The blood-brain barrier (BBB) can be temporarily disrupted with microbubble-mediated focused ultrasound (FUS). Here, we describe a step-by-step protocol for high-throughput BBB opening in vivo using a modular FUS system accessible for non-ultrasound experts.
High-grade gliomas such as diffused intrinsic pontine glioma are highly invasive tumors with a high mortality. Despite years of extensive research, the blood-brain barrier is an important aspect because chemotherapy is not able to effectively reach the brain. Fortunately, focused ultrasound is able to temporarily and locally open the blood-brain barrier for effective treatments.
We developed a small and cost-effective neuro-navigated focused ultrasound system for blood-brain barrier disruption. This system is built with commercially available components and with a high degree of automation, it allows for preclinical drug evaluation studies. Precision medicine is defined as the right drug at the right dose for the right patient at the right time.
To achieve this, many preselected drugs need to be screened. Our compact high-throughput focused ultrasound system for blood-brain barrier disruption allow us to do just that. Demonstrating the procedure will be Rianne Haumann and Elvin t Hart, two PhD students from my research group.
The workflow for image-guided stereotactic neuro-navigation focused ultrasound includes interventional imaging and acoustic coupling for sonoporation at the ultrasound platform. Afterwards, followup imaging can be performed. Select a suitable transducer based on the requirements of the designed experiment.
Place it in a 3D printed cone filled with degassed water and closed at the bottom with an acoustically transparent Mylar membrane. Connect the transducer to a function generator and a power amplifier. Mount the transducer on a motorized linear stage for automatic vertical positioning and connect it to the power amplifier.
Use a detachable stereotactic platform that includes temperature-regulated heating, bite and ear bars, anesthesia, and multi-modality fiducial markers. Attach the platform to the 2D linear stage. Before the FUS system can be used, calibrate it and determine the focus point of the transducer.
The focus point corresponds to the vertical position of the animal on the stereotactic platform. Acclimatize the animal to the facility for at least one week after arrival and weigh it regularly. On the day of the experiment, administer buprenorphine via a subcutaneous injection 30 minutes prior to FUS treatment.
After anesthetizing the mouse, apply eye ointment to prevent dry eyes. If necessary, remove the hair on the top of the animal's head with a razor and depilatory cream. Wash the skin with water to remove any residues.
For experiments with BLI tumor models, inject 150 microliters of D-luciferin intra-peritoneally with a 29 gauge insulin syringe. 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. Acoustic pressure with a mechanical index of 0.4 in combination with microbubbles provided a very sensitive and reliable means of stable cavitation detection in comparison to no detection of subharmonics when no microbubbles were injected or the observation of inertial cavitation when an MI of 0.6 was applied. While acoustic pressure with an MI of up to 0.6 resulted in no macroscopic damage, microscopic damage was evidenced histologically at an MI of 0.6.
A pressure amplitude with an MI of 0.8 resulted in a macroscopic brain hemorrhage of larger vessels and widespread tissue lysis with the extravasation of erythrocytes. Intravenous Evans blue was injected to validate the opening of the BBB in the pontine region. Extravasation of Evans blue conjugated albumin was observed at the level of the pons and the cerebellum in the mouse treated with FUS and microbubbles demonstrating precise targeting of the region of interest.
The most important steps of this protocol are the correct placement of the tail vein catheter, the positioning and targeting of the animal, and the microbubble injection with focused ultrasound with cavitation detection. This self-made focused ultrasound system can be adapted and redesigned for multiple research questions, this while being cost-effective and having an automated workflow, which makes it suitable for relatively inexperienced researchers.
