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

All procedures involving human participants have been performed in compliance with the institutional, national and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Focused Ultrasound System

NOTE: The described setup is an in-house built BBB disruption system based on commercially available components and includes a 3D-printed custom-made cone and detachable stereotactic platform. The system is designed modular, which facilitates modifications according to available equipment and specific use. The protocol describes the procedure for the sonoporation of a larger area in the pontine region of the mouse brain. By adjusting the target location, different parts of the brain could be targeted. In this study, a 1 MHz mono-element transducer with a focal length of 75 mm, an aperture of 60 mm, and a focal area of 1.5 x 1.5 x 5 mm (FWHM of peak pressure) was used. The focal plane of the transducer is positioned through the cranium of the animal in the horizontal plane intersecting with the ear bars.

1. Select an appropriate transducer for BBB opening in rodents.
   NOTE: Based on the properties of the microbubbles and the employed frequency, the acoustic settings, in particular the mechanical index (MI), are subject to change.
2. Place the transducer in the 3D-printed cone.
3. Employ an acoustically transparent mylar membrane at the bottom-end of the cone to achieve acoustic coupling of the beam propagation path, and fill the cone with degassed water.
4. Mount the transducer above the animal on a motorized linear stage as shown in Figure 1 allowing automatic vertical positioning of the transducer.
5. Design a detachable stereotactic platform based on the requirements of the study, which includes temperature regulated heating, bite and ear bars, anesthesia and multi-modality fiducial markers, as shown in Figure 1 and Figure 2. The mounting of the stereotactic platform consists of a 2D linear stage system, which allows precise automatic positioning (< 0.1 mm) of the animal under the beam.
6. Connect the transducer to the acoustic emission chain shown in Figure 1 , consisting of a transducer, a function generator and a power amplifier.
7. Devise an image-processing pipeline to detect the multi-modality fiducial markers that allow precise sonoporation targeting of the brain area of interest and collection of the cavitation data detected by the needle hydrophone.
8. Calibrate the system and determine the focus point of the transducer in correspondence to vertical positioning of the animal on the stereotactic platform.

2. Animal Preparation

NOTE: The following protocol is specified for mice but can be adapted for rats. For these experiments female athymic nude Foxn1-/- mice (6–8 week old) were used.

1. Allow the animal to acclimatize for at least one week in the animal facility and weigh the animal regularly.
2. Administer buprenorphine (0.05 mg/kg) via subcutaneous (s.c.) injection 30 min prior to FUS treatment to start analgesic treatment.
3. Anesthetize the animal with 3% isoflurane, 2 L/min O₂ and verify that the animal is deeply anesthetized. Keep the animals anesthetized during the whole procedure and monitor the breathing frequency and heart rate to adjust the concentration of isoflurane as required.
4. Apply eye ointment to prevent dry eyes and avoid possible injury.
5. Remove hair on the top of the head with a razor and depilatory cream and wash afterwards with water to remove any residues to avoid irritation to the skin.
6. For experiments with BLI tumor models, inject 150 µL of D-luciferin (30 mg/mL) intraperitoneal (i.p.) with a 29G insulin syringe for BLI image-guidance.
7. Insert a 26–30G tail vein catheter and flush the catheter and vein with a small volume of heparin solution (5 UI/mL). Fill the catheter with heparin solution to avoid blood clotting.
   NOTE: Good catheterization is seen when there is a reflux of blood into the catheter. Avoid air bubbles in the catheter to prevent emboli. To avoid excessive injection pressure, make sure the length of the catheter is as short as possible.
8. Place the animal on the temperature regulated stereotactic platform to avoid hypothermia.
   NOTE: Hypothermia reduces blood circulation, which can affect the injection/circulation of microbubbles and the pharmacokinetics of the drugs.
9. Immobilize and fix the head of the animal on the stereotactic platform using ear bars and a bite bar. Fixate the body with a strap and tape the tail of the animal to the platform.

3. In Vivo Image-guided Focused Ultrasound

NOTE: For this protocol a 1 MHz mono-element transducer with a tone-burst pulse with a 10 ms duration, a MI of 0.4 and a pulse repetition frequency of 1.6 Hz with 40 cycles for 240 s was used. The protocol is optimized for microbubbles stabilized by phospholipids containing sulphur hexafluoride (SF₆) as an innocuous gas, whereby the mean bubble diameter is 2.5 μm and more than 90% of the bubbles are smaller than 8 μm.

1. Place the stereotactic platform with the mounted animal in the imaging modality (e.g., BLI or X-ray) and take image(s) of the animal.
2. Use the multi-modality fiducial markers in combination with the image-processing pipeline to mark the position of the animal according to the focus point of the transducer.
3. Determine the target area by placing a brain outline over the acquired X-ray image or using BLI images to determine the center of the tumor (Figure 2). The position of specific parts of the brain are specified in the Paxinos Brain Atlas using the skull markings bregma and lambda as reference points. For example the pons is located x=-1.0, y=-0.8 and z=-4.5 from lambda.
4. Shield the animal’s nostrils and mouth with adhesive tape to prevent ultrasound gel interfering with breathing.
5. Apply ultrasound gel on top of the animal’s head.
6. Retract the skin of the animals’ neck, lubricate the needle hydrophone with ultrasound gel and place the needle hydrophone in the direct vicinity of the occipital bone.
7. Guide the transducer to the correct position using the image-processing pipeline and the focus point.
8. Apply the preconfigured settings to all attached devices and target the brain region of interest.
   NOTE: Depending on the research question, tumor or brain regions can be sonoporated as a single focal point or as volumetric shape, as shown in Figure 2.
9. Activate microbubbles as described by the manufacturer. Inject one bolus of 120 µL (5.4 µg) of microbubbles.
10. Flush the tail vein catheter with saline to check the opening of the catheter.
11. Inject the microbubbles and start the insonation.
12. Record microbubble cavitation with the needle hydrophone.
13. Administer an intravascular contrast agent or drug after sonoporation. The dose, timing and planning are dependent on the purpose of the study and the drug.
    NOTE: Evans blue is a common color agent to assess BBB opening.
14. Monitor the animal until the predetermined time point or before the humane endpoint.