Monique Etienne

Diabetes-Induced Mechanical Hyperalgesia Involves Spinal Mitogen-Activated Protein Kinase Activation in Neurons and Microglia via N-Methyl-D-aspartate-Dependent Mechanisms

Molecular mechanisms underlying diabetes-induced painful neuropathy are poorly understood. We have demonstrated, in rats with streptozotocin-induced diabetes, that mechanical hyperalgesia, a common symptom of diabetic neuropathy, was correlated with an early increase in extracellular signal-regulated protein kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) phosphorylation in the spinal cord and dorsal root ganglion at 3 weeks after induction of diabetes. This change was specific to hyperalgesia because nonhyperalgesic rats failed to have such an increase. Immunoblot analysis showed no variation of protein levels, suggesting a post-translational regulation of the corresponding kinases. In diabetic hyperalgesic rats, immunocytochemistry revealed that all phosphorylated mitogen-activated protein kinases (MAPKs) colocalized with both the neuronal (NeuN) and microglial (OX42) cell-specific markers but not with the astrocyte marker [glial fibrillary acidic protein (GFAP)] in the superficial dorsal horn-laminae of the spinal cord. In these same rats, a 7-day administration [5 μg/rat/day, intrathecal (i.t.)] of 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), and anthra(1,9-cd)pyrazol-6(2H)-one (SP600125), which inhibited MAPK kinase, p38, and JNK, respectively, suppressed mechanical hyperalgesia, and decreased phosphorylation of the kinases. To characterize the cellular events upstream of MAPKs, we have examined the role of the NMDA receptor known to be implicated in pain hypersensitivity. The prolonged blockade of this receptor during 7 days by (5R, 10S)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]-cyclohepten-5-10-imine hydrogen maleate (MK801; 5 μg/rat/day, i.t.), a noncompetitive NMDA receptor antagonist, reversed hyperalgesia developed by diabetic rats and blocked phosphorylation of all MAPKs. These results demonstrate for the first time that NMDA receptor-dependent phosphorylation of MAPKs in spinal cord neurons and microglia contribute to the establishment and longterm maintenance of painful diabetic hyperalgesia and that these kinases represent potential targets for pain therapy.

Endocannabinoid and serotonergic systems are needed for acetaminophen-induced analgesia.

Abstract Acetaminophen is the most used analgesic/antipyretic drug. Its unclear mechanism of action could rely on cyclooxygenase inhibition, NO synthesis blockade or reinforcement of the serotonergic system. Here we show that in thermal, mechanical and chemical pain tests, AM‐251, a specific CB1 receptor antagonist, abolished the analgesic action of acetaminophen, which was also lost in CB1 receptor knockout mice. Moreover, acetaminophen was shown unable to bind to CB1 receptors demonstrating an indirect involvement of these receptors in the analgesic effect of this compound. Accordingly with these results, we also demonstrated that the inhibition of FAAH, an enzyme involved in the cerebral metabolism of acetaminophen into AM404, known to reinforce the activity of the endocannabinoid system, suppressed the antinociceptive effect of acetaminophen. In addition, similarly to the interaction of acetaminophen with bulbospinal serotonergic pathways and spinal serotonin receptors, we observed that the antinociceptive activity of ACEA, a CB1 receptor agonist, was inhibited by lesion of bulbospinal serotonergic pathways and antagonists of spinal 5‐HT receptors. We therefore propose that acetaminophen‐induced analgesia could involve the following sequence: (1) FAAH‐dependent metabolism of acetaminophen into AM404; (2) indirect involvement of CB1 receptors by this metabolite; (3) endocannabinoid‐dependent reinforcement of the serotonergic bulbospinal pathways, and (4) involvement of spinal pain‐suppressing serotonergic receptors.

Decrease in non-selective, non-sustained attention induced by a chronic visceral inflammatory state as a new pain evaluation in rats.

