Oxygen Radicals Elicit Paralysis and Collapse of Spinal Cord Neuron Growth Cones upon Exposure to Proinflammatory Cytokines
Figure 3
TNFα stimulates ROS formation in advancing growth cones. ROS formation in advancing SC neuron growth cones was revealed by ratiometric fluorescence imaging. Dissociated E7 chick SC neurons (laminin) were loaded (30 min) with the oxidation-sensitive fluorescent indicator 2′,7′-dihydrodichlorofluorescein (10 μM DCF) and the oxidation-inert fluorescent indicator Calcein blue (4 μM CB). Images of randomly selected growth cones were acquired under FITC fluorescence illumination (DCF), DAPI fluorescence illumination (CB), and phase contrast at short time intervals before and after addition of stimuli (40x, oil, identical parameters). (a) Intracellular ROS formation is represented by a heat spectrum ranging from blue (basal ROS levels) to red (increased ROS levels). Growth cone morphology (arrow) disintegrated over a time period of 11 minutes upon acute exposure to 100 ng/mL TNFα (phase images, upper panel). Ratio imaging (middle panel) revealed a sharp increase in ROS production ( = +3 min) shortly after TNFα exposure that persisted ( = +8 min) compared to basal levels ( = −3 min) further illustrated by profile images of ratiometric images (lower panel) (Scale bar = 5 μm). Note the loss of filopodia and lamellipodia and the contraction of the growth cone body (last panel) were preceded by an increase in ROS. (b) ROS formation in advancing SC neuron growth cones was quantified by ratiometric fluorescence imaging. Maximum DCF and CB fluorescence intensities per growth cone area were determined on a pixel-by-pixel basis and DCF/CB ratios calculated for each condition and time point. All DCF/CB ratios were normalized (average DCF/CB ratio at = 0 min) and plotted against time. Exposure to 100 ng/mL TNFα (filled circles, ), 100 ng/mL IL-1β (filled squares, ), or 100 μM hydrogen peroxide (positive control, open circles, ) at = 3 min (arrow) elicited a significant ROS formation (stippled line = significance threshold) in advancing growth cones within less than 5 min upon addition compared to 10 μg/mL ovalbumin (negative control, open triangles, ).
