Sexually dimorphic effects of pexidartinib on nerve injury-induced neuropathic pain in mice.

It is well-established that spinal microglia and peripheral macrophages play critical roles in the etiology of neuropathic pain; however, growing evidence suggests sex differences in pain hypersensitivity owing to microglia and macrophages. Therefore, it is crucial to understand sex- and androgen-dependent characteristics of pain-related myeloid cells in mice with nerve injury-induced neuropathic pain. To deplete microglia and macrophages, pexidartinib (PLX3397), an inhibitor of the colony-stimulating factor 1 receptor, was orally administered, and mice were subjected to partial sciatic nerve ligation (PSL). Following PSL induction, healthy male and female mice and male gonadectomized (GDX) mice exhibited similar levels of spinal microglial activation, peripheral macrophage accumulation, and mechanical allodynia. Treatment with PLX3397 significantly suppressed mechanical allodynia in normal males; this was not observed in female and GDX male mice. Sex- and androgen-dependent differences in the PLX3397-mediated preventive effects were observed on spinal microglia and dorsal root ganglia (DRG) macrophages, as well as in expression patterns of pain-related inflammatory mediators in these cells. Conversely, no sex- or androgen-dependent differences were detected in sciatic nerve macrophages, and inhibition of peripheral CC-chemokine receptor 5 prevented neuropathic pain in both sexes. Collectively, these findings demonstrate the presence of considerable sex- and androgen-dependent differences in the etiology of neuropathic pain in spinal microglia and DRG macrophages but not in sciatic nerve macrophages. Given that the mechanisms of neuropathic pain may differ among experimental models and clinical conditions, accumulating several lines of evidence is crucial to comprehensively clarifying the sex-dependent regulatory mechanisms of pain.

Chemogenetic Regulation of CX3CR1-Expressing Microglia Using Gi-DREADD Exerts Sex-Dependent Anti-Allodynic Effects in Mouse Models of Neuropathic Pain.

Despite growing evidence suggesting that spinal microglia play an important role in the molecular mechanism underlying experimental neuropathic pain (NP) in male rodents, evidence regarding the sex-dependent role of these microglia in NP is insufficient. In this study, we evaluated the effects of microglial regulation on NP using Gi-designer receptors exclusively activated by designer drugs (Gi-DREADD) driven by the microglia-specific Cx3cr1 promoter. For the Cre-dependent expression of human Gi-coupled M4 muscarinic receptors (hM4Di) in CX3C chemokine receptor 1-expressing (CX3CR1+) cells, R26-LSL-hM4Di-DREADD mice were crossed with CX3CR1-Cre mice. Mouse models of NP were generated by partial sciatic nerve ligation (PSL) and treatment with anti-cancer agent paclitaxel (PTX) or oxaliplatin (OXA), and mechanical allodynia was evaluated using the von Frey test. Immunohistochemistry revealed that hM4Di was specifically expressed on Iba1+ microglia, but not on astrocytes or neurons in the spinal dorsal horn of CX3CR1-hM4Di mice. PSL-induced mechanical allodynia was significantly attenuated by systemic (intraperitoneal, i.p.) administration of 10 mg/kg of clozapine N-oxide (CNO), a hM4Di-selective ligand, in male CX3CR1-hM4Di mice. The mechanical threshold in naive CX3CR1-hM4Di mice was not altered by i.p. administration of CNO. Consistently, local (intrathecal, i.t.) administration of CNO (20 nmol) significantly relieved PSL-induced mechanical allodynia in male CX3CR1-hM4Di mice. However, neither i.p. nor i.t. administration of CNO affected PSL-induced mechanical allodynia in female CX3CR1-hM4Di mice. Both i.p. and i.t. administration of CNO relieved PTX-induced mechanical allodynia in male CX3CR1-hM4Di mice, and a limited effect of i.p. CNO was observed in female CX3CR1-hM4Di mice. Unlike PTX-induced allodynia, OXA-induced mechanical allodynia was slightly improved, but not significantly relieved, by i.p. administration of CNO in both male and female CX3CR1-hM4Di mice. These results suggest that spinal microglia can be regulated by Gi-DREADD and support the notion that CX3CR1+ spinal microglia play sex-dependent roles in nerve injury-induced NP; however, their roles may vary among different models of NP.

