阅读说明：本技术 神经调节技术 (Neuromodulation techniques ) 是由 克里斯托弗·迈克尔·普莱奥 拉克施米·西雷莎·卡努马勒 维多利亚·尤金妮亚·科特罗 约翰·弗莱 于 2019-08-30 设计创作，主要内容包括：本公开的主题一般地涉及以下技术：进行具有一个或多个治疗参数的治疗规程以在远端位点处引起目标生理学结果,在治疗规程完成后评价所关心的区域中基因的表达水平,和基于基因的表达水平改变一个或多个治疗参数。治疗规程可以包括对所关心的区域的一种或多种超声波能治疗。(The subject matter of the present disclosure relates generally to the following technologies: performing a treatment protocol having one or more treatment parameters to elicit a target physiological result at the distal site, assessing the expression level of the gene in the region of interest after completion of the treatment protocol, and altering the one or more treatment parameters based on the expression level of the gene. The treatment protocol may include one or more ultrasonic energy treatments of the region of interest.)

1. A method, comprising:

performing a treatment protocol having one or more treatment parameters to cause a change in the concentration of one or more molecules of interest at the distal site, the treatment protocol comprising one or more ultrasonic energy treatments of the region of interest;

assessing the level of expression of genes in the region of interest and the concentration of the one or more molecules of interest at the distal site after completion of the treatment protocol; and

altering the one or more therapeutic parameters based on the expression level of the gene, the concentration of the one or more molecules of interest, or both.

2. The method of claim 1, wherein the one or more treatment parameters comprise a frequency of application of the ultrasound energy treatment, a duration of a recovery period between ultrasound energy treatments, or a combination thereof.

3. The method of claim 1, further comprising evaluating a baseline expression level of the gene prior to performing the treatment protocol, and wherein the expression level of the gene is evaluated relative to the baseline expression level of the gene.

4. The method of claim 1, further comprising performing an altered treatment protocol having one or more altered treatment parameters, the altered treatment protocol comprising one or more altered treatments of ultrasound energy to the region of interest.

5. The method of claim 1, wherein performing the treatment protocol causes a change in the concentration of the one or more molecules of interest at the distal site and a change in the expression level of the gene in the region of interest.

6. The method of claim 1, wherein evaluating the expression level of the gene comprises evaluating the expression level of the gene from RNA transcriptome data, mRNA transcriptome data, cluster analysis of the RNA transcriptome data or the mRNA transcriptome data, or a combination thereof.

7. The method of claim 1, wherein the region of interest comprises a portion of a liver.

8. The method of claim 1, wherein the region of interest comprises a portion of the spleen.

9. The method of claim 1, wherein the region of interest comprises a portion of the pancreas.

10. The method of claim 1, wherein the region of interest comprises a portion of a peripheral nerve ganglion.

11. A method, comprising:

performing a treatment protocol having one or more treatment parameters to induce a target physiological outcome at the distal site, the treatment protocol including one or more ultrasound energy treatments of the region of interest;

assessing the expression level of genes in the region of interest after completion of the treatment protocol; and

altering the one or more treatment parameters based on the expression level of the gene.

12. The method of claim 11, wherein the one or more treatment parameters comprise an application frequency of the ultrasound energy treatment, a treatment duration of the ultrasound energy treatment, a recovery period between two or more subgroups of a series of ultrasound energy treatments, or a combination thereof.

13. The method of claim 11, further comprising performing an altered treatment protocol having one or more altered treatment parameters, the altered treatment protocol comprising one or more altered treatments of ultrasound energy to the region of interest.

14. The method of claim 11, wherein the treatment protocol is performed to cause a target physiological outcome at the distal site without causing a change in the expression level of the gene in the region of interest.

15. The method of claim 11, wherein the target physiological result comprises a change in concentration of an enzyme used for synthesis or secretion of a circulating vasodilator molecule, a circulating vasoconstrictor molecule, or both, and wherein the gene comprises a gene associated with the enzyme.

16. The method of claim 15, wherein the target physiological result comprises a change in blood pressure and the circulating vasodilator molecule comprises acetylcholine.

17. A method, comprising:

performing a treatment protocol having one or more treatment parameters to cause a change in the concentration of one or more molecules of interest at the distal site, the treatment protocol comprising one or more ultrasonic energy treatments of the region of interest;

assessing RNA transcription of the gene in the region of interest; and

altering the one or more treatment parameters based on RNA transcription of the gene in the region of interest.

18. The method of claim 17, wherein evaluating RNA transcription of genes in the region of interest comprises sequencing RNA of blood cells extracted from the region of interest.

Background

The subject matter disclosed herein relates to neuromodulation, and more particularly to techniques for modulating a physiological response using energy applied from an energy source.

Neuromodulation has been used to treat a variety of clinical conditions. For example, electrical stimulation at multiple locations along the spinal cord has been used to treat chronic back pain. The implantable device may periodically generate electrical energy that is applied to the tissue to activate certain nerve fibers, which may result in a reduction in pain sensation. For spinal cord stimulation, the stimulation electrodes are typically located in the epidural space, although the pulse generator may be located somewhat remote from the electrodes, for example, in the abdominal or hip region, but need to be connected to the electrodes by wires. In other implementations, deep brain stimulation may be used to stimulate specific regions of the brain to treat dyskinesia, and the stimulation location may be guided by neuroimaging. Such central nervous system stimulation is typically targeted to local nerve or brain cell function and is mediated by electrodes that deliver electrical pulses and are located at or near the target nerve. However, positioning the electrode at or near the target nerve is difficult. For example, these techniques may involve surgically placing electrodes that deliver energy. Furthermore, targeting of specific tissues by neuromodulation is difficult. Electrodes located at or near certain target nerves mediate neuromodulation by triggering action potentials in the nerve fibers, which in turn lead to the release of neurotransmitters at the neurite contacts and synaptic communication with the next nerve. Since current implementations of implanted electrodes stimulate multiple nerves or axons at once, this propagation can result in relatively greater or more diffuse physiological effects than desired. Since neural pathways are complex and interconnected, more targeted modulation effects may be more clinically useful.

Disclosure of Invention

The following outlines certain embodiments that match the scope of the initially claimed subject matter. These embodiments are not intended to limit the scope of the claimed subject matter, but rather these embodiments are intended only to provide a brief summary of possible embodiments. Indeed, the present disclosure may encompass a variety of forms that may be similar to or different from the embodiments described below.

In one embodiment, a method may include performing a therapeutic protocol having one or more therapeutic parameters to cause a change in concentration of one or more molecules of interest at a remote site (digital site). The treatment protocol may include one or more ultrasound energy (ultrasound) treatments of the region of interest. The method may include evaluating the expression level of the gene in the region of interest and the concentration of the one or more molecules of interest at the distal site after completion of the treatment protocol, and altering one or more treatment parameters based on the expression level of the gene, the concentration of the one or more molecules of interest, or both.

In another embodiment, a method may include performing a therapy protocol having one or more therapy parameters to cause a target physiological result at a remote site. The treatment protocol may include one or more ultrasonic energy treatments of the region of interest. The method can include evaluating the expression level of the gene in the region of interest after completion of the treatment protocol and altering one or more treatment parameters based on the expression level of the gene.

In another embodiment, a method may include performing a therapeutic protocol having one or more therapeutic parameters to cause a change in the concentration of one or more molecules of interest at a distal site. The treatment protocol may include one or more ultrasonic energy treatments of the region of interest. The method can include evaluating RNA transcription of the gene in the region of interest and altering one or more treatment parameters based on the RNA transcription of the gene in the region of interest.

Drawings

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

FIG. 1 is a schematic diagram of a neuromodulation system using a pulse generator, according to a disclosed embodiment of the invention;

FIG. 2 is a block diagram of a neuromodulation system according to embodiments of the present disclosure;

FIG. 3 is a schematic view of an apparatus for applying ultrasonic energy in operation, in accordance with a disclosed embodiment of the invention;

FIG. 4 is an example of an energy application device that may be used in conjunction with the neuromodulation system of FIG. 1, according to embodiments of the present disclosure;

FIG. 5 is a schematic illustration of an experimental apparatus for ultrasound energy application to achieve a target physiological result in accordance with a disclosed embodiment of the invention;

FIG. 6 is a flow diagram of a neuromodulation technique for optimizing therapy over time, according to a disclosed embodiment of the invention;

FIG. 7A is an experimental timeline of ultrasonic energy application over a two week period in accordance with a disclosed embodiment of the invention;

FIG. 7B is an experimental timeline of ultrasonic energy application over a one week period in accordance with a disclosed embodiment of the invention;

FIG. 7C is an experimental timeline for a sham-control application over a two week period in accordance with a disclosed embodiment of the invention;

FIG. 7D is an experimental timeline for untreated controls according to a disclosed embodiment of the invention;

FIG. 8 shows transcriptomic data for genes associated with cytokine activity (gene ontology GO:0005125), according to embodiments disclosed herein;

FIG. 9A shows RNA concentrations of Tumor Necrosis Factor (TNF) in untreated spleen, sham-control spleen, and ultrasonically-stimulated spleen, according to disclosed embodiments of the present invention;

FIG. 9B shows the RNA concentration of interleukin 1 α in untreated spleen, sham-control spleen and ultrasonically-stimulated spleen according to disclosed embodiments of the invention;

FIG. 9C shows the RNA concentration of interleukin 6 in untreated spleen, sham-control spleen and ultrasonically-stimulated spleen according to disclosed embodiments of the invention;

FIG. 9D shows RNA concentrations of C-C motif chemokine ligand 4 in untreated spleen, sham-control spleen, and sonicated spleen, in accordance with a disclosed embodiment of the invention;

FIG. 9E shows the RNA concentration of interleukin 1 β in untreated spleen, sham-control spleen and ultrasonically-stimulated spleen according to disclosed embodiments of the invention;

FIG. 9F shows RNA concentrations of C-C motif chemokine ligand 20 in untreated spleen, sham-control spleen, and sonicated spleen, in accordance with an embodiment of the present disclosure;

