Accelerated skin healing using non-invasive focused ultrasound

Abstract

The present approach generally relates to promoting wound healing, including chronic wounds that are typically associated with delayed healing times or are refractory. In one embodiment, a method for promoting wound healing includes placing an ultrasound transducer at a stimulation site in a subject having a wound. Pulsed focused ultrasound (pFUS) is noninvasively applied using the transducer to cause modulation of a target anatomical site containing resident or circulating immune cells. Modulation of the target anatomical site in the subject causes migration of one or more of monocytes, macrophages, or neutrophils to the wound surface.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 413,365, filed October 5, 2022, and entitled "Accelerated Skin Healing in a Rodent Model of Type 2 Diabetes Using Non-Invasive Focused Ultrasound of the Spleen," which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates to the use of applied energy such as pulsed focused ultrasound (pFUS) to promote wound healing, such as skin healing. More specifically, the present disclosure discloses an approach to apply pulsed focused ultrasound to target tissue, such as the spleen, to promote and accelerate healing of wounds, such as resistant or slow-healing chronic wounds. [Background technology]

[0003] The subject matter discussed in this section should not be considered prior art merely as a result of its inclusion in this section. Similarly, the problems mentioned in this section or problems related to the subject matter listed as background should not be considered previously recognized in the prior art. The subject matter of this section merely represents various approaches, which may themselves correspond to embodiments of the claimed technology.

[0004] The estimated cost of treating non-healing wounds is in the billions of dollars, affecting more than 6.5 million people in the United States and increasing with the aging population. Non-healing wounds also cause a significant number of deaths, with 5-year mortality rates higher than many common cancers, such as prostate and breast cancer. When amputation is required due to tissue death and subsequent necrosis, the 5-year mortality rate approaches 50%. This is due to the approximately 50% risk of wound infection. Once an initial amputation is required, multiple amputations are often necessary, increasing the 5-year mortality rate to over 70%. Chronic wounds, defined as wounds that do not heal within 3 months, tend to have a different microbiota than healing wounds, and although infection is not a necessary condition for impaired healing, these chronic wounds are more susceptible to infection. Chronic wounds generally fall into three categories: diabetic foot ulcers (DFUs), pressure ulcers (bed sores), and lower extremity ulcers caused by peripheral arterial disease (PAD) or post-thrombotic syndrome (PTS). Chronic inflammation, a common comorbidity in type 2 diabetes mellitus (T2DM), can adversely affect healing by increasing the systemic levels of inflammatory cytokines. Diabetic foot ulcers represent the most common complication in patients with poorly controlled disease (e.g., accelerated metabolic syndrome, chronic inflammation), affecting more than 25% of patients with type 2 diabetes. Patients with diabetic foot ulcers often cite a lack of mobility and / or flexibility as the reason for their inability to successfully adhere to foot self-care protocols. Many diabetic patients report the development of slow-healing or chronic wounds, including DFUs, resulting in an annual cost to the healthcare system of over $20 billion. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 9,585,977 Summary of the Invention

[0006] A summary of some 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 some embodiments, and that these aspects do not limit the scope of the disclosure. Indeed, the disclosure may encompass a variety of aspects that may not be set forth below.

[0007] This approach generally involves promoting wound healing, including chronic wounds that are typically associated with delayed healing times or are refractory. For example, healing is delayed in type 2 diabetes, and new therapeutic approaches would be useful. As discussed herein, daily splenic irradiation with pulsed focused ultrasound (pFUS) during wound healing can accelerate the rate of closure through altering systemic cytokine titers. For example, general energy, such as ultrasound energy, can be applied to one or more internal organs, structures, or features that modulate or affect the cholinergic anti-inflammatory pathway. Specifically, splenic pulsed focused ultrasound alters inflammatory cytokines in models of acute endotoxemia and pneumonia through modulation of the cholinergic anti-inflammatory pathway (CAP). Noninvasive ultrasound can be applied to the spleen of a type 2 diabetes mellitus (T2DM) rodent model (Zucker diabetic Sprague-Dawley (ZDSD) rat) with a full-thickness excision wound in an attempt to accelerate wound healing through modulation of aberrant cytokine expression. In the approach discussed herein, daily (1x / day, Monday through Friday) pulsed focused ultrasound pulses were applied extracorporeally to the spleen site for 3 minutes (e.g., treatment duration) over 18 courses of 15 days (e.g., treatment period). Wound diameter was measured daily, and relative cytokine levels were assessed in spleen and wound lysates. Noninvasive splenic pulsed focused ultrasound accelerated wound closure by up to 4.5 days compared to sham controls. Healing times in all treatment groups were comparable to those observed in healthy rats in previously published studies (see below), suggesting that pulsed-focused ultrasound treatment restored a normal wound-healing phenotype in ZDSD rats. IL-6 (which is pro-inflammatory at high levels but can be anti-inflammatory at low levels) was relatively lower in the stimulated spleen (-2.24 ± 0.81 Log2FC p=0.02), whereas L-selectin (an adhesion molecule important for cell migration and epithelialization) was relatively higher in the wound surface of stimulated rodents (2.53 ± 0.72 Log2FC p=0.003).Thus, splenic pulsed focused ultrasound accelerates healing in rodent models and has the potential to provide a novel, systemic, non-invasive approach for wound treatment.

[0008] In one embodiment, a method for promoting wound healing is provided. According to this embodiment, an ultrasound transducer is placed at a stimulation site in a subject having a wound. Pulsed focused ultrasound (pFUS) is non-invasively applied to a target organ of the subject using the transducer to cause modulation of the subject's cholinergic anti-inflammatory pathway. Modulation of the subject's cholinergic anti-inflammatory pathway causes migration of one or more of monocytes, macrophages, or neutrophils to the wound surface.

[0009] According to aspects of such embodiments, the stimulation site is distal to the wound.

[0010] According to aspects of such embodiments, the target organ is the subject's spleen, and non-invasively applied pulsed focused ultrasound stimulates nerve tracts within the spleen.

[0011] According to aspects of these embodiments, the wound is an acute or chronic skin wound.

[0012] According to aspects of these embodiments, modulation of the cholinergic anti-inflammatory pathway in a subject alters the concentration of systemically circulating pro-inflammatory molecules. According to yet other aspects of these embodiments, the pro-inflammatory molecules include TNFα and IL-6.

