Effect of pharmacological modulation of the kynurenine pathway on pain-related behavior and opioid analgesia in a mouse model of neuropathic pain.

Dysfunction of the central nervous system are accompanied by changes in tryptophan metabolism, with the kynurenine pathway (KP) being the main route of its catabolism. Recently, KP metabolites, which are collectively called kynurenines, have become an area of intense research due to their ability to directly and indirectly affect a variety of classic neurotransmitter systems. However, the significance of KP in neuropathic pain is still poorly understood. Therefore, we designed several experiments to verify changes in the mRNA levels of KP enzymes in parallel with other factors related to this metabolic route after chronic constriction injury of the sciatic nerve (CCI model) in mice. The analysis revealed an increase in, Kmo, Kynu and Haoo mRNA levels in the spinal cord on the 7th day after CCI, while Kat1, Kat2, Tdo2, Ido2 and Qprt mRNA levels remain unchanged. Subsequent pharmacological studies provided evidence that modulation of KP by single intrathecal administration of 1-D-MT, UPF468 or L-kynurenine attenuates mechanical and thermal hypersensitivity and increases the effectiveness of selected opioids in mice as measured on day 7 after CCI. Moreover, our results provide the first evidence that the injection of L-kynurenine preceded by UPF468 (KMO inhibitor) is more effective at reducing hypersensitivity in animals with neuropathic pain. Importantly, L-kynurenine also exerts an analgesic effect after intravenous injections, which is enhanced by the administration of minocycline, an inhibitor of microglial activation. Additionally, L-kynurenine administered intrathecally and intravenously enhances analgesia evoked by all tested opioids (morphine, buprenorphine and oxycodone). Overall, our results indicate that the modulation of KP at different levels might be a new pharmacological tool in neuropathy management.

Analgesic Effects of Fisetin, Peimine, Astaxanthin, Artemisinin, Bardoxolone Methyl and 740 Y-P and Their Influence on Opioid Analgesia in a Mouse Model of Neuropathic Pain.

Treatment of neuropathic pain remains a challenge for modern medicine due to the insufficiently understood molecular mechanisms of its development and maintenance. One of the most important cascades that modulate the nociceptive response is the family of mitogen-activated protein (MAP) kinases and phosphatidylinositol-3-kinase (PI3K), as well as nuclear factor erythroid 2-related factor 2 (Nrf2). The aim of this study was to determine the effect of nonselective modulators of MAP kinases-fisetin (ERK1/2 and NFκB inhibitor, PI3K activator), peimine (MAPK inhibitor), astaxanthin (MAPK inhibitor, Nrf2 activator) and artemisinin (MAPK inhibitor, NFκB activator), as well as bardoxolone methyl (selective activator of Nrf2) and 740 Y-P (selective activator of PI3K)-in mice with peripheral neuropathy and to compare their antinociceptive potency and examine their effect on analgesia induced by opioids. The study was performed using albino Swiss male mice that were exposed to chronic constriction injury of the sciatic nerve (CCI model). Tactile and thermal hypersensitivity was measured using von Frey and cold plate tests, respectively. Single doses of substances were administered intrathecally on day 7 after CCI. Among the tested substances, fisetin, peimine, and astaxanthin effectively diminished tactile and thermal hypersensitivity in mice after CCI, while artemisinin did not exhibit analgesic potency in this model of neuropathic pain. Additionally, both of the activators tested, bardoxolone methyl and 740 Y-P, also showed analgesic effects after intrathecal administration in mice exposed to CCI. In the case of astaxanthin and bardoxolone methyl, an increase in analgesia after combined administration with morphine, buprenorphine, and/or oxycodone was observed. Fisetin and peimine induced a similar effect on tactile hypersensitivity, where analgesia was enhanced after administration of morphine or oxycodone. In the case of 740 Y-P, the effects of combined administration with each opioid were observed only in the case of thermal hypersensitivity. The results of our research clearly indicate that substances that inhibit all three MAPKs provide pain relief and improve opioid effectiveness, especially if they additionally block NF-κB, such as peimine, inhibit NF-κB and activate PI3K, such as fisetin, or activate Nrf2, such as astaxanthin. In light of our research, Nrf2 activation appears to be particularly beneficial. The abovementioned substances bring promising results, and further research on them will broaden our knowledge regarding the mechanisms of neuropathy and perhaps contribute to the development of more effective therapy in the future.

The Kynurenine Pathway as a Potential Target for Neuropathic Pain Therapy Design: From Basic Research to Clinical Perspectives.

