The adaptation of inverse dynamics was performed to quantitatively examine the subtle locomotor changes, previously undetectable, in rodent locomotion following nervous system injury. The first experiment performed an injury with known effects, a unilateral lesion of the medial and lateral branches of the left tibial nerve of Long-Evans rats, and measured the resulting data via inverse dynamics. Special effort was made to account for skin movement artefacts using a global optimization method for marker digitization. The second experiment attempted to apply this technique to Long-Evans rats with spinal hemisections at spinal level T-10. After the peripheral nerve injury to the tibial nerve branches, the main findings were that ankle joint still produces an extensor moment and positive power without the active contraction of the gastrocnemius m. It is possible that this phenomenon is due to passive contractile elements of the muscle and tendon. In addition, the knee and hip of the lesion leg stiffen, resulting in substantial reductions in moment generation and nearly total losses of both negative and positive power production. There were also compensations made by the opposite hindlimb and contralateral forelimb. The spinal cord hemisection produced subtle, complicated intra and interlimb changes in both joint moment and joint power analysis that could not be seen by looking at joint angles alone.

Amputation is a life-changing event that results in a drastic reduction in quality of life including extreme loss of function and severe mental, emotional and physical pain. In order to mitigate these negative outcomes, there is great interest in the design of ‘advanced/robotic’ prosthetics that cosmetically and functionally mimic the lost limb. While the robotics side of advanced prosthetics has seen many advances recently, they still provide only a fraction of the natural limbs’ functionality. At the heart of the issue is the interface between the robotic limb and the individual that needs significant development. Amputees retain significant function in their nerves post-amputation, which offers a unique opportunity to interface with the peripheral nerve. Here we evaluate a relatively new approach to peripheral nerve interfacing by using microchannels, which hold the intrinsic ability to record larger neural signals from nerves than previously developed peripheral nerve interfaces. We first demonstrate that microchannel scaffolds are well suited for chronic integration with amputated nerves and promote highly organized nerve regeneration. We then demonstrate the ability to record neural signals, specifically action potentials, using microchannels permanently integrated with electrodes after chronic implantation in a terminal study. Together these studies suggest that microchannels are well suited for chronic implantation and stable peripheral nerve interfacing. As a next step toward clinical translation, we developed fully-integrated high electrode count microchannel interfacing technology capable of functioning while implanted in awake and freely moving animal models as needed for pre-clinical evaluation. Importantly, fabrication techniques were developed that apply to a broad range of flexible devices/sensors benefiting from flexible interconnects, surface mount device (SMD) integration, and/or operation in aqueous environments. Examples include diabetic glucose sensors, flexible skin based health monitors, and the burgeoning flexible wearable technology industry. Finally, we successfully utilized the fully integrated microchannel interfaces to record action potentials in the challenging awake and freely moving animal model validating the microchannel approach for peripheral nerve interfacing. In the end, the findings of these studies help direct and give significant credence to future technology development enabling eventual clinical application of microchannels for peripheral nerve interfacing.

