Neuromodulation Technology

Abstract

The subject matter of the present disclosure generally relates to techniques for neuromodulation of tissue (e.g., an organ) that involves applying energy (e.g., ultrasound energy) to the tissue to alter the activity of synapses between neurons and non-neuronal cells. In one embodiment, the energy is applied to produce competing or opposing effects for bidirectional control of physiological processes. [Selected Figure] Figure 1

Description

[Technical field]

[0001] The subject matter disclosed herein relates to neuromodulation, and more particularly, to techniques for modulating physiological responses using energy applied from an energy source. [Background technology]

[0002] Neuromodulation is used to treat a variety of conditions. For example, chronic back pain is treated by electrical stimulation at various sites along the spinal cord. Such treatment is performed by an implantable device that periodically generates electrical energy that is applied to tissue to activate specific nerve fibers. This can reduce the sensation of pain. For spinal cord stimulation, the stimulating electrode is typically placed in the epidural space. Alternatively, a pulse generator may be placed some distance away from the electrode, for example in the abdomen or buttocks, and connected to the electrode via a lead. As another example, deep brain stimulation may be performed to stimulate specific areas of the brain to treat movement disorders. Neuroimaging may provide guidance to the stimulation location. Such central nervous system stimulation may generally be performed via electrodes that target local nerve or brain cell function, deliver electrical pulses, and are placed at or near the target nerve. Summary of the Invention [Problem to be solved by the invention]

[0003] However, it is difficult to place electrodes at or near a target nerve. For example, such techniques may involve placing energy delivery electrodes through a surgical procedure. Furthermore, it is difficult to target specific tissues with neuromodulation. Electrodes placed at or near a specific target nerve mediate neuromodulation by triggering action potentials in nerve fibers. This can result in neurotransmitter release by neural synapses and synaptic transmission with adjacent nerves. Currently, implanted electrodes stimulate many nerves or axons at once. Thus, the propagation can result in larger or more diffuse physiological effects than expected. Because neural pathways are complex and interconnected, more targeted modulation effects would be more clinically effective. [Means for solving the problem]

[0004] Certain embodiments commensurate in scope with the originally claimed subject matter are summarized below. These embodiments are not intended to limit the scope of the claimed subject matter, but rather these embodiments are intended only to provide a brief summary of possible embodiments. Indeed, the invention may encompass a variety of forms that may be similar to or different from the embodiments set forth below.

[0005] In one embodiment, a regulating system is provided, comprising at least one energy applicator and a controller, the controller configured to: focus the at least one energy applicator on a first region of interest of a first tissue, the first tissue including a first plurality of axon terminals of respective neurons, one or more of the axon terminals forming synapses between individual axon terminals and corresponding non-neuronal cells, the first region of interest including a first subset of the first plurality of axon terminals; adjustably control the at least one energy applicator to apply a first energy to the first region of interest to selectively modulate a first molecule of interest; focus the at least one energy applicator on a second region of interest including a second subset of axon terminals; and adjustably control the at least one energy applicator to apply a second energy to the second region of interest to selectively modulate the first molecule of interest or a second molecule of interest.

[0006] In another embodiment, a regulation system is provided that includes at least one energy application device configured to apply energy to a first region of interest in a first tissue and a second region of interest in a second tissue in a subject, the first tissue including a plurality of axon terminals of respective neurons, the axon terminals forming synapses between individual axon terminals and corresponding non-neuronal cells. The system further includes a controller configured to spatially select the first region of interest in the first tissue and the second region of interest in the second tissue, focus the energy to the first region of interest and the second region of interest, adjustably control the application of energy to the first region of interest via the at least one energy application device, and control the application of energy to the second region of interest via the at least one energy application device to induce preferential activation of a subset of the synapses present in the first region of interest to alter a first molecular concentration of a first molecule and to alter a second molecular concentration of a second molecule.

[0007] In another embodiment, a regulation system is provided that includes an energy application device configured to apply energy to a region of interest of a pancreas in a subject, the region of interest being a sub-region of the pancreas that includes synapses between neural cells and corresponding non-neuronal cells, and further includes a controller configured to spatially select the region of interest of the pancreas, focus the energy at the region of interest, and adjustably control the application of the energy to the region of interest of the pancreas via the energy application device, thereby causing preferential activation at the synapses between neural cells and corresponding non-neuronal cells in the region of interest, thereby selectively modulating or causing a change in concentration of one or more molecules of interest.

[0008] These and other features, aspects, and advantages of the present invention will be better understood when the following detailed description is read in conjunction with the accompanying drawings, in which like characters represent like parts throughout. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 is a schematic diagram of a neuromodulation system using a pulse generator according to an embodiment of the present disclosure. [Diagram 2] FIG. 1 is a block diagram of a neuromodulation system according to an embodiment of the present disclosure. [Diagram 3] FIG. 1 is a schematic diagram of an ultrasonic energy application device in operation according to an embodiment of the present disclosure. [Figure 4A] 1 illustrates ultrasound visualization of the spleen, which may be used as spatial information to focus on a region of interest in the spleen, according to an embodiment of the present disclosure. [Figure 4B] 1 illustrates ultrasound visualization of the liver that may be used as spatial information to focus on a region of interest in the liver, according to an embodiment of the present disclosure. [Diagram 5] FIG. 1 is a flow diagram of a neuromodulation technique according to an embodiment of the present disclosure. [Figure 6] FIG. 1 is a schematic diagram of an energy application device configured as an extracorporeal device and including an ultrasound transducer. [Figure 7] FIG. 1 is a schematic diagram of an energy application device and pulse generator configured to apply high intensity focused ultrasound. [Figure 8] 8 illustrates an example of an energy application device that may be used in conjunction with the system of FIG. 7. [Figure 9] FIG. 1 is a schematic diagram of an experimental setup for application of ultrasonic energy to achieve a desired physiological effect. [Figure 10] 1 is an experimental timeline for ultrasonic energy application. [Figure 11] 4 illustrates the pulse characteristics of an applied ultrasonic energy pulse. [Figure 12] The hydrophone measurement setup is shown. [Figure 13] 1 shows an example of an ultrasonic pressure field in the xy plane. [Figure 14] We demonstrate the experimental workflow of LPS injection to generate models of inflammation and / or hyperglycemia / hyperinsulinemia and ultrasound treatment. [Figure 15A] FIG. 1 is a schematic diagram of comprehensive vagus nerve stimulation. [Figure 15B] FIG. 1 is a schematic diagram of organ-based, targeted peripheral neuromodulation. [Figure 16A] 1 is an experimental timeline for application of ultrasound energy to the rat spleen. [Figure 16B] 1 shows splenic norepinephrine, acetylcholine, and TNF-α at different applied ultrasound energy levels to a rat spleen, shown as ultrasound pressure in MPa. [Figure 16C] Circulating concentrations of TNF-α are shown under the same conditions as in FIG. 16B. [Figure 16D] Splenic IL-1α concentrations are shown under the same conditions as in FIG. 16B. [Figure 16E] The response time to induced changes in splenic TNFα concentrations compared to controls is shown. [Figure 16F] 2D ultrasound images of a rat spleen are shown, which are used to focus ultrasound stimulation to spatially select the splenic target. [Figure 16G] FIG. 16G shows a timeline of a study designed to measure the duration of the effect of stimulation on cholinergic anti-inflammatory pathway activation. [Figure 16H] 4 shows splenic TNF-α concentrations following protective ultrasound treatment. [Figure 16I] 1 shows the concentrations of activated / phosphorylated kinases as a result of splenic ultrasound modulation. [Figure 16J] 1 shows exemplary ultrasound burst durations using different ultrasound stimulation parameters and the effect on norepinephrine (NE), acetylcholine (ACh), and tissue necrosis factor alpha (TNF-α) concentrations in ultrasound-stimulated spleen (following LPS injection). [Figure 16K] Exemplary ultrasound carrier frequencies using different ultrasound stimulation parameters and their effects on norepinephrine (NE), acetylcholine (ACh), and tissue necrosis factor alpha (TNF-α) concentrations in ultrasound-stimulated spleen (following LPS injection) are shown. [Figure 17A] The effects of splenic ultrasound modulation are shown compared to standard electrodes or implanted vagus nerve stimulation (VNS) on splenic TNF-α in the presence of various inhibitors. [Figure 17B] Shown are the effects of α-bungarotoxin on splenic concentrations of (left) norepinephrine (NE) and (right) TNF-α following ultrasound stimulation in LPS-treated rodents with and without the effects of BTX or surgical vagus nerve stimulation. [Figure 17C] Data are presented comparing the effects of VNS (at several stimulation intensities and frequencies) versus splenic ultrasound stimulation (at 0.83 MPa) on heart rate. [Figure 17D] We present data supporting the previously observed side effects of VNS and the lack of side effects when using splenic ultrasound stimulation with regard to suppression of LPS-induced hyperglycemia. [Figure 18A] 2D ultrasound image of a rat liver used to focus ultrasound stimulation. [Figure 18B] 1 shows the effect of ultrasound stimulation of the liver on LPS-induced hyperglycemia. [Figure 18C]Measurements of the relative concentrations (compared to those in the absence of ultrasound stimulation) of several molecules related to either insulin sensitivity and changes in hypothalamic markers related to insulin-mediated and non-insulin-dependent glucose uptake and metabolic function in the liver are shown. [Figure 18D] Shown are cFOS immunohistochemistry images (left) and data showing the number of activated neurons in LPS control and ultrasound stimulated samples (right). [Figure 18E] Further immunohistochemistry images are shown showing cFOS expression in the brainstem of LPS controls (top) versus ultrasound stimulated samples (bottom). [Figure 18F] An exemplary MRI overlay between activation maps (over SPGR, left) and a brain atlas (over SPBR, right) is shown. [Figure 18G] FIG. 18G shows t-test values ​​from corresponding pixel comparisons between pre- and post-treatment DWI images in which a valid PVN ROI was applied to both images. [Figure 19] FIG. 19 shows reduced circulating glucose levels by pancreatic ultrasound stimulation in LPS rats. [Figure 20] FIG. 20 shows an increase in circulating insulin in pancreatically stimulated LPS SD rats. [Figure 21] FIG. 21 shows that there was no significant change in circulating glucagon concentrations as a result of ultrasound stimulation in gastrointestinal, hepatic, and pancreatic stimulated LPS SD rats. [Figure 22] FIG. 22 shows the levels of insulin in pancreatic tissue as a result of pancreatic ultrasound stimulation in LPS SD rats. [Diagram 23] FIG. 23 shows the levels of glucagon in pancreatic tissue as a result of pancreatic ultrasound stimulation in LPS SD rats. [Figure 24] FIG. 24 illustrates regions of interest within gastrointestinal tissue targeted for gastrointestinal ultrasound stimulation in LPS SD rats as described herein. [Diagram 25] FIG. 25 shows the reduction in circulating glucose in response to gastrointestinal ultrasound stimulation in a rat model. [Figure 26] FIG. 26 shows insulin levels in the pancreas following gastrointestinal ultrasound stimulation. [Figure 27] FIG. 27 shows glucagon levels in the pancreas following gastrointestinal ultrasound stimulation. [Figure 28] FIG. 28 shows hypothalamic IRS1 levels in response to gastrointestinal, hepatic, and pancreatic ultrasound stimulation compared to controls. [Figure 29] FIG. 29 shows hypothalamic phospho-Akt levels in response to gastrointestinal, hepatic, and pancreatic ultrasound stimulation compared to controls. [Diagram 30] FIG. 30 shows hypothalamic glucose-6-phosphate levels in response to gastrointestinal, hepatic, and pancreatic ultrasound stimulation compared to controls. [Diagram 31] FIG. 31 shows hypothalamic GLP-1 levels in response to gastrointestinal, hepatic, and pancreatic ultrasound stimulation compared to controls. [Diagram 32] FIG. 32 shows hypothalamic BDNF levels in response to gastrointestinal, hepatic, and pancreatic ultrasound stimulation compared to controls. [Diagram 33] FIG. 33 shows hypothalamic GABA levels in response to gastrointestinal, hepatic, and pancreatic ultrasound stimulation compared to controls. [Diagram 34] FIG. 34 shows hypothalamic NPY levels in response to gastrointestinal, hepatic, and pancreatic ultrasound stimulation compared to controls. [Diagram 35] FIG. 35 shows pancreatic GLUT2 levels and the effects of hepatic, gastrointestinal, or pancreatic ultrasound stimulation. [Diagram 36] FIG. 36 shows pancreatic phospho-Akt levels and the effect of hepatic, gastrointestinal, or pancreatic ultrasound stimulation. [Figure 37] FIG. 37 shows pancreatic IRS1 levels and the effects of hepatic, gastrointestinal, or pancreatic ultrasound stimulation. [Figure 38] FIG. 38 shows pancreatic GLP-1 levels and the effect of hepatic, gastrointestinal, or pancreatic ultrasound stimulation. [Figure 39] FIG. 39 shows pancreatic insulin levels and the effects of hepatic, gastrointestinal, or pancreatic ultrasound stimulation. [Diagram 40]FIG. 40 shows pancreatic glucagon levels and the effect of hepatic, gastrointestinal, or pancreatic ultrasound stimulation. [Diagram 41] FIG. 41 shows hepatic glucose-6-phosphate levels and the effect of hepatic, gastrointestinal, or pancreatic ultrasound stimulation. [Diagram 42] FIG. 42 shows hepatic norepinephrine levels and the effect of hepatic, gastrointestinal, or pancreatic ultrasound stimulation. [Diagram 43] FIG. 43 shows hepatic IRS-1 levels and the effect of hepatic, gastrointestinal, or pancreatic ultrasound stimulation. [Diagram 44] FIG. 44 shows hepatic phospho-Akt levels and the effect of hepatic, gastrointestinal, or pancreatic ultrasound stimulation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] One or more specific embodiments are described below. In order to provide a concise description of these embodiments, not all features of the actual form are described herein. It should be understood that the development of such an actual implementation, like any engineering or design project, requires numerous implementation-specific decisions to be made to achieve developer-specific goals, including compliance with system-related and business-related constraints that may vary from implementation to implementation. It should be understood that such development efforts may be complex and time-consuming, but are nevertheless within the routine scope of design, fabrication, and manufacturing for those of ordinary skill in the art having the benefit of this disclosure.

[0011] The examples or diagram representations shown herein should in no way be considered to limit, restrict, or define the terms used therein. Instead, these examples or diagram representations should be considered to be described with respect to various specific embodiments and should be considered to be merely exemplary. Those skilled in the art will understand that any term used with these examples or diagram representations also applies to some embodiments, even if not used elsewhere in the specification, and all of those embodiments are included within the scope of the term or terms. Language designating such non-limiting example and diagram representations includes, but is not limited to, "for example," "for example," "such as," "for example," "including," "certain embodiments," "some embodiments," and "in one embodiment."

[0012] Provided herein are techniques for neuromodulation that realize a desired physiological effect that is a result of the modulation based on direct and focused modulation of a target region of interest. One or more target regions of interest may be any tissue or structure in the body that has axon endings that form synapses with non-neuronal cells or fluids. In one example, the region of interest may be within an organ or structure such as the spleen, liver, pancreas, or gastrointestinal tissue. In another example, the region of interest may be within lymphatic tissue. Neuromodulation of a region of interest allows for localized, limited, and non-ablative application of energy only to the target region of interest, preventing energy from being applied outside the region of interest. Energy application may cause downstream effects outside the target region of interest, for example, in the same organ, tissue, or structure that contains the region of interest, or in other organs and structures that do not contain the region of interest. In some embodiments, downstream effects may occur, for example, in the region of the hypothalamus. Energy application may also produce effects along the target nerve upstream of the site of energy application. In some embodiments, effects outside the target region of interest may be achieved without applying energy directly to areas outside the region of interest where downstream or upstream effects occur. Thus, local energy application may be used to achieve or effect systemic effects, which may include local effects, downstream effects and / or upstream effects.

