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1.  Peripheral nerve injury alters spinal nicotinic acetylcholine receptor pharmacology 
European journal of pharmacology  2008;590(1-3):163-169.
Nicotinic acetylcholine receptors are widely expressed in the rat spinal cord and modulate innocuous and nociceptive transmission. The present studies were designed to investigate the plasticity of spinal nicotinic acetylcholine receptors modulating mechanosensitive information following spinal nerve ligation. A tonic inhibitory cholinergic tone mediated by dihydro-β-erythroidine- (DHβE) and methyllycaconitine- (MLA) sensitive nicotinic acetylcholine receptors was identified in the normal rat spinal cord and cholinergic tone at both populations of nicotinic acetylcholine receptors was lost ipsilateral to spinal nerve ligation. The administration of intrathecal nicotinic acetylcholine receptor agonists reduced mechanical paw pressure thresholds with a potency of epibatidine = A-85380 >> nicotine > choline in the normal rat. Following spinal nerve ligation, intrathecal epibatidine and nicotine produced an ipsilateral antinociception, but intrathecal A-85380 and choline did not. The antinociceptive response to intrathecal nicotine was blocked with the α7* and α9α10*-selective nicotinic acetylcholine receptor antagonist, MLA, and the αβ heteromeric nicotinic acetylcholine receptor antagonist, DHβE. The antinociceptive effects of both intrathecal nicotine and epibatidine were mediated by GABAA receptors. Spinal [3H]epibatidine saturation binding was unchanged in spinal nerve-ligated rats, but spinal nerve ligation did increase the ability of nicotine to displace [3H]epibatidine from spinal cord membranes. Spinal nerve ligation altered the expression of nicotinic acetylcholine receptor subunits ipsilaterally, with a large increase in the modulatory α5 subunit. Taken together these results suggest that pro- and antinociceptive populations of spinal nicotinic acetylcholine receptors modulate the transmission of mechanosensitive information and that spinal nerve ligation-induced changes in spinal nicotinic acetylcholine receptors likely result from a change in subunit composition rather than overt loss of nicotinic acetylcholine receptor subtypes.
doi:10.1016/j.ejphar.2008.06.020
PMCID: PMC2569862  PMID: 18573248
neuropathic pain; antinociception; nicotine; nociception
2.  Modulation of TNF Release by Choline Requires α7 Subunit Nicotinic Acetylcholine Receptor-Mediated Signaling 
Molecular Medicine  2008;14(9-10):567-574.
The α7 subunit-containing nicotinic acetylcholine receptor (α7nAChR) is an essential component in the vagus nerve-based cholinergic anti-inflammatory pathway that regulates the levels of TNF, high mobility group box 1 (HMGB1), and other cytokines during inflammation. Choline is an essential nutrient, a cell membrane constituent, a precursor in the biosynthesis of acetylcholine, and a selective natural α7nAChR agonist. Here, we studied the anti-inflammatory potential of choline in murine endotoxemia and sepsis, and the role of the α7nAChR in mediating the suppressive effect of choline on TNF release. Choline (0.1–50 mM) dose-dependently suppressed TNF release from endotoxin-activated RAW macrophage-like cells, and this effect was associated with significant inhibition of NF-κB activation. Choline (50 mg/kg, intraperitoneally [i.p.]) treatment prior to endotoxin administration in mice significantly reduced systemic TNF levels. In contrast to its TNF suppressive effect in wild type mice, choline (50 mg/kg, i.p.) failed to inhibit systemic TNF levels in α7nAChR knockout mice during endotoxemia. Choline also failed to suppress TNF release from endotoxin-activated peritoneal macrophages isolated from α7nAChR knockout mice. Choline treatment prior to endotoxin resulted in a significantly improved survival rate as compared with saline-treated endotoxemic controls. Choline also suppressed HMGB1 release in vitro and in vivo, and choline treatment initiated 24 h after cecal ligation and puncture (CLP)-induced polymicrobial sepsis significantly improved survival in mice. In addition, choline suppressed TNF release from endotoxin-activated human whole blood and macrophages. Collectively, these data characterize the anti-inflammatory efficacy of choline and demonstrate that the modulation of TNF release by choline requires α7nAChR-mediated signaling.
doi:10.2119/2008-00079.Parrish
PMCID: PMC2435495  PMID: 18584048
3.  An Autoradiographic Analysis Of Rat Brain Nicotinic Receptor Plasticity Following Dietary Choline Modification 
Choline is known to be involved with numerous physiological functions of the nervous system and also acts as a direct acting agonist of α7 nicotinic acetylcholine receptors (nAChRs). The purpose of this study was to conduct a brain region-specific evaluation of changes in nAChR subtype expression following dietary choline modification. In addition, we assessed changes in body weight, food/water intake, as well as changes spatial learning (Morris Water Maze) in response to dietary choline modification. Male Sprague Dawley rats were exposed to standard, choline supplemented or choline deficient diets for periods of 14 or 28 days. Choline supplemented animals gained significantly less weight over the course of the experiment, in spite of the fact that there were minimal differences in food consumption between the dietary regimens. Spatial memory did not differ between animals maintained on a standard rat diet, and the choline supplemented food. Brains of the animals kept on the diets for 14 and 28 days were used for quantitative autoradiographic analysis of nicotinic receptor subtypes using 125I-Bungarotoxin (α7) and 125I-Epibatidine (non-α7). There were no significant differences in nicotinic receptor binding or physiologic parameters measured between animals fed standard and choline deficient diets. However 2 weeks of dietary choline supplementation caused significant up-regulation of α7 receptors without significant effect on the density of non-α7 nAChRs. Increases in BTX binding predominantly occurred in cortical and hippocampal brain regions and ranged between 14 and 30 percent depending on the brain region. The results of our study suggest that choline acts as a selective agonist at α7 nicotinic cholinergic receptors in the rat central nervous system.
doi:10.1016/j.pbb.2006.04.002
PMCID: PMC1976431  PMID: 16753203
ℑ7 nAChR; Cholinergic; Acetylcholine; Morris Water Maze; Up-regulation; Bungarotoxin; Epibatidine
4.  Influence of δ-Opioid Receptors in the Behavioral Effects of Nicotine 
Neuropsychopharmacology  2012;37(10):2332-2344.