Bone marrow-derived mesenchymal stem cells (MSC) have been shown to provide neuroprotection after transplantation into the injured nervous system. The present thesis investigates whether adult human and rat MSC differentiated along a Schwann cell lineage could increase their expression of neurotrophic factors and promote regeneration after transplantation into the injured peripheral nerve and spinal cord. Human and rat mesenchymal stem cells (hMSC and rMSC) expressed characteristic stem cell surface markers, mRNA transcripts for different neurotrophic factors and demonstrated multi-lineage differentiation potential. Following treatment with a cocktail of growth factors, the hMSC and rMSC expressed typical Schwann cells markers at both the transcriptional and translational level and significantly increased production of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). Age and time in culture are of relevance for clinical settings and growth-promoting effects of hMSC from young donors (16-18 years) and old donors (67-75 years) were compared. Undifferentiated hMSC from both young and old donors increased total neurite length of cultured dorsal root ganglion (DRG) neurons. Differentiation of hMSC from the young donors, but not the eldery donors, further enhanced the neurite outgrowth. Undifferentiated hMSC were cultured for eleven weeks in order to examine the effect of in vitro expansion time on neurite outgrowth. hMSC from the young donors maintained their proliferation rate and their ability to enhance neurite outgrowth from DRG neurons. Using a sciatic nerve injury model, a 10mm gap was bridged with either an empty tubular fibrin glue conduit, or conduits containing hMSC, with and without cyclosporine treatment. Cells were labeled with PKH26 prior to transplantation. At 3 weeks after injury the conduits with cells and immunosuppression increased regeneration compared with an empty conduit. PKH26 labeled human cells survived in the rat model and the inflammatory reaction could be suppressed by cyclosporine. After cervical C4 hemisection, BrdU/GFP-labeled rMSC were injected into the lateral funiculus rostral and caudal to the spinal cord lesion site. Spinal cords were analyzed 2-8 weeks after transplantation. Transplanted MSC remained at the injection sites and in the trauma zone for several weeks and were often associated with numerous neurofilament-positive axons. Transplanted rMSC induced up-regulation of vascular endothelial growth factor in spinal cord tissue rostral to the injury site, but did not affect expression of brain-derived neurotrophic factor. Although rMSC provided neuroprotection for rubrospinal neurons and significantly attenuated astroglial and microglial reaction, cell transplantation caused aberrant sprouting of calcitonin gene-related peptide immunostained sensory axons in the dorsal horn. In summary these results demonstrate that both rat and human MSC can be differentiated towards the glial cell lineage, and show functional characteristics similar to Schwann cells. hMSC from the young donors represent a more favorable source for neurotransplantation since they maintain proliferation rate and preserve their growth-promoting effects in long-term cultures. The data also suggest that differentiated MSC increase expression of neurotrophic factors and support regeneration after peripheral nerve and spinal cord injury.

Orientador: Antonio Carlos Alessi / Banca: Rosemeri de Oliveira Vasconcelos / Banca: Roberta Ferro de Godoy / Resumo: Lesões no Sistema Nervoso Periférico (SNP) são relativamente freqüentes. Apesar do SNP apresentar capacidade regenerativa em muitas situações não ocorre regeneração nervosa adequada levando a perdas funcionais. A Catuama é a associação de quatro extratos hidroalcoólicos obtidos de plantas brasileiras (Paullinia cupana, Trichilia catigua, Ptychopetalum olacoides e Zingiber officinale) com conhecida ação neuroprotetora, antiinflamatória, anti-oxidante e antidepressiva. O bilobalide é um componente extraído das folhas de Ginkgo biloba que tem demonstrada ação neuroprotetora nos sistemas nervosos central e periférico. O presente estudo avaliou a ação da Catuama e do bilobalide na regeneração nervosa periférica de ratos submetidos à secção do nervo isquiático e reparação com implante de tubo de silicone preenchido por colágeno deixando um intervalo entre os segmentos nervosos de 10mm. A Catuama foi administrada por via oral nos primeiros 28 dias de pós-operatório, nas doses de 100, 200 e 400mg/kg e comparada ao bilobalide na dose de 200μM adicionado ao colágeno líquido do implante e ao grupo controle. Os animais foram avaliados na primeira, quinta e décima semana de pós-operatório pelo teste de marcha e na décima semana pela avaliação eletrofiosiológica e pelas análises quantitativa e qualitativa dos cortes histológicos de amostras do nervo isquiático e do músculo gastrocnêmio. Em todas as análises utilizadas não foi encontrada diferença (P>0,05) entre os grupos experimentais o que indica que a Catuama e o bilobalide não interferiram na regeneração nervosa periférica nas condições experimentais. / Abstract: Peripheral nerve lesions