The study was supported by the Ministry of Science and Technology (Project MOST 105-2221-E-400 -001) and the National Health Research Institutes (Project BN-105-PP-10), Taiwan.

The Institutional Animal Care and Use Committee of the National Health Research Institutes in Taiwan approved all animal protocols.
1. Induction of Diabetic Model in Male Adult Sprague - Dawley (SD) Rats
<ol>
	<li>Remove rat food pellets from the cage to fast male SD rats (300 - 350 g) for 6 h prior to STZ induction.</li>
	<li>Prepare sodium citrate buffer (0.1 M, pH 4.5).
	<ol>
		<li>Dissolve 1.05 g citric acid monohydrate (C6H8O7 &#183; H2O; mol. wt. 210.14) in 50 mL distilled water to make a 0.1 M citric acid solution.</li>
		<li>Dissolve 1.47 g trisodium citrate dihydrate (C8H5O7Na3 &#183; 2H2O; mol. wt. 294.12) in 50 mL distilled water to make a 0.1 M sodium citrate solution.</li>
		<li>Add 25 mL citric acid solution to 25 mL sodium citrate solution. Monitor the sodium citrate buffer pH (pH 4.5) using a pH meter. 
		NOTE: The buffer is made isotonic by addition of an appropriate volume of 0.1 M sodium citrate solution.</li>
	</ol>
	</li>
	<li>Dissolve STZ in 0.1 M sodium citrate buffer to yield a 50 mg/mL STZ solution. 
	NOTE: The STZ solution is light sensitive, therefore, cover the STZ solution with aluminum foil and use within 15 - 20 min.</li>
	<li>Draw the 50 mg/kg STZ solution into a 1 mL insulin or tuberculin syringe with 26- to 28-gauge needle.&#160;Clean the injection site with an ethanol pad and intraperitoneally inject the STZ solution into the lower right quadrant of the abdomen to avoid damaging abdominal organs. &#160;</li>
	<li>Supply rats with 10% sucrose water as the sole water source for 48 h after STZ injection to prevent hypoglycemia.</li>
</ol>
2. Confirmation of Diabetes in STZ-induced Rats
<ol>
	<li>Monitor the fasting plasma glucose concentration of all rats injected by STZ post 72 h with a glucose meter.
	<ol>
		<li>Fast the STZ-induced diabetic rats for 15 h before measuring the fasting blood glucose level.</li>
		<li>Restrain the rats in a restraint bag and expose the tails for blood collection during the blood glucose measurements.</li>
		<li>Use 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. Record the fasting plasma glucose levels. 
		NOTE: The glucose meter detects and displays the blood glucose level in units of mg/dL. Exclude the rats with fasting blood glucose levels below 150 mg/dL after 2 weeks of STZ-induction.</li>
	</ol>
	</li>
</ol>
3. Evaluation of Peripheral Diabetic Neuropathy in Diabetic Rats
<ol>
	<li>Assess the mechanical allodynia with electronic von Frey.
	<ol>
		<li>Habituate the STZ-induced diabetic rats in a cage on a 1 cm diameter metal mesh floor for 30 min before evaluating the hind paw withdrawal response.</li>
		<li>Use an electronic von Frey probe with rigid tip (0.8 mm in diameter) 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.</li>
		<li>Record the pressure that shows on the system and repeat the measurement 5 times per rat, with a 30 s interval between each measurement.</li>
	</ol>
	</li>
	<li>Assess the heat hyperalgesia with a hot plate.
	<ol>
		<li>Habituate the STZ-induced diabetic rats on the hot plate (24 &#177; 0.5 &#176;C) for 10 min before evaluating the pain response.</li>
		<li>Remove and place the rats back into their cages after habituation, heat the hot plate and maintain the hot plate&#39;s temperature at 55 &#177; 0.5 &#176;C.</li>
		<li>Place the rat on the heated hot plate while simultaneously starting the timer.</li>
		<li>When the rat exhibits distinct behaviors, such as licking the hind paw or abnormally flicking the hind paw, stop the timer, and record the withdrawal latency. 
		NOTE: If a rat does not express distinct behaviors after 20 s (20 s cut-off time), terminate the hot plate test and remove the rat from the hot plate.</li>
	</ol>
	</li>
</ol>
4. In Vivo Nerve Conduction Blockage with the HIFU Transducer
NOTE: The in vivo experiment starts on week 5 after 50 mg/kg STZ injection.
<ol>
	<li>Perform animal procedures before blocking of the CMAPs with HIFU sonication.
	<ol>
		<li>Sterilize the surgical tools (scalpel, scissors, forceps, and glass hook) in an autoclave before the surgery.</li>
