Accelerating skin healing with non-invasive focused ultrasound

Abstract

The methods of the present invention generally relate to promoting healing of wounds, including chronic wounds that are generally associated with slow healing times or persistent. In one embodiment, a method of promoting wound healing includes positioning an ultrasound transducer at a stimulation site on a subject having a wound. Pulse focused ultrasound (pFUS) is non-invasively applied using the transducer to cause modulation of a target anatomical site containing resident or circulating immune cells. Modulation of the target anatomical site of the subject results in migration of one or more of monocytes, macrophages, or neutrophils to the wound bed of the wound.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 413,365, filed on October 5, 2022, entitled “ACCELERATING CUTANEOUS HEALING ARODENT MODEL OF TYPE II DIABETES UTILIZING NON-INVASIVE FOCUSED ULTRASOUND OF THE SPLEEN,” which is incorporated herein by reference in its entirety. Technical Field

[0003] 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 methods of applying pulsed focused ultrasound to target tissues, such as the spleen, to promote and accelerate the healing of wounds, such as chronic wounds, that otherwise resist healing or heal slowly. Background Art

[0004] The subject matter discussed in this section should not be assumed to be prior art simply because it is mentioned in this section. Similarly, issues mentioned in this section or related to 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 themselves may also correspond to specific implementations of the claimed technology.

[0005] The cost of caring for non-healing wounds is estimated to reach billions, with more than 6.5 million people affected in the United States and increasing as the population ages. There is also a significant mortality rate associated with non-healing wounds, with a 5-year mortality rate higher than many common cancers such as prostate cancer and breast cancer. When amputation is required due to necrosis after tissue death, the 5-year mortality rate is close to 50%. This is attributed to a wound infection rate of about 50%. Once the first amputation is necessary, multiple amputations are usually required, and the 5-year mortality rate rises to more than 70%. Chronic wounds, which are defined as wounds that do not heal within 3 months, tend to have different microflora from healing wounds and are more susceptible to infection, but infection is not a prerequisite for impaired healing. Chronic wounds are generally divided into three categories: diabetic foot ulcers (DFU), pressure ulcers (bedsores), and leg ulcers caused by peripheral arterial disease (PAD) or post-thrombotic syndrome (PTS). Chronic inflammation, a frequent comorbidity of type 2 diabetes (T2DM), can negatively affect healing by increasing systemic levels of proinflammatory cytokines. Diabetic foot ulcers represent the most common complication in patients with poor disease control (e.g., accelerated metabolic syndrome, chronic inflammation), affecting more than 25% of patients with type 2 diabetes. Patients with diabetic foot ulcers frequently cite lack of mobility and / or flexibility as reasons for low compliance with foot self-care regimens. Many patients with diabetes report incidences of slow-healing or chronic wounds, including DFUs, which result in an annual burden of more than $20 billion on the healthcare system. Summary of the invention

[0006] The following shows an overview of certain embodiments disclosed herein. It should be understood that these aspects are provided only to provide the reader with a brief overview of these specific embodiments, and these aspects are not intended to limit the scope of the present disclosure. In fact, the present disclosure may cover various aspects that may not be shown below.

[0007] The inventive method is generally related to promoting the healing of wounds, including chronic wounds that are usually associated with slow healing time or are persistent. By way of example, the healing of type 2 diabetes is delayed, and new treatment methods may prove to be helpful. As discussed herein, during wound healing, exposing the spleen to pulsed focused ultrasound (pFUS) every day can accelerate the closure rate via the systemic cytokine titer of change. By way of example, generally speaking, energy (such as ultrasonic energy) can be applied to one or more internal organs, structures or features that regulate or otherwise affect the cholinergic anti-inflammatory pathway. In particular, spleen pulsed focused ultrasound changes the inflammatory cytokines in acute endotoxemia and pneumonia models via the regulation of the cholinergic anti-inflammatory pathway (CAP). Non-invasive ultrasound (its spleen for type 2 diabetes (T2DM) rodent model (Zucker Diabetic Sprague Dawley (ZDSD) rat) with full-thickness skin excision wounds) can be applied to attempt to accelerate wound healing via regulating abnormal cytokine expression. In the method discussed herein, pulsed focused ultrasound pulses were applied externally to the spleen area for 3 minutes (e.g., treatment duration) every day (1 time / day, Monday to Friday) over 15 days of 18 courses (e.g., treatment intervals). The wound diameter was measured every day, and the relative levels of cytokines in spleen and wound bed lysates were evaluated. Non-invasive spleen pulsed focused ultrasound accelerated wound closure by up to 4.5 days compared to sham controls. The healing times of all treatment groups were comparable to those of healthy rats in previously published studies (see below), indicating that pulsed focused ultrasound treatment restored the normal wound healing phenotype of ZDSD rats. IL-6 (high levels of pro-inflammatory, low levels may be anti-inflammatory) was lower in stimulated spleens (-2.24±0.81 Log2FC p=0.02), while L-selectin (adhesion molecule, important for cell migration and epithelialization) was higher in the wound bed of stimulated rodents (2.53±0.72 Log2FC p=0.003). In conclusion, splenic pulsed focused ultrasound accelerated healing in a rodent model and has the potential to provide a new, systemic, non-invasive approach to wound care.