are common. Functional recovery after peripheral nerve injuries is frequently poor, despite the capacity for axonal regeneration, resulting in most cases in a functional loss of the nerve. The Catuama is composed of four brasilian plants extracts (Paullinia cupana, Trichilia catigua, Ptychopetalum olacoides e Zingiber officinale). The Catuama is known as having neuroprotector, antinflamatory, antioxidant and antidepressive effects. Bilobalide, extraction of the leaves of Ginkgo biloba, is known by it's neuroprotector effect in the central and peripheral nervous system. The present study evaluates the effect of Catuama and bilobalide on peripheral nerve regeneration in rats following a sciatic nerve section. Sciatic nerve of adult rats was transected with a 10-mm gap and the proximal and distal nerve stumps were fixed in a silicone tube filled with collagen. The Catuama was oral administered along 28 days after the surgery. Three different treatment groups using doses of 100, 200 and 400mg/kg of Catuama were compared with one group using 200μM bilobalide associated with the liquid collagen in the silicone tube and the control group. Animals evaluations were done by a walk test on the first, fifth and tenth week after the surgery. Eletrophysiologic stimulation and quantitative and qualitative hystologic analysis of the sciatic nerve and gastrocnemius muscle were also realized on the tenth week after the surgery. No statistical difference was found in these variables between the five groups (P >0.05). These results suggest that there was no effect of Catuama or bilobalide on peripheral nerve regeneration regeneration. / Mestre

Made available in DSpace on 2014-06-11T19:30:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-09Bitstream added on 2014-06-13T21:00:59Z : No. of bitstreams: 1 reis_fj_dr_botfm.pdf: 9162185 bytes, checksum: 58fc2f834c62afaebb389b3271538311 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As injúrias aos nervos periféricos levam à perda parcial ou total da função sensorial e ou motora, determinando aos indivíduos, prejuízos emocionais, sociais e financeiros. Apesar de existirem diversos métodos microcirúrgicos para a reparação destas áreas desnervadas, apenas uma recuperação limitada pode ser obtida. Deste modo, nosso objetivo foi avaliar se a associação de duas técnicas de reparo nervoso, um enxerto de nervo com neurorrafia término-lateral (NTL) e neurotização muscular direta (NMD) associados a uma neurorrafia término-terminal (NTT), podem superar o reparo funcional insatisfatório obtido com as diferentes técnicas empregadas isoladamente; se a utilização de outra via de reinervação poderia prejudicar a recuperação funcional obtida apenas com a NTT; se a associação de dois enxertos poderia aumentar a taxa de recuperação funcional obtida com um enxerto. Para tanto, foram utilizados 140 ratos machos Wistar, divididos em 7 grupos: G1 - Normal; G2 - Desnervado; G3 - NTT; G4 - NTT + enxerto simples (NTL e NMD); G5 - NTT + enxerto duplo; G6 - enxerto simples; G7 - enxerto duplo. O nervo fibular foi seccionado e imediatamente após, foi realizada a NTT. Os enxertos foram realizados com segmentos do nervo sural, coletados para esse propósito. O segmento do nervo sural teve seu coto proximal suturado por NTL à lateral do nervo tibial, sem janela epineural, e seu coto distal implantado no músculo tibial cranial, através de NMD. Após 6 meses, foi realizada a avaliação funcional da marcha, estudo eletrofisiológico, avaliação da massa do músculo tibial cranial (MTC), avaliação histológica do músculo e dos segmentos do nervo fibular e dos enxertos. Os resultados foram submetidos à ANOVA e, se detectada diferença estatística, ao teste de Tukey. Na avaliação funcional da... / The injuries to peripheral nerves causes total or subtotal loss of sensory and/or motor function, with social and financial problems. In spite of different microsurgical techniques, the results of peripheral nerve repair have today reached a plateau where functional recovery is still unsatisfactory. We tested the efficacy of association of two microsurgical techniques, end-to-side neurorrhaphy (ESN) and direct muscle neurotization (DMN), with nerve graft, and end-to-end neurorrhaphy (EEN) to functional recovery of desnerved muscle; if the other offer to reinnervation could affect the functional recovery of EEN; if the association of two nerves grafts could increase the functional recovery achieved with one nerve graft. 140 rats, male, Wistar, were divided into seven groups: G1 - normal; G2 - desnerved; G3 - end-to-end neurorrhaphy (EEN); G4 - EEN + single graft (ESN and DMN); G5 - EEN + double graft; G6 - single graft; G7 - double graft. The peroneal nerve was transected and immediately re-anastomosed with EEN. The grafts were making with the sural nerve. The proximal stump of sural nerve was sutured to the tibial nerve with ESN, without epineural window. The distal stump was implanted into the lateral head of the tibial cranial muscle (TCM). After six months, the animals were subjected to Walking Tracks Analysis, electrophysiologic tests, sacrificed, and the muscles and nerves of interest were taken to examination. There aren’t differences significant to functional evaluating among groups G1 (normal), G3, G4 and G5. The group G7 had better recovery than G6 and G2 (desnerved), however worst than G1, G3, G4 and G5. In the electrophysiologic tests, there wasn´t differences among G3, G4, G5 and G7. However, were better than G2, and worst than G1. In the mass of tibial cranial muscle, there weren´t difference among... (Complete abstract click electronic access below)