FIG. 10A shows RNA and protein concentrations of mitogen-activated protein kinase 14(p38) in untreated spleen, sham-control spleen, and ultrasonically-stimulated spleen, according to disclosed embodiments of the present invention;

FIG. 10B shows RNA and protein concentrations of ribosomal protein S6 kinase B1(p70S6K) in untreated spleen, sham-control spleen and sonicated spleen, in accordance with a disclosed embodiment of the present invention;

FIG. 10C shows RNA and protein concentrations of v-Akt murine thymoma virus oncogene homolog (Akt) in untreated spleen, sham-control spleen and ultrasound-stimulated spleen, in accordance with a disclosed embodiment of the present invention;

FIG. 10D shows RNA and protein concentrations of glycogen synthase kinase 3 β (GSK3B) in untreated, sham, and sonicated spleens, according to disclosed embodiments of the present invention;

FIG. 10E shows RNA and protein concentrations of SRC proto-oncogene, non-receptor tyrosine kinase (c-SRC), in untreated spleen, sham-control spleen and ultrasound-stimulated spleen, according to the disclosed embodiments of the present invention;

FIG. 10F shows RNA and protein concentrations of the kappa light chain polypeptide gene enhancer (NF-kappa β) in B cells in untreated spleen, sham-control spleen and sonicated spleen in accordance with the disclosed embodiments;

FIG. 10G shows RNA and protein concentrations of cytokine Signal transduction inhibitory factor 3(SOCS3) in untreated spleen, sham-control spleen and ultrasound-stimulated spleen, in accordance with a disclosed embodiment of the invention;

FIG. 11 shows RNA concentrations of nicotinic cholinergic receptor alpha 4 subunit (CHRNA4) in untreated spleen, sham-control spleen and ultrasound-stimulated spleen, in accordance with a disclosed embodiment of the present invention;

FIG. 12A shows transcriptome data for genes involved in the positive regulation of activated T cell proliferation (gene ontology: GO:0042104), according to embodiments disclosed herein;

FIG. 12B shows transcriptome data for genes involved in the regulation of B cell activation (gene ontology: GO:0050864), according to embodiments disclosed herein;

FIG. 13A shows transcriptome data for genes involved in glucan biosynthesis according to embodiments disclosed herein (gene ontology: GO: 0009250);

FIG. 13B shows transcriptome data for genes encoding proteins thought to be transcription factors, according to embodiments disclosed herein;

FIG. 14 shows transcriptome data of genes involved in the ability to detect LPS according to embodiments disclosed herein;

FIG. 15 shows transcriptome data of genes involved in detecting the ability of LPS and key cytokine response genes, according to embodiments disclosed herein;

FIG. 16A shows RNA concentrations of nicotinic cholinergic receptor alpha 4 subunit (CHRNA4) in untreated spleen, sham-control spleen and ultrasound-stimulated spleen, in accordance with a disclosed embodiment of the present invention;

FIG. 16B shows RNA concentrations of choline O-transacetylase (CHAT) in untreated, sham, and sonicated spleens, in accordance with embodiments disclosed herein;

FIG. 16C shows RNA concentrations of solute carrier family 5 member 7(SLC5A7) in untreated spleen, sham-control spleen and sonicated spleen in accordance with a disclosed embodiment of the invention;

FIG. 16D shows RNA concentrations of acetylcholinesterase (Carterdt blood group) (ACHE) in untreated spleen, sham-control spleen and ultrasound-stimulated spleen according to an embodiment of the present disclosure;

FIG. 16E shows RNA concentrations of Butyrylcholinesterase (BCHE) in untreated spleen, sham-control spleen and sonicated spleen according to the disclosed embodiments of the present invention;

FIG. 16F shows RNA concentrations of solute carrier family 18 member A3(SLC18A3) in untreated spleen, sham-control spleen and sonicated spleen in accordance with a disclosed embodiment of the present invention;

FIG. 17A shows RNA concentrations of aquaporin 1 (Colton) blood type) in untreated spleen, sham-control spleen, and ultrasonically-stimulated spleen, in accordance with a disclosed embodiment of the invention;

FIG. 17B shows RNA concentrations of aquaporin 3 (Gill blood type) in untreated spleen, sham-control spleen, and ultrasonically-stimulated spleen, in accordance with a disclosed embodiment of the invention;

FIG. 18A shows the RNA concentration of transmembrane protein 176A (TMEM176A) in untreated spleen, sham-control spleen and sonicated spleen in accordance with a disclosed embodiment of the present invention;

FIG. 18B shows the RNA concentration of transmembrane protein 176B (TMEM176B) in untreated spleen, sham-control spleen and sonicated spleen in accordance with a disclosed embodiment of the present invention;

FIG. 18C shows transcriptome data for genes associated with TMEM176A and TMEM176B, according to a disclosed embodiment of the invention; and

figure 19 shows the concentration of norepinephrine, acetylcholine, and tumor necrosis factor in the blood after application of ultrasound energy to the spleen, right adrenal gland, sacral ganglia, nodose ganglia, and nucleus solitarius of an animal model of lipopolysaccharide-induced hyperglycemia, according to an embodiment of the present disclosure.

Detailed Description

One or more specific embodiments will be described below. In an effort to provide a concise description of these embodiments, not all features of an actual implementation are described in the specification. It should be appreciated that in the development of any such actual implementation, as in any engineering or design project, numerous implementation-specific decisions must be made to achieve the developers' specific goals, such as compliance with system-related and business-related constraints, which may vary from one implementation to another. Moreover, it should be appreciated that such a development effort might be complex and time consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.

Any examples or illustrations provided herein are not to be considered in any way as limitations, restrictions, or expressive definitions of any term or terms that they use. Rather, these examples or illustrations should be considered in relation to the various embodiments and are illustrative only. Those of ordinary skill in the art will understand that any one or more of the terms used in these examples or illustrations will encompass other embodiments that may or may not be given therewith or elsewhere in the specification, and all such embodiments are intended to be included within the scope of the term. Language designating such non-limiting examples and illustrations includes, but is not limited to: "for example," such as, "" like, "" including, "" in certain embodiments, "" in some embodiments, "and" in one embodiment.

When introducing elements of various embodiments of the present disclosure, the articles "a," "an," and "the" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements. Furthermore, any numerical examples in the following discussion are intended to be non-limiting, and thus other numbers, ranges, and percentages are within the scope of the disclosed embodiments.

Provided herein are neuromodulation techniques for treating a subject by applying energy to tissue or other anatomical regions at a nerve end site. The applied energy may be targeted to a sub-region of the tissue or structure such that the total energy applied is relatively low. Furthermore, by targeting a particular region of interest, a particular neural pathway can be selectively modulated while other non-targeted neural pathways outside the region of interest remain unmodulated. In this way, neuromodulation of one sub-region of the organ may achieve a different target physiological result than neuromodulation of a different sub-region of the organ. In a specific example, targeting the hepatic portal (porta hepatic) region of the liver with neuromodulatory energy may therefore cause a change in circulating glucose concentration. Other regions of the liver may not be targeted to the same change in circulating glucose concentration. In another example, targeting the spleen region with neuromodulatory energy may result in a change in norepinephrine and acetylcholine concentration. In addition, neuromodulation of peripheral nerve ganglia may modulate cholinergic anti-inflammatory pathways, dopamine production pathways, and glucose regulation and/or insulin production pathways. Thus, based on the nerves to which neuromodulation energy is directly targeted, the distribution of nerve endings (e.g., axon endings) within an organ may in turn allow selective neuromodulation to achieve a target physiological outcome. These techniques in the peripheral nerve achieve the targeted effect of propagation at the peripheral nerve pathway endings. However, the target physiological result may include activity changes in multiple and overlapping pathways.

As discussed herein, neuromodulation may be implemented to elicit a target physiological outcome, e.g., for treatment of certain disorders. Patients may benefit from a neuromodulation therapy regimen that is periodic (e.g., daily) over a period of time (e.g., weeks) to achieve a desired target physiological result. However, activity changes in multiple and overlapping pathways can lead to compensatory physiological effects over time, which make neuromodulation therapy less effective over time. Thus, the techniques of the present invention provide for the evaluation of markers of multiple pathways, so that the effectiveness of neuromodulation can be evaluated. In the case of diminishing returns, the treatment regimen may introduce a recovery period to allow the compensatory pathway to return to normal, and then resume neuromodulation. In one embodiment, the assessment is a gene expression profile of a plurality of markers, wherein some profiles are associated with physiological changes that reduce the effectiveness of neuromodulation, and other profiles are associated with a gene expression environment that is favorable for effective neuromodulation. Characteristic gene expression or marker expression profiles are indicative of complex physiological changes propagated through the application of energy to the nerve axon ends in the neuromodulation implementations provided herein. In another embodiment, the profile relates to a change or modification in the concentration of one or more molecules of interest (e.g., circulating molecules, biomarkers).

The human nervous system is a complex network of nerve cells or neurons, with the pivot being present in the brain and spinal cord and the periphery in various nerves of the body. Neurons have cell bodies, dendrites, and axons. A nerve is a group of neurons that serve a specific body part. Nerves can contain hundreds of neurons to hundreds of thousands of neurons. Nerves typically contain both afferent and efferent neurons. Afferent neurons carry signals to the central nervous system and efferent neurons carry signals to the periphery. A group of neuronal cells in one location is called a ganglion. Electrical signals generated in the nerve are conducted through neurons and nerves (e.g., by internal or external stimuli). Neurons release neurotransmitters at synapses (connections) adjacent to the receiving cell to allow electrical signals to continue and modulate.

The electrical signals of the neurons are called action potentials. When the voltage potential across the cell membrane is greater than a certain threshold, an action potential is initiated. The action potential then propagates along the length of the neuron. The action potential of a nerve is complex and represents the sum of the action potentials of individual neurons therein. The junction between the axonal end of a neuron and the recipient cell is called a synapse. The action potential travels along the axon of the neuron to its axon terminal, which is the distal end of the axon nerve branch that forms the presynaptic terminal or synaptic end of the nerve fiber. The electrical impulses of the action potential trigger the migration of vesicles containing neurotransmitters to the presynaptic membrane at the presynaptic axon terminal and eventually release the neurotransmitters into the synaptic cleft (e.g., the space formed between presynaptic and postsynaptic cells) or the axon extracellular space (axaxaxaxacellular space). Chemical synapses convert electrical signals of action potentials into chemical signals by releasing neurotransmitters. In contrast to chemical synapses, electrical synapses allow ionic currents to flow into presynaptic axonal terminals and across the cell membrane into postsynaptic cells.