[0008] In one embodiment, a method for promoting wound healing is provided. According to this embodiment, an ultrasonic transducer is positioned at a stimulation site on a subject with a wound. Pulsed focused ultrasound (pFUS) is non-invasively applied to a target organ of a subject using a transducer to cause regulation of the subject's cholinergic anti-inflammatory pathway. Regulation of the subject's cholinergic anti-inflammatory pathway causes one or more of monocytes, macrophages, or neutrophils to migrate to the wound bed of the wound.

[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 spleen of the subject, and the non-invasively applied pulsed focused ultrasound stimulates neural pathways within the spleen.

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

[0012] According to aspects of such embodiments, modulation of the subject's cholinergic anti-inflammatory pathway alters the concentration of systemic circulating pro-inflammatory molecules. According to further aspects of such embodiments, the pro-inflammatory molecules comprise TNFα and IL-6.

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

[0014] According to aspects of such embodiments, applying pulsed focused ultrasound to the target organ of the subject includes applying pulsed focused ultrasound to the spleen of the subject at least once per day during the treatment interval.

[0015] According to aspects of such an embodiment, the pulsed focused ultrasound (pFUS) applied to the target organ has the following parameters: a pulse center frequency of 1.1 MHz, a pulse repetition period of 0.5 msec, a pulse width of 300 mV, and a pulse width of 1.1 MHz. pp pulse amplitude, 150 cycle burst, and 200msec burst period.

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

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

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

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

[0020] In an additional embodiment, a system for promoting wound healing is provided. According to this embodiment, the system includes: a function generator configured to generate a pulsed sine waveform; an RF power amplifier configured to amplify the pulsed sine waveform; a matching network configured to receive the amplified pulsed sine waveform; and a transducer connected to the matching network and configured to generate focused ultrasound pulses based on the amplified pulsed sine waveform. According to this embodiment, the system is configured to direct the focused ultrasound pulses toward the target organ when the transducer is placed at the external stimulation site of the subject so as to cause regulation of the cholinergic anti-inflammatory pathway of the subject. Regulation of the cholinergic anti-inflammatory pathway of the subject causes one or more of monocytes, macrophages, or neutrophils to migrate to the wound bed of the wound.

[0021] According to aspects of such additional embodiments, the transducer comprises a high intensity focused ultrasound (HIFU) transducer.

[0022] According to aspects of such additional embodiments, the focused ultrasound pulses have a pulse center frequency of 1.1 MHz, a pulse repetition period of 0.5 msec, and a pulse amplitude of 300 mV. pp The pulse amplitude.

[0023] According to aspects of such additional embodiments, the target organ is a spleen of the subject, and the non-invasively applied pulsed focused ultrasound stimulates neural pathways within the spleen.

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

[0025] According to aspects of such additional embodiments, modulation of the cholinergic anti-inflammatory pathway modulates aberrant cytokine expression in the subject.

[0026] In another embodiment, a method for promoting wound healing is provided. According to this embodiment, a high-intensity focused ultrasound (HIFU) transducer is positioned at a stimulation site on a subject with a chronic skin wound that is away from the stimulation site. Pulsed focused ultrasound (pFUS) is non-invasively applied to the spleen of the subject at least once a day during the treatment interval using the transducer to stimulate neural pathways within the spleen and cause regulation of the subject's cholinergic anti-inflammatory pathways. The pulsed focused ultrasound applied to the spleen has a pulse center frequency of 1.1 MHz, a pulse repetition period of 0.5 msec, and a 300 mV pp Modulation of the cholinergic anti-inflammatory pathway modulates aberrant cytokine expression in the subject and alters the concentration of systemic circulating pro-inflammatory molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] These and other features, aspects and advantages of the present invention will be better understood when the following detailed description is read with reference to the accompanying drawings, in which like characters refer to like parts throughout the several views, and in which:

[0028] Figure 1 Depicted are focused ultrasound systems adapted for non-invasively applying focused ultrasound pulses to a target organ to promote a wound healing process in accordance with aspects of the present disclosure;

[0029] Figure 2 Depicts the stimulation site of a focused ultrasound pulse and the relationship between a target organ (here depicted as the spleen) and the skin through blood and lymphatic circulation according to aspects of the present disclosure;

[0030] Figure 3 Visually depicting a study timeline on a logarithmic scale (top), combined with expected wound healing stages following pulsed focused ultrasound intervention (bottom) and associated immune response times matched to the study timeline, in accordance with aspects of the present disclosure;

[0031] Figure 4 graphically depicting changes or lack of changes in body weight between study groups according to aspects of the present disclosure;

[0032] Figure 5 graphically depicting glucose levels between study groups according to aspects of the present disclosure;

[0033] Figure 6 depicts excisional wounds by day and study group according to aspects of the present disclosure;

[0034] Figure 7 graphically depicting changes in wound size (% of initial wound diameter (mm)) over time and by study group in accordance with aspects of the present disclosure;

[0035] Figure 8 Depicted are linear regressions associated with wound closure over time for each study group in accordance with aspects of the present disclosure;

[0036] Fig. 9 depicts heat maps showing relative changes in proteins for each study group and at the spleen and wound bed in accordance with aspects of the present disclosure; and

[0037] Fig.10 The negative correlation between higher aggregated protein expression and smaller wounds according to aspects of the present disclosure is graphically depicted. DETAILED DESCRIPTION

[0038] One or more specific embodiments will be described below. In order to provide a concise description of these embodiments, not all features of an actual implementation are described in the specification. It should be understood that in the development of any such actual implementation, as in any engineering or design project, many implementation-specific decisions must be made to achieve the developer's specific goals, such as complying with system-related and business-related constraints that may vary from implementation to implementation. In addition, it should be understood that such development efforts may be complex and time-consuming, but are still routine tasks for design, fabrication, and manufacturing for ordinary technicians who benefit from this disclosure.

