Radiation enhancement using focussed ultrasound-stimulated microbubbles for breast cancer: A Phase 1 clinical trial.

BACKGROUND: Preclinical studies have demonstrated that tumour cell death can be enhanced 10- to 40-fold when radiotherapy is combined with focussed ultrasound-stimulated microbubble (FUS-MB) treatment. The acoustic exposure of microbubbles (intravascular gas microspheres) within the target volume causes bubble cavitation, which induces perturbation of tumour vasculature and activates endothelial cell apoptotic pathways responsible for the ablative effect of stereotactic body radiotherapy. Subsequent irradiation of a microbubble-sensitised tumour causes rapid increased tumour death. The study here presents the mature safety and efficacy outcomes of magnetic resonance (MR)-guided FUS-MB (MRgFUS-MB) treatment, a radioenhancement therapy for breast cancer. METHODS AND FINDINGS: This prospective, single-center, single-arm Phase 1 clinical trial included patients with stages I-IV breast cancer with in situ tumours for whom breast or chest wall radiotherapy was deemed adequate by a multidisciplinary team (clinicaltrials.gov identifier: NCT04431674). Patients were excluded if they had contraindications for contrast-enhanced MR or microbubble administration. Patients underwent 2 to 3 MRgFUS-MB treatments throughout radiotherapy. An MR-coupled focussed ultrasound device operating at 800 kHz and 570 kPa peak negative pressure was used to sonicate intravenously administrated microbubbles within the MR-guided target volume. The primary outcome was acute toxicity per Common Terminology Criteria for Adverse Events (CTCAE) v5.0. Secondary outcomes were tumour response at 3 months and local control (LC). A total of 21 female patients presenting with 23 primary breast tumours were enrolled and allocated to intervention between August/2020 and November/2022. Three patients subsequently withdrew consent and, therefore, 18 patients with 20 tumours were included in the safety and LC analyses. Two patients died due to progressive metastatic disease before 3 months following treatment completion and were excluded from the tumour response analysis. The prescribed radiation doses were 20 Gy/5 fractions (40%, n = 8/20), 30 to 35 Gy/5 fractions (35%, n = 7/20), 30 to 40 Gy/10 fractions (15%, n = 3/20), and 66 Gy/33 fractions (10%, n = 2/20). The median follow-up was 9 months (range, 0.3 to 29). Radiation dermatitis was the most common acute toxicity (Grade 1 in 16/20, Grade 2 in 1/20, and Grade 3 in 2/20). One patient developed grade 1 allergic reaction possibly related to microbubbles administration. At 3 months, 18 tumours were evaluated for response: 9 exhibited complete response (50%, n = 9/18), 6 partial response (33%, n = 6/18), 2 stable disease (11%, n = 2/18), and 1 progressive disease (6%, n = 1/18). Further follow-up of responses indicated that the 6-, 12-, and 24-month LC rates were 94% (95% confidence interval [CI] [84%, 100%]), 88% (95% CI [75%, 100%]), and 76% (95% CI [54%, 100%]), respectively. The study's limitations include variable tumour sizes and dose fractionation regimens and the anticipated small sample size typical for a Phase 1 clinical trial. CONCLUSIONS: MRgFUS-MB is an innovative radioenhancement therapy associated with a safe profile, potentially promising responses, and durable LC. These results warrant validation in Phase 2 clinical trials. TRIAL REGISTRATION: clinicaltrials.gov, identifier NCT04431674.

Determining Ultrasound Parameters for Bursting Polymer Microbubbles for Future Use in Spinal Cord Injury.

