Artificial Perfusion of the Fetal Circulation of the In situ Mouse Placenta: Methodology and Validation

Here we present methodology and validation (including measurement of unidirectional maternofetal clearance (Kmf) of 45Ca and 14C-mannitol) for in situ perfusion of the mouse placenta. On day 18 of gestation (term = 19 days) mice were anaesthetised and the uterus delivered into a saline bath (40°C). A fetus was selected, the umbilical artery and vein catheterised and perfused with Krebs Ringer (pH 7.4) at 60 μl/min. 45Ca/14C-mannitol (2 μCi/5 μCi in 50 μl saline) was injected via maternal tail vein. Perfusate samples were collected every 5 min for 45 min. Maternal carotid artery pressure was monitored throughout perfusion. A terminal maternal cardiac blood sample was taken and analysed. Placentas were immersion fixed and processed for electron microscopy. Kmf for 45Ca and 14C-mannitol was calculated as perfusate [45Ca or 14C-mannitol] × perfusion rate/maternal plasma [45Ca or 14C-mannitol] × placental weight. Maternal cardiac blood chemistry at termination (n = 8–15, mean ± SEM) was as follows: pH 7.153 ± 0.016, PCO2 45.48 ± 2.06 mmHg, PO2 66.47 ± 7.10 mmHg, Na+ 151.4 ± 1.2 mmol/l, K+ 5.54 ± 0.17 mmol/l, Ca2+ 1.15 ± 0.03 mmol/l, glucose 7.2 ± 0.5 mmol/l, and lactate 1.76 ± 0.77 mmol/l. A successful 45 min perfusion in which perfusate recovery was >95% occurred in >50% of animals. Perfusion did not alter placental morphology or carotid pressure. Kmf (μl/min/g placenta) for 45Ca (66.0 ± 8.4 (n = 7)) was significantly higher than Kmf for 14C-mannitol (20.0 ± 2.4 (n = 5)) (p < 0.01). These data demonstrate physiological perfusion of the mouse placenta in situ and its usefulness for measurement of solute transfer.