Retrograde labeling in peripheral nerve research: it is not all black and white.
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.
Hayashi
Ayato
A
Division of Plastic and Reconstructive Surgery, Washington University School of Medicine, 660 South Euclid Avenue, Saint Louis 63110, USA.
Moradzadeh
Arash
A
Hunter
Daniel A
DA
Kawamura
David H
DH
Puppala
Vinay K
VK
Tung
Thomas H H
TH
Mackinnon
Susan E
SE
Myckatyn
Terence M
TM
D000578:Amidines / Q000493:pharmacokinetics; D000818:Animals; D001370:Axonal Transport / Q000502:physiology*; D003911:Dextrans / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000493:pharmacokinetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D009416:Nerve Regeneration / Q000502:physiology*; D010525:Peripheral Nerves / Q000502:physiology*; D012235:Rhodamines / Q000493:pharmacokinetics; D013266:Stilbamidines / Q000493:pharmacokinetics
C049774:2-hydroxy-4,4'-diamidinostilbene, methanesulfonate salt; D000578:Amidines; D003911:Dextrans; D005456:Fluorescent Dyes; C067661:Fluoro-Ruby; D012235:Rhodamines; D013266:Stilbamidines; C031455:diamidino compound 253-50