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Proton and sodium MRI assessment of myocardial viability Eissa Nadi Eissa Agour Proton and sodium MRI assessment of myocardial viability Over het afbeelden van de levensvatbaarheid van hartspier middels proton en natrium ion magnetische resonantie (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. G.J. van der Zwaan, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op woensdag 31 oktober 2012 des ochtends te 10.30 uur door Eissa Nadi Eissa Agour geboren op 16 January 1979, Riyadh, Saudi Arabia Promotoren: Prof. dr. P.A.F.M. Doevendans Prof. dr. C.J.A. van Echteld Co-promotoren: Dr.ir. G.J. Strijkers Dr. F. Arslan The research described in this thesis was supported by a grant of the Dutch Heart Foundation (DHF-2006B066) and by the Netherlands Heart Institute (ICIN). Financial support by the Dutch Heart Foundation for the publication of this thesis is gratefully acknowledged Financial support by the Heart & Lung Foundation Utrecht for the publication of this thesis is gratefully acknowledged ISBN: 978-90-8891-506-2 Printed & lay-out by: Proefschriftmaken.nl || Printyourthesis.com Published by: Uitgeverij BOXPress, Oisterwijk This thesis is dedicated to my parents Content Chapter 1 General introduction 9 Part I - Proton MRI Chapter 2 Quantitative T2* assessment of acute and chronic myocardial ischemia/reperfusion injury in mice Chapter 3 Quantitative changes in T2* reflect remodeling of both remote and ischemic myocardium in a murine heart failure model Chapter 4 25 47 Lack of circulating Toll-like receptor 2 promotes survival and prevents adverse remodeling after myocardial infarction 63 Part II - Sodium MRI Chapter 5 Na Chemical Shift Imaging and Gd Enhancement of 23 myocardial edema Chapter 6 81 Differentiation of myocardial injury in various infarct models with 23Na Chemical Shift Imaging and Late Gd Enhancement MRI 105 Part III Chapter 7 Summarizing discussion 131 Brief summary in Dutch - Korte samenvatting in het Nederlands 141 List of publications 143 Acknowledgements - Dankwoord 147 Curriculum vitae 151 7 Chapter 1 General Introduction This chapter provides a brief introduction of myocardial injury, a brief summary of cardiac imaging methods, followed by an overview of 1H MRI and 23Na MRI methods to assess myocardial viability. Finally, study aim and thesis outline are presented. 9 Chapter 1 1.1 Myocardial injury Ischemic heart disease is a leading cause of morbidity and mortality globally. Ischemic heart disease is characterized by partial or total lack of oxygen-rich blood supply to the myocardium. Although early reperfusion can salvage myocardium at risk, the beneficial effect after reperfusion is not always apparent. The severity of myocardial injury is influenced by many factors, not only the occlusion location within the coronary arteries, but also the ischemic duration. If the blood flow is restored (reperfusion) after a brief, mildly ischemic period [1], the myocardial cellular injury may still be reversible. In contrast, a prolonged, totally ischemic period ultimately causes irreversible cellular injury and leads to cell death. Such severe injury likely results in impairment of cardiac function. After ischemia and reperfusion, the myocardium undergoes a complex cascade of dynamic changes, both the necrotic and viable myocardium [2]. Therefore, in ischemic heart disease, distinguishing between viable and non-viable myocardium is of high clinical importance, as viable myocardium may benefit from revascularization whereas scar tissue will not. Equally important is the differentiation from non-ischemic heart disease. Sensitive and accurate characterization of the myocardial status is therefore essential for accurate diagnosis and suitable therapy planning. Ultimately, this may significantly improve survival and outcome [3;4] Rodent models of myocardial infarction To mitigate the risk of myocardial infarction and morbidity in patients, the search for alternative therapeutic interventions and in suitable experimental myocardial infarction (MI) models is ongoing. Research using animal models helps identifying novel molecular mechanisms and therapeutic targets as well as suitable intervention methods, providing valuable information for clinical translation. Many researchers have investigated the progress of myocardial infarction in well-defined, in vivo small animal models of infarction [5]. Already In 1895, Oscar Langendorff pioneered the isolated perfused rodent heart. Since then Langendorff perfused hearts have 10 General Introduction yielded valuable data, increasing our understanding of the fundamental physiology of the heart [6]., despite the difference in cardiac anatomy in these animal models, as well as the accelerated infarct development process and the higher heart beat, in comparison to humans 1.2 Imaging methods for assessing myocardial viability Many studies have shown that performing ECG tests [7] and probing specific blood biomarkers [8] are clinically valuable for the detection of myocyte death. Such tests therefore are usually performed as the first diagnostic assessment for suspected MI. However, more precise and detailed information about the extent of MI can be obtained using medical imaging. Many clinical and pre-clinical studies have demonstrated the potential of non-invasive imaging to characterize myocardium throughout the development of MI [9;10], including ultrasound [11], computed tomography CT [12], single photon emission computed tomography (SPECT) [13], positron emission tomography (PET) [14], and magnetic resonance imaging (MRI) [15]. Each of which has unique strengths and limitations. Furthermore, combining different techniques might lead to a better understanding of the disease and help in more precise and accurate estimation of the extent of infarction [9;15;16]. Echocardiography has emerged as a powerful tool for imaging of cardiac function and structure. Echocardiography techniques such as tissue Doppler and strain imaging provide interesting new readouts [17]. Still the interpretation of images remains a challenge and operator experience is critical [11], particularly for imaging small animals. Parallel to the recent advances in clinical and research fields, computed tomography (CT) has evolved into a reliable cardiac imaging modality [10]. Advanced multi slice CT is a powerful modality in cardiac imaging and is preferred above MRI for visualizing coronary arteries and quantifying calcium content. SPECT and PET are widely used nuclear medical imaging methods for assessing myocardial perfusion and metabolism. On the downside, these imaging methods require injection of radiotracers and suffer from poor spatial resolution 11 Chapter 1 [18;19]. MRI has a lot to offer in clinical and experimental cardiovascular research. It combines high spatial resolution with acceptable imaging time. Cardiovascular MRI is capable of providing a variety of anatomical, functional and physiological readouts of the heart in health and disease, which aid in clinical decision-making and provide fundamental insights in heart function. A number of these readouts are illustrated in figure 1 and detailed in the next section. 