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.

Characterization of spinal microglial activation in a mouse model of imiquimod-induced psoriasis.

Although microglia are associated with chronic pain, the role of spinal microglia in the regulation of itch remains unclear. In this study, we characterized spinal microglial activation in a mouse model of imiquimod (IMQ)-induced psoriasis. Hypertrophic (activated) microglia were observed throughout the spinal cord after the topical application of IMQ. Furthermore, the mRNA expression of microglial markers and inflammatory mediators was upregulated. Ablation of itch-related sensory neurons using resiniferatoxin decreased itch-related scratching behavior and the number of hypertrophic microglia in the spinal dorsal horn. Conclusively, sensory neuron input may partially contribute to spinal microglial activation after IMQ application.

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.

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.

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.

Epigenetic augmentation of the macrophage inflammatory protein 2/C-X-C chemokine receptor type 2 axis through histone H3 acetylation in injured peripheral nerves elicits neuropathic pain.

Although there is growing evidence showing that the involvement of chemokines in the pathogenesis of neuropathic pain is associated with neuroinflammation, the details are unclear. We investigated the C-X-C chemokine ligand type 2 [macrophage inflammatory protein 2 (MIP-2)]/C-X-C chemokine receptor type 2 (CXCR2) axis and epigenetic regulation of these molecules in neuropathic pain after peripheral nerve injury. Expression of MIP-2 and CXCR2 were up-regulated and localized on accumulated neutrophils and macrophages in the injured sciatic nerve (SCN) after partial sciatic nerve ligation (PSL). Perineural injection of MIP-2-neutralizing antibody (anti-MIP-2) or the CXCR2 antagonist N-(2-bromophenyl)-N'-(2-hydroxy-4-nitrophenyl)urea (SB225002) prevented PSL-induced tactile allodynia and thermal hyperalgesia. Perineural injection of recombinant MIP-2 elicited neuropathic pain-like behaviors. Anti-MIP-2 suppressed neutrophil accumulation in the SCN after PSL. Neutrophil depletion by intraperitoneal injection of Ly6G antibody attenuated PSL-induced neuropathic pain. Both anti-MIP-2 and SB225002 suppressed up-regulation of inflammatory cytokines and chemokines in the injured SCN. In addition, acetylation of histone H3 [lysine (Lys9)-acetylated histone H3 (AcK9-H3)] on the promoter region of MIP-2 and CXCR2 was increased in the injured SCN after PSL. Expression of AcK9-H3 was observed in the nuclei of neutrophils and macrophages surrounding the epineurium. Administration of the histone acetyltransferase inhibitor anacardic acid suppressed the up-regulation of MIP-2 and CXCR2 in the SCN after PSL and resulted in the prevention of PSL-induced neuropathic pain. Taken together, these results show that augmentation of the MIP-2/CXCR2 axis by hyperacetylation of histone H3 on the promoter region of MIP-2 and CXCR2 located in the injured peripheral nerve elicits chronic neuroinflammation through neutrophil accumulation, leading to neuropathic pain.

Quality assessment of Japanese clinical practice guidelines including recommendations for acupuncture.

Background: The quality of clinical practice guidelines (CPGs) should be extensively evaluated. This study aimed to evaluate Japanese CPGs that include recommendations for acupuncture. Methods: In a literature search, CPGs including recommendations for acupuncture published in Japan until October 2021 were sought. We assessed (1) whether the CPGs were developed in accordance with the Grading Recommendations Assessment, Development and Evaluation (GRADE) system, (2) the quality of the CPGs using the Appraisal of Guidelines for Research and Evaluation (AGREE) II, and (3) whether the strength of the recommendations for acupuncture was consistent with each CPG's predefined procedure. Results: Seventeen CPGs including 23 recommendations in total were identified and assessed. (1) Three CPGs were in accordance with the GRADE system. (2) The mean score of overall assessment using AGREE II was 4.5 on a 7-point Likert scale. The mean domain scores were 77% for domain 1 (scope and purpose), 54% for domain 2 (stakeholder involvement), 48% for domain 3 (rigor of development), 78% for domain 4 (clarity of presentation), 20% for domain 5 (applicability), and 51% for domain 6 (editorial independence). (3) The strength of the recommendations for acupuncture in two CPGs was judged to be underestimated. Some of the CPGs contained elementary problems that were not considered in AGREE II. Conclusion: The methodological quality of Japanese CPGs including recommendations for acupuncture was not necessarily high. Since technical issues exist in each field of therapy, the respective experts should be involved in developing and reviewing CPGs to disseminate accurate health information.

