La sensation du toucher permet de détecter des stimuli mécaniques via des neurones mécanosensitifs dont les corps cellulaires sont localisés dans le ganglion rachidien dorsal. Ces neurones projettent vers la peau en périphérie et font leurs synapses avec les interneurones dorsaux de la moelle épinière. L'étude de ces neurones était difficile à cause du manque de marqueurs spécifiques pour ces neurones, jusqu'à la découverte du gène MafA dans le ganglion rachidien dorsal. En effet, mon équipe a montré que MafA est un marqueur spécifique des neurones mécanosensitifs à bas seuil de type Rapidly adapting. Le gène c-Maf, de la même famille que MafA, est aussi exprimé dans ces neurones et il est l'acteur principal de leur développement et de leur fonction. Afin de comprendre comment c-Maf contrôle le développement des neurones somatosensoriels, la première partie de mon travail visait à identifier de nouveaux gènes cibles du facteur de transcription c-Maf et à savoir comment l'expression de ce dernier est régulée dans les neurones du ganglion rachidien dorsal. Concernant la recherche des gènes cibles de c-Maf, j'ai réalisé l'étude de l'expression de gènes candidats dans le contexte de perte de fonction de c-Maf chez la souris. J'ai pu identifier deux cibles : p-cadhérine et mab21/L2, parmi une liste de gènes candidats. J'ai par la suite analysé l'expression de P-cadhérine au cours du développement et j'ai observé qu'elle est exprimée dans la sous-population de neurones sensoriels myélinisés exprimant c-Maf ainsi que dans certains interneurones des laminae III/IV de la moelle épinière. Une expression particulière de P-cadherine est observée dans les cellules des capsules frontières dans les zones d'entrée et de sortie des racines rachidiennes. Suite à ces observations, nous avons émis l'hypothèse suivante : l'expression de la p-cadhérine est régulée par c-Maf dans les neurones sensoriels ainsi que dans les interneurones pour assurer la connexion synaptique entre eux. Concernant, la régulation du gène c-maf, la question reste ouverte. La deuxième partie de mon étude concernait l'analyse du rôle de facteurs de transcription MafA, c-Maf, Runx3 et Er81 dans la plasticité neuronale induite chez la souris adulte après axotomie du nerf sciatique. Ces facteurs sont impliqués dans le développement des neurones somatosensoriels. Cette analyse a montré que l'expression de MafA et Er81 diminue trois jours après axotomie du nerf périphérique, alors que celle de Runx3 et de c-Maf n'est pas affectée. On peut suggérer que chez l'adulte, la régulation de l'expression de MafA et Er81 dépend des facteurs neurotrophiques libérés par les cibles de ces neurones tandis que celle de c-Maf et Runx3 en est indépendante. / The sense of touch relies on the detection of mechanical stimuli by specialized cutaneous mechanosensory neurons whose cell bodies are located in the dorsal root ganglia. These neurons project peripherally to the skin and synapse on target interneurons in the spinal cord. Until the discovery of MafA expression in the dorsal root ganglion, the lack of molecular markers of mechanoreceptor neurons has made it difficult to analyze the development of these neurons. My team showed that MafA is a specific molecular marker for low-threshold mechanoreceptor neurons RAM. C-Maf gene is a member of the Maf family and it is expressed in the MafA sensory neurons. The transcription factor c-Maf controls touch receptor development and function.In order to understand how c-Maf controls somatosensory neurons development, the first objective of my study was to find new targets for c-Maf transcription factor and to know how c-Maf expression is regulated in the dorsal root ganglion. Therefore, I have analysed the expression of different candidate genes in a loss of function context of c-Maf in the mice. I identified two targets: p-cadherin and Mab21/L2 among a list of candidates. Then, I analysed the p-cadherin expression during development and found that this target of c-Maf is expressed in a sub-population of c-Maf sensory neurons and interneurons of the laminae III/IV of the spinal cord. A particular expression of p-cadherin was noticed in the boundary cap cells at the dorsal root entry zone and the motor exit point of the spinal cord. These observations let us put the following hypothesis: c-Maf regulates p-cadherin expression in the sensory neurons and the interneurons to enable specific connections between these neurons. No identified factors were found to regulate c-Maf expression. In the second part of my study, I focused my efforts on the analysis of the role of MafA, c-MAf, Runx3 and Er81 transcription factors in neuronal plasticity induced in the adult mice three days after sciatic nerve axotomy. These factors are involved in the development of somatosensory neurons. The analysis showed that MafA and Er81 expression are down-regulated after peripheral nerve axotomy but the c-Maf and Runx3 expression did not change. We suggest that at adult stage the regulation of MafA and Er81 expression depend on neurotrophic factors released by the targets of these neurons but it's not the case for c-Maf and Runx3 expression.

