Neuromodulation techniques for perturbation of physiological systems

Abstract

The present disclosure relates to a technique for inducing physiological perturbation in a subject through neuromodulation, for example, peripheral neuromodulation of a target region of an organ.The nature and degree of perturbation can be related to the clinical condition of the subject.Therefore, the evaluation of one or more characteristics of the perturbation can be used to determine the clinical condition of the subject.

Description

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with Government support under Contract No. HR0011-18-C-0040 awarded by the Defense Advanced Research Projects Agency (DARPA). The Government has certain rights in this invention. [Technical field]

[0002] The subject matter disclosed herein relates to techniques for evaluating physiological systems through deliberate perturbation of such systems, as well as evaluation of recovery from and / or characteristics of the perturbation. In particular, the perturbation may be the result of targeted neuromodulation. [Background technology]

[0003] Healthcare providers use a variety of methods to test and diagnose clinical conditions. In certain cases, a subject may be subjected to a condition that induces some kind of stress or perturbation, and an evaluation of the subject's response to the stress or perturbation may indicate the clinical condition or overall health. Such perturbations may be induced by exercise, drugs, fasting, and the like. For example, a glucose tolerance test may involve administering a glucose load to a subject and evaluating a blood sample taken from the subject after administration to determine how the subject responds. However, such tests are complex and may require dietary restrictions (e.g., prior fasting), which some subjects may not adhere to, which may affect the results. Furthermore, administration of drugs may be accompanied by undesirable side effects. Thus, improved techniques for diagnosing certain clinical conditions would be beneficial. Summary of the Invention

[0004] The disclosed 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, this disclosure may encompass a variety of forms that may be similar to or different from the embodiments set forth below.

[0005] In one embodiment, a regulation system is provided that includes an energy application device configured to apply energy to a region of interest to induce a physiological perturbation in a subject. The system also includes a controller configured to control the application of energy to the region of interest via the energy application device to induce the physiological perturbation to induce a change in concentration of one or more molecules of interest relative to a baseline concentration. The controller is also configured to receive information indicating the concentration of one or more molecules of interest and determine that the subject is in a category selected from two or more categories based on the change in concentration of the one or more molecules of interest relative to a baseline concentration within a period of time.

[0006] In another embodiment, a method of inducing a physiological perturbation in a subject is provided, the method including directing an energy application device to a region of interest in the subject, applying energy to the region of interest such that, as a result of the application, activity of at least one axon terminal in the region of interest is modulated, evaluating one or more characteristics of the perturbation at one or more time points following the perturbation, and providing an indication of a clinical condition of the subject based on the evaluation.

[0007] In another embodiment, a regulation system is provided that includes an energy application device configured to apply energy to a first target region of a first organ and a second target region of a second organ. The system also includes a controller configured to control a first application of energy to the first target region via the energy application device to cause a first perturbation as a result of the first application of energy, receive information indicative of the first perturbation characteristic, control a second application of energy to the second target region via the energy application device to cause a second perturbation as a result of the second application of energy, receive information indicative of the second perturbation characteristic, and determine a clinical condition of the subject based on the first perturbation characteristic and the second perturbation characteristic, and provide an indication of the clinical condition.

[0008] In another embodiment, a method of assessing a physiological perturbation in a subject is provided, the method comprising applying ultrasound energy to a region of interest in the subject to induce an approximated fasting state in the subject via neuromodulation, receiving glucose and insulin concentration data from the subject in the approximated fasting state, applying the data to a model, the model being based on a relationship between glucose and insulin concentrations in the approximated fasting state for a plurality of normal subjects, receiving an indication of insulin resistance in the subject using the model, and providing a treatment recommendation based on the indication. [Brief description of the drawings]

[0009] 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 the drawings.

[0010] [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 neuromodulation. [Figure 4] 1 is a flow diagram of a neuromodulation technique according to an embodiment of the present disclosure. [Diagram 5] 1 is a flow diagram of a neuromodulation technique according to an embodiment of the present disclosure. [Figure 6] 1 is a flow diagram of a neuromodulation technique according to an embodiment of the present disclosure. [Figure 7] FIG. 1 is a schematic diagram of an ultrasonic energy application device in operation according to an embodiment of the present disclosure. [Figure 8] FIG. 1 is a schematic diagram of an experimental setup for ultrasonic energy application to achieve targeted physiological perturbation, according to an embodiment of the present disclosure. [Figure 9] 1 is an experimental timeline of ultrasonic energy application according to an embodiment of the present disclosure. [Figure 10] FIG. 1 shows circulating glucose concentrations following application of ultrasound energy in a Zucker-HFD animal type 2 diabetes model according to an embodiment of the present disclosure. [Figure 11] FIG. 11 shows food consumption in animals during the experimental timeline of FIG. [Figure 12] FIG. 11 shows the change in percent weight gain in treated versus control animals during the experimental timeline of FIG. 10. [Figure 13] FIG. 11 illustrates modified homeostasis model assessment scores for insulin resistance using a neuromodulation technology-based model according to an embodiment of the present disclosure in animals during the experimental timeline of FIG. [Figure 14] 1 is a flow diagram of a neuromodulation technique according to an embodiment of the present disclosure. [Figure 15] 1 shows response and recovery profiles to neuromodulatory perturbations according to embodiments of the present disclosure in highly insulin resistant subjects. [Figure 16] 1 shows response and recovery profiles to neuromodulatory perturbations according to embodiments of the present disclosure in low insulin resistant subjects. [Figure 17] 1 shows response and recovery profiles to neuromodulatory perturbations according to embodiments of the present disclosure in low insulin resistant subjects. [Figure 18] 1 is a flow diagram of a neuromodulation technique according to an embodiment of the present disclosure. [Figure 19] FIG. 1 shows TNF mRNA expression in treated vs. control groups following LPS exposure. [Figure 20] FIG. 1 shows mRNA expression of various markers in treated versus control groups following LPS exposure. [Figure 21] FIG. 1 illustrates an exemplary protocol for acquiring baseline and potential post-exposure data for evaluating changes in neuromodulation-based responses following exposure to a substance or pathogen. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] One or more specific embodiments are described below. In order to provide a concise description of these embodiments, not all characteristics of an actual implementation are described herein. It should be understood that in the development of an actual implementation, such as an engineering or design project, numerous implementation-specific decisions must be made to achieve the developer's particular goals, such as addressing system-related and business-related constraints, which may vary from implementation to implementation. Moreover, it should be understood that such a development effort may be complex and time-consuming, but is nevertheless a routine undertaking of design, fabrication, and manufacture for those of ordinary skill in the art having the benefit of this disclosure.

[0012] Any examples or illustrations provided herein should not be considered in any way as expressing a restriction, limitation, or definition to any term or terms in which they are used. Instead, these examples or figures should be considered as being described with respect to various specific embodiments and should be considered as being illustrative only. Those skilled in the art will understand that any term or terms utilized in these examples or figures encompass other embodiments that may or may not be provided therewith or elsewhere in this specification, and all such embodiments are intended to be included within the scope of that term or terms. Language designating such non-limiting examples and figures includes, but is not limited to, "for example," "for instance," "such as," "eg," "including," "in certain embodiments," "in some embodiments," and "in one (an) embodiment."

[0013] Provided herein are techniques for physiological perturbation via neuromodulation of targeted regions of interest and for causing targeted physiological perturbation that is the result of the modulation. Such physiological perturbation can be useful for evaluating a subject's response to the perturbation in a controlled environment. For example, physiological perturbation can adjust metabolic pathways to the effect of the perturbation to achieve homeostasis. The quality or effectiveness of the response can be a measure of the subject's overall condition. In one embodiment, neuromodulation is used alternatively or in addition to other techniques for inducing physiological changes (e.g., exercise stress testing, drug administration). Drugs can perturb physiological systems, but their effects can vary from subject to subject due to variability in patient comorbidities, or variability in physiological processes that respond to drugs and are not connected to the system being examined. For example, certain subjects may metabolize drugs at different rates due to pharmacogenetic differences in drug metabolism pathways. Differences in metabolic rates can result in differences in drug effects. Thus, such tests using drugs may not be able to identify patients with certain clinical conditions. That is, a particular patient may have a response that is difficult to fit into existing indicators or response type models.

[0014] The neuromodulation applied to cause a physiological perturbation can be targeted, allowing the healthcare provider to induce a desired change and evaluate one or more characteristics of the perturbation. Such characteristics can include the change in concentration of one or more molecules of interest in response to the perturbation and the time to recovery (e.g., evaluating the persistence of the physiological perturbation). Thus, the evaluated characteristic of the perturbation can be the change in concentration of one or more molecules of interest over a predetermined period and / or total time to achieve a predetermined baseline concentration of one or more molecules of interest. In certain embodiments, based on the evaluation, a diagnosis of a clinical condition can be made and / or a treatment or treatment recommendation can be provided to the subject.

[0015] Neuromodulation of a target region may cause physiological perturbations in healthy or diseased subjects. In one embodiment, neuromodulation that does not cause characteristic changes in a subject may be associated with a clinical condition or disease diagnosis. In another embodiment, different clinical conditions or categories may be associated with specific characteristic responses to neuromodulation. That is, the application of energy to a target region and the lack of a predicted physiological response to perturbation may indicate a diseased organ, including metabolic pathways or inflammatory / anti-inflammatory pathways, that cannot respond to neuromodulation as in a healthy patient. In one example, neuromodulation energy applied to the pancreas without a resulting increase in circulating insulin levels indicates a non-responsive subject that can no longer produce insulin. Thus, a treatment recommendation may be provided that neuromodulation therapy does not work and insulin should be provided.

