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

Protocollo

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Microbubble Preparation

1. Preparing microbubbles for injection
   NOTE: The assistant performs this step when ‘Injection of Microbubbles (MB) into Embryos’ 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 microbubbles 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). 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.

2. 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.

Materiali

Name	Company	Catalog Number	Comments
Ice
0.9% sodium chloride (saline)	Hospira	0409-7984-11
Phosphate Buffered Saline [1x]	Sigma	D8537	1x, w/o calcium chloride & magnesium chloride
Ultrasound contrast agent, target ready and untargeted	MicroMarker; VisualSonics Inc.
Ultrasound gel (Aquasonic 100, colourless)	CSP Medical	133-1009
Ice bucket	Cole Parmer	RK 06274-01
Imaging Platform	VisualSonics Inc.	Integrated Rail System
Light source, fiber-optic	Fisher Scientific	12-562-36	Ideally has adjustable arms
Luers (12), polypropylene barbed female ¼-28 UNF thread	Cole Parmer	45500-30
Micro-ultrasound system, high-frequency	VisualSonics Inc.	Vevo2100
Needles, 21 gauge  (1”)	VWR	305165
Perforated spoon (Moria)	Fine Science Tools	MC 17 10373-17
Pins (6), black anodized minutien 0.15 mm	Fine Science Tools	26002-15
Syringes, 1 mL Normject	Fisher	14-817-25
Syringe infusion pump	Bio-lynx	NE-1000
Pipettors [2-20 μL, 20-200 μL, 100-1,000 μL]	Eppendorf	Research Plus adjustable 3120000038; 3120000054; 3120000062
Pipettor tips [2-200 μL, 50-1,000 μL]	Eppendorf	epT.I.P.S. 22491334; 022491351
Scissors
Glass capillaries, 1 x 90 mm GD-1 with filament	Narishige	GD-1
Glass needle puller	Narishige	PN-30
Tubes, Eppendorf	VWR	20170-577
Tube racks (3)	VWR	82024-462
Hot plate	VWR	89090-994
Syringes (10), 30 mL	VWR	CA64000-041
Ultrasound transducer, 20 MHz	VisualSonics Inc.	MS250