A aceleração da regeneração nervosa aliada à sua qualidade através da inoculação de células no sítio da lesão tem sido objeto de diversos estudos experimentais. Assim, o presente trabalho objetiva avaliar a regeneração nervosa associando a técnica de tubulização com a fração mononuclear autóloga de medula óssea em coelhos. Foram utilizados 28 coelhos da raça Nova Zelândia, hígidos, distribuídos em dois grupos: terapia (GT) e controle (GC), de igual número, subdivididos de acordo com o tempo de avaliação em 50 e 75 dias. Todos os animais foram anestesiados e submetidos a secção do nervo femoral direito com imediata neurorrafia utilizando tubo de silicone, deixando um intervalo de 5 mm entre os cotos nervosos, porém apenas o GT recebeu a terapia celular. A avaliação da regeneração foi realizada funcionalmente e histologicamente, sendo os dados de função obtidos por exame clínico neurológico realizado a cada dez dias de pós-operatório e eletrofisiologia nervosa realizada previamente a eutanásia aos 50 e 75 de pós-operatório. Na análise histológica constataram-se presença de ponte nervosa e boa regeneração das fibras em todos os animais. A eletrofisiologia nervosa demonstrou queda nos valores de amplitude e aumento da latência, independentemente dos grupos. Entretanto, na avaliação funcional da marcha, o GT apresentou melhor desempenho significativo nas três primeiras semanas de avaliação. Estes dados sugerem que a terapia celular associada à técnica de tubulização influencia beneficamente no início da regeneração nervosa, proporcionando retorno funcional do nervo mais precoce quando comparado a animais controle. / The acceleration of nerve regeneration combined with its quality by the inoculation of cells is the site of the injury has been the object of several experimental studies. This paper aims to evaluate the nerve regeneration using the tubulization technique associated with the transplantation of autologous mononuclear cell from bone marrow in rabbits. The study has used twenty-eight animals New Zealand, healthy, allocated in two groups with equal number: therapy group (TG) and control group (CG), divided according to the evaluation duration time at 50 and 75 days. All animals were anaesthetized and then had their right femoral nerve sectioned, followed by immediate neurorrhaphy with a silicon tube, leaving a 5mm gap between the nervous stumps, but only the GT received cell therapy. The evaluation of the regeneration was made histologically and functionally through clinical examination carried out each ten days postsurgery and through electrophysiology prior to euthanasia at 50 and 75 postsurgery in both groups. The histological analysis, showed the presence of nerve repair and good regeneration of fibers in both groups. The electrophysiology showed a reduction of the amplitude values and increased latency, irrespective of the group. However, the lameness analysis showed that the GT had significantly better performance on the first three weeks postoperatively. The results suggest that cell therapy associated with the silicon channels can be a good influence on the beginning of nerve regeneration, providing functional return of the nerve earlier when compared to control animals.

Submitted by Edineia Teixeira (edineia.teixeira@unioeste.br) on 2017-12-01T13:24:56Z No. of bitstreams: 2 JULIANA _ANTUNES2016.pdf: 2044516 bytes, checksum: 8b8259c8bf77ca4b4a753df40515ef1f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-12-01T13:24:56Z (GMT). No. of bitstreams: 2 JULIANA _ANTUNES2016.pdf: 2044516 bytes, checksum: 8b8259c8bf77ca4b4a753df40515ef1f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-03-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The research is experimental, quantitative and random character, which aimed to analyze the effect of resistance exercise climb stairs, on the regeneration of the sciatic nerve of Wistar rats. The sample consisted of 24 animals were randomly separated into four groups: GC - control; GE - only exercised; GL - injured and untreated; and GLE - injured and treated. The compressive lesion of the right sciatic nerve was performed in anesthetized animals and was to conduct a nerve impingement in 30 seconds with hemostat. From the third day after surgery, the animals of GE and GLE were submitted to resistance exercise climb stairs, and made two sets of ten climbs the ladder with overhead 100 grams adapted to its tail, with an interval of one minute between one series and another 5 days a week for 3 weeks. In the course of this period, all animals were evaluated for the presence of edema and nociceptive threshold of the right hind limb. After treatment, the animals were anesthetized and euthanized to collect a fragment of two centimeters of the right sciatic nerve, which was subjected to routine histological