Apesar do desenvolvimento da tecnologia envolvendo o campo da regeneração em nervos periféricos, ainda não existe uma técnica para recuperação do tecido nervoso que apresente resultados totalmente satisfatórios, fato que desperta o interesse de vários pesquisadores em todo mundo e representa um desafio para os cirurgiões que realizam procedimentos reconstrutivos e estéticos. Embora o enxerto autólogo de nervo seja a melhor alternativa para a recuperação de nervos periféricos lesados, as técnicas que envolvem o reparo tubular têm alcançado excelentes resultados através da utilização de materiais sintéticos e biológicos, sob a forma de tubos, no reparo de extensos segmentos nervosos. Hoje, através dos avanços da engenharia tecidual, novos materiais estão em desenvolvimento, com o objetivo de aliar características microscópicas às técnicas cirúrgicas existentes. O colágeno, que é sabidamente um elemento promotor da proliferação celular e do reparo tecidual, tem sido amplamente utilizado em estudos de regeneração nervosa. Da mesma maneira, o ácido poli-láctico-poli-glicólico (PLGA), um copolímero sintético, tem apresentado algumas características favoráveis ao processo de regeneração, como biocompatibilidade e propriedades mecânicas adequadas. Com o propósito de facilitar a regeneração nervosa quando ocorre perda tecidual, uma técnica já difundida pode ser destacada: o enxerto de veia invertida, em que a veia jugular externa é utilizada ao avesso, funcionando como um tubo, criando um microambiente para a regeneração nervosa, através da exposição de elementos fundamentais da camada mais externa do vaso (túnica adventícia). Neste trabalho, agregamos como diferencial a utilização de dois tipos de membranas, especialmente desenvolvidas para fins odontológicos, que visam neoformação óssea, em um estudo que visa à regeneração de um nervo periférico misto, o nervo isquiático. As membranas de colágeno e PLGA foram colocadas na região do enxerto, sob a forma de tubo, com o objetivo de recuperar um segmento de 10 mm do nervo referido. E, por fim, aliamos a utilização de um segmento de 5 mm da veia jugular externa como tecido de preenchimento dos tubos feitos com as membranas, na tentativa de verificar a eficácia das moléculas biológicas presentes neste vaso, porém agora sendo utilizadas diretamente dentro do enxerto. Após um período de 6 semanas, o grupo que apresentou o melhor resultado foi aquele em que foi utilizada a membrana de colágeno, na forma de tubo, preenchida com a veia jugular externa. Nesse grupo, o diâmetro médio das fibras mielínicas apresentou um valor médio de 5,8 µm e espessura média da bainha de mielina de 1,65 µm. Para o período de 12 semanas, entre os grupos analisados, o maior valor médio encontrado para o diâmetro foi de 5,64 µm e 1,31 µm para a espessura, sendo que este resultado foi apresentado pelo grupo em que se utilizou a membrana de PLGA sem preenchimento com a veia jugular. Embora os valores não sejam muito inferiores, nos dois períodos, ficaram abaixo dos valores encontrados nos grupos controle normais. Em suma, acreditamos que as membranas de colágeno e PLGA, associadas à técnica de tubulização, apresentam grande potencial para serem utilizadas na regeneração de nervos periféricos. / Despite the development of technology involved in peripheral nerve regeneration, there is no technique that presents a recovery of the nervous tissue with completely satisfactory results. This fact arouses interest of several researchers around the word. Although the autologous nerve grafting is the current gold standard for the repair of large nerve gaps, over the past decades the development of artificial nerve conduits has therefore been of great interest due to the excellent results achieved. Through the advances in tissue engineering, new materials, synthetic and biological, have been used for construct nerve guides. The collagen is one of the oldest natural polymers used as a biomaterial, and it is known as a promoter of cell proliferation and of tissue repair. In the same way, the synthetic copolymer, poly lactic-co-glycolic acid (PLGA), have been used for nerve regeneration, and it have shown some favorable characteristics to nerve repair such as stability, biocompatibility, and mechanical properties. With the purpose of facilitating the regeneration in large nerve gaps, the inside-out vein graft is a widespread technique, where the vein works as a conduit, and it provides a good microenvironment for axon regeneration through the exposition of some primordial elements of its adventitial layer. In this work, we join as a differential the use of two materials, specially developed for dental purposes, which are