Multiple studies in animal models and humans suggest that the endogenous opioid system is an important neurobiological substrate for nicotine addictive properties. In this study, we evaluated the participation of δ-opioid receptors in different behavioral responses of nicotine by using δ-opioid receptor knockout mice. Acute nicotine administration induced hypolocomotion and antinociception in wild-type mice, which were similar in knockout animals. The development of tolerance to nicotine-induced antinociception was also similar in both genotypes. In agreement, the expression and functional activity of δ-opioid receptors were not modified in the different layers of the spinal cord and brain areas evaluated after chronic nicotine treatment. The somatic manifestation of the nicotine withdrawal syndrome precipitated by mecamylamine was also similar in wild-type and δ-opioid receptor knockout mice. In contrast, nicotine induced a conditioned place preference in wild-type animals that was abolished in knockout mice. Moreover, a lower percentage of acquisition of intravenous nicotine self-administration was observed in mice lacking δ-opioid receptors as well as in wild-type mice treated with the selective δ-opioid receptor antagonist naltrindole. Accordingly, in-vivo microdialysis studies revealed that the enhancement in dopamine extracellular levels induced by nicotine in the nucleus accumbens was reduced in mutant mice. In summary, the present results show that δ-opioid receptors are involved in the modulation of nicotine rewarding effects. However, this opioid receptor does not participate either in several acute effects of nicotine or in the development of tolerance and physical dependence induced by chronic nicotine administration.
doi:10.1038/npp.2012.88
PMCID: PMC3422497  PMID: 22669166
dependence; tolerance; reward; knockout; microdialysis; self-administration; behavioral science; addiction & substance abuse; animal models; psychopharmacology; dependence; tolerance; reward; knockout; microdialysis
5.  Cannabinoid CB2 Receptors Contribute to Upregulation of β-endorphin in Inflamed Skin Tissues by Electroacupuncture 
Molecular Pain  2011;7:98.
Background
Electroacupuncture (EA) can produce analgesia by increasing the β-endorphin level and activation of peripheral μ-opioid receptors in inflamed tissues. Endogenous cannabinoids and peripheral cannabinoid CB2 receptors (CB2Rs) are also involved in the antinociceptive effect of EA on inflammatory pain. However, little is known about how peripheral CB2Rs interact with the endogenous opioid system at the inflammatory site and how this interaction contributes to the antinociceptive effect of EA on inflammatory pain. In this study, we determined the role of peripheral CB2Rs in the effects of EA on the expression of β-endorphin in inflamed skin tissues and inflammatory pain.
Results
Inflammatory pain was induced by injection of complete Freund's adjuvant into the left hindpaw of rats. Thermal hyperalgesia was tested with a radiant heat stimulus, and mechanical allodynia was quantified using von Frey filaments. The mRNA level of POMC and protein level of β-endorphin were quantified by real-time PCR and Western blotting, respectively. The β-endorphin-containing keratinocytes and immune cells in the inflamed skin tissues were detected by double-immunofluorescence labeling. The CB2R agonist AM1241 or EA significantly reduced thermal hyperalgesia and mechanical allodynia, whereas the selective μ-opioid receptor antagonist β-funaltrexamine significantly attenuated the antinociceptive effect produced by them. AM1241 or EA significantly increased the mRNA level of POMC and the protein level of β-endorphin in inflamed skin tissues, and these effects were significantly attenuated by pretreatment with the CB2R antagonist AM630. AM1241 or EA also significantly increased the percentage of β-endorphin-immunoreactive keratinocytes, macrophages, and T-lymphocytes in inflamed skin tissues, and these effects were blocked by AM630.
Conclusions
EA and CB2R stimulation reduce inflammatory pain through activation of μ-opioid receptors. EA increases endogenous opioid expression in keratinocytes and infiltrating immune cells at the inflammatory site through CB2R activation.
doi:10.1186/1744-8069-7-98
PMCID: PMC3281798  PMID: 22177137
acupuncture; inflammatory pain; β-endorphin; cannabinoid CB2 receptors; μ-opioid receptors
6.  Impaired Preneoplastic Changes and Liver Tumor Formation in Tumor Necrosis Factor Receptor Type 1 Knockout Mice 
The Journal of Experimental Medicine  2000;192(12):1809-1818.
