Effects Of Low-intensity Pulsed Ultrasound Research Articles

Protective effect of low‐intensity pulsed ultrasound on immune checkpoint inhibitor-related myocarditis via fine-tuning CD4+ T-cell differentiation

PurposeImmune checkpoint inhibitors (ICIs) have transformed traditional cancer treatments. Specifically, ICI-related myocarditis is an immune-related adverse event (irAE) with high mortality. ICIs activate CD4+ T-lymphocyte reprogramming, causing an imbalance between Th17 and Treg cell differentiation, ultimately leading to myocardial inflammatory damage. Low-intensity pulsed ultrasound (LIPUS) can limit inflammatory responses, with positive therapeutic effects across various cardiovascular inflammatory diseases; however, its role in the pathogenesis of ICI-related myocarditis and CD4+ T-cell dysfunction remains unclear. Accordingly, this study investigated whether LIPUS can alleviate ICI-related myocarditis inflammatory damage and, if so, aimed to elucidate the beneficial effects of LIPUS and its underlying molecular mechanisms.MethodsAn in vivo model of ICI-related myocarditis was obtained by intraperitonially injecting male A/J mice with an InVivoPlus anti-mouse PD-1 inhibitor. LIPUS treatment was performed via an ultrasound-guided application to the heart via the chest wall. The echocardiographic parameters were observed and cardiac function was assessed using an in vivo imaging system. The expression of core components of the HIPPO pathway was analyzed via western blotting.ResultsLIPUS treatment reduced cardiac immune responses and inflammatory cardiac injury. Further, LIPUS treatment alleviated the inflammatory response in mice with ICI-related myocarditis. Mechanistically, in the HIPPO pathway, the activation of Mst1–TAZ axis improved autoimmune inflammation by altering the interaction between the transcription factors FOXP3 and RORγt and regulating the differentiation of Treg and Th17 cells.ConclusionLIPUS therapy was shown to reduce ICI-related myocarditis inflammatory damage and improve cardiac function, representing an exciting finding for irAEs treatment.

Latest progress in low-intensity pulsed ultrasound for studying exosomes derived from stem/progenitor cells.

Stem cells have self-renewal, replication, and multidirectional differentiation potential, while progenitor cells are undifferentiated, pluripotent or specialized stem cells. Stem/progenitor cells secrete various factors, such as cytokines, exosomes, non-coding RNAs, and proteins, and have a wide range of applications in regenerative medicine. However, therapies based on stem cells and their secreted exosomes present limitations, such as insufficient source materials, mature differentiation, and low transplantation success rates, and methods addressing these problems are urgently required. Ultrasound is gaining increasing attention as an emerging technology. Low-intensity pulsed ultrasound (LIPUS) has mechanical, thermal, and cavitation effects and produces vibrational stimuli that can lead to a series of biochemical changes in organs, tissues, and cells, such as the release of extracellular bodies, cytokines, and other signals. These changes can alter the cellular microenvironment and affect biological behaviors, such as cell differentiation and proliferation. Here, we discuss the effects of LIPUS on the biological functions of stem/progenitor cells, exosomes, and non-coding RNAs, alterations involved in related pathways, various emerging applications, and future perspectives. We review the roles and mechanisms of LIPUS in stem/progenitor cells and exosomes with the aim of providing a deeper understanding of LIPUS and promoting research and development in this field.

IMMU-52. LOW-INTENSITY PULSED ULTRASOUND CONDUCTS ANTIGEN-INDEPENDENT T CELL HOMING INTO THE BRAIN PARENCHYMA WITH THE INTACT BLOOD-BRAIN BARRIER

