Ultrasound neuromodulation techniques

Abstract

The presently disclosed subject matter relates generally to techniques for neuromodulation of tissue, which includes applying energy (e.g., ultrasound energy) into the tissue to cause altered activity at synapses between neuronal and non-neuronal cells. In one implementation, the energy is applied to cause a persistent effect resulting from repeated application of the energy within a predetermined therapeutic window.

Description

Technical Field

[0001] The topics disclosed in this article relate to neural modulation, and more specifically to techniques for modulating physiological responses using energy applied from an energy source. Background Technology

[0002] Neuromodulation has been used to treat a variety of clinical conditions. For example, electrical stimulation along various sites of the spinal cord has been used to treat chronic back pain. Such treatments can be performed using implantable devices that periodically generate electrical energy, which is applied to tissue to activate certain nerve fibers, potentially leading to a reduction in pain. In the case of spinal cord stimulation, the stimulating electrodes are typically located in the epidural space, although the pulse generator may be positioned slightly away from the electrodes, such as in the abdominal or gluteal region, but connected to the electrodes via wires. In other implementations, deep brain stimulation can be used to stimulate specific areas of the brain to treat motor disorders, and the stimulation location can be guided by neuroimaging. This type of central nervous system stimulation typically targets local nerve or brain cell function and is mediated by electrodes that deliver electrical impulses and are located at or near the target nerve. However, positioning the electrodes at or near the target nerve is challenging. For example, such techniques may involve surgically placing the energy-delivering electrodes. Furthermore, targeting specific tissues through neuromodulation is challenging. Electrodes located at or near specific target nerves mediate neural modulation by triggering action potentials in nerve fibers, leading to the release of neurotransmitters at the synapse and synaptic communication with the next nerve. This propagation can result in relatively larger or more diffuse physiological effects than expected because current implantable electrodes simultaneously stimulate many nerves or axons. Due to the complexity and interconnection of neural pathways, more targeted modulation may be more useful clinically. Summary of the Invention

[0003] The following outlines some embodiments corresponding to the scope of the original claimed subject matter. These embodiments are not intended to limit the scope of the claimed subject matter, but are merely intended to provide a brief overview of possible implementations. In fact, the invention can encompass a variety of forms that may be similar to or different from the embodiments set forth below.

[0004] In one embodiment, a modulation system is provided, comprising an energy application device configured to apply energy to a target region of a subject, the target region being a subregion of an organ containing synapses between neurons and corresponding non-neuronal cells; and a controller configured to: spatially select the target region; focus the energy on the target region; and adjustably control the repeated application of energy to the target region via the energy application device to induce repeated preferential activation of a subset of synapses located in the target region for a predetermined time period, to cause sustained changes in one or more target molecules after repeated energy application.

[0005] In another embodiment, a modulation system is provided, comprising an energy application device configured to apply energy to a target region of a subject, the target region being a subregion of an organ containing synapses between neurons and corresponding non-neuronal cells; and a controller configured to: spatially select the target region; focus the energy on the target region; and repeatedly control the application of energy via the energy application device to the target region to induce preferential activation of a subset of the synapses located within the target region for a predetermined time period, to induce sustained changes in one or more target molecules after repeated energy application.

[0006] In another embodiment, a system for treating diabetes in a subject is provided, comprising an ultrasound energy application device configured to apply an ultrasound dose regimen to an internal organ; and a controller adapted to control the ultrasound energy application device to apply the ultrasound dose regimen, wherein the ultrasound dose regimen comprises multiple energy doses applied at separate time points within a time window of the ultrasound dose regimen.

[0007] In another embodiment, a modulation system is provided, comprising an energy application device configured to apply energy to a target region of a subject, the target region being a subregion of an organ containing synapses between neurons and corresponding non-neuronal cells; and a controller configured to: spatially select the target region; focus the energy on the target region; and repeatedly control the application of energy to the target region via the energy application device to apply a low duty-cycle (VCC) energy dosing scheme to the target region, wherein the low duty-cycle dosing scheme comprises a plurality of electrical stimulations spaced apart by an adjustable disconnection period of at least 4 hours, wherein the disconnection period is determined at least in part by feedback received by the controller.

[0008] In another embodiment, a method of treating a subject suffering from a metabolic disorder is provided, comprising applying an ultrasound dosing protocol to the visceral organs of the subject suffering from the metabolic disorder to treat the metabolic disorder, wherein the ultrasound dosing protocol comprises multiple ultrasound energy doses applied at individual time points. Attached Figure Description

[0009] These and other features, aspects, and advantages of the invention will be better understood when the following detailed description is read with reference to the accompanying drawings, in which the same characters represent the same parts throughout the drawings, wherein:

[0010] Figure 1 This is a schematic diagram of a neuromodulation system using a pulse generator according to an embodiment of the present disclosure;

[0011] Figure 2 This is a block diagram of a neural modulation system according to an embodiment of the present disclosure;

[0012] Figure 3 This is a schematic diagram of an ultrasonic energy application device in operation according to an embodiment of the present disclosure;

[0013] Figure 4A This is an ultrasound visualization of the spleen according to an embodiment of the present disclosure, which can be used as spatial information to focus on a target region in the spleen.

[0014] Figure 4B This is an ultrasound visualization of the liver according to an embodiment of the present disclosure, which can be used as spatial information to focus on a target area in the liver;

[0015] Figure 5 This is a flowchart of a neuromodulation technique according to an embodiment of the present disclosure;

[0016] Figure 6 This is a schematic diagram of an energy application device configured as an external device and including an ultrasonic transducer;

[0017] Figure 7 This is a schematic diagram of an energy application device and a pulse generator configured to apply high-intensity focused ultrasound;

[0018] Figure 8 It is possible to be with Figure 7 An example of an energy application device used in conjunction with a system;

[0019] Figure 9 This is a schematic diagram of the experimental setup for applying ultrasound energy to achieve the target physiological outcome.

[0020] Figure 10 It is the experimental timeline of the application of ultrasonic energy;

[0021] Figure 11 The pulse characteristics of the applied ultrasonic energy pulse are shown;

[0022] Figure 12 The hydrophone measurement settings are shown;

[0023] Figure 13 An example of an ultrasonic pressure field in the xy plane is shown;

[0024] Figure 14 The experimental workflow for LPS injection is shown to generate models of inflammation and / or hyperglycemia / hyperinsulinemia and ultrasound therapy.

[0025] Figure 15A This is a schematic diagram of wide vagus nerve stimulation;

[0026] Figure 15B This is a schematic diagram based on the peripheral nerve modulation of target organs;

[0027] Figure 16A shows the experimental timeline of applying ultrasonic energy to the spleen of rats;

[0028] Figure 16B shows the levels of norepinephrine, acetylcholine, and TNF-α in the spleen of rats when different ultrasound energy levels were applied. The applied ultrasound energy levels are shown as ultrasound pressure in MPa.

[0029] Figure 16C shows the circulating concentration of TNF-α under the same conditions as in Figure 16B;

[0030] Figure 16D shows the spleen IL-1α concentration under the same conditions as in Figure 16B;

[0031] Figure 16E shows the response time of induced changes in spleen TNFα concentration relative to the control;

[0032] Figure 16F shows a 2D ultrasound image of a rat spleen used for focused ultrasound stimulation to spatially select a spleen target.

[0033] Figure 16G shows a timeline of a study designed to measure the duration of stimulation-induced activation of the cholinergic anti-inflammatory pathway;

[0034] Figure 16H shows the concentration of TNF-α in the spleen after protective ultrasound therapy;

[0035] Figure 16I The concentrations of activated / phosphorylated kinases induced by splenic ultrasound modulation were shown;

[0036] Figure 16J Exemplary ultrasound burst duration and the effects of using alternative ultrasound stimulation parameters on the concentrations of norepinephrine (NE), acetylcholine (ACh), and tissue necrosis factor α (TNF-α) in ultrasound-stimulated spleen (after LPS injection) are shown.

[0037] Figure 16K The effects of exemplary ultrasound carrier frequencies and alternative ultrasound stimulation parameters on the concentrations of norepinephrine (NE), acetylcholine (ACh), and tissue necrosis factor α (TNF-α) in ultrasound-stimulated spleen (after LPS injection) are shown.

[0038] Figure 17A The study demonstrated the effect of splenic ultrasound modulation on splenic TNF-α compared to standard electrode or implant-based vagus nerve stimulation (VNS), and identified multiple inhibitors.

[0039] Figure 17B The effect of α-Bungarus venom on spleen concentrations of (left) norepinephrine (NE) and (right) TNF-α was shown in rodents treated with ultrasound stimulation of LPS with and without BTX or surgical vagotomy.

[0040] Figure 17C Data comparing the effects of VNS (at various stimulation intensities and frequencies) and splenic ultrasound stimulation (at 0.83 MPa) on heart rate are presented;

[0041] Figure 17D The data shown confirmed the previously observed side effect of VNS in reducing LPS-induced hyperglycemia, and this side effect was not present when using spleen ultrasound stimulation.

[0042] Figure 18A These are 2D ultrasound images of rat livers used for focused ultrasound stimulation.

[0043] Figure 18B The effect of liver ultrasound stimulation on LPS-induced hyperglycemia was shown;

[0044] Figure 18CThe study showed measurements of the relative concentrations (compared to no ultrasound stimulation) of various molecules associated with insulin sensitivity and insulin-mediated and non-insulin-dependent glucose uptake in the liver, as well as changes in hypothalamic markers related to metabolic function.

[0045] Figure 18D The image shows an immunohistochemical image of cFOS (left), and data showing the number of activated neurons in LPS control and ultrasound-stimulated samples (right).

[0046] Figure 18E Other immunohistochemical images are shown, which illustrate cFOS expression in the brainstem in LPS control (top) and ultrasound-stimulated sample (bottom);

[0047] Figure 18F An example MRI overlay is shown between an activation map (exceeding the SPGR volume; left) and a brain atlas (exceeding the SPBR volume; right);

[0048] Figure 18G The graph shows the increase of ADC in the left and right paraventricular nuclei (PVN) of the hypothalamus;

[0049] Figure 19 The study showed circulating glucose levels in diabetic rats after liver stimulation.

[0050] Figure 20 The study showed circulating triglycerides in diabetic rats after liver stimulation.

[0051] Figure 21 The study showed circulating glucagon levels in diabetic rats after liver stimulation.

[0052] Figure 22 The study showed circulating insulin levels in diabetic rats after liver stimulation.

[0053] Figure 23 The study showed circulating leptin levels in diabetic rats after liver stimulation.

[0054] Figure 24 The study showed circulating norepinephrine levels in diabetic rats following liver stimulation.

[0055] Figure 25 The study showed that hypothalamic insulin receptor substrate 1 (IRS-1) was stimulated by liver in diabetic rats.

[0056] Figure 26 The study showed hypothalamic phosphorylation of Akt in diabetic rats following liver stimulation.

[0057] Figure 27 The hypothalamus GLUT4 was shown after liver stimulation in diabetic rats;

[0058] Figure 28 The study showed hypothalamic norepinephrine levels after liver stimulation in diabetic rats.

[0059] Figure 29 The hypothalamus showed glucose-6-phosphate after liver stimulation in diabetic rats;

[0060] Figure 30 The study showed that the hypothalamic glucagon-like peptide-1 (GLP-1) in diabetic rats was stimulated by liver activity.

[0061] Figure 31 The study showed that the hypothalamus contained γ-aminobutyric acid (GABA) after liver stimulation in diabetic rats.

[0062] Figure 32 The study showed that brain-derived neurotrophic factor (BDNF) in the hypothalamus was stimulated after liver stimulation in diabetic rats.

[0063] Figure 33 The study showed hypothalamic neuropeptide Y (NPY) in diabetic rats after liver stimulation.

[0064] Figure 34 The liver IRS-1 in diabetic rats after liver stimulation was shown;

[0065] Figure 35 This shows liver phosphorylation of Akt in diabetic rats after liver stimulation;

[0066] Figure 36 The study showed hepatic glucose transporter 2 (GLUT2) after liver stimulation in diabetic rats.

[0067] Figure 37 This shows hepatic norepinephrine levels after liver stimulation in diabetic rats;

[0068] Figure 38 This shows liver glucose-6-phosphate after liver stimulation in diabetic rats;

[0069] Figure 39 The liver GLP-1 level was shown after liver stimulation in diabetic rats;

[0070] Figure 40 The study showed glucagon levels in the pancreas of diabetic rats after liver stimulation.

[0071] Figure 41 This shows pancreatic insulin in diabetic rats after liver stimulation;

[0072] Figure 42 The study showed pancreatic leptin levels in diabetic rats after liver stimulation.

[0073] Figure 43The pancreatic IRS-1 in diabetic rats after liver stimulation was shown;

[0074] Figure 44 The pancreas GLUT2 in diabetic rats after liver stimulation was shown;

[0075] Figure 45 This shows pancreatic phosphorylation of Akt after liver stimulation in diabetic rats; and

[0076] Figure 46 The sustained effect of treatment was demonstrated in the Zucker rat model. Detailed Implementation

[0077] One or more specific embodiments will be described below. To provide a concise description of these embodiments, not all features of the actual embodiments are described in the specification. It should be understood that in the development of any such actual embodiment (as in any engineering or design project), many implementation-specific decisions must be made to achieve the developer's specific goals, such as complying with system-related and business-related constraints, which may vary from embodiment to embodiment. Furthermore, it should be understood that such development work may be complex and time-consuming, but it will still be routine work for design, manufacture, and production for those skilled in the art who benefit from this disclosure.

[0078] Any examples or descriptions given herein shall not be construed in any way as a constraint, limitation, or explicit limitation thereof on the use of any one or more terms. Rather, these examples or descriptions should be considered as descriptions of various specific implementations and are for illustrative purposes only. Those skilled in the art will understand that any one or more terms used with these examples or descriptions will cover other implementations that may or may not be given with them or elsewhere in the specification, and all such implementations are intended to be included within the scope of such one or more terms. The language used to designate such non-limiting examples and descriptions includes, but is not limited to, “for example,” “for instance,” “such as,” “e.g.,” “including,” and “in one implementation.”

