasma) were analysed. KEY FINDINGS: Intense acute swimming-induced muscle mechanical hyperalgesia was dose-dependently inhibited by IL-1ra treatment. IL-1ß levels were increased in soleus, but not gastrocnemius muscle and spinal cord 2 and 4 h after the session, respectively. Intense acute swimming-induced increase of myeloperoxidase activity and reduced GSH levels in soleus muscle were reversed by IL-1ra treatment. In the spinal cord, exercise induced an increase of GSH levels, which was reduced by IL-1ra. Finally, IL-1ra treatment reduced plasma levels of CK, an indicator of myocyte damage. CONCLUSIONS: IL-1ß mediates intense acute swimming-induced muscle mechanical hyperalgesia by peripheral (soleus muscle) and spinal cord integrative mechanisms and could be considered a potential target to treat exercise-induced muscle pain.

Dipyrone metabolite 4-MAA induces hypothermia and inhibits PGE2 -dependent and -independent fever while 4-AA only blocks PGE2 -dependent fever.

BACKGROUND AND PURPOSE: The antipyretic and hypothermic prodrug dipyrone prevents PGE2 -dependent and -independent fever induced by LPS from Escherichia coli and Tityus serrulatus venom (Tsv) respectively. We aimed to identify the dipyrone metabolites responsible for the antipyretic and hypothermic effects. EXPERIMENTAL APPROACH: Male Wistar rats were treated i.p. with indomethacin (2 mg·kg(-1) ), dipyrone, 4-methylaminoantipyrine (4-MAA), 4-aminoantipyrine (4-AA) (60-360 mg·kg(-1) ), 4-formylaminoantipyrine, 4-acethylaminoantipyrine (120-360 mg·kg(-1) ) or vehicle 30 min before i.p. injection of LPS (50 µg·kg(-1) ), Tsv (150 µg·kg(-1) ) or saline. Rectal temperatures were measured by tele-thermometry and dipyrone metabolite concentrations determined in the plasma, CSF and hypothalamus by LC-MS/MS. PGE2 concentrations were determined in the CSF and hypothalamus by elisa. KEY RESULTS: In contrast to LPS, Tsv-induced fever was not followed by increased PGE2 in the CSF or hypothalamus. The antipyretic time-course of 4-MAA and 4-AA on LPS-induced fever overlapped with the period of the highest concentrations of 4-MAA and 4-AA in the hypothalamus, CSF and plasma. These metabolites reduced LPS-induced fever and the PGE2 increase in the plasma, CSF and hypothalamus. Only 4-MAA inhibited Tsv-induced fever. The higher doses of dipyrone and 4-MAA also induced hypothermia. CONCLUSIONS AND IMPLICATIONS: The presence of 4-MAA and 4-AA in the CSF and hypothalamus was associated with PGE2 synthesis inhibition and a decrease in LPS-induced fever. 4-MAA was also shown to be an antipyretic metabolite for PGE2 -independent fever induced by Tsv suggesting that it is responsible for the additional antipyretic mechanism of dipyrone. Moreover, 4-MAA is the hypothermic metabolite of dipyrone.

Lipopolysaccharide induces inflammatory hyperalgesia triggering a TLR4/MyD88-dependent cytokine cascade in the mice paw.