OBJECTIVE: We believe our poly(lactic acid) (PLA) microbubbles are well suited for therapeutic delivery to spinal cord injury (SCI) using ultrasound-triggered bursting. We investigated the feasibility of clinical ultrasound bursting in situ, the optimal bursting parameters in vitro and the loading and release of a model bio-active DNA. METHODS: Microbubbles were tested using clinical ultrasound in a rat cadaver SCI model. Burst pressure thresholds were determined using the change in enhancement after ultrasound exposure. Resonance frequency, acoustic enhancement, sizing and morphology were evaluated by comparing two microbubble porogens, ammonium carbonate and ammonium carbamate. Oligonucleotides were loaded into the shell and released using the found optimized ultrasound bursting parameters. RESULTS: In situ imaging and bursting were successful. In vitro bursting thresholds using frequencies 1, 2.25 and 5 MHz were identified between peak negative pressures 0.2 and 0.5 MPa, believed to be safe for spinal cord. The pressure threshold decreased with decreasing frequencies. PLA bursting was optimized near the resonance frequency of 2.5 to 3.0 MHz using 2.25 MHz and not at lower frequencies. PLA microbubbles, initially with a mean size of approximately 2 µm, remained in one piece, collapsed to between 0.5 and 1 µm and did not fragment. Significantly more oligonucleotide was released after ultrasound bursting of loaded microbubbles. Microbubble-sized debris was detected when using ammonium carbamate, leading to inaccurate microbubble concentration measurements. CONCLUSION: PLA microbubbles made with ammonium carbonate and burst at appropriate parameters have the potential to safely improve intrathecal therapeutic delivery to SCI using targeted ultrasound.

Super-Resolution Ultrasound Imaging for Monitoring the Therapeutic Efficacy of a Vascular Disrupting Agent in an Animal Model of Breast Cancer.

OBJECTIVE: Evaluate the use of super-resolution ultrasound (SRUS) imaging for the early detection of tumor response to treatment using a vascular-disrupting agent (VDA). METHODS: A population of 28 female nude athymic mice (Charles River Laboratories) were implanted with human breast cancer cells (MDA-MB-231, ATCC) in the mammary fat pad and allowed to grow. Ultrasound imaging was performed using a Vevo 3100 scanner (FUJIFILM VisualSonics Inc) equipped with the MX250 linear array transducer immediately before and after receiving bolus injections of a microbubble (MB) contrast agent (Definity, Lantheus Medical Imaging) via the tail vein. Following baseline ultrasound imaging, VDA drug (combretastatin A4 phosphate, CA4P, Sigma Aldrich) or control saline was injected via the placed catheter. After 4 or 24 hours, repeat ultrasound imaging along the same tumor cross-section occurred. Direct intratumoral pressure measurements were obtained using a calibrated sensor. All raw ultrasound data were saved for offline processing and SRUS image reconstruction using custom MATLAB software (MathWorks Inc). From a region encompassing the tumor space and the entire postprocessed ultrasound image sequence, time MB count (TMC) curves were generated in addition to traditional SRUS maps reflecting MB enumeration at each pixel location. Peak enhancement (PE) and wash-in rate (WIR) were extracted from these TMC curves. At termination, intratumoral microvessel density (MVD) was quantified using tomato lectin labeling of patent blood vessels. RESULTS: SRUS images exhibited a clear difference between control and treated tumors. While there was no difference in any group parameters at baseline (0 hour, P > .09), both SRUS-derived PE and WIR measurements in tumors treated with VDA exhibited significant decreases by 4 (P = .03 and P = .05, respectively) and 24 hours (P = .02 and P = .01, respectively), but not in control group tumors (P > .22). Similarly, SRUS derived microvascular maps were not different at baseline (P = .81), but measures of vessel density were lower in treated tumors at both 4 and 24 hours (P < .04). An inverse relationship between intratumoral pressure and both PE and WIR parameters were found in control tumors (R2 > .09, P < .03). CONCLUSION: SRUS imaging is a new modality for assessing tumor response to treatment using a VDA.

Sonoselective delivery using ultrasound and microbubbles combined with intravenous rAAV9 CLDN5-GFP does not increase endothelial gene expression.