processing, for morphological analysis and Western blotting technique for molecular analysis. Regarding edema evaluation results, there was only meant difference in comparing the evaluations (p <0.001), indicating reduced over time member of the volume, but there was no significant difference between groups. With regard to nociception results in the lesion site and planting region was observed difference between the ratings (p <0.001), and the nociceptive threshold increased over the study time, however between the groups, so there was difference between compared to GC and GE, GL and GLE, with the former had higher results. Regarding histological analysis, GC and G2 showed normal appearance around nerve tissue, as GL and GLE showed large amount of inflammatory infiltrate, degeneration and disruption of nerve fibers, but in GLE also met you merge nerve fibers. Regarding the morphometric analysis, no significant difference between the groups, as, fiber density, amount of axons and the quotient G; For the nerve fiber diameter and myelin sheath, GC and GE were significantly better results when compared to GL and GLE; and as the diameter of the axon GC obtained significantly higher results than the other groups. Finally, molecular analysis showed values higher brain-derived neurotrophic factor in injured groups. Therefore, it is concluded that the injury model in the present study was effective to cause significant changes in the sciatic nerve of the animal, but resisted exercise climb stairs, the proposed parameters was not significantly effective in reducing edema and pain of animals and could not accelerate morphological regeneration after sciatic nerve compression injury. / A pesquisa é de caráter experimental, quantitativa e randomizada, que teve como objetivo, analisar o efeito do exercício físico resistido de subida em escada, sobre a regeneração do nervo isquiático de ratos Wistar. A amostra foi composta por 24 animais, separados aleatoriamente em quatro grupos: GC – controle; GE – somente exercitado; GL – lesionado e não tratado; e GLE – lesionado e tratado. A lesão compressiva do nervo isquiático direito foi efetuada nos animais anestesiados e consistiu em realizar um pinçamento no nervo por 30 segundos com pinça hemostática. A partir do terceiro dia de pós-operatório, os animais de GE e GLE foram submetidos ao exercício físico resistido de subida em escada, sendo realizadas duas séries de dez subidas na escada, com sobrecarga de 100 gramas adaptada a sua cauda, com intervalo de um minuto entre uma série e outra, 5 dias por semana durante 3 semanas. Ao decorrer deste período, todos os animais foram avaliados, quanto a presença de edema e limiar nociceptivo do membro pélvico direito. Ao término do tratamento, os animais foram anestesiados e eutanasiados para a coleta de um fragmento de dois centímetros do nervo isquiático direito, o qual foi submetido a processamento histológico de rotina, para analise morfológica e à técnica de Western Blotting para análise molecular. Com relação aos resultados da avaliação do edema, observou-se diferença significava somente na comparação entre as avaliações (p<0,001), denotando redução do volume do membro ao longo do tempo, porém não houve diferença significativa entre os grupos. Quanto aos resultados da nocicepção no local da lesão e na região plantar, foi possível observar diferença entre as avaliações (p<0,001), sendo que o limiar nociceptivo aumentou com o passar do tempo de estudo, porém entre os grupos, so houve diferença entre GC e GE comparado a GL e GLE, sendo que os primeiros obtiveram resultados maiores. Referente a análise histológica, GC e GE apresentaram aspecto normal em todo tecido nervoso, já GL e GLE mostraram grande quantidade de infiltrado inflamatório, degeneração e desorganização das fibras nervosas, porém em GLE encontrou-se também fibras nervosas integras. Com relação a análise morfométrica, não ocorreu diferença significativa entre os grupos, quanto a, densidade de fibras, quantidade de axônios e ao quociente G; Para o diâmetro da fibra nervosa e da bainha de mielina, GC e GE apresentaram resultados significativamente maiores quando comparados ao GL e GLE; e quanto ao diâmetro do axônio GC obteve resultados significativamente maiores que os demais grupos. E por fim, a análise molecular, mostrou valores maiores do fator neurotrófico derivado encéfalo nos grupos lesionados. Portanto, conclui-se que o modelo de lesão realizado neste estudo foi efetivo, pois causou alterações significativas no nervo isquiático dos animais, porém o exercício físico resistido de subida em escada, nos parâmetros propostos, não foi significativamente eficiente na redução do edema e da dor dos animais, bem como não conseguiu acelerar a regeneração morfológica do nervo isquiático após lesão compressiva.