Inflammatory pain can be triggered by different stimuli, such as trauma, radiation, antigen and infection. In a model of inflammatory pain caused by infection, injection in the mice paw of lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) agonist, produces mechanical hyperalgesia. We identify here the TLR4 linked signaling pathways that elicit this response. Firstly, LPS paw injection in wild type (WT) mice produced mechanical hyperalgesia that was not altered in TRIF-/- mice. On the other hand, this response was absent in TLR4 mutant and MyD88 null mice and reduced in TNFR1 null mice. Either an IL-1 receptor antagonist, anti-KC/CXCL1 antibody, indomethacin or guanethidine injection also lessened this response. Moreover, LPS-induced time dependent increases in TNF-α, KC/CXCL1 and IL-1ß expression in the mice paw, which were absent in TLR4 mutant and MyD88 null mice. Furthermore, in TNFR1 deficient mice, the LPS-induced rises in KC/CXCL1 and IL-1ß release were less than in their wild type counterpart. LPS also induced increase of myeloperoxidase activity in the paw skin, which was inhibited in TLR4 mutant and MyD88 null mice, and not altered in TRIF-/- mice. These results suggest that LPS-induced inflammatory pain in mice is solely dependent on the TLR4/MyD88 rather than the TLR4/TRIF signaling pathway. This pathway triggers pronociceptive cytokine TNF-α release that in turn mediates rises in KC/CXCL1 and IL-1ß expression. Finally, these cytokines might be involved in stimulating production of directly-acting hyperalgesic mediators such as prostaglandins and sympathomimetic amine.

Role of TNF-α/TNFR1 in intense acute swimming-induced delayed onset muscle soreness in mice.

The injection of cytokines such as TNF-α induces muscle pain. Herein, it was addressed the role of endogenous TNF-α/TNFR1 signaling in intense acute swimming-induced muscle mechanical hyperalgesia in mice. Mice were exposed to water during 30 s (sham) or to a single session of 30-120 min of swimming. Intense acute swimming induced a dose-dependent (time of exercise-dependent) muscle mechanical hyperalgesia, which peaked after 24 h presenting characteristics of delayed onset muscle soreness (DOMS). The intense acute swimming (120 min)-induced muscle mechanical hyperalgesia was reduced in etanercept (soluble TNF receptor) treated and TNFR1 deficient ((-/-)) mice. TNF-α levels increased 2 and 4 h after intense acute swimming in soleus muscle (but not in gastrocnemius), and spinal cord, respectively. Exercise induced an increase of myeloperoxidase activity and decrease in reduced glutathione levels in an etanercept-sensitive and TNFR1-dependent manners in the soleus muscle, but not in the gastrocnemius muscle. Concluding, TNF-α/TNFR1 signaling mediates intense acute swimming-induced DOMS by an initial role in the soleus muscle followed by spinal cord, inducing muscle inflammatory hyperalgesia and oxidative stress. The knowledge of these mechanisms might contribute to improve the training of athletes, individuals with physical impairment and intense training such as military settings.

5-lipoxygenase deficiency reduces acetaminophen-induced hepatotoxicity and lethality.

5-Lipoxygenase (5-LO) converts arachidonic acid into leukotrienes (LTs) and is involved in inflammation. At present, the participation of 5-LO in acetaminophen (APAP)-induced hepatotoxicity and liver damage has not been addressed. 5-LO deficient (5-LO⁻/⁻) mice and background wild type mice were challenged with APAP (0.3-6 g/kg) or saline. The lethality, liver damage, neutrophil and macrophage recruitment, LTB4, cytokine production, and oxidative stress were assessed. APAP induced a dose-dependent mortality, and the dose of 3 g/kg was selected for next experiments. APAP induced LTB4 production in the liver, the primary target organ in APAP toxicity. Histopathological analysis revealed that 5-LO⁻/⁻ mice presented reduced APAP-induced liver necrosis and inflammation compared with WT mice. APAP-induced lethality, increase of plasma levels of aspartate aminotransferase and alanine aminotransferase, liver cytokine (IL-1ß, TNF-α , IFN- γ, and IL-10), superoxide anion, and thiobarbituric acid reactive substances production, myeloperoxidase and N-acetyl-ß-D-glucosaminidase activity, Nrf2 and gp91(phox) mRNA expression, and decrease of reduced glutathione and antioxidant capacity measured by 2,2'-azinobis(3-ethylbenzothiazoline 6-sulfonate) assay were prevented in 5-LO⁻/⁻ mice compared to WT mice. Therefore, 5-LO deficiency resulted in reduced mortality due to reduced liver inflammatory and oxidative damage, suggesting 5-LO is a promising target to reduce APAP-induced lethality and liver inflammatory/oxidative damage.

