High-Intensity Focused Ultrasound for Blocking Nerve Action Potentials in a Diabetic Rat Model

Begin with an anesthetized diabetic rat with an exposed pain-transmitting sciatic nerve that contains pre-implanted stimulating electrodes. The sciatic nerve is connected to the gastrocnemius muscle, where the recording electrodes are positioned.

Lift the sciatic nerve and position the high-intensity focused ultrasound or HIFU fixator base beneath it.

Using a connector, secure the nerve in the fixator slot and attach the HIFU transducer over it.

Fill the fixator’s central well with a degassed coupling solution for ultrasound propagation.

Deliver an electrical stimulus through the stimulating electrodes to trigger nerve action potentials that are transmitted to the muscle.

Record this baseline muscle signal.

While continuing to record, direct the HIFU beam through the transducer to the nerve.

HIFU induces mechanical stress and increases temperature that transiently blocks action potential propagation to the muscle and lowers the signal.

This correlates with diminished pain perception, demonstrating HIFU's potential for diabetic neuropathic pain relief.

Five weeks after STZ injection, after anesthetizing the rats and performing a pinch of the plantar surface of the foot according to the text protocol, use an electric clipper to remove the hair from the thigh and lower back. Apply liquid iodine to the skin on the surgical site. Then use an alcohol pad to wipe off the liquid iodine.

With a sterile scalpel, make an incision of the skin at the dorsal thigh. Use surgical scissors to spread the tissue underneath the skin. And use skin hooks to secure the skin.

With the scissors, carefully separate the muscles parallel to the femur until the mid thigh sciatic nerve fibers embedded in the muscles are visible. Carefully use a glass hook to separate the mid thigh sciatic nerve from the surrounding connective tissue and muscles. Then to position the sciatic nerve in the High-Intensity Focused Ultrasound or HIFU focal local zone using a custom made nerve fixator, use a glass hook to carefully lift the nerve. And put it in the slot of component 1 of the device.

Next, screw component 2 to component 1. Then use Ringer's solution to fill the central well of component 1 for ultrasonic propagation and nerve preservation. Screw component 3 to the HIFU housing cone. Then through the four legs of component 2, dock component 3 with component 2.

After inserting one pair of acupuncture needles into the origin of the sciatic nerve and the other pair into the gastrocnemius muscle, connect each pair of needles to the electrophysiology acquisition system through an electrical coaxial cable. Set the sampling rate and bandwidth of the electrophysiology acquisition system to 50 kilohertz and 70 hertz, 3 kilohertz, respectively. Then at the origin of the sciatic nerve, apply a supramaximal stimulus with a pulse width of 0.1 milliseconds to the stimulating electrodes. In the electrophysiology acquisition system, record the Compound Muscle Action Potentials or CMAPs from the recording electrodes. And amplify the CMAPs with the built-in amplifier.

To use a commercial 2.68 millihertz HIFU transducer to suppress the CMAPs in diabetic neuropathic rats, immerse the spherical cone, the HIFU transducer, and the cone cover in the tank filled with degassed water. Put the HIFU transducer into the spherical cone. And use six head screws to fix the cone cover to the top opening of the spherical cone.

After the bubbles in the spherical cone are expelled naturally due to the low density of the bubbles compared to the water, use transparent 0.03 millimeter thick tape to seal the front end opening of the cone. Then screw component 3 onto the spherical cone. And remove the HIFU transducer with the spherical cone and component 3 from the degassed water tank.

Carefully insert component 1 into the space between the nerve and the muscle. And position the nerve in the gap of component 1. To ensure that the nerve is inside the focal of the HIFU, as demonstrated earlier, screw component 2 to component 1. After screwing component 3 to the HIFU housing cone, through the four legs of component 2, dock component 3 with component 2 and use Ringer's solution to fill the central well of component 1 for ultrasonic propagation and nerve preservation.

Link a function generator and a radio frequency power amplifier. Connect the power amplifier to the HIFU transducer for generation of the HIFU beam. Then manually set the voltage output of the function generator to the HIFU transducer via the power amplifier. And set a timer to measure HIFU exposure time.

Manually turn off the function generator once the HIFU exposure time is up. Simultaneously deliver the stimulus via the electrophysiology acquisition system and HIFU beam via the HIFU system to the sciatic nerve while recording the CMAPs. Gradually increase the HIFU exposure on the sciatic nerve from 3 seconds, 5 seconds, then to 8 seconds until a decrease or inhibition of the amplitude of CMAPs is observed.

Record the CMAPs once per second during the delivery of the HIFU beam. After observing the change in the amplitude of CMAPs, turn off the HIFU system. And manually click on the record icon on the electrophysiology acquisition software to record CMAPs for two hours. After removing the nerve fixator components, suturing the surgical site of the diabetic rat, and allowing the rats to recover on days 7, 14, and 28 after the initial HIFU sonication, insert stimulating and recording electrodes in the anesthetized diabetic neuropathic rats as before. And record additional CMAPs.