This article describes a method for delivering fluorescently labeled antibodies to the brain of an anesthetized mouse using focused ultrasound. The technique involves the use of microbubbles to temporarily disrupt blood-brain barrier tight junctions, allowing for targeted delivery of antibodies.
Begin with an anesthetized mouse and shave its head to improve ultrasound wave transmission.
Take a mixture of fluorescently labeled antibodies and microbubbles — a gas-filled core shell surrounded by a lipid layer. Inject them retro-orbitally into the mouse.
The injected microbubbles and antibodies enter the mouse's circulation and reach the brain.
Restrain the mouse and place an ultrasound gel-filled container on the shaved area.
Position the ultrasound transducer and transmit a high-frequency focused ultrasound wave.
The ultrasound waves cause microbubbles to expand and contract repeatedly.
This disrupts the tight junctions of the blood vessels in the brain and creates temporary localized openings.
These openings allow the labeled antibodies to exit the blood vessels and enter the mouse's brain.
Perform fluorescence imaging of the brain and locate fluorescent antibodies, confirming ultrasound-assisted antibody delivery in the mouse model.
To prepare an animal for the analysis after confirming a lack of response to toe-pinch in an anesthetized mouse, use a permanent marker to label the center of the head. Then, glue plastic wrap to the bottom of a small weigh boat with the bottom cut off, and fill the weigh boat with ultrasound gel.
For focused ultrasound treatment, invert the vial of microbubbles and gently load 1 microliter of the solution per gram of the mouse's body weight into a 29-gauge insulin syringe.
Add 100 microliters of fluorescently labeled antibody to the syringe. and gently invert and roll the syringe between thumb and index finger to mix the antibody and the microbubbles within the syringe. Carefully and slowly, retro-orbitally, inject 150 microliters of the microbubble and antibody solution into the mouse, and set a timer for two minutes.
Apply ophthalmic ointment. Then, place the mouse in the head holder and fix its nose in the holder. Place the small ultrasound gel-filled weigh boat on top of the head and lower the water ballast until it sits on top of the ultrasound gel within the weigh boat.
Then, use the joystick to visually align the transducer focus within the center of the head. When the timer goes off in the motion tab of the software, select Reset Origin and select a Complete scan. When the treatment is complete, apply ophthalmic ointment to the animal's eyes, and place the mouse in a warmed recovery chamber.
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