Techniques for neural regulation

Abstract

The subject matter of this disclosure generally relates to techniques for neuromodulation of tissue, which involve applying energy (e.g., ultrasound energy) within the tissue to alter activity at synapses between neuronal and non-neuronal cells. [Solution] An energy application device configured to apply energy to a target region of tissue of a subject, the tissue including a plurality of axon terminals of respective neurons, the axon terminals forming axonal extracellular synapses between individual axon terminals and respective non-neuronal cells, and an energy application device for spatially selecting the target region of the tissue, the target region including a subset of the plurality of axon terminals and an associated subset of the axonal extracellular synapses.

Description

Cross-reference with related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 415,212, filed on 31 October 2016, which is incorporated herein by reference in its entirety for all purposes. Government support statement.

[0002] This invention was made with government support under grant number W911NF-09-1-0125, awarded by the Defense Advanced Research Projects Agency (DARPA). The government has certain rights in this invention. [Background technology]

[0003] The subject matter disclosed herein relates to neuromodulation, and more specifically, to techniques for modulating physiological responses using energy applied from an energy source.

[0004] Neuromodulation has been used to treat a variety of clinical conditions. For example, electrical stimulation at various locations along the spinal cord has been used to treat chronic back pain. Such treatment may be performed by an implantable device that periodically generates electrical energy applied to tissue to activate specific nerve fibers, which can result in a reduction of pain sensation. In the case of spinal cord stimulation, the stimulating electrodes are generally placed in the epidural space, but the pulse generator can be placed somewhat away from the electrodes, for example in the abdominal or gluteal region, but connected to the electrodes via wires. In other implementation forms, deep brain stimulation can be used to stimulate specific areas of the brain to treat motor disorders, and the stimulation site may be guided by neuroimaging. Such central nervous system stimulation generally targets a local nerve or brain cell function, is mediated by electrodes that deliver electrical pulses, and are placed on or near the target nerve. However, placing electrodes on or near the target nerve is difficult. For example, such techniques may involve the surgical placement of electrodes that deliver energy. Furthermore, targeting specific tissues via neuromodulation is difficult. Electrodes placed on or near specific target nerves mediate neuromodulation by inducing action potentials within nerve fibers, resulting in neurotransmitter release at nerve synapses and synaptic connections with subsequent nerves. Such propagation can produce relatively large or more diffuse physiological effects than desired, as current implementations of implanted electrodes stimulate many nerves or axons at once. Because neural pathways are complex and interconnected, more targeted modulating effects may be more clinically useful. [Overview of the Initiative]

[0005] The following is a summary of specific embodiments that are equivalent in subject matter and scope to those originally claimed. These embodiments are not intended to limit the scope of the claimed subject matter, but rather are intended only to provide a brief summary of possible embodiments. In fact, this disclosure may encompass a variety of forms that may be similar to or different from the embodiments described below.

[0006] In one embodiment, a system is provided comprising an energy delivery device configured to apply energy to a target region of a subject's tissue, the tissue comprising multiple axon terminals of each neuron, the axon terminals forming synapses (e.g., extra-axonal cells or other synaptic types) between individual axon terminals and their respective non-neuronal cells. The system also comprises a controller configured to spatially select a target region of the tissue, the target region comprising a subset of multiple axon terminals and a subset of associated synapses; to focus energy on the target region; and to control the application of energy to the target region via the energy delivery device to induce preferential activation of the subset of associated synapses and produce a target physiological outcome.

[0007] In another embodiment, a method is provided which includes the steps of: directing an energy application device towards an organ or peripheral tissue; focusing the energy onto a target region of the organ or peripheral tissue, the target region being a small region of peripheral tissue or organ containing synapses between nerve cells and non-nerve cells; and applying pulsed power to the energy application device via a pulse generator to deliver energy to at least one of nerve cells or non-nerve cells at a type of synapse selected to achieve a target physiological outcome, thereby modulating their activity.

[0008] Another embodiment provides a method comprising the steps of: positioning an energy delivery device in a location where it can modulate tissue; focusing the energy delivery device onto a target region within the tissue; and applying one or more energy pulses within the target region to alter the activity at synapses between nerve cells and non-nerve cells relative to baseline activity before applying one or more energy pulses in response to one or more energy pulses. [Brief explanation of the drawing]

[0009] These and other features, aspects, and advantages of the present invention will be better understood by reading the following detailed description with reference to the attached drawings, where the same letters represent the same parts.

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

[0011] [Figure 2] This is a block diagram of a neural control 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 4] This embodiment of the present disclosure is an ultrasound visualization of the spleen that can be used as spatial information to focus on a target region within the spleen.

[0014] [Figure 5] This is a flowchart of the neuromodulation technology according to the embodiments of this disclosure.

[0015] [Figure 6] This is a schematic diagram of an energy application device, configured as an external device, which includes an ultrasonic transducer.

[0016] [Figure 7] This is a schematic diagram of an energy application device and pulse generator configured to apply high-density focused ultrasound.

[0017] [Figure 8] This is an example of an energy application device that can be used with the system shown in Figure 7.

[0018] [Figure 9]Schematic diagram of an experimental setup for applying ultrasonic energy to achieve a targeted physiological result.

[0019] [Figure 10] Experimental timeline of ultrasonic energy application.

[0020] [Figure 11] Diagram showing the pulse characteristics of the applied ultrasonic energy pulses.

[0021] [Figure 12] Diagram showing the hydrophone measurement setup.

[0022] [Figure 13] Diagram showing an example of the ultrasonic pressure field in the x-y plane.

[0023] [Figure 14] Diagram showing the experimental workflow of LPS injection for generating models of inflammation and / or hyperglycemia / hyperinsulinemia and ultrasonic treatment.

[0024] [Figure 15] Diagram showing examples of T1 / DWI images in rats along with the cross-correlation coefficient for each slice.

[0025] [Figure 16] Diagram showing the results of standard electrode stimulation on the ipsilateral and contralateral popliteal lymph nodes and the distal axillary lymph nodes.

[0026] [Figure 17] Diagram showing the results of comparing the number of lymphocytes in the non-stimulated popliteal lymph nodes with respect to the directly stimulated popliteal lymph nodes after ultrasonic stimulation.

[0027] [Figure 18] Diagram showing the concentrations of norepinephrine, epinephrine, and dopamine in the lymph node dissociation tissue after ultrasonic stimulation.

[0028] [Figure 19] This figure shows the concentrations of norepinephrine, epinephrine, acetylcholine, and dopamine in the spleen after ultrasound stimulation.

[0029] [Figure 20] This figure shows the splenic concentration of TNF-α in relation to peak ultrasound pressure.

[0030] [Figure 21A] This figure shows the splenic concentration of norepinephrine in relation to peak ultrasound pressure.

[0031] [Figure 21B] This figure shows the splenic concentration of acetylcholine in relation to peak ultrasound pressure.

[0032] [Figure 21C] This figure shows the splenic concentration of TNF-α in relation to peak ultrasound pressure.

[0033] [Figure 22] This figure shows the circulating concentration of TNF-α in relation to the peak ultrasonic pressure.

[0034] [Figure 23] This figure shows the concentration of TNF-α in the liver of stimulated and LPS-treated animals compared to a control group of unstimulated but LPS-treated animals.

[0035] [Figure 24] This table shows cytokine changes compared to a control group for different time-averaged intensities of ultrasound treatment at the treatment site.

[0036] [Figure 25] This figure shows the percentage change in the phosphorylation state of secondary messenger molecules compared to an LPS-treated control for different peak pressures of spleen ultrasound treatment.

[0037] [Figure 26] This figure shows the splenic concentration of TNF-α in stimulated LPS-treated animals under the condition of pathway blockade.

[0038] [Figure 27] This figure shows the splenic concentration of norepinephrine in stimulated LPS-treated animals under the condition of pathway blockade.

[0039] [Figure 28] This figure shows the splenic concentration of TNF-α in stimulated LPS-treated animals compared to animals treated with vagus nerve transection or pathway blockade.

[0040] [Figure 29] This figure shows the splenic concentration of TNF-α at various time points after ultrasound treatment combined with LPS treatment.

[0041] [Figure 30] This is an experimental timeline for LPS treatment after ultrasound treatment.

[0042] [Figure 31] This figure shows the splenic concentration of TNF-α at various time points after ultrasound and LPS treatment, according to the timeline in Figure 30.

[0043] [Figure 32] This figure shows the splenic concentration of TNF-α in non-invasive ultrasound-stimulated animals and counter-electrode-stimulated animals.

[0044] [Figure 33] These are ultrasound images used to identify the target area and ultrasound placement for the ultrasound procedures shown in Figures 35A and 35B.

[0045] [Figure 34] This is a diagram showing the setup for a controlled electrical stimulation experiment.

[0046] [Figure 35A] This figure shows the splenic concentration of TNF-α in non-invasive ultrasound-stimulated animals versus electrode-stimulated control animals under LPS treatment conditions. Electrode stimulation was performed according to the setup shown in Figure 34.

[0047] [Figure 35B] This figure shows the circulating glucose concentration of non-invasive ultrasound-stimulated animals versus electrode-stimulated control animals under LPS treatment conditions, with electrode stimulation performed according to the setup shown in Figure 34.

[0048] [Figure 36] This figure shows the effect of VNS electrode stimulation versus ultrasound-induced splenic nerve modulation on heart rate compared to unstimulated animals.

[0049] [Figure 37A] This figure shows the effect of ultrasound-induced hepatic hilar hepatic regulation on blood glucose compared to a control group.

[0050] [Figure 37B] This figure shows the effect of ultrasound-mediated hepatic regulation at the hepatic hilum on the liver concentration of various molecules.

[0051] [Figure 37C] This figure shows the effect of ultrasound-mediated hepatic regulation at the hepatic hilum on the liver concentration of various molecules.

[0052] [Figure 38A] This figure shows the effect of ultrasound-mediated hepatic hilar regulation on the concentration of various molecules in the brain.

[0053] [Figure 38B] This figure shows the effect of ultrasound-mediated hepatic hilar regulation on the concentration of various molecules in the brain.

[0054] [Figure 38C] This figure shows the effect of ultrasound-mediated hepatic hilar regulation on the concentration of various molecules in the brain.

[0055] [Figure 39A]This figure shows the effect of ultrasound-controlled liver function at various locations on glucose concentration.

[0056] [Figure 39B] This figure shows the adjustment position of the target area in Figure 39A.

[0057] [Figure 40] This figure shows that splenic ultrasound stimulation in LPS-treated animals does not produce the same effects as those seen in the liver.

[0058] [Figure 41A] This figure shows the hypothalamic NPY concentration in control animals versus LPS-treated animals.

[0059] [Figure 41B] This figure shows the hypothalamic POMC concentration in control animals versus LPS-treated stimulated animals.

[0060] [Figure 42A] This figure shows coronal sections from unstimulated LPS pseudoanimals.

[0061] [Figure 42B] This figure shows coronal sections from animals treated with stimulated LPS.

[0062] [Figure 43] This figure shows DfMRI data from animals treated with stimulated LPS.

[0063] [Figure 44] This figure shows DfMRI data from unstimulated LPS dummy animals.

[0064] [Figure 45A] This figure shows cFOS and Glut1 staining in the control hippocampus.

[0065] [Figure 45B] This figure shows cFOS and Glut1 staining in stimulated hippocampus.

[0066] [Figure 45C] This figure shows the fluorescent staining RFU of the image in Figure 45A compared to the image in Figure 45B.

[0067] [Figure 46] This figure shows hippocampal norepinephrine levels after liver stimulation in LPS-treated animals treated with pathway blockers.

