A Esclerose Lateral Amiotrófica (ELA) é uma doença neurodegenerativa progressiva de evolução rápida, caracterizada pela perda seletiva dos neurônios motores (NM) superiores e inferiores. Recentemente, as células gliais centrais (astrócito, microglia e oligodendrócito) mostraram-se tóxicas aos NM, porém os detalhes moleculares não estão completamente elucidados. Em relação às células gliais periféricas, alterações eletrofisiológicas no nervo ciático do modelo animal da ELA na idade pré-sintomática foram reportadas pelo nosso grupo e os achados de denervação precoce tanto no modelo animal quanto em pacientes sugerem a participação das células de Schwann (CS) na morte neuronal retrógrada na ELA, teoria conhecida como dying back. Nesse contexto, as CS mostraram-se capazes de induzir a retração axonal e a denervação das junções neuromusculares, eventos precoces na doença, ocorrendo possivelmente na fase présintomática. O objetivo deste trabalho foi verificar a influência das CS do modelo experimental na fase pré-sintomática e do paciente com evolução recente da forma esporádica da ELA, na sobrevida e no tamanho dos prolongamentos dos NM in vitro e entender a natureza molecular do fenômeno. Culturas de CS altamente purificadas foram obtidas a partir do nervo ciático do camundongo modelo animal e do nervo periférico de pacientes com ELA. Os NM da medula espinal de camundongos neonatos foram co-cultivados com as CS. A neurodegeneração foi avaliada pela presença do marcador Fluoro-Jade C (FJC). Os NM também foram tratados com o meio condicionado das culturas de CS do modelo animal ou dos pacientes com ELA. Os motoneurônios tiveram os seus prolongamentos contados e a morte neuronal foi identificada pela presença do FJC. Diversos fatores neurotróficos foram quantificados no meio condicionado das culturas de CS pela técnica de ELISA. A reação em cadeia da polimerase quantitativa (do inglês, quantitative polymerase chain reaction - qPCR) foi realizada para detectar alterações nas CS e no nervo periférico que pudessem estar relacionadas com disfunção na unidade CS/NM. Os resultados mostraram que os NM cultivados na ausência das CS mostraram-se mais susceptíveis à morte. Os NM cocultivados com as CS ELA mostraram maior número de perfis neurodegenerativos em comparação com os NM co-cultivados com as CS controle. Após o tratamento com o meio condicionado das CS ELA, os NM mostraram redução no tamanho dos prolongamentos e aumento do número de células em neurodegeneração em comparação com o grupo controle. Quantidades reduzidas dos fatores neurotróficos foram encontradas no meio condicionado das culturas de CS ELA. Alterações na expressão gênica das CS e no nervo periférico evidenciaram disfunções na unidade CS/NM que podem estar contribuindo para o processo neurodegenerativo visto na ELA. Conclui-se que a falência nos mecanismos de neuroproteção pelas CS ELA é um importante mecanismo implicado na morte neuronal, com grande potencial terapêutico / Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective loss of upper and lower motor neurons (MN). Recently, central glia (astrocytes, microglias and olygodendrocytes) were toxic to the MN, but the molecular aspects have not fully described. In relation to the peripheral glia, electrophysiological changes in the sciatic nerve of ALS animal model in the presymptomatic stage have been reported by our group and early denervation findings in both animal models and patients suggests the participation of Schwann cells (SC) in the retrograde neuronal death of ALS , theory known as dying back. In this context, the SC proved to be able to induce axonal retraction and denervation of the neuromuscular junctions, early events in the disease, possibly occurring in the pre-symptomatic phase. The aim of this thesis was to investigate the influence of SC of pre-symptomatic experimental model and from patient with recent evolution of ALS sporadic form, in the survival and axonal length of MN in vitro and understand the molecular nature of the phenomenon. Highly purified SC cultures were obtained from the sciatic nerve of the animal model and from ALS patient\'s peripheral nerve. MN from the newborn mouse spinal cord were co-cultured with SC and the neurodegeneration was assessed by the presence of the marker Fluoro-Jade C (FJC). MN were also treated with conditioned medium from cultures of SC of the animal model or ALS patients. MN had their neuronal length measured and neuronal degeneration was identified by the presence of the FJC. Several neurotrophic factors were measured in conditioned medium of mice and ALS patient\'s SC cultures by ELISA. The chain reaction quantitative polymerase (qPCR) was performed to detect changes in the SC and peripheral nerve that could be related with dysfunction in the functional unit SC/MN. The MN co-cultured with ALS SC showed a greater number of neurodegenerative profiles compared with MN cocultured with control SC. After treatment with ALS SC conditioned medium, MN showed a reduction in the neuronal length and increased number of cells in neurodegeneration compared with the control group. Lower levels of neurotrophic factors were found in the conditioned medium of ALS SC cultures. Changes in the gene expression of SC and peripheral nerve showed dysfunctions in SC/MN unit, which may be contributing to the neurodegenerative process seen in ALS. In conclusion, the failure of neuroprotection by ALS SC is an important mechanism implicated in the MN cell death, with great therapeutic potential

Type 1 diabetes develops through the progressive destruction of the insulin-producing beta-cells. Regeneration or replacement of beta-cells is therefore needed to restore normal glucose homeostasis. Presently, normoglycemia can be achieved by the transplantation of whole pancreas or isolated islets of Langerhans. Islet transplantation can be performed through a simple laparoscopic procedure, but the long-term graft survival is low due to poor revascularization and early cell death. This thesis examined the possibility of using different auxiliary cells (Schwann cells, endothelial progenitor cells, and neural crest stem cells) to improve the engraftment and function of endogenous and transplanted islets. Co-transplantation of Schwann cells with islets improved islet graft function early after transplantation, and caused an increased islet mass at one month posttransplantation. However, the vascular densities of these grafts were decreased, which also related to an impaired graft function. Islet grafts containing endothelial progenitor cells had a superior vascular density, with functional chimeric blood vessels and substantially higher blood perfusion and oxygen tension than control transplants. By culturing and transplanting islets together with neural crest stem cells it was found that islets exposed to these cells had a higher beta-cell proliferation compared with control islets. At one month posttransplantation, the grafts with neural crest stem cells also had a superior vascular- and neural density. The potential of intracardially injected neural crest stem cells to home to the pancreas and ameliorate hyperglycemia in diabetic mice was investigated. During a three-week period after such cell treatment blood glucose concentrations decreased, but were not fully normalized. Neural crest stem cells were present in more than 10% of the pancreatic islets at two days postinjection, at which time the beta-cell proliferation was markedly increased when compared with islets of saline-treated diabetic animals. Three weeks later, a doubled beta-cell mass was observed in animals receiving neural crest stem cells. In summary, islets can easily be transplanted together with different auxiliary cells. Some of these cells provide the possibility of improving vascular- and neural engraftment, as well as beta-cell growth and survival. Systemic administration of neural crest stem cells holds the potential of regenerating the endogenous beta-cells.

