Indiana University-Purdue University Indianapolis (IUPUI) / ABSTRACT Yue Wang Neurofibromin, nerve growth factor and Ras: their roles in controlling the excitability of mouse sensory neurons Neurofibromin, the product of the Nf1 gene, is a guanosine triphosphatase activating protein (GAP) for p21ras (Ras) that accelerates the conversion of active Ras-GTP to inactive Ras-GDP. It is likely that sensory neurons with reduced levels of neurofibromin have augmented Ras-GTP activity. In a mouse model with a heterozygous mutation of the Nf1 gene (Nf1+/-), the patch-clamp recording technique is used to investigate the role of neurofibromin in controlling the state of neuronal excitability. Sensory neurons isolated from adult Nf1+/- mice generate more APs in response to a ramp of depolarizing current compared to Nf1+/+ mice. In order to elucidate whether the activation of Ras underlies this augmented excitability, sensory neurons are exposed to nerve growth factor (NGF) that activates Ras. In Nf1+/+ neurons, exposure to NGF increases the production of APs. To examine whether activation of Ras contributes to the NGF-induced sensitization in Nf1+/+ neurons, an antibody that neutralizes Ras activity is internally perfused into neurons. The NGF-mediated augmentation of excitability is suppressed by the Ras-blocking antibody in Nf1+/+ neurons, suggesting the NGF-induced sensitization in Nf1+/+ neurons depends on the activation of Ras. Surprisingly, the excitability of Nf1+/- neurons is not altered by the blocking antibody, suggesting that this enhanced excitability may depend on previous activation of downstream effectors of Ras. To determine the mechanism giving rise to augmented excitability of Nf1+/- neurons, isolated membrane currents are examined. Consistent with the enhanced excitability of Nf1+/- neurons, the peak current density of tetrodotoxin-resistant (TTX-R) and TTX-sensitive (TTX-S) sodium currents (INa) are significantly larger than in Nf1+/+ neurons. Although the voltage for half-maximal activation (V0.5) is not different, there is a significant depolarizing shift in the V0.5 for steady-state inactivation of INa in Nf1+/- neurons. In summary, these results demonstrate that the enhanced production of APs in Nf1+/- neurons results from a larger current amplitude and a depolarized voltage dependence of steady-state inactivation of INa that leads to more sodium channels being available for the subsequent firing of APs. My investigation supports the idea that regulation of channels by the Ras cascade is an important determinant of neuronal excitability. Grant D. Nicol, Ph.D, Chair

No description available.

A la suite d'un traumatisme nerveux les neurones périphériques ont la capacité de régénérer. La repousse est possible grâce à l'environnement permissif et les aptitudes intrinsèques des neurones périphériques à entamer un processus régénératif. Cette capacité intrinsèque se traduit par des remaniements cellulaires et moléculaires induits notamment par la modification de l'expression de nombreux gènes. Ma thèse a porté sur l'étude de l'un d'entre eux : CaMKIα (Calcium-Calmodulin-dependent kinase Iα), dont nous avons montré l'induction de l'expression dans les neurones de ganglions rachidiens dorsaux par une lésion du nerf sciatique. Cette kinase, jamais encore décrite dans le système nerveux périphérique adulte, est impliquée dans le développement neuronal au niveau central. Nous avons établi que l'expression de CaMKIα est spécifiquement induite à la suite de différents types de traumatismes mécaniques du nerf sciatique (sections, compressions chroniques ou aiguës) dans une population restreinte de neurones lésés, majoritairement myélinisés. La localisation subcellulaire de CaMKIα, à la fois dans le corps cellulaire des neurones et dans les fibres du nerf sciatique, évoque un transport axonal de la kinase vers le site de lésion. L'inhibition de la voie de signalisation de CaMKIα par traitement pharmacologique ou l'utilisation de siRNA dirigés contre CaMKIα induit in vitro une chute significative de la vitesse de pousse des neurites des neurones lésés. L'ensemble de ces résultats suggère que l'induction de CaMKIα contribue à la régénération axonale post-lésionnelle des neurones périphériques. / Peripheral neurons have the capacity to regenerate after injury. This regeneration is allowed by thefavorable environment generated by the cellular components of the system and intrinsic aptitudes ofthe peripheral neurons to enter this process. These intrinsic abilities are manifested as cellular changes and molecular alterations including transcriptional and post-transcriptional modifications. Prior to my work, our laboratory carried out transcriptomic analysis on dorsal root ganglia after nerve injury. This allowed us to highlight a set of genes induced in response to peripheral nerve lesion. My thesis focused on one of them: CaMKIα (Calcium-Calmodulin-dependent kinase Iα). This kinase, not previously described in the adult peripheral nervous system, has been shown to be involved in central nervous system neuronal development. We have shown that CaMKIα is specifically induced following different kinds of mechanical lesions of the sciatic nerve (sections and acute or chronic crush) in a restricted, predominantly myelinated, population of injured neurons. The subcellular location of CaMKIα, both in the soma and nerve fibers suggest an axonal transit of the kinase to the injury site. The inhibition of the CaMKIα signaling pathway by a pharmacological compound or RNA silencing in vitro induced a significantly decreased velocity of neurite growth in injured neurons. Taken together, these results suggest that the induction of CaMKIα contributes to the post injury axonal regeneration of peripheral neurons.

