Regulation Of Inflammatory Pain Research Articles

Inhibition of TRPM8 function by prostacyclin receptor agonists requires coupling to Gq/11 proteins.

The TRPM8 ion channel is involved in innocuous cold sensing and has a potent anti-inflammatory action. Its activation by lower temperature or chemical agonists such as menthol and icilin induces analgesic effects, reversing hypersensitivity and reducing chronic pain. On the other hand, prostacyclin (PGI2) enhances pain and inflammation by activating the IP receptors. Due to the critical roles of TRPM8 and IP receptors in the regulation of inflammatory pain, and considering their overlapping expression pattern, we analysed the functional interaction between human TRPM8 and IP receptors. We transiently expressed human TRPM8 channels and IP receptors in HEK293T cells and carried out intracellular calcium and cAMP measurements. Additionally, we cultured neurons from the dorsal root ganglia (DRGs) of mice and determined the increase in intracellular calcium triggered by the TRPM8 agonist, icilin, in the presence of the IP receptor agonist cicaprost, the IP receptor antagonist Cay10441, and the Gq/11 inhibitor YM254890. Activation of IP receptors by selective agonists (cicaprost, beraprost, and iloprost) inhibited TRPM8 channel function, independently of the Gs-cAMP pathway. The potent inhibition of TRPM8 channels by IP receptor agonists involved Gq/11 coupling. These effects were also observed in neurons isolated from murine DRGs. Our results demonstrate an unusual signalling pathway of IP receptors by coupling to Gq/11 proteins to inhibit TRPM8 channel function. This pathway may contribute to a better understanding of the role of TRPM8 channels and IP receptors in regulating pain and inflammation.

TIMP-1 as a Potential Effector of Ion Channel Expression and Inflammatory Pain

Millions of Americans are affected by chronic pain, yet treatment remains largely ineffective. Unresolved inflammation contributes to chronic pain development and understanding how inflammation resolves is integral for developing effective therapeutics. Towards this goal, we have identified tissue inhibitor of metalloproteinases (TIMP-1) as a regulator of inflammatory pain. Mice with genetic deletions of TIMP-1 (T1KO) exhibit prolonged mechanical and thermal hypersensitivity following cutaneous inflammation compared to wild-type (WT) mice. Electrophysiological recordings from nociceptive neurons revealed that T1KO nociceptors exhibit lower activation thresholds and increased incidence of spontaneous activity and after discharge in response to inflammation. These results suggest that increased nociceptor firing increases behavioral sensitivity in T1KO mice. Recent RNA sequencing of the dorsal root ganglia (DRG) has identified multiple high priority transcriptomic targets that may underlie nociceptor responsiveness in T1KO mice, including increased expression of Scn9a (Nav1.7), Scn10a (Nav1.8), Trpm3, and Piezo2, which all encode ion channels in neurons that either sense or propagate noxious stimuli. Subsequent analysis of protein and gene expression confirm increased expression of Nav1.7 in T1KO DRG, suggesting that TIMP-1 may serve to normalize Nav-dependent nociceptor action potential firing. Inhibition of one of these channels alone is insufficient to significantly reduce the multimodal aspects associated with pathological pain states, illustrating the critical need to develop intervention strategies designed to simultaneously modulate activity of multiple targets. These results suggest a novel TIMP-1-mediated mechanism that may function to attenuate inflammatory pain. NINDS R03NS096454, NINDS R21NS104789 (KMB) Rita Allen Foundation Award in Pain (KMB) KUMC Biomedical Research Training Program (OCE) Madison and Lila Self Graduate Fellowship (RNS) NIGMS P20GM103418 (KMB, OCE, EEY).

