Focused ultrasound neuromodulation of the spleen activates an anti-inflammatory response in humans.

Focused ultrasound stimulation (FUS) activates mechanosensitive ion channels and is emerging as a method of noninvasive neuromodulation. In preclinical studies, FUS of the spleen (sFUS) activates an anti-inflammatory neural pathway which suppresses acute and chronic inflammation. However, the relevance of sFUS for regulating inflammatory responses in humans is unknown. Here, we used a modified diagnostic ultrasound imaging system to target the spleen of healthy human subjects with 3 min of continuously swept or stationary focused pulsed ultrasound, delivered at three different energy levels within allowable safety exposure limits. Potential anti-inflammatory effects of sFUS were assessed by measuring sFUS-elicited changes in endotoxin-induced tumor necrosis factor (TNF) production in whole blood samples from insonified subjects. We found that stimulation with either continuously swept or focused pulsed ultrasound has an anti-inflammatory effect: sFUS lowers TNF production for >2 h, with TNF returning to baseline by 24 h following sFUS. This response is independent of anatomical target (i.e., spleen hilum or parenchyma) or ultrasound energy level. No clinical, biochemical, or hematological parameters are adversely impacted. This is the first demonstration that sFUS suppresses the normal inflammatory response in humans, with potential implications for noninvasive bioelectronic therapy of inflammatory disorders.

Accelerating cutaneous healing in a rodent model of type II diabetes utilizing non-invasive focused ultrasound targeted at the spleen.

Healing of wounds is delayed in Type 2 Diabetes Mellitus (T2DM), and new treatment approaches are urgently needed. Our earlier work showed that splenic pulsed focused ultrasound (pFUS) alters inflammatory cytokines in models of acute endotoxemia and pneumonia via modulation of the cholinergic anti-inflammatory pathway (CAP) (ref below). Based on these earlier results, we hypothesized that daily splenic exposure to pFUS during wound healing would accelerate closure rate via altered systemic cytokine titers. In this study, we applied non-invasive ultrasound directed to the spleen of a rodent model [Zucker Diabetic Sprague Dawley (ZDSD) rats] of T2DM with full thickness cutaneous excisional wounds in an attempt to accelerate wound healing via normalization of T2DM-driven aberrant cytokine expression. Daily (1x/day, Monday-Friday) pFUS pulses were targeted externally to the spleen area for 3 min over the course of 15 days. Wound diameter was measured daily, and levels of cytokines were evaluated in spleen and wound bed lysates. Non-invasive splenic pFUS accelerated wound closure by up to 4.5 days vs. sham controls. The time to heal in all treated groups was comparable to that of healthy rats from previously published studies (ref below), suggesting that the pFUS treatment restored a normal wound healing phenotype to the ZDSD rats. IL-6 was lower in stimulated spleen (-2.24 ± 0.81 Log2FC, p = 0.02) while L-selectin was higher in the wound bed of stimulated rodents (2.53 ± 0.72 Log2FC, p = 0.003). In summary, splenic pFUS accelerates healing in a T2DM rat model, demonstrating the potential of the method to provide a novel, non-invasive approach for wound care in diabetes.

Radiolabeled Aminopyrazoles as Novel Isoform Selective Probes for pJNK3 Quantification.

The c-Jun N-terminal kinase 3 (JNK3) is a stress-activated kinase primarily expressed in the brain and implicated as an early mediator of neuronal apoptosis. We sought to develop a PET tracer to visualize pathological JNK3 activation. Because regional JNK3 activation precedes apoptosis, such an imaging agent might enable the detection of "at risk" brain regions prior to neuronal death. We prepared a set of 19F-containing compounds on the basis of the reported aminopyrazoles. The candidate, F3, was tritiated and used in autoradiography experiments to demonstrate regional and temporal changes in JNK3 activation in a mouse model of Parkinson's disease. A significant increase in pJNK3 B max versus control animals in multiple brain regions was observed at 8 months, including the ventral midbrain. Pathological activation of JNK3 in these regions preceded statistically significant neuron loss. Analyses of brain concentrations of [18F]-F3 in naïve rats following intravenous injection revealed a small but detectable signal over the background, but was likely not sufficient to support PET imaging.

