Escherichia coli endotoxin

All animal procedures were in accordance with the Animal Care and Use Committee guidelines at Johns Hopkins University and the United States Department of Agriculture (reference number: RB14M324), as described previously [18 (link), 19 (link)]. Timed pregnant New Zealand white rabbits were obtained from Robinson Services Inc. (Winston-Salem, NC). Briefly, pregnant rabbits in the endotoxin/CP group underwent laparotomy at gestational day 28 (term pregnancy is 31 days) and were injected with 1 mL of saline containing Escherichia coli endotoxin (~6000 EU) (serotype O127: B8, Sigma Aldrich) along the length of the uterus. At this dose, the newborn kits have been shown to have uniform pro-inflammatory microglial activation in the periventricular region (PVR), increased expression of TNF-α, and display a phenotype of CP with predominantly hindlimb hypertonia [16 (link), 19 (link)]. The healthy control group included pregnant rabbits that had no surgery or intervention. All pregnant dams were induced on the evening of gestational day 30 (G30) to control timing of delivery, and kits were used for the experiments on postnatal day 1, corresponding to G31.

On day 11, pigs in all three groups were randomly assigned to (±LPS) treatment (Figure 1). All pigs in the LPS groups received an 8 h continuous infusion (10 µg/kg·h) of Escherichia coli endotoxin (lyophilized E. coli Serotype 0111-B4, Sigma Chemical, St. Louis, MO, USA) while the control groups received an equal volume of sterile saline solution (0.9% sodium chloride). The LPS (5 mg/mL in 0.9% saline) or 0.9% saline only was added to the respective lipid emulsion and thus continuously infused into the intravenous catheter via Y-connection along with total PN solution. Vital signs including, heart rate, SpO₂, and rectal temperature were measured each hour during the LPS or saline infusion. Jugular blood samples were collected into EDTA tubes on days 0, 11, and hourly during the 8 h continuous LPS or saline infusion, and then processed to separate plasma and red blood cells. Both fractions were frozen in separate tubes and stored at −80 °C. All pigs were euthanized at the end of the 8 h protocol.