Les pathologies du système somatosensoriel, appelées neuropathies sensitives périphériques, touchent environ 3 millions de personnes en France et causent des déficits sensoriels multiples. Parmi elles, les douleurs neuropathiques post traumatiques sont les plus fréquentes et sont souvent chroniques et résistantes aux traitements actuels. Une lésion nerveuse périphérique induit des réponses cellulaires permettant la survie et la régénération de ces neurones. Les ganglions rachidiens dorsaux (DRG) contiennent une variété de neurones sensitifs qui transmettent les stimuli somatiques. Suite à une blessure du nerf périphérique les neurones sensitifs s'adaptent à un nouvel environnement pour réussir leur élongation axonale. Parmi les mécanismes cellulaires conduisant à une croissance neuritique améliorée, il a été démontré qu'une lésion primaire in vivo du nerf augmente la régénération axonale suite à une deuxième lésion. In vitro, les neurones qui ont été conditionnés par le premier traumatisme montrent une croissance neuronale plus rapide et plus élonguée appelée croissance régénérative. L'élasticité est un paramètre déterminant des propriétés mécaniques de la membrane cellulaire. Elle donne des informations importantes sur la santé et la fonction de la cellule. Le microscope à force atomique (AFM) est devenu de nos jours un outil commun pour l'imagerie à haute résolution de matériaux biologiques puisque les cellules vivantes peuvent être imagées dans leurs conditions physiologiques. En plus du rôle des propriétés élastiques dans le processus de régénération, l'organisation structurale des tissus est en grande partie déterminante du degré et de la direction de croissance et du mouvement cellulaire. Le guidage de la croissance par la modification des surfaces ou « patterning » est possible avec la technique de « microcontact printing » qui permet la conception de circuits de protéines avec des géométries bien définies. Les protéines de la matrice extracellulaire. Dans la première partie de la thèse nous avons mis en évidence les propriétés mécaniques de la membrane de neurones sensitifs issus de DRG de souris adultes suite à une lésion du nerf sciatique gauche. Les neurones sensitifs conditionnés montrent un mode de croissance neuritique plus rapide et plus élonguée, moins de branchements neuritiques et plus de souplesse membranaire des somas et des cônes de croissance. Dans un deuxième volet du travail nous avons réussi à normaliser la pousse régénérative et l'activité électrique des neurones sensitifs et motoneurones spinaux en utilisant le patterning des protéines d'adhésion cellulaire (ECM) dans le but d'imiter la croissance longitudinale in vivo. / Peripheral nerve injuries lead to paralysis, anesthesia and lack of autonomic control of the affected body areas. The trauma results in loss of motor and sensory functions conveyed by the involved nerves. This process is referred to as Wallerian degeneration; it creates a microenviroment in the injury site that favors neurites regrowth. The increased intrinsic growth capacity of injured peripheral neurons is manifested experimentally by the conditioning lesion paradigm. Axotomy of a peripheral neuron previous to the test lesion, ‘‘primes'' the neuron, switches it on to a regenerative state and, thus, it will regenerate faster after receiving the second injury. Mechanical interactions play a key role in many processes associated with neuronal growth and development. Membrane cytoskeleton elasticity is a determining parameter of membrane mechanical properties and provides important information toward the health and function of the cell. For this reason the first objective of this thesis was to understand the conditioning injury effects on both morphology and rheological properties of live sensory neurons cell bodies and growth cones, using particularly the atomic force microscopy, and to correlate this to eventual modifications in the composition of the cytoskeletal proteins. In addition to the role of cell elastic properties and mechanical sensing in the regeneration process, the structural organization of tissues plays a major part in deciding the degree and direction of tissue growth and cell movement. The ability to guide cells and their outgrowth by modifying surfaces is possible with the microcontact printing technique which enables the design of protein pathways with experimentally defined geometries. Therefore, the second objective of the thesis was to modulate the regenerative growth of dorsal root ganglia sensory neurons and spinal motoneurons using cell adhesion proteins in order to physically mimic the in vivo longitudinal axonal growth. We used the extracellular matrix (ECM) proteins, ideal biomolecules for printing as they can guide in vitro the cellular adhesion, differentiation, migration. The patterning allowed us to normalize neurite elongation and electrical activity of sensory neurons before and after conditioning lesion.

