Low-intensity Pulsed Ultrasound Stimulation Research Articles

Low-intensity pulsed ultrasound reduces alveolar bone resorption during orthodontic treatment via Lamin A/C-Yes-associated protein axis in stem cells.

The bone remodeling during orthodontic treatment for malocclusion often requires a long duration of around two to three years, which also may lead to some complications such as alveolar bone resorption or tooth root resorption. Low-intensity pulsed ultrasound (LIPUS), a noninvasive physical therapy, has been shown to promote bone fracture healing. It is also reported that LIPUS could reduce the duration of orthodontic treatment; however, how LIPUS regulates the bone metabolism during the orthodontic treatment process is still unclear. To investigate the effects of LIPUS on bone remodeling in an orthodontic tooth movement (OTM) model and explore the underlying mechanisms. A rat model of OTM was established, and alveolar bone remodeling and tooth movement rate were evaluated via micro-computed tomography and staining of tissue sections. In vitro, human bone marrow mesenchymal stem cells (hBMSCs) were isolated to detect their osteogenic differentiation potential under compression and LIPUS stimulation by quantitative reverse transcription-polymerase chain reaction, Western blot, alkaline phosphatase (ALP) staining, and Alizarin red staining. The expression of Yes-associated protein (YAP1), the actin cytoskeleton, and the Lamin A/C nucleoskeleton were detected with or without YAP1 small interfering RNA (siRNA) application via immunofluorescence. The force treatment inhibited the osteogenic differentiation potential of hBMSCs; moreover, the expression of osteogenesis markers, such as type 1 collagen (COL1), runt-related transcription factor 2, ALP, and osteocalcin (OCN), decreased. LIPUS could rescue the osteogenic differentiation of hBMSCs with increased expression of osteogenic marker inhibited by force. Mechanically, the expression of LaminA/C, F-actin, and YAP1 was downregulated after force treatment, which could be rescued by LIPUS. Moreover, the osteogenic differentiation of hBMSCs increased by LIPUS could be attenuated by YAP siRNA treatment. Consistently, LIPUS increased alveolar bone density and decreased vertical bone absorption in vivo. The decreased expression of COL1, OCN, and YAP1 on the compression side of the alveolar bone was partially rescued by LIPUS. LIPUS can accelerate tooth movement and reduce alveolar bone resorption by modulating the cytoskeleton-Lamin A/C-YAP axis, which may be a promising strategy to reduce the orthodontic treatment process.

LIPUS regulates the progression of knee osteoarthritis in mice through primary cilia-mediated TRPV4 channels.

Osteoarthritis (OA) is a common disease in middle-aged and elderly people. An imbalance in calcium ion homeostasis will contribute to chondrocyte apoptosis and ultimately lead to the progression of OA. Transient receptor potential channel 4 (TRPV4) is involved in the regulation of intracellular calcium homeostasis. TRPV4 is expressed in primary cilia, which can sense mechanical stimuli from outside the cell, and its abnormal expression is closely related to the development of OA. Low-intensity pulsed ultrasound (LIPUS) can alleviate chondrocyte apoptosis while the exact mechanism is unclear. In this project, with the aim of revealing the mechanism of action of LIPUS, we proposed to use OA chondrocytesand animal models, LIPUS intervention, inhibition of primary cilia, use TRPV4 inhibitors or TRPV4agonist, and use Immunofluorescence (IF), Immunohistochemistry (IHC), Western Blot (WB), Quantitative Real-time PCR (QP) to detect the expression of cartilage synthetic matrix and endoplasmic reticulum stress markers.The results revealed that LIPUS altered primary cilia expression, promoted synthetic matrix metabolism in articular chondrocytes and was associated with primary cilia. In addition, LIPUS exerted a active effect on OA by activating TRPV4, inducing calcium inward flow, and facilitating the entry of NF-κB into the nucleus to regulate synthetic matrix gene transcription. Inhibition of TRPV4 altered primary cilia expression in response to LIPUS stimulation, and knockdown of primary cilia similarly inhibited TRPV4 function. These results suggest that LIPUS mediates TRPV4 channels through primary cilia to regulate the process of knee osteoarthritis in mice.

