Focused ultrasound-induced inhibition of peripheral nerve fibers in an animal model of acute pain

Abstract

Background

Moderate-to-severe acute pain is prevalent in many healthcare settings and associated with adverse outcomes. Peripheral nerve blockade using traditional needle-based and local anesthetic-based techniques improves pain outcomes for some patient populations but has shortcomings limiting use. These limitations include its invasiveness, potential for local anesthetic systemic toxicity, risk of infection with an indwelling catheter, and relatively short duration of blockade compared with the period of pain after major injuries. Focused ultrasound is capable of inhibiting the peripheral nervous system and has potential as a pain management tool. However, investigations of its effect on peripheral nerve nociceptive fibers in animal models of acute pain are lacking. In an in vivo acute pain model, we investigated focused ultrasound’s effects on behavior and peripheral nerve structure.

Methods

Focused ultrasound was applied directly to the sciatic nerve of rats just prior to a hindpaw incision; three control groups (focused ultrasound sham only, hindpaw incision only, focused ultrasound sham+hindpaw incision) were also included. For all four groups (intervention and controls), behavioral testing (thermal and mechanical hyperalgesia, hindpaw extension and flexion) took place for 4 weeks. Structural changes to peripheral nerves of non-focused ultrasound controls and after focused ultrasound application were assessed on days 0 and 14 using light microscopy and transmission electron microscopy.

Results

Compared with controls, after focused ultrasound application, animals had (1) increased mechanical nociceptive thresholds for 2 weeks; (2) sustained increase in thermal nociceptive thresholds for ≥4 weeks; (3) a decrease in hindpaw motor response for 0.5 weeks; and (4) a decrease in hindpaw plantar sensation for 2 weeks. At 14 days after focused ultrasound application, alterations to myelin sheaths and nerve fiber ultrastructure were observed both by light and electron microscopy.

Discussion

Focused ultrasound, using a distinct parameter set, reversibly inhibits A-delta peripheral nerve nociceptive, motor, and non-nociceptive sensory fiber-mediated behaviors, has a prolonged effect on C nociceptive fiber-mediated behavior, and alters nerve structure. Focused ultrasound may have potential as a peripheral nerve blockade technique for acute pain management. However, further investigation is required to determine C fiber inhibition duration and the significance of nerve structural changes.

INTRODUCTION

Acute pain is an important public health issue. The prevalence of acute pain is high across multiple healthcare settings, including inpatient wards, emergency departments, and postoperative units. Acute pain is frequently inadequately managed; yet, poorly controlled acute pain is associated with significant morbidity.¹ Both opioid and non-opioid analgesics are commonly used for managing acute pain but are limited by side effects.² Further, mu-opioid receptor agonists preferentially inhibit C-fibers over A-delta fibers and thus are better at controlling resting, not movement associated, pain.³

In some surgical populations, peripheral nerve blockade (PNB) lessens acute pain, reduces the risk of side effects from systemic analgesics (eg, nausea, pruritus, and confusion), and improves other outcomes (morbidity and mortality).^{4 5} Yet, single-shot PNB is short-lived (<24 hours) and may result in rebound pain after cessation.⁶ Continuous PNB (by indwelling catheter) necessitates specialists for administration and monitoring, increases care complexity, and may be problematic to arrange for outpatients. However, continuous PNBs are effective at managing acute pain relative to opioids, and perioperative PNB is associated with a lower risk of chronic postsurgical pain after some surgeries. Yet, adverse events can occur with the use of regional anesthesia, and are typically related to its invasiveness and side effects of local anesthetics (eg, local anesthetic systemic toxicity, infection).^{7 8} Local anesthetics block pain by inhibiting neural activity of A-delta and C fibers. However, the local anesthetics used clinically for PNB also inhibit A-alpha and A-beta fibers which underlie both motor and non-nociceptive sensory (eg, touch) function, respectively. Motor fiber inhibition from local anesthetic in PNBs may increase the risk of patient injury from falls⁹ and prevent participation in early physical therapy.¹⁰