Abstract The blood-brain barrier (BBB) protects the brain parenchyma from excessive inflammation but also leads to poor immune cell homing, hampering the success of immunotherapies, such as ones for low-grade glioma, where the BBB is intact. Low-intensity pulsed ultrasound (LIPU) combined with the application of microbubbles allows the non-invasive, transient opening of the BBB and recent studies have observed immune cell infiltration into malignant gliomas following LIPU. While these data warrant mechanistic investigations on the effects of LIPU in preclinical settings, most mouse models of malignant glioma already display BBB disruption. Therefore, to study whether LIPU affects antigen-specific immune cell trafficking through the BBB, we created a novel immunocompetent mouse model of non-tumor bearing mice that display an intact BBB and express a minigene cassette encoding for four immunogenic peptides in glia cells under the glial fibrillary acidic protein (GFAP) promotor. These peptides are either presented by major histocompatibility complex (MHC) class I: human melanoma antigen glycoprotein (gp) 100 and lymphocytic choriomeningitis virus (LCMV)gp31) or class II: LCMV gp66 and tyrosinase-related protein 1. Minigene expression in astrocytes induced antigen-specific T cell proliferation and Interferon-γ release in vitro. Intravenous adoptive T cell transfer of naïve or respectively CD3/CD28-coactivated antigen-specific CD 8 T cells targeting gp100 peptide followed by immediate LIPU treatment induced T cell homing into the CNS parenchyma, and astrocyte-T cell interaction was confirmed by ex-vivo two-photon imaging. Furthermore, some animals that underwent immediate LIPU treatment after transfer of preactivated antigen-specific T cells developed neurological deficits such as ataxia, while no deficits were observed in those animals that underwent LIPU but did not receive T cells or received activated T cells but did not undergo LIFU treatment. In conclusion, LIPU might be a novel effective tool to support antigen-independent immune cell homing with the intact BBB.

Transcriptome profile analysis revealed the potential mechanism of LIPUS treatment for Adriamycin-induced chronic kidney disease rat

BackgroundDeveloping effective therapeutic strategies to delay the progression of chronic kidney disease (CKD) remains a significant challenge. Low-intensity pulsed ultrasound (LIPUS) has demonstrated potential for treating CKD, but the underlying molecular mechanisms are still elusive. This study aimed to evaluate the therapeutic efficacy of LIPUS and to elucidate the involved genes and signaling pathways. MethodsThe CKD model was established in rats using Adriamycin (ADR). The bilateral kidneys of CKD rats were continuously stimulated with LIPUS for a period of four weeks. The therapeutic efficacy was defined by renal function and histopathological evaluation. RNA sequencing was employed to profile the transcriptome of rat kidneys in each group. Cluster analysis was utilized to identify differentially expressed genes (DEGs), followed by enrichment analysis of their associated pathways using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. ResultsLIPUS treatment improved ADR-induced renal dysfunction in the CKD group. Renal fibrosis and pathological damages were also alleviated in the ADR + LIPUS group compared to the ADR group. Cluster analysis identified 844 DEGs. GO enrichment analysis revealed enrichment in inflammatory response terms, while KEGG enrichment analysis highlighted the nuclear factor kappa B (NF-κB) signaling and ferroptosis-related pathways. ConclusionContinuous LIPUS treatment improved ADR-induced renal fibrosis and dysfunction. The therapeutic effect of LIPUS was primarily due to its ability to suppress the CKD-related inflammation, which was associated with the modulation of the NF-κB and ferroptosis signaling pathways. These findings provide a new insight into the potential molecular mechanisms of LIPUS in treating CKD. Further research is necessary to confirm these findings and to identify potential therapeutic targets within these pathways.

Increased intracellular diffusivity of macromolecules within a mammalian cell by low-intensity pulsed ultrasound

Whilst a number of studies have demonstrated that low-intensity pulsed ultrasound (LIPUS) is a promising therapeutic ultrasound technique that can be used for delivering mild mechanical stimuli to target tissue non-invasively, the underlying biophysical mechanisms still remain unclear. Most mechanism studies have focused explicitly on the effects of LIPUS on the cell membrane and mechanosensitive receptors. In the present study, we propose an additional mechanism by which LIPUS propagation through living cells may directly impact intracellular dynamics, particularly the diffusion transport of biomolecules. To support our hypothesis, human epithelial-like cells (SaOS-2 and HeLa) seeded on a confocal dish placed on a microscope stage were exposed to LIPUS with various exposure conditions (ultrasound frequencies of 0.5, 1 and 3 MHz, peak acoustic pressure of 200 and 400 kPa, a pulse repetition frequency of 1 kHz and a 20 % duty cycle), and the diffusivities of various sizes of biomolecules in the cytoplasm area were measured using fluorescence recovery after photobleaching (FRAP). Furthermore, giant unilamellar vesicles (GUVs) filled with macromolecules were used to examine the physical causal relationship between LIPUS and molecular diffusion changes. Nucleocytoplasmic transport coefficients were also measured by modified FRAP that bleaches the whole cell nuclear region. Extracellular signal-regulated kinases (ERK) activity (the phosphorylation dynamics) was monitored using fluorescence resonance energy transfer (FRET) microscopy. All the measurements were taken during, before and after the LIPUS exposure. Our experimental results clearly showed that the diffusion coefficients of macromolecules within the cell increased with acoustic pressure by 12.1 to 33.5 % during the sonication, and the increments were proportional to their molecular sizes regardless of the ultrasound frequency used. This observation in living cells was consistent with the GUVs exposed to the LIPUS, which indicated that the diffusivity increase was a passive physical response to the acoustic energy of LIPUS. Under the 1 MHz LIPUS exposure with 400 kPa, the passive nucleocytoplasmic transport of enhanced green fluorescent protein (EGFP) was accelerated by 21.4 %. With the same LIPUS exposure condition, both the diffusivity and phosphorylation of ERK induced by EGF treatment were significantly elevated simultaneously, which implied that LIPUS could also modify the kinase kinetics in the signal transduction process. Taken together, this study is the first attempt to uncover the physical link between LIPUS and the dynamics of intracellular macromolecules and related biological processes that LIPUS can possibly increase the diffusivity of intracellular macromolecules, leading to the changes in the basic cellular processes: passive nucleocytoplasmic transport and ERK. Our findings can provide a novel perspective that the mechanotransduction process that the intracellular region, in addition to the cell membrane, can convert the acoustic stimuli of LIPUS to biochemical signals.

