F. Viguier

Multiple roles of serotonin in pain control mechanisms--implications of 5-HT₇ and other 5-HT receptor types.

Among monoamine neurotransmitters, serotonin (5-HT) is known to play complex modulatory roles in pain signaling mechanisms since the first reports, about forty years ago, on its essentially pro-nociceptive effects at the periphery and anti-nociceptive effects when injected directly at the spinal cord level. The discovery of multiple 5-HT receptor subtypes allowed possible explanations to this dual action at the periphery versus the central nervous system (CNS) since both excitatory and inhibitory effects can be exerted through 5-HT activation of different 5-HT receptors. However, it also appeared that activation of the same receptor subtype at CNS level might induce variable effects depending on the physiological or pathophysiological status of the animal administered with agonists. In particular, the marked neuroplastic changes induced by nerve lesion, which account for sensitization of pain signaling mechanisms, can contribute to dramatic changes in the effects of a given 5-HT receptor agonist in neuropathic rats versus intact healthy rats. This has notably been observed with 5-HT₇ receptor agonists which exert a pronociceptive action in healthy rats but alleviate hyperalgesia consecutive to nerve lesion in neuropathic animals. Analysis of cellular mechanisms underlying such dual 5-HT actions mediated by a single receptor subtype indicates that the neuronal phenotype which expresses this receptor also plays a key role in determining which modulatory action 5-HT would finally exert on pain signaling mechanisms.

Differential effects of calcitonin gene-related peptide receptor blockade by olcegepant on mechanical allodynia induced by ligation of the infraorbital nerve vs the sciatic nerve in the rat.

Summary Calcitonin gene‐related peptide receptor blockade by olcegepant reduced mechanical allodynia induced by infraorbital nerve ligation but not that caused by sciatic nerve ligation in rats. Abstract Previous studies showed that 5‐hydroxytryptamine (5‐HT)1B/1D receptor stimulation by triptans alleviates neuropathic pain caused by chronic constriction injury to the infraorbital nerve (CCI‐ION) but not the sciatic nerve (CCI‐SN) in rats. To assess whether such differential effects in the cephalic vs extracephalic territories is a property shared by other antimigraine drugs, we used the same models to investigate the effects of olcegepant, which has an antimigraine action mediated through calcitonin gene‐related peptide (CGRP) receptor blockade. Adult male rats underwent unilateral CCI to the ION or the SN, and subsequent allodynia and/or hyperalgesia were assessed in ipsilateral vibrissal territory or hindpaw, respectively, using von Frey filaments and validated nociceptive tests. c‐Fos expression was quantified by immunohistochemistry and interleukin 6 and activating transcription factor 3 (ATF3) mRNAs by real‐time quantitative reverse transcriptase‐polymerase chain reaction. Like naratriptan (0.1 to 0.3 mg/kg, subcutaneously), olcegepant (0.3 to 0.9 mg/kg, intravenously) markedly reduced mechanical allodynia in CCI‐ION rats. In contrast, in CCI‐SN rats, mechanical allodynia was completely unaffected and hyperalgesia was only marginally reduced by these drugs. A supra‐additive antiallodynic effect was observed in CCI‐ION rats treated with olcegepant (0.3 mg/kg intravenously) plus naratriptan (0.1 mg/kg subcutaneously), whereas this drug combination remained inactive in CCI‐SN rats. Olcegepant (0.6 mg/kg, intravenously) significantly reduced the number of c‐Fos immunolabeled cells in spinal nucleus of the trigeminal nerve and upregulation of ATF3 transcript (a marker of neuron injury) but not that of interleukin‐6 in trigeminal ganglion of CCI‐ION rats. These findings suggest that CGRP receptor blockade might be of potential interest to alleviate trigeminal neuropathic pain.

GABA, but not opioids, mediates the anti-hyperalgesic effects of 5-HT7 receptor activation in rats suffering from neuropathic pain

Among receptors mediating serotonin actions in pain control, the 5-HT(7)R is of special interest because it is expressed by primary afferent fibers and intrinsic GABAergic and opioidergic interneurons within the spinal dorsal horn. Herein, we investigated whether GABA and/or opioids contribute to 5-HT(7)R-mediated control of neuropathic pain caused by nerve ligation. Acute administration of 5-HT(7)R agonists (AS-19, MSD-5a, E-55888) was found to markedly reduce mechanical and thermal hyperalgesia in rats with unilateral constriction injury to the sciatic nerve (CCI-SN). In contrast, mechanical hypersensitivity caused by unilateral constriction injury to the infraorbital nerve was essentially unaffected by these ligands. Further characterization of the anti-hyperalgesic effect of 5-HT(7)R activation by the selective agonist E-55888 showed that it was associated with a decrease in IL-1ß mRNA overexpression in ipsilateral L4-L6 dorsal root ganglia and lumbar dorsal horn in CCI-SN rats. In addition, E-55888 diminished CCI-SN-associated increase in c-Fos immunolabeling in superficial laminae of the lumbar dorsal horn and the locus coeruleus, but increased c-Fos immunolabeling in the nucleus tractus solitarius and the parabrachial area in both control and CCI-SN rats. When injected intrathecally (i.t.), bicuculline (3 μg i.t.), but neither phaclofen (5 μg i.t.) nor naloxone (10 μg i.t.), significantly reduced the anti-hyperalgesic effects of 5-HT(7)R activation (E-55888, 10 mg/kg s.c.) in CCI-SN rats. These data support the idea that 5-HT(7)R-mediated inhibitory control of neuropathic pain is underlain by excitation of GABAergic interneurons within the dorsal horn. In addition, 5-HT(7)R activation-induced c-Fos increase in the nucleus tractus solitarius and the parabrachial area suggests that supraspinal mechanisms might also be involved.