&NA; The aim of this study was to develop a new behavioral pain test based on the evaluation of cognitive capacity impairments in rats with colitis and to determine the impact of different acute analgesic treatments. Colitis was induced in rats by an enema containing 2,4,6‐trinitrobenzen sulfonic acid. Visual non‐selective, non‐sustained attentional level was assessed by a new behavioral testing procedure. Animals were familiarized on three consecutive days with an open field containing four small, similar, familiar objects. On the day of testing, one of the objects was randomly replaced by a new one. Attentional level was determined by the ability of the rat to perceive this small modification to its familiar environment. The effect of morphine, acetaminophen, aspirin or ibuprofen treatment was assessed on testing day and compared with that observed during a Von Frey test to assess referred tactile hypersensitivity of the skin of the lower back. Rats with colitis had decreased attentional level but no change in their locomotor activity, interest in the environment or memory encoding. Morphine (1 mg/kg, s.c. and 10 &mgr;g/rat, i.t.) and acetaminophen (200 mg/kg, p.o.) had a beneficial effect on attentional level and on referred tactile hypersensitivity. Testing for the latter showed that aspirin and ibuprofen (400 mg/kg, p.o.) were ineffective. The decrease in visual non‐selective, non‐sustained attention induced by chronic inflammatory painful state can be relieved by effective analgesic treatments. This finding could lead to the development of a new behavioral test to assess spontaneous pain in chronic painful subjects.

Fatty Acid Amide Hydrolase-Dependent Generation of Antinociceptive Drug Metabolites Acting on TRPV1 in the Brain

The discovery that paracetamol is metabolized to the potent TRPV1 activator N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM404) and that this metabolite contributes to paracetamol’s antinociceptive effect in rodents via activation of TRPV1 in the central nervous system (CNS) has provided a potential strategy for developing novel analgesics. Here we validated this strategy by examining the metabolism and antinociceptive activity of the de-acetylated paracetamol metabolite 4-aminophenol and 4-hydroxy-3-methoxybenzylamine (HMBA), both of which may undergo a fatty acid amide hydrolase (FAAH)-dependent biotransformation to potent TRPV1 activators in the brain. Systemic administration of 4-aminophenol and HMBA led to a dose-dependent formation of AM404 plus N-(4-hydroxyphenyl)-9Z-octadecenamide (HPODA) and arvanil plus olvanil in the mouse brain, respectively. The order of potency of these lipid metabolites as TRPV1 activators was arvanil = olvanil>>AM404> HPODA. Both 4-aminophenol and HMBA displayed antinociceptive activity in various rodent pain tests. The formation of AM404, arvanil and olvanil, but not HPODA, and the antinociceptive effects of 4-aminophenol and HMBA were substantially reduced or disappeared in FAAH null mice. The activity of 4-aminophenol in the mouse formalin, von Frey and tail immersion tests was also lost in TRPV1 null mice. Intracerebroventricular injection of the TRPV1 blocker capsazepine eliminated the antinociceptive effects of 4-aminophenol and HMBA in the mouse formalin test. In the rat, pharmacological inhibition of FAAH, TRPV1, cannabinoid CB1 receptors and spinal 5-HT3 or 5-HT1A receptors, and chemical deletion of bulbospinal serotonergic pathways prevented the antinociceptive action of 4-aminophenol. Thus, the pharmacological profile of 4-aminophenol was identical to that previously reported for paracetamol, supporting our suggestion that this drug metabolite contributes to paracetamol’s analgesic activity via activation of bulbospinal pathways. Our findings demonstrate that it is possible to construct novel antinociceptive drugs based on fatty acid conjugation as a metabolic pathway for the generation of TRPV1 modulators in the CNS.

Acetaminophen recruits spinal p42/p44 MAPKs and GH/IGF-1 receptors to produce analgesia via the serotonergic system

The mechanism of action of acetaminophen is currently widely discussed. Direct inhibition of cyclooxygenase isoforms remains the commonly advanced hypothesis. We combined behavioral studies with molecular techniques to investigate the mechanism of action of acetaminophen in a model of tonic pain in rats. We show that acetaminophen indirectly stimulates spinal 5-hydroxytryptamine (5-HT)1A receptors in the formalin test, thereby increasing transcript and protein levels of low-affinity neurotrophin receptor, insulin-like growth factor-1 (IGF-1) receptor α subunit, and growth hormone receptor and reducing the amount of somatostatin 3 receptor (sst3R) mRNA. Those cellular events seem to be important for the antinociceptive activity of acetaminophen. Indeed, down-regulation of sst3R mRNA depends on acetaminophen-elicited, 5-HT1A receptordependent increase in neuronal extracellular signal-regulated kinase 1/2 (ERK1/2) activities that mediate antinociception. In addition, spinal growth hormone (GH) and IGF-1 receptors would also be involved in the antinociceptive activity of the analgesic at different degrees. Our results show the involvement of specific 5-HT1A receptor-dependent cellular events in acetaminophen-produced antinociception and consequently indicate that inhibition of cyclooxygenase activities is not the exclusive mechanism involved. Furthermore, we propose that the mechanisms of 5-HT1A receptor-elicited antinociception and the role of the spinal ERK1/2 pathway in nociception are more intricate than suspected so far and that the GH/IGF-1 axis is an interesting new player in the regulation of spinal nociception.