[0016] Perturbations can be induced by challenges such as exercise, administration of drugs, and / or dietary changes (fasting), but such perturbations are typically broad and untargeted. In contrast, as provided herein, neuromodulation can target one or more specific structures in the body to cause targeted or predictable physiological perturbations. Furthermore, insofar as multiple organs may be involved in metabolic processes, neuromodulation of one or more target regions in a particular organ can be beneficial in identifying parts of the pathway that may be dysfunctional.

[0017] As provided herein, neuromodulation can induce physiological perturbations through modulation of activity at axon terminals in a target region. The targeted region or target region can be any tissue or structure in the body that has axon terminals that form synapses with non-neuronal cells or fluids. In one example, the target region can be in an organ or structure such as the spleen, liver, pancreas, or gastrointestinal tissue. In another example, the target region can be in lymphatic tissue. Neuromodulation of a target region allows for localized, limited, and non-ablative application of energy only to the target region without applying energy outside the target region. Energy application can induce efferent perturbations outside the targeted target region, for example, in the same organ, tissue, or structure that includes the target region, or in other organs and structures that do not include the targeted target region. In some embodiments, afferent effects can be induced in the hypothalamic region, as an example. Energy application can also induce afferent effects along the target nerve upstream of the energy application site. In some embodiments, effects outside the targeted region(s) of interest can be achieved without applying energy directly to areas outside the region(s) of interest where the effect is induced. Thus, local energy application can be used to achieve or effect systemic perturbations that may include local effects, downstream effects, and / or upstream effects.

[0018] The disclosed technology can be used to exert perturbation of physiological processes of the body using external or extracorporeal sources to cause targeted physiological perturbation in a subject.Through neuromodulation to targeted target regions, physiological processes can be altered, slowed down, stopped, or reversed.Also provided herein are technologies that can be applied to a subject to disrupt or temporarily challenge the dynamic equilibrium or homeostasis of physiological processes, such as glucose regulation, or to stimulate physiological systems back to homeostasis.Neuromodulation to targeted target regions can exert changes in physiological processes to interrupt, reduce, or enhance one or more physiological pathways in a subject, resulting in desired physiological perturbation.

[0019] The neuromodulation techniques discussed herein may be used to cause physiological perturbations in the neuromodulated subject. The physiological perturbations may include local changes in the region or tissue of interest to which energy is applied, as well as systemic changes that are the result of neuromodulation. In certain embodiments, one or more characteristics of the perturbation may be evaluated as part of the technique. These changes may include changes in one or more molecules of interest, changes in physiological parameters of the subject, displacement, enlargement, or other morphological changes in one or more tissue structures of the subject, changes in cell populations of the subject, changes in blood or other fluid flow parameters, and the like. In one embodiment, the physiological perturbation causes a change in the concentration (e.g., increase, decrease) of the molecule of interest and / or a change in the characteristics of the molecule of interest. That is, perturbation may include selective modulation of tissue production or release of one or more molecules of interest (e.g., a first molecule of interest, a second molecule of interest, etc.) and may refer to the modulation or influence of a molecule's concentration (circulation, tissue) or characteristics (covalent modification) as a result of the application of energy to one or more regions of interest (e.g., a first region of interest, a second region of interest, etc.) in one or more tissues (e.g., a first tissue, a second tissue, etc.). Modulation of a molecule of interest may include changes in molecular characteristics such as expression, secretion, protein translocation, and direct activity changes. Modulation may be driven based on the effect of applied energy to ion channels on either driving neural activity and function itself, or on the modulation of adjacent non-neuronal cells as a result of molecules derived from neural activity or direct activation within the non-neuronal cells. Modulation of a molecule of interest may also refer to maintaining a desired concentration of a molecule such that expected changes or fluctuations in concentration do not occur as a result of neural modulation. Modulation of a molecule of interest may refer to causing changes in molecular characteristics such as enzyme-mediated covalent modifications (changes in phosphorylation, acetylation, ribosylation, etc.). That is, it should be understood that the selective regulation of a molecule of interest can refer to molecular concentration and / or molecular characteristics. The molecule of interest can be a biological molecule, such as one or more of carbohydrates (monosaccharides, polysaccharides), lipids, nucleic acids (DNA, RNA), or proteins.In certain embodiments, the molecule of interest can be a signaling molecule, such as a hormone (an amine hormone, a peptide hormone, or a steroid hormone).

[0020] Certain embodiments described herein provide neuromodulation techniques for the diagnosis of metabolic dysfunction. In one embodiment, the diagnosis can be the presence or absence of glucose metabolic dysfunction and related disorders. Glucose regulation is complex and involves different local and systemic metabolic pathways. Application of energy to a targeted region of interest (single or multiple) results in characteristic changes in these metabolic pathways that affect glucose regulation. In some embodiments, modulation in one or more regions of interest can be used to identify disorders including, but not limited to, diabetes (i.e., type 1 or type 2 diabetes), hyperglycemia, hyperlipidemia, sepsis, trauma, infection, physiological stress, diabetes-related dementia, obesity, or other eating or metabolic disorders. 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 that present with hyperglycemia. Targeted perturbations via neuromodulation can induce changes in glucoregulatory hormones in blood or tissues to cause deviations from glucose concentrations. Targeted perturbations via neuromodulation may also induce changes in the activity of sensory or effector neurons in metabolic physiological control systems, and the response of these systems may be analyzed by imaging (e.g., MRI) or electrical recording (e.g., ECG) or other physiological monitoring. Changes to these systems as a result of neuromodulation may differ between healthy and diseased patients. In healthy subjects, perturbations may clear over time so that glucose concentrations return to normal. Furthermore, physiological perturbations may be used to identify subjects who do not have a disease diagnosis but are pre-diabetic. In one embodiment, physiological perturbations are used to identify subjects with insulin resistance, who may or may not be identified as diabetic.

[0021] Certain embodiments described herein provide neuromodulation techniques for the diagnosis of inflammation and immune function and related disorder control. Modulation of immune cell inflammatory and activity states involves different local and systemic neural, humoral, and cellular pathways. Application of energy to targeted target regions causes characteristic changes in these inflammatory and anti-inflammatory pathways that affect circulating cytokine and neurotransmitter concentrations and thus immune cell activity and state. In some embodiments, modulation at one or more target regions can be used to identify disorders including, but not limited to, rheumatoid arthritis, irritable bowel disease, psoriasis, or other chronic inflammatory or related disorders. Targeted perturbation via neuromodulation can induce changes in immune markers such as inflammatory cytokines, hormones, or neurotransmitters in blood or tissues to cause changes in the activity of intrinsic or circulating immune cells. Targeted perturbation via neuromodulation can also induce changes in the activity of sensory or effector neurons within the inflammatory control system, and the response of this system can be analyzed by imaging or electrical recording. Changes to these systems as a result of neuromodulation can differ between healthy and diseased patients. In healthy patients, perturbations may cause temporary or smaller changes to physiology, whereas in diseased patients, perturbations may be more persistent. Additionally, physiological perturbations can be used to identify patients who do not have a current disease diagnosis, or who are pre-symptomatic or have developed a chronic inflammatory disease.

[0022] To that end, the technology relates to targeted modulation of synapses at axon terminals in tissues via direct application of energy by an energy source to induce changes that result in measurable physiological perturbations (e.g., changes in circulating molecule concentrations or a series of concentration changes forming a characteristic physiological profile). The targeted synapses may include extra-axonal cell synapses formed between presynaptic axon terminals and postsynaptic non-neuronal cells. Additionally, while certain disclosed embodiments are discussed in the context of extra-axonal cell synapses, it should be understood that axon terminals may form axonal secretory, axonal synapses, axonal-somatic or extra-axonal cell synapses, and additionally or alternatively, these synapse types are considered to be selectively modulated as provided herein. Additionally, certain axon terminals may terminate in interstitial or bodily fluids that may also experience neurotransmitter release as a result of modulation. The disclosed synapses may be modulated to change activity at the synapse, such as neurotransmitter release from presynaptic axon terminals, as a result of energy application. Thus, changes in activity may result in local and / or non-local (e.g., systemic) effects that cause a global profile of physiological changes associated with a desired or targeted physiological perturbation. The present technology allows for targeted focusing of energy to a volume of tissue that contains specific axon terminals, preferentially directly activating the targeted axon terminals to achieve the desired physiological perturbation. In this way, in certain embodiments, targeted axon terminals within the target region are activated while axon terminals within the same organ or tissue structure but outside the target region are not activated. Because organs and tissue structures may contain different types of axon terminals that form synapses with different types of postsynaptic non-neuronal cells, a target region may be selected that contains a specific type of axon terminal that, when activated, results in the desired targeted physiological perturbation. Thus, modulation may target a specific type of axon terminal based on presynaptic neuron type, postsynaptic cell type, or both.