1.3 Cardiac Magnetic Resonance imaging (CMRI) Distinguishing viable from non-viable myocardium is essential for prognosis of patient with ischemic heart disease. The available clinical diagnostic modalities to detect myocardial viability and test sarcolemmal function include PET and MRI. However, PET scans suffer from poor spatial resolution and involve appreciable radiation exposure for the patient, in particular in serial examinations. On the other hand, MRI shows better spatial resolution and does not use ionizing radiation. Moreover, a variety of cardiovascular MRI methods is available, both clinically and pre-clinically. Most cardiovascular MR images are acquired using hydrogen nuclei (protons). However, MR images can also be obtained using other nuclei, such as 13C, 31P and 23Na. In this thesis, we will focus on proton and sodium nuclei for MRI. Proton (1H) MRI Many researchers have investigated the potential of 1H based cardiovascular MRI methods, mainly because of its relatively high signal to noise ratio. The flexibility to adjust MR pulse sequence makes it possible to obtain high spatial resolution MR images with different contrast for different soft tissues. Cine MRI is the most commonly used MRI tool which provided valuable information related to cardiac morphology and function. Cine MRI is commonly used to yield a movie of a single or multiple slices of the heart. Valuable information about heart size and function can be obtained; such as LV volume, ejection fraction (EF), and wall thickness and thickening. 12 General Introduction To monitor myocardial perfusion, a sequence of MR images are acquired directly after intravenously injected bolus of Gd-based contrast agent through the myocardium, in a so-called first pass myocardial perfusion MRI. Figure 1. Various MRI readouts of mouse hearts with (A) Cine MRI at baseline and 28 days after Ischemia Reperfusion (I/R) injury. (B)T2* weighted MRI 2 days after injection with micro RNA-55 transfected CMPCs with SPIOS labeling. The red arrows indicate SPIOS labeled CMPCs. (C) Quantitative T2* mapping of and Late Gadolinium enhancement (LGE) MRI of the same mouse at day 1 post I/R injury. Red arrows indicate infarct areas. MR longitudinal relaxation time (T1) and transverse relaxation time (T2) have different values for different components and biological structures, which can be identified using T1- weighted and T2‑weighted MRI. In addition, MR sequence parameters can be adapted to achieve specific resolution and contrast to track pathological changes and identify injured myocardium. For instance, a T1-weighted MRI sequence in combination with a gadolinium contrast agent injection is suitable to determine infarct size after a delay between contrast injection and MRI scan, in so-called Late Gadolinium Enhancement (LGE) MRI [20]. LGE MRI shows enhancement of the injured myocardium. However, the enhancement pattern in LGE MRI can be complicated, by the formation of interstitial edema, which may occur in the peri-infarct zone or in other cardiac diseases [21-23]. The sensitivity of the T2 relaxation time to myocardial water content makes T2‑weighted MRI suitable to image myocardial edema as an 13 Chapter 1 indicator of the area at risk (AAR) following myocardial infarction (MI) in mice [24] and in human [25]. T2* MRI has been used as an imaging tool to diagnose the presence of hemorrhage and myocardial iron[26]. Apparently, T1-weighted and T2‑weighted MRI show great promise to delineate injured myocardium both in patients and in animal studies[27-32]. However, both these methods depend on relative regional differences in myocardial signal intensity and are perhaps less suitable to identify globally edematous tissue associated with other disease states, such as hypertension or myocarditis. Alternatively, quantitative relaxation time measurement by MR is an emerging tool in the assessment of myocardial infarction. In quantitative MRI, relaxation times on a standard scale (in ms) are related to specific pathological conditions. Quantitative MR methods, such asT1 mapping,T2 mapping andT2* mapping, enable a quantitative measure of different pathological status of the myocardium, thus eliminating the problem of choosing threshold values. For example, cardiac T1 mapping has proven useful in differentiating healthy from infarcted myocardial tissue and enables quantification of local contrast agent concentration [33]. In addition, cardiac T1 mapping has been used to quantify diffuse myocardial fibrosis in cardiac diseases [34]. T2 mapping was used to distinguish reversible from irreversible myocardial edema [35] and has been recently used to measure the extent of myocardial fibrosis in diabetic mice [34]. Recently O’Regan et al. [36] explored the use of T2* mapping to quantify post‑reperfusion hemorrhage of the heart following percutaneous primary coronary intervention after MI. T2* mapping has also been used to correlate myocardial iron and systolic function in thalassemia patients, who are at risk for cardiomyopathy. Recently, we [37] used T2* mapping in mouse models to assess myocardial infarction (figure 1G) which will be the main subject of chapters 3 and 4 of this thesis. 14 General Introduction Sodium (23Na) MRI Sodium and cell viability Healthy cardiomyocytes maintain ionic gradients across the sarcolemma. Maintaining + this ionic homeostasis is essential to maintain cellular viability and function. The Na + K -ATPase is considered as the main force to maintain these gradients across the + + sarcolemma, This Na -K -ATPase is electrogenic and transports 2 K+ ions into the cell against 3 Na+ out of the cell. Another important exchanger involved in Na+ transport across the cell membrane is the Na+/Ca2+ exchanger, which transport 3 Na+ into the cell in exchange for 1 Ca2+. These and other transport mechanism such as Na+/H+ exchange and Na+/HCO3- are essential to maintain a stable gradients and play a role in maintain cellular volume regulation, but are also involved in dysregulation of ion gradients in myocardial disease states. Viable cell maintain a Na+ concentration gradient across the sarcolemmal with extracellular/intracellular ratio of 140 mM/10 mM. During the very first few minutes + of myocardial ischemia, Nai already starts to rise due to inhibition of the sarcolemmal + + + + + Na -K -ATPase and continued or increased influx through the Na -channel, via the + Na -H exchanger and possibly through other routes [38]. This very early rise in Nai + makes Nai a very sensitive indicator of ischemia. Reperfusion of viable myocardium + + + causes an immediate reactivation of Na -K -ATPase function and a decrease in Nai . + However, if reperfusion is delayed, Nai recovers but remains elevated above the + baseline level. Therefore, Nai is a very sensitive indicator of myocardial viability. Imaging of myocardial sodium (23Na MRI) Proton based MRI is commonly used in the clinic. However, proton based MRI methods, such as T1-weighted and T2-weighted MRI do not provide information on the pathophysiological state of the myocardium at the biochemical level [3]. Imaging of myocardial sodium (23Na) MRI is an alternative method for evaluating myocardial viability via assessment of Na+ homeostasis [39] and cell volume regulation. 15 Chapter 1 The relatively low 23Na concentrations in biological tissues (table 1) and the fast biexponential signal decay make sodium MRI a challenge. However, recent advances in MRI and the availability of higher MR field strengths enable in vivo 23Na MRI in reasonable scan times. Still, 23Na MRI has poor spatial resolution, which is the reason that it is always combined with 1H MRI (figure 2). 1 H Na 23 Nucleus Natural abundance (%) 99.99 100.00 Relative MRI sensitivity (%) 100 9.3 In vivo concentration (mM) H2O: 110 000 Intracellular: 10 Extracellular: 140 Overall in vivo signal 1 4 x1-5 Table 1 Myocardial tissue concentrations and MR sensitivity of 1H and 23Na MRI [40] Clinical 23Na MRI examinations are extremely limited and are mostly concerned with imaging of total sodium (Nat+) [41]. An elevation of total sodium Nat+ signal has been observed after myocardial infarction in a number of studies, both in humans and animals [41-43]. This elevated signal might reflect different patho-physiological conditions., For example, in acute, low-flow ischemia, the Nat+ signal is expected to increase because of unbalanced ion homeostasis and edema formation [41]. Similarly, the Nat signal is significantly elevated during chronic scar formation, because of the expansion of the extracellular space in scar [40]. Using Nat+ MRI alone would not discriminate between these two conditions. 