Sonodynamic Therapy for the Treatment of Glioblastoma Multiforme in a Mouse Model Using a Portable Benchtop Focused Ultrasound System

Sonodynamic therapy is an incision-free, tumor-specific and low intensity ultrasound treatment for glioblastoma, this fast growing and aggressive brain tumor. MR guidance allows for precision delivery of this focused ultrasound energy to specific brain areas, thereby sparing normal brain tissue and eloquent area. This protocol gives researchers a guideline for preclinical sonodynamic therapy and includes literature-backed parameters for the treatment of glioblastoma in a rodent model.

This technique has the potential to treat brain tumors and other types of central nervous system diseases using non-ionizing radiation without the need for invasive surgeries, without the need for painful chemotherapies, or without the need for radiation therapy. The reported protocol on this study can be generally deemed repeatable and reliable for researchers interested in focused ultrasound, MR-guided Focused ultrasound, sonodynamic therapy and neurosciences, while technical expertise is necessary for appropriate handling of MRI and focused ultrasound system. Demonstrating the procedure will be Griffin Mess, a research engineer and a rising star scientist from my laboratory.

To begin, prepare the MRI bed by placing the nose cone piece into its slot. Then slide the bite bar through the bite bar holes located on the nose cone and at the end of the bed, with the bite guard end hovering over the open well in the MRI bed. Place the anesthetized animal on the MRI bed with its ears lined up with the stereotactic ear bar holes and latch its teeth through the bite guard to keep it in place.

Slide the nose cone forward so that it is placed over the top of the animal's snout. Slide the ear bars through the holes on both sides of the MRI bed and raise the animal's head until both ear bars can fit into the ear canals of the mouse. Ensure that the animal is in a comfortable position.

Then, using a flathead screwdriver, attach the MRI compatible screws to lock in both ear bars, the nose cone and the bite bar. While the animal is waiting, place the MRI bed on a warm heat pad to maintain the body temperature. Insert the phantom in its corresponding MRI bed location and then place the MRI bed into the MRI cradle in its corresponding slot.

Place the MRI cradle in its corresponding location in the MRI. Take an MRI scan of the phantom. Remove the cradle from the magnet bore, but keep it on the scanner.

Remove the MRI bed containing the phantom from the cradle, and then place the bed with the phantom onto the MRGFUS system by sliding the peg on the bottom into its correct slot. Slot the registration tip onto the magnetic slots on the transducer arm so that it points downward toward the phantom. On the software, choose guided focus finding and then select jog mode on to begin guided focused finding.

After jog mode is toggled, use the left, right, up, down, page up and page down keys to manually move around the transducer arm in the left, right, forward, backward, up and down directions respectively. Adjust the pointer manually in all three dimensions until the tip of the pointer touches the middle of the cross pattern that lies on the top of the phantom. The axial phantom slices will then populate the screen on the right side.

Scroll through these slices via the computer mouse's scroll bar. Adjust the brightness by clicking and holding on the image and then moving the computer mouse up or down. Click on the middle of the phantom and a red circle will pop up.

Click and drag on the circle until it is the same diameter and lines up with the circumference of the phantom. Save this home position coordinates by clicking set LR, AP, and SI.Click jog mode off. Remove the registration tip from the transducer arm and then select exit focus finding.

Confirm the home position to complete the initialization sequence. Take the MRI bed containing the animal stereotactically fixed and place it in the MRI cradle previously connected to the MRI scanner. Attach the inlet and outlet anesthesia tubes to the corresponding tubes in the MRI machine.

slide the MRI cradle containing the animal into the MRI bore, making sure to keep the same positioning as where the phantom was placed. Perform a localizer to see the location of the animal brain and then a post-contrast T1-weighted MRI scan covering the entire brain using the MRI settings. In the software, go to the main initialization page and click on guided focus finding.

Then click on jog mode on. Next place a dab of centrifuged ultrasound gel on the animal's head, such that the gel covers the entire scalp above the skull. Then fill 80%of the water bath with deionized and degassed water and place the water bath onto its corresponding columns on the platform.

Lower the water bath until the bottom membrane touches the ultrasound gel on the animal's head, forming a coupling surface between the water and the gel. Submerge the ultrasound transducer into the water bath, ensuring no air bubbles are forming on the transducer surface. After lowering the transducer arm, align the magnetic slots with each other while the transducer surface remains submerged.

For burst mode, go to the sonication settings tab and enter the sonication time. Click on the target icon in the top middle of the page and select the proper locations on the correct MRI slice where the FUS focal region is to be aimed. Clicking the focal spot will highlight the coordinates and the corresponding focal region on the image will turn blue.

The last column on the table shows the power level to be sonicated at each focal spot. Once satisfied with all focal regions, select motion test. Once ready, select start sonication to begin the sonication protocol, moving the transducer and applying the FUS parameters at each focal region selected.

The growth rate of the tumors from pretreatment to 24 hours post-SDT treatment was calculated on the basis of the measured luminescence. The results show that the treated animals showed a reduction in tumor growth compared to controls. Further, contrast-enhanced MR imaging showed that the average grayscale intensity of the contrast agent in tumors increased in the control group to a larger magnitude than in the treated groups, indicating that a lesser amount of contrast agent entered the tumors following treatment.

While we have developed the reported protocol for the treatment of glioblastoma brain tumors, we look forward to learning how other colleagues and investigators will optimize their own parameters for their own particular research. The good news is that sonodynamic therapy is currently undergoing phase one testing in human clinical trials, and we can't wait to learn about its promises for our patients. For maximal efficiency, preclinical optimization of this reported therapy in animal models is necessary to study set and setting, including a whole variety of sonosensitizers, as well as combination therapies.