[0013] Additionally, the technology relates to bidirectional control of complex physiological processes. For example, to avoid excessive activation of one pathway and excessive changes in physiological effects as a result of energy application in a particular region of interest, energy may be applied to another region of interest associated with a competing or inactivated pathway. This induces changes in physiological effects in the opposite direction, maintaining a dynamic balance of physiological effects to achieve the desired physiological effect. In a particular example, to achieve a desired circulating glucose concentration or range of concentrations in a hyperglycemic subject, energy may be applied to a region of interest that causes a decrease in circulating glucose concentration. Meanwhile, to avoid glucose overcorrection and resulting hypoglycemia, energy may be applied to a second region of interest that causes an increase in circulating glucose concentration to actively maintain a dynamic equilibrium of circulating glucose concentration and stabilize circulating glucose concentration at a desired level. For example, if application of energy directly to the liver results in a decrease in glucose beyond a clinically acceptable level, energy may be applied to the pancreas to compensate by increasing glucagon production. As provided herein, bidirectional stimulation may be performed on a first region of interest in a first organ and a second region of interest in a second organ. In another embodiment, multi-site neuromodulation can be neuromodulation performed at different sites that enhance the same pathway. The application of energy to the first and second regions of interest can be simultaneous or at different times (e.g., separated by seconds, minutes, days, or hours) and can be performed by the same or different energy application devices.

[0014] The disclosed technology can be used to exert external control over physiological processes of the body to achieve a desired physiological effect in a subject. Through neuromodulation to a target region of interest, physiological processes can be altered, slowed down, stopped, or reversed. Also provided herein are technologies that can be applied to a subject to promote dynamic equilibrium or homeostasis of physiological processes, such as glucose regulation. Targeted neuromodulation can act against ongoing etiology or disease progression, provide treatment, and improve outcomes compared to controls (i.e., compared to untreated subjects). In some embodiments, targeted neuromodulation can be preventative and occur before a specific event occurs. For example, targeted neuromodulation can be used to prevent anorexia associated with certain medical procedures or conditions, or applied before or during a meal to change the body's response to food.

[0015] Neuromodulation to a target region of interest may produce changes in physiological processes that block, reduce, or enhance one or more physiological pathways in a subject to achieve a desired physiological effect. Furthermore, local energy application may produce systemic changes, such that different physiological pathways may be altered in different ways and at different locations in the body. This alters the overall characteristic properties of the physiological changes in the subject, as well as the characteristics of the targeted neuromodulation for a particular subject. These alterations are complex. Nevertheless, the present neuromodulation techniques produce one or more targeted, measurable physiological effects in the treated subject as a result of neuromodulation that may not be achievable without the application of energy or other intervention to the target region(s) of interest. Furthermore, other types of interventions (e.g., drug therapy) may produce a subset of the physiological changes caused by neuromodulation. However, in certain embodiments, the characteristics of the physiological effects induced as a result of neuromodulation may be specific to the neuromodulation (and associated modulation parameters) at the target region(s) of interest, and may vary from patient to patient.

[0016] Using the neuromodulation techniques discussed herein, physiological effects of changing the concentration (e.g., increase, decrease) of a molecule of interest and / or changing the properties of the molecule of interest can be achieved. That is, selective modulation of one or more molecules of interest (e.g., first molecule of interest, second molecule of interest, ...) as a result of application of energy to one or more regions of interest (e.g., first region of interest, second region of interest, ...) in one or more tissues (e.g., first tissue, second tissue, ...) can refer to modulation or influence of the concentration (circulation, tissue) or characteristics (covalent modification) of the molecule. Modulation of a molecule of interest can include changes in molecular characteristics such as protein expression, secretion, translocation, as well as changes in direct activity, either by the application of energy itself or as a result of direct effects on ion channels of the molecule. Modulation of a molecule of interest can also refer to maintaining a desired concentration of the molecule such that expected changes or fluctuations in concentration do not occur as a result of neuromodulation. Modulation of a molecule of interest can refer to causing changes in molecular properties such as enzyme-mediated covalent modifications (changes in phosphorylation, acetylation, ribosylation, etc.). That is, it should be understood that selective modulation of the molecule of interest may indicate molecular concentration and / or molecular properties. The molecule of interest may be a biomolecule, such as one or more of carbohydrates (monosaccharides, polysaccharides), lipids, nucleic acids (DNA, RNA), or proteins. In certain embodiments, the molecule of interest may be a signaling molecule, such as a hormone (amine hormone, peptide hormone, or steroid hormone).

[0017] Certain embodiments described herein provide neuromodulation techniques to achieve desired physiological effects for the treatment of glucose metabolism and related disorders. Glucose regulation is complex and involves different local and systemic metabolic pathways. Energy application to one or more targeted regions of interest causes characteristic changes in these metabolic pathways to improve glucose regulation. In some embodiments, modulation at one or more regions of interest can be utilized to treat disorders including, but not limited to, diabetes (i.e., type 1 or type 2 diabetes), hyperglycemia, sepsis, trauma, infection, physiological stress, diabetes-related dementia, obesity, or other eating or metabolic disorders. In some embodiments, neuromodulation can be utilized to promote weight loss, appetite control, cachexia treatment, or appetite enhancement. In one example, physiological stress can be medically defined to include various acute medical conditions (infection, severe injury / trauma, heart attack, bypass), as well as surgical cases with symptoms of hyperglycemia. For example, direct stimulation of the pancreas can achieve appetite enhancement, and direct stimulation of the liver can cause NPY reduction, thereby promoting satiety signals. The desired physiological effect may include regulating the subject's circulating (i.e., blood) glucose concentration to be within a desired concentration range associated with normal glucose levels, avoiding hyperglycemia or hypoglycemia. In this manner, selective regulation of a molecule of interest may be achieved. The regulation may be the result of induced changes in blood or tissue glucoregulatory hormones via targeted neuromodulation, resulting in a desired glucose concentration (i.e., a desired glucose end point). Additionally, glucose regulation may be beneficial for patients who are undiagnosed and healthy, but who are pre-diabetic or wish to maintain a healthy weight.

[0018] Advantages of targeted neuromodulation provided herein include the flexibility of adjusting or regulating the therapeutic level (i.e., the adjustment parameters can be adjusted). For example, the location, frequency, and duration of energy application can be adjusted to facilitate adjustment of the desired physiological effect in response to changes in the physiological state of the subject. For example, food intake results in changes in endocrine hormone levels. Targeted neuromodulation can be used to induce desired changes in the body's response to food intake, whether the response is healthy or pathological. Furthermore, it can be tailored to the characteristic response of a particular subject to food intake or other physiological conditions. For example, a bidirectional treatment where neuromodulation treatment induces increased circulating insulin and consequently decreased circulating glucose in some patients, and increased tissue and / or blood glucagon in others, can avoid the hypoglycemic endpoint of the treatment. That is, the concentration of circulating glucose after treatment associated with hypoglycemia may be desirable to avoid. Thus, the neuromodulation treatment provided herein can be performed at a single site or multiple sites as appropriate to achieve the desired concentration of circulating glucose. Because glucose concentrations vary in response to food, sleep, and other physiological inputs, the desired glucose concentration may fall within a range of acceptable fasting or post-prandial glucose levels appropriate for the subject.

[0019] To that end, the present technology relates to targeted modulation of synapses at axon terminals in tissues by direct application of energy from an energy source that causes changes that result in a measurable physiological effect (e.g., a change in circulating molecule concentration, or a series of concentration changes that result in a characteristic physiological characteristic). The targeted synapse may include an axon-extracellular synapse formed between a presynaptic axon terminal and a postsynaptic non-neuronal cell. Additionally, while certain disclosed embodiments are discussed with respect to axon-extracellular synapses, it should be understood that the axon terminal may form an axon-secretory, axon-synapse, axon- or axon-extracellular synapse, and / or these types of synapses are contemplated to be selectively modulated as described herein. Additionally, certain axon terminals may also be terminations in interstitial or body fluids that may also release neurotransmitters as a result of modulation. The disclosed synapses may be modulated such that the application of energy results in altered activity at the synapse, e.g., release of neurotransmitters from the presynaptic axon terminal. Thus, the altered activity may result in local and / or non-local (e.g., systemic) effects, resulting in an overall profile of physiological changes associated with a desired or targeted physiological effect. The present technique allows for the focus of energy on portions of tissue that contain specific axon terminals, thereby preferentially and directly activating the targeted axon terminals to achieve the desired outcome. In this manner, in certain embodiments, targeted axon terminals within a target region are activated without activating axon terminals outside the region of interest within the same organ or tissue structure. Organs and tissue structures may contain different types of axon terminals that form synapses with different types of postsynaptic non-neuronal cells. Thus, a region of interest may be selected that contains a particular type of axon terminal whose activation results in a desired physiological effect. Thus, modulation may be targeted to a particular type of axon terminal based on presynaptic neuron type, postsynaptic cell type, or both.

[0020] For example, in one embodiment, the type of axon terminal can be an axon terminal that forms an axon-extracellular synapse with a resident (i.e., tissue-resident or non-circulating) liver, pancreas, or gastrointestinal tissue cell. That is, an axon-extracellular synapse is formed at the junction between the axon terminal and a non-neuronal cell, interstitium, or body fluid. Thus, energy application results in modulation of function in the region of interest. However, based on the population of axon terminal types and the characteristics of the presynaptic neuron type and postsynaptic cell (e.g., immune cells, lymphocytes, mucosal cells, muscle cells, etc.) of the axon-extracellular synapse, the physiological effect of interest achieved may differ. Thus, application of energy to a region of interest in a tissue of interest may activate axon terminals and their associated axon-extracellular synapses within the region of interest without affecting non-target axon terminals (and associated synapses) outside the region of interest. However, because modulation may result in systemic effects, activation of axon terminals within the region of interest may also result in certain systemic changes in non-target axon terminals outside the region of interest. As described herein, preferential activation or direct activation may refer to cells or structures (e.g., synapses) within the region of interest to which energy is applied directly (e.g., energy is applied directly to the cells or structures). For example, the direct recipients of energy application as provided herein are axon terminals, axon-extracellular synapses, and / or postsynaptic non-neuronal cells, or interstitium or body fluids within the region of interest. Preferential or direct activation may be understood for regions outside the region of interest that are not directly applied with energy. However, such regions also undergo physiological changes as a result of energy application.

[0021] The human nervous system is a complex network of nerve cells (neurons) located at the center, such as the brain and spinal cord, and at the periphery of the body in various nerves. Neurons have a cell body, dendrites, and an axon. A nerve is a group of neurons that serves a specific part of the body. A nerve may contain hundreds to hundreds of thousands of neurons. Many nerves contain both afferent and efferent neurons. Afferent neurons carry signals to the central nervous system, and efferent neurons carry signals to the periphery. A group of nerve cell bodies in one location is called a ganglion. Electrical signals generated within a nerve (for example, via intrinsic or externally applied stimuli) are transmitted through neurons and nerves. Neurons release neurotransmitters at synapses (connections) adjacent to receptor cells to allow the electrical signal to persist and be regulated. In the periphery, synaptic transmission often occurs in ganglia.

[0022] The electrical signal of a neuron is called an action potential. When the potential across the cell membrane exceeds a certain threshold, an action potential is generated. The action potential then propagates throughout the length of the neuron. The action potential of a nerve is complex, being the sum of the action potentials of the individual neurons within it. The junction between the axon terminal of a neuron and the receptor cell is called a synapse. The action potential travels down the axon of the neuron to its axon terminal, the distal end of the axon nerve branch that forms the presynaptic or synaptic terminal of the nerve fiber. The electrical impulse of the action potential results in the movement of vesicles containing neurotransmitters to the presynaptic membrane of the presynaptic axon terminal, which ultimately results in the release of neurotransmitters into the synaptic cleft (e.g., the space formed between the presynaptic and postsynaptic cells) or outside the axon cell. The synapse that reaches the synaptic terminal and converts the electrical signal of the action potential into a chemical signal of neurotransmitter release is called a chemical synapse. Chemical synapses are contrasted with electrical synapses. An electrical synapse is one in which an ionic current flows into the presynaptic axon terminal and crosses the barrier of two cell membranes to enter the postsynaptic cell.

[0023] The physiological effects of action potentials are mediated by the movement of ions across the cell membrane. Neurons transport Na + , K + , Cl- Neurons actively maintain a resting membrane potential via ion pumps that facilitate the passage of ions such as arginine, arginine, and acetylcholine across the nerve membrane. Different types of neurons can maintain different resting potentials (e.g., from -75 mV to -55 mV). Action potentials are generated by the influx of ions, i.e., the movement of electrical charges that produces large fluctuations in the membrane potential associated with a temporary increase in the membrane potential (e.g., an increase in the range of 30 to 60 mV). Action potentials in individual neurons arise in response to the release of neurotransmitters from presynaptic (e.g., upstream) neurons, triggering receptor binding at the postsynaptic cell and a series of events that lead to ion influx and membrane depolarization. This results in an action potential that is propagated through the nerve.

[0024] A synapse may be located at the junction between two neurons, allowing the propagation of an action potential down a nerve fiber. However, axon terminals may also form synapses at junctions between neurons and non-neuronal cells, or terminate in interstitial or body fluids. Examples of types of synapses include synapses with immune cells at neuroimmune junctions, synapses with resident sensory cells in organs, or synapses with glandular cells. Release of a neurotransmitter into the synaptic cleft and binding to receptors on the postsynaptic membrane of the postsynaptic cell results in a downstream effect. This effect depends on the nature of the presynaptic neuron and the particular neurotransmitter released, as well as the nature of the postsynaptic cell (e.g., the type of available receptors on the postsynaptic cell). Furthermore, action potentials can be excitatory or inhibitory. An excitatory postsynaptic action potential is a postsynaptic potential where the postsynaptic neuron is likely to fire or release an action potential in response. On the other hand, an inhibitory postsynaptic action potential is a postsynaptic potential where the postsynaptic neuron is unlikely to fire or release an action potential in response. Furthermore, several neurons may work together to release neurotransmitters in a coordinated manner to either induce downstream action potentials or inhibit downstream action potentials.

[0025] Neuromodulation is a technique in which energy from an external source is applied to specific regions of the nervous system to activate or increase a nerve or nerve function and / or block or decrease a nerve or nerve function. In certain neuromodulation techniques, one or more electrodes are applied to or near a target nerve, and the application of energy is transmitted through the nerve (e.g., as an action potential) to elicit a physiological response in an area downstream from the site of energy application. However, due to the complexity of the nervous system, it is difficult to predict the extent and ultimate endpoint of the physiological response at a particular site of energy application.

[0026] Although techniques involving ultrasonic modulation of the central nervous system (i.e., brain tissue) have been shown to achieve desired modulation of neural activity, attempts at modulation of peripheral nerves have lagged behind. For example, ultrasonic modulation of the central nervous system (CNS) may involve stimulation of cortical regions of the brain that contain a large amount of synaptic structures, whereas attempts at ultrasonic stimulation of peripheral nerves target nerve trunks that have fewer or no synaptic structures.

