An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models

The overall goal of this ultrasound technique is to perform temporary block of peripheral nerve conduction using an ultrasound thermal effect. This method can help answer key questions in the pain management field such as analgesia of diabetic peripheral neuropathy. The main advantage of this technique is that ultrasound exposure on the target nerve is localized and repeatable.

The implications of this technique is tended toward analgesia of human neuropathic pain because ultrasound treatment is a long, very safe process. Demonstrating the procedure will be Cheng Jung-Sung and Tan Joo-Shin plus research assistants from a laboratory. After fasting male Sprague-Dawley or SD rats and preparing STZ solution according to the text protocol, use a 26 or 28 gauge needle to draw the 50 milligram per kilogram STZ solution into a one milliliter insulin or tuberculin syringe.

Use an ethanol pad to clean the injection site on the rats and inject the STZ solution into the animal's abdomen. At 72 hours post-injection, measure the fasting plasma glucose concentration of all rats injected by using a blood lancet to prick the tip of the tail to obtain a small drop of blood. Place the drop of blood on a glucose test strip and insert it into a glucose meter before recording the fasting plasma glucose levels.

To habituate a STZ-induced diabetic rat, transfer it to a cage on a one centimeter diameter metal mesh floor for 30 minutes. Then, to evaluate the hind paw withdrawal response, use an electronic Von Frey Probe with a rigid tip to manually apply pressure to the plantar surface of the hind paw of the rats and gradually increase the pressure until a paw withdrawal response is seen. Record the pressure and wait 30 seconds before repeating the measurement again.

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 zone, using a custom-made nerve fixator, use a glass hook to carefully lift the nerve and put it in the slot of component one of the device. Next, screw component two to component one.

Then use Ringer's Solution to fill the central well of component one for ultrasonic propagation and nerve preservation. Screw component three to the HIFU housing cone. Then through the four legs of component two, dock component three with component two.

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 three 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 three onto the spherical cone and remove the HIFU transducer with the spherical cone and component three from the degassed water tank. Carefully insert component one into the space between the nerve and the muscle and position the nerve in the gap of component one.

To ensure that the nerve is inside the focal of the HIFU as demonstrated earlier, screw component two to component one. After screwing component three to the HIFU housing cone, through the four legs of component two, dock component three with component two and use Ringer's Solution to fill the central well of component one 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 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 three seconds, five seconds, then to eight 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 seven, 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 as demonstrated earlier in this video. The in vivo study demonstrated in this video showed that with a high-intensity focused ultrasound, or HIFU dose of three seconds of sonication at an intensity of 2810 watts per square centimeter, the CMAPs were suppressed by 20 per cent of baseline but they were completely recovered after 30 minutes.

For the five second HIFU exposure at the same intensity, the CMAPs decreased to 65.4 per cent of baseline at four minutes and recovered to 73.7 per cent of baseline by 120 minutes. The CMAPs did not return to baseline levels until day 14. When HIFU sonication time was increased to 8 seconds under the same intensity, the CMAPs were reduced to 26 per cent of baseline at four minutes but increased to 38 per cent of baseline at 120 minutes and gradually increased to 74 per cent of baseline by day 28.

Once mastered, this technique can be done in one hour if it is performed properly. While attempting this procedure, it's important to remember to treat the nerve very carefully. Following this procedure, other methods like brand easy detection can be performed in order to answer additional questions like peripheral sensoral interaction.

After its development, this technique paved the way for researchers in the field of pain management to explore a new energistic modality for accupain in peripheral neuropathy. After watching this video, you should have a good understanding of how to graph the conduction of diabetic neuropathic nerves in a rat model by ultrasound. Don't forget that working with the power amplifier can be extremely hazardous and the precautions such as wearing insulating work gloves should always be taken while performing this procedure.