Ultrasound Neuromodulation of the Spleen Has Time-Dependent Anti-Inflammatory Effect in a Pneumonia Model.

Interfaces between the nervous and immune systems have been shown essential for the coordination and regulation of immune responses. Non-invasive ultrasound stimulation targeted to the spleen has recently been shown capable of activating one such interface, the splenic cholinergic anti-inflammatory pathway (CAP). Over the past decade, CAP and other neuroimmune pathways have been activated using implanted nerve stimulators and tested to prevent cytokine release and inflammation. However, CAP studies have typically been performed in models of severe, systemic (e.g., endotoxemia) or chronic inflammation (e.g., collagen-induced arthritis or DSS-induced colitis). Herein, we examined the effects of activation of the splenic CAP with ultrasound in a model of local bacterial infection by lung instillation of 105 CFU of Streptococcus pneumoniae. We demonstrate a time-dependent effect of CAP activation on the cytokine response assay during infection progression. CAP activation-induced cytokine suppression is absent at intermediate times post-infection (16 hours following inoculation), but present during the early (4 hours) and later phases (48 hours). These results indicate that cytokine inhibition associated with splenic CAP activation is not observed at all timepoints following bacterial infection and highlights the importance of further studying neuroimmune interfaces within the context of different immune system and inflammatory states.

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.

Targeted peripheral focused ultrasound stimulation attenuates obesity-induced metabolic and inflammatory dysfunctions.

Obesity, a growing health concern, is associated with an increased risk of morbidity and mortality. Chronic low-grade inflammation is implicated in obesity-driven metabolic complications. Peripheral focused ultrasound stimulation (pFUS) is an emerging non-invasive technology that modulates inflammation. Here, we reasoned that focused ultrasound stimulation of the liver may alleviate obesity-related inflammation and other comorbidities. After 8 weeks on a high-fat high-carbohydrate "Western" diet, C57BL/6J mice were subjected to either sham stimulation or focused ultrasound stimulation at the porta hepatis. Daily liver-focused ultrasound stimulation for 8 weeks significantly decreased body weight, circulating lipids and mitigated dysregulation of adipokines. In addition, liver-focused ultrasound stimulation significantly reduced hepatic cytokine levels and leukocyte infiltration. Our findings demonstrate the efficacy of hepatic focused ultrasound for alleviating obesity and obesity-associated complications in mice. These findings suggest a previously unrecognized potential of hepatic focused ultrasound as a possible novel noninvasive approach in the context of obesity.

Non-invasive peripheral focused ultrasound neuromodulation of the celiac plexus ameliorates symptoms in a rat model of inflammatory bowel disease.

NEW FINDINGS: What is the central question of this study? Does peripheral non-invasive focused ultrasound targeted to the celiac plexus improve inflammatory bowel disease? What is the main finding and its importance? Peripheral non-invasive focused ultrasound targeted to the celiac plexus in a rat model of ulcerative colitis improved stool consistency and reduced stool bloodiness, which coincided with a longer and healthier colon than in animals without focused ultrasound treatment. The findings suggest that this novel neuromodulatory technology could serve as a plausible therapeutic approach for improving symptoms of inflammatory bowel disease. ABSTRACT: Individuals suffering from inflammatory bowel disease (IBD) experience significantly diminished quality of life. Here, we aim to stimulate the celiac plexus with non-invasive peripheral focused ultrasound (FUS) to modulate the enteric cholinergic anti-inflammatory pathway. This approach may have clinical utility as an efficacious IBD treatment given the non-invasive and targeted nature of this therapy. We employed the dextran sodium sulfate (DSS) model of colitis, administering lower (5%) and higher (7%) doses to rats in drinking water. FUS on the celiac plexus administered twice a day for 12 consecutive days to rats with severe IBD improved stool consistency scores from 2.2 ± 1 to 1.0 ± 0.0 with peak efficacy on day 5 and maximum reduction in gross bleeding scores from 1.8 ± 0.8 to 0.8 ± 0.8 on day 6. Similar improvements were seen in animals in the low dose DSS group, who received FUS only once daily for 12 days. Moreover, animals in the high dose DSS group receiving FUS twice daily maintained colon length (17.7 ± 2.5 cm), while rats drinking DSS without FUS exhibited marked damage and shortening of the colon (13.8 ± 0.6 cm) as expected. Inflammatory cytokines such as interleukin (IL)-1ß, IL-6, IL-17, tumour necrosis factor-α and interferon-γ were reduced with DSS but coincided with control levels after FUS, which is plausibly due to a loss of colon crypts in the former and healthier crypts in the latter. Lastly, overall, these results suggest non-invasive FUS of peripheral ganglion can deliver precision therapy to improve IBD symptomology.

