Low-intensity Pulsed Ultrasound Stimulation Research Articles

Ultrasound Neuromodulation Reduces Demyelination in a Rat Model of Multiple Sclerosis.

Microglia, astrocytes, and oligodendrocyte progenitor cells (OPCs) may serve as targets for remyelination-enhancing therapy. Low-intensity pulsed ultrasound (LIPUS) has been demonstrated to ameliorate myelin loss and inhibit neuroinflammation in animal models of brain disorders; however, the underlying mechanisms through which LIPUS stimulates remyelination and glial activation are not well-understood. This study explored the impacts of LIPUS on remyelination and resident cells following lysolecithin (LPC)-induced local demyelination in the hippocampus. Demyelination was induced by the micro-injection of 1.5 μL of 1% LPC into the rat hippocampus, and the treatment groups received daily LIPUS stimulation for 5 days. The therapeutic effects of LIPUS on LPC-induced demyelination were assessed through immunohistochemistry staining. The staining was performed to evaluate remyelination and Iba-1 staining as a microglia marker. Our data revealed that LIPUS significantly increased myelin basic protein (MBP) expression. Moreover, the IHC results showed that LIPUS significantly inhibited glial cell activation, enhanced mature oligodendrocyte density, and promoted brain-derived neurotrophic factor (BDNF) expression at the lesion site. In addition, a heterologous population of microglia with various morphologies can be found in the demyelination lesion after LIPUS treatment. These data show that LIPUS stimulation may serve as a potential treatment for accelerating remyelination through the attenuation of glial activation and the enhancement of mature oligodendrocyte density and BDNF production.

Low-Intensity Pulsed Ultrasound Enhanced Adipose-Derived Stem Cell-Mediated Angiogenesis in the Treatment of Diabetic Erectile Dysfunction through the Piezo-ERK-VEGF Axis.

Objectives Erectile dysfunction is a major comorbidity of diabetes. Stem cell transplantation is a promising method to treat diabetic erectile dysfunction. In this study, we evaluated whether low-intensity pulsed ultrasound (LIPUS) could enhance the efficacy of adipose-derived stem cells (ADSCs) and investigated the underlying molecular mechanism. Materials and methods. Sixty 8-week-old male Sprague–Dawley rats were randomly divided into the normal control (NC) cohort or the streptozocin-induced diabetic ED cohort, which was further subdivided into DM, ADSC, LIPUS, and ADSC+LIPUS groups. Rats in the ADSC or ADSC+LIPUS group received ADSC intracavernosal injection. Rats in the LIPUS or ADSC+LIPUS group were treated with LIPUS. The intracavernous pressure (ICP) and mean arterial pressure (MAP) were recorded at Day 28 after injection. The corpus cavernosum tissues were harvested and subjected to histologic analysis and ELISA. The effects of LIPUS on proliferation and cytokine secretion capacity of ADSCs were assessed in vitro. RNA sequencing and bioinformatic analysis were applied to predict the mechanism involved, and western blotting and ELISA were used for verification. Results Rats in the ADSC+LIPUS group achieved significantly higher ICP and ICP/MAP ratios than those in the DM, ADSC, and LIPUS groups. In addition, the amount of cavernous endothelium and cGMP level also increased significantly in the ADSC+LIPUS group. In vitro experiments demonstrated that LIPUS promoted proliferation and cell cycle progression in ADSCs. The excretion of cytokines such as CXCL12, FGF2, and VEGF was also enhanced by LIPUS. Bioinformatic analysis based on RNA sequencing indicated that LIPUS stimulation might activate the MAPK pathway. We confirmed that LIPUS enhanced ADSC VEGF secretion through the Piezo-ERK pathway. Conclusion LIPUS enhanced the curative effects of ADSCs on diabetic erectile dysfunction through the activation of the Piezo-ERK-VEGF pathway. ADSC transplantation combined with LIPUS could be applied as a synergistic treatment for diabetic ED.

