Diagnostic Ultrasound–guided Focused Ultrasound–induced Noninvasive, Reversible Peripheral Nerve Blockade in an In Vivo Model of Acute Pain: A Proof-of-Concept Study

Moderate to severe acute pain after surgery is common and associated with worsened patient outcomes.¹ Traditional needle and local anesthetic-based peripheral nerve blocks effectively manage pain in certain patients, but various factors limit their use.² Focused ultrasound, a noninvasive method for delivering energy to tissues, holds promise as a noninvasive alternative for peripheral nerve block to manage pain.^3,4 We previously found that after invasive focused ultrasound application to the sciatic nerve in an in vivo acute pain model,⁵ animals had increased mechanical withdrawal threshold for 2 weeks, increased thermal withdrawal latency for 9 weeks, decreased hind paw plantar sensation for 2 weeks, and decreased hind paw motor response for 0.5 weeks; by microscopy, nerve structure was altered after focused ultrasound application and returned to baseline by 24 weeks.^6,7 We hypothesized that by using coaligned diagnostic ultrasound and focused ultrasound transducers, the sciatic nerve could be visualized and energy noninvasively applied, resulting in reversible inhibition of both sensory and motor fibers in an in vivo acute pain model.

Adult male and female Sprague–Dawley rats, 200 to 400 g, were randomly assigned to study arms (intervention arm: focused ultrasound application plus hind paw incision; control arm: hind paw incision only). A sham focused ultrasound group was excluded since diagnostic ultrasound alone has no physiologic effects, and the incision-only group controls for handling, anesthesia, and procedural exposure. The focused ultrasound (1.5 MHz, Sonele Inc., Canada) and diagnostic ultrasound (50 MHz, Capistrano Labs, USA) were coaligned using a needle hydrophone (HNA 0400, ONDA Corporation, USA; fig. 1A). After general anesthesia was induced, hair was removed from the skin overlying the right biceps femoris, and animals were placed in a heated water bath. The diagnostic ultrasound was utilized to visualize the sciatic nerve, and focused ultrasound was applied transcutaneously to the sciatic nerve (fig. 1, B and C). Focused ultrasound parameters (megapascals [2 to 7], duty cycle [25 to 100%], time [10 to 75 s], and burst period [50 to 100 ms]) were iteratively adjusted until a parameter set was determined, resulting in reversible nerve inhibition, as measured with behavioral tests.

Fig. 1.

(A) Alignment of dUS and FUS transducers using a needle hydrophone. (B) Position of aligned dUS and FUS transducers on the rat. (C) dUS image of a rat’s sciatic nerve before overlaying the FUS focal zone (high energy area) on it. dUS, diagnostic ultrasound; FUS, focused ultrasound.

After focused ultrasound application, a hind paw incision was performed. As described previously,⁵ a 1-cm longitudinal incision was made on the plantar aspect of the hind paw, starting 0.5 cm from the heel through the skin, fascia, and plantaris muscle. After hemostasis, the skin was closed with interrupted 5-0 nylon sutures.

Animal behavioral testing was conducted before and after focused ultrasound application and hind paw incision; testing was performed 2 h after emergence from general anesthesia on the day of focused ultrasound and hind paw incision (day 0) and for up to 24 weeks afterward (post–focused ultrasound/hind paw incision days [postoperative days] 4, 7, 11, 14, 17, 21, 24, 28, 35, 42, 56, 70, 84, 98, 112, 140, and 168). The primary outcomes were changes in hind paw nociceptive withdrawal thresholds using the Randall–Selitto test to assess mechanical hyperalgesia and the modified Hargreaves method to assess thermal hyperalgesia.⁸ The secondary outcomes were changes to hind paw motor and nonpain sensory behaviors using the hind paw grasp reflex (flexion) to assess motor and nonpain sensory function, and the startle reflex in which hind paw digit extension occurs to assess motor function.^9,10 Animals were handled daily for 2 days before test habituation, then habituated to each behavioral test for 2 days, and then baseline testing was conducted on 2 days before focused ultrasound application/hind paw incision. For Randall–Selitto testing, a gradually increasing force was applied with the pointed force applicator to the hind paw plantar surface within the sciatic nerve distribution until a withdrawal response or vocalization occurred (maximum force, 250 g).⁸ For modified Hargreaves testing, radiant heat was applied to the hind paw plantar surface, and the latency to withdrawal was measured (maximum 20 s). Hind paw flexion and extension were separately scored as 0 (no response), 0.5 (any partial response), or 1 (full response).⁶ As focused ultrasound was applied approximately 1 cm proximal to the trifurcation into its terminal branches, focused ultrasound–induced inhibition of A-alpha and A-beta fibers will result in a reduction or absence of hind paw extension and/or flexion. However, limb withdrawal, as observed in the Randall–Selitto and Hargreaves tests, is mediated by proximal hindlimb muscles that remain unaffected by the focused ultrasound application site. Blinding was not feasible due to requirements for focused ultrasound application, pain model induction, and behavioral testing.

