Decreasing infection after skin injuries utilizing non-invasive focused ultrasound

Abstract

The present approaches are generally directed to inducing an immunological response in a subject to decrease infection after incurring a skin injury, including thermal skin injuries typically associated with bacterial infection. A method for inducing an immunological response in a subject includes positioning an ultrasound energy application device (12) on the subject's skin in alignment with a target anatomic site including an internal organ. The method also includes applying pulsed focused ultrasound (pFUS) energy to the target anatomic site to treat an infection in the subject, wherein the target anatomic site is distal from a wound associated with the infection.

Description

DECREASING INFECTION AFTER SKIN INJURIES UTILIZING NON-INVASIVE FOCUSED ULTRASOUND CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to and the benefit of U.S. Provisional Application No. 63 / 755,135, filed on February 6, 2025, which is incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT

[0002] This invention was made with US Government support under contract number EGS # MT21010.007.E001 awarded by Medical Technology Enterprise Consortium (MTEC) and Naval Advanced Medical Development (NAMD). The Government has certain rights in the invention.TECHNICAL FIELD

[0003] The present disclosure is related to the use of applied energy, such as pulsed focused ultrasound (pFUS) to decrease infection that may occur after incurring a wound to the skin. More specifically, the present disclosure discloses approaches in which pulsed focused ultrasound is applied to target tissue, such as the spleen, to decrease bacteria load within a wound caused by a skin injury, thereby decreasing risk or severity of infection.BACKGROUND

[0004] The subject matter discussed in this section should not be assumed to be prior art merely as a result of its mention in this section. Similarly, a problem mentioned in this section or associated with the subject matter provided as background should not be assumed to have been previously recognized in the prior art. The subject matter in this section merely represents different approaches, which in and of themselves can also correspond to implementations of the claimed technology.

[0005] Skin injuries to the skin, such as burn wounds, are created when hot, solid objects, open flames, or hot gases or vapors (e.g., steam) contact the skin. Often burn wounds occur within an area of the skin that is not sterile / clean and / or exposure to infectious agents after wounding / during healing. The unsterile environment in the presence of the burn wound may lead to an infection (e.g., bacterial infection). When a bacterial infection is present, the body’s first response is inflammation, which causes fluid and immune cells to flood the burn wound area as a means of clearing the area of both bacteria and cellular debris. As part of that response, biochemicals are released from the immune cells to keep the immune system ramped up to fight the infection. Tn certain cases, the infection may lead to sepsis, which is a serious condition that may lead to organ failure and death. As such, bum patients are often associated with high mortality due to infection. While antibiotics are available, certain bacteria may be resistant to a particular antibiotic, or there may be irreparable damage to the body, which may have been incurred during the onset of sepsis (e.g., mild sepsis) within a patient. Accordingly, there is currently a need for treatments to efficiently clear bacteria from a wound and reduce the incidence of sepsis.BRIEF DESCRIPTION

[0006] A summary of certain embodiments disclosed herein is set forth below. It should be understood that these aspects are presented merely to provide the reader with a brief summary of these certain embodiments and that these aspects are not intended to limit the scope of this disclosure. Indeed, this disclosure may encompass a variety of aspects that may not be set forth below.

[0007] In one embodiment, a method of inducing an immunological response in a subject includes positioning an energy application device on the subject’s skin in alignment with a target anatomic site comprising an internal organ and applying pulsed focused ultrasound (pFUS) energy to the target anatomic site to treat an infection in the subject, wherein the target anatomic site is distal from a wound associated with the infection.

[0008] In one embodiment, a system of inducing an immunological response in a subject includes an energy application device. The system also includes a controller, wherein the controller is configured to communicate with the energy application device to cause acts to be performed including facilitating positioning of the energy application device on a subject’s skin in alignment with a target anatomic site using image data, wherein the target anatomic site comprises an internal organ. The controller is also configured to cause acts to be performed including applying, via the energy application device, pulsed focused ultrasound (pFUS) to energy to the target anatomic site to treat an infection in the subject, the target anatomic site is distal from a wound associated with the infection.

[0009] In one embodiment, a tangible, non-transitory, computer-readable medium, including instructions that, when executed by processing circuitry, cause the processing circuitry to perform acts including facilitating positioning of an energy application device on a subject’s skin in alignment with a target anatomic site using image data, wherein the target anatomic site comprises an internal organ. The processing circuitry may also perform acts including applying, via the energy application device, pulsed focused ultrasound (pFUS) energy to the target anatomic site to treat an infection in the subject, the target anatomic site is distal from a wound associated with the infection.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] These and other features, aspects, and advantages of the present subject matter 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:

[0011] FIG. 1 is a schematic representation of a neuromodulation system according to embodiments of the present disclosure;

[0012] FIG. 2 depicts a focused ultrasound system suitable for non-invasively applying focused ultrasound pulses to a target organ to facilitate a wound healing process, in accordance with aspects of the present disclosure;

[0013] FIG. 3 is a block diagram of a neuromodulation system according to embodiments of the present disclosure;

[0014] FIG. 4 depicts a stimulation site of focused ultrasound pulses and the relationship between a target organ, here depicted as a spleen, and skin through both blood and lymph circulation, in accordance with aspects of the present disclosure;

[0015] FIG. 5 graphically depicts change in colony-forming units (CFU) per wound by study group, in accordance with aspects of the present disclosure;

[0016] FIG. 6 is a flow diagram of a method for neuromodulation to decrease infection after skin injury, in accordance with aspects of the present disclosure; and

[0017] FIG. 7 is a flow diagram of a method of a neuromodulation technique for reducing bacteria load and occurrence of an infection corresponding to a skin injury, in accordance with aspects of the present disclosure.DETAILED DESCRIPTION

[0018] 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 wouldnevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill having the benefit of this disclosure.

[0019] Any examples or illustrations given herein are not to be regarded as restrictions on, limits to, or express definitions of, any term or terms with which they are utilized. Instead, these examples or illustrations are to be regarded as being described with respect to various particular embodiments and as illustrative only. Those of ordinary skill in the art will appreciate that any term or terms with which these examples or illustrations are utilized 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 that term or terms. Language designating such non-limiting examples and illustrations includes, but is not limited to, “for example”, “for instance”, “such as”, “e.g.”, “including”, “in certain embodiments”, “in some embodiments”, and “in one (an) embodiment.” All numerical values within the detailed description herein are modified by “about” the indicated value, and take into account experimental error and variations, manufacturing tolerances or constraints, and so forth that would be expected by a person having ordinary skill in the art. For example, “about” or “approximately” may refer to ±0.5%, ±1%, ±2, ±5%, ±10%, or ±15%.

[0020] When introducing elements of various embodiments of the present subject matter, the articles “a,” “an,” “the,” and “said” 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 nonlimiting, and thus additional numerical values, ranges, and percentages are within the scope of the disclosed embodiments.

[0021] Normal wound healing consists of four phases (hemostasis, inflammation, proliferation, and remodeling) and is similar for acute and chronic wounds, albeit with different timing. Failure at any one of these stages can lead to a non-healing wound. Hemostasis, consisting of a rapid migration of platelets, neutrophils, and fibrin deposition,causes the wound site to close by clotting. The inflammatory phase (early and late) starts with infiltration of the wound site by neutrophils (within 24-36 hours), whose main function is to prevent infection by phagocytosing bacteria / pathogens, foreign particles, damaged cells, and damaged tissue. As part of the late inflammatory phase (48 - 72 h after injury) the macrophage population becomes dominant after chemoattraction of monocytes via clotting factors, cytokines and chemokines. Macrophages also provide tissue growth factors such as Transforming Growth Factor (TGF)-P, TGF-a, epidermal growth factor (EGF), fibroblast growth factor (FGF) and collagenase which are required for activation of the cells involved in wound repair (including keratinocytes, fibroblasts, and vascular endothelial cells). Lymphocytes (T-cells) enter the wound site in the late inflammatory phase (72 h after injury) mediated by interleukin-1 (IL-1), complement system components such as C3 and C5, and immunoglobulin G (IgG) breakdown products. Classical macrophages (Ml) secrete proinflammatory cytokines which help recruit lymphocytes to the wound bed. Non-classical macrophages (M2) secrete anti-inflammatory cytokines which trigger the transition into the proliferation phase phenotype. The proliferative phase starts on or around the third day after wounding and lasts for about 2 weeks. It is characterized by migration of fibroblasts, keratinocytes, epithelial cells and vascular cells, collagen synthesis, adhesion, traction, and epithelization. Finally, the remodeling / maturation phase is responsible for development of new epithelium and scar tissue formation and can last 1-2 years. This phase also draws the wound together, much like the contraction of muscle cells, and forms a repaired extracellular matrix (ECM). Collagen is remodeled and the wound fully closes. The skin is generally at about 80% integrity until this phase is finished and is significantly weaker and prone to reinjury during this time. Any one of these stages can fail, although healing tends to become arrested in the inflammatory stage, never progressing to epithelialization and reconstruction.