Extracellular vesicles derived from human dental mesenchymal stem cells stimulated with low-intensity pulsed ultrasound alleviate inflammation-induced bone loss in a mouse model of periodontitis

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have emerged as a new mode of intercellular crosstalk and are responsible for many of the therapeutic effects of MSCs. To promote the application of MSC-EVs, recent studies have focused on the manipulation of MSCs to improve the production of EVs and EV-mediated activities. The current paper details an optimization method using non-invasive low-intensity pulsed ultrasound (LIPUS) as the stimulation for improving oral MSC-EV production and effectiveness. Stem cells from apical papilla (SCAP), a type of oral mesenchymal stem cell, displayed intensity-dependent pro-osteogenic and anti-inflammatory responses to LIPUS without significant cytotoxicity or apoptosis. The stimuli increased the secretion of EVs by promoting the expression of neutral sphingomyelinases in SCAP. In addition, EVs from LIPUS-induced SCAP exhibited stronger efficacy in promoting the osteogenic differentiation and anti-inflammation of periodontal ligament cells in vitro and alleviating oral inflammatory bone loss in vivo. In addition, LIPUS stimulation affected the physical characteristics and miRNA cargo of SCAP-EVs. Further investigations indicated that miR-935 is an important mediator of the pro-osteogenic and anti-inflammatory capabilities of LIPUS-induced SCAP-EVs. Taken together, these findings demonstrate that LIPUS is a simple and effective physical method to optimize SCAP-EV production and efficacy.

Effects of low-intensity pulsed ultrasound stimulation on cell seeded 3D hybrid scaffold as a novel strategy for meniscus regeneration: An in vitro study.

Menisci are fibrocartilaginous structures in the knee joint with an inadequate regenerative capacity, which causes low healing potential and further leads to osteoarthritis. Recently, three-dimensional (3D) printing techniques and ultrasound treatment have gained plenty of attention for meniscus tissue engineering. The present study investigates the effectiveness of low-intensity pulsed ultrasound stimulations (LIPUS) on the proliferation, viability, morphology, and gene expression of the chondrocytes seeded on 3D printed polyurethane scaffolds dip-coated with gellan gum, hyaluronic acid, and glucosamine. LIPUS stimulation was performed at 100, 200, and 300mW/cm2 intensities for 20min/day. A faster gap closure (78.08±2.56%) in the migration scratch assay was observed in the 200mW/cm2 group after 24h. Also, inverted microscopic and scanning electron microscopic images showed no cell morphology changes during LIPUS exposure at different intensities. The 3D cultured chondrocytes under LIPUS treatment revealed a promotion in cell proliferation rate and viability as the intensity doses increased. Additionally, LIPUS could stimulate chondrocytes to overexpress the aggrecan and collagen II genes and improve their chondrogenic phenotype. This study recommends that the combination of LIPUS treatment and 3D hybrid scaffolds can be considered as a valuable treatment for meniscus regeneration based on our in vitro data.

Development of a Novel Perfusion Rotating Wall Vessel Bioreactor with Ultrasound Stimulation for Mass-Production of Mineralized Tissue Constructs.

As stem cells are considered a promising cell source for tissue engineering, many culture strategies have been extensively studied to generate in vitro stem cell-based tissue constructs. However, most approaches using conventional tissue culture plates are limited by the lack of biological relevance in stem cell microenvironments required for neotissue formation. In this study, a novel perfusion rotating wall vessel (RWV) bioreactor was developed for mass-production of stem cell-based 3D tissue constructs. An automated RWV bioreactor was fabricated, which is capable of controlling continuous medium perfusion, highly efficient gas exchange with surrounding air, as well as low-intensity pulsed ultrasound (LIPUS) stimulation. Embryonic stem cells encapsulated in alginate/gelatin hydrogel were cultured in the osteogenic medium by using our bioreactor system. Cellular viability, growth kinetics, and osteogenesis/mineralization were thoroughly evaluated, and culture media were profiled at real time. The in vivo efficacy was examined by a rabbit cranial defect model. Our bioreactor successfully maintained the optimal culture environments for stem cell proliferation, osteogenic differentiation, and mineralized tissue formation during the culture period. The mineralized tissue constructs produced by our bioreactor demonstrated higher void filling efficacy in the large bone defects compared to the group implanted with hydrogel beads only. In addition, the LIPUS modules mounted on our bioreactor successfully reached higher mineralization of the tissue constructs compared to the groups without LIPUS stimulation. This study suggests an effective biomanufacturing strategy for mass-production of implantable mineralized tissue constructs from stem cells that could be applicable to future clinical practice.

