Focused ultrasound (FUS) probe set-up and characterization

A block diagram of the focused ultrasound (FUS) system is shown in Supplementary Figure 1. The system consists of a 1.1 MHz, High Intensity Focused Ultrasound (HIFU) transducer (Sonic Concepts H106), a matching network (Sonic Concepts), an RF power amplifier (ENI 350L) and a function generator (Agilent 33120A). The 70-mm-diameter HIFU transducer has a spherical face with a 65-mm radius of curvature. It has a 20-mm-diameter hole in the center into which an imaging transducer can be inserted. The transducer depth of focus is 65 mm. The numerically simulated pressure profile has a full width at half amplitude of 1.8 mm laterally and 12 mm in the depth direction. The HIFU transducer is acoustically coupled to the animal through a 6-cm-tall plastic cone filled with degassed water.

The function generator produces a pulsed sinusoidal waveform, shown schematically in Supplementary Figure 2. This pulsed sinusoidal waveform is amplified by the RF power amplifier and sent to the impedance-matching network connected to the HIFU transducer. For most of the animal experiments, the pulse center frequency was 1.1 MHz, the pulse repetition period was 0.5 ms (corresponding to a pulse repetition frequency of 2000 Hz); the pulse amplitude and pulse length varied. Supplementary Table 1 lists the combinations of pulse amplitude and length that were used in the experiments; the third column lists the peak ultrasound pressure at the focus derived from the pulse voltage amplitude.

The voltage-to-pressure calibration of the HIFU transducer was performed in degassed water using a needle hydrophone (ONDA HNA-0400). The HIFU transducer was driven by a 100-cycle sinusoidal voltage waveform. To locate the position of peak pressure, the hydrophone was scanned in a neighborhood of the nominal transducer focus point in 0.1 mm steps in the lateral plane and in 0.2 steps in the depth direction. Supplementary Figure 3 shows a scan through a plane at the depth of focus. For driving voltages below 60 V, the nonlinearity of water was small, i.e., the maximum negative pressure and the maximum positive pressure were nearly equal, and the pressure varied linearly with driving voltage.

Ultrasound targeting for organ-specific neuromodulation

A Vivid E9 ultrasound system (GE Healthcare) or an 11L probe (GE Healthcare) were used for the ultrasound scan before neuromodulation started. Supplementary Figure 4 shows the images of the rat spleen (Supplementary Figure 4A) and rat liver (Supplementary Figure 4B). The HIFU transducer was positioned on the target area based on this initial image. Another ultrasound scan was also performed using a smaller imaging probe (3S, GE Healthcare), which was placed in the opening of the HIFU transducer (Supplementary Figure 4C). The imaging beam of the 3S probe was aligned with the U/S beam. Therefore, one could confirm that the U/S beam was targeted at the region of interest using an image of the targeted organ (visualized on the Vivid E9). After the organ of interest was identified, the transducer position was marked on the animal’s skin and the HIFU transducer was positioned on the marked area, ultrasound stand-offs were utilized to adjust depth for the appropriate organ target.

Animal models and ultrasound stimulation protocol

Adult male Sprague–Dawley rats 8–12 weeks old (250–300 g; Charles River Laboratories) were housed at 25 °C on a 12-h light/dark cycle and acclimatized for 1 week, with handling, before experiments were conducted to minimize potential confounding measures due to stress response. Water and regular rodent chow were available ad libitum. Experiments were performed under protocols approved by the Institutional Animal Care and Use Committee of GE Global Research.

Endotoxin (lipopolysaccharide (LPS) from Escherichia coli, 0111: B4; Sigma–Aldrich) was used to produce a significant state of inflammation and metabolic dysfunction (e.g., hyperglycemia and insulin resistance) in naive adult Sprague–Dawley rats. LPS was administered to animals (10 mg/kg), which corresponds to an approximate LD75 dose, via intraperitoneal (IP) injection causing significant elevation in concentrations of TNF, circulating glucose, and insulin; these concentrations peak in 4 h but remain elevated as compared to control for up to 8 h post injection. After LPS administration, spleen, liver, hypothalamic, hippocampal, and blood samples were harvested at 60 min for most studies; at 1, 2, and 3 h for kinetic studies (Fig. 2e) and at 0.5, 1, 2, 4, 8, 24, and 48 h for duration studies (Fig. 2g, h). Spleen and liver samples were prepared as described below. Samples were analyzed by enzyme-linked immunosorbent assay (ELISA) for changes in cytokine (Bio-Plex Pro; Bio-Rad), tumor necrosis factor alpha (TNF) (Lifespan) and acetylcholine (Lifespan) concentration as described below. Catecholamine concentrations were assessed using high performance liquid chromatography (HPLC) detection and ELISA (Rocky Mountain Diagnostic) analysis as described below.

