Fig. 1

First signs of EAN are associated with inflammatory infiltration within the saphenous nerve. a Low-power image of dissected saphenous nerve, labeled in situ with TMRM (red) and IB4 (green), showing the outlines of nerve fibers and the connective tissues surrounding them. b High-power image of nerve fibers within the saphenous nerve in an adjuvant control animal, showing one IB4⁺ C fiber (green) and a number of elongated, polarized axonal mitochondria (red). c High-power image of nerve fibers within the saphenous nerve in a symptomatic animal on the first day of onset of EAN, showing a number of infiltrating inflammatory cells labeled with IB4⁺ (green arrows), within which mitochondria labeled with TMRM can be seen. In one axon (white arrowhead), polarized axonal mitochondria appear focally accumulated, as opposed to uniformly distributed mitochondrial in surrounding fibers. One of the inflammatory cells (dashed yellow arrow) can be seen juxtaposed to this focal accumulation (see also Additional file 1: Video S1, encircled). d, d′ After 40 min, the same inflammatory cell (dashed yellow arrow) is present at the same location (see also Additional file 2: Video S2). d′ The focal accumulation of axonal mitochondria shown from a different optical plane in order to visualize the mitochondria in the inset (see also Additional file 2: Video S2). This accumulation of mitochondria appears to have increased in comparison with the earlier time point (c). e In the symptomatic phase of EAN (n = 101 axons; 3 animals), the number of both anterogradely and retrogradely moving mitochondria in normal appearing axons was significantly decreased in comparison with adjuvant-only (n = 133 axons; 6 animals) and asymptomatic EAN animals (n = 44 axons; 3 animals) (***p < 0.001, two-way ANOVA). The systemic injection of LPS did not significantly change the number of transported mitochondria in comparison with asymptomatic EAN or adjuvant control animals and remained significantly higher than the number of anterogradely moving mitochondria in symptomatic EAN animals (***p < 0.001, two-way ANOVA). Similarly, incubation in vivo of saphenous nerve axons with macrophages activated ex vivo did not significantly change the number of moving mitochondria in comparison with controls, although the number of transported mitochondria in both directions in these animals tended to be lower than in controls and animals with systemic LPS. f The average speed of moving mitochondria was not affected by either of the studied conditions (p > 0.05, two-way ANOVA)