16 General Introduction Figure 2 A typical example of perfused rat heart with Na Chemical Shift Imaging CSI 23 representing extracellular Na (Nae) and H Late gadolinium enhanced (LGE) MRI, 1 arrows show injured myocardium in the LGE MRI. More information on the sodium gradient and cellular membrane integrity can be + + obtained by imaging Nai and Nae cellular separately. This requires a paramagnetic chemical shift agent, such as [TmDOTP]5- and a 23Na chemical shift imaging (CSI) sequence. 23Na CSI will be the main topic in the second part of this thesis. As mentioned 23 earlier, due to rapid change of Nai+ during ischemia and reperfusion, Na CSI appears to be an ideal diagnostic modality for early detection of myocardial viability. 1.4 Aim of the study and thesis outline The aim of the studies presented in this thesis was to explore new MRI methods for assessment of myocardial viability. This thesis constitutes of two main parts. Part 1 demonstrates the assessment of myocardial viability using proton MRI methods. Chapter 2 explores the utility of quantitative T2* mapping as a noninvasive technique to characterize the myocardium in the acute and chronic phases of I/R injury in an in vivo mouse model. Chapter 3 explores the utility of quantitative T2* mapping to characterize the myocardium in the acute and chronic phases of permanent coronary artery ligation in a similar in vivo mouse model. Chapter 4 gives an example of how 17 Chapter 1 cine MRI can demonstrate prevention of adverse remodeling after infarction in TLR2/- mice. Part II is dedicated to Sodium MRI in comparison to Proton MRI methods. Chapter 5, describes the development of a “non-infarct model” of extracellular edema in isolated rat hearts based on different perfusion pressures. Subsequently, the influence of myocardial edema is investigated with 23Na CSI in combination with a shift reagent and 1H MRI in combination with a contrast agent. In Chapter 6, 23Na CSI is used to study cellular integrity and ion homeostasis during and after myocardial ischemia in rat heart. Three conditions were studied: acute ischemia and reperfusion, MI 2 hours after reperfusion and chronic MI. Finally, this thesis concludes with a summarizing discussion in chapter 7. Reference List 1. Miura M, Matsuoka H, Saito T, Kanazawa T (1991) The Pathophysiology of Myocardial Stunning - Reversibility, Accumulation and Continuity of the Ischemic Myocardial Damage After Reperfusion. Japanese Circulation Journal-English Edition 55:868-877 2. 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Eur Radiol 17:610-617 26. van den Bos EJ, Baks T, Moelker AD, Kerver W, van Geuns RJ, van der Giessen WJ, Duncker DJ, Wielopolski PA (2006) Magnetic resonance imaging of haemorrhage within reperfused myocardial infarcts: possible interference with iron oxide-labelled cell tracking? Eur Heart J 27:1620-1626 27. Aletras AH, Tilak GS, Natanzon A, Hsu LY, Gonzalez FM, Hoyt RF, Arai AE (2006) Retrospective determination of the area at risk for reperfused acute myocardial infarction with T2weighted cardiac magnetic resonance imaging - Histopathological and displacement encoding with stimulated echoes (DENSE) functional validations. Circulation 113:18651870 28. Edwards NC, Routledge H, Steeds RP (2009) T2-weighted magnetic resonance imaging to assess myocardial oedema in ischaemic heart disease. Heart 95:1357-1361 29. Eitel I, Friedrich MG (2011) T2-weighted cardiovascular magnetic resonance in acute cardiac disease. J Cardiovasc Magn Reson 13:13 30. Raman SV, Simonetti OP, Winner MW, Dickerson JA, He X, Mazzaferri EL, Ambrosio G (2010) Cardiac Magnetic Resonance With Edema Imaging Identifies Myocardium at Risk and Predicts Worse Outcome in Patients With Non-ST-Segment Elevation Acute Coronary Syndrome. Journal of the American College of Cardiology 55:2480-2488 31. Ubachs JFA, Engblom H, Erlinge D, Jovinge S, Hedstrom E, Carlsson M, Arheden H (2010) Cardiovascular magnetic resonance of the myocardium at risk in acute reperfused myocardial infarction: comparison of T2-weighted imaging versus the circumferential endocardial extent of late gadolinium enhancement with transmural projection. Journal of Cardiovascular Magnetic Resonance 12: 32. Wright J, Adriaenssens T, Dymarkowski S, Desmet W, Bogaert J (2009) Quantification of Myocardial Area at Risk With T2-Weighted CMR Comparison With Contrast-Enhanced CMR and Coronary Angiography. Jacc-Cardiovascular Imaging 2:825-831 20 General Introduction 33. Coolen BF, Geelen T, Paulis LE, Nicolay K, Strijkers GJ (2011) Regional contrast agent quantification in a mouse model of myocardial infarction using 3D cardiac T1 mapping. J Cardiovasc Magn Reson 13:56 34. Bun SS, Kober F, Jacquier A, Espinosa L, Kalifa J, Bonzi MF, Kopp F, Lalevee N, Zaffran S, Deharo JC, Cozzone PJ, Bernard M (2012) Value of in vivo T2 measurement for myocardial fibrosis assessment in diabetic mice at 11.75 T. Invest Radiol 47:319-323 35. Gouya H, Vignaux O, Le RP, Chanson P, Bertherat J, Bertagna X, Legmann P (2008) Rapidly reversible myocardial edema in patients with acromegaly: assessment with ultrafast T2 mapping in a single-breath-hold MRI sequence. AJR Am J Roentgenol 190:1576-1582 36. O’Regan DP, Ahmed R, Karunanithy N, Neuwirth C, Tan Y, Durighel G, Hajnal JV, Nadra I, Corbett SJ, Cook SA (2009) Reperfusion hemorrhage following acute myocardial infarction: assessment with T2* mapping and effect on measuring the area at risk. Radiology 250:916922 37. Aguor EN, Arslan F, van de Kolk CW, Nederhoff MG, Doevendans PA, Van Echteld CJ, Pasterkamp G, Strijkers GJ (2012) Quantitative T (2) (*) assessment of acute and chronic myocardial ischemia/reperfusion injury in mice. MAGMA 38. Van Emous JG, Schreur JH, Ruigrok TJ, Van Echteld CJ (1998) Both Na+-K+ ATPase and Na +-H+ exchanger are immediately active upon post-ischemic reperfusion in isolated rat hearts. J Mol Cell Cardiol 30:337-348 39. Ouwerkerk R, Bleich KB, Gillen JS, Pomper MG, Bottomley PA (2003) Tissue sodium concentration in human brain tumors as measured with 23Na MR imaging. Radiology 227:529-537 40. Ridgway JP (2010) Cardiovascular magnetic resonance physics for clinicians: part I. J Cardiovasc Magn Reson 12:71 41. Kim RJ, Lima JA, Chen EL, Reeder SB, Klocke FJ, Zerhouni EA, Judd RM (1997) Fast 23Na magnetic resonance imaging of acute reperfused myocardial infarction. Potential to assess myocardial viability. Circulation 95:1877-1885 42. Constantinides CD, Kraitchman DL, O’Brien KO, Boada FE, Gillen J, Bottomley PA (2001) Noninvasive quantification of total sodium concentrations in acute reperfused myocardial infarction using 23Na MRI. Magn Reson Med 46:1144-1151 43. Kim RJ, Judd RM, Chen EL, Fieno DS, Parrish TB, Lima JA (1999) Relationship of elevated 23Na magnetic resonance image intensity to infarct size after acute reperfused myocardial infarction. Circulation 100:185-192 21 Part I Proton MRI 23 Chapter 2 Quantitative T2* assessment of acute and chronic myocardial ischemia/reperfusion injury in mice Eissa N. E. Aguor, Fatih Arslan, Cees W.A. van de Kolk, Marcel G.J. Nederhoff, Pieter A. Doevendans, Cees J.A. van Echteld, Gerard Pasterkamp, Gustav J. Strijkers MAGMA. 