Granulocyte-Colony Stimulating Factor (G-CSF) induces mechanical hyperalgesia via spinal activation of MAP kinases and PI(3)K in mice

Granulocyte-colony stimulating factor (G-CSF) is a current pharmacological approach to increase peripheral neutrophil counts after anti-tumor therapies. Pain is most relevant side effect of G-CSF in healthy volunteers and cancer patients. Therefore, the mechanisms of G-CSF-induced hyperalgesia were investigated focusing on the role of spinal mitogen-activated protein (MAP) kinases ERK (extracellular signal-regulated kinase). JNK (Jun N-terminal Kinase) and p38, and PI(3)K (phosphatidylinositol 3-kinase). G-CSF induced dose (30-300 ng/paw)-dependent mechanical hyperalgesia, which was inhibited by local post-treatment with morphine. This effect of morphine was reversed by naloxone (opioid receptor antagonist). Furthermore, G-CSF-induced hyperalgesia was inhibited in a dose-dependent manner by intrathecal pre-treatment with ERK (PD98059), JNK (SB600125), p38 (SB202190) or PI(3)K (wortmanin) inhibitors. The co-treatment with MAP kinase and PI(3)K inhibitors, at doses that were ineffective as single treatment, significantly inhibited G-CSF-induced hyperalgesia. Concluding, in addition to systemic opioids, peripheral opioids as well as spinal treatment with MAP kinases and PI(3)K inhibitors also reduce G-CSF-induced pain. (C) 2011 Elsevier Inc. All rights reserved.

Role of the spinal cord heme oxygenase-carbon monoxide-cGMP pathway in the nociceptive response of rats

The aim of the present study was to investigate the role of the spinal cord heme oxygenase (HO)-carbon monoxide (CO)-soluble guanylate cyclase (sGC)-cGMP pathway in nociceptive response of rats to the formalin experimental nociceptive model. Animals were handled and adapted to the experimental environment for a few days before the formalin test was applied. For the formalin test 50 mu l of a 1% formalin solution was injected subcutaneously in the dorsal surface of the right hind paw. Following injections, animals were observed for I h and flinching behavior was measured as the nociceptive response. Thirty min before the test, rats were pretreated with intrathecal injections with the HO inhibitor, zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) or heme-lysinate, which is known to induce the HO pathway. Control animals were treated with vehicles. We observed a significant increase in nociceptive response of rats treated with ZnDPBG, and a drastic reduction of flinching nociceptive behavioral response in the heme-lysinate treated animals. Furthermore, the HO pathway seems to act via cGMP, since methylene blue (a sGC inhibitor) prevented the reduction of flinching nociceptive behavioral response caused by heme-lysinate. These findings strongly indicate that the HO pathway plays a spinal antinociceptive role during the formalin test, acting via cGMP. (C) 2007 Elsevier B.V. All rights reserved.

Statines et effets indésirables musculaires [Statins and muscular side-effects]

Statins are effective in the treatment of hypercholesterolemia for primary and secondary prevention of cardiovascular disease. While most side effects of statins are mild and transient, muscular symptoms are relatively common (5 to 10% of patients), but rarely serious (myositis, rhabdomyolysis). In cases of myopathy, the severity of symptoms and the determination of CK (creatine kinase) determine whether discontinuation of statin is necessary. Alternative strategies are also suggested. This article reviews suggestions on the management of these complaints that are a challenge in clinical practice.

Chronic intrathecal cannulation enhances nociceptive responses in rats

The influence of a chronically implanted spinal cannula on the nociceptive response induced by mechanical, chemical or thermal stimuli was evaluated. The hyperalgesia in response to mechanical stimulation induced by carrageenin or prostaglandin E2 (PGE2) was significantly increased in cannulated (Cn) rats, compared with naive (Nv) or sham-operated (Sh) rats. Only Cn animals presented an enhanced nociceptive response in the first phase of the formalin test when low doses were used (0.3 and 1%). The withdrawal latency to thermal stimulation of a paw inflamed by carrageenin was significantly reduced in Cn rats but not in Nv or Sh rats. In contrast to Nv and Sh rats, injection in Cn animals of a standard non-steroid anti-inflammatory drug, indomethacin, either intraperitoneally or into the spinal cord via an implanted cannula or by direct puncture of the intrathecal space significantly blocked the intensity of the hyperalgesia induced by PGE2. Cannulated animals treated with indomethacin also showed a significant inhibition of second phase formalin-induced paw flinches. Histopathological analysis of the spinal cord showed an increased frequency of mononuclear inflammatory cells in the Cn groups. Thus, the presence of a chronically implanted cannula seems to cause nociceptive spinal sensitization to mechanical, chemical and thermal stimulation, which can be blocked by indomethacin, thus suggesting that it may result from the spinal release of prostaglandins due to an ongoing mild inflammation.