[0022] In certain cases, the wound area may be located in an unsterile area, which may leave the wound site vulnerable to bacteria exposure, thereby increasing the chance of infection (e.g., sepsis) and further complicating the healing process. For example, unhealed burn wounds may increase the risk for sepsis, which can occur at various points during thehealing cascade. Long hospitalization for burn wounds is often correlated with infections caused by drug resistant bacterial and fungal strains. Thus, close monitoring, early identification of infection, and appropriate treatment are needed throughout the healing process to prevent sepsis and its consequences. As discussed herein, techniques involving starting pulsed focused ultrasound (pFUS) treatment less than or at about 24 hours after wound creation were explored to attenuate early inflammation through cholinergic antiinflammatory pathway (CAP) modulation, which in turn alters the healing timeline and facilitates a reduction in bacteria load in a wound site.

[0023] Close monitoring, early identification of infection, and appropriate treatment are needed throughout the healing process to prevent sepsis and its consequences. However, advancement in wound treatment and corresponding infections (e.g., bacterial infections) has been lacking for decades. The development of new technology and advancement in the treatment of wounds, such as reducing bacterial load in skin wounds to reduce infections and treatments of sepsis are needed. Conventional techniques to treat bacterial infections (or sepsis) include oral / intravenous (IV) antibiotics, cytokine biologies, peptides, anti-pd-L1 antibodies, modulation of the spleen by using devices to deliver therapeutic drugs locally to a region of the spleen, and the like. All of these mitigations are directed to nursing the infection directly but not treating a root cause, such as reducing bacterial load associated with the wound site and the body’s response to the bacterial load prior to a subject becoming septic.

[0024] Bioelectronic medicine is a rapidly evolving field. In practice, electrical stimulation may be applied to nerve fibers to elicit a response and in many cases an electrode is surgically implanted to deliver energy to the vagus nerve which then has a downstream effect. Vagus nerve stimulation can modulate inflammation via (CAP) signaling. CAP is a neural pathway that inhibits tumor necrosis factor-alpha (TNFa) production (as well as other pro-inflammatory cytokines) in the spleen, liver and heart when activated and further inhibits molecule production (e.g., TNFa) in circulating immune cells. This pathway requires both the vagus nerve and a7 receptors, and if eitherare disrupted, the anti-inflammatory response is reversed. Techniques are described herein that provide for less invasive intervention (e.g., non-invasive) that can be applied in a clinical or point of care setting (i.e., non-surgical) to alter the systemic response to inflammation and occurrence of infection by facilitating a reduction in bacteria load in a wound site of a wound.

[0025] The present disclosure is directed to systems and methods to non-invasively activate the CAP by applying pulsed focused ultrasound (pFUS) directly to a target organ (e.g., spleen) as a method to decrease likelihood of bacterial infection after incurring skin injuries / wounds (e.g., bum wounds, deep partial thickness wounds, thermal injuries, diabetic foot ulcers, skin tears, impaired skin integrity, lesions). The treatment may start around 24 hours or sooner after injury, which allows for the initial immune response to initiate. The neuromodulating energy from the pFUS activates the CAP, which is innervated by the vagus nerve through the celiac plexus and splenic nerve terminating in the spleen, thereby allowing the healing cascade to progress. In this way, the applied pFUS treatment alters the immune response at a distal wound site by stimulating one or more nerve pathways within the spleen. For example, the applied pFUS may enable the skin wound to progress past the inflammation stage more quickly such that the healing progress is not halted within a prolonged inflammation phase. It is believed that application of the pFUS to the spleen promotes the release of reservoir cells (e.g., macrophages, B-cells) and biochemicals (e.g., cytokines, chemokines) from the spleen to a wound bed of a skin wound, which has downstream effects such as altering cellular content within the wound bed to facilitate reduction of bacteria load within the wound, thereby reducing occurrence of infection. In particular, following ultrasound stimulation, the pFUS may stimulate norepinephrine release from the splenic nerve terminals. This may lead to an immune cell activation, wherein norepinephrine may act on adrenergic receptors (e.g., P2 adrenergic receptors (P2AR)) of certain T-cells to stimulate them to produce and release acetylcholine (ACh). The release of ACh by T-cells ACh binds to nicotinic acetylcholine receptors (e.g., alpha-7 nicotinic acetylcholine receptor) of macrophages, thereby promoting an antiinflammatory effect. That is, the binding of the alpha-7 nicotinic acetylcholine receptorinhibits macrophages from producing pro-inflammatory cytokines such as TNF-a, thereby reducing inflammation. Additionally and / or alternatively, norepinephrine may act directly on myeloid cells to inhibit cytokine production, thereby bypassing the processes described above in relation to T-cells (e.g., norepinephrine binding to B2AR of T-cells). In particular, following ultrasound stimulation, the pFUS may stimulate norepinephrine release from the splenic nerve terminals. This may lead to an immune cell activation, wherein norepinephrine may act on adrenergic receptors (e.g., 02 adrenergic receptors (02AR)) of certain T-cells to stimulate them to produce and release acetylcholine (ACh). The release of ACh by T-cells ACh binds to nicotinic acetylcholine receptors (e.g., alpha-7 nicotinic acetylcholine receptor) of macrophages, thereby promoting an anti-inflammatory effect. That is, the binding of the alpha-7 nicotinic acetylcholine receptor inhibits macrophages from producing pro-inflammatory cytokines such as TNF-a, thereby reducing inflammation. Additionally and / or alternatively, norepinephrine may act directly on myeloid cells to inhibit cytokine production, thereby bypassing the processes described above in relation to T-cells (e.g., norepinephrine binding to 02AR of T-cells). Thus, in the event the skin wound is exposed to bacteria, neuromodulation of the spleen may alter the systemic response to inflammation at the skin wound site by mitigating the onset of an advanced bacterial infection, which advantageously enables the prevention / reduces the risk of sepsis in a subject. Furthermore, application of pFUS to the spleen enables untargeted wound healing (i.e., energy is not directly applied to the site of the wound) given wounds may be located distally (e.g., away) from the stimulation site. In this way, the present techniques enable untargeted healing of wounds (e.g., thermal wounds) that may be present anywhere on the body. Although examples discussed herein generally relate to applying ultrasound pulses to a spleen, it should be understood that the target organ may be any organ capable of modulating inflammation via the CAP signaling in response to non-invasive application of pulsed ultrasound energy.

[0026] With the preceding in mind, FIG. 1 is a schematic representation of a system 10 for neuromodulation. While the depicted elements of the system 10 are shown separately, it should be understood that some or all of the elements may be combined with one another.Further, some or all of the elements may communicate in a wired or wireless manner with one another.

