Fluoro-Ruby as a reliable marker for regenerating fiber tracts

Axon visualization techniques are important in assessing the efficacy of interventional approaches to stimulate neural regeneration. Whereas the labeling of descending tracts in the spinal cord has been well established using the intracortical injection of biotin dextran amine (BDA), the labeling of ascending sensory fibers of the dorsal funiculus is more problematic. Fluoro-Ruby (FR; dextran tetramethylrhodamine; MW 10,000) is a bidirectional permanent tracer, but the retrograde tracing of fibers is particularly prominent, and FR is a highly sensitive tracer that can be applied in discrete injection sites. In the present report, we used FR to efficiently label ascending fibers in the dorsal columns of the rat spinal cord. After transplantation of olfactory ensheathing cells into the transected dorsal funiculus, the application of FR was able to detect regenerating ascending fibers in the spinal cord. Regenerated fibers crossing the injury site were labeled and easily identified. It is likely that the tracer was taken up by damaged fibers. As additional advantages, the labeling is resistant to photobleaching and no additional tissue processing is necessary for visualization. It can be used for in vivo as well as in vitro injections. The findings indicate that FR can be used as a reliable fluorescent marker to study ascending regenerated fibers in the spinal cord axonal regeneration.