The physiological effects of action potentials are mediated by ionic movement across cell membranes. Neuronal passage favoring ions, e.g. Na⁺、K⁺And Cl^-Ion pumps moving through the neuronal membrane actively maintain a static membrane potential. Different types of neurons can maintain different resting membrane potentials (e.g., -75mV to-55 mV). The action potential is generated by ion influx (i.e. charge movement), creating a large deviation in membrane potential associated with transient increases in transmembrane voltage. For example, the increase in membrane potential may be in the range of 30mV to 60 mV. An action potential in a postsynaptic neuron may begin in response to the release of a neurotransmitter by a presynaptic (e.g., upstream) neuron. Neurotransmitters released from presynaptic neurons bind to receptors of postsynaptic neurons, which in turn lead to ion influx and subsequent transmembrane depolarization. The action potential then propagates along the nerve, as this process occurs between subsequent neurons within the nerve.

Synapses may be located at a junction between two neurons, which allows an action potential to propagate along a nerve fiber. However, axonal ends may also synapse at junctions between neurons and non-neuronal cells and may terminate at interstitial or body fluids. For example, types of synapses include synapses with immune cells at a neuro-immune junction, synapses with resident sensory cells within an organ, or synapses with glandular cells. The release of neurotransmitters into the synaptic cleft and the binding of the neurotransmitters to receptors in the postsynaptic membrane of the postsynaptic cell results in downstream effects that depend on the properties of the presynaptic neuron, the specific neurotransmitter released, and the properties of the postsynaptic cell (e.g., the available receptor type of the postsynaptic cell). In addition, the action potential may be stimulatory or inhibitory. Stimulating postsynaptic action potentials increases the likelihood that a postsynaptic neuron will fire or release a subsequent action potential. In contrast, inhibitory postsynaptic action potentials reduce the likelihood that a postsynaptic neuron will fire or release a subsequent action potential. Some neurons can work together to release neurotransmitters that trigger or inhibit downstream action potentials.

Neuromodulation applies energy from an external energy source to a specific region of the nervous system to activate and/or block one or more nerves or to increase and/or decrease nerve function. Electrical neuromodulation applies one or more electrodes at or near a target nerve, and energy application is carried out by the nerve (e.g., as an action potential) to elicit a physiological response in a region downstream of the energy application site. However, due to the complexity of the nervous system, it is difficult to predict the extent and ultimate end point of the physiological response for a given energy application site. Although strategies for ultrasound modulation of the central nervous system (i.e., brain tissue) have demonstrated successful modulation of neural activity, attempts to modulate peripheral nerves have lagged behind. For example, ultrasound modulation of the Central Nervous System (CNS) involves stimulation of areas of the cerebral cortex that have abundant synaptic structures, whereas attempts to ultrasonically stimulate peripheral nerves target the nerve trunk, which is not enriched or devoid of synaptic structures.

Provided herein are techniques for neuromodulation based on direct and focused modulation of a target region of interest and for eliciting a target physiological result as a result of the neuromodulation. Neuromodulation of a target region of interest allows for limited and non-ablative application of energy only to the target region of interest, without applying energy outside of the target region of interest. By applying energy to a volume of tissue that includes synapses, a desired result may be achieved by preferentially activating synapses to cause release of neurotransmitters or neuropeptides. In this way, synapses within the region of interest are activated, while synapses outside the region of interest are not activated. The targeted presynaptic neuronal cell may release a neurotransmitter or neuropeptide, or cause an alteration in neurotransmitter or neuropeptide release in the vicinity of a neighboring non-neuronal cell or neuronal cell to modulate cellular activity. This, in turn, can result in local and/or non-local (e.g., systemic) effects outside of the region of interest (e.g., in a tissue or structure containing the target region of interest or in an organ, tissue or structure that does not contain the target region of interest) without directly stimulating the synapse-rich region.

Neuromodulation may be used to treat glucose metabolism and related disorders, alter disease progression, and control systemic inflammation. For example, liver modulation at one or more regions of interest may be used to treat diabetes (i.e., type 1 or type 2 diabetes), hyperglycemia, sepsis, trauma, infection, diabetes-related dementia, obesity, or other dietary or metabolic disorders. Neuromodulation may also be used to promote weight loss, control appetite, treat cachexia, or enhance appetite. For example, direct pancreatic stimulation may lead to enhanced appetite, direct liver stimulation may cause a reduction in neuropeptide y (npy), which in turn promotes satiety signaling, and direct spleen stimulation may lead to reduced systemic inflammation. In addition, neuromodulation of peripheral nerve ganglia may modulate the cholinergic anti-inflammatory pathway (CAP), the dopamine production pathway, and the glucose regulation and/or insulin production pathway.

Neuromodulation of a target region of interest may result in a treatment outcome that is sustained beyond the treatment time. For example, treatment with repeated energy applications to a target region of interest over a predetermined period of time may result in sustained improvement in disease symptoms. The predetermined period of time may be a time window of hours, days, weeks, or months during which the neuromodulation therapy occurs. Additionally, the neuromodulation therapy may include one or more individual energy application events over a predetermined time period. Separate energy application events may be repeatedly applied to the same region of interest over a predetermined period of time. For example, neuromodulation therapy may occur daily, every other day, every third day, every week, or at any other suitable therapy rate over a predetermined period of time in the region of interest based on preset modulation parameters.

For example, the modulation parameters may include a variety of stimulation time patterns from continuous to intermittent. The adjustment parameters may also include the frequency and duration of the stimulus application. The frequency of application may be continuous or delivered over various time periods, for example, over a day or week. The duration of treatment may last for a variety of periods of time, including, but not limited to, minutes to hours. Additionally, the adjustment parameter may include a duration of a recovery period between ultrasound energy treatments. The duration of the recovery period may allow the compensatory pathways of the region of interest to return to normal before resuming neuromodulation therapy.

However, continuous stimulation of a synaptic within the region of interest can produce undesirable physiological consequences in the subject over time. For example, a series of energy application events applied to a subject over a period of time (as part of a neuromodulation therapy protocol) that releases a particular molecule of interest (e.g., a neurotransmitter or neuropeptide) can also cause a change in gene expression of a cellular receptor and/or channel of the molecule of interest in the subject over a period of time (e.g., relative to a decrease in baseline expression levels prior to the energy application event). Down-regulation of gene expression may occur when cells are over-activated by a particular molecule for an extended period of time, and the corresponding production of cellular receptors for that molecule may be reduced to compensate for over-stimulation of the cells by the molecule. As such, repeated stimulation of synapses over an extended period of time to release molecules of interest in a region of interest may cause down-regulation of gene expression of the corresponding cellular receptor and/or channel, thereby reducing the effectiveness of subsequent energy application events over the extended period of time. Alternatively, repeated stimulation of synapses within a tissue region may cause upregulation of genes involved in the half-life of neurotransmitters or neuropeptides by metabolizing, degrading or inhibiting them. For example, Butyrylcholinesterase (BCHE) is a gene that can be upregulated, followed by protein translation, glycosylation, and secretion. BCHE can hydrolyze the neurotransmitter acetylcholine, has been proposed as a prognostic marker for a variety of diseases (e.g., obesity, metabolic syndrome, inflammatory diseases, malignancies), and is a preventative measure for nerve agents and pesticides. BCHE is expressed in most tissues, including brain, liver and lymphoid tissues.

Accordingly, provided herein are techniques for determining and/or altering one or more treatment parameters of a neuromodulation treatment for a subject such that an acute response (e.g., release of neurotransmitters or neuropeptides) to each energy application event may be maintained while undesirable chronic effects may be minimized. The treatment parameters can include a treatment period in which neuromodulation therapy (e.g., energy application events alone) is applied to the subject, a treatment frequency of the energy application events alone within the treatment period, and/or a duration of a recovery period (e.g., a time period between subsequent neuromodulation treatments or treatment periods). For example, the convalescent phase may facilitate the reversal of any undesirable physiological effects caused by neuromodulation therapy, such as undesirable changes in gene expression levels. After the duration of the recovery period has elapsed and any changes in gene expression levels have been substantially reversed (e.g., toward the baseline threshold before neuromodulation therapy), subsequent neuromodulation therapy can be initiated. In some embodiments, the change in gene expression level may be a desired neuromodulation therapeutic effect in the subject. In these embodiments, if a previous neuromodulation therapy altered (e.g., increased or decreased) gene expression levels in the appropriate direction, the recovery phase may be omitted and a subsequent neuromodulation therapy may be initiated.

The treatment parameters (e.g., treatment period, treatment frequency, and duration of recovery period) may be determined prior to the first neuromodulation treatment of the subject. In some embodiments, the treatment parameters may be determined based on a desired physiological outcome in the subject as a result of neuromodulation therapy or historical or experimental data. After completion of the first neuromodulation therapy, the expression level of certain genes in the subject can be evaluated and it can be determined whether the expression level of the genes is desired. If the expression level of the gene is desired, a subsequent neuromodulation therapy may be performed based on the previously determined therapy parameters. If the expression level of the gene is not desired, the therapeutic parameter for subsequent neuromodulation may be altered based on factors including, but not limited to, the gene expression level evaluated in the subject, the physiological outcome desired in the subject, historical data, or experimental data. As such, the neuromodulation techniques provided herein can optimize the treatment parameters of the neuromodulation therapy for the subject over an extended period of time, such that the desired acute response to the neuromodulation therapy can be maintained while minimizing any undesirable chronic effects of the neuromodulation therapy.