[0079] This paper provides neuromodulation techniques based on direct and focused stimulation of a target region. The target region can be any tissue or structure in the body with axonal terminals of various types that form synapses with non-neuronal cells or fluid. In one example, the target region can be in an organ or structure, such as liver, pancreas, or gastrointestinal tissue. Neuromodulation of the target region allows for the limited and non-ablative application of energy to the target region without applying energy outside the target region. Energy application may trigger effects outside the target region, for example, in organs containing the target region and in other organs and structures that do not contain the target region. However, these effects can be achieved outside the target region without applying energy directly to areas outside the target region. Therefore, systemic effects can be achieved through localized energy application. As provided herein, systemic effects can also be achieved through intermittent and discontinuous energy application. Furthermore, the effects can be achieved hours and days after energy application.

[0080] In some implementations, neuromodulation, as described herein, can be used to treat chronic conditions to alter their progression, and in other implementations, to reverse the effects of chronic conditions. In one implementation, a patient diagnosed with a disease may require neuromodulation therapy. After treatment, the patient may have reached clinical benchmarks associated with healthy patients. For example, a diabetic patient's blood glucose and / or insulin levels may be outside the normal range. After treatment, the patient's blood glucose and / or insulin levels may return to the normal range. In another example, a patient with an abnormal immune response may regain characteristics of a normal immune response after treatment, including altered immune cell populations and / or altered lymphatic drainage.

[0081] Neuromodulation of a target region can produce therapeutic results that last beyond the duration of treatment. Neuromodulation can alter a patient's disease state to achieve durable results. For example, treatment involving repeated application of energy to a target region within a defined time period can produce sustained improvement in disease symptoms. In one embodiment, the improvement is relative to an untreated patient or a patient receiving conventional therapy. The predetermined time period can be a window of several hours or days during which treatment is performed. Furthermore, the treatment may include one or more individual energy application events within the predetermined time period.

[0082] This document also provides techniques for treating glucose metabolism and related conditions and for altering disease progression. In one implementation, hepatic modulation in one or more target regions can be used to treat diabetes (i.e., type 1 or type 2 diabetes), hyperglycemia, sepsis, trauma, infection, diabetes-related dementia, obesity, or other dietary or metabolic abnormalities. In one example, neural modulation can be used to promote weight loss, control appetite, treat cachexia, or increase appetite. For example, direct pancreatic stimulation can lead to increased appetite, while direct liver stimulation can lead to decreased NPY, thereby promoting satiety signals. The neural modulation provided herein can alter the glucose regulation setpoint relative to the pre-treatment state to achieve a durable therapeutic effect over days, weeks, and / or months after treatment. In one example, neural modulation in a diabetic patient can cause an initial decrease in circulating glucose relative to baseline (before neural modulation) during the treatment window (e.g., hours or days). However, after treatment, although circulating glucose may increase over the post-treatment period, this increase can stabilize at a new setpoint significantly lower than the pre-treatment setpoint. This new setpoint can be at a level relevant to clinical benefit.

[0083] As described herein, the neuromodulation therapy presented herein may involve repeatedly and individually applying energy to the same target area within a predetermined treatment period. For example, neuromodulation may be performed once daily on the target area (e.g., the porta hepatis), and this once-daily treatment may be performed according to preset modulation parameters, such as for two or more consecutive days.

[0084] This technology relates to modulating synapses at axonal terminals in tissue by applying energy from an energy source. These may include, for example, extra-axonal synapses formed between presynaptic axonal terminals and postsynaptic non-neuronal cells. Furthermore, although some disclosed embodiments are discussed in the context of extra-axonal synapses, it should be understood that axonal terminals may form axonal secretions, axonal synapses, axonosomes, or extra-axonal synapses, and additionally or alternatively, as provided herein, these synapse types are intended to be selectively modulated. Additionally, some axonal terminals may terminate in interstitial fluid or body fluids, and they may also undergo neurotransmitter release due to modulation. The disclosed synapses can be modulated to alter activity within the synapse, such as the release of neurotransmitters from presynaptic axonal terminals. In turn, the altered activity can lead to local and / or non-local (e.g., systemic) effects. This technology allows energy to be focused in a targeted manner onto a tissue volume including certain axonal terminals to preferentially and directly activate the targeted axonal terminals, thereby achieving the desired results. In this way, targeted axon terminals within the target region are activated, whereas in some implementations, axon terminals in the same organ or tissue structure but outside the target region are not activated. Because organs and tissue structures may include different types of axon terminals that form synapses with different types of postsynaptic non-neuronal cells, a target region including axon terminals can be selected, and when said axon terminals are activated, the desired target physiological outcome can be produced. Therefore, this modulation can target specific types of axon terminals based on presynaptic neuron type, postsynaptic cell type, or both.

[0085] For example, in one implementation, the type of axon terminal can be an axon terminal that forms an extra-axonal synapse with resident (i.e., tissue-resident or non-circulating) liver, pancreas, or gastrointestinal tissue cells. That is, extra-axonal synapses form at the junction between the axon terminal and a non-neuronal cell or interstitial fluid or body fluid. Therefore, the application of energy leads to the modulation of metabolic function in the target region. However, it should be understood that different target physiological effects can be achieved based on the population of axon terminal types and the characteristics of the presynaptic neuron type and postsynaptic cells (e.g., immune cells, lymphocytes, mucosal cells, muscle cells, etc.) of the extra-axonal synapses. Therefore, applying energy to a target region in a subject's tissue can activate axon terminals and their associated extra-axonal synapses within the target region, while untargeted axon terminals (and associated synapses) outside the target region may remain unaffected. However, since modulation can lead to systemic effects, untargeted axon terminals outside the target region may experience certain systemic changes due to the activation of axon terminals within the target region. As described herein, preferential activation or direct activation can refer to cells or structures within a target region that experience direct energy application. That is, axonal terminals, extracellular synapses of axons, and / or postsynaptic non-neuronal cells or interstitial fluid or body fluids directly experience the energy applied as described herein.

[0086] The human nervous system is a complex network of nerve cells or neurons located centrally in the brain and spinal cord, and peripherally surrounding various nerves throughout the body. Neurons have cell bodies, dendrites, and axons. A nerve is a group of neurons serving a specific part of the body. A nerve may contain hundreds to hundreds of thousands of neurons. Nerves typically contain both afferent and efferent neurons. Afferent neurons transmit signals to the central nervous system, while efferent neurons transmit signals to the surrounding area. A group of neuronal cell bodies at a location is called a ganglion. Electrical signals generated in a nerve (e.g., through stimulation, which can be intrinsic or externally applied) are transmitted through neurons and nerve conduction. Neurons release neurotransmitters at synapses (connections) near receiving cells to allow for the continuity and modulation of electrical signals. Peripherally, synaptic transmission often occurs in ganglia.

[0087] The electrical signal of a neuron is called an action potential. An action potential is initiated when the voltage potential across the cell membrane exceeds a certain threshold. This action potential then propagates along the length of the neuron. A neuronal action potential is complex and represents the sum of the action potentials of individual neurons within it. The connection between the axonal terminus of a neuron and the receiving cell is called a synapse. The action potential travels along the axon of the neuron to its axonal terminus, which is the presynaptic terminal or the distal end of the axonal branch that forms the nerve fiber. The electrical impulse of the action potential triggers vesicles containing neurotransmitters to migrate to the presynaptic membrane at the presynaptic axonal terminus and ultimately release the neurotransmitter into the synaptic cleft (e.g., the space between the presynaptic and postsynaptic cells) or the extracellular space of the axon. A synapse that reaches the synaptic terminus to convert the electrical signal of the action potential into a chemical signal for neurotransmitter release is a chemical synapse. Chemical synapses can be contrasted with electrical synapses, in which the ionic current flowing into the presynaptic axonal terminus can cross the barriers of both cell membranes and enter the postsynaptic cell.

[0088] The physiological effects of action potentials are mediated by the movement of ions across the cell membrane. Neurons actively maintain the resting membrane potential via ion pumps, which promote the movement of ions such as Na+. + K + and Cl - Plasma moves across the neuronal membrane. Different types of neurons can maintain different resting potentials, such as -75mV to -55mV. Action potentials are generated by the influx of ions, i.e., the movement of charge to create a large deviation in the membrane potential, which is associated with a temporary increase in transmembrane voltage, such as an increase in membrane potential to 30-60mV. Action potentials in individual neurons can be initiated in response to the release of neurotransmitters from presynaptic (e.g., upstream) neurons, which in turn leads to receptor binding on the postsynaptic cell and an event cascade, resulting in ion influx and membrane depolarization, thereby generating action potentials that propagate through the nerve.

[0089] Synapses may be located at the junction between two neurons, allowing action potentials to propagate along nerve fibers. However, axonal terminals may also form synapses at the junction between neurons and non-neuronal cells, or they may terminate in interstitial fluid or body fluids. Examples of synapse types include synapses with immune cells at the neuroimmune junction, synapses with resident sensory cells within organs, or synapses with glandular cells. The release of neurotransmitters into the synaptic cleft and their binding to receptors in the postsynaptic membrane of the postsynaptic cell leads to downstream effects, which depend on the nature of the presynaptic neuron and the specific neurotransmitter released, as well as the nature of the postsynaptic cell, such as the type of receptors available on the postsynaptic cell. Furthermore, action potentials can be excitatory or inhibitory. Excitatory postsynaptic action potentials are those that make the postsynaptic neuron more likely to generate or release subsequent action potentials, while inhibitory postsynaptic action potentials are those that make the postsynaptic neuron less likely to generate or release subsequent action potentials. In addition, multiple neurons can work together to release neurotransmitters in a coordinated manner, thereby triggering or inhibiting downstream action potentials.

[0090] Neuromodulation is a technique that applies energy from an external energy source to certain areas of the nervous system to activate or enhance nerves or nerve function and / or block or deactivate nerves or nerve function. In some neuromodulation techniques, one or more electrodes are applied at or near a target nerve, and the applied energy (e.g., as an action potential) is carried through the nerve to induce a physiological response in a region downstream of the energy application site. However, due to the complexity of the nervous system, it is difficult to predict the extent and ultimate endpoint of the physiological response at a given energy application site.

[0091] While strategies for ultrasound modulation of the central nervous system (i.e., brain tissue) have proven successful in modulating neural activity, attempts to modulate the peripheral nervous system have lagged behind. For example, ultrasound modulation of the central nervous system (CNS) involves stimulating regions of the cerebral cortex rich in synaptic structures, while attempts to stimulate the peripheral nervous system with ultrasound target neural trunks with fewer or no synaptic structures.

[0092] In this technique, peripheral nerve modulation involves targeting one or more peripheral axon terminals to influence blood glucose levels and / or glucose regulation pathways and / or insulin production pathways. In this technique, repetitive energy pulses are applied to the internal tissue of a subject containing axon terminals, which include extracellular synapses of the axon or neuronal connections to other cell types, interstitial fluid, or body fluids, such as at synapses between neuronal and non-neuronal cells. Applying energy to the synapse leads to activation of the presynaptic axon terminal and / or postsynaptic cells, resulting in the target physiological outcome. In one example, stimulation of the axon terminal releases neurotransmitters / neuropeptides, or induces altered neurotransmitter release near adjacent non-neuronal cells (e.g., secretory cells or other cells), and modulates cellular activity. Furthermore, this modulation allows for the regulation of other tissue structures or organs without direct stimulation. In one embodiment, directly applying energy to a relatively small area of ​​an organ (e.g., less than 25% of the total organ volume) may result in stimulating action potentials in afferent projecting neurons projecting to different regions of the brain (e.g., the hypothalamus). However, this result can be achieved without direct brain stimulation of synaptic-rich regions. Direct brain stimulation can lead to unintended activation of other pathways, potentially interfering with or overriding the desired physiological outcome. Furthermore, direct brain stimulation may involve invasive procedures. Therefore, this technique allows for granular activation of brain or intra-organ activity in a more targeted and specific manner compared to direct brain stimulation or electrical peripheral nerve stimulation.

[0093] The benefits of this technique include localized modulation at target areas of tissue to alter the concentration of one or more target molecules. Furthermore, this localized modulation may involve directly activating a relatively small area of ​​tissue (e.g., less than 25% of the total tissue volume) to achieve these effects. In this way, the total applied energy is relatively small to achieve the desired physiological outcome. In some embodiments, the applied energy may come from a non-invasive external energy source (e.g., an ultrasound energy source, a mechanical vibrator). For example, a focused energy probe can apply energy through the subject's skin and focus it on a target area of ​​internal tissue. Such embodiments achieve the desired physiological outcome without invasive procedures or the side effects that might be associated with other types of procedures or therapies.

[0094] This article provides a technique for neuromodulation in which energy from an energy source (e.g., an external or in vitro energy source) is applied to the axon terminal in such a manner that neurotransmitter release at the focal point of energy application (e.g., the axon terminal) is triggered in response to the energy application rather than in response to an action potential. That is, energy is applied directly to the axon terminal instead of an action potential to promote neurotransmitter release to the neuronal junction (i.e., the synapse) with a non-neuronal cell. Directly applying energy to the axon terminal further induces the release of neurotransmitters from the intrasynaptic axon terminal (e.g., the extracellular synapse) to the vicinity of adjacent non-neuronal cells. In one embodiment, the energy source is an external energy source, such as an ultrasonic energy source or a mechanical vibrator. In this way, non-invasive and targeted neuromodulation can be achieved directly at the energy focal point, rather than through modulation at an upstream site, which in turn triggers an action potential that activates the downstream target.