Accumulating evidence suggests the key role of the kynurenine pathway (KP) of the tryptophan metabolism in the pathogenesis of several diseases. Despite extensive research aimed at clarifying the mechanisms underlying the development and maintenance of neuropathic pain, the roles of KP metabolites in this process are still not fully known. Although the function of the peripheral KP has been known for several years, it has only recently been acknowledged that its metabolites within the central nervous system have remarkable consequences related to physiology and behavior. Both the products and metabolites of the KP are involved in the pathogenesis of pain conditions. Apart from the neuroactive properties of kynurenines, the KP regulates several neurotransmitter systems in direct or indirect ways. Some neuroactive metabolites are known to have neuroprotective properties (kynurenic acid, nicotinamide adenine dinucleotide cofactor), while others are toxic (3-hydroxykynurenine, quinolinic acid). Numerous animal models show that modulation of the KP may turn out to be a viable target for the treatment of diseases. Importantly, some compounds that affect KP enzymes are currently described to possess analgesic properties. Additionally, kynurenine metabolites may be useful for assessing response to therapy or as biomarkers in therapeutic monitoring. The following review describes the molecular site of action and changes in the levels of metabolites of the kynurenine pathway in the pathogenesis of various conditions, with a particular emphasis on their involvement in neuropathy. Moreover, the potential clinical implications of KP modulation in chronic pain therapy as well as the directions of new research initiatives are discussed.

Comparison of the Effects of Chemokine Receptors CXCR2 and CXCR3 Pharmacological Modulation in Neuropathic Pain Model-In Vivo and In Vitro Study.

Recent findings have highlighted the roles of CXC chemokine family in the mechanisms of neuropathic pain. Our studies provide evidence that single/repeated intrathecal administration of CXCR2 (NVP-CXCR2-20) and CXCR3 ((±)-NBI-74330) antagonists explicitly attenuated mechanical/thermal hypersensitivity in rats after chronic constriction injury of the sciatic nerve. After repeated administration, both antagonists showed strong analgesic activity toward thermal hypersensitivity; however, (±)-NBI-74330 was more effective at reducing mechanical hypersensitivity. Interestingly, repeated intrathecal administration of both antagonists decreased the mRNA and/or protein levels of pronociceptive interleukins (i.e., IL-1beta, IL-6, IL-18) in the spinal cord, but only (±)-NBI-74330 decreased their levels in the dorsal root ganglia after nerve injury. Furthermore, only the CXCR3 antagonist influenced the spinal mRNA levels of antinociceptive factors (i.e., IL-1RA, IL-10). Additionally, antagonists effectively reduced the mRNA levels of pronociceptive chemokines; NVP-CXCR2-20 decreased the levels of CCL2, CCL6, CCL7, and CXCL4, while (±)-NBI-74330 reduced the levels of CCL3, CCL6, CXCL4, and CXCL9. Importantly, the results obtained from the primary microglial and astroglial cell cultures clearly suggest that both antagonists can directly affect the release of these ligands, mainly in microglia. Interestingly, NVP-CXCR2-20 induced analgesic effects after intraperitoneal administration. Our research revealed important roles for CXCR2 and CXCR3 in nociceptive transmission, especially in neuropathic pain.

Chemokines CCL2 and CCL7, but not CCL12, play a significant role in the development of pain-related behavior and opioid-induced analgesia.

The complex neuroimmunological interactions mediated by chemokines are suggested to be responsible for the development of neuropathic pain. The lack of knowledge regarding the detailed pathomechanism of neuropathy is one reason for the lack of optimally efficient therapies. Recently, several lines of evidence indicated that expression of CCR2 is increased in spinal cord neurons and microglial cells after peripheral nerve injury. It was previously shown that administration of CCR2 antagonists induces analgesic effects; however, the role of CCR2 ligands in neuropathic pain still needs to be explained. Thus, the goal of our studies was to investigate the roles of CCL2, CCL7, and CCL12 in neuropathic pain development and opioid effectiveness. The experiments were conducted on primary glial cell cultures and two groups of mice: naive and neuropathic. We used chronic constriction injury (CCI) of the sciatic nerve as a neuropathic pain model. Mice intrathecally received chemokines (CCL2, CCL7, CCL12) at a dose of 10, 100 or 500 ng, neutralizing antibodies (anti-CCL2, anti-CCL7) at a dose of 1, 4 or 8 µg, and opioids (morphine, buprenorphine) at a dose of 1 µg. The pain-related behaviors were assessed using the von Frey and cold plate tests. The biochemical analysis of mRNA expression of glial markers, CCL2, CCL7 and CCL12 was performed using quantitative reverse transcriptase real-time PCR. We demonstrated that CCI of the sciatic nerve elevated spinal expression of CCL2, CCL7 and CCL12 in mice, in parallel with microglia and astroglial activation markers. Moreover, intrathecal injection of CCL2 and CCL7 induced pain-related behavior in naive mice in a dose-dependent manner. Surprisingly, intrathecal injection of CCL12 did not influence nociceptive transmission in naive or neuropathic mice. Additionally, we showed for the first time that intrathecal injection of CCL2 and CCL7 neutralizing antibodies not only attenuated CCI-induced pain-related behaviors in mice but also augmented the analgesia induced by morphine and buprenorphine. In vitro studies suggest that both microglia and astrocytes are an important cellular sources of the examined chemokines. Our results revealed the crucial roles of CCL2 and CCL7, but not CCL12, in neuropathic pain development and indicated that pharmacological modulation of these factors may serve as a potential therapeutic target for new (co)analgesics.