To this end, the disclosed neuromodulation techniques may be used in conjunction with neuromodulation systems. FIG. 1 is a schematic diagram of a system 10 for neuromodulation to effect neurotransmitter release and/or activation of synaptic components (e.g., presynaptic cells, postsynaptic cells) in response to energy application. The illustrated system includes a pulse generator 14 coupled to the energy application device 12 (e.g., an ultrasonic transducer). The energy application device 12 is configured to receive the energy pulse (e.g., via one or more wires or a wireless connection) and direct the energy pulse to a region of interest (e.g., a peripheral nerve ganglion or portion thereof), which in turn results in the production of a target physiological result. In certain embodiments, the pulse generator 14 and/or the energy application device 12 may be implanted at a biocompatible site (e.g., the abdomen) and the lead internally coupled to 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 ultrasonic transducer.

In certain embodiments, the energy application device 12 and/or the pulse generator 14 may communicate wirelessly, for example, through a controller 16, which in turn may provide instructions to the pulse generator 14. In other embodiments, the pulse generator 14 may be an extracorporeal device (e.g., operable to apply energy percutaneously or in a non-invasive manner from a location external to the subject) and may be integrated into the controller 16 in certain embodiments. In embodiments in which the pulse generator 14 is located outside the body, the caregiver can operate the energy application device 12 and position it at a point on or above the subject's skin to deliver the energy pulse percutaneously to the desired internal tissue. Once positioned to apply the energy pulse to the desired site, the system 10 can begin neuromodulation to achieve the target physiological result or clinical effect.

In certain embodiments, the system 10 may include an evaluation device 20 coupled to the controller 16 and configured to evaluate a characteristic indicative of whether the adjusted target physiological result has been achieved. In one embodiment, the target physiological result may be local. For example, modulation may result in changes to local tissue or changes in function, such as changes in tissue structure, local changes in the concentration of certain molecules, tissue displacement, fluid motion enhancement, and the like.

In certain embodiments, modulation may result in systemic and/or non-local changes. The target physiological result may involve a change in circulating molecular concentration or a change in tissue characteristics of the region of interest that does not include energy applied directly thereto. In one example, the shift may be a representative measure of the desired adjustment, and a shift measure that is lower than the desired shift value may result in a change in the adjustment parameter until the desired shift value is caused. Thus, in some embodiments, the evaluation device 20 may be configured to evaluate concentration variations. In some embodiments, the evaluation device 20 may be an imaging device configured to evaluate changes in tissue size and/or position. Although the display elements of system 10 are shown separately, it should be understood that some or all of the elements may be combined with each other. Further, some or all of the elements may be in wired or wireless communication with each other.

Based on the evaluation, the adjustment parameters of the controller 16 may be changed. For example, if the desired modulation is associated with a change in concentration (cyclic concentration of one or more molecules or tissue concentration) within a defined time window (e.g., 5 minutes or 30 minutes after initiation of the energy application procedure) or relative to a baseline at the beginning of the procedure, a change in a modulation parameter (e.g., pulse frequency) may be desired, which in turn may be provided to the controller 16 by an operator or by an automatic feedback loop for defining or adjusting the energy application parameter or modulation parameter of the pulse generator 14.

The system 10 as provided herein can provide energy pulses according to a variety of regulatory parameters. For example, the modulation parameters may include a variety of stimulation time patterns from continuous to intermittent. With intermittent stimulation, energy is delivered at a specific frequency for a period of time during a signal-on time (signal-on time). The signal-on time is followed by a period of no energy delivery, which is referred to as the signal-off time. The adjustment parameters may also include the frequency and duration of the stimulus application. The frequency of application may be continuous or delivered over various time periods, for example, over a day or week. The duration of treatment may last for a variety of periods of time, including, but not limited to, minutes to hours. In certain embodiments, the duration of treatment with the indicated stimulation pattern may last 1 hour, for example, it is repeated at 72 hour intervals. In certain embodiments, a shorter duration (e.g., 30 minutes) of treatment may be delivered at a higher frequency (e.g., 3 hours). Thus, depending on the adjustment parameters, such as treatment duration and frequency, the energy application can be adjustably controlled to achieve the desired result.

In certain embodiments, the adjustment parameters of the controller 16 may be varied based on one or more characteristics of the disease of the subject to achieve a desired result. The duration of treatment and/or frequency of application of energy application can be increased or decreased based on the progression of the disease over time to achieve the desired therapeutic result. For example, if the patient's disease state worsens, the treatment duration and/or frequency of application may increase over time to achieve the desired result. In another example, if the disease state of a patient improves, the duration of treatment and/or frequency of application may be maintained or reduced over time.

Fig. 2 is a block diagram of certain components of 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 the controller 16. The controller 16 includes a processor 30 for controlling the device. Software code or instructions are stored in the memory 32 of the controller 16 for execution by the processor 30 to control the various components of the device. The controller 16 and/or the pulse generator 14 may be connected to the energy application device 12 by one or more wires 33 or wirelessly.

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 adjust the procedure. Each tuning program may include one or more sets of tuning parameters including pulse amplitude, pulse width, pulse frequency, and the like. The pulse generator 14 is responsive to control signals from the controller 16 to change its internal parameters, thereby changing the stimulation characteristics of the energy pulses transmitted over the lead 33 to the subject to which the energy application device 12 is applied. Any suitable type of pulse generating circuit may be used including, but not limited to, constant current, constant voltage, multiple individual current or voltage sources, etc. The energy applied is a function of the current amplitude and the pulse width duration. The controller 16 allows for adjustable control of the energy by changing the adjustment parameters and/or starting the energy application at a particular time, cancelling the energy application at a particular time or suppressing the energy application at a particular time. In one embodiment, the adjustable control of the energy application device is based on information related to the concentration of one or more molecules (e.g., circulating molecules) in the subject. If this information comes from the evaluation device 20, a feedback loop may drive the adjustable control. For example, if the circulating glucose concentration measured by the evaluation device 20 is above a predetermined threshold or range, the controller 16 may initiate energy application to the region of interest (e.g., the peripheral nerve ganglion) and using the modulation parameter associated with circulating glucose lowering. Initiation of energy application may be initiated in vitro by a glucose concentration excursion above a predetermined (e.g., desired) threshold or range. In another embodiment, the adjustable control may be in the form of changing an adjustment parameter when the initial application of energy does not result in an expected change that produces the target physiological result (e.g., concentration of the molecule of interest) within a predetermined time range (e.g., 1 hour, 2 hours, 4 hours, 1 day). In certain embodiments, the evaluation device 20 may be a device that measures gene expression.

In one embodiment, the memory 32 stores different operating modes that are selectable by an operator. For example, the stored manner of operation may include instructions for executing a set of adjustment parameters related to a particular treatment site (e.g., liver, spleen, peripheral ganglia, or pancreas). Different sites may have different relevant regulatory parameters. The controller 16 may be configured to execute the appropriate instructions based on the selection rather than having the operator manually enter the mode. In another embodiment, memory 32 stores operating modes for different therapy types. Example (b)For example, activation may be associated with a different stimulation pressure or frequency range relative to those associated with inhibiting or blocking tissue function. In a specific example, when the energy application device is an ultrasonic transducer, the average power (instantaneous average intensity) and the peak positive pressure are 1mW/cm²To 30,000mW/cm²(instantaneous average strength) and in the range of 0.1MPa to 7MPa (peak pressure). In one example, the instantaneous average intensity in the region of interest is less than 35W/cm²To avoid levels associated with thermal damage and exfoliation or cavitation. In another specific example, when the energy application device is a mechanical actuator (activator), the amplitude is in the range of 0.1 to 10 mm. The selected frequency may be based on the energy application mode (e.g., ultrasonic actuator or mechanical actuator).

In another embodiment, the memory 32 stores a calibration or setup mode that allows adjustment or change of tuning parameters to achieve a desired result. In one example, stimulation is initiated at a lower energy parameter and gradually increased, either automatically or by receiving operator input. In this way, the operator can effect an adjustment of the effect caused as the adjustment parameter changes.

The system may also include an imaging device that facilitates focusing the energy application device 12. In certain embodiments, the imaging device may be integrated into the energy application device 12 or the imaging device may be the same device as the energy application device 12, applying different ultrasound parameters (frequency, aperture, or energy) for selecting (e.g., spatially selecting) a region of interest and for focusing energy to the selected region of interest for targeting and subsequent neuromodulation. In another embodiment, memory 32 stores one or more targeting or focusing patterns for spatially selecting a region of interest within an organ or tissue structure. The spatial selection may depend on the imaging data as provided herein. Based on the spatial selection, the energy application device 12 may focus to a selected volume in the patient corresponding to the region of interest. For example, the energy application device 12 may be configured to first operate in a targeted mode to apply targeted mode energy for capturing image data for identifying a region of interest. The targeted mode energy is not at a level and/or is not applied with a tuning parameter suitable for preferential activation. However, once the region of interest is identified, the controller 16 may then operate in a therapeutic mode according to the adjustment parameters associated with the preferential activation.

The controller 16 may also be configured to receive input related to a target physiological outcome as input to adjust parameter selection. For example, when the imaging modality is used to evaluate a tissue feature as a result of energy application to a region of interest, the controller 16 may be configured to receive a calculated index or parameter of the feature. Based on whether the index or parameter is above or below a predetermined threshold, the adjustment parameter may be changed. In one embodiment, the parameter may be a measure of tissue displacement of the affected tissue or a measure of depth of the affected tissue. Other parameters may include evaluating the concentration of one or more molecules of interest (e.g., evaluating one or more of a change in concentration, rate of change, relative to a threshold or baseline/control to determine whether the concentration is within a desired range). In addition, the energy application device 12 (e.g., an ultrasound transducer) may be operated under the control of the controller 16 to (1) acquire image data that may be used to spatially select a region of interest within the body, (2) apply modulation energy to the region of interest, and (3) acquire image data to determine that a target physiological outcome has occurred (e.g., via displacement measurements). In such an embodiment, the imaging device, the evaluation device 20 and the energy application device 12 may be the same device.

