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IN VIVO MRI OF MOUSE HEART AT 11.7 T: MONITORING OF STEM CELL THERAPY FOR MYOCARDIAL INFARCTION AND EVALUATION OF CARDIAC HYPERTROPHY DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Aditi C. Kulkarni, M. Sc. The Ohio State University 2008 Dissertation Committee Approved by Periannan Kuppusamy, PhD, Adviser Petra Schmalbrock, PhD Hiranmoy Das, PhD Adviser Graduate Program in Biophysics Copyright by Aditi C. Kulkarni 2008 ABSTRACT Cardiovascular disease (CVD) is one of the major causes of morbidity and mortality in the western world. It accounts for more than a third of the deaths in the United States and is a serious cause of concern. Early detection of the abnormal cardiovascular conditions may help in their diagnosis and treatment, thus, reducing the mortality associated with them. Visualization of the heart and the blood vessels may help in the detection and diagnosis of these diseases in their early stage of development. Many techniques such as X ray computed tomography (CT), angiography, magnetic resonance imaging (MRI), echocardiography and nuclear imaging are used for cardiac imaging in the clinic. Among these, MRI stands out due to its advantages such as noninvasiveness, high spatial and temporal resolution, and repeatability and reproducibility of the measurements. It can also be used to ii obtain functional data from the structural information of the heart, making it a valuable diagnostic tool for various cardiac pathologies. This dissertation reports the development of cardiac MR imaging methods and its application to cardiac disease models at high magnetic field (11.7 T). Mouse was chosen as the animal model for studying cardiac disease due to several reasons including anatomical similarity of mouse and human hearts, similarity of murine cardiovascular disorders to human, and ease of genetic manipulation in mouse. The development of a cardiac MRI (CMRI) method for mouse at high magnetic field (11.7 T) was the main objective of this dissertation. Optimization of hardware and software parameters was performed to obtain images of the mouse heart, which is characterized by its small size and very fast motion. Methods for data analysis were developed to obtain functional data (ejection fraction, stroke volume, cardiac output etc) from the anatomical and cine images of the mouse heart. M mode echocardiography (ECHO) is one of the most widely used methods for functional analysis of the heart. Therefore, CMRI was compared to ECHO for functional analysis of healthy and infarcted mouse heart. Although the absolute iii values of functional parameters obtained from the ECHO and CMRI were different, they showed similar trends over time. In general, there was agreement in the ejection fraction measurements made obtained from the two methods. After development and validation of the method, CMRI was employed for structural and functional evaluation of mouse heart after myocardial infarction (MI). MI was induced in the mice by permanent ligation of the left coronary artery. CMRI was able to detect the structural changes in the MI heart. It also showed a significant decrease in the function of mouse heart after MI. Another application of CMRI was to assess the cardiac hypertrophy in a transgenic mouse model of chronic hypertension. The hypothesis was that chronic hypertension, created by the transgenic model, leads to cardiac hypertrophy. Previous studies using ECHO were not able to substantially support this hypothesis. However, CMRI of these transgenic mice detected the decrease in the cardiac function and increase in the wall of the left ventricle, thus validating the model itself. Thus, CMRI proved to be a valuable and unique diagnostic tool for assessing cardiac hypertrophy. iv Cardiomyoplasty, or implantation of stem cells into the infarcted heart muscle, is emerging as a promising approach for cardiac therapy. Different types of stem cells have been extensively studied for cardiac tissue regeneration, but very few studies have investigated the fate of transplanted stem cells. In this study, CMRI was used to monitor labeled stem cells that were transplanted in the mouse heart after MI. Mouse mesenchymal stem cells were labeled with superparamagnetic iron oxide particles (SPIO), before transplantation in the mouse heart. An optimized CMRI method was developed and tested in vitro, as well as, ex vivo. CMRI enabled the detection of the cells in vivo and their visualization for up to 4 weeks. In conclusion, in this dissertation the development and validation of cardiac MRI methods for imaging mouse heart at high magnetic field (11.7 T) have been performed successfully. These methods were applied to the evaluation of myocardial infarction and cardiac hypertrophy in mouse and the monitoring of labeled stem cells after their transplantation in the infarcted heart. v To Aai-Baba For believing in me, always vi ACKNOWLEDGMENTS This dissertation work is the