Figure 2.

TNFα and IL-1β provoke a redox-sensitive collapse of growth cone motility and morphology. Advancing growth cones in SC neurons cultures (laminin) were randomly selected and images were acquired at 3 min time intervals (20x magnification, phase contrast) in the presence of 10 μM MnTBAP, 2 μM DPI, or PBS (equal volume) before and after acute exposure (t = 6 min) to TNFα (50 or 100 ng/mL), IL-1β (50 or 100 ng/mL), or ovalbumin (10 μg/mL). Growth cone advance was measured as the extension of the growth cone/neurite boundary (μm) per time interval and plotted against time (min) with slopes indicating growth rates. (a and b) Growth cones ceased motility and advance within minutes upon exposure to cytokines (open diamonds, open circles) compared to a steady growth cone advance under control (ovalbumin, closed squares). Growth cones exposed to 100 μM H₂O₂ (open triangles) mostly responded with paralysis, yet slow recovery was measured. In the presence of 50 ng/mL cytokines (open diamonds), growth cones resumed advance after a lag phase however at much slower growth rates, whereas no recovery was detected at concentrations of 100 ng/mL TNFα or IL-1β (open circles). (c and d) ROS scavenging with 5 μM MnTBAP (open triangles) or NOX inhibition with 2 μM DPI (open diamonds) rescued growth cone advance upon acute exposure to 100 ng/mL TNFα (c) or 100 ng/mL IL-1β (d), respectively (open circles). All data were obtained from at least three different dissections (duplicate cultures each, >30 growth cones total) with error bars representing SEM. (e) TNFα elicited a dose-dependent growth cone collapse at concentrations higher than 50 ng/mL. Growth cones with collapsed morphology were quantified (random fields of view) 30 min after application to allow for possible recovery of morphology. (f) Preincubation of SC neuron culture either with 10 μM MnTBAP or with 2 μM DPI provided significant protection against growth cone collapse in the presence of 100 ng/mL TNFα or 100 ng/mL IL-1β (dark grey bars; **P < 0.05) as opposed to cytokines alone (black bars), which caused substantial growth cone collapse (dark bar, *P < 0.05) compared to control (open bar). A presence of 10 μM MnTBAP (light grey bar, Mn) had no effect on basal levels of collapsed growth cones, whereas 2 μM DPI increased the percentage of collapse growth cones. All data (e and f) were obtained from at least three different dissections (duplicate cultures each). Error bars represent ±SEM.