[0039] When introducing elements of various embodiments of the present disclosure, the articles "a," "an," and "the" are intended to mean that there are one or more of such elements. The terms "comprising," "including," and "having" are intended to be inclusive, and mean that additional elements may be present in addition to the listed elements. Additionally, it should be understood that references to "one embodiment" or "an embodiment" of the present disclosure are not intended to be interpreted as excluding the existence of additional embodiments that also incorporate the referenced features.

[0040] Normal wound healing is composed of four phases (hemostasis, inflammation, proliferation and remodeling) and is similar for acute and chronic wounds, although timing is different. Failure at any stage in these phases can lead to non-healing wounds. Hemostasis composed of the rapid migration of platelets, neutrophils and fibrin deposition causes wound site closure by coagulation. The inflammatory phase (early and late stages) begins with the infiltration of neutrophils to the wound site (within 24-36 hours), and its main function is to prevent the infection of phagocytic bacteria / pathogens, foreign particles, damaged cells and tissues. As a part of the late stage of inflammation (48-72 hours after injury), after attracting monocytes via coagulation factors, cytokines and chemokines, macrophage colonies become dominant. 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 necessary for activating cells (including keratinocytes, fibroblasts and vascular endothelial cells) involved in wound repair. Lymphocytes (T-cells) enter the wound site in the late stage of inflammation (72 hours after injury) mediated by interleukin-1 (IL-1), complement system components such as C3 and C5, and immunoglobulin G (IgG) breakdown products. Classical macrophages (M1) secrete proinflammatory cytokines, which help recruit lymphocytes to the wound bed. Non-classical macrophages (M2) secrete anti-inflammatory cytokines, which trigger the transition to the proliferative phase phenotype, which is observed to be accelerated by pulsed ultrasound treatment as discussed herein. The proliferative phase begins on the third day after injury and lasts for about 2 weeks. It is characterized by the migration, collagen synthesis, adhesion, traction and epithelialization of fibroblasts, keratinocytes, epithelial cells and vascular cells. Finally, the remodeling / maturation phase is responsible for the development of new epithelium and the formation of scar tissue, and can last for 1-2 years. This period also pulls the wound together, much like the contraction of muscle cells, and forms the ECM of repair. Collagen is remodeled, and the wound is completely closed. Before the end of this period, the skin is typically about 80% intact and is significantly weaker and prone to re-injury during this time. Once a scar is formed, the skin never regains the full integrity of uninjured skin and also lacks elasticity, resulting in impaired movement. Any of these stages may fail, although healing tends to become stagnant in the inflammatory phase and never progresses to epithelialization and remodeling. As discussed herein, a technique involving pulsed focused ultrasound (pFUS) treatment initiated 24 hours after wound formation was explored to attenuate early inflammation through CAP modulation, which in turn altered the healing timeline.

[0041] For decades, chronic wound treatment (including diabetic foot ulcer) has been lacking progress. The development of new technologies and the progress of general wound (such as chronic diabetic foot ulcer wound) treatment are needed. Prior art includes negative pressure wound therapy (NPWT), and current conventional treatment consists of wound management using techniques such as wet wound healing, debridement, dressings soaked with growth factors, and walkers (walker cast) to reduce the load on the injured area, all of which are intended to care for wounds, rather than treating the root causes such as chronic inflammation. NPWT, which is more effective for acute wounds, is still used for chronic wounds, but the success rate is limited. Although targeted drug therapy is largely non-existent, local and systemic antibiotic administration is the most widely used.

[0042] Bioelectronic medicine is a rapidly developing field. In practice, electrical stimulation can be applied to nerve fibers to induce a response, and in many cases, electrodes are surgically implanted to deliver energy to the vagus nerve, which then has downstream effects. Vagus nerve stimulation can regulate inflammation through cholinergic anti-inflammatory pathway (CAP) signaling. CAP is a neural pathway that inhibits TNFα production (and other proinflammatory cytokines) in the spleen, liver, and heart when activated. This pathway requires both the vagus nerve and the 7α receptor, and if either is destroyed, the anti-inflammatory response is reversed. This article describes a technique that provides less invasive intervention and can be applied to a point-of-care setting (i.e., non-surgical). In particular, as discussed herein, a method for non-invasively activating CAP using focused ultrasound applied directly to a target organ (e.g., spleen) is described. Although the examples discussed herein generally involve applying ultrasonic pulses to the spleen, it should be understood that the target organ can be any organ capable of regulating inflammation via cholinergic anti-inflammatory pathway (CAP) signaling in response to the non-invasive application of pulsed ultrasonic energy. In the examples described herein, a single ultrasound treatment was found to reduce systemic tumor necrosis factor alpha (TNFα) within 1 hour in an in vivo LPS-induced endotoxemia model. In addition, peripheral blood collected after pulsed focused ultrasound stimulation and exposed to LPS ex vivo produced similar results. Systemic effects were also observed at sites distant from the stimulation site, such as reduced inflammation caused by S. pneumoniae challenge.