Pre regeneráciu a rast poranených nervových vlákien bolo preskúmaných mnoho postupov, no výsledný rast axónov je často náhodný až dezorganizovaný a odráža sa na zložitejšom zotavovaní pacienta. V tejto práci boli vyrobené nové skafoldy s mikroštruktúrnymi a mechanickými vlastnosťami nervového skafoldu pomocou metódy freeze-casting. Konkrétne boli vyrobené biokeramické skafoldy na báze fosforečnanov vápenatých, oxidu titaničitého alebo oxidu zirkoničitého. Pomocou kontrolovaného rastu ľadu v jednom smere bola pripravená orientovaná mikroštruktúra. Pozorovanie pomocou skenovacej elektrónovej mikroskopie potvrdilo lineárne orientované póry (lamelárny systém), v ktorých priemerná veľkosť pórov klesala so zvyšujúcou sa rýchlosťou mrazenia. Skafoldy pripravené pomocou mrazenia v tekutom dusíku vykazovali vynikajúce mechanické vlastnosti, kde pevnosť v ohybe bola získaná v rozmedzí 10–17 MPa. Tie isté skafoldy mali vzdialenosť medzilamelamelárnych priestorov 10–30 µm, ktorých parametre sú vhodné pre nervové skafoldy. Biokompatibilita bola vyhodnotená pomocou Schwannových buniek in vitro, kde bola pozorovaná adhézia a rast v lamelárnom smere. Cytotoxické testy odhalili negatívny vplyv vyššej koncentrácie vápnika na prežitie Schwannových buniek. Pripravené skafoldy mali schopnosť tvorby apatitu na povrchu v podobe embryonálnych a nukleačných centier a apatitu samotného. Skafoldy na báze fosforečnanov vápenatých a oxidu titaničitého vykazovali sľubné regeneračné vlastnosti, konkrétne adhéziu a rast prostredníctvom pórovitej štruktúry a taktiež vynikajúce mechanické vlastnosti.

Le vieillissement entraîne plusieurs changements au niveau de la fonction musculaire qui peuvent mener à une perte de la masse musculaire et de sa fonction qu’on appelle sarcopénie. La sarcopénie entraîne une augmentation du risque de chutes et d’hospitalisations qui nuit à la qualité de vie des personnes âgées. Le vieillissement de la population représente un enjeu important au sein de la société en raison de son impact socioéconomique élevé. Plusieurs facteurs contribuent à ce déclin observé au cours du vieillissement, mais un des éléments clés qui y contribue sont des altérations de la jonction neuromusculaire (JNM). La JNM est une synapse tripartite composée de la terminaison nerveuse présynaptique, de la fibre musculaire postsynaptique et des cellules de Schwann périsynaptiques (CSPs), des cellules gliales. Les CSPs jouent un rôle essentiel dans la maintenance, la modulation de la transmission et de la plasticité synaptique et la réparation de la JNM. Plusieurs études effectuées chez le murin ont démontré que la JNM présente des altérations telles que de la dénervation, de la fragmentation du postsynaptique et des signes de modulation et de réparation gliaux au cours du vieillissement. Ces altérations contribuent aux déficits de la fonction neuromusculaire observés lors du vieillissement. La JNM humaine demeure cependant sous-étudiée, particulièrement en considérant sa structure tripartite. Afin de mieux comprendre le vieillissement neuromusculaire chez l’humain, des biopsies du Vastus lateralis ont été effectuées chez 4 jeunes adultes (23-28 ans) et 5 personnes âgées (60-75 ans) sains et actifs. Un marquage immunohistochimique a été effectué sur les biopsies afin d’identifier les trois composantes de base de la JNM et le type de fibre, puis visualiser en microscopie confocale. Des mesures fonctionnelles ont également été prélevées pour chacun des participants âgés. L’analyse des JNMs a permis de démontrer qu’une instabilité de l’innervation de même qu’une relation tripartite divergente se développe avec l’âge. Ces altérations corrèlent également avec un déficit fonctionnel. Dans l’ensemble, notre étude présente des altérations de la JNM humaine au cours du vieillissement ayant un impact sur la fonction neuromusculaire. Elle pourrait permettre de mieux comprendre les mécanismes à la base du vieillissement neuromusculaire pour développer des stratégies d’intervention thérapeutiques efficaces pour limiter l’impact du vieillissement. / Several changes occur in muscular function in aging which can lead to a loss of muscle mass and function called sarcopenia. Sarcopenia can lead to an increased risk of fall and hospitalization and to a poor quality of life. Aging of the population represents an important societal issue due to its high socioeconomic impact. Many factors contribute to the decline of muscular function seen in aging, but alterations of the neuromuscular junctions are a key element leading to sarcopenia. The NMJ is a tripartite synapse composed of the presynaptic nerve terminal, the postsynaptic muscle fiber as well as perisynaptic Schwann cells (PSC), glial cells. PSCs play a key role in maintenance, modulation of synaptic transmission and plasticity as well as repair of the NMJ. Several rodent studies have shown that the NMJ present alterations such as denervation, fragmentation of the postsynaptic and glial-related signs of modulation and repair in aging. These alterations contribute to the neuromuscular deficits observed in aging. However, the NMJ remain widely understudied, particularly when considering its tripartite structure. In order to get a better understanding of neuromuscular aging in humans, biopsies form the Vastus lateralis were performed on 4 young (23-28 years old) and 5 older (60-75 years old) healthy and physically active men. Immunohistochemistry labelling of the NMJ’s main components and type of fibers was performed and then imaged using confocal microscopy. Functional assessment was also measured for each older adult. Analysis of NMJs revealed an instability in the innervation as well as a divergent tripartite relationship in older individuals. These alterations also correlated with neuromuscular deficits. Taken altogether, our study highlights alterations of the NMJ in aging leading to altered neuromuscular function. This could lead to a better understanding of the underlying mechanisms leading to sarcopenia and to develop better therapeutic strategies to limit its impact during aging.