[0068] [Figure 47] This figure shows hippocampal norepinephrine levels after treatment with agonists targeting diabetic drug targets in LPS-treated animals.

[0069] [Figure 48] This figure shows tissue displacement tracked using a reference ultrasound signal.

[0070] [Figure 49] This figure shows shear wave measurements obtained from the spleen stimulation site.

[0071] [Figure 50] This figure shows displacement measurements obtained from the spleen stimulation site.

[0072] [Figure 51] This figure shows the splenic norepinephrine concentration in animals treated with reduced ultrasound clinical efficacy.

[0073] [Figure 52] This figure shows the spleen TNF-α concentration in animals treated with reduced ultrasound clinical efficacy.

[0074] [Figure 53] This figure shows the blood norepinephrine concentration in animals treated with reduced ultrasound clinical efficacy.

[0075] [Figure 54]This figure shows the blood TNF-α concentration in animals treated with reduced ultrasound clinical efficacy.

[0076] [Figure 55] This figure shows the spleen TNF-α concentration in animals treated with a modified pulse sequence.

[0077] [Figure 56] This figure shows the splenic norepinephrine concentration in animals treated with a modified pulse sequence.

[0078] [Figure 57] This figure shows the blood TNF-α concentration in animals treated with a modified pulse sequence.

[0079] [Figure 58] This figure shows the blood norepinephrine concentration in animals treated with a modified pulse sequence.

[0080] [Figure 59] This figure shows the splenic norepinephrine concentration compared to a control after direct mechanical vibration of the spleen.

[0081] [Figure 60] This figure shows the spleen TNF-α concentration compared to a control after direct mechanical vibration of the spleen. [Modes for carrying out the invention]

[0082] One or more specific embodiments are described below. In our efforts to provide a concise description of these embodiments, not all features of actual implementations are described herein. Naturally, in developing such actual implementations, as in engineering or design projects, numerous implementation-specific decisions must be made to achieve developer-specific goals, such as compliance with system-related and business-related constraints, and these will differ from implementation to implementation. Furthermore, while such development efforts can be complex and time-consuming, it should be understood that they are nonetheless routine design, fabrication, and manufacturing tasks for those skilled in the art who benefit from this disclosure.

[0083] No example or illustration provided herein should ever be considered to represent any restriction, limitation, or definition of any term used herein. Rather, these examples or illustrations should be considered to describe various specific embodiments and should be considered merely illustrative. Those skilled in the art will recognize that the terms used herein encompass other embodiments, which may or may not be given with them or elsewhere in the specification, and that all such embodiments are intended to fall within the scope of those terms. Words designating such non-limiting examples and illustrations include, but are not limited to, “for example,” “for instance,” “etc.,” “e.g.,” “including,” and “in one embodiment.”

[0084] This technique relates to the modulation of synapses at axon terminals within tissues via the application of energy from an energy source. For example, this may include extra-axonal cell synapses formed between presynaptic axon terminals and postsynaptic nonneurons. Furthermore, while the specific embodiments disclosed are discussed in the context of extra-axonal cell synapses, it should be understood that axon terminals may form axon-secretory, axon-synaptic, inter-axonal cell body, or extra-axonal cell synapses, and in addition, or alternatively, these synaptic types are intended to be selectively modulated as provided herein. Moreover, certain axon terminals may terminate in interstitial fluid or body fluids, and neurotransmitter release may also occur as a result of the modulation. The disclosed synapses can be modulated to alter intra-synaptic activity, for example, neurotransmitter release from presynaptic axon terminals. In other words, altered activity can result in local and / or non-local (e.g., systemic) effects. This technology enables the focusing of energy in a manner that targets a volume of tissue containing specific axon terminals in order to preferentially and directly activate target axon terminals to achieve a desired outcome. In this way, target axon terminals within the region of interest are activated, while in certain embodiments, axon terminals located outside the region of interest, even if they are within the same organ or tissue structure, are not activated. Since organs and tissue structures may contain different types of axon terminals that synapse with different types of postsynaptic nonneurons, a region of interest containing axon terminals that, when activated, produce the desired target physiological outcome can be selected. Thus, the modulation can target specific types of axon terminals based on presynaptic neuron type, postsynaptic cell type, or both.

[0085] For example, in one embodiment, the type of axon terminal may be an axon terminal that forms an extra-axon cell synapse with an endogenous (i.e., tissue-endogenous or acircumcirculatory) immune cell. That is, an extra-axon cell synapse is formed at the junction between the axon terminal and a non-neuronal cell or interstitial fluid or body fluid. Therefore, the application of energy results in the modulation of immune function in the target region. However, it should be understood that different targeted physiological effects may be achieved based on the characteristics of the axon terminal population and the presynaptic neuron type and postsynaptic cells (e.g., immune cells, lymphocytes, mucosal cells, muscle cells, etc.) of the extra-axon cell synapses. Therefore, applying energy to a target region in the subject's tissue may activate the axon terminals within the target region and their associated extra-axon cell synapses, but the non-target axon terminals (and associated synapses) outside the target region will not be affected. However, since the modulation can have systemic effects, certain systemic changes may occur in the non-target axon terminals outside the target region as a result of the activation of the axon terminals within the target region. Where used herein, preferential activation or direct activation may refer to cells or structures that experience direct energy application within a region of interest, namely axon terminals, extra-axonal cell synapses, and / or postsynaptic nonneurons, or interstitial fluid or body fluids that directly experience applied energy, as provided herein.

[0086] The human nervous system is a complex network of nerve cells, or neurons, found in the central nervous system of the brain and spinal cord, and peripherally within various nerves throughout the body. Neurons have a cell body, dendrites, and axons. A nerve is a group of neurons that act on a specific part of the body. A nerve can contain hundreds or even hundreds of thousands of neurons. Nerves often contain both afferent and efferent neurons. Afferent neurons carry signals to the central nervous system, while efferent neurons carry signals to the periphery. A group of nerve cell bodies in a given location is known as a ganglion. Electrical signals generated within a nerve (e.g., via stimuli that can be applied endogenously or externally) are conducted through neurons and nerves. Neurons release neurotransmitters at synapses (junctions) adjacent to receptor cells to enable the continuation and regulation of electrical signals. Peripherally, synaptic transmission often occurs in ganglia.

[0087] The electrical signals of neurons are known as action potentials. An action potential is initiated when the potential across the cell membrane exceeds a certain threshold. This action potential then propagates along the length of the neuron. A nerve action potential is complex and represents the sum of the action potentials of the individual neurons within it. The junction between the axon terminal of a neuron and a receptor cell is called a synapse. The action potential travels down the neuron's axon to its axon terminal, i.e., the distal end of the branch of the axon nerve that forms the presynaptic end, or to the synaptic aneurysm of the nerve fiber. The electrical impulse of the action potential induces the migration of vesicles containing neurotransmitters from the presynaptic axon terminal to the presynaptic membrane, ultimately inducing the release of neurotransmitters into the synaptic cleft (e.g., the space formed between the presynaptic and postsynaptic cells) or extra-axonal cell space. The synapse that reaches the synaptic aneurysm to convert the electrical signal of the action potential into a chemical signal for neurotransmitter release is called a chemical synapse. Chemical synapses may be in contrast to electrical synapses, in which ionic currents flowing into the presynaptic axon terminal can cross the barrier between the two 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 have Na₂ ions passing through the neuronal membrane. + , K + , and Cl ‐ The resting membrane potential is actively maintained via ion pumps that facilitate the movement of ions. Different types of neurons may maintain different resting potentials, e.g., between -75mV and -55mV. Action potentials are generated by the movement of charges that produce a large deviation in membrane potential, associated with the influx of ions, i.e., a transient increase in voltage across the membrane (e.g., an increase in membrane potential within the range of 30-60mV). Action potentials in individual neurons may be initiated in response to the release of neurotransmitters from presynaptic (e.g., upstream) neurons, resulting in a cascade of events leading to receptor binding in the postsynaptic cell, as well as membrane depolarization resulting in the influx of ions and the propagation of action potentials through the nerve.

[0089] Synapses can be located at the junction between two neurons, allowing action potentials to propagate along nerve fibers. However, axon terminals can also form synapses at the junction between nerve cells and non-nerve cells, or terminate in interstitial fluid or body fluid. Examples of synaptic types include synapses with immune cells at neuroimmune junctions, synapses with sensory cells present within organs, or synapses with glandular cells. The release of neurotransmitters into the synaptic cleft and the binding of postsynaptic cells to receptors on the postsynaptic membrane result in downstream effects that depend on the properties of the presynaptic neuron and the specific neurotransmitters released, as well as the properties of the postsynaptic cell (e.g., the types of receptors available to the postsynaptic cell). Furthermore, action potentials can be excitatory or inhibitory. An excitatory postsynaptic action potential is a postsynaptic potential that makes the postsynaptic neuron more likely to fire or release the next action potential, while an inhibitory postsynaptic action potential is a postsynaptic potential that makes the postsynaptic neuron less likely to fire or release the next action potential. Furthermore, several neurons may work together to induce downstream action potentials or release neurotransmitters that inhibit downstream action potentials.

[0090] Neuromodulation is a technique that applies energy from an external energy source to specific regions of the nervous system to activate or increase nerves or nerve function, and / or block or decrease nerve function. In certain neuromodulatory techniques, one or more electrodes are applied to or near a target nerve, and the applied energy is carried through the nerve (e.g., as action potentials) to elicit a physiological response in a region downstream of the energy application site. However, because the nervous system is complex, it is difficult to predict the range and ultimate endpoint of the physiological response for a given energy application site.

[0091] While strategies for ultrasound modulation of the central nervous system (i.e., brain tissue) have demonstrated success in regulating neural activity, attempts to modulate peripheral nerves have lagged behind. For example, ultrasound modulation of the central nervous system (CNS) involves stimulating cortical regions of the brain where synaptic structures are abundant, whereas attempts at ultrasound stimulation of peripheral nerves target nerve trunks where synaptic structures are less numerous or absent. In this technique, peripheral nerve modulation involves targeting one or more peripheral axon terminals. Furthermore, instead of targeting traditional neuron-neuronal synapses in peripheral tissue where stimulation leads to the propagation of neurogenic action potentials, this technique applies one or more energy pulses to internal tissue of the subject, including axon terminals that include extra-axonal cell synapses, or neuronal junctions with other cell types, interstitial fluid, or body fluids, such as neuroimmune synapses, where stimulation of the axon terminal releases neurotransmitters / neuropeptides or induces altered neurotransmitter release in the vicinity of adjacent non-neuronal cells such as immune cells or other cells, thereby regulating cell activity. Furthermore, through such modulation, modulation of other tissue structures or organs can be achieved without direct stimulation. In one embodiment, direct energy application to a relatively small region of an organ (e.g., less than 25% of the total organ volume) can result in the stimulation of action potentials in afferent projecting neurons projecting into different regions of the brain (e.g., the hypothalamus). However, this result can be achieved without direct brain stimulation of synapse-rich regions. Direct brain stimulation can lead to undesirable activation of other pathways that may interfere with or overwhelm the desired physiological outcome. Therefore, this technique enables granular activation of either brain activity or intra-organ activity in a more targeted and specific manner than direct brain stimulation or electrical peripheral nerve stimulation.

[0092] The advantages of this technique include localized modulation of a target area of ​​tissue to achieve localized and / or systemic effects. Furthermore, localized modulation may involve the direct activation of 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 certain embodiments, the applied energy may come from a non-invasive extracorporeal energy source (e.g., an ultrasonic energy source). For example, a focused energy probe can apply energy through the skin of the subject and focus it on a target area of ​​internal tissue. Such embodiments achieve the desired physiological outcome without invasive procedures or side effects that may be associated with other types of procedures or treatments.