Lipopolysaccharide from Rhodobacter sphaeroides (TLR4 antagonist) attenuates hypersensitivity and modulates nociceptive factors.

CONTEXT: Accumulating evidence has demonstrated that Toll-like receptors (TLRs), especially TLR4 localized on microglia/macrophages, may play a significant role in nociception. OBJECTIVE: We examine the role of TLR4 in a neuropathic pain model. Using behavioural/biochemical methods, we examined the influence of TLR4 antagonist on levels of hypersensitivity and nociceptive factors whose contribution to neuropathy development has been confirmed. MATERIALS AND METHODS: Behavioural (von Frey's/cold plate) tests were performed with Wistar male rats after intrathecal administration of a TLR4 antagonist (LPS-RS ULTRAPURE (LPS-RSU), 20 µG: lipopolysaccharide from Rhodobacter sphaeroides, InvivoGen, San Diego, CA) 16 H and 1 h before chronic constriction injury (cci) to the sciatic nerve and then daily for 7 d. three groups were used: an intact group and two cci-exposed groups that received vehicle or LPS-RSU. tissue [spinal cord/dorsal root ganglia (DRG)] for western blot analysis was collected on day 7. RESULTS: The pharmacological blockade of TLR4 diminished mechanical (from ca. 40% to 16% that in the INTACT group) and thermal (from ca. 51% to 32% that in the INTACT group) hypersensitivity despite the enhanced activation of IBA-1-positive cells in DRG. Moreover, LPS-RSU changed the ratio between IL-18/IL-18BP and MMP-9/TIMP-1 in favour of the increase of antinociceptive factors IL-18BP (25%-spinal; 96%-DRG) and TIMP-1 (15%-spinal; 50%-DRG) and additionally led to an increased IL-6 (40%-spinal; 161%-DRG), which is known to have analgesic properties in neuropathy. CONCLUSIONS: Our results provide evidence that LPS-RSU influences pain through the expression of TLR4. TLR4 blockade has analgesic properties and restores the balance between nociceptive factors, which indicates its engagement in neuropathy development.

Blockade of IL-18 signaling diminished neuropathic pain and enhanced the efficacy of morphine and buprenorphine.

Currently, the low efficacy of antinociceptive drugs for the treatment of neuropathic pain is a major therapeutic problem. Here, we show the potential role of interleukin (IL)-18 signaling in this phenomenon. IL-18 is an important molecule that performs various crucial functions, including the alteration of nociceptive transmission in response to neuropathic pain. We have studied the changes in the mRNA and protein levels (qRT-PCR and Western blot analysis, respectively) of IL-18, IL-18-binding protein (IL-18BP) and the IL-18 receptor (IL-18R) over time in rats following chronic constriction injury (CCI) of the sciatic nerve. Our study demonstrated that the spinal levels of IL-18BP were slightly downregulated at days 7 and 14 in the rats subjected to CCI. In contrast, the IL-18 and IL-18R mRNA expression and protein levels were elevated in the ipsilateral spinal cord on days 2, 7 and 14. Moreover, in rats exposed to a single intrathecal administration of IL-18BP (50 and 100 ng) 7 or 14 days following CCI, symptoms of neuropathic pain were attenuated, and the analgesia pursuant to morphine and buprenorphine (0.5 and 2.5 µg) was enhanced. In summary, the restoration of the analgesic activity of morphine and buprenorphine via the blockade of IL-18 signaling suggests that increased IL-18 pathway may account for the decreased analgesic efficacy of opioids for neuropathic pain.

Microglial Inhibition Influences XCL1/XCR1 Expression and Causes Analgesic Effects in a Mouse Model of Diabetic Neuropathy.