La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative qui affecte spécifiquement les motoneurones (MNs) supérieurs et inférieurs conduisant à une paralysie musculaire. La dénervation des jonction neuromusculaires (JNMs) se produit en amont de la mort des MNs de la moelle épinière chez les patients atteint de la SLA et dans de nombreux modèles murins de la maladie. Récemment, des chercheurs ont révélé une altération de la transmission synaptique, une instabilité morphologique, et une réparation inappropriée des JNMs dans le modèle de souris SOD1 en amont de l’apparition des désordres moteurs. Tandis que notre laboratoire a étudié les trois éléments synaptiques, ce mémoire porte une attention particulière aux cellules de Schwann périsynaptiques (CSPs), les cellules gliales à la JNM, considérant leurs rôles fondamentaux dans la régulation de la structure et la fonction de la JNM. Alors que de nombreuses études ont démontré une susceptibilité à la dénervation dépendante du type d’unité motrice, où certaines serait plus vulnérables au processus de dénervation que d’autres, les propriétés altérées des CSPs ont été généralisé à tous les types de JNMs étudiés. Notamment, des études réalisées dans le laboratoire ont rapporté une capacité inappropriée des CSPs à décoder l’information basée sur une augmentation de l’activation des récepteurs muscariniques (mAChRs). Les fonctions des mAChRs des CSPs sont d’une importance particulière puisque leur activité est essentielle à la stabilité des JNMs et à leur réparation et est régulé par l’activité synaptique. De manière importante, nous avons observé que la diminution chronique in vivo de l’activation des mAChRs des CSPs chez les souris SOD1G37R favorise la réparation de la JNM et améliore les fonctions motrices chez l’animal. Ainsi, la moindre altération dans les propriétés des CSPs pourrait contribuer directement à la vulnérabilité des NMJs dans la SLA. Considérant le rôle crucial des cellules gliales dans la maintenance et la réparation des JNMs, nous avons émis l’hypothèse que les CSPs contribuent à la différence de vulnérabilité observée dans la SLA. Nous avons postulé que l’hyperactivité muscarinique des CSPs contribue à l’instabilité des JNMs vulnérables, alors qu’une activité muscarinique normale contribue à la stabilité des JNMs résistantes. Pour mieux comprendre les différences dans les propriétés des CSPs contribuant à cette différence de vulnérabilité, nous avons étudié les propriétés fonctionnelles des CSPs par imagerie calcique afin de caractériser la signature muscarinique des CSPs aux JNMs d’un muscle vulnerable, l’extensor digitorum longus (EDL). Nous avons évalué l’intégrité des JNMs par un triple marquage immunohistochimique. De manière intéressante, nos résultats ont montré que L’utilisation d’un outil chémogénétique nous a permis d’augmenter l’excitabilité des AChRs des CSPs aux JNMs résistantes des MEOs. L’évaluation de l’intégrité des JNMs par un triple marquage immunohistochimique a montré que le traitement au CNO induit de l’instabilité au niveau des JNMs et nous avons observé des signes de dénervation. Établir un potentiel rôle des CSPs dans la résistance des JNMs a permis de souligner un nouveau facteur important dans la pathophysiologie de la SLA et a fourni des connaissances dans les mécanismes de résistance sélective/vulnérabilité à la dénervation. Cela permet d’ouvrir le champ à de nouvelles cibles thérapeutiques ciblant les cellules gliales à la JNM. De plus, ce nouveau contexte conceptuel de susceptibilité des JNMs peut être transposé à d’autres maladies neuromusculaires. / Amyotrophic lateral sclerosis (ALS) is a fatal late-onset neurodegenerative disease characterized by progressive loss of upper and lower motor neurons (MNs) leading to muscular paralysis. Denervation of the neuromuscular junction (NMJ) is an early pathological event that occurs before the loss of spinal cord MNs in ALS patients and various murine models of the disease. Recently, authors revealed an alteration of synaptic transmission, morphological instability, and inappropriate repair in NMJs of SOD1 mice model prior to motor impairments. While our laboratory studied all three synaptic elements, we put a particular attention to Perisynaptic Schwann cells (PSC), glial cells at the NMJ, owing to their fundamental roles in regulating NMJ structure and function. While numerous studies demonstrated a motor-unit type dependent susceptibility to denervation where some motor units (MUs) would be more vulnerable than others, altered PSC properties were generalized among all types of NMJ studied. Notably, studies performed in the laboratory reported an inappropriate PSC decoding capability based on an enhanced activation of mAChRs. PSC mAChR functions is of particular importance since it is essential for the management of NMJ stability and repair and is regulated by synaptic activity. Importantly, we observed that chronic in vivo dampening of PSC