Identification of Plasma Biomarkers Involved in Regulation of Inflammatory Pain

Physicians are challenged in treating pain patients due to the lack of quantifiable, objective methods of measuring pain in the clinic; pain sensation is multifaceted and subjective to each individual. There is a critical need for point-of-care quantification of accessible biomarkers to provide objective analyses beyond the subjective pain scales currently employed in clinical care settings. In the present experiment, we employed an animal model to test the hypothesis that blood-born regulators of the inflammatory response directly associate with an objective behavioral response to inflammatory pain. Upon induction of localized paw inflammation, we measured various cytokines and matrix metalloproteinases (MMPs) that are known to participate in the inflammatory response and investigated their relationship to the behavioral response across a 24-hour period. Methods MaleSprague-Dawley rats (n=6/group) outfitted with indwelling jugular catheters were injected with 0.1 ml of saline or 0.1 ml of the pro-inflammatory compound λ-carrageenan (1%) into the left hindpaw. Quantification of carrageenan-evoked paw inflammation (paw thickness) and locomotor activity, as well as collection of blood (500 µl) for later biochemical analyses occurred 24 hrs prior to intraplantar injection (baseline) and at four timepoints post-injection (1-24 hrs). Plasma was aliquoted from each sample for detection of cytokines (BioPlex Rat Cytokine Panel) and MMPs. Results Intraplantar injection with 1% λ-carrageenan induced a significant increase in paw thickness across 24 hrs, with peak inflammation observed at 8 hrs (p<0.05 vs saline). Pearson's correlation analyses revealed that locomotor activity counts negatively correlated with paw inflammation at 8 hrs (r2=0.5645, p<0.05), demonstrating impaired locomotion at the peak of paw inflammation. Expression of the chemokines C-X-C motif chemokine ligand 1 (Gro KC) (r2=0.7061, p<0.05) and monocyte chemoattractant protein-1 (MCP-1) (r2=0.5050, p<0.05) positively correlated with paw inflammation and negatively correlated with locomotor activity at 8 hrs (Gro KC, r2=0.6386, p<0.05; MCP-1, r2=0.4150, p<0.05). The ratio of MMP9 to MMP2 activity negatively correlated with paw inflammation (r2=0.4759, p<0.05) at 8 hrs. No relationship between MMP9/MMP2 activity and locomotor activity was observed this timepoint. Interestingly, a mixed model ANOVA of the time course of MMP9/MMP2 activity revealed a significant increase at 1 hr (p<0.05 vs saline). Conclusions We postulate that the chemokines Gro KC and MCP-1 as well as the ratio of MMP9 to MMP2 activity may serve as predictive biomarkers for the timecourse of inflammation-associated behavioral deficits. These data define opportunities for the future development of a point-of-care device to objectively quantify biomarkers for inflammatory pain states.

Selective inhibition of adenylyl cyclase subtype 1 reduces inflammatory pain in chicken of gouty arthritis.

Lack of uricase leads to the high incidence of gout in humans and poultry, which is different from rodents. Therefore, chicken is considered to be one of the ideal animal models for the study of gout. Gout-related pain caused by the accumulation of urate in joints is one type of inflammatory pain, which causes damage to joint function. Our previous studies have demonstrated the crucial role of calcium-stimulated adenylyl cyclase subtype 1 (AC1) in inflammatory pain in rodents; however, there is no study in poultry. In the present study, we injected mono-sodium urate (MSU) into the left ankle joint of the chicken to establish a gouty arthritis model, and tested the effect of AC1 inhibitor NB001 on gouty arthritis in chickens. We found that MSU successfully induced spontaneous pain behaviors including sitting, standing on one leg, and limping after 1–3 h of injection into the left ankle of chickens. In addition, edema and mechanical pain hypersensitivity also occurred in the left ankle of chickens with gouty arthritis. After peroral administration of NB001 on chickens with gouty arthritis, both the spontaneous pain behaviors and the mechanical pain hypersensitivity were effectively relieved. The MSU-induced edema in the left ankle of chickens was not affected by NB001, suggesting a central effect of NB001. Our results provide a strong evidence that AC1 is involved in the regulation of inflammatory pain in poultry. A selective AC1 inhibitor NB001 produces an analgesic effect (not anti-inflammatory effect) on gouty pain and may be used for future treatment of gouty pain in both humans and poultry.

Neurexin-2 is a potential regulator of inflammatory pain in the spinal dorsal horn of rats.

Chronic pain is one of the serious conditions that affect human health and remains cure still remains a serious challenge as the molecular mechanism remains largely unclear. Here, we used label‐free proteomics to identify potential target proteins that regulate peripheral inflammatory pain and reveal its mechanism of action. Inflammation in peripheral tissue was induced by injecting complete Freund's adjuvant (CFA) into rat hind paw. A proteomic method was adopted to compare the spinal dorsal horn (SDH) in peripheral inflammatory pain (PIP) model rats with controls. Differential proteins were identified in SDH proteome by label‐free quantification. The role of screened target proteins in the PIP was verified by small interfering RNA (siRNA). A total of 3072 and 3049 proteins were identified in CFA and normal saline (NS) groups, respectively, and 13 proteins were identified as differentially expressed in the CFA group. One of them, neurexin‐2, was validated for its role in the inflammatory pain. Neurexin‐2 was up‐regulated in the CFA group, which was confirmed by quantitative PCR. Besides, intrathecal siRNA‐mediated knock‐down of neurexin‐2 attenuated CFA‐induced mechanical and thermal hyperalgesia and reduced the expression of SDH membrane glutamate receptors (eg mGlu receptor 1, AMPA receptor) and postsynaptic density (eg PSD‐95, DLG2). These findings increased the understanding of the role of neurexin‐2 in the inflammatory pain, implicating that neurexin‐2 acts as a potential regulatory protein of inflammatory pain through affecting synaptic plasticity in the SDH of rats.

