Efeitos neuroprotetor e regenerativo do selante de fibrina derivado da peçonha de serpente combinado com células tronco mononucleares após reparo do nervo isquiático de ratos neonatos
Carregando...
Data
2016-02-29
Autores
Biscola, Natalia Perussi [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
A realização de reparos eficientes em lesões do sistema nervoso é um desafio para a medicina. O uso de modelos experimentais bem estabelecidos permite a avaliação de diferentes tratamentos a fim de prevenir as lesões causadas. O presente estudo investigou o potencial neuroprotetor e regenerativo de células tronco mononucleares da medula óssea (CTMMO), após reparo com selante de fibrina derivado da peçonha de serpente sobre neurônios motores e sensitivos, em ratos neonatos submetidos à axotomia do nervo isquiático. Para isso, os animais foram divididos em seis grupos: Grupo I (AX): axotomia do nervo isquiático sem tratamento; Grupo II (AX+SF): axotomia do nervo isquiático, seguida de reparo término terminal com selante de fibrina produzido pelo Centro de Estudos de Venenos de Animais Peçonhentos (CEVAP); Grupo III (AX+SF+CTMMO): axotomia do nervo isquiático, seguida de reparo término terminal com selante de fibrina do CEVAP e tratamento com CTMMO; Grupo IV (AX+CTMMO): axotomia do nervo isquiático, seguida de tratamento com CTMMO; Grupo V (AX+SFC): axotomia do nervo isquiático, seguida de reparo término terminal com selante de fibrina comercial; Grupo VI: Controle sem lesão (teste motor). Os animais foram analisados 4, 8 e 12 semanas após a lesão. Foram realizadas as técnicas de citometria de fluxo para caracterização das CTMMO; imunoistoquímica para verificação da cobertura sináptica, gliose reativa e resposta microglial; coloração de Nissl para sobrevivência neuronal; morfometria do nervo isquiático, para verificar a regeneração nervosa; morfometria da medula espinal, para avaliar os inputs sinápticos, além dos testes motor (Catwalk) e sensitivo (von Frey), para avaliar a melhora funcional e nociceptiva. Testes ANOVA, seguido de pós-teste Bonferroni foram utilizados para análises estatísticas. O reparo com selante resultou em preservação da cobertura sináptica e aumento significativo da sobrevivência neuronal nos grupos em que o reparo foi realizado apenas com os selantes, ou quando foi associado com as CTMMO (aproximadamente 54%), comparativamente aos grupos em que não foi utilizado selante (aproximadamente 32%), em todos os períodos analisados. As reatividades astroglial e microglial diminuíram nos mesmos grupos (AX+SF, AX+SF+CTMMO e AX+SFC), 4 semanas após a lesão. Além disso, o reparo possibilitou a regeneração axonal, resultando em maior número de fibras nervosas, com diâmetro e espessura da bainha de mielina próximas ao controle e neurônios com preferência por terminais inibitórios no momento de recuperação. Consequentemente, os animais apresentaram recuperação funcional e sensitiva. Portanto, os resultados apresentados sugerem que o reparo do nervo isquiático com selante de fibrina em ratos neonatos resulta em neuroproteção e regeneração de neurônios motores e sensitivos, sendo que não foram detectados benefícios adicionais com a terapia celular empregada.