		<li>Anesthetize the rats with intraperitoneal injection of tiletamine/zolazepam mixture (40 mg/kg) and xylazine (10 mg/kg) or via inhalation of 1.75% of isoflurane via isoflurane vaporizer. Place the rats on a heating pad to maintain the body temperature.</li>
		<li>Position the rats for surgery in ventral recumbency. Apply the eye ointment. Extend the leg of the rat and pinch the plantar surface of the foot with fingernails to ascertain the depth of anesthesia. If the rats show withdrawal responses, apply additional anesthesia.</li>
		<li>Remove hair from the thigh and lower back of the rats with an electric clipper. Apply liquid iodine with clean gauze at the surgical site and circularly move the gauze to the outward of the surgical site. Use an alcohol pad to wipe the liquid iodine with the same circular movement. This experiment is performed on the left and right surgical site. Repeat the procedure at the other surgical site when perform the next step of procedure.</li>
		<li>Use a sterile surgical scissors or scalpel to make an incision of the skin at the dorsal thigh. Use blunt surgical scissors to carefully separate the tissue underneath the skin and secure the skin with skin hooks.&#160;The femur can be seen within the muscles.</li>
		<li>Use the scissors to carefully separate the muscles parallel to femur until the mid-thigh sciatic nerve fibers that are embedded in the muscles are visible. Carefully use a glass hook to separate the mid-thigh sciatic nerve from the surrounding connective tissues and muscles.</li>
	</ol>
	</li>
	<li>Position the sciatic nerve in the HIFU focal zone using a custom-made nerve fixator (Figure 1 and Figure 3). 
	NOTE: The custom-made nerve fixator consists of 3 components (Figure 3). All the components are made of transparent polymethylmethacrylate (PMMA). The external thread of the top structure of component I is 2.5 mm tall and M10XP0.7 (Figure 4A). The central well is a 4.0 mm-diameter hole through component I. The bottom opening of the well is sealed by a sheet of tape. The diameter of the slot is 1.2 mm and the distance between the central plane of the slot and the top surface of component I is 3.1 mm. Component II consists of a main body, four legs and the bottom structure (Figure 4B). The internal thread of the bottom structure is 2.5 mm tall and M10XP0.7 to fit the external thread of component I. The diameter of the central hole is the same as the central well of component I. The dimensions of the main body are 32 mm in diameter and 5.4 mm in thickness. Four legs are symmetrically deployed. Two identical short legs are designed for alignment and two identical long legs work for hooking on component III. The outer diameter and height of component III are 41 mm and 9.2 mm. The internal thread is M36XP1.0 and the through hole is 27.5 mm in diameter (Figure 4C). The cone is a hollow taper with the top opening of 84 mm in diameter and the bottom opening of 27.5 mm in diameter. The height is 57.5 mm (Figure 5A).
	<ol>
		<li>Before the experiment, soak the custom-made acrylic nerve fixator in bleach-solution for 30 - 60 min followed by soaking in sterile water.</li>
		<li>Using a glass hook, lift the nerve carefully and put it in the slot of component I.</li>
		<li>Screw component II to component I. Fill the central well of component I with Ringer&#39;s solution for ultrasonic propagation and nerve preservation.</li>
		<li>Screw component III to the HIFU housing cone. Dock component III with component II through the four legs of component II. 
		NOTE: The geometric center of three components and the transducer are aligned. The distance between the nerve and the transducer is equal to the focal length, which ensures the nerve is inside the HIFU focal zone.</li>
	</ol>
	</li>
	<li>Insert 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 (Figure 1). 
	NOTE: On one end of the cable are two alligator clips to clip two needles separately and on the other end of the cable is a BNC connector to link the system. The pairs of acupuncture needles work as stimulating electrodes at the sciatic nerve and the recording electrodes at the gastrocnemius muscle.
	<ol>
