Focus Ultrasound Based Microbubble Mediated Blood-Brain Barrier Opening: A Technique to Create Localized Transient Openings in Blood-Brain Barrier of Mouse by Sonoporation

The blood-brain barrier or BBB consists of closely arranged endothelial cells connected through tight junctions. This forms a compact seal that restricts the movement of molecules to the brain.  To permeate BBB, prep an anesthetized mouse by securing it in the prone position onto a stereotactic unit.

Next, insert a catheter into the tail vein and flush it with an anticoagulant solution to avoid blood clotting. Take a suspension of microbubbles - microspheres of gas enclosed within a lipid layer. Inject the microbubbles into the lateral tail vein. These microbubbles enter the bloodstream, eventually reaching the brain.

Subsequently, identify the location of the brain and apply ultrasound gel to the corresponding spot on the head for better conduction of waves. Implant an ultrasound transducer over the designated area on the mouse's head. Use the transducer to apply strong and targeted ultrasound waves.

Due to the acoustic pressure from ultrasound waves, microbubbles rapidly expand and contract several times. These oscillations cause sonopermeation - disruption of the endothelial tight junctions - generating localized openings in BBB. These openings facilitate the delivery of microbubbles to the brain.

Finally, inject the dye-containing solution intravascularly. The dye molecules extravasate from vessels through the transient openings in BBB. The presence of dye in the mouse brain indicates successful sonoporation.

For the administration of microbubbles for BBB opening, insert a 26 to 30 gauge tail vein catheter and flush the vein with a small volume of heparin solution. Secure the catheter with tissue tape to prevent detachment. If the catheterization is successful, fill the catheter with the heparin solution to avoid blood clotting.

Place the animal on the temperature-regulated stereotactic platform to avoid hypothermia. Fix the head of the animal using a bite bar and ear bars and tape the tail and catheter to the platform. Fix the body of the mouse with a strap.

Place the stereotactic platform with the mounted mouse in the imaging modality and take images of the animal. Determine whether the animal is correctly placed on the platform. Mark the position of the animal using the multi-modality fiducial markers in combination with the image processing pipeline according to the focus point of the transducer. Determine the target area by placing a brain outline over the acquired X-ray image, or by using BLI images to determine the center of the tumor.

Before positioning the transducer, protect the animal's nostrils and mouth with tape to prevent ultrasound gel from interfering with breathing. Apply ultrasound gel on top of the mouse's head for proper sound conduction. Record microbubble cavitation with the needle hydrophone, which is placed in the direct vicinity of the occipital bone.

Based on the calibration values, guide the transducer to the correct position above the animal. Apply pre-configured settings to all attached devices to target the brain region of interest. After the transducer is placed in the right position, dissolve and activate the microbubbles as described by the manufacturer.

Before microbubble injection, flush the tail vein catheter with saline to confirm tail vein access. Start the insonication trajectory and simultaneously inject the microbubbles. At the same time, the needle hydrophone will detect and verify microbubble cavitation in real-time. If desired, administer an intravascular contrast agent or drug after sonoporation. Monitor the animal until a predetermined time point.