C57bl 6j strain

The wild type (WT: strain C57BL/6J) and Cav1 knockout mice (KO: strain B6.Cg-Cav1^tm1Mls/J) were obtained from the Jackson Laboratory. Approximately 8-week old mice of both strains were used to establish timed pregnancy separately as described earlier [6 (link)]. The vaginal plug was observed to keep a record of the start of pregnancy (day 1). On day 15, the pregnant mice were euthanized, and the fetuses and placentae were collected [22 (link)]. They were washed in sterile PBS and snap frozen in liquid nitrogen. Day 15 was chosen for sample collection because the placenta becomes fully formed and functional at this time point [23 (link)]. The fetal brain also shows the development of all layers of the cerebral cortex at this time [10 (link)]. The development of the cerebral cortex is reliant on placenta function, particularly oxygen transportation to the fetus [24 (link)]. Moreover, the placenta and fetal brain express different receptor and ligand genes suggesting a robust regulation of the brain–placental axis at this developmental time [6 (link)]. The animal procedures were performed by approved procedures and protocols (Approval #25580) of the Institutional Animal Care and Use Committee of the University of Missouri following the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA).

Breeding pairs of eNOS^-/- mice, strain B6.129P2-Nos3^tm1Unc/J, stock # 002684, and wild-type (WT) mice, strain C57BL/6J, stock # 000664, were obtained from Jackson Laboratory (Bar Harbor, ME, USA) and were used to establish breeding colonies. All the animals were maintained at a room temperature of 23 ± 1°C and exposed to a 12 hour alternating light/dark cycle. The animals were fed standard rodent chow (Teklad no. 2918; Harlan Laboratories, Indianapolis, IN, USA) and given tap water ad libitum. The absence of eNOS expression was confirmed in all eNOS^-/- mice by immunoblot analysis.