Increased intracellular diffusivity of macromolecules within a mammalian cell by low-intensity pulsed ultrasound.

Whilst a number of studies have demonstrated that low-intensity pulsed ultrasound (LIPUS) is a promising therapeutic ultrasound technique that can be used for delivering mild mechanical stimuli to target tissue non-invasively, the underlying biophysical mechanisms still remain unclear. Most mechanism studies have focused explicitly on the effects of LIPUS on the cell membrane and mechanosensitive receptors. In the present study, we propose an additional mechanism by which LIPUS propagation through living cells may directly impact intracellular dynamics, particularly the diffusion transport of biomolecules. To support our hypothesis, human epithelial-like cells (SaOS-2 and HeLa) seeded on a confocal dish placed on a microscope stage were exposed to LIPUS with various exposure conditions (ultrasound frequencies of 0.5, 1 and 3 MHz, peak acoustic pressure of 200 and 400 kPa, a pulse repetition frequency of 1 kHz and a 20 % duty cycle), and the diffusivities of various sizes of biomolecules in the cytoplasm area were measured using fluorescence recovery after photobleaching (FRAP). Furthermore, giant unilamellar vesicles (GUVs) filled with macromolecules were used to examine the physical causal relationship between LIPUS and molecular diffusion changes. Nucleocytoplasmic transport coefficients were also measured by modified FRAP that bleaches the whole cell nuclear region. Extracellular signal-regulated kinases (ERK) activity (the phosphorylation dynamics) was monitored using fluorescence resonance energy transfer (FRET) microscopy. All the measurements were taken during, before and after the LIPUS exposure. Our experimental results clearly showed that the diffusion coefficients of macromolecules within the cell increased with acoustic pressure by 12.1 to 33.5 % during the sonication, and the increments were proportional to their molecular sizes regardless of the ultrasound frequency used. This observation in living cells was consistent with the GUVs exposed to the LIPUS, which indicated that the diffusivity increase was a passive physical response to the acoustic energy of LIPUS. Under the 1 MHz LIPUS exposure with 400 kPa, the passive nucleocytoplasmic transport of enhanced green fluorescent protein (EGFP) was accelerated by 21.4 %. With the same LIPUS exposure condition, both the diffusivity and phosphorylation of ERK induced by EGF treatment were significantly elevated simultaneously, which implied that LIPUS could also modify the kinase kinetics in the signal transduction process. Taken together, this study is the first attempt to uncover the physical link between LIPUS and the dynamics of intracellular macromolecules and related biological processes that LIPUS can possibly increase the diffusivity of intracellular macromolecules, leading to the changes in the basic cellular processes: passive nucleocytoplasmic transport and ERK. Our findings can provide a novel perspective that the mechanotransduction process that the intracellular region, in addition to the cell membrane, can convert the acoustic stimuli of LIPUS to biochemical signals.

Selective Cell-Cell Adhesion Regulation via Cyclic Mechanical Deformation Induced by Ultrafast Nanovibrations.

The adoption of dynamic mechanomodulation to regulate cellular behavior is an alternative to the use of chemical drugs, allowing spatiotemporal control. However, cell-selective targeting of mechanical stimuli is challenging due to the lack of strategies with which to convert macroscopic mechanical movements to different cellular responses. Here, we designed a nanoscale vibrating surface that controls cell behavior via selective repetitive cell deformation based on a poroelastic cell model. The vibrating indentations induce repetitive water redistribution in the cells with water redistribution rates faster than the vibrating rate; however, in the opposite case, cells perceive the vibrations as a one-time stimulus. The selective regulation of cell-cell adhesion through adjusting the frequency of nanovibration was demonstrated by suppression of cadherin expression in smooth muscle cells (fast water redistribution rate) with no change in vascular endothelial cells (slow water redistribution rate). This technique may provide a new strategy for cell-type-specific mechanical stimulation.