[0027] In the present technique, modulation of peripheral nerves involves applying energy to one or more targeted peripheral axon terminals to affect blood glucose levels and / or to affect glucose regulatory pathways and / or insulin production pathways. In the present technique, one or more energy pulses are applied to a subject's internal tissue, including an axon terminal. The axon terminal includes an axon extracellular synapse and a nerve junction with other cell types, interstitial fluid, or body fluids, for example, at a synapse between a neuronal cell and a non-neuronal cell. Thus, application of energy to a synapse results in direct activation of the presynaptic axon terminal and / or direct activation at the postsynaptic cell to achieve a desired physiological effect. In one example, stimulation of the axon terminal causes release of neurotransmitters / neuropeptides or altered neurotransmitter release in the vicinity of adjacent non-neuronal cells, such as secretory cells or other cells. This modulates cellular activity of adjacent or nearby non-neuronal cells, including the postsynaptic cells. Furthermore, through such modulation, other tissue structures or organs may be modulated without being directly stimulated. In one embodiment, direct energy application to a relatively small area of ​​an organ (e.g., less than 25% of the total organ volume) can result in action potentials in afferent neurons that project to different regions of the brain (e.g., the hypothalamus). However, this can be achieved without direct brain stimulation to synapse-rich regions. Direct brain stimulation can result in undesired activation of other pathways, potentially interfering with or inhibiting the desired physiological effect. Furthermore, direct brain stimulation can involve invasive procedures. Thus, the present technology allows for detailed activation of either brain activity or activity within an organ, tissue, or structure in a more targeted and specific manner than direct brain stimulation or electrical peripheral nerve stimulation. That is, detailed activation can allow for predictable and targeted activation of only certain molecules and not other molecules, and / or only certain tissues and not other tissues.

[0028] Advantages of the present technology include localized modulation of tissue at a region of interest, i.e., to achieve the effect of changing the concentration of one or more molecules of interest. Moreover, to achieve the above-mentioned effects, the localized modulation may include direct activation of a relatively small region of tissue (e.g., less than 25% of the total tissue volume). Thus, the total energy applied to achieve the desired physiological effect is relatively small. In certain embodiments, the energy applied may be from a non-invasive external energy source (e.g., ultrasonic energy source, mechanical vibrator). For example, a focused energy probe may apply energy through the subject's skin in a focused manner to a region of interest in the internal tissue. Such embodiments achieve the desired physiological effect without invasive procedures and without side effects that may be associated with other types of treatments or therapies.

[0029] Furthermore, the technology relates to multi-site control of complex physiological processes. In some embodiments, a first energy application at a first region of interest may induce a first result (e.g., increase / decrease in the concentration of one or more molecules that are outside a desired range). A second energy may be applied to a different second region of interest to produce a result in the opposite direction (e.g., decrease / increase the concentration of one or more molecules to be within a desired range). In this way, the concentration of the molecule of interest can be selectively regulated. The first and second regions of interest may be in the same organ, tissue or structure, or in different organs, tissues or structures. In some embodiments, the first effect may be related to the overactivation of one pathway and the second effect may be related to a competing or deactivated pathway, thereby providing a compensatory effect to the first result to achieve bidirectional control or regulation of a metabolic pathway or the concentration of the molecule of interest.

[0030] In a non-limiting example, to control the circulating glucose concentration of a hyperglycemic subject to a desired range, a first energy may be applied to a first region of interest causing a decrease in the circulating glucose concentration. Meanwhile, to avoid glucose overcorrection and the resulting hypoglycemia, a second energy may be applied to a second region of interest different from the first region of interest causing an increase or decrease in the circulating molecule concentration to stabilize or regulate the glucose concentration to a desired level. Thus, in one example, a first energy may be applied to a first region of interest in the liver to decrease the circulating glucose concentration, i.e., the first molecule of interest. A second energy may be applied to a second region of interest in the pancreas to increase glucagon, i.e., the second molecule of interest, thereby increasing circulating glucose, i.e., the first molecule of interest. However, the magnitude of the effect on circulating glucose based on the application of the first energy may be different from the application of the second energy. That is, the application of the first energy may decrease the glucose concentration to a greater extent than the application of the second energy increases glucose for the same subject. In this manner, treatment at the first region of interest and the second region of interest may be calibrated to achieve a desired favorable physiological effect and adjust the glucose end point.

[0031] Furthermore, to achieve the desired adjustment, the adjustment parameters of the energy application in the first region of interest may be the same or different from the adjustment parameters in the second region of interest. They may be configured for the specific site of energy application. In some embodiments, the first region of interest may have a broader range of physiological effects compared to the second region of interest. Thus, adjustably controlling the energy application in the first region of interest may result in a more diverse range of effects, while adjustably controlling the energy application in the second region of interest may result in a more limited range of effects. Furthermore, this may vary from patient to patient. Diseased organs may respond differently to energy application compared to healthy organs.

[0032] The selective regulation provided herein can be triggered by the concentration (e.g., circulating concentration, tissue concentration) of the molecule of interest deviating from a desired range. Depending on how it deviates (above or below the desired range), energy is applied to the appropriate region of interest. For example, determining that the concentration of the molecule of interest is above a desired concentration can result in the application of energy to the region of interest, resulting in a decrease in the concentration of the molecule of interest. Bidirectional control can be achieved through the application of energy to a region of interest associated with a competing pathway. Furthermore, determining that the concentration of the molecule of interest is within a target concentration range can provide instructions to not trigger the application of energy or to trigger the application of a lower level of energy.

[0033] In one embodiment, direct energy application (i.e., direct stimulation) of a region of interest in the pancreas can cause an increase in circulating insulin concentration as a result of increased pancreatic insulin production, but energy can also be applied to a region of interest in gastrointestinal tissue to competitively reduce circulating insulin concentration. In certain embodiments, energy application to three or more regions of interest can be applied, allowing for a coordinated function to control circulating insulin concentration within a desired range. For example, the target region of interest can be in the liver, pancreas, or gastrointestinal tissue.

[0034] Certain embodiments describe a technique of neuromodulation, in which energy from an energy source (e.g., an external or extracorporeal energy source) is applied to an axon terminal. This allows a physiological effect, such as neurotransmitter release, to occur at the energy application focal site, such as the axon terminal, not through an action potential, but through energy application. That is, direct energy application to the axon terminal acts in place of an action potential to promote neurotransmitter release to a neural junction (i.e., synapse) with a non-neuronal cell. Direct energy application to the axon terminal further causes changes in neurotransmitter release from the axon terminal within the synapse (e.g., axon-extracellular synapse) to nearby adjacent non-neuronal cells. In one embodiment, the energy source is an extracorporeal energy source, such as an ultrasonic energy source or a mechanical vibrator. In this way, non-invasive and targeted neuromodulation can be achieved directly at the energy focal site, rather than through stimulation at an upstream site that would generate an action potential that propagates to a downstream target site and activates the downstream site.

[0035] In certain embodiments, the target tissue is an internal tissue or organ that is difficult to reach by electrical stimulation techniques using electrodes.Possible target tissues include gastrointestinal (GI) tissues (stomach, intestine), muscle tissues (cardiac, smooth and skeletal), epithelial tissues (epidermis, organ / GI lining), connective tissue, glandular tissues (exocrine / endocrine), etc.In one example, focused application of energy at the neuromuscular junction promotes neurotransmitter release at the neuromuscular junction without upstream action potentials.In one embodiment, possible regulatory targets may include sites in the pancreas involved in controlling insulin release or sites in the liver involved in glucose regulation.

[0036] As such, the disclosed neuromodulation techniques may be used with a neuromodulation system. FIG. 1 is a schematic diagram of a system 10 for neuromodulation in which application of energy results in the release of neurotransmitters and / or activation of synaptic components (e.g., presynaptic cells, postsynaptic cells). The illustrated system includes a pulse generator 14 coupled to an energy application device 12 (e.g., an ultrasound transducer). The energy application device 12 is configured to receive energy pulses that, in use, are delivered to an area of ​​interest of an internal tissue or organ of a subject, e.g., via a wire or wireless connection. This achieves a desired physiological effect. 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), with one or more leads internally coupling 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 micromachined ultrasound transducer.

[0037] In certain embodiments, the energy application device 12 and / or the pulse generator 14 may communicate wirelessly with, for example, the controller 16, which may provide instructions to the pulse generator 14. In other embodiments, the pulse generator 14 may be an extracorporeal device, e.g., operable to apply energy transcutaneously or non-invasively from a location outside the subject's body, and in certain embodiments may be integrated within the controller 16. In embodiments in which the pulse generator 14 is extracorporeal, the energy application device 12 may be operated by a caregiver and placed in contact or non-contact on the subject's skin such that energy pulses are delivered transcutaneously to desired internal tissues. Once positioned to apply energy pulses to the desired site, the system 10 may initiate neuromodulation to achieve a desired physiological or clinical effect.

[0038] In certain embodiments, the system 10 may include an evaluation device 20 coupled to the controller 16 to evaluate a characteristic indicative of whether the desired physiological effect of the modulation has been achieved. In one embodiment, the desired physiological effect may be local. For example, the modulation may result in a local tissue or functional change, such as a change in tissue structure, a local change in the concentration of a particular molecule, tissue displacement, increased fluid movement, etc.

[0039] The modulation may result in systemic (non-local) changes. The desired physiological effect may relate to changes in the concentration of circulating molecules or changes in the properties of tissues that do not include the region of interest where the energy is directly applied. In one example, the displacement may be a surrogate measurement of the desired modulation, and if the measured displacement is less than the expected displacement value, the modulation parameters may be modified until the expected displacement value is obtained. Thus, the evaluation device 20 may be configured in some embodiments to evaluate the concentration change. In some embodiments, the evaluation device 20 may be an imaging device configured to evaluate changes in the size and / or position of the organ. Although the illustrated elements of the system 10 are shown separately, it will be understood that some or all of the elements may be combined with each other. Furthermore, some or all of the elements may be in wired or wireless communication with each other.

[0040] Based on the evaluation, adjustment parameters of the controller 16 can be altered. For example, if the desired adjustment relates to a change within a defined time frame (e.g., 5 minutes, 30 minutes after the start of the energy application procedure) or relative concentration (circulating or tissue concentration of one or more molecules), or to a baseline at the start of treatment, a change in adjustment parameters, such as pulse frequency or other parameters, may be desirable. Such parameters may be provided to the controller 16 by an operator or via an automated feedback loop, thereby defining or adjusting the energy application or adjustment parameters of the pulse generator 14.

[0041] The system 10 described herein can provide energy pulses according to various adjustment parameters. For example, the adjustment parameters can include various stimulation time patterns, from continuous to intermittent. For intermittent stimulation, the signal-on time is supplied with energy at a certain frequency for a certain period of time. The signal-on time is followed by a period during which no energy is supplied, which is referred to as the signal-off time. The adjustment parameters can also include the frequency and duration of the stimulation application. The application frequency can be continuous or the delivery can be performed for various periods of time, such as within a day or a week. The treatment period can last for various periods of time, including but not limited to minutes to hours. In certain embodiments, the treatment period with a specified stimulation pattern is repeated at intervals of 1 hour, such as 72 hours. In certain embodiments, the treatment may be performed more frequently, such as every 3 hours, for a shorter duration, such as 30 minutes. According to the adjustment parameters, such as the treatment period and frequency, the energy application can be adjustably controlled to obtain a desired effect.

[0042] 2 is a block diagram of certain components of the system 10. As described herein, the system 10 for neuromodulation may include a pulse generator 14 that is utilized to generate a plurality of energy pulses that are applied to the tissue of a target. The pulse generator 14 may be provided separately or may be integrated with an external device such as a controller 16. The controller 16 includes a processor 30 for controlling the device. Software code or instructions executed by the processor 30 to control the various components of the device are stored in a memory 32 of the controller 16. The controller 16 and / or the pulse generator 14 may be connected to the energy application device 12 via one or more leads 33 or wirelessly.

[0043] The controller 16 also includes a user interface having input / output circuitry 34 and display 36 that is utilized to allow the clinician to provide selection input or modulation parameters to the adjustment programs. Each adjustment program may include one or more sets of adjustment parameters including pulse amplitude, pulse width, pulse frequency, etc. The pulse generator 14 changes its internal parameters in response to control signals from the controller device 16 to vary the stimulation characteristics of the energy pulses transmitted via the leads 33 to the subject to which the energy application device 12 is utilized. Any suitable type of pulse generation circuitry may be used including, but not limited to, constant current, constant voltage, multiple independent current or voltage sources, etc. The energy applied is based on the current amplitude and duration of the pulse width. The controller 16 allows for adjustably controlling the energy by modifying the adjustment parameters and / or initiating energy application at a specific time or stopping / inhibiting energy application at a specific time. In one embodiment, the adjustable control of the energy application device is based on information regarding the concentration of one or more molecules (e.g., circulating molecules) in the subject. If the information is from the evaluation device 20, a feedback loop can drive the adjustable control. For example, if the circulating glucose concentration measured by the evaluation device 20 exceeds a predetermined threshold or range, the controller 16 may initiate energy application to the region of interest (e.g., liver) with adjustment parameters related to the reduction of circulating glucose. Initiation of energy application may be triggered by the glucose concentration rising above a predetermined (e.g., desired) threshold or outside a predetermined range. In another embodiment, the adjustable control may be in the form of altering the adjustment parameters if the initial application of energy does not result in an expected change in the targeted physiological effect (e.g., concentration of a molecule of interest) within a predetermined time frame (e.g., 1 hour, 2 hours, 4 hours, 1 day).

[0044] In one embodiment, the memory 32 stores different operating modes selectable by the operator. For example, the stored operating modes may include instructions for executing a set of adjustment parameters associated with a particular treatment site, such as an area of ​​interest in the liver, pancreas, gastrointestinal tract, or spleen. The associated adjustment parameters may vary depending on the site. Rather than having the operator manually enter the mode, the controller 16 may be configured to execute the appropriate instructions based on the selection. In another embodiment, the memory 32 stores multiple operating modes for different types of treatments. For example, activation may be associated with a different stimulation pressure or frequency range than that associated with inhibiting or blocking tissue function. In a particular example, if the energy application device is an ultrasound transducer, the time-averaged power (time-averaged intensity) and peak positive pressure may be greater than 1 mW / cm. 2 to 30,000mW / cm 2 (time-averaged intensity) and in the range of 0.1 MPa to 7 MPa (peak pressure). In one example, the time-averaged intensity at the region of interest is 35 W / cm to avoid levels associated with thermal damage and ablation / cavitation. 2 In another particular example, when the energy application device is a mechanical actuator, the amplitude of the vibration is in the range of 0.1 to 10 mm. The selected frequency may depend on the form of energy application, e.g., ultrasonic or mechanical actuator.

[0045] In another embodiment, the memory 32 stores a calibration or setup mode that allows for adjustment or modification of tuning parameters to achieve a desired effect. In one example, stimulation begins with a lower energy parameter and gradually increases, either automatically or in response to receiving operator input. In this manner, the operator can achieve adjustment of the resulting effect as the tuning parameters are changed.

[0046] The system may also include an imaging device to facilitate focusing by the energy application device 12. In one embodiment, the imaging device is integrated with or is the same device as the energy application device 12 such that different ultrasound parameters (frequency, aperture, or energy) are applied to select (e.g., spatially select) the region of interest and to focus and target the selected region of interest for subsequent neuromodulation. In another embodiment, the memory 32 stores one or more targeting or focusing modes used to spatially select the region of interest within an organ or tissue structure. The spatial selection may include selecting a sub-region of the organ to identify a portion of the organ corresponding to the region of interest. As described herein, the spatial selection may depend on image data. Based on the spatial selection, the energy application device 12 may focus on the selected portion corresponding to the region of interest. For example, the energy application device 12 may be configured to operate in a targeting mode first to apply targeting mode energy to capture image data used to identify the region of interest. The targeting mode energy is not at a level suitable for preferential activation and / or has not been applied with suitable modulation parameters. Once the region of interest has been identified, the controller 16 may operate in a therapeutic mode according to adjustment parameters associated with preferential activation.

