Orientador: William Dias Belangero / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T07:47:22Z (GMT). No. of bitstreams: 1 OliveiraFilho_OsvaldoMendesde_D.pdf: 2509545 bytes, checksum: 6effb16e1eedd8b6d33c41daea640b76 (MD5) Previous issue date: 2014 / Resumo: O objetivo deste trabalho foi comparar através da avaliação funcional pelo grasping test e análise histomorfométrica o tratamento da lesão do nervo mediano em ratos de linhagem Wistar através da microneurorrafia tradicional com a microneurorrafia associada à administração do monossialogangliosídeo (GM1) e avaliar especificamente se o GM1 melhora a regeneração axonal do nervo mediano e a função da musculatura por ele inervado. Material e Método: Foram empregados 32 ratos machos de linhagem Wistar. Destes, foram selecionados aleatoriamente 10 animais, grupo 0, para obtenção da força de preensão média em ratos normais, antes do procedimento cirúrgico. Esses animais foram reintegrados aos grupos. Foram criados o grupo I, com 10 animais, em que foi feita ressecção de 5 mm do nervo mediano do membro anterior direito e não foi submetido a nenhum tratamento. Nos outros grupos foi produzida uma lesão transversa do nervo mediano proximalmente ao epicôndilo medial criando-se os grupos II, tratados com microneurorrafia epineural externa e o grupo III, tratado com a microneurorrafia epineural externa associada à administração intraperitoneal de GM1. A cirurgia foi realizada imediatamente após a lesão e a técnica utilizada foi a sutura término-terminal. Foi realizada análise funcional semanal durante seis semanas através do teste de preensão da musculatura flexora dos dedos, que é específico para avaliar a ação do nervo mediano. Após esse período, os animais foram submetidos a eutanásia. As porções proximal e distal dos nervos foram coradas com azul de toluidina a 1% e realizada a análise histológica. Pela análise morfométrica obteve-se o número e diâmetro dos axônios nos cotos proximais e distais, criando-se uma nova fórmula com inclusão tanto do número como do diâmetro dos axônios para a avaliação da regeneração nervosa. Resultados: Os valores médios da força de preensão exercida pelos ratos do grupo 0 foram comparados aos animais dos grupos II e III através da análise de variância (ANOVA one way). Para a comparação dos valores médios da força realizada pelos ratos do grupo II e III foi feito o teste de Wilkoxon. Do ponto de vista funcional, o grupo III imprimiu uma maior força média com erro menor que 5% e realizou o teste de preensão mais precocemente. O grupo tratado com o GM1 apresentou um número 28% maior de axônios regenerados no segmento distal, com padrão histológico mais organizado e homogêneo e uma diferença significativa no diâmetro médio dos mesmos. Conclusão: Pode-se afirmar com erro menor que 5% que os grupos II (microneurorrafia) e III (microneurorrafia e GM1) apresentaram diferenças em relação à recuperação funcional, tendo o grupo III reagido melhor ao teste de preensão. O padrão histológico do grupo III apresentou maior grau de mielinização, tendo-se observado maior diâmetro médio nos axônios dos cotos distais (p=0,0056). Há um significativo indicio (p=0,0536) de que a utilização do GM1 nas cirurgias dos nervos periféricos melhora o padrão de regeneração axonal. Palavras Chaves: GM1, nervo mediano, regeneração axonal, ratos Wistar, avaliação funcional, morfometria / Abstract: Summary OBJECTIVES: The objective of this study was to compare the treatment of nerve median injuries in Wistar rats submitted to traditional microneurorraphy with the treatment that combined microneurorraphy and monossialoganglioside (GM1) administration while also specifically evaluating if GM1 promotes an increase in median nerve axonal regeneration, thus improving the function of the muscles in its territory of innervation. This comparison was done through functional evaluation measured by the grasping test and histomorphometric analysis. MATERIAL AND METHODS: Experiments were performed in thirty-two Wistar rats. Among them, 10 were randomly selected (group 0) to determine the average grasping strength in normal rats. These animals were then reunited with the others. There were three groups: group I (control group), submitted to a 5 mm lesion in the median nerve of the right forelimb and no treatment. Group II, submitted to lesion of the median nerve proximal to the medial epicondyle, treated with external epineural microneurorraphy, and group III, submitted to the same lesion