Noninvasive mechanical destruction of liver tissue and tissue decellularisation by pressure-modulated shockwave histotripsy.

Introduction: Boiling histotripsy (BH) is a promising High Intensity Focused Ultrasound (HIFU) technique that can be used to mechanically fractionate solid tumours at the HIFU focus noninvasively, promoting tumour immunity. Because of the occurrence of shock scattering phenomenon during BH process, the treatment accuracy of BH is, however, somewhat limited. To induce more localised and selective tissue destruction, the concept of pressure modulation has recently been proposed in our previous in vitro tissue phantom study. The aim of the present study was therefore to investigate whether this newly developed histotripsy approach termed pressure-modulated shockwave histotripsy (PSH) can be used to induce localised mechanical tissue fractionation in in vivo animal model. Methods: In the present study, 8 Sprague Dawley rats underwent the PSH treatment and were sacrificed immediately after the exposure for morphological and histological analyses (paraffin embedded liver tissue sections were stained with H&E and MT). Partially exteriorised rat's left lateral liver lobe in vivo was exposed to a 2.0 MHz HIFU transducer with peak positive (P +) and negative (P -) pressures of 89.1 MPa and -14.6 MPa, a pulse length of 5 to 34 ms, a pressure modulation time at 4 ms where P + and P - decreased to 29.9 MPa and - 9.6 MPa, a pulse repetition frequency of 1 Hz, a duty cycle of 1% and number of pulses of 1 to 20. Three lesions were produced on each animal. For comparison, the same exposure condition but no pressure modulation was also employed to create a number of lesions in the liver. Results and Discussion: Experimental results showed that a partial mechanical destruction of liver tissue in the form of an oval in the absence of thermal damage was clearly observed at the HIFU focus after the PSH exposure. With a single pulse length of 7 ms, a PSH lesion created in the liver was measured to be a length of 1.04 ± 0.04 mm and a width of 0.87 ± 0.21 mm which was 2.37 times in length (p = 0.027) and 1.35 times in width (p = 0.1295) smaller than a lesion produced by no pressure modulation approach (e.g., BH). It was also observed that the length of a PSH lesion gradually grew towards the opposite direction to the HIFU source along the axial direction with the PSH pulse length, eventually leading to the generation of an elongated lesion in the liver. In addition, our experimental results demonstrated the feasibility of inducing partial decellularisation effect where liver tissue was partially destructed with intact extracellular matrix (i.e., intact fibrillar collagen) with the shortest PSH pulse length. Taken together, these results suggest that PSH could be used to induce a highly localised tissue fractionation with a desired degree of mechanical damage from complete tissue fractionation to tissue decellularisation through controlling the dynamics of boiling bubbles without inducing the shock scattering effect.

Sonothrombolysis with an acoustic net-assisted boiling histotripsy: A proof-of-concept study.