BACKGROUND: Recent studies indicated the involvement of some chemokines in the development of diabetic neuropathy; however, participation of the chemokine-C-motif ligand (XCL) subfamily remains unknown. The goal of this study was to examine how microglial inhibition by minocycline hydrochloride (MC) influences chemokine-C-motif ligand 1 (XCL1)-chemokine-C-motif receptor 1 (XCR1)/G protein-coupled receptor 5 expression and the development of allodynia/hyperalgesia in streptozotocin-induced diabetic neuropathy. METHODS: The studies were performed on streptozotocin (200 mg/kg, intraperitoneally)-induced mouse diabetic neuropathic pain model and primary glial cell cultures. The MC (30 mg/kg, intraperitoneally) was injected two times daily until day 21. XCL1 and its neutralizing antibody were injected intrathecally, and behavior was evaluated with von Frey and cold plate tests. Quantitative analysis of protein expression of glial markers, XCL1, and/or XCR1 was performed by Western blot and visualized by immunofluorescence. RESULTS: MC treatment diminished allodynia (0.9 ± 0.1 g; n = 7 vs. 3.8 ± 0.7 g; n = 7) and hyperalgesia (6.5 ± 0.6 s; n = 7 vs. 16.5 ± 1 s; n = 7) in the streptozotocin-induced diabetes. Repeated MC administration prevented microglial activation and inhibited the up-regulation of the XCL1/XCR1 levels. XCL1 administration (10 to 500 ng/5 µl; n = 9) in naive mice enhanced nociceptive transmission, and injections of neutralizing XCL1 (4 to 8 µg/5 µl; n = 10) antibody into the mice with diabetic neuropathic pain diminished allodynia/hyperalgesia. Microglia activation evoked in primary microglial cell cultures resulted in enhanced XCL1 release and XCR1 expression. Additionally, double immunofluorescence indicated the widespread coexpression of XCR1-expressing cells with spinal neurons. CONCLUSIONS: In diabetic neuropathy, declining levels of XCL1 evoked by microglia inhibition result in the cause of analgesia. The putative mechanism corroborating this finding can be related to lower spinal expression of XCR1 together with the lack of stimulation of these XCR1 receptors, which are localized on neurons.

Blockade of Toll-Like Receptors (TLR2, TLR4) Attenuates Pain and Potentiates Buprenorphine Analgesia in a Rat Neuropathic Pain Model.

Accumulating evidence indicates that microglial TLR2 and TLR4 play a significant role in nociception. Experiments were conducted to evaluate the contribution of TLR2 and TLR4 and their adaptor molecules to neuropathy and their ability to amplify opioid effectiveness. Behavioral tests (von Frey's and cold plate) and biochemical (Western blot and qRT-PCR) analysis of spinal cord and DRG tissue were conducted after chronic constriction injury (CCI) to the sciatic nerve. Repeated intrathecal administration of LPS-RS (TLR2 and TLR4 antagonist) and LPS-RS Ultrapure (TLR4 antagonist) attenuated allodynia and hyperalgesia. Biochemical analysis revealed time-dependent upregulation of mRNA and/or protein levels of TLR2 and TLR4 and MyD88 and TRIF adaptor molecules, which was paralleled by an increase in IBA-1/CD40-positive cells under neuropathy. LPS-RS and LPS-RS Ultrapure similarly influenced opioid analgesia by enhancing the effectiveness of buprenorphine but not morphine. Summing up, in light of their upregulation over the course of pain, both TLR2 and TLR4 may indeed play a significant role in neuropathy, which could be linked to the observed activation of IBA-1/CD40-positive cells. Blockade of TLR2 and TLR4 produced analgesia and enhanced buprenorphine's effectiveness, which suggests that they may be a putative target for future pharmacological pain relief tools, especially for opioid rotation, when the effect of morphine is tolerated.

Parthenolide Relieves Pain and Promotes M2 Microglia/Macrophage Polarization in Rat Model of Neuropathy.

Neuropathic pain treatment remains a challenge because pathomechanism is not fully understood. It is believed that glial activation and increased spinal nociceptive factors are crucial for neuropathy. We investigated the effect of parthenolide (PTL) on the chronic constriction injury to the sciatic nerve (CCI)-induced neuropathy in rat. We analyzed spinal changes in glial markers and M1 and M2 polarization factors, as well as intracellular signaling pathways. PTL (5 µg; i.t.) was preemptively and then daily administered for 7 days after CCI. PTL attenuated the allodynia and hyperalgesia and increased the protein level of IBA1 (a microglial/macrophage marker) but did not change GFAP (an astrocyte marker) on day 7 after CCI. PTL reduced the protein level of M1 (IL-1ß, IL-18, and iNOS) and enhanced M2 (IL-10, TIMP1) factors. In addition, it downregulated the phosphorylated form of NF-κB, p38MAPK, and ERK1/2 protein level and upregulated STAT3. In primary microglial cell culture we have shown that IL-1ß, IL-18, iNOS, IL-6, IL-10, and TIMP1 are of microglial origin. Summing up, PTL directly or indirectly attenuates neuropathy symptoms and promotes M2 microglia/macrophages polarization. We suggest that neuropathic pain therapies should be shifted from blanketed microglia/macrophage suppression toward maintenance of the balance between neuroprotective and neurotoxic microglia/macrophage phenotypes.