Ca2+ signaling–mediated low-intensity pulsed ultrasound–induced proliferation and activation of motor neuron cells

Motor neuron diseases (MND) including amyotrophic lateral sclerosis and Parkinson disease are commonly neurodegenerative, causing a gradual loss of nerve cells and affecting the mechanisms underlying changes in calcium (Ca2+)-regulated dendritic growth. In this study, the NSC-34 cell line, a population of hybridomas generated using mouse spinal cord cells with neuroblastoma, was used to investigate the effect of low-intensity pulsed ultrasound (LIPUS) as part of an MND treatment model. After NSC-34 cells were seeded for 24 h, LIPUS stimulation was performed on the cells at days 1 and 3 using a non-focused transducer at 1.15 MHz for 8 min. NSC-34 cell proliferation and morphological changes were observed at various LIPUS intensities and different combinations of Ca2+ channel blockers. The nuclear translocation of Ca2+-dependent transcription factors was also examined. We observed that the neurite outgrowth and cell number of NSC-34 significantly increased with LIPUS stimulation at days 2 and 4, which may be associated with the treatment’s positive effect on the activation of Ca2+-dependent transcription factors, such as nuclear factor of activated T cells and nuclear factor-kappa B. Our findings suggest that the LIPUS-induced Ca2+ signaling and transcription factor activation facilitate the morphological maturation and proliferation of NSC-34 cells, presenting a promising noninvasive method to improve stimulation therapy for MNDs in the future.

Transcranial Ultrasound Stimulation Reverses Behavior Changes and the Expression of Calcium-Binding Protein in a Rodent Model of Schizophrenia.

Cognitive dysfunctions are a core feature of schizophrenia that may be linked to abnormalities in gamma-aminobutyric-acid (GABA)ergic neurons. Traditional antipsychotics show poor efficacy in treating cognitive symptoms. The purpose of this study was to investigate the restorative role of transcranial ultrasound stimulation (TUS) in counteracting dizocilpine (MK-801)-induced cognitive deficits and GABAergic interneuron dysfunction in a simulation of schizophrenia. Some rats subjected to MK-801 administration were treated with low-intensity pulsed ultrasound (LIPUS) daily for 5days, while other rats subjected to MK-801 administration received no LIPUS treatment. After LIPUS treatment, the neuroprotective effects of LIPUS in the LIPUS-treated rats were assessed through behavioral analysis, western blotting, and histological observations. Compared with the MK-801-treated group, the MK-801 plus LIPUS-treated rats revealed a preference for novel objects. The MK-801 plus LIPUS-treated rats also exhibited a significant decrease in swim times compared to the MK-801-treated rats. LIPUS stimulation significantly increased hippocampal levels of CB and PV and restored the cell densities of PV + and CB + in the cingulate cortex in the MK-801 plus LIPUS-treated group. In addition, LIPUS stimulation rebalanced the BDNF levels in the hippocampus and medial prefrontal cortex. Our findings indicate that LIPUS improves cognitive deficits and ameliorates neuropathology in MK-801-treated rats. These results suggest that LIPUS may constitute a potential novel therapeutic approach for the treatment of schizophrenia.

Non-Invasive Local Acoustic Therapy Ameliorates Diabetic Heart Fibrosis by Suppressing ACE-Mediated Oxidative Stress and Inflammation in Cardiac Fibroblasts.

