Endotoxemia Model in Mice

All mice were housed and cared for at the Rangos Research Center of the Children’s Hospital of Pittsburgh (Pittsburgh, PA), and all experiments were approved by the Institutional Review Board of the University of Pittsburgh (Permit number: 12040382). All studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Throughout the course of all experiments, mice were housed with access to food, water and standard bedding. Endotoxemia was induced in all experiments by intraperitoneal injection of LPS (Escherichia coli 0111:B4 purified by gel filtration chromatography, >99% pure, Sigma-Aldrich) at a dose of 3 mg/kg for 6 hours into 6 week old male mice. At the end of each experiment, all animals were euthanized by CO₂ and cervical dislocation. Immediately prior to injection into mice, the compounds were diluted to an experimental concentration of 100 uM in PBS, with the total concentration of DMSO in the final diluted drug at 1%. Compounds were closely examined to insure that no precipitate formed prior to injection and were stored on ice until injection. In all experiments listed, compounds were delivered to 6 week old mice 30 minutes prior to injection with LPS. Control animals not receiving compound received 1% DMSO dissolved in PBS (“vehicle controls”). Where indicated, mice were also injected with LPS along with the NFκB inhibitor Bay-11-7082 (20 mg/kg, 30 min pretreatment i.p., Cayman Chemical). In addition to assessing the effect on clinical activity of the mice in which the degree of piloerection, tachypnea and movement activity (huddled in the corner versus roaming freely) were assessed, LPS and individual compounds were also injected into NFκB-luciferase reporter mice, in which NFκB is upstream of the luciferase gene (strain NFκB-RE-luc, Taconic Farms Inc, Hudson, NY). In these studies, 6h after LPS injection, mice were administered an i.p. injection of luciferin (160 ug/kg, Caliper Life Sciences), then after 10 minutes, a whole animal image to evaluate luciferase activity was obtained using the IVIS Lumina 3D Optical in vivo imaging system (Caliper Life Sciences, Hopkinton, MA) under 1.5% isofluorane anesthesia. Prior to being euthanized, mice from the above experiments were anesthetized with 1.5% isofluorane and a retro-orbital sinus puncture was performed to obtain a blood sample; serum was obtained via centrifugation and ELISA was performed to assess IL-6 expression (R&D Biosystems). The extent of expression of the pro-inflammatory cytokines IL-6 and iNOS within the intestinal mucosa was determined by RT-PCR (see below).