Nogo-A promotes inflammatory heat hyperalgesia by maintaining TRPV-1 function in the rat dorsal root ganglion neuron.

Nogo-A is a key inhibitory molecule of axon regeneration in oligodendrocytes. However, little is known about its role in adult neurons. In this study, we showed an important function of Nogo-A on regulation of inflammatory pain in dorsal root ganglion (DRG) neurons. In adult rats with complete Freund's adjuvant (CFA) hind paw inflammation, DRG neurons showed a significant increase in Nogo-A expression. Disruption of Nogo-A signaling with Nogo-66 receptor antagonist peptide, Nogo-A blocking antibody, Nogo-A short hairpin RNA, or Nogo-A gene knockout attenuated CFA-induced inflammatory heat hyperalgesia. Moreover, disruption of Nogo-A signaling suppressed the function and expression in DRG neurons of the transient receptor potential vanilloid subfamily member (TRPV)-1 channel, which is known to be the endogenous transducer of noxious heat during inflammation. These effects were accompanied with a reduction in LIM domain kinase (LIMK)/cofilin phosphorylation and actin polymerization. Similar disruption of actin filament architecture by direct action of Latrunculin A reduced the TRPV-1 activity and up-regulation of TRPV-1 protein caused by CFA. We conclude that Nogo-A plays an essential role in the development of inflammatory heat hyperalgesia, partly through maintaining TRPV-1 function via activation of the LIMK/cofilin pathway, which regulates actin filament dynamics. These findings support a therapeutic potential of modulating Nogo-A signaling in pain management.-Hu, F., Liu, H.-C., Su, D.-Q., Chen, H.-J., Chan, S.-O., Wang, Y., Wang, J. Nogo-A promotes inflammatory heat hyperalgesia by maintaining TRPV-1 function in the rat dorsal root ganglion neuron.

Alleviation of Behavioral Hypersensitivity in Mouse Models of Inflammatory Pain with Two Structurally Different Casein Kinase 1 (CK1) Inhibitors

BackgroundThe phylogenetically highly conserved CK1 protein kinases consisting of at least seven isoforms form a distinct family within the eukaryotic protein kinases. CK1 family members play crucial roles in a wide range of signaling activities. However, the functional role of CK1 in somatosensory pain signaling has not yet been fully understood. The aim of this study was to clarify the role of CK1 in the regulation of inflammatory pain in mouse carrageenan and complete Freund’s adjuvant (CFA) models.ResultsWe have used two structurally different CK1 inhibitors, TG003 and IC261. TG003, which was originally identified as a cdc2-like kinase inhibitor, had potent inhibitory effects on CK1 isoforms in vitro and in cultured cells. Intrathecal injection of either TG003 (1-100 pmol) or IC261 (0.1-1 nmol) dose-dependently decreased mechanical allodynia and thermal hyperalgesia induced by carrageenan or CFA. Bath-application of either TG003 (1 μM) or IC261 (1 μM) had only marginal effects on spontaneous excitatory postsynaptic currents (sEPSCs) recorded in the substantia gelatinosa neurons of control mice. However, both compounds decreased the frequency of sEPSCs in both inflammatory pain models.ConclusionsThese results suggest that CK1 plays an important pathophysiological role in spinal inflammatory pain transmission, and that inhibition of the CK1 activity may provide a novel strategy for the treatment of inflammatory pain.

Sa2020 Specific Mobilization of Effector CD4+ T Lymphocytes in the Gut Reduces DSS-Induced Visceral Hypersensitivity in Mice