2012, DOI 10.1007/s10334-012-0304-0 25 Chapter 2 Abstract Object Imaging of myocardial infarct composition is essential to assess efficacy of emerging therapeutics. T2* mapping has the potential to image myocardial hemorrhage and fibrosis by virtue of its short T2*. We aimed to quantify T2* in acute and chronic myocardial ischemia/reperfusion (I/R) injury in mice. Materials and methods I/R‑injury was induced in C57BL/6 mice (n=9). Sham‑operated mice (n=8) served as controls. MRI was performed at baseline, and 1, 7 and 28 days after surgery. MRI at 9.4 T consisted of Cine, T2*‑mapping and late-gadolinium-enhancement (LGE). Mice (n=6) were histologically assessed for hemorrhage and collagen in the fibrotic scar. Results Baseline T2*‑values were 17.1±2.0 ms. At day 1, LGE displayed a homogeneous infarct enhancement. T2* in infarct (12.0±1.1 ms) and remote myocardium (13.9±0.8 ms) was lower than at baseline. On days 7 and 28, LGE was heterogeneous. T2* in the infarct decreased to 7.9±0.7 ms and 6.4±0.7 ms, whereas T2*‑values in the remote myocardium were 14.2±1.1 ms and 15.6±1.0 ms. Histology revealed deposition of iron and collagen in parallel with decreased T2*. Conclusion T2*‑values are dynamic during infarct development and decrease significantly during scar maturation. In the acute phase, T2* values in infarcted myocardium differ significantly from those in the chronic phase. T2*‑mapping was able to confirm the presence of a chronic infarction in cases where LGE was inconclusive. Hence, T2* may be used to discriminate between acute and chronic infarctions. Keywords cardiovascular magnetic resonance, myocardial infarction, T2*, LGE, mouse, fibrosis, hemorrhage 26 Quantitative T2* assessment of acute and chronic myocardial ischemia/reperfusion injury in mice 2.1 Introduction Despite recent advances in therapeutics and diagnostics, morbidity and mortality after myocardial infarction (MI) remains a great socio-economic burden in Western societies. Early identification of patients with sub-optimal cardiac recovery is of great importance to improve clinical management. For this reason, both functional and structural characterization of the infarcted myocardium is important for risk stratification of patients after MI. Technological advances in Cardiovascular Magnetic Resonance (CMR) have demonstrated different ways to track and characterize MI at different time points [1-6]. CMR has been applied to analyze inflammation, edema, necrosis, fibrosis, left-ventricular (LV) remodeling and metabolism in myocardial infarction [7-15]. Pathological changes in the injured myocardium, such as the presence of fibrosis, hemorrhage and edema, influence the signal intensity on T1‑ and T2‑weighted imaging [7,16-18]. CMR is thus considered as one of the most important imaging tools in the assessment of the heart after MI. Typically, T1-weighted imaging is performed some time after injecting a gadolinium‑based contrast agent to delineate injured myocardium in a Late Gadolinium Enhancement (LGE) scan [19]. The enhancement mechanism of infarcted tissue is related to the extent of the extracellular volume, which is associated with the delayed washout relative to the healthy myocardium. LGE is widely used and without competition considered the gold standard to assess infarct size. Nevertheless, there are certain limitations of LGE related to pathological processes after the onset of infarction. In the acute and sub-acute phase after successful reperfusion of the ischemic myocardium, the transmural infarct extent is not stable and an overestimation of infarct size has been reported [20]. In the chronic phase, excessive and heterogeneous collagen deposition during scar maturation may hamper signal enhancement by LGE. In view of the above limitations, several alternative contrast mechanisms are explored to study the pathophysiological changes in the injured myocardium. The sensitivity 27 Chapter 2 of the T2 relaxation time to myocardial water content has led to the application of T2‑weighted CMR protocols to image myocardial edema as an indicator of inflammation and necrosis in acute MI [13,15,21-24]. T2* contrast has been explored as a tool to diagnose the presence of hemorrhage and myocardial iron [1-5,25-28]. Recently, it was demonstrated that T2* has the potential to image fibrosis in the infarct zone using an ultra-short echo time (UTE) technique [29]. In this paper, we hypothesize that the structural and pathological changes in acute and chronic MI, such as the presence and development of myocardial hemorrhage and fibrotic tissue progressively influence T2* values in the infarcted myocardium. To investigate this, we quantified T2* using a T2*‑mapping protocol longitudinally in acute and chronic ischemia/reperfusion (I/R) infarcts in mice. We explored the utility of T2* mapping as a complementary tool to LGE to characterize the infarcted myocardium. 2.2 Materials and methods Experimental design C57Bl6/J mice (n=23, body weight=20–25 g) were used in this study. CMR measurements were performed at baseline (BL), 1 day (acute infarct), 7 days and 28 days (chronic infarct) after induction of myocardial infarction (MI) by 30 min transient ligation of the left coronary artery (n=9). A sham‑operated group (n=8) served as control. The CMR protocol for the infarct group consisted of a multi‑gradient‑echo quantitative T2*‑mapping sequence, traditional LGE to assess the location and size of the infarct, and gradient‑echo Cine imaging to determine cardiac function. The sham-operated (control) group underwent gradient‑echo Cine imaging only to evaluate cardiac function. In the first group of 9 mice with MI, 1 mouse died at day 28 during the CMR scan. Another 3 mice of this group presented a small infarct on LGE images at day 1, but no chronic myocardial infarct at day 7 and 28 (as exemplified in Fig. 3). A separate set of mice with myocardial infarction (n=6) were sacrificed either at day 1 (n=2), 7 (n=2), or 28 (n=2) for the histopathological characterization of myocardial tissues. All animal experiments were performed in accordance with the national guidelines 28 Quantitative T2* assessment of acute and chronic myocardial ischemia/reperfusion injury in mice on animal care and with prior approval by the Animal Experimentation Committee of Utrecht University. Mouse model of myocardial I/R injury Mice underwent myocardial I/R surgery as previously described [30]. Briefly, mice were anesthetized with a mixture of Fentanyl (Jansen-Cilag) 0.05 mg/kg, Dormicum (Roche) 5 mg/kg, and medetomidine 0.5 mg/kg through an intraperitoneal injection. A core body temperature of about 37 °C was maintained during surgery by continuous monitoring with a rectal thermometer and an automatic heating blanket. Mice were intubated and ventilated (Harvard Apparatus Inc, Holliston, Mass) with 100% oxygen. The left coronary artery (LCA) was ligated for 30 min with an 8-0 Ethilon (Ethicon) suture with a section of polyethylene-10 tubing placed over the left coronary artery. Ischemia was confirmed by bleaching of the myocardium and ventricular tachyarrhythmia. Reperfusion was initiated by release of the ligature and removal of the polyethylene-10 tubing. In sham-operated animals, the suture was placed beneath the left coronary artery without ligation. Reperfusion of the endangered myocardium was characterized by typical hyperemia in the first few minutes. The chest wall was closed, and the animals subcutaneously received Antisedan (Pfizer) 2.5 mg/kg, Anexate (Roche) 0.5 mg/kg, and Temgesic (Schering-Plough) 0.1 mg/kg. CMR protocol A vertical 9.4 T and 89-mm-diameter bore scanner was used, equipped with 1500 mT/m gradients (Bruker BioSpin GmbH, Ettlingen, Germany). Mice were anesthetized with 1.5‑3 vol% isoflurane in a 2:1 mixture of air (0.3 l/min) and oxygen (0.15 l/min). The mice were put on a custom‑built cradle and positioned inside a 3-cm‑diameter quadrature volume coil. During the CMR examination anesthesia was maintained at 1.5‑2.5 vol% isoflurane to keep the respiratory rate at 50-60 bpm. We did not use ECG leads for cardiac triggering but derived both cardiac and respiratory motion signals from a pressure pad placed under the chest of the mouse. 