Boiling histotripsy exhibits anti-fibrotic effects in animal models of liver fibrosis.

Liver fibrosis is a hallmark of chronic liver disease which could lead to liver cirrhosis or liver cancer. However, there is currently lack of a direct treatment for liver fibrosis. Boiling histotripsy (BH) is an emerging non-invasive high-intensity focused ultrasound technique that can be employed to mechanically destruct solid tumour at the focus via acoustic cavitation without significant adverse effect on surrounding tissue. Here, we investigated whether BH can mechanically fractionate liver fibrotic tissue thereby exhibiting an anti-fibrotic effect in an animal model of liver fibrosis. BH-treated penumbra and its identical lobe showed reduced liver fibrosis, accompanied by increased hepatocyte specific marker expression, compared to the BH-untreated lobe. Furthermore, BH treatment improved serological liver function markers without notable adverse effects. The ability of BH to reduce fibrosis and promote liver regeneration in liver fibrotic tissue suggests that BH could potentially be an effective and reliable therapeutic approach against liver fibrosis.

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.

Micro-ultrasonic Assessment of Early Stage Clot Formation and Whole Blood Coagulation Using an All-Optical Ultrasound Transducer and Adaptive Signal Processing Algorithm.

Abnormal formation of solid thrombus inside a blood vessel can cause thrombotic morbidity and mortality. This necessitates early stage diagnosis, which requires quantitative assessment with a small volume, for effective therapy with low risk to unwanted development of various diseases. We propose a micro-ultrasonic diagnosis using an all-optical ultrasound-based spectral sensing (AOUSS) technique for sensitive and quantitative characterization of early stage and whole blood coagulation. The AOUSS technique detects and analyzes minute viscoelastic variations of blood at a micro-ultrasonic spot (<100 µm) defined by laser-generated focused ultrasound (LGFU). This utilizes (1) a uniquely designed optical transducer configuration for frequency-spectral matching and wideband operation (6 dB widths: 7-32 MHz and d.c. ∼ 46 MHz, respectively) and (2) an empirical mode decomposition (EMD)-based signal process particularly adapted to nonstationary LGFU signals backscattered from the spot. An EMD-derived spectral analysis enables one to assess viscoelastic variations during the initiation of fibrin formation, which occurs at a very early stage of blood coagulation (1 min) with high sensitivity (frequency transition per storage modulus increment = 8.81 MHz/MPa). Our results exhibit strong agreement with those obtained by conventional rheometry (Pearson's R > 0.95), which are also confirmed by optical microscopy. The micro-ultrasonic and high-sensitivity detection of AOUSS poses a potential clinical significance, serving as a screening modality to diagnose early stage clot formation (e.g., as an indicator for hypercoagulation of blood) and stages of blood-to-clot transition to check a potential risk for development into thrombotic diseases.

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.

Laser-generated focused ultrasound transducer using a perforated photoacoustic lens for tissue characterization.

We demonstrate a laser-generated focused ultrasound (LGFU) transducer using a perforated-photoacoustic (PA) lens and a piezoelectric probe hydrophone suitable for high-frequency ultrasound tissue characterization. The perforated-PA lens employed a centrally located hydrophone to achieve a maximum directional response at 0° from the axial direction of the lens. Under pulsed laser irradiation, the lens produced LGFU pulses with a frequency bandwidth of 6-30 MHz and high-peak pressure amplitudes of up to 46.5 MPa at a 70-µm lateral focal width. Since the hydrophone capable of covering the transmitter frequency range (∼20 MHz) was integrated with the lens, this hybrid transducer differentiated tissue elasticity by generating and detecting high-frequency ultrasound signals. Backscattered (BS) waves from excised tissues (bone, skin, muscle, and fat) were measured and also confirmed by laser-flash shadowgraphy. We characterized the LGFU-BS signals in terms of mean frequency and spectral energy in the frequency domain, enabling to clearly differentiate tissue types. Tissue characterization was also performed with respect to the LGFU penetration depth (from the surface, 1-, and 2-mm depth). Despite acoustic attenuation over the penetration depth, LGFU-BS characterization shows consistent results that can differentiate the elastic properties of tissues. We expect that the proposed transducer can be utilized for other tissue types and also for non-destructive evaluation based on the elasticity of unknown materials.

Side-Polished Fiber-Optic Line Sensor for High-Frequency Broadband Ultrasound Detection.

We demonstrate a side-polished fiber-optic ultrasound sensor (SPFS) with a broad frequency bandwidth (dc⁻46 MHz at 6-dB reduction) and a wide amplitude detection range from several kPa to 4.8 MPa. It also exhibits a high acoustic sensitivity of 426 mV/MPa with a signal-to-noise ratio of 35 dB and a noise-equivalent pressure of 6.6 kPa (over 1⁻50 MHz bandwidth) measured at 7-MHz frequency. The SPFS does not require multi-layer-coated structures that are used in other high-sensitivity optical detectors. Without any coating, this uses a microscale-roughened structure for evanescent-field interaction with an external medium acoustically modulated. Such unique structure allows significantly high sensitivity despite having a small detection area of only 0.016 mm² as a narrow line sensor with a width of 8 µm. The SPFS performance is characterized in terms of acoustic frequency, amplitude responses, and sensitivities that are compared with those of a 1-mm diameter piezoelectric hydrophone used as a reference.