Whilst sonothrombolysis is a promising and noninvasive ultrasound technique for treating blood clots, bleeding caused by thrombolytic agents used for dissolving clots and potential obstruction of blood flow by detached clots (i.e., embolus) are the major limitations of the current approach. In the present study, a new sonothrombolysis method is proposed for treating embolus without the use of thrombolytic drugs. Our proposed method involves (a) generating a spatially localised acoustic radiation force in a blood vessel against the blood flow to trap moving blood clots (i.e., generation of an acoustic net), (b) producing acoustic cavitation to mechanically destroy the trapped embolus, and (c) acoustically monitoring the trapping and mechanical fractionation processes. Three different ultrasound transducers with different purposes were employed in the proposed method: (1) 1-MHz dual focused ultrasound (dFUS) transducers for capturing moving blood clots, (2) a 2-MHz High Intensity Focused Ultrasound (HIFU) source for fractionating blood clots and (3) a passive acoustic emission detector with broad bandwidth (10 kHz to 20 MHz) for receiving and analysing acoustic waves scattered from a trapped embolus and acoustic cavitation. To demonstrate the feasibility of the proposed method, in vitro experiments with an optically transparent blood vessel-mimicking phantom filled with a blood mimicking fluid and a blood clot (1.2 to 5 mm in diameter) were performed with varying the dFUS and HIFU exposure conditions under various flow conditions (from 1.77 to 6.19 cm/s). A high-speed camera was used to observe the production of acoustic fields, acoustic cavitation formation and blood clot fragmentation within a blood vessel by the proposed method. Numerical simulations of acoustic and temperature fields generated under a given exposure condition were also conducted to further interpret experimental results on the proposed sonothrombolysis. Our results clearly showed that fringe pattern-like acoustic pressure fields (fringe width of 1 mm) produced in a blood vessel by the dFUS captured an embolus (1.2 to 5 mm in diameter) at the flow velocity up to 6.19 cm/s. This was likely to be due to the greater magnitude of the dFUS-induced acoustic radiation force exerted on an embolus in the opposite direction to the flow in a blood vessel than that of the drag force produced by the flow. The acoustically trapped embolus was then mechanically destructed into small pieces of debris (18 to 60 µm sized residual fragments) by the HIFU-induced strong cavitation without damaging the blood vessel walls. We also observed that acoustic emissions emitted from a blood clot captured by the dFUS and cavitation produced by the HIFU were clearly distinguished in the frequency domain. Taken together, these results can suggest that our proposed sonothrombolysis method could be used as a promising tool for treating thrombosis and embolism through capturing and destroying blood clots effectively.

Metabolic activity of visceral adipose tissue is associated with age-related macular degeneration: a pilot 18F-FDG PET/CT study.

Background: Obesity is known to increase the risk and severity of age-related macular degeneration (AMD). Increased inflamed metabolic activity of visceral adipose tissue (VAT) is considered as a crucial underlying mechanism for the harmful effects of obesity. In this study, we aimed to investigate the inflamed metabolic activity of VAT with 18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) and their association with AMD. Materials and methods: A total of 57 elderly participants (aged ≥ 50 years) who underwent 18F-FDG PET/CT for health screening and subsequent fundoscopic exam for complaint of recently impaired vision were enrolled. The metabolic activity of VAT was measured from the maximum standardized uptake value (SUVmax) of VAT. The early AMD participant was defined as the participant with either eye satisfying AMD and without any sign of advanced AMD (neovascular AMD or geographic atrophy). The late AMD participant was defined as the participant with either eye satisfying advanced AMD. Results: VAT SUVmax was highest in participants with late AMD, intermediate in early AMD, and lowest in non-AMD participants. The levels of systemic inflammation surrogate markers were also highest in late AMD group. Furthermore, VAT SUVmax was positively correlated with systemic inflammation surrogate markers and independently associated with the late AMD. Conclusions: The metabolic activity of VAT evaluated by 18F-FDG PET/CT was associated with the severity of AMD and synchronized with the level of systemic inflammation. Thus, VAT SUVmax could be potentially employed as a surrogate marker of obesity-driven VAT inflammation associated with AMD.

Investigation of the long-term healing response of the liver to boiling histotripsy treatment in vivo.

Boiling histotripsy (BH) is a promising High-Intensity Focused Ultrasound technique that can be employed to mechanically fractionate solid tumours. Whilst studies have shown the feasibility of BH to destroy liver cancer, no study has reported on the healing process of BH-treated liver tissue. We therefore extensively investigated the evolution of the healing response of liver to BH in order to provide an insight into the healing mechanisms. In the present study, 14 Sprague Dawley rats underwent the BH treatment and were sacrificed on days 0, 3, 7, 14, and 28 for morphological, histological, serological and qPCR analyses. The area of the treated region was 1.44 cm2 (1.2 cm × 1.2 cm). A well-defined BH lesion filled with coagulated blood formed on day 0. A week after the treatment, fibroblast activation was induced at the treatment site, leading to the formation of extracellular matrix structure (ECM). The ECM was then disrupted for 7 to 28 days. Regenerated normal hepatocytes and newly formed blood vessels were found within the BH region with the absence of hepatic fibrosis. No significant morphological, histological and genetic changes around the BH lesion occurred. These results suggest that BH could be a safe and promising therapeutic tool for treating solid tumours without inducing any significant adverse effect such as the formation of liver fibrosis.

