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

Ayato Hayashi; Arash Moradzadeh; Daniel A. Hunter; David H. Kawamura; Vinay K. Puppala; Thomas H. H. Tung; Susan E. Mackinnon; Terence M. Myckatyn

doi:10.1055/s-2007-992344

Journal of Reconstructive Microsurgery, Table of Contents

J Reconstr Microsurg 2007; 23(7): 381-389
DOI: 10.1055/s-2007-992344

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

Abstract

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

Full Text

References

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
2 Kristensson K. Transport of fluorescent protein tracer in peripheral nerves. Acta Neuropathol (Berl). 1970; 16 293-300
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
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
5 Weiss P, Hiscoe H N. Experiments on the mechanism of nerve growth. J Exp Zool. 1948; 107 315-395
6 Kristensson K, Olsson Y. Retrograde axonal transport of protein. Brain Res. 1971; 29 363-365
7 Wessendorf M W. Fluoro-Gold: composition, and mechanism of uptake. Brain Res. 1991; 553 135-148
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
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
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
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
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
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
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
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
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
17 Kuypers H G, Huisman A M. Fluorescent neuronal tracers. Adv Cell Neurobiol. 1984; 5 307-340
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
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
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
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
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
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
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
25 Theriault E, Tator C H. Persistence of rubrospinal projections following spinal cord injury in the rat. J Comp Neurol. 1994; 342 249-258
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
53 Clarke P G. How inaccurate is the Abercrombie correction factor for cell counts?. Trends Neurosci. 1992; 15 211-212
54 Boyd J G, Gordon T. The neurotrophin receptors, trkB and p75, differentially regulate motor axonal regeneration. J Neurobiol. 2001; 49 314-325
55 Abercrombie M. Estimation of nuclear population from microtome sections. Anat Rec. 1946; 94 239-247
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
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
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
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
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

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