Fractalkine mediates inflammatory pain through activation of satellite glial cells.

The activation of the satellite glial cells (SGCs) surrounding the dorsal root ganglion (DRG) neurons appears to play a role in pathological pain. We tested the hypothesis that fractalkine, which is constitutively expressed by primary nociceptive neurons, is the link between peripheral inflammation and the activation of SGCs and is thus responsible for the genesis of the inflammatory pain. The injection of carrageenin into the rat hind paw induced a decrease in the mechanical nociceptive threshold (hypernociception), which was associated with an increase in mRNA and GFAP protein expression in the DRG. Both events were inhibited by anti-fractalkine antibody administered directly into the DRG (L5) [intraganglionar (i.gl.)]. The administration of fractalkine into the DRG (L5) produced mechanical hypernociception in a dose-, time-, and CX3C receptor-1 (CX3CR1)-dependent manner. Fractalkine's hypernociceptive effect appears to be indirect, as it was reduced by local treatment with anti-TNF-α antibody, IL-1-receptor antagonist, or indomethacin. Accordingly, the in vitro incubation of isolated and cultured SGC with fractalkine induced the production/release of TNF-α, IL-1ß, and prostaglandin E2. Finally, treatment with i.gl. fluorocitrate blocked fractalkine (i.gl.)- and carrageenin (paw)-induced hypernociception. Overall, these results suggest that, during peripheral inflammation, fractalkine is released in the DRG and contributes to the genesis of inflammatory hypernociception. Fractalkine's effect appears to be dependent on the activation of the SGCs, leading to the production of TNFα, IL-1ß, and prostanoids, which are likely responsible for the maintenance of inflammatory pain. Thus, these results indicate that the inhibition of fractalkine/CX3CR1 signaling in SGCs may serve as a target to control inflammatory pain.

Skin vasodilation and analgesic effect of a topical nitric oxide-releasing hydrogel.

New approaches based on topical treatments are needed for treating pain and impaired dermal blood flow. We used a topical Pluronic F127 hydrogel containing S-nitrosoglutathione (GSNO) as a prodrug to generate free NO, an effector molecule that exerts both dermal vasodilation and antinociceptive effects. GSNO-containing hydrogels underwent gelation above 12 °C and released free NO at rates that were directly dependent on the GSNO concentration in the range of 50-150 mM. The topical application of this material led to dose-response dermal vasodilation in healthy volunteers and to a reduction of up to 50 % of the hypernociception intensity in Wistar rats that were subjected to inflammatory pain. Mechanistic investigations indicated that the antinociceptive effect of the topical F127/GSNO hydrogels is produced by the local activation of the cGMP/PKG/KATP channel-signaling pathway, which was stimulated by the free NO that diffused through the skin. These results expand the scope of the biomedical applications of this material and may represent a new approach for the topical treatment of inflammatory pain.

The nitroxyl donor, Angeli's salt, inhibits inflammatory hyperalgesia in rats.

Nitric oxide modulates pain development. However, there is no evidence on the effect of nitroxyl (HNO/NO⁻) in nociception. Therefore, we addressed whether nitroxyl inhibits inflammatory hyperalgesia and its mechanism using the nitroxyl donor Angeli's salt (AS; Na2N2O3). Mechanical hyperalgesia was evaluated using a modified Randall and Selitto method in rats, cytokine production by ELISA and nitroxyl was determined by confocal microscopy in DAF (a cell permeable reagent that is converted into a fluorescent molecule by nitrogen oxides)-treated dorsal root ganglia neurons in culture. Local pre-treatment with AS (17-450 µg/paw, 30 min) inhibited the carrageenin-induced mechanical hyperalgesia in a dose- and time-dependent manner with maximum inhibition of 97%. AS also inhibited carrageenin-induced cytokine production. AS inhibited the hyperalgesia induced by other inflammatory stimuli including lipopolysaccharide, tumor necrosis factor-α, interleukin-1ß and prostaglandin E2. Furthermore, the analgesic effect of AS was prevented by treatment with ODQ (a soluble guanylate cyclase inhibitor), KT5823 (a protein kinase G [PKG] inhibitor) or glybenclamide (an ATP-sensitive K⁺ channel blocker), but not with naloxone (an opioid receptor antagonist). AS induced concentration-dependent increase in fluorescence intensity of DAF-treated neurons in a l-cysteine (nitroxyl scavenger) sensitive manner. l-cysteine did not affect the NO⁺ donor S-Nitroso-N-acetyl-DL- penicillamine (SNAP)-induced anti-hyperalgesia or fluorescence of DAF-treated neurons. This is the first study to demonstrate that nitroxyl inhibits inflammatory hyperalgesia by reducing cytokine production and activating the cGMP/PKG/ATP-sensitive K⁺ channel signaling pathway in vivo.