Mechanisms of nuclei growth in ultrasound bubble nucleation.

This paper interrogates the intersections between bubble dynamics and classical nucleation theory (CNT) towards constructing a model that describes intermediary nucleation events between the extrema of cavitation and boiling. We employ Zeldovich's hydrodynamic approach to obtain a description of bubble nuclei that grow simultaneously via hydrodynamic excitation by the acoustic field and vapour transport. By quantifying the relative dominance of both mechanisms, it is then possible to discern the extent to which viscosity, inertia, surface tension and vapour transport shape the growth of bubble nuclei through non-dimensional numbers that naturally arise within the theory. The first non-dimensional number Φ12/Φ2 is analogous to the Laplace number, representing the balance between surface tension and inertial constraints to viscous effects. The second non-dimensional number δ represents how enthalpy transport into the bubble can reduce nucleation rates by cooling the surrounding liquid. This formulation adds to the current understanding of ultrasound bubble nucleation by accounting for bubble dynamics during nucleation, quantifying the physical distinctions between "boiling" and "cavitation" bubbles through non-dimensional parameters, and outlining the characteristic timescales of nucleation according to the growth mechanism of bubbles throughout the histotripsy temperature range. We observed in our simulations that viscous effects control the process of ultrasound nucleation in water-like media throughout the 0-120 °C temperature range, although this dominance decreases with increasing temperatures. Enthalpy transport was found to reduce nucleation rates for increasing temperatures. This effect becomes significant at temperatures above 30 °C and favours the creation of fewer nuclei that are larger in size. Conversely, negligible enthalpy transport at lower temperatures can enable the nucleation of dense clusters of small nuclei, such as cavitation clouds. We find that nuclei growth as modelled by the Rayleigh-Plesset equation occurs over shorter timescales than as modelled by vapour-dominated growth. This suggests that the first stage of bubble nuclei growth is hydrodynamic, and vapour transport effects can only be observed over longer timescales. Finally, we propose that this framework can be used for comparison between different experiments in bubble nucleation, towards standardisation and dosimetry of protocols.

Control of the dynamics of a boiling vapour bubble using pressure-modulated high intensity focused ultrasound without the shock scattering effect: A first proof-of-concept study.

