Low Intensity Focused Ultrasound Increases Duration of Anti-Nociceptive Responses in Female Common Peroneal Nerve Injury Rats.

OBJECTIVE: Chronic pain affects 7%-10% of Americans, occurs more frequently and severely in females, and available treatments have been shown to have less efficacy in female patients. Preclinical models addressing sex-specific treatment differences in the treatment of chronic pain have been limited. Here we examine the sex-specific effects of low intensity focused ultrasound (liFUS) in a modified sciatic nerve injury (SNI) model. MATERIALS AND METHODS: A modified SNI performed by ligating the common peroneal nerve (CPN) was used to measure sensory, behavioral pain responses, and nerve conduction studies in female and male rats, following liFUS of the L5 dorsal root ganglion. RESULTS: Using the same dose of liFUS in females and males of the same weight, CPN latency immediately after treatment was increased for 50 min in females compared to 25 min in males (p < 0.001). Improvements in mechanical pain thresholds after liFUS lasted significantly longer in females (seven days; p < 0.05) compared to males (three days; p < 0.05). In females, there was a significant improvement in depression-like behavior as a result of liFUS (N = 5; p < 0.01); however, because males never developed depression-like behavior there was no change after liFUS treatment. CONCLUSIONS: Neuromodulation with liFUS has a greater effect in female rats on CPN latency, mechanical allodynia duration, and depression-like behavior. In order to customize neuromodulatory techniques for different patient phenotypes, it is essential to understand how they may alter sex-specific pathophysiologies.

Stimulation of the hepatoportal nerve plexus with focused ultrasound restores glucose homoeostasis in diabetic mice, rats and swine.

Peripheral neurons that sense glucose relay signals of glucose availability to integrative clusters of neurons in the brain. However, the roles of such signalling pathways in the maintenance of glucose homoeostasis and their contribution to disease are unknown. Here we show that the selective activation of the nerve plexus of the hepatic portal system via peripheral focused ultrasound stimulation (pFUS) improves glucose homoeostasis in mice and rats with insulin-resistant diabetes and in swine subject to hyperinsulinemic-euglycaemic clamps. pFUS modulated the activity of sensory projections to the hypothalamus, altered the concentrations of metabolism-regulating neurotransmitters, and enhanced glucose tolerance and utilization in the three species, whereas physical transection or chemical blocking of the liver-brain nerve pathway abolished the effect of pFUS on glucose tolerance. Longitudinal multi-omic profiling of metabolic tissues from the treated animals confirmed pFUS-induced modifications of key metabolic functions in liver, pancreas, muscle, adipose, kidney and intestinal tissues. Non-invasive ultrasound activation of afferent autonomic nerves may represent a non-pharmacologic therapy for the restoration of glucose homoeostasis in type-2 diabetes and other metabolic diseases.

Predicting ablation zones with multislice volumetric 2-D magnetic resonance thermal imaging.

BACKGROUND: High-intensity focused ultrasound (HIFU) serves as a noninvasive stereotactic system for the ablation of brain metastases; however, treatments are limited to simple geometries and energy delivery is limited by the high acoustic attenuation of the calvarium. Minimally-invasive magnetic resonance-guided robotically-assisted (MRgRA) needle-based therapeutic ultrasound (NBTU) using multislice volumetric 2-D magnetic resonance thermal imaging (MRTI) overcomes these limitations and has potential to produce less collateral tissue damage than current methods. OBJECTIVE: To correlate multislice volumetric 2-D MRTI volumes with histologically confirmed regions of tissue damage in MRgRA NBTU. METHODS: Seven swine underwent a total of 8 frontal MRgRA NBTU lesions. MRTI ablation volumes were compared to histologic tissue damage on brain sections stained with 2,3,5-triphenyltetrazolium chloride (TTC). Bland-Altman analyses and correlation trends were used to compare MRTI and TTC ablation volumes. RESULTS: Data from the initial and third swine's ablations were excluded due to sub-optimal tissue staining. For the remaining ablations (n = 6), the limits of agreement between the MRTI and histologic volumes ranged from -0.149 cm3 to 0.252 cm3 with a mean difference of 0.052 ± 0.042 cm3 (11.1%). There was a high correlation between the MRTI and histology volumes (r2 = 0.831) with a strong linear relationship (r = 0.868). CONCLUSION: We used a volumetric MRTI technique to accurately track thermal changes during MRgRA NBTU in preparation for human trials. Improved volumetric coverage with MRTI enhanced our delivery of therapy and has far-reaching implications for focused ultrasound in the broader clinical setting.