Differential anti-neuropathic pain effects of tetrodotoxin in sciatic nerve- versus infraorbital nerve-ligated rats--behavioral, pharmacological and immunohistochemical investigations

Several voltage-gated sodium channels are expressed in primary sensory neurons where they control excitability and participate in the generation and propagation of action potentials. Peripheral nerve injury-induced alterations in both tetrodotoxin (TTX)-sensitive and TTX-resistant sodium channels have been proposed to contribute to neuropathic pain caused by such lesion. We herein investigated whether the blockade of TTX-sensitive channels could reduce pain-related behaviors and evoked c-Fos immunoreactivity in rats with neuropathic pain produced by chronic unilateral constriction injury to either the sciatic nerve or the infraorbital nerve. Acute as well as subchronic administration of TTX (1-6 mug/kg s.c.) was found to suppress for up to 3 h allodynia and hyperalgesia in sciatic nerve-ligated rats. In contrast, TTX was only moderately effective in rats with ligated infraorbital nerve. In sciatic nerve-ligated rats, TTX administration prevented the increased c-Fos immunoreactivity occurring in the dorsal horn of the lumbar cord and some supraspinal areas in response to light mechanical stimulation of the nerve-injured hindpaw. The anti-allodynia/antihyperalgesia caused by TTX in these neuropathic rats was promoted by combined treatment with naloxone (0.5 mg/kg s.c.) but unaffected by the 5-HT(1B) receptor antagonist F11648 (0.5 mg/kg s.c.) and the alpha(2)-adrenergic receptor antagonist idazoxan (0.5 mg/kg i.v.). In contrast, the anti-allodynic and anti-hyperalgesic effects of TTX were significantly attenuated by co-administration of morphine (3 mg/kg s.c.) or the cholecystokinin(2)-receptor antagonist CI-1015 (0.1 mg/kg i.p.). These results indicate that TTX alleviates pain-related behaviors in sciatic nerve-lesioned rats through mechanisms that involve complex interactions with opioidergic systems.

Respective pharmacological features of neuropathic-like pain evoked by intrathecal BDNF versus sciatic nerve ligation in rats

Numerous reported data support the idea that Brain Derived Neurotrophic Factor (BDNF) is critically involved in both depression and comorbid pain. The possible direct effect of BDNF on pain mechanisms was assessed here and compared with behavioral/neurobiological features of neuropathic pain caused by chronic constriction injury to the sciatic nerve (CCI-SN). Sprague-Dawley male rats were either injected intrathecally with BDNF (3.0 ng i.t.) or subjected to unilateral CCI-SN. Their respective responses to anti-hyperalgesic drugs were assessed using the Randall-Selitto test and both immunohistochemical and RT-qPCR approaches were used to investigate molecular/cellular mechanisms underlying hyperalgesia in both models. Long lasting hyperalgesia and allodynia were induced by i.t. BDNF in intact healthy rats like those found after CCI-SN. Acute treatment with the BDNF-TrkB receptor antagonist cyclotraxin B completely prevented i.t. BDNF-induced hyperalgesia and partially reversed this symptom in both BDNF-pretreated and CCI-SN lesioned rats. Acute administration of the anticonvulsant pregabalin, the NMDA receptor antagonist ketamine, the opioid analgesics morphine and tapentadol or the antidepressant agomelatine also transiently reversed hyperalgesia in both i.t. BDNF injected- and CCI-SN lesioned-rats. Marked induction of microglia activation markers (OX42, Iba1, P-p38), proinflammatory cytokine IL-6, NMDA receptor subunit NR2B and BDNF was found in spinal cord and/or dorsal root ganglia of CCI-SN rats. A long lasting spinal BDNF overexpression was also observed in BDNF i.t. rats, indicating an autocrine self-induction, with downstream long lasting TrkB-mediated neuropathic-like pain. Accordingly, TrkB blockade appeared as a relevant approach to alleviate not only i.t. BDNF- but also nerve lesion-evoked neuropathic pain.

