Single Sonication Research Articles

Age influences therapeutic ultrasound effects: From increased inflammation in young to decreased inflammaging in adults

BACKGROUND: Therapeutic ultrasound is a controversial topic in physical therapy because outcomes varied due to different experimental designs. At the cellular level, acoustic radiation force (i.e., pressure) from ultrasound previously showed activation of sodium/calcium channels, hyper-proliferation and increased cytokine signaling. However, few have studied the immune response after ultrasound despite the tight coupling between muscle inflammation and regeneration. Additionally, limited work has assessed ultrasound in aged populations. Therefore, a deeper understanding of how therapeutic ultrasound influences the muscle microenvironment is required to use it as a regenerative therapy. The aim of this study was to evaluate inflammatory cytokine signaling and immune cell infiltration after ultrasound in young and aging murine muscle. We hypothesized that the immune response will increase with ultrasound pressure but decrease with age. METHODS: Young (6-weeks-old) and Adult (52-weeks-old) male and female non-injured gastrocnemii were treated with a single sonication of either low intensity pulsed ultrasound (LIPUS) at ~0.243 MPa or pulsed focused ultrasound (pFUS) at ~5.96 MPa. After sonication, muscles were harvested at 1-, 8-, and 24-hours for cytokine analysis using an immunology multiplex assay (138 mice) and at 1- and 24-hours for flow cytometry (120 mice). Control mice were not treated. Sexes were combined due to minimal, if any, differences. Statistical analyses were performed using an ANOVA with post-hoc Bonferroni comparisons ( p < 0.05); only differences compared to control are described. RESULTS: In Young post-LIPUS, IL-15 was upregulated at 1-hour and likely caused the increased M1 and M2 macrophages (M2 > M1) at 1-hour. In Young post-pFUS, the upregulation of VEGF at 1-hour was counteracted by MIG at 8-hours resulting in no change in endothelial cells. Increased MCP-1 at 24-hours post-pFUS corresponded with increased neutrophils, monocytes, and M1 and M2 macrophages (M1 > M2) at 24-hours. In Adult, cytokine expression throughout the 24-hour period was downregulated after LIPUS (IL-9/-15) and pFUS (IL-1α/-2/-6/-9, M-CSF, MIP-1α). Thus, there were no changes in the immune cell response over time post-LIPUS or -pFUS. Additionally, for most of these cytokines, Adult control expression was at least two times greater than Young control. CONCLUSIONS: In Young, LIPUS caused an early, temporary pro- and anti-inflammatory response while pFUS caused a late, extended pro-inflammatory response. However, this response was lost in Adult where age-induced inflammaging was decreased after sonication. In younger muscle, ultrasound could enhance regeneration by augmenting the immune response while in aging muscle it could be used to decrease inflammation induced atrophy. Overall, therapeutic ultrasound caused a pressure dependent inflammatory response that can increase or decrease intrinsic signaling and cell recruitment in adolescent or aged muscles, respectively. This work was funded by the Intramural Research Program of the National Institutes of Health, Clinical Center and National Institutes of Biomedical Imaging and Bioengineering. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Small volume blood-brain barrier opening in macaques with a 1 MHz ultrasound phased array

The use of focused ultrasound to open the blood-brain barrier (BBB) has the potential to deliver drugs to specific regions of the brain. The size of the BBB opening and ability to localize the opening determines the spatial extent and is a limiting factor in many applications of BBB opening where targeting a small brain region is desired. Here we evaluate the performance of a system designed for small opening volumes and highlight the unique challenges associated with pushing the spatial precision of this technique. To achieve small volume openings in cortical regions of the macaque brain, we tested a custom 1 MHz array transducer integrated into a magnetic resonance image-guided focused ultrasound system. Using real-time cavitation monitoring, we demonstrated twelve instances of single sonication, small volume BBB opening with average volumes of 59 ± 37 mm3 and 184 ± 2 mm3 in cortical and subcortical targets, respectively. We found high correlation between subject-specific acoustic simulations and observed openings when incorporating grey matter segmentation (R2 = 0.8577), and the threshold for BBB opening based on simulations was 0.53 MPa. Analysis of MRI-based safety assessment and cavitation signals indicate a safe pressure range for 1 MHz BBB opening and suggest that our system can be used to deliver drugs and gene therapy to small brain regions.