Inflammatory bowel diseases (IBD) are characterized by an uncontrolled inflammatory response to colonic luminal content involving both innate and adaptive immunity. Indeed, aberrant responses of CD4+ Th1 and Th17 lymphocytes against microbiota highly contribute to the development of IBD. We have previously shown that effector CD4+ T lymphocytes generated in response to microbes are responsible for inflammatory pain relief that occurs spontaneously within the few days following the initiation of the immune response. The endogenous T cell-dependent regulation of inflammatory pain is mediated by the local release of opioids that have been synthesized upon the activation of CD4+ T lymphocytes within draining lymph nodes (Boue J, et al. J. Immunol., 2011, 186:5078-5084). Given that the production of opioids is not restricted to a particular phenotype of effector CD4+ T lymphocytes (Boue J, et al. Pain, 2012, 153, 485-493), we wondered whether colitogenic CD4+ Th17 and Th1 lymphocytes that accumulate within inflamed colon also produce opioids and contribute to reduce inflammation-induced visceral pain. For this purpose, we used the colitis model induced by the transfer of Naive CD4+CD45RBhigh T lymphocytes into immune-deficient SCID mice. In this model, in which the absence of efficient regulation of the T cell response against microbiota leads to chronic inflammation of the gut, we showed that Th1 and Th17 lymphocytes isolated from inflamed colon 6 weeks after T cell transfer, expressed high level of endogenous opioids. The production of opioids by colitogenic CD4+ T lymphocytes was responsible for a significant reduction of inflammation-associated abdominal pain as assessed by colorectal distension. Indeed, visceral sensitivity was increased in mice locally treated with an opioid receptor antagonist, unable to cross the blood-brain barrier. Taken together, our results indicate that colitogenic mucosal CD4+ T lymphocytes that play a pivotal role in colitis pathogenesis reduce pain associated with inflammationinduced tissue injury.

Sa2019 Regulation of Inflammation-Associated Abdominal Pain by Colitogenic Mucosal CD4+ T Lymphocytes: The Role of Opioids

Inflammatory bowel diseases (IBD) are characterized by an uncontrolled inflammatory response to colonic luminal content involving both innate and adaptive immunity. Indeed, aberrant responses of CD4+ Th1 and Th17 lymphocytes against microbiota highly contribute to the development of IBD. We have previously shown that effector CD4+ T lymphocytes generated in response to microbes are responsible for inflammatory pain relief that occurs spontaneously within the few days following the initiation of the immune response. The endogenous T cell-dependent regulation of inflammatory pain is mediated by the local release of opioids that have been synthesized upon the activation of CD4+ T lymphocytes within draining lymph nodes (Boue J, et al. J. Immunol., 2011, 186:5078-5084). Given that the production of opioids is not restricted to a particular phenotype of effector CD4+ T lymphocytes (Boue J, et al. Pain, 2012, 153, 485-493), we wondered whether colitogenic CD4+ Th17 and Th1 lymphocytes that accumulate within inflamed colon also produce opioids and contribute to reduce inflammation-induced visceral pain. For this purpose, we used the colitis model induced by the transfer of Naive CD4+CD45RBhigh T lymphocytes into immune-deficient SCID mice. In this model, in which the absence of efficient regulation of the T cell response against microbiota leads to chronic inflammation of the gut, we showed that Th1 and Th17 lymphocytes isolated from inflamed colon 6 weeks after T cell transfer, expressed high level of endogenous opioids. The production of opioids by colitogenic CD4+ T lymphocytes was responsible for a significant reduction of inflammation-associated abdominal pain as assessed by colorectal distension. Indeed, visceral sensitivity was increased in mice locally treated with an opioid receptor antagonist, unable to cross the blood-brain barrier. Taken together, our results indicate that colitogenic mucosal CD4+ T lymphocytes that play a pivotal role in colitis pathogenesis reduce pain associated with inflammationinduced tissue injury.

Regulation of inflammatory pain by NF-κB and CX3CR1 at the spinal cord of rats

To observe the effect of intrathecal injection of nuclear factor-κB (NF-κB) inhibitor of pyrrolidine dithiocarbamate (PDTC) on pain sensitivity thresholds and the expression of spinal cord CX3C chemokine receptor 1 (CX3CR1) in monoarthritis (MA) model in rats. Forty-eight Sprague-Dawley rats were randomly divided into 4 groups (12 each) after successful intrathecal catheterization: (1) sham operation with physiological saline group (the sham group); (2) MA with normal saline group (the MA group); (3) 10 μL 100 μmol/L PDTC before MA (the PDTC pre-treatment group); (4)MA before 10 μL 100 μmol/L PDTC (the PDTC post-treatment group). Normal saline or PDTC was injected 5 d after the intrathecal catheterization. Pain sensitivity thresholds were measured in the 4 groups before and after the intrathecal injection at different time points. Rat monoarthritis model was subsequently built by injecting complete Freund's adjuvant (CFA) into the left ankle joint of the rats. On day 3 after the intrathecal injection, expression of microglia in the L₅ spinal cord segment was observed by immunohistochemical method, and the lumbar segments L₄-L₅ of spinal cord were taken to perform RT-PCR to examine the expression of NF-κB mRNA and CX3CR1 mRNA. Compared with the MA group, the pain sensitivity thresholds in the sham group, the PDTC pre-treatment group and the PDTC post-treatment group at each time point after the intrathecal injection increased significantly (P<0.05), while microglia in the L₅ spinal cord segment decreased significantly (P<0.05) and expression of CX3CR1 mRNA and NF-κB mRNA in the lumbar segments L₄-L₅ of spinal cord decreased significantly (P<0.05). The hyperalgesic effect of the CFA-induced model of monoarthritis can be relieved by intrathecal injection of NF-κB inhibitor PDTC. Its mechanism is possibly related to NF-κB signal pathway which is involved in the formation of inflammatory pain through regulating CX3CR1 expression.