Chemogenetic activation of central gastrin-releasing peptide-expressing neurons elicits itch-related scratching behavior in male and female mice.

Several lines of evidence have clarified that the key transmission pathways of itching sensation travel from the periphery to the central nervous system (CNS). Despite the functional significance of gastrin-releasing peptide (GRP) and its cognate receptor in the itch processing mechanism in the spinal dorsal horn (SDH), the roles of GRP-expressing (GRP+ ) neurons in different regions remain unclear. This study aimed to determine whether GRP+ neurons in the CNS directly modulated itch processing. To specifically activate spinal and supraspinal GRP neurons by the designer receptors exclusively activated by designer drugs (DREADDs) system, CAG-LSL-Gq-DREADD mice were crossed with GRP-Cre mice, resulting in the development of GRP-hM3Dq mice. Immunohistochemistry showed that hM3Dq was highly expressed in the SDH and brainstem closely related to sensory processing. The intraperitoneal, intrathecal, or intracerebroventricular administration of clozapine-N-oxide, an agonist of hM3Dq, strongly elicited dermatome-dependent itch-related scratching behavior, but did not change pain sensitivity. Importantly, GRP-Gq-DREADD-mediated scratching behavior in GRP-hM3Dq mice was not affected by the ablation of transient receptor potential vanilloid 1+ sensory C-fibers, and it was also observed to a similar degree under chronic itch conditions. Furthermore, there were no significant sex differences in the scratching behavior elicited by GRP-Gq-DREADD, suggesting that itch-dominant roles of central GRP+ neurons might be common in both sexes, at least under normal physiological conditions. These novel findings not only contribute to understanding the functional roles of central GRP+ neurons further, but also propose the development of future effective therapeutics for intractable itching.

Critical role of GRP receptor-expressing neurons in the spinal transmission of imiquimod-induced psoriatic itch.

AIM: Ample evidence indicates that gastrin-releasing peptide receptor (GRPR)-expressing neurons play a critical role in the transmission of acute itch. However, the pathophysiology of spinal mechanisms underlying intractable itch such as psoriasis remains unclear. In this study, we aimed to determine whether itch-responsive GRPR+ neurons contribute to the spinal transmission of imiquimod (IMQ)-induced psoriatic itch. METHODS: To generate a psoriasis model, C57BL/6J mice received a daily topical application of 5% IMQ cream on their shaved back skin for 7-10 consecutive days. GRP+ neurons were inhibited using Cre-dependent expression of Gi-designer receptors exclusively activated by designer drugs (DREADDs), while GRPR+ neurons were ablated by intrathecal administration of bombesin-saporin. RESULTS: Repeated topical application of IMQ elicited psoriasis-like dermatitis and scratching behaviors. The mRNA expression levels of GRP and GRPR were upregulated in the cervical spinal dorsal horn (SDH) on days 7 and 10 after IMQ application. Either chemogenetic silencing of GRP+ neurons by Gi-DREADD or ablation of GRPR+ neurons significantly attenuated IMQ-induced scratching behaviors. CONCLUSION: The GRP-GRPR system might be enhanced in the SDH, and itch-responsive GRPR+ neurons largely contribute to intractable itch in a mouse model of psoriasis.

Association between oxidative stress and microRNA expression pattern of ALS patients in the high-incidence area of the Kii Peninsula.