29 Chapter 2 The CMR protocol consisted of the following steps. After global shimming and RF pulse calibration, 2- and 4-chamber view scout scans were made to plan a single mid-cavity short-axis slice. Cine imaging was performed with a retrospectively‑triggered (selfgated) gradient‑echo sequence [31], with the following parameters: TR = 6.8 ms, TE = 1.9 ms, number of movie frames = 15, slice thickness = 1 mm, matrix = 256x256, field‑of‑view = 3x3 cm2. Because of the slight difference of hearts size between mice and the dilation of each heart after infarction, seven to 9 short‑axis slices with inter‑slice distance of 1 mm and no slice gap were measured to cover the heart from apex to base. Based on the Cine short‑axis images, a mid axial slice was selected that included an area of remote viable tissue as well as a substantial infarct area. T2* mapping was performed in this slice using a cardiac‑triggered multi‑gradient-echo sequence with the following parameters: TR = R‑R interval, TE = 1.22, 3.45, 5.68, 7.91, 10.14, and 12.37 ms, slice thickness = 1 mm, matrix = 128x128, field‑of‑view = 3x3 cm2. The trigger delay was chosen in the mid-diastolic rest period of the heart cycle observed in the Cine series. The acquisition window of less than 13 ms resulted in images, which were sufficiently co-registered for the different echo times to allow for a pixel‑wise T2* determination (Fig. 1) [32]. LGE scans were performed in the same slice with a cardiac‑triggered inversion‑recovery segmented gradient‑echo sequence, with the following parameters: TR = 5.8 ms, TE = 2.2 ms, number of segments = 16, slice thickness = 1 mm, matrix = 256x256, field‑of‑view = 3x3 cm2. The inversion time (TI) was optimized to null the signal from remote myocardium [33,34]. Gadobutrol (Gadovist, Bayer Schering Pharma AG, Berlin, Germany) dose of 1 mmol/ml was used. To reduce volume related variability, contrast agent was diluted 1:4 with a standard saline solution. This dilution resulted in administered weight-adapted volume of 2 µl/g body weight, resulting in an equivalent dose of 0.5 mmol/kg body weight [33]. The contrast agent was injected intravenously through the tail vein, using a 24-gauge catheter. CMR data analysis T2* maps were generated in Mathematica 7 (Wolfram Research, Champaign, IL, USA) by pixel‑wise fittings of the multi-gradient-echo signal intensities to the equation S = S0 e –TE/T2*. The LV myocardium was manually segmented by drawing epicardial and 30 Quantitative T2* assessment of acute and chronic myocardial ischemia/reperfusion injury in mice endocardial contours, excluding the papillary muscles. LGE‑based single-slice infarct size is reported as percentage of the LV myocardial cross‑sectional area. A region of interest in the infarct in the myocardial free wall (infarct ROI) was drawn based on gadolinium enhancement and presence of akinetic myocardium. A second ROI was drawn in the non‑enhancing remote tissue in the septum (remote ROI). For the control mice, ROIs were drawn in the septum and the free wall. These ROIs were drawn within the zone free from susceptibility artifacts induced by lungs. T2* values are reported as group average and standard deviation of the mean T2* in the ROIs. Cine images were analyzed with Qmass digital imaging software (Medis, Leiden, the Netherlands). Semi‑automatic segmentation was used to determine end-diastolic volume (EDV), end‑systolic volume (ESV), wall thickness (WT) and systolic wall thickening (SWT). Ejection fraction (EF) was calculated as 100%(EDV‑ESV)/EDV. Histopathology For histological evaluation a 1.5‑mm‑thick slice containing infarcted myocardium was fixed in 4% formalin and paraffin‑embedded. Hereafter, 5‑µm‑thick sections were mounted on glass slides and stained with Picrosirius red staining for collagen, Prussian blue staining for iron, and Hematoxylin-Eosin (HE) for cellular structures. The stained sections were photographed with a digital image microscopy using white or polarized light. Statistical analysis All statistical analysis was evaluated using (SPSS 17 for Windows; SPSS). Results were expressed as mean ± standard deviation for each group of animals. An independent two-tailed sample t-test and the Mann-Whitney exact two-tailed U test were performed to compare mean values in subjects for each time point post MI versus baseline. Results were considered statistically significant for a p‑value of less than 0.05. The correlation analysis of T2* values against wall thickness (WT) was performed using linear regression. 31 Chapter 2 2.3Results An image series of a mid-axial slice with varying TE for a representative mouse at baseline is shown in Fig. 1a. The LV myocardium was well co‑registered for the different images. Signal loss due to susceptibility differences was occasionally observed for the images with the longest TE. Pixel‑wise fitting of the signal intensities in the LV myocardium as function of TE resulted in a T2* map as shown in Fig. 1b. T2* values were homogeneous throughout the left-ventricular myocardial wall and consistent between mice, with a mean T2* value of 17.1±2.0 ms in the free wall. Figure 2 displays a collection of images, consisting of LGE, T2* maps, and Cine images at diastole, for a representative mouse at day 1, 7 and 28 after induction of the myocardial infarction. On day 1, LGE resulted in a homogeneous enhancement of the infarct in Figure 1. Representative series of cardiac images with varying TE and corresponding T2* map. (a) Gradient-echo CMR with varying TE for a mid‑ventricle short-axis slice of a representative mouse at baseline. (b) Top: Signal intensity S in the myocardium as function of TE. The solid line is an exponential fit to the data. Bottom: T2* map. T2* in the LV myocardium at end‑diastole is color-coded between 0 and 20 ms according to the pseudo color scale on the right. The background image is a regular gradient echo image with TE = 1.22 ms. 32 Quantitative T2* assessment of acute and chronic myocardial ischemia/reperfusion injury in mice the mid-anterior and mid-anterolateral wall, whereas on days 7 and 28 enhanced area was smaller, heterogeneous, and difficult to detect. Corresponding T2* maps revealed a different picture. At day 1, a significant change in T2* could be detected in the LGEbased infarct area. Further, at days 7 and 28, T2* dramatically decreased, particularly in the mid‑anterolateral region indicating the existence of a significant infarct. The presence of an infarct was further corroborated by the Cine images, which showed increased wall thickness (WT) in the mid-anterior and mid-anterolateral wall at day 1, followed by progressive thinning below baseline levels at days 7 and 28, respectively. Figure 2. Mid-ventricle short-axis slice of a mouse heart at 1, 7, and 28 days after I/R myocardial infarction. (left) Cine images at end‑diastole. At day 1 wall thickness in the free wall is increased, whereas at days 7 and 28 the free wall decreased in thickness. (middle) Corresponding T2* maps color-coded from 0 to 20 ms. T2* is decreased in the infarct in the free wall at day 7 and 28 (red arrows). (right) Corresponding LGE images. Yellow arrows point to the location of the infarct. Infarct location was confirmed by the presence of an akinetic area and locally reduced wall thickening in Cine imaging. Figure 3 shows LGE and T2* maps of one of three mice with a small infarct in the midanterolateral wall, as indicated by a small area of LGE at day 1. Gadolinium enhancement 33 Chapter 2 could not be detected anymore at days 7 and 28. In the first group of n=9 mice with MI, n=3 mice displayed this atypical behavior. Presumably, LGE at day 1 revealed a small area at risk that could not be detected in the chronic phase. Importantly, T2* values in the mid-anterolateral wall also remained unaffected throughout infarct healing and did not deviate from baseline values. Figure 3. T2* maps and LGE of a mouse with a small LGE positive area at day 1 but no detectable infarct at later days. LGE showed enhancement in the mid‑anterior wall at day 1 (yellow arrow). Enhancement however, was absent at 7 and 28 days. In parallel, T2* values in the infarct area were maintained at baseline levels. T2* maps displayed spurious lowering in areas with no infarct related to susceptibility artifacts (red arrows). Figure 4. Quantitative T2* values in the infarct and remote myocardium at baseline (BL) and up to 28 days after surgery. n=5 mice, * = significantly different from