[0027] The neuromodulation system 10 may be used to achieve neurotransmitter release and / or activate components (e.g., the presynaptic cell, the postsynaptic cell) of a synapse in response to an application of energy (e.g., ultrasound energy). The illustrated system 10 includes an energy application device 12 (e.g., an ultrasound therapy probe) coupled to a pulse generator 14. In certain embodiments, the pulse generator 14 may be an extracorporeal device, e.g., may operate to apply energy transdermally or in a noninvasive manner from a position outside of a subject’s body. The energy application device 12 is configured to receive energy pulses from the pulse generator 14, e.g., via leads or wireless connection. During operation, the energy pulses are directed to a region of interest 16 of an internal tissue or organ of a subject (e.g., a peripheral tissue), which in turn results in a distal physiological outcome, such as an untargeted wound healing response in a skin wound and / or reduction in bacteria load within the skin wound. The physiological outcome may include a local and / or systemic change in concentration of a biologically active molecule, process, or function. For example, for the treatment of a skin wounds including, but not limited to, burn wounds, deep partial thickness wounds, thermal injuries, skin tears, diabetic foot ulcers, lesions, impaired skin the applied energy pulses may result in changes to a concentration of pro-inflammatory molecules and / or immune cells in the wound bed of the skin wound and / or the spleen.

[0028] In certain embodiments, the energy application device 12 and / or the pulse generator 14 may communicate wirelessly, for example with a controller 18 that may in turn provide instructions to the pulse generator 14. In other embodiments, the pulse generator 14 may be integrated within the controller 18. In embodiments in which the pulse generator 14 is extracorporeal, the energy application device 12 may be operated by a caregiver (e.g., sonographer) and positioned at a spot on or above a subject’s skin such that the energy pulses are delivered transdermally to a desired internal tissue / region of interest (e.g., a peripheral tissue that includes one or more peripheral axon terminals). Forexample, the regions of interest within organs containing either blood vessels, nerves, or other anatomical / visual landmarks may be spatially selected in image data to facilitate positioning and used to identify areas with specific axon terminals and synapses. Such visual landmarks may include the hilum of the spleen that includes one or more nerve pathways and / or blood vessels. Once the energy application device 12 is positioned to apply energy pulses to the desired site (e.g., region of interest 16), the system 10 may initiate neuromodulation of one or more target internal sites (e.g., one or more nerve pathways) of any portion of the spleen and / or the hilum of the spleen to achieve a targeted physiological outcome or clinical effect(s), such as treatment of a wound on the subject’s skin (e.g., thermal wounds) to reduce bacteria load and reduce incidence of infection (e.g., treat / prevent sepsis). It should be noted that any portion of the spleen may be stimulated, as the spleen exhibits a homogenous response when stimulated via pFUS.

[0029] In certain embodiments, the system 10 may include an assessment device 20 coupled to the controller 18. The assessment device 20 may assess characteristics that are indicative of whether the targeted physiological outcome(s) of the neuromodulation have been achieved. For example, the targeted physiological outcomes may include, but are not limited to, tissue displacement, tissue structure / size changes, a change in concentration of one or more molecules (either local, non-local, or circulating concentration), a change in gene or marker expression, increased fluid movement, afferent activity, and cell migration, etc. For example, tissue displacement (e.g., a blood vessel displacement of an adjacent artery) may occur as a result of energy application to the tissue. By assessing the tissue displacement to the spleen or lymph node (e.g., via imaging), other effects may be estimated. For example, a certain displacement may be characteristic of a particular change in molecule concentration. Accordingly, the targeted physiological outcome may be a goal of the treatment. For example, the targeted physiological outcome may include secretion of reservoir cells (e.g., monocytes, macrophages, neutrophils) and / or biomarkers (e.g., chemokines, cytokines) from the spleen to the wound bed of the skin wound, cell viability, and / or cell stability.

[0030] Additionally, or alternatively, the modulation may result in systemic or non-local changes, and the targeted physiological outcome may be related to a change in concentration of circulating molecules or a change in a characteristic of a tissue that does not include the region of interest 16 to which energy was directly applied. For example, application of energy to the spleen may alter migration of immune cells (e.g., monocytes, macrophages, neutrophils) and / or biomarkers to the wound bed of a wound. In another example, the displacement may be a proxy measurement for a desired modulation, and displacement measurements below an expected displacement value may result in modification of modulation parameters until an expected displacement value is induced. Accordingly, the assessment device 20 may be configured to assess concentration changes of a molecule or molecules of interest in some embodiments. In certain embodiments, the assessment device 20 may be an imaging device configured to assess changes in organ size and / or position, as well as changes in tissue characteristics. Further, in another embodiment, the assessment device 20 may assess local temperature rise of tissue, which may be detected using a separate temperature sensor or ultrasound imaging data from the energy application device 12. Assessment of speed of sound differences may be detected through difference imaging techniques pre / during / post therapy.

[0031] Based on the assessment from the assessment device 20, the modulation parameters of the controller 18 may be altered such that an effective amount of energy is applied. For example, if a desired modulation is associated with a change in concentration of a molecule of interest (e.g., circulating concentration or tissue concentration of one or more molecules) within a defined time window (e.g., 5 minutes, 30 minutes after a procedure of energy application starts) or relative to a baseline measurement at the start or before initiation of a procedure, a change of the modulation parameters such as pulse frequency or other parameters may be indicated, which in turn may be provided to the controller 18. The desired change in modulation parameters may be provided to the controller either by an operator or via an automatic feedback loop, for defining or adjusting the energy application parameters or modulation parameters of the pulse generator 14 until the modulation parameters result in an effective amount of energy being applied.

[0032] The system 10 as provided herein may provide energy pulses according to various modulation parameters. For example, the modulation parameters may include various stimulation time patterns, ranging from continuous to intermittent. With intermittent simulation, energy is delivered for a period of time at a certain frequency during a signal-on time. The signal-on time is followed by a period of time with no energy delivery, referred to as signal-off time. The modulation parameters may also include frequency and duration of a stimulation application. The application frequency may be delivered at various time periods, for example, within a day, a week, two weeks, and so forth. The treatment duration may last for various time periods, including but not limited to, from a few minutes (e.g., three minutes), such as about three minutes, about five minutes, about 10 minutes to about several hours. In certain embodiments, treatment duration with a specified stimulation pattern may last for about 1 minute, about 3 minutes, about 5 minutes, about 7 minutes, about 10 minutes, repeated at, e.g., 24-hour intervals (i.e., daily), 48-hr intervals (i.e., every other day), and so forth. In certain embodiments, treatment may be delivered at a higher frequency, say every three hours, for shorter durations, for example, 30 minutes. The application of energy, in accordance with modulation parameters, such as treatment duration and frequency, may be adjustably controlled to achieve a desired result. It should be noted in certain embodiments, the present techniques may be utilized to facilitate and reduce infection that may be associated with thermal wounds and / or chronic wounds such as diabetic foot ulcers (DFU), pressure ulcers (bed sores), leg ulcers, or other wounds that are otherwise resistant to healing or slow to heal.

[0033] Turning to FIG. 2, cutaneous wound healing was accelerated and bacteria load within the wound was reduced by using a pulsed focused ultrasound system to stimulate nerve pathways within the spleen (as illustrated below in FIG. 4 below). It should be noted that the components and devices illustrated and discussed with respect to FIG. 2 may be incorporated into the neuromodulation system of FIG. 1, and thus, certain components of FIG. 2 may be referred to interchangeably with components of FIG. 1. Additionally and / or alternatively, the system of FIG. 2 may be modified to include one or more of components of the system of FIG. 1 described above (e.g., assessment device 20).

[0034] FIG. 2 illustrates aspects of a focused ultrasound system 50 used to perform the techniques discussed herein. In one embodiment the system 50 comprises a 1.1 MHz, High Intensity Focused Ultrasound (HIFU) transducer 52 (e.g., energy application device 12) and matching network 54, an RF power amplifier 56, and a function generator 58, which together may function as the pulse generator 14. In this example the function generator 58 produces a pulsed sinusoidal waveform. This pulsed sinusoidal waveform is amplified by the RF power amplifier 56 and sent to the impedance-matching network 108 connected to the transducer 52. In certain embodiments the pulse center frequency was 1.1 MHz. In one embodiment, controller 18, such as one or more processors configured to process executable code or one or more application specific integrated circuits (ASICs) may be provided as part of (or in communication with) one or more of the function generator 58, RF power amplifier 56, matching network 54, and / or HIFU transducer 52 so as to allow directed energy application to a target organ as described herein, such as at the intervals, energies, and / or durations described herein to stimulate wound healing.