In another implementation, the desired tuning parameter settings may also be stored by the controller 16. In this way, subject-specific parameters can be determined. Furthermore, the effectiveness of these parameters can be evaluated over time. If a particular set of parameters is not effective over time, the subject may develop insensitivity/tolerance to activation pathways or treatment parameters. If the system 10 includes an evaluation device 20, the evaluation device 20 may provide feedback to the controller 16. In certain embodiments, the feedback may originate from the user or from the evaluation device 20 indicating characteristics of the target physiological outcome. The controller 16 may be configured to cause the energy application device to apply energy according to the conditioning parameters and to dynamically adjust the conditioning parameters based on the feedback. For example, based on the feedback, the processor 16 may automatically change the adjustment parameters (e.g., the frequency, amplitude, or pulse width of the ultrasonic beam or mechanical vibrations) in real-time and in response to the feedback from the evaluation device 20.

For example, application of energy to a region of interest in accordance with the disclosed techniques may be used as part of a treatment protocol with desired results to maintain a circulating glucose concentration at a predetermined concentration, such as below about 200mg/dL and/or above about 70 mg/dL. This technique can be used to maintain glucose in the range of about 4 to 8mmol/L or about 70 to 150 mg/dL. The techniques can be used to maintain a normoglycemic range for a subject (e.g., a patient), where the normoglycemic range can be an individualized range based on patient individual factors, such as weight, age, and/or medical history. Thus, the application of energy to one or more regions of interest may be adjusted in real time based on the desired final concentration of the molecule of interest and may be adjusted in a feedback loop based on input from the evaluation device 20. For example, if the evaluation device 20 is a circulating glucose monitor or a blood glucose monitor, real-time glucose measurements may be used as input to the controller 16. As provided herein, a treatment protocol (a series of regularly or irregularly spaced energy application events) can be implemented, and circulating glucose concentrations can be evaluated during the course of treatment. In one embodiment, the patient is hyperglycemic at the beginning of treatment, but the administration of the treatment protocol reduces the concentration of circulating glucose and maintains the concentration within the desired range for a period of time (e.g., one month). After a period of time, the circulating glucose concentration may begin to gradually increase even though periodic energy application events are applied according to the treatment protocol. When the circulating glucose concentration is no longer within the desired range, a treatment recovery period may be implemented until such time as the patient again responds to the treatment protocol.

In another embodiment, the techniques of the present invention may be used to induce a characteristic physiological change profile. For example, a profile may include a set of molecules of interest that are elevated in concentration in tissue and/or blood due to energy application, and another set of molecules of interest that are reduced in concentration in tissue and/or blood due to energy application. The profile may also include a set of molecules that do not change as a result of energy application. The profile may be limited to simultaneous changes associated with a desired physiological outcome. For example, the profile may include a decrease in circulating glucose observed along with an increase in circulating insulin.

Fig. 3 is a specific example in which the energy application device 12 includes an ultrasonic transducer 42 capable of applying energy to a target tissue 43 (shown by way of non-limiting example as a spleen). The energy application device 12 may include a control circuit for controlling the ultrasonic transducer 42. The control loop of the processor 30 may be integrated into 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 44 and focusing the applied energy to the region of interest 44 of the tissue 43 or structure.

The desired target tissue 43 may be an internal tissue or organ that includes presynaptic neuronal cells with axonal ends 46 that form synapses 47 with postsynaptic cells 48 (e.g., splenic T cells). Synapses may be stimulated by applying energy directly to axon ends 46 within a focused field of an ultrasonic transducer 42 focused on a region of interest 44 of a target tissue 43 to cause release of molecules into synaptic cleft 47. In the illustrated embodiment, the axon terminals 46 synapse with spleen T cells, and release of neurotransmitters in synaptic cleft 47 and/or changes in ion channel activity in turn lead to downstream effects, such as activation of the cholinergic anti-inflammatory pathway (CAP). The region of interest may be selected to include a particular type of axon terminal 46, such as a particular neuron type of axon terminal 46 and/or an axon terminal 46 that synapses with a particular type of non-neuronal cell. Thus, the region of interest 44 may be selected to correspond to a portion of the target tissue 43 having a desired axon end 46 (and associated non-neuronal cells 48). In certain embodiments, the application of energy may be selected to preferentially trigger the release of one or more molecules (e.g., neurotransmitters or neuropeptides) from a nerve within a synapse. In certain embodiments, the application of energy may be selected to directly activate neuronal cells in the region of interest 44 by direct energy transduction (i.e., force conduction or voltage activated proteins within non-neuronal cells) to preferentially trigger the release of one or more molecules (e.g., neurotransmitters or neuropeptides). In certain embodiments, the application of energy may be selected to preferentially trigger the release of one or more molecules (e.g., neurotransmitters or neuropeptides) by causing activation within neuronal cells within the region of interest 44 that causes the desired physiological effect.

In certain embodiments, the energy may be focused or concentrated to less than about 25mm³Within the volume of (a). In certain embodiments, the energy may be focused or concentrated at about 0.5mm³To 50mm³Within the volume of (a). The focal volume and depth of focus to focus or concentrate the energy within the region of interest 44 may be influenced by the size or configuration of the energy application device 12. The focal volume of the energy application may be defined by the focusing field of the energy application device 12. As provided herein, energy may be applied substantially only to the region of interest 44 to preferentially activate the spleen 43. Thus, only a subset of the multiple axonal ends 46 within the spleen 43 are exposed to direct energy application.

The region of interest 44 containing axons 46 can be identified by imaging, with reference to anatomical landmarks or the like, to perform spatial selection. The region of interest 44 in the target tissue 43 may be selected based on factors including, but not limited to, historical or experimental data (e.g., data showing a particular location in combination with a desired or target physiological outcome). Alternatively or additionally, the system 10 may apply energy to the individual axon tips 46 until a desired target physiological effect is achieved. It should be understood that the spleen 43 is by way of example only. The disclosed selection of axonal tips 46 for preferential activation of direct energy application to the region of interest 44 by using spatial information of the imaged nerve may be used in conjunction with other organs or structures (e.g., liver, pancreas, gastrointestinal tissue, or peripheral nerve ganglia).

In other embodiments, the region of interest may be identified by the presence or absence of one or more biomarkers. These markers can be evaluated by staining the tissue and obtaining an image indicative of the staining to identify regions of the tissue that include the biomarkers. In some embodiments, biomarker information may be obtained by in vivo staining techniques, thereby obtaining location data of the biomarkers within the subject in real time. In other embodiments, biomarker information may be obtained by an in vitro staining technique to obtain location data for one or more representative images used to predict biomarker location within a subject. In some embodiments, the region of interest is selected to correspond to tissue that is enriched for a particular biomarker or that lacks a particular biomarker. For example, the one or more biomarkers can include a neuronal structure marker (e.g., a myelin marker).

The region of interest in the organ or tissue may be selected based on the operator's input space. For example, the operator may indicate a region of interest on the acquired image by directly manipulating the image (e.g., drawing or writing a 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 the image data to achieve spatial selection. In some embodiments, the spatial selection includes storing data related to the region of interest in a memory and accessing the data.

The assessment of neuromodulation may be used as an input or feedback to select or alter neuromodulation parameters. The evaluation techniques may use direct evaluation of tissue condition or function as a result of the targeted physiology. The assessment may occur 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, positron emission tomography or acoustic monitoring, thermal monitoring. The assessment techniques may also include nucleic acid, protein, and/or marker concentration assessment. Images from evaluation techniques can be received by the system for automatic or manual evaluation. Based on the image data, the adjustment parameters may be changed. For example, tissue size changes or shifts can be used as markers of local neurotransmitter concentration, and as surrogate markers of local cell exposure to phenotypically regulated neurotransmitters, and effectively as markers of predicted effect on glucose metabolism pathways or systemic inflammatory pathways. The local concentration may represent the concentration within the focal field of the energy application.

Additionally or alternatively, the system may assess the presence or concentration of one or more molecules in the tissue region or in the blood. Tissue may be obtained by fine needle puncture, and assessment of the presence or level of a molecule of interest (e.g., a metabolic molecule, a metabolic pathway marker, a neurotransmitter, a catecholamine, a cholinesterase) may be performed by any suitable technique known to those of skill in the art.

In other embodiments, the target physiological result may include, but is not limited to, tissue displacement, tissue size changes, changes in one or more molecular concentrations (e.g., local, non-local, or circulating concentrations), changes in gene or marker expression, afferent activity, cell migration, and the like. For example, tissue displacement may occur as a result of application of energy to the tissue. By evaluating tissue displacement (e.g., by imaging), other effects can be estimated. For example, a certain shift may be characterized by a specific change in the concentration of molecules. In one example, based on empirical data, a tissue shift of 5% (e.g., liver shift) may indicate or be related to a desired decrease in circulating glucose concentration. In another example, tissue displacement may be evaluated by comparing reference image data (tissue image before applying energy to the tissue) with processed image data (tissue image acquired after applying energy to the tissue) to determine a maximum or average tissue displacement value. If the shift is greater than the threshold shift, the energy application may be evaluated as likely to have caused the desired target physiological result.

Fig. 4 is an example of an energy application device 12 that may be used in conjunction with the system 10 shown in fig. 1, the system 10 including a High Intensity Focused Ultrasound (HIFU) transducer 74A and an imaging ultrasound transducer 74B arranged in a single energy application device 12 that may be controlled (e.g., by the controller 16) to apply energy and image target tissue as provided herein.

Fig. 5 shows an experimental setup for conducting certain neuromodulation experiments focused on a target tissue 43 (e.g., spleen, liver, pancreas, gastrointestinal tissue, or peripheral nerve ganglion) as provided herein. The energy application device 12 can be operated by the controller 16 to apply energy to a region of interest in the target tissue 43 according to the parameter settings. Although the experimental setup shown is shown as a 40W RF amplifier, this is merely illustrative and other amplifiers (e.g., linear amplifiers) may be used. In some devices, the rat head is inserted into a birdcage coil.

Fig. 6 is a flow diagram of a method 80 for evaluating the effect of neuromodulation therapy over a period of time, which in turn may be used as input or feedback for selecting or changing a therapy parameter. Although fig. 6 is described with respect to the evaluation of the effect of a neuromodulation therapy on gene expression in a subject, it should be understood that the evaluated effect of a neuromodulation therapy may include other desired physiological effects of a neuromodulation therapy in a subject or undesired physiological effects of a neuromodulation therapy in a subject. In some embodiments, the profile of physiological changes induced by neuromodulation therapy may be evaluated in fig. 6.