[0043] As discussed in this article, Figure 1 showed that skin wound healing was accelerated by stimulating neural pathways within the spleen using a pulsed focused ultrasound system. Figure 2, depicting the relationship between the stimulation site and the spleen and skin through the blood and lymphatic circulation. Based on the results disclosed herein, it was observed that splenic pulsed focused ultrasound altered the monocytes / macrophages / neutrophils that migrated to the wound bed, which in turn altered the wound, making it more susceptible to healing mediated by a systemic immune response, and altered the concentration of systemic circulating pro-inflammatory molecules (such as TNFα, IL-6).

[0044] ZDSD rats were tested as a model of delayed healing. The spleen of ZDSD rats with full-thickness excision skin wounds was stimulated once a day for 15 days. The test group included animals from three age classifications, which corresponded to different disease progressions of type 2 diabetes from prediabetes to late-stage diabetes with comorbidity. In order to further simulate chronic inflammation (comorbidity of type 2 diabetes), the oldest group received ultra-low doses of LPS (5ng / kg) 3 times a week starting 14 days before wound formation. Compared with sham-operated controls, the acceleration of pulsed focused ultrasound-induced wound closure was observed for all age groups. As discussed herein, in this way, ultrasound was used to stimulate the spleen CAP and regulate the immune system away from the wound site to accelerate healing.

[0045] Considering the foregoing, Figure 1 Aspects of a focused ultrasound system 100 for performing the techniques discussed herein are shown. In one embodiment, the system 100 includes a 1.1 MHz high intensity focused ultrasound (HIFU) transducer 104 and a matching network 108, an RF power amplifier 112, and a function generator 116. In this example, the function generator 116 generates a pulsed sine waveform. The pulsed sine waveform is amplified by the RF power amplifier 112 and sent to the impedance matching network 108 connected to the transducer 104. In some embodiments, the pulse center frequency is 1.1 MHz, the pulse repetition period is 0.5 msec (corresponding to a pulse repetition frequency of 2000 Hz); and the pulse amplitude is 300 mV pp In one embodiment, control circuitry, such as one or more processors or one or more application specific integrated circuits (ASICs) configured to process executable code, may be provided as part of (or in communication with) one or more of the function generator 116, the RF power amplifier 112, the matching network 108, and / or the HIFU transducer 104 to allow for direct application of energy to a target organ to stimulate wound healing, such as at the intervals, energies, and / or durations described herein.

[0046] In one specific implementation, the transducer 104 is a HIFU transducer having a diameter of 70 mm and having a spherical surface with 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 curve has a full width at half maximum of 1.8 mm in the lateral direction and 12 mm in the depth direction. The HIFU transducer 104 can be acoustically coupled to the subject via a support (such as a 6 cm high plastic cone filled with degassed water) and an acoustic coupling gel.

[0047] Steering Figure 2 , depicts an abdomen showing the location of the spleen 140 and the delivery location of pulsed focused ultrasound via the transducer 104. When non-invasive ultrasound energy is applied to the spleen 102, cytokine production is altered, as discussed herein. The splenic lymph nodes 144 and the splenic artery 148 and vein 152 are shown enlarged. The splenic lymph nodes 144 are connected to the lymphatic vasculature, which connects the entire lymphatic system, including the lymph in the skin. Skin-resident macrophages release chemoattractants to recruit systemic immune cells through extravasation. When the skin is injured (depicted as injured skin 156 distal to the stimulation site), the blood vessels around the area deliver platelets to achieve homeostasis, followed by delivery of neutrophils within 1 hour to induce inflammation and start the healing process. Monocytes then arrive and differentiate into M1 macrophages. Efferent lymphatic vessels help drain protein-rich inflammatory fluid areas from the interstitium, while afferent vessels help supply cytokines and lymphocytes to the area to help heal.

[0048] With respect to the animal studies discussed herein, thirty ZDSD rats aged 16-25 weeks were randomly divided into 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, with induced chronic inflammation (5 ng / kg LPS; 3X weeks). Each group was further divided into pulsed focused ultrasound stimulation (5 rodents, 2 wounds each) or sham control (5 rodents, 2 wounds each). During the study, one animal was removed from the Group 2 sham cohort due to hypertensive complications. Animals were obtained from Charles River Laboratories and housed on a 12:12 light-dark cycle. They were allowed free access to food and water and fed LabDiet5008.