[0047] The controller 16 may also be configured to receive inputs related to the desired physiological effect as inputs corresponding to the selection of the modulation parameters. For example, if an imaging modality is used to evaluate tissue properties, the controller 16 may be configured to receive a calculated index or parameter of the property. Based on whether the index or parameter exceeds or is below a predefined threshold, the modulation parameters may be altered. In one embodiment, the parameter may be a measurement of tissue displacement of the diseased tissue, or a measurement of the depth of the diseased tissue. Other parameters may include an evaluation of the concentration of one or more molecules of interest (e.g., change in concentration relative to a threshold or baseline / control, rate of change, evaluation of whether the concentration is within a desired range). Additionally, the energy application device 12 (e.g., an ultrasound transducer) may operate under the control of the controller 16 to a) capture tissue image data that may be used to spatially select a region of interest within the target tissue, b) apply modulation energy to the region of interest, and c) perform imaging to determine if the desired physiological effect has been achieved (e.g., by displacement measurement). In such an embodiment, the imaging device, evaluation device 20, and energy application device 12 may be the same device.

[0048] In another form, the desired set of adjustment parameters may be further stored by the controller 16. In this manner, subject-specific parameters may be determined. Furthermore, the effectiveness of such parameters may be evaluated over time. If the effect of a particular set of parameters decreases over time, the subject may become refractory to the activated pathway. If the system 10 includes an evaluation device 20, the evaluation device 20 may provide feedback to the controller 16. In certain embodiments, feedback characteristic of the desired physiological effect may be received from a user or the evaluation device 20. The controller 16 may be configured to cause the energy application device to apply energy according to the adjustment parameters and dynamically adjust the adjustment parameters based on the feedback. For example, based on the feedback, the processor 16 may automatically change the adjustment parameters (e.g., frequency, amplitude, or pulse width of the ultrasound beam or mechanical vibration) in response to the feedback from the evaluation device 20 in real time.

[0049] In one example, the technology can be used to treat subjects with metabolic disorders. The technology can also be used to regulate blood glucose levels in subjects with impaired glucose regulation. Thus, the technology can be used to promote homeostasis of a molecule of interest or promote the achievement of a desired circulating concentration or concentration range of one or more molecules of interest (e.g., glucose, insulin, glucagon, or a combination thereof). In one embodiment, the technology can be utilized to control circulating (i.e., blood) glucose levels. In one embodiment, the following thresholds can be used to maintain blood glucose levels in dynamic equilibrium in the normal range: After fasting: Less than 50 mg / dL (2.8 mmol / L): Insulin shock 50 to 70 mg / dL (2.8 to 3.9 mmol / L): Hypoglycemia / hypoglycemia 70 to 110 mg / dL (3.9 to 6.1 mmol / L): Normal 110 to 125 mg / dL (6.1 to 6.9 mmol / L): Elevated / impaired (prediabetes) 125(7mmol / L): Diabetes No fasting (about 2 hours after a meal): 70 to 140 mg / dL: Normal 140 to 199 mg / dL (8 to 11 mmol / L): Elevated or "borderline" / prediabetic 200mg / dL or more: (11mmol / L): Diabetes For example, the technique may be used to maintain circulating glucose concentrations below about 200 mg / dL and / or above about 70 mg / dL. Using this technique, glucose may be maintained in a range of about 4 to 8 mmol / L or about 70 to 150 mg / dL. This technique may be used to maintain a normal blood glucose range for a subject (e.g., a patient). The normal blood glucose range may be an individualized range based on the patient's individual factors, such as weight, age, medical history, etc. Thus, the application of energy to one or more regions of interest may be adjusted in real time based on the desired final concentration of the molecule of interest and may be adjusted in a feedback loop based on input from the evaluation device 20. For example, if the evaluation device 20 is a circulating glucose monitor or a blood glucose monitor, the real-time measurement of glucose may be used as an input to the controller 16.

[0050] In another embodiment, the present technology may be used to develop a characteristic signature of a physiological change. For example, the characteristic signature may include a group of molecules of interest that increase in concentration in tissues and / or blood as a result of energy application and another group of molecules of interest that decrease in concentration in tissues and / or blood as a result of energy application. The characteristic signature may include a group of molecules that do not change as a result of energy application. The characteristic signature may define the concomitant changes associated with a desired physiological effect. For example, the signature may include a decrease in circulating glucose seen with an increase in circulating insulin.

[0051] 3 illustrates a particular example in which the energy application device 12 includes an ultrasound transducer 42 capable of applying energy to a target tissue 43, shown as a liver as a non-limiting example. The energy application device 12 may include control circuitry for controlling the ultrasound transducer 42. The control circuitry of the processor 30 may be integrated into the energy application device 12 (e.g., via an integral controller 16) or may be a separate component. The ultrasound transducer 42 may also be configured to acquire image data to spatially select a desired or target region of interest and focus the applied energy to the region of interest of the target tissue or structure.

[0052] The desired target tissue 43 may be an internal tissue or organ that contains synapses of axon terminals 46 and non-neuronal cells 48. The synapses may be stimulated by applying energy directly to the axon terminals within the focal region of an ultrasound transducer 42 focused on a region of interest 44 of the target tissue 43, causing molecules to be released into the synaptic space 49. In the illustrated embodiment, the axon terminals 46 form synapses with hepatocytes. The release of neurotransmitters 47 and / or changes in ion channel activity result in downstream effects such as activation of glucose metabolism. The region of interest may be selected to include a particular type of axon terminals 46, for example, axon terminals 46 of a particular neuron type and / or those that form synapses with a particular type of non-neuronal cell. Thus, the region of interest 44 may be selected to correspond to a portion of the target tissue 43 that has the desired axon terminals 46 (and associated non-neuronal cells 48). Energy application may be selected to preferentially trigger the release of one or more molecules, such as neurotransmitters, from the nerves in the synapse or directly activate the non-neuronal cells themselves through direct energy transduction (i.e., mechanical transduction or voltage-activated proteins in the non-neuronal cells). Or it may activate both neural and non-neuronal cells to achieve the desired physiological effect. The region of interest may be selected as the site of neural penetration into the organ. In one embodiment, liver stimulation or modulation may refer to modulation of the region of interest 44 at or adjacent to the hepatic hilum.

[0053] The energy may be focused or substantially focused on the region of interest 44 and concentrated on only a portion of the interior tissue 43, for example, less than about 50%, 25%, 10%, or 5% of the total volume of the tissue 43. In one embodiment, the energy may be applied to two or more regions of interest 44 within the target tissue 43, and the total volume of the two or more regions of interest 44 may be less than about 90%, 50%, 25%, 10%, or 5% of the total volume of the tissue 43. In one embodiment, the energy is applied to only about 1%-50% of the total volume of the tissue 43, only about 1%-25% of the total volume of the tissue 43, only about 1%-10% of the total volume of the tissue 43, or only about 1%-5% of the total volume of the tissue 43. In certain embodiments, only the axon terminals 46 of the region of interest 44 of the target tissue 43 directly receive the applied energy and release neurotransmitters. And the unstimulated axon terminals outside the region of interest 44 do not substantially receive the energy and therefore are not similarly activated / stimulated. In some embodiments, axon terminals 46 in the portion of the tissue directly receiving the energy undergo altered neurotransmitter release. In this manner, subregions of the tissue may be targeted for neuromodulation. For example, one or more subregions may be selected. In some embodiments, energy application parameters may be selected to result in preferential activation of either neural or non-neural components within the tissue directly receiving the energy to achieve a desired composite physiological effect. In certain embodiments, the energy is delivered to a site of approximately 25 mm 3 In certain embodiments, the energy can be concentrated or focused within a volume of less than about 0.5 mm. 3 From 50mm 3 The amount of focusing and the focal depth for focusing or concentrating the energy within the region of interest 44 may be influenced by the size / configuration of the energy application device 12. The amount of focusing of the energy application may be defined by the focal range of the energy application device 12.

[0054] As described herein, energy may be applied substantially only to one or more regions of interest 44 and not substantially generally or non-specifically throughout the tissue 43 to preferentially activate synapses as desired to achieve a desired physiological effect. Thus, only a subset of a number of different types of axon terminals 46 within the tissue 43 are exposed to the direct energy application. FIG. 4 is an image of blood flow (as obtained with Doppler ultrasound) within the spleen that may be used as spatial information to spatially select a region of interest in a target organ. For example, a region of interest within the organ containing either blood vessels, nerves, or other anatomical landmarks may be spatially selected and used to identify regions with specific axon terminals and synapses. In one embodiment, the region of interest is selected by identifying the splenic artery and spatially selecting a region close to or parallel to the splenic artery. The organ structure may be segmented based on tissue function, blood vessels, and nerve stimulation transmission within the organ. A subset of axon terminals may then be selected to be included in the region of interest to which energy is directly applied. Other axon terminals may be outside the region of interest and therefore may not be exposed to the directly applied energy. One or more individual axon terminals for inclusion in the region of interest may be selected based on factors including, but not limited to, historical and experimental data, such as data showing associations between specific locations and desired or desired physiological effects. In another embodiment, the location of the 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. Additionally / alternatively, the system 10 may apply energy to the individual axon terminals until the desired physiological effect is achieved. It should be understood that the spleen image is merely an example. The selection of axon terminals for preferential activation by direct energy application to the region of interest using spatial information of visualized nerves in the present disclosure may be used in combination with other organs or structures (e.g., liver, pancreas, gastrointestinal tissue).

[0055] The disclosed techniques may be used to assess neuromodulation effects, which may be used as input or feedback to select or modify neuromodulation parameters. The disclosed techniques may use direct assessment of tissue status or function as the desired physiological effect. Assessment may be performed before (i.e., baseline assessment), during, and / or after neuromodulation.

[0056] The evaluation technique may include at least one of functional magnetic resonance imaging, diffusion tensor magnetic resonance imaging, positive emission tomography, or acoustic monitoring, thermal monitoring. The evaluation technique may also include protein and / or marker concentration evaluation. Images from the evaluation technique may be received by the system for automatic or manual evaluation. Modulation parameters may also be modified based on the image data. For example, changes in organ size or displacement may be utilized as markers of local neurotransmitter concentration and may be effectively used as surrogate markers of local cellular exposure to phenotypic modulating neurotransmitters and as markers of predicted effects on glucose metabolic pathways. Local concentration may refer to the concentration within the focal range of energy application.

[0057] Additionally / alternatively, the system may assess the presence or concentration of one or more molecules in tissue or in the blood circulation. Concentration in tissue may also be referred to as local or resident concentration. Tissue may be obtained by fine needle aspiration, and assessment of the presence or level of a molecule of interest (e.g., metabolic molecule, marker of metabolic pathway, peptide mediator, catecholamine) may be performed by any suitable technique known to those skilled in the art.

[0058] In other embodiments, the desired physiological effect may include, but is not limited to, tissue displacement, changes in tissue size, changes in concentration of one or more molecules (either local, non-local, or circulating concentration), changes in genes, marker expression, afferent activity, and cell migration. For example, tissue displacement (e.g., liver displacement) may occur as a result of the application of energy to the tissue. By assessing the tissue displacement (e.g., via imaging), other effects may be estimated. For example, a particular displacement may be characteristic of a particular change in molecular concentration. In one example, a 5% liver displacement may indicate or be associated with a desired reduction in circulating glucose concentration based on experimental data. In another example, tissue displacement may be assessed by comparing reference image data (tissue image before application of energy to the tissue) with post-treatment image data (tissue image taken after application of energy to the tissue) to determine a parameter of displacement. The parameter may be a maximum or average displacement value of the tissue. If the parameter of displacement is greater than a threshold displacement, it may be assessed that the application of energy likely achieved the desired physiological effect.

[0059] FIG. 5 is a flow diagram of a method 50 for stimulating a region of interest in a target tissue. In the method 50, a region of interest is spatially selected 52. An energy application device is positioned such that an energy pulse is focused at the desired region of interest at step 54. A pulse generator applies a plurality of energy pulses at step 56 to the region of interest in the target tissue, thereby preferentially activating a subset of synapses present in the target tissue at step 58, e.g., to stimulate axon terminals to release neurotransmitters and / or induce changes in neurotransmitter release and / or induce changes in activity in non-neuronal cells (intrasynapses) to achieve a desired physiological effect. 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 status of tissue function or condition may be used. Based on the evaluated tissue function, adjustment parameters of one or more energy pulses may be modified (e.g., dynamically or adjustably controlled) to achieve the targeted physiological effect.

[0060] In one embodiment, evaluation can be performed before and after applying the energy pulse to evaluate the change in glucose concentration as a result of modulation. If the glucose concentration exceeds or falls below a threshold, modulation parameters can be appropriately modified. For example, if the glucose concentration with the desired physiological effect is, the applied energy during neuromodulation can be reduced to a minimum level that achieves the desired effect. If the change in the characteristic relative to the threshold is associated with an insufficient change in glucose concentration, certain modulation parameters can be changed, including but not limited to modulation amplitude or frequency, pulse shape, stimulation pattern, and / or stimulation location.

[0061] Additionally, the evaluated characteristic or condition may be a value or index, e.g., flow rate, concentration, cell population, or any combination thereof, which may be analyzed by appropriate techniques. For example, a relative change over a threshold may be used to determine whether to modify a modulation parameter. The desired modulation may be evaluated via a measured clinical effect, such as the presence or absence of an increase in tissue structure size (e.g., lymph node size) or a change in concentration of one or more released molecules (e.g., compared to baseline concentration before neuromodulation). In one embodiment, the desired modulation may include an increase in concentration over a threshold, e.g., about a 50%, 100%, 200%, 400%, 1000% increase in concentration compared to baseline. In the case of a blocking treatment, the evaluation may include identifying a decrease in the concentration of the molecule over time, e.g., at least a 10%, 20%, 30%, 50%, or 75% decrease of the molecule of interest. Additionally, for a particular subject, the desired blocking treatment may include maintaining a relatively stable concentration of a particular molecule relative to other clinical events that may tend to increase the concentration of the molecule. That is, the desired blocking may be to block the possibility of an increase. The increase or decrease, or other resulting measurable effect, may be measured within a certain time frame, for example, within about 5 minutes, within about 30 minutes, from the start of treatment. In certain embodiments, if neuromodulation is determined to be desirable, the change in neuromodulation is an indication to stop the application of energy pulses. In another embodiment, if neuromodulation is undesirable, one or more parameters of the neuromodulation are altered. For example, the change in the modulation parameter may be an increase in pulse repetition frequency, such as a stepwise increase in frequency from 10 to 100 Hz and evaluation of the desired characteristics until the desired neuromodulation is achieved. In another form, the pulse width may be altered. In other embodiments, two or more parameters may be altered together, in parallel, or sequentially. If neuromodulation is not desirable after altering multiple parameters, the focus (i.e., site) of energy application may be altered.

[0062] The energy application device 12 may be configured as an external non-invasive device or an internal device (e.g., a minimally invasive device). As described above, the energy application device 12 may be an external non-invasive ultrasound transducer or a mechanical actuator. For example, FIG. 6 shows an embodiment of the energy application device 12 configured as a handheld ultrasound probe including an ultrasound transducer 74. However, it should be understood that other non-invasive implementations are also contemplated, including other methods for configuring, attaching, or positioning an ultrasound transducer probe on an anatomical target. Furthermore, in addition to a handheld configuration, the energy application device 12 may include a steering mechanism in response to commands from the controller 16. The steering mechanism may point or orient the energy application device 12 toward the target tissue 43 (or structure). The controller 16 may then focus the energy application to the region of interest 44. EXAMPLES

[0063] FIG. 7 is a block diagram of a system 10 including an energy applicator 12 and a pulse generator 14 configured to apply high intensity 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 for obtaining the experimental results provided herein, the pulse generator included a 1.1 MHz high intensity focused ultrasound (HIFU) transducer (Sonic Concepts H106), a matching network (e.g., Sonic Concepts), an RF power amplifier (ENI350L), and a function generator (Agilent 33120A). In the illustrated example, the 70 mm diameter HIFU transducer has a spherical surface with a radius of curvature of 65 mm, with a 20 mm diameter hole in the center where the imaging transducer can be inserted. The focal depth of the transducer is 65 mm. The numerically simulated pressure profile has an amplitude half-width of 1.8 mm laterally and 12 mm in depth. The HIFU transducer 12 was coupled to the animal subject via a 6 cm tall plastic cone filled with degassed water. Figure 8 is an example of an energy application device that may be used with the system 10 of Figure 7, including a HIFU transducer 74A and an imaging ultrasound transducer 74B disposed in a single energy application device 12 that may be controlled by the controller 16 to apply energy and image to the target tissue, for example, as described herein.