[0093] The local modulation of the disclosed technique was demonstrated compared to other modulation techniques, such as electrical stimulation via electrodes. For example, stimulation of neural pathways that modulate systemic inflammation was demonstrated without out-of-target stimulation of pathways involved in metabolic regulation (previous electrical stimulation experiments on peripheral nerves failed to isolate these out-of-target effects). Furthermore, the technique was demonstrated to induce a variety of clinically useful physiological outcomes, both local and systemic.

[0094] Provided herein are techniques for neuromodulation in which energy from an energy source (e.g., an external or extracorporeal energy source) is applied to an axon terminal in such a way that the neurotransmitter released at the energy application focal site, e.g., the axon terminal, is induced in response to the application of energy rather than to an action potential. That is, the direct application of energy to the axon terminal acts in place of an action potential to promote the release of neurotransmitters to neuroimmune junctions or other neuronal junctions with non-neuronal cells. The direct application of energy to the axon terminal further induces altered neurotransmitter release from the axon terminal within an extra-axonal cell synapse to the vicinity of an adjacent non-neuronal cell. In one embodiment, the energy source is an extracorporeal energy source such as an ultrasonic energy source or a mechanical transducer. In this way, non-invasive and targeted neuromodulation can be achieved directly at the energy focal site rather than through modulation at an upstream site, which then induces an action potential that activates a downstream target.

[0095] This technology can be used in conjunction with the modulation of any neuroimmuno-interface (e.g., junctions or synapses between axon terminals and immune cells), as well as with the modulation of lymph node neuromodulation or lymphocyte retention neuroimmune reflexes. Furthermore, direct modulation of immune cells themselves, such as immune cells that are not part of a junction with a neuron, is also intended. While specific embodiments of this disclosure are presented in the context of neuroimmunomodulation, it should be understood that the disclosed technology may be used with other target tissues and other types of non-neuronal cells. As described herein, non-neuronal cells may include immune cells, endothelial cells, secretory cells, and the like.

[0096] In certain embodiments, the target tissue is internal tissue or organ that is difficult to access using electrical stimulation techniques. Other intended tissue targets include gastrointestinal tissue (stomach, intestines), muscle tissue (heart, smooth and skeletal), epithelial tissue (epidermis, organ / gastrointestinal lining), connective tissue, and glandular tissue (exocrine / endocrine). In one example, concentrated energy application at the neuromuscular junction promotes neurotransmitter release at the neuromuscular junction without upstream action potentials. Intended regulatory targets may include parts of the spleen involved in regulating TNF-α release from macrophages, sites within the adrenal gland for regulating dopamine release, or local sites within the mesenteric plexus that regulate inflammation and macrophage function in the intestines. Furthermore, neuroimmune interfaces that can control antibody production or lymphocyte functional state can be regulated via ultrasound energy.

[0097] To this end, the disclosed neuromodulation techniques may be used in conjunction with a neuromodulation system. Figure 1 is a schematic diagram of a system 10 for neuromodulation to achieve neurotransmitter release and / or activation of components within extra-axonal cell synapses in response to energy application. The illustrated 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 wires or wireless connections, which, when used, are directed to a target area of ​​internal tissue or organ in the subject, thereby producing the desired physiological outcome. In certain 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 wires 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 micromachine ultrasonic transducer.

[0098] In certain embodiments, the energy delivery device 12 and / or pulse generator 14 can communicate wirelessly with a controller 16, for example, which can sequentially provide commands to the pulse generator 14. In other embodiments, the pulse generator 14 may be an external device, for example, which can be operated to deliver energy percutaneously or non-invasively from a location outside the subject's body, and in certain embodiments, it can be integrated into the controller 16. In embodiments where the pulse generator 14 is external, the energy delivery device 12 may be operated by a caregiver and positioned on or above the subject's skin so that energy pulses are delivered percutaneously to desired internal tissue. Once positioned to deliver energy pulses to the desired site, the system 10 can initiate neuromodulation to achieve a target physiological outcome or clinical effect.

[0099] In certain embodiments, the system 10 may include an evaluation device 20, which is coupled to the controller 16 and evaluates features indicating whether the target physiological outcome of the modulation has been achieved. In one embodiment, the target physiological outcome may be local. For example, modulation may result in local tissue or functional changes such as changes in tissue structure, local increases in the concentration of specific molecules, tissue displacement, and increased fluid movement. Modulation may result in systemic or non-local changes, and the target physiological outcome may relate to changes in the concentration of circulating molecules or changes in tissue features that do not involve a target area to which energy is directly applied. In one example, displacement may be a surrogate measure for the success of modulation, and a displacement measurement below the expected displacement value may result in a modification of the modulation parameter until the expected displacement value is observed.

[0100] In embodiments where energy is applied to the neuroimmune junction, the modulation can also result in changes in immune function, such as changes in the population of immune cells or changes in the presence or concentration of compounds in lymphoid tissue. Based on evaluation, the modulation parameters of the controller 16 can be modified. For example, if successful modulation is associated with a decrease in norepinephrine or tumor necrosis factor concentration within a defined time frame (e.g., 5 minutes, 30 minutes after the start of treatment) compared to the start of treatment, then changes in frequency or other parameters may be desirable, which may be provided to the controller 16 by the operator to define or adjust the energy pulses of the pulse generator 14.

[0101] The system 10 provided herein can deliver energy pulses according to various control parameters. For example, the control parameters may include various stimulation time patterns ranging from continuous to intermittent. In intermittent stimulation, energy is supplied at a constant frequency for a certain period during the signal-on time. Following the signal-on time is a period without energy supply, called the signal-off time. The control parameters may also include the frequency and duration of stimulation application. The application frequency may be continuous or delivered over various periods, for example, within a day or within a week. The duration of treatment may last over various periods, including but not limited to several minutes to several hours. In certain embodiments, the duration of treatment using a given stimulation pattern may last for one hour and be repeated, for example, at 72-hour intervals. In certain embodiments, treatment may be delivered at a higher frequency, for example, every three hours, with a shorter duration, for example, 30 minutes. The duration and frequency of treatment may be controlled to be adjustable in order to achieve the desired results.

[0102] Figure 2 is a block diagram of a specific component of system 10. As provided herein, system 10 for neuromodulation may include a pulse generator 14 adapted to generate multiple energy pulses for application to the tissue of a subject. The pulse generator 14 may be separate 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 the memory 32 of the controller 16 and executed by the processor 30 to control various components of the device. The controller 16 and / or the pulse generator 14 may be connected to an energy application device 12 via one or more wires 33.

[0103] The controller 16 also includes a user interface having input / output circuits 34 and a display 36 adapted to allow a clinician 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 control device 16 to alter the stimulating characteristics of the energy pulses transmitted to the subject via the conductors 33. Any suitable type of pulse generation circuit can be used, including constant current, constant voltage, multiple independent current, or voltage sources. The applied energy is a function of current amplitude and pulse width duration.

[0104] In one embodiment, memory 32 stores different operating modes that can be selected by the operator. For example, a stored operating mode may include instructions for executing a set of modulatory parameters associated with a particular treatment site. Different sites may have different associated modulatory parameters. Rather than requiring the operator to manually input a mode, the controller 16 may be configured to execute the appropriate instructions based on the selection. In another embodiment, memory 32 stores operating modes for different types of treatment. For example, activation may be associated with different stimulation pressures or frequency ranges for those related to tissue dysfunction or blockage. In a specific example, if the energy application device is an ultrasonic transducer, the time-averaged power and peak positive pressure may be 1 mW / cm². 2 ~30,000 mW / cm 2 The amplitude is within the range of 0.1 MPa to 7 MPa. In another specific example, if the energy application device is a mechanical actuator, the vibration amplitude is within the range of 0.1 to 10 mm. The selected frequency may depend on the mode of energy application, e.g., ultrasonic or mechanical actuator.

[0105] In another embodiment, memory 32 stores calibration or setting modes that allow adjustment or modification of control parameters to achieve a desired result. In one example, the stimulus starts with a lower energy parameter and gradually increases automatically or upon receiving operator input. In this way, the operator can observe the effect as the control parameter is changed.

[0106] The system may also include an imaging device to facilitate focusing of the energy application device 12. In one embodiment, the imaging device may be integrated with the energy application device 12, or it may be the same device as the energy application device 12, so that different ultrasound parameters (frequency, aperture, or energy) are applied for targeting and subsequent neuromodulation. In another embodiment, the memory 32 stores targeting or focusing modes used to spatially select a region of interest within an organ or tissue structure. For example, the energy application device 12 may be configured to initially operate in targeting mode to apply energy used to acquire image data used to identify a region of interest. The energy in targeting mode is not applied to, and / or at, the level of modulating parameters suitable for preferential activation. However, once a region of interest is identified, the controller 16 can operate in therapeutic mode according to modulating parameters related to preferential activation.

[0107] The controller 16 may also be configured to receive input regarding the target physiological outcome as input to the selection of the modulatory parameter. For example, if the imaging modality is used to evaluate tissue features, the controller 16 may be configured to receive a calculated index or parameter of the features. The modulatory parameter can be modified based on whether the index or parameter is above or below a threshold. In one embodiment, the parameter may be a measure of tissue displacement of the affected tissue or a measure of the depth of the affected tissue. Furthermore, the energy application device 12 (e.g., an ultrasound transducer) operates under the control of the controller 16 to a) acquire image data to spatially select a target region within the target tissue, b) apply modulatory energy to the target region, and c) acquire images to determine that the target physiological outcome has occurred (e.g., by displacement measurement). In such embodiments, the imaging device, evaluation device 20, and energy application device 12 may be the same device.

[0108] In another implementation, the controller 16 can also store a successful set of adjustment parameters. 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 not very effective over time, the subject may be experiencing a lack of sensitivity to the activation pathway. If the system 10 includes an evaluation device 20, the evaluation device may provide feedback to the controller 16. In certain embodiments, the feedback can be received from a user or the evaluation device 20 that exhibits the characteristics of the target physiological outcome. The controller 16 can be configured to cause an energy application device to apply energy according to the adjustment parameters and to dynamically change the adjustment parameters based on the feedback. For example, based on the feedback, the processor 16 can automatically change the adjustment parameters (e.g., frequency, amplitude, or pulse width of an ultrasonic beam or mechanical vibration).

[0109] Figure 3 shows a specific example in which the energy application device 12 includes an ultrasonic transducer 42 that can apply energy to a target tissue, shown as the spleen as an example. The energy application device 12 may include a control circuit for controlling the ultrasonic transducer 42. The ultrasonic transducer 42 may also be configured to acquire image data to help focus the applied energy to a desired target location.

[0110] The desired target tissue 43 may be an internal tissue or organ containing synapses of axon terminals 46 and non-neuronal cells 48, which can be stimulated by directly applying energy to the axon terminals within the focal region of an ultrasonic transducer 42 focused on a target region 44 of the target tissue 43 for release into the synaptic space 49. In the illustrated embodiment, the axon terminals 46 form synapses with immune cells, and the release of neurotransmitters triggers downstream effects such as the activation of macrophages or other endogenous or migratory immune cells. The target region can be selected to include a specific type of axon terminal 46 (e.g., splenic axons in the white pulp as shown in the illustration), such as those forming synapses with a particular neuronal type of axon terminal 46 and / or a specific type of non-neuronal cell 48 (e.g., splenic T cells as shown in the illustration). Thus, the target region 44 can be selected to correspond to a portion of the target tissue 43 having the desired axon terminals 46 (and associated non-neuronal cells 48). The application of energy can be selectively chosen to preferentially elicit the release of neurotransmitters from nerves within synapses, or to directly activate non-neuronal cells themselves via direct energy transfer (i.e., mechanotransduction or potential-activating proteins within non-neuronal cells), or to induce activation in both neuronal and non-neuronal cells to produce the desired physiological effect. In the illustrated example, the region of interest includes innervated red and / or white splenic pulp tissue extending from the splenic nerve. Where described herein, stimulation or modulation of the spleen may refer to modulation of the region of interest 44, which includes innervated white splenic pulp tissue.