The extent of myocardial fibrosis is closely related to the prognosis of diabetic cardiomyopathy (DCM). Low-intensity pulsed ultrasound (LIPUS) has been reported to have multiple biological effects. However, the effect of LIPUS on diabetic heart fibrosis remains unclear. The present study aimed to investigate the effect of LIPUS on diabetic heart fibrosis and explore its underlying mechanisms. High glucose (HG) was applied to cultured neonatal rat cardiac fibroblasts (NRCFs) to mimic the in vivo hyperglycemia microenvironment. LIPUS (19.30mW/cm2 to 77.20mW/cm2) dose-dependently inhibited HG-induced fibrotic response in NRCFs. Also, LIPUS downregulated NADPH oxidase 4 (NOX4)-associated oxidative stress and nod-like receptor protein-3 (NLRP3) inflammasome activation in NRCFs. In vivo, diabetes in mice was induced with streptozotocin (STZ). Mice in the LIPUS group and STZ + LIPUS group were treated with LIPUS (77.20mW/cm2) twice a week for 12weeks and then euthanized at 12weeks or 24weeks post-diabetes. Treatment with LIPUS significantly ameliorated the progression of cardiac fibrosis (Masson staining 6.5 ± 2.3% vs. 2.8 ± 1.5%, P< 0.001) and dysfunction (E/A ratio 1.35 ± 0.14 vs. 1.59 ± 0.11, P< 0.05), as well as NOX4-associated oxidative stress (relative expression fold of NOX4 1.43 ± 0.12 vs. 1.07 ± 0.10, P< 0.01; relative DHE fluorescence 1.51 ± 0.13 vs. 1.28 ± 0.06, P< 0.05) and NLRP3 inflammasome activation (relative expression fold of NLRP3 1.57 ± 0.12 vs. 1.05 ± 0.16, P< 0.01), at 12weeks post-diabetes. At 24weeks post-diabetes, the heart function in diabetic mice treated with LIPUS was still significantly better than untreated diabetic mice (E/A ratio 1.08 ± 0.12 vs. 1.49 ± 0.14, P< 0.001). Further exploration revealed that LIPUS significantly attenuated the upregulated angiotensin-converting enzyme (ACE) and angiotensin II (AngII), in both HG-induced NRCFs and diabetic hearts (relative expression of ACE in myocardium 3.77 ± 0.55 vs. 1.07 ± 0.13, P< 0.001; AngII in myocardium 115.5 ± 21.77ng/ml vs. 84.28 ± 9.03ng/ml, P< 0.01). Captopril, an ACE inhibitor, inhibited NOX4-associated oxidative stress and NLRP3 inflammasome activation in both HG-induced NRCFs and diabetic hearts. Our results indicate that non-invasive local LIPUS therapy attenuated heart fibrosis and dysfunction in diabetic mice and the effect could be largely preserved at least 12weeks after suspending LIPUS stimulation. LIPUS ameliorated diabetic heart fibrosis by inhibiting ACE-mediated NOX4-associated oxidative stress and NLRP3 inflammasome activation in cardiac fibroblasts. Our study may provide a novel therapeutic approach to hamper the progression of diabetic heart fibrosis.

Anti-inflammatory and Neuroprotective Effects of Transcranial Ultrasound Stimulation on Parkinson's Disease.

Low-intensity pulsed ultrasound (LIPUS) is a promising non-invasive neuromodulation tool for deep brain stimulation. Here, we investigated the impact of LIPUS, including neuroprotective effects, on the pathology of Parkinson's disease (PD) in an animal model. Sprague-Dawley rats were injected with 6-hydroxydopamine (6-OHDA) at two sites in the right striatum. LIPUS (1 MHz, 5% duty cycle, 1-Hz pulse repetition frequency, 15 min/d) stimulation was then applied to some of the rats (the 6-OHDA+LIPUS group) beginning 2 wk after the 6-OHDA administration, while the remaining rats (the 6-OHDA group) received no LIPUS stimulation. The 6-OHDA-induced inflammatory responses and expressions of neurotrophic factors were quantified with immunofluorescence activity. The safety of LIPUS was assessed using hematoxylin and eosin and Nissl staining. LIPUS treatment significantly inhibited 6-OHDA-induced glial activation and the phosphorylation of nuclear factor-κB p65 in the substantia nigra pars compacta. Further study revealed that LIPUS effectively preserved the levels of neurotrophic factors, dopamine transporter and tight junction proteins of the blood-brain barrier in the 6-OHDA+LIPUS group compared with the 6-OHDA group. These results indicate that LIPUS acts via multiple neuroprotective mechanisms in the PD rat model and suggest that LIPUS can be viewed as a potential treatment for PD.

The Attenuating Effect of Low-Intensity Pulsed Ultrasound on Hypoxia-Induced Rat Chondrocyte Damage in TMJ Osteoarthritis Based on TMT Labeling Quantitative Proteomic Analysis.

