Supplementary Material for: No short term effect of low dose nicotine on inflammation after global hypoxia in newborn piglets

Introduction: Perinatal asphyxia initiates cytokine release and complement activation with risk of brain damage. We assessed the effect of nicotine on innate immunity and hypothesized that nicotine infusion in a newborn piglet model of asphyxia would decrease the immune response and be neuroprotective. Methods: Newborn piglets (n=41) were randomized to one of three groups after hypoxia: Two groups receiving nicotine, 1) 18 µg/kg/h (n=17), 2) 46 µg/kg/h (n=15), and 3) control group receiving saline (n=9). C3a, IL-6, TNF and IL-10 were measured in plasma and IL-6 and IL-8 in microdialysis fluid from cerebral periventricular white matter, using immuno-assays. Results: Plasma C3a and IL-6 increased significantly from start to end hypoxia (mean 1.9±0.24 to 2.4±0.31 ng/mL and 1.66±1.04 to 2.68±0.71 pg/mL, respectively), while IL-10 and TNF increased significantly after four hours (mean 1.4±1.08 to 2.9±1.87 and 1.4±0.29 to 1.7±0.25 pg/mL, respectively)(p<0.001 for all). IL–6 increased significantly (p<0.001) in microdialysis samples from end hypoxia to end experiment (mean 0.65±0.88 to 2.78±1.84 ng/mL). No significant differences were observed between the nicotine groups and the control group neither in plasma nor in microdialysis samples. Conclusion: Hypoxia lead to rapid release of cytokines in plasma and cerebral microdialysis fluid, and complement activation measured on C3a. However, low dose nicotine administration did not affect the immune response.