baseline (p < 0.05), and ** = significantly different from baseline (p < 0.005). Quantitative T2* values (n=5 mice, Fig. 4) were dynamic throughout infarct development. In the infarct, quantitative T2* values decreased significantly (p < 0.05) from baseline (17.1±2.0 ms) to day 1 (12.0±1.1 ms) followed by a further decrease (p < 0.005) both 34 Quantitative T2* assessment of acute and chronic myocardial ischemia/reperfusion injury in mice at day 7 (7.9±1.0 ms) and day 28 (6.4±0.7 ms). In addition, the mid-septal (remote) area showed a significant (p < 0.05) decrease of T2* values from baseline (18.2±2.0 ms) to day 1 (13.9±0.8 ms), then slightly recovered at day 7 (14.2±1.1 ms) and day 28 (15.6±1.0 ms). Global cardiac function of both infarct and sham-operated groups was assessed by Cine imaging (Figs. 5a-c). The infarct group displayed a progressive and significant increase in end-diastolic volume (EDV) and end-systolic volume (ESV), indicative for a considerable dilation of the heart due to the infarct. For the sham-operated group no significant changes in EDV and ESV upon time after surgery were observed. The infarct group presented a decline in ejection fraction (EF) from 54±3 % at baseline to 39±2 %, 28 days after surgery (Fig. 5c). For the sham-operated group, EF was slightly elevated at day 1, however normalized at day 7 and 28 after surgery. Group-averaged (n=5) infarct sizes from LGE analysis of a mid-ventricular slice as function of days after surgery are Figure 5. Left ventricle functional parameters and infarct size for infarct (n=5) and shamoperated (n=8) groups at baseline (BL) and up to 28 days after surgery. (a) End diastolic volume. (b) End systolic volume. (c) Ejection fraction. (d) Single-slice infarct size. * = significantly different from baseline (p < 0.05) , and ** = significantly different from baseline (p < 0.005). 35 Chapter 2 presented in Fig. 5d. At day 1, LGE revealed an average infarct size of 41±6 %, dropping to 26±3 % and 22±4 % (p < 0.05, compared to day 1) of the LV area at days 7 and 28, respectively, which could be related to infarct resorption over time [35]. Figure 6. Regional wall thickness (WT) and systolic wall thickening (SWT) for infarct (n=5) and sham-operated (n=8) groups. (a) WT for infarct group in remote and infarct wall. (b) SWT for infarct group in remote and infarct wall. (c) WT for shamoperated group in septal and free wall. (d) SWT for sham‑operated group in septal and free wall. * = significantly different from baseline (p < 0.05) , and ** = significantly different from baseline (p < 0.005). In the infarct group, WT in the free wall (infarct area) showed a significant (p < 0.005) transient increase day 1 after myocardial infarction, followed by progressive thinning (Fig. 6a). Decreased WT in the infarct paralleled a decrease of the quantitative T2* values (Fig. 4). The mid-septal (remote) area in the infarct group showed no significant changes in WT. In line with the increased WT of the free wall at day 1, the systolic wall thickening (SWT), as a measure of local systolic function, also displayed a transient depression 1 day after surgery, only partially recovering at days 7 and 28 (Fig. 6b). Also, 36 Quantitative T2* assessment of acute and chronic myocardial ischemia/reperfusion injury in mice SWT of the remote area was slightly depressed at all time points compared to baseline. The sham-operated group revealed no significant changes in both WT and SWT as function of days after surgery (Figs. 6c and d). WT in the infarct correlated positively and significantly with T2* (Fig. 7). Thus, the chronic MI is characterized by a lower T2* value compared to the acute MI. Figure 7. Correlation between infarct wall thickness (WT) and T2*. Figure 8. Representative histological images of infarct myocardial tissue at day 1, 7 and 28 post MI. (a) Picrosirius red. (b) Prussian-blue. (c) Hematoxylin-Eosin. Scale bar equals 200 μm. 37 Chapter 2 Since T2* changes are related to the structural and compositional changes in the infarct myocardial tissue, we performed histology at each time point after I/R injury. Figure 8 shows images of 3 representative mice at day 1, 7 and 28 after MI. Picrosirius staining (Fig. 8a) revealed no collagen deposits in the infarct area at day 1. At day 7 substantial collagen deposition was observed, followed by a significant increase at day 28. Sections stained with Prussian Blue showed scattered spots of iron as early as 1 day after MI (Fig. 8b). Iron deposition became more prominent at day 7 and further increased at day 28. Finally, on sections stained with Hematoxylin-Eosin (Fig. 8c) the infarct area on day 1 was characterized by infiltration of inflammatory cells. 2.4Discussion In this study we aimed to systematically study quantitative changes in T2* of the mouse myocardium up to 28 days post I/R injury and to evaluate its added value in characterizing chronic infarcts in comparison to commonly applied LGE and Cine imaging. Combining different CMR techniques, if interpreted carefully, are a potential advantage in the evaluation of the pathophysiological changes in the injured myocardium. Baseline T2* values were comparable to T2 values found at 9.4 T [36] and 11.75 T [37], which suggests that decay due to static magnetic field inhomogeneities is largely absent in the healthy myocardium. We found that quantitative T2* values decreased progressively during infarct development. Throughout the observation period, the local decrease of T2* in the infarct was accompanied by globally and locally reduced ventricular function (Figs. 4-7). The reduction in T2* with infarct age could be explained by the presence of iron (Fig. 8a) [3,38]. Additionally, the formation of significant amounts of collagen (Fig. 8b) may contribute to the progressive decline of T2* with infarct age [29]. LGE imaging is currently unchallenged the gold standard technique to assess infarct size. Nevertheless, in certain cases when LGE imaging is inconclusive, additional characterization of the infarct by T2* imaging could provide complementary information. In the acute phase after I/R injury LGE‑based infarct size may be inaccurate 38 Quantitative T2* assessment of acute and chronic myocardial ischemia/reperfusion injury in mice [39], when there is loss of cell membrane integrity [16], an inflammatory response [40,41], necrosis, hemorrhage, micro‑vascular obstruction (MVO) and edema. Indeed, for the acute infarcts at day 1 we found a large LGE-based infarct size accompanied by increased wall-thickness, suggesting the presence of edema. We did not observe a transient increase in T2* at day 1 by the presence of this edema, as previously observed with T2‑weighted imaging [24]. The effect of edema on T2* is apparently lower than the decrease of T2* induced by iron or collagen (Fig. 8). This observation is critical in the clinical setting where early assessment of viability is significantly hampered by the presence of infarct-induced edema. T2*-imaging may be a tool to circumvent this inevitable problem after acute MI. In the chronic infarct, the formation of scar tissue could be responsible for a reduced distribution volume for the extracellular contrast agent. In turn, reduced distribution volume leads to an apparent reduction in infarct size and inhomogeneous enhancement (Figs. 2 and 5). Additionally, the extremely low T2* in the infarct (Fig. 2), which locally decreased down to a few milliseconds, could effectively null the signal of the inversion recovery gradient‑echo sequence with an echo time of 2.2 ms, obscuring the late gadolinium enhancement [42]. At day 1, a significant decrease of T2* (Fig. 4) and a transient depression of systolic performance (SWT; Fig. 6) were found not only in the infarct, but also to a lesser extent in the remote myocardium. This could be a secondary response in parallel to the structural changes in the infarct region. These findings are in agreement with those