As provided herein, the evaluation of whether a patient responds to a treatment protocol can be based on a profile. The profile can define simultaneous changes relative to a baseline profile evaluated prior to administration of a treatment protocol, the changes correlated with a desired physiological outcome. For example, a profile may include concentration information for a set of molecules of interest that are elevated in tissue and/or blood due to neuromodulation therapy, another set of molecules of interest that are reduced in tissue and/or blood due to neuromodulation therapy, and/or a set of molecules that are not altered due to neuromodulation therapy. The profile may include expression information for a set of genes whose expression is increased as a result of neuromodulation therapy, another set of genes whose expression is decreased as a result of neuromodulation therapy, and/or a set of genes whose expression is unchanged as a result of neuromodulation therapy. Alternatively or additionally, the profile may reflect changes in the activation or modification state, e.g., phosphorylation, acetylation, of certain proteins relative to baseline.

In the method 80, one or more neuromodulation therapy parameters in a therapy protocol may be selected or determined in step 82. In certain embodiments, a treatment parameter may be selected or determined based on a desired physiological outcome in the subject. The treatment parameters can include a treatment period in which neuromodulation therapy (e.g., a separate energy application event) is applied to the subject. For example, a treatment session for a subject may occur for 3 days, 5 days, one week, one and a half weeks, two weeks, or any other suitable period of time to achieve a desired physiological result in the subject. The treatment parameters may also include a treatment frequency of individual energy application events to the subject over the treatment period. For example, the treatment frequency can be twice daily, once daily, four times weekly, three times weekly, twice weekly, once weekly, or any other suitable frequency of energy application events to achieve a desired physiological result in the subject. In addition, the treatment parameters may include a treatment recovery period (e.g., a continuous neuromodulation treatment or a time period between treatment periods). The duration of the recovery period may be beneficial for the reversal of any undesirable physiological effects caused by neuromodulation therapy. For example, the duration of the recovery period may allow the expression level of a particular gene to return to an expression level similar to the baseline threshold prior to neuromodulation therapy. In some embodiments, the duration of the recovery period can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, one week, two weeks, or any other suitable time period between the first treatment period and the second treatment period to achieve the desired physiological result in the subject. In certain embodiments, the duration of the recovery period may not be determined until step 88 of the method 80. In some embodiments, a treatment parameter may be determined based on factors including, but not limited to, historical or experimental data (e.g., data showing a combination of a particular treatment parameter and a desired or targeted physiological outcome). In some embodiments, the treatment parameter can be determined based on the subject's current physiological state, medical history, weight, or age.

In step 84, neuromodulation therapy is applied to the subject to achieve a desired physiological result based on the treatment parameters (e.g., treatment period, frequency, and/or remaining period) determined in step 82. For example, neuromodulation therapy for a subject may include applying energy to a target tissue of the subject's body once a day for one week. In another example, neuromodulation therapy for a subject may include applying energy to a target tissue of a subject's body once a day for two weeks. After completion of a single neuromodulation therapy or series of therapies, the effect of the neuromodulation therapy on the expression level of a particular gene in the subject may be evaluated in step 86.

For example, one or more evaluation techniques for determining the expression level of a particular gene in a subject may be used. The evaluation technique can generate RNA or mRNA transcriptome data from a subset of cells in a target tissue by RNA sequencing, quantitative polymerase chain reaction (qPCR), microarray, or other suitable technique. In some embodiments, a particular gene for evaluation may be selected or determined based on its relationship to a desired physiological outcome. For example, the genes evaluated may produce molecules (e.g., proteins) associated with a desired pathway (e.g., a cholinergic anti-inflammatory pathway) that is activated by a neuromodulation therapy. In certain embodiments, the assessment of the gene expression level of a particular gene may be performed prior to the start of neuromodulation therapy of the subject to acquire a baseline level of gene expression for the particular gene. Evaluation data gene expression data can also be evaluated by group-level analysis of multiple genes, such as clustering. Clustering techniques may be used for class discovery or classification. The patient's characteristic gene expression profile may be used to identify the closest match in the categorized gene expression profiles. For example, a responsive patient may have a gene expression profile characterized by gene expression associated with therapeutic responsiveness, while a treatment-insensitive patient may have a gene expression profile characterized by gene expression associated with therapeutic non-responsiveness. Cluster analysis can be used to determine whether a patient's uncategorized characteristic gene expression profile should be classified as responsive or non-responsive to treatment.

In certain embodiments, one or more evaluations of changes in the concentration of one or more molecules of interest (e.g., glucose, neurotransmitters, neuropeptides, cholinesterase, or cytokines) may also be performed in step 86. The evaluation of the change in concentration of the molecule of interest can be used to determine whether the treatment parameters and/or modulation parameters as described herein need to be altered to achieve the desired physiological result. Once it is determined that the treatment parameters and/or modulation parameters need to be changed to achieve the desired physiological result from the neuromodulation therapy, the treatment parameters and/or modulation parameters may be changed as described herein.

Based on the gene expression level as assessed in step 86, a determination is made in step 88 whether the neuromodulation therapy has an effect on the expression level of the subject specific gene. Once the effect on gene expression levels is determined to be desired, the method 80 may continue to step 90 and a subsequent neuromodulation therapy may be applied according to the therapy parameters determined in step 82. For example, in certain embodiments, a change in the expression level of a particular gene as a result of a neuromodulation therapy may not be desirable. If the expression level of a particular gene is assessed in step 86 as not having a significant change (e.g., as compared to a baseline threshold value prior to neuromodulation therapy), the neuromodulation therapy parameters determined in step 82 may be used in further neuromodulation therapy of the subject. In another example, in certain embodiments, a change (e.g., an increase or decrease) in the expression level of a particular gene as a result of a neuromodulation therapy may be desired. If the expression level of a particular gene is assessed as significantly changed in step 86 (e.g., compared to a baseline threshold prior to neuromodulation therapy), the neuromodulation therapy parameters determined in step 82 may be used in further neuromodulation therapy of the subject.

In step 88, once the effect on the gene expression level is determined to be undesirable, the method 80 may continue to step 92 and the treatment parameter of the neuromodulation therapy may be altered based on the effect assessed on the gene expression level in the subject. For example, a change (e.g., an increase or decrease) in the expression level of a particular gene as a result of a neuromodulation therapy may not be desirable. If it is determined that the expression level of a particular gene is significantly changed (e.g., compared to a baseline threshold prior to neuromodulation therapy), the neuromodulation therapy parameters can be altered (e.g., dynamically or adjustably controlled) to achieve a desired physiological result while minimizing the effect on gene expression in the subject. In another example, a change (e.g., an increase or decrease) in the expression level of a particular gene as a result of a neuromodulation therapy may be desired. If it is determined that the expression level of a particular gene has not significantly changed (e.g., as compared to a baseline threshold prior to neuromodulation therapy), the neuromodulation therapy parameters can be altered (e.g., dynamically or adjustably controlled) to achieve a desired physiological result and a desired change in gene expression. In some embodiments, the treatment parameters can be altered based on factors including, but not limited to, historical data, experimental data, the current physiological state of the subject, medical history, weight or age, the effect assessed on gene expression in the subject, or the desired physiological outcome in the subject.

In one embodiment, a gene expression level is assessed as a significant change from a baseline threshold prior to neuromodulation therapy if the increase or decrease in the gene expression level is at least a 10%, 20%, 30%, 50%, or 75% increase or decrease in the baseline threshold for the gene expression level. In another embodiment, a gene expression level is assessed as not significantly changed from a baseline threshold prior to neuromodulation therapy if the gene expression level is at most a 10%, 5%, 3%, 2%, 1%, or 0% increase or decrease in the baseline threshold for the gene expression level.

The treatment period of any subsequent neuromodulation therapy (e.g., energy application event alone) applied to the subject may be altered in step 92. In some embodiments, the treatment period of the subject can be increased or decreased relative to the treatment period of the prior neuromodulation treatment. In some embodiments, the treatment period of the subject may not be changed according to the treatment period of the previous neuromodulation treatment. For any subsequent neuromodulation therapy, the treatment frequency of energy application events to the subject alone during the treatment period may be altered in step 92. In some embodiments, the frequency of treatment may be increased or decreased relative to the frequency of treatment of energy application events in prior neuromodulation treatments. In some embodiments, the treatment frequency may not be changed according to the treatment frequency of energy application events in a previous neuromodulation treatment.

The duration of the recovery period between subsequent treatment periods or neuromodulation treatments may be altered (or determined, if not determined in step 82). In some embodiments, the duration of the convalescent period can be determined based on the effect that neuromodulation therapy has on the level of expression of a particular gene. For example, the duration of the convalescent period can be determined as a particular time period based on the degree to which the expression levels of a set of genes are elevated and/or the expression levels of a set of genes are reduced relative to the baseline expression levels prior to the neuromodulation therapy. In some embodiments, if a change (e.g., an increase or decrease) in the expression level of a particular gene is desired but not assessed, a subsequent neuromodulation therapy of the subject may be immediately initiated without a recovery period. In some embodiments, if a change in the expression level of a particular gene is evaluated but not desired, a defined or altered recovery period can occur before the subject begins subsequent neuromodulation therapy based on the degree of change in the gene expression level. Additionally or alternatively, the duration of the recovery period may be determined based on factors including, but not limited to, historical or experimental data (e.g., data showing the relationship of a particular recovery period to the reversal of a desired or targeted gene expression change). In some embodiments, the duration of the recovery period can be determined by assessing the gene expression level in the subject at discrete time points after a previous neuromodulation therapy until it is determined that the gene expression level has returned to normal (e.g., a baseline gene expression level prior to the neuromodulation therapy).