Focused ultrasound (FUS) is widely investigated for its potential in modulating central nervous system activity, specifically to treat diseases such as essential tremor, Parkinson’s disease, and obsessive–compulsive disorder.^{11 12} There is also increasing evidence that FUS may be a tool for treating peripheral pain conditions. Studies of the effect of FUS on the peripheral nervous system (PNS) have found reversible or permanent reduction in peripheral nerve action potential (AP) amplitudes across different animal models.^{13 14} In rodent pain models, FUS application to the dorsal root ganglion temporarily increased mechanical and thermal thresholds in neuropathic animals compared with control animals.^{15 16} Histopathological signs of damage from FUS in these studies were generally minimal and reversible, or absent. Results from our laboratory indicate that increasing FUS pressures alter components of the rat sciatic nerve AP similarly to increasing concentrations of the local anesthetics bupivacaine and ropivacaine, and with nerve structural changes only at higher FUS pressures.¹⁷

Thus, prior work suggests the potential of FUS to provide reversible inhibition of nociception, but investigations of behavioral results after FUS application to peripheral nerves in acute pain models are lacking. We hypothesized that pre-injury FUS application to the rat sciatic nerve would provide reversible inhibition of nociceptive fibers with minimal or no inhibition of motor fibers and with minimal or no nerve structural changes in an in vivo model of acute incisional pain.

METHODS

Outcomes

The primary aims were to determine the change to hindpaw (HP) withdrawal thresholds (using tests for mechanical and thermal hyperalgesia) over 4 weeks after direct FUS application to the rat sciatic nerve in animals prior to an HP incision, compared with controls. Secondary aims were to determine motor (using HP extension and flexion) and histopathological (using light and electron microscopy) changes after FUS application to the rat sciatic nerve in animals prior to an HP incision, compared with sham controls.

Study arms

Adult, male Sprague-Dawley rats (Charles River Laboratories, Wilmington, Massachusetts, USA), 600–800 g were randomly assigned to study arms (intervention arm, INT: FUS application+HP incision; control arm 1, C1: FUS sham only; control arm 2, C2: HP incision only; control arm 3, C3: FUS sham+HP incision. Larger (>500 g) rats were used as they have longer peripheral nerves to facilitate direct FUS application.

FUS application to sciatic nerve

FUS was applied to the rat sciatic nerve preemptively, prior to making an HP incision. While FUS can be applied non-invasively using diagnostic ultrasound or MRI guidance, due to challenges in accurately applying FUS to a small target in animal models, many studies assessing alterations in peripheral nerve activity do so by applying FUS directly to the nerve after surgical dissection to it. Here, after inducing general anesthesia with isoflurane, shaving the surgical site, and applying an antiseptic, a lateral incision was made through skin, approximately 5 mm distal to the ischium and perpendicular to the femur, to expose the underlying biceps femoris muscle, followed by dissection to expose the sciatic nerve. A 2 cm portion of the sciatic nerve proximal to the separation into its three terminal branches was exposed and separated from the surrounding tissue. A custom designed and 3D-printed FUS cone base (printed with polylactic acid, a material with a low acoustic impedance)¹⁸ with a groove to hold the nerve in a fixed position (figure 1A) was carefully inserted under the nerve until the nerve was within the base nerve groove. The cone base fixed the nerve in place to ensure accurate FUS application to the nerve when the FUS transducer is secured to it. A fiber optic temperature probe (TS5, Micronor, Camarillo, California, USA) was positioned through an opening in the cone base so that it was just below and touching the nerve at the FUS application site; temperature was recorded continuously. After applying ultrasound transmission gel (Aquasonic 100, Parker Laboratories, Fairfield, New Jersey, USA) to the top of the nerve in the cone base groove, the FUS transducer and its coupling cone were attached to the FUS cone base (figure 1B). A FUS transducer (custom built in-house, resonant frequency 1.47 megahertz, 100 mm radius of curvature) was used. The precise FUS transducer focal distance (100 mm), focal length (approximately 10 mm), focal diameter (approximately 2 mm), and focal point pressures were previously determined using a fiber optic probe hydrophone (Precision Acoustics, Dorset, UK). The transducer was fitted with a coupling cone filled with 1% agarose (Agarose LE, GoldBio, St. Louis, Missouri, USA) in deionized water. The midpoint of the FUS focal area (focal point) is 1 mm beyond the coupling cone’s tip. The FUS application site was approximately 1 centimeter proximal to the separation of the sciatic nerve into its three terminal branches. FUS was applied in a pulse train, where each 25 ms pulse was followed by 25 ms off, for a duty cycle of 50%. FUS was applied once using two pulse trains: the first pulse train was applied with a peak-to-peak pressure of 1.86 megapascals (spatial peak pulse average intensities (Isppa)=110.4 W/cm²; mechanical index (MI)=0.786) for 8 s; 2 min later, a second FUS pulse train with a peak-to-peak pressure of 1.97 megapascals (Isppa=123.8 W/cm²; MI=0.823) was applied to the nerve for an additional 8 s. This protocol was developed after an extensive dose-finding study. After FUS application, the FUS transducer, its coupling cone, and the cone base were removed. Muscle and skin were closed in two layers, the HP incision was made (described below), and isoflurane was discontinued. For FUS sham only controls (C1), all steps above were performed, but after the FUS coupling cone was attached to the cone base, FUS was not applied to the sciatic nerve; the cone remained in place for 3 min before the cone and cone base were removed and the incision was closed as described above; no HP incision was made. For HP incision only controls (C2), only the HP incision was made. For FUS sham+HP incision controls (C4), both the FUS sham protocol (above) and the HP incision were completed.