Utilization of focused ultrasound for opening of the blood-nerve barrier

Objective. Focused ultrasound (FUS) use with and without microbubbles (MB) for investigation of the blood-nerve barrier (BNB) within the peripheral nervous system (PNS) has been performed in this study. We evaluate the feasibility of BNB opening in a rodent sciatic nerve model by direct vision FUS treatment and provide preliminary results of magnetic resonance guided FUS (MRgFUS). Approach. Twenty rodent bilateral sciatic nerves were investigated. Rodents were treated using a benchtop FUS system to directly visualize nerve FUS studies. Definity MB, Evans blue dye (EB) and latex micro beads were injected during studies. Selected animals underwent further compound muscle action potential (CMAP) studies. Sonication peak pressure (MPa), width, duty-cycle and duration as well as MB concentration were varied to investigate effective pressure threshold. Further preliminary MRgFUS studies were performed on selected animals. Immunohistochemistry and histological analysis under florescent microscopy were performed at termination of experiments to verify treatment outcomes. Main results. Three ultrasound pressures and three microbubble concentrations at a single sonication frequency (476.5 kHz) were performed under direct open targeting. Histological analysis demonstrated nerve internal architecture disruption at 1.2 MPa with 166.7 μl kg−1 while 0.3 MPa, with 40 μl kg−1 MB concentration was the lower threshold for consistently observed disruption of the BNB without anatomical microarchitecture disruption. EB leakage was confirmed at the target region in histological evaluation of nerve following MB injection and FUS sonication. Supra-harmonic emissions were detected during FUS exposures following MB injection but not at baseline reference, indicating effective MB response and stable cavitation. CMAP amplitudes showed delayed onset latency and lower amplitudes in sonicated nerves compared to control nerves without evidence of complete conduction block, suggesting a transient BNB disruption, while at lower limit pressure subtle conduction changes were observed. In MRgFUS, targeted nerves demonstrated further contrast agent leak as well as supra-harmonic frequency detection. Significance. Opening of the BNB in the PNS was achieved using FUS and MB in a rodent model. Ongoing work aims to refine FUS parameters for drug delivery into the nerve after experimental transient BNB disruption.

Technical Comparison of Treatment Efficiency of Magnetic Resonance-Guided Focused Ultrasound Thalamotomy and Pallidotomy in Skull Density Ratio-Matched Patient Cohorts.

ObjectiveMR-guided focused ultrasound (MRgFUS) is increasingly being used to treat patients with essential tremor (ET) and Parkinson's disease (PD) with thalamotomy and pallidotomy, respectively. Pallidotomy is performed off-center within the cranium compared to thalamotomy and may present challenges to therapeutic lesioning due to this location. However, the impact of target location on treatment efficiency and ability to create therapeutic lesions has not been studied. This study aimed to compare the physical efficiency of MRgFUS thalamotomy and pallidotomy.MethodsTreatment characteristics were compared between patients treated with thalamotomy (n = 20) or pallidotomy (n = 20), matched by skull density ratios (SDR). Aspects of treatment efficiency were compared between these groups. Demographic and comparative statistics were conducted to assess these differences. Acoustic field simulations were performed to compare and validate the simulated temperature profile for VIM and GPi ablation.ResultsLower SDR values were associated with greater energy requirement for thalamotomy (R2 = 0.197, p = 0.049) and pallidotomy (R2 = 0.342, p = 0.007). The impact of low SDR on efficiency reduction was greater for pallidotomy, approaching significance (p = 0.061). A nearly two-fold increase in energy was needed to reach 50°C in pallidotomy (10.9kJ) than in thalamotomy (5.7kJ), (p = 0.002). Despite lower energy requirement, the maximum average temperature reached was higher in thalamotomy (56.7°C) than in pallidotomy (55.0°C), (p = 0.017). Mean incident angle of acoustic beams was lesser in thalamotomy (12.7°) than in pallidotomy (18.6°), (p < 0.001). For all patients, a lesser mean incident angle correlated with a higher maximum average temperature reached (R2 = 0.124, p = 0.026), and less energy needed to reach 50°C (R2=0.134, p = 0.020). Greater skull thickness was associated with a higher maximum energy for a single sonication for thalamotomy (R2 = 0.206, p = 0.045) and pallidotomy (R2 = 0.403, p = 0.003). An acoustic and temperature field simulation validated similar findings for thalamotomy and pallidotomy in a single patient.ConclusionThe centrally located VIM offers a more efficient location for therapeutic lesioning compared to GPi pallidotomy in SDR matched cohort of patients. The impact on therapeutic lesioning with lower SDR may be greater for pallidotomy patients. As newer off-center targets are investigated, these findings can inform patient selection and treatment requirements for lesion production.