Hepatic stem cells (oval cells) proliferate within the liver after exposure to a variety of hepatic carcinogens and can generate both hepatocytes and bile duct cells. Oval cell proliferation is commonly seen in the preneoplastic stages of liver carcinogenesis, often accompanied by an inflammatory response. Tumor necrosis factor (TNF), an inflammatory cytokine, is also important in liver regeneration and hepatocellular growth. The experiments reported here explore the relationship among the TNF inflammatory pathway, liver stem cell activation, and tumorigenesis. We demonstrate that TNF is upregulated during oval cell proliferation induced by a choline-deficient, ethionine-supplemented diet and that it is expressed by oval cells. In TNF receptor type 1 knockout mice, oval cell proliferation is substantially impaired and tumorigenesis is reduced. Oval cell proliferation is impaired to a lesser extent in interleukin 6 knockout mice and is unchanged in TNF receptor type 2 knockout mice. These findings demonstrate that TNF signaling participates in the proliferation of oval cells during the preneoplastic phase of liver carcinogenesis and that loss of signaling through the TNF receptor type 1 reduces the incidence of tumor formation. The TNF inflammatory pathway may be a target for therapeutic intervention during the early stages of liver carcinogenesis.
PMCID: PMC2213505  PMID: 11120777
cytokines; carcinoma, hepatocellular; interleukin 6; stem cells; liver regeneration
7.  OBSERVATIONS ON THE CONDITIONS OF DIETARY HEPATIC INJURY (NECROSIS, CIRRHOSIS) IN RATS 
Experimental dietary hepatic injury (diffuse or focal necrosis and cirrhosis in rats, with or without ascites and pleural and pericardial effusion) is determined by the dietary factors instrumental also in the production of fat infiltration of the liver and thus opposed to the lipotropic activity of casein. Accordingly, rats maintained on a diet low in casein with a moderately high or high content of fat and without choline regularly exhibited hepatic injury after between 100 and 150 days. Supplements of l-cystine had an aggravating effect on the production of cirrhosis of the liver, whereas a supplement of choline alone reduced the severity and the incidence of hepatic injury, although not decisively. The combined administration of l-cystine plus choline or of dl-methionine in adequate doses, however, proved to be highly effective in preventing injury to the liver. These conclusions have been corroborated by the use of different modifications of the basal diet. Rats with dietary hepatic injury exhibit, in sequence, changes that vary from diffuse necrosis resembling human acute or subacute yellow atrophy to advanced portal cirrhosis. Diffuse necrotizing nephrosis was a frequent accompaniment of the hepatic injury. Cystine again, proved to be a factor which aggravated this condition.
PMCID: PMC2135254  PMID: 19871189
8.  Is Hippocampus Susceptible to Antinociceptive Tolerance to NSAIDs Like the Periaqueductal Grey? 
Pain Research and Treatment  2014;2014:654578.
Emotional distress is the most undesirable feature of painful experience. Numerous studies have demonstrated the important role of the limbic system in the affective-motivational component of pain. The purpose of this paper was to examine whether microinjection of nonsteroidal anti-inflammatory drugs (NSAIDs), Clodifen, Ketorolac, and Xefocam, into the dorsal hippocampus (DH) leads to the development of antinociceptive tolerance in male rats. We found that microinjection of these NSAIDs into the DH induces antinociception as revealed by a latency increase in the tail-flick (TF) and hot plate (HP) tests compared to controls treated with saline into the DH. Subsequent tests on consecutive three days, however, showed that the antinociceptive effect of NSAIDs progressively decreased, suggesting tolerance developed to this effect of NSAIDs. Both pretreatment and posttreatment with the opioid antagonist naloxone into the DH significantly reduced the antinociceptive effect of NSAIDs in both pain models. Our data indicate that microinjection of NSAIDs into the DH induces antinociception which is mediated via the opioid system and exhibits tolerance.
doi:10.1155/2014/654578
PMCID: PMC4000673  PMID: 24818020
9.  Antinociceptive Activity of Melicope ptelefolia Ethanolic Extract in Experimental Animals 
Melicope ptelefolia is a medicinal herb commonly used in Malaysia to treat fever, pain, wounds, and itches. The present study was conducted to evaluate the antinociceptive activity of the Melicope ptelefolia ethanolic extract (MPEE) using animal models of nociception. The antinociceptive activity of the extract was assessed using acetic acid-induced abdominal writhing, hot-plate, and formalin-induced paw licking tests. Oral administration of MPEE produced significant dose-dependent antinociceptive effects when tested in mice and rats using acetic acid-induced abdominal constriction test and on the second phase of the formalin-induced paw licking test, respectively. It was also demonstrated that MPEE had no effect on the response latency time to the heat stimulus in the thermal model of the hot-plate test. In addition, the antinociception produced by MPEE was not blocked by naloxone. Furthermore, oral administration of MPEE did not produce any effect in motor performance of the rota-rod test and in acute toxicity study no abnormal behaviors as well as mortality were observed up to a dose level of the extract of 5 g/kg. These results indicated that MPEE at all doses investigated which did not produce any sedative and toxic effects exerted pronounce antinociceptive activity that acts peripherally in experimental animals.
doi:10.1155/2010/937642
PMCID: PMC3022215  PMID: 21274262
10.  Phosphatidylcholine and the CDP-Choline Cycle 
Biochimica et biophysica acta  2012;1831(3):523-532.