OBJETIVO: avaliar o efeito das neuregulinas 1-alfa e 1-beta na regeneração de nervos ciáticos de camundongos C57BL/6J, adultos, machos, através da técnica de tubulização. MÉTODOS: Utilizaram-se 18 animais, divididos em 3 grupos, implantando-se prótese de polietileno em falhas de 4,0 mm no nervo ciático esquerdo: grupo 1 contendo apenas colágeno purificado (Vitrogen®); grupo 2, colágeno associado a neuregulina 1-alfa; grupo 3 com colágeno e neuregulina 1-beta. O grupo controle foi formado por 6 segmentos de nervos ciáticos direitos. Após 4 semanas, os animais foram sacrificados; extraiu-se segmento do ponto médio do nervo regenerado no interior das próteses, padronizaram-se cortes histológicos e confecção das lâminas para análise histomorfométrica. Confrontaram-se os resultados estatisticamente. RESULTADOS: Os animais tratados com neuregulinas tiveram maior número de axônios mielinizados, com diferença estatisticamente significante quando comparados ao grupo colágeno. Não houve diferença estatística entre os grupos de neuregulinas 1-alfa e 1-beta. CONCLUSÃO: a adição de neuregulinas proporcionou aumento significativo do número de fibras mielinizadas. / PURPOSE: To evaluate the effect of neuregulins 1-alpha and 1-beta on the regeneration the sciatic nerves of male adult C57BL/6J mice, using the tubulization technique. METHODS: Eighteen animals were used, divided into three groups. A polyethylene prosthesis was implanted in a 4.0 mm defect of the left sciatic nerve, as follows: group 1 containing only purified collagen (Vitrogen®); group 2, collagen with neuregulin 1-alpha; group 3, collagen with neuregulin 1-beta. The control group was formed by six segments of right sciatic nerves. Four weeks later, the animals were sacrificed. A segment from the midpoint of the nerve regenerated inside the prostheses was extracted, histological sections were standardized and slides were made up for histomorphometric analysis. The results were statistically compared using the Tukey multiple comparisons test and Students t test. RESULTS: The animals treated with neuregulins had greater numbers of myelinized axons, with a statistically significant difference in relation to the collagen-only group. There was no statistical difference between the neuregulin 1-alpha and 1- beta groups. CONCLUSION: It was concluded that the addition of neuregulins provided a significant increase in the number of myelinized fibers.

Orientador: Alexandre Leite Rodrigues de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T08:37:52Z (GMT). No. of bitstreams: 1 Cartarozzi_LucianaPolitti_M.pdf: 5081962 bytes, checksum: 794c493e497a269465a5b035175dfbcb (MD5) Previous issue date: 2013 / Resumo: A regeneração nervosa periférica é um processo complexo dependente do rearranjo e ativação das células de Schwann. O estímulo das células de Schwann pode ser alcançado através do enxerto de células tronco exógenas. Com o intuito de entender a importância do enxerto de células tronco mesenquimais (MSC) no processo regenerativo periférico, utilizamos o modelo de tubulização do nervo isquiático. As próteses tubulares foram preparadas a partir de membranas de poli-caprolactona (PCL) e preenchidas com selante de fibrina (FG), utilizado, neste caso, como substrato para as MSC. A técnica de tubulização foi feita em ratas fêmeas Lewis adultas, divididas em 4 grupos (n = 5 por grupo): normal, PCL (tubo vazio); FG (tubo preenchido com selante de fibrina) e FG+MSC (tubo preenchido com selante de fibrina e enxertado com MSC). Sessenta dias após lesão, os nervos regenerados foram processados para imunoistoquímica e microscopia de luz. A presença de MSC GFP-positivas foi detectada nos nervos dos animais que receberam enxerto de MSC, indicando que a sobrevivência, a longo prazo, das células tronco no tecido. A regeneração axonal, analisada por imunoistoquímica, revelou expressão de elementos básicos do nervo periférico, ou seja, componentes dos axônios e da lâmina basal tiveram a expressão equivalente em todos os grupos experimentais. A organização axonal foi observada através da marcação anti-neurofilamento. A presença das células de Schwann foi analisada através da marcação anti-S100 e o anticorpo anti-colágeno IV foi utilizado para detecção da lâmina basal. A imunomarcação anti-p75NTR, o receptor de baixa afinidade para neurotrofinas, foi utilizada para investigar a reatividade das células