of Bogaert et al., who observed a dysfunction in the remote area at 5±2 days after reperfusion in patients with transmural anterior MI [43]. Others have also shown that remodeling is initiated in the remote myocardium as early as 1 day after I/R injury [4447]. Comparing Figs. 2 and 3 makes an interesting case. Figure 2 presented an example of a mouse with a considerable infarct at day 1, based on a large LGE positive area. However, at days 7 and 28, the infarct was difficult to detect on LGE, from which one could jump to the conclusion that a considerable part of the area at risk at day 1 recovered at 39 Chapter 2 later time points. However, the T2* maps at day 7 and 28 revealed low T2* values in the infarct area, indicating that the infarct was not recovered. The presence of non-viable myocardium (not detected by LGE) was confirmed by akinesia of that area and locally suppressed systolic thickening (SWT) in Cine imaging. Figure 3, conversely, showed one of three mice that presented a small infarct by LGE at day 1, but displayed no positive enhancement at day 7 and 28. For this mouse T2* in the infarct region was preserved during follow-up, indicating that for this mouse the initial area at risk recovered at later time points, as confirmed by healthy LV function. Thus, T2* is able to provide additional information, complementary to LGE, about the myocardial viability after infarction. Taken together, these findings suggest that T2* can be used as a surrogate marker for myocardial recovery during follow-up after myocardial infarction. A limitation to the use of T2* as a diagnostic marker for myocardial injury is contamination of intrinsic T2* values by the presence of macroscopic susceptibility differences, mainly present at the air-lung interfaces [26,27]. Occasionally, we observed lowered T2* values at the lung-heart interface (see for example Fig. 3) in areas with no infarct and no wallmotion abnormalities. Estimations of infarct sizes on the basis of T2* reduction were therefore inaccurate. Also the orientation of the heart with respect to the magnetic field could alter absolute T2* values. Improvements in local shimming techniques might alleviate some of these susceptibility contaminations. Still, use of T2* mapping as a stand-alone technique without the complementary information from LGE is undesired. 2.5Conclusions Imaging of the myocardium with a T2* mapping technique in a mouse model of myocardial I/R injury provides useful and complementary information to LGE and Cine imaging. The changes of the T2* were associated with the infarct age, reversibility of the injury, and gave an indication of the structural variation in the myocardium throughout infarct maturation. T2* decrease in the infarcts was associated with iron and collagen depositions. In cases where LGE was inconclusive, T2* mapping was able to confirm 40 Quantitative T2* assessment of acute and chronic myocardial ischemia/reperfusion injury in mice the presence of a chronic myocardial scar in mice. Taken together, T2* mapping may provide important additional information on myocardial viability in the acute and chronic phase and scar maturation after acute MI. 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Pepe A, Positano V, Santarelli MF, Sorrentino F, Cracolici E, De Marchi D, Maggio A, Midiri M, Landini L, Lombardi M (2006) Multislice multiecho T2* cardiovascular magnetic resonance for detection of the heterogeneous distribution of myocardial iron overload. J Magn Reson Imaging 23:662-668 39. Schwitter J, Saeed M, Wendland MF, Derugin N, Canet E, Brasch RC, Higgins CB (1997) Influence of severity of myocardial injury on distribution of macromolecules: extravascular versus intravascular gadolinium-based magnetic resonance contrast agents. J Am Coll Cardiol 30:1086-1094 40. Murphy E, Steenbergen C (2008) Mechanisms underlying acute protection from cardiac ischemia-reperfusion injury. Physiol Rev 88:581-609 41. Arslan F, de Kleijn DP, Timmers L, Doevendans PA, Pasterkamp G (2008) Bridging innate immunity and myocardial ischemia/reperfusion injury: the search for therapeutic targets. Curr Pharm Des 14:1205-1216 42. Bremerich J, Colet JM, Giovenzana GB, Aime S, Scheffler K, Laurent S, Bongartz G, Muller RN (2001) Slow clearance gadolinium-based extracellular and intravascular contrast media for three-dimensional MR angiography. J Magn Reson Imaging 13:588-593 43. Bogaert J, Bosmans H, Maes A, Suetens P, Marchal G, Rademakers FE (2000) Remote myocardial dysfunction after acute anterior myocardial infarction: impact of left ventricular shape on regional function: a magnetic resonance myocardial tagging study. J Am Coll Cardiol 35:1525-1534 44 Quantitative T2* assessment of acute and chronic myocardial ischemia/reperfusion injury in mice 44. Lessick J, Sideman S, Azhari H, Marcus M, Grenadier E, Beyar R (1991) Regional threedimensional geometry and function of left ventricles with fibrous aneurysms. A cinecomputed tomography study. Circulation 84:1072-1086 45. Kramer CM, Lima JA, Reichek N, Ferrari VA, Llaneras MR, Palmon LC, Yeh IT, Tallant B, Axel L (1993) Regional differences in function within noninfarcted myocardium during left ventricular remodeling. Circulation 88:1279-1288 46. Kramer CM, Rogers WJ, Theobald TM, Power TP, Petruolo S, Reichek N (1996) Remote noninfarcted region dysfunction soon after first anterior myocardial infarction. A magnetic resonance tagging study. Circulation 94:660-666 47. Rademakers F, Van de Werf F, Mortelmans L, Marchal G, Bogaert J (2003) Evolution of regional performance after an acute anterior myocardial infarction in humans using magnetic resonance tagging. J Physiol 546:777-787 45 Chapter 3 Quantitative changes in T2* reflect remodelling of both remote and ischemic myocardium in a murine heart failure model Eissa N. E. Aguor†, Cees W.A. van de Kolk†, Gustav J. Strijkers, Marcel G.J. Nederhoff, Pieter A. Doevendans, Cees J.A. van Echteld, Fatih Arslan † These authors contributed equally to this study In preparation 47 Chapter 3 Abstract Heart failure is an increasing burden to western societies due to aging and increased survival of patients suffering from myocardial infarction. Early identification of adverse structural changes in the myocardium may optimize clinical management. Late Gadolinium enhancement (LGE)-MRI is widely used to assess infarct size and myocardial fibrosis. We have recently shown that quantitative T2* mapping can provide additional information on infarct status and changes in the infarcted myocardium in relatively small murine infarctions after ischemia/reperfusion injury, In this study, we further explored quantitative changes in T2* in both infarcted and remote myocardium in a murine heart failure model induced by severe myocardial infarction. Myocardial infarction (MI) was surgically induced in C57Bl/6J mice (n=12) by permanent ligation of the left coronary artery. A sham‑operated group (n=8) served as control. MRI at 9.4 T was performed at baseline, 1, 7 and 28 days after surgery. The protocol consisted of Cine-MRI, multi‑gradient‑echo T2*‑mapping and LGE. Histological heart sections were stained to identify hemorrhage and fibrosis. Baseline myocardial T2* was 15.0±1.1 ms in the remote myocardium (septal wall) and 14.6±1.0 ms in the free wall. At day 1, LGE displayed a homogeneous enhancement of the infarction. In addition, T2* values in the infarcts substantially decreased (5.7±0.4 ms), whereas a slight decrease was observed in the remote myocardium (13.0±1.5 ms). On days 7 and 28, LGE area of enhancement was smaller and heterogeneous compared to LGE at day 1. After 7 and 28 days, T2* values in the infarct remained low (5.5±0.5 ms and 5.0±0.5 ms, respectively). Interestingly, T2* values in the remote myocardium continued to decrease during follow-up (9.3±1.3 ms and 8.4±0.3 ms at day 7 and 28, respectively). Histological analysis revealed progressive deposition of collagen in the infarct and