[0064] FIG. 9 illustrates an experimental setup used to perform certain spleen modulation experiments 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 between 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 region of interest within the target tissue 43. As described herein, the target tissue may be the spleen, liver, pancreas, gastrointestinal tissue, etc. Although a 40W RF amplifier is shown in the illustrated experimental setup, this is merely by way of example and other amplifiers (e.g., linear amplifiers) may be used. In a particular setup, the rat's head is inserted into a birdcage coil.

[0065] FIG. 10 shows an experimental timeline of ultrasound energy application used to perform certain modulation experiments described herein. In the illustrated embodiment, ultrasound application was performed for 1 minute before and after lipopolysaccharide injection. Lipopolysaccharide (LPS) is a bacterial membrane molecule that induces a strong immune or inflammatory response. LPS from E. coli 0111:B4 (Sigma-Aldrich) was used to induce significant inflammation and metabolic dysfunction (such as hyperglycemia and insulin resistance) in drug-naive adult Sprague-Dawley (SD) rats. LPS was administered to the animals (10 mg / kg) via intraperitoneal (IP) injection and significantly increased the concentrations of TNF, circulating glucose, and insulin. These concentrations peaked at 4 hours but remained elevated for up to 8 hours after injection compared to controls. Animals were euthanized after sonication for organ harvest and processing. While the illustrated period is 1 hour, it should be understood that this is an example and that in other embodiments, the period for evaluating the resulting changes may vary.

[0066] The function generator 80 generates a pulsed sine wave waveform as shown in FIG. 11. This pulsed sine wave waveform is amplified by an RF power amplifier and sent to the matching network of the HIFU transducer. Three ultrasound parameters are adjustable during the animal experiment: pulse amplitude, pulse length, and pulse repetition frequency. The pulse amplitude ranges from 0.5V peak to 62V peak. Three pulse lengths are used: 18.2us, 136.4us, and 363.6us. In one embodiment, the pulse repetition frequency (1 / T) is 2kHz. The treatment time is 1 minute. This ultrasound adjustment parameter is an example. In one embodiment, the adjustment is achieved with an ultrasound stimulation having an ultrasound transducer frequency in the range of about 0.1MHz to about 5MHz, and the ultrasound stimulation has an ultrasound frequency pulse repetition frequency in the range of about 0.1Hz to about 10kHz. The ultrasound cycles per pulse of ultrasound energy can range from about 1 to 1000. In one embodiment, the pulse center frequency was 1.1 MHz, the pulse repetition period was 0.5 ms (corresponding to a pulse repetition frequency of 2000 Hz), and the pulse amplitude and pulse length were varied. Table 1 summarizes the HIFU ultrasound parameters. [Table 1] Pressure measurements were performed in degassed water using a HIFU hydrophone (HNA-0400) manufactured by Onda Corp. The HIFU transducer was driven by a 100-cycle sinusoidal waveform. The hydrophone was scanned with a focal spot with a grid size of 0.1 mm in the xy plane and a step size of 0.2 mm along the z axis. Figure 12 shows the hydrophone setup, and Figure 13A shows the scan results. The peak positive (negative) pressure is defined as the maximum positive (negative) pressure at the transducer focal spot in the xy plane. The input voltage was low enough to eliminate the effects of nonlinearity. Thus, the value of the positive peak pressure was equal to the value of the negative peak pressure. To estimate the peak pressure at the full operating voltage, the peak pressure was measured at several different driving voltages before cavitation occurred, and curve fitting was performed. The calculated peak pressures are shown in Table 1. Ultrasound targeting for organ-specific neuromodulation

[0067] Before the start of neuromodulation, a GE Vivid E9 ultrasound system and an 11L probe were used for ultrasound scanning. The region of interest was indicated by labeling on the animal's skin. The HIFU transducer was placed in the labeled region. Another ultrasound scan was also performed using a small imaging probe (3S) placed at the aperture of the HIFU transducer. The imaging beam of the 3S probe was aligned with the HIFU beam. Thus, an image of the organ of interest (visualized by the ultrasound scanner) could be used to confirm that the HIFU beam was aimed at the region of interest. animal procedures

[0068] Adult male Sprague-Dawley rats (250-300 g, Charles River Laboratories) aged 8-12 weeks were housed at 25°C on a 12-h light / dark cycle and allowed to acclimate for 1 week before experiments. Water and regular rodent chow were available ad libitum.

[0069] Endotoxin (LPS from E. coli 0111:B4 from Sigma-Aldrich) was used to place drug-naive adult Sprague-Dawley rats into a state of significant inflammation and metabolic dysfunction (e.g., hyperglycemia and hyperinsulinemia). LPS was administered to animals (10 mg / kg, Rosa-Ballinas PNAS, 2008) via intraperitoneal (IP) injection and significantly increased concentrations of TNF, circulating glucose, and circulating insulin. These concentrations peaked at 4 h but remained elevated for up to 8 h after injection compared to controls. Samples of spleen, liver, hypothalamus, hippocampus, and blood were taken at 60 min (for power studies) and 30, 60, 120, 240, or 480 min (for duration and kinetics studies) after LPS administration. Spleen and liver samples were homogenized in a solution of PBS containing phosphatase (0.2mM phenylmethylsulfonyl fluoride, 5ug / mL aprotinin, 1mM benzamidine, 1mM sodium orthobandate and 2uM cantharidin) and protease (1uL to 20mg tissue, by Roche Diagnostics) inhibitors. A target final concentration of 0.2g tissue per mL of PBS solution was applied to all samples. Blood samples were stored with anticoagulant (disodium) EDTA to prevent clotting of samples. Samples were analyzed by ELISA assay for changes in cytokine (Bio-Plex Pro; Bio-Rad), TNF (Lifespan) and acetylcholine (Lifespan) concentrations. Catecholamine concentrations were assessed using HPLC detection or ELISA (Rocky Mountain Diagnostic) analysis.

[0070] The effect of LPS on blood glucose and insulin levels was examined. Blood samples were taken from the tail vein at 0, 60, 90, 120, 150, 180, and 240 min after LPS injection to measure glucose and insulin levels. Circulating blood glucose concentrations were measured with a OneTouch Elite glucometer (LifeScan; Johnson & Johnson). Insulin concentrations in plasma obtained from blood were determined using an ELISA kit (Crystal Chem, Chicago, IL), thereby determining the effect of LPS and subsequent ultrasound stimulation on whole-body insulin resistance. Changes in signaling were measured by evaluation of biomarkers including p38, p7056k, Akt, GSK3B, c-Src, NF-κβ, SOCS3, IRS-1, NPY, and POMC in liver, muscle, heart, and hypothalamus tissue samples. Changes produced by ultrasound stimulation may include changes in insulin-mediated glucose uptake, as well as related molecules related to inhibition / activation of metabolic activity. Ultrasound neuromodulation may employ the following procedures. Animals may be anesthetized with 2 to 4% isoflurane. To prevent hyperthermia during the procedure, animals may be placed on a water-circulating heating pad. The area of ​​interest that is the target of ultrasound stimulation (the nerve of interest) is shaved with a disposable razor and animal clippers prior to stimulation. Diagnostic ultrasound imaging can be used to spatially select a region of interest. Liver: Hilar area demonstrated by Doppler identification of the hepatic portal vein. Spleen: Visual identification of the spleen by diagnostic ultrasound. The location of stimulation may be maintained along the identified splenic axis. The area is permanently marked for later identification. The FUS ultrasound probe or LogiQ E9 probe may be placed in a designated region of interest previously identified by diagnostic ultrasound. The ultrasound pulses may then be administered such that the total duration of a single stimulus does not exceed a single 1 minute pulse. At no point does the energy reach levels associated with thermal damage and ablation / cavitation (energy during ablation / cavitation is 35 W / cm2). That is, in certain embodiments, the time-averaged intensity in the region of interest is 35 W / cm2. 2 is less than. Thereafter, LPS (10 mg / kg) may be injected intraperitoneally (for acute / kinetic studies). Alternatively, due to the duration of effect, LPS may not be injected at this point, but instead may be injected at a specified later time point. A second 1 minute ultrasound stimulus may be applied. The animals may then be incubated under anesthesia for acute (1 h) and kinetic (changes up to 3 h post-LPS) studies, after which the animals are put to sleep and tissue and blood samples are taken. For the duration of the efficacy study, LPS is not injected at the time of ultrasound stimulation, but rather at designated time points (e.g., 0.5, 1, 2, 4, or 8 h) after ultrasound stimulation application. Animals are then placed in an anesthetic holding chamber and monitored until euthanasia and tissue / fluid collection. The incision may be made starting from the bottom of the peritoneal cavity and down to the pleural cavity. The organs may be rapidly removed and homogenized in a solution of PBS containing phosphatase (0.2 mM phenylmethylsulfonyl fluoride, 5ug / mL aprotinin, 1 mM benzamidine, 1 mM sodium orthobandate and 2uM cantharidin) and protease (1uL to 20mg tissue, by Roche Diagnostics) inhibitors. A target final concentration of 0.2g tissue per mL of PBS solution was applied to all samples. Blood samples were stored with anticoagulant (disodium) EDTA to prevent clotting of the samples. Samples are then stored at -80°C until analysis. 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 assessed using HPLC detection or ELISA (Rocky Mountain Diagnostic) analysis. Experimental procedure for controlling vagus nerve stimulation using electrodes

[0071] 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 the left cervical vagus nerve was exposed by blunt dissection. 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 received vagus nerve stimulation for 3 min before and after IP injection of 10mg / kg LPS. Rats were euthanized 60 min after LPS injection, and spleen and blood samples were taken for TNF measurements. For sham-operated rats, the vagus nerve was exposed but not touched or manipulated. HPLC analysis

[0072] Serum samples were injected directly into the machine without any pretreatment. Tissue homogenates were first homogenized in 0.1 M perchloric acid and centrifuged for 15 min, after which the supernatant was separated and the samples were injected into an HPLC (Dhir & Kulkarni, 2007).

[0073] Catecholamines (norepinephrine / epinephrine) were analyzed by high performance liquid chromatograph (HPLC) equipped with an in-line ultraviolet detector. A test column, Supelco Discovery C18 (15cmx4.6mm ID, 5um particle size), was used in this analysis. The biphasic mobile phase consisted of [A] acetonitrile:[B] 50mM KH2PO4 (set to pH 3) (containing phosphoric acid). The solution was then buffered with 100mg / L EDTA and 200mg / L 1-octanesulfonic acid. The final concentration of the mobile phase mixture was set to A:B=5:95. A flow rate of 1mL / min was used to improve overall peak resolution and minimize column pressure compression due to the viscosity of the mobile phase used, while the column was constantly kept at 20°C. The UV detector was maintained at 254nm, a wavelength known to capture the absorption of catecholamines including norepinephrine, epinephrine and dopamine. Chemical inhibition of ultrasound-regulated molecular signaling pathways

[0074] To further explore the effects of mechanical vs. direct nerve stimulation (and preferential modulation of non-neuronal components of nerve vs. axonal extracellular synapses), we used either an SRC inhibitor (a common marker of direct mechanoreceptors) or a PI3K inhibitor (a common marker of nerve signaling) before performing the ultrasound stimulation procedure described above. Tissue extraction and paraffin block conversion:

[0075] The tissue (rat brain) is immediately placed in fixative and fixed in 10% formalin at 4° C. for approximately 24 hours. Process the tissue using the following steps (and vacuum and pressure during each incubation): a. 70% ethanol, 37℃, 40 minutes b. 80% ethanol, 37°C, 40 min c. 95% ethanol, 37℃, 40 minutes d. 95% ethanol, 37℃, 40 minutes e. 100% ethanol, 37℃, 40 minutes f. 100% ethanol, 37℃, 40 minutes g. Xylene, 37℃, 40 minutes h. Xylene, 37℃, 40 minutes i. Paraffin, 65℃, 40 minutes j. Paraffin, 65℃, 40 minutes k. Paraffin, 65℃, 40 minutes l. Paraffin, 65°C, 40 minutes* Leave in this paraffin until ready to implant, but no longer than 12-18 hours. Implant in paraffin blocks for sectioning, allow blocks to cool / harden before sectioning. Sections are 5 microns thick. Recover by floating in a 50°C water bath. Use positively charged slides and attempt tissue placement in the same orientation on all slides. Air dry slides. It is best to dry overnight at room temperature, but slides can be placed on a 40°C slide warmer to dry faster, but do not leave slides on the warmer for more than an hour. Store slides at 4°C. IHC process

[0076] Formalin-fixed paraffin-embedded (FFPE) tissue samples (rat brain) were baked at 65°C for 1 h. Slides were deparaffinized in xylene, rehydrated by reducing the ethanol concentration in washes, and processed for antigen retrieval. A two-step antigen retrieval method has been developed specifically for multiplexing with FFPE tissues, allowing the simultaneous use of antibodies with different antigen retrieval conditions on the same sample. Samples were then incubated in 0.3% PBS-TritonX-100 for 10 min at ambient temperature before blocking nonspecific binding with 10% (wt / vol) donkey serum and 3% (wt / vol) BSA in 1xPBS for 45 min at room temperature. Primary antibody cFOS (santa cruz-SC52) was diluted to an optimized concentration (5 μg / mL) and applied in PBS / 3% (vol / vol) BSA for 1 h at room temperature. Samples were then washed sequentially for 10 min in PBS, PBS-TritonX-100, and PBS again, with agitation in each step. For secondary antibody detection, samples were incubated with primary antibody species-specific secondary donkey IgG conjugated with either Cy3 or Cy5. Slides were then washed as above, stained with DAPI (10 μg / mL) for 5 min, washed again with PBS, and mounted with antifade media for imaging. Whole tissue images were captured on a fluorescent Olympus IX81 microscope at 10x magnification. Image Processing

[0077] Autofluorescence (AF), typical of FFPE tissues, needs to be characterized and separated from the fluorophore signal of interest using an autofluorescence subtraction process, whereby an image of the unstained sample is acquired in addition to the stained images. The unstained and stained images are normalized with respect to their exposure time and dark pixel value (pixel intensity value at zero exposure time). Each normalized autofluorescence image is then subtracted from the corresponding normalized stained image. The AF subtracted image is composited with a registered 4',6-diamidino-2-phenylindole (DAPI) image. The same region of interest was imaged across multiple stimulated and control samples, and images were qualitatively assessed for cFOS expression to detect changes in gene expression associated with neuronal activation.