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disorder of the upper and lower motor neuron systems. The high incidence of ALS in the southern part of the Kii Peninsula of Japan (K-ALS) was reported in the 1960s, but it has gradually decreased to the worldwide average. Although causes of the high incidence of ALS in this area are unknown, our previous studies suggested that environmental factors, including essential mineral deficiency and increased metal-induced oxidative stress, play a role in its development. Recently, it has been reported that microRNAs (miRNA) contribute to the degeneration of nervous system such as ALS. The aim of this study is to explore specific miRNAs in K-ALS and evaluate relationships between oxidative stress. We comprehensively analyzed serum miRNAs and examined urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG), serum Cu/Zn superoxide dismutase (SOD) and serum Nɛ-hexanoyl lysin (HEL) as oxidative stress markers in the patients with K-ALS, sporadic ALS (S-ALS), residents in this area (K-residents) and controls from another area. The expression levels of miR-92a-3p and miR-486-5p in the patients with K-ALS were significantly higher than those in controls. The HEL levels were significantly higher in the patients with K-ALS than in those with S-ALS and controls. The expression levels of miR-92a-3p and miR-486-5p were not correlated with the levels of HEL. A set of high levels of miR-92a-3p, miR-486-5p and serum HEL may be a useful biomarker for K-ALS in the Kii Peninsula. The findings should be further studied by a large number of subjects.

GRP receptor and AMPA receptor cooperatively regulate itch-responsive neurons in the spinal dorsal horn.

Gastrin-releasing peptide (GRP) receptor-expressing (GRPR)+ neurons have a central role in the spinal transmission of itch. Because their fundamental regulatory mechanisms are not yet understood, it is important to determine how such neurons are excited and integrate itch sensation. In this study, we investigated the mechanisms for the activation of itch-responsive GRPR+ neurons in the spinal dorsal horn (SDH). GRPR+ neurons expressed the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) containing the GluR2 subunit. In mice, peripherally elicited histaminergic and non-histaminergic itch was prevented by intrathecal (i.t.) administration of the AMPAR antagonist NBQX, which was consistent with the fact that firing of GRPR+ neurons in SDH under histaminergic and non-histaminergic itch was completely blocked by NBQX, but not by the GRPR antagonist RC-3095. Because GRP+ neurons in SDH contain glutamate, we investigated the role of GRP+ (GRP+/Glu+) neurons in regulating itch. Chemogenetic inhibition of GRP+ neurons suppressed both histaminergic and non-histaminergic itch without affecting the mechanical pain threshold. In nonhuman primates, i.t. administration of NBQX also attenuated peripherally elicited itch without affecting the thermal pain threshold. In a mouse model of diphenylcyclopropenone (DCP)-induced contact dermatitis, GRP, GRPR, and AMPAR subunits were upregulated in SDH. DCP-induced itch was prevented by either silencing GRP+ neurons or ablation of GRPR+ neurons. Altogether, these findings demonstrate that GRP and glutamate cooperatively regulate GRPR+ AMPAR+ neurons in SDH, mediating itch sensation. GRP-GRPR and the glutamate-AMPAR system may play pivotal roles in the spinal transmission of itch in rodents and nonhuman primates.

Leptin enhances CC-chemokine ligand expression in cultured murine macrophage.

Despite accumulating evidence, the role of leptin in chemokine expression is poorly understood. In this study, we evaluated the effects of leptin on CC-chemokine ligands (CCLs), CCL3, CCL4, and CCL5 gene expression in cultured murine macrophage, J774A.1 cells. Expression of all these CCLs mRNA was gradually increased and significant up-regulation was observed for 3-12 h exposure to leptin (1 microM). The phosphorylated signal transducer and activator of transcription 3 (pSTAT3) was significantly increased for 5-20 min exposure to leptin, and it was localized in leptin receptor-positive macrophage. Pretreatment with AG490 (100 microM), a janus kinase 2 (JAK2) inhibitor, significantly suppressed leptin-induced pSTAT3 increases and the up-regulation of CCLs mRNA expression. In conclusion, leptin enhances CCLs expression in cultured murine macrophage, through activation of a JAK2-STAT3 pathway. Therefore, a new paradigm of leptin-mediated chemokine expression may lead to the clarification of complex immune systems in future.

Activation of extracellular signal-regulated kinase in sciatic nerve contributes to neuropathic pain after partial sciatic nerve ligation in mice.