[0013] According to aspects of such embodiments, modulation of the cholinergic anti-inflammatory pathway regulates aberrant cytokine expression in a subject.

[0014] According to aspects of such embodiments, applying pulsed focused ultrasound to a target organ of the subject includes applying pulsed focused ultrasound to the subject's spleen at least once per day for a treatment period.

[0015] According to aspects of this embodiment, the pulsed focused ultrasound (pFUS) applied to the target organ has a pulse center frequency of 1.1 MHz, a pulse repetition period of 0.5 ms, and a pulse amplitude of 300 mV. pp , a burst of 150 cycles, and a burst period of 200 milliseconds.

[0016] According to aspects of such embodiments, applying pulsed focused ultrasound (pFUS) to a subject's spleen accelerates wound closure by 3 to 5 days relative to no treatment.

[0017] According to aspects of such embodiments, the target organ is the spleen and the level of IL-6 is decreased in the spleen in response to application of pulsed focused ultrasound (pFUS) to the spleen.

[0018] According to aspects of such embodiments, levels of L-selectin are increased at the wound surface in response to application of pulsed focused ultrasound (pFUS) to the target organ.

[0019] According to aspects of such embodiments, a first pulsed focused ultrasound (pFUS) is applied to the subject's target organ within 24 hours of wound formation.

[0020] In another embodiment, a system for promoting wound healing is provided. According to this embodiment, the system includes a waveform generator configured to generate a pulsed sinusoidal waveform, an RF power amplifier configured to amplify the pulsed sinusoidal waveform, a matching network configured to receive the amplified pulsed sinusoidal waveform, and a transducer connected to the matching network and configured to generate focused ultrasound pulses based on the amplified pulsed sinusoidal waveform. According to this embodiment, the system is configured to, after placement of the transducer at an external stimulation site in a subject, direct the focused ultrasound pulses toward a target organ to cause modulation of the subject's cholinergic anti-inflammatory pathway. Modulation of the subject's cholinergic anti-inflammatory pathway causes migration of one or more of monocytes, macrophages, or neutrophils to the wound surface.

[0021] According to aspects of this alternative embodiment, the transducer includes a high intensity focused ultrasound (HIFU) transducer.

[0022] According to aspects of this other embodiment, the focused ultrasound pulses have a pulse center frequency of 1.1 MHz, a pulse repetition period of 0.5 milliseconds, and a pulse amplitude of 300 mV. pp It has.

[0023] According to aspects of this alternative embodiment, the target organ is the subject's spleen, and non-invasively applied pulsed focused ultrasound stimulates nerve tracts within the spleen.

[0024] According to aspects of such another embodiment, modulation of the cholinergic anti-inflammatory pathway in a subject alters the concentration of systemically circulating pro-inflammatory molecules.

[0025] According to aspects of such another embodiment, modulation of the cholinergic anti-inflammatory pathway regulates aberrant cytokine expression in the subject.

[0026] In yet another embodiment, a method for promoting wound healing is provided. According to this embodiment, a high intensity focused ultrasound (HIFU) transducer is placed at a stimulation site in a subject having a chronic skin wound distal to the stimulation site. Pulsed focused ultrasound (pFUS) is non-invasively applied to the subject's spleen using the transducer at least once per day for a treatment period to stimulate nerve pathways in the spleen, resulting in modulation of the subject's cholinergic anti-inflammatory pathway. The pulsed focused ultrasound applied to the spleen has a pulse center frequency of 1.1 MHz, a pulse repetition period of 0.5 ms, and a pulse amplitude of 300 mV. pp Modulation of the cholinergic anti-inflammatory pathway modulates aberrant cytokine expression in a subject, altering the levels of circulating pro-inflammatory molecules. [Brief explanation of the drawings]

[0027] These and other features, aspects, and advantages of the present invention will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings, in which like characters represent like parts throughout. [Figure 1] FIG. 1 illustrates a focused ultrasound system suitable for non-invasively applying focused ultrasound pulses to a target organ to promote the wound healing process in accordance with aspects of the present disclosure. [Figure 2] FIG. 1 illustrates a stimulation site for focused ultrasound pulses according to aspects of the present disclosure and the relationship between a target organ, illustrated here as the spleen, and the skin through both blood and lymphatic circulation. [Figure 3] FIG. 1 is a visual representation of a test schedule (top panel) on a logarithmic scale according to aspects of the present disclosure, along with expected stages of wound healing (bottom panel) and associated immune response times aligned to the test schedule following pulsed focused ultrasound intervention. [Figure 4] FIG. 1 is a graph showing change or no change in body weight among study groups according to aspects of the present disclosure. [Figure 5] FIG. 1 is a graph showing blood glucose levels among study groups according to aspects of the present disclosure. [Figure 6]FIG. 1 shows excision wounds by day and test group according to aspects of the present disclosure. [Figure 7] FIG. 1 is a graph showing the change in wound size (% (mm) of initial wound diameter) over time for each study group according to each aspect of the present disclosure. [Figure 8] FIG. 10 shows linear regressions for each test group relating to wound closure over time according to aspects of the present disclosure. [Figure 9] FIG. 10 is a heat map showing the relative changes in proteins in the spleen and wound area for each study group according to each aspect of the disclosure. [Figure 10] FIG. 10 is a graphical representation showing the negative correlation between higher agrin protein expression and smaller wounds according to aspects of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0028] One or more specific embodiments will be described below. In an attempt to provide a concise description of these embodiments, not all features of an actual implementation will be described herein. It will be appreciated that the development of any such actual implementation, like any engineering or design project, will involve many implementation-specific decisions to achieve the developer's specific goals, such as adhering to system-related and business-related constraints that may vary from implementation to implementation. It will also be appreciated that such a development effort may be complex and time-consuming, but would nevertheless be a routine undertaking of design, fabrication, and manufacture for those of ordinary skill in the art having the benefit of this disclosure.

[0029] When describing elements of various embodiments of the presently disclosed subject matter, the terms "singular," "definite," "the," "said," and the like are intended to mean that there are one or more of the elements in question. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements. Additionally, references to "one embodiment" or "an embodiment" in the present disclosure should not be interpreted as excluding the existence of additional embodiments that also incorporate the listed features.

