HIFU-induced cavitation and heating in ex vivo porcine subcutaneous fat

The present study is motivated by the fact that there are no published studies quantifying cavitation activity and heating induced by ultrasound in adipose tissue and that there are currently no reliable techniques for monitoring successful deposition of ultrasound energy in fat in real time. High-intensity focused ultrasound (HIFU) exposures were performed in excised porcine fat at four different frequencies (0.5, 1.1, 1.6 and 3.4 MHz) over a range of pressure amplitudes and exposure durations. The transmission losses arising from reflection at the skin interface and attenuation through skin and fat were quantified at all frequencies using an embedded needle hydrophone. A 15 MHz passive cavitation detector (PCD) coaxial to the HIFU transducer was used to capture acoustic emissions emanating from the focus during HIFU exposures, while the focal temperature rise was measured using minimally invasive needle thermocouples. Repeatable temperature rises in excess of 10°C could be readily instigated across all four frequencies for acoustic intensities (Ispta) in excess of 50 W/cm² within the first 2 s of exposure. Even though cavitation could not be initiated at 1.1, 1.6 and 3.4 MHz over the in situ peak rarefactional (p_-) pressure range 0-3 MPa explored in the present study, inertial cavitation activity was always initiated at 0.5 MHz for pressures greater than 1.6 MPa (p_-) and was found to enhance focal heat deposition. A good correlation was identified between the energy of broadband emissions detected by the PCD and the focal temperature rise at 0.5 MHz, particularly for short 2 s exposures, which could be exploited as a tool for noninvasive monitoring of successful treatment delivery. (E-mail: zoe.kyriakou@eng.ox.ac.uk)     

Temporal and Spatial Detection of HIFU-Induced Inertial and Hot-Vapor Cavitation with a Diagnostic Ultrasound System

The onset and presence of inertial cavitation and near-boiling temperatures in high-intensity focused ultrasound (HIFU) therapy have been identified as important indicators of energy deposition for therapy guidance. Passive cavitation detection is commonly used to detect bubble emissions, where a fixed-focus single-element acoustic transducer is typically used as a passive cavitation detector (PCD). This technique is suboptimal for clinical applications, because most PCD transducers are tightly focused and afford limited spatial coverage of the HIFU focal region. A Terason 2000 Ultrasound System was used as a PCD array to expand the spatial detection region for cavitation by operating in passive mode, obtaining the radiofrequency signals corresponding to each scan line and filtering the contribution from scattering of the HIFU signal harmonics. This approach allows for spatially resolved detection of both inertial and stable cavitation throughout the focal region. Measurements with the PCD array during sonication with a 1.1-MHz HIFU source in tissue phantoms were compared with single-element PCD and thermocouple sensing. Stable cavitation signals at the harmonics and superharmonics increased in a threshold fashion for temperatures >90°C, an effect attributed to high vapor pressure in the cavities. Incorporation of these detection techniques in a diagnostic ultrasound platform could result in a powerful tool for improving HIFU guidance and treatment. (E-mail: cfarny@bwh.harvard.edu)     

Cavitation-enhanced extravasation for drug delivery

A flow-through tissue-mimicking phantom composed of a biocompatible hydro-gel with embedded tumour cells was used to assess and optimize the role of ultrasound-induced cavitation on the extravasation of a macromolecular compound from a channel mimicking vessel in the gel, namely a non-replicating luciferase-expressing adenovirus (Ad-Luc). Using a 500 KHz therapeutic ultrasound transducer confocally aligned with a focussed passive cavitation detector, different exposure conditions and burst mode timings were selected by performing time and frequency domain analysis of passively recorded acoustic emissions, in the absence and in the presence of ultrasound contrast agents acting as cavitation nuclei. In the presence of Sonovue, maximum ultraharmonic emissions were detected for peak rarefactional pressures of 360 kPa, and maximum broadband emissions occurred at 1250 kPa. The energy of the recorded acoustic emissions was used to optimise the pulse repetition frequency and duty cycle in order to maximize either ultraharmonic or broadband emissions while keeping the acoustic energy delivered to the focus constant. Cell viability measurements indicated that none of the insonation conditions investigated induces cell death in the absence of a therapeutic agent (i.e. virus). Phase contrast images of the tissue-mimicking phantom showed that short range vessel disruption can occur when ultra-harmonic emissions (nf0/2) are maximised whereas formation of a micro-channel perpendicular to the flow can be obtained in the presence of broadband acoustic emissions. Following Ad-Luc delivery, luciferase expression measurements showed that a 60-fold increase in its bioavailability can be achieved when broadband noise emissions are present during insonation, even for modest contrast agent concentrations. The findings of the present study suggest that drug delivery systems based on acoustic cavitation may help enhance the extravasation of anticancer agents, thus increasing their penetration distance to hypoxic regions and poorly vascularised tumour regions.     