culmination of twenty odd years of educational journey. I would like to begin by thanking all who have been directly or indirectly associated it and have influenced me. Although I may not be able to mention all the names, I would like to thank you all for helping me work towards my goal. Firstly, I would like to express my sincere gratitude towards my adviser Dr. P Kuppusamy for giving me the opportunity to work on this project. Despite his myriad responsibilities, he was always available for his students and ensured that we received all the resources and support needed for our projects. I have learnt a lot from him, on professional as well as personal levels. It s a testimony to his deep sense of responsibility, genuine caring attitude and confidence in his students that I am completing my dissertation work in the field of magnetic resonance imaging. I would like to thank my dissertation committee members Prof. Petra Schmalbrock and Prof. Hiranmoy Das for their help and support. I am thankful to Prof. Jay L Zweier and Prof. Chandan K Sen for being a part of my candidacy examination committee. Special thanks to Prof. Schmalborck for her staunch support during some rough patches which would have been difficult to cross without her on my side. vii I am thankful to Dr. Mahmood Khan for his help with the animal models. He helped me focus my project work and always had a few words of encouragement. His enthusiasm is infectious. I would also like to thank our collaborators, Dr. Hamdy Hassanain and Mahmood Awad for their help with the transgenic animal model. Dr. Anna Bratasz has the sole credit for making me animal and EPR literate. She is the best lab instructor ever! Thanks to Dr. Surya Gnyawali for guiding me when I was lost in the jungle of cardiac MRI optimization. Nancy Trigg and Brian Rivera were my genies who provided me with whatever I needed whenever I needed it. I am especially grateful to M. Lakshmi Kuppusamy for making Columbus home away from home. Her love and care, not to forget the scrumptious idli dosas, have saved my mother countless hours of worry. Overall, I was fortunate to be a part of a happy and nourishing workgroup that helped me grow as a researcher and as a person. Thank you all! This journey would have been very lonely without my friends. I am thankful to all my friends for their love and support. Thanks to Deepti, Guru, Jonathan, Shabnam, Simi, Soma and Vinh for making Columbus and grad school an enjoyable experience. I owe a huge debt of gratitude to Deepti, Anamika, Subhangi and Aparajita. Even after being with me physically and (more often than not) telephonically for over a decade, they still consider me a close friend. If that is not the proof of their courage and patience, I don t know what is! viii I would also like to thank my mentors back home who helped shape my mind. I am grateful to Prof. K. C. Sharma, Prof. P. N. Sen and Dr. A. P. Kesarkar for taking me under their wing and easing me into research. I am blessed with a wonderful family. Their unconditional love and unwavering support has often humbled me. They were with me every step of the way, especially when the road got a bit too bumpy. I am grateful to be a part of their life. Abhijit has been the lighthouse of this journey. No part of this would ever have happened without him in my life. Thank you, for not letting me give up when I was tempted to take the easy way out, and giving me the strength and courage to continue. And lastly, Too karta, mee tar karan; tujhech dene, tulach arpan. ix VITA December Born Pune, India B.Sc., Physics, University of Pune, Pune, India M.Sc., Physics, University of Pune, Pune, India 2005 present...graduate Research Associate, The Ohio State University, Columbus, Ohio PUBLICATIONS Kulkarni AC, Bratasz A, Rivera B, Krishna MC, Kuppusamy P. Redox Mapping of Biological Samples Using EPR Imaging. Israel J. Chem. 2008; 48(1): Kulkarni AC, Kuppusamy P, Parinandi N. Oxygen, the lead actor in the pathophysiologic drama: enactment of the trinity of normoxia, hypoxia, and hyperoxia in disease and therapy. Antioxid Redox Signal Oct;9(10): Bratasz A, Kulkarni AC, Kuppusamy P.A highly sensitive biocompatible spin probe for imaging of oxygen concentration in tissues. Biophys J Apr 15;92(8): Raut JS, Bhattad P, Kulkarni AC, Naik VM. ʺMicro potteryʺ marangoni effect driven assembly of amphiphilic fibers. Langmuir Jan 18;21(2): FIELDS OF STUDY Major Field: Biophysics x TABLE OF CONTENTS Abstract...ii Acknowledgments.....vii Vita...x List of Tables......xvi List of Figures...xvii Chapters: 1. Introduction Heart: Structure and function Choosing mouse model Need for cardiac imaging Cardiac imaging techniques Literature review for CMRI in mouse List of abbreviations Overview of the dissertation Cardiac magnetic resonance imaging Introduction Protocol for cardiac imaging of mouse MR scanner used for mouse cardiac imaging...17 xi 2.2.2 Animal preparation and set up Gating strategies for triggered cardiac