Role of Cavitation During Bulk Ultrasound Ablation: Ex Vivo and in Vivo Studies
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Role of Cavitation during Bulk Ultrasound Ablation: Ex vivo and In Vivo Studies A dissertation submitted to Graduate School at the University of Cincinnati in the partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Engineering at the University of Cincinnati by Chandrapriya Karunakaran July 2012 Dissertation Advisor T. Douglas Mast, PhD Abstract Bubble activity can complicate ultrasound treatment by shielding ultrasound energy from the focus or by increasing local ultrasound absorption, rendering the treatment unpredictable. In this thesis, the role of bubble activity was evaluated for ex vivo and in vivo bulk ultrasound ablation experiments. Overpressure was used to suppress cavitation and tissue vaporization in the ex vivo studies. Ex vivo bovine liver was ablated with unfocused ultrasound (3.1 MHz or 4.8 MHz) at 31 W/cm2 for 10 or 20 minutes. A passive cavitation detector (PCD) was used to record acoustic emissions throughout the treatment. Subharmonic, broadband and low-frequency emissions were quantified by processing signals recorded by the PCD. The treated liver was sliced and stained with 2% triphenyl tetrazolium chloride (TTC) to evaluate lesion geometry. Multivariate multiple regression models were computed to predict lesion areas and depths based on the three acoustic emission levels. Results confirm that the three acoustic emissions were significant in predicting lesion dimensions. It was concluded that subharmonic activity may increase lesion area by redistributing ultrasound energy locally, while tissue vaporization may decrease lesion area and depth by shielding energy from the treatment zone. TTC uptake of ultrasound treated tissue was compared to TTC uptake of thermally heated tissue, with comparable thermal doses, to evaluate the contribution of nonthermal bioeffects in bulk ultrasound ablation. Logistic regression modeling was performed to estimate and compare the probability of tissue coagulation, for range of thermal doses, among the two experimental groups (ultrasound and thermally heated). Results suggested no significant differences between TTC uptake levels of thermally heated and ultrasound treated tissue, suggesting that cavitation during bulk ultrasound ablation may translate to thermal bioeffects evident in the tissue. 2 To validate TTC staining as a method to evaluate ultrasound treatment success, tissue from multiple TTC uptake regions of ultrasound ablated liver and VX2 tumor were stained with 4', 6-diamidino-2-phenylindole (DAPI). Nuclear size distributions among the three TTC uptake regions were quantified and compared to evaluate changes in histology among the three regions. Results suggest that TTC uptake levels correspond to distinguishable differences in nuclear size distribution. Cells in the region of partial TTC uptake may undergo apoptosis. The role of cavitation in ultrasound ablation in vivo was investigated by employing passive cavitation imaging to record and monitor cavitation during treatment of swine liver with ultrasound (2700-6000 W/cm2, 20 sec-2 minutes). Passive cavitation imaging can spatially and temporally resolve cavitation signals including subharmonic, broadband and harmonic emissions. Spatial localization of subharmonic, broadband, low-frequency and harmonic activity was compared to local tissue ablation through receiver operating characteristic (ROC) curve analysis. The results suggest that for these in vivo experiments, with no significant subharmonic or broadband emissions, harmonic emissions were the most significant in predicting tissue ablation. Results from this thesis suggest that cavitation can play opposing roles in bulk ultrasound ablation. Cavitation detection and imaging can be used to predict thermal lesion formation. TTC uptake levels correlate to thermal dose and thermal bioeffects in ultrasound ablation and can be used to evaluate treatment success. The results from this thesis can be extended to aid in planning and monitoring of ultrasound treatments and increase the efficiency of ultrasound ablation. 