[0078] Spleen histology evaluation: Spleens from stimulated and control rats were processed into paraffin blocks as described above. Paraffin-embedded sections were washed, stained for H&E, and scanned on a brightfield Olympus scanner according to standard procedures described in the literature. H&E images were qualitatively evaluated for differences in morphology, and no significant differences were identified between stimulated and control samples. Heart rate monitoring and analysis

[0079] Heart rate (during ultrasound or electrode stimulation experiments) was observed 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 animals' footpads during the stimulation procedure. Animals were allowed to acclimate to the environment for at least 5 min before measurements, a time that was confirmed to be sufficient for the animals to recover to normal heart rate activity measured physiologically under control. Measurements were recorded before (2 min recording period), during, and after (2 min recording period) stimulation with electrical microelectrodes or ultrasound probes, respectively. Diffusion functional MRI measurements of ultrasound-induced activation

[0080] Neuronal activation can be detected using blood oxygen level-dependent (BOLD) fMRI. Brain regions with increased metabolic demands lead to increased cerebral blood flow, an increased supply of oxygenated blood, and a decreased gradient echo signal. Sensitivity to the BOLD effect requires fast gradient echo acquisition. This causes unwanted signal loss in brain regions adjacent to air pockets, such as the sinuses and ear canal, preventing the detection of neuronal activation near these specific brain regions. Alternatively, spin-echo (or double spin-echo) diffusion-weighted imaging (DWI) may be used to minimize signal loss in regions characterized by extensive heterogeneity. In DWI-fMRI, both an increase in the volume of the slowly diffusing (presumably intracellular) water pool, or an increase in the diffusion (or apparent diffusion coefficient (ADC)) of water, were mapped to cell swelling and membrane expansion caused by neuronal activation.

[0081] Ten rats underwent brain MRI scans using the paradigm in Figure 14. Six of them received both LPS injections and ultrasound treatment (as described above). The other four received only LPS injections. Ten Sprague-Dawley rats were anesthetized using 3% isoflurane, placed supine, and had their heads inserted into a birdcage coil. The abdominal region was coupled to an MR-compatible ultrasound probe (f=1.47MHz) via a gel / water-filled cone. The probe was focused on the hilum of the liver, an area of ​​the liver that contained glucose-sensitive neurons. Scanning was performed on a 3T GE DV scanner (Waukesha, WI) using a Doty Scientific quadrature birdcage coil. The scan began with a T1 acquisition using a spoiled gradient echo sequence with an in-plane / out-of-plane spatial resolution of 0.4 / 1 mm, a TE / TR of 10 / 1475 ms, and a total acquisition time of 3:22 min. Six blocks of double spin-echo diffusion-weighted imaging (DWI) images (referred to as forward polarity gradient, or FPG) were acquired with an in-plane / out-of-plane spatial resolution of 0.6 / 1 mm, a TE / TR of 82 / 3400 ms, and a total acquisition time of 1:49 min per block, using 3 / 4 averages for b=0 / b=1000 s / mm2, respectively. Upon completion of the six pre-injection DWI acquisitions, another DWI acquisition was performed with the gradient direction reversed for the purpose of correcting for distortion. This acquisition is referred to as a reverse polarity gradient (RPG) acquisition. Another six blocks of FPG DWI images were acquired following the LPS injection, the first ultrasound treatment, a 5-minute wait period, and the second ultrasound treatment. For control rats that received only LPS injection, the last six DWI blocks were taken immediately after LPS injection.

[0082] Ultrasound treatments were performed using an MR-compatible 1.47 MHz focused ultrasound transducer coupled to the region of interest (e.g., gastrointestinal tissue, pancreas, liver, etc.) using a water-filled cone. Each ultrasound treatment lasted for 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 rat's abdomen was outside the imaging coil. Placing the animal in a supine position allowed the ultrasound probe to be easily coupled through the skin to the liver using coupling gel. A cross-correlation coefficient (ccc) between the T1 image and the (distortion-corrected) b = 0 DWI image of at least 0.5 was used to identify slices to be used for further analysis. Apparent diffusion coefficients (ADCs) were calculated for pre- and post-treatment images. Pre- and post-treatment image data were pooled together for statistical analysis. Rigorous registration between the T1 images and the rat atlas was utilized to identify regions that showed significant changes using pixel-wise t-tests. The registration transformation from the T1 and atlas images was applied to the distortion-corrected DWI and ADC images. Cholinergic anti-inflammatory pathway

[0083] This example presents a non-invasive method to stimulate specific axonal projections in an organ using ultrasound energy application to achieve physiological effects corresponding to the simulation. Ultrasound was first applied to the spleen. This confirmed that axons associated with the cholinergic anti-inflammatory pathway (CAP) were stimulated to regulate systemic cytokine concentrations. When this pathway was blocked chemically or mechanically, the ultrasound-induced effect was suppressed. Changes in ultrasound parameters differentially affected extracellular neurotransmitter concentrations, intracellular kinase activity, and CAP-related cytokines. Thus, it was shown that desirable physiological responses can be produced by modifying ultrasound parameters. Next, ultrasound stimulation of the liver was shown to modulate a sensory pathway that regulates blood glucose. This effect was found to depend on the stimulation of a specific anatomical site in the liver. Taken together, these data indicate that ultrasound neuromodulation in organs can provide a precise neuromodulation method that facilitates stimulating a small subset of neurons in an organ or tissue to affect specific physiological functions (e.g., blood glucose regulation by liver neuromodulation and systemic cytokines by splenic neuromodulation).

[0084] Within peripheral nerves, individual axons are tightly bundled into groups (fascicles) and encased within protective tissue. This makes it difficult to selectively stimulate a subset of axons that terminate in a specific organ and uniquely modulate the function of communicating cells within that organ. Clinical implementation of precise peripheral nerve stimulation remains complex. Figure 15A shows a partial schematic of the complex system of projecting efferent and afferent neurons within the vagus nerve, an exemplary innervated organ, and the approximate location of the stimulator used for cervical VNS. Afferent neurons arise from the dorsal motor nucleus of the vagus nerve (DMV), and afferent neurons enter the brain via the nucleus tractus solitarius (NTS). Peripheral nerves leading to visceral organs contain both efferent and afferent neurons, which are difficult to stimulate independently using distal cervical VNS implants.

[0085] FIG. 15B is a schematic diagram showing targeted organ-based peripheral neuromodulation, where a subset of axons terminating within the organ are preferentially stimulated using focused pulsed ultrasound. The subjects investigated here include axon terminals in the spleen, which are associated with the cholinergic anti-inflammatory pathway, and sensory terminals in the liver, which are associated with transmitting metabolic information to the brain to contribute to glucose homeostasis. The focused pulsed ultrasound provided herein stimulates a subset of axons terminating within the organ (FIG. 15B). In the specific example provided herein, ultrasound energy was focused on axon projections in the spleen (to affect systemic inflammation via the cholinergic anti-inflammatory pathway (CAP)) and liver (to transmit metabolic information to the brain and maintain glucose homeostasis).

[0086] Each cell type involved in the CAP was observed under different ultrasound stimulation parameters in a rodent LPS-induced inflammation model (Figure 16A and B; CAP (Figure 15B)), which consists of three major cell types. The three major cell types are postsynaptic axon terminals projecting from the splenic ganglion, intermediate T cells, and macrophages that regulate circulating cytokine levels. We observed the CAP response to local ultrasound stimulation by measuring the splenic concentrations of CAP-associated neurotransmitters and cytokines such as norepinephrine (NE), acetylcholine (ACh), and tumor necrosis factor (TNF-α).

[0087] Ultrasound stimulation was performed as described herein and as shown in the timeline of FIG. 16A to show the targeted physiological effects that occurred compared to controls. One minute of ultrasound stimulation was given before and after LPS injection. Samples were collected to measure the induced changes that occurred in local (i.e., tissue) as well as systemic neurotransmitters and cytokines (FIGS. 16B-E). This showed the effect of ultrasound-induced CAP neuromodulation on the response to the LPS model. The response time for all data except FIG. 16E was one hour. However, it should be understood that the response times shown are merely exemplary. That is, the changes that occur as a result of neuromodulation in the region of interest may occur within one hour and, in some embodiments, may persist for several hours or days. Thus, as provided herein, evaluation of measurable induced changes caused by neuromodulation may occur at baseline (at or before neuromodulation) and at one or more time points (minutes, hours, days) after neuromodulation. As part of a treatment procedure, certain subjects are monitored continuously or intermittently to evaluate one or more molecules or concentrations of interest (or other measurable effects such as organ displacement).

[0088] Sham controls were achieved by placing the ultrasound transducer on the target organ and not applying ultrasound stimuli. Figure 16B shows the measured CAP responses in drug-naïve rats, sham controls (rats that received LPS but no ultrasound stimulation), and animals that received LPS at various ultrasound stimulation pressures. Concentrations of norepinephrine (i), acetylcholine (ii), and TNF-α (iii) are shown for drug-naïve animals, sham controls, and animals with ultrasound stimulation pressures ranging from 0.03 to 1.72 MPa. It should be understood that the x-axis labels indicating drug-naïve animals, sham controls, and ultrasound stimulation pressures in (iii) apply to all three graphs. Splenic norepinephrine levels averaged 140 nmol / L in drug-naïve animals. Meanwhile, LPS-induced inflammation reduced norepinephrine levels to nearly zero, indicating suppression of CAP signaling in early inflammation.

[0089] As shown, ultrasound stimulation attenuated the LPS response to levels measured in drug-naïve animals (Figure 16B.i.). Similar to the CAP signaling process, the increase in norepinephrine in ultrasound-stimulated animals correlated with an increase in splenic acetylcholine. At an ultrasound pressure of 0.83 MPa, the mean acetylcholine concentration was nearly three times that measured in sham control animals (Figure 16B.ii). Figure 16C shows circulating concentrations of TNF-α in the same conditions as Figure 16B. Note that the x-axis labels indicating drug-naïve animals, sham controls, and ultrasound stimulation pressure in Figure 16C also apply to Figure 16B. Figure 16D shows splenic IL-1α concentrations in the same conditions as Figure 16B. Both splenic (Figure 16B.iii) and circulating TNF-α (Figure 16C) levels were significantly reduced compared to sham control animals. The response to treatment was dependent on ultrasound pressure (0.83 MPa was used in subsequent experiments). As further evidence that splenic ultrasound stimulation specifically caused changes in CAP, FIG. 16D shows that ultrasound stimulation affected the concentration of other proteins regulated by TNF-α specific pathways, such as interleukin-1-alpha (IL-1α). Thus, this data shows that the concentration of a molecule of interest can be precisely controlled by controlling an adjustable regulation parameter, such as ultrasound pressure. By varying the ultrasound pressure applied to the region of interest, a desired physiological effect (e.g., a desired concentration change in one or more molecules of interest) can be achieved. The pressure applied may be empirically determined on a patient-by-patient basis, but in some embodiments, an ultrasound stimulation pressure of 0.03 to 1.72 MPa is used for targeted neuromodulation. As described herein, ultrasound pressure can be a regulation parameter that is changed or adjusted as described herein to achieve a desired physiological response. Other adjustable parameters can be treatment timelines (e.g., duration of treatment, separation between treatments, and delay times of other clinical events or assessment via assessment devices).

[0090] FIG. 16F shows the splenic target indicated by the arrow. FIG. 16G shows a non-limiting embodiment of a timeline of a study designed to measure the duration of the effect of stimulation on CAP activation. In this study, ultrasound stimulation was applied before LPS injection, and the delay between ultrasound stimulation and LPS injection was varied from 0.5 to 48 hours. FIG. 16H shows the concentration of splenic TNF-α as a percentage (%) of the splenic TNF-α concentration measured in sham controls after ultrasound treatment (i.e., ultrasound stimulation performed before LPS injection) after variable delay times from 0.5 to 48 hours. FIG. 16I shows the concentrations of activated and / or phosphorylated kinases (p38, p70S6K, Akt, GSK3B, c-SRC, NF-kβ, SOCS3) in the presence (shaded lines) and absence (black bars) of ultrasound stimulation with ultrasound stimulation pressures ranging from 0.13 to 1.72 MPa.

[0091] Similar to previous implantable VNS studies, the peak ultrasound-mediated response occurred 1 to 2 hours after treatment (Figure 16E). Furthermore, splenic ultrasound could be applied prior to LPS injection to obtain a protective effect (Figures 16G and 16H), again consistent with previous invasive VNS studies. Figure 16H shows that the protective effect lasted for 48 hours after treatment. To further identify the protective effect, we measured ultrasound activation of specific intracellular kinases associated with LPS-, CAP-, or TNF-α-mediated signaling (Figure 16I). These data indicate that the activation of some kinases (e.g., p38 and p70S6K) was significantly promoted by ultrasound. Furthermore, we show that the ultrasound pressure-dependent response of some kinases (e.g., p38) roughly correlates with the ultrasound pressure dependence previously observed (Figures 16B to D).

[0092] Figures 16J and 16K show data for norepinephrine (NE), acetylcholine (ACh), and tissue necrosis factor alpha (TNF-α) concentrations in ultrasound stimulated spleens (post-LPS injection) using alternative ultrasound stimulation parameters for burst duration and carrier frequency. Data are shown as a comparison to the 0.83 MPa data in Figure 16B for spleen samples taken after stimulation with alternative burst durations (left) or ultrasound carrier frequencies (right).

[0093] The effect of splenic ultrasound modulation was compared with standard electrodes or implanted vagus nerve stimulation (VNS) performed as described herein. In Figures 17A and 17B, "+" indicates the presence of the indicated event or inhibitor, and "-" indicates the absence of the indicated event or inhibitor. In Figure 17A, the X-axis represents the induced changes in rat spleen under different conditions (ultrasound vs. VNS, with different inhibitors), and the Y-axis represents the relative concentration change of splenic TNF-α (vs. LPS-treated control) in percent. The relative concentrations are shown for VNS stimulation and ultrasound stimulation (with ultrasound stimulation pressure of 0.83 MPa) with and without PP2 (partially selective Src kinase inhibitor), LY294002 (PI3-kinase selective inhibitor), PD98059 (MEK1 and MEK2 selective MAPK selective inhibitor), and α-bungarotoxin (BTX; a known antagonist of α7nAChR in the CAP pathway). Figure 17B shows the effect of BTX on splenic concentrations of (left) norepinephrine (NE) and (right) TNF-α after ultrasound stimulation in LPS-treated rodents with and without the effects of BTX or surgical vagus nerve stimulation. Figure 17A shows that invasive cervical VNS and non-invasive splenic ultrasound stimulation have roughly equivalent effects on TNF-α production. Furthermore, Figure 17B shows that splenic injection of α-bungarotoxin (BTX; a known antagonist of α7nAChR) suppressed the effect of ultrasound stimulation on TNF-α concentrations. This indicates that the desired CAP regulation by ultrasound involves splenic α7nAChR signaling (as does VNS-based CAP activation). Similar to the CAP model (Figure 15B), NE concentrations were not affected by BTX (i.e., BTX blocked the effect of elevated NE through the α7nAChR pathway). Vagotomy also inhibited ultrasound-induced CAP regulation, providing additional evidence that the ultrasound effect on CAP occurs via the nerve (Figure 17B). Finally, the kinase inhibitors PP2 (partially selective for Src kinase) and LY294002 (PI3-kinase selective) were shown to inhibit the ultrasound effect, whereas PD98059 (MEK1 and MEK2 selective MAPK inhibitor) had no effect (Figure 17A). These results support those in Figure 16I.Here, ultrasound stimulation caused changes in kinase activation within the PI3 (i.e., Akt, P70S6K), c-Src, and p38-MAPK pathways, but not in kinases involved in bacterial antigen responses (i.e., NFKB, GSK3B). Such changes may be part of the desired signature achieved through targeted neuromodulation and indicative of targeted physiological effects.

[0094] The physiological specificity of focused ultrasound stimulation was investigated by measuring some known side effects of invasive VNS. Figure 17C shows data comparing the effects of VNS (at several stimulation intensities and frequencies) versus splenic ultrasound stimulation (with an ultrasound stimulation pressure of 0.83 MPa) on heart rate. Figure 17C shows the changes in heart rate caused by cervical VNS or splenic ultrasound stimulation. At VNS intensities of 1 and 5 volts (known to activate the CAP), heart rate was significantly reduced. However, local splenic ultrasound stimulation had no effect on heart rate.

