RESUMO
Peripheral-nerve injuries are a common clinical problem and often result in long-term functional deficits. Reconstruction of peripheral-nerve defects is currently undertaken with nerve autografts. However, there is a limited availability of nerves that can be sacrificed and the functional recovery is never 100% satisfactory. We have previously shown that gene therapy with vascular endothelial growth factor (VEGF) significantly improved nerve regeneration, neuronal survival, and muscle activity. Our hypothesis is that granulocyte colony-stimulating factor (G-CSF) synergizes with VEGF to improve the functional outcome after sciatic nerve transection. The left sciatic nerves and the adjacent muscle groups of adult mice were exposed, and 50 or 100 µg (in 50 µl PBS) of VEGF and/or G-CSF genes was injected locally, just below the sciatic nerve, and transferred by electroporation. The sciatic nerves were transected and placed in an empty polycaprolactone (PCL) nerve guide, leaving a 3-mm gap to challenge nerve regeneration. After 6 weeks, the mice were perfused and the sciatic nerve, the dorsal root ganglion (DRG), the spinal cord and the gastrocnemius muscle were processed for light and transmission electron microscopy. Treated animals showed significant improvement in functional and histological analyses compared with the control group. However, the best results were obtained with the G-CSF+VEGF-treated animals: quantitative analysis of regenerated nerves showed a significant increase in the number of myelinated fibers and blood vessels, and the number of neurons in the DRG and motoneurons in the spinal cord was significantly higher. Motor function also showed that functional recovery occurred earlier in animals receiving G-CSF+VEGF-treatment. The gastrocnemius muscle showed an increase in weight and in the levels of creatine phosphokinase, suggesting an improvement of reinnervation and muscle activity. These results suggest that these two factors acted synergistically and optimized the nerve repair potential, improving regeneration after a transection lesion.
Assuntos
Fator Estimulador de Colônias de Granulócitos/uso terapêutico , Regeneração Nervosa/fisiologia , Recuperação de Função Fisiológica/fisiologia , Neuropatia Ciática/terapia , Fator A de Crescimento do Endotélio Vascular/uso terapêutico , Análise de Variância , Animais , Creatina Quinase/metabolismo , Modelos Animais de Doenças , Feminino , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Gânglios Espinais/ultraestrutura , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Locomoção/genética , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Regeneração Nervosa/genética , Recuperação de Função Fisiológica/genética , Neuropatia Ciática/patologia , Medula Espinal/patologia , Medula Espinal/ultraestrutura , Transplante , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
AIMS: Recent studies have emphasized the beneficial effects of the vascular endothelial growth factor (VEGF) on neurone survival and Schwann cell proliferation. VEGF is a potent angiogenic factor, and angiogenesis has long been recognized as an important and necessary step during tissue repair. Here, we investigated the effects of VEGF on sciatic nerve regeneration. METHODS: Using light and electron microscopy, we evaluated sciatic nerve regeneration after transection and VEGF gene therapy. We examined the survival of the neurones in the dorsal root ganglia and in lumbar 4 segment of spinal cord. We also evaluated the functional recovery using the sciatic functional index and gastrocnemius muscle weight. In addition, we evaluated the VEGF expression by immunohistochemistry. RESULTS: Fluorescein isothiocyanate-dextran (FITC-dextran) fluorescence of nerves and muscles revealed intense staining in the VEGF-treated group. Quantitative analysis showed that the numbers of myelinated fibres and blood vessels were significantly higher in VEGF-treated animals. VEGF also increased the survival of neurone cell bodies in dorsal root ganglia and in spinal cord. The sciatic functional index and gastrocnemius muscle weight reached significantly higher values in VEGF-treated animals. CONCLUSION: We demonstrate a positive relationship between increased vascularization and enhanced nerve regeneration, indicating that VEGF administration can support and enhance the growth of regenerating nerve fibres, probably through a combination of angiogenic, neurotrophic and neuroprotective effects.