[0030] Normal wound healing consists of four phases (hemostasis, inflammation, proliferation, and repair), which are similar in timing for both acute and chronic wounds. Inadequate performance of any one of these phases can result in non-healing wounds. Hemostasis involves the rapid migration of platelets and neutrophils and the deposition of fibrin, resulting in closure of the wound site through coagulation. The inflammatory phase (early and late) begins with the infiltration of the wound site by neutrophils (within 24 to 36 hours), whose primary function is to prevent infection by phagocytosing bacteria / pathogens, foreign particles, damaged cells, and tissue. As part of the late inflammatory phase (48 to 72 hours after wounding), a population of macrophages becomes dominant after chemoattraction of monocytes via clotting factors, cytokines, and chemokines. Macrophages also provide tissue growth factors such as transforming growth factor (TGF)-β, TGF-α, epidermal growth factor (EGF), fibroblast growth factor (FGF), and collagenase, which are required for activating cells involved in wound repair (including keratinocytes, fibroblasts, and vascular endothelial cells). During the late inflammatory phase (72 hours after wounding), lymphocytes (T cells) enter the wound site mediated by interleukin-1 (IL-1), complement system components such as C3 and C5, and immunoglobulin G (IgG) degradation products. Classical macrophages (M1) secrete proinflammatory cytokines that help recruit lymphocytes to the wound surface. Nonclassical macrophages (M2) secrete anti-inflammatory cytokines that trigger a transition to a proliferative phenotype, which has been observed to be accelerated by pulsed ultrasound treatment as discussed herein. The proliferative phase begins three days after wounding and lasts approximately two weeks. This phase is characterized by migration of fibroblasts, keratinocytes, epithelial cells, and vascular cells, collagen synthesis, adhesion, traction, and epithelialization. Finally, the repair / maturation phase is responsible for new epithelial growth and scar tissue formation and lasts one to two years. This phase also tightens the wound, much like muscle cell contraction, forming a repaired ECM. Collagen is repaired and the wound closes completely. Skin is typically about 80% healthy by the end of this phase, during which time it is significantly weaker and more susceptible to re-injury.Once scarring occurs, the skin never regains its full pre-injury integrity, becoming inflexible and restricting movement. While any of these stages can be incomplete, healing tends to remain in the inflammatory phase and not progress to epithelialization and remodeling. As discussed herein, approaches involving pulsed focused ultrasound (pFUS) treatment initiated 24 hours after wounding have been explored to attenuate early inflammation through CAP modulation, thereby altering the healing timeline.

[0031] Progress in chronic wound treatment (including diabetic foot ulcers) has stagnated for decades. There is a widespread need for the development of new techniques and advances in the treatment of wounds such as chronic diabetic foot ulcer wounds. Conventional approaches include negative pressure wound therapy (NPWT), and current conventional treatments consist of wound management using techniques such as moist wound healing, debridement, growth factor-soaked dressings, and the use of walker casts to offload the injured area. While these palliative measures are intended to care for the wound, they do not address the underlying cause, such as chronic inflammation. While NPWT has shown greater efficacy in acute wounds, its use in chronic wounds remains limited. Targeted drug treatments are often lacking, with antibiotics being the most widely used, both topical and systemic.

[0032] Bioelectronic medicine is a rapidly developing field. Practically, electrical stimulation can be applied to nerve fibers to elicit a response; often, electrodes are surgically implanted to deliver energy to the vagus nerve, which then produces downstream effects. Vagus nerve stimulation can modulate inflammation through cholinergic anti-inflammatory pathway (CAP) signaling. The CAP is a neural pathway that, when activated, inhibits TNFα production (and other inflammatory cytokines) in the spleen, liver, and heart. This pathway requires both the vagus nerve and α-7 receptors; disruption of either reverses the anti-inflammatory response. This paper describes a technique that provides a less invasive intervention and can be applied in a clinical setting (i.e., nonsurgical). Specifically, as discussed herein, we describe a method for noninvasively activating the CAP using focused ultrasound applied directly to a target organ (e.g., the spleen). While the examples discussed herein generally relate to the application of ultrasound pulses to the spleen, it should be understood that the target organ can be any organ capable of modulating inflammation via cholinergic anti-inflammatory pathway (CAP) signaling in response to noninvasive application of pulsed ultrasound energy. In the examples described herein, a single ultrasound treatment was found to reduce systemic tumor necrosis factor alpha (TNFα) levels within one hour in an in vivo LPS-induced model of endotoxemia. Additionally, similar results were achieved in peripheral blood collected after pulsed focused ultrasound stimulation and exposed to LPS ex vivo. Systemic effects, such as attenuation of inflammation resulting from a Streptococcus pneumoniae challenge, were also observed at sites distal to the stimulation site.

[0033] As discussed herein, skin wound healing was accelerated by stimulating nerve pathways within the spleen using a pulsed focused ultrasound system, as shown in Figure 1. Figure 2 illustrates the stimulation site and the relationship between the spleen and skin through the blood and lymphatic circulation. Based on the results disclosed herein, it was observed that pulsed focused ultrasound in the spleen alters monocytes / macrophages / neutrophils that migrate to the wound surface, which in turn modulates the wound to be more cooperative with healing through mediation of a systemic immune response, and also alters the concentration of systemically circulating pro-inflammatory molecules (e.g., TNFα, IL-6).

[0034] ZDSD rats were tested as a model of slow healing. The spleens of ZDSD rats with full-thickness excisional skin wounds were stimulated up to once daily for 15 days. Test groups included animals from three age groups corresponding to different stages of type 2 diabetes disease progression, from prediabetes to advanced diabetes with comorbidities. To further model the chronic inflammation comorbidity of type 2 diabetes, the oldest group received ultra-low doses of LPS (5 ng / kg) three times a week, starting 14 days before wound creation. Pulsed-focused ultrasound-induced acceleration of wound closure was observed compared to sham controls in all age groups. As discussed herein, ultrasound was used in this manner to stimulate splenic CAPs and modulate the immune system distal to the wound site to accelerate healing.