muscarinic activation in SOD1G37R fostered NMJ repair and improved motor function in the ALS mouse model. Hence, any alteration of PSC properties may directly contribute to NMJ vulnerability in ALS. Owing to the critical roles of glial cells for the maintenance and repair of NMJs, we hypothesized that PSC contribute to the differential vulnerability observed in ALS. We proposed that the hyperactive muscarinic excitation of PSCs contributes to NMJ instability at vulnerable NMJs while the normal muscarinic activity contributes to their stability in resistant ones. To better understand the distinctions in PSCs properties contributing to a difference in NMJ vulnerability, we studied the PSC functional properties by calcium imaging to characterize the muscarinic signature of PSCs at the NMJ of a vulnerable muscle, the extensor digitorum longus (EDL). We assessed the integrity of the NMJ by a triple immunostaining. Interestingly, our data revealed that altering PSC properties at resistant NMJs by enhancing the muscarinic excitation of PSCs using a viral strategy created NMJ instability with signs of denervation. Determining the potential role of PSC in the resistance of NMJs highlighted a novel important factor underlying the pathophysiology of ALS and provided significant insights into the mechanisms of selective resistance/vulnerability to denervation. This could pave the way to novel therapeutic targets and strategies targeting glial cells at the NMJ. Furthermore, this novel conceptual context may be carried over to NMJ susceptibility for other neuromuscular diseases.

Les cellules de Schwann jouent un rôle crucial dans la réparation et la régénération des nerfs périphériques en soutenant la croissance axonale et en libérant des facteurs neurotrophiques essentiels. La capacité de convertir des fibroblastes en cellules de Schwann est particulièrement intéressante dans le contexte de lésion d’un nerf périphérique, où la restauration de la fonction nerveuse est un objectif critique. Cette étude examine la reprogrammation directe des fibroblastes en cellules de Schwann, en utilisant les facteurs de transcription SOX10 et EGR2 via la transduction lentivirale. Nous avons testé divers milieux de culture connus pour identifier les conditions de conversion optimales, et avons établi qu'une multiplicité d'infection de 300 assurait une reprogrammation robuste. Cependant, maintenir la viabilité cellulaire au-delà de dix jours a présenté un défi significatif. Pour résoudre ce problème, nous avons développé un nouveau milieu de culture, que nous avons appelé Schwann Cell Medium 4 (SCM4), incorporant de petites molécules connues pour être impliquées dans le développement des cellules de Schwann. SCM4 a considérablement amélioré l'expression des marqueurs clés des cellules de Schwann, y compris SOX10, EGR2, Growth Associated Protein 43, le récepteur neurotrophique P75, et la protéine zéro de la myéline, tout en améliorant la survie globale des cellules. De plus, SCM4 a favorisé une libération plus élevée de BDNF, un facteur neurotrophique crucial pour le soutien et le développement neuronal. Les résultats obtenus avec les cellules converties dans le SCM4 sont comparables à ceux obtenus avec des cellules de Schwann dérivées de cellules souches pluripotentes induites et des cellules de Schwann humaines primaires, démontrant que notre protocole produit des cellules s’apparentant aux cellules de Schwann. Ces résultats soulignent l'importance de conditions de culture optimisées pour la reprogrammation des cellules de Schwann et offrent des perspectives prometteuses pour de futures applications cliniques dans le traitement des maladies neurodégénératives et des lésions nerveuses périphériques. / Schwann cells play a crucial role in the repair and regeneration of peripheral nerves by providing support for axonal growth and releasing essential neurotrophic factors. The ability to convert fibroblasts into Schwann cells is particularly relevant in the context of peripheral nerve injury, where the restoration of nerve function is a critical goal. This study investigates the direct reprogramming of fibroblasts into Schwann cells, employing the transcription factors SOX10 and EGR2 through lentiviral transduction. We tested various culture media described in the literature to identify the optimal reprogramming conditions, and have determined that a multiplicity of infection of 300 ensured robust reprogramming. However, maintaining cell viability beyond ten days presented a significant challenge. To address this issue, we developed a new culture medium, which we termed Schwann Cell Medium 4 (SCM4), incorporating small molecules known to be involved in Schwann cell development. SCM4 markedly enhanced the expression of key Schwann cell markers, including SOX10, EGR2, Growth Associated Protein 43, P75 Neurotrophin Receptor, and Myelin Protein Zero, and also improved overall cell survival. Furthermore, SCM4 promoted a higher release of BDNF, a critical neurotrophic factor for neuronal survival and development. The results obtained with SCM4 were compared to those obtained from Schwann cells derived from induced pluripotent stem cells and primary human Schwann cells, demonstrating that our protocol produced a comparable cell product. These findings underscore the importance of optimized culture conditions for Schwann cell reprogramming and offer promising prospects for future clinical applications in the treatment of neurodegenerative diseases and peripheral nerve injuries.

