Abstract: Acupuncture is a therapeutic procedure that has been practiced for thousands of years in South East Asia to treat a variety of pathological symptoms and conditions. In 1997, the NIH released a consensus statement concluding that acupuncture is effective or at least useful for the treatment of 13 conditions including nausea and vomiting induced by surgery or chemotherapy, dental pain, and lower back pain. Although the beneficial aspects of acupuncture have been widely accepted, the mechanisms by which acupuncture acts are only partially understood. This review highlights the pharmacological mechanisms underlying the neurological (sensory), cardiovascular and gastrointestinal effects of electroacupuncture.

Abstract: Nitric oxide (NO) plays pivotal roles in controlling physiological functions, participates in pathophysiological intervention, and is involved in mechanisms underlying beneficial or untoward actions of therapeutic agents. Endogenous nitric oxide is formed by three isoforms of nitric oxide synthase: endothelial, neurogenic and inducible. The former two are constitutively present mainly in the endothelium and nervous system, respectively, and the latter one is induced by lipopolysaccharides or cytokines mainly in mitochondria and glial cells. Constitutively formed nitric oxide modulates the actions of morphine and related analgesics by either enhancing or reducing antinociception. Tolerance to and dependence on morphine or its withdrawal syndrome are likely prevented by nitric oxide synthase inhibition. Information concerning modulation of morphine actions by nitric oxide is undoubtedly useful in establishing new strategies for efficient antinociceptive treatment and for minimizing noxious and unintended reactions.

Abstract: Nerve injury may result in neuropathic pain, characterized by allodynia and hyperalgesia. Accumulating evidence suggests the existence of a molecular substrate for neuropathic pain produced by neurons, glia, and immune cells. Here, we show that leptin, an adipokine exclusively produced by adipocytes, is critical for the development of tactile allodynia through macrophage activation in mice with partial sciatic nerve ligation (PSL). PSL increased leptin expression in adipocytes distributed at the epineurium of the injured sciatic nerve (SCN). Leptin-deficient animals, ob/ob mice, showed an absence of PSL-induced tactile allodynia, which was reversed by the administration of leptin to the injured SCN. Perineural injection of a neutralizing antibody against leptin reproduced this attenuation. Macrophages recruited to the perineurium of the SCN expressed the leptin receptor and phosphorylated signal transducer and activator of transcription 3 (pSTAT3), a transcription factor downstream of leptin. PSL also up-regulated the accepted mediators of neuropathic pain--namely, cyclooxygenase-2, inducible nitric oxide synthase, and matrix metalloprotease-9--in the injured SCN, with transcriptional activation of their gene promoters by pSTAT3. This up-regulation was partly reproduced in a macrophage cell line treated with leptin. Administration of peritoneal macrophages treated with leptin to the injured SCN reversed the failure of ob/ob mice to develop PSL-induced tactile allodynia. We suggest that leptin induces recruited macrophages to produce pronociceptive mediators for the development of tactile allodynia. This report shows that adipocytes associated with primary afferent neurons may be involved in the development of neuropathic pain through adipokine secretion.

Abstract: Aminopeptidase N (APN), belonged to metalloproteinase, is an essential peptidase involved in the process of tumor invasion and metastasis. A series of tripeptide analogs with the scaffold 3-phenylpropane-1,2-diamine were designed, synthesized and evaluated for their ability to inhibit APN. Preliminary activity evaluation showed that most of target compounds possessed potent inhibitory activities against APN. With in this series, compound A6 and B6 exhibited good potency with the IC(50) values of 8.8+/-1.3 microM and 8.6+/-1.1 microM, respectively.

Abstract: 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.

Abstract: 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.