[0035] With the above in mind, Figure 1 illustrates various aspects of a focused ultrasound system 100 that may be used to perform the techniques discussed herein. In one embodiment, the system 100 includes a 1.1 MHz high intensity focused ultrasound (HIFU) transducer 104 and matching circuit 108, an RF power amplifier 112, and a waveform generator 116. In this example, the waveform generator 116 generates a pulsed sinusoidal waveform that is amplified by the RF power amplifier 112 and sent to the impedance matching circuit 108, which is connected to the transducer 104. In some embodiments, the pulse center frequency is 1.1 MHz, the pulse repetition period is 0.5 milliseconds (corresponding to a pulse repetition frequency of 2000 Hz), and the pulse amplitude is 300 mV. ppIn one embodiment, control circuitry, 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 waveform generator 116, the RF power amplifier 112, the matching circuit 108, and / or the HIFU transducer 104 to enable directed energy application to a target organ as described herein, such as for periods, energies, and / or durations as described herein to stimulate wound healing.

[0036] In one implementation, the transducer 104 is a 70 mm diameter HIFU transducer with a spherical surface having a radius of curvature of 65 mm. In one such embodiment, the transducer focal depth is between 50 mm and 80 mm, such as 65 mm. In one embodiment, the numerically simulated pressure profile has an amplitude half-width of 1.8 mm laterally and 12 mm in depth. The HIFU transducer 104 may be acoustically coupled to the subject through a standoff, such as a 6 cm tall plastic cone filled with degassed water and acoustic coupling gel.

[0037] Turning to Figure 2, the abdomen is shown showing the location of the spleen 140 and the delivery location of pulsed focused ultrasound via the transducer 104. When noninvasive ultrasound energy is applied to the spleen 102, cytokine production is modulated as discussed herein. The splenic lymph nodes 144 are shown enlarged, along with the splenic artery 148 and splenic vein 152. The splenic lymph nodes 144 are connected to lymphatic vessels, which in turn connect to the entire lymphatic system, including the cutaneous lymphatic vessels. Skin-resident macrophages release chemoattractants that recruit systemic immune cells through extravasation. When the skin is injured (shown as the injured skin 156 distal to the site of injury), within an hour, blood vessels around the wound deliver platelets to achieve hemostasis, followed by neutrophils to initiate inflammation and the healing process. Monocytes then arrive and differentiate into M1 macrophages. Efferent lymphatic vessels help drain the area of ​​protein-rich inflammatory fluid from the interstitium, and afferent vessels help supply the area with cytokines and lymphocytes to aid in healing.

[0038] For the animal studies discussed herein, 30 ZDSD rats aged 16 to 25 weeks were randomly assigned to the following groups: Group 1 (N = 10): 16 weeks old, Group 2 (N = 10): 22 weeks old, and Group 3 (N = 10): 25 weeks old, and underwent induced chronic inflammation (5 ng / kg LPS, 3 × weeks). Each group was further divided into those receiving pulsed focused ultrasound stimulation (5 rodents with 2 wounds each) or sham control (5 rodents with 2 wounds each). One animal was removed from the sham cohort in Group 2 during the study due to complications from hypertension. Animals were obtained from Charles River Laboratories and housed under a 12:12 light / dark cycle. Animals had free access to both food and water and were fed LabDiet 5008.

[0039] Two full-thickness excision wounds were created on the head of each rat under 3% isoflurane anesthesia. After hair removal and cleaning of the area with betadine and 70% ethanol, an 8-mm biopsy needle was used to create the wound, and the skin was carefully removed. Painkillers were withheld due to the effects of each drug class on immune responses. No physical signs of pain or discomfort were observed throughout the study. The wounds were left uncovered postoperatively, and each animal was housed singly to avoid complications from cage-mate activity. Animals in Group 3 received 5 ng / kg LPS intraperitoneally three times a week, starting 14 days before wound creation and continuing throughout the study, to induce chronic inflammation. Starting on postoperative day 1, pulsed focused ultrasound energy was applied to the spleen after coupling a HIFU transducer 104 to the depilated skin using coupling gel, while using isoflurane for restraint. The wounds were photographed and their diameter was recorded using a digital caliper, measured from anterior to posterior. For the ultrasound stimulation group, energy was applied for three 1-minute sessions with a 30-second break between sessions. The following ultrasound parameters were used: 1.1 MHz, 300 mV pp , 150-cycle bursts, burst period 200 ms. For sham controls, a transducer was placed over the spleen, but no energy was applied. Stimulation was performed on postoperative days 1 to 4, 7 to 11, and 14 to 15. Animals were euthanized on day 16 regardless of wound progression.

[0040] Blood was drawn weekly and analyzed for both complete blood count and blood chemistry. Blood chemistry measurements, including blood glucose, were performed at 9:00 AM. Residual plasma was frozen for biochemical analysis. At the completion of each study, animals were euthanized by CO2 asphyxiation. Terminal blood draws were completed via cardiac puncture, and the spleens and wound beds were partially excised, snap-frozen, and stored at -80°C until cytokines were extracted and analyzed.

[0041] With the above in mind, Figure 3 visually illustrates each aspect of this test schedule in relation to the wound healing timeline. In this figure, Figure 3 visually depicts the test schedule (top panel) on a logarithmic scale, including LPS injection for Group 3 and pulsed focused ultrasound stimulation for all groups. Additionally, Figure 3 visually depicts the expected wound healing phases (bottom panel) following pulsed focused ultrasound intervention with associated immune response times consistent with the test timeline shown above. As evident in the wound healing profile, phenotypic changes are evident in response to pulsed ultrasound treatment, such as the accelerated transition from the appearance of M1 macrophages to the appearance of M2 macrophages in the wound profile.

[0042] For protein extraction for biochemical analysis, frozen tissues were mashed and added to ice-cold PBS supplemented with protease inhibitors. Samples were homogenized using an IKA T18 Ultra-Turrax set at 24,000 RPM until the tissue was dispersed. Samples were kept on ice until all samples were processed, and then the tissue homogenate was clarified by centrifugation at 4°C. The OD280 of each sample was assessed prior to each assay using a NanoDrop instrument as outlined in the assay protocol.

