A Methodological Protocol and Considerations for Transcranial Ultrasonic Stimulation in Exploratory Clinical Human Studies

Our neuromodulation and the Stroke Recovery Lab focus on using various neuromodulation tools to enhance post-stroke recovery, including motor recovery. This particular project involves using low-intensity transcranial ultrasound stimulation, a new non-invasive brain stimulation tool to modulate the motor cortex excitability and promote motor learning in stroke patients. To begin, prepare a water tank with degassed deionized water to prevent potential damage to the hydrophone.

Fixate the ultrasound transducer onto the fixture on the water tank, and mount the hydrophone onto the three-axis positioning system. Connect the hydrophone to an oscilloscope and then connect the needle tip along with the cable. Switch on the hydrophone power only after it is fully immersed in water.

Drive the ultrasound transducer using a function generator. Then position the hydrophone tip at the approximate focal region of the ultrasound transducer using the three-axis positioning system. Record the oscilloscope readings at each location to obtain the acoustic pressure distribution and document the hydrophone location corresponding to the focal region.

Assess the transmitting sensitivity of the ultrasound transducer at the focal point by applying different input voltages. Connect the transcranial ultrasound stimulation hardware and start oscilloscope recording. Turn on the oscilloscope.

Set it to run and adjust both vertical and horizontal axes to the scale of interest. Flip the main power switch of the amplifier to the On position. Dial the power output of the amplifier to zero.

Configure both function generators to the desired waveform, ensuring ramping is added to each pulse duration. Now press Output on the function generator. Dial the amplifier output to 100%using the adjust wheel and monitor the gain display.

Press the amplifier power button to start its output. Confirm the drive signal waveform on the oscilloscope, and allow the drive system to run for 15 minutes to stabilize the output. Check for ramping at the beginning and end of each pulse duration on the drive signal waveform.

Dial the amplifier output to zero. Fully immerse the transducer in water, positioning the exit plane approximately one inch below the surface facing upward. Then dial the amplifier output back to 100%and observe whether the ramping is successful.

Now mount a neuro navigation tool tracker onto the transducer holder. In the software, navigate to Window and Tool Calibrations. Click New Calibration.

Enter a name and select the correct tool tracker. Hold the transducer with the tracker against the calibration block, such that the reference indicator pin touches and is perpendicular to the center of the transducer's exit plane. Click Begin Calibration Countdown.

Hold steady for five seconds and repeat if necessary until success appears. Then close both Edit Calibration and Tool Calibrations windows. Press the amplifier power button to turn off its output when the participant is ready for transcranial ultrasound stimulation.

Disconnect the oscilloscope from the amplifier and connect the transducer to the amplifier output. If using the simulation-based target, load the target into the neuro navigation software. In the Sessions tab, open the new dropdown and select Online Session.

Under the Targets tab, click on the Trajectory-x-mod target and press Add to add it for the session. Bring up the bullseye tool centric or bullseye target centric view in the Perform window. Set the driver to the correct TUS transducer and select the correct target in the Targets to sample list on the upper left.

Comb the participant's hair to expose the scalp at the TUS transducer site and create an anterior/posterior parting line. Apply the gassed ultrasound gel along it. Create one to three additional parting lines approximately 1.5 centimeters away on each side, and repeat the same process.

Finally, return to the initial parting line and repeat the process four times. Now, strap the TUS transducer onto the participant's head and align it with the target using the bullseye view. Adjust the transducer to minimize distance, tilting, and rotational deviations to within operational limits.

If safety monitoring is required, insert a thermocouple wire into the ultrasound gel close to the scalp. Tape the thermocouple wire on the head using paper tape to prevent movement. Press the amplifier's power button to start output and begin TUS.

Simultaneously press Sample Now on the neuro navigation software to record initial transducer placement. Continuously monitor the three alignment metrics in the bullseye view. Monitor the scalp temperature using the thermocouple wire if installed.

To finish TUS, press the amplifier power button to stop the output. Remove the transducer and all head-mounted equipment from the participant. Inspect the scalp for any signs of redness, and ask the participant about any discomfort or adverse effects.

If no issues are reported or after addressing concerns, gently wipe off the ultrasound gel from the scalp. Click Finish on the neuro navigation window and save the project by selecting File and pressing Save Project. From the sessions list, select the current session and click Review.

In the Sessions Review window, choose brain site text file from the Export dropdown menu. Ensure Orientation is selected and set Snap to to Nothing. Configure other export settings as needed, then click Save to export the stimulation data.

A satisfactory simulation based targeting result was achieved where the acoustic focus reached the intended cortical stimulation region. A scenario in simulation-based targeting that requires additional iterations due to misalignment between the acoustic focus and the intended stimulation target. Adjusting the Distance TX out plane to skin parameter to a more realistic value to correct the visualization scaling issue in the simulation interface.

A clean motor evoked potential was recorded, indicating good transcranial magnetic stimulation, coil placement, and orientation. A waveform was observed that might not represent a motor evoked potential. A successful MT one millivolt determination was made at 52%maximum stimulator output, with five positive and five negative responses at that level.

A potentially inconclusive MT one millivolt determination was recorded at 87%maximum stimulator output due to consecutive, rather than alternating positive and negative responses. A steady, even oscilloscope reading of the transducer driving signal was obtained, indicating proper equipment function. Good alignment was achieved between the physical transducer placement and the predefined target using neuro navigation.

A misaligned transducer placement was observed, indicating the need for adjustment to match the target. We established TUS safety and effective intensity in stroke patients showing motor cortex TUS can enhance motor learning and cortical excitation. There's no methodological article focusing on the use of TUS in neurologically disease adults.

This protocol addresses this gap. We hope this protocol can reduce the methodological variability and improve the reliability and reproducibility of future human ultrasound studies.