Pilot study on the effects of low intensity focused ultrasound in a swine model of neuropathic pain

Abstract

Objective: The authors' laboratory has previously demonstrated beneficial effects of noninvasive low intensity focused ultrasound (liFUS), targeted at the dorsal root ganglion (DRG), for reducing allodynia in rodent neuropathic pain models. However, in rats the DRG is 5 mm below the skin when approached laterally, while in humans the DRG is typically 5-8 cm deep. Here, using a modified liFUS probe, the authors demonstrated the feasibility of using external liFUS for modulation of antinociceptive responses in neuropathic swine.

Methods: Two cohorts of swine underwent a common peroneal nerve injury (CPNI) to induce neuropathic pain. In the first cohort, pigs (14 kg) were iteratively tested to determine treatment parameters. liFUS penetration to the L5 DRG was verified by using a thermocouple to monitor tissue temperature changes and by measuring nerve conduction velocity (NCV) at the corresponding common peroneal nerve (CPN). Pain behaviors were monitored before and after treatment. DRG was evaluated for tissue damage postmortem. Based on data from the first cohort, a treatment algorithm was developed, parameter predictions were verified, and neuropathic pain was significantly modified in a second cohort of larger swine (20 kg).

Results: The authors performed a dose-response curve analysis in 14-kg CPNI swine. Specifically, after confirming that the liFUS probe could reach 5 cm in ex vivo tissue experiments, the authors tested liFUS in 14-kg CPNI swine. The mean ± SEM DRG depth was 3.79 ± 0.09 cm in this initial cohort. The parameters were determined and then extrapolated to larger animals (20 kg), and predictions were verified. Tissue temperature elevations at the treatment site did not exceed 2°C, and the expected increases in the CPN NCV were observed. liFUS treatment eliminated pain guarding in all animals for the duration of follow-up (up to 1 month) and improved allodynia for 5 days postprocedure. No evidence of histological damage was seen using Fluoro-Jade and H&E staining.

Conclusions: The results demonstrate that a 5-cm depth can be reached with external liFUS and alters pain behavior and allodynia in a large-animal model of neuropathic pain.

Keywords: noninvasive modulatory ultrasound; pain; peripheral nerve; pigs; preclinical.

FIG. 1.

liFUS probe and thermocouple placement (SonixMDP, Ultrasonix). Relative placement of liFUS applicator probe and thermocouple probe during liFUS treatment.

FIG. 2.

Mechanical and thermal thresholds of the dorsal aspect of the left hind hoof. A: VFF (force in grams) pre- and post-liFUS treatment. One-way repeated-measure ANOVA revealed a main effect of liFUS treatment (p < 0.001; n = 6). Tukey post hoc comparison was used to determine the significance of individual time points. VFF scores significantly increased for 4 days following liFUS compared to the neuropathic state (3.93 ± 1.02 g, p < 0.001). Effects were no longer significant on days 5–7. B: One-way ANOVA of the effect of liFUS on VFF thresholds in larger animals (n = 4) revealed a similar significant main effect of liFUS (p < 0.001) when compared to post-CPNI baseline scores (0.99 ± 0.19 g) for up to 5 days after treatment. C: Thermal thresholds were found to significantly decrease following CPNI ligation surgery: 33.17°C ± 2.00°C (p = 0.03; n = 4) compared to baseline scores. Threshold scores increased (p = 0.02) 24 hours after liFUS treatment. Seven days post-liFUS treatment, thermal thresholds were no longer significantly different from baseline. *p < 0.05; ***p < 0.01.

FIG. 3.

L5 DRG histology. A: Fluoro-Jade staining of left DRG 7 days post-liFUS at ×20 with DAPI counterstain showing no signs of neurodegeneration. B: H&E stain of left DRG at ×20 shows morphologically normal DRG cells, with intact Nissl substance without signs of pyknosis or edema.