Peripheral nerve injury induces a robust regenerative state in sensory neurons that includes elevated expression of injury/regeneration-associated genes. The molecular signal(s) underlying the transition to the regenerating state are largely unknown. Brain-derived neurotrophic factor (BDNF) is the sole identified neurotrophin that is upregulated in sensory neurons following peripheral nerve injury. As members of the neurotrophin family exert a profound influence on the intact phenotype of sensory neurons, I hypothesize that injury-associated alterations in BDNF expression play a similar role in the injured/regenerating response. Antagonizing endogenous BDNF with a function-blocking antibody prevented increases in injury/regeneration-associated gene expression and decreased the growth capabilities of the injured sensory neurons. However, BDNF was not important for maintaining this cell body response in injured neurons. The elevation of BDNF expression in injured sensory neurons either through intrathecal infusion or electrical stimulation was associated with increased injury/regeneration-associated gene expression in a dose dependent manner and the latter corresponded to increased sensory axonal regeneration. Though BDNF was able to induce and enhance the intrinsic cell body response of injured sensory neurons, exogenous BDNF was not sufficient to induce an injury phenotype in intact sensory neurons. Thus, additional signals are likely induced by the injury response. In conclusion, BDNF plays a critical role in inducing the regenerative state in sensory neurons following injury and strategies aimed at elevating levels of BDNF available to the injured sensory neuron during the inductive phase improve the cell body response.

Peripheral nerve injury induces a robust regenerative state in sensory neurons that includes elevated expression of injury/regeneration-associated genes. The molecular signal(s) underlying the transition to the regenerating state are largely unknown. Brain-derived neurotrophic factor (BDNF) is the sole identified neurotrophin that is upregulated in sensory neurons following peripheral nerve injury. As members of the neurotrophin family exert a profound influence on the intact phenotype of sensory neurons, I hypothesize that injury-associated alterations in BDNF expression play a similar role in the injured/regenerating response. Antagonizing endogenous BDNF with a function-blocking antibody prevented increases in injury/regeneration-associated gene expression and decreased the growth capabilities of the injured sensory neurons. However, BDNF was not important for maintaining this cell body response in injured neurons. The elevation of BDNF expression in injured sensory neurons either through intrathecal infusion or electrical stimulation was associated with increased injury/regeneration-associated gene expression in a dose dependent manner and the latter corresponded to increased sensory axonal regeneration. Though BDNF was able to induce and enhance the intrinsic cell body response of injured sensory neurons, exogenous BDNF was not sufficient to induce an injury phenotype in intact sensory neurons. Thus, additional signals are likely induced by the injury response. In conclusion, BDNF plays a critical role in inducing the regenerative state in sensory neurons following injury and strategies aimed at elevating levels of BDNF available to the injured sensory neuron during the inductive phase improve the cell body response.