The link between LPS and insulin resistance is well detailed in the literature with hyperglycemia and hyperinsulinemia frequently observed during bacterial infection as an indicator of poor clinical outcome in patients. Thus, to follow the effects of LPS and US treatment on blood glucose and insulin levels, blood samples were obtained from the tail vein at 0, 15, 30, and 60 min after LPS injection. Blood glucose concentrations were measured by a OneTouch Elite glucometer (LifeScan; Johnson & Johnson). Insulin concentrations in plasma, obtained from blood, were determined using an ELISA kit (Crystal Chem). Signal transduction changes were measured by assessment of key biomarkers including: p38, p7056k, Akt, GSK3B, c-Src, NF-κβ, SOCS3, IRS-1, NPY, and POMC in liver, muscle, cardiac, and hypothalamic tissue samples.

Mice

All mice experiments were performed under protocols approved by the Institutional Animal Care and Use Committee of the Feinstein Institute for Medical Research, Northwell Health System. Animals were housed at 25 °C on a 12-h light/dark cycle, and acclimatized for at least 1 week before conducting experiments. Water and regular rodent chow were available ad libitum. Wild type C57black/6 mice, nude (nu/nu) mice, α7 nicotinic receptor knock out mice (B6.129S7-Chrna7tm1Bay, number 003232), ChAT-floxed (B6.129-Chattm1Jrs/J), and mice expressing Cre recombinase under the control of the endogenous CD4 promoter (CD4-Cre), 8–12 weeks old (20–25 g) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). ChAT-floxed and CD4-Cre mice were crossed to generate mice genetically devoid of ChAT in the CD4+ population.

Catecholamine depletion

Reserpine (Sigma–Aldrich) dissolved in glacial acetic acid (Sigma–Aldrich) was diluted with sterile saline to a final glacial acetic acid concentration of 0.5%. Mice received intraperitoneal administration of reserpine (10 mg/kg) reserpine 24 h before the beginning of experiments.

Ultrasound stimulation protocol

The protocol used for ultrasound neuromodulation was as follows. Animals were anesthetized with 2–4% isoflurane. The animal was laid on a water circulating warming pad to prevent hyperthermia during the procedure. The region above the designated point for U/S stimulus (nerve of interest) was shaved with a disposable razor and animal clippers prior to stimulation. A Vivid E9 Diagnostic imaging ultrasound system was used to identify the region of interest as follows. Liver: the porta hepatis as indicated by Doppler identification of the hepatic portal vein. Spleen: visual identification of the spleen by diagnostic ultrasound. Location of stimuli was maintained along the splenic axis as identified. The area was marked with a permanent marker for later identification. Ultrasound stimulation was applied using a research FUS system. The U/S probe was placed at the designated area of interest identified by the diagnostic ultrasound probe (Supplementary Figures 1 and 4). An U/S stimulus was then applied with total duration of a single stimulus not surpassing a single 1 min pulse. At no point was the energy allowed to reach levels associated with thermal damage and ablation/cavitation (35 W/cm2 for ablation/cavitation). LPS (10 mg/kg) was then injected IP (for acute/kinetic studies). Alternatively, for duration of effect, LPS was not injected here and was instead injected at a later designated time point. Second 1-min US stimuli may then be applied.

The animal was then allowed to incubate under anesthesia, due to the concentration of LPS being equivalent to an LD75 dose, for acute (1-h) and kinetic (varying up to a maximum of 3 h post LPS) studies. After incubation, the animal was euthanized and tissue, blood samples are collected as described below. For duration of effect studies, LPS was not injected at the time of U/S stimulus but rather at a designated delay after the U/S stimuli have been applied (e.g., 0.5, 1, 2, 4, 8, 24, or 48 h). After the delay, the animal was placed into an anesthetic holding chamber and monitored until euthanasia and tissue/fluid collection.