L-Type Calcium Channel Modulates Low-Intensity Pulsed Ultrasound-Induced Excitation in Cultured Hippocampal Neurons.

As a noninvasive technique, ultrasound stimulation is known to modulate neuronal activity both in vitro and in vivo. The latest explanation of this phenomenon is that the acoustic wave can activate the ion channels and further impact the electrophysiological properties of targeted neurons. However, the underlying mechanism of low-intensity pulsed ultrasound (LIPUS)-induced neuro-modulation effects is still unclear. Here, we characterize the excitatory effects of LIPUS on spontaneous activity and the intracellular Ca2+ homeostasis in cultured hippocampal neurons. By whole-cell patch clamp recording, we found that 15min of 1-MHz LIPUS boosts the frequency of both spontaneous action potentials and spontaneous excitatory synaptic currents (sEPSCs) and also increases the amplitude of sEPSCs in hippocampal neurons. This phenomenon lasts for > 10min after LIPUS exposure. Together with Ca2+ imaging, we clarified that LIPUS increases the [Ca2+]cyto level by facilitating L-type Ca2+ channels (LTCCs). In addition, due to the [Ca2+]cyto elevation by LIPUS exposure, the Ca2+-dependent CaMKII-CREB pathway can be activated within 30 min to further regulate the gene transcription and protein expression. Our work suggests that LIPUS regulates neuronal activity in a Ca2+-dependent manner via LTCCs. This may also explain the multi-activation effects of LIPUS beyond neurons. LIPUS stimulation potentiates spontaneous neuronal activity by increasing Ca2+ influx.

P079 Low-intensity Pulsed Ultrasound reflects the status of mucosal injury and repair through stage-specific miRNAs in experimental model of Ulcerative Colitis

Abstract Background Diagnosis and therapeutic efficacy monitoring of Ulcerative Colitis (UC) are based on a multidisciplinary approach comprising symptomatology and invasive endoscopic assessment coupled to the histopathological characterization of gut tissue biopsies. New technologies that non-invasively reflect tissue-specific changes remains an unmet need. Here we exploited Low Intensity Pulsed Ultrasound (LIPUS) to induce tissue stage-specific miRNAs in circulating blood as a non-invasive approach to reflect the status of the colonic mucosa in experimental model of colitis. Methods Based on our previous study, LIPUS was applied at a frequency of 38 kHz and at an intensity of 0 (as control) or 150 mW/cm2 for 3 min, using a device dedicated for in vivo studies. Acute and chronic colitis were induced in C57BL/6N mice (5 animals/group) by administration of 2 and 2.5% ad libitum dextran sodium sulfate (DSS) respectively in their drinking water for 7 days for the acute and three cycles of 7 days for the chronic model. miRNA array profiling was performed on extracellular vesicles (EVs) isolated from plasma of both acute and chronic models, at both active and remission state, 2 h after LIPUS stimulation. miRNA were correlated to the mucosal status by multiplexed immunofluorescence staining using Opal Multiplex IHC (CD4, CD8, CD68, pan-cytokeratin and CD31 markers) and EVs characterized by MACSplex surface epitope detection. Results An enrichment of EVs characterized mainly by stromal and immune cell surface markers was found in both acute and chronic inflammatory states, LIPUS stimulation significantly modulated miRNA profiles and clearly discriminated between active acute and chronic phases. 61 miRNAs in acute and 28 in chronic colitis were specifically and significantly upregulated by LIPUS in the active phase, with a fold change ranging from 2 to 1500 compared to control. Both acute and chronic disease showed a miRNA profile coherent with the active inflammatory state of intestinal mucosa. Only 10 and 23 miRNAs were upregulated in acute and chronic remission respectively. The enhancement of miR-18a-3p and miR-301b-3p in the recovery phases correlated to instetin with reduced immune cell infiltration and epithelial layer regeneration. Conclusion An optimal LIPUS dosage that non-invasively targets the colon induces the tissue to release specific miRNAs that reflect the mucosal status. LIPUS may open a new era of non-image ultrasound diagnostics that works as disruptive liquid biopsy allowing the detection of tissue biomarkers for monitoring IBD disease progression.

