Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

The overall goal of this procedure is to inject microbubbles into isolated living mouse embryos. For contrast enhanced ultrasound imaging. This is accomplished by first harvesting living E 16.5 or E 17.5 embryos from a pregnant mouse.

After preparing the targeting microbubbles, the microbubbles are injected in the living embryo via the placental vascular network. The final step is to image the microbubbles using ultrasound imaging. Ultimately, contrast enhanced ultrasound imaging may be used to measure vascular perfusion parameters and assess targeted microbubble binding in embryos.

Generally, individuals new to this method will struggle with the embryo handling and injection portions of the protocol until they've had sufficient practice in applying the needle and forceps pressures required for a successful experiment demonstrating the procedure will be Brian Nixon, a co-op student from our lab To reconstitute the target ready vials begin by diluting 20 micrograms of the antibody solution into a final volume of one milliliter of saline. Keep the vials on ice. Next, fill a syringe with one milliliter of saline.

Removing all air bubbles. Attach a 21 gauge needle and inject the solution slowly into the microbubble vial. Slowly withdraw the plunger, removing one milliliter of air, then withdraw the needle.

Gently agitate the vial and let it stand on ice for five minutes. After five minutes, fill a new syringe with the antibody dilution and add it to the appropriate vial. After agitating, gently let the mixture now two milliliters.

Stand for 10 minutes on ice. To reconstitute the untargeted vials, fill a syringe with one milliliter of saline. Removing all air bubbles.

Attach a 21 gauge needle and inject the solution slowly into a new microbubble vial. Slowly withdraw the plunger, removing one milliliter of air, then withdraw the needle. Gently agitate and let it stand for five minutes.

After adding an additional one milliliter of saline to bring the volume up to two milliliters, gently agitate and let it stand for 10 minutes on ice. After gently agitating the desired microbubble vial, use a 21 gauge needle on a one milliliter syringe to draw up approximately 50 microliters of the solution. Then inject the microbubbles into an empty two milliliter micro centrifuge tube.

Next pipette a 10 microliter sample of Microbubble stock solution into 10 milliliters of diluent. After gently mixing, use Coulter counting to assess the concentration and size distributions of the microbubble population. Count a minimum of three 50 microliter measurements per vial sample.

Using a 30 micrometer aperture. This step is performed in parallel with the injection of microbubbles into the embryos. While the surgeon is injecting the embryos, the assistant prepares the next injection to ensure that the surgeon is blind to the type of bubble being injected.

Determine the volume of stock bubble solution required to produce a final concentration of 100 million microbubbles per milliliter in a volume of 400 microliters. Aliquot the corresponding volume of saline into an empty micro centrifuge tube. After gently agitating the selected microbubble vial, use a one milliliter syringe with a 21 gauge needle to draw up an excess volume of the solution.

Then inject the microbubbles into an empty micro centrifuge tube. Next, pipette the necessary volume of Microbubble stock solution and add to the Ali quad 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 lure 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 micro bubbles 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 sack 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 pinned down the yolk sack and edges of the placenta, avoiding any major vessels using a syringe containing prewarm. 45 degrees Celsius.

PBS wash the embryo until blood can be seen flowing into the embryo. The umbilical artery is identified by the pulsitile 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 prewarm ultrasound gel, use fine forceps to delicately remove any air bubbles from around the embryo and top up the dish with prewarm 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 six 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.

This B mode ultrasound image shows an exteriorized E 17.5 embryo immediately prior to the injection of targeted microbubbles. This nonlinear contrast ultrasound image shows the embryo before the injection of microbubbles, when only the strongly reflecting interfaces such as bone are visible with minimal signal from the soft tissues. The injection of the microbubbles enhances the contrast throughout the animal, including the inferior vena cava, heart and brain.

An intensity plot of microbubbles as a function of time within a single region of interest in the embryonic brain allows the identification of various perfusion parameters, including peak enhancement or pe wash in rate shown by the slope and time to peak or TTP. The average contrast mean power ratios from endoglin targeted microbubbles were significantly higher than those collected for control in untargeted microbubbles. Furthermore, endoglin targeted microbubble binding was found to be significantly higher in ogly plus plus embryos compared to ogly plus minus embryos.

Once mastered, each embryo can be injected and imaged in 15 minutes if the technique is performed properly.