CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A riparina II, uma alcamida isolada do fruto nÃo maduro de Aniba riparia, foi avaliada em modelos animais para avaliaÃÃo de drogas com atividade antinociceptiva e antiinflamatÃria, tais como contorÃÃes abdominais induzidas por Ãcido acÃtico; teste da formalina; placa quente; teste da nocicepÃÃo induzida por capsaicina; teste da nocicepÃÃo induzida por glutamato; edemas de pata induzido por carragenina, dextrano, histamina e serotonina e peritonite induzida por carragenina e fMLP. Foram utilizados camundongos Swiss (20-30g) ou ratos Wistar (150-250g). A riparina II foi administrada de forma aguda em todos os testes, nas doses de 25 e 50 mg/kg, por via oral. Os resultados mostraram que a riparina II demonstrou possuir atividade antinociceptiva no modelo de nocicepÃÃo visceral induzida por Ãcido acÃtico. O prà tratamento com a riparina II reduziu significativamente a nocicepÃÃo inflamatÃria induzida pela segunda fase da formalina, mas nÃo alterou a nocicepÃÃo neurogÃnica induzida pela primeira fase do teste da formalina. TambÃm reduziu a nocicepÃÃo induzida pela administraÃÃo intraplantar de glutamato e capsaicina. A riparina II nÃo demonstrou atividade no modelo de nocicepÃÃo tÃrmica da placa quente. Nos modelos de edema de pata induzidos por carragenina e dextrano, os animais tratados com a riparina II exibiram edemas menores em comparaÃÃo com os animais tratados apenas com o veÃculo. Riparina II foi capaz de reduzir os edemas de pata induzidos por histamina e bradicinina, mas nÃo o edemasinduzido por serotonina. No modelo de peritonite induzida por carragenina (agente quimiotÃtico indireto) e por fMLP (agente quimiotÃtico direto), a riparina II foi capaz de reduzir o influxo de leucÃcitos, a atividade da enzima mieloperoxidase e os nÃveis de TNF-α e IL-1β no fluido peritoneal. Em sÃntese, esses resultados indicam que a riparina II possui atividade antinociceptiva e antiinflamatÃria em modelos quÃmicos de dor e inflamaÃÃo. AlÃm disso, a riparina II pode estar inibindo direta ou indiretamente a aÃÃo, produÃÃo ou liberaÃÃo de mediadores prà inflamatÃrios envolvidos na geraÃÃo da dor e da inflamaÃÃo. A aÃÃo antiinflamatÃria foi confirmada pelos achados histopatolÃgicos, com a reduÃÃo do edema e infiltrado inflamatÃrio. / Riparin II, an alcamide isolated from the green fruit of Aniba riparia, was tested in animal models, such as acetic acid-induced abdominal writhing, formalin test, hot plate test, capsaicin and glutamate-induced nociception, paw edema induced by carrageenan, dextran, histamine and serotonin and peritonitis induced by carrageenan and fMLP, in order to evaluate its antinociceptive and antiinflamatory activities. Male Swiss mice (20-30g) and male Wistar rats (150-250g) were treated by gavage with riparin II at the doses of 25 and 50 mg/kg. The results show that riparin II demonstrated an antiinflammatory activity at the animal model of acetic acid-induced abdominal writhing. The pretreatment with riparin II reduced significantly the nociception induced by glutamate and capsaicin, and also the inflammatory nociception induced at the second phase of the formalin test, but was not able to reduce the neurogenic nociception induced at the first phase of the test. Riparin II did not show any activity at the hot plate test. At the animal models of paw edema induced by carrageenan and dextran, the animals treated with riparin II showed lower edema when compared to the control group. The treatment with Riparin II was able to reduce the paw edemas induced by histamine and bradykinin, but not the edema induced by serotonin. At the model of peritonitis induced by carrageenan (indirect chemotactic agent) and by fMLP (direct chemotactic agent), the treatment with riparin II reduced the influx of leukocytes, the activity of the enzyme myeloperoxidase and also the levels of TNF-α and IL-1β in the peritoneal fluid. In summary, these results indicate that riparin II has an antinociceptive and antiinflammatory activity in chemical models of nociception and inflammation. Besides that, riparin II might be inhibiting directly or indirectly the activity, production or releasing of pro-inflammatory mediators involved at the generation of pain and inflammation. The antiinflammatory activity was confirmed through the histopathological findings, with the reduction of the edema and the inflammatory infiltrate.