[0023] For example, in one embodiment, the type of axon terminal may be an axon terminal that forms an axon-extracellular synapse with an endogenous (i.e., tissue-resident or non-circulating) liver, pancreas, or gastrointestinal tissue cell. That is, the axon-extracellular synapse is formed at the junction between the axon terminal and a non-neuronal cell or interstitial or bodily fluid. Thus, the application of energy results in the modulation of function in the target area. However, it should be understood that different targeted physiological effects can be achieved based on the characteristics of the population of axon terminal types and the presynaptic neuron type and postsynaptic cell (e.g., immune cells, lymphatic cells, mucosal cells, muscle cells, etc.) of the axon-extracellular synapse. Furthermore, as described above, the axon terminal may terminate in an interstitial or bodily fluid that may also experience neurotransmitter release as a result of the modulation. Thus, application of energy to a target region in a subject's tissue may activate axon terminals (and their associated extra-axonal cell synapses, if applicable) within the target region, but may not affect non-target axon terminals (and associated synapses) outside the target region. However, because modulation may have a systemic effect, non-target axon terminals outside the target region may experience certain systemic changes as a result of activation of axon terminals within the target region. As provided herein, preferential activation or direct activation may refer to cells or structures (e.g., synapses) within the target region that receive direct application of energy (e.g., energy is applied directly to the cells or structures). For example, axon terminals, extra-axonal cell synapses, and / or postsynaptic non-neuronal cells within the target region, or interstitial or bodily fluids that may directly receive the applied energy provided herein. Even regions that receive physiological changes as a result of energy application may be considered preferentially or directly activated, as opposed to regions outside the target region that do not receive direct energy application.

[0024] Neuromodulation is a technique in which energy from an external energy source is applied to a specific region 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 carried through the nerve (e.g., as an action potential) to cause a physiological response in a region downstream of the site of energy application. However, because the nervous system is complex, it is difficult to predict the extent and ultimate endpoint of the physiological response to a given site of energy application. In one example, stimulation of an axon terminal releases neurotransmitters / neuropeptides or induces altered neurotransmitter release in the vicinity of adjacent non-neuronal cells, such as secretory cells or other cells, modulating the cellular activity of adjacent or nearby non-neuronal cells, including postsynaptic cells.

[0025] Advantages of the present technology include localized modulation at targeted regions of tissue to achieve physiological perturbations that can be used to assess the condition of a subject. Furthermore, localized modulation can include direct activation of relatively small regions of tissue (e.g., less than 25% of the total tissue volume) to achieve these effects. In this manner, the total applied energy to achieve the desired physiological perturbation is relatively small. In certain embodiments, the applied energy can be from a non-invasive external energy source (e.g., ultrasonic energy source, mechanical transducer). For example, a focused energy probe can apply energy through the skin of the subject and is focused to a targeted region of internal tissue. Such embodiments achieve the desired physiological perturbation without invasive procedures or without side effects that may be associated with other types of treatments or therapies.

[0026] In certain embodiments, a technique for neuromodulation is provided in which energy from an energy source (e.g., an external or extracorporeal energy source) is applied to an axon terminal such that induced physiological perturbations, e.g., neurotransmitter release, at the focal site of energy application, e.g., the axon terminal, are triggered in response to the energy application, rather than in response to an action potential. That is, the direct application of energy to the axon terminal acts in place of an action potential to promote the release of neurotransmitters to the neural junctions (i.e., synapses) with non-neuronal cells. The direct application of energy to the axon terminal further induces the release of altered neurotransmitters from the axon terminals in the synapses (e.g., axon-extracellular synapses) to the vicinity of adjacent non-neuronal cells. In one embodiment, the energy source is an extracorporeal energy source, such as an ultrasonic energy source or a mechanical transducer. In this way, non-invasive and targeted neuromodulation can be achieved directly at the energy focal site, rather than via stimulation at an upstream site, which induces action potentials to propagate to downstream site targets and activate downstream targets.

[0027] In certain embodiments, the target tissue is an internal tissue or organ that is difficult to access using electrical stimulation techniques with electrodes. Contemplated tissue targets include gastrointestinal (GI) tissue (stomach, intestine), muscle tissue (cardiac, smooth and skeletal), epithelial tissue (epidermis, organ / GI lining), connective tissue, glandular tissue (exocrine / endocrine), organ tissue, and the like. In one example, focused application of energy at a neuromuscular junction promotes the release of neurotransmitters at the neuromuscular junction without an upstream action potential. In one embodiment, contemplated targets or target regions for modulation may include a portion of the pancreas responsible for controlling insulin release or a portion of the liver responsible for glucose regulation. In another embodiment, contemplated target regions may be located in the liver. In another embodiment, contemplated target regions may be located in the spleen. However, it should be understood that these embodiments are by way of example only.

[0028] To that end, the disclosed neuromodulation techniques may be used with a neuromodulation system. FIG. 1 is a schematic diagram of a system 10 for neuromodulation to achieve neurotransmitter release and / or activate components of a synapse (e.g., presynaptic cells, postsynaptic cells) in response to the application of energy. 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, for example, via a lead or wireless connection, and in use is directed to a target region of an internal tissue or organ of a subject, resulting in a targeted physiological perturbation. 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.

[0029] In certain embodiments, the energy application device 12 and / or the pulse generator 14 can be in wireless communication with a controller 16, which can in turn provide instructions, for example, to the pulse generator 14. In other embodiments, the pulse generator 14 can 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, can be incorporated within the controller 16. In embodiments in which the pulse generator 14 is extracorporeal, the energy application device 12 can be operated by a caregiver and positioned at a point on or above the subject's skin such that energy pulses are delivered transcutaneously to the desired internal tissue. Once positioned to apply energy pulses to the desired site, the system 10 can initiate neuromodulation to achieve a targeted physiological perturbation or clinical effect.

[0030] In certain embodiments, the system 10 may include an evaluation device 20 coupled to the controller 16 to evaluate a characteristic indicative of whether a targeted physiological perturbation of modulation has been achieved. In one embodiment, the targeted physiological perturbation may be local. For example, modulation may result in a localized tissue or functional change, such as a change in tissue structure, a localized change in the concentration of a particular molecule, tissue displacement, increased fluid movement, etc.

[0031] Modulation may result in systemic or non-localized changes, and targeted physiological perturbations may be related to changes in the concentration of circulating molecules or changes in tissue characteristics that do not include the region of interest where energy was directly applied. In one example, tissue displacement may be a surrogate measurement of the desired modulation, and displacement measurements less than an expected displacement value may result in modification of modulation parameters until the expected displacement value is induced. Thus, the evaluation device 20 may be configured in some embodiments to evaluate changes in molecular concentrations or activities. For example, the evaluation device 20 may include chemical sensors for the molecules of interest. In some embodiments, the evaluation device 20 may be an imaging device configured to evaluate changes in organ size and / or position, changes in bodily fluid flow, changes in RNA or protein expression, or other indicators of physiological perturbations. In another embodiment, the evaluation device 20 may be a circulating glucose monitor and / or a continuous glucose monitor that measures interstitial fluid glucose. The evaluation device 20 may be an MRI device or other device capable of acquiring image data of a subject to identify systemic changes. The evaluation device 20 may be an ECG or other physiological monitor. Although the illustrated elements of the system 10 are shown separately, it should be understood that some or all of the elements may be combined with each other. Additionally, some or all of the elements may be in wired or wireless communication with one another.

[0032] Based on the evaluation, adjustment parameters of the controller 16 can be altered. For example, if the desired adjustment relates to a change in concentration (circulating or tissue concentration of one or more molecules) within a defined time window (e.g., 5 minutes, 30 minutes after starting the energy application treatment) or relative to a baseline at the start of the treatment, a change in adjustment parameters, such as pulse frequency or other parameters, may be desired, which in turn can be provided by an operator or via an automated feedback loop to the controller 16 to define or adjust the energy application or adjustment parameters of the pulse generator 14.

[0033] The system 10 provided herein can provide energy pulses according to various modulation parameters. For example, modulation parameters can include various stimulation time patterns ranging from continuous to intermittent. In intermittent stimulation, energy is delivered for a period of time at a certain frequency during the signal on time. The signal on time is followed by a period of no energy delivery, called the signal off time. Modulation parameters can also include the frequency and duration of stimulation application. The frequency of application can be continuous or delivered for various periods of time, for example, within a day or within a week. In one example, modulation can be specified for time of day (morning, evening, night) or meals (fasting, non-fasting). The treatment period to cause a physiological perturbation can last for various periods of time, including, but not limited to, minutes to hours. In certain embodiments, the duration of treatment with a specified stimulation pattern can last for one hour and can be repeated at intervals of, for example, 72 hours. In certain embodiments, energy can be delivered more frequently, for example every three hours, for a shorter duration, for example, for 30 minutes. According to adjustment parameters such as duration and frequency of treatment, the application of energy can be adjustably controlled to achieve desired results.

[0034] 2 is a block diagram of certain components of the system 10. As provided herein, the system 10 for neuromodulation may include a pulse generator 14 adapted to generate a plurality of energy pulses for application to tissue of a subject. The pulse generator 14 may be separate or may be integrated into an external device such as a controller 16. The controller 16 includes a processor 30 for controlling the device. Software code or instructions are stored in a memory 32 of the controller 16 and executed by the processor 30 to control various components of the device. The controller 16 and / or the pulse generator 14 may be connected to the energy application device 12 via one or more leads 33 or wirelessly.