Boiling histotripsy is a promising High-Intensity Focused Ultrasound (HIFU) technique that can be used to induce mechanical tissue fractionation at the HIFU focus via cavitation. Two different types of cavitation produced during boiling histotripsy exposure can contribute towards mechanical tissue destruction: (1) a boiling vapour bubble at the HIFU focus and (2) cavitation clouds in between the boiling bubble and the HIFU source. Control of the extent and degree of mechanical damage produced by boiling histotripsy is necessary when treating a solid tumour adjacent to normal tissue or major blood vessels. This is, however, difficult to achieve with boiling histotripsy due to the stochastic formation of the shock scattering-induced inertial cavitation clouds. In the present study, a new histotripsy method termed pressure-modulated shockwave histotripsy is proposed as an alternative to or in addition to boiling histotripsy without inducing the shock scattering effect. The proposed concept is (a) to generate a boiling vapour bubble via localised shockwave heating and (b) subsequently control its extent and lifetime through manipulating peak pressure magnitudes and a HIFU pulse length. To demonstrate the feasibility of the proposed method, bubble dynamics induced at the HIFU focus in an optically transparent liver tissue phantom were investigated using a high speed camera and a passive cavitation detection systems under a single 10, 50 or 100 ms-long 2, 3.5 or 5 MHz pressure-modulated HIFU pulse with varying peak positive and negative pressure amplitudes from 5 to 89 MPa and -3.7 to -14.6 MPa at the focus. Furthermore, a numerical simulation of 2D nonlinear wave propagation with the presence of a boiling bubble at the focus of a HIFU field was conducted by numerically solving the generalised Westervelt equation. The high speed camera experimental results showed that, with the proposed pressure-modulated shockwave histotripsy, boiling bubbles generated by shockwave heating merged together, forming a larger bubble (of the order of a few hundred micron) at the HIFU focus. This coalesced boiling bubble then persisted and maintained within the HIFU focal zone until the end of the exposure (10, 50, or 100 ms). Furthermore, and most importantly, no violent cavitation clouds which typically appear in boiling histotripsy occurred during the proposed histotripsy excitation (i.e. no shock scattering effect). This was likely because that the peak negative pressure magnitude of the backscattered acoustic field by the boiling bubble was below the cavitation cloud intrinsic threshold. The size of the coalesced boiling bubble gradually increased with the peak pressure magnitudes. In addition, with the proposed method, an oval shaped lesion with a length of 0.6 mm and a width of 0.1 mm appeared at the HIFU focus in the tissue phantom, whereas a larger lesion in the form of a tadpole (length: 2.7 mm, width: 0.3 mm) was produced by boiling histotripsy. Taken together, these results suggest that the proposed pressure-modulated shockwave histotripsy could potentially be used to induce a more spatially localised tissue destruction with a desired degree of mechanical damage through controlling the size and lifetime of a boiling bubble without the shock scattering effect.

Simultaneous measurements of acoustic emission and sonochemical luminescence for monitoring ultrasonic cavitation.

In the present study, a novel hybrid method was considered to identify and measure inertial cavitation activity using acoustic and optical emissions from violent bubble collapses. A photomultiplier (PMT) tube and a calibrated cylindrical needle hydrophone were used to simultaneously detect sonochemical luminescence (SCL) signals and acoustic emissions, respectively, during sonication. A cylindrical focusing ultrasound transducer operating at 398.4 kHz was employed to produce a dense cavitation bubble cloud at the focus. The results clearly showed that a similar trend between the PMT output (i.e., the SCL results) and the broad band acoustic emissions started to appear at the frequencies considered above the fourth harmonic of the sonication frequency. The experimental observation suggests that the occurrence of inertial cavitation can be monitored using the high pass spectral acoustic power and the cut-off frequency can be effectively chosen with the aid of sonochemical luminescence measurement. The hybrid method is expected to be useful for cavitation dosimetry in various medical and industrial applications.

Modeling the Physics of Bubble Nucleation in Histotripsy.

This work aims to establish a theoretical framework for the modeling of bubble nucleation in histotripsy. A phenomenological version of the classical nucleation theory was parametrized with histotripsy experimental data, fitting a temperature-dependent activity factor that harmonizes theoretical predictions and experimental data for bubble nucleation at both high and low temperatures. Simulations of histotripsy pressure and temperature fields are then used in order to understand spatial and temporal properties of bubble nucleation at varying sonication conditions. This modeling framework offers a thermodynamic understanding on the role of the ultrasound frequency, waveforms, peak focal pressures, and duty cycle on patterns of ultrasound-induced bubble nucleation. It was found that at temperatures lower than 50 °C, nucleation rates are more appreciable at very large negative pressures such as -30 MPa. For focal peak-negative pressures of -15 MPa, characteristic of boiling histotripsy, nucleation rates grow by 20 orders of magnitude in the temperature interval 60 °C-100 °C.

The interaction of shockwaves with a vapour bubble in boiling histotripsy: The shock scattering effect.