Evidence of Long-range nerve pathways connecting and coordinating activity in secondary lymph organs.

Background: Peripheral nerve reflexes enable organ systems to maintain long-term physiological homeostasis while responding to rapidly changing environmental conditions. Electrical nerve stimulation is commonly used to activate these reflexes and modulate organ function, giving rise to an emerging class of therapeutics called bioelectronic medicines. Dogma maintains that immune cell migration to and from organs is mediated by inflammatory signals (i.e. cytokines or pathogen associated signaling molecules). However, nerve reflexes that regulate immune function have only recently been elucidated, and stimulation of these reflexes for therapeutic effect has not been fully investigated. Methods: We utilized both electrical and ultrasound-based nerve stimulation to activate nerve pathways projecting to specific lymph nodes. Tissue and cell analysis of the stimulated lymph node, distal lymph nodes and immune organs is then utilized to measure the stimulation-induced changes in neurotransmitter/neuropeptide concentrations and immune cellularity in each of these sites. Results and conclusions: In this report, we demonstrate that activation of nerves and stimulated release of neurotransmitters within a local lymph node results in transient retention of immune cells (e.g. lymphocytes and neutrophils) at that location. Furthermore, such stimulation results in transient changes in neurotransmitter concentrations at distal organs of the immune system, spleen and liver, and mobilization of immune cells into the circulation. This report will enable future studies in which stimulation of these long-range nerve connections between lymphatic and immune organs can be applied for clinical purpose, including therapeutic modulation of cellularity during vaccination, active allergic response, or active auto-immune disease.

Peripheral Focused Ultrasound Neuromodulation (pFUS).

BACKGROUND: A fundamental limit to the study of the peripheral nervous system and its effect on organ function is the lack of tools to selectively target and stimulate specific neurons. Traditional implant and electrode-based systems remain too large and invasive for use at the organ or sub-organ level (without stimulating or effecting neighboring organs and tissues). Recent progress in optical and genetic tools (such as optogenetics) has provided a new level of molecular specificity and selectivity to the neurons that are stimulated by bioelectronic devices. However, the modified neurons that result from use of these tools (that can be selectively activated based on expression of light, heat, or stimuli sensitive ion channels) often still require stimulation by implantable devices and face difficult scientific, technical, and regulatory hurdles for clinical translation. NEW METHOD: Herein, we present a new tool for selective activation of neuronal pathways using anatomical site-specific, peripheral focused ultrasound neuromodulation (pFUS). RESULTS: We utilize three experimental models to expand upon and further characterize pFUS beyond data outlined to our initial report (Cotero et al., 2019a), and further demonstrate its importance as a new investigative and translational tool. First, we utilized an interconnected microporous gel scaffold to culture isolated dorsal root ganglion (DRG) neurons in an interconnected, three-dimensional in vitro culture. (Griffin et al., 2015, Tay et al., 2018) Using this system, we directly applied ultrasound (US) stimuli and confirmed US activation of peripheral neurons at pressures consistent with recent in vivo observations. (Cotero et al., 2019a, Zachs, 2019, Gigliotti et al., 2013) Next, we tested the capability of pFUS to activate previously reported nerve pathways at multiple locations within the neural circuit, including primary sensory ganglia (i.e. inferior ganglion of the vagus nerve), peripheral ganglia (i.e. sacral ganglia), and within target end-organs. In addition, we compared selective activation of multiple anatomically overlapping neural pathways (i.e. activation of the cholinergic anti-inflammatory pathway (Tracey, 2009, Pavlov and Tracey, 2012) vs. metabolic sensory pathways (O'Hare and Zsombok, 2015, Roh et al., 2016, Pocai et al., 2005) after stimulation of each separate target site. Finally, we utilized an established model of metabolic dysfunction (the LPS-induced inflammation/hyperglycemia model) to demonstrate pFUS capability to stimulate and assess alternative therapeutic stimulation sites (i.e. liver, pancreas, and intestines) in a simple and clinically relevant manner. This is demonstrated by ultrasound induced attenuation of LPS-induced hyperglycemia by stimulation at all three anatomical targets, and mapping of the effect to a specific molecular product of excitable cell types within each stimulus site. COMPARISON WITH EXISTING METHODS: The ease-of-use and non-invasive nature of pFUS provides a solution to many of the challenges facing traditional toolsets, such as implantable electrodes and genetic/optogenetic nerve stimulation strategies. CONCLUSIONS: The pFUS tool described herein provides a fundamental technology for the future study and manipulation of the peripheral nervous and neuroendocrine systems.