[0035] The controller 16 also includes a user interface having input / output circuitry 34 and a display 36 adapted to allow the clinician to provide selection inputs or adjustment parameters to the adjustment programs. Each adjustment program can 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 change the stimulation characteristics of the energy pulses delivered via the leads 33 to the subject to which the energy application device 12 is applied. Any suitable type of pulse generation circuit can be used including, but not limited to, constant current, constant voltage, multiple independent current or voltage sources, etc. The applied energy is a function of current amplitude and pulse width duration. The controller 16 allows for adjustably controlling the energy by modifying the adjustment parameters and / or initiating energy application at certain times or canceling / inhibiting energy application at certain times. In one embodiment, the adjustable control of the energy application device is based on information regarding the concentration of one or more molecules in the subject (e.g., circulating molecules). If the information is from the evaluation device 20, a feedback loop can drive the adjustable control. For example, a diagnosis may be made based on circulating glucose concentrations measured by evaluation device 20 in response to neuromodulation. If the concentration exceeds a predetermined threshold or range, controller 16 may initiate a treatment protocol of energy application to a region of interest (e.g., liver) and use modulation parameters associated with decreasing circulating glucose. The treatment protocol may use modulation parameters (e.g., higher energy levels, more frequent applications) that differ from those used in the diagnostic protocol. The controller may initiate a treatment protocol of energy application to a region of interest (e.g., spleen or immune tissue) and use modulation parameters associated with increasing or decreasing circulating cytokines or immune cell numbers, activity, or phenotype.

[0036] In one embodiment, the memory 32 stores different operating modes selectable by the operator. For example, the stored operating modes may include instructions to execute a set of adjustment parameters associated with a particular treatment site, such as a target region in the liver, pancreas, digestive tract, or spleen. Different sites may have different associated adjustment parameters. 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 operating modes for different types of treatments. For example, activation may be associated with a different stimulation pressure or frequency range compared to one associated with inhibition or blocking of tissue function. In a particular example, where the energy application device is an ultrasound transducer, the time-averaged power (time-averaged intensity) and peak positive pressure are in the range of 1 mW / cm2 to 30,000 mW / cm2 (time-averaged intensity) and 0.1 MPa to 7 MPa (peak pressure). In one example, the time-averaged intensity is less than 35 W / cm2 at the target region to avoid thermal damage and levels associated with ablation or cavitation. In another specific example, where the energy application device is a mechanical actuator, the amplitude of vibration is in the range of 0.1-10 mm. The selected frequency may depend on the mode of energy application, e.g., ultrasonic or mechanical actuator. The controller 16 may operate in a diagnostic protocol mode, where the results of the diagnosis may trigger a change to a treatment mode of operation. For example, the diagnostic mode may apply neuromodulation energy once or repeatedly within a relatively short time window (e.g., 1 hour), and the treatment mode of operation may include repeated neuromodulation events (e.g., daily, hourly) during the treatment protocol.

[0037] The system may also include an imaging device to facilitate focusing of the energy application device 12. In one embodiment, the imaging device may be integrated with the energy application device 12 or may be 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) a region of interest and to focus energy to the selected region of interest for targeting and subsequent neuromodulation. In another embodiment, the memory 32 stores one or more targeting or focusing modes used to spatially select a region of interest within an organ or tissue structure. The spatial selection may include selecting a sub-region of the organ to identify a volume of the organ that corresponds to the region of interest. The spatial selection may depend on the image data provided herein. Based on the spatial selection, the energy application device 12 may be focused to a selected volume that corresponds to the region of interest. For example, the energy application device 12 may be configured to operate initially in a targeting mode to apply a targeting mode energy that is used 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 is not applied with modulation parameters. However, once a region of interest has been identified, the controller 16 can operate in a treatment mode according to adjustment parameters associated with preferential activation.

[0038] The controller 16 may also be configured to receive inputs related to the targeted physiological perturbation as inputs for the selection of the modulation parameters. For example, if an imaging modality is used to evaluate a tissue characteristic, the controller 16 may be configured to receive a calculated index or parameter of the characteristic. Based on whether the index or parameter is above or below a predetermined threshold, a diagnosis may be made and an indication of the diagnosis may be provided (e.g., via a display). In one embodiment, the parameter may be a measure of tissue displacement of the diseased tissue or a measure of the depth of the diseased tissue. Other parameters may include evaluating the concentration of one or more molecules of interest (e.g., evaluating one or more of the change in concentration relative to a threshold or baseline / control, the rate of change, determining 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) acquire 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) acquire images to determine (e.g., via displacement measurements) that a targeted physiological perturbation has occurred. In such an embodiment, the imaging device, the evaluation device 20, and the energy application device 12 may be the same device.

[0039] 3 is a specific example in which the energy application device 12 includes an ultrasound transducer 42 capable of applying energy to a target tissue 43, e.g., liver, spleen, pancreas. The energy application device 12 can include control circuitry for controlling the ultrasound transducer 42. The control circuitry of the processor 30 (FIG. 2) may be integral with the energy application device 12 (e.g., via the integrated controller 16) or may be a separate component. The ultrasound transducer 42 may also be configured to acquire image data to aid in spatially selecting a desired or targeted region of interest and focusing the applied energy to the region of interest of the target tissue or structure.

[0040] The desired target tissue 43 may be an internal tissue or organ that contains synapses of axon terminals and non-neuronal cells. Synapses may be stimulated by direct application of energy to axon terminals within the focal region of an ultrasound transducer 42 focused on a target region 44 of the target tissue 43, causing release of molecules, e.g., neurotransmitter release, into the synaptic space, and / or changes in ion channel activity may in turn cause downstream effects. Sensory synapses (or transmitting cells) may also be stimulated by direct application of energy to peripheral terminals and neurons within the focal region of an ultrasound transducer focused on a target region of the tissue, causing direct neural signaling back to the CNS (i.e., driving or mimicking sensory input to CNS control centers, ganglia, and nuclei). Target regions may be selected to contain axon terminals of specific neuron types and / or specific types of axon terminals, such as those that form synapses with certain types of non-neuronal cells. Thus, target regions 44 may be selected to correspond to a portion of the target tissue 43 that has the desired axon terminals (and associated non-neuronal cells). The energy application may be selected to preferentially induce the release of one or more molecules, such as neurotransmitters, from the nerves in the synapse, or directly activate the non-neuronal cells themselves via direct energy transduction (i.e., mechanotransduction or voltage-activated proteins in the non-neuronal cells), or to cause activation in both neuronal and non-neuronal cells that induces a desired physiological effect. The target region may be selected as a site of neural entry into the organ. In one embodiment, liver stimulation or modulation may refer to modulation of a target region 44 at or adjacent to the hepatic hilum.

[0041] The energy can be focused or substantially concentrated on the target region 44 and only a portion of the internal tissue 43, for example, less than about 50%, less than about 25%, less than about 10%, or less than about 5% of the total volume of the tissue 43. In one embodiment, energy can be applied to two or more target regions 44 within the target tissue 43, and the total volume of the two or more target regions 44 can be less than about 90%, less than about 50%, less than about 25%, less than about 10%, or less than about 5% of the total volume of the tissue 43. In one embodiment, 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 axon terminals in the region of interest 44 of the target tissue 43 directly receive the applied energy and release neurotransmitters, while unstimulated axon terminals outside of the region of interest 44 do not receive substantial energy and are therefore not activated / stimulated in the same manner. In some embodiments, axon terminals in the portion of the tissue that directly receives the energy are induced to have altered neurotransmitter release. In this manner, tissue sub-regions can be targeted for neuromodulation in a granular manner, for example, one or more sub-regions can be selected. In some embodiments, energy application parameters can be selected to induce preferential activation of either neural or non-neural components in the tissue that directly receives the energy to induce the desired composite physiological effect. In certain embodiments, the energy is applied over a range of about 25 mm 3 In certain embodiments, the energy may be focused or concentrated within a volume of less than about 0.5 mm. 3 ~50mm 3 The focal volume and focal depth for focusing or concentrating energy within the target region 44 may be affected by the size / configuration of the energy application device 12. The focal volume of the energy application may be defined by the focal region of the energy application device 12.

[0042] As provided herein, energy can be applied substantially only to the region of interest 44, and not substantially in a general or unspecified manner throughout the tissue 43, to preferentially activate synapses in a targeted manner to achieve a targeted physiological perturbation. Thus, only a subset of multiple different types of axon terminals within the tissue 43 are directly exposed to the energy application. The disclosed techniques can be used to assess the condition of a subject as a result of perturbations caused by neuromodulation. The disclosed techniques can use direct assessment of tissue condition or function as a targeted physiological perturbation. These techniques can also be used to detect deviations from or to homeostatic states or set points of physiological neuroimmune, neurohormonal, and / or neuroreflex systems. Assessments can be performed before (i.e., baseline assessment), during, and / or after neuromodulation. The evaluation technique may include at least one of functional magnetic resonance imaging, diffusion tensor magnetic resonance imaging, positive emission tomography, acoustic monitoring, thermal monitoring, or chemical sensing (e.g., immunochemical sensing). The evaluation technique may also include protein and / or molecular concentration evaluation. Images from the evaluation technique may be received by the system for automatic or manual evaluation. Based on the image data, the regulation parameters may also be modified. 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 regulating neurotransmitters and as markers of predicted effects on glucose metabolic pathways. Local concentration may refer to the concentration within the focal region of energy application.

[0043] Additionally or alternatively, the system can assess the presence or concentration of one or more molecules circulating in tissue or blood. Concentration in tissue can be referred to as local or resident concentration. Tissue can be obtained by fine needle aspirate, and assessment of the presence or level of target molecule (e.g., metabolic molecule, metabolic pathway marker, peptide mediator, catecholamine) can be performed by any suitable technique known to those skilled in the art.