La douleur est perçue par des neurones spécialisés, les nocicepteurs, dont le corps cellulaire est localisé, au niveau du tronc, dans les ganglions de la racine dorsale (DRG). Ces neurones détectent les informations sensorielles en périphérie (peau, muscles ou viscères) et les transmettent aux neurones spinaux qu'ils connectent au niveau de la corne dorsale de la moelle épinière. D'un point de vue morphologique, anatomique, physiologique, mais également moléculaire, une hétérogénéité importante de ces neurones est observée. Le but principal du laboratoire est de trouver de nouvelles molécules impliquées dans les mécanismes moléculaires qui spécifient les différentes sous-populations neuronales des DRG. Dans ce cadre, il a été possible d'identifier et valider plusieurs gènes présentant un profil d'expression très particulier et spécifiant des populations neuronales bien distinctes au sein des DRG. Parmi ces gènes, tafa4 est principalement exprimé dans des neurones non-peptidergiques de type C. Tafa4 est une petite protéine sécrétée proche des chemokines de type CC dont la fonction est jusqu'à présent inconnue, et dont l'expression dans les DRG n'a encore jamais été décrite. Au cours de ce travail, j'ai pu identifier Tafa4 comme un nouveau marqueur d'une sous-population de neurones sensoriels des DRG : les C-LTMRs (C-Low Threshold MechanoReceptor). La génération d'une lignée de souris Tafa4 KO dans laquelle le gène tafa4 a été remplacé par la protéine fluorescente Vénus, nous a permis de mettre en évidence que la population de neurones tafa4+ projette en central dans la lamina II interne de la moelle épinière et en périphérique exclusivement au niveau de la peau poilue. / The perception of pain is initiated by the detection of noxious stimuli by the peripheral endings of primary nociceptive neurons. They are a specialized group of small-diameter pseudounipolar neurons with cell bodies in the dorsal roots ganglia (DRG). They give rise to thinly myelinated (Ad-fibers) or unmyelinated (C-fibers) afferent fibers, which convey the signal from the periphery to the dorsal horn of the spinal cord. Our laboratory is interested in molecular mechanisms which underlie the specification of somatic sensory neurons and their properties. In order to find novel molecular factors involved in this process, we identified several new nociceptor subtype specific genes by microarray experiments. Among these genes, tafa4 which encodes a small secreted protein distantly related to CC chemokine with unknown function, appears to have a DRG-specific expression from early developmental stages and becomes restricted to a subset of C-fibers non-peptidergic nociceptors in adult DRG. By using transgenic mice, we show that Tafa4 neurons specifically project to the dorsal horn lamina IIi and innervate the hairy skin. They have electrophysiological signature of C-Low-threshold mechanoreceptors (C-LTMRs), a population of sensory neurons implicated in the injury-induced mechanical hyper-sensitivity as well as in the affective component of touch. Mutant mice lacking Tafa4 do not present developmental defects and specify Tafa4 population correctly. However, despite no obvious molecular changes in Tafa4 mutants, these mice display significant increase in tissue injury induced hyper-sensitivity which could be reduced by intrathecally applied Tafa4 protein.

