These data have also been illustrated as repeated acute events superimposed upon longitudinal decline (Fig. 7e and f) to illustrate the influence of repeated anti-viral responses on disease course. We have demonstrated that the primary response to systemic poly I:C (i.e. peripheral induction of IFNβ) was not significantly different after one, two or three systemic challenges with poly I:C (12 mg/kg i.p.). These data are shown Caspase inhibitor in Supplementary data (S3). We observed
small numbers of activated caspase-3-positive cells and larger numbers of TUNEL-positive cells in ME7 animals 15 h after treatment with saline or poly I:C. Examples of both activated caspase-3 and TUNEL-positive cells are shown in Fig. 8 (a and b). The larger number and smaller size of TUNEL-positive cells reflects the later stage of cell-degeneration, as we have previously
shown after LPS treatment of ME7 animals (Cunningham et al., 2005a and Cunningham et al., 2005b). TUNEL-positive apoptotic cells (positive labelling plus condensed nucleus) were counted in the areas of pathology (the hippocampus and thalamus) in 10 μm sections of animals 15 h post-challenge with poly I:C or saline. ME7 + poly I:C animals had significantly higher Y-27632 clinical trial numbers of apoptotic cells per 10 μm section than ME7 + saline (12 ± 3 versus 6 ± 1; p < 0.05 by one-way ANOVA with Bonferroni post hoc test). NBH + poly I:C animals showed very low number of oxyclozanide apoptotic cells (1 ± 1 per 10 μm section). These data are also shown in Table 2. We examined expression of pro-apoptotic genes PKR, Fas and Bax (Fig. 8c–e) and found a clear poly I:C-induced increase in PKR and Fas mRNA expression. Bax was induced somewhat in ME7 animals, but not elevated further by poly I:C treatment. Two time-points are
provided to provide temporal information but post hoc comparisons have only been performed on the 4 h data. Disease and poly I:C influence PKR expression (F = 13.53, df 5, 20, p < 0.0001) and Bonferroni post hoc comparisons revealed that while NBH and ME7 were not significantly different, NBH + poly I:C was significantly lower than ME7 + poly I:C at 4 h (p < 0.05). Similar analysis of Bax revealed that NBH was significantly different to ME7 but that no further changes were induced by poly I:C treatment. Analysis of Fas data revealed a significant one-way ANOVA (F = 38.3, df 5, 20, p < 0.0001) and Bonferroni post hoc tests showed that NBH was significantly different to ME7 (p < 0.001) and that ME7 + poly I:C was significantly higher than both ME7 (p < 0.001) and NBH + poly I:C (p < 0.01). Thus there was increased apoptosis and amplified expression of pro-apoptotic genes in ME7 + poly I:C animals.