Download PDF
J Reconstr Microsurg 2007; 23(7): 381-389
DOI: 10.1055/s-2007-992344
© Thieme Medical Publishers

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

Ayato Hayashi1 , Arash Moradzadeh2 , Daniel A. Hunter1 , David H. Kawamura1 , Vinay K. Puppala1 , Thomas H.H Tung1 , Susan E. Mackinnon1 , 2 , Terence M. Myckatyn1
  • 1Division of Plastic and Reconstructive Surgery, Washington University, Saint Louis, Missouri
  • 2Department of Otolaryngology-Head & Neck Surgery, Washington University, Saint Louis, Missouri
Further Information

Publication History

Publication Date:
02 November 2007 (online)

Also available at
    Permissions and Reprints

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.

KEYWORDS

Retrograde labeling - neural tracer - Fast Blue - Fluoro-Gold - peripheral nerve

REFERENCES

  • 1 Kobbert C, Apps R, Bechmann I, Lanciego J L, Mey J, Thanos S. Current concepts in neuroanatomical tracing.  Prog Neurobiol. 2000;  62 327-351
    CrossrefPubMedGoogle Scholar
  • 2 Kristensson K. Transport of fluorescent protein tracer in peripheral nerves.  Acta Neuropathol (Berl). 1970;  16 293-300
    CrossrefPubMedGoogle Scholar
  • 3 Novikova L, Novikov L, Kellerth J O. Persistent neuronal labeling by retrograde fluorescent tracers: a comparison between Fast Blue, Fluoro-Gold and various dextran conjugates.  J Neurosci Methods. 1997;  74 9-15
    CrossrefPubMedGoogle Scholar
  • 4 Puigdellivol-Sanchez A, Prats-Galino A, Ruano-Gil D, Molander C. Efficacy of the fluorescent dyes Fast Blue, Fluoro-Gold, and Diamidino Yellow for retrograde tracing to dorsal root ganglia after subcutaneous injection.  J Neurosci Methods. 1998;  86 7-16
    CrossrefPubMedGoogle Scholar
  • 5 Weiss P, Hiscoe H N. Experiments on the mechanism of nerve growth.  J Exp Zool. 1948;  107 315-395
    CrossrefPubMedGoogle Scholar
  • 6 Kristensson K, Olsson Y. Retrograde axonal transport of protein.  Brain Res. 1971;  29 363-365
    CrossrefPubMedGoogle Scholar
  • 7 Wessendorf M W. Fluoro-Gold: composition, and mechanism of uptake.  Brain Res. 1991;  553 135-148
    CrossrefPubMedGoogle Scholar
  • 8 van der Kooy D, Kuypers H G. Fluorescent retrograde double labeling: axonal branching in the ascending raphe and nigral projections.  Science. 1979;  204 873-875
    CrossrefPubMedGoogle Scholar
  • 9 Choi D, Li D, Raisman G. Fluorescent retrograde neuronal tracers that label the rat facial nucleus: a comparison of Fast Blue, Fluoro-ruby, Fluoro-emerald, Fluoro-Gold and DiI.  J Neurosci Methods. 2002;  117 167-172
    CrossrefPubMedGoogle Scholar
  • 10 Puigdellivol-Sanchez A, Valero-Cabre A, Prats-Galino A, Navarro X, Molander C. On the use of fast blue, fluoro-gold and diamidino yellow for retrograde tracing after peripheral nerve injury: uptake, fading, dye interactions, and toxicity.  J Neurosci Methods. 2002;  115 115-127
    CrossrefPubMedGoogle Scholar
  • 11 Richmond F J, Gladdy R, Creasy J L, Kitamura S, Smits E, Thomson D B. Efficacy of seven retrograde tracers, compared in multiple-labelling studies of feline motoneurones.  J Neurosci Methods. 1994;  53 35-46
    CrossrefPubMedGoogle Scholar
  • 12 Bentivoglio M, Kuypers H G, Catsman-Berrevoets C E, Loewe H, Dann O. Two new fluorescent retrograde neuronal tracers which are transported over long distances.  Neurosci Lett. 1980;  18 25-30
    CrossrefPubMedGoogle Scholar
  • 13 Bharos T B, Kuypers H G, Lemon R N, Muir R B. Divergent collaterals from deep cerebellar neurons to thalamus and tectum, and to medulla oblongata and spinal cord: retrograde fluorescent and electrophysiological studies.  Exp Brain Res. 1981;  42 399-410