[0035] In one implementation the HIFU transducer 52 is a 70-mm diameter HIFU transducer having a spherical face with a 65-mm radius of curvature. In one such embodiment the transducer depth of focus is between 50 mm and 80 mm, such as 65 mm. In one embodiment the numerically simulated pressure profile has a full width at half amplitude of 1.8 mm laterally and 12 mm in the depth direction. The HIFU transducer 52 may be acoustically coupled to the subject through a standoff, such as a 6-cm-tall plastic cone filled with degassed water, and acoustical coupling gel.

[0036] With the foregoing in mind, FIG. 3 is a block diagram of certain components of the system 10 and focused ultrasound system 50 of FIGS. 1 and 2, respectively. As provided herein, the system 10 and / or focused ultrasound system 50 for neuromodulation may include a pulse generator 14 that is adapted to generate a plurality of energy pulses for application to a tissue of a subject. The pulse generator 14 may be separate or may be integrated into an external device, such as the controller 18. The controller 18 includes a processor 82 for controlling the device. Software code or instructions are stored in thememory 84 of the controller 18 for execution by the processor 82 to control the various components of the device (e.g., pulse generator 14). The controller 18 and / or the pulse generator 14 may be connected to the energy application device 12 via one or more leads 80 or wirelessly.

[0037] The controller 18 also includes a user interface with input / output (I / O) circuitry 86 and a display 88 that are adapted to let a clinician provide selection inputs or modulation parameters to modulation programs. Each modulation program may include one or more sets of modulation parameters including pulse amplitude, pulse width, pulse frequency, etc. The pulse generator 14 modifies its internal parameters in response to the control signals from the controller 18 to vary the stimulation characteristics of energy pulses transmitted through lead 80 to a subject to which the energy application device 12 is applied. Any suitable type of pulse generating circuitry may be employed, including but not limited to, constant current, constant voltage, multiple-independent current or voltage sources, etc. The energy applied is a function of the current amplitude and pulse width duration. The controller 18 permits adjustably controlling the energy by changing the modulation parameters and / or initiating energy application at certain times or cancelling / suppressing energy application at certain times.

[0038] In one embodiment, the adjustable control of the energy application device 12 is based on information about the concentration of one or more molecules in the subject (e.g., a circulating molecule such as a biomarker associated with immune function or a population of immune cells and / or wound bed markers). For example, change in wound be markers (e.g., wound bed cells) may be determined using techniques such as flow cytometry and / or changes in cytokines as measured using antibody -based detections (e.g., enzyme-linked immunosorbent assay (ELISA) or other biochemical analyses). If the information is from the assessment device 20, a feedback loop may drive the adjustable control. For example, if pro-inflammatory markers (e.g., TNFa, TL-6), as measured by the assessment device 20, is above a predetermined threshold or range, the controller 18 may initiate energy application to a region of interest and with modulation parameters that areassociated with a change in the biomarker. The initiation of energy application may be triggered by the biomarker drifting above or below a predetermined (e.g., a pre-configured or protocol-specified) threshold or outside a predefined range. In another embodiment, the adjustable control may be in the form of altering modulation parameters when an initial application of energy does not result in an expected change in a targeted physiological outcome (e.g., concentration of a molecule of interest) within a predetermined time frame (e.g., 1 hour, 2 hours, 4 hours, 1 day).

[0039] In one embodiment, the memory 84 stores different operating modes that are selectable by the operator. For example, the stored operating modes may include instructions for executing a set of modulation parameters associated with a particular treatment site. Different sites may have different associated modulation parameters. Rather than having the operator manually input the modes, the controller 18 may be configured to execute the appropriate instruction based on the selection of the operator.

[0040] In another embodiment, the memory 84 stores operating modes for different types of procedures or treatment. For example, activation may be associated with a different stimulating pressure or frequency range relative to those associated with depressing or blocking tissue function. In a specific example, when the energy application device 12 is an ultrasound transducer, the time-averaged power (temporal average intensity) less than about 500 mW / cm2in the region of interest to avoid levels associated with thermal damage and ablation / cavitation (i.e., non-thermal and non-ablative). The selected frequencies may depend on the mode of energy application (e.g., ultrasound or mechanical actuation). The controller 18 may be capable of operating in a validating mode to acquire a treatment position, and the treatment position may be implemented as part of a treatment operating mode that executes a treatment protocol (e.g., energy application) when the energy application device 12 is positioned at the treatment position. In another embodiment, the memory 84 stores a calibration or setting mode that permits adjustment or modification of the modulation parameters to achieve a desired result. In one example, the stimulation starts at a lower energy parameter and increases incrementally, either automatically or uponreceipt of an operator input. In this manner, the operator may achieve tuning of the induced effects as the modulation parameters are being changed.

[0041] The system 10 may also include an imaging device that facilitates focusing the energy application device 12. In one embodiment, the imaging device may be integrated with or the same device as the energy application device 12 such that different ultrasound parameters (frequency, aperture, or energy) are applied 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, the memory 84 stores one or more targeting or focusing modes that is used to spatially select the region of interest within an organ or tissue structure. Spatial selection may include selecting a subregion of an organ to identify a volume of the organ that corresponds to a region of interest. Spatial selection may rely on image data as provided herein. Based on the spatial selection, the energy application device 12 may be focused on the selected volume corresponding to the region of interest. For example, the energy application device 12 may be configured to first operate in the targeting mode and / or the validating mode to capture image data to be used for identifying the region of interested and associated treatment position (e g., a position of the energy application device 12 configured to apply energy to the area of interest). The targeting mode and / or validating mode energy is not at levels and / or applied with modulation parameters suitable for preferential activation at the region of interest. However, once the region of interest is identified and targeted, the controller 18 may then operate in a treatment mode according to the modulation parameters associated with preferential activation or achieving other targeted physiological outcomes.

[0042] The controller 18 may also be configured to receive inputs related to the targeted physiological outcomes as an input to the selection of modulation parameters. For example, when an imaging modality is used to assess a tissue characteristic, the controller 18 may be configured to receive or generate a calculated index or parameter of the characteristic. Based on whether the index or parameter is above or below a predefined threshold, a diagnosis may be made, and an indication of the diagnosis may be provided(e.g., via display 88). Additionally, or alternatively, the modulation parameters may be modified based on whether the index or parameter is above or below a predefined threshold. In one embodiment, the parameter can be a measure of tissue displacement of the affected tissue or a measure of depth of the effected tissue. Other parameters may include assessing a concentration of one or more molecules of interest (e.g., assessing one or more of a change in concentration relative to a threshold or a baseline / control, a rate of change, determining whether concentration is within a desired range, etc.).

[0043] In another implementation, a desired modulation parameter set may also be stored by the controller 18 (e.g., via memory 84). In this manner, subject-specific parameters may be determined. Further, the effectiveness of such parameters may be assessed over time. If a particular set of parameters is less effective over time, the subject may be developing insensitivity to activated pathways. If the system 10 includes an assessment device 20, the assessment device 20 may provide feedback to the controller 18. In certain embodiments, the feedback may be received from a user of an assessment device 20 indicative of a characteristic of the target physiological outcome. The controller 18 may be configured to cause the energy application device to apply energy according to modulation parameters and to dynamically adjust the modulation parameters based on the feedback. For example, based on the feedback, the processor 82 may automatically alter the modulation parameters (e g., the frequency, amplitude, or pulse width of an ultrasound beam or mechanical vibration) in real time and responsive to feedback from the assessment device 20.

[0044] The disclosed techniques may be used in assessment of neuromodulation effects, which in turn may be used as an input or a feedback for selecting or modifying neuromodulation parameters. The disclosed techniques may use direct assessments of tissue condition or function as the targeted physiological outcomes. The assessment may occur before (i.e., baseline assessment), during, and / or after the neuromodulation.

[0045] Images from the assessment techniques may be received by the system for automatic or manual assessment. Based on the image data, the modulation parameters may also be modified. For example, a change in organ size or displacement may be utilized asa marker of local neurotransmitter concentration, and used as a surrogate marker for exposure of local cells to phenotype modulating neurotransmitters, and effectively as a marker of predicted effect on immune pathways. The local concentration may refer to a concentration within a field of focus of the energy application.