		<li>Set the sampling rate and bandwidth of the electrophysiology acquisition system to 50 kHz and 70 Hz-3 kHz, respectively. Apply a supra-maximal stimulus with a pulse width of 0.1 ms to the stimulating electrodes at the origin of the sciatic nerve.</li>
		<li>Record the CMAPs from the recording electrodes and amplify the CMAPs with the built-in amplifier in the electrophysiology acquisition system. 
		NOTE: Use the built-in amplifier in the electrophysiology acquisition system to amplify the nerve signals and record the CMAPs from the recording electrodes with the electrophysiology acquisition system.</li>
	</ol>
	</li>
	<li>Use a commercial 2.68 MHz HIFU transducer to suppress the CMAPs in diabetic neuropathic rats. 
	NOTE: The specifications of the transducer are described as follows: a single-element spherical bowl with aperture diameter of 6 cm and focal length of 5 cm, and an ellipsoid focal zone of 4 mm in depth and 0.8 mm in width in the free field.
	<ol>
		<li>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 fix the cone cover to the top opening of the spherical cone by 6 head screws (Figure 5B). After bubbles in the spherical cone are expelled naturally due to the low density of bubbles compared with water, seal the front-end opening of the cone by a transparent 0.03 mm thick tape. Screw component III onto the spherical cone.</li>
		<li>Take out the HIFU transducer with the spherical cone and component III from the degassed water tank. 
		NOTE: The reverse osmosis water used in the study is the water purified by the process of reverse osmosis. The reverse osmosis water is boiled to expel the gas. After cooling, the degassed water is obtained in an individual sealed tank.</li>
	</ol>
	</li>
	<li>Put component I into the space between the nerve and the muscle carefully, and position the nerve in the gap of component I. Perform steps 4.2.3 and 4.2.4 to ensure that the nerve is inside the focal zone of the HIFU (Figure 3A).</li>
	<li>Link a function generator and a radiofrequency power amplifier. Connect the power amplifier to the HIFU transducer for generation of the HIFU beam. Manually set the voltage output of the function generator to the HIFU transducer via the power amplifier. Manually turn off the function generator once the HIFU-exposure time is up. Observe time using a timer. 
	NOTE: The intensity and energy of HIFU beam used in this study are 2,810 W/cm2 and 84 J/mm2, respectively.</li>
	<li>Simultaneously, deliver the stimulus via the electrophysiology acquisition system (step 4.3) and HIFU beam via HIFU system (step 4.6) to the sciatic nerve while recording the CMAPs. Gradually increase the HIFU exposure on the sciatic nerve from 3 s, 5 s to 8 s until decrease or inhibition of the amplitude of CMAPs is observed.
	<ol>
		<li>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 every 2 min in the early 10 min, every 5 min in the consecutive 30 min, and every 10 min in the last phase until the recording time reaches 2 h.</li>
	</ol>
	</li>
	<li>Separate component II and III of the nerve fixator (Figure 3) to remove the HIFU transducer from the incision site. Separate component II and I to release the secured sciatic nerve. Suture the surgical site of the diabetic rat by 4-0 chromic catgut sutures after recording the CMAPs. Apply liquid iodine to the surgical site to prevent infection.
	<ol>
		<li>Place the cages on the heating pad and allow the rats to recover in their cages before returning them to the animal facility. Provide the rats with ibuprofen in drinking water for 3 days or intraperitoneal injection of buprenorphine (0.05 - 0.1 mg/kg).</li>
	</ol>
	</li>
	<li>Insert stimulating and recording electrodes in the origin of the sciatic nerve and the gastrocnemius muscles of anesthetized diabetic neuropathic rats as described in steps 4.1.2 and 4.3 on days 7, 14, and 28 after the initial HIFU sonication. Repeat steps 4.3.1 to 4.3.2.
	<ol>
		<li>Place the cages on the heating pad and allow the rats to recover in their cages before moving the cages to the animal facility.</li>
	</ol>
	</li>
	<li>Euthanize the rats after the experiment. Place the rat in a carbon dioxide chamber. Wait about 5 min for the rats to stop breathing. Ensure that the heart has stopped beating.</li>
</ol>

