Thoracic Dorsal Root Ganglion Application of Resiniferatoxin Reduces Myocardial Ischemia-Induced Ventricular Arrhythmias.

BACKGROUND: A myocardial ischemia/reperfusion (IR) injury activates the transient receptor potential vanilloid 1 (TRPV1) dorsal root ganglion (DRG) neurons. The activation of TRPV1 DRG neurons triggers the spinal dorsal horn and the sympathetic preganglionic neurons in the spinal intermediolateral column, which results in sympathoexcitation. In this study, we hypothesize that the selective epidural administration of resiniferatoxin (RTX) to DRGs may provide cardioprotection against ventricular arrhythmias by inhibiting afferent neurotransmission during IR injury. METHODS: Yorkshire pigs (n = 21) were assigned to either the sham, IR, or IR + RTX group. A laminectomy and sternotomy were performed on the anesthetized animals to expose the left T2-T4 spinal dorsal root and the heart for IR intervention, respectively. RTX (50 µg) was administered to the DRGs in the IR + RTX group. The activation recovery interval (ARI) was measured as a surrogate for the action potential duration (APD). Arrhythmia risk was investigated by assessing the dispersion of repolarization (DOR), a marker of arrhythmogenicity, and measuring the arrhythmia score and the number of non-sustained ventricular tachycardias (VTs). TRPV1 and calcitonin gene-related peptide (CGRP) expressions in DRGs and CGRP expression in the spinal cord were assessed using immunohistochemistry. RESULTS: The RTX mitigated IR-induced ARI shortening (-105 ms ± 13 ms in IR vs. -65 ms ± 11 ms in IR + RTX, p = 0.028) and DOR augmentation (7093 ms2 ± 701 ms2 in IR vs. 3788 ms2 ± 1161 ms2 in IR + RTX, p = 0.020). The arrhythmia score and VT episodes during an IR were decreased by RTX (arrhythmia score: 8.01 ± 1.44 in IR vs. 3.70 ± 0.81 in IR + RTX, p = 0.037. number of VT episodes: 12.00 ± 3.29 in IR vs. 0.57 ± 0.3 in IR + RTX, p = 0.002). The CGRP expression in the DRGs and spinal cord was decreased by RTX (DRGs: 6.8% ± 1.3% in IR vs. 0.6% ± 0.2% in IR + RTX, p < 0.001. Spinal cord: 12.0% ± 2.6% in IR vs. 4.5% ± 0.8% in IR + RTX, p = 0.047). CONCLUSIONS: The administration of RTX locally to thoracic DRGs reduces ventricular arrhythmia in a porcine model of IR, likely by inhibiting spinal afferent hyperactivity in the cardio-spinal sympathetic pathways.

The effects of low intensity focused ultrasound on neuronal activity in pain processing regions in a rodent model of common peroneal nerve injury.

BACKGROUND: Non-invasive, external low intensity focused ultrasound (liFUS) offers promise for treating neuropathic pain when applied to the dorsal root ganglion (DRG). OBJECTIVE: We examine how external liFUS treatment applied to the L5 DRG affects neuronal changes in single-unit activity from the primary somatosensory cortex (SI) and anterior cingulate cortex (ACC) in a common peroneal nerve injury (CPNI) rodent model. METHODS: Male Sprague Dawley rats were divided into two cohorts: CPNI liFUS and CPNI sham liFUS. Baseline single-unit activity (SUA) recordings were taken 20 min prior to treatment and for 4 h post treatment in 20 min intervals, then analyzed for frequency and compared to baseline. Recordings from the SI and ACC were separated into pyramidal and interneurons based on waveform and principal component analysis. RESULTS: Following CPNI surgery, all rats (n = 30) displayed a significant increase in mechanical sensitivity. In CPNI liFUS rats, there was a significant increase in pyramidal neuron spike frequency in the SI region compared to the CPNI sham liFUS animals beginning at 120 min following liFUS treatment (p < 0.05). In the ACC, liFUS significantly attenuated interneuron firing beginning at 80 min after liFUS treatment (p < 0.05). CONCLUSION: We demonstrate that liFUS changed neuronal spiking in the SI and ACC regions 80 and 120 min after treatment, respectively, which may in part correlate with improved sensory thresholds. This may represent a mechanism of action how liFUS attenuates neuropathic pain. Understanding the impact of liFUS on pain circuits will help advance the use of liFUS as a non-invasive neuromodulation option.

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.

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.

YAP and TAZ regulate Schwann cell proliferation and differentiation during peripheral nerve regeneration.

YAP and TAZ are effectors of the Hippo pathway that controls multicellular development by integrating chemical and mechanical signals. Peripheral nervous system development depends on the Hippo pathway. We previously showed that loss of YAP and TAZ impairs the development of peripheral nerve as well as Schwann cell myelination. The role of the Hippo pathway in peripheral nerve regeneration has just started to be explored. After injury, Schwann cells adopt new identities to promote regeneration by converting to a repair-promoting phenotype. While the reprogramming of Schwann cells to repair cells has been well characterized, the maintenance of such repair phenotype cannot be sustained for a very long period, which limits nerve repair in human. First, we show that short or long-term myelin maintenance is not affected by defect in YAP and TAZ expression. Using crush nerve injury and conditional mutagenesis in mice, we also show that YAP and TAZ are regulators of repair Schwann cell proliferation and differentiation. We found that YAP and TAZ are required in repair Schwann cells for their redifferentiation into myelinating Schwann cell following crush injury. In this present study, we describe how the Hippo pathway and YAP and TAZ regulate remyelination over time during peripheral nerve regeneration.