normally used to facilitate the osteogenesis, in a nerve regeneration study. We performed the surgical procedures in the sciatic nerve, with two types of membranes (PLGA and collagen), which were used as tubes, in order to promote the regeneration of this nerve. The collagen and PLGA membranes were used with the objective of a recovery in a 10-mm sciatic nerve gap model. These tubular implants were filled with a 5 mm segment of the jugular vein in order to verify the effectiveness of some biological molecules of the adventitia layer in the nerve regeneration. After 6 and 12 weeks the graft and the distal stump were observed under light microscopy. In each studied period, the diameters of the myelin fibers, and the thickness of the myelin sheaths of the regenerated axons were measured. After 6 weeks, the group which presented the best result was the group with the collagen membrane tube filled with the external jugular vein. In this group the diameter of the myelin fibers presented an average value of 5.8 µm and an average value of the thickness of the myelin sheaths of 1.65 µm. After 12 weeks, between all the groups, the best results were found in the group where the PLGA membrane unfilled with the external jugular vein was used as a nerve guide, which presented the average diameter of 5.64 µm, and the average thickness of 1.31 µm. Although the average values werent much lower than the values found in the controls, they were beneath than the normal groups. These results proved that the collagen and the PLGA membranes have a great potential to be used as artificial nerve conduits for promoting peripheral nerve regeneration.

Schwann cells are the principal glial cell of the peripheral nervous system and are responsible for axon maintenance, regeneration and increasing saltatory conduction of neurons. Schwann cell differentiation and myelination is mediated by a core network of transcription factors and signalling pathways, which have been divided into two groups; positive and negative regulators. Sox10, NFATc4, Oct6, Krox20 and the ERK 1/2 signalling pathway have been characterised as positive regulators of Schwann cell differentiation and myelination; with Sox10 and Krox20 also playing critical roles in myelin maintenance. On the other hand, transcription factors cJun, Pax3, Id2 and signalling pathways Notch and p38 mitogen activated protein (MAP) kinases (MAPK) have been identified as negative regulators of Schwann cell differentiation and myelin formation. Recently, the HMG transcription factor Sox2 was identified as a negative regulator of Schwann cell myelination in vitro, however its role in Schwann cell myelination in vivo has not yet been studied. This study therefore aimed to examine the role of Sox2 overexpression in Schwann cells and how it effects Schwann cell differentiation and myelination during development and after injury. In addition, we aimed to investigate for the first time the specific role of p38α (the major isoform of p38 MAPK) in Schwann cell myelination in vivo, by generating Schwann cell specific p38α conditional knockout mice. Sox2 is highly expressed in immature Schwann cells, but is downregulated as Schwann cells being to mature and differentiate. This study shows that continued expression of Sox2 during development and after injury, impairs Schwann cell differentiation and myelination by directly downregulating the expression of two core transcription factors; Sox10 and Krox20, as well as myelin proteins, P0 and MBP. In addition, we observe that continued Sox2 expression significantly increases Schwann cell proliferation and maintains Schwann cells in an immature state. Unexpectedly, we also observed that continued Sox2 expression significantly increases the number of macrophages present in the nerves of Sox2 overexpressing mice at both P60 and 21 days post injury. Phenotypically, Sox2 overexpressing mice 6 show signs of a peripheral neuropathy and animals have impaired motor and sensory function. These findings confirm that Sox2 is a negative regulator of Schwann cell myelination and suggests that continued Sox2 expression is sufficient to drive the progressive development of a peripheral nerve disorder which may resemble Charcot-Marie-Tooth type 1 demyelinating neuropathy and congenital hypomyelinating neuropathy. As a negative regulator of Schwann cell myelination, activity of the p38 MAPK pathway has been shown to inhibit myelin formation in vitro and to also induce the Schwann cell injury response; by driving Schwann cell dedifferentiation and demyelination following injury. Here we show that specific removal of the p38α isoform in Schwann cells leads to an increase in myelin thickness at early developmental time-points, along with an elevated expression of myelin proteins, P0 and MBP. Further analysis following nerve injury revealed that removal of p38α results in an initial decrease in Schwann cell demyelination, yet improves axon remyelination at 21 days post injury. These results demonstrate the specific role of p38α in regulating Schwann cell myelination, and how it could be a direct therapeutic target for improving nerve repair after injury.