Multiscale simulation of the effect of low-intensity pulsed ultrasound on the mechanical properties distribution of osteocytes

Low-intensity pulsed ultrasound (LIPUS) is a potential effective means for the prevention and treatment of disuse osteoporosis. In this paper, the effect of LIPUS exposure on the mechanical properties distribution of the osteocyte system (osteocyte body contains nucleus, osteocyte process, and primary cilia) is simulated. The results demonstrate that the mechanical micro-environment of the osteocyte is significantly improved by ultrasound exposure, and the mean von Mises stress of the osteocyte system increases linearly with the excitation sound pressure amplitude. The mechanical effect of LIPUS on osteocytes is enhanced by the stress amplification mechanism of the primary cilia and osteocyte processes.

Therapeutic Myogenesis Induced by Ultrasound Exposure in a Volumetric Skeletal Muscle Loss Injury Model

Background: Low-intensity pulsed ultrasound (LIPUS) irradiation has been shown to induce various responses in different cells. It has been shown that LIPUS activates extracellular signal–regulated kinase 1/2 (ERK1/2) through integrin. Purpose: To study the effects of LIPUS on myogenic regulatory factors and other related myogenesis elements in a volumetric skeletal muscle loss injury model. Study Design: Controlled laboratory study. Methods: C57BL/6J mice were subjected to full-thickness muscle defect injury of the quadriceps and treated with direct application of LIPUS 20 min/d or non-LIPUS treatment (control) for 3, 7, and 14 days. LIPUS was also applied to C2C12 cells in culture in the presence of low and high doses of lipopolysaccharides. The expression levels of myogenic regulatory factors and the expression levels of myokine-related and angiogenic-related proteins of the control and LIPUS groups were analyzed. Results: Muscle volume in the injury site was restored at day 14 with LIPUS treatment. Paired-box protein 7, myogenic factor 5, myogenin, and desmin expressions were significantly different between control and LIPUS groups at days 7 and 14. Myokine and angiogenic cytokine-related factors were significantly increased in the LIPUS group at day 3 and decreased with no significant difference between the groups by day 14. LIPUS induced different responses of myogenic regulatory factors in C2C12 cells with low and high doses of lipopolysaccharides. LIPUS promoted myogenesis through short-lived increase in interleukin-6 and heme oxygenase 1, together with activation of ERK1/2. Conclusion: LIPUS had a constant effect on the variables of tissue damage, from macrotrauma to microtrauma, leading to efficient muscle regeneration. Clinical Relevance: The focus of therapeutic strategies with LIPUS has been not only for microvascular regeneration but also for skeletal muscle and related local tissue recovery from acute or chronic damage.

