OGD-Rep increases retrograde transport of axonal mitochondria. (A–D) Primary cultured cortical neurons were cultured in a microfluidic platform. After staining with 10 nM MTG, the cultured neurons were subjected to OGD alone for 1 h or OGD-Rep for various time periods as indicated in DIV8. Live cell fluorescent images in axon compartments were captured by confocal microscopy. (A) Images show representative kymographs from three independent experiments. (B) Columns represent the proportion of mitochondria showing different transportation patterns. (C) Curves represent the number of mitochondria either anterogradely or retrogradely transported per 100-µm axon in 5 min. (D) Columns represent the velocity of mitochondria anterogradely or retrogradely transported in axons. (B–D) n = 18 (control, OGD alone, and reperfusion after 10-min OGD), n = 17 (reperfusion after 20-min OGD), and n = 14 (reperfusion after 40-min OGD) cells from three independent experiments for each group. (E and F) Primary cultured cortical neurons from the indicated germline mice were cultured in a microfluidic platform. After staining with 10 nM MTG, the cultured neurons were subjected to 20 min of OGD plus 40 min of reperfusion. Live cell fluorescent images in axon compartments were captured by confocal microscopy. (E) Images show representative kymographs from three independent experiments. (F) Columns represent the proportion of mitochondria showing different transportation patterns. n = 13 cells from three independent experiments for each group. The data are expressed as means ± SEM. Statistical comparisons were performed with one-way ANOVA with Sidak’s multiple-comparisons test. *P < 0.05, ***P < 0.001 versus the indicated group. Ctrl, control; Mito, mitochondria; Num, number; O-R, OGD-Rep.