Figure 1.

(A) FUS transducer cone base with a rat sciatic nerve in its groove to hold the nerve in a fixed position for FUS application; (B) FUS transducer and FUS coupling cone secured to the cone base with a nerve in its groove. (C) Schematic diagram of a cross section of the FUS coupling cone attached to the cone base. FUS, focused ultrasound.

HP incision

This acute incisional pain model has been previously described.¹⁹ Briefly, under general anesthesia with inhaled isoflurane, the plantar aspect of the HP was prepared in a sterile manner, and a 1 cm longitudinal incision was made through skin and fascia of the plantar aspect of the foot, starting 0.5 cm from the proximal edge of the heel and extending toward the toes. The plantaris muscle was elevated and incised longitudinally. The skin was opposed with non-absorbable sutures; sutures were removed on postsurgical day 14.

Behavioral testing

As described below, baseline testing occurred on 2 days prior to the day of surgery, on the day of FUS application/HP incision (week 0.0), and postoperatively (weeks 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0). All testing was performed by two individuals (TA, author; DZ, from the Acknowledgements section) who were blinded, when possible, to allocation during testing (no blinding was possible for animals without HP incision or flank incisions).

Mechanical hyperalgesia: Randall Selitto test

Nociceptive withdrawal thresholds was assessed using the Randall Selitto paw pressure test (IITC Life Science, Woodland Hills, California, USA).²⁰ The test consists of the application of slowly increasing mechanical force, in which the tip of the device is applied onto the proximal portion of the plantar surface of an HP until a withdrawal response was seen or vocalization was heard (increased at 5–10 g/s). The device uses calibrated forceps to induce quantifiable mechanical stimulation in the animal on a linear scale. The maximum force applied is limited to 250 g to avoid skin damage. Prior to the first testing session, animals were handled daily for 5–10 min for 3 days and then habituated to the restraint cloth for 2 days without testing, then baseline testing occurred for 2 days prior to FUS application. Each day of testing, animals were handled for 5 min before testing to habituate them to being held. The animals were then held in a soft cloth for immobilization. The test was repeated three times with an interval of 1 min between applications; the values were averaged.

Thermal hyperalgesia: modified Hargreaves test

Changes in thermal hyperalgesia were determined using an HP withdrawal response to noxious radiant thermal stimulation similarly to the method described by Hargreaves et al²¹ using a heated glass stand (IITC Life Science). Animals were placed on a plexiglass plate inside an opened bottom plexiglass testing cage and allowed to habituate for 15 min. Prior to the first testing session, animals were habituated to the testing cage for 15 min on three consecutive days without testing, then baseline testing occurred for two consecutive days prior to FUS application. After 15 min of habituation, radiant heat was focused on the proximal 1 cm area of the rear heel plantar surface, and the latency to reflexive withdrawal measured using an electronic timer. The test was repeated five times with an interval of 3 min between applications; the values were averaged.

