Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

NOTE: The experimental procedures performed in this study were approved by the Animal Care Committee at Sunnybrook Research Institute (Toronto, Ontario, Canada). Procedures for the humane treatment of animals must be observed at all times. It is assumed that the investigator is trained in the basic operation of an ultrasound imaging system. This protocol works best with two people.

1. Animal Models

1. Mate CD-1 male and female Mus musculus to obtain wild type embryos for perfusion studies.
2. For molecular imaging studies, generate Eng+/- mice by homologous recombination using embryonic stem cells of 129/Ola origin as described by Bourdeau et al.¹⁴.
   1. Backcross B6- Eng^+/- mice, kindly acquired from Dr. Michelle Letarte, to the CD-1 background. Use Eng^+/- CD-1 backcrossed embryos, as well as their Eng^+/+ littermate controls. Mate the mice to produce staged embryos.

2. Experimental Preparation

1. Initiate mating of mice and check plugs daily. Embryonic day 0.5 is defined as noon of the day a vaginal plug is observed.
2. Prepare embryo media by adding 50 ml of fetal bovine serum, 5 ml of 1 M HEPES and 5 ml of Penicillin-Streptomycin (10,000 units penicillin, 10,000 µg streptomycin) to 500 ml Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (4,500 mg/L). Wrap the bottle in foil and keep chilled at 4 °C.
3. Centrifuge clear ultrasound gel in 50 ml conical tubes at 140 x g for 20 min. Draw gel up into 30 ml syringes. Fill additional (marked) 30 ml syringes with phosphate buffered saline (PBS) and set aside. One litter of embryos will generally require between 2-3 syringes of ultrasound gel and 4 syringes of PBS.
4. Pull approximately 30 glass needles and gently fasten to a strip of plasticine in a 100 x 20 mm cell culture dish. This ensures that the needles are separated and secure during handling. Using a glass puller, use the following settings on 1 x 90 mm glass capillaries with filament: Heater 77, Sub magnet 55, Main magnet 70.
5. Prepare ten to twelve dissection plates (enough to ensure each embryo within a litter will receive its own dish), using a 1:8 volume ratio of curing agent to base. Pour 40 ml of base into a 50 ml conical tube. Add 5-6 ml curing agent and stir with wooden stick.
   1. Pour into 60 x 15 mm culture dishes, filling each to approximately half. Let stand O/N to set.
6. Attach female luers to 400 mm long pieces of polyvinyl chloride (PVC) tubing (inner diameter: 0.79 mm). The tubing must reach from the syringe pump to the ultrasound stage comfortably.

3. Experimental Set-up

1. Arrange the imaging platform under the surgical microscope with a 3D motor and linear array 21 MHz transducer.
   1. Orient the platform so that the rail mount is on the left, with the stage positioned directly underneath the microscope.
   2. Attach the 3D motor to the ball joint of the extending arm of the platform. Screw the quick release post of the transducer clamp into the quick release mount on the motor, facing forward, and position the transducer head in the clamp, fastening the latch.
   3. Secure the transducer connector in the active port on the front panel of the cart. Turn the machine on.
2. Initiate the 21 MHz ultrasound transducer, selecting the ‘cardiology’ setting for molecular imaging studies, and ‘general’ for perfusion studies. Shift all time-gain-compensation sliders to the exact middle position. For ‘Contrast Mode’, set the following parameters: Frequency: 18 MHz, transmit power: 4%, (0.39 MPa), Contrast Gain: 30 dB, Foci: 6 & 10 mm, Contrast Mode: Nonlinear, Burst time: 100% at 0.1 sec, Beam width: Wide.
3. Aliquot 40 ml embryo media each into four 50 ml conical tubes. Place on ice.
4. Heat 1 L of water in a 2 L beaker on a hot plate. Maintain at 45 °C. Add one syringe of ultrasound gel and PBS to the beaker to preheat. Maintain this temperature by monitoring with a thermometer at all times.
5. Set aside ten to twelve 1 ml syringes and ten to twelve 21 G needles for the injection of microbubbles.

