Figure 7.

TNFα and IL-1β impede neurite outgrowth in a redox-sensitive manner. Dissociated SC neurons were grown on laminin past the onset of neurite initiation and then incubated with 10 μM MnTBAP, 2 μM DPI, or PBS (10 μL) for 1 h prior to bath application of cytokines or ovalbumin (6 to 8 h) followed by fixation (2% glutaraldehyde). The longest neurite per neuron was measured of randomly selected neurons and the percentage of neurons with a given neurite length was plotted against neurite length. (a and b) Persistent presence of TNFα (a) or IL-1β (b) significantly reduced neurite outgrowth in a dose-dependent manner (40 ng/mL, open triangles; and 100 ng/mL, open squares) indicated by the shift of the neurite length distribution to shorter neurites compared to control (10 μg/mL ovalbumin, filled circles). (c and d) Despite a continuous presence of 100 ng/mL TNFα ((c), open squares) or 100 ng/mL IL-1β ((d), open squares), scavenging ROS with 10 μM MnTBAP (open triangles) rescued neurite outgrowth compared to controls (10 μg/mL ovalbumin, filled circles), whereas inhibiting NOX activity with 2 μM DPI (open diamonds) was only partially protective. (Neurite number measured ≥ 60 for each condition, two experiments, and duplicate cultures). (e) In the presence of 20 μM MnTBAP (open diamonds), neurite outgrowth was significantly decreased compared to control (PBS, filled circles). However, concentrations of 10 μM MnTBAP (open triangles) did not significantly alter neurite outgrowth and 5 μM MnTBAP (open circles) on the contrary causes a significant increase in neurite length. (Neurite number measured > 75 for each condition, two experiments, and duplicate cultures). (f) Lastly, an overabundance of the radical scavenger NAC (2 mM, open squares) dramatically reduced neurite outgrowth compared to control (PBS, filled circles) indicated by the shift towards shorter neurites.