Neuropathic pain represents a possible outcome of neural tissue injury; it occurs also as a concomitant symptom of different diseases or as a side effect of several treatments. Up to date, it constitutes a great challenge in clinical practice, as currently available treatments are still unsatisfactory. Mechanism-based treatment approaches are promising strategy in neuropathic pain management. However, there is still a lack of information about the exact mechanisms involved in the development and/or maintenance of neuropathic pain. This Doctoral Thesis is aimed to explore the mechanisms underlying the development of neuropathic pain states in different models. The principal part of this work is focused on the study of anti-inflammatory effect of Angiotensin II receptor type 1 (AT1R) blocker, losartan, in two different models of peripheral neuropathy: paclitaxel-induced peripheral neuropathy (PIPN) and spinal nerve ligation (SNL). The work also aimed to access the involvement of spinal transient receptor potential vanilloid type 1 (TRPV1) channels in the process of neuronal activation induced by paclitaxel (PAC) and chemokine CCL2 treatment. In order to fulfil the abovementioned aims, behavioral, immunohistochemical and molecular methods were used. For every model of peripheral neuropathy, the...

Antibody coated microprobes, inserted into the spinal cord at the L4-5 level, were used to detect whether endomorphin-2 (Endo2) was released from spinal dorsal horns in anesthetized rats in response to formalin injected into the hindpaw footpads. Saline injections were used as a control and substance P (SP) was measured to verify activation of nociceptive afferent fibers. SP but not Endo2 was released during pre-stimulation periods. Saline injections did not cause the release of either Endo2 or SP from the spinal cord. Formalin injections caused an increase in Fos expression as well as a release of SP, but not Endo2 from the ipsilateral side dorsal horn in L4-5. We conclude that Endo2 does not play a role in mediating the in vivo responses to acute inflammatory nociceptive signals at the spinal level in the anesthetized rat model.

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Les fuseaux de sommeil sont générés par le noyau réticulaire thalamique puis transmis dans la boucle thalamo-corticale durant le sommeil lent. Ils sont considérés comme ayant un rôle protecteur du sommeil en inhibant les entrées sensorielles. L'objectif de notre travail était de tester ce rôle inhibiteur sur les réactions d'éveil et les réponses évoquées par des stimulations nociceptives chez l'homme en menant trois études électrophysiologiques au cours de nuits entières. Les deux premières études ont utilisé des stimuli laser thermo-nociceptifs délivrés pendant ou en dehors de fuseaux. Les réponses cérébrales étaient obtenues par enregistrements de surface chez des sujets sains, ou intracérébraux chez des patients épileptiques. Les résultats n'ont pas montré de différence significative des réactions d'éveil ou des réponses évoquées, que les stimuli aient été délivrés pendant ou en dehors de fuseaux. Ceci était le cas dans l'étude de surface, mais également dans celle en intracérébral dans laquelle les fuseaux étaient détectés dans le thalamus et les réponses analysées dans l'insula, connue pour répondre systématiquement aux stimuli nociceptifs. Dans la troisième étude, afin d'augmenter la quantité de stimuli, des stimulations électriques ont été utilisées à intensité nociceptive. La relation temporelle entre fuseau et traitement sensoriel a ainsi été étudiée avec des enregistrements de surface à haute densité chez des sujets sains. Les réponses évoquées, présentes dans tous les cas, étaient de plus grande amplitude lors des stimuli délivrés autour du début du fuseau. Ainsi, l'effet inhibiteur du fuseau de sommeil ne semble pas s'appliquer au traitement des informations nociceptives et la modulation des réponses corticales selon le moment du fuseau pourrait refléter l'influence de l'onde lente corticale / Sleep spindles are generated by thalamic reticular nuclei and transmitted into the thalamo-cortical network during nonREM sleep. They are commonly thought to have a sleep-protecting role by inhibiting sensory inputs. The aim of our work was to test their inhibitory effect on behavioural and evoked responses to nociceptive inputs in humans by conducing three electrophysiological experiments during a whole night of sleep. The first two experiments used thermo-nociceptive laser stimuli delivered during or apart from sleep spindles. Cerebral responses were obtained with surface recordings in healthy subjects, or intracerebral ones in epileptic patients. Results showed no significant difference in arousal reactions and cortical evoked responses to stimuli delivered during or apart from sleep spindles. This was the case on surface recordings as well as on intracerebral ones in which spindles were detected within the thalamus while responses were analysed in the insula, known to systematically respond to nociceptive stimuli. In the third experiment, in order to increase the rate of stimuli, electrical ones were used at nociceptive intensities. The relationship between spindle activity and sensory processing was then investigated with surface high-density recordings in healthy subjects. Evoked responses were present in any case, but of higher amplitude around the initiation of spindle activity. Thus, the spindles inhibitory effect of sensory processing does not seem to apply to nociceptive inputs and the modulation of cortical responses according to the timing of spindle might reflect the influence of the slow oscillation