The role of PAF/PAFR signaling in zymosan-induced articular inflammatory hyperalgesia.

Platelet-activating factor (PAF) and its receptor (PAFR) have been shown to be involved in several inflammatory events, including neutrophil chemoattraction and nociception. The present study addressed the role of PAF in the genesis of articular hyperalgesia in a model of joint inflammation. Zymosan-induced articular hyperalgesia, oedema and neutrophil migration were dose-dependently reduced following pretreatment with selective PAFR antagonists, UK74505 (5, 10 and 20 mg/kg) and PCA4248 (3, 10, 30 mg/kg). These parameters were also reduced in PAF receptor-deficient mice (PAFR(-/-)). The hyperalgesic action of PAF was further confirmed by the demonstration that joint injection of PAF induces a dose- (0.3, 1 and 3 µg/joint), time- and PAFR-dependent articular hyperalgesia and oedema. The PAF hyperalgesic mechanisms were dependent on prostaglandins, leukotrienes and neutrophils, as PAF-induced articular hyperalgesia was inhibited by indomethacin (COX inhibitor), MK886 (leukotrienes synthesis inhibitor) or fucoidan (leukocyte rolling inhibitor). Furthermore, PAF-induced hyperalgesia was reduced in 5-lypoxigenase-null mice. In corroboration of these findings, intra-articular injection of PAF promotes the production of LTB(4) as well as the recruitment of neutrophils to the joint. These results suggest that PAF may participate in the cascade of events involved in the genesis of articular inflammatory hyperalgesia via stimulation of prostaglandins, leukotrienes and neutrophil migration. Finally, targeting PAF action (e.g., with a PAFR antagonist) might provide a useful therapeutic approach to inhibit articular inflammatory hyperalgesia.

Kaurenoic acid from Sphagneticola trilobata Inhibits Inflammatory Pain: effect on cytokine production and activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway.

Kaurenoic acid [ent-kaur-16-en-19-oic acid (1)] is a diterpene present in several plants including Sphagneticola trilobata. The only documented evidence for its antinociceptive effect is that it inhibits the writhing response induced by acetic acid in mice. Therefore, the analgesic effect of 1 in different models of pain and its mechanisms in mice were investigated further. Intraperitoneal and oral treatment with 1 dose-dependently inhibited inflammatory nociception induced by acetic acid. Oral treatment with 1 also inhibited overt nociception-like behavior induced by phenyl-p-benzoquinone, complete Freund's adjuvant (CFA), and both phases of the formalin test. Compound 1 also inhibited acute carrageenin- and PGE(2)-induced and chronic CFA-induced inflammatory mechanical hyperalgesia. Mechanistically, 1 inhibited the production of the hyperalgesic cytokines TNF-α and IL-1ß. Furthermore, the analgesic effect of 1 was inhibited by l-NAME, ODQ, KT5823, and glybenclamide treatment, demonstrating that such activity also depends on activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway, respectively. These results demonstrate that 1 exhibits an analgesic effect in a consistent manner and that its mechanisms involve the inhibition of cytokine production and activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway.