MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

The overall goal of this procedure is to locally disrupt the blood-brain barrier, allowing passage of molecules into the tissue, which are normally confined to the brain vasculature. This is accomplished by first preparing the animal and placing its supine on a water bath above a focused ultrasound transducer, and three axis transducer positioning system. The second step of the procedure is to acquire MRI images in order to provide targeting information to the positioning system.

The third step of the procedure is to prepare and inject the microbubble solution. The final step of the procedure is to set the ultrasound parameters and deliver the ultrasound. Ultimately, results can be obtained that show permeability of the blood-brain barrier at the Sonicated locations through contrast enhanced T one weighted MRI.

The implications of this technique extend toward therapy of brain and CNS disorders because this local and repeatable disruption of the blood-brain barrier allows investigation of pharmacological interventions for conditions which previously have had poor response to drug therapy. Generally, people new to this method will struggle because handling and injecting the bubbles requires patience and care in order to avoid breaking the bubbles before they reach the target. Demonstrating the animal preparation will be shown a ride out.

A technician from our lab Select an appropriate calibrated ultrasound transducer. Next plays a water bath filled with Degas deionized water on the bed of a 1.5 Tesla or three Tesla MRI mount the ultrasound transducer in the tank on an MRI compatible three axis positioning stage or system facing the water surface. The water bath should have a top plate for holding the animals after anesthetizing with isoflurane gas, shave the fur from the top of the head and neck.

Then remove the remaining fur using a depilatory cream. Insert a 22 gauge catheter with three-way stop cock into the tail vein and flush with a heparin saline mixture to prevent clot formation in the catheter. Deliver injectable anesthetics via intramuscular injection and remove the animal from the isof fluorine.

10 minutes prior to the start of the experiment, apply a small amount of ultrasound gel to the top of the animal's head to minimize the chance of trapped air bubbles. Then place the anesthetized animal supine on the ultrasound positioning system with the top of the head. Contacting the water bath through a hole in the top plate.

The head may be held in place using either a bite bar if available or tape placed firmly across the chin. Then tape the legs to the positioning system. Cover the animal with a towel or blanket to keep it warm.

Over the course of the experiment. Acquire baseline axial T two weighted and T one weighted MR images of the brain using the appropriate scan parameters. Select the target from the T two weighted scans, avoiding the ventricles and the brain midline and selecting a midbrain depth.

Move the transducer focus to the target location. Activate definitive microbubbles according to the manufacturer's protocol, and slowly draw up a small volume into a one milliliter syringe. Using an 18 gauge needle, remove trapped air from the syringe by gently moving the plunger back and forth instead of tapping.

As this can break the microbubbles, dilute the microbubbles in normal saline in a ratio of 10 to one saline to microbubbles by slowly injecting the required volume of microbubbles into a syringe of saline. Gently invert the syringe to thoroughly mix the microbubbles and saline until an even appearance is achieved. Calculate the required dose volume based on 0.02 milliliters per kilogram of microbubbles, or 0.2 milliliters per kilogram of the solution.

At 10 to one dilution. Set the sonication parameters using low duty cycle bursts rather than continuous wave sonication. Check that the animal's head is still coupled to the water.

Begin the sonication while simultaneously injecting the microbubbles slowly into the tail vein catheter. Then flush with 0.5 milliliters of normal saline following the sonication. Inject an MRI contrast agent via the tail vein catheter, followed by a 0.5 milliliter saline flush.

Perform T one weighted imaging until the contrast peak has passed.Sonication. Sites that have been successfully disrupted will show greater enhancement than the surrounding tissue. Perform T two weighted imaging to check for high signal at the sonication sites, which is indicative of edema here.

Typical pre and post FUST one weighted MR images are shown here. A contrast enhanced T one weighted image with distinct focal openings. Four sonicated locations with locations one and two showing particularly bright contrast enhancement is shown.

Locations one and two also show high signal strength using T two weighted imaging post FUS indicating edema. The extent of T two weighted edema can sometimes be more easily visualized on sagittal slices here, CET one weighted and T two weighted sagittal slices through sunation. Locations one and three are shown post FUS edema is visible at location one, but not location three.

Spectral analysis of captured acoustic emissions data may show harmonic emissions and or sub and ultra harmonic emissions when stable cavitation is occurring. Data captured during a single 10 millisecond burst at 551.5 kilohertz shows the fundamental frequency as well as harmonics and both sub and ultra harmonics. The use of higher sonication frequencies results in more localized openings due to the smaller focal spot size as shown here.

Higher frequencies can be used to create smaller regions of opening, which allows for midbrain investigation with fewer near skull effects. While attempting this procedure, it's important to remember that careful handling of the microbubbles is critical for achieving good results. Once this technique is refined, investigators can start to answer other questions like, what is the efficacy of delivering a therapeutic to a specific target in the brain?