Effects of ultrasonic exposure parameters on myocardial lesions induced by high-intensity focused ultrasound.

OBJECTIVE: This study evaluated variables relevant to creating myocardial lesions using high-intensity focused ultrasound (HIFU). Without an effective means of tracking heart motion, lesion formation in the moving ventricle can be accomplished by intermittent delivery of HIFU energy synchronized by electrocardiographic triggering. In anticipation of future clinical applications, multiple lesions were created by brief HIFU pulses in calf myocardial tissue ex vivo. METHODS: Experiments used f-number 1.1 spherical cap HIFU transducers operating near 5 MHz with in situ spatial average intensities of 13 and 7.4 kW/cm2 at corresponding depths of 10 and 25 mm in the tissue. The distance from the HIFU transducer to the tissue surface was measured with a 7.5-MHz A-mode transducer coaxial and confocal with the HIFU transducer. After exposures, fresh, unstained tissue was dissected to measure visible lesion length and width. Lesion dimensions were plotted as functions of pulse parameters, cardiac structure, tissue temperature, and focal depth. RESULTS: Lesion size in ex vivo tissue depended strongly on the total exposure time but did not depend strongly on pulse duration. Lesion width depended strongly on the pulse-to-pulse interval, and lesion width and length depended strongly on the initial tissue temperature. CONCLUSIONS: High-intensity focused ultrasound creates well-demarcated lesions in ex vivo cardiac muscle without damaging intervening or distal tissue. These initial studies suggest that HIFU offers an effective, noninvasive method for ablating myocardial tissues to treat several important cardiac diseases.     

Measurements of bubble-enhanced heating from focused, MHz-frequency ultrasound in a tissue-mimicking material.

Time-resolved measurements of the temperature field in an agar-based tissue-mimicking phantom insonated with a large aperture 1-MHz focused acoustic transducer are reported. The acoustic pressure amplitude and insonation duration were varied. Above a critical threshold acoustic pressure, a large increase in the temperature rise during insonation was observed. Evidence for the hypothesis that cavitation bubble activity in the focal zone is the cause of enhanced heating is presented and discussed. Mechanisms for bubble-assisted heating are presented and modeled, and quantitative estimates for the thermal power generated by viscous dissipation and bubble acoustic radiation are given.     

Sonolysis of Albunex-supplemented, 40% hematocrit human erythrocytes by pulsed 1-MHz ultrasound: pulse number, pulse duration and exposure vessel rotation dependence.

The hypotheses tested were that sonolysis of erythrocytes in the presence of a gas-based ultrasound contrast agent in vitro will be related quantitatively to the duration and number of ultrasound pulses applied using a constant pulse repetition period and, at least qualitatively, to the total exposure duration (i.e., the product of pulse number x pulse duration). An objective was to determine the influence of sample rotation during insonation on the amount of hemolysis produced under these conditions. Human erythrocytes, suspended to 40% hematocrit in autologous plasma containing 3.6% (V:V) Albunex, were exposed/sham-exposed to 1-100 pulses of 1-MHz ultrasound (6.2 MPa peak positive, 3.6 MPa peak negative acoustic pressures; I(SPTP) approximately 800 W/cm2) using a 1-s pulse repetition period. Pulse durations ranged from 20-20,000 micros; samples were either stationary or rotated (200 rpm) during insonation. Hemolysis was independent of vessel rotation treatment at all tested pulse durations and pulse numbers. Levels of hemolysis statistically greater than in sham-exposed samples were obtained with > or = 50 pulses of 20 micros duration, and > or = 1 pulse of 200, 2000 or 20,000 micros duration. Hemolysis increased with increasing pulse number and pulse duration. Approximately equivalent levels of hemolysis were produced by different pulse number x pulse duration combinations, yielding the same total exposure duration.     

