Fig. 5

Expression of cell death related proteins in cortical neurons. CNPase+/Cleaved Casp-3+ oligodendrocytes are illustrated at 28 dpi in the cortical layers of MOG immunised animals injected with cytokine viral vectors, indicating apoptosis (a), whereas no NeuN+/Cleaved Casp-3+ neurons were found (b). QRT-PCR analysis showed increased gene expression for the necroptosis genes RIPK3 and MLKL in cortical tissue from 28 dpi cytokine viral vector injected animals compared to GFP controls, with no significant difference between IFA or MOG immunised animals (c). Levels of endogenous rat IFNγ (d) and TNF (e) genes were upregulated at 28 and 56 dpi in both MOG and IFA immunised animals, with highest levels at 28dpi. Statistics C-E: t-tests on Ct values, *P < 0.05. Laminar distribution of pMLKL staining in naïve and MOG animals shows very minimal expression in naïve animals and significant expression in layers II/III and V in a MOG immunised animal at 56 dpi (f). This pattern was not different between MOG and IFA immunised animals (not shown). pMLKL expression was greatest in layers II-III in closest proximity to the subpial surface, with similar numbers of cells were present in IFA and MOG immunised animals (g,h). Co-staining for NeuN and pMLKL showed the absence of pMLKL in naïve cortex (i) and identified pMLKL-expressing cells as neurons in 56 dpi cytokine vector injected MOG immunised animals (j; from region in dashed box). Confocal imaging of neurons from the subpial cortical layers showed that pMLKL was upregulated in cytokine vector injected animals within the nucleus of neurons, whilst the unphosphorylated form was located in the cytoplasm (k). Although pMLKL was largely constrained to the nucleus in the majority of neurons (k,l), pMLKL could also be seen in the cytoplasm/membrane compartment in some neurons (m). Scale bars = 10 μm (a,b), 200 μm (f-h), 5 μm (k-m)