The CDP-choline pathway of phosphatidylcholine (PtdCho) biosynthesis was first described more than 50 years ago. Investigation of the CDP-choline pathway in yeast provides a basis for understanding the CDP-choline pathway in mammals. PtdCho is considered as an intermediate in a cycle of synthesis and degradation, and the activity of a CDP-choline cycle is linked to subcellular membrane lipid movement. The components of the mammalian CDP-choline pathway include choline transport, choline kinase, phosphocholine cytidylyltransferase, and choline phosphotransferase activities. The protein isoforms and biochemical mechanisms of regulation of the pathway enzymes are related to their cell and tissue-specific functions. Regulated PtdCho turnover mediated by phospholipases or neuropathy target esterase participates in the mammalian CDP-choline cycle. Knockout mouse models define the biological functions of the CDP-choline cycle in mammalian cells and tissues. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.
doi:10.1016/j.bbalip.2012.09.009
PMCID: PMC3562404  PMID: 23010477
11.  The effect of intrathecal gabapentin on mechanical and thermal hyperalgesia in neuropathic rats induced by spinal nerve ligation. 
Journal of Korean Medical Science  2002;17(2):225-229.
Gabapentin decreases the level of glutamate and elevates that of alpha-amino-butyric acid in the central nervous system. Gabapentin was shown to have antinociceptive effects in several facilitated pain models. Intrathecal gabapentin was also known to be effective in reducing mechanical allodynia in animals with neuropathic pain. In this study, we investigated to see whether intrathecal gabapentin produces antihyperalgesic effects on thermal and mechanical hyperalgesia in neuropathic rats and whether its effects are associated with motor impairment. To induce neuropathic pain in Sprague-Dawley rats, left L5 and L6 spinal nerves were ligated. After a week, lumbar catheterization into subarachnoid space was performed. Then, paw withdrawal times to thermal stimuli and vocalization thresholds to paw pressure were determined before and up to 2 hr after intrathecal injection of gabapentin. Also, motor functions including performance times on rota-rod were determined. Intrathecal gabapentin attenuated significantly thermal and mechanical hyperalgesia in neuropathic rats, but did not block thermal and mechanical nociception in sham-operated rats. Intrathecal gabapentin of antihyperalgesic doses inhibited motor coordination performance without evident ambulatory dysfunction. This study demonstrates that intrathecal gabapentin is effective against thermal and mechanical hyperalgesia, in spite of moderate impairment of motor coordination.
PMCID: PMC3054857  PMID: 11961308
12.  INTRATHECAL INJECTION OF AN ALPHA 7 NICOTINIC ACETYLCHOLINE RECEPTOR AGONIST ATTENUATES GP120-INDUCED MECHANICAL ALLODYNIA AND SPINAL PRO-INFLAMMATORY CYTOKINE PROFILES IN RATS 
Brain, behavior, and immunity  2010;24(6):959-967.
Nicotinic acetylcholine receptors (nAchR) are key receptors in the autonomic nervous system, but also are present on immune cells. The alpha seven subunit of nAchR (α7nAchR) suppresses pro-inflammation in peripheral monocytes by decreasing proinflammatory cytokine production. In spinal cord, α7nAchR are found on microglia, which are known to induce and maintain pain. We predicted that α7nAchR agonists might attenuate intrathecal HIV-1 gp120-induced, proinflammatory cytokine- and microglia-dependent mechanical allodynia. Choline, a precursor for acetylcholine and selective agonist for α7nAchR, was administered intrathecally either with, or 30 min after, intrathecal gp120. Choline significantly blocked and reversed gp120 induced mechanical allodynia for at least 4 hr after drug administration. In addition, intrathecal choline, delivered either with or 30 min after gp120, reduced gp120-induced IL-1β protein and pro-inflammatory cytokine mRNAs within the lumbar spinal cord. A second α7nAchR agonist, GTS-21, also significantly reversed gp120-induced mechanical allodynia and lumbar spinal cord levels of proinflammatory cytokine mRNAs and IL-1β protein. A role of microglia is suggested by the observation that intrathecal choline suppressed the gp120-induced expression of, cd11b, a macrophage/microglial activation marker. Taken together, the data support that α7nAchR may be a novel target for treating pain where microglia maintain the proinflammatory state within the spinal cord.
doi:10.1016/j.bbi.2010.03.008
PMCID: PMC2902784  PMID: 20353818
pain; choline; intrathecal; rats; tumor necrosis factor
13.  Toll like receptor (TLR)-4 as a regulator of peripheral endogenous opioid-mediated analgesia in inflammation 
Molecular Pain  2014;10:10.
Background
Leukocytes containing opioid peptides locally control inflammatory pain. In the early phase of complete Freund’s adjuvant (CFA)-induced hind paw inflammation, formyl peptides (derived e.g. from Mycobacterium butyricum) trigger the release of opioid peptides from neutrophils contributing to tonic basal antinociception. In the later phase we hypothesized that toll-like-receptor-(TLR)-4 activation of monocytes/macrophages triggers opioid peptide release and thereby stimulates peripheral opioid-dependent antinociception.
Results
In Wistar rats with CFA hind paw inflammation in the later inflammatory phase (48–96 h) systemic leukocyte depletion by cyclophosphamide (CTX) or locally injected naloxone (NLX) further decreased mechanical and thermal nociceptive thresholds. In vitro β-endorphin (β-END) content increased during human monocyte differentiation as well as in anti-inflammatory CD14+CD16- or non-classical M2 macrophages. Monocytes expressing TLR4 dose-dependently released β-END after stimulation with lipopolysaccharide (LPS) dependent on intracellular calcium. Despite TLR4 expression proinflammatory M1 and anti-inflammatory M2 macrophages only secreted opioid peptides in response to ionomycin, a calcium ionophore. Intraplantar injection of LPS as a TLR4 agonist into the inflamed paw elicited an immediate opioid- and dose-dependent antinociception, which was blocked by TAK-242, a small-molecule inhibitor of TLR4, or by peripheral applied NLX. In the later phase LPS lowered mechanical and thermal nociceptive thresholds. Furthermore, local peripheral TLR4 blockade worsened thermal and mechanical nociceptive pain thresholds in CFA inflammation.