Low intensity pulsed ultrasound ameliorates Adriamycin-induced chronic renal injury by inhibiting ferroptosis

ABSTRACTObjectiveIt is very important to develop a new therapeutic strategy to cope with the increasing morbidity and mortality of chronic kidney disease (CKD). As a kind of physical therapy, low intensity pulsed ultrasound (LIPUS) has remarkable anti-inflammatory and repair-promoting effects and is expected to become a new therapeutic method for CKD. This study aims to clarify the treatment effect of LIPUS on CKD-related renal inflammation and fibrosis, and to further explore the potential signal network of LIPUS treatment for ameliorating chronic renal injury.MethodsA rat model simulating the progress of CKD was established by twice tail-vein injection of Adriamycin (ADR). Under anesthesia, bilateral kidneys of CKD rats were continuously stimulated by LIPUS for four weeks. The parameters of LIPUS were 1.0 MHz, 60 mW/cm2, 50% duty cycle and 20 min/d.ResultsLIPUS treatment effectively inhibited ADR-induced renal inflammation and fibrosis, and improved CKD-related to oxidative stress and ferroptosis. In addition, the therapeutic effect of LIPUS is closely related to the regulation of TGF-β1/Smad and Nrf2/keap1/HO-1 signalling pathways.DiscussionThis study provides a new direction for further mechanism research and lays an important foundation for clinical trials.

Low-intensity pulsed ultrasound ameliorates glia-mediated inflammation and neuronal damage in experimental intracerebral hemorrhage conditions

BackgroundIntracerebral hemorrhage (ICH) is a condition associated with high morbidity and mortality, and glia-mediated inflammation is a major contributor to neurological deficits. However, there is currently no proven effective treatment for clinical ICH. Recently, low-intensity pulsed ultrasound (LIPUS), a non-invasive method, has shown potential for neuroprotection in neurodegenerative diseases. This study aimed to investigate the neuroprotective effects and potential mechanisms of LIPUS on glia-mediated inflammation in ICH.MethodsThis study used 289 mice to investigate the effects of LIPUS on ICH. ICH was induced by injecting bacterial collagenase (type VII-S; 0.0375 U) into the striatum of the mice. LIPUS was applied noninvasively for 3 days, including a 2-h-delayed intervention to mimic clinical usage. The study evaluated neurological function, histology, brain water content, hemoglobin content, MRI, and protein expression of neurotrophic factors, inflammatory molecules, and apoptosis. In vitro studies investigated glia-mediated inflammation by adding thrombin (10 U/mL) or conditioned media to primary and cell line cultures. The PI3K inhibitor LY294002 was used to confirm the effects of PI3K/Akt signaling after LIPUS treatment.ResultsLIPUS treatment improved neurological deficits and reduced tissue loss, edema, and neurodegeneration after ICH. The protective effects of LIPUS resulted from decreased glia-mediated inflammation by inhibiting PI3K/Akt-NF-κB signaling, which reduced cytokine expression and attenuated microglial activation-induced neuronal damage in vitro.ConclusionsLIPUS treatment improved neurological outcomes and reduced glia-mediated inflammation by inhibiting PI3K/Akt-NF-κB signaling after ICH. LIPUS may provide a non-invasive potential management strategy for ICH.

Low-intensity pulsed ultrasound at ST36 improves the gastric motility by TNF-α/IKKβ/NF-κB signaling pathway in diabetic rats.

Low-intensity pulsed ultrasound (LIPUS) can effectively regulate the central and peripheral nervous system. However, whether LIPUS could act on acupuncture points to modulate the activity of peripheral nervous has rarely been studied. Our study aimed to investigate whether LIPUS at ST36 could improve gastric emptying in diabetic gastroparesis rats. Sprague-Dawley male rats were divided into three groups: control group (CON), diabetic gastroparesis group (DM), and diabetic gastroparesis LIPUS treated group (LIPUS). The body weight and blood glucose were recorded every week. Glucose tolerance, gastric emptying rate, and gastric motility were measured before and after treatment. Gastric motility was assessed by ultrasonic examination and Muscle strip experiment. The expression level of c-Kit was assessed by immunohistochemistry staining. Levels of TNF-α, p-NF-κB p-65, NF-κB p-65, and p-IKKβ, IKKβ were measured by western blot. We reported LIPUS at an intensity of 0.88W/cm2 exhibited significant differences in functional recovery of gastric delayed emptying in diabetic rats. Through ultrasound gastric motility functional testing and analysis of gastric antral smooth muscle strips indirectly and directly proved the effectiveness of LIPUS for the recovery of gastric delayed emptying. Pathological analysis and western blot indicated that the mechanism by which LIPUS applied to ST36 improved gastric motility may be partially attributed to the inhibition of the TNF-α/IKKβ/NF-κB signaling pathway, thereby rescuing the damaged interstitial cells of Cajal network. LIPUS at ST36 improved the gastric motility and rescued the damaged networks of interstitial cells of Cajal. LIPUS may have a promising therapeutic potential for diabetic gastroparesis.