Durant le vieillissement, l’ensemble des fonctions de l’organisme se détériore, que ce soit aussi bien au niveau moteur que cognitif. Le vieillissement s’accompagne d’une diminution de la force, ainsi que de la masse musculaire. Des études récentes tendent à montrer que cette perte de masse musculaire que l’on appelle sarcopénie aurait pour origine un dérèglement de la jonction neuromusculaire. Les changements au niveau du présynaptique et du post synaptiques lors du vieillissement normal font l’objet de plusieurs études, mais les changements relatifs aux cellules de Schwann périsynaptique sont très peu connus. Le but de cette étude est donc d’analyser les modifications des interactions neurone-glie à la jonction neuromusculaire. Dans cette étude, nous montrons que certaines fonctions des cellules gliales de la synapse âgée sont déréglées, en particulier, le type de récepteurs activés par une stimulation nerveuse à haute fréquence. D’autre part, nos résultats montrent que les mécanismes responsables de l’augmentation de la transmission synaptique suite à cette stimulation nerveuse à haute fréquence sont altérés à la synapse âgée. Enfin, outre les modifications de la terminaison axonale et de la fibre musculaire, les cellules gliales montrent des signes de réorganisation structurelle propre à une synapse en réparation. Ces résultats montrent que le fonctionnement de la jonction neuromusculaire et a fortiori les interactions neurones-glie sont altérées lors du vieillissement normal. / Aging comes with an alteration and organism functions including cognitive and motor functions. Major weakening of the neuromuscular system occurs which includes muscle weight loss, difficulties in initiating voluntary movement and reduced muscle strength. The possible role of the alteration of the neuromuscular junction has been examined but always only considering the pre- and postsynaptic elements. However, perisynaptic Schwann cells (PSCs), glial cells at the neuromuscular junction (NMJ), play fundamental roles in the regulation of the synaptic efficacy of the NMJ as well as in its maintenance and stability. Hence, we analysed NMJ properties and their glial cells in aging. This study shows that PSCs function at the old NMJ are dysregulated. Indeed, PSCs ability to detect synaptic transmission, determined using imaging of intracellular Ca2+, was maintained in PSCs at NMJs from old mice, but the contribution of the muscarinic component was greatly reduced. On the other hand, our results using synaptic recordings are showing that a number of synaptic plasticity events known to be regulated by PSCs are reduced at NMJs of old mice. Finally, morphological NMJ reorganisation and sprouting of PSCs were also observed. These data suggest that PSC properties are consistent with the repair of the NMJ that may also result in their reduced ability in regulating synaptic efficacy.

INTRODUÇÃO: O nervo facial desempenha um papel importante em diversas funções fisiológicas no organismo, no entanto, distúrbios funcionais desse nervo podem também afetar a psique do indivíduo, provocando mudanças significativas na autoimagem, interferindo no rendimento profissional e piorando a qualidade de vida. Lesões graves do nervo facial (neurotmeses) mesmo quando tratadas precocemente apresentam resultados funcionais pobres. Com a recente descoberta das células-tronco, as células multipotentes do estroma mesenquimal indiferenciadas (CMEMi) ou diferenciadas em células com fenótipo de células de Schwann (CMEMd) podem ser uma alternativa melhor para o tratamento de lesões graves do nervo facial. OBJETIVOS: Avaliar a melhora funcional e histológica do ramo mandibular do nervo facial após neurotmese e implantação das CMEMi e CMEMd. MÉTODOS: Em 48 ratos Wistar realizou-se a neurotmese do ramo mandibular direito do nervo facial com a formação de um hiato de 3mm e a tubulização (conduíte de silicone) da região do nervo lesada. Foram criados quatro grupos de acordo com o método de reparo: conduíte de silicone vazio (grupo A, grupo controle); conduíte de silicone com gel acelular (grupo B); conduíte de silicone com gel acelular e CMEMi (grupo C), e conduíte de silicone com gel acelular e CMEMd (grupo D). Um quinto grupo, grupo N, foi criado a partir de segmentos do nervo normal para a avaliação histológica. Os resultados funcionais foram avaliados com o estudo de condução nervosa e os histológicos por avaliação qualitativa e quantitativa dos segmentos proximais e distais. RESULTADOS: Na avaliação funcional, após 6 semanas, os grupos C e D apresentaram amplitudes maiores que os grupos A e B (p<0,001). O grupo C apresentou duração menor que os grupos A, B e D (p<0,001). Na avaliação qualitativa dos segmentos proximais, houve pouca diferença entre os grupos, já nos segmentos distais, as diferenças dos grupos A e B em relação aos grupos C e D foram bem evidentes, no entanto, em ambos os segmentos, o grupo C foi o que mais se aproximou do nervo normal. Na avaliação histológica quantitativa do segmento proximal, não houve diferença no número total e na densidade axonal entre os grupos (p0,169), somente nos diâmetros axonais dos grupos A e B quando comparados ao nervo normal (p<0,001). No segmento distal, o número e a densidade axonal do grupo C foram maiores que os do grupo A e B (p=0,001) e iguais as do grupo D (p=0,711), porém, em