LPS endotoxemia challenge

Lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (Sigma-Aldrich, St. Louis, MO) was dissolved in saline and sonicated for 30 min before administration. LPS doses were determined empirically to produce physiological levels of TNF as described in Caravaca et al., 2019. In one set of experiments, performed by the Tracey group (Feinstein Institutes), 8-week-old mice (n = 12) were implanted with a left VN cuff. On day 9–17 post-surgery, VNS or sham stimulation was delivered twice (once in the morning and once in the evening) under light anesthesia using 1 mA intensity at 250 μs PW and 30 Hz frequency for 5 min. On the following day, mice were administered LPS (0.7 mg/kg, i.p.) 5 hr after receiving a third dose of VNS or sham stimulation. In another set of experiments, performed by the Zanos group (Feinstein Institutes), 8-week-old mice were implanted with a left VN cuff. HRT was determined at least 5 days before endotoxemia to avoid any long-lived VNS effects. 2–6 weeks post-implantation, animals were anesthetized and received either sham stimulation or VNS at HRT intensity using 250 μs PW and 10 Hz frequency for 5 min. LPS was administered to mice (0.1 mg/kg, i.p.) 3 hr after stimulation. In both sets of experiments, blood was collected by cardiac puncture 90 min post-LPS injection and left to clot for 1 hr at room temperature. The blood samples were then centrifuged at 2000 xg for 10 min and serum collected for TNF determination by ELISA (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Serum samples were assayed in duplicate for each animal.