BACKGROUND: The mitogen-activated protein kinase family plays an important role in several types of pain. However, the detailed role of phosphorylated extracellular signal-regulated kinase (pERK) in the region of injured peripheral nerve is poorly understood. In this study, we investigated whether pERK in injured sciatic nerve contributes to neuropathic pain induced by partial sciatic nerve ligation (PSL) in mice. METHODS: Mice received PSL; pERK1/2 (p44/42) in sciatic nerve was measured by both Western blotting and immunohistochemistry. U0126 (an ERK kinase inhibitor) was injected twice, an intraneural injection (20 nmol/2 microL) 30 min before PSL, and a perineural injection (20 nmol/10 microL) on Day 1 after PSL. Thermal hyperalgesia and tactile allodynia induced by PSL were evaluated by the thermal paw withdrawal test and the von Frey test, respectively. RESULTS: As measured by Western blotting, in sham-operated mice, the levels of pERK1/2 in sciatic nerve were constant and the same as those in naive mice across Days 1-14. In PSL-operated mice, a significant increase in pERK1/2 was observed on Day 1 after PSL and persisted until Day 3. As measured by immunohistochemistry, immunoreactivity of pERK1/2 in PSL-operated sciatic nerve was markedly increased in comparison with that in sham-operated sciatic nerve on Day 1 after PSL. In the sciatic nerve on Day 1 after PSL, as indicated by double immunostaining, the increased immunoreactivity of pERK1/2 was colocalized with glial fibrillary acidic protein (GFAP), a marker of Schwann cells, but not F4/80, a marker of macrophages. PSL-induced thermal hyperalgesia was significantly attenuated by treatment with U0126 on Days 3, 7, and 14 after PSL. The PSL-induced tactile allodynia was also significantly attenuated by treatment with U0126 on Days 7 and 14 after PSL. CONCLUSION: Activation of ERK in Schwann cells of the injured peripheral nervous system may play an important role in the development of neuropathic pain. Our results suggest that pERK itself and ERK-related mediators are potential therapeutic targets for the treatment of neuropathic pain.

Upregulation of CCL7 and CCL2 in reward system mediated through dopamine D1 receptor signaling underlies methamphetamine-induced place preference in mice.

We previously showed that the CC-chemokine ligand 2 (CCL2)-CC-chemokine receptor 2 (CCR2) system is responsible for conditioned place preference (CPP) by methamphetamine (Meth). In this study, we investigated the roles for other chemokines mediating Meth-induced CPP and the upstream factors upregulating chemokines in mice. We found that CCL7 mRNA level was upregulated in the prefrontal cortex (PFC) after Meth administration (3mg/kg, subcutaneous), and increased CCL7 immunoreactivity was localized to the PFC NeuN-positive neurons. Meth-induced CPP was blocked by the dopamine D1 receptor antagonist SCH 23390 but not by the D2 receptor antagonists raclopride or haloperidol. The D1 receptor agonist SKF 81297 alone elicited CPP, suggesting a critical role of D1 receptor signaling in Meth-induced reward. Consistent with these results, the Meth-induced upregulation of CCL7 and CCL2 were attenuated by SCH 23390, and a single administration of SKF 81297 upregulated mRNA expression levels of CCL7 and CCL2 in the PFC. Furthermore, Meth-induced CPP was prevented by INCB 3284, a selective antagonist of CCR2, a receptor that binds both CCL7 and CCL2. Collectively, we identified two CC-chemokines (i.e., CCL7 and CCL2) as key regulatory factors in Meth-induced reward. Pharmacological inhibitors of these chemokines may warrant development as novel therapeutics for ameliorating Meth addiction.

Peroxisome proliferator-activated receptor gamma activation relieves expression of behavioral sensitization to methamphetamine in mice.

