Oxygen Radicals Elicit Paralysis and Collapse of Spinal Cord Neuron Growth Cones upon Exposure to Proinflammatory Cytokines
Figure 1
TNFα and IL-1β impair motility and morphology of neuronal growth cones. (a) Acute exposure of dissociated E7 SC neurons (laminin) to 100 ng/mL TNFα (top panel) or IL-1β (middle panel) provoked paralysis of growth cone advance and rapid degeneration of the morphology of growth cones and neurites as opposed to 10 μg/mL ovalbumin (bottom panel). Stars indicate location of the growth cone/neurite border with respect to first image in each panel, respectively (scale bar = 10 μm). (b) To restrict cytokine exposure exclusively to advancing growth cones, polystyrene beads (2.5 × 105 beads/mL, 4 μm in diameter) coated with TNFα or IL-1β were applied to SC neuron cultures and growth cone-bead encounters observed under phase contrast (63x oil, phase contrast). Following physical contact of growth cones to cytokine-coated beads (TNFα—top panel, IL-1β—middle panel), growth cone motility ceased followed by the progressive degeneration of growth cone morphology upon reaching complete collapse. Contact with ovalbumin-coated beads had no influence on growth cone morphology and advance (bottom panel). (c) E7 SC neurons grown on laminin for 2 days were fixed in paraformaldehyde. Cytokine receptors were revealed by indirect immunocytochemistry and analyzed by confocal microscopy (Zeiss LSM510, 40x oil, NA 1.30). TNFα receptor 1 (TNF-R1) and IL-1β receptor (IL-1R) were expressed on cell bodies (arrows) and neurites were expressed as well as on growth cones and filopodia (arrowheads). In contrast, TNFα receptor 2 (TNF-R2) expression was restricted to cell bodies. (Scale bars: upper panel, 20 μm; lower panel 10 μm). Western blots of whole spinal cord (E7 chick) extracts revealed immunoreactivity (stars) against avian cytokine receptors TNF-R1 (48 kDa), TNF-R2 (70 kDa), and IL-1R (76 kDa) as determined in spinal cord lysates (50 μg total protein per lane).
