Full modelling of high-intensity focused ultrasound and thermal heating in the kidney using realistic patient models

Objective: High-intensity focused ultrasound (HIFU) therapy can be used for non-invasive treatment of kidney (renal) cancer, but the clinical outcomes have been variable. In this study, the efficacy of renal HIFU therapy was studied using nonlinear acoustic and thermal simulations in three patients. Methods: The acoustic simulations were conducted with and without refraction in order to investigate its effect on the shape, size and pressure distribution at the focus. The values for the attenuation, sound speed, perfusion and thermal conductivity of the kidney were varied over the reported ranges to determine the effect of variability on heating. Furthermore, the phase aberration was studied in order to quantify the underlying phase shifts using a second order polynomial function. Results: The ultrasound field intensity was found to drop on average 11.1 dB with refraction and 6.4 dB without refraction. Reflection at tissue interfaces was found to result in a loss less than 0.1 dB. Focal point splitting due to refraction significantly reduced the heating efficacy. Perfusion did not have a large effect on heating during short sonication durations. Small changes in temperature were seen with varying attenuation and thermal conductivity, but no visible changes were present with sound speed variations. The aberration study revealed an underlying trend in the spatial distribution of the phase shifts. Conclusion: The results show that the efficacy of HIFU therapy in the kidney could be improved with aberration correction. Significance: A method is proposed by which patient specific pre-treatment calculations could be used to overcome the aberration and therefore make ultrasound treatment possible.Comment: Journal paper, IEEE Transactions on Biomedical Engineering (2018

How sonoporation disrupts cellular structural integrity: morphological and cytoskeletal observations

Posters: no. 1Control ID: 1672429OBJECTIVES: In considering sonoporation for drug delivery applications, it is essential to understand how living cells respond to this puncturing force. Here we seek to investigate the effects of sonoporation on cellular structural integrity. We hypothesize that the membrane morphology and cytoskeletal behavior of sonoporated cells under recovery would inherently differ from that of normal viable cells. METHODS: A customized and calibrated exposure platform was developed for this work, and the ZR-75-30 breast carcinoma cells were used as the cell model. The cells were exposed to either single or multiple pulses of 1 MHz ultrasound (pulse length: 30 or 100 cycles; PRF: 1kHz; duration: up to 60s) with 0.45 MPa spatial-averaged peak negative pressure and in the presence of lipid-shelled microbubbles. Confocal microscopy was used to examine insitu the structural integrity of sonoporated cells (identified as ones with exogenous fluorescent marker internalization). For investigations on membrane morphology, FM 4-64 was used as the membrane dye (red), and calcein was used as the sonoporation marker (green); for studies on cytoskeletal behavior, CellLight (green) and propidium iodide (red) were used to respectively label actin filaments and sonoporated cells. Observation started from before exposure to up to 2 h after exposure, and confocal images were acquired at real-time frame rates. Cellular structural features and their temporal kinetics were quantitatively analyzed to assess the consistency of trends amongst a group of cells. RESULTS: Sonoporated cells exhibited membrane shrinkage (decreased by 61% in a cell’s cross-sectional area) and intracellular lipid accumulation (381% increase compared to control) over a 2 h period. The morphological repression of sonoporated cells was also found to correspond with post-sonoporation cytoskeletal processes: actin depolymerization was observed as soon as pores were induced on the membrane. These results show that cellular structural integrity is indeed disrupted over the course of sonoporation. CONCLUSIONS: Our investigation shows that the biophysical impact of sonoporation is by no means limited to the induction of membrane pores: e.g. structural integrity is concomitantly affected in the process. This prompts the need for further fundamental studies to unravel the complex sequence of biological events involved in sonoporation.postprin

Developmental delays and subcellular stress as downstream effects of sonoporation