Peroxisome proliferator-activated receptor (PPAR) is a ligand-activated transcriptional factor that regulates lipid metabolism and inflammation. Behavioral sensitization is an experimental model of psychostimulant psychosis; it is elicited by repeated administration of psychostimulants and has recently been implicated in brain inflammation. We examined the involvement of PPARgamma, one of the isotypes of PPAR, in development of behavioral sensitization to the stimulant effect of methamphetamine (METH) (1 mg/kg, subcutaneously) in mice. Repeated administration of METH (once daily for 5 days) enhanced the locomotor-activating effect of METH, which was reproduced by METH challenge on withdrawal day 7 (test day 12). The protein level and the activity of PPARgamma were significantly increased in the nuclear fraction of whole brain after 5 days of METH administration (test day 5) and on withdrawal day 7 (test day 12). Both pioglitazone and ciglitazone (PPARgamma agonists; 0.5-5.0 microg, intracerebroventricularly (i.c.v.), once daily) prevented the expression of behavioral sensitization to METH challenge on withdrawal day 7, but not the sensitization that occurred during repeated administration of METH. In addition, the magnitude of expression of behavioral sensitization was augmented by treatments with GW9662 (a PPARgamma antagonist; 0.5-5.0 microg i.c.v., once daily) during the withdrawal period. The pioglitazone-induced alleviation of behavioral sensitization was synergistically facilitated by simultaneous i.c.v. injection of 9-cis-retinoic acid (1.0 microg), an agonist for the retinoid X receptor which is a ligand-activated nuclear receptor that forms heterodimers with PPAR. These results suggest that PPARgamma has a significant role in the expression of behavioral sensitization to METH in mice.

Involvement of serine/threonine protein phosphatases sensitive to okadaic acid in restraint stress-induced hyperlocomotion in cocaine-sensitized mice.

We used okadaic acid (OA), a potent preferential inhibitor of PP2A and PP5 but not PP1 (PP subfamilies), to examine the involvement of serine/threonine protein phosphatase (PP) in behavioral sensitization stimulated by treatment with cocaine in mice. Repeated administration of cocaine (10 mg kg(-1)) once a day for five consecutive days produced a progressive increase in locomotor activity that was maintained after the cessation of cocaine treatment, as revealed by the fact that a challenge dose of cocaine given on day 7 of withdrawal reproduced an enhanced stimulant effect. On the seventh day of withdrawal, OA-sensitive PP activity and expression of PP2A and PP5, but not PP1gamma, were increased in whole-cell extract of the nucleus accumbens and the ventral tegmental area in cocaine-sensitized mice, compared to saline-treated mice. Restraint stress increased locomotor activity in cocaine-sensitized mice on day 7 after drug administration was ceased. The locomotor activity was more susceptible to restraint-elicited enhancement in cocaine-sensitized mice than in saline-treated mice. The restraint-induced hyperlocomotion was suppressed by a single intracerebroventricular injection of OA immediately before restraint in cocaine-sensitized mice, but this suppression did not occur in saline-treated mice. The membrane fraction of the whole brain in cocaine-sensitized mice showed that OA-sensitive activity levels rise after mice are subjected to restraint, and this is concomitant with an increase in expression levels of PP2A and PP5, but not PP1gamma. These results suggest that the upregulated OA-sensitive PPs are involved in stress-induced hyperlocomotion in cocaine-sensitized mice. There may be intracellular mechanisms mediating psychostimulant cross-sensitization to stress underlying the spontaneous recurrence of its psychosis.

P-glycoprotein ATPase activating effect of opioid analgesics and their P-glycoprotein-dependent antinociception in mice.

It is well known that opioid analgesics exert central antinociceptive actions. However, in vivo and in vitro studies have shown that some opioid analgesics given systemically have limited access to the central nervous system because of the blood-brain barrier (BBB). P-glycoprotein (P-gp), an ATP-dependent drug efflux transporter, is one component of the BBB. In this report, we assessed the antinociceptive effect of morphine, fentanyl, and meperidine in P-gp deficient (mdr1a KO) mice, and compared these effects with those in wild type (WT) mice. The antinociceptive effects of morphine and fentanyl in mdr1a KO mice were significantly greater than those in WT mice. However, there was no clear difference in the antinociceptive effects of meperidine in the two genotypes. In addition, we determined the effect of opioid analgesics on P-gp ATPase activity, which is requisite for drug transport, using mouse brain capillary endothelial cells. In our observations, morphine and fentanyl, but not meperidine, significantly increased P-gp ATPase activity, and the drugs' concentration-response curves were bell-shaped, reaching a peak at a concentration of 1 muM. These results suggest that P-gp ATPase activity may be, at least in part, involved in the antinociceptive potencies of those opioid analgesics that are substrates for P-gp.