4. Surgical Procedure

NOTE: Have an assistant prepare the bubbles (stage 5) while the surgeon commences the surgical procedure. The protocol described here has been adapted from Whiteley et al.¹⁵

1. Collect E16.5 or E17.5 embryos from the pregnant female mouse following cervical dislocation.
   NOTE: The effects of anesthesia on embryos have not yet been studied in detail¹⁶. Additional methods, including decapitation, may be appropriate options for the sacrifice of the pregnant mice.
   1. Cut open the abdomen to reveal the uterus & embryos. Carefully and quickly remove the uterus by cutting at the tips of the uterine horns (severing the ovarian and uterine vessels) and severing at the vagina and bladder.
   2. Using splinter forceps, transfer the uterus to a tube of 40 ml of ice-cold embryo media as quickly as possible without damaging the embryos. Discard the mouse carcass.
2. Relocate to microscope and stage. Deposit the uterus into a 100 x 20 mm cell culture dish. Transfer the uterus into a new dish filled with 50 ml fresh embryo media.
3. Using sterilized (70% ethanol spray) fine forceps, gently tear and pull away the outer membranes of the uterus starting at one end. Expose the placental decidua, parietal yolk sac and Reichert’s membrane to separate and remove from the underlying visceral yolk sac.
4. Dissect out the embryos one by one, keeping the visceral yolk sacs intact and handling the placenta as gently as possible¹⁷. Take care to avoid rupture of the yolk sac as the embryos pop out immediately.
   1. Use the perforated spoon to move the embryos to another dish kept on ice with fresh embryo media. Keep the embryos chilled until needed. Change the media in the embryo dish every 1-1.5 hr. With these conditions embryos are viable for up to 4 hr after dissection.

5. Microbubble Preparation

NOTE: Perform molecular imaging with ultrasound using both targeted and untargeted microbubbles. A single experiment can require as many as 3 separate vials of microbubbles: i) antibody targeted microbubbles, ii) control isotype antibody targeted microbubbles and iii) untargeted microbubbles. The contrast agent comes as a dry-freeze powder and must be reconstituted with saline before injection. There are ~2 x 10⁹ microbubbles in each untargeted microbubble vial and ~8.8 x 10⁸ microbubbles in the target ready vials. The microbubbles are stable for up to 3 hr after reconstitution.

