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
Although the majority of peripheral-nerve regeneration studies are carried out on the sciatic nerve, lesions of the upper extremities are more common in humans and usually lead to significant physical disabilities. The present study was driven by the hypothesis that a combination of strategies, namely grafts of mesenchymal stem cells (MSC) and resorbable polycaprolactone (PCL) conduits would improve median-nerve regeneration after transection. Mouse median nerves were transected and sutured to PCL tubes that were filled with either green fluorescent protein (GFP(+)) MSC in DMEM or with DMEM alone. During the post-operative period, animals were tested weekly for flexor digitorum muscle function by means of the grasping test. After 8 weeks, the proximal and middle portions of the PCL tube and the regenerating nerves were harvested and processed for light and electron microscopy. The flexor digitorum muscle was weighed and subjected to biochemical analysis for creatine phosphokinase (CK) levels. Scanning electron microscopy of the PCL tube 8 weeks after implantation showed clear signs of wall disintegration. MSC-treated animals showed significantly larger numbers of myelinated and unmyelinated nerve fibers and blood vessels compared with DMEM-treated animals. The flexor digitorum muscle CK levels were significantly higher in the MSC-treated animals, but muscle weight values did not differ between the groups. Compared with the DMEM-treated group, MSC-treated animals showed, by the grasping test, improved functional performance throughout the period analyzed. Immunofluorescence for S-100 and GFP showed, in a few cases, double-labeled cells, suggesting that transplanted cells may occasionally transdifferentiate into Schwann cells. Our data demonstrate that the polycaprolactone conduit filled with MSC is capable of significantly improving the median-nerve regeneration after a traumatic lesion.