Flexion-extension testing

Rodents have a startle reflex in which acoustic and tactile stimuli evoke contraction of major muscles, resulting in HP digit extension. This reflex requires both an intact specific neural pathway and extensor muscle activity. Rodents also have a hindlimb grasp reflex; when the plantar surface of the HP touches a surface, the HP flexes in response. This reflex requires intact non-nociceptive sensory fiber activity and plantar flexor muscle activity. Animals were observed for intact HP startle extension reflexes and HP grasp reflexes. Prior to the first testing session, animals were handled daily for 5–10 min for 3 days and then baseline testing occurred for two consecutive days prior to FUS application and the incision. Rats were gently grasped around the body and tail, lifted above the counter-top, and observed for HP digit extension. HP digit extension was graded as 0 (no response), 0.5 (any partial response), or 1 (full response). Next, rats were gently grasped around the body and tail and lifted so that the plantar surface of their bilateral HPs touched a metal grip bar. HP digit flexion was scored as 0 (no response), 0.5 (any partial response), or 1 (full response). Rats need to have HP plantar sensation intact to retain the HP grasp reflex. During each testing session, we assessed both hind paws to ensure the full extension and grasp reflexes were noted with the contralateral (non-intervention/control) HP. Using the contralateral HP extension and grasp for comparison, a score of full, partial, or no extension and grasping were given for the intervention/control HP.^{22 23}

Data analysis and sample size

At each time point, for continuous outcomes, test results are presented as mean ±95% CI. Results from tests with continuous outcomes were compared using a two sample t-test. Findings from the intervention group were compared with the three control groups. For all performed statistical tests, the significance level was set to <0.05. With a significance level of p<0.05 and a power of β=0.8, a standardized effect (difference between means divided by SD) of 1.25 is detectable with a group size of n=6.

Histopathology

Histopathological assessment of peripheral nerve anatomy using light microscopy and transmission electron microscopy was performed on a separate cohort of nerves.

Nerves to which FUS had been applied (identical to the INT arm described above) were excised either on the day of surgery (3–4 hours after FUS application) or 14 days after FUS application. Control nerves did not have FUS applied (from ‘normal’ animals without interventions). For light microscopy, a 2 cm section of nerve was placed in fixative (4% paraformaldehyde in 1 × PBS (145 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.4)) for 48–72 hours at room temperature, transferred to 70% ethanol at room temperature, and sent to the Stanford Medicine Animal Histology Services core for routine processing, paraffin embedding, sectioning, and staining (H&E, Luxol Fast Blue (LFB), Masson’s Trichrome, and Bielschowsky silver). Tissue sections were visualized with an Olympus BX43 upright brightfield microscope (Olympus Corporation, Tokyo, Japan), and digital images captured with the Olympus cellSens software.

For electron microscopy, a 2 mm section of nerve was placed in fixative (2% glutaraldehyde, 4% paraformaldehyde, 0.1M sodium cacodylate, pH 7.4). The fixative was kept at room temperature for 30 min, then transferred to ice and sent to the Stanford Medicine Cell Sciences Imaging Facility Electron Microscopy Core for processing (serial dehydration, embedding, and sectioning) as previously described.²⁴ Tissue sections were visualized with a JEOL 1400 TEM (JEOL, Tokyo, Japan), and digital images captured with a Gatan Orius 832 4k × 2.6k digital camera (Gatan, Pleasanton, California, USA). Quantification of electron microscopy images to determine axon diameter, myelin thickness, g-ratio (the ratio of the inner diameter of an axon to its total outer diameter), the number of myelinated axons per 7381.836 mm2 (area of a 500 × micrograph), the number of degenerating axons per 7381.836 mm2 (area of a 500 × micrograph), and the number of double myelin/myelin whorls per 7381.836 mm2 (area of a 500 × micrograph) was performed manually using Fiji/Image-J (open source software: https://imagej.net/software/fiji/). Mean±SEM was calculated. Study arms were compared using one-way-analysis of variance and Tukey tests.

RESULTS

Study arm characteristics

Twelve rats were included in the intervention study arm, INT: FUS application+HP incision (median weight on the day of surgery, 708 g); six rats were included in control arm 1, C1: FUS sham only (784 g); six rats were included in control arm 2, C2: HP incision only (730 g); six rats were included in control arm 3, C3: FUS sham+HP incision (767 g). A total of 100% of rats were male; the left limb was used in 100% of rats (table 1).

Table 1.