Characterized nano-size curcumin and rosemary oil for the limitation microbial spoilage of rainbow trout fillets

Curcumin and rosemary oil nanoemulsions (CUR and RON), having 184.3 nm and 158.3 nm average diameters, were successfully produced by a single sonication technique. Effectiveness of nanoemulsions against P. aeruginosa, E. coli, and S. typhimurium were tested by agar diffusion method. Total mesophilic bacteria (TMABc) growth in fish fillets treated with RON and CUR nanoemulsions was acceptable for human consumption up to the 9th day. Total psychrophilic bacteria counts (TPBc) in the fish fillets treated with RON and CUR was found to be lower (26% and 17%) than the control group samples, respectively. While TMAB growth in fish fillets treated with RON was effectively declined from 5.59 to 3.48 log CFU/g, TMAB count of the fish fillets treated with CUR was decreased from 7.42 to 6.53 log CFU/g during the analysis period. Therefore, the obtained novel nanomaterial could be effectively used to limit the rapid increase in bacterial spoilage of fish meat in the food industry.

Validation of hybrid angular spectrum acoustic and thermal modelling in phantoms

In focused ultrasound (FUS) thermal ablation of diseased tissue, acoustic beam and thermal simulations enable treatment planning and optimization. In this study, a treatment-planning methodology that uses the hybrid angular spectrum (HAS) method and the Pennes’ bioheat equation (PBHE) is experimentally validated in homogeneous tissue-mimicking phantoms. Simulated three-dimensional temperature profiles are compared to volumetric MR thermometry imaging (MRTI) of FUS sonications in the phantoms, whose acoustic and thermal properties are independently measured. Additionally, Monte Carlo (MC) uncertainty analysis is performed to quantify the effect of tissue property uncertainties on simulation results. The mean error between simulated and experimental spatiotemporal peak temperature rise was +0.33°C (+6.9%). Despite this error, the experimental temperature rise fell within the expected uncertainty of the simulation, as determined by the MC analysis. The average errors of the simulated transverse and longitudinal full width half maximum (FWHM) of the profiles were –1.9% and 7.5%, respectively. A linear regression and local sensitivity analysis revealed that simulated temperature amplitude is more sensitive to uncertainties in simulation inputs than in the profile width and shape. Acoustic power, acoustic attenuation and thermal conductivity had the greatest impact on peak temperature rise uncertainty; thermal conductivity and volumetric heat capacity had the greatest impact on FWHM uncertainty. This study validates that using the HAS and PBHE method can adequately predict temperature profiles from single sonications in homogeneous media. Further, it informs the need to accurately measure or predict patient-specific properties for improved treatment planning of ablative FUS surgeries.

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles.

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising approach to cancer therapy. The glycosaminoglycan placental chondroitin sulfate A (plCSA) is expressed on a wide range of cancer cells and placental trophoblasts, and malarial protein VAR2CSA can specifically bind to plCSA. A reported placental chondroitin sulfate A binding peptide (plCSA-BP), derived from malarial protein VAR2CSA, can also specifically bind to plCSA on cancer cells and placental trophoblasts. Hence, plCSA-BP-conjugated nanoparticles could be used as a tool for targeted drug delivery to human cancers and placental trophoblasts. In this protocol, we describe a method to synthesize plCSA-BP-conjugated lipid-polymer nanoparticles loaded with doxorubicin (plCSA-DNPs); the method consists of a single sonication step and bioconjugate techniques. In addition, several methods for characterizing plCSA-DNPs, including determining their physicochemical properties and cellular uptake by placental choriocarcinoma (JEG3) cells, are described.