[0049] Two full-thickness skin excision wounds were produced on the head of each rat under anesthesia with 3% isoflurane. After shaving the head and cleaning the area with povidone-iodine and 70% ethanol, wounds were produced using an 8mm biopsy punch instrument and the skin was carefully removed. Analgesics were discontinued due to the effects of each class of drugs on the immune response; there were no physical signs of pain or discomfort throughout the study. Wounds were left uncovered after surgery, and animals were housed individually to avoid complications caused by cage companion activities. Animals in Group 3 received 5ng / kg LPS 3X / week by intraperitoneal injection starting 14 days before injury and continued throughout the study to induce chronic inflammation. Starting on postoperative day 1, after coupling the HIFU transducer 104 to the shaved skin with coupling gel, while restrained with isoflurane, pulsed focused ultrasound energy was applied to the spleen. The wounds were photographed and measured from front to back using a digital caliper, and the diameter was recorded. For the group receiving U / S stimulation, energy was applied 3 times, one minute each, with a 30-second rest between each. The following ultrasound parameters were used: 1.1 MHz, 300 mV pp , 150 cycle burst, 200 msec burst period. In the case of sham control, the transducer was placed on the spleen but no energy was applied. Stimulation occurred on postoperative days 1-4, 7-11, 14-15. Animals were euthanized on day 16 regardless of wound progression.

[0050] Blood was drawn weekly and analyzed for complete blood counts and blood chemistry. Blood chemistry measurements, including glucose, were performed before 9:00 AM. Remaining plasma was frozen for biochemical analysis. After completion of each study, animals were euthanized by CO2 asphyxiation. Final blood draws were completed by cardiac puncture, and the spleen and wound bed were removed, snap frozen, and stored at -80°C until extraction and analysis of cytokines.

[0051] Considering the foregoing, Figure 3 Aspects of this study timeline are shown visually in relation to the wound healing timeline. In this illustration, Figure 3 The study timeline is visually represented (top), including LPS injections for Group 3 and pulsed focused ultrasound stimulation for all groups on a logarithmic scale. Figure 3 The expected stages of wound healing following pulsed focused ultrasound intervention are visually represented (bottom), and the associated immune response times match the study timeline shown above. It is evident in the wound healing curves that there are phenotypic changes in response to pulsed ultrasound treatment, such as an accelerated shift from the presence of M1 macrophages to the presence of M2 macrophages in the wound curves.

[0052] For protein extraction for biochemical analysis, frozen tissues were minced 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 tissue was dispersed. Samples were kept on ice until all samples were processed, and tissue homogenates were then cleared by centrifugation at 4°C. Prior to each assay as outlined in the assay protocol, a NanoDrop instrument was used to assess the OD280 of each sample.

[0053] Relative to cytokine screening, 34 cytokines, chemokines and growth factor panels (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, VEGFA) are assessed using sandwich-based antibody arrays (C2 antibody arrays). One hundred OD units of spleen or wound bed protein lysates are incubated with membrane arrays, and processed according to the manufacturer's instructions, except that streptavidin-cy5 secondary antibodies are used to replace streptavidin-HRP to utilize fluorescence scanning. Typhoon scanner is used to image the film, and the median fluorescence intensity value is extracted using ImageJ software using microarray curves. All sample blocks were normalized to the reference block using the positive control spot, and the background was subtracted using the blank spot.

[0054] In terms of statistical analysis, the results described herein are expressed as mean ± SEM. Multiple unpaired Student's t-tests (healing rate and cytokine data) or repeated measures two-way ANOVA, followed by Tukey's multiple comparison test (body weight, glucose level and wound size over time) were used to assess the differences between the groups. Data were considered significant when P≤0.05. The time of complete wound closure was inferred using linear regression analysis of normalized wound diameter (% mm of initial diameter). The x-intercept (day) when y=0 (wound size) was defined as the predicted closure time. Cytokine arrays were compared by calculating the log2 multiple change (Log2FC) of the median fluorescence intensity of each pair of samples (pFUS vs. sham surgery). Heat maps were generated and hierarchical cluster analysis was performed to show the differences in the groups. Pearson correlation was used to determine the significant correlation between wound size and relative cytokine expression.

[0055] In view of the foregoing methodological and analytical discussions, the results described below were obtained. Physiological characteristics were assessed to track the general health of the animals throughout the study. Animals were weighed every weekday (i.e., Monday through Friday). Glucose was measured on Day 14 (Group 3, when LPS administration was initiated), Day 0 (all groups), and Day 16 (all groups). In terms of physiological characteristic results, although there were no differences in body weight between animals within the groups receiving pulsed focused ultrasound or sham surgery controls, they were different between age groups, as shown in Table 1. Figure 4 In particular, Figure 4 As shown, Group 1 (16 week ZDSD prediabetic rats) did not exhibit significant weight changes at 15 weeks. Similarly, Group 2 (22 week ZDSD diabetic rats) did not exhibit significant weight changes. Group 3 (25 week +10 ng / kg LPS) lost 4.6% body weight, which was significant from day 0 (*p<0.05) and may be due to the response to LPS. In particular, both pulsed focused ultrasound and sham controls exhibited significant weight loss during the study period relative to Group 3, 5.7% (P<0.05). This was expected due to the low-grade chronic inflammation induced by LPS injection.

[0056] Random glucose levels were assessed on day 0 (before surgery) and day 16. Glucose levels in each group differed according to age but remained constant between day 0 and day 16, as Figure 5 In particular, no significant changes in blood glucose levels were found in Group 1, Group 2 or Group 3 during the study.

[0057] Although complete blood counts (CBC) were within normal ranges for each group, significant differences in white blood cells (WBC) were observed over time and between pulsed focused ultrasound and sham treated animals in Groups 1 and 2. Both monocytes and lymphocytes were lower in Group 1 at day 7, and both neutrophils and monocytes were reduced in Group 2 at day 16. No significant differences were observed in neutrophil, lymphocyte, or monocyte populations. Throughout the study, blood chemistry in each group was within normal ranges in all parameters measured, except for glucose as described above.