Plusieurs variables biologiques, psychologiques, ainsi que des différences culturelles, ont été mises en cause afin d'expliquer la différence de perception de la douleur existante entre les hommes et les femmes. Il est connu que les hormones gonadiques influencent la réponse nociceptive chez l’animal et chez l’humain. Le cerveau a aussi la capacité de synthétiser ses propres "hormones sexuelles", les neurostéroïdes. L'objectif de cette thèse était: 1) évaluer les facteurs physiologiques et psychologiques influençant de perception de la douleur chez les hommes et les femmes, 2) évaluer l'implication des androgènes et du cortisol sur les symptômes cliniques et la perception de la douleur chez des sujets atteints de fibromyalgie et sains, et 3) évaluer l'implication des hormones gonadiques et de leurs métabolites 3α5α-réduits dans la transmission et la modulation de la douleur chez animaux les mâles et les femelles par l'utilisation de modèles de douleur comportementaux. / Several biological, psychological, and cultural differences can explain the difference in pain perception between men and women. It is known that gonadal hormones influence the nociceptive response in animals and humans. The brain also has the ability to synthesize its own "sex hormones", also named neurosteroids. The aims of this thesis were: 1) to assess the physiological and psychological factors influencing the difference in pain perception between men and women, 2) to relate the levels of androgens and cortisol with clinical symptoms and pain perception in healthy volunteers and patients with fibromyalgia, and 3) to evaluate the involvement of gonadal hormones and of their 3α5α-reduced metabolites in the transmission of pain and the effectiveness of descending pain modulation systems (DPMS) in males and females using behavioral pain model in rats and mice.

La douleur est un problème de santé publique majeur qui réduit la qualité de vie des patients et engendre un coût élevé pour la société. Malgré les efforts fournis pour développer de nouveaux analgésiques, les opiacés restent le moyen le plus efficace pour réduire la douleur moyenne à intense. Cependant, leur utilisation prolongée est responsable du développement d’une tolérance à leurs effets analgésiques et d’une hypersensibilité à la douleur (hyperalgésie). Il a été proposé que ces phénomènes pourraient résulter de l'activation d’un système anti-opioïde tel que celui des récepteurs RF-amide, mais leurs mécanismes d’action sont encore mal compris. L'objectif de ce projet a ainsi été d'étudier l'implication des récepteurs RF-amide NPFFR1, NPFFR2 et GPR103a dans le développement de l'hyperalgésie induite par les opiacés. Allant du niveau cellulaire, en s’intéressant à l’expression des ARNm, à la modulation de l’activité neuronale induite par le 26RFa, jusqu’à un niveau plus intégré via une approche in vivo et l’étude des seuils nociceptifs et de la douleur chez la souris ; ce travail a permis d’avoir une vision globale des effets du système RF-amide et principalement de GPR103a. En abordant la question des effets secondaires associés aux traitements chroniques opiacés, ce projet pourrait conduire à l'élaboration de stratégies de traitement de la douleur prometteuses. / Pain is a major health problem that reduces quality of life and imparts high social and economic costs. Despite efforts to develop new analgesics, opiates remain the most effective way to reduce severe pain. However, their prolonged use is associated with the development of analgesic tolerance and hypersensitivity to pain (hyperalgesia). It has been proposed that these phenomena would result from the activation of anti-opioid system like RF-amide receptors system, but their mechanism of action is still poorly understood. The objective of this project was to study the involvement of NPFFR1, NPFFR2 and GPR103a receptors in the development of opioidinduced hyperalgesia. This work gave an overall view of the effects of the RF-amide system and mainly of GPR103a, ranging from the cellular level, to the expression of mRNAs, to the modulation of the 26RFa-induced neuronal activity, up to an integrated level via an in vivo approach and the study of nociceptive thresholds and pain in mice. By addressing the issue of side effects associated with opioid chronic treatments, this project may lead to the development of promising strategies for pain treatment.

