Focused ultrasound effects on nerve action potential in vitro

Abstract

Minimally invasive applications of thermal and mechanical energy to selective areas of the human anatomy have led to significant advances in treatment of and recovery from typical surgical interventions. Image-guided focused ultrasound allows energy to be deposited deep into the tissue, completely noninvasively. There has long been interest in using this focal energy delivery to block nerve conduction for pain control and local anesthesia. In this study, we have performed an in vitro study to further extend our knowledge of this potential clinical application. The sciatic nerves from the bullfrog (Rana catesbeiana) were subjected to focused ultrasound (at frequencies of 0.661 MHz and 1.986 MHz) and to heated Ringer's solution. The nerve action potential was shown to decrease in the experiments and correlated with temperature elevation measured in the nerve. The action potential recovered either completely, partially or not at all, depending on the parameters of the ultrasound exposure. The reduction of the baseline nerve temperature by circulating cooling fluid through the sonication chamber did not prevent the collapse of the nerve action potential; but higher power was required to induce the same endpoint as without cooling. These results indicate that a thermal mechanism of focused ultrasound can be used to block nerve conduction, either temporarily or permanently.

Figure 1

Experimental setup. A. Schematic vertical cross section of the sonication setup. B. Photograph of the nerve chamber, from above, showing the nerve mounted on stimulating and recording electrodes in the lateral pools.

Figure 2

An example of the reduction of the action potential after a 30 s sonication at the frequency of 0.661 MHz and at the acoustic power of 16 W. The ringer solution temperature was maintained at room temperature (20–23°C).

Figure 3

Mean action potential change of the nerves exposed to ultrasound as a function of the focal peak intensity. A. The frequency was 0.661 MHz. B. The frequency was 1.981 MHz. The ringer solution temperature was maintained at room temperature (20–23°C).

Figure 4

Recovery of action potential after a complete conduction block induced by a 30 s sonication at the frequency of 0.661 MHz at the acoustic power of 16 W. The ringer solution temperature was maintained at room temperature (20–23°C).

Figure 5

Normalized action potential recovery after a complete sonication induced conduction block as a function of time after the 30 s sonications at the frequency of 0.661 MHz. One of the nerves (sonicated at approximately 370 W/cm²) recovered completely, in approximately 90 minutes, another (sonicated at approximately 400 W/cm²) showed only partial recovery of the action potential, and in the third nerve (sonicated at approximately 650 W/cm²), the action potential was completely and permanently eliminated. The ringer solution temperature was maintained at room temperature (20–23°C).

Figure 6

Cumulative percentage of nerves having detectable action potential after sonication that induced a complete conduction block. Only the nerves that recovered completely were included in this graph. The nerves were sonicated for 30 s at or above 370 W/cm² with the frequency of 0.668 MHz. The ringer solution temperature was maintained at room temperature (20–23°C).

Figure 7

Action potential as a function of nerve temperature for the sonicated and water bath heated nerves (one nerve each). A similar result was observed in a repeat experiment with different nerves.

Figure 8

Normalized action potential as a function of the peak intensity for the two surrounding liquid temperatures for both (FL and FH) frequencies.

Figure 9

Normalized action potential as a function of the measured nerve temperature for the burst sonications. The burst length was 10 ms and the pulse repetition frequency was 10 or 20 Hz and the total sonication duration 30 s. The open and closed symbols are for the FL and FH frequencies, respectively.

Figure 10

Microphotographs (pattern 1, described in the results) of the longitudinal sections of the frog sciatic nerve treated with FUS: (A) Low magnification of the nerve; the treated segment is shown in the boxed area; (B)–(E) Untreated nerve segment demonstrates parallel nerve fibers and normal Schwann cell nuclei (arrows in C); (F)–(H) Small area (arrow in A), in which the nerve structure is disorganized; with some myelin sheaths are fragmented into globules (G) and Schwann cell nuclei appear pyknotic; axis cylinders are mostly undamaged (H). Staining: A, B, C, F, G – H& E; D, E, H: Silver impregnation. Bars: (A)-1mm; (B) – (H) −100 μm.

Figure 11

Microphotographs (pattern 2, described in the results) of the longitudinal sections of the frog sciatic nerve treated with FUS: (A) The low magnification irreversibly damaged nerve; the treated nerve segment appears swollen and torn apart; (B)–(F) The degenerated axis cylinders are swollen and vacuolated. Many of them develop ovoid and spherical forms assuming the appearance of a string of beads; (D)–(E) Some fibers are completely dissociated into free spheres; (F) The myelin sheaths are fragmented into pale barely visible globules. Bars: (A)-1 mm; (B)–(F) −100 μm.