A novel method for simultaneous anterograde and retrograde labeling of spinal cord motor tracts in the same animal

Examination of repaired spinal cord tracts has usually required separate groups of animals for anterograde and retrograde tracing owing to the incompatibility of techniques such as tissue fixation. However, anterograde and retrograde labeling of different animals subjected to the same repair may not allow accurate examination of that repair strategy because widely variable results can occur in animals subjected to the same strategy. We have developed a reliable method of labeling spinal cord motor tracts bidirectionally in the same animal using Dil, a lipophilic dye, to anterogradely label the corticospinal tract and Fluoro-Gold (FG) to retrogradely label cortical and brainstem neurons of several spinal cord motor tracts in normal and injured adult rats. Other tracer combinations (lipophilic dyes or fluorescent dextrans) were also investigated but were less effective. We also developed methods to minimize autofluorescence with the Dil/FG technique, and found that the Dil/FG technique is compatible with decalcification and immunohistochemistry for several markers relevant for studies of spinal cord regeneration. Thus, the use of anterograde Dil and retrograde FG is a novel technique for bidirectional labeling of the motor tracts of the adult spinal cord with fluorescent tracers and should be useful for demonstrating neurite regeneration in studies of spinal cord repair.