Virus production: adeno-associated virus (AAV).

BACKGROUND: Adeno-associated virus (AAV) vector-mediated transgene expression is a promising therapeutic to change the intrinsic state of neurons and promote repair after central nervous system injury. Given that numerous transgenes have been identified as potential candidates, the present study demonstrates how to determine whether their expression by AAV has a direct intrinsic effect on axon regeneration. METHODS: Serotype 2 AAV-enhanced green fluorescent protein (EGFP) was stereotaxically injected into the brainstem of adult rats, followed by a complete transection of the thoracic spinal cord and Schwann cell (SC) bridge implantation. RESULTS: The expression of EGFP in brainstem neurons labeled numerous axons in the thoracic spinal cord and that regenerated into the SC bridge. The number of EGFP-labeled axons rostral to the bridge directly correlated with the number of EGFP-labeled axons that regenerated into the bridge. Animals with a greater number of EGFP-labeled axons rostral to the bridge exhibited an increased percentage of those axons found near the distal end of the bridge compared to animals with a lesser number. This suggested that EGFP may accumulate distally in the axon with time, enabling easier visualization. By labeling brainstem axons with EGFP before injury, numerous axon remnants undergoing Wallerian degeneration may be identified distal to the complete transection up to 6 weeks after injury. CONCLUSIONS: Serotype 2 AAV-EGFP enabled easy visualization of brainstem axon regeneration. Rigorous models of axonal injury (i.e. complete transection and cell implantation) should be used in combination with AAV-EGFP to directly assess AAV-mediated expression of therapeutic transgenes as intrinsic treatments to improve axonal regeneration.