[0095] FIG. 17D shows data supporting the previously observed side effects of VNS on suppression of LPS-induced hyperglycemia and the lack of side effects when using splenic ultrasound stimulation. The graph shows the relative blood glucose concentrations (compared to pre-injection concentrations) at 5, 15, 30, and 60 minutes after LPS injection for LPS control (no ultrasound stimulation), or LPS injection combined with splenic ultrasound stimulation or cervical VNS stimulation. FIG. 17D shows that ultrasound stimulation did not result in changes in glucose concentrations compared to the LPS-only control, whereas the VNS experiment had the side effect of attenuating hyperglycemia. This metabolic side effect of CAP-targeted VNS may be due to non-targeted VNS of the second (non-CAP) vagus nerve pathway. Such non-targeted effects are a result of the broader and less specific (i.e., less targeted) effects of VNS compared to the targeted peripheral neuromodulation provided herein. Advantages of the described targeted neuromodulation include avoiding undesired non-targeted effects. We further used high-precision ultrasound stimulation technology to investigate whether VNS-induced reduction in hyperglycemia was associated with direct stimulation of axons derived from metabolic sensory neurons.

[0096] In some embodiments, ultrasound images may be used to guide ultrasound stimulation to spatially select a region of interest for targeted ultrasound stimulation delivery. As described herein, spatial selection or a spatial selection step may include acquiring an image of a tissue or organ (or a portion of a tissue or organ) and identifying a region of interest within the organ based on the image (e.g., an ultrasound image). In some embodiments, the tissue or organ may have anatomical features that are used to guide the selection of the region of interest within the organ. Such features may, in some embodiments, include, as non-limiting examples, sites of vascular or nerve entry into the organ, tissue types within the organ, interiors or edges of the organ, or sub-organ structures. In certain embodiments, the anatomical features may include the hilum of the liver, sub-organs of the gastrointestinal tract (stomach, small intestine, large intestine), pancreatic duct, or white pulp of the spleen. By identifying anatomical features in the image, a region of interest may be selected that overlaps, includes, or is adjacent to the anatomical feature. In other embodiments, the anatomical feature may be excluded from the region of interest. For example, intestinal tissue may be selected as the region of interest rather than stomach tissue. Identification of anatomical features can be done via morphological features visible in the image (e.g., visible in an ultrasound image) or by structure recognition features of the imaging modality used to acquire the image. As disclosed herein, the system 10 can be configured such that the energy application device 12 operates in an imaging mode to acquire an image, and operates in an energy application mode after the image is acquired and a region of interest is spatially selected based on the image.

[0097] In other embodiments, the region of interest may be identified by the presence or absence of one or more biological markers. Such markers may be assessed by staining the organ or tissue and acquiring images showing the staining to identify regions of the organ or tissue containing one or more biological markers. In some embodiments, the biological marker information may be obtained by a vital staining technique, which may provide real-time location data of the biological markers in a subject-specific tissue or organ. In other embodiments, the biological marker information may be obtained by an in vitro staining technique, which may provide location data of one or more representative images that are used to predict the location of the biological markers in the subject's tissue or organ. In some embodiments, the region of interest is selected to correspond to a site of the tissue or organ that is enriched in a particular biological marker or that lacks a particular biological marker. For example, the one or more biological markers may include a marker of neuronal structure (e.g., a myelin sheath marker).

[0098] The region of interest of an organ or tissue may be spatially selected based on operator input. For example, an operator may specify a region of interest on an acquired image by directly manipulating the image (i.e., drawing or writing the region of interest on the image) or by providing image coordinate information corresponding to the region of interest. In another embodiment, the region of interest may be automatically selected based on image data to achieve spatial selection. In some embodiments, the spatial selection includes storing data associated with the region of interest in a memory and accessing the data.

[0099] Once spatial selection is performed, the system 10 is configured to apply energy to the region of interest as described herein. For example, as shown in FIG. 18A, a 2D ultrasound image of a rat liver is used to guide ultrasound stimulation and selectively focus on the target hepatic hilar region. The white arrow indicates the liver outline and the central arrow indicates the region of interest. FIG. 18A shows that ultrasound image guidance allows spatial selection of the hepatic hilar region of the liver and allows ultrasound stimulation to be directed to the selected hepatic hilar region containing glucose-sensitive neurons for local targeted ultrasound neuromodulation.

[0100] FIG. 18B shows a non-limiting example of selectively applying ultrasonic stimulation to various regions of the liver of an LPS-induced hyperglycemia animal model to achieve the desired regulation of blood glucose concentrations. The graph in FIG. 18B shows the relative blood glucose concentrations (compared to LPS pre-injection concentrations) at 5, 15, 30, and 60 minutes after LPS injection. In the group that received only LPS injection without ultrasonic stimulation, LPS-induced hyperglycemia is observed. The data further show that ultrasonic stimulation of the distal lobes of the liver does not significantly affect blood glucose concentrations. In contrast, selective application of ultrasonic stimulation to the hepatic hilar region can be utilized to reverse LPS-induced hyperglycemia and regulate blood glucose concentrations. Thus, the site of the region of interest produces different outcomes, and broad or non-targeted liver treatment to the lobes does not achieve the same effect as ultrasonic treatment targeted to the hepatic hilar region (using a region of interest adjacent to or including the hepatic hilar region). As shown in the embodiment of FIG. 18B, applying ultrasound stimulation to regions of interest in the liver according to the procedure shown in FIG. 16G provides protection against LPS-induced hyperglycemia in the model and / or limits the increase in blood glucose concentration and regulates it below postprandial concentrations. Furthermore, regulation can be specific to the anatomical site. FIG. 18B shows that by directing ultrasound stimulation to the right or left lobe of the liver, the ultrasound-induced effect on blood glucose was attenuated. That is, not all regions of the liver responded in the same way to ultrasound energy application. In some embodiments, energy application may be used as a protective treatment or as a treatment applied prior to an expected systemic attack or destruction.

[0101] Selective modulation of one or more molecules of interest may be achieved at a site directly exposed to ultrasound stimulation (i.e., the organ containing the target region of interest). For example, targeted ultrasound stimulation of the liver at the region of interest includes changes in hepatic concentrations of signaling molecules in the liver tissue, where those related to glucose metabolism may remain unchanged (FIG. 18C, grey bars). Additionally / alternatively, selective modulation of one or more molecules of interest may also be achieved at distal sites not directly exposed to ultrasound stimulation. For example, FIG. 18C shows that application of ultrasound stimulation to a site of the liver induces significant changes in hypothalamic concentrations of NPY and NE, as well as increased phosphorylation of ion channels indicative of increased activity of the insulin signaling pathway, as indicated by improved insulin sensitivity and improved glucose utilization.

[0102] FIG. 18C shows the measurement of relative concentrations (compared to no ultrasound stimulation) of several molecules related to either insulin sensitivity and changes in hypothalamic markers related to insulin-mediated and non-insulin-dependent glucose uptake and metabolic function in the liver. In the liver, epinephrine was reduced compared to the changes in norepinephrine. Epinephrine can cause a rapid increase in blood glucose by promoting the release of stores in the liver. However, norepinephrine does not contribute significantly to hepatic glucose production, in contrast to the effects of norepinephrine on glucose uptake by skeletal muscle and fat. Increased norepinephrine in the hypothalamus may indicate increased hyperinsulinemia (indicating decreased insulin sensitivity) and impaired glucose tolerance / hyperglycemia. Ultrasound stimulation may selectively modulate or change the concentration of one or more molecules of interest at distal sites not directly stimulated. For example, as shown in FIG. 18C, ultrasound stimulation may be utilized to modulate the concentration of molecules such as norepinephrine (NE), protein kinase B (pAkt), insulin receptor substrate 1 (IRS-1), and neuropeptide Y (NPY) in distal hypothalamic sites by applying ultrasound stimulation directly to the liver sites. In certain embodiments, ultrasound stimulation is selectively applied to spatially selected regions of interest of tissue to achieve a desired (i.e., targeted) physiological effect. Tissues may be selected from liver, pancreas, gastrointestinal tract, spleen, and the like. The desired effect may be a change in the concentration of a molecule or a clinically significant marker. In certain embodiments, ultrasound-induced neuromodulation can be quantified by the degree of activity-dependent expression of the immediate early gene cFOS within defined hypothalamic and brainstem sub-nuclei (Figures 18D and 18E, respectively). Figure 18D shows cFOS immunohistochemistry images (left) and data showing the percentage of activated neurons in LPS control (left) and ultrasound-stimulated samples (right). In the control images, the percentage of activated neurons is about 7.7%, while in the stimulated images, the percentage of stimulated neurons is about 4.9%. Images and data are segmented in the paraventricular nucleus (PVN). The reduction in neural activity, represented by the reduction in the percentage of activated neurons after ultrasound stimulation, further supports (in addition to the data in Figure 18C) the effect of hepatic ultrasound neuromodulation on whole-body glucose utilization and metabolic signaling. Figure 18E shows additional immunohistochemistry images showing cFOS expression in the brainstem of LPS control (top) versus ultrasound-stimulated samples (bottom). The image is segmented at the nucleus of the solitary tract (NTS), showing increased expression in ultrasound-stimulated samples. Figure 18F shows an example MRI overlay between the activation map (weighted spoiled gradient-reduced cold-echo volume, left) and the brain atlas (weighted spoiled gradient-reduced cold-echo volume, right). This example shows increased ADC in both the left and right paraventricular nuclei of the hypothalamus (PVN) (arrows, left image). This is consistent with neuronal deactivation. Figure 18G is a bar graph summarizing the results. Three of six rats showed significant deactivation of the PVN. None of the control animals showed such deactivation. Furthermore, the hyperglycemia observed in non-sonicated animals was not observed in the ultrasound-treated animals.

[0103] Table 2 shows the t-test values ​​obtained comparing ADC values ​​within the validated PVN regions of interest between pre- and post-treatment scans. [Table 2] For 3 of 6 rats, the increase in PVN ADC in response to ultrasound stimulation was generally consistent with the c-Fos expression data, indicating ultrasound-induced deactivation of LPS-activated pathways communicating with the hypothalamus, although only a subset of LPS + ultrasound animals showed this effect (rats 1, 4, and 5 in Table S2).

[0104] Compared to controls, a significant decrease was observed in cFOS-positive (c-Fos+, Figure 18D) cells in the paraventricular nucleus (PVN), suggesting ultrasound-induced modulation of LPS-induced neural signaling. These data support that the arcuate nucleus (ARC) modifies signals to the PVN based on peripheral sensory information via NPY-expressing neurons, so that concentrations of NPY and GABA are significantly decreased after ultrasound stimulation. Furthermore, altered hypothalamic c-Fos expression was accompanied by a significant increase in c-Fos expression in the nucleus of the solitary tract (NTS; Figure 18E), indicating modulation by ultrasound via signaling through the nucleophilic pathway.

[0105] Apparent diffusion coefficients (ADC) from diffusion-weighted functional magnetic resonance imaging (DfMRI) images in hypothalamic subnuclei were compared before and after ultrasound stimulation of the liver. Upon ultrasound stimulation, ADC increased within the PVN. This corroborated both chemical (POMC, NPY, NE) and c-Fos expression data, demonstrating ultrasound-induced deactivation of LPS-activated pathways that communicate to the hypothalamus. These results are consistent with activation of pathways regulating the effects of LPS on energy metabolism via the NPY system and its effects on PVN signaling to the outside.

[0106] The disclosed technology described herein allows for precise neuromodulation with a simple non-invasive technique, which has been demonstrated for two specific neural pathways (CAPs in the spleen and metabotropic sensory neurons in the liver) and can be applied to modulate other peripheral neural circuits. Hyperglycemia model

[0107] Neuromodulation via application of energy to a region of interest as described herein can be used to treat a subject and / or induce desired metabolic changes in a subject (e.g., a diseased subject, a healthy subject). In some embodiments, energy (e.g., ultrasonic energy, mechanical energy) can be applied to one or more of a liver region of interest, a gastrointestinal region of interest, or a pancreatic region of interest, by way of example. Application of energy induces a desired metabolic outcome via selective neuromodulation of synapses in the region of interest, resulting in local effects at or near the site of energy application, as well as systemic effects in distal tissues and circulating molecules.

[0108] 19-44 are embodiments of ultrasonic energy application in LPS rats. Insulin and glucagon are synthesized in pancreatic islet cells and are involved in the regulation of blood glucose. LPS was used to generate acute insulin resistance and transient hyperglycemia in Sprague Dawley rats. In certain embodiments, ultrasonic stimulation may be applied to a region of interest in the pancreas. This may regulate insulin production and induce a significant increase in circulating insulin concentration, thereby resulting in a rapid decrease in circulating glucose concentration. As shown in FIG. 19, circulating glucose concentration decreased in response to pancreatic ultrasonic stimulation to a selected region of interest in the pancreas. Pancreatic ultrasonic stimulation resulted in a decrease in circulating glucose concentration compared to control LPS SD rats. In comparison, ultrasonic stimulation may also be applied to a region of interest at or near the hepatic hilum in accordance with the techniques described herein to achieve similar results of decreasing circulating glucose concentration. Thus, in certain embodiments, a subject may be treated by applying energy to pancreatic tissue in accordance with embodiments disclosed herein to induce a decrease in circulating glucose. The desired therapeutic outcome of energy application to pancreatic tissue may be assessed by ascertaining circulating glucose concentration.

[0109] FIG. 20 shows that the regulation of circulating insulin concentration is achieved by selectively applying ultrasonic stimulation to a region of interest in the pancreas, for example, a significant increase in circulating insulin concentration is achieved by selectively applying ultrasonic stimulation to a region of interest in the pancreatic tissue of LPS SD rats. On the other hand, ultrasonic stimulation with the modulation parameters described herein applied to a region of interest selected to correspond to a site of the liver or gastrointestinal tissue of LPS SD rats does not produce a significant change compared to control LPS SD rats. This suggests that although regulation of circulating glucose concentration can be achieved by directing ultrasonic stimulation to pancreatic or liver tissue, the mechanism of reducing circulating glucose concentration may be different for pancreatic stimulation compared to liver stimulation. In the case of ultrasonic stimulation of the pancreas, the induction of an increase in circulating insulin seen after direct ultrasonic stimulation may contribute to the induction of a decrease in circulating glucose concentration, as well as, for example, early insulin resistance. FIG. 21 shows that there is no significant change in circulating glucagon concentration as a result of ultrasonic stimulation in gastrointestinal, hepatic, and pancreatic stimulated LPS SD rats. Thus, in certain embodiments, a subject can be treated by applying energy to pancreatic tissue in accordance with embodiments disclosed herein to induce an increase in circulating insulin. The desired therapeutic outcome of applying energy to pancreatic tissue can be assessed by ascertaining circulating insulin concentrations, either alone or in conjunction with circulating glucose concentrations.

[0110] Figures 22-23 show the concentration of molecules in pancreatic tissue as a result of applying ultrasonic stimulation to regions of interest in pancreatic tissue of LPS SD rats. As shown in Figure 22, pancreatic stimulation does not induce a significant change in insulin concentration in pancreatic tissue. The lack of observable changes in insulin in pancreatic tissue may be related to an increase in circulating insulin by releasing insulin stored in the pancreas. That is, an increase in pancreatic insulin may lead to a subsequent increase in circulating insulin. This may then be followed by a return of pancreatic insulin levels to baseline via consumption during the refractory period when insulin levels recover after release. In contrast, applying pancreatic ultrasonic stimulation can induce a significant increase in glucagon concentration in pancreatic tissue compared to control LPS SD rats not subjected to ultrasonic stimulation (Figure 23). Glucagon is a protein involved in the regulation of glucose production in the liver (by glycogenolysis), independent of the current hyperglycemia in the described animal model. 22 and 23, the data indicates that application of ultrasound stimulation, e.g., image-guided application / delivery of ultrasound stimulation to a region of interest in the pancreas, can be used to modulate glucagon concentrations (i.e., induce changes, e.g., increase glucagon secretion by pancreatic alpha cells) and regulate the concentration of circulating glucose. In certain embodiments, application of ultrasound stimulation to a region of interest in pancreatic tissue can be used to treat recurrent hypoglycemia, e.g., to treat type 1 diabetes, as a stand-alone treatment or in combination with other treatment options.