Comparison of Purification Processes for Recombinant Human Growth Hormone Produced in E. coli

In this study, purification of recombinant human growth hormone (rhGH) with FLAG tag from E. coli inclusion bodies was described. The step-by-step process carried out was as follows: bacterial cells were suspended in lysis buffer and disrupted in a single sonication step. Then, inclusion bodies were isolated and solubilized in different buffers to obtain the best one which was found to be Tris buffer containing 2% deoxycholate with a satisfyingly adequate capacity to dissolve inclusion bodies at pH 12.5. In the third step, the proteins in solubilizing buffer were refolded by being diluted in refolding buffer. This was carried out with lowering the pH value to 8 using a direct dilution (to five volumes) process. Following a specific enterokinase cleavage for removing the tag, the rhGH was purified by ion-exchange chromatography. Following with the research, for the first time, a comparative study was performed on two weak ion-exchangers, namely DEAE and CM-Sepharose (two most commonly used resins), so as to investigate their pH dependence and resolution. DEAE-Sepharose at pH 8.25 exhibited the best overall performance and efficiency for rhGH purification as measured by either yield or purity. The overall process was reproducible and easy to scale up, making it a suitable choice for large-scale production of therapeutic proteins.

Determination of Polycyclic Aromatic Hydrocarbons (PAH4) in the Traditional Lebanese Grilled Chicken: Implementation of New, Rapid and Economic Analysis Method

Polycyclic aromatic hydrocarbons (PAH4) (benzo[a]anthracene, chrysene, benzo[b]fluoranthene, and benzo[a]pyrene) are evaluated in a traditional and widely consumed staple in Lebanon “Lebanese Grilled Chicken.” Forty samples with different local additives grilled by charcoal were purchased from different restaurants located in different regions in Lebanon. For this purpose, a simple and a reliable analytical method based on sonication technique and gas chromatography coupled to mass spectrometer (GC-MS) has been developed and validated. Several parameters affecting the extraction efficiency, such as the nature and the volume of the extract solvent, as well as other factors were investigated and optimized. The developed method is environmentally and economically friendly with a minimized consuming time, consisting of a single sonication step for 15 min using 12 mL of non-carcinogenic solvent acetonitrile (ACN) and without any further concentration prior to analysis. Following extraction, the cleanup step was based on freezing and d-SPE by C18 addition. Under optimized conditions, the method performances were evaluated; the limits of quantification (LOQs) achieved were lower than 0.689 μg kg−1, and these values fit the performance criteria for the method given by the EC that defined the LOQ values of PAH lower than 0.9 μg kg−1. In addition, the recovery values of the analyzed PAHs ranged from 88.9 to 119.3% with relative standard deviations (RSDs) less than 8% (n = 15). The levels of PAH4 were in the range from 1.52 to 49.9 μg kg−1, where about 40% of the Lebanese grilled chicken exceeded the EU commission MRLs of 12 μg kg−1.

Blood‐Brain Barrier Closure Time After Controlled Ultrasound‐Induced Opening Is Independent of Opening Volume

Microbubble-mediated focused ultrasound (US) opening of the blood-brain barrier (BBB) has shown promising results for the treatment of brain tumors and conditions such as Alzheimer disease. Practical clinical implementation of focused US treatments would aim to treat a substantial portion of the brain; thus, the safety of opening large volumes must be investigated. This study investigated whether the opened volume affects the time for the BBB to be restored after treatment. Sprague Dawley rats (n = 5) received bilateral focused US treatments. One hemisphere received a single sonication, and the contralateral hemisphere was targeted with 4 overlapping foci. Contrast-enhanced T1-weighted magnetic resonance imaging was used to assess the integrity of the BBB at 0, 6, and 24 hours after focused US. At time 0, there was no significant difference in the mean enhancement between the single- and multi-point sonications (mean ± SD, 29.7% ± 18.4% versus 29.7% ± 24.1%; P = .9975). The mean cross-sectional area of the BBB opening resulting from the multi-point sonication was approximately 3.5-fold larger than that of the single-point case (14.2 ± 4.7 versus 4.1 ± 3.3 mm2 ; P < .0001). The opened volumes in 9 of 10 hemispheres were closed by 6 hours after focused US. The remaining treatment location had substantially reduced enhancement at 6 hours and was closed by 24 hours. Histologic analysis revealed small morphologic changes associated with this location. T2-weighted images at 6 and 24 hours showed no signs of edema. T2*-weighted images obtained at 6 hours also showed no signs hemorrhage in any animal. The time for the BBB to close after focused US was independent of the opening volume on the time scale investigated. No differences in treatment effects were observable by magnetic resonance imaging follow-up between larger- and smaller-volume sonications, suggesting that larger-volume BBB opening can be performed safely.

Renal ablation using magnetic resonance-guided high intensity focused ultrasound: Magnetic resonance imaging and histopathology assessment.