Influence of Low-Intensity Pulsed Ultrasound Parameters on the Bone Mineral Density in Rat Model: A Systematic Review.

Bone recovery typically depends on the age of organisms or the prevalence of metabolic disorders such as osteoporosis, which is a metabolic condition characterized by decreased bone strength and bone mineral density (BMD). Therefore, low-intensity pulsed ultrasound (LIPUS), a non-invasive method for osteogenic stimulation, presents promising results. However, heterogeneity in animal study designs is a typical characteristic. Hence, we conducted a systematic review to evaluate the effectiveness of LIPUS in the recovery of experimental bone defects using rat models. We examined the areal and volumetric BMD to identify LIPUS doses to be applied and evaluated the accuracy reported by previous studies. The Virtual Health Library regional portal, PubMed, Embase, EBSCOhost, Scopus and CAPES were reviewed for animal studies that compared fracture treatments based on LIPUS with sham or no treatments using rat models and reported BMD as an outcome. The tool provided by the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) and the Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES) checklist were used to assess the bias and quality of such studies. Of the six studies reviewed, the most frequently used LIPUS dose had an ultrasonic frequency of 1.0MHz, repetition rate of 0.1kHz and pulse duration of 2000μs. An intensity (ISATA) of 30mW/cm2 was the most preferred for bone recovery. However, the BMD could not solely irrefutably evaluate the effectiveness of LIPUS in bone recovery as the results were discordant with each other. The discrepancies in experimental methodologies, low-quality classifications and high risk of bias in the selected studies, however, did not validate the undertaking of a meta-analysis. On the basis of the BMD results, no sufficient evidence was found to recommend the use of LIPUS for bone recovery in rat models. Thus, this systematic review indicates that the accuracy of such reports must be improved to improve their scientific quality to facilitate a transition of LIPUS applications from pre-clinical research to clinic use.

Low-intensity pulsed ultrasound alleviates doxorubicin-induced cardiotoxicity via inhibition of S100a8/a9-mediated cardiac recruitment of neutrophils.

Doxorubicin (DOX)-induced cardiotoxicity limits its broad use as a chemotherapy agent. The development of effective and non-invasive strategies to prevent DOX-associated adverse cardiac events is urgently needed. We aimed to examine whether and how low-intensity pulsed ultrasound (LIPUS) plays a protective role in DOX-induced cardiotoxicity. Male C57BL/6J mice were used to establish models of both acute and chronic DOX-induced cardiomyopathy. Non-invasive LIPUS therapy was conducted for four consecutive days after DOX administration. Cardiac contractile function was evaluated by echocardiography. Myocardial apoptosis, oxidative stress, and fibrosis were analyzed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining, dihydroethidium (DHE) staining, and picrosirius red staining assays. RNA-seq analysis was performed to unbiasedly explore the possible downstream regulatory mechanisms. Neutrophil recruitment and infiltration in the heart were analyzed by flow cytometry. The S100a8/a9 inhibitor ABR-238901 was utilized to identify the effect of S100a8/a9 signaling. We found that LIPUS therapy elicited a great benefit on DOX-induced heart contractile dysfunction in both acute and chronic DOX models. Chronic DOX administration increased serum creatine kinase and lactate dehydrogenase levels, as well as myocardial apoptosis, all of which were significantly mitigated by LIPUS. In addition, LIPUS treatment prevented chronic DOX-induced cardiac oxidative stress and fibrosis. RNA-seq analysis revealed that LIPUS treatment partially reversed alterations of gene expression induced by DOX. Gene ontology (GO) analysis of the downregulated genes between DOX-LIPUS and DOX-Sham groups indicated that inhibition of neutrophil chemotaxis might be involved in the protective effects of LIPUS therapy. Flow cytometry analysis illustrated the inhibitory effects of LIPUS on DOX-induced neutrophil recruitment and infiltration in the heart. Moreover, S100 calcium binding protein A8/A9 (S100a8/a9) was identified as a potential key target of LIPUS therapy. S100a8/a9 inhibition by ABR-238901 showed a similar heart protective effect against DOX-induced cardiomyopathy to LIPUS treatment. LIPUS therapy prevents DOX-induced cardiotoxicity through inhibition of S100a8/a9-mediated neutrophil recruitment to the heart, suggesting its potential application in cancer patients undergoing chemotherapy with DOX.

Ultrasound reduces inflammation by modulating M1/M2 polarization of microglia through STAT1/STAT6/PPARγ signaling pathways.

