A successful application of these techniques will allow for the kinetic evaluation of pharmacological trials or the passage of xenobiotic contaminants through a morphologically intact placenta. The rodent placenta perfusion technique allows a higher throughput of pharmacological or toxicological studies, as multiple compounds may be assessed using the tissue of a single dam. Demonstrating the technique will be Jeanine D'Errico, a senior graduate student from my laboratory.
Before beginning the procedure, gently fill all of the chambers, needles, glass micropipettes, tubing and reservoirs with warmed physiological salt solution or PSS, carefully removing any air bubbles with a fine tipped transfer pipette as necessary. Turn all of the three way stopcocks to the off position to secure the fluid within the pipettes. Place a single tie on each of the two glass micropipettes prepared for uterine cannulation and on the two blunt tip needles designated for umbilical cannulation.
Then secure the ties to prevent loss during the chamber movements. After confirming a lack of response to toe pinch in an anesthetized gestational day 20 female pregnant rat, lift one uterine horn from the abdomen and spread the horn out longways, external to the animal. Use braided silk sutures to tie off the uterine artery at the vaginal and ovarian ends of the horn, including the ovary inside the suture with the uterine horn.
Using surgical scissors, make incisions on the proximal side of the ovary tie and the distal side of the vaginal tie. Transfer the uterine horn into a dissecting dish lined with silicone rubber and filled with cold PSS. With the ovary side to the left and the vaginal side to the right, gently push a dissecting pin through the uterine horn into the silicone rubber to stabilize the uterus.
Select a maternal placenta fetal unit central to the horn. Using fine forceps and scissors, remove the amniotic membrane from the fetal surface of the placenta taking care to avoid the umbilical cord. Unravel and ligate the umbilical cord to separate the fetal pup.
After identifying the umbilical artery and vein, gently separate and ligate the umbilical vessels. Then cut the umbilical vein slight shorter than the umbilical artery for ease of identification. And ligate the uterine artery and vein with surgical scissors.
Maintaining the correct orientation of the uterine artery anatomical blood flow, place the placental unit into the modified isolated vessel chamber filled with warm oxygenated PSS under a dissecting microscope. Using one pair of fine forceps in each hand, cannulate the proximal and distal ends of the uterine artery onto the glass micropipettes. Tightly secure the artery with the previously placed sterile nylon suture.
Cannulate and secure the umbilical artery onto the 23 gauge blunt needle. Then cannulate and secure the umbilical vein onto the 25 gauge blunt needle and backfill all of the stopcocks and tubes to prevent air bubbles. Remember to check all of the stopcocks, cannulas, and tubing for air bubbles, as an air bolus in the system will lead to cellular distress and eliminate the counter current flow within the placenta.
Connect all the tubing according to the figure. Place small weigh boats under the distal cannulation of the maternal uterine artery and under the needle cannulation of the fetal umbilical vein to catch the effluent that will emerge during the course of the procedure. Prepare the collection plate for the effluent for later analysis.
Open the stopcock to permit fluid flow through the tubing toward the placenta and turn on the peristaltic pump, pressure control and pressure monitor. Slowly increase the pressure to 80 millimeters mercury read on the pressure monitor. Slowly open the stopcock to permit fluid flow to the uterine artery.
Refill all of the reservoirs to maintain the fluid volume throughout the experiment. After the tissues have equilibrated for 30 minutes to allow the vasculature to adjust to the new fluid flow, establish the baseline perfusion by collecting effluents for 10 minutes from both of the weigh boats. Measure the volume of fluid that emerges through the uterine artery and umbilical vein.
At the end of the baseline collection period, administer the experimental treatment into the uterine artery. Then collect samples from the distal uterine artery and fetal umbilical vein effluent every 10 minutes for a total of 180 minutes after the infusion. Measure the contaminants within the fluid samples.
Perfusion with Evans blue dye to test the system and to visualize the appropriate fluid and placental barrier function for preventing contaminant transfer to the fetal compartment reveals that the dye reached and perfused the placental tissue within this system. Upon closer investigation, it is clear that the Evans blue dye did not enter the fetal umbilical vein, which is expected, as the Evans blue dye is bound to albumin. In this representative experiment, a reduced fluid transfer to the fetal compartment within 10 minutes of polystyrene infusion was identified.
The polystyrene transfer to the fetal compartment then peaked at 20 minutes and continued for 90 minutes. The most important thing to remember during the cannulation is to go slowly. The pipettes and the tissue are very delicate and can easily become broken or torn.
By weighing the effluence, this methodology could be used to investigate placental physiology as well as material translocation.