Posters: no. 2Control ID: 1672434OBJECTIVES: The biological impact of sonoporation has often been overlooked. Here we seek to obtain insight into the cytotoxic impact of sonoporation by gaining new perspectives on anti-proliferative characteristics that may emerge within sonoporated cells. We particularly focused on investigating the cell-cycle progression kinetics of sonoporated cells and identifying organelles that may be stressed in the recovery process. METHODS: In line with recommendations on exposure hardware design, an immersion-based ultrasound platform has been developed. It delivers 1 MHz ultrasound pulses (100 cycles; 1 kHz PRF; 60 s total duration) with 0.45 MPa peak negative pressure to a cell chamber that housed HL-60 leukemia cells and lipid-shelled microbubbles at a 10:1 cell-tobubble ratio (for 1e6/ml cell density). Calcein was used to facilitate tracking of sonoporated cells with enhanced uptake of exogenous molecules. The developmental trend of sonoporated cells was quantitatively analyzed using BrdU/DNA flow cytometry that monitors the cell population’s DNA synthesis kinetics. This allowed us to measure the temporal progression of DNA synthesis of sonoporated cells. To investigate whether sonoporation would upset subcellular homeostasis, post-exposure cell samples were also assayed for various proteins using Western blot analysis. Analysis focus was placed on the endoplasmic reticulum (ER): an important organelle with multi-faceted role in cellular functioning. The post-exposure observation time spanned between 0-24 h. RESULTS: Despite maintaining viability, sonoporated cells were found to exhibit delays in cell-cycle progression. Specifically, their DNA synthesis time was lengthened substantially (for HL-60 cells: 8.7 h for control vs 13.4 h for the sonoporated group). This indicates that sonoporated cells were under stress: a phenomenon that is supported by our Western blot assays showing upregulation of ER-resident enzymes (PDI, Ero1), ER stress sensors (PERK, IRE1), and ER-triggered pro-apoptotic signals (CHOP, JNK). CONCLUSIONS: Sonoporation, whilst being able to facilitate internalization of exogenous molecules, may inadvertently elicit a cellular stress response. These findings seem to echo recent calls for reconsideration of efficiency issues in sonoporation-mediated drug delivery. Further efforts would be necessary to improve the efficiency of sonoporation-based biomedical applications where cell death is not desirable.postprin

A study on the change in plasma membrane potential during sonoporation

Posters: no. 4Control ID: 1680329OBJECTIVES: There has been validated that the correlation of sonoporation with calcium transients is generated by ultrasound-mediated microbubbles activity. Besides calcium, other ionic flows are likely involved in sonoporation. Our hypothesis is the cell electrophysiological properties are related to the intracellular delivery by ultrasound and microbubbles. In this study, a real-time live cell imaging platform is used to determine whether plasma membrane potential change is related to the sonoporation process at the cellular level. METHODS: Hela cells were cultured in DMEM supplemented with 10% FBS in Opticell Chamber at 37 °C and 5% CO2, and reached 80% confluency before experiments. The Calcein Blue-AM, DiBAC4(3) loaded cells in the Opticell chamber filled with PI solution and Sonovue microbubbles were immerged in a water tank on a inverted fluorescence microscope. Pulsed ultrasound (1MHz freq., 20 cycles, 20Hz PRF, 0.2-0.5MPa PNP) was irradiated at the angle of 45° to the region of interest for 1s.The real-time fluorescence imaging for different probes was acquired by a cooled CCD camera every 20s for 10min. The time-lapse fluorescence images were quantitatively analyzed to evaluate the correlation of cell viability, intracellular delivery with plasma membrane potential change. RESULTS: Our preliminary data showed that the PI fluorescence, which indicated intracellular delivery, was immediately accumulated in cells adjacent to microbubbles after exposure, suggesting that their membranes were damaged by ultrasound-activated microbubbles. However, the fluorescence reached its highest level within 4 to 6 minutes and was unchanged thereafter, indicating the membrane was gradually repaired within this period. Furthermore, using DIBAC4(3), which detected the change in the cell membrane potential, we found that the loss of membrane potential might be associated with intracellular delivery, because the PI fluorescence accumulation was usually accompanied with the change in DIBAC4 (3) fluorescence. CONCLUSIONS: Our study suggests that there may be a linkage between the cell membrane potential change and intracellular delivery mediated by ultrasound and microbubbles. We also suggest that other ionic flows or ion channels may be involved in the cell membrane potential change in sonoporation. Further efforts to explore the cellular mechanism of this phenomenon will improve our understanding of sonoporation.postprin

Real-time imaging of cellular dynamics during low-intensity pulsed ultrasound exposure