[0058] Considering the above timeline, an excisional wound was created on day 0. Figure 6 The leftmost column of Figure 1 shows a representative longitudinal image of the progression of wound healing in ZDSD rats (scale bar = 10 mm (lower right panel)). As described above, ultrasound stimulation or sham stimulation was initiated approximately 24 hours after the wounding procedure. Although an 8 mm biopsy punch was used to create a defined wound, there was still variation in wound diameter (≤ 1.5 mm; ≈ 14%) due to skin laxity. By day 7, Group 1 demonstrated a 4.68 ± 4.9% (ns) reduction in the percentage of initial wound diameter with pFUS compared to sham surgery ( Figure 6 The difference increased to 12.16±4.7% (p<0.05) on day 15. Figure 6 Rightmost column). A decrease of 15.9 ± 4.1% (p < 0.05) on day 7 (pFUS vs. sham) and 10.97 ± 4.2% (p < 0.05) on day 15 was observed for Group 2. A decrease of 14.83 ± 6% (p < 0.05) on day 7 (pFUS vs. sham) and 15.12 ± 2.2% (p < 0.001) on day 15 were revealed for Group 3. Sham controls (33%-41% of original wound size on day 15) were similar to the ZDSD from previously published reports (≈33% of original wound size). These results are in Figure 7 As shown in the figure. Figure 7 , wound diameters were normalized to their respective day 0 diameters (E / B). By day 7, Group 2 and Group 3 pFUS treated rats were significantly smaller compared to sham controls (Δ15.94±4.12% (p=0.003) and Δ14.83±6.02 (p=0.024)). On day 15, all pFUS treatments were significantly smaller than shams (20±4 vs. 33±3; ​​p<0.05), and Group 3 treated was also significantly smaller than sham controls (24±2 vs. 38±2; p<0.001). N=10 wounds / cohort.

[0059] When the wound closed over time in all groups, the rate of closure (% change in diameter mm / day) was significantly accelerated during the first 24 hours after the first stimulation in all three pulsed focused ultrasound cohorts (Table 1). In particular, the rate of % diameter reduction / day was calculated. The data indicate that the degree of effect of pulsed focused ultrasound on the first 24 hours of healing was greater than that on the remaining time course. This may indicate that early wound bed inflammation was altered in a way that promoted healing. The rate of closure remained accelerated until day 15, although not significantly. Since many wounds were not allowed to progress to complete closure, the predicted healing time (X-intercept) was determined by linear regression of daily wound diameter measurements, as Figure 8 . In particular, linear regression analysis was used to predict the number of days to healing reduced with pulsed focused ultrasound stimulation in each group. The height of the slope and the intercept 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). On day 15, regression analysis indicated that for all pulsed focused ultrasound stimulation cohorts, the wound would be completely closed between days 17 and 23 (similar to healthy SD rats), as determined by the 95% CI of the X intercept (days) when Y = 0 (wound size). Table 2 shows Figure 8The regression analysis depicted in Figure 2 was performed. The X-intercept of the linear regression line was designated as the healing days, and the days that FUS was faster were the difference between sham and FUS at the X-intercept. As shown, FUS stimulation resulted in healing times 2.6-4.5 days earlier.

[0060] Table 1

[0061]

[0062] Table 2

[0063]

[0064] Protein expression was also examined in spleen and wound bed lysates. In particular, the association between spleen stimulation and distal wound sites was examined by assaying spleen and wound bed lysates for a panel of chemokines, cytokines, and growth factors. At 16 days post-wounding, relative changes (log2FC) in proteins were determined and plotted as heat maps using hierarchical cluster analysis, as shown in Figure 2. Fig. 9 Specifically, a panel of cytokines, chemokines, and growth factors was evaluated in spleen and wound bed lysates at day 16 post-injury. A heat map was generated ( Fig. 9 ), which depicts relative changes (log2 fold change in median fluorescence intensity) comparing pulsed focused ultrasound to sham controls. Hierarchical cluster analysis was performed with rows as the center; unit variance scaling was applied to rows. Rows were clustered using correlation distance and average connectivity. Columns were grouped first by position and then by group. Asterisks indicate significant differences in relative expression (p<0.05).

[0065] Of the 34 proteins examined, spleen lysates from Group 1 had four proteins with significantly different expression levels (aggregin, 0.87±0.24 (p=0.004); ciliated neutrophil factor (CNTF), -0.58±0.25 (p=0.04), and IL-1R6, -1.20±0.38 (p=0.01)); Group 2 had 11 proteins that were significantly different from sham controls (aggregin, 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); IL-1 R6, 2.32±1.03 (p=0.047); 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, -2.93±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 ( and Group 3 had 8 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 different expression levels of five proteins (aggregin, 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 metalloproteinases (TIMP)-1, -0.43±0.18 (p=0.03)); Group 2 wound bed lysates were not found to have any significant differences, and Group 3 had different levels of three proteins (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 with significantly altered expression in both spleen and wound bed (aggregin, increased in both, IL-1R6, decreased in spleen and increased in wound), and Group 3 had one protein (RAGE, decreased in both spleen and wound bed). The dendrogram including all 3 test groups as well as spleen and wound bed lysate data revealed 3 distinct clusters of tightly associated proteins, each 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 2 proteins with the tightest connections were Fas ligand and Fractalkine. Cluster 2. MIP-3α, monocyte chemoattractant 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 reveals the tightest connections between CINC proteins. Cluster 3. Prolactin R, IFN-γ, activin A, RAGE, IL-1α, B7-2 / CD86, PDFG-AA, IL-2, and IL-4. In cluster 3, the 2 closest proteins are B7-2 / CD86 and PDGF-AA.

