Double-labeling of saphenous nerve neuron pools: a model for determining the accuracy of axon regeneration at the single neuron level.

We have developed a labeling procedure which accurately and consistently labels the original sensory pools projecting to their respective nerve branches as a model to quantify the accuracy of nerve regeneration at the single neuron level. Adult and juvenile rats had the saphenous branch of the femoral nerve transected just distal to the bifurcation of the nerve into a sensory branch (saphenous nerve) and a motor branch (nerve to the quadriceps muscle) and exposed to a 3% solution of 1,1'-di-octadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) for 1 h, and then reanastomosed. Two weeks later the sensory branch was redivided proximal to the previous site and labeled with a second tracer (5% fluorogold) using similar procedures. Five days later the animals were killed and cryostat sections were prepared and analyzed with a fluorescence microscope to score single- and double-labeled primary sensory neurons. The results show that the primary sensory neurons which project into the saphenous nerve can be reliably prelabeled by exposure of the saphenous nerve to DiI, and two weeks later approximately 99% of the same population of neurons can be labeled by exposure of the nerve to a second dye, fluorogold. This model system will be very powerful for future studies concerning target reinnervation following nerve regeneration.