[0043] For cytokine screening, a panel of 34 cytokines, chemokines, and growth factors (gene symbols: AGER, AGRN, CCL2, CCL20, CD86, CNTF, CSF2, CX3CL1, CXCL1, CXCL2, CXCL3, CXCL5, FASLG, ICAM1, IFNG, IL10, IL13, IL1A, IL1B, IL1RL2, IL2, IL4, IL6, INHBA, LEP, MMP8, NGF, PDGFA, PPBP, PRLR, SELL, TIMP1, TNF, and VEGFA) was evaluated using a sandwich antibody array (C2 antibody array). One hundred OD units of spleen or wound surface protein lysate were incubated with the membrane array and processed according to the manufacturer's instructions, except that streptavidin-cy5 secondary antibody was used instead of streptavidin-HRP for fluorescence scanning. Membranes were imaged using a Typhoon scanner, and median fluorescence intensity was extracted using ImageJ software and Microarray Profile. All specimen blocks were normalized to a reference block using positive control spots, and background was subtracted using blank spots.

[0044] For statistical analysis, results as described herein are expressed as mean ± SEM. Differences between groups were assessed using a multiple unpaired Student's t-test (healing rate and cytokine data) or a repeated-measures two-way analysis of variance (2-way ANOVA) followed by Tukey's multiple comparison test (body weight, blood glucose levels, and wound size over time). Data were considered significant at P ≤ 0.05. Linear regression analysis of normalized wound diameter (% mm of initial diameter) was used to extrapolate the time at which the wound was considered completely closed. The x-intercept (days) at y = 0 (wound size) was defined as the predicted closure time. Cytokine arrays were compared by calculating the log2 fold change (Log2FC) of median fluorescence intensity for each pair of specimens (pFUS vs. sham). Heat maps were generated, and hierarchical clustering analysis was performed to demonstrate differences within each group. Pearson correlation was used to determine significant correlations between wound size and relative cytokine expression.

[0045] With the above methodological and analytical discussion in mind, the results were obtained as follows. Physiological characteristics were assessed to track the overall health of the animals throughout the study. Animals were weighed every day of the week (i.e., Monday through Friday). Blood glucose levels were measured on Day -14 (Group 3, at the start of LPS administration), Day 0 (all groups), and Day 16 (all groups). Regarding physiological characteristics, body weight did not differ between animals within each pulsed-focused ultrasound or sham-control group, but did differ between age groups, as shown graphically in Figure 4. Specifically, as shown in Figure 4, Group 1 (16-week ZDSD prediabetic rats) did not exhibit significant changes in body weight at Week 15. Similarly, Group 2 (22-week ZDSD diabetic rats) also did not have significant changes in body weight. Group 3 (25 weeks + 10 ng / kg LPS) experienced a 4.6% weight loss, which was significant from day 0 (*p<0.05) and was attributed to a response to LPS. Specifically, for Group 3, both pulsed-focused ultrasound and sham controls experienced significant weight loss over the course of the study, i.e., 5.7% (P<0.05), which was predicted to be due to the mild chronic inflammation elicited by the LPS injection.

[0046] Random blood glucose levels were assessed on Day 0 (pre-surgery) and Day 16. Blood glucose levels differed in each group according to age, as shown graphically in Figure 5, but remained stable between Day 0 and Day 16. Specifically, no significant changes were observed in blood glucose levels in Groups 1, 2, or 3 over the course of the study.

[0047] Complete blood counts (CBCs) for each group were within normal limits, but significant differences in white blood cells (WBCs) were observed over time in Groups 1 and 2 and between animals treated with pulsed focused ultrasound and sham-treated animals. Both monocytes and lymphocytes were lower in Group 1 on Day 7, and neutrophils and monocytes were reduced in Group 2 by Day 16. No significant differences were observed in neutrophil, lymphocyte, or monocyte populations. Blood chemistries for each group were within normal limits for all measured parameters throughout the study, with the exception of blood glucose levels, as noted above.

[0048] With the aforementioned timeline in mind, excision wounds were created on day 0 and are shown in the left column of Figure 6, which shows representative longitudinal images of wound healing progression in ZDSD rats (scale bar = 10 mm (lower right panel)). As previously described, ultrasound or sham stimulation began approximately 24 h after the wounding procedure. Although an 8 mm biopsy needle was used to create the desired wound, there was still variation in wound diameter due to skin laxity (≤1.5 mm, ≈14%). In Group 1, there was a 4.68 ± 4.9% (ns) reduction in pFUS vs. sham by day 7 (middle column of Figure 6), as a percentage of the initial wound diameter, and by day 15, the difference increased to 12.16 ± 4.7% (p < 0.05) (right column of Figure 6). Group 2 experienced a 15.9 ± 4.1% (p<0.05) reduction (pFUS vs. sham) at day 7 and a 10.97 ± 4.2% (p<0.05) reduction at day 15. Group 3 experienced a 14.83 ± 6% (p<0.05) reduction (pFUS vs. sham) at day 7 and a 15.12 ± 2.2% (p<0.001) reduction at day 15. The sham control (33% to 41% of the original wound size at day 15) was similar to the ZDSD (approximately 33% of the original wound size) from a previously published study. These results are graphically presented in Figure 7. For Figure 7, wound diameters were normalized to their respective day 0 diameters (E / B). Wounds in both pFUS-treated rats in Groups 2 and 3 significantly shrank by Day 7 compared to sham controls (Δ15.94 ± 4.12% (p = 0.003) and Δ14.83 ± 6.02% (p = 0.024)). At Day 15, all pFUS-treated groups shrank significantly more than sham controls (20 ± 4 vs. 33 ± 3; p < 0.05), and Group 3 treatment also shrank significantly more than sham controls (24 ± 2 vs. 38 ± 2; p < 0.001). N = 10 wounds per cohort.

[0049] While wounds closed over time in all groups, the rate of closure (% mm diameter change / day) was significantly accelerated in all three pulsed-focused ultrasound cohorts during the first 24 hours after the first stimulation (Table 1). Specifically, the % diameter reduction / day was calculated. The data suggest that pulsed-focused ultrasound affected healing during the first 24 hours to a greater extent than over the remaining time course. This may indicate that early wound inflammation was altered in a manner that promoted healing. The rate of closure continued to accelerate through day 15, but not significantly. Because many wounds did not progress to complete closure, the predicted time to healing (x-intercept) was determined by linear regression of daily wound diameter measurements, as shown graphically in Figure 8. Specifically, linear regression analysis was used to predict the number of days to healing reduced by pulsed-focused ultrasound stimulation in each group. The slopes and intercepts of the gradients were significantly different within each group (Group 1, p = 0.0032; Group 2, p = 0.0005; Group 3, p = 0.0012) and between groups (p < 0.001). At day 15, regression analysis suggested that wounds completely closed between days 17 and 23 for all pulsed-focused ultrasound stimulation cohorts (similar to healthy SD rats), as determined by the 95% CI of the x-intercept (days) at Y = 0 (wound size). Table 2 demonstrates the regression analysis shown in Figure 8. The x-intercept of the linear regression line represents the number of days to healing, and the number of days saved by pulsed-focused ultrasound is the difference in the x-intercept between sham and pulsed-focused ultrasound. As shown, FUS stimulation accelerated healing time by 2.6 to 4.5 days.