[0111] Energy can be focused or substantially concentrated on only a portion of the target region 44 and the internal tissue 43, for example, less than 50%, less than 25%, less than 10%, or less than 5% of the total volume of the tissue 43. In one embodiment, energy can be applied to two or more target regions 44 within the target tissue 43, and the total volume of the target regions 44 may be less than 90%, less than 50%, less than 25%, less than 10%, or less than 5% of the total volume of the tissue 43. In one embodiment, energy is applied to only about 1% to 50% of the total volume of the tissue 43, only about 1% to 25% of the total volume of the tissue 43, only about 1% to 10% of the total volume of the tissue 43, or only about 1% to 5% of the total volume of the tissue 43. In certain embodiments, only axon terminals 46 within the target region 44 of the target tissue 43 directly receive energy and release neurotransmitters, while unstimulated axon terminals outside the target region 44 receive virtually no energy and are therefore not activated / stimulated. In some embodiments, the axon terminals 46 in the portion of the tissue that directly receives energy will induce modified neurotransmitter release. In this way, tissue compartments can be targeted for neuromodulation in a granular manner. In some embodiments, the energy application parameter can be selected to induce preferential activation of either neuronal or non-neuronal components within the tissue that directly receives energy, thereby inducing a desired combined physiological effect. In certain embodiments, the energy is applied at approximately 25 mm 3 It can be focused or concentrated within a volume of less than 0.5 mm. In certain embodiments, energy can be concentrated within about 0.5 mm. 3 ~50mm 3 The energy can be focused or concentrated within a certain volume. The focal volume and depth of focus for focusing or concentrating energy within the target region 44 may be affected by the size / configuration of the energy application device 12. The focal volume of the energy application may be defined by the focal region of the energy application device 12.

[0112] As provided herein, energy can be applied substantially only to the target region 42 to preferentially activate synapses in a targeted manner to achieve the desired physiological outcome, and not substantially applied in an unspecified manner throughout or across the entire tissue 43. Thus, only a subset of multiple different types of axon terminals 46 within the tissue 43 is exposed to direct energy application. Figure 4 is an image of blood flow (Doppler ultrasound) within the spleen, which can serve as spatial information for selecting a target region in the spleen. For example, it can be used to select a target region within an organ that includes any of the blood vessels, nerves, or other anatomical landmarks and to identify regions with specific axon terminals and synapses. In one embodiment, the target region is selected by identifying the splenic artery and spatially selecting a region close to or parallel to the splenic artery. The organ structure is typically segmented based on lower organ tissue function, blood vessels, and innervation, and a subset of axon terminals can be selected so that they are included in the target region to which energy is directly applied. Other axon terminals are outside the target region and are not exposed to the directly applied energy. The individual axon terminals to be included in the target region may be based on historical or experimental data (e.g., based on the association of a specific location with a desired target physiological outcome). In another embodiment, the location of axon terminals and their adjacent tissues or structures may be used to select individual axon terminals from the entire set of axon terminals for preferential activation. Alternatively or additionally, system 10 may apply energy to individual axon terminals until the desired target physiological effect is achieved. It should be understood that the spleen image is for illustrative purposes only. The disclosed selection of axon terminals for preferential activation by applying energy directly to the target region using visualized spatial information of nerves can be used in conjunction with other organs or structures (e.g., liver, lymph nodes).

[0113] The disclosed techniques can be used to evaluate neuromodulatory effects and, consequently, as inputs or feedback for selecting or modifying neuromodulatory parameters. The disclosed techniques can be used for direct assessment of tissue state or function as a target physiological outcome. Assessments may occur before (i.e., baseline assessment), during, and / or after neuromodulation. For example, for subjects requiring increased lymphatic drainage, such drainage may be monitored before, during, and / or after neuromodulation to determine whether the selected parameters have achieved a sufficient increase in drainage. Thus, lymphatic drainage can be assessed by one or more in vivo techniques for assessing lymphatic drainage. In one embodiment, an exogenous contrast agent is administered directly into the lymphoid tissue or indirectly via intradermal injection. For example, a gadolinium-based contrast agent can be used. Depending on the desired clinical outcome, either local or systemic flow can be addressed. For example, lymphatic angiography (MR) can be used to assess lymphatic drainage of the extremities. In another embodiment, fluorescent microlymphangiography (FML) may also be used to assess lymphatic drainage. FML uses intradermal administration of a fluorescent dye, FITC conjugated to dextran (FITC-dextran), and video fluorescence microscopy techniques to acquire high-resolution images. In yet another embodiment, imaging may include dyes or indicators that target lymphoid-specific markers such as LYVE-1, Prox-1, podoplanin, and VEGFR3.

[0114] The evaluation techniques may include at least one of the following: functional magnetic resonance imaging, diffusion tensor magnetic resonance imaging, positive emission tomography, or acoustic monitoring or thermal monitoring. The evaluation techniques may also include protein and / or marker concentration assessment.

[0115] Images from the evaluation technique may be received by the system for automated or manual evaluation. Based on the image data, the modulating parameters may also be modified. For example, if lymphatic drainage increases in the presence of stable vital signs and other health indicators, the stimulation may be stepped back to the minimum energy required to maintain the desired increased lymphatic drainage. In other embodiments, changes in lymphatic drainage can be used as a marker of local neurotransmitter concentration and can be effectively used as a surrogate marker for exposing local immune (immune interaction) cells to phenotypic modulating neurotransmitters and as a marker of predictive effects on immune function. Local concentration may refer to the concentration within the focal region of energy application.

[0116] Additionally or alternatively, the system can assess the presence or concentration of neurotransmitters or cells in lymphatic tissue, lymph fluid, or blood. Lymph fluid or tissue can be obtained by fine-needle aspiration, and the assessment of the presence or level of neurotransmitters (e.g., peptide neurotransmitters, catecholamines) can be carried out by any suitable technique known to those skilled in the art.

[0117] In another embodiment, changes in the types and / or numbers of cells in lymph nodes or lymphoid tissue may indicate lymphoid tissue function. Cell populations can be evaluated by ex vivo techniques such as flow cytometry. In another embodiment, lymphatic cell populations can be evaluated by laser scanning in vivo confocal microscopy (IVCM) with an endogenous contrast agent.

[0118] In other embodiments, the target physiological outcomes may include, but are not limited to, tissue displacement, changes in tissue size, changes in the concentration of one or more molecules (local, nonlocal, or cyclic), gene changes, or marker expression, afferent activity, and cell migration.

[0119] Figure 5 is a flowchart of method 50 for stimulating a target region of target tissue. In this method, in step 54, an energy delivery device is positioned so that an energy pulse is focused on the desired target region; in step 56, a pulse generator delivers multiple energy pulses to the target tissue to activate a subset of synapses in the target tissue, for example, stimulating axon terminals to release neurotransmitters and / or inducing alterations in neurotransmitter release and / or inducing alterations in activity in non-neuronal cells (within the synapses), thereby producing the desired physiological outcome in step 58. In certain embodiments, the method may include a step of evaluating the effect of the stimulation. For example, one or more direct or indirect assessments of the state of tissue function or condition can be used. Based on the assessed tissue function, one or more modulo parameters of the energy pulses can be modified (for example, dynamically or tadulatorily controlled) to achieve the desired physiological outcome.

[0120] In one embodiment, evaluations can be performed before and after modulation to assess changes in lymphatic function as a result of modulation. If the desired change in the evaluated characteristic state of lymphatic function is above or below a threshold, appropriate modifications can be made to the modulation parameters. For example, if the change in characteristic relative to the threshold is associated with successful activation of lymphatic tissue, the energy applied during neuromodulation can be stepped back to the minimum level supporting the desired outcome. If the change in characteristic relative to the threshold is associated with insufficient activation of lymphatic tissue, specific modulation parameters, including but not limited to modulation amplitude or frequency, pulse shape, stimulation pattern, and / or stimulation site, can be modified. It should also be understood that a particular desired clinical outcome may instead be associated with blockade of activation. In such embodiments, evaluations of decreased neuronal and / or lymphatic function are relevant to maintaining the modulation parameters, and if undesirable levels of lymphatic activity persist, the modulation parameters can be modified.

[0121] Furthermore, the evaluated characteristics or states may be values ​​or indicators, e.g., flow rate, concentration, cell population, or any combination thereof, which can then be analyzed by appropriate techniques. For example, relative changes above a threshold can be used to determine whether the regulatory parameters have been modified. The success of the regulation 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 the releasing molecule (e.g., relative to the baseline concentration before neuromodulation). In one embodiment, the success of the regulation may include an increase in concentration above a threshold, e.g., an increase of approximately 50%, 100%, 200%, 400%, or 1000% of the concentration compared to baseline. In blockade therapy, the assessment may include tracking a decrease in molecular concentration over time, e.g., a decrease of at least 10%, 20%, 30%, 50%, or 75% of the target molecule. Furthermore, for a particular subject, the success of blockade therapy may include the maintenance of a relatively stable concentration of the particular molecule in the context of other clinical events that may tend to increase the molecular concentration. In other words, if blockade is successful, a potential increase may be prevented. An increase, decrease, or other observable effect may be measured within a certain time frame from the start of treatment, for example, within about 5 minutes or 30 minutes. In certain embodiments, if neuromodulation is determined to be successful, the change in neuromodulation is a command to stop the application of energy pulses. In other embodiments, if neuromodulation is unsuccessful, one or more parameters of neuromodulation are changed. For example, changes in the modulation parameters may be an increase in pulse repetition frequency, such as a stepwise increase in frequency from 10 to 100 Hz, and an evaluation of desired features until successful neuromodulation is achieved. In other implementations, the pulse width may be changed. In other embodiments, two or more parameters may be changed together. If neuromodulation is unsuccessful after changing multiple parameters, the focus of energy application may be changed.

[0122] The energy application device 12 can be configured as an extracorporeal, non-invasive device or an internal device, such as a minimally invasive device. As stated above, the energy application device 12 may be an extracorporeal, non-invasive ultrasound transducer or a mechanical actuator. For example, Figure 6 shows one embodiment of the energy application device configured as a handheld ultrasound probe including an ultrasound transducer 74. However, it should be understood that other non-invasive implementation forms are also intended, including other methods for configuring, attaching, or positioning the ultrasound transducer probe on an anatomical target. Furthermore, in addition to a handheld configuration, the energy application device 12 may include a control mechanism that responds to commands from a controller 16. The control mechanism can orient or direct the energy application device 12 toward a target tissue 43 (or structure), and the controller 16 can then focus the energy application onto the target area 44. [Examples]

[0123] 1. Experimental Method Figure 7 is a block diagram of a system 10 including an energy application device 12 and a pulse generator 14 configured to apply high-density focused ultrasound (HIFU). In one embodiment, the system 10 includes a pulse generator including, for example, a function generator 80, a power amplifier 82, and a matching network 84. In one embodiment used to generate the experimental results provided herein, the pulse generator includes a 1.1 MHz high-density focused ultrasound (HIFU) transducer (Sonic Concepts H106), a matching network (Sonic Concepts), an RF power amplifier (ENI350L), and a function generator (Agilent 33120A). The HIFU transducer 12 was connected to an animal subject through a 6 cm high plastic cone filled with degassed water. Figure 8 shows an example of an energy application device that may be used with the system 10 of Figure 7, and includes a HIFU transducer 74A and an imaging ultrasound transducer 74B located in a single energy application device 12, which can be controlled, for example, by a controller 16, to apply energy and image target tissue as provided herein.