Concurrent nociceptive stimulation impairs the anxiolytic effect of midazolam injected into the periaqueductal gray in mice

This study investigated whether the opportunity to avoid or escape the open arms of an elevated plus-maze (EPM) affects the antinociceptive response observed when mice are subjected to open arm confinement. Furthermore, in order to better characterize the relationship between emotion and antinociception in the EPM, we examined the behavioral effects of midazolam injection into the midbrain periaqueductal gray matter (PAG). As our main aim was to evaluate the relevance of different levels of approach-avoid conflict (i.e. The presence of open and closed arms) to maze-induced antinociception, mice were exposed to one of three types of EPM-a standard EPM (sEPM), an open EPM (oEPM: four open arms) or, as a control condition, an enclosed EPM (eEPM: four enclosed arms). Nociception was assessed using the formalin test. Twenty minutes after formalin injection (50 mu l, 2.5% formalin) into the dorsal right hind paw, mice received an intra-PAG injection of saline or midazolam (10-20 nmol). Five minutes later, they were individually exposed to one of the mazes for 10 min (25-35 min after formalin injection). Videotapes of the test sessions were scored for a variety of behavioral measures including time spent licking the formalin-injected paw. To examine whether the effects of midazolam on anxiety-like behavior may have been influenced by concurrent nociceptive stimulation (i.e. formalin pretreatment), naive mice were submitted to a similar procedure to that described above for the sEPM test but without formalin pretreatment. Results showed that mice exposed to the oEPM spent significantly less time licking the injected paw compared to groups exposed to either the sEPM or eEPM. Although exposure to the sEPM induced anxiety-like behaviors (i.e. open arm avoidance), it did not result in antinociception. Intra-PAG infusions of midazolam failed to block oEPM-induced antinociception or to alter sEPM-induced anxiety in mice that had received formalin injection. However, under normal test conditions (i.e. in the absence of formalin-induced nociceptive stimulation), intra-PAG midazolam produced clear anti-anxiety effects in mice exposed to the sEPM. Findings are discussed in terms of different emotional states induced by the oEPM and sEPM and the influence of concurrent nociceptive stimulation on the anti-anxiety effect of intra-PAG midazolam. (c) 2005 Elsevier B.V. All rights reserved.

Activation of 5-HT2C receptors in the dorsal periaqueductal gray increases antinociception in mice exposed to the elevated plus-maze

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Evaluation of antinociceptive and antiinflammatory effects of Croton pullei var. glabrior Lanj. (Euphorbiaceae)

Croton pullei var. glabrior Lanj. (Euphorbiaceae) é uma liana, amplamente distribuída na Floresta Amazônica. Na medicina popular, diversas plantas do gênero Croton têm sido utilizadas com fins terapêuticos em patologias que envolvem dor e inflamação, o que justifica este trabalho. O objetivo deste estudo foi investigar as atividades antinociceptiva e do extrato metanólico das folhas de C. pullei (MECP). O MECP reduziu, de forma dose-dependente, o número de contorções abdominais (1,2 %) em camundongos, sugerindo uma atividade antinociceptiva da planta. Por outro lado, o MECP não alterou significativamente a reatividade ao estímulo térmico no teste da placa quente e a reatividade à estimulação química na primeira fase do teste da formalina, indicando um mecanismo não-opioidérgico. O MECP reduziu a nocicepção na segunda fase do teste da formalina, inibiu o edema de orelha induzido pelo óleo de croton e reduziu a migração leucocitária no teste da peritonite induzida por carragenina, indicando uma atividade antiinflamatória. Apesar dos mecanismos responsáveis pelos efeitos da planta ainda não estarem completamente esclarecidos, estes resultados parecem justificar o uso medicinal potencial de Croton pullei var. glabrior Lanj. em patologias que envolvam dor e inflamação.