[0050] [Table 1]

[0051] [Table 2]

[0052] Protein expression in spleen and wound lysates was also examined. Specifically, both spleen and wound lysates were assayed against a panel of chemokines, cytokines, and growth factors to examine the association between splenic stimulation and distal wound sites. At 16 days post-wounding, relative protein changes were determined (log2FC) and plotted as heat maps using hierarchical clustering analysis, as shown in Figure 9. Specifically, spleen and wound lysates at 16 days post-wounding were assayed against a panel of cytokines, chemokines, and growth factors. A heat map (Figure 9) was generated showing relative changes (log2 fold change in median fluorescence intensity) comparing pulsed-focused ultrasound versus sham controls. Hierarchical clustering was performed row-centered, and rows were subjected to unit variance scaling. Rows were clustered using correlation distance and average linkage. Columns were grouped first by location and then by group. Asterisks indicate significant differences in relative expression (p<0.05).

[0053] Of the 34 proteins tested, spleen lysates from Group 1 had four proteins with significantly different expression levels (agrin, 0.87 ± 0.24 (p = 0.004); ciliary neurotrophic factor (CNTF), -0.58 ± 0.25 (p = 0.04); and IL-1 R6, -1.20 ± 0.38 (p = 0.01)). Group 2 had 11 proteins with significant differences from sham controls (agrin, 3.10 ± 0.78 (p = 0.003); cytokine-induced neutrophil chemoattractant (CINC)-2α, 1.59 ± 0.67 (p = 0.046); CINC-3, 1.64 ± 0.68 (p = 0.04); Fas ligand, -0.85 ± 0.28 (p = 0.01); and IL-1 R6, -1.20 ± 0.38 (p = 0.01)). R6, 2.32±1.03(p=.047);IL-13, -0.71±0.28(p=0.03);IL-6, -2.24±0.81(p=0.02);Leptin, -1.22±0.42(p=0.02);Prolactin R, -2.9 3±1.24(p=0.046);RAGE (receptor for advanced glycation end products), -2.70±0.95(p=0.02);TNFα, -0.85±0.31(p=0.02);Vascular endothelial growth factor (VEGF), -0.60±0.24(p=0. 03)), Group 3 had eight significantly altered protein levels (CINC-1, -1.98 ± 0.70 (p = 0.02); CINC-2α, -1.61 ± 0.71 (p = 0.049); IL-13, -1.29 ± 0.50 (p = 0.03); IL-2, -1.45 ± 0.47 (p = 0.01); IL-4, -1.53 ​​± 0.62 (p = 0.03); prolactin R, 0.14 ± 0.06 (p = 0.04); RAGE, -2.95 ± 0.31 (p = 0.00001)). Wound lysates from group 1 had five proteins with different expression levels (agrin, 0.68 ± 0.28 (p = 0.03); IL-1 R6, 1.64 ± 0.42 (p = 0.001); IL-10, -0.57 ± 0.24 (p = 0.03); L-selectin, 2.53 ± 0.72 (p = 0.003); tissue inhibitor of metalloproteinase (TIMP)-1, -0.43 ± 0.18 (p = 0.03)).Wound lysates from Group 2 were found to have no significant differences, while Group 3 had three distinct protein levels (CNTF, 2.02 ± 0.79 (p = 0.03); macrophage inflammatory protein (MIP)-3α, -1.93 ± 0.67 (p = 0.02); RAGE, -2.14 ± 0.90 (p = 0.04)). Group 1 had two proteins whose expression was significantly altered in both the spleen and wound (agrin was increased in both; IL-1 R6 was decreased in the spleen and elevated in the wound), while Group 3 had one (RAGE was decreased in both the spleen and wound). Dendrograms including data from all three study groups and both the spleen and wound lysates revealed three distinct clusters of closely related proteins in each group, consisting of the following proteins: Cluster 1: Intercellular adhesion molecule (ICAM)-1, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), TIMP-1, IL-10, fractalkine, Fas ligand, IL-13, CNTF, leptin, lipopolysaccharide-induced CXC chemokine (LIX), TNFα, and VEGF. Within this cluster, the two proteins with nearest neighbor connections were Fas ligand and fractalkine. Cluster 2: MIP-3α, monocyte chemotactic protein (MCP)-1, CINC-1, CINC-2α, CINC-3, IL-1β, matrix metalloproteinase (MMP)-8, agrin, thymic chemokine-1, β-NGF, L-selectin, and IL-1R6. This cluster revealed nearest neighbor connections among the CINC proteins. Cluster 3: Prolactin R, IFN-γ, activin A, RAGE, IL-1α, B7-2 / CD86, PDGF-AA, IL-2, and IL-4. In cluster 3, the two closest neighbor proteins were B7-2 / CD86 and PDGF-AA.

[0054] Table 3 summarizes both the proteins involved in wound healing and their role in wound healing, along with the major clusters into which they fall. Specifically, the analytes tested in the test panel are listed along with their role in wound healing. Proteins that were significantly different between pulsed-focused ultrasound treatment and sham control at 16 days post-wounding are shown in bold italics. Samples from each healing stage were significantly altered in the tissues tested (spleen (S); wound surface (W); both (B); neither (N)), and many processes within each stage were similarly affected. Superscripts indicate the cluster to which the protein was associated in the hierarchical cluster analysis.

[0055] The proteins measured in this study were associated with individual steps and processes in the wound healing cascade, and many of these had significant changes. Among the notable features shown in Table 3, the results demonstrate the upregulation of reparative and proliferative proteins known to be affected by M2 macrophage cells, corresponding to phenotypic changes in the wound healing profile in response to pulsed ultrasound treatment as described herein.