ULTRASOUND COUNTERACTS INFLAMMATION AND INDUCES CHONDROGENIC DIFFERENTIATION OF ASCS IN 3D PIEZOELECTRIC HYDROGELS

In cartilage tissue engineering (TE),new solutions are needed to effectively drive chondrogenic differentiation of mesenchymal stromal cells in both normal and inflammatory milieu. Ultrasound waves represent an interesting tool to facilitate chondrogenesis. In particular, low intensity pulsed ultrasound (LIPUS)has been shown to regulate the differentiation of adipose mesenchymal stromal cells. Hydrogels are promising biomaterials capable of encapsulating MSCs by providing an instructive biomimetic environment, graphene oxide (GO) has emerged as a promising nanomaterial for cartilage TE due to its chondroinductive properties when embedded in polymeric formulations, and piezoelectric nanomaterials, such as barium titanate nanoparticles (BTNPs),can be exploited as nanoscale transducers capable of inducing cell growth/differentiation. The aim of this study was to investigate the effect of dose-controlled LIPUS in counteracting inflammation and positively committing chondrogenesis of ASCs embedded in a 3D piezoelectric hydrogel.ASCs at 2*106 cells/mL were embedded in a 3D VitroGel RGD® hydrogel without nanoparticles (Control) or doped with 25 µg/ml of GO nanoflakes and 50 µg/ml BTNPs.The hydrogels were exposed to basal or inflammatory milieu (+IL1β 10ng/ml)and then to LIPUS stimulation every 2 days for 10 days of culture. Hydrogels were chondrogenic differentiated and analyzed after 2,10 and 28 days. At each time point cell viability, cytotoxicity, gene expression and immunohistochemistry (COL2, aggrecan, SOX9, COL1)and inflammatory cytokines were evaluated.Ultrasound stimulation significantly induced chondrogenic differentiation of ASCs loaded into 3D piezoelectric hydrogels under basal conditions: COL2, aggrecan and SOX9 were significantly overexpressed, while the fibrotic marker COL1 decreased compared to control samples. LIPUS also has potent anti-inflammatory effects by reducing IL6 and IL8 and maintaining its ability to boost chondrogenesis.These results suggest that the combination of LIPUS and piezoelectric hydrogels promotes the differentiation of ASCs encapsulated in a 3D hydrogel by reducing the inflammatory milieu, thus representing a promising tool in the field of cartilage TE.Acknowledgements: This work received funding from the European Union's Horizon 2020 research and innovation program, grant agreement No 814413, project ADMAIORA (AdvanceD nanocomposite MAterIals for in situ treatment and ultRAsound-mediated management of osteoarthritis).

Daily low-intensity pulsed ultrasound stimulation mitigates joint degradation and pain in a post-traumatic osteoarthritis rat model

ObjectivesThe aim of this study was to investigate the effects of low-intensity pulsed ultrasound (LIPUS) in a post-traumatic osteoarthritis (OA) rat model and in vitro. MethodsThirty-eight male, four-month-old Sprague Dawley rats were randomly assigned to Sham, Sham ​+ ​US, OA, and OA ​+ ​US. Sham surgery was performed to serve as a negative control, and anterior cruciate ligament transection was used to induce OA. Three days after the surgical procedures, Sham ​+ ​US and OA ​+ ​US animals received daily LIPUS treatment, while the rest of the groups received sham ultrasound (US) signals. Behavioral pain tests were performed at baseline and every week thereafter. After 31 days, the tissues were collected, and histological analyses were performed on knees and innervated dorsal root ganglia (DRG) neurons traced by retrograde labeling. Furthermore, to assess the activation of osteoclasts by LIPUS treatment, RAW264.7 ​cells were differentiated into osteoclasts and treated with LIPUS. ResultsJoint degradation in cartilage and bone microarchitecture were mitigated in OA ​+ ​US compared to OA. OA ​+ ​US showed improvements in behavioral pain tests. A significant increase of large soma-sized DRG neurons was located in OA compared to Sham. In addition, a greater percentage of large soma-sized innervated neurons were calcitonin gene-related peptide-positive. Daily LIPUS treatment suppressed osteoclastogenesis in vitro, which was confirmed via histological analyses and mRNA expression. Finally, lower expression of netrin-1, a sensory innervation-related protein, was found in the LIPUS treated cells. ConclusionOur findings demonstrate that early intervention using LIPUS treatment has protective effects from the progression of knee OA, including reduced tissue degradation, mitigated pain characteristics, improved subchondral bone microarchitecture, and less sensory innervation. Furthermore, daily LIPUS treatment has a suppressive effect on osteoclastogenesis, which may be linked to the suppression of sensory innervation in OA. The translational potential of this articleThis study presents a new potential for early intervention in treating OA symptoms through the use of LIPUS, which involves the suppression of osteoclastogenesis and the alteration of DRG profiles. This intervention aims to delay joint degradation and reduce pain.

