Novel Uses for Ultrasound as Both an Imaging and Therapeutic Tool in the Characterization and Percutaneous Revascularization of Chronic Total Occlusion By Amandeep Singh Thind A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Medical Biophysics University of Toronto Amandeep Singh Thind Doctor of Philosophy Thesis Department of Medical Biophysics, University of Toronto Sunnybrook Health Sciences Centre, S639-2075 Bayview Avenue Toronto, Ontario, M4N 3M5 Canada © Copyright Amandeep Thind 2011 Abstract Thesis Title: Novel Uses for Ultrasound as Both an Imaging and Therapeutic Tool in the Characterization and Percutaneous Revascularization of Chronic Total Occlusion Amandeep Singh Thind, Doctor of Philosophy, Department of Medical Biophysics, University of Toronto, 2011. Revascularization of Chronic Total Occlusions (CTO) by percutaneous coronary interventions is limited by low success rates, primarily due to difficulty in guidewire crossing. There are a number of contributing factors that make guidewire crossing challenging. Two of the most significant impediments are: a) inability to adequately visualize the CTO to appropriately plan a pathway to the distal lumen, and b) difficulty in physically crossing the rigid endcap at the proximal end of CTO without using stiff wires. Moreover, there is a significant knowledge gap in the composition of CTOs, and the consequent impact of that composition on crossability. This thesis presents tools and techniques to help mitigate the current shortcomings, while shedding new light on CTO composition and maturation. The tools and techniques presented herein are based upon ultrasound approaches with the intent of eventually developing these strategies into catheter based solutions. Recent studies have suggested that the presence of microvessels in CTO may provide a preferred pathway for guidewire crossing. However, due to limited resolution and a lack of soft tissue contrast in angiography, microvessels within CTO cannot generally be detected by in-vivo angiographic techniques, and when they are visualized, it is unknown whether or not they are intraluminal. In this thesis, high frequency ultrasound with Power Doppler overlays is shown to be capable of detecting and tracking ii transluminal recanalization channels using an in vivo porcine model of CTO. It is also shown that ultrasound is a more sensitive technique to detect and map these channels than MRI. Furthermore, features of microvasculature in CTOs that had not previously been seen are presented. A technique was then developed to facilitate guidewire crossing through the proximal endcap, also known as the proximal fibrous cap (PFC). In order to assess the ease with which a probe is able to perforate the PFC, a system was designed and to measure the force required for PFC puncture. This system was validated by examining the required puncture forces for CTOs of different ages. It was shown that CTOs less than 6 weeks in age are significantly easier to puncture than those greater than 12 weeks. This coincides with differences in composition, with the presence of softer materials at the earlier time point, such as thrombus and proteoglycans compared to stiffer fibrotic materials which predominate at late timepoints. After development and validation of a reliable technique to measure ease of PFC puncture, the efficacy of therapies designed to modify PFC compliance could be assessed. The use of ultrasound mediated microbubble (UMM) disruption to act as an adjuvant to accelerate collagenase therapy in CTO was examined. A significant reduction in puncture force and an increase in the amount of collagen degraded was achieved using a combined UMM + collagenase treatment compared with collagenase therapy alone and UMM treatment alone. iii To Jeevan, whose arrival gave me the inspiration to complete this work iv Acknowledgements The work presented in this thesis would not have been possible without the help of many people who provided a combination of guidance, support, motivation, compassion and entertainment. First I would like to thank my supervisor, Dr. F. Stuart Foster for providing me with an opportunity to complete this work with the freedom to make mistakes, learn from them, and ultimately become an independent scientist. My supervisory committee members, Drs. Graham Wright, Alex Vitkin, and Bradley Strauss provided me with a great deal of guidance in a constructive and productive manner. I was very fortunate to be able to make numerous extremely fruitful scientific collaborations. Without a doubt, the work presented in this thesis would not have been possible without the Strauss lab. Starting with Dr. Strauss, who despite his unbelievably busy schedule, was always willing to have a friendly discussion. Within the lab, Michelle Ladouceur-Wodzak was absolutely vital to the animal experiments, and always brought a cheery attitude. Aaron Teitelbaum was a patient teacher, and taught me how to perform a number of procedures for which I am grateful. I also had excellent collaborations with Drs. Raffi Karshafian and David Goertz, who provided expertise in therapeutic ultrasound, as well as excellent insights on academia and research as a whole. Dr. Brian Courtney proved to be an excellent mentor and provided me with the opportunity for a very exciting side project to my thesis work, and I feel fortunate to have him as a colleague and friend. I would also like to acknowledge the members of the CTO team for helping to guide me through the early stages of my graduate career. In particular, I appreciate the discussions and support I received from Nigel Munce, General Leung, and Kevan Anderson. v I would like to very emphatically thank all of the members of the Foster and Burns labs. I can’t think of a better environment and group of colleagues to spend almost 6 years of my career with. The ultrasound lab environment very much starts with Kasia Harasiewicz. She is truly the heart and soul of the lab, and was always the first person I went to talk to about anything – good or bad. The members of the ultrasound group are really what kept me sane through my time at Sunnybrook, and I’d particularly like to thank in no particular order: John Hudson, Kogee Leung, Shawn Stapleton, Robin Castelino, and Toby Lam. You were a blast to be around from the beginning to the end, were instrumental in keeping the lab fun, and making sure that I don’t stop believing. I will miss our daily chats about anything and everything when I leave the lab. I have also been very lucky to have such a supportive family. My brother, sister, and parents have been there for me since the day I was born, never doubting me in any endeavours I have embarked upon. I’ve also recently seen many additions to the family, and I am privileged to have in-laws that treat me like I’ve been part of the family my whole life. Most importantly, I’d like to thank my wife and best friend, Loveleen. I met her during the first year of graduate school, and she has turned my life upside down. She has been with me every step of the way to provide me support and encouragement to pick me up no matter how down I was. I look forward to the next chapter in our lives together, and all the joys and challenges that come along with being a husband and father. vi Table of Contents Abstract ......................................................................................................................................................... ii Acknowledgements ....................................................................................................................................... v Table of Contents ........................................................................................................................................ vii List of Figures ................................................................................................................................................ x List of Tables ............................................................................................................................................... xv List of Acronyms & Abbreviations ............................................................................................................... xvi 1 Introduction .......................................................................................................................................... 