Effect of First-Line Ziconotide Intrathecal Drug Therapy for Neuropathic Pain on Disability, Emotional Well-Being, and Pain Catastrophizing.

BACKGROUND: Previous studies have shown decreased pain scores with ziconotide as a first-line agent for intrathecal drug therapy (IDT). Subset analysis suggests that patients with neuropathic pain have greater improvement. We prospectively examine the role of first-line ziconotide IDT on the tridimensional pain experience in ziconotide IDT-naive patients with neuropathic pain. METHODS: We included patients who underwent a successful ziconotide trial and were scheduled for standard-of-care IDT pump placement. Scores were collected at baseline and latest follow-up for the following measures: Short-Form 36 (SF-36), Oswestry Disability Index (ODI), Beck Depression Inventory, and Pain Catastrophizing Scale (PCS). Numeric rating scale (NRS) scores were also collected at each follow-up visit to monitor patients' pain levels and to guide ziconotide dose titration. Responders were identified as patients who had a previously established minimum clinically important difference of a ≥1.2-point reduction in NRS current scores. RESULTS: Eleven of 14 patients completed long-term follow-up. There were 7 responders based on NRS minimum clinically important difference. At a mean (±standard error of the mean) follow-up of 10.91 ± 0.70 months, SF-36 emotional well-being (P = 0.04), SF-36 pain (P = 0.02), and ODI (P = 0.03) significantly improved for the entire cohort and in responders (SF-36 emotional well-being, P = 0.01; SF-36 pain, P = 0.04; ODI, P = 0.02). PCS-Rumination (P = 0.02), PCS-Helplessness (P = 0.02), and PCS-Total (P = 0.003) scores improved significantly for responders only. CONCLUSIONS: We show that ziconotide IDT improves pain as well as emotional components and function. Our study adds prospective evidence to the literature on IDT for neuropathic pain, specifically its role in improving disability, emotional well-being, and catastrophizing.

Efficacy of Simultaneous Usage of Spinal Cord Stimulation and Intrathecal Therapy for Nonmalignant Chronic Neuropathic Pain.

BACKGROUND: Some patients with chronic pain and implanted spinal cord stimulators or intrathecal (IT) pumps fail to obtain significant pain relief. The use of dual modality treatment with both therapies is understudied. This study evaluated comprehensive outcomes in this patient population and reported outcomes primarily using IT ziconotide. METHODS: We retrospectively analyzed 11 patients with chronic pain treated with both spinal cord stimulation and IT therapy. When a primary treatment failed to achieve significant pain relief, a secondary device was trialed and implanted. Pain severity (measured by a numeric rating scale) was assessed by the change from baseline to after the first and second intervention. In a subset of patients (n = 6), quality-of-life metrics were also assessed. Outcome measures were analyzed closest to the 1-year follow-up date after implantation of the first modality and then at the most recent follow-up after implantation of the second modality. RESULTS: Spinal cord stimulation leads were percutaneous (n = 2) or paddles (n = 9) and commonly covered T8-10. IT medication included ziconotide (n = 8), baclofen (n = 1), hydromorphone (n = 1), and morphine/clonidine (n = 1). There was a mean of 19.64 ± 3.17 months between primary and secondary intervention. There was a significant improvement in pain severity from baseline to implantation of the second modality (P = 0.032) at a mean follow-up of 50.18 ± 11.83 months. CONCLUSIONS: Dual modality therapy is a potential treatment option in patients who have lost efficacy with a single neuromodulation modality. Further study is required to identify potential responders and nonresponders.

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.

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.

High-Intensity Ultrasound Treatment for Vincristine-Induced Neuropathic Pain.

BACKGROUND: Vincristine is a commonly used chemotherapeutic agent that results in debilitating untreatable peripheral neuropathy. OBJECTIVE: To determine the effects of pulsed high-intensity focused ultrasound (HIFU) on sensory thresholds in a validated vincristine-induced neuropathy (VIN) rodent model. METHODS: VIN was induced and mechanical allodynia was confirmed by nociceptive testing. von Frey fibers and Randall-Sellito test were used as measures of innocuous and noxious mechanical thresholds, respectively, and the hot plate test for thermal thresholds. Tests were performed before VIN, after 2 wk of vincristine, at 24, 48, 72, and 120 h after HIFU applied to the left L5 dorsal root ganglia at 3 Watts for 3 min. Comparisons were made between a VIN cohort who underwent HIFU, a VIN cohort who underwent sham HIFU, and naïve rodents who underwent HIFU. RESULTS: VIN HIFU rats had significantly increased mechanical thresholds at 24 h (P < .001), 48 h (P = .008), 72 h (P = .003), and 120 h (P = .03) after treatment, when compared to pre-HIFU thresholds. Furthermore, at 24 and 48 h following treatment, VIN HIFU rats had significantly higher innocuous and noxious mechanical thresholds and thermal thresholds than VIN sham HIFU rats (P < .001). Thresholds were not altered in naïve rodents who underwent HIFU. Histological data of L5 dorsal root ganglia of VIN HIFU rats suggest that transient cellular edema resolves by 48 h. CONCLUSION: Our data suggest that HIFU increases mechanical and thermal thresholds in VIN rodents. Whether HIFU can preclude the development of reduced thresholds in the VIN model warrants further study.