Tissue harvesting and sample preparation

An incision was made starting at the base of the peritoneal cavity extending up and through to the pleural cavity. Organs (including spleen and liver) were rapidly removed and homogenized in a solution of phosphate-buffered saline (PBS), containing phosphatase (0.2-mM phenylmethylsulfonyl fluoride, 5-µg/mL aprotinin, 1-mM benzamidine, 1-mM sodium orthovanadate, and 2-µM cantharidin) and protease (1-µL to 20 mg of tissue as per Roche Diagnostics) inhibitors. A targeted final concentration of 0.2-g tissue per mL PBS solution was applied in all samples. Blood samples were stored with the anti-coagulant disodium (ethylenedinitrilo)tetraacetic acid (EDTA) to prevent coagulation of samples. Samples were then stored at −80 °C until analysis.

ELISA analyses

A detailed protocol for ELISA Assays was provided by the respective supplier of the kits: https://www.lsbio.com/elisakits/manualpdf/ls-f24977.pdf, Acetylcholine: http://www.abcam.com/ps/products/65/ab65345/documents/ab65345%20Choline%20Acetylcholine%20Assay%20Kit%20protocol%20v11%20(website).pdf, PI3K Activation Profile (Akt/GSK/p70S6K): https://www.thermofisher.com/order/catalog/product/85-86048-11, SRC: https://www.lsbio.com/elisakits/manualpdf/ls-f11230.pdf, P38 (MAPK): https://www.thermofisher.com/order/catalog/product/85-86022-11.

HPLC analyses

Serum samples were injected directly into the machine with no pre-treatment. Tissue homogenates were initially homogenized with 0.1-M perchloric acid and centrifuged for 15 min, after which the supernatant was separated, and the sample injected into the HPLC. Catecholamines norepinephrine and epinephrine were analyzed by HPLC with inline ultraviolet detector. The test column used in this analysis was a Supelco Discovery C18 (15-cm × 4.6-mm inside diameter, 5-µm particle size). A biphasic mobile phase comprised of [A] acetonitrile: [B] 50 = mM KH 2 PO 4 , set to pH 3 (with phosphoric acid). The solution was then buffered with 100-mg/L EDTA and 200-mg/L 1-octane-sulfonic acid. Final concentration of mobile phase mixture was set to 5:95, A:B. A flow rate of 1 mL/min was used to improve overall peak resolution while the column was held to a consistent 20 °C to minimize pressure compaction of the column resulting from the viscosity of the utilized mobile phase. The UV detector was maintained at a 254-nm wavelength, which is known to capture the absorption for catecholamines including norepinephrine, epinephrine, and dopamine.

Immunohistochemistry and histology protocols

Tissue extraction and paraffin block conversion performed as follows. Put tissue (rat brain) into fixative immediately and fix ~24 h in 10% formalin at 4 °C. Process tissue with the following protocol (with vacuum and pressure during each incubation): 70% ethanol, 37 °C, 40 min, 80% ethanol, 37 °C, 40 min, 95% ethanol, 37 °C, 40 min, 95% ethanol, 37 °C, 40 min, 100% ethanol, 37 C, 40 min, 100% ethanol, 37 °C, 40 min, xylene, 37 °C, 40 min, xylene, 37 °C, 40 min, paraffin, 65 °C, 40 min, paraffin, 65 °C, 40 min, paraffin, 65 °C, 40 min. Sample is then left in this paraffin until ready for embedding (not to exceed ~12–18 h).

Embed into Paraffin block for sectioning, allow block to cool/harden before sectioning. Section 5-µm thick, float on 50 °C water bath for collection. Use positively charged slides and try to position the tissue in the same orientation for every slide. Air dry slides. Overnight at room temperature seems to be the best for drying but the slides can be placed on a 40 °C slide warmer to speed up the drying process, but do not leave slides more than an hour on the warmer. Store slides at 4 °C.

Formalin-fixed paraffin-embedded (FFPE) tissue samples (rat brains) were baked at 65 °C for 1 h. Slides were deparaffinized with xylene, rehydrated by decreasing ethanol concentration washes, and then processed for antigen retrieval. A two-step antigen retrieval method was developed specifically for multiplexing with FFPE tissues, which allowed for the use of antibodies with different antigen retrieval conditions to be used together on the same samples. Samples were then incubated in PBS with 0.3% Triton X-100 for 10 min at ambient temperature before blocking against nonspecific binding with 10% (wt/vol) donkey serum and 3% (wt/vol) bovine serum albumin (BSA) in 1× PBS for 45 min at room temperature. Primary antibody c-Fos (Santa Cruz-SC52; sc-166940) was diluted to optimized concentration (5 μg/mL) and applied for 1 h at room temperature in PBS/3% (vol/vol) BSA. Samples were then washed sequentially in PBS, PBS-TritonX-100, and then PBS again for 10 min, each with agitation. In the case of secondary antibody detection, samples were incubated with primary antibody species-specific secondary Donkey IgG conjugated to either Cy3 or Cy5. Slides were then washed as above and stained in DAPI (10 μg/mL) for 5 min, rinsed again in PBS, and then mounted with antifade media for image acquisition. Whole-tissue images were acquired on fluorescence microscope (Olympus IX81) at ×10 magnification. Autofluorescence, which is typical of FFPE tissues, needs to be properly characterized and separated from target fluorophore signals. We used autofluorescence removal processes, wherein an image of the unstained sample is acquired in addition to the stained image. The unstained and stained images are normalized with respect to their exposure times and the dark pixel value (pixel intensity value at zero exposure time). Each normalized autofluorescence image is then subtracted from the corresponding normalized stained image. We ensured that the same region in the stimulated and control samples were imaged.

