*4.2. Placental Ischemia Induction*

On GD 14, rats were anesthetized using isoflurane and an abdominal incision was made. The utero-placental unit was exteriorized and a silver clip (0.203 mm) was placed on the abdominal aorta (below the kidneys and above the bifurcation). Silver clips (0.1 mm) were also placed on both uterine artery branches between the ovaries and the first pup. This procedure induces placental ischemia by reducing utero-placental perfusion pressure (RUPP). Pregnant rats in the sham group were treated similarly, in that, an abdominal incision was made, the uterine horn was exteriorized, and vessels were manipulated without clip placement. Carprofen (5 mg/kg) was used as an analgesic in both groups.

#### *4.3. Carotid Surgery and Blood Pressure Measurement*

On GD18, rats were anesthetized using isoflurane anesthesia and the left carotid artery was isolated and cannulated using pre-made saline-filled catheters. Catheters were secured and exteriorized at the nape of the neck. The incision was closed and secured using Vetbond. The following morning, rats were placed in restrainer cages and catheters were connected to a pressure transducer. After

30 min of acclimation, blood pressure was recorded for 30 min using LabChart software. The mean arterial pressure was calculated for the duration of 30 min.

### *4.4. Harvest and Collection of Tissues*

On GD 19, pregnan<sup>t</sup> rats were anesthetized using isoflurane, and an abdominal incision was made. After exteriorization of the fetal-placental unit, maternal blood was collected from the abdominal aorta. Dams were euthanized by removal of the heart. The number of live and resorbed pups were counted. Fetuses were euthanized by decapitation. Trunk blood was collected from fetuses using micro-capillary tubes and hematocrit was noted. Fetal heads were processed di fferently depending on endpoints. For brain water content, brains were removed, weighed, and then dried for 48 h at 60 ◦C. Brain water content was calculated as a percentage ((wet weight − dry weight)/ wet weight). Brains for molecular analyses were removed and flash frozen in liquid nitrogen, followed by storage in a −80 ◦C freezer until processing. Brains for immunofluorescence staining or histology were kept within the skull and placed in 4% paraformaldehyde at 4 ◦C overnight. The following day, brains were removed from the skull and placed back in 4% paraformaldehyde overnight. Brains were then transferred to a 30% sucrose solution at 4 ◦C for 72 h and then embedded in Cryogel, and frozen at −80 ◦C until sectioning (1–2 fetal brains per mold). Brains were sectioned at 20 μm thickness, transferred directly to slides, and stored at −20 ◦C until staining.