Luman (CREB3, LZIP) is a basic leucine zipper transcription factor involved in regulation of the unfolded protein response (UPR), dendritic cell maturation, and cell migration. But despite reported expression in primary sensory neurons, little is known about its role in the nervous system. Luman mRNA from rat sensory neurons was amplified and its coding sequence was determined. The rat Luman cDNA contains a full-length open reading frame encoding 387 amino acids, and the recombinant protein generated from this clone activated transcription from UPR elements. Quantitative RT-PCR revealed rat Luman transcripts in a variety of rat tissues with the highest levels in nervous system tissue. In situ hybridization confirmed the findings and demonstrated that the Luman mRNA hybridization signal localizes to neurons and satellite glial cells in dorsal root ganglia (DRG), the cytoplasm of hepatocytes in liver, and the hippocampal pyramidal cell layers in CA1 and CA3 and the granular cell layer of the dentate gyrus. Luman protein localizes with axonal endoplasmic reticulum (ER) components along the axon length within the sciatic nerve and is activated by sciatic nerve injury. Adult sensory axons also contain Luman mRNA which is translated within the axon and transported to the cell body via the importin-mediated retrograde transport system in response to nerve injury. Further, creation of an N-terminal, C-terminal dual fluorescence-tagged Luman adenoviral construct allowed visualization of the cleavage and retrograde translocation of the N-terminal portion of Luman to the nucleus in real time in vivo and in vitro. Neuronal or subcellular axonal knockdown of Luman significantly impaired the intrinsic ability of injury-conditioned, but not naïve, sensory neurons to extend the regeneration-associated elongating form of neurites. Sciatic nerve crush injury also induced activation of the UPR in axotomized DRGs, including genes linked to cholesterol biosynthesis. Knockdown of Luman decreased the activation of UPR and cholesterol biosynthesis, and axotomy-inducted increases in neurite outgrowth, which could be largely rescued with either mild UPR inducer treatment or cholesterol supplementation. Together these findings provide novel insights linking remote injury-associated axonal ER responses to the regenerative growth capacity of adult sensory neurons via axonal activation and synthesis of Luman and reveal a role for the UPR in regulation of axotomy-induced neurite outgrowth that is critically dependent on Luman.

Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 161 pages. Includes Vita. Includes bibliographical references.

A significant component of diffuse brain injury (DBI) is diffuse axonal injury (DAI) which is responsible for the morbidity and mortality associated with this condition. DAI and its experimental counterpart traumatic axonal injury (TAI) result in scattered microscopic pathology characterized by focal impairment of axonal transport leading to progressive swelling and delayed axotomy. DBI-mediated perisomatic axotomy does not result in acute neuronal death suggesting that delayed axotomy was responsible for this unanticipated response. To evaluate this hypothesis, we examined the spatiotemporal progression of DBI-mediated perisomatic TAI. LM / TEM identified impaired axonal transport within 15 - 30 min post-injury. Perisomatic TAI revealed somata and related proximal / distal axonal segments with normal ultrastructural detail continuous with axonal swellings. In other cases, axotomy was confirmed by loss of axonal continuity distal to the swelling. By 60 - 180 min post-injury, somatic, proximal segment, and swelling ultrastructure were comparable to earlier time points although swelling diameter increased. Distal segment ultrastructure revealed the initial stages of Wallerian degeneration. Axotomy sites did not internalize pre-injury administered dextran suggesting pathogenesis independent of altered axolemmal permeability. Given the rapidity of perisomatic axotomy, absence of axolemmal permeability may constitute the more significant finding in terms of somatic protection.DBI-mediated neuroinflammatory reactions were then examined to see if this non-lethal neuronal pathology evoked responses comparable to those following focal injury. Microglia / macrophage responses within diffusely injured loci uncomplicated by focal pathology were explored using LM, TEM, and confocal evaluations as was albumin immunoreactivity to assess injury-induced blood-brain barrier (BBB) alterations. Initially, microglial activation was observed within injured loci while microglia within adjoining regions maintained resting phenotypes. Scattered activated microglia were observed among injured axons though no clear associations were seen. Later, activated microglia contained myelin debris while only limited microglial aggregations were recognized. Macrophages also localized to injured loci with select cells approximating somata of axotomized neurons. Immune cell observations correlated with altered BBB permeability. These data indicated rapid, yet initially uncoordinated, and persistent immune cell reactivity to DBI pathology. Taken together, these responses suggest that histopathological evaluation following DBI may include non-lethal neuronal injury with unique neuroinflammatory findings.