Study arm characteristics

Study arm	No of animals	DOS weight (g, median (IQR))
INT: FUS+HP incision	12	708 (667–736)
C1: FUS sham only	6	784 (756–805)
C2: HP incision only	6	730 (711–757)
C3: FUS sham+HP incision	6	767 (745–792)

C1, control arm 1; C2, control arm 2; C3, control arm 3; DOS, day of surgery; FUS, focused ultrasound; HP, hindpaw; INT, intervention arm.

The mean peak temperature during the first FUS pulse train was 57.1°C (95% CI 52.1°C to 62.1°C), and the mean peak temperature during the second FUS pulse train was 61.4°C (95% CI 54.7°C to 68.2°C) (online supplemental figure 1). The thermal dose was 21,005 CEM43 °C.²⁵

Behavioral tests

Mechanical hyperalgesia, Randall Selitto test

Change in individual study arm values over time: Qualitatively, compared with the baseline values before the day of surgery, the INT (FUS+HP incision) group had no change to the withdrawal threshold on the day of surgery (week 0.0), with a decrease in withdrawal threshold over the first 1 week, and a stable withdrawal threshold from week 1 to week 4. The C1 group (FUS sham, no HP incision) had no change in withdrawal threshold on the day of surgery (week 0.0) compared with the baseline values before the day of surgery and over the subsequent 4 weeks. Both the C2 (HP incision only) and C3 (FUS sham+HP incision) groups had an immediate decreased withdrawal threshold on the day of surgery (week 0.0) with only a small increase in withdrawal threshold over the subsequent 4 weeks (figure 2A).

Figure 2.

Behavioral hyperalgesia and motor test results from the intervention group and control arm groups. (A) Randall Selitto testing; (B). Hargreaves testing. (C). HP extension; D. HP flexion. ‘*’ Denotes a two-sample t-test between the control and intervention arm p<0.05 int, intervention arm; C1, control arm 1; C2, control arm 2; C3, control arm 3; FUS, focused ultrasound; HP, hindpaw.

Comparison of INT group to control groups

Using the Randall Selitto test, there was no significant difference between the INT group and the C1 group on the day of surgery (week 0.0), but there were significant differences on weeks 0.5 to 4.0. There was a significant difference between the INT group and the C2 group on weeks 0.0 to 2.0 and on week 3.5. There was a significant difference between the INT group and the C3 group on the day of surgery (week 0.0) to week 2.5 and on week 3.5.

Thermal hyperalgesia: modified Hargreaves test

Change in individual study arm values over time

Qualitatively, the INT (FUS+HP incision) group had an increased withdrawal threshold on the day of surgery (week 0.0) compared with the baseline values before the day of surgery; this increased withdrawal threshold was sustained over 4 weeks after surgery. The C1 group (FUS sham, no HP incision) had no change in withdrawal threshold on the day of surgery (week 0.0) compared with the baseline values before the day of surgery or over the subsequent 4 weeks. Both the C2 (HP incision only) and C3 (FUS sham+HP incision) groups had an immediate decreased withdrawal threshold on the day of surgery (week 0.0), followed by increased withdrawal threshold on subsequent testing timepoints and a return to presurgery baseline values by week 2 (figure 2B).

Comparison of INT group to control groups

Using the modified Hargreaves test, there was a significant difference between the INT group and all three control groups (C1, C2, C3) on the day of surgery (week 0.0) to week 4.0.

Motor, Extension: Startle reflex

Change in individual study arm values over time

Qualitatively, the INT (FUS+HP incision) group had a decrease in HP motor (extension) function compared with the baseline values before the day of surgery. On the day of surgery (week 0.0), the mean decrease in function was approximately 10%; HP extension was decreased by approximately 30% on weeks 0.5 –1.5 and then remained decreased by about 10% to week 4.0 (figure 2C).

Comparison of INT group to control groups

Using the startle reflex to test HP extension, there was a significant difference between the INT group and all three control groups (C1, C2, C3) on week 0.5 only.

Motor, flexion: hindlimb grasp reflex

Change in individual study arm values over time

Qualitatively, the INT (FUS+HP incision) group had a decrease in HP flexion compared with the baseline values before the day of surgery. On the day of surgery (week 0.0) and week 0.5, the mean decrease in function was approximately 80%; HP flexion improved toward presurgery values over the 4 weeks after surgery, but remained about 10% decreased on week 4.0 (figure 2D).