[0056] [Table 3]

[0057] One observation was that agrin, which is associated with angiogenesis, ECM formation, and ECM repair and is more highly expressed in fibroblasts, monocytes, and T cells, was moderately correlated with wound diameter. Specifically, agrin was identified as having a significant (P<0.05) correlation with wound size. Figure 10 graphically illustrates the negative correlation (Pearson coefficient -0.5266) between agrin protein expression and wound size, indicating that higher agrin protein expression correlates with smaller wounds. Specifically, the scatter plot shows a significant correlation between agrin protein expression at the wound surface and the percentage of wounds present at 15 days after wounding. Smaller (closer) wounds exhibited higher agrin expression, which is associated with agrin's role in ECM formation and repair. Most of the smaller wounds were also present in the pulsed-focused ultrasound group, suggesting that they were progressing toward proliferation and repair in the wound healing cascade.

[0058] With this in mind, these results support several points. The ZDSD rat model is a cross between Zucker diabetic obese (ZDFfa / fa) rats and SD rats. This model mimics the stages of type 2 diabetes, from prediabetes to overt diabetes and diabetic complications. These rats have comorbidities similar to those of human type 2 diabetes, including nephropathy, neuropathy, fatty liver, hypertension, metabolic syndrome, cardiac dysfunction, and chronic inflammation. Non-fasting blood glucose levels are generally defined as <140 mg / dL for non-diseased individuals, >200 mg / dL for diabetes, and an intermediate level for prediabetes. Starting at week 21, non-fasting blood glucose levels naturally rise above 200 mg / dL in this animal model. A characteristic delay in wound healing was observed in all three groups with elevated levels above normal, consistent with previous studies of the ZDSD model.

[0059] The rate of wound closure was observed to be significantly faster in animals treated with pulsed focused ultrasound during the 24 hours following the first pulsed focused ultrasound treatment (day 2 of the study). This corresponds to the time in a healthy healing cascade when the classical macrophage (M1) population is at its peak and neutrophils are at their post-peak. Pulsed focused ultrasound may promote progression through the inflammatory phase of healing through alterations in systemic cytokine levels. Measurement of splenic and whole blood levels of TNFα and IL-1α demonstrated that splenic pulsed focused ultrasound modulated systemic CAP and NF-κB signaling in a rat model of endotoxemia. In addition, this modulation can be linked via long-distance neural pathways between locally stimulated lymph nodes and the spleen, and pulsed focused ultrasound modulation of CAP attenuated the immune response in a mouse model of pneumonia. The lack of progression from a pro-inflammatory to an anti-inflammatory phenotype is one of the major drivers of delayed wound healing. The method and associated data described here suggest that splenic-targeted pulsed focused ultrasound stimulation accelerates wound closure in ZDSD rats, resulting in wound healing responses similar to those in healthy rats. It also suggests that stimulation alters the systemic immune response through changes in healing rate, driving a more rapid shift from a pro-inflammatory to an anti-inflammatory (M1 to M2) response than observed in sham controls. This may be explained by the continuous replenishment of wound macrophages from activated circulating monocytes. Furthermore, the total blood volume of a 500-g rat (average starting weight in this study) is approximately 31 mL, and the splenic blood volume is approximately 5% of this total volume, or 1.5 mL, resulting in a blood flow rate to the spleen of 0.63 mL / min. During the 3-minute ultrasound stimulation, an additional 1.9 mL of blood volume circulated through the spleen in addition to the resident volume, resulting in approximately 11% of the blood volume being exposed to neurotransmitters and signaling molecules released in the spleen as a result of the ultrasound energy, which may explain the differences in immune cell activation and cytokine expression.

[0060] Injury-induced splenic contracture, occurring simultaneously with the first ultrasound administration described herein, results in the release of stored immune cells into the circulation. Follow-up studies have shown that these stored cells migrate to the injury site. Furthermore, blocking acetylcholine receptor 7a (the binding site for acetylcholine released during CAP activation) in monocytes attenuates CAP activation, and TNFα expression remains elevated even after LPS challenge. These findings, combined with the differential cytokine expression observed in the studies described herein, provide an indication of how splenic stimulation by ultrasound may have systemic effects on peripheral organs (skin) via CAP activation. Stored monocytes can be activated (reducing TNFα and other proinflammatory cytokines) via acetylcholine binding to 7α receptors after pulsed focused ultrasound in the spleen. As the wound heals and the spleen is stimulated daily, net monocyte infiltration into the wound surface may increase relative to unstimulated sham controls. Pulsed focused ultrasound treatment may also allow these cells to be more effectively recruited from the splenic monocyte reservoir after their release into the circulation.

[0061] Cytokines, chemokines, and growth factors are central to orchestrating wound healing responses, and as discussed herein, some are modulated by pulsed-focused ultrasound treatment of the spleen. Specifically, high expression of RAGE is linked to inflammation, hyperglycemia, Alzheimer's disease, cancer, and aging. When blocked or downregulated, RAGE suppresses inflammatory cell influx, NF-κB signaling, and cytokine production. RAGE was significantly lower in the spleens of Groups 2 and 3 and in the wound surface of Group 3. It is involved in the inflammatory phase of healing and is highly expressed in T and B lymphocytes and macrophages. L-selectin, which is expressed in leukocytes and mediates the capture and binding of lymphocytes and neutrophils to the vascular endothelium to control their trafficking to inflammatory sites, was significantly higher in the wound surface of Group 1. Ciliary neurotrophic factor (CNTF) has been shown to protect against LPS-induced endotoxemia, reducing TNFα production and also increasing M2 macrophage differentiation. Cytokines that were altered in the spleen 2 weeks after ultrasound stimulation included IL-6, IL-13, CNTF, TNFα, CINC-1, CINC-2α, CINC-3, IL-1 R6, and RAGE, all of which are expressed in the spleen and secreted into the blood by leukocytes. Because these proteins are secreted, they likely contribute to the systemic response to wounding. IL-1 R6 is primarily expressed in the skin, but also in CD4 +It is also expressed in T cells and monocytes, but not in neutrophils. IL-1R6 activation triggers NF-κB and MAPK signaling, both of which are essential for healing progression. Surprisingly, IL-10, a potent anti-inflammatory cytokine, was lower in both spleens and wound lysates in some groups. A possible explanation is that by day 16, wounds have progressed past the inflammatory phase, in which IL-10 dominates. Both agrin and IL-1R6, which are involved in ECM repair, were increased in pulsed-focused ultrasound vs. sham controls, while TIMP-1, which is linked to scarring / fibrosis, was decreased. Differences in expression of any particular cytokine between groups are related to the severity of type 2 diabetes modeled by each group and may be due to differential wound healing progression. Taken together, cytokines, chemokines, and growth factors play a role in many steps in the wound healing cascade, and aberrant expression impedes progression through the healing cascade. Pulsed focused ultrasound stimulation of splenic CAPs alters proteins involved in each stage of wound healing and many processes within each stage, including a decrease in the expression of several pro-inflammatory proteins and an increase in the expression of several anti-inflammatory proteins.