Repair of peripheral nerve injury often results in poor functional motor recovery. This deficit has previously been attributed to the failure of axons to regenerate into the muscle. However, we have recently reported that following nerve injury in mice, axons have regenerated to the motor end plate in animals with poor recovery. We proposed that following axonal injury, there is a critical period during which the axon must reach the muscle in order to form a functional neuromuscular junction. We have developed a mouse model of prolonged denervation, in which the sciatic nerve is crushed repeatedly every few days, preventing regenerating axons from reaching the muscle. This multiple crush model allows us to vary the period of denervation by modifying the number of crushes. Motor recovery as assessed using the toe-spreading score occurs after 3 or 4 multiple crushes every 7 days (24 or 31 days of denervation) but not after 5 crushes (38 days). Immunostaining for alpha-bungarotoxin and neurofilament confirmed end plate reinnervation. Thus following denervation > 38 days, a motor deficit persists despite end plate reinnervation. Although the mechanism for the deficit requires investigation, these results suggest that functional neuromuscular junction reestablishment more than end plate reinnervation and that there is a time limit for functional synapse reformation.

This project focused on the design of a cellular scaffold applicable for the promotion of peripheral nerve regeneration. Firstly, we established a correlation between the organization of HA/PEG co-polymeric networks to their mechanical and degradability properties; cell adhesion was conferred to all gels by the incorporation of RGD peptides. Three families of hydrogels were produced using different procedures to permit an increasing physical incorporation of HA into a PEGDA-based network. From a comparative study of rheological properties and enzymatic degradability, co-networks obtained using thiolated HA as chain transfer agent during PEGDA photo-polymerization were selected for further biological investigations, aiming to link the cellular response of L929 murine fibroblasts (phenotype, proliferation rate, metabolic activity) to the composition and the consistency of selected hydrogels. Our findings showed that there is a clear relation between increasing hardness and increasing cell spreading/proliferation rate. This study illustrated the possibility to fine tune cell/material interactions with appropriate reactive processing techniques. As a spin-off of this study, we become interested in the interplay of cellular interactions in the use of materials that contain both HA and RGD peptides, which can bind at the same time to HA receptors such as CD44 and av integrins. We focused on soluble HA derivatives, with or without dandling RGD peptides. The kinetics and the mechanistic details of both HA and HA-RGD internalization were studied in a phagocytic model (J774.2 murine macrophages). HA-RGD showed a form of synergic binding to integrins and CD44 (HA receptor), whereas its uptake remained solely regulated by CD44 dwell-time on the cell membrane. This study demonstrated that the knowledge of the rate-determining steps of the uptake of a carrier is necessary for assessing its efficiency. In this case, the presence of multiple ligands on a carrier was beneficial in some respect, but may not be optimal to overcome internalization limitations that arise from the slow turnover of the determining receptor. Finally, we studied the relation between the regulation of the expression of CD44 / RHAMM (HA receptor mediated motility) and the motility of Schwann cells (peripheral glial cells) and stem cells differentiated into a glial phenotype. Rt-PCR and immuno-assay experiments suggested that RHAMM up-regulation is associated with glial differentiation and we speculate that in the future this HA receptor could be considered as a differentiation marker. We also illustrated the importance of HA / RHAMM interaction for the motility of glial cells. These results indicate the importance of HA in mediating glial cell function during peripheral nerve regeneration and have implications for therapeutic repair strategies.