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The authors have nothing to disclose.

Partial and temporary suppression of action potentials of the neuropathic nerves from diabetic rats in vivo and instant occurrence of the blocking effect after HIFU treatment both were observed. The 28-day follow-up study on CMAPs demonstrated that a safe blockade of the nerve conduction could be carried out at an appropriate HIFU exposure. As a result, the above protocol of the HIFU treatment can provide an alternative solution for the reversible conduction block of sciatic nerves in diabetic rats.
In this method, there is no neural degeneration, and the sensory nerves can recover completely over a period of hours to several days in mild nerve injuries14. For severe injuries, the temporal course of sensory recovery typically takes several months, if it occurs at all. Furthermore, the peripheral neural fibers regenerate more completely when the endoneurial tubes and Schwann cell basal lamina are intact following crushing injuries15. Therefore, we infer that the HIFU caused mild but reversible nerve injuries for the case because the suppressed CMAPs after the HIFU treatment returned to baseline over time. For severe nerve injuries, the CMAPs only recovered partially, even after 28 days.
The technique of this study provided an experimental platform for animal study of HIFU effects on peripheral nerves prior to clinical study. The HIFU focal zone can precisely aim at the target nerve because of the structural components and the protocol developed in this study, which solves the previous problem of positioning. Besides the normal nerve, the HIFU blocking technique can be also applied to the diseased nerve. However, the limitations of the current technique include the lack of temperature monitoring (leading to damage of the surrounding tissue), and the short blocking effect, although repeating the HIFU exposure may extend the analgesia. To translate the HIFU blocking technique to clinical trials, a noninvasive guidance of the HIFU focal zone is required, like ultrasound imaging or MR imaging, to identify the position of the target tissue and monitor the temperature in real time.
The existing oral medications result in only one in four patients with neuropathic pain experiences of over 50% pain relief and pose several significant side effects such as drowsiness, dizziness and somnolence16. Physical modalities are developed to hopefully improve analgesic efficacy like acupuncture, electrical and magnetic stimulations. However, the efficacy of acupuncture relies highly on the clinician experience, and the procedure is invasive. The efficacy of noninvasive electrical stimulation or magnetic stimulation is approximately 40% for pain freedom at 2 h. Both stimulations are not focused on the local site, which produces some adverse events17. Therefore, to satisfy clinical unmet needs for peripheral pain relief, the HIFU blocking technique is a promising tool because of instant effectiveness, reversible effect, physical therapy, non-invasive treatment, and potential home-use.
It is critical to aim the HIFU focal zone at the sciatic nerve accurately. Figure 3 illustrates the schematic procedure for positioning the nerve in the focal zone. The first step is to use a glass hook to slightly lift the sciatic nerve and put in the component I below the nerve, and then put down the nerve into the slot of component I (Figure 3A). The nerve passes the central site of component I through the first step. The second step is to assemble component II with component I via the screw cap structure. The assembly of components I and II is the nerve fixator shown in Figure 2B. Component II plays the role of linking components I and III. Before combining components II and III, they are filled with degassing Ringer&#39;s solution to transmit the ultrasound and preserve the nerve. The last step is to insert component III front-end structure of the HIFU transducer cone coupler into component II. Two pairs of flexible long and short pillars of component II provide sufficient fixing force. Assembly of components II and III is designed based on the mortise-tenon principle, which can ensure that the central point of the three components is in axis. The focal length of the HIFU transducer is equal to the distance between the central point of the transducer and the central point of component I. As a result, the nerve is certainly inside the focal zone, which is an ellipsoid with a width of 0.8 mm and a depth of 4 mm.

Oral medications, acupuncture1, and electrical nerve stimulation2 have been used for the treatment of painful diabetic polyneuropathy. However, the side effects of the oral medications, invasive operation of acupuncture, and electrical nerve stimulation hamper the therapeutic efficacy and patient&#39;s adherence. Ultrasound block of peripheral nerves in animal models has been investigated for decades3,4,5. The conduction of in vitro sciatic nerves of the large green frog was inhibited reversibly after the treatment of 10 - 20 pulses of ultrasound exposure for 0.4 - 1.0 s6. One factor to block nerve conduction is the temperature rise induced by ultrasound7. For patients with polyneuropathy, the suppression of compound muscle action potentials (CMAPs) was performed in the peroneal nerve exposed to low-intensity ultrasound for 2 min8. The full recovery time was within 5 min.
Recently, the Food and Drug Administration of the United States approved HIFU as a non-invasive treatment for uterine fibroid tumors9, pain palliations of bone metastases10, and prostate cancer11. An HIFU transducer emits acoustic beams outside the body, and the beams transmit in various tissue mediums and converge on the target tumor at the focus. The focal zone is immediately formed to generate localized effects on target tumors without damaging the surrounding tissues. HIFU has also been applied to inhibit nerve conduction or cause nerve denervation in in vivo experiments of normal Sprague-Dawley (SD) rats12. In addition, the short-term and long-term effects of HIFU on neuropathic nerves have been investigated13. Previous outcomes demonstrated that the reversible or permanent block of sensory nerve conduction could be achieved by HIFU with appropriate parameters. Besides analgesic applications, HIFU might be used as a tool to investigate the relative contribution of peripheral and central components to nerve conduction blockade for basic research of neurology and development of pain medication. Therefore, a HIFU blocking technology platform specific for peripheral nerves in animal models is needed. The purpose of this article is to demonstrate the procedures for partially or completely blocking the action potentials of peripheral nerves in diabetic neuropathic rats by HIFU. Diabetic rat models and evaluation of peripheral neuropathic symptoms were established. An HIFU platform and experimental processes specific for treating rat sciatic nerves are presented.

