Figure 7.

ER channel blockers inhibit the increase in oxidative stress triggered by axotomy. Superoxide levels in neurites were measured using DHE (5 μm). Embryonic DRGs were cultured for 7 d, axotomized, and immediately treated with Rya (5 μm), U73122 (2 μm), Ru360 (5 μm), Trolox (10 μm), or CsA (20 μm). After 6 h, superoxide levels were assayed by culturing neurites with DHE and observed by phase-contrast bright-field (BF; left) and fluorescence microscopy (right). A, DHE fluorescence is almost not detectable in non-injured neurites (0 h). At 6 hpa, the levels of DHE fluorescence strongly increase in axotomized neurites [vehicle (Veh)]. This increase in DHE fluorescence is inhibited by treatment with the ER channel inhibitors Rya and U73122, the mitochondrial uniporter inhibitor Ru360, the antioxidant Trolox, and the mPTP inhibitor CsA. Notice that, in all cases, neurites appear relatively conserved at 6 hpa by bright field. Scale bar, 50 μm. B, The DHE signal in neurites was calculated as the ratio of the total area of DHE signal in neurites normalized by the total neurite length in the corresponding field (n = 3 samples, 2 images per sample; *p < 0.05 by Student's t test compared with vehicle; error bars indicate SEM).