All procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and with the approval of the Institutional Animal Care and Use Committee at Boston Children’s Hospital. Under isoflurane anesthesia, a laparotomy was performed on pregnant Sprague–Dawley rats on E18 (Figure 1A). For TSHI, uterine arteries were occluded (Figure 1B) and after 60 minutes the clips were removed (Figure 1C). For the combined injury, following 60 minutes of TSHI, 4 μg sterile LPS (LPS 0111:B4, Sigma, St. Louis, MO) mixed with diluted Evans blue dye (Sigma) was injected into each amniotic sac (Figure 1D). For LPS alone, LPS was injected without transient uterine artery occlusion. For sham controls, the laparotomy was performed and uterine horns were exposed for 60 minutes, without artery occlusion or LPS injection. Thus, all dams experienced an equivalent time of laparotomy under anesthesia. Pups were born at term (E22) and matured with their respective dams. Litter size was recorded. Pups were weaned at P21. Pups were weighed at the ages noted, and gender was recorded. Overall, 12 sham, 16 TSHI, 7 LPS and 18 TSHI + LPS dams were used.
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