Dose response studies have previously been performed

(Cunningham et al., 2007) and this dose produces hypothermia and weight loss in normal mice that is comparable with that induced by infection with influenza virus at 0.1 of its LD50 (McKinstry et al., 2009). The choice of 18 weeks post-ME7 inoculation as the point www.selleckchem.com/products/epz015666.html for systemic challenge for mRNA transcriptional analysis was based on our previous finding that robust priming of microglia occurs at this time (Cunningham et al., 2005a). Animals were initially perfused at 6 h post-poly I:C to capture the time point at which qualitative and quantitative differences were apparent in the hypothermic response (from preliminary data) and subsequently, further animals were perfused at 4 h to examine earlier gene expression. In a subset of animals repeated systemic challenges were made at 14, 16 and 18 weeks to examine the effect of

repeated “viral stimulation” on the progression of neurodegenerative disease. In these animals, temperature responses and acute neurological deficits were measured after each of the three challenges. The data have been presented for temperature at 14 weeks and neurological changes at 16 weeks since these were the APO866 concentration earliest time points in disease at which robust changes were evident. The two weeks interim period allowed full recovery from each systemic inflammatory response before initiation of subsequent challenges. Tissue for analysis of histological changes was taken at 3 or 15 h post-poly I:C to examine microglial and inflammatory markers and neurodegenerative changes, respectively. Under terminal anaesthesia the thoracic cavity was opened and blood collected into heparinised tubes directly from the right atrium of the heart. This procedure was carried out 6 h post-poly I:C challenge. Whole blood was centrifuged to remove cells and the remaining plasma aliquoted and stored at −20 °C before assay. Urease These

samples were then analysed for IL-6, TNF-α and IFN-β (IL-1β levels were previously determined to be considerably lower after poly I:C challenge). Samples were serially diluted to verify linear responses and were quantified only if the absorbance fell on the linear portion of the standard curve. The IFNβ assay kit was supplied by Biosource (Nivelles, Belgium) and mouse IL-6, and TNF-α were measured using R&D systems “duo-set” kits. Protocols followed for these assays were as previously published (Cunningham et al., 2007). The reliable quantitation limit of all assays was 15.6 pg/ml. ME7 and NBH animals were deeply anaesthetised and transcardially perfused with heparinised saline followed by 10% formal-saline at 18 weeks post-inoculation and at 3, 6 or 15 h post-poly I:C or saline for histology experiments.