Retrograde Labeling in Peripheral Nerve Research: It Is Not All Black and White

Abstract

Retrograde labeling has become an important method of evaluation for peripheral nerve regeneration after injury. We review the features of the commonly used retrograde tracers Fast Blue, Fluoro-Gold, and Fluoro Ruby in addition to the various application methods (conduit reservoir, intramuscular injection, and crystal powder application) and the techniques used to count stained neurons. Upon application of the staining techniques and dyes in a rat and mouse nerve injury model, Fluoro-Gold was found to stain the greatest number of neurons with all application methods. However, due to variability of staining intensity, neuron size, and background staining, it is difficult to count the stained neurons accurately. Fast Blue stains consistently using intramuscular injection in the mouse but fails to provide adequate staining using the muscle injection method in the rat model and shows high failure rates using the conduit reservoir technique. However, crystal dye application with Fast Blue to the cut nerve end provides excellent results. We believe that it is imperative to use the various tracers and application methods prior to their experimental use to develop a consistent standardized approach to retrograde labeling.

Efficacy of fluorescent tracers in retrograde labeling of cutaneous afferent neurons in the rat

“…However, based on our results and the results of the studies in which the efficacy of retrograde labeling of motor neurons was assessed by the same technique (Haase and Payne, 1990;Richmond et al, 1994;Novikova et al, 1997;Choi et al, 2002;Hayashi et al, 2007), we believe that the tracers FG, TB and DiI are the most efficient and suitable for retrograde labeling also in experiments examining preferential motor or sensory reinnervation after peripheral nerve injury.…”

“…The labeling efficacy of different fluorescent tracers when applied either individually (single labeling) or in combination with other tracer or tracers (double or multiple labeling) has been shown to be highly variable (Haase and Payne, 1990;Richmond et al, 1994;Novikova et al, 1997;Puigdellívol-Sánchez et al, 1998Choi et al, 2002;Byers et al, 2002;Hayashi et al, 2007). This was found to be related to intrinsic properties of the tracers, the method of its application, and the simultaneous use of additional tracers, as well as to the survival time of experimental animals after tracer application (Richmond et al, 1994;Novikova et al, 1997;Puigdellívol-Sánchez et al, 2002;Byers et al, 2002;Hayashi et al, 2007). In addition, a failure to obtain 100% double labeling in the experiments, in which the same total number of neurons was labeled by two tracers, suggests selective labeling of different populations of neurons (Puigdellívol-Sánchez et al, 1998;Messina et al, 2000).…”

“…The efficacy of different fluorescent tracers in retrograde labeling of motor neurons has been extensively investigated in various experimental conditions (Haase and Payne, 1990;Richmond et al., 1994;Novikova et al, 1997;Messina et al, 2000;Choi et al, 2002;Hayashi et al, 2007). On the other hand, there are only a few studies regarding the efficacies of different fluorescent tracers in retrograde labeling of sensory neurons (Harsh et al, 1991;Byers et al, 2002;Puigdellívol-Sánchez et al, 1998.…”

Motoneuron survival after chronic and sequential peripheral nerve injuries in the rat

“…These techniques were equally reliable, rather than injecting the tracer into the muscle close to nerve insertion. 20 The left side of each rat served as the experimental side for single and sequential nerve injuries (Fig. 1).…”

Characterization of Neuronal Death and Functional Deficits following Nerve Injury during the Early Postnatal Developmental Period in Rats

“…Sciatic nerve crush injury in rodents is an excellent experimental model of axonotmetic type injuries and provides a framework for evaluating functional recovery following this kind of injury [12,52,53] . Fluorogold was chosen for these studies because this fluorescent dye can remain inside the cell bodies of both motor and sensory neurons for prolonged periods of time [54][55][56] . Consistent with previous research, we believe that reduction in Fluorogold-positive neurons represents an accurate assessment of neuronal death.…”