The continuous outcomes from the Randall–Selitto and modified Hargreaves tests are reported as a mean (95% CI) at each time point. Data were analyzed using a two-way mixed-model ANOVA (factors: group [intervention vs. control] and time) in GraphPad Prism version 10 (GraphPad Software, USA). Post hoc analysis was corrected for multiple comparisons using the Bonferroni correction. Statistical significance was defined as P < 0.05. A priori power analysis determined that six animals per group would provide 80% power (β = 0.8) to detect an effect size of 1.25 at α = 0.05.

Focused ultrasound was applied to 44 animals to determine a set of parameters that achieved reversible motor and sensory inhibition (6.65 megapascals, 100% duty cycle, 50 ms burst period, 60 s; spatial peak temporal average intensity, 1,436 W cm⁻²). The intervention study arm (focused ultrasound plus hind paw incision) included 12 rats (during iterative optimization of the focused ultrasound parameters, 12 animals ultimately received focused ultrasound using the final parameter set), 100% female, median weight (interquartile range) 223 (218 to 256) g. The control arm (hind paw incision only) included six rats, 50% female, and had a median weight (interquartile range) of 268 (239 to 280) g.

A significant group by time interaction was observed for both mechanical withdrawal thresholds and thermal withdrawal latencies (P < 0.001). Mechanical thresholds were significantly higher in the intervention group than in controls (P < 0.001) from week 0.0 to 3.5 (Randall–Selitto test, fig. 2A; Supplemental Digital Content tables 1 and 2, https://links.lww.com/ALN/E531). Thermal withdrawal latencies differed significantly between the intervention and control groups (P < 0.001) from week 0.0 through 3.0 (Hargreaves test, fig. 2B; Supplemental Digital Content tables 3 and 4, https://links.lww.com/ALN/E531).

Fig. 2.

Behavioral test results from the intervention and control arms. (A) Thermal withdrawal latency (modified Hargreaves). (B) Mechanical withdrawal threshold (Randall–Selitto). (C) HP flexion. (D) HP extension. (A and B) Data are presented as mean (95% CI). Statistical comparisons were performed using a two-way mixed-model ANOVA with fixed effects for group and time. Post hoc analysis was Bonferroni-corrected for multiple comparisons. Significant differences between groups are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. (C and D) Data are presented descriptively without inferential statistical testing due to the absence of variance in control animals. dUS, diagnostic ultrasound; FUS, focused ultrasound; HP, hind paw; INT, intervention arm.

Hind paw flexion and extension outcomes were summarized descriptively due to the absence of variance in control animals, and no inferential statistical testing was performed for these measures. For hind paw flexion and extension (fig. 2, C and D; Supplemental Digital Content tables 5 and 6, https://links.lww.com/ALN/E531), in the intervention group, hind paw flexion scores were reduced from week 0.0 to 4.0 and hind paw extension scores were reduced from week 0.0 to 6.0, with gradual recovery thereafter. In contrast, control animals demonstrated invariant full flexion and extension responses across all time points (mean ± SD, 1 ± 0).

In a rat model of acute incisional pain, using coaligned diagnostic ultrasound and focused ultrasound transducers to noninvasively apply energy to the sciatic nerve, we determined a set of focused ultrasound parameters that resulted in reversible inhibition of sensory and motor behaviors. The study was limited by unequal sex distribution and partial blinding, although no sex-related differences were observed in behavioral outcomes. While behavioral test results show that the focused ultrasound parameters utilized resulted in relatively long-lasting inhibition of both nociceptive and nonnociceptive nerve fibers, all behavioral changes were reversible. While this proof-of-concept study provides preliminary evidence that diagnostic ultrasound–guided focused ultrasound can induce peripheral nerve inhibition to manage pain, these findings should be interpreted cautiously. Additional work is required to better define parameter sets and determine whether selective, reversible nociceptive fiber inhibition can be achieved reliably before considering clinical translation. Future investigations will (1) determine focused ultrasound parameters that achieve shorter duration or selective nociceptive reversible inhibition with minimal motor and non–pain sensory effects, and (2) employ microscopy to evaluate peripheral nerve structural changes over time after focused ultrasound application.

Supplementary Material

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Supplemental tables, https://links.lww.com/ALN/E531