[0124] Figure 9 shows an experimental setup used to carry out a specific spleen regulation experiment as provided herein. While the illustrated embodiment shows a spleen target tissue 43, it should be understood that certain elements of the experimental setup may be common among different target tissues 43. For example, the energy application device 12 may operate according to parameters set by the controller 16 to apply energy to a target area within the target tissue 43.

[0125] Figure 10 shows an experimental timeline for ultrasound energy application used to perform a specific controlled experiment as provided herein. In the illustrated embodiment, ultrasound is applied for one minute before and after lipopolysaccharide injection. Lipopolysaccharides (LPS) are bacterial membrane molecules that elicit a strong immune or inflammatory response. Therefore, LPS-treated animals can serve as a model for disease or condition in which the immune response is active. In the experimental timeline, organ harvesting and measurement of various target physiological outcomes may occur after a certain period has elapsed since treatment.

[0126] The function generator 80 generates a pulsed sinusoidal waveform as shown in Figure 11. This pulsed sinusoidal waveform is amplified by an RF power amplifier and transmitted to the matching network of the HIFU transducer. During animal experiments, three ultrasonic parameters—pulse amplitude, pulse length, and pulse repetition frequency—can be adjusted. The pulse amplitude ranges from a 0.5V peak to a 62V peak. Three pulse lengths are used: 18.2us, 136.4us, and 363.6us. The pulse repetition frequency (1 / T) is 2kHz. The treatment time is 1 minute. Table 1 summarizes the HIFU ultrasonic parameters. The ultrasonic adjustment parameters are examples. In one embodiment, adjustment is provided using an ultrasonic stimulus having an ultrasonic transducer frequency in the range of approximately 0.1MHz to approximately 5MHz, and the ultrasonic stimulus has an ultrasonic frequency pulse repetition frequency in the range of approximately 0.1Hz to approximately 10kHz. The ultrasonic cycles per pulse of ultrasonic energy may be in the range of approximately 1 to approximately 1000. JPEG0007885063000001.jpg51127

[0127] Pressure measurements were performed in degassed water using an Onda Corp. HIFU hydrophone (HNA-0400). The HIFU transducer was driven by a 100-cycle sinusoidal waveform. The hydrophone scanned through the focus with a grid size of 0.1 mm in the x-y plane and a step size of 0.2 mm along the z axis. Figure 12 shows the hydrophone setup, and Figure 13 shows the scan results. Peak positive (negative) pressure is defined as the maximum positive (negative) pressure at the transducer focus in the x-y plane. The input voltage was low enough to eliminate the effects of nonlinearity. Therefore, the peak positive pressure value was identical to the peak negative pressure value. To estimate the peak pressure at all operating voltages, the peak pressure was measured at several different drive voltages before cavitation occurred and curve fitting was performed. The calculated peak pressures are shown in Table 1. Ultrasound targeting for organ-specific neuromodulation

[0128] Before neural modulation began, an ultrasound scan was performed using a GE Vivid E9 ultrasound system and an 11L probe. The target region was labeled on the animal's skin. A HIFU transducer was positioned over the labeled region. Another ultrasound scan was performed using a smaller imaging probe (3S), which was positioned at the aperture of the HIFU transducer. The imaging beam of the 3S probe was aligned with the HIFU beam. Thus, using images of the target organ (visualized on the ultrasound scanner), it was possible to confirm that the HIFU beam was targeting the target region. Animal Protocol

[0129] Before conducting the experiment, adult male Sprague-Dawley rats (250-300g, Charles River Laboratories), 8-12 weeks old, were reared at 25°C with a 12-hour light / dark cycle for one week to acclimate them. They had free access to water and normal rodent food.

[0130] Endotoxin (LPS derived from E. coli, 0111:B4; Sigma-Aldrich) was used to induce significant inflammation and metabolic dysfunction (e.g., hyperglycemia and hyperinsulinemia) in untreated adult Sprague-Dawley rats. Intraperitoneal (IP) injection of LPS (10 mg / kg; Rosa-Ballinas PNAS, 2008) induced significant increases in TNF, circulating glucose, and insulin concentrations, peaking at 4 hours but remaining elevated up to 8 hours after injection compared to the control group. Spleen, liver, hypothalamus, hippocampus, and blood samples were collected 60 minutes after LPS administration (for efficacy studies) and at 30, 60, 120, 240, or 480 minutes after LPS administration (for duration and kinetic studies). Spleen and liver samples were homogenized in PBS solution containing phosphatase (0.2 mM phenylmethylsulfonyl fluoride, 5 μg / mL aprotinin, 1 mM benzamidine, 1 mM sodium orthovanadate, and 2 μM cantharidin) and protease (Roche Diagnostics, 1 μL to 20 mg tissue) inhibitors. A target final concentration of 0.2 g of tissue per 1 mL of PBS solution was applied to all samples. Blood samples were stored with an anticoagulant (disodium EDTA) to prevent coagulation. Samples were analyzed by ELISA assay for changes in cytokine (Bio-Plex Pro; Bio-Rad), TNF (Lifespan), and acetylcholine (Lifespan) concentrations. Catecholamine concentrations were evaluated using HPLC detection or ELISA (Rocky Mountain Diagnostics) analysis.

[0131] The effects of LPS on blood glucose and insulin levels were investigated. Blood samples were collected from the tail vein at 0, 60, 90, 120, 150, 180, and 240 minutes after LPS injection, and glucose and insulin levels were measured. Circulating blood glucose concentration was measured using a OneTouch Elite glucometer (LifeScan, Johnson & Johnson). Plasma insulin concentrations obtained from blood samples were determined using an ELISA kit (Crystal Chem, Chicago, IL) to determine the effects of LPS and subsequent ultrasound stimulation on systemic insulin resistance. Changes in signaling were measured by evaluating key biomarkers, including IRS-1 / 2, PI3K, Akt, GLUT2, GLUT4, GLUTamate, GABA, and AMPK, in liver, muscle, cardiac, and hypothalamic tissue samples. The protocol used for ultrasound nerve conditioning was as follows: The animals are anesthetized with 2-4% isoflurane. To prevent hyperthermia during the procedure, the animal is placed face down on a water-circulating warming pad. Before stimulation, the area on the designated site (target nerve) for ultrasonic stimulation may be shaved with a disposable razor and animal clippers. The target area is identified using diagnostic imaging ultrasound. The liver is defined as the hepatic hilum, as indicated by Doppler identification of the hepatic portal vein. The spleen is visually identified using diagnostic ultrasound. The stimulation location is maintained along the identified splenic axis. You may add permanent markers to the areas so that they can be identified later. Place either the FUS ultrasound probe or the LogiQ E9 probe over the designated target area previously identified with diagnostic ultrasound. Next, an ultrasound pulse is administered for a total duration of a single stimulus that does not exceed one minute. At any point, the energy does not reach levels associated with thermal damage or ablation / cavitation. Subsequently, LPS (10 mg / kg) may be administered by intraperitoneal injection (for acute / pharmacokinetic studies). Alternatively, LPS may not be administered at this time, but instead at a later specified time, for the duration of the effect. Apply a second 1-minute ultrasound stimulation. Next, the animals can be incubated under anesthesia for acute (1 hour) and kinetic (variable up to 3 hours after LPS) studies. After that, the animals are sacrificed and tissue and blood samples are collected. For studies on the duration of effect, LPS is not injected during ultrasound stimulation, but rather at specified time points after the application of ultrasound stimulation (e.g., 0.5, 1, 2, 4, or 8 hours). The animals are then placed in an anesthetic holding chamber and monitored until euthanasia and tissue / fluid collection. An incision was made extending from the base of the peritoneal cavity to the pleural cavity. Organs (including the spleen and liver) were promptly removed and homogenized in a PBS solution containing phosphatase (0.2 mM phenylmethylsulfonyl fluoride, 5 μg / mL aprotinin, 1 mM benzamidine, 1 mM sodium orthovanadate, and 2 μM cantharidin) and protease (Roche Diagnostics, 1 μL to 20 mg of tissue) inhibitors. A target final concentration of 0.2 g of tissue per 1 mL of PBS solution was applied to all samples. Blood samples were stored with an anticoagulant (disodium EDTA) to prevent coagulation. The samples were then stored at -80°C until analysis. The samples were analyzed by ELISA assay for changes in cytokine (Bio-Plex Pro; Bio-Rad), TNF (Lifespan / Abcam / ThermoFisher), and acetylcholine (Lifespan) concentrations. Catecholamine concentrations were evaluated using HPLC detection or ELISA (Rocky Mountain Diagnostic) analysis. Electrode-based vagus nerve stimulation control experimental protocol

[0132] Male Sprague-Dawley rats were anesthetized with 2% isoflurane. A single incision was made along the neck to expose the trapezius, sternocleidomastoid, and masseter muscles, and a blunt incision was made to expose the left cervical vagus nerve. Microelectrodes were placed along the main trunk of the exposed cervical vagus nerve. Electrical stimulation (5V, 30Hz, 2ms; 5V, 5Hz, 2ms; 1V, 5Hz, 2ms) was generated using a BIOPAC MP150 module under the control of AcqKnowledge software (Biopac Systems). Rats were given 3 minutes of vagus nerve stimulation before and after IP injection of 10 mg / kg of LPS. Rats were euthanized 60 minutes after LPS injection, and spleen and blood samples were collected for TNF measurement. In rats that underwent sham surgery, the vagus nerve was exposed but not touched or manipulated. HPLC analysis

[0133] Serum samples were injected directly into the machine without pretreatment. Tissue homogenates were first homogenized with 0.1 M perchloric acid, centrifuged for 15 minutes, the supernatant was separated, and the samples were injected into HPLC (Dhir & Kulkarni, 2007).