Author Correction: Noninvasive sub-organ ultrasound stimulation for targeted neuromodulation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Noninvasive Neuromodulation of Peripheral Nerve Pathways Using Ultrasound and Its Current Therapeutic Implications.

This review describes work from several research groups in which ultrasound is being used to target the peripheral nervous system and perform neuromodulation noninvasively. Although these techniques are in their infancy compared to implant-based and electrical nerve stimulation, if successful this new noninvasive method for neuromodulation could solve many of the challenges facing the field of bioelectronic medicine. The work outlined herein shows results in which two different (potentially therapeutic) targets are stimulated, a neuroimmune pathway within the spleen and a nutrient/sensory pathway within the liver. Both data and discussion are provided that compare this new noninvasive technique to implant-based nerve stimulation.

Peripheral Focused Ultrasound Stimulation (pFUS): New Competitor in Pharmaceutical Markets?

A new study published in Nature Communications outlines our group's results using focused ultrasound stimulation within peripheral organs to precisely activate autonomic nerve circuits. The concept is demonstrated by modulating two different (and potentially therapeutic) targets in animal models, a neuroimmune connection in the spleen (that modulates blood cytokine concentrations) and a nutrient sensory pathway within the liver (that modulates metabolism). Connected to this work is a companion Nature Communications publication that utilizes an ultrasound stimulus focused on the spleen to reduce disease severity in a serum-transferred rodent model of inflammatory arthritis. These reports highlight the growing evidence that ultrasound energy (previously shown to enable activation or modulation of central nervous system pathways) may be used to perform peripheral neuromodulation. In this commentary, we highlight the main findings and discuss their implications for new forms of ultrasound-based therapy. Though challenges remain, a new noninvasive method for precision neuromodulation could solve many of the challenges facing the nascent field of bioelectronic medicine. That is, the use of ultrasound to directly modulate neurophysiological systems therapeutically may provide alternatives to traditional pharmaceuticals. However, to alter the current pharmaceutical paradigm, the field will need to develop a new understanding of how traditional drug concepts (such as dose and pharmacokinetics-pharmacodynamics) relate to the parameters, protocols, and outcomes of this new stimulation technology.

Noninvasive sub-organ ultrasound stimulation for targeted neuromodulation.

Tools for noninvasively modulating neural signaling in peripheral organs will advance the study of nerves and their effect on homeostasis and disease. Herein, we demonstrate a noninvasive method to modulate specific signaling pathways within organs using ultrasound (U/S). U/S is first applied to spleen to modulate the cholinergic anti-inflammatory pathway (CAP), and US stimulation is shown to reduce cytokine response to endotoxin to the same levels as implant-based vagus nerve stimulation (VNS). Next, hepatic U/S stimulation is shown to modulate pathways that regulate blood glucose and is as effective as VNS in suppressing the hyperglycemic effect of endotoxin exposure. This response to hepatic U/S is only found when targeting specific sub-organ locations known to contain glucose sensory neurons, and both molecular (i.e. neurotransmitter concentration and cFOS expression) and neuroimaging results indicate US induced signaling to metabolism-related hypothalamic sub-nuclei. These data demonstrate that U/S stimulation within organs provides a new method for site-selective neuromodulation to regulate specific physiological functions.

