Análise dos receptores de acetilcolina e proteínas associadas no reparo de nervo periférico após uso de selante de fibrina e neurorrafia: estudo experimental em ratos
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Data
2019-02-27
Autores
Pinto, Carina Guidi [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
As lesões nervosas periféricas (LNPs) levam a uma perda da conexão entre o axônio e o músculo, ocasionandointerrupção na transmissão do impulso nervoso e alterações nas junções neuromusculares (JNMs). O reparo das LNPs através da neurorrafia é um método amplamente utilizado, porém a recuperação tanto morfológica e funcional é incompleta, sendo um desafio para a clínica médica. Diante disso métodos alternativos como uso de selante de fibrina vêm sendo utilizados para minimizar danos e acelerar a recuperação nervosa e muscular, havendo poucas referências em relação às JNMs. O objetivo deste estudo foi verificar se o uso do selante de fibrina associado a um ponto de sutura atinge ou supera os resultados da sutura convencional após LNP com foco nos receptores de acetilcolina (nAChRs) e proteínas associadas às JNMs no músculo sóleo. Foram utilizados 40 ratos Wistar machos adultos (CEUA: 1173/2016), divididos em 4 grupos: Controle-Sham (CS), Controle-Desnervado (CD), Lesão Sutura (LS) e Lesão Sutura + Selante de Fibrina (LSS). No grupo CS foi realizada incisão, afastamento da musculatura e localização do nervo isquiático direito. No grupo CD foi realizada neurotmese (gap de 6 mm).Nos grupos CD, LS e LSS foi realizadafixação dos cotos na musculaturaapós a neurotmese. Após 7 dias foi realizada reconexão dos cotos com três pontos de sutura no grupo LS e no grupo LSS um ponto de sutura foi associada ao selante de fibrina(CEVAP). Após 60 dias os animais foram eutanasiados, os músculos sóleos removidos e analisados através de microscopia confocal para obter a morfologia e morfometria dos nAChRs(α-bungaratoxina) e a morfologia do terminal nervoso (NF200) e através do Western Blotting para obter a quantificação proteica dos nAChRs (α1, ε e γ), S100, Agrina, MMP-3, MuSK e Rapsina. A análise qualitativa da microscopia confocal no grupo CS mostrou nAChRs com distribuição compatível com a normalidade com braços contínuos e terminais nervosos íntegros, preservados e sem descontinuidade. No grupo CD foi observado achatamento na placa motora, fragmentação dos nAChRs, com distribuição em “ilhas” e terminais nervosos emaranhados. Nos grupos LS e LSS a morfologia mostrou-se intermediária entre os grupos CS e CD, com terminais nervosos mais delgados. Os dados morfométricos foram compatíveis com a morfologia, nos grupos LS e LSS apresentaram valores intermediários entre os grupos CS e CD e houve aumento da área relativa planar dos grupos LS e LSS, evidenciando uma menor fragmentação dos nAChRs. Os valores encontrados na expressão proteica nos grupos LS e LSS foram semelhantes ao CS. Neste estudo foi observado que após reconstrução nervosa, nos grupos LS e LSS houve um retorno dos nAChRs ao estágio maduro, justificado pela expressão proteica da subunidade ε (madura). Valores inversos foram encontrados para a subunidade γ (imatura, desnervada). Desse modo pode-se concluir que houve semelhança entre os grupos com reconstrução nervosaevidenciando a recuperação após reconexão dos cotos nervosos, considerando que houve retorno da estrutura morfológica com menor fragmentação dos nAChRs e retorno das proteínas associadas à JNM ao seu padrão maduro e o selante de fibrina com a redução do número de pontos de sutura mostrou-se promissor com um retorno acentuado da subunidadeγrelacionado ao padrão imaturo-desnervado do receptor.