Effects of external low intensity focused ultrasound on inflammatory markers in neuropathic pain.

BACKGROUND: Changes in inflammatory cytokine levels contribute to the induction and maintenance of neuropathic pain. We have shown that external low intensity focused ultrasound (liFUS) reduces allodynia in a common peroneal nerve injury (CPNI). Here, we investigate an underlying mechanism of action for this treatment and measure the effect of liFUS on inflammatory markers. METHODS: Male rats were divided into four groups: CPNI/liFUS, CPNI/shamliFUS, shamCPNI/liFUS, and shamCPNI/shamliFUS. Mechanical nociceptive thresholds were measured using Von Frey filaments (VFF) to confirm the absence/presence of allodynia at baseline, after CPNI, and after liFUS. Commercial microarray and ELISA assays were used to assess cytokine expression in the treated L5 dorsal root ganglion (DRG) and dorsal horn (DH) tissue 24 and 72 h after liFUS. RESULTS: VFF thresholds were significantly reduced following CPNI in both groups that received the injury (p < 0.001). After liFUS, only the CPNI/liFUS cohort showed a significant increase in mechanical thresholds (p < 0.001). CPNI significantly increased TNFa, IL6, CNTF, IL1b (p < 0.05 for all) levels in the DRG and DH, compared to baseline, consistent with previous work in sciatic nerve injury. LiFUS in CPNI rats resulted in a decrease in these cytokines in DRG 72 h post-therapy (TNFa, IL6, CNTF and IL1b, p < 0.001). In the DH, IL1b, CNTF, and TNFa (p < 0.05 for all) decreased 72 h after liFUS. CONCLUSION: We have demonstrated that liFUS modifies inflammatory cytokines in both DRG and DH in CPNI rats. These data provide evidence that liFUS, reverses the allodynic phenotype, in part, by altering inflammatory cytokine pathways.

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

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.

Development of a common peroneal nerve injury model in domestic swine for the study of translational neuropathic pain treatments.

OBJECTIVE: To date, muscular and bone pain have been studied in domestic swine models, but the only neuropathic pain model described in swine is a mixed neuritis model. Common peroneal nerve injury (CPNI) neuropathic pain models have been utilized in both mice and rats. METHODS: The authors developed a swine surgical CPNI model of neuropathic pain. Behavioral outcomes were validated with von Frey filament testing, thermal sensitivity assessments, and social and motor scoring. Demyelination of the nerve was confirmed through standard histological assessment. The contralateral nerve served as the control. RESULTS: CPNI induced mechanical and thermal allodynia (p < 0.001 [n = 10] and p < 0.05 [n = 4], respectively) and increased pain behavior, i.e., guarding of the painful leg (n = 12). Myelin protein zero (P0) staining revealed demyelination of the ligated nerve upstream of the ligation site. CONCLUSIONS: In a neuropathic pain model in domestic swine, the authors demonstrated that CPNI induces demyelination of the common peroneal nerve, which the authors hypothesize is responsible for the resulting allodynic pain behavior. As the anatomical features of domestic swine resemble those of humans more closely than previously used rat and mouse models, utilizing this swine model, which is to the authors' knowledge the first of its kind, will aid in the translation of experimental treatments to clinical trials.