Le récepteur opioïde mu est responsable des propriétés analgésiques et addictives puissantes de la morphine et de l’héroïne, mais son mode d’action à l’échelle des circuits neuronaux est mal connu et a été peu étudié par des approches génétiques. Le récepteur mu est largement exprimé dans le système nerveux, essentiellement dans des neurones GABAergiques. Le premier objectif de mon projet a été d’inactiver le gène codant pour le récepteur mu dans les neurones GABAergiques du cerveau antérieur et d’en étudier les conséquences comportementales. Notre étude montre que ces récepteurs ne sont pas impliqués dans l’analgésie et la dépendance physique à la morphine, mais qu’ils sont essentiels à l’effet hyperlocomoteur de l’héroïne. De plus, nos résultats indiquent que ces récepteurs limitent la motivation à consommer de l’héroïne et du chocolat, révélant un rôle entièrement nouveau pour cette population particulière de récepteurs (Manuscrit 1 : Mu opioid receptors in GABAergic forebrain neurons are necessary for heroin hyperlocomotion and reduce motivation for heroin and palatable food). Aussi, cette population de récepteurs mu n’est pas responsable du syndrome autistique décrit chez les souris knockout totales (Manuscrit 2 : Mu opioid receptors in GABAergic forebrain neurons are not involved in autistic-like symptoms). Enfin, nous avons développé un nouveau modèle transgénique visant l’inactivation génétique du récepteur mu dans les neurones glutamatergiques, mais qui n’a pas abouti à un knockout conditionnel détectable. Nous avons aussi initié la création d’une lignée transgénique Cre pour l’inactivation de gènes d’intérêt dans l’amygdale étendue, qui permettra notamment d’étudier le rôle du récepteur mu dans ce microcircuit. / Mu opioid receptors mediate the strong analgesic and addictive properties of morphine and heroin;however mu receptor function at circuit levels is not well understood and has been poorly studied by genetic approaches. These receptors are widely expressed throughout the nervous system, essentially in GABAergic neurons. The first aim of my project was to genetically inactivate the mu receptor gene in GABAergic forebrain neurons and study the behavioral consequences. Our study shows that these mu receptors are not implicated in morphine-induced analgesia and physical dependence, but are essential for locomotor effects of heroin. Moreover, our data show that these receptors inhibit motivation to consume heroin and chocolate, revealing an entirely new role for this particular population of mu receptors (Manuscript 1: Mu opioid receptors in GABAergic forebrain neurons are necessary for heroin hyperlocomotion and reduce motivation for heroin and palatable food). Also, mu receptors expressed in forebrain GABAergic neurons are not responsible for the autistic syndrome described in total mu receptor knockout mice (Manuscript 2: Mu opioid receptors in GABAergic forebrain neurons are not involved in autistic-like symptoms). Finally, we developed a new transgenic model targeting the mu receptor gene in glutamatergic neurons, but receptor deletion was not detectable in conditional mice. We also initiated the creation of a transgenic Cre driver line to knockout genes of interest in the extended amygdala, and this tool will enable us to study mu receptor function within this microcircuit.