Boiling histotripsy is a High Intensity Focused Ultrasound (HIFU) technique which uses a number of short pulses with high acoustic pressures at the HIFU focus to induce mechanical tissue fractionation. In boiling histotripsy, two different types of acoustic cavitation contribute towards mechanical tissue destruction: a boiling vapour bubble and cavitation clouds. An understanding of the mechanisms underpinning these phenomena and their dynamics is therefore paramount to predicting and controlling the overall size of a lesion produced for a given boiling histotripsy exposure condition. A number of studies have shown the effects of shockwave heating in generating a boiling bubble at the HIFU focus and have studied its dynamics under boiling histotripsy insonation. However, not much is known about the subsequent production of cavitation clouds that form between the HIFU transducer and the boiling bubble. The main objective of the present study is to examine what causes this bubble cluster formation after the generation of a boiling vapour bubble. A numerical simulation of 2D nonlinear wave propagation with the presence of a bubble at the focus of a HIFU field was performed using the k-Wave MATLAB toolbox for time domain ultrasound simulations, which numerically solves the generalised Westervelt equation. The numerical results clearly demonstrate the appearance of the constructive interference of a backscattered shockwave by a bubble with incoming incident shockwaves. This interaction (i.e., the reflected and inverted peak positive phase from the bubble with the incoming incident rarefactional phase) can eventually induce a greater peak negative pressure field compared to that without the bubble at the HIFU focus. In addition, the backscattered peak negative pressure magnitude gradually increased from 17.4 MPa to 31.6 MPa when increasing the bubble size from 0.2 mm to 1.5 mm. The latter value is above the intrinsic cavitation threshold of -28 MPa in soft tissue. Our results suggest that the formation of a cavitation cloud in boiling histotripsy is a threshold effect which primarily depends (a) the size and location of a boiling bubble, and (b) the sum of the incident field and that scattered by a bubble.

A local difference in blood-brain barrier permeability in the caudate putamen and thalamus of a rat brain induced by focused ultrasound.

A blood-brain barrier (BBB) opening induced by focused ultrasound (FUS) has been widely studied as an effective way of treating brain diseases. We investigate the effect of ultrasound's incidence angle at caudate putamen (Cp) and thalamus (Th) of the rat brain by inducing the same power of focused ultrasound that corresponds to the acoustic pressure of 0.65 MPa in free field. The BBB permeability (Ktrans) was quantitatively evaluated with dynamic contrast-enhanced magnetic resonance imaging. The group averaged (n = 11) maximum Ktrans at Cp (0.021 ± 0.012 min-1) was 1.39 times smaller than the Ktrans of Th (0.029 ± 0.01 min-1) with p = 0.00343. The group averaged (n = 6) ultrasound's incidence angles measured using the computed tomography image of rat skulls were compared with the maximum Ktrans and showed a negatively linear relation R2 = 0.7972). The maximum acoustic pressure computed from the acoustic simulation showed higher average acoustic pressures at Th (0.37 ± 0.02 MPa) compared to pressures at Cp (0.32 ± 0.01 MPa) with p = 0.138 × 10-11. More red blood cell were observed at the Th region compared to the Cp region in the tissue staining. These results indicate that localized characteristics of the sonication target within the subject should be considered for safer and more efficient BBB disruption induced by FUS.

Method to optimize the placement of a single-element transducer for transcranial focused ultrasound.