Transcranial ultrasound combined with intravenous microbubbles can be used to increase blood-brain barrier permeability or, at lower pressures, to mediate sonoselective gene delivery to endothelial cells. Previously, sonoselective gene delivery with plasmid-coated microbubbles as gene carriers resulted in transient transgene expression in the brain endothelium. We investigated the potential of recombinant adeno-associated virus 9 (rAAV9), a serotype known for its efficient transduction and long-term transgene expression, for sonoselective gene delivery to endothelial cells of the brain. We found that rAAV9 led to gene delivery to brain endothelial cells following intravenous administration at a dosage of 1 × 1011 GC/g. However, the sonoselective gene delivery approach with intravenous rAAV9, using the same parameters as previously used for plasmid delivery, did not increase transgene expression in brain endothelial cells targeted. These results suggest that intravenous rAAV9 are using mechanisms of entry into the cerebrovasculature that are not significantly influenced by sonoselective treatments known to facilitate endothelial cell entry of plasmids coated onto microbubbles.

Diagnostic ultrasound-mediated microbubble cavitation dose-dependently improves diabetic cardiomyopathy through angiogenesis.

Ultrasound-mediated microbubble cavitation (UMMC) induces therapeutic angiogenesis to treat ischemic diseases. This study aimed to investigate whether diagnostic UMMC alleviates diabetic cardiomyopathy (DCM) and, if so, through which mechanisms. DCM model was established by injecting streptozocin into rats to induce hyperglycemia, followed by a high-fat diet. The combined therapy of cation microbubble with low-intensity diagnostic ultrasound (frequency = 4 MHz), with a pulse frequency of 20 Hz and pulse length (PL) of 8, 18, 26, or 36 cycles, was given to rats twice a week for 8 consecutive weeks. Diagnostic UMMC therapy with PL at 8, 18, and 26 cycles, but not 36 cycles, dramatically prevented myocardial fibrosis, improved heart functions, and increased angiogenesis, accompanied by increased levels of PI3K, Akt, and eNOS proteins in the DCM model of rats. In cultured endothelial cells, low-intensity UMMC treatment (PL = 3 cycles, sound pressure level = 50%, mechanical index = 0.82) increased cell viability and activated PI3K-Akt-eNOS signaling. The combination of diagnostic ultrasound with microbubble destruction dose-dependently promoted angiogenesis, thus improving heart function through PI3K-Akt-eNOS signaling in diabetes. Accordingly, diagnostic UMMC therapy should be considered to protect the heart in patients with diabetes.

Nanomaterials as Ultrasound Theragnostic Tools for Heart Disease Treatment/Diagnosis.

A variety of different nanomaterials (NMs) such as microbubbles (MBs), nanobubbles (NBs), nanodroplets (NDs), and silica hollow meso-structures have been tested as ultrasound contrast agents for the detection of heart diseases. The inner part of these NMs is made gaseous to yield an ultrasound contrast, which arises from the difference in acoustic impedance between the interior and exterior of such a structure. Furthermore, to specifically achieve a contrast in the diseased heart region (DHR), NMs can be designed to target this region in essentially three different ways (i.e., passively when NMs are small enough to diffuse through the holes of the vessels supplying the DHR, actively by being associated with a ligand that recognizes a receptor of the DHR, or magnetically by applying a magnetic field orientated in the direction of the DHR on a NM responding to such stimulus). The localization and resolution of ultrasound imaging can be further improved by applying ultrasounds in the DHR, by increasing the ultrasound frequency, or by using harmonic, sub-harmonic, or super-resolution imaging. Local imaging can be achieved with other non-gaseous NMs of metallic composition (i.e., essentially made of Au) by using photoacoustic imaging, thus widening the range of NMs usable for cardiac applications. These contrast agents may also have a therapeutic efficacy by carrying/activating/releasing a heart disease drug, by triggering ultrasound targeted microbubble destruction or enhanced cavitation in the DHR, for example, resulting in thrombolysis or helping to prevent heart transplant rejection.

A review of bioeffects induced by focused ultrasound combined with microbubbles on the neurovascular unit.