de Schwann. A marcação basal deste, em nervos não lesionados, foi aumentada pelo processo regenerativo, sendo estatisticamente maior no grupo FG+MSC (77% em relação ao nervo contralateral; p<0.001). Além disso, houve colocalização de MSC GFP-positivas e imunomarcação anti-BDNF, evidenciando uma possível via de atuação das células sobre o comportamento das células de Schwann. A partir da análise das secções semi-finas dos nervos pudemos avaliar que a área dos nervos regenerados no interior das próteses tubulares foi estatisticamente igual nos diferentes grupos experimentais. Quando quantificamos o número de axônios mielinizados por uma área fixa, o grupo FG+MSC apresentou maior densidade de axônios em relação ao grupo controle (25%, p<0,05). Da mesma maneira, quando analisamos os parâmetros morfométricos nos diferentes grupos experimentais, o grupo FG+MSC apresentou uma tendência a apresentar axônios de maior calibre e bainha de mielina mais espessa, em relação aos demais grupos, sendo que, a EBM, no intervalo de 1,46 a 2,25?m, foi significantemente maior em relação aos grupos PCL e FG (p<0,05). Como consequência, os animais do grupo FG+MSC mostraram recuperação motora significativamente maior na sétima e oitava semana de análise do índice funcional do nervo fibular. Os achados deste estudo mostram que as MSC enxertadas conjuntamente com selante de fibrina influenciam positivamente o processo regenerativo, modulando a reatividade das células de Schwann / Abstract: Peripheral nerve regeneration is a complex process that is dependent on the rearrangement and activation of Schwann Cells (SC). Such stimulation of SCs may be achieved by the use of exogenous stem cells. In order to better understand the importance of mesenchymal stem cell (MSC) grafting in the peripheral regeneration process we have used the model of sciatic nerve tubulization. Tubular prostheses were prepared from polycaprolactone (PCL) membranes and filled with fibrin sealant (FS), which was used as a substrate for the MSC. The technique of tubulization was applied in adult Lewis female rats that were divided into four groups (n = 5 per group): normal, PCL (empty tube), FS (tube filled with fibrin sealant) and FS + MSC (tube filled with fibrin sealant and grafted with MSC). Sixty days after injury, the regenerated nerves were processed for imunohistochemistry and observed under fluorescence microscopy. The presence of GFP positive stem cells was detected in the nerves of the animals that received MSC grafts, indicating the long term survival of such cells. The axonal regeneration process was studied by immunohistochemistry and revealed the presence of the basic elements of the peripheral nerve, namely axons and basal lamina components that were equivalent in all experimental groups. The axonal organization was observed with anti-neurofilament immunostaining. The presence of SCs was analyzed with anti-S100 immunostaining and anti-type IV collagen was used to detect the basal lamina. Anti-p75NTR, the low affinity receptor for neurotrophins, was used to investigate the reactivity of the SCs. A basal positive labeling in uninjured nerves was detected, which was upregulated by the regenerative process, being statistically higher in FS + MSC group (77% relative to uninjured nerve; p<0.001). Moreover there was colocalization between GFP-positive MSC and anti-BNDF immunolabeling, showing a possible pathway that these cells induce the reactivity of SCs. From sciatic nerve semi-thin sections we were able to evaluate that the areas of regenerated nerves were statistically the same in the different experimental groups. When we quantified the number of myelinated axons in 50.000?m2, the FG+MSC group showed higher density of axons when compared with PCL group (25%, p<0,05). In the same way, the analysis of morphometric parameters showed that the FG+MSC group have a tendency to present higher caliber axons and ticker myelin sheath when compared with other groups, being that the myelin sheath thickness, in the interval between 1,46 to 2,25?m, was significantly higher in FG+MSC group when compared to PCL and FG (p<0,05). As the functional result of the findings above, the FG+MSC animals showed higher motor function recovery, analyzed by FFI, at seventh and eighth weeks after lesion. The findings herein show that MSC associated with the FS scaffold improve the regeneration process by positively modulating the reactivity of SCs / Mestrado / Biologia Celular / Mestra em Biologia Celular e Estrutural