Label-free independent quantitation of viable and non-viable cells using a multivariable multi-resonant sensor.

Biological cells are utilized for diverse biotechnological and bioengineering purposes ranging from the production of biopharmaceuticals, to cell therapy, "human-on-a-chip" drug and toxicology assays, and drug-resistance tests. In these and other applications, it is critical to quantify the levels of not only viable but also non-viable cells. While traditional off-line cell-staining methods are available for counting of non-viable cells, many applications cannot periodically remove cells for their off-line analysis because of the risk of contamination or workflow logistics. Here we show in-situ label-free quantitation of viable and non-viable cells with multivariable multi-resonant sensors. We used Chinese hamster ovary (CHO) cells in suspension culture in single-use bioreactors as a representative example. The resonant sensor design strategy permitted enhanced sensor sensitivity versus conventional non-resonant measurements and probed the spectral dispersion of viable and non-viable cells with multiple resonances. These capabilities of label-free in-situ analysis of cell viability can be attractive in diverse cell applications such as cell suspensions, adhered cells, and their 3D assemblages.

In vitro and in vivo DFO-chelatable labile iron release profiles among commercially available intravenous iron nanoparticle formulations.

Intravenous (IV) iron formulations are complex colloidal suspensions of iron oxide nanoparticles. Small changes in formulation can allow more labile iron to be released after injection causing toxicity. Thus, bioequivalence (BE) evaluation of generic IV iron formulations remains challenging. We evaluated labile iron release in vitro and in vivo using a high performance liquid chromatography chelatable iron assay to develop a relational model to support BE. In vitro labile iron release and in vivo labile iron pharmacokinetics were evaluated for Venofer®, Ferrlecit®, generic sodium ferric gluconate complex, InFeD®, Feraheme® and a pre-clinical formulation GE121333. Labile iron release profiles were studied in vitro in 150 mM saline and a biorelevant matrix (rat serum) at 0.952 mgFe/mL. In vivo plasma labile iron concentration-time profiles (t0-240 min) were studied in rats after a 40 mgFe/kg IV dose. In vitro labile iron release in saline was significantly higher compared to rat serum, especially with InFeD®. An in vitro release constant (iKr) was calculated which correlated well with maximal plasma concentrations in the in vivo rat PK model (R2 = 0.711). These data suggest an in vitro to in vivo correlation model of labile iron release kinetics could be applied to BE. Other generic IV iron formulations need to be studied to validate this model.

Fluorescence Phenomena in Nerve-Labeling Styryl-Type Dyes.

Several classes of diversely substituted styryl type dyes have been synthesized with the goal of extending their expected fluorescent properties as much towards red as possible given the constraint that they maintain drug-like properties and retain high affinity binding to their biological target. We report on the synthesis, optical properties of a series of styryl dyes ( d1-d14 ), and the anomalous photophysical behavior of several of these Donor-Acceptor pairs separated by long conjugated π-systems ( d7-d10 ). We further describe an unusual dual emission behavior with two distinct ground state conformers which could be individually excited to locally excited (LE) and twisted intramolecular charge transfer (TICT) excited state in push-pull dye systems ( d7 , d9 and d10 ). Additionally, unexpected emission behavior in dye systems d7 and d8 wherein the amino- derivative d7 displayed a dual emission in polar medium while the N,N-dimethyl derivative d8 and other methylated derivatives d12-d14 showed only LE emission but did not show any TICT emission. Based on photophysical and nerve binding studies, we down selected compounds that exhibited the most robust fluorescent staining of nerve tissue sections. These dyes ( d7 , d9 , and d10 ) were subsequently selected for in-vivo fluorescence imaging studies in rodents using the small animal multispectral imaging instrument and the dual-mode laparoscopic instrument developed in-house.