Focused ultrasound combined with circulating microbubbles (FUS+MB) can transiently enhance blood-brain barrier (BBB) permeability at targeted brain locations. Its great promise in improving drug delivery to the brain is reflected by a rapidly growing number of clinical trials using FUS+MB to treat various brain diseases. As the clinical applications of FUS+MB continue to expand, it is critical to have a better understanding of the molecular and cellular effects induced by FUS+MB to enhance the efficacy of current treatment and enable the discovery of new therapeutic strategies. Existing studies primarily focus on FUS+MB-induced effects on brain endothelial cells, the major cellular component of BBB. However, bioeffects induced by FUS+MB expand beyond the BBB to cells surrounding blood vessels, including astrocytes, microglia, and neurons. Together these cell types comprise the neurovascular unit (NVU). In this review, we examine cell-type-specific bioeffects of FUS+MB on different NVU components, including enhanced permeability in endothelial cells, activation of astrocytes and microglia, as well as increased intraneuron protein metabolism and neuronal activity. Finally, we discuss knowledge gaps that must be addressed to further advance clinical applications of FUS+MB.

Applications of Ultrasound-Mediated Drug Delivery and Gene Therapy.

Gene therapy has continuously evolved throughout the years since its first proposal to develop more specific and effective transfection, capable of treating a myriad of health conditions. Viral vectors are some of the most common and most efficient vehicles for gene transfer. However, the safe and effective delivery of gene therapy remains a major obstacle. Ultrasound contrast agents in the form of microbubbles have provided a unique solution to fulfill the need to shield the vectors from the host immune system and the need for site specific targeted therapy. Since the discovery of the biophysical and biological effects of microbubble sonification, multiple developments have been made to enhance its applicability in targeted drug delivery. The concurrent development of viral vectors and recent research on dual vector strategies have shown promising results. This review will explore the mechanisms and recent advancements in the knowledge of ultrasound-mediated microbubbles in targeting gene and drug therapy.

Ultrasound-targeted microbubble destruction mediated miR-492 inhibitor suppresses the tumorigenesis in non-small cell lung cancer.

BACKGROUND: Ultrasound-targeted microbubble destruction (UTMD) is a novel adjuvant tumor therapeutic method by enhancing exogenous gene transfection to target tissues. This study aims to investigate the role of microRNA-492 (miR-492) in non-small cell lung cancer (NSCLC) and further analyze the effects of UTMD-mediated miR-492 inhibitor on tumorigenesis. METHODS: The expression of miR-492 was detected by qRT-PCR. Co-transfection of microbubbles and miR-492 inhibitor with Lipofectamine 3000 was performed to achieve UTMD-mediated miR-492 inhibition in NSCLC cells. CCK-8 and Transwell assay were used to determine NSCLC cell proliferation, and the migration and invasion. RESULT: High expression of miR-492 was associated with poor prognosis in NSCLC patients. miR-492 inhibitor suppressed tumor cell proliferation, migration and invasion, and UTMD not only increased the transfection efficiency of miR-492 inhibitor, but also enhance the inhibitory effects on cell biological behaviors. CONCLUSION: The results showed that the expression level of miR-492 was up-regulated in NSCLC tissue samples and cells. Silencing of miR-492 inhibited NSCLC cell proliferation, migration and invasion, and UTMD-mediated miR-492 inhibitor could promote more significant inhibition, which indicated that UTMD-mediated miR-492 inhibitor might provide a novel strategy for the treatment of NSCLC.KEY MESSAGESmiR-492 inhibitor inhibited cell proliferation, migration and invasion.UTMD-mediated miR-492 inhibitor can promote more significant inhibition.UTMD-mediated miR-492 inhibitor provide a new strategy for NSCLC.

CMBs carrying PTX and CRISPR/Cas9 targeting C­erbB­2 plasmids interfere with endometrial cancer cells.