todos os grupos, número e a densidade axonal foram menores que do grupo N (p0,003). Não houve diferença na média dos diâmetros entre os grupos operados (p0,007), somente quando comparados com o grupo N (p<0,001). CONCLUSÕES: As CMEMi assim como as CMEMd beneficiaram a regeneração do ramo mandibular do nervo facial de ratos Wistar, contudo, as CMEMi apresentaram resultados funcionais e histológicos melhores que as CMEMd / INTRODUCTION: Facial nerve performs an important function in different physiological activities in the organism, however, functional disturbances of such nerve may also attack a persons mind, causing expressive changes in their self-image, interfering in professional life and aggravating their quality of life. Severe lesions in the facial nerve (neurotmesis) present poor functional results even when early treated. With recent discovering of the stem cells, undifferentiated multipotent stem cell (uMSC) from mesenchymal stroma or differentiated to Schwann cell-like (dMSC) can be a better perspective to treat severe lesion of the facial nerve. OBJECTIVES: The objective of this study is to evaluate the functional and histological improvement of the mandibular branch after neurotmesis and implantation of the uMSC and dMSC. METHODS: The neurotmesis of the right mandibular branch of the facial nerve with a 3mm gap formation and tubulization (silicone tubing) of the wounded nerve area was performed in 48 Wistar rats. Four groups were divided according to the restoration method: empty silicone tubing (group A, control group); silicone tubing with non-cell gel (group B); silicone tubing with non-cell gel and uMSC (group C) and silicone tubing with non-cell gel and dMSC (group D). A fifth group (N) was created from the normal nerve segments to perform histological evaluation. The nerve conduction study evaluated the functional results; quantity and quality evaluation of the distal and proximal segment evaluated the histological results. RESULTS: After six weeks, regarding functional evaluation, groups C and D presented larger amplitude than groups A and B (p<0.001). Group C presented lesser duration than groups A, B and D (p<0.001). There was little difference among the groups in the quality evaluation of the proximal segments; on the other hand, the differences in groups A and B in relation to groups C and D were quite expressive in the distal segments. However, group C, in both segments, was the one that came closer to the normal nerve. Regarding quantity histological evaluation of the proximal segment, there was no difference in the total number and in the axonal density among the groups (p0.169); there was difference only in the axonal diameters in groups A and B when compared to normal nerve (p<0,001). Regarding distal segment, axonal density and number, in group C, were higher than in group A and B (p=0.001) and the same as in group D (p=0,711), but number and axonal density were lesser than in group N (p0,003). There was no difference in the diameter average among the operated groups (p0.007), when only compared to group N (p<0.001). CONCLUSION: Both uMSC and dMSC benefited regeneration of the mandibular branch of the facial nerve in Wistar rats, although uMSC presented better functional and histological results

INTRODUÇÃO: As lesões traumáticas graves do nervo facial, relativamente frequentes no cotidiano do otorrinolaringologista, mesmo que reparadas com as melhores técnicas microcirúrgicas, apresentam recuperação funcional extremamente limitada acarretando grande impacto na motricidade facial e, consequentemente, na qualidade de vida dos pacientes. OBJETIVOS: Avaliar a recuperação funcional (eletroneuromiografia), histológica (quantitativa e qualitativa) e imunohistoquímica do ramo mandibular do nervo facial de ratos obtidas após autoenxerto combinado a transplante de células-tronco multipotentes de polpa dentária indiferenciadas (CTPD). Seguiu-se comparação com os resultados anteriormente obtidos pelos mesmos pesquisadores e utilizando mesma técnica cirúrgica após transplante de células-tronco de medula óssea indiferenciadas (CTMOi) e diferenciadas em Schwann-like (CTMOd). Realizou-se autoenxerto no ramo mandibular do nervo facial de ratos para reparo de GAP de 5mm provocado por duas neurotmeses sequenciais, reimplantação do próprio fragmento retirado, sendo o mesmo envolvido por tubo de ácido poliglicólico (Grupo A) e preenchido com gel acelular de lâmina basal purificada, com transplante de CTPD (grupo B). Após seis semanas, os animais foram sacrificados e as análises realizadas. RESULTADOS: Observou-se que, seis semanas após a cirurgia, os animais do grupo tratado com células-tronco apresentaram valores médios de amplitude do potencial de ação muscular composto (PAMC) (CTPD 3,79±1,74mV; CTMOd 2,7±0,53mV; CTMOi 1,81±0,77mV) estatisticamente superiores ao grupo controle (0,75±0,46mV, p < 0,001). Os diâmetros axonais médios, também, foram significativamente maiores nos grupos tratados com células-tronco (CTPD 3,04±0,49?m; CTMOd 3,5±0,16?m; CTMOi 3,15±0,32?m) do que no grupo controle (2,13±0,07?m), com valor de p < 0,001. A densidade axonal foi estatisticamente superior no grupo controle (0,021±0,003axônios/um2) quando comparada aos grupos tratados (CTPD 0,014±0,004 axônios/um2; CTMOd 0,017±0,003axônios/?m2; CTMOi 0,015±0,002axônios/um2, p=0,004).Ao ensaio