[0095] In some implementations, the target tissue is an internal tissue or organ that is difficult to access using electrical stimulation techniques. Anticipated tissue targets include gastrointestinal (GI) tissue (stomach, intestine), muscle tissue (heart, smooth tissue, and bone), epithelial tissue (epidermis, organ / GI lining), connective tissue, glandular tissue (exocrine / endocrine), etc. In one example, focused application of energy at the neuromuscular junction can promote neurotransmitter release at the neuromuscular junction without upstream action potentials. Anticipated regulatory targets may include portions of the pancreas responsible for controlling insulin release or portions of the liver responsible for glucose regulation.

[0096] Neuromodulation of a target region can alter physiological processes, thereby disrupting, reducing, or enhancing one or more physiological pathways in a subject to produce the desired physiological outcome. Furthermore, because local energy application can lead to systemic changes, different physiological pathways can be altered in different ways and at different locations within the body to elicit a general profile of physiological changes characteristic of a subject and induced by targeted neuromodulation. While these changes are complex, this neuromodulation technique provides one or more measurable targeted physiological outcomes that are the result of neuromodulation in the treated subject and would not be available without energy application to the target region or other interventions. Additionally, while other types of interventions (e.g., pharmacological treatment) may produce a subset of physiological changes induced by neuromodulation, in some implementations, the profile of physiological changes induced by neuromodulation may be specific to the neuromodulation of the target region (and its associated regulatory parameters) and may vary from patient to patient.

[0097] The neuromodulation techniques discussed in this article can be used to induce physiological outcomes of changes in the concentration (e.g., increase, decrease) and / or properties of target molecules. That is, selective modulation of one or more target molecules (e.g., a first target molecule, a second target molecule, etc.) can refer to regulating or influencing the concentration (circulation, tissue) or properties (covalent modification) of molecules by applying energy to one or more target regions (e.g., a first target region, a second target region, etc.) in one or more tissues (e.g., a first tissue, a second tissue, etc.). Modulation of target molecules can include alterations in molecular properties (such as protein expression, secretion, transport) and direct changes in activity based on ion channel effects derived from the energy application itself or from molecules directly affecting ion channels. Modulation of target molecules can also refer to maintaining a desired concentration of molecules so that the expected concentration changes or fluctuations do not occur due to neural modulation. Modulation of target molecules can refer to inducing changes in molecular properties, such as enzyme-mediated covalent modifications (changes in phosphorylation, acetylation, riboylation, etc.). In other words, it should be understood that selective modulation of target molecules can refer to both molecular concentration and / or molecular properties. The target molecule can be a biomolecule, such as one or more of carbohydrates (monosaccharides, polysaccharides), lipids, nucleic acids (DNA, RNA), or proteins. In some embodiments, the target molecule can be a signaling molecule, such as a hormone (amine hormone, peptide hormone, or steroid hormone).

[0098] The disclosed neuromodulation technology can be used in conjunction with a neuromodulation system. Figure 1 This is a schematic diagram of system 10 for neuromodulation in response to energy application to achieve neurotransmitter release and / or activation of synaptic components (e.g., presynaptic and postsynaptic cells). The depicted system includes a pulse generator 14 coupled to an energy application device 12 (e.g., an ultrasonic transducer). The energy application device 12 is configured to receive energy pulses, for example via leads or a wireless connection, which are directed in use to a target area of ​​a subject's internal tissue or organ, thereby leading to a target physiological outcome. In some embodiments, the pulse generator 14 and / or the energy application device 12 may be implanted in a biocompatible site (e.g., the abdomen), and one or more leads internally couple the energy application device 12 and the pulse generator 14. For example, the energy application device 12 may be a MEMS transducer, such as a capacitive micromechanical ultrasonic transducer.

[0099] In some embodiments, the energy application device 12 and / or pulse generator 14 may be wirelessly connected, for example, to a controller 16, which in turn may provide instructions to the pulse generator 14. In other embodiments, the pulse generator 14 may be an external device, for example, operable to apply energy percutaneously or non-invasively from a location outside the subject's body, and in some embodiments, may be integrated within the controller 16. In embodiments where the pulse generator 14 is external, the energy application device 12 may be operated by a caregiver and positioned at a point on or above the subject's skin, such that energy pulses are delivered percutaneously to the desired internal tissue. Once positioned to apply energy pulses to the desired site, the system 10 may initiate neuromodulation to achieve the target physiological outcome or clinical effect.

[0100] In some embodiments, system 10 may include an evaluation device 20 coupled to controller 16 and evaluating characteristics indicating whether the target physiological outcome of the modulation has been achieved. In one embodiment, the target physiological outcome may be localized. For example, modulation may result in local tissue or functional changes, such as changes in tissue structure, localized changes in the concentration of certain molecules, tissue displacement, increased fluid movement, etc.

[0101] Modulation can lead to systemic or non-local changes, and the target physiological outcome may be related to changes in circulating molecular concentrations or tissue properties, excluding the target area to which energy is directly applied. In one example, the displacement may be a proxy measurement of the desired modulation, and a displacement measurement below the expected displacement value may lead to a modification of the modulation parameters until the expected displacement value is achieved. Therefore, in some embodiments, the assessment device 20 may be configured to assess concentration changes. In some embodiments, the assessment device 20 may be an imaging device configured to assess changes in organ size and / or location. Although the depicted elements of system 10 are shown separately, it should be understood that some or all elements may be combined with each other. Furthermore, some or all elements may communicate with each other in a wired or wireless manner.

[0102] Based on the assessment, the adjustment parameters of controller 16 can be changed. For example, if the desired adjustment is associated with a change in concentration (circulating concentration or tissue concentration of one or more molecules) within a defined time window (e.g., 5 minutes or 30 minutes after the start of the energy application program) or relative to the baseline at the start of the program, it may be desirable to change the adjustment parameters, such as the pulse frequency or other parameters, which can in turn be provided to controller 16 by the operator or via an automatic feedback loop to define or adjust the energy application parameters or adjustment parameters of pulse generator 14.

[0103] The system 10 provided herein can provide energy pulses according to various adjustment parameters. For example, adjustment parameters can include various stimulation time patterns ranging from continuous to intermittent. Through intermittent stimulation, energy is delivered at a certain frequency over a period of time during the signal-on time. A period of time without energy delivery follows the signal-on time, referred to as the signal-off time. Adjustment parameters can also include the frequency and duration of stimulation application. The application frequency can be continuous or delivered at various time intervals, such as within a day or week. The treatment duration can last for various time periods, including but not limited to minutes to hours. In some embodiments, a treatment duration with a specified stimulation pattern can last for one hour, repeated at intervals such as 72 hours. In some embodiments, a shorter duration (e.g., 30 minutes) of treatment can be performed at a higher frequency (e.g., every three hours). The application of energy can be controlled and adjusted according to adjustment parameters such as treatment duration and frequency to achieve the desired result.

[0104] Figure 2 This is a block diagram of some components of system 10. As provided herein, system 10 for neuromodulation may include a pulse generator 14 adapted to generate multiple energy pulses to be applied to the tissues of a subject. The pulse generator 14 may be standalone or integrated into an external device, such as a controller 16. The controller 16 includes a processor 30 for controlling the device. Software code or instructions are stored in memory 32 of the controller 16 for execution by the processor 30 to control the components of the device. The controller 16 and / or the pulse generator 14 may be connected to the energy application device 12 via one or more leads 33 or wirelessly.

[0105] The controller 16 also includes a user interface with input / output circuitry 34 and a display 36, adapted to allow clinicians to provide selection inputs or adjustment parameters to the adjustment program. Each adjustment program may include one or more sets of adjustment parameters, including pulse amplitude, pulse width, pulse frequency, etc. The pulse generator 14 modifies its internal parameters in response to control signals from the controller device 16 to alter the stimulation characteristics of the energy pulse transmitted to the subject via lead 33 to the energy application device 12. Any suitable type of pulse generation circuit may be employed, including but not limited to constant current, constant voltage, multiple independent current or voltage sources, etc. The applied energy is a function of the current amplitude and pulse width duration. The controller 16 allows for adjustable control of the energy by changing the adjustment parameters at specific times and / or initiating energy application or canceling / inhibiting energy application at specific times. In one embodiment, the adjustable control of the energy application device is based on information about the concentration of one or more molecules (e.g., circulating molecules) in the subject. If this information comes from the assessment device 20, a feedback loop can drive the adjustable control. For example, if the circulating glucose concentration measured by the evaluation device 20 is higher than a predetermined threshold or range, the controller 16 can initiate energy application to a target region (e.g., the liver) or initiate energy application with a regulating parameter that reduces circulating glucose. The initiation of energy application can be triggered by drifting to a glucose concentration above or outside a predetermined (e.g., desired) threshold. In another embodiment, the adjustable control can take the form of changing the regulating parameter when the initial energy application does not result in the expected change in the target physiological outcome (e.g., the concentration of the target molecule) within a predetermined time frame (e.g., 1 hour, 2 hours, 4 hours, 1 day).

[0106] In one embodiment, memory 32 stores different operating modes selectable by the operator. For example, the stored operating modes may include instructions for executing a set of adjustment parameters associated with a specific treatment site (e.g., a target area in the liver, pancreas, gastrointestinal tract, or spleen). Different sites may have different associated adjustment parameters. Instead of requiring the operator to manually input the mode, controller 16 can be configured to execute appropriate instructions based on the selection. In another embodiment, memory 32 stores operating modes for different types of treatments. For example, activation may be associated with a different range of stimulation pressure or frequency relative to the range of stimulation pressure or frequency that suppresses or blocks tissue function. In a specific example, when the energy application device is an ultrasound transducer, the time-averaged power (time-averaged intensity) and peak positive pressure are 1 mW / cm². 2 -30,000mW / cm 2 (Time-averaged intensity) and 0.1 MPa to 7 MPa (peak pressure). In one example, the time-averaged intensity is less than 35 W / cm² within the target area. 2This is to avoid levels associated with thermal damage and ablation / cavitation. In another specific example, when the energy application device is a mechanical actuator, the vibration amplitude is in the range of 0.1 to 10 mm. The selected frequency can depend on the mode of energy application, such as ultrasound or a mechanical actuator.

[0107] In another embodiment, memory 32 stores calibration or setting modes, which allow adjustment or modification of regulation parameters to achieve the desired results. In one example, the stimulus begins with a low energy parameter and is increased automatically or gradually upon receiving operator input. In this way, the operator can tune the induced effect as the regulation parameters are changed.

[0108] The system may also include an imaging device that helps focus the energy application device 12. In one embodiment, the imaging device may be integrated with or identical to the energy application device 12, allowing different ultrasound parameters (frequency, aperture, or energy) to select (e.g., spatially select) a target region and focus energy onto the selected target region for targeting and subsequent neuromodulation. In another embodiment, memory 32 stores one or more targeting or focusing modes for spatially selecting target regions within an organ or tissue structure. Spatial selection may include selecting sub-regions of an organ to identify the volume of the organ corresponding to the target region. Spatial selection may rely on image data provided herein. Based on spatial selection, the energy application device 12 may focus on the selected volume corresponding to the target region. For example, the energy application device 12 may be configured to initially operate in a targeting mode to apply targeting mode energy used to capture image data to be used for identifying the target region. The targeting mode energy does not reach and / or does not apply modulation parameters suitable for preferential activation. However, once the target region is identified, the controller 16 may operate in a treatment mode according to modulation parameters associated with preferential activation.

[0109] The controller 16 can also be configured to receive inputs related to the target physiological outcome as inputs for selecting adjustment parameters. For example, when the imaging mode is used to assess tissue properties, the controller 16 can be configured to receive a calculated index or parameter of said property. The adjustment parameter can be modified based on whether the index or parameter is higher or lower than a predetermined threshold. In one embodiment, the parameter can be a measure of tissue displacement of the affected tissue or a measure of the depth of the affected tissue. Other parameters may include assessing the concentration of one or more target molecules (e.g., assessing the change in concentration relative to a threshold or baseline / control, the rate of change, determining whether the concentration is within a desired range, or one or more of these). Additionally, the energy application device 12 (e.g., an ultrasound transducer) can operate under the control of the controller 16 to a) acquire image data of tissue that can be used to spatially select a target region within a target tissue, b) apply adjustment energy to said target region, and c) acquire images to determine that a target physiological outcome has occurred (e.g., by displacement measurement). In this embodiment, the imaging device, the assessment device 20, and the energy application device 12 can be the same device.

[0110] In another embodiment, the required set of regulation parameters can also be stored by the controller 16. In this way, subject-specific parameters can be determined. Furthermore, the effectiveness of such parameters can be evaluated over time. If a particular set of parameters is less effective over time, the subject may develop insensitivity to the activated pathway. If the system 10 includes an assessment device 20, the assessment device 20 can provide feedback to the controller 16. In some embodiments, feedback indicative of characteristics of the target physiological outcome can be received from the user or the assessment device 20. The controller 16 can be configured to cause the energy application device to apply energy according to the regulation parameters and to dynamically adjust the regulation parameters based on feedback. For example, based on feedback, the controller 16 can automatically change the regulation parameters (e.g., the frequency, amplitude, or pulse width of an ultrasound beam or mechanical vibration) in real time and in response to feedback from the assessment device 20.