The effect of interleukin-1 beta (IL-1β) on the electrical properties of sensory neurons was assessed at comparable levels and exposure times to those found in animal models of neuropathic pain. Experiments involved whole cell current- or voltage-clamp recordings from rat dorsal root ganglion (DRG) neurons in defined medium, neuron enriched cultures. 5-6 days exposure to 100 pM IL-1β produced neuron specific effects. These included an increase in the excitability of medium diameter and small diameter isolectin B4 (IB4)-positive neurons that was comparable to that found after peripheral nerve injury. By contrast, a reduction in excitability was observed in large diameter neurons, while no effect was found in small diameter IB4-negative neurons. Further characterization of changes in medium and small IB4-positive neurons revealed that some, but not all, effects of IL-1β were mediated through its receptor, IL-1RI. Using appropriate voltage protocols and/or ion substitutions, it was found that neuron specific changes in several ionic currents, including alterations in hyperpolarization activated inward current (IH) and decreases in various K+ currents contribute to the increased excitability produced by IL-1β. Overall, these studies revealed that: 1. The effects of long-term exposure of DRG neurons to IL-1β are reflective of the enduring increase in primary afferent excitability reported after peripheral nerve injury. This expands the recognized role of IL-1β in acute inflammatory pain to neuropathic pain. 2. Hyperexcitability in medium neurons exposed to IL-1β likely includes mixed populations of neurons corresponding to nociceptive and non-nociceptive primary afferent fibres and, therefore, has relevance to hyperalgesia and allodynia, respectively. 3. The responsiveness of small IB4-positive neurons, but not IB4-negative, to prolonged IL-1β exposure is consistent with the suggestion that small IB4-negative afferents are involved in inflammatory pain, while small IB4-positive afferents are involved neuropathic pain. 4. The identification of receptor mediated effects and several contributing ionic mechanisms, may have relevance to the development of new therapeutic approaches to neuropathic pain. 5. IL-1β can contribute to increased neuronal excitability by mechanisms that are independent of IL-1RI signalling. This should be taken into account when targeting IL-1β, or more specifically IL-1RI, in the management of neuropathic pain.

No description available.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Nos gânglios sensitivos, os corpos celulares dos neurônios encontram-se circundados por células gliais denominadas células satélites. Estudos recentes apontam para uma possível comunicação entre neurônios e células satélites através da liberação de ATP e ativação de receptores P2X7 presentes nas células gliais. Além disto, células satélites adjacentes podem estar conectadas através de junções comunicantes (“gap junctions”). Até o presente, a comunicação entre células satélites e neurônios tem sido implicada na cronificação da dor e em processos inflamatórios. Nesse estudo buscamos avaliar o papel da comunicação entre neurônios e células satélites através da ativação dos receptores P2X7 assim como das junções comunicantes em modelos de dor aguda. Em culturas primárias de gânglios da raiz dorsal, verificamos que a administração de capsaicina leva a um aumento de cálcio em neurônios e em seguida em células satélites sendo que a resposta das células satélites foi bloqueada por A740003, um antagonista seletivo para receptores P2X7, indicando que os nociceptores quando ativados liberam ATP que, por sua vez, ativa receptores P2X7 nas células gliais. Para avaliar o papel desta comunicação celular in vivo, o antagonista P2X7 ou o bloqueador de junções comunicantes, carbenoxolona, foram administrados por via intraganglionar (L5) e foram avaliados os efeitos das injeções intraplantares de capsaicina, mentol e formalina em ratos. Tanto o A740003 quanto a carbenoxolona reduziram a nocicepção induzida por capsaicina e mentol. No teste da formalina, ambas as substâncias afetaram apenas a segunda fase do teste, considerada a fase inflamatória. Capsaicina ativa seletivamente receptores TRPV1 e mentol