[0062] With the above discussion and explanation in mind, it will be appreciated that the technical advantages of the techniques disclosed herein include, but are not limited to, promoting wound healing, including healing of chronic wounds, through the use of applied energy such as ultrasound in general, and pulsed focused ultrasound (pFUS) in particular. In some such embodiments, wound healing time may be reduced, or non-healing wounds (e.g., recalcitrant wounds) may heal in response to the application of such applied energy. To apply such focused ultrasound therapy, a device or system may be used that includes, among other components, a waveform generator configured to generate a pulsed sinusoidal waveform, a power amplifier (e.g., an RF power amplifier), a matching circuit, 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 body organs, features, or structures capable of modulating inflammation via cholinergic anti-inflammatory pathway (CAP) signaling.

[0063] This written description uses examples to disclose the invention, including the best mode, and 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 considered to be within the scope of the claims if they have structural elements that do not differ from the literal wording of the claims, or if they include equivalent structural elements that have insubstantial differences from the literal wording of the claims. [Explanation of symbols]

[0064] 100 Focused Ultrasound System 102 Spleen 104 HIFU transducer 108 Matching circuit 112 RF Output Amplifier 116 Waveform Generator 144 Splenic Lymph Nodes 148 Splenic artery 152 Splenic vein 156 Damage to the skin

Claims

1. placing an ultrasound transducer at a stimulation site in a subject having a wound; non-invasively applying pulsed focused ultrasound (pFUS) with the transducer to cause modulation of a target anatomical site containing resident or circulating immune cells; A method for promoting wound healing comprising: The method, wherein modulation of the target anatomical site in the subject results in migration of one or more of monocytes, macrophages, or neutrophils to the wound surface.

2. The method of claim 1 , wherein the stimulation site is distal to the wound.

3. 10. The method of claim 1, wherein the target anatomical site is the subject's spleen and the non-invasively applied pulsed focused ultrasound stimulates nerve tracts within the spleen.

4. 10. The method of claim 1, wherein modulation of the target anatomical site alters the concentration of systemically circulating pro-inflammatory molecules.

5. The method of claim 4, wherein the pro-inflammatory molecules include TNFα and IL-6.

6. 10. The method of claim 1, wherein applying pulsed focused ultrasound to the target anatomical location of the subject comprises applying pulsed focused ultrasound to the spleen of the subject at least once per day for a treatment period.

7. 10. The method of claim 1, wherein the pulsed focused ultrasound (pFUS) applied to the target anatomical site has the following parameters: a pulse center frequency of 1.1 MHz, a pulse repetition period of 0.5 ms, a burst of 150 cycles, a burst period of 200 ms, and a total duration of 3 minutes or more.

8. 10. The method of claim 1, wherein applying pulsed focused ultrasound (pFUS) to the spleen of the subject accelerates wound closure by 3 to 5 days versus no treatment.

9. 2. The method of claim 1, wherein the target anatomical site is the spleen, and IL-6 levels are decreased in the spleen in response to applying the pulsed focused ultrasound (pFUS) to the spleen.

10. 10. The method of claim 1, wherein in response to applying the pulsed focused ultrasound (pFUS) to the target anatomical site, levels of L-selectin are increased at the wound surface of the wound.

11. 2. The method of claim 1, wherein one or both of anti-inflammatory cytokines or proteins associated with wound healing are increased in one or both of the spleen or the wound surface.

12. 10. The method of claim 1, wherein modulation of the target anatomical site in the subject modulates the invasiveness of one or more types of circulating immune cells.

13. 10. The method of claim 1, wherein a first pulsed focused ultrasound (pFUS) is applied to the target anatomical site of the subject within 24 hours of wound formation.

14. a waveform generator configured to generate a pulsed sinusoidal waveform; an RF power amplifier configured to amplify the pulsed sinusoidal waveform; a matching circuit configured to receive the amplified pulsed sinusoidal waveform; a transducer connected to the matching circuit and configured to generate focused ultrasound pulses based on the amplified pulsed sinusoidal waveform; A system for promoting wound healing comprising: The system is configured to direct focused ultrasound pulses toward a target anatomical site containing resident or circulating immune cells after placement of the transducer at an external stimulation site in a subject; The system, wherein modulation of the target anatomical site in the subject results in migration of one or more of monocytes, macrophages, or neutrophils to the wound surface.

15. 15. The system of claim 14, wherein the focused ultrasound pulses have a pulse center frequency of 1.1 MHz, a pulse repetition period of 0.5 milliseconds, a burst of 150 cycles, a burst period of 200 milliseconds, and a total duration of 3 minutes or more.

16. 15. The system of claim 14, wherein the target anatomical site is the subject's spleen and the non-invasively applied pulsed focused ultrasound stimulates nerve tracts within the spleen.

17. 15. The system of claim 14, wherein modulation of the target anatomical site alters the concentration of systemically circulating pro-inflammatory molecules.

18. The system of claim 14, wherein one or both of anti-inflammatory cytokines or proteins associated with wound healing are increased in one or both of the spleen or the wound surface.

19. 15. The system of claim 14, wherein modulation of the target anatomical site in the subject modulates invasiveness of one or more types of circulating immune cells.

20. placing an ultrasound transducer at a stimulation site in a subject having a wound; non-invasively applying pulsed focused ultrasound (pFUS) with the transducer to cause modulation of a target anatomical site containing resident or circulating immune cells; A method for promoting wound healing comprising: The method, wherein modulation of neurons within the site results in a phenotypic change in one or more of leukocytes, monocytes, macrophages, or neutrophils that alters healing of distal surfaces of the wound.

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