[0111] In one example, this technology can be used to treat subjects with metabolic disorders. This technology can also be used to regulate blood glucose levels in subjects with glucose regulation disorders. Therefore, this technology can be used to promote the homeostasis of target molecules or to promote desired circulating concentrations or concentration ranges of one or more target molecules (e.g., glucose, insulin, glucagon, or combinations thereof). In one embodiment, this technology can be used to control circulating (i.e., blood) glucose levels. In one embodiment, the following thresholds can be used to maintain blood glucose levels in a dynamic equilibrium within a normal range:

[0112] fasting:

[0113] Below 50 mg / dL (2.8 mmol / L): Insulin shock

[0114] 50-70 mg / dL (2.8-3.9 mmol / L): Hypoglycemia / Hypoglycemia

[0115] 70-110 mg / dL (3.9-6.1 mmol / L): Normal

[0116] 110-125 mg / dL (6.1-6.9 mmol / L): Elevated / Impaired (Prediabetes)

[0117] 125 (7 mmol / L): Diabetes

[0118] Non-fasting (approximately 2 hours after a meal):

[0119] 70-140 mg / dL: Normal

[0120] 140-199 mg / dL (8-11 mmol / L): Elevated or "borderline" / prediabetes

[0121] Above 200 mg / dL (11 mmol / L): Diabetes

[0122] For example, the technology can be used to maintain circulating glucose concentrations below approximately 200 mg / dL and / or above approximately 70 mg / dL. The technology can also be used to maintain glucose in the range of approximately 4-8 mmol / L or approximately 70-150 mg / dL. The technology can be used to maintain a normal blood glucose range in a subject (e.g., a patient), where the normal blood glucose range can be an individualized range based on individual patient factors such as weight, age, and clinical history. Therefore, energy application to one or more target regions can be adjusted in real time based on the desired final concentration of the target molecule, and energy application to one or more target regions can be adjusted in a feedback loop based on input from the assessment device 20. For example, if the assessment device 20 is a circulating glucose monitor or a blood glucose monitor, real-time glucose measurements can be used as input to the controller 16.

[0123] In another embodiment, this technique can be used to profile physiological changes. For example, the profile may include a set of target molecules whose concentration increases in tissues and / or blood due to energy application, and another set of target molecules whose concentration decreases in tissues and / or blood due to energy application. The profile may also include a set of molecules that do not change with energy application. The profile may define concurrent changes associated with the desired physiological outcome. For example, the profile may include decreased circulating glucose and increased circulating insulin.

[0124] Figure 3This is a specific example in which the energy application device 12 includes an ultrasound transducer 42 capable of applying energy to a target tissue 43, which, as a non-limiting example, is shown as the liver. The energy application device 12 may include control circuitry for controlling the ultrasound transducer 42. The control circuitry of the processor 30 may be integrated with the energy application device 12 (e.g., via an integrated controller 16) or may be a separate component. The ultrasound transducer 42 may also be configured to acquire image data to assist in spatial selection of desired or target regions and to focus the applied energy onto target regions of target tissues or structures.

[0125] The desired target tissue 43 can be internal tissue or organ, including axonal terminals 46 and synapses of non-neuronal cells 48. Synapses can be stimulated by applying energy directly to the axonal terminals within the focusing region of an ultrasound transducer 42 focused on a target region 44 of the target tissue 43, causing molecules to be released into the synaptic space 49. In the described embodiment, the axonal terminals 46 form synapses with hepatocytes, and the release of neurotransmitters 47 and / or changes in ion channel activity induce downstream effects, such as activation of glucose metabolism. The target region can be selected to include a certain type of axonal terminal 46, such as axonal terminals 46 of a specific neuronal type and / or axonal terminals 46 that form synapses with a certain type of non-neuronal cell. Therefore, the target region 44 can be selected to correspond to a portion of the target tissue 43 having the desired axonal terminals 46 (and associated non-neuronal cells 48). Energy can be selectively applied to preferentially trigger the release of one or more molecules (e.g., neurotransmitters) from the synapse, or to directly activate the non-neuronal cell itself via direct energy transduction (i.e., mechanotransduction or voltage-activating proteins within non-neuronal cells), or to induce activation within both neuronal and non-neuronal cells, thereby generating the desired physiological effect. The target region can be selected as the site of nerve entry into the organ. In one embodiment, liver stimulation or modulation may refer to the modulation of the target region 44 at or near the porta hepatis.

[0126] Energy may be focused or substantially concentrated on target region 44 and concentrated only in a portion of the internal tissue 43, for example, less than about 50%, 25%, 10%, or 5% of the total volume of tissue 43. In one embodiment, energy may be applied to two or more target regions 44 within the target tissue 43, and the total volume of the two or more target regions 44 may be less than about 90%, 50%, 25%, 10%, or 5% of the total volume of tissue 43. In one embodiment, energy may be applied only to about 1%–50% of the total volume of tissue 43, only to about 1%–25% of the total volume of tissue 43, only to about 1%–10% of the total volume of tissue 43, or only to about 1%–5% of the total volume of tissue 43. In some embodiments, only the axonal terminals 46 in the target regions 44 of the target tissue 43 will directly receive the applied energy and release neurotransmitters, while unstimulated axonal terminals outside the target regions 44 do not receive a significant amount of energy and are therefore not activated / stimulated in the same manner. In some embodiments, the axonal terminal 46 in the tissue portion directly receiving energy will induce altered neurotransmitter release. In this way, tissue subregions can be targeted in a granular manner for neuromodulation, for example, one or more subregions can be selected. In some embodiments, energy application parameters can be selected to induce preferential activation of neural or non-neuronal components within the tissue directly receiving energy, thereby inducing desired combined physiological effects. In some embodiments, energy can be focused or concentrated below approximately 25 mm. 3 Within a certain volume. In some implementations, energy can be focused or concentrated to approximately 0.5 mm. 3 -50mm 3 Within the volume. The size / configuration of the energy application device 12 can affect the focal volume and focal depth used to focus or concentrate energy within the target region 44. The focal volume of energy application can be defined by the focal field of the energy application device 12.

[0127] As provided herein, energy can be applied substantially only to one or more target regions 44 to preferentially activate synapses in a targeted manner to achieve the target physiological outcome, rather than being applied substantially in a general or nonspecific manner to the entire tissue 43. Therefore, only a subset of multiple different types of axonal terminals 46 in tissue 43 are exposed to direct energy application. Figure 4 is a blood flow image in the spleen (obtained by Doppler ultrasound), which can be used as spatial information to spatially select target regions of a target organ. For example, target regions within an organ containing blood vessels, nerves, or other anatomical landmarks can be spatially selected and used to identify regions with specific axonal terminals and synapses. In one embodiment, target regions are selected by identifying the splenic artery and spatially selecting regions adjacent to or parallel to the splenic artery. Organ structures can be subdivided based on sub-organ tissue function, vascular and nerve innervation, and a subset of axonal terminals can be selected to include in the target regions where direct energy is applied. Other axonal terminals may not be within the target regions and may not be exposed to directly applied energy. The selection of single or multiple axon terminals to be included in the target region can be based on factors including, but not limited to, historical or experimental data (e.g., data showing the association of a specific location with a desired or target physiological outcome). In another embodiment, the location of the axon terminal and its adjacent tissues or structures can be used to select individual axon terminals for preferential activation from the overall set of axon terminals. Alternatively or additionally, system 10 can apply energy to individual axon terminals until the desired target physiological effect is achieved. It should be understood that the spleen image is merely an example. The disclosed method of using spatial information of visualized nerves to select axon terminals for preferential activation by directly applying energy to the target region can be used in conjunction with other organs or structures (e.g., liver, pancreas, gastrointestinal tissue).

[0128] The disclosed techniques can be used to assess the effects of neuromodulation, and can subsequently be used as inputs or feedback for selecting or modifying neuromodulation parameters. The disclosed techniques can use direct assessments of tissue condition or function as the target physiological outcome. This assessment can be performed before (i.e., baseline assessment), during, and / or after neuromodulation.

[0129] Assessment techniques may include at least one of functional magnetic resonance imaging (fMRI), diffusion tensor magnetic resonance imaging (DTI), ortho-emission computed tomography (PET), acoustic monitoring, or thermal monitoring. Assessment techniques may also include protein and / or biomarker concentration assessment. The system can receive images from the assessment techniques for automated or manual assessment. Based on the image data, regulatory parameters can also be modified. For example, changes in organ size or displacement can be used as biomarkers of local neurotransmitter concentrations, and as surrogate biomarkers of local cellular exposure to phenotypic regulatory neurotransmitters, and effectively as biomarkers predicting effects on glucose metabolism pathways. Local concentration may refer to the concentration within the focal field where energy is applied.

[0130] Additionally or alternatively, the system can assess the presence or concentration of one or more molecules in tissue or circulating in blood. The concentration in tissue may be referred to as local concentration or resident concentration. Tissue can be obtained via fine-needle aspiration, and the presence or level of the target molecule (e.g., metabolic molecules, biomarkers of metabolic pathways, peptide neurotransmitters, catecholamines) can be assessed using any suitable technique known to those skilled in the art.

[0131] In other implementations, the target physiological outcome may include, but is not limited to, changes in tissue displacement, tissue size, concentration changes (local, non-local, or circulating) of one or more molecules, changes in gene or biomarker expression, afferent activity, and cell migration. For example, energy application to tissue may result in tissue displacement (e.g., liver displacement). Other effects can be estimated by assessing tissue displacement (e.g., through imaging). For example, a certain displacement may be characteristic of a specific change in molecular concentration. In one example, based on empirical data, 5% liver displacement may indicate or be associated with a desired decrease in circulating glucose concentration. In another example, tissue displacement can be determined by comparing reference image data (tissue images before energy application) with post-treatment image data (tissue images acquired after energy application). The parameter may be a maximum or average displacement value of the tissue. If the displacement parameter is greater than a threshold displacement, the application of energy can be assessed as potentially leading to the desired target physiological outcome.

[0132] Figure 5 This is a flowchart of a method 50 for stimulating a target region of a target tissue. In method 50, a target region 52 is spatially selected. In step 54, an energy application device is positioned such that energy pulses are focused on the desired target region, and in step 56, a pulse generator applies multiple energy pulses to the target region of the target tissue to preferentially activate a subset of synapses in the target tissue, such as stimulating axonal terminals to release neurotransmitters and / or inducing altered neurotransmitter release and / or inducing altered activity in non-neuronal cells (within the synapse), thereby producing the target physiological outcome as provided herein in step 58. In some embodiments, the method may include a step of evaluating the effect of stimulation. For example, one or more direct or indirect assessments of the state of tissue function or condition may be used. Based on the evaluated tissue function, the modulatory parameters of one or more energy pulses may be modified (e.g., dynamically or adjustablely controlled) to achieve the target physiological outcome.

[0133] In one implementation, assessments can be performed before and after the application of the energy pulse to evaluate changes in glucose concentration due to modulation. If the glucose concentration is above or below a threshold, the modulation parameters can be appropriately modified. For example, if the glucose concentration has the desired physiological outcome, the energy applied during neural modulation may be reduced to the minimum level supporting the desired outcome. If the change in said characteristic relative to the threshold is not sufficiently correlated with changes in glucose concentration, certain modulation parameters can be altered, including but not limited to modulation amplitude or frequency, pulse shape, stimulation pattern, and / or stimulation location.

[0134] Furthermore, the assessed characteristic or condition can be a value or index, such as flow rate, concentration, cell population, or any combination thereof, which can be analyzed using appropriate techniques. For example, relative changes exceeding a threshold can be used to determine whether a modulating parameter has been modified. The desired modulation can be assessed by measured clinical outcomes, such as the presence or absence of an increase in tissue structure size (e.g., lymph node size) or a change in the concentration of one or more released molecules (e.g., relative to baseline concentration prior to neural modulation). In one implementation, the desired modulation may involve an increase in concentration exceeding a threshold, such as an increase relative to baseline concentration exceeding approximately 50%, 100%, 200%, 400%, or 1000%. For blocking therapy, assessment may involve tracking a decrease in molecule concentration over time, such as a decrease in the target molecule of at least 10%, 20%, 30%, 50%, or 75%. Additionally, for some subjects, the desired blocking therapy may involve maintaining a relatively stable concentration of a specific molecule against the backdrop of other clinical events that might predispose to an increase in molecule concentration. That is, the desired blocking may prevent a potential increase. Increases or decreases, or other induced and measurable effects, can be measured within a specific time window from the start of treatment, such as within approximately 5 minutes or approximately 30 minutes. In some embodiments, if neuromodulation is determined to be needed, the change in neuromodulation is an instruction to stop applying energy pulses. In another embodiment, if neuromodulation is not needed, one or more parameters of the neuromodulation are changed. For example, a change in the modulatory parameters could be an increase in the pulse repetition frequency, such as a gradual increase of 10-100 Hz, and the desired characteristics are evaluated until the desired neuromodulation is achieved. In other embodiments, the pulse width can be changed. In other embodiments, two or more parameters can be changed simultaneously, in parallel, or in series. If neuromodulation is not desired after multiple parameter changes, the focus (i.e., the site) of energy application can be changed.

[0135] The energy application device 12 can be configured as an external non-invasive device or an internal device, such as a minimally invasive device. As described above, the energy application device 12 can be an external non-invasive ultrasound transducer or a mechanical actuator. For example, Figure 6An embodiment of an energy application device 12 configured to include a handheld ultrasound probe 74 is shown. However, it should be understood that other non-invasive embodiments are also contemplated, including other methods of configuring, adhering to, or placing the ultrasound transducer probe on a anatomical target. Furthermore, in addition to a handheld configuration, the energy application device 12 may include a steering mechanism responsive to commands from a controller 16. This steering mechanism may orient or guide the energy application device 12 toward the target tissue 43 (or structure), and the controller 16 may then focus the energy application onto the target region 44.

[0136] Example

[0137] Figure 7 This is a block diagram of system 10, which includes an energy application device 12 configured to apply high-intensity focused ultrasound (HIFU) and a pulse generator 14. In one embodiment, system 10 includes, for example, a pulse generator comprising a function generator 80, a power amplifier 82, and a matching network 84. In one embodiment provided herein for generating experimental results, the pulse generator includes a 1.1 MHz high-intensity focused ultrasound (HIFU) transducer (Sonic Concepts H106), a matching network (e.g., Sonic Concepts), an RF power amplifier (ENI 350L), and a function generator (Agilent 33120A). In the example shown, the 70-mm diameter HIFU transducer has a spherical surface with a radius of curvature of 65 mm and a 20-mm diameter aperture in the center into which an imaging transducer can be inserted. The focal depth of the transducer is 65 mm. The pressure curve in the numerical simulation has a full width of 1.8 mm laterally and a depth of 12 mm at half amplitude. The HIFU transducer 12 is coupled to the animal subject through a 6 cm high plastic cone filled with degassed water. Figure 8 It is possible to be with Figure 7 The system 10 uses an energy application device that includes a HIFU transducer 74A and an imaging ultrasound transducer 74B arranged in a single energy application device 12, as provided herein, which can be controlled, for example, by a controller 16 to apply energy and image target tissue.