Comparison of INT group to control groups

Using the hindlimb grasp reflex to test HP flexion, there was a significant difference between the INT group and all three control groups (C1, C2, C3) on the day of surgery (week 0.0) to week 2.0 and on week 3.0.

The difference in means, 95% CI for the difference in means, and p values for comparison of each individual control group with the intervention group for each behavioral test are shown in online supplemental table 1.

Histology

Behavioral test results from the animals to which FUS was applied (identical to the INT arm) were similar to those described above.

Light microscopy

In 1() control nerves (no FUS application), (2) nerves on day 0 at the site of FUS application, and (3) nerves on day 14 at non-FUS application sites, the nerve structure is intact. The myelin sheaths surrounding nerve fibers are sharply defined, as seen in LFB and Trichrome-stained sections. In these regions, the nerve fibers are evident, as seen in the Bielschowsky stained sections. In contrast, at the site of FUS application, 14 days afterwards, the myelin sheaths at the FUS application site are multifocally expanded by clear spaces (H&E-stained section), and the architecture of the myelin sheaths that was seen in control animals is disrupted or absent (LFB and Masson’s trichrome-stained sections), and the nerve fibers are not readily identifiable by the Bielschowsky silver stain. These findings suggest alterations to both the myelin sheaths and the nerve fibers (figure 3A).

Figure 3.

Histological morphology of control nerves and nerves to which FUS was applied. (A) Light microscopy of control nerve (first row, no FUS applied to any part of the nerve), FUS treated nerve on the day of FUS application (second row, FUS application site on day 0), FUS treated nerve 14 days afterwards (third row, non-FUS application site on day 14), and FUS treated nerve 14 days afterwards (fourth row, FUS application site on day 14). Sections of rat sciatic nerves are stained with H&E, LFB, Masson’s Trichrome, and Bielschowsky silver. Magnification: 40x; scale bars: 20 μm. (B) Electron microscopy of control (top row) and FUS treated (bottom row) sections of rat sciatic nerves. 500x magnification scale bar is 20 μm; 1000x magnification scale bar is 10 μm; 2000x magnification scale bar is 5 μm; 4000x magnification scale bar is 2 μm. FUS, focused ultrasound; LFB, Luxol Fast Blue.

Electron microscopy

In control nerves (no FUS application), the nerve structure is intact. Myelinated axons are abundant, of normal thickness, and without disruption. Microtubules and microfilaments within the myelinated axons and the endoneurium are apparent and uniform in appearance. The endoneurium itself also has a uniform appearance. Unmyelinated axon and Remak bundles are normally distributed, apparent and intact. On day 0, at the site of FUS application, while the myelin looks normal, there is a reduction in the density of myelinated nerve fibers, suggesting tissue edema. The axons within some of the myelin sheaths have ‘shrunk’ or retracted. The endoneurium is non-uniform with decreased unmyelinated axon density. On day 14, at the site of FUS application, the number of myelinated axons is decreased and those present are thinly myelinated, suggestive of myelin loss and remyelination. The axons within the myelin sheaths are abnormal and non-uniform in appearance. There is a decreased microtubule and microfilament density within myelinated axons and the endoneurium. There are frequent electron dense abnormalities consistent with myelin whorls (‘onion bulbs’) (figures 3B and 4, online supplemental table 2).

Figure 4.

(A–C) Quantification of axon diameter, myelin thickness, and g-ratio (the ratio of the inner diameter of an axon to its total outer diameter). Gray circles, measurement of a single axon; colored circles, the average for each nerve). Mean±SEM is shown for nerves. No significant differences were detected in a one-way-ANOVA and Tukey test. (D) Percent frequency of the diameter of myelinated axons in the sciatic nerve. No significant differences were detected in a two-way-ANOVA and Tukey test. (E) Number of myelinated axons per 7381.836 mm² (area of a ×500 micrograph). Gray circles, the number of myelinated axons per micrograph; colored circles, the average of the quantified micrographs in each nerve. Mean±SEM is shown for each nerve compared in a one-way ANOVA and Tukey test. (F) g-ratio and axon diameter of analyzed axons are shown. 96–149 axons per rat were analyzed, n=2 nerves per study arm. (G, H) Number of degenerated axons or myelin whorls/double myelinated axons per 7381.836 mm² (area of a 500 micrograph). Gray circles, the number of observations per micrograph; colored circles, the average of the quantified micrographs in each nerve. Mean±SEM is shown for nerves. P values compare nerves in a one-way ANOVA and Tukey test, *p<0.05. ANOVA, analysis of variance; FUS, focused ultrasound.

