Fig. 4

Neuronal loss in the cortical parenchyma. Loss of neuronal NeuN staining is seen in layer II along the sagittal sulcus and in layer V/VI bordering the corpus callosum at 28 days following cytokine vector injection (illustrated for IFA animals in a). No neuronal loss was seen in GFP vector animals (not shown). A decreased density of NeuN+/HuCD+ expressing neurons was seen at 56 days after cytokine vector injection in cortical layers II/III in both IFA (c) and MOG (d,e) immunised animals compared to naïve (b). Extensive regions spreading down from the pial surface in some animals displayed loss of NeuN/HuC/D+ cells (e). A reduction in NeuN+/HuCD+ neurons was present in cortical layer V (white lines) in IFA and MOG cytokine vector injected animals at 56 dpi (f-h). Images of co-staining with HuC/D and NeuN reveal the loss of NeuN expression in some neurons (i). Neuronal numbers were decreased in midline regions (layers I-IV) in both MOG and IFA immunised animals at 28 and 56 dpi (j). Neuronal loss in the upper layers II/III developed more gradually in both MOG and IFA animals and only became significant at 56 dpi (k). In layer V neuronal loss was significant at 28 and 56 dpi in MOG animals but only at 56 dpi in IFA animals (l). Levels of neuronal loss were similar for MOG or IFA animals at 56 dpi (k,l). Neuronal loss only became significant in the lateral cortex at 56 dpi (m-o). Neuronal loss could also be seen 1 mm anterior to the injection site (illustrated for a MOG animal at 56 dpi in p). Immunostaining for NFil and MAP2 in upper cortical layers revealed a reduction of staining in the apical dendritic tufts and the descending dendritic bundles that extended into layer II/III (illustrated for a MOG animal at 28 dpi in q,r). 1-way ANOVA with Tukey post multiple comparisons test, ***P < 0.001, **P < 0.01, *P < 0.05. Data represents mean ± SEM n = 5–7 animals per group. Scale bars = 30 μm (a), 100 μm (b-g,n-p), 20 μm (q,r)