[0138] Figure 9An experimental setup for performing some of the spleen regulation experiments presented herein is shown. Although the depicted embodiment shows a spleen target tissue 43, it should be understood that certain elements of the experimental setup may be common across different target tissues 43. For example, the energy application device 12 may be operated according to parameters set by the controller 16 to apply energy to a target region within the target tissue 43. As discussed herein, target tissues may include the spleen, liver, pancreas, gastrointestinal tissue, etc. Although the depicted experimental setup is shown with a 40W RF amplifier, this is merely an example, and other amplifiers (e.g., linear amplifiers) may be used. In some setups, a rat's head is inserted into a birdcage coil.

[0139] Figure 10 An experimental timeline of ultrasound energy application used to perform some of the modulation experiments provided herein is shown. In the depicted embodiment, ultrasound application was performed for 1 minute before and after lipopolysaccharide (LPS) injection. LPS is a bacterial membrane molecule that can induce a strong immune or inflammatory response. The use of LPS from *E. coli* O111:B4 (Sigma–Aldrich) induced significant inflammatory and metabolic dysfunction (e.g., hyperglycemia and insulin resistance) in juvenile adult Sprague Dawley (SD) rats. Intraperitoneal (IP) injection of LPS (10 mg / kg) resulted in significant increases in TNF, circulating glucose, and insulin concentrations; these concentrations peaked within 4 hours but remained elevated for up to 8 hours post-injection compared to controls. Animals were sacrificed some time after ultrasound treatment for organ harvesting and processing. Although the time period shown by way of example is 1 hour, it should be understood that in other embodiments, the time period for assessing induced changes may be variable.

[0140] Function generator 80 generates pulse sine waves, such as Figure 11As shown. The pulsed sinusoidal waveform is amplified by an RF power amplifier and sent to the matching network of the HIFU transducer. In animal experiments, three ultrasound parameters can be adjusted: pulse amplitude, pulse length, and pulse repetition frequency. The pulse amplitude ranges from 0.5V peak to 62V peak. Three pulse lengths are used: 18.2µs, 136.4µs, and 363.6µs. In one embodiment, the pulse repetition frequency (1 / T) is 2kHz. The treatment time is 1 minute. The ultrasound adjustment parameters are exemplary. In one embodiment, adjustment is provided with ultrasound stimulation having an ultrasound transducer frequency in the range of about 0.1MHz to about 5MHz, and said ultrasound stimulation having an ultrasound frequency pulse repetition frequency in the range of about 0.1Hz to about 10kHz. The ultrasound cycle of each pulse of ultrasound energy can be in the range of about 1 to about 1000. In one embodiment, the pulse center frequency is 1.1MHz, the pulse repetition period is 0.5ms (corresponding to a pulse repetition frequency of 2000Hz); the pulse amplitude and pulse length are varied. Table 1 summarizes the HIFU ultrasound parameters.

[0141]

[0142] Pressure measurements were performed in degassed water using a HIFU hydrophone (HNA-0400) manufactured by Onda Corp. The HIFU transducer was driven by a 100-cycle sine wave. The hydrophone was scanned by a focal spot with a grid size of 0.1 mm in the xy plane and a step size of 0.2 mm along the z-axis. Figure 12 The hydrophone settings are displayed, and Figure 13 The scan results are shown. The peak positive (negative) pressure is defined as the maximum positive (negative) pressure at the transducer focus in the xy plane. The input voltage is low enough to eliminate nonlinear effects. Therefore, the peak positive pressure value is the same as the peak negative pressure value. To estimate the peak pressure at full operating voltage, the peak pressure was measured at various drive voltages before cavitation occurred and curve fitting was performed. The calculated peak pressures are shown in Table 1.

[0143] Organ-specific neural modulation ultrasound targeting

[0144] Prior to the initiation of neuromodulation, an ultrasound scan was performed using a GE Vivid E9 ultrasound system and an 11L probe. The target area was marked on the animal's skin. The HIFU transducer was placed over the marked area. A second ultrasound scan was also performed using a smaller imaging probe (3S), positioned within the opening of the HIFU transducer. The imaging beam of the 3S probe was aligned with the HIFU beam. Thus, images of the target organ (visualized on the ultrasound scanner) could be used to confirm that the HIFU beam had been targeted to the target area.

[0145] Animal Agreement

[0146] Prior to the experiments, 8- to 12-week-old adult male Sprague–Dawley rats (250–300 g; Charles River Laboratories) were acclimatized for one week at 25°C with a 12-hour light / dark cycle. They were allowed free access to water and a standard rodent diet. Eight-week-old obese Zucker rats (Charles River Laboratories) were also acclimatized at 25°C with a 12-hour light / dark cycle and fed a high-calorie diet (Purina #5008) as maintained by the supplier to promote insulin resistance and hyperglycemia. They were also allowed free access to water and a standard rodent diet.

[0147] Endotoxin (LPS from *E. coli*, 0111:B4; Sigma–Aldrich) was used to induce significant inflammatory and metabolic dysfunction (e.g., hyperglycemia and hyperinsulinemia) in juvenile adult Sprague Dawley rats. Intraperitoneal (IP) administration of LPS (10 mg / kg; Rosa-Ballinas PNAS, 2008) resulted in significant increases in TNF, circulating glucose, and circulating insulin concentrations, peaking at 4 hours but remaining elevated up to 8 hours post-injection compared to the control group. Spleen, liver, hypothalamus, hippocampus, and blood samples were collected at 60 minutes post-injection (for power studies) and at 30, 60, 120, 240, or 480 minutes post-injection (for duration and kinetic studies). Spleen and liver samples were homogenized in PBS containing phosphatase (0.2 mM benzyl sulfonyl fluoride, 5 μg / mL aprotinin, 1 mM benzylamidin, 1 mM sodium orthovanadate, and 2 μM cantharidin) and protease inhibitors (1 μL to 20 mg tissue, according to Roche Diagnostics). The target final concentration of 0.2 g tissue / mL PBS was applied to all samples. Blood samples were stored with the anticoagulant (disodium) EDTA to prevent coagulation. Changes in cytokine (Bio-Plex Pro; Bio-Rad), TNF (Lifespan), and acetylcholine (Lifespan) concentrations in the samples were analyzed by ELISA. Catecholamine concentrations were assessed using HPLC or ELISA (Rocky Mountain Diagnostic).

[0148] The effects of LPS on blood glucose and insulin levels were examined. Blood samples were collected from the tail vein at 0, 60, 90, 120, 150, 180, and 240 minutes after LPS injection to measure glucose and insulin levels. Circulating blood glucose concentrations were measured using a OneTouch Elite glucometer (LifeScan; Johnson & Johnson). Plasma insulin concentrations obtained from blood were determined using an ELISA kit (Crystal Chem, Chicago, IL) to determine the effects of LPS and subsequent ultrasound stimulation on systemic insulin tolerance. Changes in signal transduction were measured by assessing biomarkers in liver, muscle, heart, and hypothalamic tissue samples, including p38, p7056k, Akt, GSK3B, c-Src, NF-κβ, SOCS3, IRS-1, NPY, and POMC. Induced changes by ultrasound stimulation may include insulin-mediated glucose uptake and changes in related molecules associated with inhibition / activation of metabolic activity.

[0149] The protocol for ultrasound neuromodulation can be as follows:

[0150] Animals can be anesthetized with 2-4% isoflurane.

[0151] Animals can be placed face down on a water-circulating heated mat to prevent their body temperature from getting too high during surgery.

[0152] Before stimulation, the area above the target area (target nerve) for ultrasound stimulation can be shaved off with a disposable razor and animal hair trimmer.

[0153] Diagnostic imaging ultrasound can be used to spatially select the target area.

[0154] Liver: Doppler imaging of the portal vein identifies the portal vein as the porta hepatis.

[0155] Spleen: The spleen is visually identified using diagnostic ultrasound. The location of the stimulus can be maintained along the identified splenic axis.

[0156] The area can be marked with a permanent marker for subsequent identification.

[0157] The FUS ultrasound probe or LogiQ E9 probe can be placed in the designated target area previously identified by diagnostic ultrasound.

[0158] Then, ultrasonic pulses are performed using a single stimulation with a total duration not exceeding one minute. The energy is never reached levels associated with thermal damage and ablation / cavitation (ablation / cavitation at 35 W / cm²). 2 In other words, in some implementations, the time-averaged intensity in the target region is less than 35 W / cm². 2 .

[0159] LPS (10 mg / kg) can then be administered intraperitoneally (for acute / kinetic studies). Alternatively, for the duration of action, LPS may not be administered at this time, but may be administered at a subsequent specified time point.

[0160] A second 1-minute ultrasound stimulation can be applied.

[0161] The animals can then be incubated under anesthesia for acute (1 hour) and kinetic (changes within 3 hours after LPS) studies. Afterwards, the animals are undressed and tissue and blood samples are collected.

[0162] For studies on the duration of effect, LPS was injected not during ultrasound stimulation, but at specified time points after ultrasound stimulation (e.g., 0.5, 1, 2, 4, or 8 hours). Animals were then placed in an anesthetic container and monitored until euthanasia and tissue / fluid collection.

[0163] An incision can be made starting at the bottom of the peritoneal cavity, extending upwards and into the pleural cavity. Organs can be rapidly removed and homogenized in PBS containing phosphatase (0.2 mM benzyl sulfonyl fluoride, 5 μg / mL aprotinin, 1 mM benzylamidin, 1 mM sodium orthovanadate, and 2 μM cantharidin) and protease (according to Roche Diagnostics, 1 μL to 20 mg tissue) inhibitors. The target final concentration of 0.2 g tissue / mL PBS solution was applied to all samples. Blood samples were stored with the anticoagulant (disodium) EDTA to prevent coagulation. Samples were then stored at -80°C until analysis. Changes in cytokine (Bio-Plex Pro; Bio-Rad), TNF (Lifespan / Abcam / ThermoFisher), and acetylcholine (Lifespan) concentrations in the samples were analyzed by ELISA. Catecholamine concentrations were assessed using HPLC detection or ELISA (Rocky Mountain Diagnostic).

[0164] Electrode-based vagal nerve stimulation controlled trial protocol

[0165] Male Sprague-Dawley rats were anesthetized with 2% isoflurane. A cervical incision was made to expose the trapezius, sternocleidomastoid, and masseter muscles on the lateral side of the neck for blunt dissection, exposing the left cervical vagus nerve. Microelectrodes were placed along the exposed trunk of the cervical vagus nerve. Electrical stimulation was generated using a BIOPACMP150 module under the control of AcqKnowledge software (Biopac Systems) (5V, 30Hz, 2ms; 5V, 5Hz, 2ms; 1V, 5Hz, 2ms). Vagus nerve stimulation was performed for 3 minutes before and after intraperitoneal injection of 10 mg / kg LPS. The rats were euthanized 60 minutes after LPS injection, and spleen and blood samples were obtained for TNF determination. In rats undergoing sham surgery, the vagus nerve was exposed but not touched or manipulated.

[0166] HPLC analysis

[0167] Serum samples can be directly injected into the instrument without pretreatment. First, the tissue homogenate is homogenized with 0.1M perchloric acid and centrifuged for 15 minutes. Then, the supernatant is separated and the sample is injected into HPLC (Dhir & Kulkarni, 2007).

[0168] Catecholamines (norepinephrine / epinephrine) were analyzed by high-performance liquid chromatography (HPLC) using a built-in ultraviolet detector. The test column used in this analysis was a Supelco Discovery C18 (15 cm x 4.6 mm ID, 5 μm particle size). The biphase mobile phase consisted of [A] acetonitrile:[B] 50 mM KH₂PO₄, set to pH 3 (with phosphoric acid). The solution was then buffered with 100 mg / L EDTA and 200 mg / L 1-octanesulfonic acid. The final concentration of the 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 maintaining the column at a constant 20 °C to minimize column compaction due to the viscosity of the mobile phase used. The UV detector was maintained at 254 nm, a wavelength known to capture the absorption of catecholamines, including norepinephrine, epinephrine, and dopamine.

[0169] Chemical inhibition of ultrasound-regulated molecular signaling pathways

[0170] To further investigate the effects of mechanical and direct neural stimulation (and the preferential regulation of neural and non-neural components of extraaxonal synapses), SRC inhibitors (common markers of direct mechanoreceptors) or PI3K inhibitors (common markers of neural signal transduction) should be administered before the aforementioned ultrasound stimulation procedure.

[0171] Tissue extraction and paraffin block conversion:

[0172] Immediately place the tissue (mouse brain) in a fixative and fix it in 10% formalin at 4°C for 24 hours.

[0173] The tissue was treated using the following protocol (using vacuum and pressure during each incubation):

[0174] a. 70% ethanol, 37℃, 40 min

[0175] b. 80% ethanol, 37°C, 40 min

[0176] c. 95% ethanol, 37°C, 40 min

[0177] d. 95% ethanol, 37℃, 40 min

[0178] e. 100% ethanol, 37°C, 40 min

[0179] f. 100% ethanol, 37°C, 40 min

[0180] g. xylene, 37℃, 40min

[0181] h. xylene, 37℃, 40min

[0182] i. Paraffin wax, 65℃, 40min

[0183] j. Paraffin wax, 65℃, 40min

[0184] k. Paraffin wax, 65℃, 40min

[0185] l. Paraffin, 65°C, 40 min* Leave in the paraffin until ready for embedding, but do not exceed ~12-18 hours.