Activation of 5-HT2C receptors in the dorsal periaqueductal gray increases antinociception in mice exposed to the elevated plus-maze

Several findings have pointed to the role of the dorsal periaqueductal gray (dPAG) serotonin 5-HT1A and 5-HT2(A-C) receptor subtypes in the modulation of defensive behavior in animals exposed to the elevated plus-maze (EPM). Besides displaying anxiety-like behavior, rodents also exhibit antinociception in the EPM. This study investigated the effects of intra-dPAG injections of 5-HT1A and 5-HT2B/2C receptor ligands on EPM-induced antinociception in mice. Male Swiss mice received 0.1 mu l intra-dPAG injections of vehicle, 5.6 and 10 nmol of 8-OHDPAT, a 5-HT1A receptor agonist (Experiment 1), or 0.01, 0.03 and 0.1 nmol of mCPP, a 5-HT2B/2C receptor agonist (Experiment 2). Five minutes later, each mouse received an intraperitoneal injection of 0.6% acetic acid (0.1 ml/10 g body weight; nociceptive stimulus) and was individually confined in the open (OA) or enclosed (EA) arms of the EPM for 5 min, during which the number of abdominal writhes induced by the acetic acid was recorded. While intra-dPAG injection of 8-OHDPAT did not change open-arm antinociception (OAR). mCPP (0.01 nmol) enhanced it. Combined injections of ketanserin (10 nmol/0.1 mu l), a 5-HT2A/2C receptor antagonist, and 0.01 nmol of mCPP (Experiment 3), selectively and completely blocked the OAR enhancement induced by mCPP. Although intra-dPAG injection of mCPP (0.01 nmol) also produced antinociception in EA-confined mice (Experiment 2), this effect was not confirmed in Experiment 3. Moreover, no other compound changed the nociceptive response in EA-confined animals. These results suggest that the 5-HT2C receptors located within the PAG play a role in this type of environmentally induced pain inhibition in mice. (c) 2012 Elsevier B.V. All rights reserved.

Development and pharmacokinetic characterization of pulmonal and intravenous delta-9-tetrahydrocannabinol (THC) in humans

The aim of the present study was to develop a physiologically compatible inhalation solution of delta-9-tetrahydrocannabinol (THC), and to compare the pharmacokinetic and analgesic properties of pulmonal THC versus pulmonal placebo and intravenous (iv) THC, respectively. Eight healthy volunteers were included in this randomized, double-blind, crossover study. The aqueous THC formulations were prepared by using a solubilization technique. iv THC (0.053 mg/kg body weight), pulmonal THC (0.053 mg/kg), or a placebo inhalation solution was administered as single dose. At defined time points, blood samples were collected, and somatic and psychotropic side effects as well as vital functions monitored. An ice water immersion test was performed to measure analgesia. Using a pressure-driven nebulizer, the pulmonal administration of the THC liquid aerosol resulted in high THC peak plasma levels within minutes. The bioavailability of the pulmonal THC was 28.7 +/- 8.2% (mean +/- SEM). The side effects observed after pulmonal THC were coughing and slight irritation of the upper respiratory tract, very mild psychotropic symptoms, and headache. The side effects after iv THC were much more prominent. Neither pulmonal nor iv THC significantly reduced experimentally induced pain.