Innervation of an Ultrasound-Mediated PVDF-TrFE Scaffold for Skin-Tissue Engineering.

In this work, electrospun polyvinylidene-trifluoroethylene (PVDF-TrFE) was utilized for its biocompatibility, mechanics, and piezoelectric properties to promote Schwann cell (SC) elongation and sensory neuron (SN) extension. PVDF-TrFE electrospun scaffolds were characterized over a variety of electrospinning parameters (1, 2, and 3 h aligned and unaligned electrospun fibers) to determine ideal thickness, porosity, and tensile strength for use as an engineered skin tissue. PVDF-TrFE was electrically activated through mechanical deformation using low-intensity pulsed ultrasound (LIPUS) waves as a non-invasive means to trigger piezoelectric properties of the scaffold and deliver electric potential to cells. Using this therapeutic modality, neurite integration in tissue-engineered skin substitutes (TESSs) was quantified including neurite alignment, elongation, and vertical perforation into PVDF-TrFE scaffolds. Results show LIPUS stimulation promoted cell alignment on aligned scaffolds. Further, stimulation significantly increased SC elongation and SN extension separately and in coculture on aligned scaffolds but significantly decreased elongation and extension on unaligned scaffolds. This was also seen in cell perforation depth analysis into scaffolds which indicated LIPUS enhanced perforation of SCs, SNs, and cocultures on scaffolds. Taken together, this work demonstrates the immense potential for non-invasive electric stimulation of an in vitro tissue-engineered-skin model.

Computational model to address lens-based acoustic field aperture in the in vitro ultrasonic cell stimulation

Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) is a therapeutic modality used for bone tissue regeneration and healing. Its clinical efficacy is still debated, as the underlying physical phenomena remain poorly understood. The interaction between ultrasonic waves and cells, likely to trigger mechanotransduction inducing bone regeneration, is at the center of scientific concerns on the subject. In order to get new insights into these phenomena, the development of in vitro experiments is a key step but special attentions should be paid concerning to the actual acoustic area covered that has to be sufficiently large and homogeneous. To address this issue, an acoustic lens can be placed on the transducer to improve the homogeneity of the acoustic field over the entire cell culture area. A computational model is developed to test several shapes and heights of acoustic lenses and compare their effectiveness in order to find a compromise between the surface covered, the homogeneity of the intensity distribution and the acoustic pressure loss. All the lenses studied improve the enlargement of the field and its homogeneity but they all generate pressure acoustic loss. The best performing lens in terms of field homogeneity is the one that minimizes pressure acoustic loss but covers only 22% of the target surface. The best enlargement (68% of the surface covered) is obtained for a lens that produces a field that is 4 times less homogeneous and 3 times less efficient in terms of pressure acoustic loss. As no one lens is ideal, the choice of the lens should be the result of a compromise taking into account the prioritization of criteria.

Low-intensity pulsed ultrasound enhances neurite growth in serum-starved human neuroblastoma cells.