[0186] Embedded in paraffin blocks for sectioning, allowing the blocks to cool / harden before sectioning. Collect 5-micron-thick sections by floating them in a 50°C water bath. Use positively charged slides and attempt to orient the tissue on each slide in the same direction. Air dry the slides. Overnight at room temperature seems to be the best option for drying, but slides can be placed on a 40°C slide heater to speed up the drying process, but do not leave the slides on the heater for more than 1 hour. Store the slides at 4°C.

[0187] IHC treatment

[0188] Formalin-fixed paraffin-embedded (FFPE) tissue samples (rat brains) were baked at 65°C for 1 hour. Slides were dewaxed with xylene, rehydrated by washing with progressively decreasing ethanol concentrations, and then subjected to antigen recovery. A two-step antigen recovery method specifically developed for multiplexing with FFPE tissues allows antibodies with different antigen recovery conditions to be used together on the same sample. The sample was then incubated in PBS with 0.3% Triton X-100 at ambient temperature for 10 minutes, followed by nonspecific binding blocking for 45 minutes at room temperature with 10% (wt / v) donkey serum and 3% (wt / v) BSA in 1×PBS. The primary antibody cFOS (Santa Cruz-SC52) was diluted to the optimal concentration (5 μg / mL) and applied to PBS / 3% (v / v) BSA at room temperature for 1 hour. The sample was then washed sequentially in PBS, PBS-Triton X-100, and PBS again for 10 minutes, with agitation during each wash. In cases involving secondary antibody detection, the sample was incubated together with primary antibody species-specific di-antibody IgG conjugated to Cy3 or Cy5. The slides were then washed as described above, stained with DAPI (10 μg / mL) for 5 minutes, rinsed again in PBS, and fixed with an anti-fading medium for image acquisition. Whole tissue images were acquired at 10X magnification using a fluorescence Olympus IX81 microscope.

[0189] Image processing

[0190] Autofluorescence (AF) signatures of FFPE tissue should be characterized and isolated from the target fluorophore signal using an autofluorescence removal method, where images of the unstained sample are acquired in addition to stained images. The unstained and stained images are normalized relative to exposure time and dark pixel values ​​(pixel intensity values ​​at zero exposure time). Each normalized AF image is then subtracted from the corresponding normalized stained image. AF-removed images are merged with registered 4',6-diamidindole-2-benzylindole (DAPI) images. The same target region in both stimulus and control samples is imaged, and the cFOS expression in the images is qualitatively assessed to detect changes in the expression of neural activation-related genes.

[0191] Histological evaluation of the spleen: As described above, spleens from stimulated and control rats were processed into paraffin blocks. Paraffin-embedded sections were removed according to standard protocols reported in the literature and stained with H&E, then scanned on a bright-field Olympus scanner. Morphological differences in the H&E images were qualitatively assessed, and no significant differences were found between the stimulated and control samples.

[0192] Heart rate monitoring and analysis

[0193] Heart rate was monitored using a commercial infrared pulse oximeter and physiological monitoring system (Starr Lifesciences) according to the manufacturer's instructions (during ultrasound or electrode stimulation experiments). In the stimulation protocol, a foot clip sensor (provided by the manufacturer) was placed on the animal's paw pad. The animal was allowed at least 5 minutes to acclimatize before measurement, a time point sufficient for the animal to return to normal heart rate activity and physiological readings in the control group. Measurements were recorded before (2-minute recording time), during, and after stimulation with the electronic microelectrode or ultrasound probe, respectively.

[0194] Ultrasound-induced activation of diffusion function MRI measurement

[0195] Neuronal activation can be detected using blood oxygenation level-dependent (BOLD) fMRI; increased metabolic demand in brain regions leads to increased cerebral blood flow, increased oxygenated blood supply, and decreased gradient echo signal. Sensitivity to the BOLD effect requires rapid gradient echo acquisition; this can cause undesirable signal loss in brain regions near air sacs (such as the sinuses and ear canals), hindering the detection of neuronal activation in these specific brain regions. Alternatively, to minimize signal loss in regions characterized by large field inhomogeneities, spin echo (or dual spin echo) diffusion-weighted imaging (DWI) can be used. In DWI-fMRI, the slow diffusion is likely due to an increase in the volume of intracellular water pools or an increase in water diffusion (or apparent diffusion coefficient (ADC)), both attributed to cell swelling and membrane expansion caused by neuronal activation.

[0196] use Figure 14 For example, ten rats underwent brain MRI scans. Six of them received LPS injections (as described above) and ultrasound therapy; four of them received only LPS injections. Ten Sprague-Dawley rats were anesthetized with 3% isoflurane, supine, and their heads inserted into birdcage coils. The abdominal region was coupled to an MR-compatible ultrasound probe (f = 1.47 MHz) via a gel / water-filled cone, which was focused on the hepatic hilum (the liver region previously containing glucose-sensitive neurons).

[0197] Scanning was performed using a Doty Scientific orthogonal birdcage coil on a 3T GE DV scanner (Waukesha, WI). Scanning began at T1 acquisition, using a differential gradient echo sequence with an in-plane / out-of-plane spatial resolution of 0.4 / 1 mm and a TE / TR of 10 / 1475 ms, for a total acquisition time of 3:22 minutes. Six dual-spin echo diffusion-weighted imaging (DWI) images (referred to as positive polar gradient or FPG) were acquired with an in-plane / out-of-plane spatial resolution of 0.6 / 1 mm and a TE / TR of 82 / 3400 ms, averaging 3 / 4 for b = 0 / b = 1000 s / mm², with a total acquisition time of 1:49 minutes for each image. After completing six pre-injection DWI acquisitions, another DWI acquisition was performed using the reverse gradient direction for distortion correction purposes; this acquisition is called the reverse polar gradient (RPG) acquisition. Following LPS injection, rats underwent a first ultrasound treatment with a 5-minute waiting period, followed by a second ultrasound treatment, during which six additional FPG DWI images were acquired. For control rats injected with only LPS, the final six DWI images were acquired immediately after LPS injection.

[0198] Ultrasound therapy was performed using an MR-compatible 1.47 MHz focused ultrasound transducer coupled to the target region (e.g., gastrointestinal tissue, pancreas, liver, etc.) with a water-filled cone. Each ultrasound treatment lasted 60 seconds, during which a pulsed sinusoidal ultrasound shape was applied. The pulse on-time was 150 μs, and the pulse off-time was 350 μs. The rat's abdomen was outside the imaging coil; supine animal positioning ensured easy coupling of the ultrasound probe to the liver through the skin using a coupling gel. The cross-correlation coefficient (ccc) between T1 images with a minimum of 0.5 and (distortion-corrected) b=0 DWI images was used to identify slices to be used for further analysis. The apparent diffusion coefficient (ADC) of the pre- and post-treatment images was calculated; the pre- and post-treatment image data were pooled together for statistical analysis. Tight registration between the T1 images and the rat atlas was used to identify regions where a pixel-by-pixel t-test indicated significant change. The registration transformation from the T1 and atlas images was applied to the distortion-corrected DWI and ADC images.

[0199] Cholinergic anti-inflammatory pathway

[0200] This example illustrates a non-invasive approach that uses ultrasound energy application to stimulate specific axonal synapses within an organ to achieve simulated and associated physiological outcomes. Ultrasound was first applied to the spleen and found to stimulate axons associated with the cholinergic anti-inflammatory pathway (CAP) to modulate systemic cytokine concentrations. When this pathway was chemically or mechanically blocked, the ultrasound-induced effect was suppressed. Extracellular neurotransmitter concentrations, intracellular kinase activity, and CAP-related cytokines were affected differently when ultrasound parameters were varied, demonstrating the ability to produce desired physiological responses by modifying ultrasound parameters. Next, it was shown that ultrasound stimulation of the liver modulates sensory pathways regulating blood glucose, and this effect was found to depend on stimulation of specific anatomical sites within the liver. Overall, these data suggest that intra-organ ultrasound neuromodulation can provide a precise approach to neuromodulation that helps stimulate multiple small subsets of neurons within an organ or tissue to influence specific physiological functions, such as blood glucose regulation (via hepatic neuromodulation) and systemic cytokine regulation (via splenic neuromodulation).

[0201] Within peripheral nerves, individual axons are tightly clustered together and encased in protective tissue. This makes it difficult to selectively stimulate a subset of axons that terminate in a specific organ and uniquely modulate the function of communicating cells within that organ. The clinical implementation of precise peripheral nerve stimulation remains complex. Figure 15A This is a partial schematic diagram showing a complex system of afferent and efferent neurons projecting into the vagus nerve, an exemplary innervated organ, and the approximate location of a stimulator for the cervical VNS. Efferent neurons originate from the dorsal motor nucleus (DMV) of the vagus nerve and enter the brain via the nucleus of the solitary tract (NTS). The peripheral nerves guiding visceral organs contain both efferent and afferent neurons, making them difficult to stimulate individually with a distal cervical VNS implant.

[0202] Figure 15B This is a descriptive schematic diagram of peripheral neural modulation based on target organs, where focused pulsed ultrasound is used to preferentially stimulate a subset of axons terminating within the organ. The targets studied in this paper include axonal terminals in the spleen associated with cholinergic anti-inflammatory pathways and sensory terminals in the liver involved in transmitting metabolic information to the brain to help maintain glucose homeostasis. As shown in this paper, focused pulsed ultrasound stimulation terminates on a subset of axons within the organ (…). Figure 15B In some of the examples presented in this article, ultrasound energy is focused on axonal projections within the spleen (to influence systemic inflammation via the cholinergic anti-inflammatory pathway (CAP)) and axonal projections within the liver (to transmit metabolic information to the brain and maintain glucose homeostasis).

[0203] In a rodent LPS-induced inflammation model, each cell type involved in CAP was monitored under different ultrasound stimulation parameters (Fig. 16A and B). Figure 15BThe splenic endothelial cell (CAP) is composed of three main cell types: postsynaptic axonal terminals projecting from the splenic ganglion, intermediate T cells, and macrophages that regulate circulating cytokine levels. The CAP response to local ultrasound stimulation was monitored by measuring splenic concentrations of CAP-related neurotransmitters and cytokines, including norepinephrine (NE), acetylcholine (ACh), and tumor necrosis factor (TNF-α).

[0204] Ultrasound stimulation was performed as provided herein and according to the timeline shown in Figure 16A to demonstrate the induced target physiological outcomes relative to the control. Ultrasound stimulation was administered for one minute before and after LPS injection; samples were collected and the resulting local (i.e., tissue) changes and systemic neurotransmitter and cytokine changes were measured (Figures 16B-E) to demonstrate the effect of ultrasound-induced CAP neural modulation on the LPS model response. Except for Figure 16E, the response time for all data was 1 hour. However, it should be understood that the depicted response times are merely exemplary. That is, the induction of changes due to neural modulation in the target region can occur within one hour, and in some embodiments, may last for hours or days. Therefore, as provided herein, the assessment of measurable induced changes due to neural modulation can be performed at baseline (at or before neural modulation) and at one or more time points (intervals of minutes, hours, or days) after neural modulation. As part of the treatment regimen, certain subjects may be monitored continuously or intermittently to assess the concentration of one or more target molecules (or other measurable effects, such as organ displacement).

[0205] A sham-operated control was performed by placing an ultrasound transducer on the target organ without applying ultrasound stimulation. Figure 16B shows the CAP response measured in naive rats, sham-operated controls (rats receiving LPS but not ultrasound stimulation), and animals receiving LPS and various ultrasound stimulation pressures. The concentrations of norepinephrine (i), acetylcholine (ii), and TNF-α (iii) are shown in naive animals, sham-operated controls, and animals with ultrasound stimulation pressures of 0.03–1.72 MPa. It is noteworthy that the x-axis labels in (iii) representing naive animals, sham-operated controls, and ultrasound stimulation pressures apply to all three plots. The average spleen norepinephrine level in naive animals was 140 nmol / L, while LPS-induced inflammation reduced norepinephrine levels to near zero, indicating that CAP signaling was suppressed at the onset of inflammation.

[0206] As shown in the figures, ultrasound stimulation reduced the LPS response toward levels measured in juvenile animals (Fig. 16B.i.). Consistent with CAP signaling, the increase in norepinephrine in ultrasound-stimulated animals was associated with an increase in splenic acetylcholine; at an ultrasound pressure of 0.83 MPa, the mean acetylcholine concentration was almost three times that found in sham-operated animals (Fig. 16B.ii.). Fig. 16C shows the circulating concentration of TNF-α under the same conditions as in Fig. 16B. Note that the x-axis labels in Fig. 16C representing juvenile animals, sham-operated controls, and ultrasound stimulation pressure also apply to Fig. 16B. Fig. 16D shows the spleen IL-1α concentration under the same conditions as in Fig. 16B. Compared to sham-operated animals, both spleen (Fig. 16B.iii) and circulating TNF-α (Fig. 16C) levels showed reductions. The response to treatment depended on the ultrasound pressure (0.83 MPa used in subsequent experiments). As further evidence that ultrasound stimulation of the spleen specifically induces changes in CAP, Figure 16D shows that ultrasound stimulation affects the concentrations of other proteins regulated by the TNF-α-specific pathway, such as interleukin-1α (IL-1α). Therefore, the data suggest that controlling adjustable regulatory parameters (such as ultrasound pressure) can achieve targeted control of target molecule concentrations. By varying the ultrasound pressure applied to the target region, desired physiological outcomes (e.g., desired concentration changes of one or more target molecules) can be achieved. While the applied pressure can be determined empirically based on the patient's condition, in some implementations, ultrasound stimulation pressures of 0.03–1.72 MPa are used for targeted neuromodulation. As described herein, ultrasound pressure can be a regulatory parameter that can be altered or adjusted to achieve the target physiological response. Other adjustable parameters can be the treatment schedule (e.g., treatment duration, intervals between treatments, and delays for other clinical events or assessments via an evaluation device).