A tubulização tem sido considerada uma alternativa eficaz no reparo de lesões de nervos por permitir a aplicação local de fatores neurotróficos e facilitar estudos experimentais. A compreensão do mecanismo de ação no processo regenerativo é intrinsecamente dependente da identificação dos componentes moleculares ao longo dos processos degenerativo e regenerativo. A presente tese visa o estudo sequencial da identificação do perfil molecular da resposta imune no processo de regeneração de nervo entre duas técnicas microcirúrgicas: a tubulização látero-terminal (TLT) e a tubulização término-terminal (TTT). Ratas Wistar adultas foram submetidas à transecção bilateral do nervo fibular comum. Na técnica TLT: o nervo fibular foi seccionado e retirado 5mm de sua extensão, o coto proximal foi suturado na musculatura e um tubo de silicone vazio (6mm) foi interposto entre a lateral do nervo tibial (doador) e o coto distal do fibular. Na técnica TTT: o nervo fibular foi seccionado e retirado 5mm de sua extensão, e ambos os cotos foram suturados às extremidades do tubo de silicone vazio. Foram realizados os grupos experimentais: TLT convencional (TLT padrão), TLT com tubo fechado suturado a porção proximal (TLT F-P), TLT com tubo fechado suturado na porção distal (TLT FD), TTT convencional (TTT padrão), TTT com tubo fechado suturado ao coto proximal (TTT F-P), TTT com tubo fechado suturado ao coto distal (TTT F-D). O controle normal (grupo GN) foi o segmento de nervo retirado para a interposição do tubo. As análises foram realizadas em três tempos pós-cirurgia: 24 horas, 96 horas e 8 dias. As amostras foram processadas em teste comercial para identificação de anticorpos com intuito de determinar a intensidade de expressão de citocinas, quimiocinas e fatores de crescimento presentes no cone de crescimento dos diferentes grupos. Em 24h a expressão de citocinas foi similar entre os grupos, entretanto, o TLT apresentou maior número de quimiocinas expressas em relação ao TTT. O tempo de 96h foi o período com maior número de elementos que apresentaram aumento da expressão em ambos os grupos TLT e TTT, sendo o grupo TLT a apresentar número maior de quimiocinas e fatores de crescimento expressos. Em 8 dias, houve redução dos níveis de expressão de todos os elementos estudados em ambos os grupos, com exceção do CNTF que apresentou aumento de sua expressão no grupo TTT. Os grupos fechados proximais (TLT F-P e TTT F-P) apresentaram perfis de expressão distintos no tempo de 24h, não houve mudanças dos níveis de expressão de nenhum dos elementos do grupo TLT, sendo que, inversamente, o grupo TTT apresentou aumento da expressão de grande parte das proteínas estudadas. Em ambos os grupos TLT F-P e TTT F-P, os elementos investigados retornaram aos níveis basais de expressão em 96h e 8 dias. Os grupos fechados distais (TLT F-D e TTT F-D) apresentaram expressão mínima ou nenhuma dos elementos estudados. O presente estudo demonstrou que há diferenças quanto ao perfil molecular da resposta imune induzida por lesão de nervo entre as técnicas TLT e TTT. / Nerve tubulization has been considered an effective alternative to repair nerve damages due to allow the local application of growth factors and to facilitate experimental studies. The action mechanisms comprehension in nerve regeneration is intrinsically dependente of the molecular components identification over the degenerative and regenerative processes. The aim of this study was the sequencial analysis of the molecular identification of the imune response in nerve regeneration process between two microsurgical repair techniques: the end-to-side tubulization (EST) and the end-to-end tubulization (ETE). Bilateral transection of the common fibular nerve was done in adults female Wistar rats. EST model: the fibular nerve was sectioned and 5mm from his extension was removed, the proximal stump was sutured in the musculature and an empty silicone tube (6mm) was interposed between the lateral portion of the intact tibial donor nerve and the fibular nerve distal stump. ETE model: the fibular nerve