Thermal ablation of biological tissues in disease treatment: A review of computational models and future directions

Percutaneous thermal ablation has proved to be an effective modality for treating both benign and malignant tumors in various tissues. Among these modalities, radiofrequency ablation (RFA) is the most promising and widely adopted approach that has been extensively studied in the past decades. Microwave ablation (MWA) is a newly emerging modality that is gaining rapid momentum due to its capability of inducing rapid heating and attaining larger ablation volumes, and its lesser susceptibility to the heat sink effects as compared to RFA. Although the goal of both these therapies is to attain cell death in the target tissue by virtue of heating above 50 oC, their underlying mechanism of action and principles greatly differs. Computational modelling is a powerful tool for studying the effect of electromagnetic interactions within the biological tissues and predicting the treatment outcomes during thermal ablative therapies. Such a priori estimation can assist the clinical practitioners during treatment planning with the goal of attaining successful tumor destruction and preservation of the surrounding healthy tissue and critical structures. This review provides current state-of- the-art developments and associated challenges in the computational modelling of thermal ablative techniques, viz., RFA and MWA, as well as touch upon several promising avenues in the modelling of laser ablation, nanoparticles assisted magnetic hyperthermia and non- invasive RFA. The application of RFA in pain relief has been extensively reviewed from modelling point of view. Additionally, future directions have also been provided to improve these models for their successful translation and integration into the hospital work flow

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

Advancements and Breakthroughs in Ultrasound Imaging

Ultrasonic imaging is a powerful diagnostic tool available to medical practitioners, engineers and researchers today. Due to the relative safety, and the non-invasive nature, ultrasonic imaging has become one of the most rapidly advancing technologies. These rapid advances are directly related to the parallel advancements in electronics, computing, and transducer technology together with sophisticated signal processing techniques. This book focuses on state of the art developments in ultrasonic imaging applications and underlying technologies presented by leading practitioners and researchers from many parts of the world

Interstitial laser photocoagulation as a treatment for breast cancer

Conservative surgery is a safe alternative to mastectomy for some patients with breast cancer. A survey of surgeons in this thesis has shown that more surgeons would now undertake conservative surgery than they have done in the past. Recently a new technique, interstitial laser photocoagulation(ILP) has been described which is capable of in situ tissue necrosis with safe healing. The idea of ILP takes the concept of conservative surgery for breast cancer a step further. The main purpose of this thesis was to investigate the potential value of ILP as a future method of destroying breast cancers in situ leaving the area to heal via resorption and fibrosis. The aims of this thesis were to study the biology of laser interactions with breast cancers scheduled for surgery(and not to completely destroy the tumour), to optimise the laser parameters of power and exposure for a particular tumour and to find an imaging technique which will accurately predict the extent of laser damage. Forty five patients were treated with ILP prior to surgery(median 7 days). Tumour necrosis varied from 2-25mm. No laser damage was noted in 4 patients. Two patients developed minor complications and treatment was abandoned early due to pain in a further 4 patients. The presence of charring within the tumour was associated with larger diameters of necrosis than when charring was absent(median 13 vs 6 mm, p=0.002) and use of a precharred fibre produced similar lesions(median 14mm) which were more predictable.The histological features in the tumour following ILP were of coagulative necrosis which appeared to heal by the formation of fibrous tissue. An area of heat fixed, morphologically preserved tissue was noted within the zone of coagulative necrosis which was thought to be non-viable. Ultrasonography, Com puterised Tomography(CT) and M agnetic Resonace Imaging(MRI) were all used to monitor necrosis. Ultrasound was unable to predict the extent of necrosis as measured in the resected specimen(r=0.3, p=N.S.) but was reasonable at predicting tumour size(r=0.6, p=0.001). CT and MRI show some promise but were only investigated in small numbers of patients. This study has shown that ILP is simple and safe and when using a pre-charred fibre, predictable. If the initial results of imaging using CT and MRI are confirmed in larger studies then ILP could possibly have a role in the treatment of small breast cancers

Medical Robotics

The first generation of surgical robots are already being installed in a number of operating rooms around the world. Robotics is being introduced to medicine because it allows for unprecedented control and precision of surgical instruments in minimally invasive procedures. So far, robots have been used to position an endoscope, perform gallbladder surgery and correct gastroesophogeal reflux and heartburn. The ultimate goal of the robotic surgery field is to design a robot that can be used to perform closed-chest, beating-heart surgery. The use of robotics in surgery will expand over the next decades without any doubt. Minimally Invasive Surgery (MIS) is a revolutionary approach in surgery. In MIS, the operation is performed with instruments and viewing equipment inserted into the body through small incisions created by the surgeon, in contrast to open surgery with large incisions. This minimizes surgical trauma and damage to healthy tissue, resulting in shorter patient recovery time. The aim of this book is to provide an overview of the state-of-art, to present new ideas, original results and practical experiences in this expanding area. Nevertheless, many chapters in the book concern advanced research on this growing area. The book provides critical analysis of clinical trials, assessment of the benefits and risks of the application of these technologies. This book is certainly a small sample of the research activity on Medical Robotics going on around the globe as you read it, but it surely covers a good deal of what has been done in the field recently, and as such it works as a valuable source for researchers interested in the involved subjects, whether they are currently “medical roboticists” or not