[0111] FIG. 24 illustrates a region of interest in gastrointestinal tissue targeted by gastrointestinal ultrasound stimulation in LPS SD rats as described herein. The region of interest in the gastrointestinal tissue may be spatially selected as described herein. For example, the region of interest may be selected using image analysis to identify sections of the stomach and intestine and their location relative to other organs. For example, the stomach may be relatively close to the pancreas, while a particular portion of the intestine may be located away or distant from other organs (i.e., the pancreas). Targeting the intestine provides a relatively simple spatial selection while avoiding the pancreas and thus the relatively complex spatial selection of the stomach tissue. FIG. 25 illustrates the reduction in circulating glucose in response to gastrointestinal ultrasound stimulation in a rat model. After ultrasound stimulation, a small but significant reduction was observed in insulin in the pancreas (FIG. 26). A small but significant change was observed in glucagon in the pancreas (FIG. 27). Thus, as described herein, neuromodulation via application of energy (e.g., ultrasound energy) to gastrointestinal tissue in a region of interest may be used to control circulating glucose in a subject. Subjects with hyperglycemia may be treated by applying energy to gastrointestinal tissues in accordance with embodiments disclosed herein to induce a reduction in circulating glucose. After treatment, circulating glucose concentrations may be continuously or intermittently evaluated to determine additional treatment parameters. In certain embodiments, effects may last for hours and / or days. Additionally, neuromodulation via application of energy to gastrointestinal tissues of interest may be used to control metabolic processes that affect insulin levels in the pancreas. Subjects may be treated by applying energy to gastrointestinal tissues in accordance with embodiments disclosed herein to induce a reduction in circulating glucose. The desired therapeutic outcome of applying energy to gastrointestinal tissues may be assessed by ascertaining one or more of circulating glucose concentrations, concentrations of pancreatic insulin, and concentrations of glucagon in the pancreas. For example, a reduction in circulating glucose without a significant change in pancreatic glucagon may indicate a desired characteristic feature of glucose metabolism in the treated subject.

[0112] Figures 28-33 show hypothalamic markers (IRS1, phospho-AKt, GLP-1, hypothalamic NPY, or glucose-6-phosphate) in response to gastrointestinal, hepatic, and pancreatic ultrasound stimulation compared to controls. In pancreatic sonicated animals, protein subunits involved in the insulin signaling / insulin-mediated glucose uptake pathway were significantly increased. This pathway is known to be dysfunctional in the hypothalamus in rodents and humans, and insulin resistance represents a common feature of type 2 diabetes. The combination of an increase in this signaling pathway and an increase in hypothalamic glucose-6-phosphate concentrations is indicative of insulin signaling and whole-body insulin sensitivity. Limited insulin signaling-related responses were identified following gastrointestinal stimulation. Increases in GLP-1 and related receptor activation are known to promote weight loss and improve glucose tolerance, something that is currently targeted in pharmacological interventions in diabetic patients (e.g., GLP-1 agonists, exenatide). Increases in GLP-1 were identified as a result of pancreatic and gastrointestinal ultrasound stimulation (Figure 31). Increases in GABA in the hypothalamus (as seen in liver stimulated rats as a result of FIG. 33) affect known hypothalamic markers (e.g., NPY) and modulate the magnitude of glucagon and sympathoadrenal responses to glucose, thereby achieving peripheral glucose control. Hypothalamic markers include markers of energy balance, such as NPY, POMC, and BDNF, among others. Hypothalamic NPY has been shown to decrease with liver stimulation (FIG. 34). As disclosed herein, signature-induced changes or lack thereof in hypothalamic markers occurring simultaneously with other changes (e.g., changes in glucose or insulin concentrations as a result of gastrointestinal, hepatic, and / or pancreatic ultrasound stimulation) can result in a set of changes or signatures associated with a targeted physiological effect. The system can be configured to evaluate one or more of these changes as a proxy for a desired physiological effect or as a signature of a desired glucose metabolism in a treated subject.

[0113] Figures 35-37 show pancreatic markers of the insulin-mediated glucose uptake pathway in pancreatic β cells, and the effect of hepatic, gastrointestinal, or pancreatic ultrasound stimulation on these markers. No changes were observed in IRS-1 and pAKT, but GLUT2 was increased. This likely indicates that the application of ultrasound energy increases glucose uptake by GLUT2 in an insulin-independent manner in the pancreas. Figures 38-40 show pancreatic hormones involved in glucose regulation by the pancreas, and the effect of hepatic, gastrointestinal, or pancreatic ultrasound stimulation on these markers. GLUT2 showed a significant decrease in response to pancreatic stimulation and a significant increase in response to gastrointestinal stimulation. Thus, pancreatic and hepatic stimulation may activate opposing or bidirectional pathways involved in glucose regulation. To avoid overstimulation of glucose regulatory pathways in the liver, both pancreatic and liver tissues can be targeted for ultrasound (or other energy) application in a subject. Thus, not only are glucose regulatory pathways activated, but also signaling molecules associated with downregulation are affected. By stimulating the bidirectional pathway, a stable glucose concentration can be achieved for subjects at risk for hyperglycemia or hypoglycemia. It is understood that GLUT2 is an example of a marker for a bidirectional pathway, and other mechanisms or pathways can also be targeted. As provided herein, bidirectional stimulation can be performed on a first region of interest in a first organ or tissue and a second region of interest in a second organ or tissue. In another embodiment, multi-site neuromodulation can be performed at different sites that enhance the same pathway. The energy application to the first region of interest and the second region of interest can be performed in parallel or sequentially. In one embodiment, the first region of interest is in the liver and the second region of interest is in the pancreas. In another embodiment, the first region of interest is in the gastrointestinal tissue and the second region of interest is in the liver.

[0114] Figures 41 to 44 show hepatic markers and the effect of hepatic, gastrointestinal, or pancreatic ultrasound stimulation on these markers. Increases in glucose-6-phosphate indicate modulation of hepatic glycolytic and gluconeogenic pathways. As disclosed herein, characteristic changes or lack thereof in hepatic markers occurring simultaneously with other changes (e.g., changes in glucose or insulin concentrations as a result of ultrasound stimulation) can result in a set of changes or characteristic features associated with a targeted physiological effect. The system can be configured to evaluate one or more of these changes in lieu of the desired physiological effect.

[0115] The written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any device or system, and performing the incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims to the extent that there are structural elements that do not contradict the language of the claims, or if they include equivalent structural elements that do not differ materially from the language of the claims.

Claims

1. At least one energy application device; A controller; an evaluation device coupled to the controller for evaluating a characteristic indicative of whether a desired physiological effect has been achieved by adjusting the at least one energy application device; Equipped with The controller: focusing the at least one energy application device on a first region of interest of a first tissue, the first tissue including a first plurality of axon terminals of respective neurons, one or more of the plurality of axon terminals forming synapses between respective axon terminals and corresponding non-neuronal cells, the first region of interest including a first subset of the first plurality of axon terminals; adjustably controlling the at least one energy application device to apply a first energy to the first region of interest to activate the first subset to selectively modulate a first molecule of interest; adjustably controlling the at least one energy application device to apply a second energy to a second region of interest comprising a second subset of axon terminals to activate the second subset, to selectively modulate a second molecule of interest that modulates a physiological effect produced by the first molecule of interest; the first molecule of interest is different from the second molecule of interest; The controller further comprises: determining, via the evaluation device, measurements of the change in the property of the first tissue and a second tissue in the second region of interest; modifying the adjustment parameter of the first energy or the second energy based on the measured change in the property being below a first threshold value related to a change in concentration of a first molecule of interest; and modifying the adjustment parameter of the second energy based on the measured change in the property being below a second threshold related to a change in concentration of a second molecule of interest. Regulation system.

2. The controller: A processor; 2. The system of claim 1, further comprising: a memory storing instructions configured to be executed by the processor to spatially select the first region of interest and the second region of interest, focus the energy on the first region of interest and the second region of interest, control the application of the energy, or a combination thereof.

3. The system of claim 1 , wherein the controller is configured to receive an input from an evaluation device indicative of a concentration of a first molecule of interest, a concentration of a second molecule of interest, or both.

4. The system of claim 3 , wherein the evaluation device is a blood glucose monitor.

5. The system of claim 1 , wherein the first region of interest is in a first organ and the second region of interest is in a second organ different from the first organ.

6. The system of claim 5 , wherein the first organ is a pancreas and the second organ is a liver.

7. The system of claim 5 , wherein the first organ is a pancreas and the second organ is gastrointestinal tissue.

8. 2. The system of claim 1, wherein selective modulation of the first molecule of interest via the first energy or the second energy comprises causing a change in a property of the first molecule of interest or causing a change in a concentration of the first molecule of interest relative to a baseline concentration before the energy is applied to the first region of interest, the second region of interest, or both.

9. 9. The system of claim 8, wherein selective modulation of the second molecule of interest via the second energy comprises causing a change in a property of the second molecule of interest or causing a change in a concentration of the second molecule of interest relative to a baseline concentration before the energy is applied to the second region of interest.

10. 10. The system of claim 9, wherein the concentration of the first molecule of interest is a glucose concentration and the concentration of the second molecule of interest is an insulin concentration.

11. The system of claim 10, wherein the glucose concentration is a blood glucose concentration and the insulin concentration is a pancreatic tissue insulin concentration.

12. The system of claim 9 , wherein the concentration of the first molecule of interest is a glucose concentration and the concentration of the second molecule of interest is a GLP-1 concentration.

13. The system of claim 12, wherein the glucose concentration is a blood glucose concentration and the GLP-1 concentration is a hypothalamic GLP-1 concentration.

14. 10. The system of claim 9, wherein the selective modulation of the first molecule of interest comprises a change in glucose concentration and the selective modulation of the second molecule of interest comprises a change in GLUT2 concentration, phosphorylation, translocation, or a combination thereof.

15. 15. The system of claim 14, wherein the glucose concentration is a blood glucose concentration and the GLUT2 concentration is a pancreatic tissue GLUT2 concentration.

16. 16. The system of claim 1 , wherein the first molecule of interest produces an excessive physiological effect and the second molecule of interest produces a physiological effect opposite to the excessive physiological effect produced by the first molecule of interest.

17. The system of claim 1 , wherein selective modulation of one or both of the first molecule of interest or the second molecule of interest comprises producing a change in circulating concentration.

18. 2. The system of claim 1, wherein selectively modulating the first molecule of interest via the first energy comprises causing a change in pancreatic glucagon concentration, and selectively modulating the second molecule of interest via the second energy comprises causing a change in circulating glucagon concentration.

19. The system of claim 1 , wherein the first molecule of interest is glucose or insulin.

20. 2. The system of claim 1, wherein selectively modulating one or both of the first molecule of interest or the second molecule of interest comprises maintaining a concentration of the first molecule of interest or a concentration of the second molecule of interest within a predetermined range.

21. The controller: A processor; 21. The system of claim 20, comprising: a memory storing instructions configured to be executed by the processor to determine that a concentration of the first molecule of interest, a concentration of the second molecule of interest, or both are outside of the predetermined range, and to control application of the first energy, the second energy, or both based on the determination.

22. 21. The system of claim 20, wherein the controller is configured to adjustably control the application of the first energy and the second energy to maintain a concentration of the first molecule of interest, a concentration of the second molecule of interest, or both within a predetermined range.

23. 21. The system of claim 20, wherein the predetermined range is between 70 mg / dL and 110 mg / dL.

24. 21. The system of claim 20, wherein the predetermined range is a normal blood glucose range for a subject.

25. 21. The system of claim 20, wherein the controller is configured to adjustably control the application of the first energy or the second energy to maintain a dynamic equilibrium of one or both of a concentration of the first molecule of interest or a concentration of the second molecule of interest.

26. 2. The system of claim 1, wherein the controller is configured to control application of the first energy to the first region of interest and application of the second energy to the second region of interest such that application of the first energy to the first region of interest is not simultaneous with application of the second energy to the second region of interest.

27. 10. The system of claim 1, wherein the controller is configured to control the application of the first energy to the first region of interest and the application of the second energy to the second region of interest to be applied simultaneously.

28. The system of claim 1 , wherein the instructions configured to be executed by the processor to cause the at least one energy application device to apply the first energy or the second energy comprise a set of adjustment parameters.

29. 30. The system of claim 28, wherein the adjustment parameters are selected to achieve a time-averaged intensity of ultrasound stimulation in the first region of interest or the second region of interest that is below a predetermined threshold.

30. 30. The system of claim 28, wherein the controller is configured to cause the at least one energy application device to apply the first energy or the second energy in accordance with adjustment parameters and to dynamically adjust one or more of the adjustment parameters based on feedback received from a user or an evaluation device indicative of a concentration of a first molecule of interest, a concentration of a second molecule of interest, or both.

31. At least one energy application device; and a controller. The controller: focusing the at least one energy application device on a first region of interest of a first tissue, the first tissue including a first plurality of axon terminals of respective neurons, one or more of the plurality of axon terminals forming synapses between respective axon terminals and corresponding non-neuronal cells, the first region of interest including a first subset of the first plurality of axon terminals; adjustably controlling the at least one energy application device to apply a first energy to the first region of interest to activate the first subset to selectively modulate a first molecule of interest; adjustably controlling the at least one energy application device to apply a second energy to a second region of interest comprising a second subset of axon terminals to activate the second subset, to selectively modulate a second molecule of interest that modulates a physiological effect produced by the first molecule of interest; the first molecule of interest is different from the second molecule of interest; the at least one energy application device is an ultrasound probe, and the controller is configured to operate the ultrasound probe in an imaging mode configured to acquire reference image data of the first tissue and / or a second tissue in the second region of interest prior to application of the first energy or the second energy, and to acquire post-treatment image data of the first tissue and / or the second tissue after application of the first energy or the second energy; the controller is configured to determine displacement measurements of the first tissue and / or the second tissue based on a comparison of the reference image data and the post-treatment image data; the controller is configured to modify an adjustment parameter of the first energy or the second energy based on the displacement measurement; the controller is configured to modify the adjustment parameter of the first energy or the second energy based on the displacement measurement being below a threshold displacement associated with a change in concentration of a first molecule of interest; The adjustment system, wherein the controller is configured to modify the adjustment parameter of the second energy based on the displacement measurement being below a threshold displacement associated with a change in concentration of a second molecule of interest.

32. The system of claim 1 , wherein the at least one energy applicator includes an ultrasonic transducer.

33. 2. The system of claim 1, wherein the at least one energy application device comprises a first ultrasound transducer configured to apply the first energy and the second energy, and a second ultrasound transducer configured to acquire spatial information of the first region of interest and / or the second region of interest.

34. 2. The system of claim 1, wherein the controller is configured to spatially select the first region of interest by identifying a neural or vascular entry site into the first tissue, the first region of interest at least partially overlapping the entry site.

35. The system of claim 1 , wherein the controller is configured to spatially select the first region of interest according to organ morphology.

36. The system of claim 1 , wherein the first region of interest is less than or equal to 5% of a total volume of the first tissue.

37. 37. The system of claim 36, wherein the second region of interest is less than or equal to 5% of a total volume of the second tissue.

38. The system of claim 1 , wherein the at least one energy applicator comprises a mechanical vibrator.

39. The system of claim 1 , wherein the at least one energy applicator does not apply the energy to a portion of the first tissue that is outside of the first region of interest.

40. The system of claim 1 , wherein the at least one energy applicator does not apply the energy to a portion of the second tissue that is outside of the second region of interest.

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