Activated microglia can be polarized to the pro-inflammatory M1 phenotype and the anti-inflammatory M2 phenotype. Low-intensity pulsed ultrasound (LIPUS) can attenuate pro-inflammatory responses in activated microglia. This study aimed to investigate the effects of LIPUS on M1/M2 polarization of microglial cells and the regulatory mechanisms associated with signaling pathways. BV-2 microglial cells were stimulated by lipopolysaccharide (LPS) to an M1 phenotype or by interleukin-4 (IL-4) to an M2 phenotype. Some microglial cells were exposed to LIPUS, while others were not. M1/M2 marker mRNA and protein expression were measured using real-time polymerase chain reaction and western blot, respectively. Immunofluorescence staining was performed to determine inducible nitric oxide synthase (iNOS)-/arginase-1 (Arg-1)- and CD68-/CD206-positive cells. LIPUS treatment significantly attenuated LPS-induced increases in inflammatory markers (iNOS, tumor necrosis factor-α, interleukin-1β, and interleukin-6) as well as the expression of cell surface markers (CD86 and CD68) of M1-polarized microglia. In contrast, LIPUS treatment significantly enhanced the expression of M2-related markers (Arg-1, IL-10, and Ym1) and membrane protein (CD206). LIPUS treatment prevented M1 polarization of microglia and enhanced or sustained M2 polarization by regulating M1/M2 polarization through the signal transducer and activator of transcription 1/STAT6/peroxisome proliferator-activated receptor gamma pathways. Our findings suggest that LIPUS inhibits microglial polarization and switches microglia from the M1 to the M2 phenotype.

Low-intensity pulsed ultrasound regulates osteoblast-osteoclast crosstalk via EphrinB2/EphB4 signaling for orthodontic alveolar bone remodeling.

Background: The limited regenerative potential of periodontal tissue remains a challenge in orthodontic treatment, especially with respect to alveolar bone remodeling. The dynamic balance between the bone formation of osteoblasts and the bone resorption of osteoclasts controls bone homeostasis. The osteogenic effect of low-intensity pulsed ultrasound (LIPUS) is widely accepted, so LIPUS is expected to be a promising method for alveolar bone regeneration. Osteogenesis is regulated by the acoustic mechanical effect of LIPUS, while the cellular perception, transduction mode and response regulation mechanism of LIPUS stimuli are still unclear. This study aimed to explore the effects of LIPUS on osteogenesis by osteoblast-osteoclast crosstalk and the underlying regulation mechanism. Methods: The effects of LIPUS on orthodontic tooth movement (OTM) and alveolar bone remodeling were investigated via rat model by histomorphological analysis. Mouse bone marrow mesenchymal stem cells (BMSCs) and bone marrow monocytes (BMMs) were purified and used as BMSC-derived osteoblasts and BMM-derived osteoclasts, respectively. The osteoblast-osteoclast co-culture system was used to evaluate the effect of LIPUS on cell differentiation and intercellular crosstalk by Alkaline phosphatase (ALP), Alizarin Red S (ARS), tartrate-resistant acid phosphatase (TRAP) staining, real-time quantitative PCR, western blotting and immunofluorescence. Results: LIPUS was found to improve OTM and alveolar bone remodeling in vivo, promote differentiation and EphB4 expression in BMSC-derived osteoblasts in vitro, particularly when cells were directly co-cultured with BMM-derived osteoclasts. LIPUS enhanced EphrinB2/EphB4 interaction between osteoblasts and osteoclasts in alveolar bone, activated the EphB4 receptor on osteoblasts membrane, transduced LIPUS-related mechanical signals to the intracellular cytoskeleton, and gave rise to the nuclear translocation of YAP in Hippo signaling pathway, thus regulating cell migration and osteogenic differentiation. Conclusions: This study shows that LIPUS modulates bone homeostasis by osteoblast-osteoclast crosstalk via EphrinB2/EphB4 signaling, which benefits the balance between OTM and alveolar bone remodeling.