[0044] In other embodiments, the targeted physiological perturbation 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 concentrations), changes in gene or 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 molecule concentration. In one example, a 5% liver displacement may indicate or be associated with a desired reduction in circulating glucose concentration based on empirical 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 caused the desired targeted physiological perturbation.

[0045] In one example, the technology can be used to diagnose subjects with metabolic disorders.The technology can also be used to diagnose and / or treat subjects with glucose regulation disorders.Thus, the technology can be used to promote homeostasis of a target molecule or promote a desired circulating concentration or concentration range of one or more target molecules (e.g., glucose, insulin, glucagon, or a combination thereof).In one embodiment, the technology is used to evaluate clinical conditions related to circulating (i.e., blood) glucose levels.

[0046] In one embodiment, a glucose concentration threshold can be used to distinguish between blood glucose levels outside or within the normal range as part of a diagnosis of metabolic dysfunction. Fasting: Less than 50 mg / dL (2.8 mmol / L): Insulin shock 50~70mg / dL (2.8~3.9mmol / L): Hypoglycemia / hypoglycemia 70~110mg / dL (3.9~6.1mmol / L): Normal 110-125mg / dL (6.1-6.9mmol / L): Elevated / Disordered (Prediabetes) 125(7mmol / L): Diabetes Non-fasting (approximately 2 hours after a meal): 70-140mg / dL:Normal 140-199mg / dL (8-11mmol / L): Elevated or "borderline" / diabetes More than 200mg / dL: (11mmol / L): Diabetes

[0047] However, as provided herein, the present technology allows for the assessment of perturbations as a result of neuromodulation even in the absence of fasting or non-fasting glucose information. In this way, patient non-compliance or misinformation can be eliminated as a confounding factor for the assessment of metabolic dysfunction. That is, a subject with an unknown fasting or non-fasting state can be perturbed to an approximate or pseudo-fasting state via neuromodulation even if the subject has not fasted, e.g., eaten recently. As provided herein, an approximate fasting state can refer to the state of a patient after neuromodulation energy has been applied (e.g., within 1-6 hours, within 30 minutes after) to a metabolically relevant region of interest, e.g., liver, pancreas, GI. The presence or absence of metabolic dysfunction can be identified based on the subject's profile or response to neuromodulation.

[0048] For example, the normal glucose ranges above can be used as recovery targets to track the time to recovery after changes in glucose levels following perturbations caused by neuromodulation. In another example, subjects with elevated fasting glucose levels who do not experience changes in glucose levels following neuromodulation are diagnosed with insufficient insulin production (e.g., pancreatic disorders). In contrast, if the same subject significantly lowers glucose levels (more than expected amounts) following neuromodulation, it may indicate a problem in the brain control center (e.g., glucose set point). Thus, in one embodiment, the response to the perturbation allows subjects to be classified based on their particular response profile (e.g., change in glucose vs. lack of change in glucose concentration). In one embodiment, a perturbation is induced and the concentration of one or more molecules of interest is evaluated. Based on the concentration being above or below a threshold, subjects are classified into a first category or a second category. Furthermore, their response profile can be classified relative to their baseline state. Subjects with normal glucose levels who do not experience a change in glucose after a neuromodulation-induced perturbation are classified differently (e.g., placed in the first category) than subjects with high glucose levels who do not experience a change in glucose after a neuromodulation-induced perturbation (e.g., placed in the second category). Thus, the level of change relative to the baseline is used to classify the subjects. Changes relative to the baseline less than a predefined threshold are associated with the first category, and changes greater than a predefined threshold are associated with the second category. It should be understood that the disclosed categories may include additional subcategories for subjects.

[0049] In another example, the present technology induces perturbation in both fasting and non-fasting subjects.Therefore, subjects may prefer to undergo diagnostic neuromodulation without changing dietary content.Furthermore, in certain embodiments, subjects do not need to receive intravenous administration of drugs (e.g., glucose, insulin).

[0050] In another example, as provided herein, neuromodulation of lymphatic tissues can result in perturbation of immune activity or function. In certain embodiments, neuromodulation of lymph nodes results in local enlargement of lymph nodes compared to contralateral lymph nodes. Hypertrophy or abnormal development can be associated with changes in muscle cell tone around lymphatic vessels, long-term recruitment and reorganization of lymphatic vessels around lymph nodes, and / or molecular changes in key barrier tissues (such as high endothelial venules (HEV) in lymph nodes), including changes in key transport proteins (e.g., aquaporins (water / liquid transport) or CCL 21 / CXCL 13 secretion (cellular chemokines)). Perturbations can result in dramatic changes in the entire lymph node tissue environment, including cell density, cell number, and expansion. Furthermore, lymph node activation can result in an activation chain that expands or amplifies the local activation to activate the lymphatic system systemically. That is, local stimulation can result in downstream and upstream activation of the lymphatic system. Thus, local perturbations can be used to activate a systemic immune response. Further perturbations may include changes in lymphatic flow, immune cell trafficking to / from lymphatic tissues, changes in immune cell phenotype or local immune responses, and / or antigen trafficking to / from lymphatic tissues. Local stimulation may allow tissue- or location-specific increases in lymphatic or immune cell recruitment. Thus, in one embodiment, assessment of perturbations to lymphatic tissue neuroregulation and immune activity (or lack thereof) may be used to diagnose immune dysfunction or disorders.

[0051] 4 is a flow diagram of a method 50 for causing a perturbation via neuromodulation that may be used as part of a diagnostic protocol. In the method 50, a region of interest is spatially selected 52. An energy application device is positioned to focus energy pulses at step 54 on the desired region of interest, and a pulse generator applies a plurality of energy pulses at step 56 to the region of interest in the target tissue to preferentially activate a subset of synapses in the target tissue, e.g., stimulating axon terminals to release neurotransmitters and / or induce changes in neurotransmitter release and / or induce changes in activity of non-neuronal cells (intrasynapses) to cause a targeted physiological perturbation as provided herein at step 58. In certain embodiments, the method may include a step of evaluating the effect of the perturbation. For example, one or more direct or indirect assessments of tissue function or status may be used.

[0052] In one embodiment, the evaluation is performed before and after applying the energy pulse to evaluate the change in glucose concentration as a result of the modulation. Furthermore, the feature or condition evaluated may be a value or index, such as flow rate, concentration, cell population, or any combination thereof, which may be analyzed by appropriate techniques. For example, a relative change above a threshold is used to diagnose the subject. The diagnosis is evaluated via a measured perturbation, 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., relative to baseline concentration before neuromodulation). In one embodiment, the perturbation includes an increase in concentration above a threshold, e.g., greater than about 50%, 100%, 200%, 400%, 1000% compared to baseline. The evaluation includes tracking a decrease in the concentration of the molecule over time, e.g., at least a 10%, 20%, 30%, 50%, or 75% decrease in the molecule of interest. Furthermore, for a particular subject, the diagnosis may relate to a relatively stable concentration of a particular molecule in the context of other clinical events that may tend to increase the concentration of the molecule. That is, normal subjects may respond to glucose administration, either concurrently or following neuromodulation, in a manner that is distinguishable from insulin resistant subjects. An increase or decrease or other induced and measurable effect is measured within a certain time frame, for example within about 5 minutes, about 30 minutes, from the start of treatment.

[0053] FIG. 5 is a flow diagram of a method 60 for evaluating perturbation of one or more molecules of interest relative to a baseline to diagnose a subject or part of a diagnostic protocol. For example, the disclosed techniques can be used to classify subjects into first, second, third, etc. categories based on the level of perturbation response and / or recovery back to baseline. A baseline concentration (block 62) can be determined based on the molecule concentration at or before the perturbation and can be a single baseline concentration or an average over several time points before the perturbation. The diagnostic protocol can also include a step of causing a physiological perturbation (e.g., as in method 50 of FIG. 4) via neuromodulation of a region of interest in a target tissue of the subject (block 64). The target tissue can be selected based on the diagnostic protocol. For example, a diagnosis of a glucose metabolism disorder can involve neuromodulation of the liver and / or pancreas, while a diagnosis of an immune dysfunction can involve neuromodulation of the spleen and / or lymphatic tissue. The region of interest is selected based on the target tissue disclosed with respect to FIG. 4. Deviations in the concentration of one or more molecules of interest relative to the baseline are determined at one or more time points (block 66). Based on the evaluation at one or more time points after application of energy to the region of interest, the subject's clinical condition can be determined (block 68). The time of change can be determined based on empirical evidence. For example, a particular subject can see a change in the concentration of the molecule of interest by 1 minute, 5 minutes, 10 minutes, or 60 minutes after neuromodulation. Furthermore, such changes can dissipate toward recovery after a period of time (e.g., 3 hours, 12 hours, 24 hours). Thus, the evaluation of the change is performed during a period of time during which physiological perturbations are expected to be observed, at least in a particular subject population or for a particular diagnosis. In addition, it is understood that concentrations at additional time points within the recovery period can be evaluated. Furthermore, the concentration of the molecule of interest over a period of time can be evaluated for a predicted increase or decrease (or lack of change) associated with one or more clinical conditions.