Temporomandibular joint osteoarthritis (TMJOA) is a degenerative disease with a complex and multifactorial etiology. An increased intrajoint pressure or weakened penetration can exacerbate the hypoxic state of the condylar cartilage microenvironment. Our group previously simulated the hypoxic environment of TMJOA in vitro. Low-intensity pulsed ultrasound (LIPUS) stimulation attenuates chondrocyte matrix degradation via a hypoxia-inducible factor (HIF) pathway-associated mechanism, but the mode of action of LIPUS is currently poorly understood. Moreover, most recent studies investigated the pathological mechanisms of osteoarthritis, but no biomarkers have been established for assessing the therapeutic effect of LIPUS on TMJOA with high specificity, which results in a lack of guidance regarding clinical application. Here, tandem mass tag (TMT)-based quantitative proteomic technology was used to comprehensively screen the molecular targets and pathways affected by the action of LIPUS on chondrocytes under hypoxic conditions. A bioinformatic analysis identified 902 and 131 differentially expressed proteins (DEPs) in the <1% oxygen treatment group compared with the control group and in the <1% oxygen + LIPUS stimulation group compared with the <1% oxygen treatment group, respectively. The DEPs were analyzed by gene ontology (GO), KEGG pathway and protein-protein interaction (PPI) network analyses. By acting on extracellular matrix (ECM)-associated proteins, LIPUS increases energy production and activates the FAK signaling pathway to regulate cell biological behaviors. DEPs of interest were selected to verify the reliability of the proteomic results. In addition, this experiment demonstrated that LIPUS could upregulate chondrogenic factors (such as Sox9, Collagen Ⅱ and Aggrecan) and increase the mucin sulfate content. Moreover, LIPUS reduced the hydrolytic degradation of the ECM by decreasing the MMP3/TIMP1 ratio and vascularization by downregulating VEGF. Interestingly, LIPUS improved the migration ability of chondrocytes. In summary, LIPUS can regulate complex biological processes in chondrocytes under hypoxic conditions and alter the expression of many functional proteins, which results in reductions in hypoxia-induced chondrocyte damage. ECM proteins such as thrombospondin4, thrombospondin1, IL1RL1, and tissue inhibitors of metalloproteinase 1 play a central role and can be used as specific biomarkers determining the efficacy of LIPUS and viable clinical therapeutic targets of TMJOA.

In Vitro Study on the Piezodynamic Therapy with a BaTiO3-Coating Titanium Scaffold under Low-Intensity Pulsed Ultrasound Stimulation.

To solve the poor sustainability of electroactive stimulation in clinical therapy, a strategy of combining a piezoelectric BaTiO3-coated Ti6Al4V scaffold and low-intensity pulsed ultrasound (LIPUS) was unveiled and named here as piezodynamic therapy. Thus, cell behavior could be regulated phenomenally by force and electricity simultaneously. First, BaTiO3 was deposited uniformly on the surface of the three-dimensional (3D) printed porous Ti6Al4V scaffold, which endowed the scaffold with excellent force-electricity responsiveness under pulsed ultrasound exposure. The results of live/dead staining, cell scanning electron microscopy, and F-actin staining showed that cells had better viability, better pseudo-foot adhesion, and more muscular actin bundles when they underwent the piezodynamic effect of ultrasound and piezoelectric coating. This piezodynamic therapy activated more mitochondria at the initial stage that intervened in the cell cycle by promoting cells' proliferation and weakened the apoptotic damage. The quantitative real-time polymerase chain reaction data further confirmed that the costimulation of the ultrasound and the piezoelectric scaffolds could trigger adequate current to upregulated the expression of osteogenic-related genes. The continuous electric cues could be generated by the BaTiO3-coated scaffold and intermittent LIPUS stimulation; thereon, more efficient bone healing would be promoted by piezodynamic therapy in future treatment.

Low intensity pulsed ultrasound in orthodontic tooth movement

Orthodontic treatment that takes a long time carries a large risk of complications such as caries or root resorption. Various surgical and nonsurgical interventions have been performed, but non-surgical interventions are currently not widely accepted by orthodontists. Low Intensity Pulsed Ultrasound (LIPUS) is a form of non-invasive technology that has been used in the medical field for more than six decades. In the field of dentistry, LIPUS stimulation has been studied, among others, to improve bone remodelling, regeneration of cementum, gingiva and periodontal ligament. This article aims to review the mechanism of LIPUS in orthodontic tooth movement. This review is expected to become a clinical practice guide to shorten the duration of orthodontic treatment using LIPUS in the future.

Development and validation of low-intensity pulsed ultrasound systems for highly controlled in vitro cell stimulation

This work aims to describe the development and validation of two low-intensity pulsed ultrasound stimulation systems able to control the dose delivered to the biological target. Transducer characterization was performed in terms of pressure field shape and intensity, for a high-frequency range (500 kHz to 5 MHz) and for a low-frequency value (38 kHz). This allowed defining the distance, on the beam axis, at which biological samples should be placed during stimulation and to exactly know the intensity at the target. Carefully designed retaining systems were developed, for hosting biological samples. Sealing tests proved their impermeability to external contaminants. The assembly/de-assembly time of the systems resulted ~3 min. Time-domain acoustic simulations allowed to precisely estimate the ultrasound beam within the biological sample chamber, thus enabling the possibility to precisely control the pressure to be transmitted to the biological target, by modulating the transducer's input voltage. Biological in vitro tests were also carried out, demonstrating the sterility of the system and the absence of toxic and inflammatory effects on growing cells after multiple immersions in water, over seven days.