BACKGROUND AND OBJECTIVE: Transcranial focused ultrasound (tFUS) is a promising neuromodulation technique because of its non-invasiveness and high spatial resolution (within millimeter scale). However, the presence of the skull can lead to disrupting and shifting the acoustic focus in the brain. In this study, we propose a computationally efficient way to determine the optimal position of a single-element focused ultrasound transducer which can effectively deliver acoustic energy to the brain target. We hypothesized that the placement of a single element transducer with the lowest average reflection coefficient would be the optimal position. METHODS: The reflection coefficient is defined by the ratio of the amplitude of the reflected wave to the incident wave. To calculate the reflection coefficient, we assumed ultrasound waves as straight lines (beam lines). At each beam line, the reflection coefficient was calculated from the incidence angle at the skull interface (outer/inner skull surfaces). The average reflection coefficient (ARC) was calculated at each possible placement of the transducer using a custom-built software. For comparison purposes, acoustic simulations (k-Wave MATLAB toolbox) which numerically solved the linear wave equation were performed with the same transducer positions used in the ARC calculation. In addition, the experimental validation of our proposed method was also performed by measuring acoustic wave propagation through the calvaria skull phantom in water. The accuracy of our method was defined as the distance between the two optimal transducer placements which were determined from the acoustic simulations and from the ARC method. RESULT: Simulated acoustic pressure distribution corresponding to each ARC showed an inverse relationship with peak acoustic pressures produced in the brain. In comparison to the acoustic simulations, the accuracy of our method was 5.07 ± 4.27 mm when targeting the cortical region in the brain. The computing time of ARC calculations were 0.08% of the time required for acoustic pressure simulations. CONCLUSION: We calculated the ARC to find the optimal position of the tFUS transducer used in the present study. The optimal placement of the transducer was found when the ARC was the lowest. Our numerical and experimental results showed that the proposed ARC method can effectively be used to find the optimal position of a single-element tFUS transducer for targeting the cortex region of the brain in a computationally inexpensive way.

Development of a subject-specific guide system for Low-Intensity Focused Ultrasound (LIFU) brain stimulation.

Low-Intensity Focused Ultrasound (LIFU) has recently been considered as a promising neuromodulation technique because it can noninvasively stimulate the brain with a high spatial resolution. As spatial resolution is improved, there is a growing demand for developing more accurate and convenient guide systems. Therefore, in the present study, we have developed and prototyped a 3D printed wearable subject-specific helmet for LIFU stimulation that is guaranteed to be accurate. The spatial relationship between the target position and the full-width at half-maximum (FWHM) of acoustic pressure of the transducer, i.e. focal volume, was compared using the conventional image-guided navigation system. According to the distribution of positional errors, the target position was located well within the focal volume.

Boiling Histotripsy-induced Partial Mechanical Ablation Modulates Tumour Microenvironment by Promoting Immunogenic Cell Death of Cancers.

Boiling histotripsy is a promising non-invasive High-Intensity Focused Ultrasound (HIFU) technique that employs HIFU mechanical effects to fractionate solid tumours without causing any significant thermal damage. It has been suggested that boiling histotripsy may induce a strong immune response due to the absence of denatured antigenic protein at the HIFU focus. The underlying immunological mechanisms of this technique are, however, poorly understood. In this study, we demonstrated the feasibility of using boiling histotripsy to mechanically fractionate human breast adenocarcinoma cells (MDA-MB-231) and the potential immunological effects induced by boiling histotripsy, for the first time. Our results showed that mechanical stresses produced by boiling histotripsy promote immunogenic cell death of cancer cells via TNF-induced necrosis signaling pathway. This immunogenic cell death significantly increases secretions of damage-associated molecular patterns (CRT, HSP70, HMGB-1), pro-inflammatory cytokines (IFN-γ, IL-1α, IL-1ß, IL-18) and chemokines (IL-8) which are related to M1 macrophage activation. Furthermore, the levels of these signaling proteins increase with the degree of mechanical damage induced by boiling histotripsy. Together, the results presented can suggest that boiling histotripsy could be a potential therapeutic approach for not only mechanically destroying solid tumours (e.g., breast cancer) but also promoting immunogenic cell death via TNF-induced necrosis to trigger antitumour immunity.

The effects of ultrasound pressure and temperature fields in millisecond bubble nucleation.