ativa receptores TRPM8, e possivelmente receptores TRPA1, que são expressos majoritariamente em neurônios nociceptivos associados a fibras C. Além disto, estudos de outros autores indicam a primeira fase do teste da formalina envolve principalmente a ativação de fibras do tipo Aδ enquanto que a segunda fase envolve a ativação de fibras Aδ e C. Considerando estes dados juntamente como os presentes resultados, podemos sugerir que a comunicação entre células satélites e neurônios ocorre também na dor aguda, mas apenas quando esta depende da ativação de fibras C. Deste modo, a comunicação entre neurônios e células satélites, via liberação de ATP e ativação de receptores P2X7, assim como uma comunicação entre células satélites adjacentes através de junções comunicantes parecem estar envolvidos em um processamento rápido do sinal doloroso no gânglio da raiz dorsal. / In sensory ganglia, the cellular bodies of neurons are surrounded by glial cells called satellite cells. Recent studies point to a possible communication between neurons and satellite cells through the release of ATP and activation of P2X7 receptors present in glial cells. In addition, adjacent satellite cells may be connected through gap junctions. Still today, the communication between satellite cells and neurons has been implicated in chronic pain and in inflammatory processes. In this study we sought to evaluate the role of communication between neurons and satellite cells through the activation of the P2X7 receptors as well as of the communicating junctions in acute pain models. In primary cultures of dorsal root ganglia, we found that the administration of capsaicin leads to an increase of calcium in neurons and then in satellite cells. The response of satellite cells was blocked by A740003, a selective antagonist for P2X7 receptors, indicating that nociceptors when activated release ATP, which in turn activates P2X7 receptors in the glial cells. To assess the role of this in vivo cellular communication, the P2X7 antagonist or the gap junction blocker, carbenoxolone, were administered by intraganglionar injection (L5) and the effects of intraplantar injections of capsaicin, menthol or formalin in rats were evaluated. Both A740003 and carbenoxolone reduced nociception induced by capsaicin and menthol. In the formalin test, both substances affected only the second phase of the test, considered the inflammatory phase. Capsaicin selectively activates TRPV1 receptors while menthol activates TRPM8 receptors, and possibly TRPA1 receptors, which are expressed mainly in nociceptive neurons associated with C fibers. In addition, studies by other authors indicate that the first phase of the formalin test involves primarily the activation of Aδ fibers whereas the second phase involves the activation of Aδ and C fibers. Considering these data together with the present results, we can suggest that the communication between satellite cells and neurons also occurs in acute pain, but only, when it depends on the activation of C fibers. Thus, communication between neurons and satellite cells, via release of ATP and activation of P2X7 receptors, as well as communication between adjacent satellite cells through gap junctions seems to be involved in a rapid processing of the pain signal in the dorsal root ganglion. / Dissertação (Mestrado)

Orientador: Alexandre Leite Rodrigues de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T12:05:48Z (GMT). No. of bitstreams: 1 Benitez_SuzanaUlian_M.pdf: 7544701 bytes, checksum: 2e552c7219f0c635a4b0afecffd3204d (MD5) Previous issue date: 2013 / Resumo: Lesões nas raízes dorsais da medula espinal são frequentes e muitas vezes decorrentes de acidentes automobilísticos. Devido à possibilidade de geração de dor neuropática, os procedimentos cirúrgicos não priorizam o reparo do componente aferente, sendo reparado apenas o componente motor. Adicionalmente, a perda das informações sensoriais gera parestesia ou anestesia do membro lesado, bem como descoordenação motora. Nesse contexto, novas terapias precisam ser desenvolvidas para o reparo das raízes dorsais. Uma substância capaz de conectar tecidos por adesão e que promova a hemostase e estabilidade do tecido, como o selante de fibrina (SF), pode ser uma