<table><tbody><tr><td>streptozotocin</td><td>Sigma</td><td>85882</td><td></td></tr><tr><td>citric acid monohydrate&amp;nbsp;</td><td>Sigma</td><td>C1909</td><td></td></tr><tr><td>trisodium citrate dihydrate</td><td>Sigma</td><td>W302600</td><td></td></tr><tr><td>glucose meters</td><td>Roche Accu-Check Active</td><td>GC</td><td></td></tr><tr><td>electronic von Frey device</td><td>IITC Life Science</td><td>2390</td><td></td></tr><tr><td>hot plate</td><td>IITC Life Science</td><td></td><td></td></tr><tr><td>Biopac MP36 acquisition system</td><td>Biopac Systems, Inc.</td><td></td><td></td></tr><tr><td>HIFU transducer</td><td>Sonic Concepts</td><td>H108</td><td></td></tr><tr><td>function generator</td><td>Agilent</td><td>33250A</td><td></td></tr><tr><td>power amplifier</td><td>Electronics &amp;amp; Innovation</td><td>1040L</td><td></td></tr><tr><td>Rats&amp;nbsp;</td><td>Biolasco taiwan</td><td>Sprague-Dawley</td><td></td></tr><tr><td>Puralube vet ointment</td><td>Dechra</td><td></td><td></td></tr><tr><td>isoflurane vaporizer</td><td>Parkland Scientific</td><td>V3000PS</td><td></td></tr><tr><td>Isoflurance</td><td>Attane</td><td></td><td></td></tr><tr><td>Restraint bag (Decapicones)</td><td>Braintree Scientific</td><td>DC 200</td><td></td></tr></tbody></table>

an ultrasonic tool for nerve conduction block in diabetic rat models

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU can reversibly block the conduction of peripheral nerves without damaging the nerves while using an appropriate ultrasonic parameter. Temporary and partial block of the action potentials of nerves shows that HIFU has the potential to be a useful clinical treatment for pain relief. This work demonstrates the procedures for suppressing the action potentials of neuropathic nerves in diabetic rats in vivo using an HIFU transducer. The first step is to generate adult male diabetic neuropathic rats by streptozotocin (STZ) injection. The second step is to evaluate the peripheral diabetic neuropathy in STZ-induced diabetic rats by an electronic von Frey probe and a hot plate. The final step is to record in vivo extracellular action potentials of the nerve exposed to HIFU sonication. The method showed here may benefit the study of ultrasound analgesic applications.