[0207] Figure 16F shows a 2D ultrasound image of the rat spleen used to spatially select a splenic target and focus ultrasound stimulation onto that target. Arrows indicate the outline of the spleen used for ultrasound stimulation and the target focus (i.e., the target region of the spleen). Figure 16G shows a non-limiting implementation of the study's timeline, designed to measure the duration of the effect of stimulation on CAP activation. In this study, ultrasound stimulation was applied prior to LPS injection, and the delay between ultrasound stimulation and LPS injection ranged from 0.5 to 48 hours. Figure 16H shows the concentration of TNF-α in the spleen after ultrasound treatment (i.e., ultrasound stimulation prior to LPS injection) following a variable delay time of 0.5 to 48 hours, representing the percentage (%) of TNF-α concentration in the spleen as measured in the sham-operated control. Figure 16IThe concentrations of activated and / or phosphorylated kinases (p38, p70S6K, Akt, GSK3B, c-SRC, NF-kβ, SOCS3) are shown when ultrasound stimulation is performed at pressures of 0.13–1.72 MPa with or without the use of (shaded bars).

[0208] Peak ultrasound-mediated response was observed 1–2 hours post-treatment (Fig. 16E), similar to previous implant-based VNS studies. Additionally, when applied prior to LPS injection, splenic ultrasound could provide a protective effect (Figs. 16G and 16H), also consistent with previous invasive VNS studies. Fig. 16H shows that the protective effect persisted for 48 hours post-treatment. To further characterize the protective effect, ultrasound activation of specific intracellular kinases associated with LPS, CAP, or TNF-α-mediated signaling was measured. Figure 16I These data indicate that ultrasound strongly enhances the activation of some kinases (e.g., p38 and p70S6K), and that the ultrasound-dependent response of some kinases (e.g., p38) is largely correlated with previously observed ultrasound-dependent responses (Figure 16B–D).

[0209] Figure 16J and Figure 16K Data on the concentrations of norepinephrine (NE), acetylcholine (ACh), and tissue necrosis factor-α (TNF-α) in the spleen (after LPS injection) after ultrasound stimulation with alternative ultrasound stimulation parameters (burst duration and carrier frequency) are shown. The data are presented in a manner that compares to the 0.83 MPa data in the spleen sample of Figure 16B, which were acquired after stimulation with either the alternative burst duration (left) or the ultrasound carrier frequency (right).

[0210] The effects of splenic ultrasound modulation were compared with those of standard electrode or implant-based vagus nerve stimulation (VNS), as described in this paper. Figure 17A and 17B In this context, "+" indicates the presence of the indicated event or inhibitor, while "-" indicates the absence of the indicated event or inhibitor. Figure 17AIn the figure, the X-axis represents the induced changes in rat spleen under different conditions (ultrasound versus VNS and the use of different inhibitors), and the Y-axis shows the relative concentration of TNF-α in the spleen (relative to the LPS-treated control) as a percentage change. The relative concentrations of TNF-α in the spleen under ultrasound stimulation (ultrasound stimulation pressure of 0.83 MPa) and VNS stimulation are shown with and without PP2 (a partially selective Src kinase inhibitor), LY294002 (a selective PI3-kinase inhibitor), PD98059 (a selective MAPK inhibitor of MEK1 and MEK2), and α-clad krait venom (BTX; an antagonist of α7nAChR known in the CAP pathway). Figure 17B This study shows the effect of BTX on spleen concentrations of (left) norepinephrine (NE) and (right) TNF-α after ultrasound stimulation in rodents treated with LPS and those not affected by BTX or surgical vagotomy. Figure 17A The results showed that invasive cervical VNS and non-invasive splenic ultrasound stimulation had almost the same effect on TNF-α production. Additionally, Figure 17B The results showed that splenic injection of α-banded krait venom (BTX; a known α7nAChR antagonist) inhibited the effect of ultrasound stimulation on TNF-α concentration, indicating that (as with VNS-based CAP activation) the desired CAP regulation via ultrasound involves splenic α7nAChR signaling. This contrasts with the CAP model ( Figure 15B Consistent with the findings, NE concentration was not affected by BTX (i.e., BTX blocked the effect of elevated NE via the α7nAChR pathway). Vagotomy also inhibited ultrasound-mediated modulation of CAP, providing additional evidence that the effects of ultrasound on CAP are neurally mediated. Figure 17B Finally, the kinase inhibitors PP2 (partially selective for Src kinase) and LY294002 (selective for PI3 kinase) showed inhibitory effects on sonication, while PD98059 (a selective MAPK inhibitor for MEK1 and MEK2) had no effect. Figure 17A These results confirm that Figure 16I The results showed that ultrasound stimulation altered the activation of kinases within the PI3 (Akt, P70S6K), c-Src, and p38-MAPK pathways, but did not affect kinases involved in bacterial antigen responses (NFKB, GSK3B). This change may be part of a desired profile obtained through targeted neural modulation and indicates the target physiological outcome.

[0211] The physiological specificity of focused ultrasound stimulation was examined by measuring a number of known side effects of invasive VNS. Figure 17C The data shown compare the effects of VNS (at various stimulation intensities and frequencies) and spleen ultrasound stimulation (ultrasound stimulation pressure of 0.83 MPa) on heart rate. Figure 17C The changes in heart rate induced by cervical VNS or splenic ultrasound stimulation are shown. At 1 volt and 5 volt VNS intensities (known to activate CAP), heart rate decreased significantly. However, local splenic ultrasound stimulation had no effect on heart rate.

[0212] Figure 17D The data presented confirm the previously observed side effect of VNS on LPS-induced hyperglycemia, and this side effect was absent when using splenic ultrasound stimulation. The figure shows the relative blood glucose concentrations (compared to pre-injection concentrations) at 5, 15, 30, and 60 minutes after LPS injection in LPS control (without ultrasound stimulation), or LPS injection combined with splenic ultrasound stimulation or neck VNS stimulation. Figure 17D The results showed that, compared to the LPS-only control, ultrasound stimulation did not lead to changes in glucose concentration, while VNS experiments showed a reduction in the side effect of hyperglycemia. This metabolic side effect of CAP targeting the VNS may be due to off-target VNS effects via a second (non-CAP) vagal pathway. This off-target effect is a result of the broader and less specific (i.e., less targeted) effect of the VNS compared to the peripheral nerve modulation presented in this paper. The benefit of the provided targeted neuromodulation is the avoidance of undesirable off-target effects. Precise ultrasound stimulation techniques were also used to investigate whether VNS-mediated reduction in hyperglycemia is related to direct stimulation of axons originating from metabolic sensory neurons.

[0213] In some embodiments, ultrasound images can be used to guide ultrasound stimulation to spatially select a target region for targeted delivery of ultrasound stimulation. As provided herein, spatial selection or spatially selecting may include acquiring an image of a tissue or organ (or a portion of a tissue or organ) and, based on the image (e.g., an ultrasound image), identifying a target region within the organ. In some embodiments, the tissue or organ may have anatomical features for guiding the selection of a target region within the organ. In some embodiments, as a non-limiting example, such features may include the site of a blood vessel or nerve entering the organ, the type of tissue within the organ, or internal or peripheral or sub-organ structures of the organ. In some embodiments, anatomical features may include the hepatic hilum of the liver, gastrointestinal sub-organs (stomach, small intestine, large intestine), pancreatic duct, or white pulp of the spleen. By identifying anatomical features in the image, a target region can be selected to overlap with, include, or be adjacent to the anatomical feature. In other embodiments, anatomical features may be excluded from the target region. For example, intestinal tissue may be selected instead of stomach tissue as the target region. Identification of anatomical features can be performed by morphological features visible in the image (e.g., visible in an ultrasound image) or by structural identification features of the imaging modality used to acquire the image. As disclosed herein, system 10 can be configured such that energy application device 12 is configured to operate in imaging mode to acquire an image, and then operate in energy application mode after acquiring the image and spatially selecting a target region based on the image.

[0214] In other embodiments, target regions can be identified by the presence or absence of one or more biomarkers. Such biomarkers can be assessed by staining organs or tissues and obtaining images indicating the staining to identify organ or tissue regions containing the biomarkers. In some embodiments, biomarker information can be obtained using in vivo staining techniques to obtain location data of biomarkers in tissues or organs specific to the subject in real time. In other embodiments, biomarker information can be obtained using in vitro staining techniques to obtain location data of one or more representative images, which are then used to predict the location of biomarkers within the subject's tissues or organs. In some embodiments, target regions are selected to correspond to portions of tissues or organs rich in or lacking specific biomarkers. For example, one or more biomarkers may include markers of neuronal structure (e.g., myelin markers).

[0215] Target regions within organs or tissues can be spatially selected based on operator input. For example, an operator can specify a target region on an acquired image by directly manipulating the image (i.e., drawing or writing the target region onto the image) or by providing image coordinate information corresponding to the target region. In another embodiment, target regions can be automatically selected based on image data to achieve spatial selection. In some embodiments, spatial selection includes storing and accessing data related to the target region in memory.

[0216] Once spatially selected, system 10 is configured to apply energy to the target region provided herein. For example, as Figure 18A As shown, 2D ultrasound images of rat livers were used to guide ultrasound stimulation to selectively focus on the target hepatic hilum. White arrows indicate the outline of the liver and the middle arrow indicates the target area. Figure 18A The results show that ultrasound image guidance enables spatial selection of the hepatic hilum region of the liver and directing ultrasound stimulation toward the selected hepatic hilum region, which contains glucose-sensitive neurons, for localized targeted ultrasound neuromodulation.

[0217] Figure 18B Non-limiting examples are provided of selectively applying ultrasound stimulation to various regions of the liver in an animal model of LPS-induced hyperglycemia to achieve targeted regulation of blood glucose concentration. Figure 18B The figure shows the relative blood glucose concentrations (compared to pre-LPS concentrations) at 5, 15, 30, and 60 minutes after LPS injection. LPS-induced hyperglycemia was observed in the group receiving only LPS injection without ultrasound stimulation. The data further indicate that ultrasound stimulation of the distal lobes of the liver does not significantly affect blood glucose concentrations. In contrast, selective application of ultrasound stimulation at the hepatic hilum can be used to reverse LPS-induced hyperglycemia and regulate blood glucose concentrations. Therefore, the location of the target region produces different results, and extensive or non-targeted liver treatment of the lung lobes will not achieve the same targeted effect as ultrasound treatment targeting the hepatic hilum region (using areas adjacent to or including the hepatic hilum). Figure 18B As shown in the implementation, according to the protocol illustrated in Figure 16G, ultrasound stimulation is applied to a target region in the liver, providing protection against LPS-induced model hyperglycemia, limiting and / or controlling the increase in blood glucose concentration and regulating it below postprandial levels. Furthermore, this regulation can be anatomically specific. Figure 18B The results showed that directing ultrasound stimulation towards the right or left lobe of the liver reduced the ultrasound-induced effect on blood glucose. That is, not all areas of the liver respond to ultrasound energy application in the same way. In some implementations, energy application can be used as a protective therapy or as treatment administered before anticipated systemic challenges or disruptions.

[0218] Selective modulation of one or more target molecules can be achieved at sites directly stimulated by ultrasound, i.e., in organs including the target region. For example, targeted ultrasound stimulation of the liver in the target region induces changes in the liver concentration of signaling molecules within the liver tissue. Furthermore, molecules related to glucose metabolism can remain unchanged. Figure 18C (Gray bar). Additionally or alternatively, selective modulation of one or more target molecules can be achieved at distal sites not directly subjected to ultrasound stimulation. For example, Figure 18C The results showed that applying ultrasound stimulation to the liver could induce significant changes in the concentrations of NPY and NE in the hypothalamus, and further induce increased phosphorylation of ion channels that indicate increased activity of the insulin signaling pathway, which may indicate improved insulin sensitivity and improved glucose utilization.

[0219] Figure 18C Measurements of the relative concentrations (compared to no ultrasound stimulation) of various molecules associated with insulin sensitivity and insulin-mediated and non-insulin-dependent glucose uptake in the liver were displayed, along with changes in hypothalamic markers related to metabolic function. In the liver, epinephrine decreased relative to norepinephrine. Epinephrine can cause a rapid rise in blood glucose by driving the release of reserves in the liver. However, unlike the effect of norepinephrine on glucose uptake by skeletal muscle and fat, norepinephrine does not significantly contribute to hepatic glucose production. Increased norepinephrine in the hypothalamus can indicate increased hyperinsulinemia (indicating decreased insulin sensitivity) and glucose intolerance / hyperglycemia. Ultrasound stimulation can be used to selectively modulate or induce changes in the concentration of one or more target molecules in distal sites that are not directly stimulated. For example, as... Figure 18C As shown, when direct ultrasound stimulation is applied to a site in the liver, the stimulation can be used to modulate the concentrations of molecules such as norepinephrine (NE), protein kinase B (pAkt), insulin receptor substrate 1 (IRS-1), and neuropeptide Y (NPY) in the distal hypothalamus. In some embodiments, ultrasound stimulation is selectively applied to spatially selected tissue target regions to achieve desired (i.e., target) physiological outcomes. This tissue may be selected from the liver, pancreas, gastrointestinal tract, spleen, etc. The desired outcome may be a change in the concentration of clinically relevant molecules or biomarkers. In some embodiments, ultrasound-induced neural modulation can be quantified by the activity-dependent expression levels of the immediate early gene cFOS in the defined hypothalamus and brainstem subnuclear regions (respectively...). Figure 18D and 18E ). Figure 18DThe image shows a cFOS immunohistochemical image (left) and data showing the percentage of activated neurons in an LPS control (left) and an ultrasound-stimulated sample (right). In the control image, the percentage of activated neurons is approximately 7.7%, while in the stimulated image, the percentage of stimulated neurons is approximately 4.9%. Images and data are segmented on the paraventricular nucleus (PVN). The reduction in neural activity after ultrasound stimulation (represented by the decreased percentage of activated neurons) further corroborates this (except...). Figure 18C (In addition to the data, the effects of hepatic ultrasound neuromodulation on systemic glucose utilization and metabolic signal transduction were also examined.) Figure 18E Additional immunohistochemical images are shown, illustrating cFOS expression in the brainstem in an LPS control (top) and an ultrasound-stimulated sample (bottom). Images are segmented on the nucleus tractus solitarius (NTS), showing increased expression in the ultrasound-stimulated sample. Figure 18F This image shows an example MRI overlay between an activation map (exceeding the perturbation gradient recall echo volume; left) and a brain atlas (exceeding the perturbation gradient recall echo volume; right). The example shows increased ADCs in both the left and right paraventricular nuclei (PVN) of the hypothalamus (arrows, left image), consistent with neuronal inactivation. Figure 18G The results are summarized in a bar graph. Three out of six rats showed significant PVN inactivation; none of the control animals showed this inactivation. Furthermore, the hyperglycemia observed in U / S-treated animals, which was also observed in untreated animals, was not observed in U / S-treated animals.