[0134] Catecholamines (norepinephrine / epinephrine) were analyzed using high-performance liquid chromatography (HPLC) with an in-line UV detector. The test column used for this analysis was a Supelco Discovery C18 (15 cm inner diameter × 4.6 mm, particle size 5 μm). The two-phase mobile phase contained [A]acetonitrile:[B]50 mM KH2PO4 and was set to pH 3 (using 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. The column was kept at a constant 20°C while improving overall peak separation using a flow rate of 1 mL / min to minimize column pressure compression resulting from the viscosity of the mobile phase used. The UV detector was maintained at a wavelength of 254 nm, which is known to capture absorption for catecholamines including norepinephrine, epinephrine, and dopamine. Chemical inhibition of ultrasonic modulation molecular signaling pathways

[0135] Before performing the ultrasound stimulation procedure described above, further investigate the effects of mechano-to-direct nerve stimulation (and preferential modulation of the nerve-to-non-nerve component of extraaxonal cell synapses), SRC inhibitors (common markers of direct mechanoreceptors), or PI3K inhibitors (common markers of nerve signaling). Tissue extraction and paraffin block conversion:

[0136] The tissue (rat brain) is immediately placed in the fixative solution and fixed in 10% formalin at 4°C for approximately 24 hours. The tissue is processed using the following protocol (under vacuum and pressure during each incubation). a. 70% ethanol, 37°C, 40 minutes b. 80% ethanol, 37°C, 40 minutes c. 95% ethanol, 37°C, 40 minutes d. 95% ethanol, 37°C, 40 minutes e. 100% ethanol, 37°C, 40 minutes f. 100% ethanol, 37°C, 40 minutes g. Xylene, 37°C, 40 minutes h. Xylene, 37°C, 40 minutes i. Paraffin, 65°C, 40 minutes j. Paraffin, 65°C, 40 minutes k. Paraffin, 65°C, 40 minutes l. Place in paraffin, 65°C for 40 minutes, and leave in the paraffin until ready for embedding. However, do not exceed 12-18 hours. Embed the tissue in a paraffin block for dissection, and allow the block to cool / solidify before dissection. Dissection into 5-micron thick sections and suspend them in a 50°C water bath for collection. Use positively charged slides and ensure that all slides are oriented in the same direction. Air dry the slides. Leaving them at room temperature overnight seems to be best for drying, but the drying process can be accelerated by placing the slides on a 40°C slide warmer. However, do not leave the slides on the warmer for more than one hour. Store the slides at 4°C. IHC process

[0137] Formalin-fixed, paraffin-embedded (FFPE) tissue samples (rat brain) were calcined at 65°C for 1 hour. The glass slides were deparaffinized with xylene, rehydrated by washing with reduced ethanol concentration, and then processed for antigen detection. In particular, a two-step antigen detection method was developed for redundancy with FFPE tissue, allowing the use of antibodies with different antigen detection conditions on the same sample (2). Next, the samples were incubated in PBS containing 0.3% Triton X-100 at ambient temperature for 10 minutes, and then nonspecific binding was blocked at room temperature for 45 minutes using 10% (wt / vol) donkey serum and 3% (wt / vol) BSA in 1×PBS. The primary antibody cFOS (santa cruz-SC52) was diluted to the optimal concentration (5 μg / mL) and applied in PBS / 3% (vol / vol) BSA at room temperature for 1 hour. Next, the samples were washed with PBS, PBS-Triton X-100, and then again with PBS for 10 minutes each, with agitation. For secondary antibody detection, the sample was incubated with primary antibody species-specific secondary donkey IgG conjugated to either Cy3 or Cy5. The slide glass was then washed as described above, stained in DAPI (10 μg / mL) for 5 minutes, rinsed again in PBS, and then covered with a bleed-resistant medium for image acquisition. Whole tissue images were obtained at 10x magnification using a fluorescence Olympus IX81 microscope. Image processing

[0138] Typical autofluorescence in FFPE tissue should be characterized and separated from the target fluorophore signal using an autofluorescence removal process, yielding images of the unstained sample in addition to the stained image. The unstained and stained images are normalized with respect to their exposure time and dark pixel values ​​(pixel intensity values ​​at zero exposure time). Then, each normalized autofluorescence image is subtracted from the corresponding normalized stained image. The AF-removed images merged with the registered DAPI images are shown photographically. The same regions in the stimulated and control samples were imaged, and the images were qualitatively evaluated for cFOS expression to detect changes in the expression of neuronal activation-related genes.

[0139] Histological evaluation of the spleen: Spleens from stimulated rats and control rats were prepared as paraffin blocks as described above. The paraffin-embedded sections were cleared according to standard protocols reported in the literature, stained for hematology and erythrology (H&E), and scanned with a bright-field Olympus scanner. Qualitative evaluation of the H&E images for morphological differences revealed no significant differences between the stimulated and control samples. Heart rate monitoring and analysis

[0140] Heart rate (during ultrasound or electrode stimulation experiments) was monitored using a commercially available infrared oximeter and physiological monitoring system (Starr Lifesciences) according to the manufacturer's instructions. Foot clip sensors (provided by the manufacturer) were placed on the animal's foot during the stimulation protocol. Animals were acclimatized for at least 5 minutes before measurement to determine the time and control physiological readings sufficient for the animals to recover to normal heart rate activity. Measurements were recorded before (2-minute recording period), during, and after stimulation (2-minute recording period) using either an electroelectrode or ultrasound probe. Ultrasound-induced activation by diffusion functional MRI measurement

[0141] Neuronal activation is typically detected using blood oxygen-dependent (BOLD) fMRI, where brain regions with increased metabolic demands exhibit increased cerebral blood flow, increased oxygenated blood supply, and decreased gradient echo signal. Sensitivity to the BOLD effect requires the use of fast gradient echo acquisition, which causes undesirable signal loss in brain regions adjacent to air pockets, such as the sinuses and external auditory canal, hindering the detection of neuronal activation near these specific brain regions. Alternatively, spin-echo (or double-spin-echo) diffusion-weighted imaging (DWI) can be used to minimize signal loss in regions characterized by large magnetic field heterogeneity. In DWI-fMRI, both slow-diffusing (presumably intracellular) water pool volume increases, or water diffusion (or apparent diffusion coefficient (ADC) increases), were attributed to cell swelling and membrane expansion due to neuronal activation.

[0142] Using the paradigm shown in Figure 14, brain MRI scans were performed on 10 rats. Of these, 6 received both LPS injection (as described above) and ultrasound treatment, while 4 received LPS injection only.

[0143] The scan was performed using a 3T GE DV scanner (Waukesha, WI) with a Doty Scientific orthogonal phase birdcage coil. The scan started with T1 acquisition, using a spoil 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 blocks of double-spin echo DWI images (called forward polar gradient, or FPG) were acquired, each with an in-plane / out-of-plane spatial resolution of 0.6 / 1 mm and a TE / TR of 82 / 3400 ms, using the 3 / 4 average for b=0 / b=1000 s / mm2, for a total acquisition time of 1:49 minutes per block. Upon completion of the six pre-injection DWI acquisitions, another DWI acquisition was performed with the gradient direction reversed for distortion correction purposes; this acquisition is called reverse polar gradient (RPG) acquisition. After LPS injection, the first ultrasound, a 5-minute waiting period, and the second ultrasound, FPG DWI images of the remaining six blocks were acquired. For control rats receiving only LPS injection, the last six DWI blocks followed immediately after the LPS injection.

[0144] Ultrasonography was performed using an MR-compatible 1.47 MHz focused ultrasound transducer coupled to the hepatic hilum region (including afferent nerves; as described above) of the liver using a water-filled cone. Each ultrasound procedure lasted 60 seconds, during which a pulsed sinusoidal ultrasound waveform was applied. The pulse-on time was 150 μs, and the pulse-off time was 350 μs. The abdomen of the rats was outside the imaging coil, the animals were positioned supine, and coupling gel was used to ensure easy coupling of the ultrasound probe to the liver through the skin. Data analysis and results

[0145] Based on the observation that traversing k-space in the opposite direction results in a reversal of the distortion (i.e., compression is converted to expansion), a field map that overlaps the FPG image and the RPG image can be estimated. This field map estimation is performed by setting a least-squares cost function that explicitly depends on all voxel displacements, and the smoothness of the deformation field is controlled by introducing a regularization parameter that limits the sum of the squares of the gradients of the displacements at each voxel. The numerical task of efficiently minimizing this cost function is achieved by starting from the highly blurred FPG and RPG images and gradually narrowing the width of the blurring Gaussian kernel. In all distortion corrections performed by the inventors in this work, this blurring schedule starts from a kernel width of 25 pixels, decreases in two steps to the original resolution of the input image, repeats a total of 13 times, and the regularization parameters were λ1 = 0, λ2 = 1100. In summary, the estimated field map is first obtained using each of the 12 FPGs and the b = 0 acquisition of a single RPG acquisition, and the 12 corrected FPG DWI images are obtained by applying a shift to the distorted DWI images of the FPG series.

[0146] The results of the distortion correction operation were evaluated by setting a brain boundary box for the T1 image and estimating the cross-correlation coefficient for each slice between the anatomical T1 image and the functional DWI image. Only brain slices with a cross-correlation coefficient exceeding 0.5 were left for activation region analysis.

[0147] Figure 15 shows an example of the corrected T1 / DWI images in one rat together with the cross-correlation coefficient for each slice, and the slices retained for activation analysis are highlighted in red.

[0148] For each of the 12 corrected DWI data sets, the apparent diffusion coefficient was determined for each pixel as follows. JPEG0007885063000002.jpg936Here, b1 = 0, b2 = 1000 s / mm 2 and S(b i ) represents the signal intensity at the i-th b value.

[0149] For each of the two conditions (pre-treatment and post-treatment), six ADC maps were grouped and binned, and t-tests were performed pixel by pixel to identify regions where the mean signal significantly increased or decreased as a result of ultrasound energy application (corresponding to deactivation changes in neuronal activation).

[0150] To identify anatomically activated regions, we manually registered anatomical T1 images of each rat against a rat brain atlas using the ITKSnap registration tool, which aligns brain boundaries using only translation, rotation, and scaling (9 degrees of freedom). These corrected and anatomically aligned images were then used to determine brain regions activated in response to hepatic ultrasound neuromodulation in LPS-treated rats. 2. Experimental Results

[0151] As a control, nerves from the sciatic nerve entering the popliteal lymph nodes were stimulated in rats. Using bipolar electrodes, stimulation was performed for 5 minutes with a constant range of applied currents using a 20 Hz stimulation frequency (equilibrium biphase pulse) for 200 microseconds. After stimulation, the lymph nodes were excised, dissociated into single-cell suspensions, and then analyzed using a Hemavet cell counter. The number of lymphocytes per cubic milliliter of dissociated tissue was plotted for the directly stimulated popliteal lymph node, the contralateral popliteal lymph node, and the axillary lymph node. The lymphocyte count was statistically increased only in the stimulated lymph node. The results are shown in Figure 16. As provided herein, applying energy, such as ultrasonic energy, to a target area within tissue in a targeted manner can achieve more targeted results compared to direct electrical stimulation, as shown in Figure 16.

[0152] In contrast to electrical stimulation, cells and nerve endings within the popliteal lymph nodes were stimulated by exposing the lymph nodes to ultrasound energy in the form of pulsed sinusoidal waveforms. The ultrasound parameters used were as follows: Frequency: 1.1MHz Pulse length: 136 μs Pulse repetition interval: 500 μs (27% duty cycle) Total ultrasound irradiation time: 1 minute Estimated positive pressure at the focal point: 5 MPa Estimated negative pressure at the focal point: -2 MPa Geometric shape of the transducer: Focusing spherically using a central opening. Transducer outer diameter: Approximately 70 mm Transducer central opening diameter: Approximately 20mm Focal length: Approximately 54.8mm (F-number approximately 1) Focus spot area: Approximately elliptical, with a diameter of 1.8 mm and a length of 11.7 mm.

[0153] After stimulation, the stimulated (and other) lymph nodes were excised and examined using a cell counter. As shown in Figure 17, only the stimulated lymph nodes showed an increase in lymphocytes. Neurotransmitter levels in ultrasound-stimulated lymph nodes differed from those in electricalally stimulated lymph nodes. However, cell count data for lymphocytes were similar in both ultrasound-stimulated and electricalally stimulated lymph nodes. In addition to local regulation of lymphocyte outflow (via efferent nerve fibers), electrical stimulation of nerves entering the lymph nodes was found to have distal effects on immune cell concentrations in the liver and spleen.

[0154] Figure 18 shows the results of neurotransmitter concentration measurements in ultrasound-stimulated lymph nodes. Epinephrine and dopamine levels were elevated in ultrasound-stimulated lymph nodes.