The in vivo study demonstrated that, with an HIFU dose of 3 s sonication at an intensity of 2,810 W/cm2, the CMAPs were suppressed by 20% of baseline, but they were completely recovered after 30 min (Figure 2A, diamonds) and were almost constant in the period of 28 days (Figure 2B, diamonds). For the 5 s HIFU exposure at the same intensity, the CMAPs decreased to 65.4% (9.5%) of baseline at 4 min and recovered to 73.7% (12.6%) of baseline by 120 min (Figure 2A, squares). The CMAPs did not return to baseline levels until day 14 (Figure 2B, squares). When HIFU sonication time was increased to 8 s under the same intensity, the CMAPs were reduced to 26.0% (14.1%) of baseline at 4 min (Figure 2A, triangles), but increased to 38.0% (12.0%) of baseline at 120 min, and gradually increased to 74% of baseline by day 28 (Figure 2B, triangles). See Lee, Y.F.,13 for additional details.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55675/55675fig1.jpg" /> 
Figure 1: The Experimental Setup for In Vivo Nerve Conduction Block with High Intensity Focused Ultrasound (HIFU). (A) A custom-made acrylic spherical cone that is filled with reverse osmosis degassed water was combined with the nerve fixator to ensure the focal zone of HIFU transducer was at the focal plane of the nerves. (B) Stimulating and recording electrodes are shown in the figure. The nerve fixator positioned the sciatic nerve in the focal plane of the HIFU beam. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55675/55675fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55675/55675fig2.jpg" /> 
Figure 2: The CMAPs Responses in In Vivo Diabetic Neuropathic Rats after 3 s, 5 s, and 8 s of HIFU Sonication. (A) The temporal courses of the CMAPs during day 1 recording. After 3 s, 5 s, and 8 s of HIFU sonication, the CMAPs were decreased during the early 10 min and recovered after 120 min. (B) The temporal courses of the CMAPs during day 7, 14, and 28 recordings. The CMAPs were fully recovered by day 14 after 3 s and 5 s HIFU sonication on day 1 and partially increased by day 28 after 8 s HIFU sonication. Diamonds: 3 s of HIFU sonication, squares: 5 s of HIFU sonication, and triangles: 8 s of HIFU sonication. n= 6 for each HIFU parameter. *Signi&#64257;cantly different from day 1. The data are expressed as the median (range), where the error bar is half of the range. This figure is modified from Lee, Y.F., 201513. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55675/55675fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55675/55675fig3.jpg" /> 
Figure 3: The Assembling Process to Ensure the Sciatic Nerve in the HIFU Hot Spot. (A) Three assembling steps are shown: (1) carefully settle the nerve in the slot of component I, (2) assemble component II and component I, (3) insert the front-end of component III with the HIFU transducer cone structure into component II. (B) A schematic drawing of the HIFU transducer integrated with the rat nerve. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55675/55675fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55675/55675fig4.jpg" /> 
Figure 4: The Drawings of Components I (A), II (B) and III (C). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55675/55675fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55675/55675fig5.jpg" /> 
Figure 5: The Drawings of the Spherical Cone (A) and the Cone Cover (B). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55675/55675fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models at JoVE.com

An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models

This work presents the methodology of applying high intensity-focused ultrasound to block the action potentials of diabetic neuropathic nerves.

Nerve conduction block with a high intensity-focused ultrasound (HIFU) transducer has been performed in normal and diabetic animal models recently. HIFU ...

an-ultrasonic-tool-for-nerve-conduction-block-in-diabetic-rat-models

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.3K Views.  National Health Research Institutes. Dit werk presenteert de methodologie van de toepassing van hoge intensiteit gerichte echografie om te blokkeren van de mogelijkheden van de actie van diabetische neuropathische zenuwen.

Video: An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

Neuroscience

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the &#34;cuff model&#34; of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around the main branch of the sciatic nerve. It induces a long-lasting mechanical allodynia, i.e., a nociceptive response to a normally non-nociceptive stimulus that can be evaluated by using von Frey filaments. Besides the detailed surgery and testing procedures, the interest of this model for the study of neuropathic pain mechanism, for the study of neuropathic pain sensory and anxiodepressive aspects, and for the study of neuropathic pain treatments are also discussed.

Neuropathic pain is a consequence of a lesion or disease affecting the somatosensory system. The &#8220;cuff model&#8221; of neuropathic pain in mice consists of the implantation of a polyethylene cuff around the main branch of the sciatic nerve. Mechanical allodynia is tested using von Frey filaments.

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in ...

Medicine

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and latency detect chronic axon loss and demyelination, respectively. Axonal excitability techniques &#34;by threshold tracking&#34; expand upon these measures by providing information regarding the activity of ion channels, pumps and exchangers that relate to acute function and may precede degenerative events. As such, the use of axonal excitability in animal models of neurological disorders may provide a useful in vivo measure to assess novel therapeutic interventions. Here we describe an experimental setup for multiple measures of motor axonal excitability techniques in the rat ulnar nerve.
The animals are anesthetized with isoflurane and carefully monitored to ensure constant and adequate depth of anesthesia. Body temperature, respiration rate, heart rate and saturation of oxygen in the blood are continuously monitored. Axonal excitability studies are performed using percutaneous stimulation of the ulnar nerve and recording from the hypothenar muscles of the forelimb paw. With correct electrode placement, a clear compound muscle action potential that increases in amplitude with increasing stimulus intensity is recorded. An automated program is then utilized to deliver a series of electrical pulses which generate 5 specific excitability measures in the following sequence: stimulus response behavior, strength duration time constant, threshold electrotonus, current-threshold relationship and the recovery cycle.
Data presented here indicate that these measures are repeatable and show similarity between left and right ulnar nerves when assessed on the same day. A limitation of these techniques in this setting is the effect of dose and time under anesthesia. Careful monitoring and recording of these variables should be undertaken for consideration at the time of analysis.

