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. 2022 Mar 18;15:852181. doi: 10.3389/fnmol.2022.852181

FIGURE 4.

FIGURE 4

Mitochondrial depolarization induced by axotomy is dependent on loss of NAD+ but not on ROS accumulation. (A) Intact or cut DRG axons treated with NAD+ (5 mM-30 min treatment) and immunostained for β-III tubulin (scale bar indicates 1,000μm). (B) Axon density of intact or axotomized (Ax) DRGs in radial bins of increasing distance from the center (n = 7 for each group). Values are mean ± SEM. Statistical significance was assessed using a two-factor ANOVA followed by Dunnett’s test relative to Ax + DMSO. (C) Intact or cut axons treated with either NAD+ (5 mM) or NAC (20 mM), loaded with Calcein-AM and TMRE (scale bar indicates 10 μm) and imaged 3.5 h after axotomy. (D) Mitochondrial polarization readout calculated as TMRE-positive area divided by Calcein-positive area and normalized to intact control [n = 10 (control), 5 (NAC), 4 (NAD+)]. Boxplots are minimum, 1st quartile, median, 3rd quartile, maximum. Statistical significance was assessed using a two-factor ANOVA followed by Tukey’s post hoc comparison. (E) Micrographs of intact or cut axons treated with NAD+ (5 mM) and loaded with Fluo-4-AM (scale bar indicates 25 μm). (F) Fluo-4-AM intensity in axons, normalized to intensity of intact controls [n = 10 (control), 7 (NAD+)]. Boxplots are minimum, 1st quartile, median, 3rd quartile, maximum. Statistical significance was assessed using a two-factor ANOVA followed by Tukey’s post hoc comparison, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.