Results from quantitative analysis are shown in figure 4 and online supplemental table 2. No statistical differences were found in the axon diameter, myelin thickness, g-ratio, number of myelinated axons, axon diameter g-ratio, or number of myelin whorls between control nerves and nerves on day 0 after FUS application or day 14 after FUS application. A difference was found in the number of degenerated axons per area between control nerves and nerves on day 0 after FUS application and nerves on day 0 after FUS application and nerves on day 14 after FUS application.

DISCUSSION

Previous studies in which FUS was applied to peripheral nerves in animal models found FUS conditions resulting in both reversible and permanent changes to nerve conduction and behavioral changes in chronic pain models, suggesting potential clinical applications for pain management. Thus, we applied FUS to a peripheral nerve in an animal model of acute pain and conducted behavioral testing for 4 weeks afterwards. Using a distinct parameter set, direct FUS application to the rodent sciatic nerve prior to an HP incision was found to reversibly inhibit A-delta peripheral nerve nociceptive, A-beta non-nociceptive sensory, and motor fiber-mediated behaviors, but had a prolonged (at least 4 week) inhibitory effect on C nociceptive fiber-mediated behavior. With these FUS parameters, we also observed altered peripheral nerve structure.

Using mechanical withdrawal testing, a test of both A delta and C fibers,^{20 26} FUS application increased the withdrawal threshold for 2 weeks compared with controls. However, with thermal withdrawal testing, primarily a test of C fiber nociception,^{21 27} FUS application increased the withdrawal threshold compared with controls for the duration of this study, 4 weeks. After FUS application, HP flexion was also decreased considerably for approximately 1.5–2 weeks. HP flexion was measured using the grasp reflex, which requires intact sensory and motor pathways. Given the Randall Selitto test results, the HP flexion results suggest that A-beta touch sensory fibers are reversibly inhibited for roughly the same duration of time as A-delta nociceptive fibers. After FUS application, HP extension was qualitatively decreased for the 4 weeks of testing (significantly different from controls only for 0.5 weeks), but with a greater effect during the first 1.5 weeks after FUS application. The combined results of behavioral testing suggest that after FUS application there was (1) A delta and A beta fiber inhibition for 1.5–2 weeks; (2) A-alpha motor fiber inhibition for the duration of the study, but with greatest effect during the first 1.5 weeks; and (3) C fiber inhibition for at least 4 weeks.

A-delta fibers are responsible for ‘sharp’ pain and pain with movement, while C fibers are responsible for ‘dull’ pain and pain at rest. Acute postoperative pain associated with movement or dressing change is likely mediated by A-delta nociceptors, which respond strongly to transient strong stimuli but rapidly accommodate to that stimulus.²⁸C fiber activation on the other hand is continuous with a continuous stimulus, such as an incision or wound.²⁹ The greatest amount of pain occurs in the first 2 weeks after trauma and surgery, but there is typically continued pain for an extended period of time afterwards, which often requires opioids to treat. Thus, it may be optimal to inhibit both A-delta and C fibers for approximately 2 weeks after injury and inhibit C fibers for weeks to months afterwards. While speculative, such a reduction in nociceptive fiber activities may optimize pain and reduce pain-related complications and the need for opioid analgesics.

Changes to peripheral nerve structure were noted by light and electron microscopies 2 weeks after FUS application. The calculated CEM43°C is above that found to result in injury to many tissues²⁵; thus, it is not surprizing that we found changes to PN structure after FUS application. While quantitative analysis did not demonstrate a statistical difference between most of the variables assessed, this is likely due to the lower number of nerves assessed per study arm (n=2) and difficulty accurately determining where FUS was applied for excision and analysis. We are currently conducting a long-term follow-up study assessing structural changes after FUS application, and we are including a greater number of nerves at each time point in order to potentially improve comparisons. Yet, it is apparent from both visualization of nerves from each study arm (figure 3) and qualitative assessment of variables (figure 4) that differences between nerves from each study arm exist. For example, demyelination and alterations to both the myelinated and unmyelinated axons were evident. However, it is unclear what the clinical significance of these changes may be. Similar nerve structural changes occur within 2 weeks after cryotreatment of peripheral nerves in rats, including demyelination and axonal degeneration, but without disruption of epineural or perineural structures. Yet, there is complete axonal regeneration, remyelination, and recovery of normal motor function by 8–24 weeks after cryotreatment.^{30 31} Furthermore, cryoneurolysis has been used clinically in humans with osteoarthritis or undergoing total knee arthroplasty and appears to reduce pain and opioid consumption, improve functional outcomes, and have an acceptable safety profile.³²