Low-intensity pulsed ultrasound (LIPUS) is a recognized tool for promoting nerve regeneration and repair; however, the intracellular mechanisms of LIPUS stimulation remain underexplored. The present study delves into the effects of varying LIPUS parameters, namely duty cycle, spatial average-temporal average (SATA) intensity, and ultrasound amplitude, on the therapeutic efficacy using SK-N-SH cells cultured in serum-starved conditions. Four distinct LIPUS settings were employed: (A) 50 mW/cm2, 40%, (B) 25 mW/cm2, 10%, (C) 50 mW/cm2, 20%, and (D) 25 mW/cm2, 10%. Immunochemistry analysis exhibited neurite outgrowth promotion in all LIPUS-treated groups except for Group D. Further, LIPUS treatment was found to successfully promote brain-derived neurotrophic factor (BDNF) expression and enhance the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, protein kinase B (Akt), and mammalian target of rapamycin (mTOR) signaling pathways, as evidenced by western blot analysis. The study suggests that the parameter combination of LIPUS determines the therapeutic efficacy of LIPUS. Future investigations should aim to optimize these parameters for different cell types and settings and delve deeper into the cellular response mechanism to LIPUS treatment. Such advancements may aid in tailoring LIPUS treatment strategies to specific therapeutic needs.

Effect of Novel Low-intensity Pulsed Ultrasound Stimulation on Accelerated Implant Osteointegration in Canine.

To evaluate the effect of low-intensity pulsed ultrasound (LIPU) application on dental implant accelerated osseointegration in the two-stage implant protocol. A total of 20 implants were placed in 10 mature mongrel dogs, two implants for each dog replacing the lower 3rd premolars bilaterally. After 3 months of extraction, implants were placed. After 24 hours of implantation, the right sides received LIPU for 20 mins/day, continuously for 20 days. The left sides didn't receive LIPU (control). Standardized radiographs were taken before LIPU and after 10 and 20 days for both sides. After 20 days of LIPU application, second-stage surgery was performed and provisional crowns were placed on each implant. Implants were subjected to functional occlusal loading for 4 weeks. Standardized radiographs were taken after 2 and 4 weeks of loading and analyzed to assess the peri-implant bone density changes. All data were collected, tabulated, and statistically analyzed. All tissues appeared clinically normal, with the absence of inflammation and peri-implant radiolucency. The survival rate was 100%. The LIPU group showed a statistically significantly higher percentage increase in mean bone density after 10 LIPU sessions, 20 LIPU sessions, and 2 and 4 weeks of loading, than a control group. The LIPU stimulation radiographically increased the bone density around implant area and accelerated osseointegration in the two-stage implant protocol. Low-intensity pulsed ultrasound stimulation could be beneficial in accelerating osseointegration and thus shortening the waiting period for final prosthetic delivery.

Ultrasound-Responsive Nanobubbles for Combined siRNA-Cerium Oxide Nanoparticle Delivery to Bone Cells.

This study aims to present an ultrasound-mediated nanobubble (NB)-based gene delivery system that could potentially be applied in the future to treat bone disorders such as osteoporosis. NBs are sensitive to ultrasound (US) and serve as a controlled-released carrier to deliver a mixture of Cathepsin K (CTSK) siRNA and cerium oxide nanoparticles (CeNPs). This platform aimed to reduce bone resorption via downregulating CTSK expression in osteoclasts and enhance bone formation via the antioxidant and osteogenic properties of CeNPs. CeNPs were synthesized and characterized using transmission electron microscopy and X-ray photoelectron spectroscopy. The mixture of CTSK siRNA and CeNPs was adsorbed to the surface of NBs using a sonication method. The release profiles of CTSK siRNA and CeNPs labeled with a fluorescent tag molecule were measured after low-intensity pulsed ultrasound (LIPUS) stimulation using fluorescent spectroscopy. The maximum release of CTSK siRNA and the CeNPs for 1 mg/mL of NB-(CTSK siRNA + CeNPs) was obtained at 2.5 nM and 1 µg/mL, respectively, 3 days after LIPUS stimulation. Then, Alizarin Red Staining (ARS) was applied to human bone marrow-derived mesenchymal stem cells (hMSC) and tartrate-resistant acid phosphatase (TRAP) staining was applied to human osteoclast precursors (OCP) to evaluate osteogenic promotion and osteoclastogenic inhibition effects. A higher mineralization and a lower number of osteoclasts were quantified for NB-(CTSK siRNA + CeNPs) versus control +RANKL with ARS (p < 0.001) and TRAP-positive staining (p < 0.01). This study provides a method for the delivery of gene silencing siRNA and CeNPs using a US-sensitive NB system that could potentially be used in vivo and in the treatment of bone fractures and disorders such as osteoporosis.