was sectioned and 5mm from his extension was removed, both stumps were sutured to the silicone tube extremities. The experimentals groups done: EST conventional (EST standard), EST with closed tube sutured on the proximal portion (EST C-P), EST with closed tube sutured on the distal portion (EST C-D), ETE conventional (ETE standard), ETE with closed tube sutured on the proximal stump (ETE C-P), ETE with closed tube sutured on the distal stump (ETE C-D). The normal control (NG group) was the nerve extension removed to the tube interposition. The analyzes were performed in three times: 24 hours, 96 hours and 8 days. The samples were precessed on an antibody arrays to determine the expression intensity of cytokines, chemokines ans growth factors in the growth cone of the diferente groups. On 24h the cytokine expression was similar between groups, however, the ETE group presented a higher number of the expressed chemokines than ETS. The time 96h was the period with higher number of elements that presented increased expression in both groups EST and ETE, being the group ETS to presents higher number of the chemokines and growth factors expressed. In 8 days there was expression levels reduced of the all studied elements in both groups, with the exception of CNTF that presented increased expression on ETE group. The proximals closed groups (ETS C-P and ETE CP) presented different expression profiles on the time of 24h, there were no changes in the expression levels of any of the elements of the ETS group, whereas, conversely, the ETE group showed increased expression of a large part of the proteins studied. In both groups ETS C-P and ETE C-P, the investigated elements returned to baseline levels of expression at the 96h and 8 days dates. The distal closed groups (ETS C-D and ETE CD) presented minimal expression or no expression of the studied elements. This study demonstrates that there are differences referring to the molecular profile of the immune response induced by nerve damage between the ETS and ETE models.

A tubulização tem sido considerada uma alternativa eficaz no reparo de lesões de nervos por permitir a aplicação local de fatores neurotróficos e facilitar estudos experimentais. A compreensão do mecanismo de ação no processo regenerativo é intrinsecamente dependente da identificação dos componentes moleculares ao longo dos processos degenerativo e regenerativo. A presente tese visa o estudo sequencial da identificação do perfil molecular da resposta imune no processo de regeneração de nervo entre duas técnicas microcirúrgicas: a tubulização látero-terminal (TLT) e a tubulização término-terminal (TTT). Ratas Wistar adultas foram submetidas à transecção bilateral do nervo fibular comum. Na técnica TLT: o nervo fibular foi seccionado e retirado 5mm de sua extensão, o coto proximal foi suturado na musculatura e um tubo de silicone vazio (6mm) foi interposto entre a lateral do nervo tibial (doador) e o coto distal do fibular. Na técnica TTT: o nervo fibular foi seccionado e retirado 5mm de sua extensão, e ambos os cotos foram suturados às extremidades do tubo de silicone vazio. Foram realizados os grupos experimentais: TLT convencional (TLT padrão), TLT com tubo fechado suturado a porção proximal (TLT F-P), TLT com tubo fechado suturado na porção distal (TLT FD), TTT convencional (TTT padrão), TTT com tubo fechado suturado ao coto proximal (TTT F-P), TTT com tubo fechado suturado ao coto distal (TTT F-D). O controle normal (grupo GN) foi o segmento de nervo retirado para a interposição do tubo. As análises foram realizadas em três tempos pós-cirurgia: 24 horas, 96 horas e 8 dias. As amostras foram processadas em teste comercial para identificação de anticorpos com intuito de determinar a intensidade de expressão de citocinas, quimiocinas e fatores de crescimento presentes no cone de crescimento dos diferentes grupos. Em 24h a expressão de citocinas foi similar entre os grupos, entretanto, o TLT apresentou maior número de quimiocinas expressas em relação ao TTT. O tempo de 96h foi o período com maior número de