2.1. Intrauterine inflammation (IUI).

All animals were cared for according to the guidelines of both the University of Cincinnati and the University of Pennsylvania Institutional Animal Care and Use Committees and all procedures were in compliance with the NIH guidelines for the Care and Use of Animals. Details of the intrauterine inflammation model have been published [16], and will be briefly summarized here. CD-1 out-bred mice (Charles River, Wilmington, MA) were used in the present study. These mice have a gestational period of 19 days, and the preterm period is defined as ~70% of gestation which would be embryonic day 15 (E15). To induce intrauterine inflammation, timed-pregnant CD-1 dams were placed under isoflurane anesthesia on E15. A mini-laparotomy was performed and the lower right uterine horn was identified. In separate groups of dams, lipopolysaccharide (LPS; 50 μg/100 μl/mouse, serotype 055:B5, Sigma-Aldrich, St. Louis, MO) or sterile saline (100 μl) was infused between the first two gestational sacs, with care not to inject into the amniotic cavity. Dams were exposed to LPS or saline on E15 and allowed to deliver at term (any dams that delivered prior to E18 were not used). Litters were culled to equal sizes on postnatal day 2 (PN2). At weaning (P21), animals were weighed, and then group housed (4–5/cage) with house chow and water available ad libitum. Only one animal/sex/litter was used in any one experiment.

To collect matched placenta and embryo tissue, dams were killed 48 hr following intrauterine LPS (n=10) or saline exposure (n=5) (E17) (see Figure 1 for experimental timeline). The frontal pole was dissected from the embryonic brain, and DNA and RNA were extracted using Qiagen AllPrep DNA/RNA Mini kit (Qiagen, Valencia, CA). Sex determination was done by assessment of the presence or absence of SRY (sex-determining region Y, expressed only on the Y chromosome) using embryo DNA.

Experimental timeline. In all groups, timed-pregnant dams received injections of either LPS or saline into the right uterine horn at E15. The offspring in experiment 1 were culled at E17 (48 hr following LPS or saline injection) to assess cytokine mRNA expression. The offspring in experiments 2–5 were used to assess mRNA expression of immune factors, anxiety-like behaviors and MPB protein expression. Only one offspring per sex per litter was used for each assessed endpoint.

Adult offspring (n=5/group, 50 weeks of age) were given intraperitoneal injections (at 10am) of either saline or LPS (10 μg/mouse). Animals were killed 2 hr later and brains were removed and placed immediately into RNA later. The prefrontal cortex (PFC), hypothalamus (HYP) and amygdala (AMYG) were dissected as previously described [17, 18] and RNA was extracted using Qiagen AllPrep DNA/RNA Mini kit (Qiagen, Valencia, CA).