Neuroprotective Effect of Low-Intensity Pulsed Ultrasound on the Mouse MPTP/MPP+ Model of Dopaminergic Neuron Injury

Ultrasound mediated neuromodulation has been demonstrated to a safe treatment strategy in the field of neuroscience. In this study, low-intensity pulsed ultrasound (LIPUS) was used to treat Parkinson's disease (PD) models induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium (MPP+) to explore the possibility of ultrasound neuroprotective effect on PD. The results demonstrated that LIPUS treatment can attenuate the central neurotoxicity of MPTP in mice, reduce the loss of tyrosine hydroxylase positive neurons in the substantia nigra pars compacta and decrease the apoptosis in the section of substantia nigra. The movement and balance dysfunctions in PD mice were improved with LIPUS treatment. In addition, we demonstrated that LIPUS can inhibit the decreased activity and increased apoptosis of dopaminergic neurons induced by MPP+, restrain the accumulation of reactive oxygen species (ROS) and decrease of mitochondrial membrane potential caused by MPP+. Moreover, LIPUS stimulation alone did not cause any cytotoxicity and tissue damage in our study. Taken together, the protective and regulatory effects of LIPUS on dopaminergic neurons make it possible as a new, safe and noninvasive treatment for PD.

Osteocytes as main responders to low-intensity pulsed ultrasound treatment during fracture healing

Ultrasound stimulation is a type of mechanical stress, and low-intensity pulsed ultrasound (LIPUS) devices have been used clinically to promote fracture healing. However, it remains unclear which skeletal cells, in particular osteocytes or osteoblasts, primarily respond to LIPUS stimulation and how they contribute to fracture healing. To examine this, we utilized medaka, whose bone lacks osteocytes, and zebrafish, whose bone has osteocytes, as in vivo models. Fracture healing was accelerated by ultrasound stimulation in zebrafish, but not in medaka. To examine the molecular events induced by LIPUS stimulation in osteocytes, we performed RNA sequencing of a murine osteocytic cell line exposed to LIPUS. 179 genes reacted to LIPUS stimulation, and functional cluster analysis identified among them several molecular signatures related to immunity, secretion, and transcription. Notably, most of the isolated transcription-related genes were also modulated by LIPUS in vivo in zebrafish. However, expression levels of early growth response protein 1 and 2 (Egr1, 2), JunB, forkhead box Q1 (FoxQ1), and nuclear factor of activated T cells c1 (NFATc1) were not altered by LIPUS in medaka, suggesting that these genes are key transcriptional regulators of LIPUS-dependent fracture healing via osteocytes. We therefore show that bone-embedded osteocytes are necessary for LIPUS-induced promotion of fracture healing via transcriptional control of target genes, which presumably activates neighboring cells involved in fracture healing processes.

Antenatal low-intensity pulsed ultrasound reduces neurobehavioral deficits and brain injury following dexamethasone-induced intrauterine growth restriction.

Intrauterine growth restriction (IUGR) is a leading cause of perinatal mortality and morbidity, and IUGR survivors are at increased risk of neurodevelopmental deficits. No effective interventions are currently available to improve the structure and function of the IUGR brain before birth. This study investigated the protective effects of low‐intensity pulsed ultrasound (LIPUS) on postnatal neurodevelopmental outcomes and brain injury using a rat model of IUGR induced by maternal exposure to dexamethasone (DEX). Pregnant rats were treated with DEX (200 μg/kg, s.c.) and LIPUS daily from gestational day (GD) 14 to 19. Behavioral assessments were performed on the IUGR offspring to examine neurological function. Neuropathology, levels of neurotrophic factors, and CaMKII‐Akt‐related molecules were assessed in the IUGR brain, and expression of glucose and amino acid transporters and neurotrophic factors were examined in the placenta. Maternal LIPUS treatment increased fetal weight, fetal liver weight, and placental weight following IUGR. LIPUS treatment also increased neuronal number and myelin protein expression in the IUGR brain, and attenuated neurodevelopmental deficits at postnatal day (PND) 18. However, the number of oligodendrocytes or microglia was not affected. These changes were associated with the upregulation of brain‐derived neurotrophic factor (BDNF) and placental growth factor (PlGF) protein expression, and enhancement of neuronal CaMKII and Akt activation in the IUGR brain at PND 1. Additionally, LIPUS treatment promoted glucose transporter (GLUT) 1 production and BDNF expression in the placenta, but had no effects on GLUT3 or amino acid transporter expression. Our findings suggest that antenatal LIPUS treatment may reduce IUGR‐induced brain injury via enhancing cerebral BDNF/CaMKII/Akt signaling. These data provide new evidence that LIPUS stimulation could be considered for antenatal neuroprotective therapy in IUGR.