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.

Neuroprotection by Abdominal Ultrasound in Lipopolysaccharide-Induced Systemic Inflammation.

Systemic inflammation is associated with intestinal inflammation and neuroinflammation by imbalancing the gut-brain axis. Low-intensity pulsed ultrasound (LIPUS) has neuroprotective and anti-inflammatory effects. This study explored LIPUS's neuroprotective effects against lipopolysaccharide (LPS)-induced neuroinflammation through transabdominal stimulation. Male C57BL/6J mice were intraperitoneally injected with LPS (0.75 mg/kg) daily for seven days, and abdominal LIPUS was applied to the abdominal area for 15 min/day during the last six days. One day after the last LIPUS treatment, biological samples were collected for microscopic and immunohistochemical analysis. Histological examination showed that LPS administration leads to tissue damage in the colon and brain. Transabdominal LIPUS stimulation attenuated colonic damage, reducing histological score, colonic muscle thickness, and villi shortening. Furthermore, abdominal LIPUS reduced hippocampal microglial activation (labeled by ionized calcium-binding adaptor molecule-1 [Iba-1]) and neuronal cell loss (labeled by microtubule-associated protein 2 [MAP2]). Moreover, abdominal LIPUS attenuated the number of apoptotic cells in the hippocampus and cortex. Altogether, our results indicate that abdominal LIPUS stimulation attenuates LPS-induced colonic inflammation and neuroinflammation. These findings provide new insights into the treatment strategy for neuroinflammation-related brain disorders and may facilitate method development through the gut-brain axis pathway.

Transabdominal ultrasound alleviates LPS-induced neuroinflammation by modulation of TLR4/NF-κB signaling and tight junction protein expression

AimInflammatory bowel disease (IBD) may be a risk factor in the development of brain inflammation. It has been demonstrated noninvasive neuromodulation through sub-organ ultrasound stimulation. The purpose of this study was to investigate whether abdominal low-intensity pulsed ultrasound (LIPUS) alleviates lipopolysaccharide (LPS)-induced cortical inflammation via inhibition of colonic inflammation. Materials and methodsColonic and cortical inflammation was induced in mice by LPS (0.75 mg/kg, i.p. injection) for 7 days, followed by application of LIPUS (0.5 and 1.0 W/cm2) to the abdominal area for 6 days. Biological samples were collected for Western blot analysis, gelatin zymography, colon length measurement, and histological evaluation. Key findingsLIPUS treatment significantly attenuated LPS-induced increases in IL-6, IL-1β, COX-2, and cleaved caspase-3 expression in the colon and cortex of mice. Moreover, LIPUS significantly increased the levels of tight junction proteins in the epithelial barrier in the mouse colon and cortex with LPS-induced inflammation. Compared to the group treated only with LPS, the LIPUS-treated groups showed decreased muscle thickness and increased crypt length and colon length. Furthermore, LIPUS treatment reduced inflammation by inhibiting the LPS-induced activation of TLR4/NF-κB inflammatory signaling in the brain. SignificanceWe found that LIPUS alleviated LPS-induced colonic and cortical inflammation through abdominal stimulation of mice. These results suggest that abdominal LIPUS stimulation may be a novel therapeutic strategy against neuroinflammation via enhancement of tight junction protein levels and inhibition of inflammatory responses in the colon.

LIPUS-SCs-Exo promotes peripheral nerve regeneration in cavernous nerve crush injury-induced ED rats via PI3K/Akt/FoxO signaling pathway.

