Stereological evaluation of post-traumatic peripheral nerve regenerating fibers and denervated-related muscle fibers after VEGF gene therapy.

After peripheral nerve injury it is necessary to improve post-traumatic nerve repair and to prevent the fiber atrophy of the denervated related skeletal muscle. In this study we applied the gene therapy based on Adeno-Associated Virus (AAV) to determine the over-expression of vascular endothelial growth factor (VEGF) in both regenerating peripheral nerve and in the denervated skeletal muscle. Rat median nerve defect was reconstructed by a vein segment filled with muscle fibers previously injected with either AAV2-VEGF or AAV2-LacZ, and the morphological outcome of nerve regeneration was assessed three months after surgery. A second experimental group was used to study the prevention of muscle atrophy evaluating the effect of VEGF over-expression on denervated muscles. One semi-thin section from each nerve and from each muscle was randomly selected and used for the quantitative analysis. We used a design-based quantitative morphology to evaluated some of the most important geometrical parameters that can be used for the assessment of nerve regeneration: number of fibers, density of fibers, axon and fiber diameter and myelin thickness. For the stereological evaluation of muscle fibers size we measured the cross sectional area of each muscle fiber inside sampling fields randomly selected. Results showed that over-expression of VEGF in the muscle-vein-combined guide interfered with the normal muscle degeneration inside the scaffold leading to a worse nerve regeneration. On the other hand, a positive effect was observed in the muscle treated with AAV2-VEGF that showed a significantly lower progression of atrophy in comparison to muscles treated with AAV2-LacZ. In conclusion, while local delivery of VEGF by AAV2-VEGF-injected muscle fibers did not represent a rational approach to promote axon regeneration along a nerve guide, AAV2-VEGF in a denervated skeletal muscle significantly prevents denervation-related atrophy thus creating a promising new strategy for improving the outcome of posttraumatic neuromuscular recovery. Keywords: peripheral nerve, skeletal muscle, gene therapy, VEGF, AAV This research was supported by grants MOVAG from the Compagnia di San Paolo and the European Community's Seventh Framework Programme (FP7-HEALTH-2011) under grant agreement n°278612".