To use magnetic resonance-guided high intensity focused ultrasound (MRg-HIFU), magnetic resonance imaging (MRI) and histopathology for noninvasively ablating, quantifying and characterizing ablated renal tissue. Six anesthetized/mechanically-ventilated pigs underwent single/double renal sonication (n = 24) using a 3T-MRg-HIFU (1.1 MHz frequency and 3000J-4400J energies). T2-weighted fast spin echo (T2-W), perfusion saturation recovery gradient echo and contrast enhanced (CE) T1-weighted (T1-W) sequences were used for treatment planning, temperature monitoring, lesion visualization, characterization and quantification, respectively. Histopathology was conducted in excised kidneys to quantify and characterize cellular and vascular changes. Paired Student's t-test was used and a P-value < 0.05 was considered statistically significant. Ablated renal parenchyma could not be differentiated from normal parenchyma on T2-W or non-CE T1-W sequences. Ablated renal lesions were visible as hypoenhanced regions on perfusion and CE T1-W MRI sequences, suggesting perfusion deficits and necrosis. Volumes of ablated parenchyma on CE T1-W images in vivo (0.12-0.36 cm(3) for single sonication 3000J, 0.50-0.84 cm(3), for double 3000J, 0.75-0.78 cm(3) for single 4400J and 0.12-2.65 cm(3) for double 4400J) and at postmortem (0.23-0.52 cm(3), 0.25-0.82 cm(3), 0.45-0.68 cm(3) and 0.29-1.80 cm(3), respectively) were comparable. The ablated volumes on 3000J and 4400J double sonication were significantly larger than single (P < 0.01), thus, the volume and depth of ablated tissue depends on the applied energy and number of sonication. Macroscopic and microscopic examinations confirmed the locations and presence of coagulation necrosis, vascular damage and interstitial hemorrhage, respectively. Contrast enhanced MRI provides assessment of MRg-HIFU renal ablation. Histopathology demonstrated coagulation necrosis, vascular damage and confirmed the volume of damage seen on MRI.

Kinetic analysis of mechanochemical chain scission of linear poly(phthalaldehyde).

The kinetics of mechanochemical chain scission of poly(phthalaldehyde) (PPA) are investigated. Ultrasound-induced cavitation is capable of causing chain scission in the PPA backbone that ultimately leads to rapid depolymerization of each resulting polymer fragment when above the polymer's ceiling temperature (Tc ). An interesting feature of the mechanochemical breakdown of PPA is that "half-chain" daughter fragments are not observed, since the depolymerization is rapid following chain scission. These features facilitate the determination of rate constants of activation for multiple molecular weights from a single sonication experiment. Additionally, the degradation kinetics are modified with chain-end trapping agents through variation of the nature and amount of small molecule nucleophile or electrophile.

Evaluation of Dose Distribution of Molecular Delivery After Blood-Brain Barrier Disruption by Focused Ultrasound with Treatment Planning

The permeability of the blood-brain barrier (BBB) can be enhanced by focused ultrasound (FUS) in localized regions with applications of ultrasound contrast agent (UCA). The purpose of this study was to evaluate the dose distribution of Evans blue (EB) in the targeted brain by sonication with treatment strategy. FUS exposure was applied with an ultrasound frequency of 1 MHz, a 5% duty cycle and a repetition frequency of 1 Hz. Single sonication with two doses of UCA and two sonications at the same location or an interval of 3 mm to induce BBB disruption for assessing dose distribution. The permeability of the BBB was measured quantitatively based on EB extravasation. Gadolinium deposition was monitored by contrast enhanced MR imaging for dose distribution of the focal plane. Hematoxylin and eosin staining was performed for histologic observation. No significant difference was found for EB in the focal regions between the single sonication with UCA at a dose of 300 μL/kg and repeated sonication with UCA at a lower dose of 150 μL/kg. There was a sharper dose distribution in the brain with repeated sonication at the same location, compared with the brain receiving two sonications at an interval of 3 mm. Compared with a single sonication with UCA at a dose of 150 μL/kg, the histologic evaluation of the sonicated regions indicated that more erythrocytes were seen in the brain treated with single sonication at a higher dose of 300 μL/kg or repeated sonication at a dose of 150 μL/kg. This study demonstrated that the dose distribution of molecular delivery could be regulated by sonication with treatment planning.

