Figure 8.

H₂O₂ levels in retinas of homozygous nox2/cybb^−/− mutants. A, Ratiometric images depicting H₂O₂ levels in the head of a 48 hpf WT zebrafish embryo expressing roGFP2-orp1, before (left), 10 min after 20 mm H₂O₂ (middle), and 10 min after 2.5 mm DTT (right) addition. Dashed white line demarks retina, in which H₂O₂ levels were quantified in B and C. Scale bar: 100 μm. B, Graph showing the normalized 405/480 ratios of WT embryos at 1 min intervals in response to either H₂O₂/DTT (red line; n = 5 embryos) or H₂O (gray line; n = 7 embryos) addition. Data are presented as mean ± SEM. p-values are indicated for paired t tests between H₂O and H₂O₂ (p < 0.0001) and H₂O₂ and DTT (p = 0.002). C, Bar graph showing the basal H₂O₂ levels in retinas of WT (n = 16) and nox2/cybb^−/− mutant (n = 12) embryos. nox2/cybb^−/− mutant embryos do not have significantly different H₂O₂ levels compared with WT embryos (p = 0.2675, unpaired t test with Welch's correction). D, Representative ratiometric images of cultured WT RGC neurons treated with serum-free control medium (left), treated for 30 min with 100 μm H₂O₂ (middle), and cultured nox2/cybb^−/− mutant RGC neuron (right). Scale bar, 10 μm. E, Quantification of normalized 405/480 ratio in growth cones of WT RGCs before and 30 min after treatment with 100 μm H₂O₂, which caused significant increase in growth cone H₂O₂ levels. Data are presented as mean ± SEM; n = 35 indicates number of growth cones; p = 0.0009; Wilcoxon matched pairs test. F, Growth cone H₂O₂ levels are not significantly different between WT (n = 33) and nox2/cybb^−/− (n = 25) RGC neurons (p = 0.46; Mann–Whitney test).