Alleviating Pain Hypersensitivity through Activation of Type 4 Metabotropic Glutamate Receptor

Hyperactivity of the glutamatergic system is involved in the development of central sensitization in the pain neuraxis, associated with allodynia and hyperalgesia observed in patients with chronic pain. Herein we study the ability of type 4 metabotropic glutamate receptors (mGlu4) to regulate spinal glutamate signaling and alleviate chronic pain. We show that mGlu4 are located both on unmyelinated C-fibers and spinal neurons terminals in the inner lamina II of the spinal cord where they inhibit glutamatergic transmission through coupling to Cav2.2 channels. Genetic deletion of mGlu4 in mice alters sensitivity to strong noxious mechanical compression and accelerates the onset of the nociceptive behavior in the inflammatory phase of the formalin test. However, responses to punctate mechanical stimulation and nocifensive responses to thermal noxious stimuli are not modified. Accordingly, pharmacological activation of mGlu4 inhibits mechanical hypersensitivity in animal models of inflammatory or neuropathic pain while leaving acute mechanical perception unchanged in naive animals. Together, these results reveal that mGlu4 is a promising new target for the treatment of chronic pain.

Spinal cord plasticity and acid-sensing ion channels involvement in a rodent model of irritable bowel syndrome

Irritable bowel syndrome (IBS) is a common functional gastro‐intestinal disorder characterized by intractable chronic abdominal pain. In this study, we examined the possible spinal mechanisms underlying colonic hypersensitivity (CHS) using a non‐inflammatory rat model of IBS induced by rectal enemas of butyrate, a short‐chain fatty acid. We hypothesized that spinal plasticity could be responsible for CHS and that ASIC channels, which are known to support pain‐elicited currents in the spinal cord, could contribute to central sensitization in our model of IBS. First, in order to determine if visceral pain relies on changes in spinal activity, we analyzed Fos expression in the spinal cord of rats treated with butyrate following a challenge with repetitive noxious colorectal distension. We found that Fos immunoreactivity was increased in thoracic T10–11–12, lumbar L1–2–6 and sacral S1 spinal segments. In control rats treated with saline, noxious repetitive colorectal distensions evoked Fos expression only in L1–2–6 and S1 spinal segments. Secondly, intrathecal injection of PcTx1, a specific ASIC1A antagonist, in the lumbar spinal cord completely prevented the development of CHS induced by butyrate. ASIC1 and 2 mRNAs, especially ASIC1A, were upregulated in the lumbar spinal cord. ASIC1A could thus contribute to spinal sensitization in our model of IBS, as it is supported by spinal colocalization of ASIC1A and Fos proteins. The whole data pinpoint a potential critical role of thoracic spinal cord in non‐inflammatory pain states such as IBS and suggest that ASIC channels are part of the molecular effectors of central sensitization leading to visceral pain.Irritable bowel syndrome (IBS) is a common functional gastro-intestinal disorder characterized by intractable chronic abdominal pain. In this study, we examined the possible spinal mechanisms underlying colonic hypersensitivity (CHS) using a non-inflammatory rat model of IBS induced by rectal enemas of butyrate, a short-chain fatty acid. We hypothesized that spinal plasticity could be responsible for CHS and that ASIC channels, which are known to support pain-elicited currents in the spinal cord, could contribute to central sensitization in our model of IBS. First, in order to determine if visceral pain relies on changes in spinal activity, we analyzed Fos expression in the spinal cord of rats treated with butyrate following a challenge with repetitive noxious colorectal distension. We found that Fos immunoreactivity was increased in thoracic T10-11-12, lumbar L1-2-6 and sacral S1 spinal segments. In control rats treated with saline, noxious repetitive colorectal distensions evoked Fos expression only in L1-2-6 and S1 spinal segments. Secondly, intrathecal injection of PcTx1, a specific ASIC1A antagonist, in the lumbar spinal cord completely prevented the development of CHS induced by butyrate. ASIC1 and 2 mRNAs, especially ASIC1A, were upregulated in the lumbar spinal cord. ASIC1A could thus contribute to spinal sensitization in our model of IBS, as it is supported by spinal colocalization of ASIC1A and Fos proteins. The whole data pinpoint a potential critical role of thoracic spinal cord in non-inflammatory pain states such as IBS and suggest that ASIC channels are part of the molecular effectors of central sensitization leading to visceral pain.

