title: Double gene therapy with granulocyte colony-stimulating factor and vascular endothelial growth factor acts synergistically to improve nerve regeneration and functional outcome after sciatic nerve injury in mice.
abstract: 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.
authors.affiliation: Programa de Neurociência Básica e Clínica, Instituto de Ciências Biomédicas, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, RJ, Brazil.
authors.forename: F R
authors.initials: FR
authors.lastname: Pereira Lopes
authors.forename: P K M
authors.initials: PK
authors.lastname: Martin
authors.forename: F
authors.initials: F
authors.lastname: Frattini
authors.forename: A
authors.initials: A
authors.lastname: Biancalana
authors.forename: F M
authors.initials: FM
authors.lastname: Almeida
authors.forename: M A
authors.initials: MA
authors.lastname: Tomaz
authors.forename: P A
authors.initials: PA
authors.lastname: Melo
authors.forename: R
authors.initials: R
authors.lastname: Borojevic
authors.forename: S W
authors.initials: SW
authors.lastname: Han
authors.forename: A M B
authors.initials: AM
authors.lastname: Martinez
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D003402:Creatine Kinase / Q000378:metabolism; D004195:Disease Models, Animal; D005260:Female; D005727:Ganglia, Spinal / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D016179:Granulocyte Colony-Stimulating Factor / Q000235:genetics / Q000627:therapeutic use*; D006801:Humans; D008124:Locomotion / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron; D018482:Muscle, Skeletal / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D009416:Nerve Regeneration / Q000235:genetics / Q000502:physiology*; D020127:Recovery of Function / Q000235:genetics / Q000502:physiology*; D020426:Sciatic Neuropathy / Q000473:pathology / Q000628:therapy*; D013116:Spinal Cord / Q000473:pathology / Q000648:ultrastructure; D014180:Transplantation; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000627:therapeutic use*
chemical_list: D042461:Vascular Endothelial Growth Factor A; D016179:Granulocyte Colony-Stimulating Factor; D003402:Creatine Kinase