[0066] Table 3 summarizes the proteins associated with wound healing and their role in wound healing and which main clusters they are divided into. In particular, the analytes tested in the test group are listed with their role in wound healing. 16 days after wound generation, the proteins significantly different in pulsed focused ultrasound treatment and sham control are represented in bold italics. Analytes from each healing period are significantly changed in the tested tissues (spleen (S); wound bed (W); both do not have (B); (N)), and many processes in this period are also affected. Superscripts indicate clusters associated with proteins in hierarchical cluster analysis.

[0067] The proteins measured in this study were associated with each stage and process of the wound healing cascade, and many of them had significant changes. Among the notable features shown in Table 3, the results describe the upregulation of remodeling and proliferation phase proteins known to be affected by M2 macrophages, corresponding to phenotypic changes relative to the wound healing curve in response to pulsed ultrasound treatment as described herein.

[0068] Table 3

[0069]

[0070]

[0071] One observation was that aggregate proteins, which are associated with angiogenesis, ECM formation and ECM remodeling and are more highly expressed in fibroblasts, monocytes and T cells, were moderately correlated with wound diameter. In particular, aggregate proteins were identified as having a significant correlation with wound size (P<0.05). Fig.10 The negative correlation between higher aggregin expression and smaller wounds is graphically demonstrated (Pearson coefficient -0.5266). In particular, the scatter plot shows a significant correlation between aggregin expression in the wound bed and the % of wounds present at day 15 after injury. Higher aggregin expression is shown in smaller (more closed) wounds, which is related to its role in ECM formation and remodeling. Most of the smaller wounds are also in the pulsed focused ultrasound stimulation group and may develop towards proliferation and remodeling in the wound healing cascade.

[0072] Considering the foregoing, these results support certain discussion points. The ZDSD rat model is a hybrid of Zucker diabetic obesity (ZDF fa / fa) rats and SD. This model recapitulates the stages of type 2 diabetes from prediabetes to overt diabetes to diabetic complications, and these rats have comorbidities similar to human type 2 diabetes, including nephropathy, neuropathy, fatty liver, hypertension, metabolic syndrome, cardiac dysfunction, and chronic inflammation. Non-fasting blood glucose levels are usually defined as <140 mg / dL in non-ill conditions, defined as >200 mg / dL in the case of diabetes, and the middle ground is classified as prediabetes. In this animal model, non-fasting glucose levels spontaneously increased to more than 200 mg / dL starting from the 21st week. In all 3 groups, as the blood glucose levels increased to above normal, a characteristic delay in wound healing was observed, which is similar to previous studies of the ZDSD model.

[0073] It was observed that during the 24-hour period after the first pulsed focused ultrasound treatment (the second day of the study), the wound closure rate of animals treated with pulsed focused ultrasound was significantly faster. This corresponds to the time in the healthy healing cascade when the classical macrophage (M1) population is at its peak and neutrophils are after the peak. Pulsed focused ultrasound can promote the progress of the healing inflammatory phase via changes in systemic cytokine levels. Measurements of spleen and whole blood TNFα levels and spleen IL-1α levels showed that spleen pulsed focused ultrasound regulates systemic signaling of CAP and NF-KB in the endotoxemia rat model. In addition, the regulation can be connected between the locally stimulated lymph nodes and spleen via long-range neural pathways, and pulsed focused ultrasound regulation of CAP weakens the immune response in the pneumonia mouse model. The lack of progression from pro-inflammatory to anti-inflammatory phenotypes is one of the main drivers of delayed wound healing. The currently described technology and associated data show that spleen-targeted pulsed focused ultrasound stimulation accelerates wound closure in ZDSD rats, making the wound healing response similar to that of healthy rats. It is also shown that, via the healing rate that changes, the systemic immune response changes with stimulation, driving the response from pro-inflammatory to anti-inflammatory (M1 to M2 phenotype) faster than observed in the sham control. This can be explained by continuously replenishing wound macrophages from activated circulating monocytes. In addition, the total blood volume (average starting weight in this study) of 500g rats is about 31ml, and the blood volume of the spleen is about 5% of the total volume, or 1.5ml, and the blood flow rate to the spleen is 0.63ml / min. During 3 minutes of ultrasonic stimulation, in addition to the resident volume, 1.9ml of blood volume has circulated through the spleen, causing about 11% of the blood volume to be exposed to neurotransmitters and signaling molecules released in the spleen due to ultrasonic energy, and the differences in immune cell activation and cytokine expression can be explained.