Improved Intraoperative Visualization of Nerves through a Myelin-Binding Fluorophore and Dual-Mode Laparoscopic Imaging.

The ability to visualize and spare nerves during surgery is critical for avoiding chronic morbidity, pain, and loss of function. Visualization of such critical anatomic structures is even more challenging during minimal access procedures because the small incisions limit visibility. In this study, we focus on improving imaging of nerves through the use of a new small molecule fluorophore, GE3126, used in conjunction with our dual-mode (color and fluorescence) laparoscopic imaging instrument. GE3126 has higher aqueous solubility, improved pharmacokinetics, and reduced non-specific adipose tissue fluorescence compared to previous myelin-binding fluorophores. Dosing and kinetics were initially optimized in mice. A non-clinical modified Irwin study in rats, performed to assess the potential of GE3126 to induce nervous system injuries, showed the absence of major adverse reactions. Real-time intraoperative imaging was performed in a porcine model. Compared to white light imaging, nerve visibility was enhanced under fluorescence guidance, especially for small diameter nerves obscured by fascia, blood vessels, or adipose tissue. In the porcine model, nerve visualization was observed rapidly, within 5 to 10 minutes post-intravenous injection and the nerve fluorescence signal was maintained for up to 80 minutes. The use of GE3126, coupled with practical implementation of an imaging instrument may be an important step forward in preventing nerve damage in the operating room.

Identification of the protein target of myelin-binding ligands by immunohistochemistry and biochemical analyses.

The ability to visualize myelin is important in the diagnosis of demyelinating disorders and the detection of myelin-containing nerves during surgery. The development of myelin-selective imaging agents requires that a defined target for these agents be identified and that a robust assay against the target be developed to allow for assessment of structure-activity relationships. We describe an immunohistochemical analysis and a fluorescence polarization binding assay using purified myelin basic protein (MBP) that provides quantitative evidence that MBP is the molecular binding partner of previously described myelin-selective fluorescent dyes such as BMB, GE3082, and GE3111.

Dual-mode laparoscopic fluorescence image-guided surgery using a single camera.

Iatrogenic nerve damage is a leading cause of morbidity associated with many common surgical procedures. Complications arising from these injuries may result in loss of function and/or sensation, muscle atrophy, and chronic neuropathy. Fluorescence image-guided surgery offers a potential solution for avoiding intraoperative nerve damage by highlighting nerves that are otherwise difficult to visualize. In this work we present the development of a single camera, dual-mode laparoscope that provides near simultaneous display of white-light and fluorescence images of nerves. The capability of the instrumentation is demonstrated through imaging several types of in situ rat nerves via a nerve specific contrast agent. Full color white light and high brightness fluorescence images and video of nerves as small as 100 µm in diameter are presented.

Intraoperative fluorescence imaging of peripheral and central nerves through a myelin-selective contrast agent.

PURPOSE: Patients suffer from complications as a result of unintentional nerve damage during surgery. We focus on improving intraoperative visualization of nerves through the use of a targeted fluorophore and optical imaging instrumentation. PROCEDURE: A myelin-targeting fluorophore, GE3111, was synthesized, characterized for its optical and myelin-binding properties using purified myelin basic protein, and evaluated in mice. Additionally, a compact instrument was adapted to visualize nerves. RESULTS: GE3111 was synthesized using a versatile methodology. Its optical properties were sensitive to the local environment both in vitro and in vivo. Following intravenous injection, central and peripheral nerves were visualized, with the kinetics of nerve uptake modifiable depending on the formulation. Fluorescence polarization showed specific and strong binding to purified myelin basic protein. Nerves were visualized in vivo using a dedicated compact imaging device requiring less than 2.5 mW/cm(2) of illumination at 405 nm. CONCLUSIONS: Fluorescence imaging of nerves through myelin showed a potential for use in image-guided surgery. Intraoperative nerve imaging is an example where contrast agent and instrument development come together as a result of clinical need.