Peripheral contribution of NGF and ASIC1a to colonic hypersensitivity in a rat model of irritable bowel syndrome

Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder associated with idiopathic colonic hypersensitivity (CHS). However, recent studies suggest that low‐grade inflammation could underlie CHS in IBS. The pro‐inflammatory mediator nerve growth factor (NGF) plays a key role in the sensitization of peripheral pain pathways and several studies have reported its contribution to visceral pain development. NGF modulates the expression of Acid‐Sensing Ion Channels (ASICs), which are proton sensors involved in sensory neurons sensitization. This study examined the peripheral contribution of NGF and ASICs to IBS‐like CHS induced by butyrate enemas in the rat colon.

Memantine, a promising drug for the prevention of neuropathic pain in rat

N-methyl-D-aspartate (NMDA) receptor antagonists are used for post-surgery neuropathic pain but severe side-effects limit their clinical use. Memantine, when given after surgery, shows conflicting results as regard to neuropathic pain alleviation. Here memantine is administered in animals before or after spinal nerve ligation (SNL) in order to evaluate the induced antinociceptive/cognitive effects and associated molecular events, including the phosphorylation of several tyrosine (pTyr(1336), pTyr(1472)) and serine (pSer(1303)) residues in the NR2B subunit of the NMDA receptor. Spinal nerve ligated and sham animals received memantine (20mg/kg/day) or vehicle (1ml/kg/day) by intraperitoneal route. Pre-emptive protocol started 4 days before surgery and continued for 2 days post-surgery. In the post-operative protocol, the 7 day-treatment began on the day of surgery. Tests were done before and after surgery. Tactile allodynia, mechanical hyperalgesia and spatial memory were evaluated by von Frey, Randall & Selitto and Y-maze-tests respectively, and molecular events by western-blot analysis. Spinal nerve ligated animals displayed nociception, impaired memory and increased expression of the 3 phosphorylated residues. Post-operative memantine had no beneficial effect. Pre-emptive memantine prevented the development of post-surgical nociception, impairment of spatial memory and did not increase the expression of pTyr(1472)NR2B at spinal, insular and hippocampal levels. Memantine administered a few days before surgery is a promising strategy to alleviate neuropathic pain development and impairment of cognitive function in animals. The pivotal role of pTyr(1472)NR2B must be studied further, and these findings will now be challenged in patients for the prevention of postsurgical neuropathic pain.

Monoarthritis-induced emotional and cognitive impairments in rats are sensitive to low systemic doses or intra-amygdala injections of morphine

Chronic pain is a multidimensional experience that not only includes changes in nociception but also impairments in emotional and cognitive functions, not often taken into account in preclinical research. The present study investigated emotional and cognitive impairments in an animal model of persistent inflammatory pain as well as the involvement of the basolateral complex (BLC) of the amygdala in these components. Monoarthritis was induced by intra-articular injection of complete Freund׳s adjuvant. Mechanical hypersensitivity, anxiety and depressive-like behaviours as well as cognitive capacities were assessed using several tests, such as von Frey, social interaction, open field, saccharin preference, spatial and social recognition memory tests. The effects of morphine administered systemically or into the BLC of the amygdala were also studied. Monoarthritic rats exhibited mechanical hypersensitivity, anxiety and depressive-like behaviours as well as cognitive impairments. Whereas low systemic doses and intra-BLC infusion of morphine failed to reduce mechanical hypersensitivity, they reversed monoarthritis-induced anxiety-like behaviours and cognitive impairments. Our findings further support a crucial role of amygdala in the effect of morphine on emotional/cognitive components of pain and not on mechanical hypersensitivity. Finally, our study highlights the interest of a multi-behavioural approach in the assessment of pain and the analgesic effect of drugs.