A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

Focused Ultrasound in combination with circulating micro-bubbles can be used to provide temporary millimeter size openings in the blood-brain barrier. This enables non-invasive delivery of systemically circulating agents to target brain regions. To begin, fill the catheter plug with saline and warm the rat's tail with a lamp, taking care not to overheat the animal.

Insert a 24 gauge tail vein catheter that will be used to deliver micro-bubbles, Evans blue dye, gadobutrol MRI contrast if using MRI, and the experimental agent of interest. Blood will fill the sheath when the vein is hit. Slowly, remove the inner needle while pushing the sheath further into the vein.

Screw the catheter plug into the end of the catheter port, as soon as the port has filled with blood. Carefully wrap lab tape around the catheter and the tail to keep it in place, starting with a small piece at the top and working in the caudal direction. Leave the very end of the catheter plug exposed.

Plug the anesthesia line onto the anesthesia connector on the stereotactic frame. Then fix the animal's head into the frame by placing the mouth onto the bite bar, and by guiding the ear bars into both ear canals and tightening the set screws. Move the animal to the MRI bed.

Using parameters described in the text manuscript collect coronal and axial T2 weighted images that capture the whole brain as well as the MRI fiducial for coordinate measurements. On the coronal images, find the image in which the fiducial is the largest, indicating the center of the fiducial. Record the distance from the top of the fiducial to the brain region of interest in millimeters, in both the medial-lateral direction and in the dorsal-ventral direction.

On the axial images, find the image that shows the very top of the fiducial and record the X and Y coordinates for the center of the fiducial and the X and Y coordinates of the brain region of interest. Calculate the distance from the center of the fiducial to the target brain region in both the rostral-caudal and medial-lateral directions. After gathering the coordinates, collect the pre-scan images.

Keeping the animal in this stereotaxic frame, quickly transport it from the MRI bed to the bench top FUS set up. Ensure that the animal remains asleep under the effect of anesthesia. Slide the frame into the frame holder and firmly snap it into place.

Use clippers to shave the animal's head, then brush away excess hair and apply hair remover cream to the scalp. Let it sit for three minutes and wipe it away with water and gauze. If using MRI guidance, attach the pointer and move the pointer to the location of the MRI fiducial.

Position the pointer at the very top and center of the MRI fiducial then, which is the point from which all distances in the MRI image were calculated. Remove the pointer and move the positioner to the medial-lateral coordinates and the rostral-caudal coordinates. Lick the null position button and raise the positioner up by pressing the up 50 button, to allow for the placement of the water bath and ultrasound gel.

Apply ultrasound gel to the animal's scalp and place the water bath over the animal, with the polyimide tape window pressed onto the gel, making sure that there are no air bubbles in the gel. Fill the water bath with degassed water. If using the high power transducer, lower the positioner so that the magnet is just above the water.

Attach the transducer to the positioner by carefully lowering the transducer into the water at an angle and connecting the magnets. Lower the positioner to the dorsal-ventral coordinate and turn on the RF power amp. Inject one milliliter per kilogram of 3%Evans blue dye by sticking a needle tip into the catheter plug and injecting.

The whole animal should become blue within seconds, indicating the catheter is properly positioned in the tail vein. Allow the dye to circulate for five minutes, then activate the micro-bubbles by shaking them violently with the bubble shaker. Invert this syringe several times to get a uniform distribution of micro-bubbles, then attach and fill the winged infusion set.

Position the syringe on the infusion pump and set the infusion pump to deliver 0.2 milliliters at a rate of six milliliters per hour, providing slow infusion of the micro-bubbles over the two minute FUS exposure. Insert the winged needle into the catheter plug. First run the infusion pump, wait three seconds, then start the FUS treatment by pressing the on button and on the function generator.

Repeat this twice per region with five minutes in between to allow the micro-bubbles to clear. Press the on button on the function generator again to stop the FUS treatment when the infusion pump stops at two minutes. Wait for five minutes for the micro bubbles to clear, then start the infusion and the second FUS treatment.

Immediately after the second FUS treatment, inject gadobutrol contrast and the agent of interest. Total delivered volume of all agents should not exceed five milliliters per kilogram. To confirm BBB opening, place the animal back onto the MRI bed at the exact same location and plug in the anesthesia line.

Collect MRI post-scans with the same imaging parameters as the pre-scan images to confirm gadobutrol MRI enhancement in the region of BBB opening. This protocol was used to induce localized blood-brain barrier opening with both the low power immersion transducer and the high power focused ultrasound transducer. First, the low power immersion transducer was targeted to either the anterior or medial brain hemisphere.

The animals were sacrificed with or without perfusion, and the BBB opening was visualized via Evans blue dye auto-fluorescence. In later experiments, the FUS transducer was targeted to either the hippocampus or the anterior cingulate cortex. In addition to Evans blue dye, the MRI contrast agent gadobutrol was used to verify the targeted opening of the BBB in VIVO.

After the animals were sacrificed, Evans blue dye auto-fluorescence confirmed the opening location. To assess whether this technique could be used for targeted gene delivery, AAV nine expressing green fluorescent protein and gadobutrol contrast were injected immediately after blood brain barrier opening in the hippocampus. The animal was imaged to verify the opening with gadobutrol contrast.

Then, gene delivery was confirmed by GFP expression. This protocol offers a bench-top approach to MRI guided Focus Ultrasound mediated blood-brain barrier opening, that can be easily adapted by other laboratories to provide a non-invasive translational alternative to stereotactic surgery.