alternativa a ser empregada no reparo de raízes lesadas. Além disso, o emprego conjunto do SF com células-tronco mononuclares de medula óssea (CTMMO) pode potencializar uma eventual regeneração tecidual. Assim, o presente estudo avaliou a resposta glial, a reorganização sináptica, a morfologia das fibras sensoriais e a coordenação motora após reparo com SF e terapia celular. Para isso, foram empregados ratos Lewis fêmeas (6-8 semanas), sendo divididos em três grupos: rizotomia (RZ, n=20), rizotomia reparada com SF (RZ+SF; n=22) e rizotomia reparada com SF e CTMMO (RZ+SF+CT; n=20). O tempo de sobrevida pós-cirúrgico foi de até 8 semanas. Para imunoistoquímica, foram utilizados anticorpos anti-VGLUT1 (terminais pré-sinápticos glutamatérgicos), GAD65 (terminais pré-sinápticos gabaérgicos), sinaptofisina (terminais sinápticos), GFAP (astrócitos), Iba1 (microglia) e BDNF (fator neurotrófico). Além disso, foram realizados citoquímica com Sudan black (coloração para lipídeos) e os testes comportamentais von-Frey eletrônico e walking track test (sistema CatWalk). Os resultados demonstraram regeneração das aferências nos grupos RZ+SF e RZ+SF+CT. Porém, apenas no segundo grupo, houve crescimento axonal até lâminas mais profundas da medula espinal, o que resultou em melhor desempenho nos testes comportamentais. Concluímos que o reimplante de raízes sensitivas com SF e CTMMO pode ser uma alternativa terapêutica para o reparo de lesões dorsais na interface do SNC e SNP / Abstract: Dorsal root lesions are common and often occur in automobile accidents. Due to the possibility of generating neuropathic pain, surgical procedures do not prioritize the repair of the afferent component, focusing on the motor output instead. Moreover, the loss of sensory inputs triggers paresthesis or anesthesia of the injured limb, and motor impairments. In this context, new therapies have to be developed for dorsal root repair. A substance that can promote tissue adhesion and stability and tissue haemostasis, such as fibrin sealant (FS), could be an alternative for the repair of damaged roots. Furthermore, the combined use of FS plus bone marrow mononuclear stem cells (BMSC) may enhance tissue regeneration. Thus, the present study evaluated the glial response, synaptic changes, the cytoarchitecture of the sensory fibers and motor coordination with FS with or without cell therapy for root replantation. Female Lewis rats (6-8 weeks old) were divided into three groups: rhizotomy (RZ, n = 20), rhizotomy repaired with FS (RZ+FS, n = 22) and rhizotomy repaired with SF and BMSC (RZ+FS+SC, n = 20). The survival time after surgery was up to 8 weeks. For immunohistochemistry VGLUT 1 (presynaptic glutamatergic terminals), GAD65 (GABAergic presynaptic terminals), synaptophysin (synaptic terminals), GFAP (astrocytes), Iba1 (microglia) and BDNF (neurotrophic factor) antibodies were used. Also, cytochemistry with Sudan black (lipid staining) and the behavioral tests electronic von-Frey and Walking track test (CatWalk system) were carried out. The results showed regeneration of afferent inputs in groups RZ+FS and RZ+FS+SC. However, only in the group with BMSC, the axonal growth was able to reach deeper laminae of the spinal cord, resulting in a better performance in behavioral tests. We conclude that the sensory root replantation with FS and BMSC may be an alternative therapy for the repair of dorsal root injuries in the CNS and PNS interface / Mestrado / Biologia Celular / Mestra em Biologia Celular e Estrutural

Orientador: Carlos Amilcar Parada / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T20:52:22Z (GMT). No. of bitstreams: 1 Araldi_Dioneia_D.pdf: 2812057 bytes, checksum: ba0136e00ebc1ab271d4e1ed908bcd8c (MD5) Previous issue date: 2012 / Resumo: A liberação de Interleucina-1? (IL-1?) no tecido periférico estimula a síntese de Prostaglandinas (PGs), especialmente, da Prostaglandina-E2 (PGE2), que leva a sensibilização dos nociceptores aferentes primários induzindo a hiperalgesia inflamatória. Recentemente demonstramos que a IL-1? pode ativar diretamente o receptor de Interleucina-1 (IL-1R) do nociceptor aferente periférico e levar a liberação de PGE2 associada ao desenvolvimento da hiperalgesia. A IL-1? também é liberada no