Conclusion
Endogenous opioids from monocytes/macrophages mediate endogenous antinociception in the late phase of inflammation. Peripheral TLR4 stimulation acts as a transient counter-regulatory mechanism for inflammatory pain in vivo, and increases the release of opioid peptides from monocytes in vitro. TLR4 antagonists as new treatments for sepsis and neuropathic pain might unexpectedly transiently enhance pain by impairing peripheral opioid analgesia.
doi:10.1186/1744-8069-10-10
PMCID: PMC3922964  PMID: 24499354
Toll like receptors; Analgesia; Inflammatory pain; Endogenous opioids
14.  Deletion of Nardilysin Prevents the Development of Steatohepatitis and Liver Fibrotic Changes 
PLoS ONE  2014;9(5):e98017.
Nonalcoholic steatohepatitis (NASH) is an inflammatory form of nonalcoholic fatty liver disease that progresses to liver cirrhosis. It is still unknown how only limited patients with fatty liver develop NASH. Tumor necrosis factor (TNF)-α is one of the key molecules in initiating the vicious circle of inflammations. Nardilysin (N-arginine dibasic convertase; Nrd1), a zinc metalloendopeptidase of the M16 family, enhances ectodomain shedding of TNF-α, resulting in the activation of inflammatory responses. In this study, we aimed to examine the role of Nrd1 in the development of NASH. Nrd1+/+ and Nrd1−/− mice were fed a control choline-supplemented amino acid-defined (CSAA) diet or a choline-deficient amino acid-defined (CDAA) diet. Fatty deposits were accumulated in the livers of both Nrd1+/+ and Nrd1−/− mice by the administration of the CSAA or CDAA diets, although the amount of liver triglyceride in Nrd1−/− mice was lower than that in Nrd1+/+ mice. Serum alanine aminotransferase levels were increased in Nrd1+/+ mice but not in Nrd1−/− mice fed the CDAA diet. mRNA expression of inflammatory cytokines were decreased in Nrd1−/− mice than in Nrd1+/+ mice fed the CDAA diet. While TNF-α protein was detected in both Nrd1+/+ and Nrd1−/− mouse livers fed the CDAA diet, secretion of TNF-α in Nrd1−/− mice was significantly less than that in Nrd1+/+ mice, indicating the decreased TNF-α shedding in Nrd1−/− mouse liver. Notably, fibrotic changes of the liver, accompanied by the increase of fibrogenic markers, were observed in Nrd1+/+ mice but not in Nrd1−/− mice fed the CDAA diet. Similar to the CDAA diet, fibrotic changes were not observed in Nrd1−/− mice fed a high-fat diet. Thus, deletion of nardilysin prevents the development of diet-induced steatohepatitis and liver fibrogenesis. Nardilysin could be an attractive target for anti-inflammatory therapy against NASH.
doi:10.1371/journal.pone.0098017
PMCID: PMC4029810  PMID: 24849253
15.  Analgesic effects of stem bark extracts of Trichilia monadelpha (Thonn.) JJ De Wilde 
Indian Journal of Pharmacology  2012;44(6):765-773.
Objectives:
Various parts of Trichilia monadelpha (Thonn) JJ De Wilde (Fam. Meliaceae) are used in Ghanaian traditional medicine for the treatment of painful and inflammatory conditions. The present study examined the analgesic properties of the petroleum ether (PEE), ethyl acetate (EAE), and the hydro-ethanolic (HAE) extract of the stem bark of the plant in murine models.
Materials and Methods:
PEE, EAE, and HAE were assessed in chemical (acetic acid-induced abdominal writhing and formalin tests), thermal (hot plate test), and mechanical (Randall-Selitto paw pressure test) pain models. The possible mechanisms of the antinociceptive action were also examined with various antagonists in the formalin test.
Results:
HAE, EAE, and PEE, each at doses of 10–100 mg/kg orally, and the positive controls (morphine and diclofenac) elicited significant dose-dependent antinociceptive activity in the chemical (acetic acid abdominal writhing and formalin tests), thermal (hot plate test), and mechanical (Randall-Selitto paw pressure test) pain models in rodents. The antinociceptive effect of HAE was partly or wholly reversed by systemic administration of atropine, naloxone, and glibenclamide. The antinociceptive effects of EAE and PEE were inhibited by atropine.
Conclusion:
The extracts HAE, EAE, and PEE caused dose-related antinociception in chemical, thermal, and mechanical models of pain in animals. The mechanism of action of HAE involves an interaction with muscarinic cholinergic, adenosinergic, opioidergic pathways, and ATP-sensitive K+ channels while that of EAE and PEE involve the muscarinic cholinergic system.
doi:10.4103/0253-7613.103299
PMCID: PMC3523507  PMID: 23248409
Formalin test; hot plate; pain; randall-selitto test; writhing test
16.  Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease 
Nature  2011;472(7341):57-63.
