Enhancing spinal cord repair with connexin43 antisense oligodeoxynucleotides

Abstract

Spinal cord injury is comprised of the initial injury and the secondary effects including injury spread, swelling, inflammation, and scarring. Connexin43 (Cx43) gap junctions and hemichannels contribute significantly to spinal cord injury by remaining open in dying cells, spreading toxic substances to neighbouring cells and the surrounding extracellular environment, and exacerbating inflammation. Peripheral nerve grafts and self-assembling IKVAV-PA nanoparticles have been used successfully to promote axon outgrowth from damaged spinal cord but such regeneration is limited by new scarring resulting from the intervention itself. We have investigated the effect of Cx43 downregulation in preventing the onset of secondary injury spread and enhancing these spinal cord injury repair strategies by promoting axon regeneration and behavioural improvements. An ex vivo spinal cord segment culture model was established using rat spinal cord segments cultured for five days. The segments were treated with Cx43 antisense oligodeoxynucleotides (Cx43 AsODN) resulting in viable, organotypic, airliquid interface cultures that were then used for repair studies. Fresh peripheral nerves were grafted into the spinal cord segments with Cx43AsODN applied concomitantly, followed by five days of culture and subsequent processing. Results showed that spinal cord segments cultured in the presence of Cx43 AsODN had significantly less swelling than control spinal cords, indicating the efficacy of this treatment in reducing initial cellular and tissue swelling. Improved neuronal survival, tissue preservation and axon regeneration from the injured spinal cord segments into the grafted peripheral nerve was seen in this Cx43 AsODN treated organotypic culture model. In vivo peripheral nerve grafting and Cx43 AsODN treatment was then carried out following complete transection injury in adult rats; axonal regeneration and behavioural improvement was assessed. Cx43 AsODN promoted rostral axon regeneration and behavioural improvement, and reduced the extent of the lesion. The physical properties of selfassembling IKVAV-PA nanoparticles were then investigated in vitro. The nanoparticles were found to be suitable as a stable and injectable implant material. Cx43 AsODN was combined with the IKVAV-PA gel implant and applied following an in vivo complete transection injury and behavioural and axon regeneration assessments undertaken. Although the complete transection model proved difficult, long term behavioural improvement was evidenced with signs of enhanced axon regeneration. In conclusion, ii Cx43 AsODN treatment may offer an innovative combination therapy to enhance other spinal cord repair strategies that are applied at later time points following injury.