Local anesthetics are direct neurotoxins, and in animal models, exposure of peripheral nerves to local anesthetics (including bupivacaine and ropivacaine) results in nerve structural changes very similar to those seen in this study after FUS application. Direct application of local anesthetics to intact rat sciatic nerves results in Wallerian degeneration (demyelination and axonal loss), subperineurial and endoneurial edema, mast cell degranulation, proliferation of endoneurial fibroblasts, and Schwann cell necrosis.^{33 34} However, in these prior investigations of local anesthetic-induced rat sciatic nerve structural changes, behavioral testing did not occur to correlate with the structural changes. Persistent weakness and numbness rarely occurs after PNB in humans, suggesting that if similar local anesthetic-induced peripheral nerve structure changes occur in humans after PNB, they do not commonly result in clinically-relevant adverse effects. Further investigation is necessary to determine the duration and/or permanence of behavioral (primarily C fiber inhibition) and histological changes noted here after FUS application.

While the precise mechanism of FUS’s effects on the PNS are unknown, FUS has both thermal and mechanical effects on tissues, and has been shown to effect cell membranes and mechanosensitive ion channels, likely as a result of membrane capacitance changes, sonoporation, and/or temperature changes.^{35 36} It is postulated that FUS-induced neuromodulation may be, in part, due to changes in activity of certain mechanosensitive and thermosensitive ion channels. Some channels exhibit sensitivity to FUS; Nav1.5, Piezo1, and TRESK are mechanosensitive ion channels and TRPV1 is a thermosensitive ion channel that have been implicated in FUS-induced peripheral nerve conduction blockade.^{35 36}

Only using male rats and left-sidedness limits generalizability. For histological analysis, only nerves from ‘normal’ animals were used as controls; thus, potential effects from surgical dissection to the sciatic nerve, insertion of the cone base underneath it, pressure from the FUS cone attached to the cone base with the nerve in the groove, and/or from making an ipsilateral HP incision would not be detected. Future studies will include both right and left sidedness and both male and female animals. While there are clinical scenarios where FUS could be applied directly to nerves intraoperatively (eg, to exposed nerves during limb amputations and/or to intercostal nerves during thoracic surgery), transcutaneous (non-invasive) FUS application would have much wider clinical applications. FUS is currently applied clinically, non-invasively using MRI and diagnostic ultrasound guidance; thus, it should be possible to translate the findings reported here to transcutaneous FUS application to peripheral nerves.

CONCLUSIONS

Using a distinct parameter set, direct FUS application to the sciatic nerve just prior to an HP incision was found to reversibly inhibit A delta, A-beta, and motor fiber mediated behavior as well as prolonged inhibition of C fiber mediated responses and changes to nerve structure. FUS may have potential as a peripheral nerve blockade technique for acute pain management by reversibly inhibiting nociception with minimal and reversible motor blockade. However, further investigation is required to determine the duration of C fiber inhibition and the reversibility as well as the clinical significance of nerve structural changes.

WHAT IS ALREADY KNOWN ON THIS TOPIC

• Moderate-to-severe acute pain is prevalent in many healthcare settings and associated with adverse outcomes. Peripheral nerve blockade using traditional needle-based and local anesthetic-based techniques improves pain outcomes for some patient populations but has shortcomings limiting use. Focused ultrasound is capable of inhibiting the peripheral nervous system and has potential as a pain management tool.

WHAT THIS STUDY ADDS

• In a rodent model of acute pain, focused ultrasound, using a distinct parameter set, reversibly inhibits A-delta peripheral nerve nociceptive, motor, and non-nociceptive sensory fiber-mediated behaviors, has a prolonged effect on C nociceptive fiber-mediated behavior, and alters nerve structure.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

• Focused ultrasound may have potential as a peripheral nerve blockade technique for acute pain management.

Data availability statement

Data are available on reasonable request.