The kinetics of blood brain barrier permeability and targeted doxorubicin delivery into brain induced by focused ultrasound

Focused ultrasound (FUS) combined with a circulating microbubble agent is a promising strategy to non-invasively disrupt the blood–brain barrier (BBB) and could enable targeted delivery of therapeutics that normally do not leave the brain vasculature. This study investigated the kinetics of the BBB permeability using dynamic contrast-enhanced MRI (DCE-MRI) and the resulting payload of the chemotherapy agent, doxorubicin (DOX). We also investigated how the disruption and drug delivery were affected by a double sonication (DS) with two different time intervals (10 or 120min). Two locations were sonicated transcranially in one hemisphere of the brain in 20 rats using a 690kHz FUS transducer; the other hemisphere served as a control. For BBB disruption, 10ms bursts were applied at 1Hz for 60s and combined with IV injection of a microbubble ultrasound contrast agent (Definity; 10μl/kg). DOX was injected immediately after the second location was sonicated. The transfer coefficient (Ktrans) for an MRI contrast agent (Gd-DTPA) was estimated serially at 4–5 time points ranging from 30min to 7.5hrs after sonication using DCE-MRI. After a single sonication (SS), the mean Ktrans was 0.0142±0.006min−1 at 30min and was two or more orders of magnitude higher than the non-sonicated targets. It decreased exponentially as a function of time with an estimated half-life of 2.22hrs (95% confidence intervals (CI): 1.06–3.39hrs). Adding a second sonication increased Ktrans, and with a 120min interval between sonications, prolonged the duration of the BBB disruption. Mean Ktrans estimates of 0.0205 (CI: 0.016–0.025) and 0.0216 (CI: 0.013–0.030) min−1 were achieved after DS with 10 and 120min delays, respectively. The half-life of the Ktrans decay that occurred as the barrier was restored was 1.8hrs (CI: 1.20–2.41hrs) for a 10min interval between sonications and increased to 3.34hrs (CI: 0.84–5.84hrs) for a 120min interval. DOX concentrations were significantly greater than in the non-sonicated brain for all experimental groups (p<0.0001), and 1.5-fold higher for DS with a 10min interval between sonications. A linear correlation was found between the DOX concentration achieved and the Ktrans measured at 30min after sonication (R: 0.7). These data suggest that one may be able to use Gd-DTPA as a surrogate tracer to estimate DOX delivery to the brain after FUS-induced BBB disruption. The results of this study provide information needed to take into account the dynamics BBB disruption over time after FUS.

Separation of light and heavy organic matter fractions in soil — Testing for proper density cut-off and dispersion level

Density fractionation is frequently applied to separate soil organic matter according to the degree and the mode of interaction with minerals. Density fractions are operationally defined by density cut-off and sonication intensity, which determine the nature of the separated material. However, no tests or general agreements exist on the most appropriate density cut-off as well as on method and intensity of dispersion. Numerous variants have been proposed and applied, with results often contrasting each other and being hard to interpret. Here, we aimed at separating two light fractions (free and occluded into aggregates) composed of almost pure organic material, and one heavy fraction comprising the organic–mineral associations. We tested effects of different density cut-offs and sonication intensities, in combination and separately, on fraction yields, as well as on the fractions' organic carbon, total nitrogen and lignin-derived phenols. We tried to find optimum density cut-offs and sonication intensities, providing light fractions with maximum organic material and minimum contamination by mineral material. Under the test conditions, a density of 1.6gcm−3 gave best results for all test soils, allowing for separation of maximums amounts of almost pure organic material. The density cut-off at 1.6gcm−3 is well in line with previous studies and theoretical considerations, therefore we recommend the use of this density as most suitable for separation of organic debris. Sonication levels for aggregate disruption to achieve complete separation of occluded light organic matter varied amongst soils. The necessary intensity of dispersion relates to the type of soil, depending on the stability of contained aggregates. The application of one single dispersion energy level to different soils may result either in mineral contamination or in incomplete separation of light and heavy fractions as well as in redistribution of organic material amongst fractions. This means there is no single sonication level that can be applied to all soils. Thus, obtaining a meaningful light fraction residing within aggregates (occluded light fraction) requires assessment of the dispersion energy necessary to disrupt the aggregate system of a given soil without dispersion of organic–mineral associations. This can be done in pre-experiments where the soil is fractionated at different sonication levels. The appropriate dispersion is determined by mass yields and OC content of the obtained occluded fractions.