Inflammatory Macrophages in the Sciatic Nerves Facilitate Neuropathic Pain Associated with Type 2 Diabetes Mellitus.

Despite the requirement for effective medication against neuropathic pain associated with type 2 diabetes mellitus (T2DM), mechanism-based pharmacotherapy has yet to be established. Given that long-lasting neuroinflammation, driven by inflammatory macrophages in the peripheral nerves, plays a pivotal role in intractable pain, it is important to determine whether inflammatory macrophages contribute to neuropathic pain associated with T2DM. To generate an experimental model of T2DM, C57BL/6J mice were fed a high-fat diet (HFD) ad libitum. Compared with control diet feeding, obesity and hyperglycemia were observed after HFD feeding, and the mechanical pain threshold evaluated using the von Frey test was found to be decreased, indicating the development of mechanical allodynia. The expression of mRNA markers for macrophages, inflammatory cytokines, and chemokines were significantly upregulated in the sciatic nerve (SCN) after HFD feeding. Perineural administration of saporin-conjugated anti-Mac1 antibody (Mac1-Sap) improved HFD-induced mechanical allodynia. Moreover, treatment of Mac1-Sap decreased the accumulation of F4/80+ macrophages and the upregulation of inflammatory mediators in the SCN after HFD feeding. Inoculation of lipopolysaccharide-activated peritoneal macrophages in tissue surrounding the SCN elicited mechanical allodynia. Furthermore, pharmacological inhibition of inflammatory macrophages by either perineural or systemic administration of TC-2559 [4-(5-ethoxy-3-pyridinyl)-N-methyl-(3E)-3-buten-1-amine difumarate], a α4ß2 nicotinic acetylcholine receptor-selective agonist, relieved HFD-induced mechanical allodynia. Taken together, inflammatory macrophages that accumulate in the SCN mediate the pathophysiology of neuropathic pain associated with T2DM. Inhibitory agents for macrophage-driven neuroinflammation could be potential candidates for novel pharmacotherapy against intractable neuropathic pain.

Chemokine CXCL1 is responsible for cocaine-induced reward in mice.

AIM: We have previously demonstrated that upregulation of CC chemokines through dopamine receptor signaling in the prefrontal cortex (PFC) underlies methamphetamine (Meth)-induced reward. Given the common pharmacological property of Meth and cocaine (Coca), which are highly addictive psychostimulants, we hypothesized that chemokines may also contribute to Coca-induced reward. The aim of this study was to identify a key chemokine-mediating Coca-induced reward in mice. METHODS: The mRNA expression levels of chemokines were measured by reverse transcription-quantitative polymerase chain reaction. Coca-induced reward was evaluated by conditioned place preference test. RESULTS: We found that mRNA expression levels of CC chemokine ligand 2 (CCL2), CCL7, and CXC chemokine ligand 1 (CXCL1) were upregulated in the PFC after a single administration of Coca (20 mg/kg, s.c.). Upregulation of CXCL1, but not CCL2 and CCL7, mRNA in the PFC was also observed after repeated administration of Coca. A single administration of dopamine D1 receptor agonist SKF 81297 (10 mg/kg, s.c.), but not D2 receptor agonist sumanirole, upregulated CXCL1 mRNA in the PFC. Coca-induced reward was attenuated by the pretreatment of SB 225002 (5 mg/kg, s.c.), a selective antagonist of CXC chemokine receptor 2 (CXCR2, cognate receptor for CXCL1). CONCLUSIONS: Collectively, we identified CXCL1 as a key regulator in Coca-induced reward and propose that pharmacological approach targeting CXCL1 could be a novel pharmacotherapy for Coca-induced reward.

Inhibition of peripheral macrophages by nicotinic acetylcholine receptor agonists suppresses spinal microglial activation and neuropathic pain in mice with peripheral nerve injury.

