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

Abstract

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.

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

“…However, even though the possibility of "re-uptake" has been suggested previously for FB (Innocenti et al, 1986;Rende et al, 1991;Popratiloff et al, 2001), it has not been demonstrated, and no such information seems to be available for DY. The relatively small number of FG labelled cells as compared to the other tracers, found in our material, could be explained by a less significant re-uptake phenomenon combined with fading, which has previously demonstrated for this tracer (Novikova et al, 1997;Puigdellívol et al, 2002).…”

“…However, the reported FG fading (Novikova et al, 1997), for up to a 75% of the previously labelled population after three months of follow-up (Puigdellívol et al, 2002), limits its use as a first tracer. Here, DY would appear to be a better option.…”

“…It is also important to control for the compatibility of the two tracers that have to be used in combination in each area of study (Puigdellívol et al, 1998(Puigdellívol et al, , 2000a) and for 11 their efficacy in labelling the population of regenerating neurones studied (Puigdellívol et al, 2002).…”

“…However, the cell counts in this model must be corrected due to at least two types of confounding factors. First, late detection of cell numbers may be compromised by altered uptake, fading and cell death (Puigdellívol et al, 2002). Second, the first tracer injected in the target may remain available in the tissue for "re-uptake".…”

Relationship of Axonal Voltage-gated Sodium Channel 1.8 (NaV1.8) mRNA Accumulation to Sciatic Nerve Injury-induced Painful Neuropathy in Rats

“…In this study, ϳ10% of small and medium DRG cells were found positive for the reporter GFP. Puigdellívol-Sánchez et al (46) estimated only 20 -30% of DRG neurons to innervate the hindpaw. Therefore, the relevant transfection efficiency of the present method may exceed 40%.…”