Metabolomics studies hold promise for discovery of pathways linked to disease processes. Cardiovascular disease (CVD) represents the leading cause of death and morbidity worldwide. A metabolomics approach was used to generate unbiased small molecule metabolic profiles in plasma that predict risk for CVD. Three metabolites of the dietary lipid phosphatidylcholine, namely choline, trimethylamine N-oxide (TMAO), and betaine, were identified and then shown to predict risk for CVD in an independent large clinical cohort. Dietary supplementation of mice with choline, TMAO or betaine promoted up-regulation of multiple macrophage scavenger receptors linked to atherosclerosis, and supplementation with choline or TMAO promoted atherosclerosis. Studies using germ-free mice confirmed a critical role for dietary choline and gut flora in TMAO production, augmented macrophage cholesterol accumulation and foam cell formation. Suppression of intestinal microflora in atherosclerosis-prone mice inhibited dietary choline-enhanced atherosclerosis. Genetic variations controlling expression of flavin monooxygenases (FMOs), an enzymatic source of TMAO, segregated with atherosclerosis in hyperlipidemic mice. Discovery of a relationship between gut flora-dependent metabolism of dietary phosphatidylcholine and CVD pathogenesis provides opportunities for development of both novel diagnostic tests and therapeutic approaches for atherosclerotic heart disease.
doi:10.1038/nature09922
PMCID: PMC3086762  PMID: 21475195
metabolomics; atherosclerosis; intestinal microbiota; diet; phospholipid; choline
17.  Mast cell stabilization promotes antinociceptive effects in a mouse model of postoperative pain 
Journal of Pain Research  2013;6:161-166.
Background
Nerve injury and consequent inflammatory responses produced by surgical incision result in a complicated pain status which still affects half of all surgical patients. Therefore, it is essential for anesthesiologists to identify the mechanisms of postoperative pain. Mast cells are resident cells of connective tissue and the mucosa that participate in the immune response. Degranulation of mast cells is involved in the development of postoperative pain and can be induced by surgical incision. The aim of this study was to investigate whether stabilization of mast cells causes an antinociceptive effect in a mouse model of postoperative pain.
Methods
Postoperative pain was induced by making an incision in the hind paw of BALB/c mice. The mast cell membrane stabilizer cromoglycate (200 μg/20 μL) was injected before incision of the paw, and postoperative pain responses were measured by assessing guarding behavior, withdrawal threshold to mechanical stimuli, and latency of heat pain behavior 1, 2, and 7 days after the incision.
Results
The incision produced guarding pain, mechanical allodynia, and heat hypersensitivity. Cromoglycate decreased the guarding pain score (day 1) and the withdrawal threshold to mechanical stimuli (days 1, 2, and 7). However, the withdrawal latency to heat was not affected by cromoglycate treatment.
Conclusion
Cromoglycate significantly attenuated the pain response expressed as guarding pain and mechanical allodynia in a mouse model of postoperative pain. Thus, mast cell activation is likely a mechanism of postoperative pain and is an interesting target for the development of new therapies.
doi:10.2147/JPR.S41527
PMCID: PMC3594917  PMID: 23503682
postoperative pain; mast cells; cromoglycate
18.  Antinociceptive and Anti-Inflammatory Effects of Orally Administrated Denatured Naja Naja Atra Venom on Murine Rheumatoid Arthritis Models 
To investigate the antinociceptive and anti-inflammatory activities of the denatured Naja Naja atra venom (NNAV) in rheumatoid arthritis-associated models, the denatured NNAV (heat treated; 30, 90, 270 μg/kg), the native NNAV (untreated with heat; 90 μg/kg), and Tripterygium wilfordii polyglycoside (TWP, 15 mg/kg) were administrated orally either prophylactically or therapeutically. We measured time of licking the affected paw in formaldehyde-induced inflammatory model, paw volume in egg-white-induced inflammation, and granuloma weight in formalin-soaked filter paper-induced granuloma. For adjuvant-induced arthritis (AIA) rats, paw edema, mechanical withdrawal threshold, serum levels of TNF-α and IL-10, and histopathological changes of the affected paw were assessed. We found that the denatured NNAV (90, 270 μg/kg) significantly reduced time of licking paw, paw volume, and granuloma weight in above inflammatory models and also attenuated paw edema, mechanical hyperalgesia, and histopathology changes in AIA rats. Additionally, the increase in serum TNF-α and the decrease in serum IL-10 in AIA rats were reversed by the denatured NNAV. Although the native NNAV and TWP rendered the similar pharmacological actions on the above four models with less potency than that of the denatured NNAV, these findings demonstrate that oral administration of the denatured NNAV produces antinociceptive and anti-inflammatory activities on rheumatoid arthritis.
doi:10.1155/2013/616241
PMCID: PMC3619627  PMID: 23634171
19.  Metabolic crosstalk between choline/1-carbon metabolism and energy homeostasis 
There are multiple identified mechanisms involved in energy metabolism, insulin resistance and adiposity, but there are here-to-fore unsuspected metabolic factors that also influence these processes. Studies in animal models suggest important links between choline/1-carbon metabolism and energy homeostasis. Rodents fed choline deficient diets become hypermetabolic. Mice with deletions in one of several different genes of choline metabolism have phenotypes that include increased metabolic rate, decreased body fat/lean mass ratio, increased insulin sensitivity, decreased ATP production by mitochondria, or decreased weight gain on a high fat diet. In addition, farmers have recognized that the addition of a metabolite of choline (betaine) to cattle and swine feed reduces body fat/lean mass ratio. Choline dietary intake in humans varies over a >three-fold range, and genetic variation exists that modifies individual requirements for this nutrient. Although there are some epidemiologic studies in humans suggesting a link between choline/1-carbon metabolism and energy metabolism, there have been no controlled studies in humans that were specifically designed to examine this relationship.