Effects of external low intensity focused ultrasound on electrophysiological changes in vivo in a rodent model of common peroneal nerve injury.

Non-invasive treatment methods for neuropathic pain are lacking. We assess how modulatory low intensity focused ultrasound (liFUS) at the L5 dorsal root ganglion (DRG) affects behavioral responses and sensory nerve action potentials (SNAPs) in a common peroneal nerve injury (CPNI) model. Rats were assessed for mechanical and thermal responses using Von Frey filaments (VFF) and the hot plate test (HPT) following CPNI surgery. Testing was repeated 24 h after liFUS treatment. Significant increases in mechanical and thermal sensory thresholds were seen post-liFUS treatment, indicating a reduction in sensitivity to pain (p < 0.0001, p = 0.02, respectively). Animals who received CPNI surgery had significant increases in SNAP latencies compared to sham CPNI surgery animals (p = 0.0003) before liFUS treatment. LiFUS induced significant reductions in SNAP latency in both CPNI liFUS and sham CPNI liFUS cohorts, for up to 35 min post treatment. No changes were seen in SNAP amplitude and there was no evidence of neuronal degeneration 24 h after liFUS treatment, showing that liFUS did not damage the tissue being modulated. This is the first in vivo study of the impact of liFUS on peripheral nerve electrophysiology in a model of chronic pain. This study demonstrates the effects of liFUS on peripheral nerve electrophysiology in vivo. We found that external liFUS treatment results in transient decreased latency in common peroneal nerve (CPN) sensory nerve action potentials (SNAPs) with no change in signal amplitude.

Low Intensity Focused Ultrasound Modulation of Vincristine Induced Neuropathy.

Previously, we showed internal low intensity focused ultrasound (liFUS) improves nociceptive thresholds in rats with vincristine-induced neuropathy (VIN) for 48-h post-treatment. Here, we perform more rigorous behavioral testing with the internal device and introduce external liFUS treatment. Behavioral testing confirmed VIN (Von Frey fibers, VFF; hot plate, HPT; locomotion, OFT). This was followed by internal or external liFUS treatment (2.5 W or 8 W, for 3 min, respectively) to the left L5 dorsal root ganglia (DRG). A thermocouple placed at the DRG documented temperature changes during treatment, to confirm the modulatory nature of our treatment. Behavioral testing was performed pre-liFUS, and for five consecutive days post-liFUS. Groups included: (1) VIN/liFUS, (2) saline/liFUS, (3) VIN/sham liFUS, and (4) saline/sham liFUS. Significant improvements in mechanical (VFF) and thermal (HPT) nociceptive thresholds were seen in the VIN/liFUS group following both internal and external treatment. Hematoxylin and Eosin, and Fluorojade staining showed no histological damage to the DRG. Internal liFUS treatment produced a mean temperature rise of 3.21 ±â€¯0.30 °C, whereas external liFUS resulted in a mean temperature rise of 1.78 °C ±â€¯0.21 °C. We demonstrate that, in a VIN rat model, external liFUS treatment of the L5 DRG significantly reduces nociceptive sensitivity thresholds without causing tissue damage.

Effects of subthalamic deep brain stimulation with gabapentin and morphine on mechanical and thermal thresholds in 6-hydroxydopamine lesioned rats.