Improved visualization of high-intensity focused ultrasound lesions

Spectral parameter imaging in both the fundamental and harmonic of backscattered radio-frequency (RF) data were used for immediate visualization of high-intensity focused ultrasound (HIFU) lesion sites. A focused 5-MHz HIFU transducer with a coaxial 9-MHz focused single-element diagnostic transducer was used to create and scan lesions in chicken breast and freshly excised rabbit liver. B-mode images derived from the backscattered RF signal envelope were compared with midband fit (MBF) spectral parameter images in the fundamental (9-MHz) and harmonic (18-MHz) bands of the diagnostic probe. Images of HIFU-induced lesions derived from the MBF to the calibrated spectrum showed improved contrast (approximately 3 dB) of tumor margins versus surround compared with images produced from the conventional signal envelope. MBF parameter images produced from the harmonic band showed higher contrast in attenuated structures (core, shadow) compared with either the conventional envelope (3.3 dB core; 11.6 dB shadow) or MBF images of the fundamental band (4.4 dB core; 7.4 dB shadow). The gradient between the lesion and surround was 3.4 dB/mm, 6.9 dB/mm and 17.2 dB/mm for B-mode, MBF-fundamental mode and MBF-harmonic mode, respectively. Images of threshold and "popcorn" lesions produced in freshly excised rabbit liver were most easily visualized and boundaries best-defined using MBF-harmonic mode.     

In vitro ablation of cardiac valves using high-intensity focused ultrasound

The purpose of this study was to evaluate the possibility of using high-intensity focused ultrasound (US), or HIFU, to create lesions in cardiac valves in vitro. Calf mitral valves and aortic valves were examined. Focused US energy was applied with an operating frequency of 4.67 MHz at a nominal acoustic power of 58 W for 0.2, 0.3 and 0.4 s at 4-s intervals. Mitral valve perforation was achieved with 20.8 +/- 3.7 exposures of 0.2 s, 15.4 +/- 2.1 exposures of 0.3 s or 11.2 +/- 2.3 exposures of 0.4 s. Aortic valve perforation was achieved with 13.3 +/- 2.4 exposures of 0.2 s, 10.3 +/- 2.2 exposures of 0.3 s or 8.4 +/- 1.8 exposures of 0.4 s. The mean diameter of the perforated area was 1.09 +/- 0.11 mm. The lesions were slightly discolored and coagulation of tissue around the perforation was observed. HIFU was successful in perforating cardiac valves. With further refinement, HIFU may prove useful for valvulotomy or valvuloplasty.     

An adaptive spectral estimation technique to detect cavitation in HIFU with high spatial resolution

In ultrasound-guided high-intensity focused ultrasound (HIFU) therapy, the changes observed on tissue are subtle during treatment; some ultrasound-guided HIFU protocols rely on the observation of significant brightness changes as the indicator of tissue lesions. The occurrence of a distinct hyperechogenic region (“bright-up”) around the focus is often associated with acoustic cavitation resulting in microbubble formation, but it may indicate different physical events such as larger bubbles from boiling (known to alter acoustic impedance) or sometimes lesion formation. A reliable method to distinguish and spatially localize these causes within the tissue would assist the control of HIFU delivery, which is the subject of this paper. Spectral analysis of the radio frequency (RF) signal underlying the B-mode image provides more information on the physical cause, but the usual techniques that are methods on the Fourier transform require a long series for good spectral resolution and so they give poor spatial resolution. This paper introduces an active spectral cavitation detection method to attain high spatial resolution (0.15 × 0.15 mm per pixel) through a parametric statistical method (ARMA modeling) used on finite-length data sets, which enables local changes to be identified more easily. This technique uses the characteristics of the signal itself to optimize the model parameters and structure. Its performance is assessed using synthesized cavitation RF data, and it is then demonstrated in ex vivo bovine liver during and after HIFU exposure. The results suggest that good spatial and spectral resolution can be obtained by the design of suitable algorithms. In ultrasound-guided HIFU, the technique provides a useful supplement to B-mode analysis, with no additional time penalty in data acquisition.     