Clinical treatment of erectile dysfunction (ED) caused by cavernous nerve (CN) injury during pelvic surgery is difficult. Low-intensity pulsed ultrasound (LIPUS) can be a potential strategy for neurogenic ED (NED). However, whether Schwann cells (SCs) can respond to LIPUS stimulation signals is unclear. This study aims to elucidate the signal transmission between SCs paracrine exosome (Exo) and neurons stimulated by LIPUS, as well as to analyze the role and potential mechanisms of exosomes in CN repair after injury. The major pelvic ganglion (MPG) neurons and MPG/CN explants were stimulated with LIPUS of different energy intensities to explore the appropriate LIPUS energy intensity. The exosomes were isolated and purified from LIPUS-stimulated SCs (LIPUS-SCs-Exo) and non-stimulated SCs (SCs-Exo). The effects of LIPUS-SCs-Exo on neurite outgrowth, erectile function, and cavernous penis histology were identified in bilateral cavernous nerve crush injury (BCNI)-induced ED rats. LIPUS-SCs-Exo group can enhance the axon elongation of MPG/CN and MPG neurons compared to SCs-Exo group in vitro. Then, the LIPUS-SCs-Exo group showed a stronger ability to promote the injured CN regeneration and SCs proliferation compared to the SCs-Exo group in vivo. Furthermore, the LIPUS-SCs-Exo group increased the Max intracavernous pressure (ICP)/mean arterial pressure (MAP), lumen to parenchyma and smooth muscle to collagen ratios compared to the SCs-Exo group in vivo. Additionally, high-throughput sequencing combined with bioinformatics analysis revealed the differential expression of 1689 miRNAs between the SCs-Exo group and the LIPUS-SCs-Exo group. After LIPUS-SCs-Exo treatment, the phosphorylated levels of Phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) and forkhead box O (FoxO) in MPG neurons increased significantly compared to negative control (NC) and SCs-Exo groups. Our study revealed that LIPUS stimulation could regulate the gene of MPG neurons by changing miRNAs derived from SCs-Exo, then activating the PI3K-Akt-FoxO signal pathway to enhance nerve regeneration and restore erectile function. This study had important theoretical and practical significance for improving the NED treatment.

Low-intensity pulsed ultrasound promotes mesenchymal stem cell transplantation-based articular cartilage regeneration via inhibiting the TNF signaling pathway

BackgroundMesenchymal stem cell (MSC) transplantation therapy is highly investigated for the regenerative repair of cartilage defects. Low-intensity pulsed ultrasound (LIPUS) has the potential to promote chondrogenic differentiation of MSCs. However, its underlying mechanism remains unclear. Here, we investigated the promoting effects and mechanisms underlying LIPUS stimulation on the chondrogenic differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) and further evaluated its regenerative application value in articular cartilage defects in rats.MethodsLIPUS was applied to stimulate cultured hUC-MSCs and C28/I2 cells in vitro. Immunofluorescence staining, qPCR analysis, and transcriptome sequencing were used to detect mature cartilage-related markers of gene and protein expression for a comprehensive evaluation of differentiation. Injured articular cartilage rat models were established for further hUC-MSC transplantation and LIPUS stimulation in vivo. Histopathology and H&E staining were used to evaluate the repair effects of the injured articular cartilage with LIPUS stimulation.ResultsThe results showed that LIPUS stimulation with specific parameters effectively promoted the expression of mature cartilage-related genes and proteins, inhibited TNF-α gene expression in hUC-MSCs, and exhibited anti-inflammation in C28/I2 cells. In addition, the articular cartilage defects of rats were significantly repaired after hUC-MSC transplantation and LIPUS stimulation.ConclusionsTaken together, LIPUS stimulation could realize articular cartilage regeneration based on hUC-MSC transplantation due to the inhibition of the TNF signaling pathway, which is of clinical value for the relief of osteoarthritis.