253 ANTI-HYPERALGESIC EFFECTS OF 5-HT7 RECEPTOR ACTIVATION IN RATS SUFFERING FROM NEUROPATHIC PAIN: ROLE OF GABAA RECEPTORS

Apart from glycaemia control, many drugs have been developed for the control of diabetes and its complications, but are not satisfactory in neuropathy control and limited by adverse events. Genistein, a soy isoflavone, is therapeutic for obesity, diabetes and cardiovascular diseases. Recently, we showed its timeand doserelated therapeutic effect consisting in relief of neuropathic pain caused by the mouse sciatic nerve chronic constriction injury, a mononeuropathy model. We explored the genistein capacity to attenuate diabetic neuropathic pain after its onset. The isoflavone (3 and 6mg/kg, subcutaneously), once a day for 21 days, starting two weeks after streptozotocin (STZ), relieved mechanical allodynia in STZ-induced diabetic mice, without affecting hyperglycaemia and body weight decrease. Hyperglycaemia leads to free radicals generation, which consequently creates oxidative stress that is an imbalance between radicalic and antioxidant compounds. Tissue antioxidant status is a key factor in determining the deterioration of diabetic stage and is important determinant of degenerative and painful pathological conditions in peripheral nerve fibers. Genistein increased the liver reduced glutathione, restored or increased the activities of some antioxidant enzymes and decreased the ROS and lipid peroxide overproduction in liver and brain. Hyperglycaemia leads to cytokine control deregulation. The TNF-a, IL-1b and IL-6 overproduction in the sciatic nerve but not the TNF-a increase in plasma of diabetic mice were reversed by genistein. These findings suggested genistein ability to reverse diabetic neuropathic pain and simultaneously to provide the protection against both oxidative damage and peripheral nerve inflammation.

242 CGRP‐RECEPTOR BLOCKADE BY BIBN4096BS REDUCES ALLODYNIA, FOS EXPRESSION AND ATF3 UPREGULATION IN A RAT MODEL OF TRIGEMINAL NEUROPATHIC PAIN

blunt pointer. Nevertheless, the animal has to be restrained during the test, thereby generating a stress which can interfere with pain evaluation. We have used a new device developed as an alternative to the Randall and Selitto test. The apparatus consists of a pair of large blunt forceps instrumented with a strain gauge which is placed around the hind paw of the tested animal. The force is incremented by hand until the paw withdrawal response. The device automatically records the force inducing paw-withdrawal. We evaluated the usefulness and reliability of the forceps using four pain models: an acute model of inflammatory pain (carrageenan), the Brennan model of post-operative pain and two neuropathic pain models (Chung and Bennett). In all four models the forceps test demonstrated clear differences between the response of the painful and non-painful paws and showed clear dose-response effects for morphine. Our results demonstrate that the forceps test allows a rapid and sensitive evaluation of the pain threshold in different pain models and that tactile hyperalgesia is significantly reversed by an intraperitoneal injection of morphine.

TO19 Ligands des récepteurs 5-HT7 et manifestations comportementales de douleurs neuropathiques chez le rat

Introduction Les douleurs neuropathiques sont les plus difficiles a soulager, et de nouveaux traitements, plus efficaces et bien toleres, doivent etre developpes. Dans ce contexte, nous avons recherche si le blocage ou la stimulation des recepteurs 5-HT7 de la serotonine pouvait moduler les manifestations d’allodynie/hyperalgesie dans deux modeles valides chez le rat. De fait, ces recepteurs sont situes a des relais strategiques dans les voies de la nociception. Methodes Deux semaines apres la pose unilaterale de ligatures au niveau du nerf infraorbitaire (modele cephalique) ou du nerf sciatique (modele extra-cephalique) chez le rat, les manifestations comportementales d’allodynie/hyperalgesie sont quantifiees a l’aide de tests appropries [filaments de von Frey dans le modele cephalique ; mesure des seuils de retrait de la patte et de vocalisation dans le test de Randall-Selitto applique a la patte au nerf sciatique lese]. Resultats Le blocage des recepteurs 5-HT7 (SB-269970, 3 mg/kg i.p.) exerce au mieux (1h post-injection) un effet antihyperalgesique modere dans le modele extra-cephalique (elevation du seuil de vocalisation compensant de moitie la baisse induite par la ligature du nerf sciatique), mais n’a aucune incidence sur les manifestations de douleurs dans le modele cephalique. En revanche, la stimulation des recepteurs 5-HT7 (AS-19, 2,5-10 mg/kg s.c.) entraine, dans les deux modeles, une diminution dose-dependante de l’allodynie/hyperalgesie. Cet effet est de tres longue duree (5 h dans le modele cephalique et plus de 18 h dans le modele extra-cephalique) et de forte intensite (retour aux seuils initiaux), et peut etre prevenu par l’administration prealable de l’antagoniste specifique SB-269970. Conclusions Les effets de l’agoniste AS-19 pourraient resulter de la mise en jeu d’interneurones inhibiteurs, ou de la desensibilisation rapide des recepteurs 5-HT7. Quoi qu’il en soit, ces resultats laissent a penser que la stimulation de recepteurs 5-HT7 constitue une nouvelle piste pour le traitement des douleurs neuropathiques rebelles.