Injection of Microbubbles into Isolated Mouse Embryos: A Technique to Deliver Microbubble Contrast Agents into the Vasculature of Living Murine Embryos

Pipette the necessary volume of microbubbles stock solution and add to the aliquot of saline. Mix by stirring gently with the tip of the pipette. Then, use a 21-gauge needle to draw the diluted microbubble solution into a clean syringe. After removing the needle, eliminate any air bubbles from the syringe and attach the luer and tubing. Slowly push the solution to the end of the tubing, making sure not to generate any air bubbles.

Finally, insert the syringe into a syringe infusion pump set to dispense the microbubbles at a rate of 20 microliters per minute for a total volume of 20 microliters. Attach a pulled glass needle to the end of the tubing. Move the pump close to the injection stage. Use a perforated spoon to randomly select and remove one embryo from the chilled media dish. Place the embryo in a dissection dish located on the ultrasound stage under the stereoscope.

Next, locate the side of the embryo that appears the least vascularized. Here's an area of low vascularization, generally adjacent to the head region. Then, use fine forceps to cut or tear just enough of the yolk sac and amniotic membranes to be able to remove the embryo from within, but no more. Next, gently maneuver the sac from around the embryo. Then, position the embryo on its side with the placenta and umbilical vessels in front.

Stabilize the dissection dish using small pieces of plasticine. Using four insect pins, pin down the yolk sac and edges of the placenta, avoiding any major vessels. Using a syringe containing pre-warmed 45 degrees Celsius PBS, wash the embryo until blood can be seen flowing into the embryo. The umbilical artery is identified by the pulsatile, bright red blood. The umbilical vein and its associated vascular network have darker blood and usually overlay those from the umbilical artery on the placental side.

At this point, position the dish so the injection can be done comfortably. Once the embryo has revived, cover it, but not the placenta, with pre-warmed ultrasound gel. Use fine forceps to delicately remove any air bubbles from around the embryo and top up the dish with pre-warmed PBS. Throughout the procedure, keep an eye on the level of solution in the PBS and gel syringes and add backup syringes to the heating beaker as needed.

Next, mount the glass needle on a large ball of plasticine at the edge of the dissection dish and insert the needle end into the PBS. Choose a vein on the chorionic surface of the placental disc as far from the main branch as reasonable. Then, use scissors to trim the needle tip to match the vessel size, cutting the tip at a slight angle. Use the edge of the spring scissors to remove any jagged edges of glass.

Now, using the syringe pump, slowly inject the microbubble solution at 20 microliters per minute into the glass needle until all of the air is expelled from the needle tip and the microbubbles can be seen to flow freely into the PBS. Stop the pump and reset it for an injection volume of 20 microliters. Do not allow air to enter the injection needle so as to avoid injecting air into the embryo's vascular system.

Insert the glass needle tip gently into the selected vein in the placenta and ensure that it is immobile. Swing away the stereoscope head and position the ultrasound transducer so that the embryo is situated evenly between the foci at 6 and 10 millimeters. Next, initiate imaging and start the bolus injection of the microbubbles. After a minute, when the injection is complete, start the timer for the data acquisition step.