The histological assessment of spinal cord tissue in three dimensions has previously been very time consuming and prone to errors of interpretation. Advances in tissue clearing have significantly improved visualization of fluorescently labelled axons. While recent proof-of-concept studies have been performed with transgenic mice in which axons were pre-labeled with GFP, investigating axonal regeneration requires stringent axonal tracing methods as well as the use of animal models in which transgenic axonal labeling is not available. Using rodent models of spinal cord injury, we labeled axon tracts of interest using both adeno-associated virus and chemical tracers and performed tetrahydrofuran-based tissue clearing to image multiple axon types in spinal cords using light sheet and confocal microscopy. Using this approach, we investigated the relationships between axons and scar-forming cells at the injury site as well as connections between sensory axons and motor pools in the spinal cord. In addition, we used these methods to trace axons in non-human primates. This reproducible and adaptable virus-based approach can be combined with transgenic mice or with chemical-based tract-tracing methods, providing scientists with flexibility in obtaining axonal trajectory information from transparent tissue.
Significance Statement: Recent advances in tissue clearing techniques have provided a promising method of visualizing axonal trajectories with unprecedented accuracy and speed. While previous studies have utilized transgenic labeling in mice, the use of virus or chemical neuronal tracers will provide additional spatiotemporal control as well as the ability to use animal models in which transgenic axonal labeling is not available. We used adeno-associated viruses (AAVs) and chemical tracers and performed tetrahydrofuran-based tissue clearing to image multiple axon types in the rodent and non-human primate spinal cord using light sheet and confocal microscopy. This approach will provide scientists with a simple and flexible method of obtaining axonal trajectory information from transparent tissue.
↵1 Authors report no conflict of interest.
↵3 This study was funded by NINDS R01NS081040, R21NS082835, NEI, NICHD RO1HD057632, Craig Neilsen Foundation, Department of Defense, International Spinal Research Trust, State of Florida Specific Appropriation 538, The Miami Project to Cure Paralysis, and the Buoniconti Fund.