In vivo MR acoustic radiation force imaging in the porcine liver

High intensity focused ultrasound (HIFU) in the abdomen can be sensitive to acoustic aberrations that can exist in the beam path of a single sonication. Having an accurate method to quickly visualize the transducer focus without damaging tissue could assist with executing the treatment plan accurately and predicting these changes and obstacles. By identifying these obstacles, MR acoustic radiation force imaging (MR-ARFI) provides a reliable method for visualizing the transducer focus quickly without damaging tissue and allows accurate execution of the treatment plan. MR-ARFI was used to view the HIFU focus, using a gated spin echo flyback readout-segmented echo-planar imaging sequence. HIFU spots in a phantom and in the livers of five live pigs under general anesthesia were created with a 550 kHz extracorporeal phased array transducer initially localized with a phase-dithered MR-tracking sequence to locate microcoils embedded in the transducer. MR-ARFI spots were visualized, observing the change of focal displacement and ease of steering. Finally, MR-ARFI was implemented as the principle liver HIFU calibration system, and MR-ARFI measurements of the focal location relative to the thermal ablation location in breath-hold and breathing experiments were performed. Measuring focal displacement with MR-ARFI was achieved in the phantom and in vivo liver. In one in vivo experiment, where MR-ARFI images were acquired repeatedly at the same location with different powers, the displacement had a linear relationship with power [y = 0.04x + 0.83 μm (R(2) = 0.96)]. In another experiment, the displacement images depicted the electronic steering of the focus inside the liver. With the new calibration system, the target focal location before thermal ablation was successfully verified. The entire calibration protocol delivered 20.2 J of energy to the animal (compared to greater than 800 J for a test thermal ablation). ARFI displacement maps were compared with thermal ablations during seven breath-hold ablations. The error was 0.83 ± 0.38 mm in the S/I direction and 0.99 ± 0.45 mm in the L/R direction. For six spots in breathing ablations, the mean error in the nonrespiration direction was 1.02 ± 0.89 mm. MR-ARFI has the potential to improve free-breathing plan execution accuracy compared to current calibration and acoustic beam adjustment practices. Gating the acquisition allows for visualization of the focal spot over the course of respiratory motion, while also being insensitive to motion effects that can complicate a thermal test spot. That MR-ARFI measures a mechanical property at the focus also makes it insensitive to high perfusion, of particular importance to highly perfused organs such as the liver.

Reversible blood–brain barrier disruption by repeated transcranial focused ultrasound allows enhanced extravasation

The permeability of blood–brain barrier (BBB) for albumin can be enhanced by focused ultrasound (FUS) in a targeted region when this is applied in the presence of ultrasound contrast agent (UCA). In this study, we demonstrate that, using this noninvasive treatment, Evans Blue (EB) extravasation can be enhanced by repeated sonication. Sonications were applied at an ultrasound frequency of 1 MHz with a 5% duty cycle, and a repetition frequency of 1 Hz. The brains of male Sprague–Dawley rats were subjected to FUS exposure at the same targeted site. At the same acoustic power, the extravasation caused by leakage of EB into the brain was found to be dependent on the applied sonication time. In vivo, both single and repeated sonications increased the extravasation of the albumin binding EB, especially for the repeated sonication group. In the retreatment experiment, there was a nearly twofold increase in EB extravasation in groups with a second sonication compared with the single sonication group. BBB disruption can be prolonged by repeated FUS sonication and the duration is dependent on the time point of the resonication after the first sonication. Compared to a single sonication, the MR imaging analysis and histological examination of the affected brains indicated that the pattern of contrast enhancement was changed and that vacuolation occurred after repeated sonication. This noninvasive technology offers the possibility of controlling the extent of drug delivery by means of repeated treatment and adjusting the duration and interval between sonications.