Synergistic Ablation of Liver Tissue and Liver Cancer Cells with High-Intensity Focused Ultrasound and Ethanol

We investigated the combined effect of ethanol and high-intensity focused ultrasound (HIFU), first, on heating and cavitation bubble activity in tissue-mimicking phantoms and porcine liver tissues and, second, on the viability of HepG2 liver cancer cells. Phantoms or porcine tissues were injected with ethanol and then subjected to HIFU at acoustic power ranging from 1.2 to 20.5 W (HIFU levels 1–7). Cavitation events and the temperature around the focal zone were measured with a passive cavitation detector and embedded type K thermocouples, respectively. HepG2 cells were subjected to 4% ethanol solution in growth medium (v/v) just before the cells were exposed to HIFU at 2.7, 8.7 or 12.0 W for 30 s. Cell viability was measured 2, 24 and 72 h post-treatment. The results indicate that ethanol and HIFU have a synergistic effect on liver cancer ablation as manifested by greater temperature rise and lesion volume in liver tissues and reduced viability of liver cancer cells. This effect is likely caused by reduction of the cavitation threshold in the presence of ethanol and the increased rate of ethanol diffusion through the cell membrane caused by HIFU-induced streaming, sonoporation and heating.     

Effect of high-intensity focused ultrasound on whole blood with and without microbubble contrast agent.

Using human whole blood samples with and without contrast agent (CA), we evaluated the effect of exposures to focused, continuous wave (CW) 1.1-MHz ultrasound for durations of 10 ms to 1 s at spatial average intensities of 560 to 2360 W/cm2. Cavitation was monitored with a passive cavitation detector and hemolysis was determined with spectroscopy. In whole blood alone, no significant cavitation, heating or hemolysis was detected at any exposure condition. Conversely, cavitation and hemolysis, but not heating, were detected in whole blood with CA. A CA concentration as low as 0.28 microL CA per mL whole blood at an intensity of 2360 W/cm2 for 1 s resulted in measurable cavitation and a 6-fold increase in hemolysis compared to shams. Cavitation and hemolysis increased proportional to the concentration of CA and duration of exposure. In samples containing 4.2 microL CA per mL whole blood exposed for 1 s, a threshold was seen at 1750 W/cm2 where cavitation and hemolysis increased 10-fold compared to exposures at lower intensities. HIFU exposure of whole blood containing CA leads to significant hemolysis in vitro and may lead to clinically significant hemolysis in vivo.     

一种用于HIFU聚焦性能评价的仿组织透明体模

目的建立一种用于高强度聚焦超声(HIFU)聚焦性能评价的仿组织透明体模。方法仿组织透明体模主要由聚丙烯酰胺和作为温度敏感指示剂的蛋清混合而成。在B超的监控下使用声功率160W的HIFU在不同的辐照时间下定点辐照体模和新鲜离体牛肝脏,肉眼观察HIFU在体模和新鲜离体牛肝脏中形成的生物学焦域(BFR)形态并测量BFR的长短轴。结果可用肉眼观察HIFU在仿组织透明体模中产生的BFR,其形态呈椭球体,实时超声监控为强回声,BFR的长、短轴随辐照时间的增加而增大。但在相同的辐照参数下,HIFU在仿组织透明体模中产生的BFR的长、短轴小于HIFU在新鲜离体牛肝脏中形成的BFR的长、短轴。结论该仿组织透明体模在用于HIFU聚焦性能的评价方面展示出良好的前景。     

Noninvasive in vivo measurements of hematocrit.

OBJECTIVE: To develop a clinically applicable method for noninvasive acoustic determination of hematocrit values in vivo. METHODS: The value of hematocrit was determined initially in vitro from the pulseecho measurements of acoustic attenuation. The testing was carried out in a laboratory setup with an ultrasonic transducer operating at 20 MHz and with the use of human blood samples at 37 degrees C. The attenuation coefficient measurements in blood in vivo were implemented by multigated, 20-MHz pulsed Doppler insonation. The Doppler signal was recorded in the brachial and radial arteries. Both in vitro and in vivo hematocrit data were compared with those obtained by the centrifuge method. RESULTS: The attenuation coefficient in vitro was determined from the measurements of 168 samples with hematocrit values varying between 23.9% and 51.6%. The attenuation from 20-MHz data was equal to 3.66 + 0.089 hematocrit (decibels per centimeter). The uncertainty of in vivo measurements in the brachial artery was determined to be within +/- 5% hematocrit. However, the measurements in the radial artery resulted in a clinically unacceptable uncertainty of +/- 20% hematocrit. CONCLUSIONS: The method proposed appears to be promising for in vivo determination of hematocrit, because 5% hematocrit error is adequate for monitoring changes in patients in shock or during dialysis. It was found that the multigate system largely simplified placement of an ultrasonic probing beam in the center of the blood vessel. Current work focuses on enhancing the method's applicability to arbitrarily selected vessels and to reducing the hematocrit measurement error to much less than 5% hematocrit.     