[0155] Figure 19 shows the results of the norepinephrine, epinephrine, dopamine, and acetylcholine concentrations in the spleen as distal effects of lymph node ultrasound stimulation.

[0156] Figure 20 shows the cytokine (TNF) concentrations in the spleen of ultrasound-stimulated animals compared to a control group of unstimulated animals. Figure 21A shows the norepinephrine concentration, Figure 21B shows the acetylcholine concentration, and Figure 21C shows the dose-dependent TNF-α concentration in the spleen after ultrasound stimulation. Figure 22 shows the circulating (i.e., blood) TNF-α concentration in the spleen after ultrasound stimulation.

[0157] Figure 23 shows the results from the livers of LPS-treated animals, comparing the control group with ultrasound stimulation.

[0158] Figure 24 is a table showing changes in splenic cytokines compared to controls for different time-averaged intensities of splenic ultrasound treatment. The cytokines evaluated are those that may be indicators of the cholinergic anti-inflammatory pathway, and other unrelated cytokines. For TNF-α (cholinergic anti-inflammatory pathway (CAP)-related cytokine), a decrease in change compared to the sham control was observed across all time-averaged intensities. In contrast, IFN-γ (CAP-unrelated cytokine) showed a decrease observed at higher time-averaged intensities. The results suggest that ultrasound modulation of the spleen at a certain range of time-averaged intensities can result in a corresponding decrease in cytokine concentrations specifically associated with CAP activation. Therefore, as provided herein, the target physiological outcome is a decrease in cytokine concentration, e.g., at least 1 mW / cm². 2 The time-averaged intensity was 30,000 mW / cm². 2 This may be a decrease in TNF-α or IL-1 concentration as a result of adjustment to the temporal intensity. In certain embodiments, chronic inflammation, such as arthritis, can be treated using energy applied to a target area of ​​the spleen in relation to the corresponding decrease in TNF-α or IL-1 concentration.

[0159] Figure 25 shows the percentage change in the phosphorylation status of secondary messenger molecules compared to LPS-treated controls for different peak pressures of spleen ultrasound treatment. The results indicate that ultrasound treatment in the spleen modulates multiple pathways across different cell types within the tissue. Nevertheless, modulatory parameters with more targeted effects on one (or different sets of) of these pathways may be found. As shown, for spleen ultrasound modulation, p-p38 phosphorylation across different peak pressures changes with changes in tTNF concentration in the response, while other pathways tend to increase or decrease monotonically with respect to the applied ultrasound pressure.

[0160] Figure 26 shows the splenic concentration of TNF-α in a group of stimulated LPS-treated animals under the condition of pathway blockade. BTX is a chemoblocker of acetylcholine receptors (blocking the neural pathway in integrator cells), PP2 is a Src kinase inhibitor, LY is a PI3 kinase inhibitor, and PD98059 is a MAPK kinase inhibitor. Figure 27 shows the splenic concentration of norepinephrine in a group of stimulated LPS-treated animals under the condition of pathway blockade. Figure 28 shows the splenic concentration of TNF-α in a group of stimulated LPS-treated animals compared to animals treated with vagus nerve transection or pathway blockade. Figure 29 shows the splenic concentration of TNF-α at various time points after ultrasound treatment combined with LPS treatment. The effect begins to reverse after 3 hours.

[0161] Figure 30 is an experimental timeline of LPS treatment after ultrasound treatment used to demonstrate the protective effect of ultrasound treatment against future immune / inflammatory injury. Ultrasonography appears to provide at least partial protection against LPS-induced immune induction for several hours post-treatment by protecting against LPS-related decreases in norepinephrine and subsequent activation of CAP, with the protective effect being maximal at 30-minute intervals but stabilizing at 1-2 hours. Figure 31 shows splenic TNF-α concentrations at various time points after ultrasound and LPS treatment, following the timeline in Figure 30. The protective effect of ultrasound treatment against immune injury by protection against LPS-related increases in TNF-α was greater than in untreated animals (i.e., animals without LPS or inflammatory injury), and TNF production was reduced compared to LPS-controls (i.e., no ultrasound). Therefore, as provided herein, non-invasive or minimally invasive energy application to the spleen may be used to protect against inflammatory injury or chronic inflammation (since protection from a single ultrasound adjustment is protective over a long period).

[0162] Figure 32 shows the splenic concentration of TNF-α in animals stimulated by non-invasive ultrasound versus invasive cervical vagal nerve electrode stimulation, and Figure 33 is an ultrasound image used to identify the target area and ultrasound placement for the ultrasound treatment in Figure 32 by centering the image transducer directly within the FUS cone. Stimulation and LPS treatment were performed as commonly provided herein. Non-invasive ultrasound stimulation was performed entirely non-invasively and yielded similar results (reversal of LPS-related TNF-α increase) compared to invasive ultrasound treatment (results above) or invasive electrode treatment (as shown).

[0163] Figure 34 shows the experimental setup for controlled electrical stimulation. Figure 35A shows the splenic TNF-α concentration in non-invasive ultrasound-stimulated animals versus electrode-stimulated control animals under LPS treatment conditions, with electrode stimulation performed according to the setup in Figure 34. Figure 35B shows the blood glucose concentration in non-invasive ultrasound-stimulated animals versus electrode-stimulated control animals under LPS treatment conditions, with electrode stimulation performed according to the setup in Figure 34. Ultrasound and electrical stimulation had the same effect on LPS-induced TNF concentration. LPS is also known to cause hyperglycemia (LPS control, see Figure 35B). LPS-induced hyperglycemia has been previously shown to be neutralized by the VNS (by an unknown pathway), which is an "off-target" effect from the perspective of attempting to stimulate the VNS cholinergic anti-inflammatory pathway. However, ultrasound cholinergic anti-inflammatory pathway stimulation does not produce an off-target effect on glucose. Therefore, cholinergic anti-inflammatory pathway ultrasound stimulation may be beneficial in certain clinical situations compared to VNS (or other inaccurate electroneurostimulation techniques), and organ-based extra-axonal cell synaptic ultrasound modulation is more precisely described herein.

[0164] Figure 36 shows the effect of VNS electrode stimulation versus ultrasound-induced splenic nerve modulation on heart rate compared to unstimulated animals, as measured using a commercially available non-invasive optical heart rate probe placed on the animal's leg during the experiment. The percentage change in heart rate represents the change in heart rate compared to unstimulated animals. Again, electrical stimulation is shown to have a potential off-target effect (i.e., regulation of heart rate), while organ-based extra-axonal cell synaptic ultrasound modulation does not have this off-target effect (i.e., an effect other than CAP activation).

[0165] Figure 37A shows the effect of subhilar ultrasound-mediated hepatic regulation on blood glucose compared to a control. The hepatic hilum is a region known to contain glucose-sensing neurons and was hypothesized to be the origin of the aforementioned off-target VNS effect on glucose (i.e., the VNS stimulated afferent nerves from this region in addition to the CAP pathway). Figures 37B and 37C show the effect of ultrasound-mediated hepatic regulation on the hepatic concentrations of various molecules involved in metabolism and glucose utilization. Only changes in insulin were observed in the liver. However, insulin is not produced in the liver but is transported there from other sites, indicating systemic effects on insulin production and glucose metabolism as a result of local hilar hepatic stimulation.

[0166] Figures 38A–38C show the effects of ultrasound-induced hepatic hilar modulation on the brain concentrations of various molecules. The hypothalamus is known to have regions involved in glucose regulation throughout the body. Hepatic afferent nerves are considered to be the PNS regulatory pathway to these sites (and neural modulation of sensory nerve / hepatic fluid extraaxonal cell synapses causes changes in signaling via neural pathways). Therefore, as provided herein, the direct application of energy (e.g., ultrasound energy) to the liver may be used to treat disorders related to glucose regulation. Such disorders may include obesity, diabetes-related dementia, eating disorders, diabetes, hypoglycemia, or hyperglycemia. In certain embodiments, hyperglycemia is defined as a blood glucose concentration greater than 300 mg / dL, and a successful target physiological outcome is a blood glucose concentration greater than 220 mg / dL. Treatment may involve applying energy to a target region within the liver to induce the target physiological outcome in the hypothalamic tissue. In one embodiment, the target physiological outcome is a change in pIRS-1, pAKT1 / 2, norepinephrine, epinephrine, and / or NPY in the hypothalamic tissue compared to baseline (e.g., before energy application).

[0167] Figure 39A shows the effect of ultrasound-mediated hepatic modulation at various locations within the liver on glucose concentration, and Figure 39B shows the modulation locations of the target region in Figure 39A. The ultrasound-mediated modulation effect on glucose concentration depends on the location of the target region, i.e., the stimulation site. The change in glucose was most pronounced after stimulation at the hepatic hilum, while stimulation of the left lobe showed the lowest observed change. Therefore, in certain embodiments, applying energy to a target region within the liver to induce a desired physiological outcome may include applying energy to the hepatic hilum rather than the left lobe of the liver. Figure 40 shows that splenic ultrasound stimulation in LPS-treated animals does not produce the observed effect on glucose concentration observed after hepatic ultrasound stimulation, indicating that this effect is specific to the stimulation site.

[0168] Figure 41A shows hypothalamic NPY concentrations in LPS-treated and ultrasound-stimulated animals compared to controls. Figure 41B shows hypothalamic POMC concentrations in LPS-treated and ultrasound-stimulated animals compared to controls. The data indicate that hepatic ultrasound stimulation does not activate or modulate all metabolic regulatory pathways within the hypothalamus, but is specific to particular pathways and neuronal types.

[0169] Figure 42A shows coronal sections and immunohistochemistry / staining images from unstimulated LPS dummy animals, and Figure 42B shows coronal sections from liver-stimulated LPS-treated animals. Compared to unstimulated LPS dummy animals, stimulated LPS-treated animals showed a significant decrease in cFos-positive cells in the ventromedial hypothalamus (VMH) and arcuate (ARC) nucleus, and a moderate decrease in the lateral hypothalamus (LH). Compared to unstimulated LPS dummy animals, stimulated LPS-treated animals also showed a significant increase in cFos-positive cells in the hippocampus (Hc), with a clear increase in hippocampal-specific regions (CA1, CA2, CA3). Diffusion MRI shows an increase in ADC signal in the hippocampus after ultrasound (not shown). The opposite occurs in the hypothalamus, where diffusion is greater before ultrasound stimulation compared to unstimulated animals (Figure 44) (Figure 43). cFos staining appears to support the conclusions of the dMRI study. Therefore, as provided herein, the target physiological outcomes may be alterations in neural signaling to the brain, as well as effects on gene expression within the communication and / or effect regions. Figure 45A shows cFOS and Glut1 staining in the control hippocampus, and Figure 45B shows cFOS and Glut1 staining in the stimulated hippocampus, demonstrating that hepatic ultrasound stimulation can affect upstream regions beyond the hypothalamus and have further effects (beyond glucose regulation and metabolism), including effects on cognition. Figure 45C shows the image in Figure 45A versus the image in Figure 45B with fluorescent staining RFU.

[0170] Figure 46 shows hippocampal norepinephrine after liver stimulation in LPS-treated animals treated with pathway blockers (unlike spleen blockade experiments, it does not show a blocking effect on hepatic ultrasound neuromodulation). These results also indicate that ultrasound modulation has specific effects on different extraaxonal cell synaptic targets. Figure 47 shows hippocampal norepinephrine after treatment with an agonist targeting a diabetes drug (AICAR; AMP kinase activator) in LPS-treated animals, which acts as a positive control and shows a similar effect to hepatic ultrasound treatment. Therefore, hepatic ultrasound treatment may be used to target diabetes-related pathways.