    PubMedGoogle Scholar
  • 14 Gordon D C, Richmond F J. Topography in the phrenic motoneuron nucleus demonstrated by retrograde multiple-labelling techniques.  J Comp Neurol. 1990;  292 424-434
    PubMedGoogle Scholar
  • 15 Illert M, Fritz N, Aschoff A, Hollander H. Fluorescent compounds as retrograde tracers compared with horseradish peroxidase (HRP). II. A parametric study in the peripheral motor system of the cat.  J Neurosci Methods. 1982;  6 199-218
    CrossrefPubMedGoogle Scholar
  • 16 Innocenti G M, Clarke S, Kraftsik R. Interchange of callosal and association projections in the developing visual cortex.  J Neurosci. 1986;  6 1384-1409
    PubMedGoogle Scholar
  • 17 Kuypers H G, Huisman A M. Fluorescent neuronal tracers.  Adv Cell Neurobiol. 1984;  5 307-340
    PubMedGoogle Scholar
  • 18 Popratiloff A S, Neiss W F, Skouras E, Streppel M, Guntinas-Lichius O, Angelov D N. Evaluation of muscle re-innervation employing pre- and post-axotomy injections of fluorescent retrograde tracers.  Brain Res Bull. 2001;  54 115-123
    CrossrefPubMedGoogle Scholar
  • 19 Katada A, Vos J D, Swelstad B B, Zealear D L. A sequential double labeling technique for studying changes in motoneuronal projections to muscle following nerve injury and reinnervation.  J Neurosci Methods. 2006;  155 20-27
    CrossrefPubMedGoogle Scholar
  • 20 Puigdellivol-Sanchez A, Prats-Galino A, Ruano-Gil D, Molander C. Persistence of tracer in the application site-a potential confounding factor in nerve regeneration studies.  J Neurosci Methods. 2003;  127 105-110
    CrossrefPubMedGoogle Scholar
  • 21 Vince G H, Bouterfa H, Goldbrunner R, Roosen K, Tonn J C. Fast blue, a fluorescent tracer in glioma cell culture, affects cell proliferation and motility.  Neurosci Lett. 1997;  233 148-150
    CrossrefPubMedGoogle Scholar
  • 22 Schmued L C, Fallon J H. Fluoro-Gold: a new fluorescent retrograde axonal tracer with numerous unique properties.  Brain Res. 1986;  377 147-154
    CrossrefPubMedGoogle Scholar
  • 23 Akintunde A, Buxton D F. Differential sites of origin and collateralization of corticospinal neurons in the rat: a multiple fluorescent retrograde tracer study.  Brain Res. 1992;  575 86-92
    CrossrefPubMedGoogle Scholar
  • 24 Fernandes K J, Fan D P, Tsui B J, Cassar S L, Tetzlaff W. Influence of the axotomy to cell body distance in rat rubrospinal and spinal motoneurons: differential regulation of GAP-43, tubulins, and neurofilament-M.  J Comp Neurol. 1999;  414 495-510
    PubMedGoogle Scholar
  • 25 Theriault E, Tator C H. Persistence of rubrospinal projections following spinal cord injury in the rat.  J Comp Neurol. 1994;  342 249-258
    PubMedGoogle Scholar
  • 26 Harsh C, Archibald S J, Madison R D. Double-labeling of saphenous nerve neuron pools: a model for determining the accuracy of axon regeneration at the single neuron level.  J Neurosci Methods. 1991;  39 123-130
    CrossrefPubMedGoogle Scholar
  • 27 Madison R D, Archibald S J, Brushart T M. Reinnervation accuracy of the rat femoral nerve by motor and sensory neurons.  J Neurosci. 1996;  16 5698-5703
    PubMedGoogle Scholar
  • 28 Zhang L, McClellan A D. Fluorescent tracers as potential candidates for double labeling of descending brain neurons in larval lamprey.  J Neurosci Methods. 1998;  85 51-62
    CrossrefPubMedGoogle Scholar
  • 29 Chang H T, Kuo H, Whittaker J A, Cooper N G. Light and electron microscopic analysis of projection neurons retrogradely labeled with Fluoro-Gold: notes on the application of antibodies to Fluoro-Gold.  J Neurosci Methods. 1990;  35 31-37
