Figure 6.

Na_v channel blockers reduce DCF fluorescence induced by ACM-SOD1^G93A, ACM-SOD1^G86R, and ACM-TDP43^A315T. (A) Flow diagram of experiment. Spinal cultures (4 DIV) were exposed for 30 (ACM-SOD1^G93A or ACM-TDP43^A315T) or 60 (ACM-SOD1^G86R) minutes (see Figure 2 for peak of DCF fluorescence), alone or together with Na_v channel blockers mexiletine (25 nM), spermidine (10 μ M), or riluzole (100 nM). Next, cultures were incubated with the membrane permeable ROS/RNS probe CM-H₂DCF-DA and DCF fluorescence was measured 30 min later. (B–E) Graphs showing the percentage of DCF fluorescent cells after being treated with the diverse Na_v channel blockers and Na_v channel blockers and ACM-SOD1^G93A (B), ACM-SOD1^G86R (C), ACM-SOD^WT (D), ACM-TDP43^A315T (E). In all experiment, H₂O₂ (200 μM for 20 min) served as positive control and to normalize the number of DCF-positive cells after ACM application. The graphs indicate statistics relative to motoneurons from sister cultures treated with control medium (indicated with^*) or with only the ACM (indicated with^#). Values represent means ± s.e.m. from at least 3 independent experiments, analyzed by One-Way ANOVA followed by a Tukey post-hoc test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 relative to DCF fluorescence with control media at 7 DIV; ^##P < 0.01 and ^###P < 0.001 compared to DCF fluorescence with ALS-causing ACM at 7 DIV.