BACKGROUND: Neuro-immune interaction underlies chronic neuroinflammation and aberrant sensory processing resulting in neuropathic pain. Despite the pathological significance of both neuroinflammation-driven peripheral sensitization and spinal sensitization, the functional relationship between these two distinct events has not been understood. METHODS: In this study, we determined whether inhibition of inflammatory macrophages by administration of α4ß2 nicotinic acetylcholine receptor (nAChR) agonists improves neuropathic pain and affects microglial activation in the spinal dorsal horn (SDH) in mice following partial sciatic nerve ligation (PSL). Expression levels of neuroinflammatory molecules were evaluated by RT-qPCR and immunohistochemistry, and PSL-induced mechanical allodynia was defined by the von Frey test. RESULTS: Flow cytometry revealed that CD11b+ F4/80+ macrophages were accumulated in the injured sciatic nerve (SCN) after PSL. TC-2559, a full agonist for α4ß2 nAChR, suppressed the upregulation of interleukin-1ß (IL-1ß) in the injured SCN after PSL and attenuated lipopolysaccharide-induced upregulation of IL-1ß in cultured macrophages. Systemic (subcutaneous, s.c.) administration of TC-2559 during either the early (days 0-3) or middle/late (days 7-10) phase of PSL improved mechanical allodynia. Moreover, local (perineural, p.n.) administration of TC-2559 and sazetidine A, a partial agonist for α4ß2 nAChR, during either the early or middle phase of PSL improved mechanical allodynia. However, p.n. administration of sazetidine A during the late (days 21-24) phase did not show the attenuating effect, whereas p.n. administration of TC-2559 during this phase relieved mechanical allodynia. Most importantly, p.n. administration of TC-2559 significantly suppressed morphological activation of Iba1+ microglia and decreased the upregulation of inflammatory microglia-dominant molecules, such as CD68, interferon regulatory factor 5, and IL-1ß in the SDH after PSL. CONCLUSION: These findings support the notion that pharmacological inhibition of inflammatory macrophages using an α4ß2 nAChR agonist exhibit a wide therapeutic window on neuropathic pain after nerve injury, and it could be nominated as a novel pharmacotherapy to relieve intractable pain.

Pharmacological Regulation of Neuropathic Pain Driven by Inflammatory Macrophages.

Neuropathic pain can have a major effect on quality of life but current therapies are often inadequate. Growing evidence suggests that neuropathic pain induced by nerve damage is caused by chronic inflammation. Upon nerve injury, damaged cells secrete pro-inflammatory molecules that activate cells in the surrounding tissue and recruit circulating leukocytes to the site of injury. Among these, the most abundant cell type is macrophages, which produce several key molecules involved in pain enhancement, including cytokines and chemokines. Given their central role in the regulation of peripheral sensitization, macrophage-derived cytokines and chemokines could be useful targets for the development of novel therapeutics. Inhibition of key pro-inflammatory cytokines and chemokines prevents neuroinflammation and neuropathic pain; moreover, recent studies have demonstrated the effectiveness of pharmacological inhibition of inflammatory (M1) macrophages. Nicotinic acetylcholine receptor ligands and T helper type 2 cytokines that reduce M1 macrophages are able to relieve neuropathic pain. Future translational studies in non-human primates will be crucial for determining the regulatory mechanisms underlying neuroinflammation-associated neuropathic pain. In turn, this knowledge will assist in the development of novel pharmacotherapies targeting macrophage-driven neuroinflammation for the treatment of intractable neuropathic pain.

Peripheral administration of interleukin-13 reverses inflammatory macrophage and tactile allodynia in mice with partial sciatic nerve ligation.

Inflammatory macrophages play a fundamental role in neuropathic pain. In this study, we demonstrate the effects of peripheral interleukin-13 (IL-13) on neuropathic pain after partial sciatic nerve (SCN) ligation (PSL) in mice. IL-13 receptor α1 was upregulated in accumulating macrophages in the injured SCN after PSL. Treatment with IL-13 reduced inflammatory macrophage-dominant molecules and increased suppressive macrophage-dominant molecules in cultured lipopolysaccharide-stimulated peritoneal macrophages and ex vivo SCN subjected to PSL. Moreover, the perineural administration of IL-13 relieved tactile allodynia after PSL. These results suggest that IL-13 reverses inflammatory macrophage-dependent neuropathic pain via a phenotype shift toward suppressive macrophages.

TC-2559, an α4ß2 nicotinic acetylcholine receptor agonist, suppresses the expression of CCL3 and IL-1ß through STAT3 inhibition in cultured murine macrophages.