de imunofluorescência, no grupo tratado com CTPD, observou-se células positivas para lamina humana A/C e para S100, evidenciando, assim, a presença de células humanas com fenótipo de Schwann no segmento distal do nervo analisado. No grupo tratado com CTMOi houve marcação em beta-galactosidade, mas não em S100, confirmando a presença de células exógenas, porém não diferenciadas em Schwann. Já no grupo tratado com CTMOd demonstrou a presença de células exógenas com fenótipo de Schwann ao observar a comarcação pelos marcadores beta-galactosidase e S100, mantendo, assim, o mesmo fenótipo do observado in vitro. CONCLUSÃO: Conclui-se que, segundo critérios funcionais e histológicos, a regeneração do ramo mandibular do nervo facial de ratos foi superior quando associada ao transplante de CTPD comparativamente ao controle. O grupo tratado com CTPD apresentou melhores resultados funcionais e parâmetros histológicos similares aos obtidos com CTMOi e CTMOd. Nos três grupos tratados com células-tronco, as células exógenas foram observadas após 6 semanas de experimento, com evidência de integração ao tecido neural e evidência de diferenciação in vivo para o fenótipo de Schwann apenas no grupo tratado com CTPD / INTRODUCTION: Traumatic lesions of the facial nerve, relatively frequent of the daily routine of the otorhinolaryngologist, even if when repaired with the best microsurgical techniques, have limited functional recovery causing great impact on facial motricity, consequently, on patients\' quality of life. OBJECTIVES: The purpose of this study was to evaluate the functional and histological recovery (quantitative and qualitative) and immunohistochemistry of the mandibular branch of the facial nerve of rats obtained after autograft combined with transplantation of multipotent undifferentiated dental pulp stem cells (DPSC). We compared the results obtained previously by the same researchers and using the same surgical technique after transplantation of undifferentiated bone marrow stem cells (uBMSC) and differentiated to Schwann cell-like (dBMSC). METHODS: A 5mm gap in the mandibular branch of the facial nerve was perfomed by two sequencial neurotmesis, followed by autograft with reimplantation of the removed fragment itself, tubulization with a polyglycolic acid tube (Group A) and transplantation with DPSC (group B). After six weeks, the animals were sacrificed and analyzes performed. RESULTS: Six weeks after surgery, the animals in the stem cells group had mean values of the amplitude of the compound muscle action potential (CMAP) (DPSC 3.79±1.74mV; dBMSC 2.7±0,53mV; uBMSC 1.81±0.77mV) statistically higher than the control group (0.75±0.46mV, p < 0.001). Medium axon diameters were also significantly higher in the stem cells treated groups (DPSC 3.04±0.49um, dBMSC 3.5±0.16um, uBMSC 3.15± 0.32um) than in the control group (2.13±0.07um), with a value of p < 0.001. The axonal density was statistically higher in the control group (0.021±0.003axons/um2) when compared to the treated groups (DPSC 0.014±0.004 axons/?m2, dBMSC 0.017±0.003 axons/?m2, uBMSC 0.015±0.002 axons/um2, p=0.004). In the immunofluorescence assay, cells positive for human laminA/C and for S100 were observed in the DPSC-treated group, thus evidencing the presence of human cells with Schwann cells phenotype in the distal segment of the nerve analyzed. In the group treated with uBMSC there was beta- galactosidase, but not in S100, confirming the presence of exogenous but undifferentiated cells. In the group treated with dBMSC, the presence of exogenous cells with Schwann cells phenotype was observed by observing the comarcation by beta-galactosidase and S100 markers, thus maintaining the same phenotype as that observed in vitro. CONCLUSION: According to functional and histological criteria, the regeneration of the mandibular branch of the facial nerve of rats was superior when associated with the DPSC transplant compared to the control. The DPSC treated group had better functional results and histological parameters similar to those obtained with uBMSC and dBMSC. In the stem cells-treated groups, exogenous cells were observed after 6 weeks of experiment with evidence of neural tissue integration and evidence of in vivo differentiation for the Schwann cells phenotype only in the DPSC-treated group

INTRODUÇÃO: As lesões traumáticas graves do nervo facial, relativamente frequentes no cotidiano do otorrinolaringologista, mesmo que reparadas com as melhores técnicas microcirúrgicas, apresentam recuperação funcional extremamente limitada acarretando grande impacto na motricidade facial e, consequentemente, na qualidade de vida dos pacientes. OBJETIVOS: Avaliar a recuperação funcional (eletroneuromiografia), histológica (quantitativa e qualitativa) e imunohistoquímica do ramo mandibular do nervo facial de ratos obtidas após autoenxerto combinado a transplante de células-tronco multipotentes de polpa dentária indiferenciadas (CTPD). Seguiu-se comparação com os resultados anteriormente obtidos pelos mesmos pesquisadores e utilizando mesma técnica cirúrgica após transplante de células-tronco de medula óssea indiferenciadas (CTMOi) e diferenciadas em Schwann-like (CTMOd). Realizou-se autoenxerto no ramo mandibular do nervo facial de ratos para reparo de GAP de 5mm provocado por duas neurotmeses sequenciais, reimplantação do próprio