doi:10.1515/cclm-2012-0518
PMCID: PMC3624053  PMID: 23072856
betaine; choline; insulin sensitivity; obesity; phosphatidylcholine
20.  Participation of cannabinoid receptors in peripheral nociception induced by some NSAIDs 
Nonsteroidal anti-inflammatory drugs (NSAIDs) have been used extensively to control inflammatory pain. Several peripheral antinociceptive mechanisms have been described, such as opioid system and NO/cGMP/KATP pathway activation. There is evidence that the cannabinoid system can also contribute to the in vivo pharmacological effects of ibuprofen and indomethacin. However, there is no evidence of the involvement of the endocannabinoid system in the peripheral antinociception induced by NSAIDs. Thus, the aim of this study was to investigate the participation of the endocannabinoid system in the peripheral antinociceptive effect of NSAIDs. All experiments were performed on male Wistar rats (160-200 g; N = 4 per group). Hyperalgesia was induced by a subcutaneous intraplantar (ipl) injection of prostaglandin E2 (PGE2, 2 µg/paw) in the rat's hindpaw and measured by the paw pressure test 3 h after injection. The weight in grams required to elicit a nociceptive response, paw flexion, was determined as the nociceptive threshold. The hyperalgesia was calculated as the difference between the measurements made before and after PGE2, which induced hyperalgesia (mean = 83.3 ± 4.505 g). AM-251 (80 µg/paw) and AM-630 (100 µg/paw) were used as CB1 and CB2 cannabinoid receptor antagonists, respectively. Ipl injection of 40 µg dipyrone (mean = 5.825 ± 2.842 g), 20 µg diclofenac (mean = 4.825 ± 3.850 g) and 40 µg indomethacin (mean = 6.650 ± 3.611 g) elicited a local peripheral antinociceptive effect. This effect was not antagonized by ipl CB1 cannabinoid antagonist to dipyrone (mean = 5.00 ± 0.9815 g), diclofenac (mean = 2.50 ± 0.8337 g) and indomethacin (mean = 6.650 ± 4.069 g) or CB2 cannabinoid antagonist to dipyrone (mean = 1.050 ± 6.436 g), diclofenac (mean = 6.675 ± 1.368 g) and indomethacin (mean = 2.85 ± 5.01 g). Thus, cannabinoid receptors do not seem to be involved in the peripheral antinociceptive mechanism of the NSAIDs dipyrone, diclofenac and indomethacin.
doi:10.1590/S0100-879X2012007500153
PMCID: PMC3854224  PMID: 22983178
Nonsteroidal anti-inflammatory drugs (NSAIDs); Peripheral antinociception; Cannabinoid system
21.  Plant Derived Aporphinic Alkaloid S-(+)-Dicentrine Induces Antinociceptive Effect in Both Acute and Chronic Inflammatory Pain Models: Evidence for a Role of TRPA1 Channels 
PLoS ONE  2013;8(7):e67730.
S-(+)-Dicentrine is an aporphinic alkaloid found in several plant species, mainly from Lauraceae family, which showed significant antinociceptive activity in an acute model of visceral pain in mice. In this work, we extended the knowledge on the antinociceptive properties of S-(+)-dicentrine and showed that this alkaloid also attenuates mechanical and cold hypersensitivity associated with cutaneous inflammation induced by Complete Freund’s Adjuvant in mice. Given orally, S-(+)-dicentrine (100 mg/kg) reversed CFA-induced mechanical hypersensitivity, evaluated as the paw withdrawal threshold to von Frey hairs, and this effect lasted up to 2 hours. S-(+)-Dicentrine also reversed CFA-induced cold hypersensitivity, assessed as the responses to a drop of acetone in the injured paw, but did not reverse the heat hypersensitivity, evaluated as the latency time to paw withdrawal in the hot plate (50°C). Moreover, S-(+)-dicentrine (100 mg/kg, p.o.) was effective in inhibit nociceptive responses to intraplantar injections of cinnamaldehyde, a TRPA1 activator, but not the responses induced by capsaicin, a TRPV1 activator. When administered either by oral or intraplantar routes, S-(+)-dicentrine reduced the licking time (spontaneous nociception) and increased the latency time to paw withdrawal in the cold plate (cold hypersensitivity), both induced by the intraplantar injection of cinnamaldehyde. Taken together, our data adds information about antinociceptive properties of S-(+)-dicentrine in inflammatory conditions, reducing spontaneous nociception and attenuating mechanical and cold hypersensitivity, probably via a TRPA1-dependent mechanism. It also indicates that S-(+)-dicentrine might be potentially interesting in the development of new clinically relevant drugs for the management of persistent pain, especially under inflammatory conditions.
doi:10.1371/journal.pone.0067730
PMCID: PMC3701576  PMID: 23861794
22.  Evaluation of Anti-Nociceptive and Anti-Inflammatory Activities of a Heterofucan from Dictyota menstrualis 
Marine Drugs  2013;11(8):2722-2740.
