Figure 6: Mechanoporation is the primary source of intra-axonal calcium after contusion.

(a) Subdurally loaded dye (10 kD dextran) was excluded from uninjured axons, which were revealed by ‘negative’ staining (left, maximum intensity projection of 20 optical planes). After contusion, a number of axons took up the dye (centre, same sample as left, maximum intensity projection). Without injury, labelling is cleared from extracellular space and becomes restricted to blood vessels (asterisks) and some superficial cells (right, maximum intensity projection). Images are individually adjusted to best reveal patterns of dye distribution. (b) Axonal dye uptake after delayed loading (0.8 kD cadaverine) correlated with elevations in axonal Ca²⁺ level (dye-positive axons, orange arrows; dye-negative axons, cyan arrows; white arrowheads indicate presumptive myelin sheaths that are also dye-positive; single optical plane from an in vivo time-lapse experiment). For colour look-up table cf. Fig. 2a. (c) Percentage of Ca²⁺-elevated axons that are cadaverine-positive or -negative at the first time point (19–22-min p.i.) following dye-loading at 15-min p.i. (202 axons, 3 Thy1-TNXXL mice). (d,e) Time course of dye uptake (0.8-kD cadaverine) in axons with normal Ca²⁺ levels (d, 565 axons, 15 Thy1-TNXXL mice) and in Ca²⁺-elevated axons (e, 226 axons, 15 Thy1-TNXXL mice). (f) Ca²⁺ (left) and morphological (right) changes in axons with elevated Ca²⁺ at 15-min p.i., subdivided into the dye-negative (top; 34 axons, 3 mice) and -positive (lower; 33 axons, 3 mice) axon populations. (g) Ca²⁺ (left) and morphological (right) changes in axons with elevated Ca²⁺ at 60-min p.i., subdivided into the dye-negative (top; 47 axons, 5 mice) and -positive (lower; 16 axons, 5 mice) axon populations. Scale bars in a,b, 20 μm. Note that in d,c some error bars are hidden behind data symbols.