Figure 7: Recovered axons can survive long term and maintain presynaptic boutons after spinal contusion.

(a) Schematic representation of retrograde tracing experiments. Contusions were performed in the presence of dextran (3 kD, orange; ‘Contusion’) to retrogradely label somata in DRGs caudal to the contusion. One day p.i. axons were backtraced from the midthoracic spinal cord with differentially conjugated dextran (3 kD, green; ‘Backtrace’); 1 week p.i. caudal DRGs were collected for analysis. (b) Co-labelling of somata with both dyes was evident (arrows; maximum intensity projections of whole-mount DRGs, outlined by dashed line in ‘Merge’). Right inset is indicated by white box in ‘Merge’. (c) Schematic representation of anterograde tracing experiments. One week before contusion, an AAV driving CFP expression (blue; ‘AAV’) was injected into the L5 or L6 DRG. Lesions were performed rostrally in the presence of dextran (10 kD, orange; ‘Contusion’), and tissue was collected 1 week injury p.i. (d) AAV-labelled axon terminals (left and blue in ‘Merge’) in the gracile nucleus, which have survived the contusion, as they are also labelled with a dye (centre; orange; rotated 3D renderings). The inset shows that the varicosities (arrows) on this projection could be immunolabelled with the presynaptic marker Vglut1/2 (magenta in inset; maximum intensity projection of two optical planes spaced by 0.5 μm). (e) Quantification of Vglut1/2 staining in dextran-negative and -positive axons (186 varicosities, 2 mice). (f) Numerous AAV-positive axons (left, blue) persist in the thoracic spinal cord and also contain dye-labelling (10 kD dextran; centre, orange; maximum intensity projections with dye-positive axons indicated with orange arrows, and dye-negative axons with blue arrows, degree of dye-labelling was individually confirmed for each axon in single planes). Scale bar in b, 100 μm; in d, 5 μm and in f, 20 μm.