Development of combination therapy to decrease side effects of chemotherapeutic drugs and increase their utilization rate in combination with gene editing is a key research topic in tumor treatment. The present study aimed to investigate the effect of cationic microbubbles (CMBs) carrying paclitaxel (PTX) and C­erbB­2 knockout plasmid on the endometrial cancer cell line HEC­1A and to determine how C­erbB­2 regulates the function of endometrial cancer cells. Cells were treated with CMB, PTX, PTX­CMBs, cationic plasmid­carrying or cationic PTX­carrying plasmid groups. After verifying the most effective combination of PTX­CMBs and plasmids, HEC­1A cells were transfected. Reverse transcription­quantitative (RT­q)PCR and western blotting were used to measure C­erbB­2 and protein expression. After verifying C­erbB­2 knockout, invasion, healing, clone formation and proliferation of HEC­1A cells were assessed. Simultaneously, expression levels of the genes for P21, P27, mammalian target of rapamycin (mTOR), and Bcl­2 associated death promoter (Bad) were measured by RT­qPCR. Compared with the PTX group, CMBs significantly enhanced the absorption efficiency of PTX by HEC­1A cells. C­erbB­2 knockout had an inhibitory effect on the proliferation, migration and invasion of HEC­1A cells; cell proliferation and invasion of the group carrying PTX and plasmids simultaneously were significantly weakened. The C­erbB­2­knockout group exhibited increased expression of P21 and P27. Simultaneously loading PTX and plasmid may be novel combination therapy with great potential. C­erbB­2 may regulate the proliferation of HEC­1A cells by downregulating expression of P21 and P27.

Ultrasonic technologies in imaging and drug delivery.

Ultrasonic technologies show great promise for diagnostic imaging and drug delivery in theranostic applications. The development of functional and molecular ultrasound imaging is based on the technical breakthrough of high frame-rate ultrasound. The evolution of shear wave elastography, high-frequency ultrasound imaging, ultrasound contrast imaging, and super-resolution blood flow imaging are described in this review. Recently, the therapeutic potential of the interaction of ultrasound with microbubble cavitation or droplet vaporization has become recognized. Microbubbles and phase-change droplets not only provide effective contrast media, but also show great therapeutic potential. Interaction with ultrasound induces unique and distinguishable biophysical features in microbubbles and droplets that promote drug loading and delivery. In particular, this approach demonstrates potential for central nervous system applications. Here, we systemically review the technological developments of theranostic ultrasound including novel ultrasound imaging techniques, the synergetic use of ultrasound with microbubbles and droplets, and microbubble/droplet drug-loading strategies for anticancer applications and disease modulation. These advancements have transformed ultrasound from a purely diagnostic utility into a promising theranostic tool.

Ultrasound Microbubbles Enhance the Efficacy of Insulin-Like Growth Factor-1 Therapy for the Treatment of Noise-Induced Hearing Loss.

The application of insulin-like growth factor 1 (IGF-1) to the round window membrane (RWM) is an emerging treatment for inner ear diseases. RWM permeability is the key factor for efficient IGF-1 delivery. Ultrasound microbubbles (USMBs) can increase drug permeation through the RWM. In the present study, the enhancing effect of USMBs on the efficacy of IGF-1 application and the treatment effect of USMB-mediated IGF-1 delivery for noise-induced hearing loss (NIHL) were investigated. Forty-seven guinea pigs were assigned to three groups: the USM group, which received local application of recombinant human IGF-1 (rhIGF-1, 10 µg/µL) following application of USMBs to the RWM; the RWS group, which received IGF-1 application alone; and the saline-treated group. The perilymphatic concentration of rhIGF-1 in the USM group was 1.95- and 1.67- fold of that in the RWS group, 2 and 24 h after treatment, respectively. After 5 h of 118 dB SPL noise exposure, the USM group had the lowest threshold shift in auditory brainstem response, least loss of cochlear outer hair cells, and least reduction in the number of synaptic ribbons on postexposure day 28 among the three groups. The combination of USMB and IGF-1 led to a better therapeutic response to NIHL. Two hours after treatment, the USM group had significantly higher levels of Akt1 and Mapk3 gene expression than the other two groups. The most intense immunostaining for phosphor-AKT and phospho-ERK1/2 was detected in the cochlea in the USM group. These results suggested that USMB can be applied to enhance the efficacy of IGF-1 therapy in the treatment of inner ear diseases.