Histological assessment of stimulated tissue

Spleen from stimulated rats and control rats were processed into paraffin blocks as described above. Paraffin-embedded sections were cleared and stained for H&E following standard protocol reported in the literature and scanned on a bright field scanner (Olympus). H&E images were qualitatively assessed for morphological difference and no significant difference was noticed between stimulated and control samples (Supplementary Figure 7).

Alternate pulse repetition periods and pulse amplitude

Data for blood glucose and/or splenic NE, ACH, and TNF concentrations in ultrasound-stimulated animals (with (Supplementary Figures 8–10) or without (Supplementary Figure 7) LPS injection) using alternative ultrasound-stimulation parameters were compared to those presented in the main text. We evaluated the effect of ultrasound neuromodulation in naive/non-LPS treated animals (Supplementary Figure 7) and animals treated with alternative pulse repetition periods and pulse lengths (summary of results in Supplementary Figures 8–10).

Electrode-based vagal nerve stimulation (VNS) protocol

Male Sprague–Dawley rats were anesthetized with 2–4% isoflurane. A single incision was made along the neck exposing the cervical portion of the trapezius, sternocleidomastoid and masseter muscles for blunt dissection exposing the left cervical vagus nerve. The microelectrode was placed along the main trunk of the exposed cervical vagus nerve. Electrical stimulation using three settings (5 V, 30 Hz, 2 ms; 5 V, 5 Hz, 2 ms; and 1 V, 5 Hz, 2 ms) was generated using a BIOPAC MP150 module under the control of the AcqKnowledge software (Biopac Systems). Rats underwent 3 min of VNS before and after IP injection of 10-mg/kg LPS. Following injection of 10 mg/kg LPS, a saline-soaked pad was used to hydrate the area and the cervical region was sutured closed to maintain integrity of the physiologic site. Rats were euthanized 60 min after LPS injection as described above, and spleen and blood samples were obtained for TNF determination as described above. In rats subjected to sham surgery, the vagus nerve was exposed, but not touched or manipulated.

Heart rate monitoring and analysis

Heart rate (during either ultrasound or electrode stimulation experiments) was monitored using a commercial infrared oximeter and physiological monitoring system (Starr Lifesciences) using the manufacturer’s instructions. During the stimulation protocols, the foot clip sensor (provided by the manufacturer) was placed on the footpad of the animal. The animal was allowed to acclimate for at least 5 min prior to measurement, a time point found sufficient for animals to recover to normal heart rate activities and physiological reading in controls. Measurement was recorded before (2-min recording periods), during, and after (2-min recording periods) the stimulation with either the electrical microelectrode or ultrasound probe.

cFos analysis and other measures of US-induced activation

LPS-U/S stimulated and sham animals were rapidly euthanized, and brains removed and transferred to 10% paraformaldehyde for 24 h, after which they were transferred to a 30% sucrose solution and stored for 4 °C prior to paraffin embedding (detailed in the IHC section above). Coronal section (5–10 µm) were cut by cryostat. Structures were anatomically defined according to an anatomical atlas. Quantification of c-Fos positive cells was counted with a fixed sample window across at least four sections by an experimenter blinded to the treatment conditions associated with each distinct coronal section. Regions of interest were as follows: paraventricular hypothalamic nucleus, ARC, VMN, DMN, LH, and mammillothalamic tract (all structures visible in coronal slices taken between Bregma −2.56 to −3.60 mm). The number of c-Fos positive cells in each group were expressed as a % of cFos+ cells as compared to Sham-stimulated control littermates.