A phenomenological implementation of Classical Nucleation Theory (CNT) is employed to investigate the connection between high intensity focused ultrasound (HIFU) pressure and temperature fields with the energetic requirements of bubble nucleation. As a case study, boiling histotripsy in tissue-mimicking phantoms is modelled. The physics of key components in the implementation of CNT in HIFU conditions such as the derivation of nucleation pressure thresholds and approximations regarding the surface tension of the liquid are reviewed and discussed. Simulations show that the acoustic pressure is the ultimate trigger for millisecond bubble nucleation in boiling histotripsy, however, HIFU heat deposition facilitates nucleation by lowering nucleation pressure thresholds. Nucleation thus occurs preferentially at the regions of highest heat deposition within the HIFU field. This implies that bubble nucleation subsequent to millisecond HIFU heat deposition can take place at temperatures below 100 °C as long as the focal HIFU peak negative pressure exceeds the temperature-dependent nucleation threshold. It is also found that the magnitude of nucleation pressure thresholds decreases with decreasing frequencies. Overall, results indicate that it is not possible to separate thermal and mechanical effects of HIFU in the nucleation of bubbles for timescales of a few milliseconds. This methodology provides a promising framework for studying time and space dependencies of the energetics of bubble nucleation within a HIFU field.

Mechanical damage induced by the appearance of rectified bubble growth in a viscoelastic medium during boiling histotripsy exposure.

In boiling histotripsy, the presence of a boiling vapour bubble and understanding of its dynamic behaviour are crucially important for the initiation of the tissue fractionation process and for the control of the size of a lesion produced. Whilst many in vivo studies have shown the feasibility of using boiling histotripsy in mechanical fractionation of solid tumours, not much is known about the evolution of a boiling vapour bubble in soft tissue induced by boiling histotripsy. The main objective of this present study is therefore to investigate the formation and dynamic behaviour of a boiling vapour bubble which occurs under boiling histotripsy insonation. Numerical and experimental studies on the bubble dynamics induced in optically transparent tissue-mimicking gel phantoms exposed to the field of a 2.0 MHz High Intensity Focused Ultrasound (HIFU) transducer were performed with a high speed camera. The Gilmore-Zener bubble model coupled with the Khokhlov-Zabolotskaya-Kuznetsov and the Bio-heat Transfer equations was used to simulate bubble dynamics driven by boiling histotripsy waveforms (nonlinear-shocked wave excitation) in a viscoelastic medium as functions of surrounding temperature and of tissue elasticity variations. In vivo animal experiments were also conducted to examine cellular structures around a freshly created lesion in the liver resulting from boiling histotripsy. To the best of our knowledge, this is the first study reporting the numerical and experimental evidence of the appearance of rectified bubble growth in a viscoelastic medium. Accounting for tissue phantom elasticity adds a mechanical constraint on vapour bubble growth, which improves the agreement between the simulation and the experimental results. In addition the numerical calculations showed that the asymmetry in a shockwave and water vapour transport can result in rectified bubble growth which could be responsible for HIFU-induced tissue decellularisation. Strain on liver tissue induced by this radial motion can damage liver tissue while preserving blood vessels.

A novel numerical approach to stimulation of a specific brain region using transcranial focused ultrasound.

One of the biggest challenges associated with transcranial application of focused ultrasound is the presence of skull in wave propagation resulting in the attenuation and diffraction of sound waves, which leads to disruption and shifting of the acoustic focus in the brain. In this study, we were motivated to find an optimal transducer position to effectively deliver sufficient acoustic energy to the target in the brain whilst minimizing the effects of skull on wave propagation in a computationally inexpensive way. We hypothesized that the placement of a single-element focused ultrasound transducer which gives the lowest reflection coefficient would be the optimal position. We tested our hypothesis by conducting numerical investigations of wave propagation through the skull targeting the primary motor hand representation (M1) and the supplementary motor area (SMA). The optimal transducer positions were determined using our method (reflection coefficients were lowest), whereby the simulated magnitudes of the acoustic pressure values at the M1 and the SMA were respectively 91% and 92% greater than those when the reflection coefficients were greatest.

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.