Fucan is a term that defines a family of homo- and hetero-polysaccharides containing sulfated l-fucose in its structure. In this work, a heterofucan (F2.0v) from the seaweed, Dictyota menstrualis, was evaluated as an antinociceptive and anti-inflammatory agent. F2.0v (20.0 mg/kg) inhibits 100% of leukocyte migration into the peritoneal cavity after chemical stimulation. However, F2.0v does not alter the expression of interleukin-1 beta (IL-1β) and interleukin-6 (IL-6), as well as tumor necrosis factor alpha (TNF-α). F2.0v (20.0 mg/kg) has peripheral antinociceptive activity with potency similar to dipyrone. On the other hand, it had no effect on pain response on the hot plate test. Confocal microscopy analysis and flow cytometry showed that F2.0v binds to the surface of leucocytes, which leads us to suggest that the mechanism of action of anti-inflammatory and antinociceptive F2.0v is related to its ability to inhibit the migration of leukocytes to the site of tissue injury. In summary, the data show that F2.0v compound has great potential as an antinociceptive and anti-inflammatory, and future studies will be performed to further characterize the mechanism of action of F2.0v.
doi:10.3390/md11082722
PMCID: PMC3766861  PMID: 23917068
fucan; brown seaweed; pain; analgesic; dictyotales
23.  Role of Teichoic Acid Choline Moieties in the Virulence of Streptococcus pneumoniae▿  
Infection and Immunity  2009;77(7):2824-2831.
In recent reports it was shown that genetically modified choline-free strains of Streptococcus pneumoniae (D39Cho−licA64 and D39ChiplicB31) expressing the type II capsular polysaccharide were virtually avirulent in the murine sepsis model, in sharp contrast to the isogenic and highly virulent strains D39Cho− and D39Chip, which have retained the choline residues at their surface. We now demonstrate that this choline-associated virulence is independent of Toll-like receptor 2 recognition. Also, despite the lack of virulence, choline-free strains of S. pneumoniae were able to activate splenic dendritic cells, induce secretion of proinflammatory cytokines, and produce specific protective immunity against subsequent challenge. However, after this transient engagement of the immune system the choline-free bacteria were rapidly cleared from the blood, while the isogenic virulent strain D39Cho− continued to grow, accompanied by prolonged expression of cytokines, eventually killing the experimental animals. The critical contribution of choline residues to the virulence potential of pneumococci appears to be the role that these amino alcohol residues play in a pneumococcal immune evasion strategy, the mechanism of which is unknown at the present time.
doi:10.1128/IAI.00986-08
PMCID: PMC2708541  PMID: 19433549
24.  IL-4 Deficiency Is Associated with Mechanical Hypersensitivity in Mice 
PLoS ONE  2011;6(12):e28205.
Interleukin-4 (IL-4) is an anti-inflammatory and analgesic cytokine that induces opioid receptor transcription. We investigated IL-4 knockout (ko) mice to characterize their pain behavior before and after chronic constriction injury (CCI) of the sciatic nerve as a model for neuropathic pain. We investigated opioid responsivity and measured cytokine and opioid receptor gene expression in the peripheral and central nervous system (PNS, CNS) of IL-4 ko mice in comparison with wildtype (wt) mice. Naïve IL-4 ko mice displayed tactile allodynia (wt: 0.45 g; ko: 0.18 g; p<0.001), while responses to heat and cold stimuli and to muscle pressure were not different. No compensatory changes in the gene expression of tumor necrosis factor-alpha (TNF), IL-1β, IL-10, and IL-13 were found in the PNS and CNS of naïve IL-4 ko mice. However, IL-1β gene expression was stronger in the sciatic nerve of IL-4 ko mice (p<0.001) 28 days after CCI and only IL-4 ko mice had elevated IL-10 gene expression (p = 0.014). Remarkably, CCI induced TNF (p<0.01), IL-1β (p<0.05), IL-10 (p<0.05), and IL-13 (p<0.001) gene expression exclusively in the ipsilateral spinal cord of IL-4 ko mice. The compensatory overexpression of the anti-inflammatory and analgesic cytokines IL-10 and IL-13 in the spinal cord of IL-4 ko mice may explain the lack of genotype differences for pain behavior after CCI. Additionally, CCI induced gene expression of μ, κ, and δ opioid receptors in the contralateral cortex and thalamus of IL-4 ko mice, paralleled by fast onset of morphine analgesia, but not in wt mice. We conclude that a lack of IL-4 leads to mechanical sensitivity; the compensatory hyperexpression of analgesic cytokines and opioid receptors after CCI, in turn, protects IL-4 ko mice from enhanced pain behavior after nerve lesion.
doi:10.1371/journal.pone.0028205
PMCID: PMC3229527  PMID: 22164245
25.  Persistent exercise attenuates nicotine- but not clonidine-induced antinociception in female rats. 
Exercise decreases the antinociceptive effects of opiate drugs. It has been hypothesized that the exercise-induced attenuation of opiate drug action is the result of the development of cross-tolerance between endogenous opioids released during exercise and exogenous opiates. The present study was designed to evaluate the role of exercise on non-opiate antinociception. Female Long-Evans rats were allowed ad lib access to running wheels. After three weeks, antinociceptive responses of animals were measured using the tail flick test following the administration of clonidine or nicotine. Nicotine and clonidine both produced dose-dependent increases in antinociceptive responses. Active animals were significantly less sensitive to nicotine-induced antinociception than inactive animals. There was no difference between the two groups in clonidine-induced antinociception. The results of these experiments suggest that exercise does not attenuate non-opioid, clonidine-induced antinociception. However, exercise does attenuate nicotine-induced antinociception. Therefore, the effect of persistent exercise on analgesic drugs is not specific to opiates.
doi:10.1016/j.pbb.2006.11.011
PMCID: PMC1945163  PMID: 17197014
exercise; opioids; antinociception; nicotine; clonidine; female rats

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