Efficacy of the fluorescent dyes Fast Blue, Fluoro-Gold, and Diamidino Yellow for retrograde tracing to dorsal root ganglia after subcutaneous injection

Journal of Neuroscience Methods

Prats-Galino

Ruano-Gil

et al. 2003

Selective reinnervation of peripheral targets after nerve injury might be assessed by injecting a first tracer in a target before nerve injury to label the original neuronal population, and applying a second tracer after the regeneration period to label the regenerated population. However, altered uptake of tracer, fading, and cell death may interfere with the results. Furthermore, if the first tracer injected remains in the target tissue, available for "re-uptake" by misdirected regenerating axons, which originally innervated another region, then the identification of the original population would be confused. With the aim of studying this problem, the sciatic nerve of adult rats was sectioned and sutured. After three days, to allow the distal axon to degenerate avoiding immediate retrograde transport, one of the dyes: Fast Blue (FB), Fluoro-Gold (FG) or Diamidino Yellow (DY), was injected into the tibial branch of the sciatic nerve, or in the skin of one of the denervated digits. Rats survived 2-3 months. The results showed labelled dorsal root ganglion cells and motoneurones, indicating that late re-uptake of a first tracer occurs. This phenomenon must be considered when the model of sequential labelling is used for studying the accuracy of peripheral reinnervation.3

Section: Introductionmentioning

confidence: 68%

Persistence of tracer in the application site?a potential confounding factor in nerve regeneration studies

Journal of Neuroscience Methods

Prats-Galino

Ruano-Gil

et al. 2003

“…Neuronal profiles with an identifiable nucleus were counted in every tenth DRG section and in every fourth spinal cord section (see Figure 2 and Puigdellívol et al,1998Puigdellívol et al, , 2000, for more information on the identification of labelled neurones). The three tracers can be easily differentiated by the ultraviolet filter; FB is blueish, FG is reddish, and DY is pale yellow.…”

Section: Fixation Sectioning Microscopic Examinationmentioning

confidence: 99%

“…In this paper we dissolved the dyes in water according to the recommendations in several previous publications (Bentivoglio, 1980;Keyzer, 1983;Schmued and Fallon, 1986) and because we had successfully used them obtaining similar numbers of labelled neurones after subcutaneous application (Puigdellívol et al, 1998). We used a higher concentration of FG (10%) as we had found in preliminary experiments that this concentration seems to reduce the problem of fading in the DRG at 3 months after tracer injection in the toes (unpublished).…”

Section: Technical Considerationsmentioning

confidence: 99%

“…Furthermore, the difficulties in visualizing double DY-FG cells through the same filter (Puigdellívol et al, 1998) makes the DY-FB combination more suitable for a double labelling model. In that case, a certain degree of DY fading is to be accounted for which means that only a sample of the original population will be studied.…”

Section: Use Of Fb Fg or Dy In Regeneration Studiesmentioning

confidence: 99%

“…We have recently suggested that sequential application of Fast Blue (FB), FG and Diamidino Yellow (DY) in peripheral innervation targets as outlined above may be useful to study topographical accuracy of sensory reinnervation (Puigdellívol et al, 1998). However, in preparatory experiments, we noted that previous injection of FB reduced subsequent uptake of DY applied by capsule in both sensory and motor axons (Puigdellívol et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

On the use of fast blue, fluoro-gold and diamidino yellow for retrograde tracing after peripheral nerve injury: uptake, fading, dye interactions, and toxicity

Journal of Neuroscience Methods

Valero‐Cabré

Prats-Galino

et al. 2002

Self Cite

The usefulness of three retrograde fluorescent dyes for tracing injured peripheral axons was investigated. The rat sciatic was transected bilaterally and the proximal end briefly exposed to either Fast Blue (FB), Fluoro-Gold (FG) or to Diamidino Yellow (DY) on the right side, and to saline on the left side, respectively. The nerves were then resutured and allowed to regenerate.Electrophysiological tests three months later showed similar latencies and amplitudes of evoked muscle and nerve action potentials between tracer groups. The nerves were then cut distal to the original injury and exposed to a second (different) dye. Five days later, retrogradely labelled neurones were counted in the dorsal root ganglia (DRGs) and spinal cord ventral horn. The number of neurones labelled by the first tracer was similar for all three dyes in the DRG and ventral horn except for FG, which labelled fewer motoneurones. When used as second tracer, DY labelled fewer neurones than FG and FB in some experimental situations. The total number of neurones labelled by the first and/or second tracer was reduced by about 30% compared to controls. The contributions of cell death as well as different optional tracer combinations for studies of nerve regeneration are discussed.

Representations of hindlimb digits in rat dorsal root ganglia

Prats‐Galino

Ruano-Gil

et al. 1999

J. Comp. Neurol.

Self Cite

The distribution in dorsal root ganglia of neurones that innervate the distal tips of the hindlimb digits in the rat were mapped after subcutaneous injections of the fluorescent tracers Fast Blue, Diamidino Yellow, and Fluoro‐Gold into different digits. Three‐dimensional reconstruction was used to describe the intraganglionic distribution of neurones labelled from different digits. Labelled neurones were found mainly in the L3–L5 ganglia. The distribution in ganglia and the number of neurones labelled from each digit varied considerably between cases, but mean numbers of labelled neurones were similar for the different digits. Neurones in L3 tended to innervate medial digits and neurones in L5 tended to innervate lateral digits, but most neurones from any digit were found in L4. Although overlap was considerable, the three‐dimensional reconstruction showed tendencies of neurones to be distributed in restricted territories within the dorsal root ganglia. This was especially clear in ganglion L5, where digit IV was found to be represented more rostrally than digit V. The results indicate that primary afferent neurones that innervate the hindlimb digits are represented by a crude rostrocaudal somatotopic organisation both among and within lumbar dorsal root ganglia. J. Comp. Neurol. 408:137–145, 1999. © 1999 Wiley‐Liss, Inc.