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2003, Traynor et al., 2006, Cunningham et al., 2007 and Konat et al., 2009). TLR3 stimulation induces a much more robust anti-viral response than TLR4 stimulation (Doyle et al., 2003) and this is characterised by high expression of type I interferons. In the current study, we hypothesized that the neurodegenerating brain is primed with respect to stimulation by systemic anti-viral mimetics. Thus, we predicted that ME7 prion-diseased animals would show similar systemic cytokine responses but amplified CNS inflammatory and sickness behavioural responses to systemic poly I:C stimulation, with respect to normal animals given the same stimulus. We have examined the CNS inflammatory profile and in particular, have focussed on type I interferons PI3K Inhibitor Library datasheet and downstream pathways. We Apitolisib ic50 also predicted that poly I:C would accelerate disease progression but have no lasting consequences for

normal animals. Female C57BL/6 mice (Harlan, Bicester, UK), were housed in groups of five and given access to food and water ad libitum. We used females in order to avoid fighting and injury, which has significant effects on behaviour. Animals were kept in a temperature-controlled room (21 °C) with a 12:12 h light–dark cycle. The mice were anaesthetised intraperitoneally (i.p.) with Avertin (2,2,2-tribromoethanol) and positioned in a stereotaxic frame. Two small holes were drilled in the skull either side of the midline to allow for bilateral injection of 1 μl of a 10% w/v ME7-infected C57BL/6 brain Dolutegravir homogenate made in sterile PBS. Injections were made into the dorsal hippocampus (co-ordinates from bregma: anteroposterior, – 2.0 mm; lateral, – 1.6 mm; depth,

– 1.7 mm) using a microsyringe (Hamilton, Reno, Nevada) with a 26 gauge needle. Control animals were injected with a 10% w/v normal brain homogenate (NBH) in PBS, derived from a naive C57BL/6 mouse. All procedures were performed in accordance with United Kingdom Home Office and Republic of Ireland Department of Health & Children licenses and all efforts were made to minimise both the suffering and number of animals used. Poly I:C was obtained from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). It was prepared for injection by resuspending in sterile saline, heating to 50 °C at a concentration of 2 mg/ml to ensure complete solubilisation and then allowing to cool naturally to room temperature to ensure proper annealing of double-stranded RNA. Poly I:C was stored at −20 °C until use. Experimental groups at 18 weeks post-inoculation with ME7 or NBH were challenged intraperitoneally (i.p.) with either poly I:C (12 mg/kg) or sterile saline to examine systemic and CNS inflammatory responses to systemic poly I:C.