1. Reconstitution of Target Ready Vials
   NOTE: Examples of target ready preparations: endoglin targeted microbubbles (MB_E, with biotinylated rat MJ 7/18 antibody to mouse endoglin; in house hybridoma facility); vascular endothelial growth factor receptor 2 (VEGFR2) targeted microbubbles (MB_V, with biotin anti-mouse CD309); rat isotype IgG₂ control antibody targeted microbubbles (MB_C, biotin rat isotype IgG₂ control targeting mouse IgG2a).
   1. Dilute the antibody solution (20 µg) into 1 ml (final volume) of saline. Keep on ice.
   2. Fill syringe with 1 ml saline, removing all air bubbles. Attaching a 21 G needle, inject the solution slowly into the microbubble vial.
   3. Slowly withdraw the plunger, removing 1 ml of air, and withdraw the needle. Gently agitate. Let stand 5 min on ice.
      NOTE: Ultrasound microbubbles can be destroyed under high-pressure environments.
   4. After the elapsed time, fill a new syringe with the antibody dilution and add to the appropriate vial. Gently agitate. Let the mixture (now 2 ml) stand for 10 min. Keep on ice. Assuming complete surface conjugation, the average number of bound ligand for the microbubbles is approximately¹⁸ 7,600 ligands/µm².
2. Reconstitution of Untargeted Vials
   1. Fill syringe with 1 ml saline, removing all air bubbles. Attaching a 21 G needle, inject the solution slowly into the microbubble vial.
   2. Slowly withdraw the plunger, removing 1 ml of air, and withdraw the needle. Gently agitate. Let stand 5 min. Add an additional 1 ml saline, as described above. Gently agitate and let stand 10 min on ice.
3. Coulter Counting
   1. Gently agitate the desired microbubble vial and draw ~50 µl of the solution into a 1 ml syringe using the 21 G needle. Inject the microbubbles into an empty 2 ml microcentrifuge tube.
   2. Pipette a 10 µl sample of microbubble stock solution into 10 ml of diluent.
   3. After gentle mixing, assess the concentration and size distributions of the microbubble population by using coulter counting (see List of Materials). Count a minimum of three 50 µl measurements (per vial sample) using a 30 µm aperture.
4. Preparing Microbubbles for Injection
   NOTE: The assistant performs this step when ‘Injection of Microbubbles into Embryos’ (step 6) is commenced. Each embryo is injected once, with the type of microbubble chosen for each injection to be randomly selected by the assistant such that all types of microbubble are evenly distributed throughout the procedure but given in a random order. Ensure the surgeon is blind to the type of bubble being injected.
   1. Determine the volume of stock bubble solution required to produce a final concentration of 1 x 10⁸ MB/ml in a volume of 400 µl (calculate the volume using the stock concentrations measured in 5.3.3). Aliquot the corresponding volume of saline into an empty microcentrifuge tube.
   2. Gently agitate the selected microbubble vial and draw an excess volume (~50 µl greater than that calculated above) of the solution into a 1 ml syringe using the 21 G needle. Inject the microbubbles into an empty microcentrifuge tube.
   3. Pipette the necessary volume of microbubble stock solution and add to the aliquot of saline. Mix by stirring gently with the tip of the pipette.
   4. Draw the diluted microbubble solution into a clean syringe using the 21 G needle. 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.
   5. Insert the syringe into a syringe infusion pump set to dispense the microbubbles at a rate of 20 µl/min for a total volume of 20 µl. Attach a pulled glass needle to the end of the tubing. Move the pump close to the injection stage.

6. Injection of Microbubbles into Embryos

1. Randomly select and remove (with perforated spoon) one embryo from the chilled media dish. Place in dissection dish located on the ultrasound stage under the stereoscope and remove the yolk sac and amniotic membranes with the fine forceps, cutting/tearing from the side that appears least vascularized (the antimesometrial side). This is most easily achieved by piercing an area adjacent to the head region.
   1. Cut enough to remove the embryo from within, but no more. Stabilize the dissection dishes using small pieces of plasticine.
2. Gently maneuver the sac from around the embryo. Position the embryo on its side, with the placenta and umbilical vessels in front. Using the insect pins, pin down the yolk sac (4 pins recommended) and edges of the placenta as necessary to affix in place. Avoid major vessels.
3. Wash the embryo with pre-warmed 45 °C PBS until the embryo revives. Identify the umbilical vein and its associated vascular network. Position the dish so that injection can be done comfortably.
   NOTE: Once warmed, blood in the embryo will begin to flow, visibly pumping in the umbilical artery. When flow first begins, the veins will be a bright red, with the blood in both vessels quickly appearing identical. The branches arising from the umbilical vein usually overlay those from the umbilical artery on the placental surface.
4. Once the embryo has revived, cover it (but not the placenta) with pre-warmed US gel, being sure to delicately remove any air bubbles (using the fine forceps) from around the embryo. Top up the dish with pre-warmed PBS.
   NOTE: Keep an eye on the level of solution in the PBS and gel syringes and add backup syringes to the heating beaker as needed.
5. 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. Choosing a vein on the chorionic surface of the placental disc, as far from the main branch as reasonable, trim the tip to size using scissors. Injection is easiest if the tip is cut at a slight angle. Remove any jagged edges of the glass using the edge of the spring scissors.
6. Using the syringe pump, slowly inject the microbubble solution at 20 µl/min into the glass needle until all of the air is expelled from the needle tip and microbubbles can be seen to flow freely into the PBS. Stop the pump and reset for an injection volume of 20 µl. Do not allow air to enter the embryo’s vascular system during injection.
7. Insert the glass needle tip gently into one of the veins in the placenta and ensure it is immobile. Swing away the stereoscope head and position the transducer above the embryo. Initiate imaging.