Control ID: 1671584Oral Session 5 - Bioeffects of therapeutic ultrasoundOBJECTIVE: Although the therapeutic potential of low-intensity pulsed ultrasound is unquestionable, the wave-matter interactions involved in the process remain to be vaguely characterized. Here we seek to undertake a series of in-situ cellular imaging studies that aim to analyze the mechanical impact of low-intensity pulsed ultrasound on attached fibroblasts from three different aspects: membrane, cytoskeleton, and nucleus. METHODS: Our experimental platform comprised an in-house ultrasound exposure hardware that was coupled to a confocal microscopy system. The waveguided ultrasound beam was geometrically aligned to the microscope’s fieldof-view that corresponds to the center of a polystyrene dish containing fibroblasts. Short ultrasound pulses (5 cycles; 2 kHz PRF) with 0.8 MPa peak acoustic pressure (0.21 W/cm2 SPTA intensity) were delivered over a 10 min period. Live imaging was performed on both membrane (CellMask) and cytoskeleton (actin-GFP, tubulin-RFP) over the entire observation period (up to 30 min after end of exposure). Also, pre- and post-exposure fixed-cell imaging was conducted on the nucleus (Hoechst 33342) and two cytoskeleton components related to stress fibers: F-actin (phalloidin-FITC) and vincullin (Alexa Fluor 647 conjugated). To study whether mechanotransduction was responsible in mediating ultrasound-cell interactions, some experiments were conducted with the addition of gadolinium that blocks stretch-sensitive ion channels. RESULTS: Cell shrinkage was evident over the course of low-intensity pulsed ultrasound exposure. This was accompanied with contraction of actin and tubulin. Also, an increase in central stress fibers was observed at the end of exposure, while the nucleus was found to have decreased in size. Interestingly, after the exposure, a significant rebound in cell volume was observed over a 30 min. period. These effects were not observed in cases with gadolinium blockage of mechanosensitive ion channels. CONCLUSIONS: Our results suggest that low-intensity pulsed ultrasound would transiently induce remodeling of a cell’s membrane and cytoskeleton, and it will lead to repression of nucleus. This indicates that ultrasound after all represents a mechanical stress on cellular membrane. The post-exposure outgrowth phenomenon is also of practical relevance as it may be linked to the stimulatory effects that have been already observed in low-intensity pulsed ultrasound treatments.postprin

Theoretical investigation of transgastric and intraductal approaches for ultrasound-based thermal therapy of the pancreas.

BackgroundThe goal of this study was to theoretically investigate the feasibility of intraductal and transgastric approaches to ultrasound-based thermal therapy of pancreatic tumors, and to evaluate possible treatment strategies.MethodsThis study considered ultrasound applicators with 1.2 mm outer diameter tubular transducers, which are inserted into the tissue to be treated by an endoscopic approach, either via insertion through the gastric wall (transgastric) or within the pancreatic duct lumen (intraductal). 8 patient-specific, 3D, transient, biothermal and acoustic finite element models were generated to model hyperthermia (n = 2) and ablation (n = 6), using sectored (210°-270°, n = 4) and 360° (n = 4) transducers for treatment of 3.3-17.0 cm3 tumors in the head (n = 5), body (n = 2), and tail (n = 1) of the pancreas. A parametric study was performed to determine appropriate treatment parameters as a function of tissue attenuation, blood perfusion rates, and distance to sensitive anatomy.ResultsParametric studies indicated that pancreatic tumors up to 2.5 or 2.7 cm diameter can be ablated within 10 min with the transgastric and intraductal approaches, respectively. Patient-specific simulations demonstrated that 67.1-83.3% of the volumes of four sample 3.3-11.4 cm3 tumors could be ablated within 3-10 min using transgastric or intraductal approaches. 55.3-60.0% of the volume of a large 17.0 cm3 tumor could be ablated using multiple applicator positions within 20-30 min with either transgastric or intraductal approaches. 89.9-94.7% of the volume of two 4.4-11.4 cm3 tumors could be treated with intraductal hyperthermia. Sectored applicators are effective in directing acoustic output away from and preserving sensitive structures. When acoustic energy is directed towards sensitive structures, applicators should be placed at least 13.9-14.8 mm from major vessels like the aorta, 9.4-12.0 mm from other vessels, depending on the vessel size and flow rate, and 14 mm from the duodenum.ConclusionsThis study demonstrated the feasibility of generating shaped or conformal ablative or hyperthermic temperature distributions within pancreatic tumors using transgastric or intraductal ultrasound

