A Novel Method for Simultaneous Anterograde and Retrograde Labeling of Spinal Cord Motor Tracts in the Same Animal

Abstract

S U M M A R Y Examination of repaired spinal cord tracts has usually required separate groups of animals for anterograde and retrograde tracing owing to the incompatibility of techniques such as tissue fixation. However, anterograde and retrograde labeling of different animals subjected to the same repair may not allow accurate examination of that repair strategy because widely variable results can occur in animals subjected to the same strategy. We have developed a reliable method of labeling spinal cord motor tracts bidirectionally in the same animal using DiI, a lipophilic dye, to anterogradely label the corticospinal tract and Fluoro-Gold (FG) to retrogradely label cortical and brainstem neurons of several spinal cord motor tracts in normal and injured adult rats. Other tracer combinations (lipophilic dyes or fluorescent dextrans) were also investigated but were less effective. We also developed methods to minimize autofluorescence with the DiI/FG technique, and found that the DiI/FG technique is compatible with decalcification and immunohistochemistry for several markers relevant for studies of spinal cord regeneration. Thus, the use of anterograde DiI and retrograde FG is a novel technique for bidirectional labeling of the motor tracts of the adult spinal cord with fluorescent tracers and should be useful for demonstrating neurite regeneration in studies of spinal cord repair.

Acute transplantation of olfactory ensheathing cells or Schwann cells promotes recovery after spinal cord injury in the rat

“…With a fine forceps (Dumont No. 5), two to four DiI crystals (D-3911; Molecular Probes Europe, Leiden, The Netherlands) were inserted into the depth of the cortical incision such that the cortical edges became approximated and covered the crystals (Tsai et al, 2001). Finally, the skin was sutured and disinfected.…”

Evaluation of corticospinal axon loss by fluorescent dye tracing in mice with experimental autoimmune encephalomyelitis

“…Although it is possible that DiI can cross "immature synapses" that are present between developing neurons (Vannucchi and FaussonePellegrini, 2000), DiI transcellular labeling has been found in the adult rodent spinal cord after DiI applied into the sensorimotor cortex (Tsai et al, 2001). In mouse, as early as five to seven days after DiI is taken up into the pyramidal neurons, selective CST tract labeling was present from the internal capsule to the lumbar cord.…”

“…Other spinal tracts such as the reticulospinal tract in the brainstem and the propriospinal tracts in the dorsal column, did not show DiI labeling. Moreover, the DiI labeling can not traverse a transection lesion in the spinal cord (Tsai et al, 2001). Therefore, using this approach for the EAE model, only the intact CST fibers and the segments rostral to the disruption site can be labeled.…”

Triple-Labeling Whole-Mount In Situ Hybridization Method for Analysis of Overlapping Gene Expression in Brain Tissue with High Level of Autofluorescence

“…Autofluorescence of neural tissue often complicates the use of the green fluorescent protein (GFP) [18]. More importantly for our research, animals with spinal cord transection showed significant autofluorescence of cortical, brainstem, and spinal cord neurons, which interferes with identification of labeling [19,20]. In our own research, we have encountered unacceptable levels of autofluorescence in lamprey brain and spinal cord neurons after injury ( Figure 1).…”