The anti-inflammatory properties of TC-2559, an α4ß2 nicotinic acetylcholine receptor (nAChR) agonist, on cultured murine macrophages was investigated. TC-2559 suppressed the upregulation of CC-chemokine ligand 3 (CCL3) and interleukin-1ß (IL-1ß) following lipopolysaccharide (LPS) treatment in J774A.1 cells. TC-2559 inhibited the phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) but not nuclear factor-κB p65 after LPS. Blockade of pSTAT3 by AG490 inhibited the upregulation of CCL3 and IL-1ß after LPS. In conclusion, TC-2559-driven α4ß2 nAChR signaling suppressed the upregulation of CCL3 and IL-1ß by inhibiting pSTAT3 in inflammatory macrophages, resulting in the suppression of neuropathic pain.

Macrophage-T cell interactions mediate neuropathic pain through the glucocorticoid-induced tumor necrosis factor ligand system.

Peripheral neuroinflammation caused by activated immune cells can provoke neuropathic pain. Herein, we investigate the actions of macrophages and T cells through glucocorticoid-induced tumor neurosis factor receptor ligand (GITRL) and its receptor (GITR) in neuropathic pain. After partial sciatic nerve ligation (PSL) in enhanced green fluorescent protein (eGFP) chimeric mice generated by the transplantation of eGFP(+) bone marrow cells, eGFP(+) macrophages, and T cells markedly migrated to the injured site after PSL. Administration of agents to deplete macrophages (liposome-clodronate and Clophosome-A(TM)) or T cells (anti-CD4 antibody and FTY720) could suppress PSL-induced thermal hyperalgesia and tactile allodynia. The expression levels of co-stimulatory molecules GITRL and GITR were increased on infiltrating macrophages and T cells, respectively. The perineural injection of a GITRL neutralizing antibody that could inhibit the function of the GITRL-GITR pathway attenuated PSL-induced neuropathic pain. Additionally, the induction of inflammatory cytokines and the accumulation of GITR(+) T cells in the injured SCN were abrogated after macrophage depletion by Clophosome-A(TM). In conclusion, GITRL expressed on macrophages drives cytokine release and T cell activation, resulting in neuropathic pain via GITR-dependent actions. The GITRL-GITR pathway might represent a novel target for the treatment of neuropathic pain.

Peripheral interleukin-4 ameliorates inflammatory macrophage-dependent neuropathic pain.

There is increasing evidence that inflammatory (M1-polarized) macrophages drive the nonresolving neuroinflammation that causes neuropathic pain after nerve injury. As interleukin-4 (IL-4) promotes the suppressive (M2-polarized) state in macrophages, we examined whether exploiting an IL-4-mediated pathway could ameliorate M1 macrophage-dependent neuropathic pain. The mRNA and protein expression of IL-4 receptor α chain (IL-4Rα) were upregulated in accumulating F4/80 macrophages in injured sciatic nerve (SCN). In mouse macrophage cell line J774A.1, IL-4 downregulated the mRNA expression of M1 macrophage-specific molecules (IL-1ß, CC chemokine ligand 3, and CD86) normally provoked by lipopolysaccharide, while increasing the mRNA expression of M2 macrophage-specific molecules (arginase-1, IL-10, and CD206) through a STAT6-mediated pathway. In ex vivo SCN culture, M1 molecules were highly expressed in the injured SCN on day 7 after partial SCN ligation (PSL) but were decreased by IL-4 treatment. In contrast, M2 molecules were upregulated by IL-4. IL-4 also increased phosphorylated STAT6 (pSTAT6) expression and shifted IL-1ß M1 macrophages toward a CD206 M2 phenotype. Perineural administration of IL-4 in mice subject to PSL ameliorated development and maintenance of tactile allodynia and thermal hyperalgesia. These effects of IL-4 were based on that IL-4 treatment increased the proportions of pSTAT6 and CD206 macrophages in injured SCN on day 14 after PSL. We found that neuropathic pain can be ameliorated by IL-4 treatment, which exerts its therapeutic effect on accumulating macrophages through a STAT6-dependent pathway. A shift in macrophage phenotype from the inflammatory to the suppressive phenotype, driven by IL-4R signaling, may have benefits in the treatment of neuropathic pain.