Descending pain modulation in a Kaolin-induced hydrocephalus rat model

Pain transmission at the spinal cord is modulated by descending actions that arise from supraspinal areas which collectively form the endogenous pain control system. Two key areas involved of the endogenous pain control system have a circunventricular location, namely the periaqueductal grey (PAG) and the locus coeruleus (LC). The PAG plays a crucial role in descending pain modulation as it conveys the input from higher brain centers to the spinal cord. As to the LC, it is involved in descending pain inhibition by direct noradrenergic projections to the spinal cord. In the context of neurological defects, several diseases may affect the structure and function of the brain. Hydrocephalus is a congenital or acquired disease characterized by an enlargement of the ventricles which leads to a distortion of the adjacent tissues, including the PAG and LC. Usually, patients suffering from hydrocephalus present dysfunctions in learning and memory and also motor deficits. It remains to be evaluated if lesions of the periventricular brain areas involved in pain control during hydrocephalus may affect descending pain control and, herein, affect pain responses. The studies included in the present thesis used an experimental model of hydrocephalus (the rat injected in the cisterna magna with kaolin) to study descending modulation of pain, focusing on the two circumventricular regions referred above (the PAG and the LC). In order to evaluate the effects of kaolin injection into the cisterna magna, we measured the degree of ventricular dilatation in sections encompassing the PAG by standard cytoarquitectonic stanings (thionin staining). For the LC, immunodetection of the noradrenaline-synthetizing enzyme tyrosine hydroxylase (TH) was performed, due to the noradrenergic nature of the LC neurons. In general, rats with kaolin-induced hydrocephalus presented a higher dilatation of the 4th ventricle, along with a tendency to a higher area of the PAG. Due to the validated role of detection the c-fos protooncogene as a marker of neuronal activation, we also studied neuronal activation in the several subnuclei which compose the PAG, namely the dorsomedial, dorsolateral, lateral and ventrolateral (VLPAG) parts. A decrease in the numbers of neurons immunoreactive for Fos protein (the product of activation of the c-fos protooncogene) was detected in rats injected with kaolin, whereas the remaining PAG subnuclei did not present changes in Fos-immunoreactive nuclei. Increases in the levels of TH in the LC, namely at the rostral parts of the nucleus, were detected in hydrocephalic animals. The following pain-related parameters were measured, namely 1) pain behavioural responses in a validated pain inflammatory test (the formalin test) and 2) the nociceptive activation of spinal cord neurons. A decrease in behavioral responses was detected in rats with kaolin-induced hydrocephalus was detected, namely in the second phase of the test (inflammatory phase). This is the phase of the formalin test in which the motor behaviour is less important, which is important since a semi-quantitative analysis of the motor performance of rats injected with kaolin indicates that these animals may present some motor impairments. Collectively, the results of the behavioral studies indicate that rats with kaolin-induced hydrocephalus exhibit hypoalgesia. A decrease in Fos expression was detected at the superficial dorsal layers of the spinal cord in rats with kaolin-induced hydrocephalus, further indicating that hydrocephalus decreases nociceptive responses. It remains to be ascertained if this is due to alterations in the PAG and LC in the rats with kaolin-induced hydrocephalus, which may affect descending pain modulation. It remains to be evaluated what are the mechanisms underlying the increased pain inhibition at the spinal dorsal horn in the hydrocephalus rats. Regarding the VLPAG, the decrease in neuronal activity may impair descending modulation. Since the LC has higher levels of TH in rats with kaolininduced hydrocephalus, which also appears to increase the noradrenergic innervation in the spinal dorsal horn, it is possible that an increase in the release of noradrenaline at the spinal cord accounts for pain inhibition. Our studies also determine the need to study in detail patients with hydrocephalus namely in what concerns their thresholds to pain and to perform imaging studies focused on the structure and function of pain control areas in the brain.

Open elevated plus maze-induced antinociception in rats: A non-opioid type of pain inhibition?

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Acetic acid- and phenyl-p-benzoquinone-induced overt pain-like behavior depends on spinal activation of MAP kinases, PI3K and microglia in mice

The acetic acid and phenyl-p-benzoquinone are easy and fast screening models to access the activity of novel candidates as analgesic drugs and their mechanisms. These models induce a characteristic and quantifiable overt pain-like behavior described as writhing response or abdominal contortions. The knowledge of the mechanisms involved in the chosen model is a crucial step forward demonstrating the mechanisms that the candidate drug would inhibit because the mechanisms triggered in that model will be addressed. Herein, it was investigated the role of spinal mitogen-activated protein (MAP) kinases ERK (extracellular signal-regulated kinase), JNK (Jun N-terminal Kinase) and p38, PI3K (phosphatidylinositol 3-kinase) and microglia in the writhing response induced by acetic acid and phenyl-p-benzoquinone, and flinch induced by formalin in mice. Acetic acid and phenyl-p-benzoquinone induced significant writhing response over 20 min. The nociceptive response in these models were significantly and in a dose-dependent manner reduced by intrathecal pre-treatment with ERK (PD98059), JNK (SB600125), p38 (SB202190) or PI3K (wortmannin) inhibitors. Furthermore, the co-treatment with MAP kinase and PI3K inhibitors, at doses that were ineffective as single treatment, significantly inhibited acetic acid- and phenyl-p-benzoquinone-induced nociception. The treatment with microglia inhibitors minocycline and fluorocitrate also diminished the nociceptive response. Similar results were obtained in the formalin test. Concluding. MAP kinases and PI3K are important spinal signaling kinases in acetic acid and phenyl-p-benzoquinone models of overt pain-like behavior and there is also activation of spinal microglia indicating that it is also important to determine whether drugs tested in these models also modulate such spinal mechanisms. (C) 2012 Elsevier Inc. All rights reserved.

Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection

Nerve injury leads to a neuropathic pain state that results from central sensitization. This phenomenom is mediated by NMDA receptors and may involve the production of nitric oxide (NO). In this study, we investigated the expression of the neuronal isoform of NO synthase (nNOS) in the spinal cord of 3-month-old male, Wistar rats after sciatic nerve transection (SNT). Our attention was focused on the dorsal part of L3-L5 segments receiving sensory inputs from the sciatic nerve. SNT resulted in the development of neuropathic pain symptoms confirmed by evaluating mechanical hyperalgesia (Randall and Selitto test) and allodynia (von Frey hair test). Control animals did not present any alteration (sham-animals). The selective inhibitor of nNOS, 7-nitroindazole (0.2 and 2 µg in 50 µL), blocked hyperalgesia and allodynia induced by SNT. Immunohistochemical analysis showed that nNOS was increased (48% by day 30) in the lumbar spinal cord after SNT. This increase was observed near the central canal (Rexed’s lamina X) and also in lamina I-IV of the dorsal horn. Real-time PCR results indicated an increase of nNOS mRNA detected from 1 to 30 days after SNT, with the highest increase observed 1 day after injury (1469%). Immunoblotting confirmed the increase of nNOS in the spinal cord between 1 and 15 days post-lesion (20%), reaching the greatest increase (60%) 30 days after surgery. The present findings demonstrate an increase of nNOS after peripheral nerve injury that may contribute to the increase of NO production observed after peripheral neuropathy.

Serotonin receptors are involved in the spinal mediation of descending facilitation of surgical incision-induced increase of Fos-like immunoreactivity in rats

Background: Descending pronociceptive pathways may be implicated in states of persistent pain. Paw skin incision is a well-established postoperative pain model that causes behavioral nociceptive responses and enhanced excitability of spinal dorsal horn neurons. The number of spinal c-Fos positive neurons of rats treated intrathecally with serotonin, noradrenaline or acetylcholine antagonists where evaluated to study the descending pathways activated by a surgical paw incision. Results: The number of c-Fos positive neurons in laminae I/II ipsilateral, lamina V bilateral to the incised paw, and in lamina X significantly increased after the incision. These changes: remained unchanged in phenoxybenzamine-treated rats; were increased in the contralateral lamina V of atropine-treated rats; were inhibited in the ipsilateral lamina I/II by 5-HT(1/2B/2C) (methysergide), 5-HT(2A) (ketanserin) or 5-HT(1/2A/2C/5/6/7) (methiothepin) receptors antagonists, in the ipsilateral lamina V by methysergide or methiothepin, in the contralateral lamina V by all the serotonergic antagonists and in the lamina X by LY 278,584, ketanserin or methiothepin. Conclusions: We conclude: (1) muscarinic cholinergic mechanisms reduce incision-induced response of spinal neurons inputs from the contralateral paw; (2) 5-HT(1/2A/2C/3) receptors-mediate mechanisms increase the activity of descending pathways that facilitates the response of spinal neurons to noxious inputs from the contralateral paw; (3) 5-HT(1/2A/2C) and 5-HT(1/2C) receptors increases the descending facilitation mechanisms induced by incision in the ipsilateral paw; (4) 5-HT(2A/3) receptors contribute to descending pronociceptive pathways conveyed by lamina X spinal neurons; (5) alpha-adrenergic receptors are unlikely to participate in the incision-induced facilitation of the spinal neurons.

Antinociceptive and anti-inflammatory properties of 7-hydroxycoumarin in experimental animal models: potential therapeutic for the control of inflammatory chronic pain

Objectives In the present study we investigated the anti nociceptive, anti-inflammatory and antipyretic effects of 7-hydroxycoumarin (7-HC) in animal models. Methods The effects of oral 7-HC were tested against acetic acid-induced writhing, formalin test, tail flick test, complete Freund`s adjuvant (CFA)-induced hypemociception, carrageenan-induced paw oedema, lipopolysaccharide-induced fever and the rota rod test. Key findings 7-HC (3-60 mg/kg) produced a dose-related antinociception against acetic acid-induced writhing in mice and in the formalin test. In contrast, treatment with 7-HC did not prevent thermal nociception in the tail flick test. A single treatment with 7-HC, 60 mg/kg, produced a long-lasting antinociceptive effect against CFA-induced hypernociception, a chronic inflammatory pain stimulus. Notably, at 60 mg/kg per day over 4 days the administration of 7-HC produced a continuous antinociceptive effect against CFA-induced hypernociception. 7-HC (30-120 mg/kg) produced anti-inflammatory and antipyretic effects against carrageenan-induced inflammation and lipopolysaccharide-induced fever, respectively. Moreover, 7-HC was found to be safe with respect to ulcer induction. In the rota rod test, 7-HC-treated mice did not show any motor performance alterations. Conclusions The prolonged antinociceptive and anti-inflammatory effects of 7-HC, in association with its low ulcerogenic activity, indicate that this molecule might be a good candidate for development of new drugs for the control of chronic inflammatory pain and fever.