In Vivo Electrophysiological Measurement of the Rat Ulnar Nerve with Axonal Excitability Testing

Axonal excitability techniques provide a powerful tool to examine pathophysiology and biophysical changes that precede irreversible degenerative events. This manuscript demonstrates the use of these techniques on the ulnar nerve of anesthetized rats.

Electrophysiology enables the objective assessment of peripheral nerve function in vivo. Traditional nerve conduction measures such as amplitude and ...

Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to degeneration and injury of small-diameter sensory neurons, referred to as small fiber neuropathy. Present animal models of small fiber neuropathy affect both large- and small-diameter sensory fibers and thus create a neuropathology too complex to properly assess the effects of injured small-diameter sensory fibers. Therefore, it is necessary to develop an experimental model of pure small fiber neuropathy to adequately examine these issues. This protocol describes an experimental model of small fiber neuropathy specifically affecting small-diameter sensory nerves with resiniferatoxin (RTX), an ultrapotent agonist of transient receptor potential vanilloid type 1 (TRPV1), through a single dose of intraperitoneal injection, referred to as RTX neuropathy. This RTX neuropathy showed pathological manifestations and behavioral abnormalities that mimic the clinical characteristics of patients with small fiber neuropathy, including intraepidermal nerve fiber (IENF) degeneration, specifically injury in small-diameter neurons, and induction of thermal hypoalgesia and mechanical allodynia. This protocol tested three doses of RTX (200, 50, and 10 &#181;g/kg, respectively) and concluded that a critical dose of RTX (50 &#181;g/kg) is required for the development of typical small fiber neuropathy manifestations, and prepared a modified immunostaining procedure to investigate IENF degeneration and neuronal soma injury. The modified procedure is fast, systematic, and economic. Behavioral evaluation of neuropathic pain is critical to reveal the function of small-diameter sensory nerves. The evaluation of mechanical thresholds in experimental rodents is particularly challenging and this protocol describes a customized metal mesh that is suitable for this type of assessment in rodents. In summary, RTX neuropathy is a new and easily established experimental model to evaluate the molecular significance and intervention underlying neuropathic pain for the development of therapeutic agents.

This study establishes an experimental model of pure small fiber neuropathy with resiniferatoxin (RTX). A unique dose of RTX (50 &#181;g/kg) is optimal for developing a small fiber neuropathy model that mimics patient characteristics and could help investigate the nociceptive molecular significance underlying neuropathic pain.

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular ...

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. This work describes the preparation of rat sciatic nerves for ex vivo neurophysiology, including buffer preparation, animal procedures, equipment setup and neurophysiological recording. This work provides an overview of the different types of experiments possible with this method. The outlined method aims to provide 6 h of stimulation and recording on extracted peripheral nerve tissue in tightly controlled conditions for optimal consistency in results. Results obtained using this method are A-fibre compound action potentials (CAP) with peak-to-peak amplitudes in the millivolt range over the entire duration of the experiment. CAP amplitudes and shapes are consistent and reliable, making them useful to test and compare new electrodes to existing models, or the effects of interventions on the tissue, such as the use of chemicals, surgical alterations, or neuromodulatory stimulation techniques. Both conventional commercially available cuff electrodes with platinum-iridium contacts and custom-made conductive elastomer electrodes were tested and gave similar results in terms of nerve stimulus strength-duration response.

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

This protocol describes the preparation of rat whole sciatic nerve tissue for ex vivo electrophysiological stimulation and recording in an environmentally-regulated, two-compartment, perfused saline bath.

Ex vivo preparations enable the study of many neurophysiological processes in isolation from the rest of the body while preserving local tissue structure. ...