Expression profiling of genes modulated by minocycline in a rat model of neuropathic pain.

BACKGROUND: The molecular mechanisms underlying neuropathic pain are constantly being studied to create new opportunities to prevent or alleviate neuropathic pain. The aim of our study was to determine the gene expression changes induced by sciatic nerve chronic constriction injury (CCI) that are modulated by minocycline, which can effectively diminish neuropathic pain in animal studies. The genes associated with minocycline efficacy in neuropathic pain should provide insight into the etiology of neuropathic pain and identify novel therapeutic targets. RESULTS: We screened the ipsilateral dorsal part of the lumbar spinal cord of the rat CCI model for differentially expressed genes. Out of 22,500 studied transcripts, the abundance levels of 93 transcripts were altered following sciatic nerve ligation. Percentage analysis revealed that 54 transcripts were not affected by the repeated administration of minocycline (30 mg/kg, i.p.), but the levels of 39 transcripts were modulated following minocycline treatment. We then selected two gene expression patterns, B1 and B2. The first transcription pattern, B1, consisted of 10 mRNA transcripts that increased in abundance after injury, and minocycline treatment reversed or inhibited the effect of the injury; the B2 transcription pattern consisted of 7 mRNA transcripts whose abundance decreased following sciatic nerve ligation, and minocycline treatment reversed the effect of the injury. Based on the literature, we selected seven genes for further analysis: Cd40, Clec7a, Apobec3b, Slc7a7, and Fam22f from pattern B1 and Rwdd3 and Gimap5 from pattern B2. Additionally, these genes were analyzed using quantitative PCR to determine the transcriptional changes strongly related to the development of neuropathic pain; the ipsilateral DRGs (L4-L6) were also collected and analyzed in these rats using qPCR. CONCLUSION: In this work, we confirmed gene expression alterations previously identified by microarray analysis in the spinal cord and analyzed the expression of selected genes in the DRG. Moreover, we reviewed the literature to illustrate the relevance of these findings for neuropathic pain development and therapy. Further studies are needed to elucidate the roles of the individual genes in neuropathic pain and to determine the therapeutic role of minocycline in the rat neuropathic pain model.

Mirogabalin Decreases Pain-like Behaviors by Inhibiting the Microglial/Macrophage Activation, p38MAPK Signaling, and Pronociceptive CCL2 and CCL5 Release in a Mouse Model of Neuropathic Pain.

Neuropathic pain is a chronic condition that significantly reduces the quality of life of many patients as a result of ineffective pain relief therapy. For that reason, looking for new analgesics remains an important issue. Mirogabalin is a new gabapentinoid that is a specific ligand for the α2σ-1 and α2σ-2 subunits of voltage-gated calcium channels. In the present study, we compared the analgesic effect of pregabalin and mirogabalin in a neuropathic pain chronic constriction injury (CCI) of the sciatic nerve in a mouse model. The main purpose of our study was to determine the effectiveness of mirogabalin administered both once and repeatedly and to explain how the drug influences highly activated cells at the spinal cord level in neuropathy. We also sought to understand whether mirogabalin modulates the selected intracellular pathways (p38MAPK, ERK, JNK) and chemokines (CCL2, CCL5) important for nociceptive transmission, which is crucial information from a clinical perspective. First, our study provides evidence that a single mirogabalin administration diminishes tactile hypersensitivity more effectively than pregabalin. Second, research shows that several indirect mechanisms may be responsible for the beneficial analgesic effect of mirogabalin. This study reports that repeated intraperitoneally (i.p.) mirogabalin administration strongly prevents spinal microglia/macrophage activation evoked by nerve injury, slightly suppresses astroglia and neutrophil infiltration, and reduces the p38MAPK levels associated with neuropathic pain, as measured on Day 7. Moreover, mirogabalin strongly diminished the levels of the pronociceptive chemokines CCL2 and CCL5. Our results indicate that mirogabalin may represent a new strategy for the effective pharmacotherapy of neuropathic pain.