In step 94, neuromodulation therapy is applied to the subject based on the altered treatment parameter (e.g., treatment period, treatment frequency, and/or duration of the recovery period) in step 92 to achieve the desired physiological result and the desired gene expression level in the subject. After administering the neuromodulation therapy, steps 86, 88, 92, and 94 may be repeated until the effect of the neuromodulation therapy on the evaluation of gene expression is determined to be desired in step 88. Once it is determined that the effect of neuromodulation therapy on gene expression is desired, the altered neuromodulation therapy parameters (e.g., the treatment period, the treatment frequency, and/or the duration of the recovery period) can be used in any subsequent neuromodulation therapy applied to the subject. As such, the treatment parameters of the neuromodulation therapy for a subject may be optimized to achieve a desired physiological result in the subject while achieving a desired level of gene expression.

Examples

Determination of physiological effects of neuromodulation therapy over time

Figures 7A to 7D show experimental timelines for neuromodulation therapy for practicing certain modulation experiments as provided herein. In FIG. 7A, ultrasound energy was applied to the spleen of female Sprague Dawley (SD) rats, approximately 300g and 11 weeks old at the start of the conditioning experiment. The treatment session of the ultrasound energy application event is two weeks at a treatment frequency of once a day. In figure 7B, ultrasound energy was also applied to the spleen of female SD rats, which were approximately 300g and 11 weeks old at the beginning of the conditioning experiment. The treatment session of the ultrasound energy application event is one week at a treatment frequency of once a day. In fig. 7C and 7D, sham controls were performed by placing ultrasound transducers on the spleens of female SD rats without applying ultrasound stimulation. In fig. 7C, the treatment period for the sham control was performed at a once-daily treatment frequency for two weeks. In fig. 7D, the treatment period for the sham control was one week at a once-daily treatment frequency.

In the embodiment shown in fig. 7A and 7B, each ultrasonic energy application event 100 is performed for 1 minute before and after a 30 second rest period. Standard anesthesia (e.g., 2% to 4% isoflurane) was used prior to each application of ultrasound energy to the treatment. As shown in figures 7A and 7B, Lipopolysaccharide (LPS) was administered to female SD rats by Intraperitoneal (IP) injection 4 hours after the last application of ultrasonic energy during the treatment period. LPS is a bacterial molecule that elicits a strong immune or inflammatory response. Female SD rats were sacrificed for organ harvest and handling during the time period following LPS injection. Although the time period shown is, for example, 1 hour after LPS injection, it is understood that in other embodiments, the time period over which the resulting change is assessed may be variable.

In the embodiment shown in fig. 7C and 7D, each sham-control application event 102 was performed by placing an ultrasound transducer on the spleen of female SD rats without applying ultrasound stimulation. As shown in fig. 7C, LPS was administered to female SD rats by IP injection 4 hours after the last sham-control application event during the treatment period. Female SD rats were sacrificed for organ harvest and handling during the time period following LPS injection. Although the time period shown is, for example, 1 hour after LPS injection, it is understood that in other embodiments, the time period over which the resulting change is assessed may be variable. As shown in fig. 7D, LPS was not administered to female SD rats by IP injection, and female SD rats were sacrificed at a time period after the last sham control application event. Although the time period shown is, for example, 5 hours after the last sham-control application event, it should be understood that in other embodiments, the time period over which the resulting change is evaluated may be variable. Spleen samples were harvested from sacrificed female SD rats.

The ultrasound stimulation was examined over an extended period of time (e.g., once daily for a 1 week period, once daily for a 2 week period). The splenomegaly fraction was obtained to measure the expression level of a specific gene. The splenomegaly fraction was treated in RNAlater for 24 hours and stored at-80 ℃ in RNA-free tubes. RNA was extracted from the splenomegaly fraction, prepared using TruSeq Stranded mRNA library preparation for sequencing, and sequenced for use in assessing gene expression levels.

Embodiments of the invention demonstrate non-invasive methods of stimulating axonal ends of neurons in a target tissue to achieve a desired physiological outcome (e.g., release of one or more molecules of interest, such as neurotransmitters or neuropeptides) and evaluating the expression level of a particular gene over time that is directly or indirectly related to the desired physiological outcome. Expression levels of specific cytokine-active genes associated with systemic inflammation were monitored by RNA transcriptome sequencing of stimulated and sham-control spleens. In addition, blood concentrations of various systemic inflammation-related cytokines and proteins were measured. Following a single ultrasound energy application event (e.g., a single dose) to the spleen, ultrasound neuromodulation therapy was found to affect (e.g., increase or decrease) the concentration of a particular protein in the blood and/or spleen. One week after the ultrasound energy application event to the spleen, ultrasound neuromodulation therapy was found to affect (e.g., increase or decrease) the concentration of a particular protein in the blood and/or spleen, but the gene expression level in the spleen was not significantly altered (e.g., compared to the baseline level prior to neuromodulation therapy). Two weeks after the application of the event to spleen ultrasound energy, ultrasound neuromodulation therapy was found to affect the expression levels of specific genes in the spleen. In general, the data indicates that the treatment parameters of the ultrasound neuromodulation therapy within the target tissue or structure can be optimized such that the ultrasound neuromodulation therapy can achieve the desired physiological result while causing the desired expression level of the particular gene within the subject based on one or more adjustable treatment parameters (e.g., treatment period, treatment frequency, or duration of recovery period between subsequent treatment periods).

Figure 8 shows the transcriptome data of specific genes associated with cytokine activity in untreated rats, sham controls (i.e., rats that received LPS but not sonicated) and rats that received LPS and sonicated over a one-week period and a two-week period. Expression levels of specific cytokine activity genes (gene ontology GO:0005125) were found to correlate with untreated expression levels of the cytokine activity genes after a two-week period of ultrasound stimulation. That is, the ultrasound treatment protocol is capable of reversing the effects of the immune response elicited by LPS. In one embodiment, the techniques of the present invention can track the expression of cytokine-active genes in tissues or samples collected from circulating fluids, and can use the expression of one or more cytokine-active genes as a representative marker of effective spleen neuromodulation to reduce inflammation or treat immune-related disorders as part of a treatment protocol.

Figures 9A to 9F show the RNA concentrations of various cytokines over one and two week periods in untreated rats, sham controls (i.e., rats that received LPS but not ultrasound stimulation) and rats that received LPS and ultrasound stimulation. Figure 9A shows Tumor Necrosis Factor (TNF) concentrations in untreated spleen, sham-control spleen, and sonicated spleen over a one-week period and a two-week period. FIG. 9B shows the concentration of interleukin 1 α in untreated spleen, sham-control spleen and ultrasonically-stimulated spleen over a one-week period and a two-week period. FIG. 9C shows the concentration of interleukin 6 in untreated spleen, sham-control spleen and ultrasonically-stimulated spleen over a one-week period and a two-week period. FIG. 9D shows the concentrations of C-C motif chemokine ligand 4 in untreated spleen, sham-control spleen and sonicated spleen over a one-week period and a two-week period. Figure 9E shows the concentration of interleukin 1 β in untreated spleen, sham-control spleen and sonicated spleen over a one-week period and a two-week period. FIG. 9F shows the concentrations of C-C motif chemokine ligand 20 in untreated spleen, sham-control spleen and sonicated spleen over a one-week period and a two-week period. As shown in fig. 9A to 9F, the ultrasonic stimulation over the two-week period showed lower RNA concentrations of the various cytokines compared to the concentrations of the various cytokines after the ultrasonic stimulation over the one-week period. As such, ultrasound stimulation over a two week period may alter gene expression levels as opposed to ultrasound stimulation over a one week period.

FIGS. 10A to 10G show RNA concentrations of various genes over one-week and two-week periods in untreated rats, sham controls (i.e., rats that received LPS but not sonicated), and LPS and sonicated rats. Figures 10A to 10G also show the ultrasound activation levels of various proteins at different ultrasound pressures. FIG. 10A shows RNA concentrations of mitogen-activated protein kinase 14(p38) in untreated spleen, sham-control spleen and sonicated spleen, as well as the level of sonication activation of the corresponding gene protein p38 with increasing amounts of sonication pressure, over one and two week periods. FIG. 10B shows the RNA concentration of ribosomal protein S6 kinase B1(p70S6K) in untreated spleen, sham spleen and sonicated spleen over a one-week period and a two-week period, and the level of sonication activation of the corresponding gene protein p70S6K with increasing amount of sonication pressure. FIG. 10C shows the RNA concentration of v-Akt murine thymoma virus oncogene homolog 1(Akt) in untreated spleen, sham spleen and sonicated spleen over a one week period and a two week period, and the level of sonication activation of the corresponding gene protein Akt with increasing amount of sonication pressure. FIG. 10D shows RNA concentrations of glycogen synthase kinase 3 β (GSK3B) in untreated spleen, sham-control spleen and sonicated spleen over one and two week periods, and the sonication activation level of the corresponding gene protein GSK3B with increasing amount of sonication pressure. FIG. 10E shows the RNA concentration of SRC proto-oncogene non-receptor tyrosine kinase (c-SRC) in the untreated spleen, sham-controlled spleen and sonicated spleen, as well as the level of sonication activation of the corresponding gene protein c-SRC with increasing amount of sonication pressure, over a one-week period and a two-week period. FIG. 10F shows RNA concentrations of nuclear factor of kappa light chain polypeptide gene enhancer (NF-. kappa.beta.) in B cells in untreated spleen, sham-control spleen and sonicated spleen, and the level of sonication activation of the corresponding gene protein NF-. kappa.beta.with increasing amount of sonication pressure over one and two week periods. FIG. 10G shows the RNA concentration of cytokine Signal transduction inhibitory factor 3(SOCS3) in the untreated spleen, sham-control spleen and sonicated spleen, and the sonication activation level of the corresponding gene protein SOCS3 with increasing amount of sonication pressure, for one-week and two-week periods. As shown in fig. 10A through 10G, the level of ultrasound activation (e.g., phosphorylation) of second messenger molecules is important to achieve the desired physiological results of ultrasound neuromodulation. However, the RNA expression levels of the various proteins do not appear to change based on the treatment period (e.g., one week period compared to a two week period).