Resonant ultrasonic bone penetrating needles

Bone biopsy is an invasive clinical procedure where a bone sample is recovered for analysis during the diagnosis of a medical condition. The procedure is performed while the patient is under either local or general anaesthesia as the patient can experience significant discomfort and possibly large haematoma due to the large axial and rotational forces applied through the needle to penetrate bone. It is well documented that power ultrasonic surgical devices offer advantages of low cutting force, high accuracy and preservation of soft tissues. This thesis details a study of the design, analysis and evaluation of a class of novel power ultrasonic needles for bone penetration, particularly biopsy. Micrometric vibrations generated at the distal tip of a full-wavelength resonant ultrasonic device are used to penetrate the bone. Both ultrasonic longitudinal (L) and longitudinal-torsional (L-T) coupled vibration have proven successful in several applications including ultrasonic surgical devices. Interest in ultrasonic bone cutting has grown since it was first introduced commercially as Piezosurgery in the 1990s. More recent studies have focused on precision cutting of bone, reducing the risk of damage to surrounding delicate tissues in comparison with manual and other powered instruments. Finite element analysis (FEA) is used to design full wavelength ultrasonic needle devices, where the geometry of the device is systematically modified to deter modal coupling by monitoring the frequency spacing between the longitudinal mode of interest and the neighbouring parasitic modes. FEA is further exploited to predict the achievable torsional displacement in a composite mode device tuned to vibrate in a longitudinal-torsional motion through degeneration of the longitudinal motion. While the L-mode device requires the operator to apply a slow backward and forward rotation and a small forward force, to maintain a forward motion and avoid imprinting, a L-T motion at the tip device could avoid this, simplifying the procedure, increasing precision and resulting in a cylindrical, less damaged hole surface. The dynamic behaviours predicted by FEA are validated through experimental modal analysis (EMA) demonstrating the effectiveness of FEA for the design of these devices. EMA is performed by exciting the ultrasonic needle device with a low power random excitation over a predetermined frequency range and measuring the vibration response using a 3D laser Doppler vibrometer (LDV) across a grid of points on the surface of the device. Harmonic analysis was used to investigate the behaviour of the devices at high excitation levels to capture the inherent nonlinearity of the tuned device. The response is captured using bi-directional frequency sweeps across the tuned mode of interest at increasing excitation levels. Ultrasonic surgical instruments typically require to be driven at high excitation levels to generate sufficient vibration amplitude to cut or aspirate tissue or seal vessels. The nonlinearities of the instrument and load presented by the target tissue result in resonance frequency shift, variation in the electric impedance and instability in the vibrational response which can negatively affect the efficacy of the instrument. A resonance tracking system was developed to monitor the voltage and current and adjust the frequency in real time to compensate for the frequency shift. Additional functionality was incorporated to allow modifications to the excitation signal shape and to enable power modulation techniques to be tested in a study of their effects on the rate of progression of the device in its target tissue. Prototype ultrasonic needle devices were evaluated in penetration tests conducted in bone mimic materials and animal bones. The devices recovered trabecular bone from the metaphysis of an ovine femur, and the biopsy samples were architecturally comparable to samples extracted using a trephine biopsy needle. The resonant needle device extracted a cortical bone sample from the central diaphysis, which is the strongest part of the bone, and the biopsy was of superior quality to the sample recovered by a trephine bone biopsy needle. The biopsy sample extracted by the resonant needle was architecturally uniform and cylindrical with an absence of chipping on the surface, suggesting that the biopsy was extracted with precision and control. To penetrate with the L mode device, the operator had to apply a slow backward and forward rotation and the small forward force, to maintain a forward motion. The rotation had to avoid imprinting of the needle tip in the bone, which otherwise resulted in the device stalling. However the L-T mode device, realised by incorporating helical cuts along the axial length, could penetrate the same animal bone sample only requiring the small forward force, hence simplifying the procedure for the operator. The L-T device also provided increased precision, resulting in a cylindrical, less damaged hole surface. Finally, a case study related to skull-based surgery is presented. The petrous apex is a pyramidal shaped structure at the anterior superior portion of the temporal bone and can be the location of tumours, cysts and lesions requiring diagnostic investigation. The petrous apex is challenging to access due to its medial location in the skull base and closeness to important neurovascular structures. An extended surgical approach removes the subject but is associated with morbidity and hence a minimally invasive procedure to access this site to retrieve a biopsy provides a valuable test case for the ultrasonic needle. Guided by the expertise and experience of an ear, nose and throat surgeon, the ultrasonic needle devices were modified and demonstrated in lab-based studies as a new technology for this bone penetration procedure

Dynamic Chemical Shift Imaging for Image-Guided Thermal Therapy

Magnetic resonance temperature imaging (MRTI) is recognized as a noninvasive means to provide temperature imaging for guidance in thermal therapies. The most common method of estimating temperature changes in the body using MR is by measuring the water proton resonant frequency (PRF) shift. Calculation of the complex phase difference (CPD) is the method of choice for measuring the PRF indirectly since it facilitates temperature mapping with high spatiotemporal resolution. Chemical shift imaging (CSI) techniques can provide the PRF directly with high sensitivity to temperature changes while minimizing artifacts commonly seen in CPD techniques. However, CSI techniques are currently limited by poor spatiotemporal resolution. This research intends to develop and validate a CSI-based MRTI technique with intentional spectral undersampling which allows relaxed parameters to improve spatiotemporal resolution. An algorithm based on autoregressive moving average (ARMA) modeling is developed and validated to help overcome limitations of Fourier-based analysis allowing highly accurate and precise PRF estimates. From the determined acquisition parameters and ARMA modeling, robust maps of temperature using the k-means algorithm are generated and validated in laser treatments in ex vivo tissue. The use of non-PRF based measurements provided by the technique is also investigated to aid in the validation of thermal damage predicted by an Arrhenius rate dose model