In addition to the hypothalamic specific cFos-staining analysis performed in the main Bregma −11.3 to −14.08 mm, text/figures, the team sectioned and analyzed ultrasound induced activation within the NTS. The NTS is a brainstem nucleus known to harbor purely sensory nuclei (including fibers from the vagus nerve), and project to areas of the brain involved with autonomic regulation (including the hypothalamus; see Supplementary Figure 11 for a description of these neural pathways). The increased staining of the activation specific marker in the ultrasound stimulated animals (Fig. 7) agrees with the other findings (i.e., hypothalamic neurotransmitter and protein data, and MRI data) and suggests ultrasound induction of a nerve-mediated modulation of hypothalamic metabolic control centers. This additional supplemental data (Supplementary Figure 12) suggests that this ultrasound effect is at least in part mediated through direct sensory pathways.

Diffusion functional MRI

Neuronal activation is typically detected using blood-oxygenation-level-dependent (BOLD) fMRI54; brain regions with increased metabolic demand lead to higher cerebral blood flow, an increased supply of oxygenated blood, and decreased gradient echo signal. Sensitivity to the BOLD effect requires the use of fast gradient echo acquisitions; this causes undesired signal loss in brain areas next to air pockets, such as sinuses and ear canals, and hinders detection of neuronal activation near those specific brain areas. Alternatively, to minimize signal loss in areas characterized by large field inhomogeneities, spin echo (or double spin echo) diffusion-weighted imaging (DWI) can be used for detecting neuronal activation44,45,46 or nerve activation driven by external neuromodulators47. In DWI-fMRI, a volume increase in the slow-diffusing, presumably intracellular, water pool or an increase in water diffusion (or ADC) are assigned physical changes caused by neuronal activation.

Ten Sprague–Dawley rats were anesthetized using 3% Isoflurane and placed supine, with their heads inserted in a birdcage coil. The abdomen region was coupled through a gel/water filled cone to an MR-compatible U/S probe (f = 1.47 MHz), focusing on the porta hepatis, a liver region known to contain glucose sensitive neurons. Supplementary Figure 13a depicts a schematic of the experimental setup, including the US probe connected to RF amplifier/signal generator.

Data were acquired on a 3T scanner (MR750, GE Healthcare). An SPGR T1 acquisition was followed by six blocks of DWI images, with a TE/TR of 82/3400 ms, using 3/4 averages for the b = 0/b = 1000 s/mm2 and 0.6/1-mm in-plane/out-of-plane spatial resolution. An additional reverse polarity DWI acquisition was acquired for distortion correction purposes55. Following the LPS injection, the first US treatment, a wait time and the second US treatment, another 6 blocks of DWI images were acquired. Each ultrasound treatment lasted 60 s, during which square wave pulses were applied at 150/350-μs on/off periods. The sound pressure at the focal point was approximately 3.2 MPa. Supplementary Figure 13b depicts a summary of the experimental timing. This protocol was applied to 6 rats; for the remaining 4, the last DWI blocks immediately followed the LPS injection, with no US treatment.

A cross-correlation coefficient (ccc) between the T1 images and the (distortion-corrected) b = 0 DWI images of at least 0.5 was used to identify slices to be used for further analysis. ADCs were calculated for the pre- and post-treatment images; pre- and post-treatment image data were pooled together for statistical analysis. A rigid registration between the T1 images and a rat atlas was used to determine regions in which pixel-by-pixel t-tests indicated significant changes. The registration transformation from the T1 and atlas images was applied to the distortion-corrected DWI and ADC images.

Supplementary Figure 14 shows an example of the T1/b = 0 DWI acquisition in one rat after distortion correction; only red-highlighted slices met ccc > 0.5 and were kept for statistical analysis. To follow the effects of LPS and US treatment on blood glucose level, blood samples were obtained from the tail vein immediately before the first MRI scan and at 30 min after LPS injection; blood glucose concentration was measured by a OneTouch Elite glucometer (LifeScan; Johnson & Johnson).

Figure 7b shows an example overlay between the activation maps/SPGR volume (left) and the atlas/SPGR volume (right). Note the ADC change in both PVNs of the hypothalamus (red arrows, left image), consistent with ultrasound-induced neuromodulation. Figure 7d summarizes the results in a bar graph and Supplementary Table 2 details the explicit results in all 10 animals. Three of six rats showed significant neuromodulation in the PVNs; none of the control animals showed such change in neural activity. Furthermore, the hyperglycemia observed in the non-US-treated animals was not observed in the US-treated animals.