An Acoustic Backscatter-Based Method for Localization of Lesions Induced by High-Intensity Focused Ultrasound

Ultrasound B-mode visualization of lesions produced in soft tissues using high-intensity focused ultrasound (HIFU) has been shown to be challenging when there is no cavitation activity and, therefore, no hyperechogenecity in the focal region. We investigated a method for the visualization and localization of HIFU-induced lesions after HIFU delivery was complete based on the change in backscattered radio-frequency (RF) signals. A HIFU transducer was used with focal dimension of 8 mm by 2 mm working at 5 MHz. HIFU was applied at different intensities to produce lesions in ex vivo chicken breast, with or without the generation of hyperecho in B-mode images. We compared lesion locations obtained from our RF-processing method, from measurement of physical lesions after exposure and from the B-mode images, if exposures had resulted in hyperecho. The results showed that the RF amplitude decreased as a function of time immediately after stopping the HIFU exposure. The lesions were clearly visualized in two-dimensional (2-D) images of the decay rate of RF amplitude, no matter with or without hyperecho. In experiments with hyperecho, when comparing to physical lesion locations, there was no statistically significant difference in the localization accuracy between the RF-based and the hyperecho-based method (p = 0.76). In cases without hyperecho, the distance between RF-based locations and measured lesion locations was 3.37 ± 1.59 mm (mean ± standard deviation). The axial and lateral difference were 2.00 ± 2.31 mm and 0.85 ± 2.15 mm, respectively, and no statistically significant difference was found between lesion coordinates (axial: p = 0.37 and lateral: p = 0.15). We demonstrated the feasibility of our proposed RF-based method for the localization of HIFU-induced lesions immediately after HIFU treatment. Using the decay rate in RF amplitude as the signature of lesion formation, our method can detect lesion locations even without the appearance of hyperecho. (E-mail: xlzheng@u.washington.edu)     

Real-time monitoring of high-intensity focused ultrasound lesion formation using acousto-optic sensing

High-intensity focused ultrasound (HIFU) is a promising modality that is used to noninvasively ablate soft tissue tumors. Nevertheless, real-time treatment monitoring with diagnostic ultrasound still poses a significant challenge since tissue necrosis, in the absence of cavitation or boiling, provides little acoustic contrast with normal tissue. In comparison, the optical properties of tissue are significantly altered accompanying lesion formation. A photorefractive crystal-based acousto-optic (AO) sensing system that uses a single HIFU transducer to simultaneously generate tissue necrosis and pump the AO interaction is used to monitor the real-time optical changes associated with thermal lesions induced in chicken breast ex vivo. It is found that the normalized change in AO response increases proportionally with the volume of necrosis. This study demonstrates AO sensing can identify the onset and growth of lesion formation in real time and, when used as feedback to guide exposures, results in more predictable lesion formation. (E-mails: laipuxiang@gmail.com or ronroy@bu.edu)     

Confocal dual‐frequency enhances the damaging effect of high‐intensity focused ultrasound in tissue‐mimicking phantom

The volume of the lesions created by conventional single‐frequency high‐intensity focused ultrasound (HIFU) is small, which leads to long treatment duration in patients who are undergoing tumor ablation. In this study, the lesions induced by confocal dual‐frequency HIFU in an optically transparent tissue‐mimicking phantom were investigated and compared with the lesions created by conventional single‐frequency HIFU. The results show that using different exposure times resulted in lesions of different sizes in both dual‐frequency and single‐frequency HIFU modes at the same spatially averaged intensity level (ISAL = 4900?W?cm^?2), but the lesion dimensions made in dual‐frequency mode were significantly larger than those made in single‐frequency mode. Difference frequency acoustic fields that exist in the confocal region of dual‐frequency HIFU may be the reason for the enlargement of the lesions' dimensions. The dual‐frequency HIFU mode may represent a new technique to improve the ablation efficiency of HIFU. The total time for the ablation of a tumor can be reduced, thus requiring less therapy time and reducing possible patient complications.     