[0220] Table 2 shows the t-test values ​​obtained from the comparison of ADC values ​​in validated PVN ROIs between pre- and post-treatment scans.

[0221]

[0222] Table 2

[0223] In 3 out of 6 rats, the increase in PVN ADC in response to ultrasound stimulation was largely consistent with c-Fos expression data, indicating the inactivation of the ultrasound-induced LPS activation pathway communicating with the hypothalamus. Only a few LPS+ultrasound mice showed this effect (rats 1, 4, and 5 in Table S2).

[0224] Compared with the control, cFOS was positive (c-Fos+) in the paraventricular nucleus (PVN); Figure 18D The significant reduction in NPY and GABA levels indicates ultrasound-induced modulation of LPS-induced neural signals. These data corroborate the significant decrease in NPY and GABA concentrations after ultrasound stimulation, as the arcuate nucleus (ARC) alters the signaling to the PVN via NPY-expressing neurons in response to peripheral sensory information. Furthermore, the altered expression of c-Fos in the hypothalamus was accompanied by a significant increase in c-Fos expression in the nucleus of the solitary tract (NTS). Figure 18EThis indicates the modulation mediated by signal transmission of ultrasound through the transmission pathway.

[0225] The apparent diffusion coefficient (ADC) of diffusion-weighted functional magnetic resonance imaging (DfMRI) images of the hypothalamic subnuclear area (PVN) was compared before and after hepatic ultrasound stimulation. ADC increased in response to ultrasound stimulation, confirming chemical data (POMC, NPY, and NE) and c-Fos expression data, which demonstrated the inactivation of the ultrasound-induced LPS activation pathway communicating with the hypothalamus. These results are consistent with the activation of this pathway, which modulates LPS-induced effects on energy metabolism and its influence on outgoing PVN signaling via the NPY system.

[0226] Chronic liver irritation

[0227] Results of liver stimulation in a diabetic Zucker (fa / fa) rat model are presented. Zucker rats are a model of type 2 diabetes and / or insulin resistance. Liver stimulation and biomarker analysis (i.e., circulatory and tissue) were performed on SD rats, as is typically provided herein, with the target region, as provided herein, located at or near the hepatic hilum. Circulatory non-terminal biomarkers were assessed by tail vein measurements. Figure 19 The image shows circulating non-fasting glucose levels in diabetic rats following liver stimulation. The time period shown is days 55–75. Ultrasound treatment began on day 56. Treatment was initiated after a one-day isolation period when the animals were in a pre-diabetic state. Ultrasound stimulation reduced circulating non-fasting blood glucose levels to levels consistent with those in non-diabetic rats. Figure 20 The study showed circulating triglycerides in diabetic rats after liver stimulation. Figure 21 The study showed circulating glucagon levels in diabetic rats after liver stimulation. Figure 22 The study showed circulating insulin in diabetic rats after liver stimulation. Figure 23 The study showed circulating leptin in diabetic rats after liver stimulation. Figure 24 The study shows circulating norepinephrine levels in diabetic rats following liver stimulation. Insulin levels in diabetic rats can range from approximately 3–10 μg / L. Normal non-fasting insulin levels typically range from 0.8–1.5 μg / L. Zucker rats are leptin receptor KOs but can still produce leptin spontaneously. Administration of leptin to patients with lipodystrophy-related diabetes (leptin deficiency) enhances insulin responsiveness and reduces hyperglycemia (by reducing gluconeogenesis). No changes were observed in glucagon, indicating a lack of alpha cell function in the pancreas. Liver ultrasound appears to induce beneficial changes in contrast to previous metformin treatment. For example, metformin treatment is generally not associated with a decrease in circulating insulin. In contrast, targeted liver ultrasound treatment resulted in observable changes in circulating glucose, insulin, and triglycerides. The absence of changes in norepinephrine suggests changes originating from local effects.

[0228] Figure 25-33 The concentrations of various terminal hypothalamic markers relative to controls were shown after liver ultrasound treatment. Figure 25 The hypothalamic insulin receptor substrate 1 (IRS-1) was shown after liver stimulation in diabetic rats. Figure 26 The study showed hypothalamic phosphorylation of Akt in diabetic rats following liver stimulation. Figure 27 The hypothalamus GLUT4 was shown after liver stimulation in diabetic rats. Figure 28 The study showed hypothalamic norepinephrine levels after liver stimulation in diabetic rats. Figure 29 The hypothalamus showed glucose-6-phosphate after liver stimulation in diabetic rats. Figure 30 The hypothalamic glucagon-like peptide (GLP-1) in diabetic rats after liver stimulation was shown. Figure 31 The study showed that the hypothalamus contained γ-aminobutyric acid (GABA) after liver stimulation in diabetic rats. Figure 32 The study showed that brain-derived neurotrophic factor (BDNF) in the hypothalamus was stimulated in diabetic rats after liver stimulation. Figure 33The study shows the hypothalamic neuropeptide Y (NPY) in diabetic rats following liver stimulation. IRS-1, phosphorylated-Akt, and GLUT4 signaling were significantly increased in the ultrasound-stimulated hypothalamus, consistent with previous results in LPS-induced hyperglycemia models. Glucagon-1 signaling was unchanged; these compounds typically increase insulin secretion, inhibit glucagon, and slow gastric emptying. Norepinephrine levels were not significantly changed in the treated animals compared to sham-stimulated diabetic animals. The conversion of glucose to glucose-6-phosphate via glucokinase in the hypothalamus may help regulate the first phase of glucose-responsive insulin secretion. The significant increase in IRS-PI3K-GLUT4 signaling in the ultrasound-stimulated hypothalamus is consistent with previous results in LPS-induced hyperglycemia models. Glucagon-1 signaling was altered. These compounds typically increase insulin secretion, inhibit glucagon, and slow gastric emptying. Norepinephrine levels were not significantly changed in the treated animals compared to sham-stimulated diabetic animals. The conversion of glucose to glucose-6-phosphate via glucokinase in the hypothalamus may help regulate the first phase of insulin secretion in response to glucose. Ultrasound stimulation is associated with a significant increase in the inhibitory neurotransmitter GABA. The observed elevation of BDNF, coupled with increased GABA expression, may explain the observed decrease in hypothalamic NPY. Neurons in non-diabetic rodents respond to glucose by increasing cellular uptake (through mechanisms including insulin-dependent and insulin-independent GLUT transporters) and through downstream mechanisms, which may include the conversion of glucose to glucose-6-phosphate for oxidative phosphorylation and ATP production, in which ATP-sensitive K+ channels are shut down, inhibiting GE neurons. Independent activation of the IRS-1 subunit or downstream mediators (e.g., Akt) may act as a “bypass” of the native glucose sensing mechanism, which could explain the sudden recovery of glucose sensing.

[0229] Figures 34-39 The concentrations of various terminal liver markers relative to controls were shown after liver ultrasound treatment. Figure 34 The liver IRS-1 in diabetic rats after liver stimulation is shown. Figure 35 The study showed liver phosphorylation of Akt in diabetic rats after liver stimulation. Figure 36 The liver glucose transporter 2 (GLUT2) was shown after liver stimulation in diabetic rats. Figure 37 The study showed hepatic norepinephrine levels after liver stimulation in diabetic rats. Figure 38 The liver glucose-6-phosphate after liver stimulation was shown in diabetic rats. Figure 39The study showed liver GLP-1 levels in diabetic rats after liver stimulation. A significant decrease in hepatic norepinephrine was found, independent of any observed changes in insulin-mediated glucose uptake, glucagon release, or glycolytic activity.

[0230] Figures 41-45 The concentrations of various terminal pancreatic markers relative to controls were shown after liver ultrasound treatment. Figure 40 The study showed glucagon levels in the pancreas after liver stimulation in diabetic rats. Figure 41 The study showed pancreatic insulin levels following liver stimulation in diabetic rats. Figure 42 The study showed pancreatic leptin levels after liver stimulation in diabetic rats. Figure 43 The pancreatic IRS-1 in diabetic rats after liver stimulation is shown. Figure 44 The pancreas GLUT2 in diabetic rats after liver stimulation is shown. Figure 45 The study showed pancreatic phosphorylation of Akt in diabetic rats following liver stimulation.

[0231] Figure 46 The results of repeated treatment in the Zucker rate model are shown. Rats were confirmed to have prediabetes by age (~8 weeks / 56 days). The initial treatment group consisted of ultrasound stimulation once daily for three 1-minute cycles using the parameters provided in this paper. Treatment was initiated upon arrival of prediabetes and continued for 15 days before discontinuation. Animals (grey circles) were maintained under observation for 21 days, and changes in circulating glucose and insulin were tracked.

[0232] While treatment was discontinued in the prediabetic group, animals exhibiting severe glucose concentrations (≥500 mg / dL) who had undergone sham ultrasound stimulation were initiated with ultrasound therapy (once daily, three 1-minute cycles) and glucose and insulin changes were monitored over the same 21-day period. Results showed that circulating glucose in animals that had previously received ultrasound stimulation increased slowly. However, 8 days after the termination of ultrasound stimulation, circulating glucose in these animals stabilized at ~400 mg / dL. Although these values ​​are consistent with severe diabetes, they were still significantly lower than in age-matched Zucker rats compared to diabetic controls. Therefore, the repeated treatment presented in this study can produce a sustained effect in adjusting the glucose regulation set point.

[0233] The techniques disclosed herein utilize the natural hierarchical structure and organization of the nervous system, thereby allowing for precise neural modulation through simple, non-invasive techniques. Although demonstrated for two specific neural pathways (CAP in the spleen and metabolic sensory neurons in the liver), the technique can be used to modulate other peripheral neural circuits.

[0234] This written specification uses examples to disclose the invention and also enables any person skilled in the art to practice the invention, including making and using any apparatus or system and implementing any incorporated methods. The patentable scope of the invention is defined by the claims, but may include other examples that would occur to those skilled in the art. Such other examples are intended to fall within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements that do not substantially differ from the literal language of the claims.

Claims

1. A regulating system, comprising: An energy application device configured to apply energy to a first target region of a subject, the first target region being a subregion of an organ containing synapses between neurons and corresponding non-neuronal cells, the organ being the liver, and the first target region including the hepatic hilum; and The controller is configured to: Select the first target area in space; The energy is focused on the first target area; and The energy can be tunably controlled to be repeatedly applied to the first target region via the energy application device to induce repeated preferential activation of a subset of the synapses located in the first target region over a predetermined time period, to cause a sustained change in one or more target molecules, including blood glucose, after the repeated application of the energy, and the sustained change includes a decrease in the subject's blood glucose concentration.

2. The system according to claim 1, wherein, The controller includes: processor; and The memory stores instructions configured to be executed by the processor to spatially select the first target region, focus the energy on the first target region and the second target region, or control the application of the energy, or combinations thereof.

3. The system according to claim 1, wherein, The controller is configured to receive an input indicating the concentration of a first molecule from the evaluation device.

4. A regulating system, comprising: An energy application device configured to apply energy to a target region of a subject, the target region being a subregion of an organ containing synapses between neurons and corresponding non-neuronal cells, the organ being the liver, and the target region including the hepatic hilum; and The controller is configured to: Select the target area spatially; The energy is focused on the target area; and Repeatedly controlling the application of energy to the target region via the energy application device to induce preferential activation of a subset of the synapses located in the target region for a predetermined time period, to cause a sustained change in one or more target molecules, including blood glucose, after repeated application of the energy, the sustained change including a decrease in the subject's blood glucose concentration.

5. A system for treating diabetes in a subject, the system comprising: An ultrasonic energy application device configured to apply an ultrasonic dosing scheme to a target region of an internal organ, the internal organ being the liver, and the target region being the porta hepatis; and A controller adapted to control the ultrasound energy application device to apply the ultrasound dosage scheme, wherein the ultrasound dosage scheme includes multiple energy doses applied at individual time points within a time window of the ultrasound dosage scheme to induce a sustained change in one or more target molecules, including blood glucose, after the application of the multiple energy doses, and the sustained change includes a decrease in the blood glucose concentration of the subject.

6. A regulating system, comprising: An energy application device configured to apply energy to a target region of a subject, the target region being a subregion of an organ containing synapses between neurons and corresponding non-neuronal cells, the organ being the liver, and the target region being the hilum of the liver; and The controller is configured to: Select the target area spatially; The energy is focused on the target area; and Repeatedly controlling the application of energy to the target region via the energy application device to apply a low duty cycle energy dosing regimen to the target region, wherein the low duty cycle dosing regimen includes multiple electrical stimulations spaced apart by an adjustable disconnection period of at least 4 hours to induce a sustained change in one or more target molecules, including blood glucose, after repeated application of the multiple electrical stimulations, the sustained change including a decrease in the subject's blood glucose concentration, wherein the disconnection period is determined at least in part based on feedback received by the controller.

Images