[0046] The assessment techniques may include protein and / or marker concentration assessment. For example, the system may assess the presence or concentration of one or more molecules or cells in the tissue or circulating in the blood (e.g., with or without the assessment device 20). The concentration in the tissue may be referred to as a local concentration or resident concentration. For example, circulating biomarkers in the blood may be acquired by an intravenous (IV) puncture, and the assessment of the presence or levels of molecules / markers of interest may be performed by any suitable technique known to one of ordinary skilled in the art. As provided herein, the assessment may be based on a total white blood cell counts (WBC) along with specific cell counts for neutrophils (NE), lymphocytes (LV), monocytes (MO), and basophils (BA) representing subsets of the white blood cell populations. Immune markers of interest may include, but are not limited to, cytokines (e.g., TNF-a, IL-ip, IL-6, IL-10). In certain embodiments, tissue biomarkers may be collected directly from the wound to assess for wound bed markers (e.g., immune cells) via fine needle aspiration. For example, samples may be taken at the terminal time point in which the wound bed is harvested / resected for either biochemical analysis or histology.

[0047] As discussed herein, the energy application device 12 (e.g., an ultrasound transducer) may operate under control of the controller 18 to: (a) acquire image data of a tissue that may be used to spatially select a region of interest within the target tissue, (b) apply the modulating energy to the region of interest, and (c) acquire an image to determine that the targeted physiological outcome associated with a change in a characteristic of interest has occurred (e.g., determine that a change in a CAP molecule has occurred via a displacement measurement). In such an embodiment, the imaging device, the assessment device 20, and the energy application device 12 may be the same device.

[0048] The desired target tissue that includes the region of interest 16 (see FIG. 1) may be an internal tissue or an organ that includes synapses of axon terminals and non-neuronal cells. The synapses may be stimulated by direct application of energy to the axon terminals within a field of focus or focal zone of the energy application device 12 (e.g., ultrasound transducer) focused on the region of interest 16 of the target tissue to cause release of molecules into the synaptic space, e.g., the release of neurotransmitters and / or the change in ion channel activity in turn causes downstream effects. The region of interest 16 may be selected to include a certain type of axon terminal, such as an axon terminal of a particular neuron type and / or one that forms a synapse with a certain type of non-neuronal cell. Accordingly, the region of interest 16 may be selected to correspond to a portion of the target tissue with the desired axon terminals (and associated non-neuronal cells). The energy application may be selected to preferentially trigger a release of one or more molecules such as neurotransmitters from the nerve within the synapse or directly activate the non-neuronal cell itself through direct energy transduction (i.e., mechanotransduction or voltage-activated proteins within the non-neuronal cells), or cause an activation within both the neural and non-neuronal cells that elicits a desired physiological effect. The region of interest 16 may be selected as the site of nerve entry into the organ. In one embodiment, splenic stimulation or modulation may refer to a modulation of the region of interest 16 (e.g., spleen).

[0049] The energy may be focused or substantially concentrated on a region of interest 16 and to only part of the internal tissue, e.g., less than about 50%, 25%, 10%, or 5% of the total volume of the tissue. That is, the region of interest 16 may be a sub-region of the internal tissue or organ in question. In one embodiment, the energy is applied to only about l%-50% of the total volume of the tissue, to only about 1 %-25% of the total volume of the tissue, to only about 1%- 10% of the total volume of the tissue, or to only about l%-5% of the total volume of the tissue. In certain embodiments, only an axon terminal in the region of interest 16 of the target tissue would directly receive the applied energy and release neurotransmitters while the unstimulated axon terminals outside of the region of interest 16 do not receive substantial energy and, therefore, are not activated / stimulated in the samemanner. In some embodiments, axon terminals in the portions of the tissue directly receiving the energy would induce an altered neurotransmitter release. In this manner, tissue subregions may be targeted for neuromodulation in a granular manner, e.g., one or more subregions may be selected. In some embodiments, the energy application parameters may be chosen to induce preferential activation of either neural or non-neuronal components within the tissue directly receiving energy to induce a desired combined physiological effect. In other embodiments, the energy application device 12 may be swept across the region of interest 16. In certain embodiments, the energy may be focused or concentrated within a volume of about 25 mm3-500 mm3. However, other focal volumes are also contemplated based on desired physiological outcomes. A focal volume and a focal depth for focusing or concentrating the energy within the region of interest 16 may be influenced by the size and / or configuration of the energy application device 12. The focal volume of the energy application may be defined by the field of focus or focal zone of the energy application device 12. It should be noted that the disclosed embodiments may be performed using with a large ultrasound abdominal probe, small parts ultrasound probe with a shallow focus, or a small parts ultrasound probe with a deep focus, respectively.

[0050] As provided herein, the energy may be substantially applied only to the region or regions of interest 16 to preferentially activate the synapse in a targeted manner to achieve targeted physiological outcomes. Accordingly, in certain embodiments, only a subset of a plurality of different types of axon terminals in the tissue is exposed to the direct energy application.

[0051] In certain embodiments, the pFUS energy may exhibit a carrier frequency ranging from about 0.1 to about 10.0 MHz, such as about 0.5 to about 9.5 MHz, about 1 to about 9 MHz, about 1.5 to about 8.5 MHz, about 2 to about 8 MHz, about 2.5 to about 7.5 MHz, about 3 to about 7 MHz, about 3.5 to about 6.5 MHz, about 4 to about 6 MHz, about 4.5 to about 5.5 MHz, about 0.5 to about 1.5 MHz, such as about 0.5, about 1, about 1.5, about2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10.0 MHz.

[0052] In certain embodiments, the pFUS energy may exhibit a pulse duration ranging from about 100 to about 500 ps, about 125 to about 475 ps, about 150 to about 450 ps, about 175 to about 425 ps, about 200 to about 400 ps, about 225 to about 375 ps, about 250 to about 350 ps, about 275 to about 325 ps, about 190 to about 210 ps, such as about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, or about 500 ps.In certain embodiments, the pFUS energy may exhibit a pulse repetition interval ranging from about 50 to about 1000 ms, about 100 to about 900 ms, about 200 to about 800 ms, about 300 to about 700 ms, about 400 to about 600 ms, about 190 to about 210 ms, such as about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, about 525, about 550, about 575, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1000 ms.

[0053] In certain embodiments, the pFUS energy may exhibit a pulse repetition frequency ranging from about 1.0 to about 20 Hz, about 1 to about 19 Hz, about 2 to about 18 Hz, about 3 to about 17 Hz, about 4 to about 16 Hz, about 5 to about 15 Hz, about 6 to about 14 Hz, about 7 to about 13 Hz, about 8 to about 12 Hz, about 9 to about 11 Hz, about 3 to about 7 Hz, such as about 1, about 3, about 5, about 7, about 9, about 11, about 13, about 15, about 17, or about 19 Hz.

[0054] In certain embodiments, the pFUS energy may exhibit a derating factor ranging of about -0.3 dB / MHz / cm. In certain embodiments, the pFUS energy may exhibit a derated peak negative pressure ranging from about 0.1 to about 7 MPa, about 1 to about 6 MPa, about 2 to about 5 MPa, about 3 to about 4 MPa, about 1.5 to about 2.5 MPa, such as about0.1, about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, or about 7 MPa.

[0055] In certain embodiments, the pFUS energy may exhibit a mechanical index less than or equal to 1.9. In certain embodiments, the pFUS energy may exhibit a derated pulse intensity integral ranging from about 20 to about 200 mJ / cm2, about 50 to about 175 mJ / cm2, about 75 to about 150 mJ / cm2, about 100 to about 125 mJ / cm2, about 20 to about 30 mJ / cm2, such as about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 150, about 175, or about 200 mJ / cm2.

[0056] In certain embodiments, the pFUS energy may exhibit a derated spatial peak pulse average intensity ranging from about 50 to about 700 W / cm2, about 100 to about 650 W / cm2, about 150 to about 600 W / cm2, about 200 to about 400 W / cm2, about 250 to about 350 W / cm2, about 125 to about 150 W / cm2, such as about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, or about 700 W / cm2.