Bubble dynamics in boiling histotripsy

Boiling histotripsy is a non-invasive, cavitation-based ultrasonic technique which uses a number of millisecond pulses to mechanically fractionate tissue. Though a number of studies have demonstrated the efficacy of boiling histotripsy for fractionation of solid tumours, treatment monitoring by cavitation measurement is not well studied because of the limited understanding of the dynamics of bubbles induced by boiling histotripsy. The main objectives of this work are to (a) extract qualitative and quantitative features of bubbles excited by shockwaves and (b) distinguish between the different types of cavitation activity for either a thermally or a mechanically induced lesion in the liver. A numerical bubble model based on the Gilmore equation accounting for heat and mass transfer (gas and water vapour) was developed to investigate the dynamics of a single bubble in tissue exposed to different High Intensity Focused Ultrasound fields as a function of temperature variation in the fluid. Furthermore, ex vivo liver experiments were performed with a passive cavitation detection system to obtain acoustic emissions. The numerical simulations showed that the asymmetry in a shockwave and water vapour transport are the key parameters which lead the bubble to undergo rectified growth at a boiling temperature of 100°C. The onset of rectified radial bubble motion manifested itself as (a) an increase in the radiated pressure and (b) the sudden appearance of higher order multiple harmonics in the corresponding spectrogram. Examining the frequency spectra produced by the thermal ablation and the boiling histotripsy exposures, it was observed that higher order multiple harmonics as well as higher levels of broadband emissions occurred during the boiling histotripsy insonation. These unique features in the emitted acoustic signals were consistent with the experimental measurements. These features can, therefore, be used to monitor (a) the different types of acoustic cavitation activity for either a thermal ablation or a mechanical fractionation process and (b) the onset of the formation of a boiling bubble at the focus in the course of HIFU exposure.     

Ultrastructural Analysis of Volumetric Histotripsy Bio-effects in Large Human Hematomas

Large-volume soft tissue hematomas are a serious clinical problem, which, if untreated, can have severe consequences. Current treatments are associated with significant pain and discomfort. It has been reported that in an in vitro bovine hematoma model, pulsed high-intensity focused ultrasound (HIFU) ablation, termed histotripsy, can be used to rapidly and non-invasively liquefy the hematoma through localized bubble activity, enabling fine-needle aspiration. The goals of this study were to evaluate the efficiency and speed of volumetric histotripsy liquefaction using a large in vitro human hematoma model. Large human hematoma phantoms (85 cc) were formed by recalcifying blood anticoagulated with citrate phosphate dextrose/saline–adenine–glucose–mannitol solution. Typical boiling histotripsy pulses (10 or 2 ms) or hybrid histotripsy pulses using higher-amplitude and shorter pulses (0.4 ms) were delivered at 1% duty cycle while continuously translating the HIFU focus location. Histotripsy exposures were performed under ultrasound guidance with a 1.5-MHz transducer (8-cm aperture, F# = 0.75). The volume of liquefied lesions was determined by ultrasound imaging and gross inspection. Untreated hematoma samples and samples of the liquefied lesions aspirated using a fine needle were analyzed cytologically and ultrastructurally with scanning electron microscopy. All exposures resulted in uniform liquid-filled voids with sharp edges; liquefaction speed was higher for exposures with shorter pulses and higher shock amplitudes at the focus (up to 0.32, 0.68 and 2.62 mL/min for 10-, 2- and 0.4-ms pulses, respectively). Cytological and ultrastructural observations revealed completely homogenized blood cells and fibrin fragments in the lysate. Most of the fibrin fragments were less than 20 μm in length, but a number of fragments were up to 150 μm. The lysate with residual debris of that size would potentially be amenable to fine-needle aspiration without risk for needle clogging in clinical implementation.     

Application of High-Intensity Focused Ultrasound to the Study of Mild Traumatic Brain Injury

Though intrinsically of much higher frequency than open-field blast overpressures, high-intensity focused ultrasound (HIFU) pulse trains can be frequency modulated to produce a radiation pressure having a similar form. In this study, 1.5-MHz HIFU pulse trains of 1-ms duration were applied to intact skulls of mice in vivo and resulted in blood-brain barrier disruption and immune responses (astrocyte reactivity and microglial activation). Analyses of variance indicated that 24 h after HIFU exposure, staining density for glial fibrillary acidic protein was elevated in the parietal and temporal regions of the cerebral cortex, corpus callosum and hippocampus, and staining density for the microglial marker, ionized calcium binding adaptor molecule, was elevated 2 and 24 h after exposure in the corpus callosum and hippocampus (all statistical test results, p < 0.05). HIFU shows promise for the study of some bio-effect aspects of blast-related, non-impact mild traumatic brain injuries in animals.