    CrossrefPubMedGoogle Scholar
  • 30 Van Bockstaele E J, Wright A M, Cestari D M, Pickel V M. Immunolabeling of retrogradely transported Fluoro-Gold: sensitivity and application to ultrastructural analysis of transmitter-specific mesolimbic circuitry.  J Neurosci Methods. 1994;  55 65-78
    CrossrefPubMedGoogle Scholar
  • 31 Garrett W T, McBride R L, Williams Jr J K, Feringa E R. Fluoro-Gold's toxicity makes it inferior to True Blue for long-term studies of dorsal root ganglion neurons and motoneurons.  Neurosci Lett. 1991;  128 137-139
    CrossrefPubMedGoogle Scholar
  • 32 Naumann T, Hartig W, Frotscher M. Retrograde tracing with Fluoro-Gold: different methods of tracer detection at the ultrastructural level and neurodegenerative changes of back-filled neurons in long-term studies.  J Neurosci Methods. 2000;  103 11-21
    CrossrefPubMedGoogle Scholar
  • 33 Sims T J, Gilmore S A. Regeneration of dorsal root axons into experimentally altered glial environments in the rat spinal cord.  Exp Brain Res. 1994;  99 25-33
    PubMedGoogle Scholar
  • 34 Tsai E C, van Bendegem R L, Hwang S W, Tator C H. A novel method for simultaneous anterograde and retrograde labeling of spinal cord motor tracts in the same animal.  J Histochem Cytochem. 2001;  49 1111-1122
    PubMedGoogle Scholar
  • 35 Schmued L, Kyriakidis K, Heimer L. In vivo anterograde and retrograde axonal transport of the fluorescent rhodamine-dextran-amine, Fluoro-Ruby, within the CNS.  Brain Res. 1990;  526 127-134
    CrossrefPubMedGoogle Scholar
  • 36 Fritzsch B, Sonntag R. Sequential double labelling with different fluorescent dyes coupled to dextran amines as a tool to estimate the accuracy of tracer application and of regeneration.  J Neurosci Methods. 1991;  39 9-17
    CrossrefPubMedGoogle Scholar
  • 37 Puigdellivol-Sanchez A, Prats-Galino A, Ruano-Gil D, Molander C. Fast blue and diamidino yellow as retrograde tracers in peripheral nerves: efficacy of combined nerve injection and capsule application to transected nerves in the adult rat.  J Neurosci Methods. 2000;  95 103-110
    CrossrefPubMedGoogle Scholar
  • 38 Keizer K, Kuypers H G, Huisman A M, Dann O. Diamidino yellow dihydrochloride (DY. 2HCl); a new fluorescent retrograde neuronal tracer, which migrates only very slowly out of the cell.  Exp Brain Res. 1983;  51 179-191
    PubMedGoogle Scholar
  • 39 Haase P, Payne J N. Comparison of the efficiencies of true blue and diamidino yellow as retrograde tracers in the peripheral motor system.  J Neurosci Methods. 1990;  35 175-183
    CrossrefPubMedGoogle Scholar
  • 40 Horikawa K, Powell E W. Comparison of techniques for retrograde labeling using the rat's facial nucleus.  J Neurosci Methods. 1986;  17 287-296
    CrossrefPubMedGoogle Scholar
  • 41 Hayashi A, Yanai A, Komuro Y, Nishida M, Inoue M, Seki T. Collateral sprouting occurs following end-to-side neurorrhaphy [see comment].  Plast Reconstr Surg. 2004;  114 129-137
    CrossrefPubMedGoogle Scholar
  • 42 Akintunde A, Buxton D F. Quadruple labeling of brain-stem neurons: a multiple retrograde fluorescent tracer study of axonal collateralization.  J Neurosci Methods. 1992;  45 15-22
    CrossrefPubMedGoogle Scholar
  • 43 Haenggeli C, Kato A C. Rapid and reproducible methods using fluorogold for labelling a subpopulation of cervical motoneurons: application in the wobbler mouse.  J Neurosci Methods. 2002;  116 119-124
    CrossrefPubMedGoogle Scholar