Peripherical nerve lesions (PNLs) lead to a loss of the connection between axons and muscle, resulting in the interruption of the transmission of nerve impulses and alterations in the neuromuscular junctions (NMJs). The repair of PNLs through neurorrhaphy is a widely utilized method, however the recuperation of both morphology and function is incomplete, challenging medical clinic. Thus, alternative methods such as the usage of fibrin sealants have been utilized to minimize damages and accelerate nerve and muscular recuperation, with few references in relation to NMJs. The objective of this study was to verify if the use of fibrin sealants associated to a stitch of suture reaches or surpasses the results of traditional suture after PNLs with focus in the acetylcholine receptors (nAChRs) and proteins associated to the NMJs in the soleus muscle. Forty male adult Wistar rats were utilized (CEUA: 1173/2016), divided into4 groups: Sham-Control (SC), Denervated-Control (DC), Suture Lesion (SL) and Suture Lesion + Fibrin Sealant (SFS). In SC group,it was performed incision, muscle spacing and identification of the right sciatic nerve. In DC group,it was performed neurotmesis (gap of 6 mm). In DC, SL and SFS groups,it wascarried out fixation of stumps on the musculature after neurotmesis. After 7 days the reconnection of the stumps with suture was accomplished in SL group and in SFS group the suture was associated with the fibrin sealant (CEVAP). After 60 days the animals were euthanized,soleus muscles were removed and analyzed by confocal microscopy to obtain the morphology and morphometry of the nAChRs (α-bungaratoxin) and the morphology of the nerve terminal (NF200) and by western blotting to obtain the quantification of nAChRs (α1, ε e γ), S100, Agrin, MMP-3, MuSK and Rapsin. The qualitative assessment showed in SC group that nAChRs presented a normal morphology with continuous branches and preserved nerve terminals. In DC group it was observed flattening of the neuromuscular junction, fragmentation of nAChRs, with distribution in “islands” and tangled nerve terminals. In SL and SFS groups, the morphology was in-between groups SC and DC, with thinner nerve terminals. The morphometric data were compatible with the morphology and the majority of the parameters of SL and SFS groups presented values in-between the SC and DC groups. There was an increase of relative planar area in the reconstruction groups, highlighting that there was less nAChRs fragmentation. The values found in the protein expression in SL and SFS groups were similar to SC group and showed a return of the nAChRs mature pattern justified for the ε subunit expression. Inverse values were found in γ subunit. Based on the similarity of observed results in reconstruction groups, it was possible to conclude that there was a return of both nAChRs morphology and associated proteins to the mature pattern. The fibrin sealant associated with reduced number of stitches was viable due to the smaller values of immature receptor (γ) expression compared to the suture group.
Peripherical nerve lesions (PNLs) lead to a loss of the connection between axons and muscle, resulting in the interruption of the transmission of nerve impulses and alterations in the neuromuscular junctions (NMJs). The repair of PNLs through neurorrhaphy is a widely utilized method, however the recuperation of both morphology and function is incomplete, challenging medical clinic. Thus, alternative methods such as the usage of fibrin sealants have been utilized to minimize damages and accelerate nerve and muscular recuperation, with few references in relation to NMJs. The objective of this study was to verify if the use of fibrin sealants associated to a stitch of suture reaches or surpasses the results of traditional suture after PNLs with focus in the acetylcholine receptors (nAChRs) and proteins associated to the NMJs in the soleus muscle. Forty male adult Wistar rats were utilized (CEUA: 1173/2016), divided into4 groups: Sham-Control (SC), Denervated-Control (DC), Suture Lesion (SL) and Suture Lesion + Fibrin Sealant (SFS). In SC group,it was performed incision, muscle spacing and identification of the right sciatic nerve. In DC group,it was performed neurotmesis (gap of 6 mm). In DC, SL and SFS groups,it wascarried out fixation of stumps on the musculature after neurotmesis. After 7 days the reconnection of the stumps with suture was accomplished in SL group and in SFS group the suture was associated with the fibrin sealant (CEVAP). After 60 days the animals were euthanized,soleus muscles were removed and analyzed by confocal microscopy to obtain the morphology and morphometry of the nAChRs (α-bungaratoxin) and the morphology of the nerve terminal (NF200) and by western blotting to obtain the quantification of nAChRs (α1, ε e γ), S100, Agrin, MMP-3, MuSK and Rapsin. The qualitative assessment showed in SC group that nAChRs presented a normal morphology with continuous branches and preserved nerve terminals. In DC group it was observed flattening of the neuromuscular junction, fragmentation of nAChRs, with distribution in “islands” and tangled nerve terminals. In SL and SFS groups, the morphology was in-between groups SC and DC, with thinner nerve terminals. The morphometric data were compatible with the morphology and the majority of the parameters of SL and SFS groups presented values in-between the SC and DC groups. There was an increase of relative planar area in the reconstruction groups, highlighting that there was less nAChRs fragmentation. The values found in the protein expression in SL and SFS groups were similar to SC group and showed a return of the nAChRs mature pattern justified for the ε subunit expression. Inverse values were found in γ subunit. Based on the similarity of observed results in reconstruction groups, it was possible to conclude that there was a return of both nAChRs morphology and associated proteins to the mature pattern. The fibrin sealant associated with reduced number of stitches was viable due to the smaller values of immature receptor (γ) expression compared to the suture group.
Descrição
Palavras-chave
Junção Neuromuscular, Lesão Nervosa Periférica, Selante de Fibrina