Fig. 9

HC toxicity to mouse CNS cells requires activation of the classical complement cascade. a Simplified diagram of the complement cascade depicting both the classical and alternative pathways. Red lines indicate the location of blocks in the complement pathway resulting from either C4 or C5 depletion. b C3d staining in pure neuronal monocultures. Cultures were treated with medium only (CTRL), 5% C4-depeleted, or C5-depleted human complement (C4depHC or C5depHC) for 30 min followed by live staining with anti-C3d antibody and DAPI. c C3d staining in neuroglial mixed cultures. Mixed cultures prepared from PLP-eGFP mouse pups were incubated with medium only (CTRL), 5% C4depHC, or 5% C5depHC in the presence/absence of NMO rAb #53 for 30 min followed by live staining of C3d. Cells were then fixed and stained with neuronal marker β-Tubulin and DAPI. Note: In mixed cultures treated with C5depHC alone, C3d was present on the surface of neurons (N, arrows) and differentiated OLs (OL, arrow heads), which were sensitive to HC. In cell cultures treated with NMO rAb #53 plus C5depHC, C3d was detected on the surface of neurons (arrows), OLs (arrow heads), and surrounding astrocytes (stars). No C3d staining was observed on OPCs in mixed cultures. d C3d and MAC staining in slice culture. PLP-eGFP slices were treated with 10% C5depHC or human complement (HC) for 48 h and stained with C3d and MAC, respectively. Neurofilament was visualized with NF-H staining. Deposits of C3d or MAC were detected along the NF-H+ processes (arrows) and surrounding the oligodendrocyte cell bodies (arrow heads). Scale bars 50 μm