Epigenetic regulation of CC-chemokine ligand 2 in nonresolving inflammation.

Inflammation mediated by the crosstalk between leukocytes and resident tissue cells is crucial for the maintenance of homeostasis. Because chemokine ligands and receptors, which recruit a variety of leukocytes, are widely distributed among tissues, it is important to understand the mechanisms regulating inflammatory disease. Chemokines such as CC-chemokine ligand 2 (CCL2) amplify and maintain inflammation through chemokine-cytokine networks after the recruitment of circulating leukocytes. Chemokine-dependent nonresolving inflammation occurs in the peripheral and central nervous systems, and underlies several intractable diseases, including cancer and neuropathic pain. The chronic upregulation of chemokines is often mediated by epigenetic mechanisms consisting of DNA methylation, histone modification, and nucleosome positioning. In particular, histone acetylation and methylation have been shown to play important roles in the upregulation of chemokine expression. In addition to CCL2, several other chemokines strongly contribute to neuropathic pain through epigenetic induction. Consequently, targeting epigenetic changes may have therapeutic potential for nonresolving inflammatory diseases such as neuropathic pain. Further research into the epigenetics of inflammatory diseases should promote the development of novel and effective treatment strategies for intractable inflammatory diseases.

Nicotine effects and the endogenous opioid system.

Nicotine (NIC) is an exogenous ligand of the nicotinic acetylcholine receptor (nAChR), and it influences various functions in the central nervous system. Systemic administration of NIC elicits the release of endogenous opioids (endorphins, enkephalins, and dynorphins) in the supraspinal cord. Additionally, systemic NIC administration induces the release of methionine-enkephalin in the spinal dorsal horn. NIC has acute neurophysiological actions, including antinociceptive effects, and the ability to activate the hypothalamic-pituitary-adrenal (HPA) axis. The endogenous opioid system participates in NIC-induced antinociception, but not HPA axis activation. Moreover, NIC-induced antinociception is mediated by α4ß2 and α7 nAChRs, while NIC-induced HPA axis activation is mediated by α4ß2, not α7, suggesting that the effects of NIC on the endogenous opioid system are mediated by α7, not α4ß2. NIC has substantial physical dependence liability. The opioid-receptor antagonist naloxone (NLX) elicits NIC withdrawal after repeated NIC administration, and NLX-induced NIC withdrawal is inhibited by concomitant administration of an opioid-receptor antagonist. NLX-induced NIC withdrawal is also inhibited by concomitant administration of an α7 antagonist, but not an α4ß2 antagonist. Taken together, these findings suggest that NIC-induced antinociception and the development of physical dependence are mediated by the endogenous opioid system, via the α7 nAChR.

[Development of physical dependence on nicotine and endogenous opioid system--participation of α7 nicotinic acetylcholine receptor].

Nicotine (NIC) regulates various cellular functions acting on the nicotinic acetylcholine receptor (nAChR). And nAChR consists of ligand-gated cation channels with pentameric structure and composed of α and ß subunits. In the central nervous system, α 4 ß 2 and α 7 nAChRs are the most abundantly expressed as nAChR subtypes. There are several lines of evidence indicating that systemic administration of NIC elicits the release of endogenous opioids, such as, endorphins, enkephalins and dynorphins, in the brain. NIC exerts numerous acute effects, for example, antinociceptive effects and the activating effects of the hypothalamic-pituitary-adrenal (HPA) axis. In these effects, NIC-induced antinociception, but not HPA axis activation, was inhibited by opioid receptor antagonist, naloxone (NLX), and was also suppressed in morphine tolerated mice, indicating the participation of the endogenous opioid system in NIC-induced antinociception, but not HPA axis activation. Moreover, NIC-induced antinociception was antagonized by both α 4 ß 2 and α 7 nAChR antagonists, while NIC-induced HPA axis activation was antagonized by α 4 ß 2 nAChR antagonist, but not by α 7 nAChR antagonist. These results suggest that the endogenous opioid system may not be located on the downstream of α 4 ß 2 nAChR. On the other hand, NIC has substantial physical dependence liability. NLX elicits NIC withdrawal after repeated NIC administration evaluated by corticosterone increase as a withdrawal sign, and NLX-precipitated NIC withdrawal is inhibited by concomitant administration of other opioid receptor antagonist, naltrexone, indicating the participation of endogenous opioid system in the development of physical dependence on NIC. NLX-precipitated NIC withdrawal was also inhibited by concomitant administration of an α 7 nAChR antagonist, but not an α 4 ß 2 nAChR antagonist. Taken together, these findings suggest that the endogenous opioid system may be located on the downstream of α 7 nAChR and participates in the development of physical dependence on NIC.