1 1.1 Motivation ..................................................................................................................................... 1 1.2 Occlusive Vascular Disease ........................................................................................................... 1 1.2.1 Anatomy of a healthy artery ................................................................................................. 1 1.2.2 Atherosclerosis ...................................................................................................................... 2 1.2.3 Clinical Presentation and Treatment .................................................................................... 5 1.3 Chronic Total Occlusion ................................................................................................................ 7 1.3.1 Clinical presentation & Benefits to revascularizing CTOs ..................................................... 7 1.3.2 Progression to CTO ................................................................................................................ 9 1.3.3 Maturation of CTO ................................................................................................................ 9 1.3.4 Impediments to PCI ............................................................................................................. 11 1.3.5 Animal Models of Chronic Total Occlusion ......................................................................... 12 1.3.6 CTO Crossing Strategies ...................................................................................................... 16 1.4 Collagen and Collagenase ........................................................................................................... 18 1.4.1 The Collagen Molecule ........................................................................................................ 18 1.4.2 Collagenase ......................................................................................................................... 19 1.5 Ultrasound .................................................................................................................................. 20 1.5.1 Imaging ................................................................................................................................ 20 1.5.2 Blood flow imaging ............................................................................................................. 25 1.5.3 Ultrasound mediated microbubbles (UMM) ...................................................................... 26 1.6 Thesis aims .................................................................................................................................. 28 1.6.1 Specific Aims of the Thesis .................................................................................................. 30 2 Microvascular Study of Chronic Total Occlusion in a Porcine Model ................................................. 32 vii 2.1 Introduction ................................................................................................................................ 32 2.2 Methods ...................................................................................................................................... 33 2.2.1 CTO Model .......................................................................................................................... 33 2.2.2 High Frequency Ultrasound (Micro-ultrasound) ................................................................. 34 2.2.3 MRI ...................................................................................................................................... 36 2.2.4 MicroCT (μCT) ..................................................................................................................... 36 2.2.5 Histological Processing ........................................................................................................ 37 2.3 Results ......................................................................................................................................... 38 2.3.1 Histology ............................................................................................................................. 38 2.3.2 Micro-ultrasound (μUS) ..................................................................................................... 39 2.3.3 MRI ...................................................................................................................................... 42 2.3.4 Intraluminal Microvessel CTO Features .............................................................................. 43 2.4 Discussion .................................................................................................................................... 47 2.5 Conclusions ................................................................................................................................. 50 3 A Novel Method for Measurement of Proximal Fibrous Cap Puncture Force in Chronic Total Occlusions and its application ..................................................................................................................... 52 3.1 Introduction ................................................................................................................................ 52 3.2 Materials & Methods .................................................................................................................. 53 3.2.1 CTO Model .......................................................................................................................... 53 3.2.2 Removal of CTO for Ex-Vivo Testing .................................................................................... 53 3.2.3 Puncture Force Testing ....................................................................................................... 54 3.2.4 Histology ............................................................................................................................. 58 3.2.5 Statistical Analysis ............................................................................................................... 58 3.3 Results ......................................................................................................................................... 59 3.4 Discussion .................................................................................................................................... 62 3.4.1 Potential applications ......................................................................................................... 65 3.5 Conclusions ................................................................................................................................. 66 4 The use of Ultrasound Mediated Contrast agents as an adjuvant for collagenase therapy in Chronic Total Occlusion ............................................................................................................................................ 67 4.1 Introduction ................................................................................................................................ 67 4.2 Materials and methods ............................................................................................................... 68 4.2.1 CTO Model .......................................................................................................................... 68 viii 4.2.2 Sample collection ................................................................................................................ 