Gânglio da Raiz Dorsal (GRD), entretanto a função que a IL-1? desempenha no GRD para o desenvolvimento da hiperalgesia inflamatória ainda não está clara. Portanto, o objetivo deste estudo foi investigar se a liberação de IL-1? e a ativação do Receptor de Interleucina-1 Tipo I (IL-1RI) no GRD estão envolvidos no desenvolvimento da hiperalgesia inflamatória. A administração de IL-1Ra (antagonista natural de receptor IL- 1, 6 ?g) no GRD de ratos preveniu a hiperalgesia mecânica (avaliada por meio do von Frey Eletrônico) induzida pela administração intraplantar (i.pl) de Adjuvante Completo de Freund (CFA, 100 ?L), Carragenina (Cg, 100 ?g) ou IL-1? (0,5 pg), mas não pela administração i.pl de PGE2 (100 ng), avaliadas 3 horas após suas administrações. Além disso, a administração i.pl periférica de CFA ou Cg aumentaram as concentrações de IL-1? (avaliadas por ELISA) no GRD. O tratamento ganglionar (GRD-L5) com oligonucleotídeo (ODN) antisense contra IL-1RI (30 ?g/dia durante 4 dias) reduziu de maneira significativa a expressão de IL-1RI no GRD-L5 e a hiperalgesia mecânica induzida por CFA, Cg e IL-1?, mas não pela PGE2, administradas no tecido periférico da pata. Também verificamos a hipótese de que a prévia ativação do receptor neuronal, IL-1RI, no tecido periférico é importante para a liberação de IL-1? no GRD e para a subsequente hiperalgesia induzida por PGE2. A IL-1? (0,5 pg/pata) co-administrada com a dose sub-limiar de PGE2 (10 ng/pata) em patas traseiras tratadas com indometacina induziu uma proeminente hiperalgesia, que foi prevenida pelo prétratamento com ODN antisense contra IL-1RI ou IL-1Ra (6 ?g) administrados no GRD. Além disso, o IL-1Ra reduziu a expressão de COX-2 em células do GRD. Para confirmar a ativação do IL-1RI em células do GRD, administramos Cg ou CFA no tecido periférico o que levou ao aumentou da expressão de IRAK-1 e IRAK-4 em células do GRD. Os resultados deste estudo sugerem que o desenvolvimento da hiperalgesia inflamatória depende da ativação do receptor IL-1RI neuronal no tecido periférico que, em partes, induz a liberação de IL-1? no GRD e subsequente ativação da COX-2. Os dados aqui apresentados oferecem novas perpectivas sobre a participação das células do GRD nos mecanismos envolvidos na hiperalgesia inflamatória e revelam novos e interessantes alvos para o controle das hiperalgesias inflamatórias / Abstract: The release of Interleukin-1? (IL-1?) in the peripheral tissue stimulates the synthesis of Prostaglandins, specially, Prostaglandin-E2 (PGE2) that ultimately sensitize the peripheral afferent nociceptor inducing inflammatory hyperalgesia. We have recently demonstrated that IL-1? can directly activate IL-1R receptor of peripheral afferent nociceptor to induce release of PGE2 associated to development of hyperalgesia. IL-1? is also released in Dorsal Root Ganglion (DRG), however the role that IL-1? in DRG plays to development of inflammatory hyperalgesia is not yet elucidated. Therefore, the aim of this study was to investigate whether IL-1? released in the DRG and the activation of Interleukin-1 Receptor Type I (IL-1RI) is involved in the development of the inflammatory hyperalgesia. Administration of IL-1Ra (IL-1 receptor antagonist, 6 ?g) in the DRG of rats prevented the mechanical hyperalgesia (measured with Electronic von Frey) induced by intraplantar (i.pl) administration of Complete Freund's Adjuvant (CFA, 100 ?L), Carrageenan (Cg, 100 ?g) or IL- 1? (0.5 pg), but not by PGE2 (100 ng), measured 3 hours after their administrations. Also, peripheral i.pl administration of CFA or Cg induced an increase in IL-1? concentrations (measured by ELISA) in the DRG. Ganglionar (DRG-L5) treatment with oligonucleotides (ODN) antisense against IL-1RI (30 ?g/day for four days) reduced the expression of IL-1RI in the DRG-L5 and the mechanical hyperalgesia induced by CFA, Cg, and IL-1?, but not by PGE2 administered in peripheral tissue. We also verified the hypothesis that previous activation of neuronal IL-1RI in the peripheral tissue is important to the release of IL-1? in the DRG and to the subsequent PGE2-induced hyperalgesia. IL-1? (0.5 pg/paw) co-administrated with a sub-threshold dose of PGE2 (10 ng/paw), in hind paws treated with indomethacin, greatly induces