Abstract: Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factor belonging to a nuclear hormone receptor superfamily, containing three isoforms (alpha, beta/delta, and gamma). PPARs play a critical physiological role as a primary lipid sensor and regulator of lipid metabolism. Thus, its ligands are clinically used for treatment of type 2 diabetes and hyperlipidemia. On the other hand, PPAR ligands exert the antineuroinflammatory activity through preventing upregulation of inflammatory mediators in animal models for neurodegenerative disease and autoimmune disease. Neuropathic pain and inflammatory pain, clinically important one, are chronically progressed and underlain by neuroinflammation. In a few years, some studies using experimental models emerge that administration of PPAR ligands reduces inflammatory pain and neuropathic pain. PPAR ligands repress expression of genes for inflammatory mediators involved in both pains, such as proinflammatory cytokines, by a molecular mechanism termed ligand-dependent direct transrepression. Alternative mechanism is independent of transcriptional regulation of target genes, such as inhibition of activity of ion channels involved in the development of inflammatory pain and neuropathic pain, and therefore the analgesic effect occurs with rapid onset. The effects of PPAR ligands on neuroinflammation in animal models suggest their possible use for treating human inflammatory pain and neuropathic pain.

Abstract: 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.

Abstract: In order to explore the mechanism of myoglobinuric renal toxicity, detection and identification of free radicals was performed for the reaction mixtures of bovine kidney microsomes. EPR measurements showed prominent signals for the control reaction mixture containing 2.0 mg protein/ml bovine kidney microsomes, 5 mM NADPH, 0.1 M 4-POBN and 29 mM phosphate buffer (pH 7.4). Addition of myoglobin (Mb) to the control reaction mixture resulted in increase of EPR peak height. The result indicates that Mb enhances the radical formation. An HPLC-EPR measurement showed three peaks with retention times of 29.4 min (P(1)), 32.4 min (P(2)) and 46.6 min (P(3)). HPLC-EPR-MS analyses of the P(1) and P(2) gave ions at m/z 282. The results show that 4-POBN/hydroxypentyl radical adducts form in the reaction mixture. An HPLC-EPR-MS analysis of the P(3) gave ions at m/z 266, indicating that 4-POBN/pentyl radical adduct forms in the reaction mixture.

Abstract: Nicotine is neuronal stimulating drug in the central nervous system and elicits various effects through nicotinic acetylcholine receptors. As previously reported, nicotine has an antinociceptive effect through activation of endogenous opioid neurons. However, detailed mechanisms of nicotine-induced antinociception are uncertain. In this study, we focused on spinal cord and investigated the involvement of endogenous opioidergic neurons in nicotine-induced antinociception in mice. In the tail-pinch test, subcutaneously administered nicotine (5 mg/kg) produced maximal antinociception 0.5 h after nicotine administration; this was attenuated by mecamylamine (MEC, 3 mg/kg, s.c.) or naloxone (NLX, 1 mg/kg, s.c.) administration. Intrathecal nicotine (10 mug) produced maximal antinociception at 2 min and this was also attenuated by MEC (3 mg/kg, s.c.) or NLX (1 mg/kg, s.c.) administration. The preproenkephalin (ppENK) mRNA level in spinal cord, but not dorsal root ganglion, was significantly increased 2 h following nicotine administration and recovered to control level 4 h after nicotine (5 mg/kg, s.c.) administration. This increase in ppENK mRNA level was inhibited by MEC (3 mg/kg, s.c.). The mRNA levels of preprodynorphin and preproopiomelanocortin were not increased by nicotine (5 mg/kg, s.c.). In the dorsal horn of the lumbar spinal cord, methionine-enkephalin (Met-ENK)-like IR was remarkably reduced at 0.5 h following nicotine administration and recovered to control levels by 2 h after nicotine (3 mg/kg, s.c.) administration. These results suggest that nicotine has an antinociceptive effect by promoting the release of Met-ENK, but not dynorphins and endorphins, from activated opioidergic neurons in spinal cord.

Abstract: The activities of the novel aminopeptidase N inhibitor (APNI), beta-Amino-alpha-Hydroxyl-Phenyl butanic acid-Valine (AHPA-Val), were compared with APNI (amastatin). AHPA-Val and amastatin produced competitive inhibition of the hydrolysis of Tyr-Gly in the guinea-pig striatal membrane preparation, with K(i) equal to 14.06 microM and 12.48 microM respectively. Met-enkephalin-induced twitch inhibition of the guinea-pig ileum preparation was enhanced by AHPA-Val and amastatin with pA(1/2) values (the negative logarithm concentration of APNI that decreased the IC(50) of Met-enkephalin by half), of 7.08 and 7.79 respectively. These results suggest that AHPA-Val has good activity as an APNI and that these two assay systems are useful for evaluating the potency of novel APNIs.