Regulation of Inflammatory Pain by Inhibition of Fatty Acid Amide Hydrolase

Although cannabinoids are efficacious in laboratory animal models of inflammatory pain, their established cannabimimetic actions diminish enthusiasm for their therapeutic development. Conversely, fatty acid amide hydrolase (FAAH), the chief catabolic enzyme regulating the endogenous cannabinoid N-arachidonoylethanolamine (anandamide), has emerged as an attractive target for treating pain and other conditions. Here, we tested WIN 55212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de)-1,4-benzoxazin-6-yl]-1-napthalenylmethanone], a cannabinoid receptor agonist, and genetic deletion or pharmacological inhibition of FAAH in the lipopolysaccharide (LPS) mouse model of inflammatory pain. WIN 55212-2 significantly reduced edema and hot-plate hyperalgesia caused by LPS infusion into the hind paws, although the mice also displayed analgesia and other central nervous system effects. FAAH(-/-) mice exhibited reduced paw edema and hyperalgesia in this model without apparent cannabimimetic effects. Transgenic mice expressing FAAH exclusively on neurons continued to display the antiedematous, but not the antihyperalgesic, phenotype. The CB(2) cannabinoid receptor (CB(2)) antagonist SR144528 [N-[(1S)-endo-1,3,3-trimethyl bicyclo [2.2.1] heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide] blocked this non-neuronal, anti-inflammatory phenotype, and the CB(1) cannabinoid receptor (CB(1)) antagonist rimonabant [SR141716, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] blocked the antihyperalgesic phenotype. The FAAH inhibitor URB597 [cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester] attenuated the development of LPS-induced paw edema and reversed LPS-induced hyperalgesia through the respective CB(2) and CB(1) mechanisms of action. However, the transient receptor potential vanilloid type 1 antagonist capsazepine did not affect either the antihyperalgesic or antiedematous effects of URB597. Finally, URB597 attenuated levels of the proinflammatory cytokines interleukin-1beta and tumor necrosis factor alpha in LPS-treated paws. These findings demonstrate that simultaneous elevations in non-neuronal and neuronal endocannabinoid signaling are possible through inhibition of a single enzymatic target, thereby offering a potentially powerful strategy for treating chronic inflammatory pain syndromes that operate at multiple levels of anatomical integration.

Thrombin receptor: An endogenous inhibitor of inflammatory pain, activating opioid pathways

Serine proteases such as thrombin, trypsin and mast cell tryptase can act on different cell types through protease-activated receptors (PARs). These receptors have been shown to be implicated in several phenomena such as inflammation, platelet activation, immune response and atherosclerosis. Several studies recently reported PARs expression on neurons and some of them demonstrated that these receptors could interfere with nociception. The contribution of PAR 1 to inflammatory pain and the mechanism involved in this phenomenon were investigated. Intraplantar injection of PAR 1 agonist increased withdrawal latency and reduced response frequency to von Frey filaments, thus inhibiting nociceptive response to both mechanical and thermal stimuli in mice. PAR 1 agonist also reduced carrageenan-induced inflammatory hyperalgesia. The anti-nociceptive effects of PAR 1 agonist were mediated by endogenous opioids, as this effect was inhibited by local injection of naloxone methiodide, and because intraplantar injection of PAR 1 agonist increased mRNA expression of the endogenous opioid precursor proenkephalin. However, PAR 1 agonist was not able to inhibit calcium signals in isolated sensory neurons exposed to pro-nociceptive agents. Finally, despite similar inflammatory parameters, PAR 1-deficient mice showed a strong potentiation of inflammatory hyperalgesia induced by the intraplantar injection of either formalin or carrageenan, or in the chronic model of collagen-induced arthritis, compared to wild-type mice. This study highlights a previously unknown endogenous mechanism of analgesia, showing a central role for the thrombin receptor PAR 1 in the regulation of inflammatory pain and as an activator of opioid pathways.