Exploratory study on neurochemical effects of low-intensity pulsed ultrasound in brains of mice.

There is now a relatively large body of evidence suggesting a relationship between dysfunction of myelin and oligodendrocytes and the etiology of several neuropsychiatric disorders, including depression and schizophrenia, and also suggesting that ultrasound methods may alleviate some of the symptoms of depression. We have applied low-intensity pulsed ultrasound (LIPUS) to the brains of mice treated with the demyelinating drug cuprizone, a drug that has been used as the basis for a rodent model relevant to a number of psychiatric and neurologic disorders including depression, schizophrenia, and multiple sclerosis. Prior to conducting the studies in mice, preliminary studies were carried out on the effects of LIPUS in vitro in neuron-like SH-SY5Y cells and primary glial cells. In subsequent studies in mice, female C57BL/6 mice were restrained in plastic tubes for 20 min daily with the ultrasound transducer near the end of the tube directly above the mouse's head. LIPUS was used at an intensity of 25 mW/cm2 once daily for 22 days in control mice and in mice undergoing daily repetitive restraint stress (RRS). Behavioral or neurochemical studies were done on the mice or the brain tissue obtained from them. The studies in vitro indicated that LIPUS stimulation at an intensity of 15 mW/cm2 delivered for 5 min daily for 3 days in an enclosed sterile cell culture plate in an incubator increased the viability of SH-SY5Y and primary glial cells. In the studies in mice, LIPUS elevated levels of doublecortin, a marker for neurogenesis, in the cortex compared to levels in the RRS mice and caused a trend in elevation of brain levels of brain-derived neurotrophic factor in the hippocampus relative to control levels. LIPUS also increased sucrose preference (a measure of the attenuation of anhedonia, a common symptom of several psychiatric disorders) in the RRS model in mice. The ability of LIPUS administered daily to rescue damaged myelin and oligodendrocytes was studied in mice treated chronically with cuprizone for 35 days. LIPUS increased cortex and corpus callosum levels of myelin basic protein, a protein marker for mature oligodendrocytes, and neural/glial antigen 2, a protein marker for oligodendrocyte precursor cells, relative to levels in the cuprizone + sham animals. These results of this exploratory study suggest that future comprehensive time-related studies with LIPUS on brain chemistry and behavior related to neuropsychiatric disorders are warranted. Exploratory Study on Neurochemical Effects of Low Intensity Pulsed Ultrasound in Brains of Mice. Upper part of figure: LIPUS device and in-vitro cell experimental set-up. The center image is the LIPUS generating box; the image in the upper left shows the cell experiment set-up; the image in the upper right shows a zoomed-in sketch for the cell experiment; the image in the lower left shows the set-up of repetitive restraint stress (RRS) with a mouse; the image in the lower middle shows the set-up of LIPUS treatment of a mouse; the image in the lower right shows a zoomed-in sketch for the LIPUS treatment of a mouse.

Low-Intensity Pulsed Ultrasound Stimulation Inhibits Cortical Spreading Depression.

Cortical spreading depression (CSD), which is closely correlated with migraine aura, cerebral ischemia, seizure, and brain injury, is a spreading wave of neuronal and glial depolarization. The purpose of this study is to investigate whether low-intensity pulsed ultrasound stimulation (PUS) inhibits CSD by modulating neural activity and hemodynamics. Behavioral test, intrinsic signal optical imaging and western blot analysis were used for evaluating the inhibition effect of PUS on CSD in rat. We found that: 1) 30min of PUS can significantly improve motor activity of rat with CSD. 2) Both 30s and 30min of PUS can significantly reduce count and propagation speed of CSD in rat and the inhibitory effect was enhanced with increase of ultrasound intensity. 3) 30min of PUS significantly enhanced levels of brain-derived neurotrophic factor protein in brain tissue with CSD. These results suggest that PUS has the potential to treat brain disorders associated with CSD.