[0057] In certain embodiments, the pFUS energy may exhibit a derated spatial peak temporal average intensity ranging from about 50 to about 720 W / cm2, about 100 to about 650 W / cm2, about 150 to about 600 W / cm2, about 200 to about 400 W / cm2, about 250 to about 350 W / cm2, about 125 to about 150 W / cm2, such as about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, or about 700 W / cm2. For example, the derated spatial peak temporal average intensity may be less than about 720 mW / cm2.

[0058] In certain embodiments, the pFUS energy may exhibit a radiation force ranging from about 5 to about 100 mN / cm3, about 10 to about 90 mN / cm3, about 20 to about 80 mN / cm3, about 30 to about 70 mN / cm3, about 40 to about 60 mN / cm3, about 10 to about 15 mN / cm3, such as about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, or about 80 mN / cm3.

[0059] In certain embodiments, the pFUS energy may be focused or concentrated within a volume of about 25 mm3to about 500 mm3, about 30 to about 400 mm3, about 40 to about 300 mm3, about 50 to about 200 mm3, about 60 to about 100 mm3, about 30 to about 50 mm3, such as about 25, about 30, about 35, about 40, about 45, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, or about 400 mm3.

[0060] Turning to FIG. 4, the stimulation site and the relationship between the spleen and skin through both blood and lymph circulation are depicted. In general, splenic pulsed focused ultrasound alters immune cell (e g., monocytes / macrophages / neutrophils) migration to the wound bed, and in turn, modifies the wound so that it is more amendable to healing through systemic immune response mediation and altered concentrations of systemic circulating pro-inflammatory molecules (such as TNFa, IL-6).

[0061] FIG. 4 illustrates an abdomen showing the location of the spleen 100 and delivery location of pulsed focused ultrasound via energy application device 12 is depicted. When non-invasive ultrasound energy is applied to the spleen 100 cytokine production is modified, as discussed herein. Splenic lymph nodes 102, and splenic arteries 104 and veins 106 are shown in the expansion. Splenic lymph nodes 102 connect to the lymphatic vasculature, which connects the entire lymphatic system, including lymphatics in the skin. Skin resident macrophages release chemoattractant to recruit systemic immune cells through extravasation. When skin (which may be understood herein to be the epidermis, or the epidermis and dermis in conjunction) is wounded (depicted as wounded skin 108 distal to the stimulation site), vessels surrounding the area deliver platelets to achieve homeostasis followed by neutrophils within 1 hour to initiate inflammation and start the healing process. Monocytes arrive next and differentiate into Ml macrophage. Efferent lymph vessels help to drain the area of protein rich inflammatory fluid from the interstitium while afferent vessels help supply the area with cytokines and lymphatic leukocytes to aid in healing.

[0062] The energy application device 12 may be used to apply / deliver ultrasound push pulses to activate the CAP in the spleen 100. In general, the CAP is part of a feedback loop that helps prevent the immune system from overreacting. Physiologically, this pathway is innervated by the vagus nerve through the celiac plexus and splenic nerve terminating in the spleen 100. Accordingly, application of energy to the spleen 100 elicits a systemic response (e.g., peripheral response, distal response) such that biomarkers and / or reservoir cells may migrate from the spleen 100 to a distal wound bed of a wound to reduce bacterial load within the wound bed of the bum wound.

[0063] Ultrasound treatment was applied daily or every other day for three minutes per treatment. In some embodiments, the ultrasound treatment was applied consecutively for five days per week (10 min / day) and repeated as indicated by a respective treatment protocol. However, in other implementations, different schedules may be employed. By way of example, the treatment may be applied every day, every other day, every three days, etc. In certain embodiments, the energy application device 12 may be used to target the region of interest (e.g., any portion of the spleen 100) in an unfocused manner by sweeping the energy application device 12 across the spleen 100 during the treatment protocol. In other embodiments, the energy application device 12 may be used to target the region of interest (e.g., a portion of the spleen 100) such that the energy application device 12 is focused on the portion of the spleen 100 during the treatment protocol. Accordingly, the disclosed systems and methods described herein advantageously keep the immune response in balance.

[0064] Splenic CAP stimulation was performed via pFUS to modulate the immune system distally from the burn wound site, and its effects were evaluated in a rat model using Sprague Dawley rats. Two groups of eight animals (e.g., each group had four sham controls (sham) and four ultrasound stimulation (US)) were utilized for the study. Burn wounds were created via a burn exposure to create scab-like wounds. The burns were created with a brass bar heated in boiling water. The bar was heated to about 87°C to about 100°C. The bar was subsequently removed from the water and blotted so that the skin wasnot scalded with water or steam. The burn wounds were created by exposing the bar to the rats for 20 seconds to cause a deep partial-full thickness wound. Upon tissue harvest at two days post injury and / or 24 hours after first pFUS treatment, injuries were observed on the hypodermis or subcutaneous tissue. Staphylococcus aureus (e.g., S. aureus bacteria, staph) was cultured and utilized as the bacteria for the experiment given its prevalence on the skin. Each wound bed of the bum wounds were exposed to 20,000 colony-forming units (CFU) following the wounding procedure, which was delivered via a subcutaneous injection. Ultrasound stimulation (US) or sham controls (sham) began approximately 24 hours ± 1 hour (e.g., about 23 to about 25 hours) following the wounding procedure and exposure to the bacteria. The spleens 100 were exposed to US energy for three minutes each weekday for a total of nine treatments. In the case of sham controls, the transducer was placed over the spleens 100, but no energy was applied. On day 14, wound beds were harvested and incubated in 2 milliliters (mL) of phosphate-buffer saline (PBS) buffer to release the bacteria. A small amount of the PBS was plated on bacteria culture media and incubated overnight. The colonies on each culture plate were counted, the results of which are discussed in FIG. 5 below.

[0065] The experimental studies described herein with the Sprague Dawley rats were performed using GE Healthcare L8- 18i with the following pFUS parameters:Carrier frequency: 1.1 MHzPulse duration: 200 nsPulse repetition interval: 200 ms (Pulse repetition frequency of 5 Hz)Derating factor: -0.3 dB / MHz / cmDerated peak negative pressure: 1.95 MPaMechanical Index: 1.9Derated Pulse Intensity Integral: 28.1 mJ / cm2Derated Spatial Peak Pulse Average Intensity: 141 W / cm2Derated Spatial Peak Temporal Average Intensity: 141 mW / cm2Radiation force: 13.9 mN / cm3Axial -3dB Beamwidth: 12 mmAzimuth -3dB Beamwidth: 2 mmElevation -3dB Beamwidth: 2 mmFocus volume: 40 mm3

[0066] Preclinical studies demonstrate that non-invasive splenic PFUS reduced bacteria at the wound site 14 days after skin injury and exposure to 20,000 CFU S. aureus bacteria. FIG. 5 graphically depicts change in colony-forming units (CFU) per wound by study group. There were significantly less bacterial colonies (p value < 0.05) on the wounds in animals that received ultrasound stimulation to spleens 100 compared to the sham controls. In particular, the graph demonstrates that there is surprisingly an 8X reduction in bacteria in the US group 14 days after injury / inoculum (e.g., 9 PFUS treatments) compared to sham skin injuries that had similar bacterial load. It should be noted that no bacteria counts were observed in the blood for the US group and the sham group, and bacteria was not introduced directly to circulating blood. Without being bound by theory, it is believed that activation of CAP may suppress the immune system such that it would be unable to fight against a potential bacteria strain, thereby increasing likelihood of a bacterial infection. However, these results demonstrate that splenic stimulation via ultrasound can reduce the occurrence of infections or prevent the onset of sepsis that may occur due to wounds.