hyperalgesia, which was prevented by pre-treatment with ODN antisense against IL-1RI or IL-1Ra (6 ?g) administrated in DRG. Also, IL-1Ra administrations reduced the COX-2 expression in DRG cells. To confirm IL-1RI activation in DRG cells, it was observed that IRAK-1 and IRAK-4 expression was increased in DRG neurons after administration of Cg or CFA in the peripheral tissue. These findings suggest that the development of inflammatory hyperalgesia depends on neuronal IL-RI activation in the peripheral tissue that, in turn, induces the release of IL- 1? in the DRG and subsequent COX-2 activation. These data provide new insights about the participation of DRG cells in the mechanisms underlying inflammatory hyperalgesia and reveal new interesting targets to control inflammatory hyperalgesia / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Des cellules souches dérivées des crêtes neurales ont été trouvées dans divers tissus adultes comme le ganglion de la racine dorsale (GRD). Ce projet de thèse vise à identifier et caractériser la cellule souche de ce tissu. Premièrement, nous avons étudié le potentiel souche de l’ensemble des cellules du GRD. In vitro, certaines sont capables de proliférer pour former des sphères multipotentes qui génèrent des neurones, des glies et des myofibroblastes. In vivo, selon le contexte dans lequel les cellules issues des sphères sont transplantées, elles génèreront différent types cellulaires. Dans le funiculus dorsal démyélinisé de la souris Nude, elles se différencient en cellule de Schwann alors que dans un cerveau de souris nouveau-né Shiverer, elles produisent des péricytes qui s’intègrent aux capillaires sanguins. Bien que le GRD possède une population cellulaire au potentiel souche, son identité et son rôle restent à découvrir. Afin d’identifier cette cellule, nous avons combiné plusieurs techniques et souris transgéniques pour éliminer les diverses cellules candidates. Nous avons découvert plusieurs cellules avec une plasticité intéressante. Deux progéniteurs unipotents ayant la morphologie et la signature moléculaire de péricyte et de fibroblaste de l’endonèvre ont été trouvés dans le nerf sciatique et le GRD adulte. Enfin la cellule souche du GRD correspond de par sa morphologie à une cellule satellite (SGC). Elle prolifère et est bi-potente in vitro. Elle génère, in vivo, des SGC mais également des neurones en condition pathologique. Mieux comprendre ses mécanismes de régulations pourrait ouvrir la voie à de nouvelles stratégies thérapeutiques pour les maladies du SNP. / Neural crest-derived stem cells have been identified in various adult tissues including the dorsal root ganglia (DRG). This thesis project aims to identify and characterize the putative adult DRG stem cell. First, we studied the stemness potential of global DRG cell populations. In vitro, within the adult DRG, some cells were able to form multipotent spheres that gave rise to neurons, glia and myofibroblasts. The graft of the DRG cell forming spheres proved their differentiation plasticity in vivo. Depending upon their graft environment; they generate different cell types. In the demyelinating dorsal funiculus of adult Nude mice, they formed myelinating Schwann cells while in the brain of new born Shiverer mice, they produced pericytes integrated within capillaries. Although, the DRG cells seemed to have an interesting stemness potential, their identity and their physiopathological role remain unknown. In order to characterize this stem cell and study its fate within the DRG, we combined several technics with transgenic mouse lines to exclude the diverse DRG candidate cells. We discovered different cells with interesting plasticity. Two types of unipotent progenitors that have the morphology and molecular characteristics of pericyte and endoneurial fibroblast in the adult sciatic nerve and DRG. But most of all, we found that the DRG stem cell has the phenotype of the satellite glial cell (SGC). They proliferate and are bipotente in vitro. In vivo these stem cells generate SGC under normal condition and produce glia more neurons when necessary in pathological condition. Understanding these regulation mechanisms could open the way to new therapeutic strategies for PNS diseases.