[0074] The spleen contracture caused by injury is consistent with the timing of the first ultrasound dose described herein, and causes the stored immune cells to be released into the circulation. Tracking studies have shown that these stored cells relocate to the site of injury. It has also been shown that blocking the acetylcholine receptor 7a on monocytes (the binding site of acetylcholine released during CAP activation) weakens CAP activation, and TNFα expression remains high after LPS attack. These findings, together with the differential cytokine expression observed in the study described in the present invention, provide instructions on how ultrasound spleen stimulation produces systemic effects on peripheral organs (skin) via activation of CAP. After spleen pulsed focused ultrasound (reducing TNFα and other proinflammatory cytokines), reservoir monocytes can be activated via acetylcholine binding to 7α receptors. As wounds heal, and the spleen is stimulated every day, the net infiltration of monocytes to the wound bed may increase relative to unstimulated sham control. Pulsed focused ultrasound treatment can also allow these cells to be more effectively recruited from spleen reservoir monocytes after they are released into the circulation.

[0075] Cytokines, chemokines and growth factors are central factors in coordinating wound healing responses, and as discussed herein, several of these factors are regulated by splenic pulsed focused ultrasound therapy. Specifically, high expression of RAGE is associated with inflammation, hyperglycemia, Alzheimer's disease, cancer and aging. When blocked or downregulated, inflammatory cell influx, NF-κB signaling and cytokine production are inhibited. Significantly lower RAGE in group 2 and group 3 spleens and group 3 wound beds is involved in the inflammatory phase of healing, and is highly expressed in T and B lymphocytes and macrophages. L-selectins expressed on leukocytes and responsible for mediating capture and tethering to the vascular endothelium to transport lymphocytes and neutrophils to the site of inflammation are significantly higher in the wound bed of group 1. Ciliary neutrophil factor (CNTF) has been shown to protect against LPS-induced endotoxemia and reduce TNFα production, and can also increase M2 macrophage differentiation. Cytokines that were altered in the spleen after 2 weeks of ultrasound stimulation included IL-6, IL-13, CNTF, TNFα, CINC-1, CINC-2α, CINC-3, IL-1R6, and RAGE, and all are expressed in the spleen and secreted into the blood via leukocytes. Because these proteins are secreted, they may affect the systemic response to injury. IL-1R6 is primarily expressed in the skin, but is also expressed in CD4 + It is expressed in T cells and monocytes, but not in neutrophils. 11-1R6 activation induces NF-kB and MAPK signaling, both of which are necessary for healing progression. Surprisingly, in some groups, IL-10 (a potent anti-inflammatory cytokine) was low in both spleen and wound bed lysates. The possible reason is that by day 16, the wound had developed through an inflammatory phase, in which IL-10 was the most prevalent. Compared with sham controls, pulsed focused ultrasound increased aggregated proteins and IL-1 R6 involved in ECM remodeling, while TIMP-1 involved in scarring / fibrosis decreased. The difference in expression of any specific cytokine between the groups may be due to different wound healing progressions because it is related to the severity of type 2 diabetes simulated in each group. In summary, cytokines, chemokines, and growth factors are responsible for many processes in the wound healing cascade, and abnormal expression disrupts progression through the healing cascade. Pulsed focused ultrasound stimulation of spleen CAP changes proteins associated with each period of wound healing and many processes within that period. These changes include reduced expression of some pro-inflammatory proteins and increased expression of some anti-inflammatory proteins.

[0076] In view of the foregoing discussion and explanation, it can be understood that the technical advantages of the technology disclosed in the present invention include, but are not limited to, the use of applied energy, such as general ultrasound and special pulsed focused ultrasound (pFUS), to promote wound healing, including the healing of chronic wounds. In certain such embodiments, wound healing time can be shortened or unhealed wounds (e.g., persistent wounds) can be healed in response to the application of such applied energy. Such focused ultrasound treatment can be applied using a device or system, and in addition to other components, the device or system also includes a function generator configured to generate a pulsed sinusoidal waveform, 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) can be applied to the spleen or other internal organs, features, or structures that can regulate inflammation via cholinergic anti-inflammatory pathway (CAP) signaling.

[0077] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any included methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to fall 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 insignificant differences from the literal language of the claims.

Claims

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

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

3. The method of claim 1, wherein the target anatomical site is the spleen of the subject, and wherein the non-invasively applied pulsed focused ultrasound stimulates neural pathways within the spleen.

4. 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. The method of claim 1, wherein applying pulsed focused ultrasound to the target anatomical site of the subject comprises applying pulsed focused ultrasound to the spleen of the subject at least once per day during a treatment interval.

7. 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 msec, a 150 cycle burst, a burst period of 200 msec, and a total duration of 3 minutes or more.

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

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

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

11. 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 bed.

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

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 msec, a 150 cycle burst, a burst period of 200 msec, and a total duration of 3 minutes or more.

16. The system of claim 14, wherein the target anatomical site is the spleen of the subject, and wherein the non-invasively applied pulsed focused ultrasound stimulates neural pathways within the spleen.

17. 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 bed.

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

20. A method for promoting wound healing, the method comprising: positioning an ultrasound transducer at a stimulation site on a subject having a wound; non-invasively applying pulsed focused ultrasound (pFUS) using the transducer to cause modulation of a target anatomical site containing resident or circulating immune cells; and Wherein modulation of neurons within the site causes a phenotypic change in one or more of leukocytes, monocytes, macrophages or neutrophils, which alters healing of a wound bed distal to the wound.

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