In Vivo Low-Intensity Pulsed Ultrasound (LIPUS) Following Tendon Injury Promotes Repair During Granulation but Suppresses Decorin and Biglycan Expression During Remodeling

Bench research, cross-sectional. To determine if the effects of low-intensity pulsed ultrasound (LIPUS) on matrix synthesis change at different stages of tendon healing. LIPUS is effective in promoting tendon healing by stimulation of matrix synthesis. The timing of initiation and duration of LIPUS treatment have been shown to affect its effectiveness to promote tendon healing, suggesting a change of tissue responses to LIPUS stimulation. Understanding how the cellular responses to LIPUS stimulation change during tendon healing is thus important. In a rat model of patellar tendon donor site injury, a single sonication of LIPUS or mock sonication was delivered to the injured knee of the rats on the fourth, 14th or 28th day postinjury. Tendon samples were harvested at 4 hours and 24 hours after sonication and the mRNA expression of COL1A1, COL3A1, decorin, biglycan, and TGF-beta 1 was analyzed. The results showed that a single sonication of LIPUS increased COL1A1 and COL3A1 mRNA in healing patellar tendons when administered on the fourth or 14th day postinjury, but not when administered on the 28th day postinjury. Both decorin and biglycan mRNA were decreased by treatment with LIPUS on the 28th day postinjury. Our results showed that LIPUS enhanced collagen synthesis in vivo only during the granulation phase. Matrix remodeling may be affected by LIPUS with the suppressed expression of decorin and biglycan. Our findings suggest that LIPUS should be applied during the granulation phase but not during the remodeling phase, to promote tendon healing.

Expression of Heat Shock Proteins after Ultrasound Exposure in HL-60 Cells

One of the important cellular defense mechanisms against stress is the induction of heat shock proteins (HSPs). We have recently demonstrated that a low frequency electromagnetic field is unable to induce the heat shock response (HSR). In the present study, we expanded our investigations to the induction of HSPs, particularly Hsp72, by ultrasound (US). Human promyelocytic leukemia HL-60 cells were exposed in suspension to US at 1, 3 and 10 MHz, as well as combinations of two of these frequencies. The ability of US to induce Hsp72 was tested for different frequencies, intensities and exposure times. In addition, the water bath temperature was varied from 30 to 36°C. The Hsp72 protein expression was determined 4 and 24 h after treatment. We found that the amount of Hsp72 increased with increasing US frequency, reaching its highest level of about 1800%, induced by 10 MHz. After increasing the temperature of the water bath, the amount of Hsp72 in the treated cells was also increased, whereas no induction was observed at 30°C. For all treatment conditions, ultrasound of 1 MHz was unable to significantly induce Hsp72. At 10 MHz, the exposure time was varied from 0 to 20 min. We found that the induction of Hsp72 took place after 5 min of exposure. For a fixed level of absorbed US energy, the continuous regime, as well as a pulsation of 1:2 (5 ms on and 5 ms off) induced the same Hsp72 level. Pulsation of 1:5 (2 ms on and 8 ms off) and 1:10 (1 ms on and 9 ms off) did not show any effect. A single sonication of 20 min, as well as a fractionated sonication of two 10 min exposures induced the same level of Hsp72, whereas four exposures of 5 min reduced the Hsp72 level. At the optimum exposure conditions (10 MHz, 10 min), the concentration of other HSPs was also determined. Hsp27 showed no effect but Hsp32, Hsp40 and Hsp72 were induced. Taken together, these results suggest a synergistic interaction between heat and US. (E-mail: werner.sontag@ibg.fzk.de)

Magnetic resonance guided focused ultrasound surgery. Ablation of soft tissue at bone–muscle interface in a porcine model

Pain management treatments of patients with bone metastases have either efficacy problems or significant side effects. Percutaneous radiofrequency ablation has recently proved to be of palliative value. Magnetic resonance guided focused ultrasound surgery (MRgFUS) uses focused ultrasonic energy to non-invasively create a heat-coagulated lesion deep within the body in a controlled, accurate manner. The surgeon can monitor and control energy deposition in real time. This technology represents a potential treatment modality in oncological surgery. We investigated the ability of two MRgFUS methods to accurately and safely target and ablate soft tissue at its interface with bone. Heat-ablated lesions were created by MRgFUS at the bone-muscle interface of 15 pigs. Two different methods of energy delivery were used. Temperature rise at the target adjacent to bone was monitored by real time MR thermal images. Results were evaluated by MRI (magnetic resonance imaging), nuclear scanning and by histopathological evaluation. Soft tissue lesion sizes by both methods were in the range of 1-2 cm in diameter. Targeting the focus 'behind' the bone, achieved the same result with a single sonication only. Follow up MRI and histopathological examination of all lesions showed focal damage at its interface with bone and localized damage to the outer cortex on the side closer to the targeted tissue. There was no damage to non-targeted tissue. MRgFUS by both energy deposition methods can be used to produce controlled well-localized damage to soft tissue in close proximity to bone, with minimal collateral damage.