[0054] The method 60 may be used to diagnose glucose metabolism disorders. In one embodiment, the technology may cause physiological perturbations that result in an overall decrease in circulating glucose after neural modulation of the liver (e.g., at or near the hilum of the liver) or changes in the concentration or rate of change of glucose relative to circulating hormone concentrations (such as insulin or glucagon). Such changes may be related to the clinical condition of the subject. For example, in a diabetic or prediabetic subject, the baseline circulating glucose level may be at a level above 140 mg / dL. After perturbations caused by hepatic neural modulation, the perturbed glucose level may be reduced to a level consistent with a nondiabetic subject, e.g., below 140 mg / dL. Thus, high baseline circulating glucose and perturbed deviations from baseline caused by hepatic neural modulation may be diagnostic of a diabetic or prediabetic subject. In contrast, normal baseline circulating glucose and no perturbation or small perturbations of circulating glucose caused by hepatic neural modulation may be diagnostic of a healthy or nondiabetic subject. Furthermore, high baseline circulating glucose and no or only slightly perturbed deviations from the baseline caused by hepatic neuromodulation may be diagnostic of organ dysfunction, with or without the presence of diabetes. Thus, such subjects are considered non-responsive to neuromodulation and alternative therapies are recommended.

[0055] Changes in circulating glucose can be evaluated in the context of additional molecules of interest. For example, the relationship between glucose and insulin and the resulting perturbations can indicate the clinical status of a subject. In one embodiment, physiological perturbations via targeted liver ultrasound cause observable changes in circulating glucose, insulin, cortisol and triglycerides compared to baseline in diabetic or prediabetic subjects. Perturbations that cause a decrease in glucose without a concomitant change in insulin can indicate a first type of glucose metabolic disorder, and perturbations that cause a decrease in glucose with a concomitant change in insulin can indicate a second type of glucose metabolic disorder. That is, the magnitude of the delta, which can be the glucose and / or insulin concentration change (or lack thereof) observed over time, can be a marker of a glucose metabolic disorder. In one example, insulin resistance is characterized by subjects with elevated circulating insulin. In response to neural modulation of the pancreas by the present technology, there is an initial increase in circulating insulin because the mechanical wave likely causes the release of pancreatic insulin stores. However, following the release of existing pancreatic insulin stores that causes the initial increase, there is a subsequent overall decrease in circulating insulin compared to both the levels seen in the initial increase and the initial or baseline circulating insulin before pancreatic neural modulation. Observing the response to pancreatic neural modulation in subjects with elevated circulating insulin and / or elevated circulating glucose above a threshold, either simultaneously or separately from hepatic neural modulation, and tracking the initial increase, followed by a decrease in circulating insulin, can be used to classify subjects as insulin resistant without the need to fast the subject. Furthermore, the lack of such a characteristic response can be used to identify subjects who exhibit elevated circulating insulin and / or elevated circulating glucose above a threshold, but who are not insulin resistant, and may exhibit such concentrations as a result of other dysfunctional metabolic pathways. By observing one or more types of perturbations caused by neural modulation, subjects can be organized or grouped into different categories based on a particular profile of changes.Follow-up diagnostic protocols can track deviations from previous classifications and can then be used to follow the prognosis of the subject.

[0056] Lymphatic tissue neuromodulation can be used to perturb the population of immune cells produced by lymphatic structures. In one embodiment, neuromodulation of lymph nodes can result in an increase in the population of lymphocytes circulating in the lymph. Thus, the local concentration profile of type 1 (proinflammatory) cytokines (e.g., IL-12, TNF-α, IFN-γ, IL-2, TNF-β) and type 2 (anti-inflammatory) cytokines (e.g., IL-4, IL-10, IL-13, IL-6) can be evaluated. In one embodiment, neuromodulation of lymph nodes can result in an increase or decrease in B cells or T cells circulating on the lymph, or an increase or decrease in B cells or T cells or dendritic cells recruited to lymphatic tissue. Thus, neuromodulation of lymphatic tissue can provide an opportunity for cell migration patterns. Such migration patterns can be observed using in vivo bioluminescence imaging. Other features can include changes in lymphatic drainage patterns. In certain embodiments, these changes can be perturbation features used to diagnose the subject. Healthy subjects, immunocompromised or immunodeficient subjects, and subjects with hyperactive immune responses may experience different perturbations as a result of neuromodulation of lymphatic tissue. The characteristics of these perturbations can be evaluated to create a profile. Subjects with unknown clinical conditions can be diagnosed according to the closest match to the characteristic profile. For example, immunodeficient subjects may have a characteristic lack of response or low level of perturbation in response to targeted splenic neuromodulation. In certain embodiments, occipital lymph nodes, auricular lymph nodes, cervical lymph nodes, axillary lymph nodes, inguinal lymph nodes, pulmonary lymph nodes, mediastinal lymph nodes, intraperitoneal lymph nodes, or superior trochlear lymph nodes can be targeted.

[0057] FIG. 6 is a method 69 for identifying individual portions of a disordered metabolic pathway based on perturbation patterns. Energy can be applied to individual target regions (e.g., first target region, second target region) in different organs (e.g., first organ, second organ) to induce neuromodulation and the resulting perturbation (Block 70). Energy application can be sequential (e.g., at different times) to allow for evaluation of perturbation characteristics in each organ (Block 71). Perturbation characteristics include measurable physiological changes that are evaluated. Based on the evaluation, a clinical condition can be diagnosed (Block 72). For example, energy can be applied to the liver and pancreas at different times to identify a time point of dysfunction in glucose metabolism. Subjects with diseased pancreas may respond differently than subjects who are insulin resistant. That is, subjects with insulin resistance may respond to increased levels of circulating insulin as a result of targeted pancreatic stimulation. However, subjects with diseased pancreas may be unable to produce insulin and therefore may not exhibit the characteristic of increased circulating insulin as a result of pancreatic neuromodulation. Insulin-resistant patients may also respond with a decrease in blood glucose that is not consistent with an increase in insulin concentration following stimulation of peripheral sensory sites (such as the liver or GI tract) because these sites act on glucose sensors and other CNS-based sensory sites that are deficient in regulation. Other techniques may not distinguish between these scenarios.

[0058] 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. 7 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, adhering, or positioning the ultrasound transducer probe on an anatomical target. Furthermore, in addition to a handheld configuration, the energy application device 12 may include a steering mechanism responsive to commands from the controller 16. The steering mechanism may orient or direct the energy application device 12 toward the target tissue 43 (or structure), and the controller 16 may then focus the energy application to the target region 44. The ultrasound transducer 74 may include an imaging transducer 74A used for spatial selection of the target region 44 within the target tissue 43. The ultrasound transducers 74 may include a treatment transducer 74B that applies neuromodulation energy.

[0059] In some embodiments, ultrasound images can be used to guide ultrasound stimulation to spatially select a region of interest for targeted delivery of ultrasound stimulation. As provided herein, spatial selection or spatially selecting can 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., ultrasound image). In some embodiments, the tissue or organ can have anatomical features that are used to guide the selection of a region of interest within the organ. Such features can include, in some embodiments, 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 can include the hilum of the liver, lower organs of the digestive 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 can be selected that overlaps with, includes, or is adjacent to the anatomical feature. In other embodiments, the anatomical feature can be excluded from the region of interest. For example, intestinal tissue can be selected as a region of interest rather than stomach tissue. Identification of anatomical features can be 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 is configured to operate in an imaging mode to acquire an image, and then operate in an energy application mode after the image is acquired and a region of interest is spatially selected based on the image.

[0060] 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 evaluated by staining an organ or tissue, obtaining an image showing the staining, and identifying the region of the organ or tissue that contains the biological marker. In some embodiments, the biological marker information may be obtained by in vivo staining techniques to obtain position data of the biological marker in a tissue or organ specific to the subject in real time. In other embodiments, the biological marker information may be obtained by in vitro staining techniques to obtain position data of one or more representative images, which are then used to predict the location of the biological marker in the tissue or organ of the subject. In some embodiments, the region of interest is selected to correspond to a portion of a tissue or organ that is rich in a particular biological marker or lacks a particular biological marker. For example, the one or more biological markers may include a marker of a neural structure (e.g., a myelin sheath marker).

[0061] The region of interest of an organ or tissue can be spatially selected based on operator input. For example, an operator can specify the 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, spatial selection includes storing data associated with the region of interest in memory and accessing the data.

[0062] Once spatially selected, system 10 is configured to apply energy to the region of interest as provided herein.

[0063] Working Example Targeted physiological perturbations Before starting neuromodulation, a GE Vivid E 9 ultrasound system and an 11L probe were used for ultrasound scanning. A focal region corresponding to the internal region of interest was marked on the animal's skin. The HIFU transducer was placed over the marked region. Another ultrasound scan was also performed using a smaller 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 targeted organ (visualized with the ultrasound scanner) could be used to confirm that the HIFU beam was targeted to the region of interest.

[0064] Animal protocols Adult male obese Zucker rats aged 8–12 weeks (250–300 g, Charles River Laboratories) were fed a high-fat diet according to conditions maintained by the supplier to promote the development of insulin resistance and hyperglycemia. The rats were fed a high-fat diet. Neuromodulation using applied ultrasound energy was performed on the liver. Ultrasound application was performed for 1 min. Blood samples were taken 15 min after the last ultrasound treatment to analyze changes in circulating catecholamine concentrations (e.g., norepinephrine and dopamine). Terminal blood samples were collected 60–90 min after the last ultrasound treatment to analyze changes in circulating molecule concentrations. Blood samples were stored with anticoagulant (disodium) EDTA to prevent clotting of the samples. The protocol used for ultrasound neuromodulation may be as follows.