Preclinical study to improve microbubble-mediated drug delivery in cancer using an ultrasonic probe with an interchangeable acoustic lens.

Focused ultrasound with microbubbles (FUS-MBs) has shown that it can lead to an efficient drug delivery system (DDS) involving the oscillation and destruction of the MB but is limited in drug delivery due to its narrow pressure field. However, unfocused ultrasound with MBs (UUS-MBs) and an interchangeable acoustic lens can tune and enhance the pressure field for MB destruction to overcome the disadvantages of FUS-MB DDSs. We designed a lens suitable for an ultrasound-phased array probe and studied the optimal treatment conditions for MB destruction in vitro through an optical imaging setup. The DDS effects were evaluated in a rat hepatoma model using doxorubicin (DOX) treatment. A concave lens with a radius of curvature of 2.6 mm and a thickness of 4 mm was selected and fabricated. UUS-MBs with the acoustic lens at 60 Vpp for 32 cycles and a PRF of 1 kHz could induce MB destruction, promoting the DDS even under fluidic conditions. In the animal experiment, the UUS-MBs in the acoustic lens treatment group had a higher concentration of DOX in the tumor than the control group. Our system suggests uses an acoustic lens to increase DDS effectiveness by providing sufficient ultrasound irradiation to the MBs.

Sonoporation generates downstream cellular impact after membrane resealing.

Sonoporation via microbubble-mediated ultrasound exposure has shown potential in drug and gene delivery. However, there is a general lack of mechanistic knowledge on sonoporation-induced cellular impact after membrane resealing, and this issue has made it challenging to apply sonoporation efficiently in practice. Here, we present new evidence on how sonoporation, without endangering immediate cell viability, may disrupt downstream cellular hemostasis in ways that are distinguished from the bioeffects observed in other sonicated and unsonoporated cells. Sonoporation was realized on HL-60 leukemia cells by delivering pulsed ultrasound (1 MHz frequency, 0.50 MPa peak negative pressure; 10% duty cycle; 30 s exposure period; 29.1 J/cm2 acoustic energy density) in the presence of lipid-shelled microbubbles (1:1 cell-to-bubble ratio). Results showed that 54.6% of sonoporated cells, despite remaining initially viable, underwent apoptosis or necrosis at 24 h after sonoporation. Anti-proliferation behavior was also observed in sonoporated cells as their subpopulation size was reduced by 43.8% over 24 h. Preceding these cytotoxic events, the percentages of sonoporated cells in different cell cycle phases were found to be altered by 12 h after exposure. As well, for sonoporated cells, their expressions of cytoprotective genes in the heat shock protein-70 (HSP-70) family were upregulated by at least 4.1 fold at 3 h after exposure. Taken altogether, these findings indicate that sonoporated cells attempted to restore homeostasis after membrane resealing, but many of them ultimately failed to recover. Such mechanistic knowledge should be taken into account to devise more efficient sonoporation-mediated therapeutic protocols.

Ultrasound-Targeted Microbubble Destruction Enhances the Inhibitive Efficacy of miR-21 Silencing in HeLa Cells.

