Matt Harmon

103 Chapter five such as vasoconstriction and shivering. 11 To counteract shivering and thermal discomfort during cooling, 30mg of buspirone was given orally at the initiation of induced normothermia. In addition, the subjects received clonidine (75mcg bolus followed by a continuous infusion of 1-2mcg/kg/h) and magnesium sulphate (4g bolus followed by a maximum continuous infusion of 2g/hour for 150 min). These doses were adapted from previous studies on cooling awake subjects. 12,13 To prevent nausea, ondansetron 4mg was given intravenously. Prior to LPS and at 1, 3, 6, and 8 hours after LPS-infusion, subjects scored their own thermal discomfort on a scale ranging from 0 - 10, with 0 meaning extreme cold discomfort, 5 neutral, and 10 extreme warmth discomfort. Simultaneously, shivering was measured hourly using the bedside shivering assessment scale. 14 Sample collection and analysis and outcomes Blood samples were taken from the arterial catheter just before and at 1, 3, 6, and 8 hours after LPS administration for measurement of full blood count, chemistry and blood gas analysis. EDTA whole blood was centrifugated at 1500g and the supernatant was stored at – 80°C for later analysis of cytokines. ELISA Standard sandwich enzyme-linked immunosorbent assays (ELISA) were used to measure plasma levels of interleukin (iL)-6, iL-10, and tumor necrosis factor alpha (TNF α ) according manufacturer’s protocols (eBioscience, San Diego, CA, USA). ELISAs were performed on the supernatant of the EDTA anticoagulated samples. To account for interplate variability, two samples were included on each plate. Statistical analysis Statistical analyses were performed using Rstudio version 1.2. Depending on normality of the data, differences in baseline characteristics between groups were calculated with either the students T-test or the Wilcoxon ranked sums test. Linear mixed models were used to analyze differences in continuous variables between groups over time, using timepoint and group as fixed effects and subject ID as random effect. If data was non-parametric, it was log transformed prior to statistical testing. For the cytokine measurements an area under the curve (AUC) was calculated based on the absolute change. Subsequently, the AUC between groups was compared using the Wilcoxon ranked sums test. Normally distributed data was presented a mean ± standard deviation (SD). Non-parametric data was presented