Vascular endothelial growth factor signaling in injured nerves underlies peripheral sensitization in neuropathic pain.

Chronic neuroinflammation may be a critical component of intractable inflammatory diseases, including neuropathic pain. Because angiogenesis as a result of vascular endothelial growth factor (VEGF) signaling plays a pivotal role in inflammation, we focused on the mechanisms of VEGF-regulated neuropathic pain in mice. The mRNA and protein expression of VEGFA were up-regulated in the injured sciatic nerve after partial sciatic nerve ligation (PSL). VEGFA was localized to accumulated macrophages and neutrophils derived from bone marrow. Up-regulation of VEGFA was mediated by histone H3 acetylation and trimethylation in its promoter region. VEGF receptors (VEGFR1 and VEGFR2) were localized to vascular endothelial cells or macrophages. By ex vivo fluorescence imaging and immunohistochemistry using DiI fluorescence, progression of angiogenesis was observed in the injured sciatic nerve after PSL. Perineural administration of pharmacological inhibitors of VEGFA and VEGFR tyrosine kinases prevented tactile allodynia and thermal hyperalgesia caused by PSL. Moreover, we determined the contribution of VEGF- and CXC-chemokine receptor 4-expressing angiogenic macrophages to neuropathic pain. Taken together, VEGFA is up-regulated in injured peripheral nerves and participates in angiogenesis and prolonged pain behaviors through its receptors. We propose that VEGFA-related components may underlie peripheral sensitization leading to neuropathic pain. Angiogenesis due to VEGF signaling is a key component of chronic inflammation. VEGFA up-regulation and pathological angiogenesis were observed in the injured nerves in mice. Pharmacological inhibition of VEGF signaling suppressed neuropathic pain behaviors. Therefore, VEGFA-related components may underlie peripheral neuroinflammation leading to neuropathic pain.

Possible involvement of endogenous opioid system located downstream of α7 nicotinic acetylcholine receptor in mice with physical dependence on nicotine.

We previously reported that nicotine (NIC)-induced analgesia was elicited in part by activation of the endogenous opioid system. Moreover, it is well known that NIC has physical-dependence liability, but its mechanism is unclear. Therefore, we examined whether physical dependence on NIC was mediated by activation of the endogenous opioid system in ICR mice. We evaluated increased serum corticosterone (SCS) as an indicator of NIC withdrawal, as it is a quantitative indicator of naloxone (opioid receptor antagonist, NLX)-precipitated morphine withdrawal in mice. In this study, NLX precipitated an SCS increase in mice receiving repeated NIC, by a dose-dependent mechanism, and correlated with the dose and number of days of repeated NIC administration. When an opioid receptor antagonist (naltrexone) was concomitantly administered with repeated NIC, the NLX-precipitated SCS increase was not elicited. Concomitant administration of the α7 nicotinic acetylcholine receptor (nAChR) antagonist (methyllycaconitine) with repeated NIC, but not the α4ß2 nAChR antagonist (dihydro-ß-erythroidine), did not elicit an SCS increase by NLX. Thus, a physical dependence on NIC was in part mediated by the activation of the endogenous opioid system, located downstream of α7 nAChR.

Epigenetic upregulation of CCL2 and CCL3 via histone modifications in infiltrating macrophages after peripheral nerve injury.