Figure 11 shows the RNA concentration of the nicotinic cholinergic receptor alpha 4 subunit (CHRNA4) over a one-week period and a two-week period for untreated rats, sham controls and rats receiving LPS and ultrasound stimulation. As shown in fig. 11, it was found that the concentration of CHRNA4 indicated a decrease in cholinergic anti-inflammatory pathway (CAP) response (e.g., a decrease in systemic inflammation) over a one week period in rats receiving LPS in combination with ultrasound stimulation. However, it was found that in rats receiving LPS as well as ultrasound stimulation, the concentration of CHRNA4 correlated with the concentration of CHRNA4 present in sham controls over a two-week period. Thus, it was found that ultrasound stimulation over a two week period caused elevated CHRNA4 gene expression.

FIGS. 12A and 12B show transcriptome data for genes involved in the up-regulation of activated T cell proliferation (gene ontology: GO:0042104) and genes involved in the regulation of B cell activation (gene ontology: GO:0050864) over a one-week and two-week period in untreated rats, sham controls and rats receiving LPS and ultrasound stimulation. As shown in fig. 12A and 12B, it was found that the ultrasonic stimulation over the one-week period was insufficient to suppress the proliferative response to LPS, but it was found that the ultrasonic stimulation over the two-week period was sufficient to suppress the proliferative response to LPS.

FIGS. 13A and 13B show the transcriptome data for genes involved in glucan biosynthesis processes (e.g., formation of glucan and polysaccharides consisting of only glucose residues (gene ontology: GO:0009250)) and genes encoding transcription factor proteins over a one-week period and a two-week period in untreated rats, sham controls, and rats receiving LPS and ultrasound stimulation. As shown in fig. 13A and 13B, it was found that the ultrasonic stimulation over the one-week period was insufficient to suppress the response to LPS between multiple systems and/or molecular pathways, but that the ultrasonic stimulation over the two-week period was sufficient to suppress the response to LPS between multiple systems and/or molecular pathways.

FIG. 14 shows transcriptome data of genes involved in the ability to detect LPS over a one-week period and a two-week period in untreated rats, sham controls and rats receiving LPS and ultrasound stimulation. As shown in fig. 14, it was found that the ultrasonic stimulation over the one-week period was insufficient to change the expression of genes related to the ability to detect LPS (e.g., CEBPA, CEBPB, TLR4, CD14, and LBP), but that the ultrasonic stimulation over the two-week period was sufficient to change the expression of genes related to the ability to detect LPS.

Figure 15 shows the transcriptome data of genes related to the innate immune system, specifically the ability to detect and respond to LPS, over a one-week period and a two-week period in untreated rats, sham controls and rats receiving both LPS and ultrasound stimulation. As shown in FIG. 15, ultrasound stimulation over a one week period was found to be insufficient to alter expression of pro-inflammatory genes associated with the innate immune system (e.g., TNF, IL-1A, IL-1B), but ultrasound stimulation over a two week period was found to be sufficient to alter expression of genes associated with the innate immune system.

Fig. 16A to 16G show the RNA concentrations of acetylcholine-related genes for a one-week period and a two-week period in untreated rats, sham controls and rats that received LPS and ultrasound stimulation. FIG. 16A shows RNA concentrations of nicotinic cholinergic receptor alpha 4 subunit (CHRNA4) in untreated spleen, sham-control spleen and sonicated spleen over a one-week period and a two-week period. FIG. 16B shows choline O-transacetylase (CHAT) concentrations in untreated, sham-control, and sonicated spleens over one and two week periods. Figure 16C shows the concentration of solute carrier family 5 member 7(SLC5a7) in untreated spleen, sham control spleen and sonicated spleen over a one week period and a two week period. Figure 16D shows the concentration of acetylcholinesterase (catheteria type) (ACHE) in untreated spleen, sham spleen and sonicated spleen over a one week period and a two week period. Figure 16E shows the concentration of Butyrylcholinesterase (BCHE) in untreated spleen, sham-control spleen and sonicated spleen over a one-week period and a two-week period. Figure 16F shows the concentration of solute carrier family 18 member A3(SLC18A3) in untreated spleen, sham control spleen and sonicated spleen over a one week period and a two week period. As shown in fig. 16A, the expression level of acetylcholine receptor subunit CHRNA4 was found to initially decrease after one week of sonication, but then unexpectedly started to increase after a two week period of sonication. Interestingly, the cholinesterase BCHE shown in figure 16E hydrolyzes acetylcholine and rises after one week of stimulation and remains elevated for two weeks. As such, it was found that there was a difference in the effect and modulation of neural signal transduction, including CHRNA4 and BCHE, such as the cholinergic anti-inflammatory pathway (CAP) by ultrasound stimulation between stimulation over a two week period relative to stimulation over a one week period.

Figure 17A shows the RNA concentration of aquaporin 1 (korton blood type) over a one-week period and a two-week period in untreated rats, sham controls and rats receiving LPS and ultrasound stimulation. Figure 17B shows the concentration of aquaporin 3 (gill blood type) in untreated rats, sham controls and rats receiving LPS and ultrasound stimulation over a one-week period and a two-week period. Figure 18A shows the concentration of transmembrane protein 176A (TMEM176A) over a one-week period and a two-week period in untreated rats, sham controls and rats receiving LPS and ultrasound stimulation. Figure 18B shows the concentration of transmembrane protein 176B (TMEM176B) over a one-week period and a two-week period in untreated rats, sham controls and rats receiving LPS and ultrasound stimulation. TMEM176A and TMEM176B were associated with the immature state of dendritic cells. Figure 18C shows the transcriptome data for genes associated with TMEM176A and TMEM176B grouped according to cluster analysis over one and two week periods in untreated rats, sham controls and rats receiving LPS and ultrasound stimulation. As shown, the two week group was clustered with the control/untreated group, while the one week group was clustered with the control/sham group. Thus, based on recent matches, cluster analysis (e.g., rank order, k-means) can be used to identify gene expression profile features for one-week responses, two-week responses, untreated responses, and/or no responses (similar to control/sham groups).

Figure 19 shows the average concentration of circulating transient molecules (e.g., norepinephrine, acetylcholine, and tumor necrosis factor) relative to controls following ultrasound stimulation of the spleen, right adrenal gland, sacral ganglia, nodose or nucleus solitarius. As shown in figure 19, the increase in norepinephrine concentration produced an increase in acetylcholine concentration in the blood due to spleen ultrasound stimulation.

Modulating acetylcholine levels in the blood can affect a number of physiological processes. Intravenous injection of acetylcholine has been shown to reduce heart rate and cardiac output. Acetylcholine injection can cause vasodilation and lower blood pressure through stimulation of muscarinic acetylcholine M3 receptors on endothelial cells and smooth muscle cells lining the blood vessels. In the intestine, elevated levels of acetylcholine can stimulate intestinal motility and secretion. Endogenous acetylcholine is synthesized from choline by choline o-acetylase encoded by the CHAT gene. CHAT expression in the gastrointestinal tract is moderately high and is due to high innervation by sympathetic nerves. Within the spleen, RNA expression of the CHAT gene was below the detection limit and virtually zero, as shown in fig. 17B. The source of acetylcholine in plasma is mainly attributed to circulating mononuclear leukocytes (Fujii et al, Expression and Function of the Cholinergic System in Immune Cells, Frontiers in Immunology 8 (2017)). These circulating mononuclear leukocytes are in constant exchange with the Spleen (Adams et al, Exercise and Leukocyte exchange amino Central Circulation, Lung, Spleen, and Muscle, Brain, Behavior, and Immunity 25: 658-. For example, secretion of the protein C-C motif chemokine ligand 4(CCL4) in the spleen was used to transport T cells, a class of mononuclear leukocytes, through C-C chemokine receptor type 5(CCR5) expressed primarily on T cells, macrophages and dendritic cells. FIG. 9D shows that there is a difference in RNA expression of C-C motif chemokine ligand 4(CCL4) between one and two weeks of sonication, inferring a corresponding change in trafficking and exchange between spleen and circulating mononuclear leukocytes. These transport modulations will have a corresponding modulation of acetylcholine concentrations in plasma due to these circulating mononuclear leukocytes and the eventual downstream physiological changes due to changes in plasma acetylcholine levels.

In another embodiment, changes in gene expression associated with the profile resulting from neuromodulation may be used to alter the responsiveness of a patient to one or more other therapies. For example: the profile may be associated with an increase or decrease in expression of one or more cell surface receptors.

Technical effects of the invention include providing neuromodulation therapy to a subject over an extended period of time such that a desired acute response (e.g., release of a neurotransmitter or neuropeptide) to the neuromodulation therapy may be maintained while any undesirable chronic effects of the neuromodulation therapy may be minimized. One or more treatment parameters of the neuromodulation treatment of the subject may be determined and/or altered based on any desired effect on the subject after the first series of energy application events. For example, the treatment period of an energy application event, the treatment frequency of an energy application event within a treatment period, or the duration of a recovery period between subsequent neuromodulation treatment periods may be determined or altered based on the assessed effect of a first series of energy application events of the neuromodulation treatment. In certain embodiments, the desired effect is a change in the expression level of certain genes in the subject. In certain embodiments, the desired effect is no or minimal change in the expression levels of certain genes in the subject. As such, the neuromodulation techniques provided herein can optimize the treatment parameters of the neuromodulation therapy for the subject over an extended period of time, such that the desired acute response to the neuromodulation therapy can be maintained while minimizing any undesirable chronic effects of the neuromodulation therapy.

One skilled in the art recognizes that RNA sequencing can be performed to assess RNA expression of all transcripts, transcripts of messenger RNA, or target RNA transcripts. Furthermore, RNA expression can be measured by RNA sequencing, by reverse transcription polymerase chain reaction (rt-PCR) and even by microarray technology. In addition, RNA sequencing of individual cells isolated from a tissue sample may be performed with or without fluorescence activated cell sorting.

RNA expression measurements may be made at multiple times and at different times defined by the individual's biological cycle and rhythm, such as the circadian rhythm. However, RNA expression measurements may be limited and may be performed to confirm indirect measurements indicative of changes in tissue characteristics.

This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any 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 with insubstantial differences from the literal languages of the claims.