imaging Cardiac MR data acquisition choice of pulse sequence Procedure for getting short axis images of mouse heart MR Image processing to get cardiac functional parameters Statistical methods used for data analysis Results and discussion Summary Comparison of MRI and echocardiography to assess cardiac function in a mouse model of myocardial infarction Introduction Motivation for the study Cardiac functional analysis by echocardiography Study design Materials and Methods Animals Induction of myocardial infarction Cardiac MR imaging Image processing Echocardiography measurements Statistical analysis Results Discussion...49 xii 3.5 Summary and Conclusion Structural and functional evaluation of mouse heart after myocardial infarction Introduction Myocardial infarction Left coronary artery ligation model for MI Study design Materials and Methods Animals Induction of myocardial infarction Oxygen measurement (po2) using EPR oximetry Cardiac MR imaging Image processing Visualization of fibrosis Statistical analysis Results Discussion Summary ad Conclusion Noninvasive assessment of cardiac function in a transgenic model of cardiac hypertrophy Introduction Cardiac hypertrophy CMRI as a tool for the detection and diagnosis of cardiac hypertrophy...69 xiii 5.1.3 Transgenic animal model of cardiac hypertrophy Study design Materials and Methods Animals Cardiac MR imaging Image processing Echocardiography studies Statistical analysis Results Discussion Summary and Conclusion In vivo monitoring of SPIO labeled stem cells transplanted in infarct mouse heart Introduction Stem cell therapy for cardiac repair and regeneration Stem cell tracking using MRI Study design Materials and Methods Animals Culturing of MSCs Labeling of MSCs with SPIOs Myocardial infarction and cell transplantation In vitro MR imaging Ex vivo imaging...89 xiv 6.2.7 In vivo imaging Prussian blue staining Results Discussion Summary and Conclusion Summary...99 Bibliography xv LIST OF TABLES Table 1.1 Comparison of cardiac imaging modalities...9 Table 1.2 List of frequently used abbreviations...14 Table 3.1 Cardiac functional parameters obtained from CMRI and ECHO...46 Table 5.1: Types of cardiac hypertrophy and their effects on the cardiac tissue...69 Table 5.2: Cardiac functional parameters obtained from CMRI and echocardiography..77 xvi LIST OF FIGURES Figure 1.1 Anatomy of human heart...2 Figure 1.2 Anatomical similarities of postnatal mouse and human heart...4 Figure 1.3 Cardiac imaging modalities...8 Figure 2.1 Steps in the development and optimization of CMR imaging protocol...16 Figure 2.2 MR scanner used for mouse cardiac imaging...17 Figure 2.3 Monitoring of heart rate and respiration of the mouse...19 Figure 2.4 Small animal monitoring and gating system used for cardiac imaging...21 Figure 2.5 Additional measures to improve signal to noise ratio of the ECG signal...22 Figure 2.6 Quality of ECG signal and optimization of detection parameters...23 Figure 2.7 Timing diagram of FLASH sequence used for cardiac imaging...26 Figure 2.8 Acquisition of cine loop images of the heart...27 Figure 2.9 Unsegmented k space filling of an ECG triggered GRE sequence...28 Figure 2.10 Protocol for obtaining short axis cardiac images of mouse...30 Figure 2.11 Regions of interests (ROIs) with and without papillary muscles...32 Figure 2.12 Manual planimetry of mouse heart for functional analysis...33 Figure 2.13 Choice of gating for image acquisition...35 Figure 2.14 Bright blood images of mouse heart at end diastolic phase...36 Figure 3.1 Typical M mode echocardiogram of a mouse heart...39 Figure 3.2 Slice positioning in CMRI and ECHO...40 xvii Figure 3.3 CMR images of mouse hearts at 4 weeks post MI...44 Figure 3.4 M mode echocardiograms of Control and MI mice at 4 weeks post MI...45 Figure 3.5 Comparison of EF of control and MI groups...45 Figure 3.6 Correlations between EDV and ESV measurements of ECHO and MRI...46 Figure 3.7 Comparison of mid papillary MRI with ECHO...47 Figure 3.8 Comparison of global MRI with ECHO...48 Figure 3.9 Reproducibility of EF measurements by ECHO and MRI...48 Figure 4.1 Myocardial infarction as a result of occlusion in the left coronary artery...53 Figure 4.2 Ligation of the left coronary artery in mice Figure 4.3 Measurement of myocardial oxygenation using EPR oximetry...58 Figure 4.4 Bright blood images of control and MI heart as a function of distance from the apex...59 Figure 4.5 Structural changes in mouse heart after myocardial infarction...59 Figure 4.6 LV remodeling in the mouse heart 4 weeks after the surgery...60 Figure 4.7 Cine images of the cardiac cycle of control and MI heart...61 Figure 4.8 Progressive loss of cardiac function after MI...62 Figure 4.9 Variability in the extent of remodeling in MI...63 Figure 4.10 Histological assessment of myocardial infarction in mouse heart...63 Figure 5.1 Transgenic mouse model of cardiac hypertrophy used in this study...70 Figure 5.2 MR images of transgenic (mutant RacD overexpressed) and