3 Acknowledgements This work was supported by NIH grants R43 CA124283, R21 EB008483, University of Cincinnati College of Medicine Dean’s Bridge Fund and University of Cincinnati Summer Graduate Research Fellowship. First and foremost I would like to thank my advisor Dr. T. Douglas Mast who has always encouraged and supported me in pursuing new ideas. He has taught me to be an independent researcher starting from forming an idea, to planning experiments and problem solving. I have learnt a lot about medical ultrasound and signal processing from him. I appreciate his time, effort and financial support through out my doctoral research experience. I would also like to thank Dr. Christy K. Holland who was always been available to provide her insightful thoughts and suggestions to improve the project. I would like to thank Dr. Daria Narmoneva for help and guidance with histology techniques and processing. I appreciate and thank Dr. Marepalli B. Rao who was always available to provide support with statistical methods and processing relevant to the project in spite of his busy schedule. The scientifically critical comments and suggestions provide by all the committee members have been instrumental in improving the project and streamlining the outcome of the research. I am grateful to all my colleagues and friends in biomedical engineering. I appreciate the assistance of my lab mates Dr. Vasant Salgaonkar, Swetha Subramanian, Anna Nagle, Kyle Rich for all the discussions and suggestions. I would like to appreciate Mark Burgess, Amel Alqaddah and Molly Perdix for their help with the experiments. A special thanks to Mr. Ronald Burrage for help in construction of overpressure chamber. A special thanks to all my friends in Cincinnati who have made this experience memorable by offering support and affection even without me asking for it. 4 Finally I would like to dedicate this thesis to my family including my parents Mr. & Mrs. Karunakaran, my brother and my sister for their sacrifice, love and encouragement. 5 CONTENTS 1. Introduction 1.1 Background…………………………………………………………………………………..19 1.1.1 Liver cancer incidence…………………………………………………………...............19 1.1.2 Liver cancer treatment options…………………………………………………………...20 1.1.2.1 Surgical resection ……………………………………………………........................20 1.1.2.2 Percutaneous ethanol injection (PEI) ………………………………………………..20 1.1.2.3 Electrolytic ablation………………………………………………………………….20 1.1.2.4 Cryosurgery …………………………………………………………..……………...21 1.1.2.5 Liver transplantation ………………………………………………………………...21 1.1.2.6 Thermal ablation ……………………………………………………………………21 1.1.3 Ultrasound ablation ……………………………………………………………………..23 1.1.3.1 Medical application of ultrasound ……………………………………………..……23 1.1.3.2 Bubble activity (cavitation and boiling) in ultrasound ablation …….…………….…24 1.1.3.3 Bioeffects of ultrasound ……………………………………………………………25 Thermal bioeffects Cavitation bioeffects 1.1.3.4 Role of cavitation in bulk ultrasound ablation ………………………………………27 1.1.3.5 Histology of ultrasound treated tissue ……………………………………………….28 1.2 Research objectives…………………………………………………………………………..28 1.3 Hypothesis and specific aims………………………………………………………………...29 1.4 Thesis organization…………………………………………………………………………..30 6 2. Effect of overpressure on acoustic emissions and lesion histology in bulk ultrasound ablation: ex vivo 2.1 Objectives …………………………………………………………………………………...32 2.2 Materials and methods ………………………………………………………………............33 2.2.1 Design of overpressure chamber……………………………………………………..…..33 2.2.2 Acoustic properties of the chamber ……………………………………………………..34 2.2.3 Ultrasound image-treat probes…………………………………………………………...38 2.2.4 Tissue preparation and handling ………………………………………………………...40 2.2.5 Experimental setup……………………………………………………………………….40 2.2.6 Ultrasound ablation ……………………………………………………………………...41 2.2.7 Data acquisition………………………………………………………………………….42 2.2.8 Histology evaluation …………………………………………………………………….42 2.3 Data processing………………………………………………………………………………43 2.3.1 Spectral leakage processing ……………………………………………………………..44 2.4 Statistical analysis …………………………………………………………………………...47 2.5 Results ……………………………………………………………………………………….48 2.5.1 Experimental results at 3.1 MHz ………………………………………………………….48 2.5.2 Experimental results at 4.8 MHz ………………………………………………………….56 2.6 Discussion …………………………………………………………………………………...67 2.6.1 Experiments at 3.1 MHz………. ………………………………………………………….67 2.6.2 Experiments at 4.8 MHz………..………………………………………………………….70 2.6.3 Comparing 3.1 MHz vs. 4.8 MHz experiments …………………………………………...72 2.7 Conclusion …………………………………………………………………………………..73 7 3. Role of thermal and non-thermal mechanisms in tissue bioeffects for bulk ultrasound ablation 3.1 Objective …………………………………………………………………………………….75 3.2 Materials and methods ………………………………………………………………………77 3.2.1 TTC Vs. thermal dose …………………………………………………………………...77 3.2.2 Calibration of analog water heater ………………………………………………………79 3.2.3 Replicating ultrasound thermal dose……………………………………………………..82 3.2.4 TTC uptake scores ………………………………………………………………………84 3.2.5 Proportional odds model ………………………………………………………………...85 3.3 Results………………………………………………………………………..………………87 3.3.1 TTC uptake vs. cumulative thermal dose ……………………………………………….87 3.3.2 Matching ultrasound thermal dose……………………………………………………….89 3.3.3 Proportional odds model ………………………………………………………………...94 3.4 Discussion …………………………………………………………………………………...98 3.4.1 TTC as an indicator of thermal dose …………………………………………………….98 3.4.2 Role of cavitation in altering tissue bioeffects …………………………………………..99