New insights into the analgesic properties of the XCL1/XCR1 and XCL1/ITGA9 axes modulation under neuropathic pain conditions - evidence from animal studies.

Recent studies have indicated the involvement of chemokine-C-motif ligand 1 (XCL1) in nociceptive transmission; however, the participation of its two receptors, canonical chemokine-C-motif receptor 1 (XCR1) and integrin alpha-9 (ITGA9), recently recognized as a second receptor, has not been clarified to date. The aim was to explore by which of these receptors XCL1 reveals its pronociceptive properties and how the XCL1-XCR1 and XCL1-ITGA9 axes blockade/neutralization influence on pain-related behavior and opioid analgesia in the model of neuropathic pain. In our studies we used Albino Swiss mice which were exposed to the unilateral sciatic nerve chronic constriction injury (CCI) as a neuropathic pain model. Animals received single intrathecal (i.t.) injection of XCL1, XCL1 neutralizing antibodies, antagonist of XCR1 (vMIP-II) and neutralizing antibodies of ITGA9 (YA4), using lumbar puncture technique. Additionally we performed i.t. co-administration of abovementioned neutralizing antibodies and antagonists with single dose of morphine/buprenorphine. To assess pain-related behavior the von Frey and cold plate tests were used. To measure mRNA and protein level the RT-qPCR and Western Blot/Elisa/immunofluorescence techniques were performed, respectively. Statistical analysis was conducted using ANOVA with a Bonferroni correction. Presented studies have shown time-dependent upregulation of the mRNA and/or protein expression of XCL1 in the spinal cord after nerve injury as measured on day 1, 4, 7, 14, and 35. Our immunofluorescence study showed that XCL1 is released by astroglial cells located in the spinal cord, despite the neural localization of its receptors. Our results also provided the first evidence that the blockade/neutralization of both receptors, XCR1 and ITGA9, reversed hypersensitivity after intrathecal XCL1 administration in naive mice; however, neutralization of ITGA9 was more effective. In addition, the results proved that the XCL1 neutralizing antibody and, similarly, the blockade of XCR1 and neutralization of ITGA9 diminished thermal and mechanical hypersensitivity in nerve injury-exposed mice after 7 days. Additionally, neutralization of XCL1 improves morphine analgesia. Moreover, blockade of XCR1 positively influences buprenorphine effectiveness, and neutralization of ITGA9 enhances not only buprenorphine but also morphine analgesia. Therefore, blockade of the XCL1-ITGA9 interaction may serve as an innovative strategy for the polypharmacotherapy of neuropathic pain in combination with opioids.

Mirogabalin Decreases Pain-like Behaviours and Improves Opioid and Ketamine Antinociception in a Mouse Model of Neuropathic Pain.

Neuropathic pain remains a difficult clinical challenge due to its diverse aetiology and complex pathomechanisms, which are yet to be fully understood. Despite the variety of available therapies, many patients suffer from ineffective pain relief; hence, the search for more efficacious treatments continues. The new gabapentinoid, mirogabalin has recently been approved for clinical use. Although its main mechanism of action occurs at the α2σ-1 and α2σ-2 subunits of calcium channels and is well documented, how the drug affects the disturbed neuropathic interactions at the spinal cord level has not been clarified, which is crucial information from a clinical perspective. The findings of our study suggest that several indirect mechanisms may be responsible for the beneficial analgesic effect of mirogabalin. This is the first study to report that mirogabalin enhances the mRNA expression of spinal antinociceptive factors, such as IL-10 and IL-18BP, and reduces the concentration of the pronociceptive substance P. Importantly, mirogabalin improves the morphine-, buprenorphine-, oxycodone-, and ketamine-induced antinociceptive effects in a neuropathic pain model. Our findings support the hypothesis that enhancing opioid and ketamine analgesia by combining these drugs with mirogabalin may represent a new strategy for the effective pharmacotherapy of neuropathic pain.

Blockade of CC Chemokine Receptor Type 3 Diminishes Pain and Enhances Opioid Analgesic Potency in a Model of Neuropathic Pain.