Morphine has an antinociceptive effect through activation of the okadaic-acid-sensitive Ser/Thr protein phosphatases PP 2 A and PP5 estimated by tail-pinch test in mice.

Although the serine/threonine protein kinases involved in the pharmacological action of morphine are well recognized, the critical contribution of serine/threonine protein phosphatase (PP) has been appreciated on to a slight degree. We examined the involvement of subtypes of serine/threonine protein phosphatase (PP) in the antinociceptive effect of morphine in mice. The antinociceptive effect of subcutaneously administered morphine was attenuated by simultaneously intracerebroventricular (i.c.v.) or intrathecal (i.t.) injection of okadaic acid (OA), a PP inhibitor. To reveal which subtypes of PPs participated in the antinociceptive effect of morphine, mice received i.c.v. or i.t. injections of antisense oligodeoxynucleotide (AS-ODN) directed against either the PP 2 A or PP5 subtypes of PPs before assessment of morphine-induced antinociception. Pretreatment with AS-ODN against PP 2 A or PP5 via each route weakened the antinociceptive effect of morphine, accompanied by reduction of expression levels of PP in the periaqueductal gray (PAG) and the spinal cord. Subcutaneously administered morphine increased activity of OA-sensitive PPs in the PAG and the spinal cord in a dose-dependent manner; this was prevented by concurrent administration of naloxone. These results suggest that PP 2 A and PP5 are involved in the antinociceptive effect of morphine in mice.

Electrophysiological characteristic of corticoaccumbens synapses in rat mesolimbic system reconstructed using organotypic slice cultures.

The nucleus accumbens (NAc) is a component of the mesolimbic system involved in drug dependence. Activity of nucleus accumbens neurons is modulated by glutamatergic afferents from the prefrontal cortex and by dopaminergic afferents from the ventral tegmental area (VTA). In the present study, we reconstructed the mesolimbic system using organotypic slice cultures and examined the effects of dopaminergic agents on synaptic activity in the prefrontal cortex-nucleus accumbens synapses. A slice of each of the prefrontal cortex, nucleus accumbens and ventral tegmental area in newborn rat, was arranged on a multi-electrode dish (MED) filled with culture medium so that they contacted each other, termed a 'triple culture'. Extracellular recording using microelectrodes on the multi-electrode dish showed that a single electrical stimulation of the prefrontal cortex slice evoked field excitatory postsynaptic potential, and that population spikes occurred spontaneously in the nucleus accumbens area of the triple culture. The amplitude of evoked field excitatory postsynaptic potentials and the frequency of spontaneous population spikes were decreased by glutamatergic antagonists, D(-)-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione. The D1-like receptor agonist SKF38393, but not the D2-like receptor agonist quinpirole, reduced both the amplitude of field excitatory postsynaptic potential and frequency of spontaneous population spikes. Cocaine depressed field excitatory postsynaptic potential and this depression was reversed by D1-like receptor antagonist SCH23390, but not by D2-like receptor antagonist sulpiride. These results suggest that evoked field excitatory postsynaptic potentials and spontaneous population spikes were driven by glutamatergic neurons and were subject to exogenous and endogenous dopaminergic modulation in the triple culture that was similar to that shown in in vivo.

Effects of diltiazem and MK-801 on morphine analgesia and pharmacokinetics in mice.

The effects of diltiazem, an L-type calcium channel blocker, and MK-801, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist on morphine analgesia and pharmacokinetics were examined in mice. Mice received a subcutaneous injection of morphine (3.2 mg/kg) 30 min after a subcutaneous injection of diltiazem or MK-801. Diltiazem (20-60 mg/kg) potentiated morphine analgesia and increased serum morphine levels in a dose-dependent manner. MK-801 (0.3 mg/kg) significantly attenuated morphine analgesia but had no significant effect on serum or brain morphine levels. These results suggest that a modification of morphine metabolism is involved, at least in part, in the ability of diltiazem to enhance morphine analgesia, whereas MK-801 attenuates morphine analgesia without affecting morphine pharmacokinetics.