Abstract: To clarify the role of peroxisome proliferator activated receptor gamma (PPARgamma) in neuropathic pain, we examined the effect of pioglitazone, a PPARgamma agonist, on tactile allodynia and thermal hyperalgesia in a neuropathic pain model. Mice were subjected to partial sciatic nerve ligation (PSL) and given pioglitazone (1 - 25 mg/kg, p.o.) once daily. PPARgamma was distributed in the neurons of the dorsal root ganglion and the dorsal horn of the spinal cord and in the adipocytes at the epineurium of the sciatic nerve in naive mice. PSL elicited tactile allodynia and thermal hyperalgesia for two weeks. Administration of pioglitazone for the first week after PSL attenuated thermal hyperalgesia and tactile allodynia, which was dose-dependent and blocked by GW9662 (2 mg/kg, i.p.), a PPARgamma antagonist. Administration of pioglitazone for the second week also relieved tactile allodynia, but administration one week before PSL had no effect. A single administration of pioglitazone to mice on day 7 of PSL did not alter tactile allodynia and thermal hyperalgesia. PSL-induced upregulation of tumor necrosis factor-alpha and interleukin-6, which are essential for neuropathic pain, was suppressed by pioglitazone for the first week. This suggests that pioglitazone alleviates neuropathic pain through attenuation of proinflammatory cytokine upregulation by PPARgamma stimulation.

Abstract: Although the clinical use of vincristine is limited by its adverse effect, neuropathic pain, the mechanism of this effect is poorly understood. Recently, reports demonstrated that inflammatory and immune responses play an important role in the neuropathic pain that follows peripheral nerve injury. In this study, we examined the role of macrophage-derived interleukin (IL)-6 in vincristine-induced mechanical allodynia. Vincristine sulfate (0.01-0.1 mg/kg, i.p.) was administered to male ICR mice and BALB/c mice once per day for 7 or 14 days. Mechanical allodynia was evaluated by withdrawal responses, using von Frey filaments from day 0 to day 28. In both ICR mice and BALB/c mice, significant dose-dependent increases in the percentage of withdrawal responses were observed from day 3 to day 28 following repeated administration of vincristine (0.1 mg/kg). As determined by immunohistochemistry, the number of macrophages in the region of the sciatic nerve and lumbar dorsal root ganglion was significantly increased on day 7 of vincristine administration. The expression of IL-6 was increased by vincristine administration and was co-localized in the invading macrophage. Moreover, a neutralizing antibody of IL-6, which was injected into areas surrounding the sciatic nerve on day 0, 3, and 6, significantly attenuated vincristine-induced mechanical allodynia from day 7 to day 28. In addition, the incidence of vincristine-induced mechanical allodynia in IL-6 knockout mice was lower than that in wild type mice from day 3 to day 28. These results suggest that the invading peripheral macrophage-derived IL-6 plays a critical role in vincristine-induced mechanical allodynia.

Abstract: Chronic treatment with vincristine (VCR) causes mechanical allodynia as an adverse effect. We previously reported that peripheral macrophage-derived interleukin-6 played a critical role in VCR-induced allodynia. However, the involvement of glial cell activation and central sensitization in VCR-induced allodynia is still unclear. In this study, we focused on tumor necrosis factor-alpha (TNF-alpha) in spinal cord, and investigated the role of TNF-alpha in VCR-induced allodynia in mice. VCR (0.1mg/kg, i.p.) was administered to mice once per day for 7 days. The expression of TNF-alpha mRNA and the protein in spinal cord was evaluated by quantitative real-time PCR and immunohistochemistry, respectively. In VCR-treated mice, TNF-alpha mRNA gradually increased and was significantly up-regulated on day 7. As measured by immunohistochemistry, microglia and astrocytes were activated in the spinal dorsal horn on day 7 of VCR administration. The immunoreactivity of TNF-alpha was co-localized in some of the activated microglia and astrocytes. In behavioral analysis, a neutralizing antibody of TNF-alpha, which was injected intrathecally on days 0, 3, and 6, significantly attenuated VCR-induced mechanical allodynia on days 4 and 7. These results suggest that VCR treatments elicited the activation of glial cells in spinal cord, and up-regulated TNF-alpha in these cells may play an important role in VCR-induced mechanical allodynia.