control mouse hearts...74 xviii Figure 5.3 Short axis images of hearts of control and transgenic mouse hearts at enddiastolic and end systolic states...75 Figure 5.4 Mid ventricular short axis images of control and transgenic mouse hearts...76 Figure 5.5 Cardiac functional parameters computed from MRI and ECHO of mouse hearts...78 Figure 5.6 Comparison of LV width and EF obtained from MRI and echocardiography of mouse hearts...79 Figure 5.7 Effect of age on ejection fraction, LV mass and body weight...80 Figure 6.1 Confirmation of labeling of MSCs with SPIOs...91 Figure 6.2 In vitro images of SPIO labeled stem cells...91 Figure 6.3 Ex vivo images of mouse heart after transplantation of the labeled cells...92 Figure 6.4 Locating the SPIO labeled stem cells in mouse heart...93 Figure 6.5 Monitoring the SPIO labeled stem cells in mouse heart (Short axis images).94 Figure 6.6 Monitoring the SPIO labeled stem cells in mouse heart (Long axis images).95 Figure 6.7 Prussian blue staining 4 weeks after stem cell transplantation...96 xix CHAPTER 1 INTRODUCTION 1. Introduction The overall goal of this dissertation was to develop cardiac magnetic resonance methods for imaging mouse heart at 11.7 T. This chapter provides the general introduction to the work done as a part of this dissertation. It begins with an account of heart structure and function. It explains the choice of mouse as an animal model for studying cardiac pathologies. Further, it discusses the need for cardiac imaging and provides a brief review of currently used noninvasive cardiac imaging methods. This is followed by a report on the current status of cardiac MRI in studying mouse heart. An overview of the dissertation is given at the end of this chapter. 1.1 HEART: STRUCTURE AND FUNCTION Human heart is a four chambered muscular organ that pumps blood throughout the body. Heart is situated in the thoracic cavity. It is covered by pericardium and surrounded by lungs. Figure 1.1 shows the anatomy of the human heart, illustrating the four chambers of the heart along with the major blood vessels and valves. The right side of the heart has deoxygenated blood while the left side handles oxygenated blood. The atria pump blood into the ventricles while the ventricles pump blood to the lungs (right ventricles) and to the body (left ventricle). The flow of the blood into and out of the ventricles is controlled by various unidirectional valves. 1 In humans, heart beats about times per minute. This motion is involuntary and is controlled by the cardiac electrical conduction system and neural input. The perpetual contraction relaxation of the heart is responsible for sustaining a positive blood pressure in the arteries, ensuring adequate supply of blood and nutrients to body organs. The efficiency of cardiac function is measured using many parameters termed as cardiac functional parameters. Figure 1.1 Anatomy of human heart Human heart consists of four chambers (two atria and two ventricles) that are supplied by major blood vessels. The valves (mitral and tricuspid) regulate the blood flow between the chambers and into the blood vessels. Cardiac functional parameters are mainly used for quantifying the pumping capacity of the heart. Since left ventricle (LV) is responsible for blood perfusion to body organs, unless otherwise specified, these parameters are used for describing LV function. The main parameters used for describing function of the heart are obtained from the end diastolic volume (EDV) and end systolic volume (ESV) of the LV. 2 The functional parameters are: End diastolic volume (EDV): Volume of the LV chamber when heart is completely relaxed End systolic volume (ESV): Volume of the LV chamber when the heart is completely contracted Stroke volume (SV): Amount of blood pumped out of the LV in one heart cycle Cardiac output (CO): Volume of the blood being pumped by the LV in a minute Ejection fraction (EF): Fraction of the blood pumped out of the ventricle per heart cycle The normal values of these parameters vary with the measurement techniques and the health of the individual. However, there are some general guidelines to help the physicians determine the efficiency of cardiac function. For instance, the normal value of EF is considered between %. Most of the cardiac pathologies can be diagnosed on the basis of decreased efficiency of the heart that is reflected in the functional parameters. To study cardiac disease and their effect on the function of the heart, many animal models, such as sheep, pigs, dogs, have been used. Recently small animal models such as rats and mice are being used for these studies. For this dissertation work, mouse model was chosen over the others. 1.2 CHOOSING MOUSE MODEL Mouse model has many advantages over other small and large animal models that have been used to study cardiovascular disease [1]. Mice are relatively inexpensive, have small gestation period and
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