fragmento retirado, sendo o mesmo envolvido por tubo de ácido poliglicólico (Grupo A) e preenchido com gel acelular de lâmina basal purificada, com transplante de CTPD (grupo B). Após seis semanas, os animais foram sacrificados e as análises realizadas. RESULTADOS: Observou-se que, seis semanas após a cirurgia, os animais do grupo tratado com células-tronco apresentaram valores médios de amplitude do potencial de ação muscular composto (PAMC) (CTPD 3,79±1,74mV; CTMOd 2,7±0,53mV; CTMOi 1,81±0,77mV) estatisticamente superiores ao grupo controle (0,75±0,46mV, p < 0,001). Os diâmetros axonais médios, também, foram significativamente maiores nos grupos tratados com células-tronco (CTPD 3,04±0,49?m; CTMOd 3,5±0,16?m; CTMOi 3,15±0,32?m) do que no grupo controle (2,13±0,07?m), com valor de p < 0,001. A densidade axonal foi estatisticamente superior no grupo controle (0,021±0,003axônios/um2) quando comparada aos grupos tratados (CTPD 0,014±0,004 axônios/um2; CTMOd 0,017±0,003axônios/?m2; CTMOi 0,015±0,002axônios/um2, p=0,004).Ao ensaio de imunofluorescência, no grupo tratado com CTPD, observou-se células positivas para lamina humana A/C e para S100, evidenciando, assim, a presença de células humanas com fenótipo de Schwann no segmento distal do nervo analisado. No grupo tratado com CTMOi houve marcação em beta-galactosidade, mas não em S100, confirmando a presença de células exógenas, porém não diferenciadas em Schwann. Já no grupo tratado com CTMOd demonstrou a presença de células exógenas com fenótipo de Schwann ao observar a comarcação pelos marcadores beta-galactosidase e S100, mantendo, assim, o mesmo fenótipo do observado in vitro. CONCLUSÃO: Conclui-se que, segundo critérios funcionais e histológicos, a regeneração do ramo mandibular do nervo facial de ratos foi superior quando associada ao transplante de CTPD comparativamente ao controle. O grupo tratado com CTPD apresentou melhores resultados funcionais e parâmetros histológicos similares aos obtidos com CTMOi e CTMOd. Nos três grupos tratados com células-tronco, as células exógenas foram observadas após 6 semanas de experimento, com evidência de integração ao tecido neural e evidência de diferenciação in vivo para o fenótipo de Schwann apenas no grupo tratado com CTPD / INTRODUCTION: Traumatic lesions of the facial nerve, relatively frequent of the daily routine of the otorhinolaryngologist, even if when repaired with the best microsurgical techniques, have limited functional recovery causing great impact on facial motricity, consequently, on patients\' quality of life. OBJECTIVES: The purpose of this study was to evaluate the functional and histological recovery (quantitative and qualitative) and immunohistochemistry of the mandibular branch of the facial nerve of rats obtained after autograft combined with transplantation of multipotent undifferentiated dental pulp stem cells (DPSC). We compared the results obtained previously by the same researchers and using the same surgical technique after transplantation of undifferentiated bone marrow stem cells (uBMSC) and differentiated to Schwann cell-like (dBMSC). METHODS: A 5mm gap in the mandibular branch of the facial nerve was perfomed by two sequencial neurotmesis, followed by autograft with reimplantation of the removed fragment itself, tubulization with a polyglycolic acid tube (Group A) and transplantation with DPSC (group B). After six weeks, the animals were sacrificed and analyzes performed. RESULTS: Six weeks after surgery, the animals in the stem cells group had mean values of the amplitude of the compound muscle action potential (CMAP) (DPSC 3.79±1.74mV; dBMSC 2.7±0,53mV; uBMSC 1.81±0.77mV) statistically higher than the control group (0.75±0.46mV, p < 0.001). Medium axon diameters were also significantly higher in the stem cells treated groups (DPSC 3.04±0.49um, dBMSC 3.5±0.16um, uBMSC 3.15± 0.32um) than in the control group (2.13±0.07um), with a value of p < 0.001. The axonal density was statistically higher in the control group (0.021±0.003axons/um2) when compared to the treated groups (DPSC 0.014±0.004 axons/?m2, dBMSC 0.017±0.003 axons/?m2, uBMSC 0.015±0.002 axons/um2, p=0.004). In the immunofluorescence assay, cells positive for human laminA/C and for S100 were observed in the DPSC-treated group, thus evidencing the presence of human cells with Schwann cells phenotype in the distal segment of the nerve analyzed. In the group treated with uBMSC there was beta- galactosidase, but not in S100, confirming the presence of exogenous but undifferentiated cells. In the group treated with dBMSC, the presence of exogenous cells with Schwann cells phenotype was observed by observing the comarcation by beta-galactosidase and S100 markers, thus maintaining the same phenotype as that observed in vitro. CONCLUSION: According to functional and histological criteria, the regeneration of the mandibular branch of the facial nerve of rats was superior when associated with the DPSC transplant compared to the control. The DPSC treated group had better functional results and histological parameters similar to those obtained with uBMSC and dBMSC. In the stem cells-treated groups, exogenous cells were observed after 6 weeks of experiment with evidence of neural tissue integration and evidence of in vivo differentiation for the Schwann cells phenotype only in the DPSC-treated group