As lesões dos nervos periféricos, que inervam os músculos esqueléticos, evoluem para perdas da propriocepção e alterações na morfologia e função das fibras musculares, causando um impacto negativo na qualidade de vidas dos indivíduos. Tais lesões implicam em alteração na expressão de genes específicos do músculo, como por exemplo, na MyoD e Miogenina, atuantes na ativação de células satélites e reguladores da massa muscular A técnica cirúrgica de tubulização é um recurso empregado na prática clínica para tratamento de músculos que sofreram desnervação. O objetivo do presente estudo foi analisar se a técnica de tubulização com o preenchimento de gordura altera a expressão de Myod e Miogenina, a morfometria do músculo sóleo de ratos e localização da Myod e Miogenina. Para isso, 57 ratos Wistar foram separados em grupos: controle inicial (GCI); final 45 (GCF45), final 150 (GCF150), desnervado 45 dias (GD45), desnervado 150 dias (GCD150) e grupos experimentais com veia vazia 45 dias (GESP45) e 150 dias (GESP150) e com veia preenchida de gordura 45 dias (GEG45) e 150 dias (GEG150). Para os procedimentos cirúrgicos de desnervação e reinervação e coleta do músculo os animais foram profundamente anestesiados. Após os devidos tempos experimentais, os animais foram sacrificados, o músculo sóleo foi dissecado, envolvido em meio de criopreservação e estocado a -80°C. A quantificação de mRNA do MyoD e Miogenina foi realizada por amplificação por reação em cadeia de polimerase (PCR) em tempo real (RealTimePCR) e a localização da produção de Myod e Miogenina foi realizada por microscopia confocal a laser e imunofluorescência. A morfometria foi realizada em lâminas coradas com HE, observadas em microscópio ótico e calculadas pelo software Image Pro-Plus 6.2. Os resultados do presente estudo mostraram que houve aumento da expressão do Myod e Miogenina nos grupos experimentais 45 dias quando comparados ao grupo controle inicial e um decréscimo da expressão de Myod e Miogenina para os grupos experimentais com 150 dias. A área da secção transversa nos grupos experimentais com 45 dias (GESP45 e GEG45) não apresentaram diferença estatística, quando comparado com grupo desnervado 45 dias (GCD45), enquanto que o grupo experimental com preenchimento de gordura 150 dias (GEG150) obteve os melhores resultados na medida da área da secção transversal do músculo sóleo. As lâminas observadas no microscópio confocal mostram a MyoD e Miogen localizadas no mionúcleo. Concluiu-se que o uso da gordura na técnica de tubulização do nervo ciático de ratos, interfere na regeneração do músculo sóleo. / Peripheral nerve injuries can result in the loss of propioception, morphological and functional alterations of muscle fibers which causes a negative impact on the quality of life. These injuries elicit an alteration on the expression of muscle specific genes, like MyoD and Myogenin, involved in the satellite cell activation and muscle mass regulation. The vein graft tubulization is a well known technique for treatment of denervated muscle. The aim of this work was to investigate if vein graf tubulization filled with fat tissue changes the expression and localization of MyoD and Myogenin and to study if it can modify the morphometry of soleus muscle. Fifty seven Wistar rats were divided in initial control group (ICG), final control group 45 days and 150 days (FCG45; FCG150), denervated 45 days and 150 days (D45; D150) and experimental groups with vein graft 45 days and 150 days (VG45; VG150). and vein graft filled with fat tissue 45 days and 150 days (VF45; VF150). For denervation and reinervation procedures and muscle biopsy the animals were submitted to anaesthesia and after the experimental time they were euthanized. Soleus muscle was dissected, involved in criopreservation medium and stored at -80oC. It was performed RealTime polymerase chain reaction (RealTimePCR) for MyoD and Myogenin mRNA quantification. The localization of its production was analysed by laser confocal microscopy and immunofluorescence staining. The morphometric analysis were done by Hematoxilin-Eosin staining and examined at optical microscopy using the Image ProPlus 6.2 software. There was an upregulation on the expression of MyoD and Myogenin for the experimental groups at 45 days when compared to the initial control group. On the other hand, we found a downregulation on the MyoD and Myogenin expression in the same groups with 150 days. The area of transversal section in the 45 days experimental groups (VF45, VG45) did not show statistical difference compared with denervated group with 45 days (D45). Moreover, the group filled with fat tissue at 150 days (VF150) presented the best results in the transversal section area of soleus muscle. In addition, the slides analysed under confocal microscopy showed the localization of MyoD and Myogenin in the mionuclei. In conclusion, the application of vein graft filled fat tissue improves the soleus muscle regeneration.