Neuropathic pain is a serious clinical issue, and its treatment remains a challenge in contemporary medicine. Thus, dynamic development in the area of animal and clinical studies has been observed. The mechanisms of neuropathic pain are still not fully understood; therefore, studies investigating these mechanisms are extremely important. However, much evidence indicates that changes in the activation and infiltration of immune cells cause the release of pronociceptive cytokines and contribute to neuropathic pain development and maintenance. Moreover, these changes are associated with low efficacy of opioids used to treat neuropathy. To date, the role of CC chemokine receptor type 3 (CCR3) in nociception has not been studied. Similarly, little is known about its endogenous ligands (C-C motif ligand; CCL), namely, CCL5, CCL7, CCL11, CCL24, CCL26, and CCL28. Our research showed that the development of hypersensitivity in rats following chronic constriction injury (CCI) of the sciatic nerve is associated with upregulation of CCL7 and CCL11 in the spinal cord and dorsal root ganglia (DRG). Moreover, our results provide the first evidence that single and repeated intrathecal administration of the CCR3 antagonist SB328437 diminishes mechanical and thermal hypersensitivity. Additionally, repeated administration enhances the analgesic properties of morphine and buprenorphine following nerve injury. Simultaneously, the injection of SB328437 reduces the protein levels of some pronociceptive cytokines, such as IL-6, CCL7, and CCL11, in parallel with a reduction in the activation and influx of GFAP-, CD4- and MPO-positive cells in the spinal cord and/or DRG. Moreover, we have shown for the first time that an inhibitor of myeloperoxidase-4-aminobenzoic hydrazide may relieve pain and simultaneously enhance morphine and buprenorphine efficacy. The obtained results indicate the important role of CCR3 and its modulation in neuropathic pain treatment and suggest that it represents an interesting target for future investigations.

Comparison of the beneficial effects of RS504393, maraviroc and cenicriviroc on neuropathic pain-related symptoms in rodents: behavioral and biochemical analyses.

The latest research highlights the role of chemokine signaling pathways in the development of nerve injury-induced pain. Recent studies have provided evidence for the involvement of CCR2 and CCR5 in the pathomechanism underlying neuropathy. Thus, the aim of our study was to compare the effects of a selective CCR2 antagonist (RS504393), selective CCR5 antagonist (maraviroc) and dual CCR2/CCR5 antagonist (cenicriviroc) and determine whether the simultaneous blockade of both receptors is better than blocking only one of them selectively. All experiments were performed using Wistar rats/Swiss albino mice subjected to chronic constriction injury (CCI) of the sciatic nerve. To assess pain-related reactions, the von Frey and cold plate tests were used. The mRNA analysis was performed using RT-qPCR. We demonstrated that repeated intrathecal administration of the examined antagonists attenuated neuropathic pain in rats 7 days post-CCI. mRNA analysis showed that RS504393 did not modulate the spinal expression of the examined chemokines, whereas maraviroc reduced the CCI-induced elevation of CCL4 level. Cenicriviroc significantly lowered the spinal levels of CCL2-4 and CCL7. At the dorsal root ganglia, strong impacts of RS504393 and cenicriviroc on chemokine expression were observed; both reduced the CCI-induced elevation of CCL2-5 and CCL7 levels, whereas maraviroc decreased only the CCL5 level. Importantly, we demonstrated that a single intrathecal/intraperitoneal injection of cenicriviroc had greater analgesic properties than RS504393 or maraviroc in neuropathic mice. Additionally, we demonstrated that cenicriviroc enhanced opioid-induced analgesia. Based on our results, we suggest that targeting CCR2 and CCR5 simultaneously, is an interesting alternative for neuropathic pain pharmacotherapy.

Metamizole relieves pain by influencing cytokine levels in dorsal root ganglia in a rat model of neuropathic pain.

BACKGROUND: Treatment of neuropathic pain is still challenging. Recent studies have suggested that dorsal root ganglia (DRG), which carry sensory neural signals from the peripheral nervous system to the central nervous system, are important for pathological nociception. A proper understanding of the significance and function of DRG and their role in pharmacotherapy can help to improve the treatment of neuropathic pain. Metamizole, also known as sulpyrine or dipyrone, is a non-opioid analgesic commonly used in clinical practice, but it is not used for neuropathic pain treatment. METHODS: Chronic constriction injury (CCI) of the sciatic nerve was induced in Wistar rats. Metamizole was administered intraperitoneally (ip) preemptively at 16 and 1 h before CCI and then twice a day for 7 days. To evaluate tactile and thermal hypersensitivity, von Frey and cold plate tests were conducted, respectively. RESULTS: Our behavioral results provide evidence that repeated intraperitoneal administration of metamizole diminishes the development of neuropathic pain symptoms in rats. Simultaneously, our findings provide evidence that metamizole diminishes the expression of pronociceptive interleukins (IL-1beta, IL-6, and IL-18) and chemokines (CCL2, CCL4, and CCL7) in DRG measured 7 days after sciatic nerve injury. These assays indicate, for the first time, that metamizole exerts antinociceptive effects on nerve injury-induced neuropathic pain at the DRG level. CONCLUSIONS: Finally, we indicate that metamizole-induced analgesia in neuropathy is associated with silencing of a broad spectrum of cytokines in DRG. Our results also suggest that metamizole is likely to be an effective medication for neuropathic pain.