Enhanced bone formation of calvarial bone defects by low-intensity pulsed ultrasound and recombinant human bone morphogenetic protein-9: a preliminary experimental study in rats.

The aim of this study was to evaluate the combined effects of recombinant human bone morphogenetic protein- 9 (rhBMP-9) loaded onto absorbable collagen sponges (ACS) and low-intensity pulsed ultrasound (LIPUS) on bone formation in rat calvarial defects. Circular calvarial defects were surgically created in 18 Wistar rats, which were divided into LIPUS-applied (+) and LIPUS-non-applied (-) groups. The 36 defects in each group received ACS implantation (ACS group), ACS with rhBMP-9 (rhBMP-9/ACS group), or surgical control (control group), yielding the following six groups: ACS (+/-), rhBMP-9/ACS (+/-), and control (+/-). The LIPUS-applied groups received daily LIPUS exposure starting immediately after surgery. At 4 weeks, animals were sacrificed and their defects were investigated histologically and by microcomputed tomography. Postoperative clinical healing was uneventful at all sites. More new bone was observed in the LIPUS-applied groups compared with the LIPUS-non-applied groups. Newly formed bone area (NBA)/total defect area (TA) in the ACS (+) group (46.49 ± 7.56%) was significantly greater than that observed in the ACS (-) (34.31 ± 5.68%) and control (-) (31.13 ± 6.74%) groups (p < 0.05). The rhBMP-9/ACS (+) group exhibited significantly greater bone volume, NBA, and NBA/TA than the rhBMP-9/ACS (-) group (2.46 ± 0.65 mm3 vs. 1.76 ± 0.44 mm3, 1.25 ± 0.31 mm2 vs. 0.88 ± 0.22 mm2, and 62.80 ± 11.87% vs. 42.66 ± 7.03%, respectively) (p < 0.05). Furthermore, the rhBMP-9/ ACS (+) group showed the highest level of bone formation among all groups. Within their limits, it can be concluded that LIPUS had osteopromotive potential and enhanced rhBMP-9-induced bone formation in calvarial defects of rats. The use of rhBMP-9 with LIPUS stimulation can be a potential bone regenerative therapy for craniofacial/peri-implant bone defects.

Piezo1 channel activation in response to mechanobiological acoustic radiation force in osteoblastic cells

Mechanobiological stimuli, such as low-intensity pulsed ultrasound (LIPUS), have been shown to promote bone regeneration and fresh fracture repair, but the fundamental biophysical mechanisms involved remain elusive. Here, we propose that a mechanosensitive ion channel of Piezo1 plays a pivotal role in the noninvasive ultrasound-induced mechanical transduction pathway to trigger downstream cellular signal processes. This study aims to investigate the expression and role of Piezo1 in MC3T3-E1 cells after LIPUS treatment. Immunofluorescence analysis shows that Piezo1 was present on MC3T3-E1 cells and could be ablated by shRNA transfection. MC3T3-E1 cell migration and proliferation were significantly increased by LIPUS stimulation, and knockdown of Piezo1 restricted the increase in cell migration and proliferation. After labeling with Fluo-8, MC3T3-E1 cells exhibited fluorescence intensity traces with several high peaks compared with the baseline during LIPUS stimulation. No obvious change in the fluorescence intensity tendency was observed after LIPUS stimulation in shRNA-Piezo1 cells, which was similar to the results in the GsMTx4-treated group. The phosphorylation ratio of ERK1/2 in MC3T3-E1 cells was significantly increased (P < 0.01) after LIPUS stimulation. In addition, Phalloidin-iFluor-labeled F-actin filaments immediately accumulated in the perinuclear region after LIPUS stimulation, continued for 5 min, and then returned to their initial levels at 30 min. These results suggest that Piezo1 can transduce LIPUS-induced mechanical signals into intracellular calcium. The influx of Ca2+ serves as a second messenger to activate ERK1/2 phosphorylation and perinuclear F-actin filament polymerization, which regulate the proliferation of MC3T3-E1 cells.