Respiratory organ motion in interventional MRI : tracking, guiding and modeling

Respiratory organ motion is one of the major challenges in interventional MRI, particularly in interventions with therapeutic ultrasound in the abdominal region. High-intensity focused ultrasound found an application in interventional MRI for noninvasive treatments of different abnormalities. In order to guide surgical and treatment interventions, organ motion imaging and modeling is commonly required before a treatment start. Accurate tracking of organ motion during various interventional MRI procedures is prerequisite for a successful outcome and safe therapy. In this thesis, an attempt has been made to develop approaches using focused ultrasound which could be used in future clinically for the treatment of abdominal organs, such as the liver and the kidney. Two distinct methods have been presented with its ex vivo and in vivo treatment results. In the first method, an MR-based pencil-beam navigator has been used to track organ motion and provide the motion information for acoustic focal point steering, while in the second approach a hybrid imaging using both ultrasound and magnetic resonance imaging was combined for advanced guiding capabilities. Organ motion modeling and four-dimensional imaging of organ motion is increasingly required before the surgical interventions. However, due to the current safety limitations and hardware restrictions, the MR acquisition of a time-resolved sequence of volumetric images is not possible with high temporal and spatial resolution. A novel multislice acquisition scheme that is based on a two-dimensional navigator, instead of a commonly used pencil-beam navigator, was devised to acquire the data slices and the corresponding navigator simultaneously using a CAIPIRINHA parallel imaging method. The acquisition duration for four-dimensional dataset sampling is reduced compared to the existing approaches, while the image contrast and quality are improved as well. Tracking respiratory organ motion is required in interventional procedures and during MR imaging of moving organs. An MR-based navigator is commonly used, however, it is usually associated with image artifacts, such as signal voids. Spectrally selective navigators can come in handy in cases where the imaging organ is surrounding with an adipose tissue, because it can provide an indirect measure of organ motion. A novel spectrally selective navigator based on a crossed-pair navigator has been developed. Experiments show the advantages of the application of this novel navigator for the volumetric imaging of the liver in vivo, where this navigator was used to gate the gradient-recalled echo sequence

High-intensity focused ultrasound therapy in the uterine fibroid: a clinical case study of poor heating efficacy

A clinical case study of high-intensity focused ultrasound (HIFU) treatment in the uterine fibroid was conducted. During the therapy, poor heating efficacy was observed which could be attributed to several factors such as the local perfusion rate, patient-specific anatomy or changes in acoustic parameters of the ultrasound field. In order to determine the cause of the diminished heating, perfusion analyses and ultrasound simulations were conducted using the magnetic resonance imaging (MRI) data from the treatment. The perfusion analysis showed high local perfusion rate in the myoma (301.0 +- 25.6 mL/100 g/min) compared to the surrounding myometrium (233.8 +- 16.2 mL/100 g/min). The ultrasound simulations did not show large differences in the focal point shape or the acoustic pressure (2.07 +- 0.06 MPa) when tilting the transducer. However, a small shift (-2.2 +- 1.3 mm) in the axial location of the focal point was observed. The main causes for the diminished heating were likely the high local perfusion and ultrasound attenuation due to the deep location of the myoma.Comment: Conference Proceedin

Treatment of brain cancer and ischaemic stroke utilising High Intensity Focus Ultrasound (HIFU) guide with MRI

In this thesis high intensity focused ultrasound (HIFU) is utilized for cancer treatment (thermal mode) and treatment of ischaemic stroke (mechanical mode). These two applications were investigated in vitro and in vivo models. MRI was utilized to monitor the lesions created by HIFU either in thermal or cavitation mode in freshly excised lamb brain tissue in vitro, and in rabbit brain in vivo. Additionally, MRI was used to monitor lesions deep in tissue for both in vitro and in vivo exposures. All three MRI sequences used (T1-W FSE, T2-W FSE and FLAIR) were able to detect lesions. Both thermal and bubbly lesions were best monitored using T1-W FSE with excellent contrast, proving the potential of HIFU to treat reliably tumours in the brain. A HIFU system was also used to assist thrombolysis in cooperation with a thrombolytic drug such as recombinant tissue plasminogen activator (rt-PA) in vitro and in vivo. It was shown that higher intensity results to higher volume of dissolved clot, but there is a limit of the intensity to be used in order to avoid heating of the clot and the surrounding tissue. The goal in this study was to achieve temperature elevation not exceeding 1ºC (called safe temperature). It was found that the larger the beam area the larger the dissolved clot volume. Also, the lower the frequency, the larger the volume of the dissolved clot. The results reported herein point to the use of frequency around 0.5 MHz and pulsing to optimize thrombolysis and skull penetration and at the same time avoiding unwanted heating. Finally, an Acrylonitrile Butadiene Styrene (ABS) phantom skull model was developed in order to evaluate the propagation of ultrasound using a single element transducer. The skull model was appropriately designed so that it has the same attenuation as a human skull. It was demonstrated that using a frequency of 0.5 MHz versus 1 MHz, ultrasound propagation through the phantom skull was higher. Therefore, higher frequency has poor skull penetration and a small beam size at the focus, while low frequencies have better skull penetration but with the risk of reaching the unpredictable effect of cavitation. The developed system has proven to successfully create large lesions in the brain and at the same time, these lesions are successfully monitored with excellent contrast using MRI (T1-W FSE) enabling the accurate determination of the margins of these lesions. The results reported in this study point to the use of frequency around 0.5 MHz and pulsing to optimize thrombolysis and skull penetration and at the same time avoiding unwanted heating. For treating tumours located deep in the brain and for dissolving thrombus causing an acute ischaemic stroke, further extensive clinical studies will be needed before this technology is applied to humans