68 4.2.3 Treatment groups ............................................................................................................... 70 4.2.4 Standard Treatment ............................................................................................................ 72 4.2.5 Treatment Duration ............................................................................................................ 72 4.2.6 Modified Acoustic Setup ..................................................................................................... 72 4.2.7 Collagenase ......................................................................................................................... 73 4.2.8 Acoustic parameters ........................................................................................................... 73 4.2.9 Biochemical Assays ............................................................................................................. 75 4.2.10 Puncture Force Test ............................................................................................................ 77 4.2.11 Statistical Analysis ............................................................................................................... 77 4.3 Results ......................................................................................................................................... 77 4.3.1 Standard treatment duration .............................................................................................. 77 4.3.2 Extended treatment duration ............................................................................................. 80 4.3.3 Modified acoustic setup ...................................................................................................... 80 4.4 Discussion .................................................................................................................................... 81 4.4.1 Clinical Relevance of Study ................................................................................................. 84 4.5 Conclusions ................................................................................................................................. 84 5 Summary and Future work ................................................................................................................. 86 5.1 Summary and Discussion ............................................................................................................ 86 5.2 Future work: In vivo collagenase studies .................................................................................... 87 5.2.1 Materials & Methods .......................................................................................................... 87 5.2.2 Results & Discussion ........................................................................................................... 89 5.3 Future work: Microspheres loaded with VEGF ........................................................................... 90 5.4 Future work: Collagenase dynamics studies ............................................................................... 93 5.5 Future work: Catheter based technology ................................................................................... 94 5.5.1 Imaging ................................................................................................................................ 95 5.5.2 Therapy ............................................................................................................................... 97 5.5.3 Image Guided Therapeutics ................................................................................................ 98 5.5.4 Compliance Testing ............................................................................................................. 98 5.6 Concluding remarks .................................................................................................................. 100 6 References ........................................................................................................................................ 102 ix List of Figures Figure 1-1 - The anatomy of a normal artery shown in cross- section. An artery is a three-layered structure. ................................................................................................................................... 2 Figure 1-2 - Histology of OVD. Panel A shows a Hemotoxylin & Eosin stained slide with intraplaque haemorhage (Hem) and a calcium deposit (Ca2+). Panel B shows an example of Thin Cap Fibrous Atheroma. The fibrous cap is labelled FC, and the necrotic core is labelled NC. Figure adapted from(12). ..................................................................................................................... 5 Figure 1-3 - Angiogram of a CTO before and after revascularization. Panel A shows CTO in Left Anterior Descending (LAD) artery marked with white arrow. Panel B shows result of successful recanalization. Figure adapted from (30). ................................................................................. 8 Figure 1-4 - Cross-sectional views of human coronary CTOs. Panel A shows an elastic van Gieson stain of a lipid rich lesion, with the solid black arrow showing significant cholesterol deposit. These are generally considered to be soft and strong candidates for successful PCI. Panel B shows a complex CTO stained with a Movat Pentachrome, containing Microvessels (MV), a Necrotic Core (NC), as well as significant amounts of collagen (stained yellow). Panel C shows an H&E stain of a CTO with a large calcium deposit (curved solid arrow). Panels A & C adapted from (32) and Panel B adapted from (34). Bars in A & C represent 1266µm. ................................ 11 Figure 1-5 - Longitudinal views of the Proximal Fibrous Cap. Panel A shows an Elastic Trichrome stain of a 12 week old rabbit femoral artery occlusion. The residual Lumen (L) is shown along with the Proximal Fibrous Cap (PFC). Panel B shows a Movat Pentachrome stain of a similar rabbit CTO. The Lumen (L) is shown with a thrombus formed at the site of the plaque. The PFC is shown in purple. The Media (M), IEL and EEL are also shown. ............................................... 12 Figure 1-6 - Schematic of a focused ultrasound transducer and associated beam. Depending on the strength of focus, or F-number, the width of the focus will change. This directly affects the lateral resolution. .................................................................................................................... 24 Figure 2-1 - Angiograms of right leg of porcine subject. The vessel that was occluded is the superficial branch of the femoral artery. A) shows the region before deposition of polymer. Vessel to be occluded located in yellow ellipse. B) shows the region immediately after the deposition of the polymer, with yellow ellipse indicating region of occlusion. ............................................ 35 Figure 2-2 - Histological images of CTO arteries containing intraluminal microvessels. A) shows an H&E stained vessel containing a single large microvessel (MV). This vessel was perfused with Microfil (MF) just prior to sacrifice, which can be seen as black portions within microvessels. The plaque (P) and media (M) are also marked. Bar represents 500μm. B) shows an Elastic Trichrome stain of an occluded artery with a blood filled microvessel (MV). Here, the plaque (P), media (M), and Internal (IEL) and External (EEL) Elastic Lamina regions are clearly shown for easier delineation of layer boundaries. Bar represents 500μm. C) shows the vessel in B with a PE-Cy5 fluorescent labelled CD31 stained antibody imaged at 5x. The microvessel (MV) has endothelialized and consequently fluoresces. The elastic layers (IEL and EEL) autofluoresce and are also seen. D) shows a 20x image of the same sample shown in C), showing in more detail the microvessel (MV) and the IEL. ..................................................... 40 x