[0067] In general, the anti-inflammatory response modulates pro-inflammatory molecules such as TNFa similarly to biologic anti-inflammatory treatments that are used to treat diseases such as rheumatoid arthritis, Chron’s disease, and irritable bowel syndrome (IBS). Ostensibly, the administration of biological anti-inflammatory treatments is oftendiscouraged or halted in patients exhibiting an infection or recently having received a vaccination to reduce risk of immunosuppression. In any case, the present data demonstrates that pFUS can be applied to the spleen in spite of the presence of a high bacterial load, thereby demonstrating that non-invasive application of pFUS may advantageously reduce the occurrence of skin or systemic infections coinciding with bum wounds in patients without compromising the immune system. While this experiment was initially designed as a safety study to ensure that activating the anti-inflammatory pathway did not lead to more bacteria per bum wound (e.g., make widespread infection worse), these results surprisingly demonstrate that the application of pFUS to the spleen 100 can be utilized to reduce incidence of infections corresponding with bum wounds. Without being bound by theory, it is believed that application of energy to the spleen 100 promotes reduction in bacteria at the wound site by altering concentration of systemic circulating pro-inflammatory molecules (e.g., TNFa, IL-6) and encouraging migration of reservoir cells (e.g., immune cells) and / or biomarkers from the spleen 100 to the wound bed of the burn wound. Put differently, a reduction in TNF-a and IL-6 often correlates with lower bacterial load, and daily activation of the CAP can help achieve a reduction in TNF-a and IL-6 by modulating the immune response — reducing harmful inflammation while preserving the ability to fight infection.

[0068] Moreover, infected wounds typically exhibit more than 100,000 CFU / g wound tissue. In the foregoing experiments as described in FIG. 5, wounds were harvested and minced in a buffer to release the bacteria, after which a sample of the bacteria were plated for counting. In general, the CFU per volume plated was multiplied with the total volume of the buffer to determine CFU / wound. The sham wound exhibited about 122,800 CFUs / wound, which exceeds what is otherwise considered as an infected wound. Thus the results of FIG. 5 provide several advantages. Given severe burns are often associated with high mortality rates, the present techniques advantageously enable a non-invasive method to clear bacteria from a wound site, thereby facilitating better outcomes in patients with skin or systemic infections and enabling a reduction in mortality rates.

[0069] Accordingly, the present techniques may be utilized to treat infections / reduce incidence of infections associated with a burn wound, thereby enabling downstream advantages such as reducing the incidence of sepsis in a patient. It should be noted while present embodiments were evaluated in the context of burn wounds, the techniques described herein may be suitable for reducing bacteria at a wound site associated with various types of injuries or wounds (e.g., burn injuries, osteomyelitis resulting from diabetic foot ulcer) or as a therapy to treat drug-resistant bacteria strains (e.g., methicillin-resistant Staphylococcus aureus (MRSA)). Furthermore, while the preclinical studies were evaluated with respect to S. aureus, the presented embodiments may be utilized to treat bacterial infections with other bacteria strains (e.g., Streptococcus pneumoniae for pneumonia, Escherichia coli for urinary tract infections (UTIs)).

[0070] With the preceding in mind, FIG. 6 is a flow diagram of a method for neuromodulation to decrease infection after skin injury. It should be noted that the method 150 is not limiting, and the method 150 may include additional steps than those illustrated or certain steps, illustrated as being performed concurrently, may be performed at different time periods. In certain embodiments, certain steps of the method 150 may be performed in a parallel manner or need not to be performed in a serial manner.

[0071] Referring to the method 150, at block 152, a region of interest is spatially selected. Spatial selection may include identifying an organ and / or a portion of an organ, such as a spleen or lymph node. In certain embodiments, the energy application device and / or HIFU transducer may operate in an imaging mode to acquire image data to identify the region of interest (e.g., target anatomic site) within the image data. For example, the region of interest may include a portion of a peripheral nerve that includes a junction of a nerve with a nonneuronal cell in a lymph node or spleen. Preferably, the region of interest is associated with CAP, which is innervated by the vagus nerve through the celiac plexus and splenic nerve terminating in the spleen. At block 154, the energy application device 12 is positioned on a subject’s skin in alignment with the region of interest. In this way, at block 156, the energy application device 12 is used to apply energy to the region of interest in a treatmentmode such that the applied energy promotes reduction in bacteria load and reduction of the occurrence of an infection that may be associated with a skin injury. Put differently, the energy pulses are focused at the desired region of interest to preferentially activate a subset of synapses in the target tissue, e.g., to stimulate the axon terminal to release neurotransmitters and / or induce altered neurotransmitter release and / or induce altered activity in the non-neuronal cell (within the synapse) to cause a systemic or distal immune system activation as a result of the neuromodulating energy application to the region of interest at block 158 as provided herein.

[0072] FIG. 7 is a flow diagram of a method of a neuromodulation technique for reducing bacteria load and occurrence of an infection corresponding to a skin injury. It should be noted that the method 200 is not limiting, and the method 200 may include additional steps than those illustrated or certain steps, illustrated as being performed concurrently, may be performed at different time periods. In certain embodiments, certain steps of the method 200 may be performed in a parallel manner or need not to be performed in a serial manner.

[0073] Referring to the method, at block 202, a baseline concentration of one or more markers may be determined at or before application of ultrasound energy to a subject. For example, the baseline concentration may be a single baseline concentration or an average over several time points before application of energy for a particular marker, such as concentration of immune cells (e.g., monocytes, macrophages, neutrophils), immune markers (e.g., biomarkers) such as chemokines, cytokines (e.g., TNF-a, IL-1 , IL-6, IL-10), and the like. Accordingly, a treatment protocol may include the step of controlling delivery of a first neuromodulating energy to a region of interest, as described at block 204. For example, the neuromodulating energy may applied to the spleen (or portions of the spleen) to cause a systemic or distal immune system activation such that the applied energy promotes reduction in bacteria load and reduction of the occurrence of an infection that may be associated with a skin injury. Accordingly, at block 206, a change in concentration of one or more markers is determined relative to the baseline concentration as a result of the neuromodulating energy. That is, a deviation of the concentration of one or moremolecules of interest relative to baseline may be determined at one or more time points / number of days after the applied energy. The time point or time points at which the change in may be determined based on empirical evidence. For example, certain subjects may see changes in the concentration of the molecule of interest by 1, 5, 10, or 60 minutes, one day, two days, three days, etc. after neuromodulation. Further, such changes may dissipate towards recovery after a certain period of time (e.g., after 3 hours, 12 hours, 24 hours). For example, if the deviation indicates that a concentration of one or more cytokines (e.g., TNF-a, IL-ip, IL-6, IL-10) is greater than the baseline concentration, the treatment protocol may be modified such that a second neuromodulating energy is applied to the region of interest, as described at block 210. In this way, the treatment protocol may be repeated or adjusted based on deviations in the concentrations of one or more markers.Examples

[0074] Technical effects of the disclosed embodiments include, but are not limited to the use of applied energy, such as ultrasound in general and pulsed focused ultrasound (pFUS) in particular, to decrease bacteria load within skin injuries (e.g., burn wounds, deep partial thickness wounds, thermal injuries, skin tears, diabetic foot ulcers, lesions, impaired skin) to decrease a corresponding infection. In certain embodiments, a bacterial load may be decreased within a wound bed of a wound, thereby causing a reduction in the occurrence of an infection to such applied energy applications. Accordingly, the present embodiments may be used to reduce the incidence of sepsis in a subject. A device or system may be employed to apply such focused ultrasound treatments and that comprises, among other components, a function generator configured to produce pulsed sinusoidal waveforms, a power amplifier (e.g., an RF power amplifier), a matching network, and a transducer (e.g., a high intensity focused ultrasound (HIFU) transducer). Energy (e.g., focused ultrasound pulses) may be applied to the spleen or other internal organs, features, or structures capable of modulating inflammation via cholinergic anti-inflammatory pathway (CAP) signaling.

[0075] The subject matter described in detail above may be defined by one or more clauses, as set forth below.

[0076] A method of inducing an immunological response in a subject, the method including positioning an energy application device on the subject’s skin in alignment with a target anatomic site including an internal organ, and applying pulsed focused ultrasound (pFUS) energy to the target anatomic site to treat an infection in the subject, target anatomic site is distal from a wound associated with the infection. The method of the preceding claim, wherein the pFUS energy applied to the target anatomic site causes a reduction in bacterial load in a wound bed of the wound.

