Oxygen Radicals Elicit Paralysis and Collapse of Spinal Cord Neuron Growth Cones upon Exposure to Proinflammatory Cytokines
Figure 6
TNFα and IL-1β stimulate a Rac1-mediated ROS formation in SC neurons. Dissociated SC neurons grown on laminin were infected with recombinant, replication deficient adenovirus (200 moi, infection at the time of plating) carrying FLAG-tagged constitutively active , FLAG-tagged dominant negative , or lacZ. Three days after infection, SC neurons were loaded with 10 μM DCF (30 min), incubated with NAC, MnTBAP, or DPI, and exposed to cytokines (100 ng/mL). Random images were acquired (20x) under FITC fluorescence illumination, relative maximum DCF fluorescence intensity was quantified, and all values normalized to lacZ-expressing SC neurons exposed to 10 μg/mL Ovalbumin (lacZ), our control. (a) Expression of completely abolished ROS formation in SC neurons upon exposure to 100 ng/mL TNFα or IL-1β (TNFα-N17 and IL-1β-N17, resp.) compared to lacZ-expressing SC neurons (TNFα and IL-1β, resp., ) without altering basal levels of ROS formation (N17). (b) Expression of was sufficient to induce ROS formation in SC neurons, which was negated by 2 mM NAC, 10 μM MnTBAP, or 10 μM DPI. Peroxide (200 μMol/L H2O2) served as a DCF loading control. (c) DCF-loaded SC neurons expressing (V12) revealed increases in ROS formation both in cell bodies (arrowhead) as well as in neuronal growth cones (arrow) and distal neurites (left panel). In contrast, SC neurons expressing (N17) exhibited basal DCF fluorescence intensity compared to lacZ (Con) expressing SC neurons (right panel). Note the intense DCF fluorescence in a degenerating cell (asterisk). A heat spectrum ranging from blue (basal ROS levels) to red (increased ROS levels) indicates ROS production (scale bar = 40 μm).