[0065] (A) Animals may be anesthetized with 2-4% isoflurane. (B) To prevent hyperthermia during the procedure, animals may be placed on a water-circulating heating pad. (C) The area above the targeted region of interest for ultrasound stimulation (e.g., target nerve) can be shaved with a disposable razor and animal clippers prior to stimulation. (D) Diagnostic ultrasound can be used to spatially select regions of interest. (E) The region may be marked with a permanent marker for later identification. (F) Either the FUS ultrasound probe or the LogiQ E 9 probe can be placed into the designated area of ​​interest previously identified by diagnostic ultrasound. (G) Ultrasound pulses can then be administered with a total duration of a single stimulus not exceeding a single 1 minute pulse. The energy of the ultrasound pulses can be adjusted to produce thermal damage and ablation or cavitation (e.g., 35 W / cm). 2 ) does not reach the level associated with (H) A second 1 min ultrasound pulse can be applied. (I) The animals can then be incubated under anesthesia for acute studies (e.g., 1 hour) and kinetic studies, after which the animals are sacrificed and tissue and blood samples are taken.

[0066] The incision may begin at the base of the peritoneal cavity and extend to the pleural cavity. The organs may be rapidly removed and homogenized in a PBS solution containing phosphatase (0.2 mM phenylmethylsulfonyl fluoride, 5 μg / mL aprotinin, 1 mM benzamidine, 1 mM sodium orthovanadate and 2 μM cantharidin) and protease (1 μL to 20 mg of tissue by Roche Diagnostics) inhibitors. A targeted final concentration of 0.2 g of 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. The samples are then stored at -80 °C until analysis.

[0067] Targeted tissue stimulation and physiological perturbation for the diagnosis of glucose metabolic disorders This example demonstrates a non-invasive method for achieving physiological perturbations to assess metabolic dysfunction in a patient. The disclosed technique offers advantages compared to techniques for assessing metabolic dysfunction that involve several hours of patient time, including fasting, infusion, and subsequent response / blood sampling steps. For example, in assessing a patient's insulin resistance by certain procedures, the patient may undergo a fasting period, which allows the patient's physiological system to achieve a set point relative to the population. After fasting, the patient may be administered glucose or a metabolically active compound (e.g., orally or by injection). The patient's response is then measured for a period of time, often over several hours, by monitoring changes in glucose and / or insulin or other hormones that support changes in glucose concentration. If either fasting blood glucose or insulin is determined to be above range (as determined by measuring a previous subject population), the subject may be diagnosed with diabetes. Glucose tolerance tests measure how quickly a bolus of glucose is cleared from the blood in a fasting state (compared to a population of subjects) and are used to diagnose diabetes in subjects with higher (not pathologically high) fasting glucose concentrations. Clamp techniques are used to quantify how a subject metabolizes glucose. Clamps can include hyperglycemic clamps (continuous infusion of glucose, quantifying insulin secretory capacity) or hyperinsulinemic clamps (continuous infusion of insulin, quantifying insulin resistance), which pose problems regarding the cost of evaluation and excessive complexity that would affect their use in the clinic.

[0068] The Homeostatic Model Assessment (HOMA) calculator uses a mathematical model to estimate insulin sensitivity and B-cell function from plasma insulin and glucose concentrations. This interaction between glucose and insulin in the basal state provides information about the balance between hepatic glucose output and insulin secretion, which is maintained by a feedback loop maintained by B cells in the liver and pancreas, allowing HOMA to serve as a surrogate measure of steady-state beta cell function (%B) and insulin sensitivity (%S) as a percentage of a normal reference population. The predictions used in the model arise from experimental data in humans and animals.

[0069] The disclosed techniques may replace or be used in conjunction with other techniques for the diagnosis of glucose metabolism disorders to eliminate or shorten fasting and subsequent evaluation times. In one embodiment, the modified HOMA calculator can more closely approximate the fasting state of diabetic subjects based on the disclosed neuromodulation-induced perturbations. That is, rather than requiring subjects to fast for a period of time, subjects (e.g., diabetic, normal, or unknown) may be perturbed by neuromodulation to lower circulating glucose to a state resembling a fasting state for the purpose of assessing glucose response. Furthermore, neuromodulation induces stabilization of glucose levels independent of feeding state, as indicated by the lack of fluctuation in glucose levels. Taken together, this neuromodulation-induced glucose stabilization reduces potential confounding results derived from feeding state assessments and allows for the assessment of insulin sensitivity in the absence of feeding-derived influences.

[0070] As provided herein, ultrasound stimulation was performed according to the timeline shown in FIG. 9 to demonstrate induced targeted physiological perturbations versus controls. FIG. 10 shows the results of ultrasound stimulation of the liver (portion of the liver) of obese Zucker rats compared to control rats that underwent sham ultrasound treatment. Ultrasound-treated rats were protected from increases in circulating glucose in control rats, even though both groups of rats were kept in conditions associated with the development of insulin resistance and hyperglycemia. Thus, the results demonstrate that ultrasound treatment causes perturbations in the hyperglycemic rat population that are detectable via changes in circulating glucose. FIG. 11 and FIG. 12 show that food consumption and weight were similar between the treatment and control groups. While food intake remained constant (FIG. 11), weight gain in treated animals slowed versus the control group (FIG. 12) over the experimental timeline.

[0071] As provided herein, hepatic neuromodulation induces changes in glucose metabolism to provide controlled / precise stimulation, as shown in FIG. 13, and modifies the relationship between organs to affect or change the current clinical score of homeostasis (i.e., HOMA score). The disclosed technology can be used to generate an index reflecting the neuromodulation and the resulting perturbation. Typically, the HOMA score indicates the relationship between fasting glucose levels and fasting insulin measurements. Generally, a high HOMA score reflects a high level of insulin required for glucose disposal. As provided herein, changes in insulin and glucose concentrations can be evaluated in response to hepatic neuromodulation as part of a diagnostic protocol to derive a homeostasis index that can be used to diagnose and track insulin resistance, even in subjects who have glucose levels in the normal range or are not on a controlled meal plan.

[0072] The steady-state HOMA model is based on circulating glucose and insulin levels with a population-based formula. It is assumed that the subject is fasting. Furthermore, typical assessments are based on "stimulated" responses such as glucose clamps, insulin clamps, or glucose tolerance tests. In certain embodiments, neuromodulation replaces typical fasting and stimulation (e.g., glucose clamps, insulin clamps, glucose tolerance). In one embodiment, the technology can be used to replace glucose and / or insulin infusion with neuromodulation so that testing can be performed without administration of glucose and / or insulin. However, it should be understood that neuromodulation followed by administration of glucose and / or insulin is also contemplated. In another embodiment, the technology can be used to eliminate fasting from the measurement protocol (by using neuromodulation) to provide an immediate deviation from baseline organ dynamics / interactions.

[0073] As provided herein, the technology can be used to build a population-based model of the relationship between changes in glucose and insulin concentration responses to neuromodulation. The model can be based on the glucose-insulin relationship of a population of healthy individuals perturbed to an approximated fasting state via neuromodulation. In certain embodiments, the model can be further based on the glucose-insulin relationship for a population of individuals with different types of metabolic function perturbed to an approximated fasting state via neuromodulation. The model can be applied to the glucose and insulin concentration data obtained from the neuromodulated subjects perturbed to an approximated fasting state to generate a score, the score indicating the level of insulin resistance in the subject.

[0074] In one embodiment, FIG. 14 is a flow diagram of a method 90 illustrating a metabolic dysfunction assessment technique. The technique obtains a baseline concentration of glucose and / or insulin at step 92, which may be immediately prior (within 1-6 hours) or simultaneous with the initiation of neuromodulation of an area of ​​interest within a target tissue (e.g., liver, GI, pancreas) at step 94. As described herein, a continuous glucose monitor may be used to monitor the subject so that glucose concentrations are continuously available. Based on the observed perturbations, which may be reflected in the observation of changes in glucose and / or insulin concentrations over time as a result of neuromodulation at step 96, the patient's clinical status may be determined at step 98.

[0075] The present technology may be used to organize subject populations based on the level of glucose and / or insulin change following stimulation, or to diagnose diabetes type and / or level of resistance. For example, subjects may be classified as "responders" or "strong responders" or "poor responders" or "non-responders" depending on the level of glucose and / or insulin change. For example, a non-responder may exhibit a concentration change below a predetermined threshold relative to the baseline concentration, while a responder experiences a concentration change above a predetermined threshold. In one example, a non-responder would exhibit less than about 5%, less than about 10%, or less than about 15% glucose change relative to baseline. A responder would exhibit more than about 10-15% glucose change relative to baseline, while a strong responder would exhibit more than about 50% glucose change relative to baseline. Responsiveness may be a time point associated with expected changes in a healthy or responsive population.