Chronic pain is the most common non-motor symptom among Parkinson's disease (PD) patients, with 1.85 million estimated to be in debilitating pain by 2030. Subthalamic deep brain stimulation (STN DBS) programmed for treating PD motor symptoms has also been shown to significantly improve pain scores. However, even though most patients' pain symptoms improve or disappear, 74% of patients treated develop new pain symptoms within 8 years. Previously we have shown that duloxetine and STN high frequency stimulation (HFS) significantly increase mechanical thresholds more than either alone. The current project specifically investigates the effects of gabapentin and morphine alone and with high (150 Hz; HFS) and low (50 Hz; LFS) frequency stimulation in the 6-hydroxydopamine rat model for PD. We found that HFS, LFS, gabapentin 15 mg/kg and morphine 1 mg/kg all independently improve von Frey (VF) thresholds. Neither drug augments the HFS response significantly. Morphine at 1 mg/kg showed a trend to increasing thresholds compared to LFS alone (p = 0.062). Interestingly, gabapentin significantly reduced (p = 0.019) the improved VF thresholds and Randall Selitto thresholds seen with LFS. Thus, though neither drug augments DBS, we found effects of both compounds independently increase VF thresholds, informing use of our model of chronic pain in PD. Gabapentin's reversal of LFS effects warrants further exploration.

The use of focused ultrasound for the treatment of cutaneous allodynia associated with chronic migraine.

Chronic migraines (CM) are the third most common disease and are refractory to medical treatment in 15% of patients. Currently, temporary relief is achieved with steroid blocks or pulsed radiofrequency ablation, which have short-term benefits. Our project aims to develop a non-invasive treatment for medically refractory chronic migraine, which does not require a permanent implant. This project investigates the safety and effectiveness of pulsed focused ultrasound (FUS) in a validated rodent headache model of cutaneous allodynia associated with chronic migraine (CM) as compared to sumatriptan and ablative lesioning. We demonstrate a significant reduction in mechanical thresholds as measured through Von Frey filaments in CM in the forepaw and periorbital region (p < 0.001). Sumatriptan and pulsed FUS both significantly improve thresholds at day 3 after treatment in the periorbital region. Ablative lesioning has no effect. This study provides initial evidence that FUS may provide an important therapeutic option for patients suffering from CM.

External focused ultrasound treatment for neuropathic pain induced by common peroneal nerve injury.

Neuropathic pain caused by nerve injury or compressive lesions is a debilitating condition lacking effective, long-term treatments. Our objective was to assess the effects of external focused ultrasound on sensory thresholds utilizing a common peroneal injury rat model. CPNI was induced by ligating the CPN of the left hind paw. Neuropathic phenotype was confirmed using the Von Frey Fibers (VFF) with a 50% mechanical detection threshold below 4.0. The Place Escape Avoidance Paradigm (PEAP) was employed as a behavioral correlate. External FUS treatment was applied to the left L4,5 DRG at 8 W for 3-min. There were two treatment groups; one received a single FUS treatment, while the other received two. Control groups consisted of one sham CPNI group that received FUS treatment and a CPNI group that received sham FUS treatment. Behavioral tests were conducted pre-CPNI surgery, 1-week post-surgery, and for 1-week post-FUS treatment(s). CPNI surgery resulted in lower VFF mechanical thresholds in the left hind paw compared to baseline (p < 0.0001) and increased proportion of time spent on bright side compared to baseline values on PEAP (p = 0.0473), indicating neuropathic state. FUS treatment increased VFF thresholds after 24-hours (p < 0.0001), 48-h (p = 0.0079), and 72-h (p = 0.0164). VFF returned to baseline values from day 4-7. Following a second FUS treatment on day 8, increased mechanical thresholds were similarly observed after 24-h (p = 0.0021), 48-h (p < 0.0001), and 72-h (p = 0.0256). Control group analysis showed (1) CPNI rats experienced no change in mechanical thresholds following sham FUS treatment and (2) Sham CPNI rats receiving FUS did not experience significantly different mechanical thresholds compared to baseline and post-CPNI values. Post-FUS histological analysis demonstrated healthy ganglion cells without chromatolysis. Our results demonstrate changes in VFF and PEAP in rats who underwent CPNI. Single and multiple doses of external FUS increase mechanical thresholds without inducing histological damage. Based on our results, we have demonstrated the potential of FUS to serve as a non-pharmacological and non-ablative neuromodulatory approach for the treatment of allodynia and neuropathic pain.