To gain insight into the epigenetic regulation of CC-chemokine ligand (CCL) 2 and CCL3, key players in the peripheral sensitization leading to neuropathic pain, we examined the relationship between histone H3 modification and the upregulation of these molecules using a mouse model of neuropathic pain after partial sciatic nerve ligation (PSL). We found that circuiting bone marrow (BM)-derived macrophages infiltrated into the injured sciatic nerve (SCN) using enhanced green fluorescent protein chimeric mice. The mRNA levels of CCL2, CCL3 and their receptors (CCR2 and CCR1/CCR5, respectively) were increased in the injured SCN. Chromatin immunoprecipitation assay revealed that levels of lysine 9-acetylated histone H3 (H3K9Ac) and lysine 4-trimethylated H3 (H3K4me(3)) in the promoter regions of the CCL2 and CCL3 genes were increased in the injured SCN after PSL, indicating the enhancement of gene expression. Immunoreactivity for H3K9Ac and H3K4me(3) was localized in the nuclei of infiltrating BM-derived cells and CCL-expressing cells in the injured SCN. We observed H3K9Ac and H3K4me(3) mainly in the nuclei of recruited macrophages on day 7 after PSL. Furthermore, upregulation of CCLs and CCRs were suppressed by histone acetyltransferase inhibitor, anacardic acid. Taken together, our findings demonstrate that CCL2 and CCL3 are upregulated in the injured peripheral nerve through epigenetic histone modification in infiltrating immune cells such as macrophages. These chemokine cascades may subsequently elicit chronic neuroinflammation following nerve injury.

Involvement of inflammatory mediators in neuropathic pain caused by vincristine.

Elucidation of the mechanism of neuropathic pain caused by vincristine is required because long-term treatment with this anticancer agent often causes neuropathic pain. We refer to the involvement of inflammatory mediators in vincristine-induced neuropathic pain in this review. Several reports using rodents have shown that long-lasting neuropathic pain (mechanical allodynia) is caused by repeated systemic injection of vincristine. Vincristine damaged Schwann cells and DRG neurons in this model. Vincristine-induced macrophage infiltration in the peripheral nervous system (PNS) and macrophage-derived IL-6 elicited mechanical allodynia. These findings proved that inhibition of IL-6 function prevented neuropathic pain caused by vincristine. In the central nervous system (CNS), activation of microglia and astrocytes in the spinal cord were demonstrated after long-term vincristine treatment. TNF-alpha was upregulated in activated microglia and astrocytes, and inhibition of TNF-alpha function attenuated neuropathic pain caused by vincristine. These results suggest that vincristine induces macrophage infiltration to the damaged PNS, and that macrophage-derived inflammatory cytokines such as IL-6 elicits neuroinflammation. Signal transduction of pain from the PNS to the CNS activates microglia and astrocytes, and these activated glial cells release inflammatory cytokines such as TNF-alpha. In the CNS, these inflammatory cytokines have an important role in the neuropathic pain caused by vincristine. Immune-modulating agents that prevent activation of immune cells and/or the inhibitory agents of inflammatory cytokines could prevent the neuropathic pain caused by vincristine. These agents could increase the tolerability of vincristine when used for the treatment of leukemia and lymphoma.

Suppressive effect of imipramine on vincristine-induced mechanical allodynia in mice.

Because chronic vincristine (VCR) treatment causes neuropathic pain, as demonstrated by mechanical allodynia, effective therapeutic strategy is required. In this study, we investigated a suppressive effect of imipramine (IMI) on VCR-induced mechanical allodynia in mice. VCR (0.1 mg/kg, intraperitoneally (i.p.)) was administered once per day for 7 d in ICR male mice. Mechanical allodynia was evaluated by withdrawal response using von Frey filaments. In VCR-treated mice, mechanical allodynia was observed on day 3, 7, and 14. On day 14, morphine (3 mg/kg, subcutaneously) slightly but significantly suppressed VCR-induced mechanical allodynia. The percent inhibition by morphine of VCR-induced mechanical allodynia was less than that of the lambda-carrageenan-induced inflammatory pain and was similar to that of nerve injury-induced neuropathic pain. Although single administration of IMI (30 mg/kg, i.p.) had no effect on VCR-induced mechanical allodynia, repeated administration of IMI (30 mg/kg, i.p.) for 7 d significantly suppressed VCR-induced mechanical allodynia. Suppressive effects by repeated IMI administration were observed in both early phase (day 0-6) and late phase (day 7-13) of VCR-induced mechanical allodynia. These results suggest that chronic VCR administration induces opioid analgesics-resistant mechanical allodynia, and repeated IMI administration may be an effective therapeutic approach for the treatment of VCR-induced mechanical allodynia.