[0076] FIG. 15 is an example of a neuromodulation response profile for a highly insulin resistant animal subject fed a diet that drives insulin resistance and high glucose levels. The profile shows glucose concentrations 100 over several days prior to the initiation of neuromodulation, with relatively stable high glucose concentrations above a threshold shown as a glucose concentration of 225 mg / dL. Neuromodulation is initiated at baseline 102, and the glucose response to neuromodulation is tracked over time. The illustrated subject is characterized by a rapid glucose decrease of long duration and magnitude without fasting or glucose administration. As provided herein, the response profile may be evaluated or characterized by one or more measures of response. In one embodiment, the magnitude of the change from peak baseline concentration 102 to trough 106 may be evaluated. Alternatively or additionally, the slope 104 may be evaluated (e.g., a steep slope may indicate insulin resistance). Additional metrics may include the slope from the trough 106 to the recovery peak 110, the difference between the baseline concentration and the first recovery peak 110, the time to the first recovery peak 101, and / or the area under the curve 112 relative to the set point. For example, an insulin resistant subject may have a relatively short period of lower glucose concentrations below a predetermined threshold (e.g., less than 225 mg / dL, less than 200 mg / dL) before recovering to concentrations above the predetermined threshold, which is reflected in the area under the curve 112 below the empirically determined threshold. The subject may be monitored during normal activities such as eating and sleeping, and the feature profile may reflect changes in response to these activities relative to the baseline. While previous techniques may have involved complex glucose dose response tracking over several hours in addition to fasting prior to the test, the present technique allows for the observation of changes over a relatively short period of time, which is less burdensome to the tested subject. Thus, in one embodiment, the subject is assessed using glucose concentration changes observed within 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour following neuromodulation to achieve an approximate fasting state in the subject.Furthermore, because glucose monitoring can be accomplished using a glucose monitor that allows the subject to be in an ambulatory and non-clinical setting, the present technology provides a more convenient and rapid assessment of metabolic dysfunction that does not require fasting or administration of a glucose bolus.

[0077] FIG. 16 is an example of a neuromodulation response profile of an insulin resistant subject with a starting glucose concentration lower than that of the subject of FIG. 15. The profile shows a pre-treatment glucose concentration 124 above a threshold (e.g., about 250 mg / dL) and a higher baseline concentration 122 or area under the curve 112 for the set point that declines to a trough 126 in response to neuromodulation and a recovery peak 128. For example, an insulin resistant subject may have a relatively short period of lower glucose concentration below a predetermined threshold (e.g., less than 225 mg / dL, less than 200 mg / dL) before recovering to a concentration above the predetermined threshold. The response profile shows a smaller magnitude and duration, but nevertheless a sharp decrease in glucose concentration. Thus, in certain embodiments, the response profiles in FIG. 15 and FIG. 16 can both be classified as being insulin resistant, but in different bands or categories. FIG. 17 is a response profile of a subject with a starting glucose concentration 130 below diabetic levels and little or no response to neuromodulation. Thus, subjects may be considered non-responders and may be classified differently relative to the profiles of FIGS.

[0078] In another embodiment, the disclosed technology can be used to detect differences in subject populations for various perturbations and their associated clinical conditions, and / or optimal treatment plans. Differences in subject populations can result in categorizing subjects into one of several categories and initiating treatment options based on categorization. For example, drug response can be correlated with responsiveness to neuromodulation (e.g., degree of perturbation). Furthermore, the disclosed technology can provide categorical information (insulin resistant, non-insulin resistant, responder, non-responder) as well as additional data as part of a report to caregivers.

[0079] Targeted tissue stimulation and physiological perturbation for identification of pathogen or toxin exposure As provided herein, the neuromodulation response may indicate pathogen, toxin, or environmental exposure prior to the time when the pathogen itself is detectable or disease symptoms appear (i.e., fever, etc.). That is, the present technology provides improved and more rapid assessment of pathogen exposure. Such assessment may improve outcomes for hospital-associated pathogens that may be more effectively contained if detected more quickly. While certain techniques for tracking immune or systemic responses require continuous monitoring to detect exposure to pathogens, the present technology eliminates the need for baseline monitoring by comparing pre- and post-neuromodulation changes in the expression of certain markers.

[0080] FIG. 18 is a flow diagram of a method 150 illustrating an immune response assessment technique for identifying pathogen exposure by assessing the response of an area to neuromodulation when immune tissues or structures of interest include lymphatic organs, lymphatic vessels that extend throughout the body and provide flow and drainage, the spleen, lymph nodes, Peyer's patches, and accessory lymphatic tissues (including tonsils and appendix).

[0081] The technique involves obtaining a baseline concentration of one or more markers, such as immune markers, at step 152, which may be immediately prior (within 1-6 hours) or simultaneous with the initiation of neuromodulation of a region of interest in a target tissue (e.g., spleen or immune tissue) at step 154. Based on the observed perturbation, which may be reflected in an observation of a change in the one or more markers relative to the baseline at step 156, pathogen exposure may be identified at step 158. Changes in the concentration of the one or more markers in response to neuromodulation associated with pathogen exposure or lack of pathogen exposure may be identified according to the techniques provided herein (blood concentrations, tissue concentrations, system changes identified by sensor and / or image data).

[0082] FIG. 19 shows ultrasound dose-dependent tumor necrosis factor mRNA expression changes for animal subjects exposed to endotoxin or lipopolysaccharide (LPS) and treated with neuromodulation energy applied to the spleen. High dose treated animals experienced the greatest decrease in tumor necrosis factor mRNA expression compared to controls. FIG. 20 shows mRNA expression array data for several markers for control and LPS exposed animals and high and low dose ultrasound treated animals. FIG. 21 illustrates an exemplary protocol for obtaining baseline and potential post-exposure data to assess changes in neuromodulation-based responses following exposure to a substance or pathogen. A baseline or characteristic pre-exposure response profile may be obtained and compared to a potential post-exposure response profile to assess changes caused by the exposure.

[0083] The disclosed techniques provided herein take advantage of targeted physiological outcomes achievable through neuromodulation. In addition to implementations in which targeted physiological outcomes are used to treat a subject, these outcomes can be used as part of a diagnostic protocol. For example, application of energy to a region of interest within a target tissue can result in a predictable and targeted physiological perturbation. One or more characteristics of the perturbation itself or the subject's response to the perturbation can indicate the subject's clinical condition. For example, a rapid return to homeostasis (e.g., return to baseline pre-regulatory concentrations of the molecule of interest) within a given period of time can indicate a healthy response to the perturbation. In contrast, a slow response can indicate a metabolic pathway that is underperforming compared to that of a healthy subject. Although certain embodiments of the present disclosure have been discussed in the context of glucose regulation, it should be understood that the present techniques can be used to cause perturbations in other systems (including acute or chronic inflammatory conditions and associated sensory and neuro-immune effector systems) and thus assess associated clinical conditions.

[0084] This specification uses examples to disclose the invention, including the best mode, and to enable those skilled in the art to practice the invention, including making and using any devices or systems and performing any 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 if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements that do not differ substantially from the literal language of the claims.

Claims

1. 1. A regulation system comprising: an energy application device configured to apply energy to a region of interest to cause a physiological perturbation in a subject; A controller, controlling the application of energy to the region of interest via the energy application device to induce the physiological perturbation to cause a change in concentration of one or more molecules of interest in the subject relative to a baseline concentration; receiving information indicative of the concentration of the one or more molecules of interest at one or more time points after the application of the energy; determining that the subject is in a category selected from the first category or the second category based on a change in the concentration of the one or more molecules of interest relative to the baseline concentration within a period from a first time point to a second time point. It is configured as follows: the first category being a response of neuromodulation of the target region based on a change in concentration greater than a predetermined threshold, and the second category being a non-response of neuromodulation of the target region based on a change in concentration less than a predetermined threshold; and Equipped with The target area includes the pancreas, and determining that the subject is in a category selected from the first category or the second category includes determining that the subject is insulin resistant based on a change in concentration that includes an initial increase in circulating insulin above a predetermined insulin increase threshold at the first time point, followed by a decrease in circulating insulin at the second time point compared to the initial value.

2. The system of claim 1 , wherein the concentration is a glucose concentration received from an evaluation device that includes a glucose monitor.

3. 2. The system of claim 1, wherein the first category reflects exposure to a pathogen and the second category reflects a lack of exposure to a pathogen, and the region of interest is a spleen or immune tissue.

4. The system of claim 1 , wherein the energy applicator is an ultrasound probe.

5. The system of claim 1 , wherein the system is configured to provide instructions for a neuromodulation treatment protocol upon determining that the subject belongs to the first category.

6. The system of claim 1 , wherein the controller is configured to initiate a neuromodulation treatment protocol via the energy application device upon determining that the subject belongs to the first category.

7. The system of claim 6 , wherein the neuromodulation treatment protocol comprises application of energy to the target area.

8. The system of claim 7 , wherein the one or more molecules of interest include glucose.

9. The system of claim 7 , wherein the region of interest is the spleen and the one or more molecules of interest include cytokines.

10. The system of claim 1 , further comprising an imaging transducer configured to generate image data of the region of interest.

11. The system of claim 10 , wherein the energy application device comprises an ultrasound probe, the ultrasound probe including the imaging transducer.

12. The system of claim 1 , wherein the subject has elevated circulating glucose or elevated circulating insulin at baseline.

13. The method of claim 1 , wherein the subject is not fasting.

14. The system of claim 1 , wherein the target area is tissue that contains axon terminals that form synapses with non-neuronal cells.

15. The system of claim 1 , wherein the region of interest is gastrointestinal tissue.

16. 2. The system of claim 1, wherein the controller is configured to control application of energy to the region of interest via the energy application device to induce physiological perturbations causing changes in gene expression, tissue displacement, tissue size changes, cell migration, or any combination thereof.

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