Low-Intensity Pulsed Ultrasound Attenuates Postoperative Neurocognitive Impairment and Salvages Hippocampal Synaptogenesis in Aged Mice

Postoperative neurocognitive impairment is an urgent problem with global aging accelerating. The prevention and treatment of postoperative neurocognitive impairment have been widely investigated but lack effective strategies. Low-intensity pulsed ultrasound (LIPUS), a non-invasive tool, has shown an effect on neuroprotection, but whether it could attenuate the postoperative neurocognitive impairment and the underlying mechanisms remains unknown. An experimental setup for LIPUS stimulation of the hippocampus was well established. A laparotomy model in aged mice was applied, and a Morris water maze was used to assess cognitive function. RT-qPCR and western blotting were used to detect levels of Piezo1, synapse-associated proteins in the hippocampus, respectively. Immunofluorescent staining was also used to determine the neural activation and Piezo1 expression. The results showed that LIPUS increased synapse-related proteins of the hippocampus and attenuated cognitive impairment in aged mice. Meanwhile, LIPUS suppressed the overexpression of Piezo1 in the hippocampus. We further found that LIPUS promoted Calpain1 activity and increased extracellular regulated protein kinases (Erk) phosphorylation. Our results suggested that LIPUS could improve cognitive impairment and increase hippocampal synaptogenesis through the Piezo1-mediated Calpain1/ Erk pathway. LIPUS could be used as an effective physical intervention to alleviate postoperative cognitive dysfunction in the aged population.

Low-intensity pulsed ultrasound increases neurotrophic factors secretion and suppresses inflammation in in vitro models of peripheral neuropathies

Objective. In this study, we aimed to verify the beneficial effects of low-intensity pulsed ultrasound (LIPUS) stimulation on two cell types: H2O2-treated RSC96 Schwann cells and THP-1 macrophages, used to model neuropathic inflammation. Approach. Using a set-up guaranteeing a fine control of the ultrasound dose at the target, different frequencies (38 kHz, 1 MHz, 5 MHz) and different intensities (20, 100, 500 mW cm−2) were screened to find the most effective experimental conditions for triggering beneficial effects on metabolic activity and release of neurotrophic cytokines (β-nerve growth factor, brain-derived neurotrophic factor, glial cell-derived neurotrophic factor) of RSC96 cells. The combination of parameters resulting the optimal one was applied to evaluate anti-inflammatory effects in terms of reactive oxygen species (ROS) and tumor necrosis factor-α (TNF-α) production, also investigating a possible anti-oxidant activity and mechanotransduction pathway for the anti-inflammatory process. The same optimal combination of parameters was then applied to THP-1 cells, differentiated into M1 and M2 phenotypes, to assess the effect on the expression and release of pro-inflammatory markers (TNF-α, interleukin (IL)-1β, IL-6, IL-8) and anti-inflammatory ones (IL-10 and CD206). Main results. 5 MHz and 500 mW cm−2 were found as the optimal stimulation parameters on RSC96 cells. Such parameters were also found to suppress ROS and TNF-α in the same cell line, thus highlighting a possible anti-inflammatory effect, involving the NF-kB pathway. An anti-oxidant effect induced by LIPUS was also observed. Finally, the same LIPUS parameters did not induce any differentiation towards the M1 phenotype of THP-1 cells, whereas they decreased TNF-α and IL-8 gene expression, reduced IL-8 cytokine release and increased IL-10 cytokine release in M1-polarized THP-1 cells. Significance. This study represents the first step towards the use of precisely controlled LIPUS for the treatment of peripheral neuropathies.

Design of bioglasses/PDLLA scaffolds with responsive drug delivery in ultrasonic-assisted bone repair

Low-intensity pulsed ultrasound (LIPUS) assisted bone repair is confirmed effective in clinic. Here, a 3D-printed composite poly(DL-lactic acid)/mesoporous bioactive glass scaffold was constructed for particular use in LIPUS-assisted bone tissue engineering. The scaffolds contain dimethyloxallyl glycine (DMOG) loaded microbubbles in pores, which can be released after implanting via LIPUS stimulation. Local DMOG concentrations are modulated through ultrasound power and processing time. The rat bone marrow-derived mesenchymal stem cells (rBMSCs) on these scaffolds with ultrasound treatment show improved proliferation and early osteogenic differentiation.