Open-Source Real-Time Closed-Loop Electrical Threshold Tracking for Translational Pain Research

Nociceptors are a class of primary afferent neurons that signal potentially harmful noxious stimuli. An increase in nociceptor excitability occurs in acute and chronic pain conditions. This produces abnormal ongoing activity or reduced activation thresholds to noxious stimuli. Identifying the cause of this increased excitability is required for the development and validation of mechanism-based treatments. Single-neuron electrical threshold tracking can quantify nociceptor excitability. Therefore, we have developed an application to allow such measurements and demonstrate its use in humans and rodents. APTrack provides real-time data visualization and action potential identification using a temporal raster plot. Algorithms detect action potentials by threshold crossing and monitor their latency after electrical stimulation. The plugin then modulates the electrical stimulation amplitude using an up-down method to estimate the electrical threshold of the nociceptors. The software was built upon the Open Ephys system (V0.54) and coded in C++ using the JUCE framework. It runs on Windows, Linux, and Mac operating systems. The open-source code is available (<a data-saferedirecturl="https://www.google.com/url?q=https://github.com/Microneurography/APTrack&amp;source=gmail&amp;ust=1680789829274000&amp;usg=AOvVaw0nMk9J_EI2jUn8SlmC5JJm" href="https://github.com/Microneurography/APTrack" target="_blank">https://github.com/Microneurography/APTrack</a>). The electrophysiological recordings were taken from nociceptors in both a mouse skin-nerve preparation using the teased fiber method in the saphenous nerve and in healthy human volunteers using microneurography in the superficial peroneal nerve. Nociceptors were classified by their response to thermal and mechanical stimuli, as well as by monitoring the activity-dependent slowing of the conduction velocity. The software facilitated the experiment by simplifying the action potential identification through the temporal raster plot. We demonstrate real-time closed-loop electrical threshold tracking of single-neuron action potentials during&#160;in vivo&#160;human microneurography, for the first time, and during&#160;ex vivo&#160;mouse electrophysiological recordings of C-fibers and A&#948;-fibers. We establish proof of principle by showing that the electrical threshold of a human heat-sensitive C-fiber nociceptor is reduced by heating the receptive field. This plugin enables the electrical threshold tracking of single-neuron action potentials and allows the quantification of changes in nociceptor excitability.

APTrack is a software plugin developed for the Open Ephys platform that enables real-time data visualization and the closed-loop electrical threshold tracking of neuronal action potentials. We have successfully used this in microneurography for human C-fiber nociceptors and mouse C-fiber and A&#948;-fiber nociceptors.

Nociceptors are a class of primary afferent neurons that signal potentially harmful noxious stimuli. An increase in nociceptor excitability occurs in ...

Improving FSN Therapy in the Rat Model&#58; Minimizing Discomfort and Maximizing Efficiency

Efficacy of Fu's Subcutaneous Needling on Sciatic Nerve Pain: Behavioral and Electrophysiological Changes in a Chronic Constriction Injury Rat Model

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the mechanisms of action are still not fully understood. During FSN treatment, the FSN needle is inserted and retained in the subcutaneous tissues for a long duration with a swaying movement. However, challenges arise from maintaining a posture while manipulating FSN in animal models (e.g., rats) for researchers. Uncomfortable treatment can lead to fear and resistance to FSN needles, increasing the risk of injury and may even affect research data. Anesthesia may also affect the study results too. Hence, there is a need for strategies in FSN therapy on animals that minimize injury during the intervention. This study employs a chronic constriction injury model in Sprague-Dawley rats to induce neuropathic pain. This model replicates the pain induced by nerve injury observed in humans through surgical constriction of a peripheral nerve, mimicking the compression or entrapment seen in conditions such as nerve compression syndromes and peripheral neuropathies. We introduce an appropriate manipulation for easily inserting an FSN needle into the subcutaneous layer of the animal&#39;s body, including needle insertion and direction, needle retention, and swaying movement. Minimizing the rat&#39;s discomfort prevents the rat from being tense, which causes the muscle to contract and hinder the entry of the needle and improves the study efficiency.

Author Spotlight: Unveiling the Therapeutic Effects of FSN Treatment &#8211; Bridging Research and Clinical Applications in Neuropathic Pain

We present a protocol for using Fu's subcutaneous needling in a chronic constriction injury model to induce sciatic nerve pain in rats.

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the ...

An Ultrasonic Tool for Nerve Conduction Block in Diabetic Rat Models

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