BACKGROUND Previous studies have shown that miR-21 upregulation is related to the aggressive development of cervical cancer. Ultrasound-targeted microbubble destruction (UTMD) is a method that increases the absorption of targeted genes or drugs by cells. We focus on the role of UTMD-mediated miR-21 transfection in HeLa cells, a cervical cancer cell line. MATERIAL AND METHODS The effects of different ultrasound intensities on the transfection efficiency of miR-21-enhanced green fluorescent protein (EGFP) and miR-21 inhibitor-EGFP plasmids were determined by flow cytometry. The effects of UTMD-mediated miR-21 transfection on HeLa cell proliferation, apoptosis, migration, and invasion were measured by CCK-8, flow cytometry, wound healing experiments, and transwell migration assay, respectively. Western blot and real-time quantitative PCR were used to detect the expression of tumor-related genes. RESULTS When the ultrasound intensity was 1.5 W/cm², the miR-21 plasmid had the highest transfection efficiency. Exogenous miR-21 promoted cell proliferation, migration, and invasion, and inhibited cell apoptosis in HeLa cells. Treatment of cells with UTMD further enhanced the effects of miR-21-EGFP and miR-21 inhibitor-EGFP. In addition, miR-21 overexpression significantly increased the expression of p-Akt, Akt, Bcl-2, Wnt, ß-catenin, matrix metalloprotein-9 (MMP-9), and epidermal growth factor (EGFR) levels, and decreased Bax expression. The regulatory role of miR-21 inhibitor-EGFP was opposite to that of miR-21-EGFP. After UTMD, miR-21-EGFP and miR-21 inhibitor-EGFP had more significant regulatory effects on these genes. CONCLUSIONS Our research revealed that an ultrasound intensity of 1.5 W/cm² is the best parameter for miR-21 transfection. UTMD can enhance the biological function of miR-21 in HeLa cells, and alter the effect of miR-21 on apoptosis, metastasis, and phosphorylation genes.

Using ultrasound-targeted microbubble destruction to enhance radiotherapy of glioblastoma.

OBJECTIVE: To investigate the efficacy and mechanism of ultrasound-targeted microbubble destruction (UTMD) combined with radiotherapy (XRT) on glioblastoma. METHODS: The enhanced radiosensitization by UTMD was assessed through colony formation and cell apoptosis in Human glioblastoma cells (U87MG). Subcutaneous transplantation tumors in 24 nude mice implanted with U87MG cells were randomly assigned to 4 different treatment groups (Control, UTMD, XRT, and UTMD + XRT) based on tumor sizes (100-300 mm3). Tumor growth was observed for 10 days after treatment, and then histopathology stains (HE, CD34, and γH2AX) were applied to the tumor samples. A TUNEL staining experiment was applied to detect the apoptosis rate of mice tumor samples. Meanwhile, tissue proteins were extracted from animal specimens, and the expressions of dsDNA break repair-related proteins from animal specimens were examined by the western blot. RESULTS: When the radiotherapy dose was 4 Gy, the colony formation rate of U87MG cells in the UTMD + XRT group was 32 ± 8%, lower than the XRT group (54 ± 14%, p < 0.01). The early apoptotic rate of the UTMD + XRT group was 21.1 ± 3% at 48 h, higher than that of the XRT group (15.2 ± 4%). The tumor growth curve indicated that the tumor growth was inhibited in the UTMD + XRT group compared with other groups during 10 days of observation. In TUNEL experiment, the apoptotic cells of the UTMD + XRT group were higher than that of the XRT group (p < 0.05). The UTMD + XRT group had the lowest MVD value, but was not significantly different from other groups (p > 0.05). In addition, γH2AX increased due to the addition of UTMD to radiotherapy compared to XRT in immunohistochemistry (p < 0.05). CONCLUSIONS: Our study clearly demonstrated the enhanced destructive effect of UTMD combined with 4 Gy radiotherapy on glioblastoma. This could be partly achieved by the increased ability of DNA damage of tumor cells.

Ultrasound-Mediated Microbubble Cavitation Transiently Reverses Acute Hindlimb Tissue Ischemia through Augmentation of Microcirculation Perfusion via the eNOS/NO Pathway.