7. Ultrasound Molecular Imaging

NOTE: Using the contrast nonlinear imaging conditions set previously, position the transducer so that the embryo is situated evenly between the foci at 6 and 10 mm. Once positioned, start the bolus injection of the microbubbles. When the injection is complete, start the timer.

1. Perfusion Imaging
   1. Ensure that the maximum number of frames for nonlinear contrast mode is selected by pressing the ‘study management’ button and clicking the ‘Prefs’ tab at the top of the study browser. Check the appropriate box in the ‘Preferences’ window.
   2. Adjust the imaging settings by initiating nonlinear imaging. Press the ‘contrast mode’ button on the control panel. Refine the 2D gain by adjusting the ‘gain’ dial to 30 dB, reduce the power to 4% using the ‘power’ dial, decrease the frequency to 1 Hz using the ‘frequency’ dial and set the beam width (bottom left corner) to wide using the roller ball/mouse.
      NOTE: All controls are located on the control panel of the ultrasound system.
   3. Capture the entire wash-in and dissipation of the microbubble bolus by recording a 20 min cine loop at a frame rate of 1 Hz. Press scan/freeze to start the data acquisition.
   4. Once the full sequence of images has been captured, press the ‘cine store’ button located on the control panel to save the sequence of images in the system buffer. The system creates a date-stamped cine loop of the nonlinear contrast model data acquired.
2. Targeted Microbubble Imaging
   1. Under ‘Prefs’, set the burst time to 0.1 sec. Set the appropriate imaging parameters using the controls described above (7.1.2.).
   2. Initiate nonlinear contrast imaging by pressing the ‘contrast mode’ button on the control panel. Ensure the embryo is properly situated under the transducer and that the flow of microbubble contrast agent through the embryo is visible in the nonlinear contrast mode image.
   3. To assess targeted microbubble binding during embryonic molecular imaging studies, allow the microbubbles to circulate undisturbed for 3 min and 40 sec after completion of injection. This time is to allow retention of the circulating microbubbles. Press ‘scan/freeze’ to halt imaging during this time.
   4. At 3 min and 40 sec, resume imaging to confirm that the embryo is still visible, is adequately covered with PBS, and that microbubbles continue to circulate. Press ‘scan/freeze’ to initiate imaging.
   5. Acquire the disruption/replenishment sequence at 3 min and 50 sec post injection by recording a ‘pre-destruction’ acoustic response sequence at 29 Hz. Press ‘scan/freeze’ to start the data acquisition. At 4 min post injection^18,19, initiate a 0.1 sec high-frequency acoustic burst. Press the ‘burst’ button to implement a burst.
   6. Continue to record frames for the remainder of the sequence (until the buffer line is full) and save the cine loop by pressing ‘cine store’.
   7. Collect an additional cine loop of circulating microbubbles. This subsequent ‘post-destruction’ imaging sequence is assumed to contain only circulating bubble signals that have replenished the beam. Press ‘scan/freeze’ to initiate imaging and ‘cine store’ to collect the images.
   8. Label the cine loops after imaging each embryo. Press ‘study management’, highlight the file using the roller ball and the ‘select’ button, press ‘image label’ and name appropriately.

8. Handling of Embryos after Injection

1. Post imaging, move the transducer, unpin the embryo and euthanize (via decapitation, cervical dislocation, carbon dioxide asphyxiation, or anesthesia followed by rapid freezing or fixation) as desired.
2. For each subsequent embryo injection a fresh dissection dish, needle, syringe and tubing segment must be used, with preparation of the next batch of microbubble injection solution taking place during dissection and revival.
3. Save tissue samples (e.g., tail, brain) for additional analyses (e.g., genotyping determined by PCR analysis of isolated tail DNA, lacz staining, or semi-quantitative measures of biomarker expression using western blots²¹).