and treated with external epineural microneurorraphy associated with intraperitoneal administration of GM1. Surgery was undertaken immediately after the damage to the nerve and end-to-end suture was used. Functional analysis through the grasping test of the flexor muscles of the fingers was assessed weekly; this test is specific to evaluate the action of the median nerve. In this experimental model, the animal is lifted by the tail and is stimulated to grasp a bar with its paw; the bar is located on the top of a conventional digital balance. While grasping the bar with its paw, the rat continued to be held by the tail until it releases the bar and the number on the scale is registered. After the functional evaluation the animals were euthanized. The proximal and distal portions of the nerves were colored with 1% toluidine blue dye. After the histologic exam, morphometric analysis was done by counting the number and diameter of the axons in the proximal and distal stumps. A new formula was designed including the number and diameter of the axons to evaluate nerve regeneration. RESULTS: The mean values of grasping strength exerted by rats in group I (control), were compared with group II (only microneurorraphy) and group III (microneurorraphy and GM1) through the analysis of variance (ANOVA one way). To compare the mean values of the strength sustained by rats in groups II and III, the Wilkoxon test was applied. From the functional perspective, the group that received GM1 performed the grasping test earlier, exerting a greater mean strength (error inferior to 5%). The microscopic analysis demonstrated that the group treated with GM1 showed a higher number of regenerated axons better organized and homogenous. And also that this group had a slightly thicker myelin sheath. There was a significant difference in the mean diameter of the axons of the distal segment and a number 28% higher of regenerated axons in the group treated with GM1. CONCLUSIONS: The authors can state with error inferior to 5% that the groups II and III showed differences in relation to functional recovery, group III performing better when submitted to the grasping test. Histological pattern of the group that received GM1 showed a higher degree of myelination. It was observed a greater mean diameter in the axons of distal stumps (p=0,0056). There is a significant indication (p=0,0536) that the use of GM1 in peripheral nerve surgery improves the pattern of axonal regeneration. Key Words: GM1, median nerve, axonal regeneration, Wistar rats, functional evaluation, morphometry / Doutorado / Medicina Interna / Doutor em Ciências Médicas

GM1 gangliosidosis is a lysosomal storage disorder caused by a deficiency in the catabolizing enzyme β-galactosidase (βgal). This leads to accumulation of GM1-ganglioside (GM1) in the lysosome inducing ER stress and cell death. GM1 gangliosidosis is primarily a disorder of the central nervous system (CNS) with peripheral organ involvement. In this work we report two major findings, 1) systemic treatment of GM1 gangliosidosis with an adenoassociated virus (AAV9) encoding mouse-βgal (mβgal) in a GM1 gangliosidosis mouse model (βGal-/-), and 2) an investigation into an intracranial injection of a therapeutic AAVrh8 encoding mβgal. Systemic treatment of GM1 gangliosidosis with AAV9 resulted in a moderate expression of enzyme in the CNS, reduction of GM1 storage, significant retention of motor function and a significant increase in lifespan. Interestingly, the therapeutic effect was more robust in females. Intracranial injections of AAVrh8 vector expressing high levels of βgal resulted in enzyme spread throughout the brain, significant retention of motor function and a significant increase in lifespan. Histological alterations were also found at the injection site in both βGal-/- and normal animals. We constructed a series of vectors with a range of decreasing enzyme expression levels to investigate the cause for the unanticipated result. Microarrays were performed on the injection site and we showed that a lower expressing AAVrh8-mβgal vector mitigated the negative response. Intracranial injection of this newly developed vector was shown to clear lysosomal storage throughout the CNS of βGal-/- mice. Taken together, these studies indicate that a combined systemic and fine-tuned intracranial approach may be the most effective in clearing lysosomal storage completely in the CNS while providing therapeutic benefit to the periphery.