Ultrasound-mediated microbubble cavitation improves perfusion in chronic limb and myocardial ischemia. The purpose of this study was to determine the effects of ultrasound-mediated microbubble cavitation in acute limb ischemia and investigate the mechanism of action. The animal with acute hindlimb ischemia was established using male Sprague-Dawley rats. The rats were randomly divided into three groups: intermittent high-mechanical-index ultrasound pulses combined with microbubbles (ultrasound [US] + MB group), US alone (US group) and MB alone (MB group). Both hindlimbs were treated for 10 min. Contrast ultrasound perfusion imaging of both hindlimbs was performed immediately and 5, 10, 15, 20 and 25 min after treatment. The role of the nitric oxide (NO) pathway in increasing blood flow in acutely ischemic tissue was evaluated by inhibiting endothelial nitric oxide synthase (eNOS) with Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME). In the US + MB group, microvascular blood volume and microvascular blood flow of the ischemic hindlimb were significantly increased after treatment (both p values <0.05), while the microvascular flux rate (ß) increased, but not significantly (p > 0.05). The increases were observed immediately after treatment, and had dissipated by 25 min. Changes in the US and MB groups were minimal. Inhibitory studies indicated cavitation increased phospho-eNOS concentration in ischemic hindlimb muscle tissue, and the increase was significantly inhibited by L-NAME (p < 0.05). Ultrasound-mediated microbubble cavitation transiently increases local perfusion in acutely ischemic tissue, mainly by improving microcirculatory perfusion. The eNOS/NO signaling pathway appears to be an important mediator of the effect.

Innovative Applications of MR-Guided Focused Ultrasound for Neurological Disorders.

Magnetic resonance-guided focused ultrasound (MRgFUS) is a cutting-edge technology that is changing the practice of movement disorders surgery. Given the noninvasive and innovative nature of this technology, there is great interest in expanding the use of MRgFUS to additional diseases and applications. Current approved applications target the motor thalamus to treat tremor, but clinical trials are exploring or plan to study noninvasive lesions with MRgFUS to ablate tumor cells in the brain as well as novel targets for movement disorders and brain regions associated with pain and epilepsy. Although there are additional potential indications for lesioning, the ability to improve function by destroying parts of the brain is still limited. However, MRgFUS can also be applied to a brain target after intravenous delivery of microbubbles to create cavitations and focally open the blood-brain barrier (BBB). This has already proven to be safe and technically feasible in human patients with Alzheimer's disease, and this action alone has potential to clear extracellular pathology associated with this and other neurodegenerative disorders. This also provides a foundation for noninvasive intravenous delivery of therapeutic molecules to precise brain targets after transient disruption of the BBB. Certain chemotherapies for brain tumors, immunotherapies, gene, and cell therapies are all examples of therapeutic or even restorative agents that normally will not enter the brain without direct infusion but which have been shown in preclinical studies to effectively traverse the BBB after transient disruption with MRgFUS. Here we will review these novel applications of MRgFUS to provide an overview of the extraordinary potential of this technology to expand future neurosurgical treatments of brain diseases.

Advances of Contrast-Enhanced Ultrasonography and Elastography in Kidney Transplantation: From Microscopic to Microcosmic.

Kidney transplantation is the best choice for patients with end-stage renal disease. To date, allograft biopsy remains the gold standard for revealing pathologic changes and predicting long-term outcomes. However, the invasive nature of transplant biopsy greatly limits its application. Ultrasound has been a first-line examination for evaluating kidney allografts for a long time. Advances in ultrasound in recent years, especially the growing number of studies in elastography and contrast-enhanced ultrasonography (CEUS), have shed new light on its application in kidney transplantation. Elastography, including strain elastography and shear wave elastography, is used mainly to assess allograft stiffness and, thus, predict renal fibrosis. CEUS has been used extensively in evaluating blood microperfusion, assessing acute kidney injury and detecting different complications after transplantation. Requiring the use of microbubbles also makes CEUS a novel method of gene transfer and drug delivery, enabling promising targeted diagnosis and therapy. In this review, we summarize the advances of elastography and CEUS in kidney transplantation and evaluate their potential efficiency in becoming a better complement to or even substitute for transplant biopsy in the future.