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.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23056号 / 医博第4683号 / 新制||医||1048(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 戸口田 淳也, 教授 森本 尚樹, 教授 伊佐 正 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

6-hydroxydopa (6-OHDOPA) was synthesized with the expectation that it would be able to cross the blood-brain barrier to be enzymatically decarboxylated to 6-hydroxydopamine (6-OHDA), the newly discovered neurotoxin for noradrenergic and dopaminergic neurons. In part, 6-OHDOPA fulfilled these criteria. When administered experimentally to rodents, 6-OHDOPA destroyed peripheral sympathetic noradrenergic nerves and did exert neurotoxicity to noradrenergic nerves in brain—in large part, from its conversion to 6-OHDA. However, the efficacy of 6-OHDOPA was less than that of 6-OHDA; also, 6-OHDOPA was relatively selective for noradrenergic neurons; near-lethal doses of 6-OHDOPA were required to damage dopaminergic nerves; and ultimately, 6-OHDOPA was found to be an agonist at AMPA receptors, thus accounting for more non-specificity. Nevertheless, 6-OHDOPA was found to be a particularly valuable tool in uncovering processes and mechanisms associated with noradrenergic nerve regeneration and sprouting, particularly when administered to perinatal rodents. Also, 6-OHDOPA was a good tool for selective mapping of noradrenergic nerve tracts in brain, since dopaminergic tracts were unaffected and did not interfere with the histofluorescent methodology used for this purpose in the early 1970s. As an experimental research tool, 6-OHDOPA was valuable in a short time-window, but its utility is largely limited because of newer research technologies that provide better means today for nerve tract mapping, and for experimental approaches engaged toward study of processes and mechanisms attending nerve regeneration. AMPA actions of 6-OHDOPA have not been extensively studied, so this avenue may enliven use of 6-OHDOPA in the future.

The use of autografts is the gold standard for peripheral nerve regeneration (PNR) while biomedical engineering made some contributions to improve PNR. A next generation of nerve guidance conduits (NGC) is required to transmit topographical and biochemical signals towards severed nerves. In this thesis, the gelatin hydrolyzate Collagel® (COL) and anhydride-containing cross-linkers (oPNMA, oPDMA) were used to fabricate crosslinked hydrogels (cGEL) for PNR. At first, established cGEL formulations were adjusted towards an injection-molding tool with static mixer. Therefore, the gelation kinetic was modified by variation of the gelation base. Hence, high reactive oPNMA was available for fabrication of robust cGEL based NGC. Secondly, novel cGEL and molding technique were adapted towards the fabrication of cGEL-based filler for polymer-derived braided NGC. Shear-thinning filler was developed that allowed direct application inside the conduit lumen with minimal mechanical stiffness but sufficient scaffolding properties. Besides pristine filler, chemically modified filler was designed with a small mimetic of the nerve growth factor, LM11A-31, that was grafted to oPNMA. In a rat sciatic nerve model, the performance of this derivatized filler was comparable to the control and underlined the potential of chemical cues in PNR. A number of small diamines were further integrated into oPNMA and oPDMA to modify cGEL bulk. In addition to chemical feasibility, the cytocompatibility and cellular response were tested on L929 mouse fibroblasts and human adipose-derived stem cells. The functionalization showed an impact on the cell behavior with differences in cell proliferation, migration and spreading. Finally, modified oPNMA-derived hydrogels were tested on neonatale Schwann cells. The cell viability and extension was maintained in all hydrogels while the impact of LM11A-31 was not as pronounced. This thesis emphasizes the potential of cGEL hydrogels in nerve implants as fillers or conduits and, thus, is a promising building block for a new generation of NGC.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19398号 / 医博第4049号 / 新制||医||1012(附属図書館) / 32423 / 京都大学大学院医学研究科医学専攻 / (主査)教授 戸口田 淳也, 教授 妻木 範行, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM