Fig. 2

Complement-mediated neuron injury in astrocyte-neuron cocultures. a Complement-dependent cytotoxicity in pure neuron and pure astrocyte cultures following incubation with 20 μg/ml AQP4-IgG and 2% human complement for 2 h, with fixable dead cell marker (reactive amine dye, labeled “dead cell”). Cultures were immunostained for GFAP and MAP2, with dead cells stained red. b Cocultures were incubated as in a and immunostained for GFAP (green) and MAP2 (gray), with dead cells red. Expanded images (lower panels) showing dead astrocytes (filled yellow arrowheads) and nearby dead neurons (open yellow arrowheads) in representative fields. Bar graph at the right shows percentage of dead neurons at different distances from the center of dead astrocytes (mean ± S.E.M., n = 6 cultures, total 32 astrocytes imaged, *P < 0.01 comparing AQP4-IgG vs. control IgG). c Cocultures were incubated with 1% NMO patient serum and 5% human complement and immunostained as in panel b. d Panels from time-lapse image sequence (see Additional file 1: Movie 1) showing astrocytes and neurons in coculture before and at 0.5 and 2 h after addition of 20 μg/ml AQP4-IgG and 2% human complement containing dead cell marker ethidium homodimer-1. Labels in the left panel: n, neuron; a, astrocyte. Filled arrowheads point to early damage of neurons in contact with astrocytes, and red color is due to uptake of dead cell marker. Open arrowheads indicate sites of neurite blebbing and degeneration