elementos que apresentaram aumento da expressão em ambos os grupos TLT e TTT, sendo o grupo TLT a apresentar número maior de quimiocinas e fatores de crescimento expressos. Em 8 dias, houve redução dos níveis de expressão de todos os elementos estudados em ambos os grupos, com exceção do CNTF que apresentou aumento de sua expressão no grupo TTT. Os grupos fechados proximais (TLT F-P e TTT F-P) apresentaram perfis de expressão distintos no tempo de 24h, não houve mudanças dos níveis de expressão de nenhum dos elementos do grupo TLT, sendo que, inversamente, o grupo TTT apresentou aumento da expressão de grande parte das proteínas estudadas. Em ambos os grupos TLT F-P e TTT F-P, os elementos investigados retornaram aos níveis basais de expressão em 96h e 8 dias. Os grupos fechados distais (TLT F-D e TTT F-D) apresentaram expressão mínima ou nenhuma dos elementos estudados. O presente estudo demonstrou que há diferenças quanto ao perfil molecular da resposta imune induzida por lesão de nervo entre as técnicas TLT e TTT. / Nerve tubulization has been considered an effective alternative to repair nerve damages due to allow the local application of growth factors and to facilitate experimental studies. The action mechanisms comprehension in nerve regeneration is intrinsically dependente of the molecular components identification over the degenerative and regenerative processes. The aim of this study was the sequencial analysis of the molecular identification of the imune response in nerve regeneration process between two microsurgical repair techniques: the end-to-side tubulization (EST) and the end-to-end tubulization (ETE). Bilateral transection of the common fibular nerve was done in adults female Wistar rats. EST model: the fibular nerve was sectioned and 5mm from his extension was removed, the proximal stump was sutured in the musculature and an empty silicone tube (6mm) was interposed between the lateral portion of the intact tibial donor nerve and the fibular nerve distal stump. ETE model: the fibular nerve was sectioned and 5mm from his extension was removed, both stumps were sutured to the silicone tube extremities. The experimentals groups done: EST conventional (EST standard), EST with closed tube sutured on the proximal portion (EST C-P), EST with closed tube sutured on the distal portion (EST C-D), ETE conventional (ETE standard), ETE with closed tube sutured on the proximal stump (ETE C-P), ETE with closed tube sutured on the distal stump (ETE C-D). The normal control (NG group) was the nerve extension removed to the tube interposition. The analyzes were performed in three times: 24 hours, 96 hours and 8 days. The samples were precessed on an antibody arrays to determine the expression intensity of cytokines, chemokines ans growth factors in the growth cone of the diferente groups. On 24h the cytokine expression was similar between groups, however, the ETE group presented a higher number of the expressed chemokines than ETS. The time 96h was the period with higher number of elements that presented increased expression in both groups EST and ETE, being the group ETS to presents higher number of the chemokines and growth factors expressed. In 8 days there was expression levels reduced of the all studied elements in both groups, with the exception of CNTF that presented increased expression on ETE group. The proximals closed groups (ETS C-P and ETE CP) presented different expression profiles on the time of 24h, there were no changes in the expression levels of any of the elements of the ETS group, whereas, conversely, the ETE group showed increased expression of a large part of the proteins studied. In both groups ETS C-P and ETE C-P, the investigated elements returned to baseline levels of expression at the 96h and 8 days dates. The distal closed groups (ETS C-D and ETE CD) presented minimal expression or no expression of the studied elements. This study demonstrates that there are differences referring to the molecular profile of the immune response induced by nerve damage between the ETS and ETE models.