[0077] The method of the preceding clause, wherein the internal organ includes the spleen.

[0078] The method of any of the preceding clauses, wherein pFUS energy is first applied to the target anatomic site of the subject about 24 hours after formation of the wound.

[0079] The method of any of the preceding clauses, wherein the bacterial load comprises staphylococcus aureus.

[0080] The method of any of the preceding clauses, wherein the wound is a thermal wound.

[0081] The method of any of the preceding clauses, wherein the application of pFUS energy causes a change in concentration of one or more circulating markers in one or both of the spleen or the thermal wound.

[0082] The method of any of the preceding clauses, wherein the one or more circulating markers includes one or both of TNF-a or interleukin-6 (IL-6).

[0083] The method of any of the preceding clauses, wherein the application of pFUS energy prevents the onset of sepsis in the subject.

[0084] A system of inducing an immunological response in a subject, the system including an energy application device, and a controller, wherein the controller is configured to communicate with the energy application device to cause acts to beperformed including facilitating positioning of the energy application device on a subject’s skin in alignment with a target anatomic site using image data, wherein the target anatomic site includes an internal organ and applying, via the energy application device, pulsed focused ultrasound (pFUS) energy to the target anatomic site to treat an infection in the subject, target anatomic site is distal from a wound associated with the infection. The system of the preceding claim, wherein the application of pFUS energy activates the cholinergic anti-inflammatory pathway (CAP) within the target anatomic site, wherein the target anatomic site comprises the spleen.

[0085] The system of the preceding clause, wherein the controller is further configured to perform acts including determining a change in concentration of one or more circulating markers and / or a change in concentration of one or more wound bed markers in response to the applied pFUS energy, wherein the change in concentration is compared to a predetermined threshold, and in response to the change in concentration exceeding the predetermined threshold, applying additional pFUS energy to the target anatomic site.

[0086] The system of any of the preceding clauses, wherein the applied pFUS energy or the applied additional pFUS energy causes the one or more circulating markers to change in concentration in one or both of the spleen or the wound.

[0087] The system of any of the preceding clauses, wherein the pFUS energy is applied daily as part of a treatment protocol.

[0088] The system of any of the preceding clauses, wherein application of pFUS energy to the target anatomic site causes a reduction in bacterial load in a wound bed of the wound.

[0089] The system of any of the preceding clauses, wherein a pFUS energy is first applied to the target anatomic site of the subject within or at about 24 hours after formation of the thermal wound.

[0090] The system of any of the preceding clauses, wherein the bacterial load comprises staphylococcus aureus.

[0091] The system of any of the preceding clauses, wherein application of pFUS energy prevents the onset of sepsis in the subject.

[0092] A tangible, non-transitory, computer-readable medium, including instructions that, when executed by processing circuitry, cause the processing circuitry to perform acts including facilitating positioning of an energy application device on a subject’s skin in alignment with a target anatomic site using image data, wherein the target anatomic site includes an internal organ, and applying, via the energy application device, pulsed focused ultrasound (pFUS) energy to the target anatomic site to treat an infection in the subject, target anatomic site is distal from a wound associated with the infection. The tangible, non-transitory, computer-readable medium of claim 19, wherein the instructions, when executed by processing circuitry, cause the processing circuitry to perform further acts including determining a change in concentration of one or more circulating markers and / or a change in concentration of one or more wound bed markers in response to the applied pFUS energy, wherein the change in concentration is compared to a predetermined threshold, and in response to the change in concentration exceeding the predetermined threshold, applying additional pFUS energy to the target anatomic site.

[0093] The techniques presented and claimed herein are referenced and applied to material objects and concrete examples of a practical nature that demonstrably improve the present technical field and, as such, are not abstract, intangible or purely theoretical. Further, if any claims appended to the end of this specification contain one or more elements designated as “means for [perform]ing [a function]...” or “step for [perform]ing [a function]...”, it is intended that such elements are to be interpreted under 35 U.S.C.112(f). However, for any claims containing elements designated in any other manner, it is intended that such elements are not to be interpreted under 35 U.S.C. 112(f).

[0094] This written description uses examples to disclose the present subject matter, including the best mode, and also to enable any person skilled in the art to practice the subject matter, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the subject matter 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.

Claims

CLAIMS:

1. A method of inducing an immunological response in a subject, the method comprising:positioning an energy application device on the subject’s skin in alignment with a target anatomic site comprising an internal organ; andapplying pulsed focused ultrasound (pFUS) energy to the target anatomic site to treat an infection in the subject, wherein the target anatomic site is distal from a wound associated with the infection.

2. The method of claim 1, wherein the pFUS energy applied to the target anatomic site causes a reduction in bacterial load in a wound bed of the wound.

3. The method of claim 1, wherein the internal organ comprises the spleen.

4. The method of claim 1, wherein pFUS energy is first applied to the target anatomic site of the subject about 24 hours after formation of the wound.

5. The method of claim 2, wherein the bacterial load comprises staphylococcus aureus.

6. The method of claim 1, wherein the wound is a thermal wound.

7. The method of claim 1, wherein the application of pFUS energy causes a change in concentration of one or more circulating markers in one or both of the spleen or the wound.

8. The method of claim 7, wherein the one or more circulating markers comprises one or both of TNF-a or interleukin-6 (IL-6).

9. The method of claim 1, wherein the application of pFUS energy prevents the onset of sepsis in the subject.

10. A system of inducing an immunological response in a subject, the system comprising:an energy application device; anda controller, wherein the controller is configured to communicate with the energy application device to cause acts to be performed comprising:facilitating positioning of the energy application device on a subject’s skin in alignment with a target anatomic site using image data, wherein the target anatomic site comprises an internal organ; andapplying, via the energy application device, pulsed focused ultrasound (pFUS) energy to the target anatomic site to treat an infection in the subject, wherein the target anatomic site is distal from a wound associated with the infection.

11. The system of claim 10, wherein the application of pFUS energy activates the cholinergic anti-inflammatory pathway (CAP) within the target anatomic site, wherein the target anatomic site comprises the spleen.

12. The system of claim 10, wherein the controller is further configured to perform acts comprising:determining a change in concentration of one or more circulating markers and / or a change in concentration of one or more wound bed markers in response to the applied pFUS energy, wherein the change in concentration is compared to a predetermined threshold; andin response to the change in concentration exceeding the predetermined threshold, applying additional pFUS energy to the target anatomic site.

13. The system of claim 12, wherein the applied pFUS energy or the applied additional pFUS energy causes the one or more circulating markers to change in concentration in one or both of the spleen or the wound.

14. The system of claim 10, wherein the pFUS energy is applied daily as part of a treatment protocol.

15. The system of claim 10, wherein application of pFUS energy to the target anatomic site causes a reduction in bacterial load in a wound bed of the wound.

16. The system of claim 10, wherein a pFUS energy is first applied to the target anatomic site of the subject within or at about 24 hours after formation of the wound.

17. The system of claim 15, wherein the bacterial load comprises staphylococcus aureus.

18. The system of claim 10, wherein application of pFUS energy prevents the onset of sepsis in the subject.

19. A tangible, non-transitory, computer-readable medium, comprising instructions that, when executed by processing circuitry, cause the processing circuitry to perform acts comprising:facilitating positioning of an energy application device on a subject’s skin in alignment with a target anatomic site using image data, wherein the target anatomic site comprises an internal organ; andapplying, via the energy application device, pulsed focused ultrasound (pFUS) energy to the target anatomic site to treat an infection in the subject, wherein the target anatomic site is distal from a wound associated with the infection.

20. The tangible, non-transitory, computer-readable medium of claim 19, wherein the instructions, when executed by processing circuitry, cause the processing circuitry to perform further acts comprising:determining a change in concentration of one or more circulating markers and / or a change in concentration of one or more wound bed markers in response to the applied pFUS energy, wherein the change in concentration is compared to a predetermined threshold; andin response to the change in concentration exceeding the predetermined threshold, applying additional pFUS energy to the target anatomic site.

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