Fig. 5

Complement-associated neuronal damage and loss. Large pyramidal neurons (Smi32+) of the deeper laminae of the demyelinated MS cortex displayed surface and cytoplasmic immunoreactive for C3b (a, arrows indicate surface-associated immunoreactivity visualised in a single 0.5-μm optical section acquired by confocal microscopy), some of which (b, projected z-stack image) displayed a dysmorphic nucleus (arrow). Twice as many neurons were immunolabelled with anti-C3b in the MS cortex in comparison to control tissues (c) and C3b+ neurons in MS (Smi32+) displayed a markedly altered nuclear area and aspect ratio (d, e; Box and whiskers plot of median, interquartile and minimum to maximum data range). C3b+ neurons expressed aberrant neurofilaments in the perikarya (f; hypo-phosphorylated neurofilaments, Smi34+) were positive for the pro-apoptotic kinase phosphorylated PKR (g) and the numbers of pPKR+ neuronal nuclei were elevated in MS cortex (g′) in comparison to control. Complement-labelled neurons occasionally expressed the apoptosis-associated markers (h) activated caspase3 (p17 subunit) and (i–i′′′) DNA strand breaks (TUNEL reaction positive, C3b+ neuron). The density of NeuN+ neurons was reduced in normal appearing (18 % reduction) and lesioned (25 % reduction) layer V grey matter, in comparison to non-neurological controls (j–j′′). Group medians compared by the non-parametric Mann-Whitney test (c–e) or Kruskal-Wallis and Dunn’s multiple comparison post-test (j′′). Scale bars: a, b, 50 μm; f–i, 25 μm; j, j′, 200 μm