Abstract: 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.

Abstract: Identification of a free radical is performed for the reaction mixture of rat brain homogenate with a ferrous ion/ascorbic acid system using EPR, high performance liquid chromatography-electron paramagnetic resonance spectrometry (HPLC-EPR) and high performance liquid chromatography-electron paramagnetic resonance-mass spectrometry (HPLC-EPR-MS). EPR measurements of the reaction mixtures showed prominent signals with hyperfine coupling constants (alpha(N) = 1.58 mT and alpha(H)beta = 0.26 mT). No EPR spectrum was detectable without rat brain homogenate, suggesting that the radical is derived from rat brain homogenate. An HPLC-EPR analysis of the reaction mixture showed a peak with retention time of 33.7 min. An HPLC-EPR-MS analysis of the peak gave two ions at m/z 224 and 137, suggesting that alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN)/ethyl radical adduct forms in the reaction mixture.

Abstract: We previously demonstrated that electroacupuncture (EA) stimulation both produced antinociception and attenuated intrathecal (i.t.) morphine analgesia, suggesting that EA is capable of inducing two opposing systems, that is, opioid and anti-opioid mechanisms. This study examined the involvement of cholecystokinin (CCK) in the anti-opioid effects following EA in the spinal cord. EA was applied to commonly used acupoints for antinociception, ST-36 located 5-mm lateral to the anterior tubercle of the tibia, and analgesia was assessed by the hind-paw pressure test in male Sprague-Dawley rats. I.t. administration of CCK (0.01 - 10 microg) attenuated i.t. morphine analgesia (10 microg) dose-dependently. The attenuation of morphine analgesia following EA was reversed by i.t. proglumide, a CCK-receptor antagonist (0.01 microg). CCK-like immunoreactivity was increased in lamina I and II in the dorsal horn, and expression of spinal CCK mRNA increased after EA. Moreover, i.t. pretreatment with the neurokinin-1 (NK1)-receptor antagonist L-703,606 (18 microg) reversed both EA- and CCK-induced attenuation of morphine analgesia. These results suggest that CCK-mediated neural systems in the spinal cord may be involved in the attenuation of morphine analgesia following EA and that substance P-induced activation of NK1 receptors may be responsible for the downstream neuronal transmission of the CCK-mediated neuronal system.

Abstract: It is known that opioid analgesics given systemically have limited distribution into the brain because of their interaction with P-glycoprotein (P-gp), an ATP-dependent efflux pump acting at the blood-brain barrier (BBB). We previously found that morphine and fentanyl showed higher analgesic potencies in P-gp-deficient mice compared with those in wild-type mice, suggesting that their analgesic effects are considerably dependent on P-gp expression. In this study, we focused on individual differences in the analgesic effectiveness of morphine, in cortical P-gp expression, and in basal P-gp ATPase activity in male ICR mice. We found that there were 3- to 10-fold differences between the magnitude of morphine analgesia (3 mg/kg, s.c.; tail-pinch method) in mice. Furthermore, there was a significant negative correlation between morphine's analgesic effects and individual P-gp expression in the cortex as estimated by western blot analysis. In addition, basal P-gp ATPase activities in isolated membrane preparations of brain capillary endothelial cells (BCECs) were negatively correlated with the magnitude of the analgesic effect of morphine. These results indicate that the individual differences in morphine analgesia may be due to some functional or quantitative differences in individual P-gp in BCECs, acting at the BBB.

Abstract: 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.