The efficient repair in nervous system related lesions is an ancient challenge for medicine. The use of well-established experimental models allows to evaluate the potential of different treatments in order to prevent the induced changes. The aim of this study was to evaluate the neuroprotective and regenerative potential of bone marrow mononuclear stem cells (BMMSC) after neurorrhaphy with fibrin sealant derived from snake venom on spinal motoneurons and DRG sensory neurons of neonatal rats subjected to sciatic nerve axotomy. Therefore, the animals were divided into six groups: Group I (AX): axotomy of the sciatic nerve without treatment; Group II (AX+FS): axotomy of the sciatic nerve, followed by end-to-end neurorrhaphy with fibrin sealant produced by CEVAP; Group III (AX+FS+BMMSC): axotomy of sciatic nerve, followed by treatment with BMMSC and endto- end neurorrhaphy with fibrin sealant from CEVAP; Group IV (AX+BMMSC): sciatic nerve axotomy and treatment with BMMSC; Group V(AX+CFS): axotomy of the sciatic nerve, followed by end-toend neurorrhaphy with commercial fibrin sealant; Group VI: Control group without lesion (motor tests). The samples were analyzed 4, 8 and 12 weeks post lesion. Flow cytometry was used to characterize the BMMSC. Immunohistochemistry to evaluate reactive gliosis, microglial reaction and synapse preservation. Nissl staining was employed to investigate neuronal survival; axonal regeneration by sciatic nerve morphometry; synaptic inputs, by spinal cord morphometry. Additionally, motor (walking track test) and sensitive (von Frey) behavior recovery was studied and correlated with morphological data. ANOVA and Bonferroni post-test was used for statistical analyses. Neurorrhaphy indicated preservation of synaptic covering on lesioned motoneurons and neuronal survival, in the groups repaired with both sealant alone or when treated with BMMSC (around 54%), as compared to axotomy alone (around 32%) in all analyzed survival times. Reactive gliosis and microglial reaction decreased in the same groups (AX+FS, AX+FS+BMMSC, AX+CFS) with 4 weeks. Regarding axonal regeneration, neurorrhaphy allowed recovery of greater number of myelinated fibers, that presented also improved morphometric parameters. In addition, preferential preservation of inhibitory synaptic terminals was detected in neurorrhaphy groups. Such results were accompanied by significant improvement in the motor as well as in the nociceptive recovery. Overall, the present data suggest that acute repair of neonatal peripheral nerves with fibrin sealant result in neuroprotection and regeneration of both motor and DRG neurons, not being further improved by BMMSC therapy.
The efficient repair in nervous system related lesions is an ancient challenge for medicine. The use of well-established experimental models allows to evaluate the potential of different treatments in order to prevent the induced changes. The aim of this study was to evaluate the neuroprotective and regenerative potential of bone marrow mononuclear stem cells (BMMSC) after neurorrhaphy with fibrin sealant derived from snake venom on spinal motoneurons and DRG sensory neurons of neonatal rats subjected to sciatic nerve axotomy. Therefore, the animals were divided into six groups: Group I (AX): axotomy of the sciatic nerve without treatment; Group II (AX+FS): axotomy of the sciatic nerve, followed by end-to-end neurorrhaphy with fibrin sealant produced by CEVAP; Group III (AX+FS+BMMSC): axotomy of sciatic nerve, followed by treatment with BMMSC and endto- end neurorrhaphy with fibrin sealant from CEVAP; Group IV (AX+BMMSC): sciatic nerve axotomy and treatment with BMMSC; Group V(AX+CFS): axotomy of the sciatic nerve, followed by end-toend neurorrhaphy with commercial fibrin sealant; Group VI: Control group without lesion (motor tests). The samples were analyzed 4, 8 and 12 weeks post lesion. Flow cytometry was used to characterize the BMMSC. Immunohistochemistry to evaluate reactive gliosis, microglial reaction and synapse preservation. Nissl staining was employed to investigate neuronal survival; axonal regeneration by sciatic nerve morphometry; synaptic inputs, by spinal cord morphometry. Additionally, motor (walking track test) and sensitive (von Frey) behavior recovery was studied and correlated with morphological data. ANOVA and Bonferroni post-test was used for statistical analyses. Neurorrhaphy indicated preservation of synaptic covering on lesioned motoneurons and neuronal survival, in the groups repaired with both sealant alone or when treated with BMMSC (around 54%), as compared to axotomy alone (around 32%) in all analyzed survival times. Reactive gliosis and microglial reaction decreased in the same groups (AX+FS, AX+FS+BMMSC, AX+CFS) with 4 weeks. Regarding axonal regeneration, neurorrhaphy allowed recovery of greater number of myelinated fibers, that presented also improved morphometric parameters. In addition, preferential preservation of inhibitory synaptic terminals was detected in neurorrhaphy groups. Such results were accompanied by significant improvement in the motor as well as in the nociceptive recovery. Overall, the present data suggest that acute repair of neonatal peripheral nerves with fibrin sealant result in neuroprotection and regeneration of both motor and DRG neurons, not being further improved by BMMSC therapy.
Descrição
Palavras-chave
Axotomia, Nervo isquiático, Neurorrafia, Regeneração nervosa, Selante de fibrina, Reparo