[0171] Figure 48 shows a schematic diagram illustrating how tissue displacement during neuromodulation can be tracked with a reference ultrasound imaging signal before and after ultrasound neuromodulation. The ultrasound probe may be used for 1) imaging, 2) displacement measurement, and 3) stimulation. Tissue displacement can be used to assess the level of tissue movement induced by ultrasound stimulation (and thus the probability of activating stretch-activating proteins or ion channels at target extra-axonal cell synapses). That is, for subjects with different body mass indices (and different tissue thicknesses between the ultrasound probe and the neuromodulation target, which can attenuate the applied ultrasound energy), the likelihood of successful neuromodulation can be assessed using direct measurement of tissue displacement (at the neuromodulation site), and ultrasound parameters can be adjusted accordingly. For example, tissue displacement can be measured using a reference pulse.

[0172] Figure 49 shows shear wave measurements obtained from the spleen stimulation site, and Figure 50 shows displacement measurements obtained from the spleen stimulation site. Displacement measurements can be performed and used for feedback to reach the target physiological outcome. For example, the feedback may be reaching a minimum threshold within at least one imaging frame and achieving a specific total displacement value based on the area under the displacement curve, etc.

[0173] Figure 51 shows the splenic norepinephrine concentration in animals treated with reduced ultrasound clinical efficacy. Figure 52 shows the splenic TNF-α concentration in animals treated with reduced ultrasound clinical efficacy. Figure 53 shows the serum norepinephrine concentration in animals treated with reduced ultrasound clinical efficacy. Figure 54 shows the serum TNF-α concentration in animals treated with reduced ultrasound clinical efficacy. The reduced clinical efficacy is proportional to 100% clinical efficacy.

[0174] Figures 55-58 show the results of spleen ultrasound stimulation using a modified pulse sequence of 800 pulses performed for 30 or 15 seconds at a time, showing the same effect as 800 cycles performed all at once. Figure 55 shows spleen TNF-α concentration in animals treated with the modified pulse sequence. Figure 56 shows spleen norepinephrine concentration in animals treated with the modified pulse sequence. Figure 57 shows serum TNF-α concentration in animals treated with the modified pulse sequence. Figure 58 shows serum norepinephrine concentration in animals treated with the modified pulse sequence.

[0175] Figure 59 shows the splenic norepinephrine concentration compared to a control after mechanical vibration energy was applied to the spleen, and Figure 60 shows the splenic TNF-α concentration compared to a control after mechanical vibration energy was applied to the spleen. Mechanical vibration energy was generated using the TIRA Vibration System Model TV50018 from TIRA GmbH, consisting of a shaker (Model S504) and an amplifier (BAA60). The tip of the shaker was in direct contact with the spleen surface. Figures 59 and 60 demonstrate that mechanical vibration energy modulates specific target extraaxonal cell synapses in a similar manner to ultrasonic energy.

[0176] Provided herein are techniques for neuromodulation based on direct and focused stimulation of a target region. The target region may be any tissue or structure in the body that has multiple types of axonal terminals that form extra-axonal cell synapses with non-neuronal cells or body fluids. In one example, the target region may be within an immune organ or structure such as the spleen or lymph nodes. Neuromodulation of lymphoid tissue may alter drainage velocity and / or the cell population in the drainage fluid. Because immune cells and nerves innervating the lymphatic compartments of lymph nodes coexist, neurotransmitter release may simultaneously affect both lymphoid and immune function, and thus observable changes in lymphoid function (i.e., easily observable changes in the size and / or flow of lymphoid tissue using non-invasive imaging techniques) can be used as a target physiological outcome and as a surrogate measure of immune cell neuromodulation (i.e., simultaneous changes in the immune cell phenotype due to local neurotransmitter release).

[0177] Provided herein are techniques that may be applied to “neuroimmune synapses” or “neuroimmune interfaces.” The depicted example relates to the application of energy to lymph nodes adjacent to the exit lymphatic vessels. These nerves can alter the passage of lymphocytes through the lymph nodes, and when stimulated, result in increased lymphocyte retention (i.e., more lymphocytes in the lymph nodes). One molecular mechanism for reducing lymphocyte release from the exit lymphatic vessels is the modulation of CCR7 chemoreceptor activity via β-adrenergic receptor coupling and signaling.

[0178] This specification also provides techniques that may be applied to the treatment of glucose metabolism and related disorders. In one embodiment, hepatic regulation in one or more target regions can be used to treat diabetes, hyperglycemia, diabetes-related dementia, obesity, or other eating or metabolic disorders.

[0179] This specification discloses the present invention and uses examples to enable those skilled in the art to carry out the invention, including manufacturing and using any apparatus or system, and performing any incorporated method. The patentable scope of the present invention is defined by the claims and may include other examples that a person skilled in the art can conceive. Such other examples are intended to be within the scope of the claims if they have structural elements that are not different from the literal language of the claims, or if they include equivalent structural elements that are slightly different from the literal language of the claims.

Claims

1. It is a neural regulatory system, An ultrasonic energy application device configured to apply ultrasonic energy of 1 mW / cm² to 30,000 mW / cm² and a peak ultrasonic positive pressure in the range of 0.1 MPa to 7 MPa to a target area of ​​a test subject, and A controller having memory, the memory includes instructions, The target region is a synapse with the non-nerve tissue of the subject, and a synaptic space exists between the nerve tissue and the synapse. When the command is executed by the controller, it causes the controller to control the ultrasonic energy application device. The ultrasonic energy application device applies ultrasonic energy used to acquire image data of the non-nerve tissue to acquire image data, and identifies the non-nerve tissue in the image data. A system that, guided by image processing of the aforementioned image data, applies ultrasonic energy to the target region using an ultrasonic energy application device, focuses the ultrasonic energy on the target region, induces tissue displacement of the non-nerve tissue within the target region with the ultrasonic energy, and causes the release of synapse-related neurotransmitters, thereby producing the target physiological result.

2. The system according to claim 1, wherein the controller is configured to cause the ultrasonic energy application device to apply the ultrasonic energy according to a set of adjustment parameters, and to dynamically change the set of adjustment parameters based on feedback received from an evaluation device or user that exhibits the characteristics of the target physiological result.

3. The system according to claim 2, wherein the feedback indicating the characteristics of the target physiological outcome is a change in the concentration of glucose or insulin, or glucose and insulin, in the subject's blood relative to the concentration measured before the application of the ultrasonic energy.

4. The system according to claim 2, wherein the feedback indicating the characteristics of the target physiological outcome is a change in the concentration of TNF-α or interleukin-1, or TNF-α and interleukin-1, in the subject's blood relative to the concentration measured before the application of the ultrasonic energy, and the non-nerve tissue is spleen tissue.

5. The system according to claim 2, wherein the feedback indicating the characteristics of the target physiological outcome is a change in the concentration of one or both of norepinephrine and acetylcholine in the subject's blood relative to the concentration measured before the application of the ultrasonic energy.

6. When the memory is executed by the controller, the controller is instructed to control the ultrasonic energy application device. To obtain additional image data, The system according to claim 1, further comprising a command to measure the tissue displacement caused by the application of the ultrasonic energy using the additional image data.

7. The system according to claim 1, wherein the non-nerve tissue is liver tissue and the target region is the hepatic portal vein.

8. The system according to claim 1, wherein the non-nerve tissue is spleen tissue.

9. The system according to claim 6, wherein the probability of activating ion channels in the non-nerve tissue is evaluated using the tissue displacement.

10. A software code that causes a controller to control an ultrasonic energy application device, wherein the controller and the ultrasonic energy application device are included in an ultrasonic system, the ultrasonic energy application device has an ultrasonic energy of 1 mW / cm² to 30,000 mW / cm² and a peak ultrasonic positive pressure in the range of 0.1 MPa to 7 MPa, and is configured to apply the ultrasonic energy to a target area of ​​a subject to modulate nerve function, and the controller has a memory for storing the software code. The target region is a synapse with the non-nerve tissue of the subject, and a synaptic space exists between the nerve tissue and the synapse. The aforementioned software code is transmitted to the controller, The steps include: acquiring image data of the non-nerve tissue using the ultrasonic energy application device; Identifying the non-nerve tissue in the image data, wherein the target region includes the non-nerve tissue; The steps include controlling the ultrasonic energy application device to apply ultrasonic energy to the target region in order to achieve a target physiological result through guidance by image processing of the aforementioned image data, Steps to acquire additional image data, A step of measuring the tissue displacement of the non-nerve tissue using the additional image data, A step of determining whether the target physiological outcome has been achieved based on the measured tissue displacement, Make it run, Software code in which the aforementioned target physiological outcome is achieved by releasing neurotransmitters associated with the synapse.

11. The software code according to claim 10, wherein the ultrasonic energy has an ultrasonic frequency in the range of approximately 0.1 MHz to approximately 5 MHz.

12. The software code according to claim 10, wherein the non-nerve tissue is liver tissue, and the target physiological outcome is a change in blood glucose or insulin concentration.

13. The software code according to claim 10, wherein the release of a neurotransmitter or neuropeptide is induced in order to achieve the aforementioned target physiological outcome.

14. The software code according to claim 13, wherein the non-nerve tissue is splenic tissue and the target physiological outcome is a change in TNFα secretion.

15. The software code according to claim 13, wherein the non-nerve tissue is liver tissue, and the target physiological outcome is activation of afferent nerves.

16. The aforementioned software code further, A step of applying one or more ultrasonic energy pulses to the target region, wherein a change is induced in the activity at the synapse between nerve cells and non-nerve cells in response to the one or more ultrasonic energy pulses, with respect to the activity measured before the application of the one or more ultrasonic energy pulses. The software code according to claim 10, which causes the controller to execute the above.

17. The software code according to claim 10, wherein the non-nerve tissue is liver tissue or spleen tissue.

18. It is a neural regulatory system, Includes an ultrasonic energy application device and a controller, The controller includes memory, and when the controller is executed, the memory is provided to the controller. Using the aforementioned ultrasonic energy application device, image data of non-nerve tissue is acquired. To identify the target region of the image data that includes the non-nerve tissue, The target region is a synapse with the non-nerve tissue of the subject, and a synaptic space exists between the nerve tissue and the synapse. A therapeutic energy of 1 mW / cm² to 30,000 mW / cm² and a peak ultrasonic positive pressure in the range of 0.1 MPa to 7 MPa, wherein the therapeutic energy is applied to the target region using adjustment parameters via the ultrasonic energy application device guided by image processing of the image data, thereby causing tissue displacement of the non-nerve tissue in the target region. The ultrasonic energy application device is used to acquire additional image data. Using the additional image data, measure the tissue displacement caused by the application of the therapeutic energy. Includes a controller that stores commands to change one or more of the adjustment parameters based on the measured tissue displacement, A system that, in response to the application of the therapeutic energy to the target region, causes the release of synapse-related neurotransmitters, thereby producing a target physiological outcome.

19. The system according to claim 18, wherein the image data is acquired before the therapeutic energy is applied, and the additional image data is acquired after the therapeutic energy is applied.

20. When an instruction is executed by the controller, the controller, Additional image data is acquired via the ultrasonic energy application device. The system according to claim 1, wherein tissue displacement caused by the application of the therapeutic energy is measured using the additional image data.

21. When an instruction is executed by the controller, the controller, The system according to claim 20, wherein the measured tissue displacement is compared with a threshold.

22. When an instruction is executed by the controller, the controller, The system according to claim 21, wherein the parameters of the therapeutic energy are changed based on the results of the comparison.

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