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Doctoral Thesis
DOI
https://doi.org/10.11606/T.25.2013.tde-11042015-092834
Document
Author
Full name
Farooque Jamaluddin Ahmed
Institute/School/College
Knowledge Area
Date of Defense
Published
Bauru, 2013
Supervisor
Committee
Rodrigues, Antonio de Castro (President)
Carreira, Daniela Gamba Garib
Matsumoto, Mariza Akemi
Pegoraro, Camila de Oliveira Rodini
Silva, Alan Roger dos Santos
Title in Portuguese
Análise da expressão de neurotrofinas durante a regeneração de nervo periférico de rato por enxerto venoso
Keywords in Portuguese
Enxerto de veia
Nervo isquiático
Neurotrofinas
Abstract in Portuguese
Análise da expressão de neurotrofinas durante a regeneração de nervo periférico de rato por enxerto venoso Enxertos de veias têm sido empregados para preencher lacunas em nervos periféricos transeccionados para melhor recuperação funcional. No entanto, vários inconvenientes, como a constrição do enxerto secundário foram observados. Uma nova alternativa para esta técnica foi desenvolvida. Simplesmente invertendo a veia de dentro para fora, chamado do Inside- out vein graft. As neurotrofinas são uma família de fatores neurotróficos conhecidos por desempenhar um papel significativo na regeneração de nervos periféricos. A família da neurotrofina é constituído por fator de crescimento nervoso (NGF), fator neurotrófico derivado do cérebro (BDNF), Neurotrofina-3 (NT-3) e Neurotrofina-4 (NT-4). No campo da neurobiologia, vários autores têm utilizado a técnica de PCR a fim de obter mais informações sobre os nervos regenerados. Neste estudo, foi utilizada a técnica de biologia molecular para explorar o papel e o nível das neurotrofinas durante a regeneração de nervos periféricos com enxerto de veia. O nervo isquiático de ratos foi seccionado e reparado com enxerto de veia invertida (IOVG) e técnicas de enxerto de veia padrão (SVG). No grupo controle, os ratos foram operados e o nervo isquiático foi mantido intacto. Os animais foram sacrificados após 6 e 12 semanas e os enxertos foram colhidos para observar o nível das neurotrofinas. Músculos EDL e Sóleo foram excisados e pesados para determinar a diferença de peso entre os grupos. Um pequeno segmento dos cotos distais de ambos os grupos SVG e IOVG também foram excisados e foram processados histologicamente para examinar a quantidade de axónios regenerados. Além disso, um outro pequeno segmento do coto distal foi processado para RT-PCR para analisar o nível das neurotrofinas nesta área.A tecnica do walk track analysis foi realizada para determinar o índice funcional do nervo isquiático nos grupos. Em 6 semanas, não ocorreu crescimento neuronal significativo no coto distal dos dois tipos de enxertos, porém um crescimento foi observado em 12 semanas. Não houve diferença significativa na massa muscular entre IOVG e SVG em ambos os períodos de tempo. No entanto, um aumento significativo na massa muscular foi observado a partir de 6 a 12 semanas nos grupos IOVG e SVG. Um aumento significativo na produção de NT-3 foi observado no grupo de SVG em ambos, enxerto e o coto distal quando comparados a partir de 6 a 12 semanas, no entanto, não houve aumento observado no nível de neurotrofinas dos outros tipos (NGF e NT-4) . Surpreendentemente, não houve aumento significativo da NT-3 no grupo IOVG. Conclui-se que, entre as neurotrofinas avaliadas neste estudo, não há nenhuma diferença significativa no seu nível de RNAm entre os dois grupos, exceto NT-3. Finalmente, uma vez que o nível de RNAm de NT-3 aumenta significativamente entre 6 e 12 semanas no grupo SVG e não no IOVG, observado por estas duas técnicas de nível molecular, estudos adicionais necessitam serem feitos para decifrar o mecanismo exato.
Title in English
Analysis of the expression of neurotrophins during regeneration of peripheral nerves in rats with vein graft
Keywords in English
Neurotrophins
Sciatic nerve
Vein graft
Abstract in English
Vein grafts have been employed to bridge the gap in transected peripheral nerves to produce better functional recovery. However several disadvantages such as secondary graft constriction were observed and a new alternative to this technique was developed by simply reversing the vein inside out. Both inside out and standard vein grafts were successfully used in recovering the sensory segmental defect in humans. Neurotrophins are a family of eurotrophic factors known to play an important role in the regeneration of peripheral nerves. The neurotrophin family consists of Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3) and Neurotropinh-4 (NT-4). In the neurobiology field, several authors have been using PCR technique in order to gain more information regarding regenerated nerves. In this study, we employed this molecular biology technique to explore the role and level of the neurotrophins during the peripheral nerve regeneration with vein graft. The sciatic nerve of rats were sectioned and repaired with Inside out vein graft (IOVG) and standard vein graft techniques (SVG). In the control group the rats were sham operated wherein the sciatic nerve was kept intact. The animals were euthanized at 6 and 12 weeks and the grafts were harvested to observe the level the neurotrophins. EDL and Sol muscles were excised and measured to determine any weight difference between the groups. A small segment of the distal stumps from both the SVG and IOVG groups were also excised and were subjected to histological process to examine the amount of regenerated axon. In addition, another small segment of the distal stump was processed for RT-PCR to further examine the level of the neurotrophins in this area. At 6 weeks, no significant neuronal growth was observed in the distal stump of both graft types but a distinct growth was seen at 12 weeks. Walk track analysis showed poor motor function recovery in the experimental groups during both time intervals. Morphometric analysis demonstrated no significant differences in the amount of myelination between both the groups. There was no significant difference in the muscle mass between IOVG and SVG in both time periods. However, a significant increase in both the muscle mass was observed from 6 to 12 weeks in the IOVG and SVG groups. A significant increase in the production of NT-3 was observed in SVG group in both the distal stump and graft segment when compared from 6 to 12 weeks; however there was no observed increase in the level of other neurotrophins (NGF and NT-4). Surprisingly, no significant increase of NT-3 was noticed in the IOVG group. We conclude that amongst the neurotrophins evaluated in this study, there is no significant difference in their mRNA level between both groups except NT-3. Also, since mRNA level of NT-3 increases significantly between 6 and 12 weeks in SVG and not in IOVG, it suggests that the mechanism by which these two techniques operate at a molecular level may differ and further studies need to be done to decipher the exact mechanism.
 
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Publishing Date
2015-04-15
 
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