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 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.
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'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.
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
2. Confirmation of Diabetes in STZ-induced Rats
3. Evaluation of Peripheral Diabetic Neuropathy in Diabetic Rats
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.
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.
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. Please click here to view a larger version of this figure.
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. *Significantly 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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 4: The Drawings of Components I (A), II (B) and III (C). Please click here to view a larger version of this figure.
Figure 5: The Drawings of the Spherical Cone (A) and the Cone Cover (B). Please click here to view a larger version of this figure.
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'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.
The authors have nothing to disclose.
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.
streptozotocin | Sigma | 85882 | |
citric acid monohydrate | Sigma | C1909 | |
trisodium citrate dihydrate | Sigma | W302600 | |
glucose meters | Roche Accu-Check Active | GC | |
electronic von Frey device | IITC Life Science | 2390 | |
hot plate | IITC Life Science | ||
Biopac MP36 acquisition system | Biopac Systems, Inc. | ||
HIFU transducer | Sonic Concepts | H108 | |
function generator | Agilent | 33250A | |
power amplifier | Electronics & Innovation | 1040L | |
Rats | Biolasco taiwan | Sprague-Dawley | |
Puralube vet ointment | Dechra | ||
isoflurane vaporizer | Parkland Scientific | V3000PS | |
Isoflurance | Attane | ||
Restraint bag (Decapicones) | Braintree Scientific | DC 200 |