La netrine-1 joue un rôle important en tant qu’un élément de guidance pour la croissance axonale au début du développement du système nerveux central. Des études récentes ont démontré une expression de la netrin-1 dans le cerveau antérieur adulte, où elle régule la fonction synaptique et la plasticité dans les neurones corticaux. Cependant, la contribution de la netrine-1 dans la rétine adulte reste encore inconnue. Le but de cette étude est donc de caractériser l'expression de la netrine-1 dans la rétine des souris adultes sauvages (rétine intacte) et malades (rétine blessée). L'expression rétinéene de la netrine-1 et de son récepteur, supprimée dans le cancer colorectal (DCC), a été déterminée, à partir des immunobuvardages, chez des souris post-natales de jour 0 (P0), P14 et adultes. Le recours au double marquage de la netrine-1 avec un anticorps spécifique contre RBPMS, un marqueur sélectif pour les cellules ganglionnaires de la rétine (RGC), a permis l’identification de sa localisation sur les sections transversales de rétine. De plus, les niveaux de netrin-1 ont également été quantifiés à trois et sept jours après l'axotomie du nerf optique. Nous avons démontré que la netrine-1 et DCC sont fortement exprimés dans la rétine à P0, toutefois l’expression de netrin-1 est relativement stable pendant le développent alors que l’expression de DCC est remarquablement réduite à l'âge adulte. De plus, ces expériences ont conclu une expression robuste de la netrin-1 dans le soma RGC adulte et, une expression des récepteurs DCC autour du corps cellulaire. Fait important, nous avons aussi pu démontrer que les niveaux d’expressions de la netrine-1 et DCC sont réduits à trois et sept jours suivant l'axotomie du nerf optique. Cependant, la surexpression de la protéine netrin-1 n'a pas eu un effet significatif sur la survie du RGC par rapport aux témoins injectés par un véhicule. Les résultats obtenus suggèrent que : (i) la netrine-1 est abondamment exprimée dans la rétine néonatale et subit une diminution importante à l'âge adulte, (ii) la netrine-1 et son récepteur DCC sont présents dans les RGC et, (iii) l'expression de la netrine-1 diminue considérablement suite à une lésion axonale. Ensemble, ces résultats suggèrent un rôle pour la netrin-1 dans le système visuel chez les adultes. / Netrin-1 plays a highly-conserved role as a guidance cue directing axonal growth during the early stages of central nervous system development. Recent data has shown that netrin-1 is continued to be expressed in the adult forebrain where it regulates synaptic function and plasticity in cortical neurons. However, the contribution of netrin-1 in the adult retina remains unknown. The purpose of this study was to characterize the expression of netrin-1 in the intact and injured adult mouse retina. The retinal expression of netrin-1 and its receptor, deleted in colorectal cancer (DCC), was examined at postnatal day 0 (P0), P14, and adult mice using western blots. Double labeling of netrin-1 with an antibody against RBPMS, a selective marker for retinal ganglion cells (RGCs), was used to determine its location on retinal cross-sections. Netrin-1 levels were also quantified at 3 and 7 days after optic nerve axotomy. We demonstrate that netrin-1 and DCC are abundantly and widely expressed in the retina at P0, but although the expression level of netrin-1 remains relatively stable during development, the expression level of DCC is markedly downregulated in adulthood. Adult RGC soma were shown to be endowed with robust netrin-1 expression. DCC receptors were also found to be expressed around the cell body. Importantly, we show that netrin-1 and DCC levels are further downregulated at 3 and 7 days after optic nerve axotomy. However, the over-expression of netrin-1 protein failed to exert any significant effect on RGC survival in comparison to vehicle-injected controls. Our data support that: 1) netrin-1 is abundantly expressed in the neonatal retina and undergoes marked downregulation in adulthood, 2) netrin-1 and DCC are present in RGCs, 3) netrin-1 expression decreases rapidly after axonal injury. Together, these results suggest a role for netrin-1 in the mature visual system.