A novel therapeutic approach of ultrasound stimulation to restore forelimb functions following cervical cord injury in rats

Low-intensity pulsed ultrasound (LIPUS) stimulation has shown promising results in neurorehabilitation following a traumatic injury in brain and peripheral nerves. However, the effects of LIPUS stimulation in the injured neural circuit after spinal cord injury (SCI) are still unknown. We investigated the effects of LIPUS on forelimb functions in chronic cervical cord injured rats with and without a serotonergic agonist drug, Buspirone treatment. Twenty-six rats were trained for forelimb reaching and grasping followed by C4 dorsal funiculi crush injury. To deliver LIPUS, a silicon-coated ultrasound disc was implanted above the cervical cord and electromyography electrodes were implanted into forelimb muscles. In two cohorts (LIPUS and LIPUS + Buspirone combined) rats were tested pre-, with- and post-ultrasound stimulation. In LIPUS group rats, fore-limb reaching and grasping success rates first increased and then dropped after 3 weeks while for combination of drug and LIPUS stimulation the score continued to increase. Furthermore, LIPUS stimulation alone did not result in any significant improvement of grip strength compared to the control and combined groups. The findings of this study indicated the potential of LIPUS in SCI recovery and offer a future research direction of a new neuromodulation method.

Focused Ultrasound Stimulates the Prefrontal Cortex and Prevents MK-801-Induced Psychiatric Symptoms of Schizophrenia in Rats.

Treatment of schizophrenia remains a major challenge. Recent studies have focused on glutamatergic signaling hypoactivity through N-methyl-D-aspartate (NMDA) receptors. Low-intensity pulsed ultrasound (LIPUS) improves behavioral deficits and ameliorates neuropathology in dizocilpine (MK-801)-treated rats. The aim of this study was to investigate the efficacy of LIPUS against psychiatric symptoms and anxiety-like behaviors. Rats assigned to 4 groups were pretreated with or without LIPUS for 5 days. The open field and prepulse inhibition tests were performed after saline or MK-801 (0.3 mg/kg) administration. Then, the neuroprotective effects of LIPUS on the MK-801-treated rats were evaluated using western blotting and immunohistochemical staining. LIPUS stimulation of the prefrontal cortex (PFC) prevented deficits in locomotor activity and sensorimotor gating and improved anxiety-like behavior. MK-801 downregulated the expression of NR1, the NMDA receptor, in rat medial PFC (mPFC). NR1 expression was significantly higher in animals receiving LIPUS pretreatment compared to those receiving only MK-801. In contrast, a significant increase in c-Fos-positive cells in the mPFC and ventral tegmental area was observed in the MK-801-treated rats compared to those receiving only saline; this change was suppressed by pretreatment with LIPUS. This study provides new evidence for the role of LIPUS stimulation in regulating the NMDA receptor and modulating c-Fos activity, which makes it a potentially valuable antipsychotic treatment for schizophrenia.

Low-intensity pulsed ultrasound reduces lymphedema by regulating macrophage polarization and enhancing microcirculation.

Background: Conventional therapies reduce lymphedema but do not cure it because they cannot modulate the pathophysiology of secondary lymphedema. Lymphedema is characterized by inflammation. We hypothesized that low-intensity pulsed ultrasound (LIPUS) treatment could reduce lymphedema by enhancing anti-inflammatory macrophage polarization and microcirculation. Methods: The rat tail secondary lymphedema model was established through the surgical ligation of lymphatic vessels. The rats were randomly divided into the normal, lymphedema, and LIPUS treatment groups. The LIPUS treatment (3min daily) was applied 3days after establishing the model. The total treatment period was 28days. Swelling, fibro adipose deposition, and inflammation of the rat tail were evaluated by HE staining and Masson's staining. The photoacoustic imaging system and laser Doppler flowmetry were used to monitor microcirculation changes in rat tails after LIPUS treatment. The cell inflammation model was activated with lipopolysaccharides. Flow cytometry and fluorescence staining were used to observe the dynamic process of macrophage polarization. Results: After 28days of treatment, compared with the lymphedema group, the tail circumference and subcutaneous tissue thickness of rats in the LIPUS group were decreased by 30%, the proportion of collagen fibers and the lymphatic vessel cross-sectional area was decreased, and tail blood flow was increased significantly. Cellular experiments revealed a decrease in CD86+ macrophages (M1) after LIPUS treatment. Conclusion: The transition of M1 macrophage and the promotion of microcirculation could be responsible for the beneficial effect of LIPUS on lymphedema.