CCR4 antagonist (C021) influences the level of nociceptive factors and enhances the analgesic potency of morphine in a rat model of neuropathic pain.

Neuropathic pain is a chronic condition which significantly reduces the quality of life and serious clinical issue that is in general resistant to available therapies. Therefore looking for new analgesics is still critical issue. Recent, studies have indicated that chemokine signaling pathways are crucial for the development of neuropathy; however, the role of CC chemokine receptor 4 (CCR4) in this process has not yet been studied. Therefore, the aim of our research was to investigate the influence of C021 (a CCR4 antagonist) and CCR4 CC chemokine ligands 17 and 22 (CCL17 and CCL22) on the development of hypersensitivity and the effectiveness of morphine induced analgesia in naive animals and/or animals exposed to chronic constriction injury (CCI) of the sciatic nerve. Firstly, we demonstrated that the intrathecal administration of CCL17 and CCL22 induced pain-related behavior in naive mice. Secondly, we revealed that the intrathecal injection of C021 significantly reduced CCI-induced hypersensitivity after nerve injury. In parallel, C021 reduced microglia/macrophages activation and the level of some pronociceptive interleukins (IL-1beta; IL-18) in the spinal cord 8 days after CCI. Moreover, C021 not only attenuated mechanical and thermal hypersensitivity but also enhanced the analgesic properties of morphine. Our research indicates that CCR4 ligands might be important factors in the early stages of neuropathy, when we observe intense microglia/macrophages activation. Moreover, pharmacological blockade of CCR4 may serve as a potential new target for better understanding the mechanisms of neuropathic pain development.

Neuropathic Pain Dysregulates Gene Expression of the Forebrain Opioid and Dopamine Systems.

Disturbances in the function of the mesostriatal dopamine system may contribute to the development and maintenance of chronic pain, including its sensory and emotional/cognitive aspects. In the present study, we assessed the influence of chronic constriction injury (CCI) of the sciatic nerve on the expression of genes coding for dopamine and opioid receptors as well as opioid propeptides in the mouse mesostriatal system, particularly in the nucleus accumbens. We demonstrated bilateral increases in mRNA levels of the dopamine D1 and D2 receptors (the latter accompanied by elevated protein level), opioid propeptides proenkephalin and prodynorphin, as well as delta and kappa (but not mu) opioid receptors in the nucleus accumbens at 7 to 14 days after CCI. These results show that CCI-induced neuropathic pain is accompanied by a major transcriptional dysregulation of molecules involved in dopaminergic and opioidergic signaling in the striatum/nucleus accumbens. Possible functional consequences of these changes include opposite effects of upregulated enkephalin/delta opioid receptor signaling vs. dynorphin/kappa opioid receptor signaling, with the former most likely having an analgesic effect and the latter exacerbating pain and contributing to pain-related negative emotional states.

Kynurenic acid and zaprinast diminished CXCL17-evoked pain-related behaviour and enhanced morphine analgesia in a mouse neuropathic pain model.

BACKGROUND: The G protein-coupled receptor 35 (GPR35), is considered important for nociceptive transmission, as suggested by accumulating evidence. This receptor was discovered in 1998; however, a lack of pharmacological tools prevented a complete understanding of its function and how to exploit it therapeutically. We studied the influence of CXCL17, kynurenic acid and zaprinast on nociceptive transmission in naïve and neuropathic mice. Additionally, we investigated the influence of kynurenic acid and zaprinast on morphine effectiveness in neuropathic pain. METHODS: The chronic constriction injury (CCI) of the sciatic nerve in Swiss mice was performed. The CXCL17, kynurenic acid, zaprinast and morphine were injected intrathecally into naive and CCI-exposed mice at day 14. To evaluate tactile and thermal hypersensitivity, the von Frey and cold plate tests were used, respectively. RESULTS: Our results have shown, for the first time, that administration of CXCL17 in naïve mice induced strong pain-related behaviours, as measured by von Frey and cold plate tests. Moreover, we demonstrated that kynurenic acid and zaprinast diminished CXCL17-evoked pain-related behaviours in both tests. Kynurenic acid and zaprinast reduced thermal and tactile hypersensitivity developed by sciatic nerve injury and strongly enhanced the effectiveness of morphine in neuropathy. CONCLUSIONS: Our study highlights the importance of GPR35 as a receptor involved in neuropathic pain development. Therefore, these results suggest that the modulation of GPR35 could become a potential strategy for the treatment of neuropathic pain.