  • 44 Sagot Y, Rosse T, Vejsada R, Perrelet D, Kato A C. Differential effects of neurotrophic factors on motoneuron retrograde labeling in a murine model of motoneuron disease.  J Neurosci. 1998;  18 1132-1141
    PubMedGoogle Scholar
  • 45 O'Brien C, Woolf C J, Fitzgerald M, Lindsay R M, Molander C. Differences in the chemical expression of rat primary afferent neurons which innervate skin, muscle or joint.  Neuroscience. 1989;  32 493-502
    CrossrefPubMedGoogle Scholar
  • 46 Honig M G, Hume R I. Dil and diO: versatile fluorescent dyes for neuronal labelling and pathway tracing.  Trends Neurosci. 1989;  12 333-335
    CrossrefPubMedGoogle Scholar
  • 47 Honig M G, Hume R I. Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures.  J Cell Biol. 1986;  103 171-187
    CrossrefPubMedGoogle Scholar
  • 48 Conde F. Further studies on the use of the fluorescent tracers fast blue and diamidino yellow: effective uptake area and cellular storage sites.  J Neurosci Methods. 1987;  21 31-43
    CrossrefPubMedGoogle Scholar
  • 49 Darian-Smith C, Darian-Smith I, Cheema S S. Thalamic projections to sensorimotor cortex in the macaque monkey: use of multiple retrograde fluorescent tracers.  J Comp Neurol. 1990;  299 17-46
    PubMedGoogle Scholar
  • 50 Taylor D C, Pierau F K, Schmid H. The use of fluorescent tracers in the peripheral sensory nervous system.  J Neurosci Methods. 1983;  8 211-224
    CrossrefPubMedGoogle Scholar
  • 51 Hoke A, Redett R, Hameed H et al.. Schwann cells express motor and sensory phenotypes that regulate axon regeneration.  J Neurosci. 2006;  26 9646-9655
    CrossrefPubMedGoogle Scholar
  • 52 Tarasidis G, Watanabe O, Mackinnon S E, Strasberg S R, Haughey B H, Hunter D A. End-to-side neurorrhaphy resulting in limited sensory axonal regeneration in a rat model.  Ann Otol Rhinol Laryngol. 1997;  106 506-512
    PubMedGoogle Scholar
  • 53 Clarke P G. How inaccurate is the Abercrombie correction factor for cell counts?.  Trends Neurosci. 1992;  15 211-212
    CrossrefPubMedGoogle Scholar
  • 54 Boyd J G, Gordon T. The neurotrophin receptors, trkB and p75, differentially regulate motor axonal regeneration.  J Neurobiol. 2001;  49 314-325
    CrossrefPubMedGoogle Scholar
  • 55 Abercrombie M. Estimation of nuclear population from microtome sections.  Anat Rec. 1946;  94 239-247
    CrossrefPubMedGoogle Scholar
  • 56 Smolen A J, Wright L L, Cunningham T J. Neuron numbers in the superior cervical sympathetic ganglion of the rat: a critical comparison of methods for cell counting.  J Neurocytol. 1983;  12 739-750
    CrossrefPubMedGoogle Scholar
  • 57 Williams R W, Rakic P. Three-dimensional counting: an accurate and direct method to estimate numbers of cells in sectioned material.  J Comp Neurol. 1988;  278 344-352
    PubMedGoogle Scholar
  • 58 Hendry I A. A method to correct adequately for the change in neuronal size when estimating neuronal numbers after nerve growth factor treatment.  J Neurocytol. 1976;  5 337-349
    CrossrefPubMedGoogle Scholar
  • 59 Gundersen H J, Jensen E B. The efficiency of systematic sampling in stereology and its prediction.  J Microsc. 1987;  147(Pt 3) 229-263
    CrossrefPubMedGoogle Scholar
  • 60 Sterio D C. The unbiased estimation of number and sizes of arbitrary particles using the disector.  J Microsc. 1984;  134(Pt 2) 127-136
    CrossrefPubMedGoogle Scholar

Terence M MyckatynM.D. 

Division of Plastic and Reconstructive Surgery, Washington University School of Medicine

660 South Euclid Avenue, Campus Box 8238, Saint Louis, MO 63110