Low-Intensity Pulsed Ultrasound Attenuates Periodontal Ligament Cells Apoptosis by Activating Yes-Associated Protein-Regulated Autophagy

The goal of the work described here was to determine if low-intensity pulsed ultrasound (LIPUS) has an anti-inflammatory effect on lipopolysaccharide (LPS)-induced inflammation in periodontal ligament cells (PDLCs). The mechanism underlying this effect remains to be explored and is likely related to PDLC apoptosis regulated by Yes-associated protein (YAP) and autophagy. To verify this hypothesis, we used a rat model of periodontitis and primary human PDLCs. We examined alveolar bone resorption in rats and apoptosis, autophagy and YAP activity in LPS-treated PDLCs with and without application of LIPUS by cellular immunofluorescence, transmission electron microscopy and Western blotting. Then, siRNA transfection was used to decrease YAP expression to confirm the regulatory role of YAP in the anti-apoptotic effect of LIPUS on PDLCs. We found that LIPUS attenuated alveolar bone resorption in rats and this was accompanied by YAP activation. LIPUS inhibited hPDLC apoptosis by YAP activation, and promoted autophagic degradation to help autophagy completion. These effects were reversed after YAP expression was blocked. LIPUS attenuates PDLC apoptosis by activating Yes-associated protein-regulated autophagy.

Effect of low-intensity pulsed ultrasound on osteogenic differentiation of human induced membrane-derived cells in Masquelet technique

IntroductionThe Masquelet technique is a relatively new method for large bone defect treatment. In this technique, grafted bone tissue is used, and after the cement is removed, the induced membrane (IM; that form around the cement spacers placed in the bone defect region) is thought to play an important role in promoting bone formation. On the other hand, low-intensity pulsed ultrasound (LIPUS) is known to promote fracture healing and angiogenesis through mechanical stimulation. This study aimed to investigate the in vitro effects of LIPUS on the osteogenic differentiation of human induced membrane-derived cells (IMCs). MethodsSeven patients who had been treated using the Masquelet technique were enrolled. The IM was harvested during the second stage of the technique. IMCs were isolated, cultured in growth medium, and then divided into two groups: (1) control group, IMCs cultured in osteogenic medium without LIPUS, and (2) LIPUS group, IMCs cultured in osteogenic medium with LIPUS treatment. Adherent cells from the IM samples were harvested after the first passage and evaluated for cell surface protein expression using immunostaining. A cell proliferation assay was used to count the number of IMCs using a hemocytometer. Osteogenic differentiation capability was assessed using an alkaline phosphatase (ALP) activity assay, Alizarin Red S staining, and real-time reverse transcription-polymerase chain reaction. ResultsCell surface antigen profiling revealed that the IMCs contained cells positive for the mesenchymal stem cell–related markers CD73, CD90, and CD105. No significant difference in cell numbers was found between the control and LIPUS groups. The ALP activity of IMCs in the LIPUS group was significantly higher than that in the control group on days 7 and 14. Alizarin red S staining intensity was significantly higher in the LIPUS group than in the control group on day 21. Runx2 and VEGF expression was significantly upregulated on days 7 and 14, respectively, compared with levels in the control group. ConclusionWe demonstrated the significant effect of LIPUS on the osteogenic differentiation of human IMCs. This study indicates that LIPUS can be used as an additional tool for the enhancement of the healing process of the Masquelet technique.

Application of low-intensity pulsed ultrasound on tissue resident stem cells: Potential for ophthalmic diseases.

Tissue-resident stem cells (TRSCs) have the ability to self-renew and differentiate throughout an individual's lifespan, and they utilize both mechanisms to maintain homeostasis and regenerate damaged tissues. Several studies suggest that these stem cells can serve as a potential source for cell-replacement-based therapy by promoting differentiation or expansion. In recent years, low-intensity pulsed ultrasound (LIPUS) has been demonstrated to effectively stimulate stem cell proliferation and differentiation, promote tissue regeneration, and inhibit inflammatory responses. To present a comprehensive overview of current application and mechanism of LIPUS on tissue resident stem cells. We searched PubMed, Web of Science for articles on the effects of LIPUS on tissue resident stem cells and its application. The LIPUS could modulate cellular activities such as cell viability, proliferation and differentiation of tissue resident stem cells and related cells through various cellular signaling pathways. Currently, LIPUS, as the main therapeutic ultrasound, is being widely used in the treatment of preclinical and clinical diseases. The stem cell research is the hot topic in the biological science, while in recent years, increasing evidence has shown that TRSCs are good targets for LIPUS-regulated regenerative medicine. LIPUS may be a novel and valuable therapeutic approach for the treatment of ophthalmic diseases. How to further improve its efficiency and accuracy, as well as the biological mechanism therein, will be the focus of future research.