to a lesser degree in the remote myocardium, in parallel with decreased T2*. Quantitative T2* values changed dynamically in this murine heart failure model. T2* in infarcted areas exhibited a significant decrease starting from day 1, and further decreased with scar maturation. In contrast to our previous observations in relatively 48 Quantitative changes in T2* reflect remodelling of both remote and ischemic myocardium in a murine heart failure model small murine infarctions, T2* in remote tissue also decreased significantly from baseline, most likely as ,a result of adverse ventricular remodeling of non-infarcted areas after MI. Serial LGE scans revealed merely changes in the infarct area, whereas the remote myocardium did not exhibit any dynamics in LGE assessments. In conclusion, quantitative T2* assessment may provide a valuable readout of both the infarct and remote areas in heart failure after MI. 49 Chapter 3 3.1Background Over the past decade, optimized reperfusion therapy has resulted in improved survival of patients after myocardial infarction (MI). However, morbidity and mortality of surviving patients is significantly increased due to aging and heart failure development [1]. Heart failure is one of the most common complications after infarction and an increasing socio-economic burden to Western societies. Early characterization of structural changes within the infarcted and non-ischemic remote myocardium may be of great importance for clinical management. In this respect, magnetic resonance imaging (MRI) is a powerful tool to visualize, characterize and follow the injured myocardium after an initial ischemic insult. Traditionally, MRI has been applied to assess functional and pathological changes of the injured myocardium. Typically, T1weighted MRI is performed some time after injecting a Gadolinium‑based contrast agent to delineate injured myocardium in a late Gadolinium enhancement (LGE) scan [2]. The signal enhancement in LGE-MRI is influenced by many factors, such as the used MRI protocol, MR sequence parameters, scan timing, the presence of edema [your paper and others], and wash-in and wash-out kinetics of the contrast agent [2-4]. Thus, LGE-MRI accuracy especially in acute MI has been subject to debate. After the onset of myocardial ischemia, the post-injured myocardium undergoes dynamic changes. Necrosis, increased extracellular volume, decreased perfusion, microvascular obstruction, hemoglobin degradation products and the presence of scar tissue influence the T1 and T2(*) tissue relaxation times [5]. T2*-weighted imaging has been used before as a tool to diagnose the presence of hemorrhage [6;7] and myocardial iron [8]. Also, T2*‑weighted imaging was used to track targeted superparamagnetic iron oxide contrast agents in the myocardium [6] and to localize iron oxide-labeled stem cells [9]. In general, quantitative relaxation time mapping MRI might be preferred over relaxation time weighted imaging, because it is less subject to variations in protocol and physiological parameters. Several quantitative T1-mapping [10], T2-mapping [11] and T2*-mapping [5;12] protocols were found useful in the assessment of infarcts. 50 Quantitative changes in T2* reflect remodelling of both remote and ischemic myocardium in a murine heart failure model Specifically, T2* values may provide important information on the condition of the post-injured myocardium related to the presence of edema [12;13], hemorrhage [12], and the formation of collagenous scar tissue [14]. Previously, we have shown the benefit of cardiac T2* mapping in mice with relatively small infarcts[15]. Subjecting mice to 30 minutes of ischemia followed by reperfusion, we observed that T2* values in the infarct decreased during scar maturation, suggesting that T2* values can differentiate acute from chronic MI. However, we did not observe a change in T2* values in the remote myocardium. This may be due to the fact that the relatively small infarct size and only 30 minutes ischemia followed by reperfusion does not result in significant structural alterations within the remote myocardium. In the present study, we used a heart failure model to address whether T2* mapping has any added value in assessing non-ischemic remote myocardium after MI. 3.2Methods Experimental design C57Bl/6J mice (n=12, body weight=20-25 g) were used in this study. Cardiac MRI measurements were performed at baseline, 1, 7 and 28 days after myocardial infarction (MI) induced by permanent ligation of the left coronary artery (LCA). The MRI protocol consisted of a multi‑gradient‑echo quantitative T2*‑mapping sequence, traditional LGE‑MRI to assess the location and size of the infarction, and gradient‑echo Cine imaging to determine cardiac function. All animal experiments were performed in accordance with the national guidelines on animal care and with prior approval by the Animal Experimentation Committee of Utrecht University. Myocardial infarction Mice underwent permanent LCA ligation as previously described [16]. Briefly, mice were anesthetized with a mixture of Fentanyl (Jansen-Cilag) 0.05 mg/kg, Dormicum (Roche) 5 mg/kg, and medetomidine 0.5 mg/kg through an intraperitoneal injection. 51 Chapter 3 A core body temperature of about 37 °C was maintained during surgery by continuous monitoring with a rectal thermometer and an automatic heating blanket. Mice were intubated and ventilated (Harvard Apparatus Inc, Holliston, Mass) with 100% oxygen. The left coronary artery was ligated permanently with an 8-0 Ethilon (Ethicon) suture. Ischemia was confirmed by bleaching of the myocardium and transient tachycardia. The chest wall was closed, and the animals subcutaneously received Antisedan (Pfizer) 2.5 mg/kg, Anexate (Roche) 0.5 mg/kg, and Temgesic (Schering-Plough) 0.1 mg/kg. Magnetic resonance imaging A vertical 9.4 T and 89-mm-diameter bore scanner was used, equipped with 1500 mT/m gradients (Bruker BioSpin GmbH, Ettlingen, Germany). Mice were anesthetized with 1.5‑3 vol% isoflurane in a 2:1 mixture of air (0.3 l/min) and oxygen (0.15 l/min). The mice were put on a custom‑built cradle and positioned inside a 3-cm‑diameter quadrature volume coil. During the MRI examination anesthesia was maintained between 1.5‑2.5 vol% isoflurane to keep the respiratory rate at 50-60 bpm. For cardiac triggering and respiratory gating, a pressure balloon was placed under the chest, at the level of the heart. The MRI protocol consisted of the following steps. Two- and 4-chamber view scout scans were made to plan a single mid-ventricular short-axis slice. Cine imaging was performed with a retrospectively‑triggered gradient‑echo sequence, with the following parameters: TR = 6.8 ms, TE = 1.9 ms, number of movie frames = 15, slice thickness = 1 mm, matrix = 256x256, field‑of‑view = 3x3 cm2. Seven to 9 short‑axis slices with inter‑slice distance of 1 mm were measured to cover the heart from apex to base. Based on the Cine axial images, a position of the axial midventricle lower papillary muscle slice was imported to include an area of remote viable tissue as well as a substantial infarct area. T2* mapping was performed using a cardiac‑triggered multi‑gradient-echo sequence with the following parameters: TR = R‑R interval, TE = 1.22, 3.45, 5.68, 7.91, 10.14, and 12.37 ms, slice thickness = 1 mm, matrix = 128x128, field‑of‑view = 3x3 cm2. The trigger delay was chosen according to the mid-diastolic rest period of the heart cycle observed in the Cine series. LGE‑MRI scans were performed in the same slice with a cardiac‑triggered inversion‑recovery 52 Quantitative changes in T2* reflect remodelling of both remote and ischemic myocardium in a murine heart failure model segmented gradient‑echo sequence, with the following parameters: TR = 5.8 ms, TE = 2.2 ms, number of segments = 16, slice thickness = 1 mm, matrix = 256x256, field‑of‑view = 3x3 cm2. The inversion time (TI) was 160 ms to null the signal from remote myocardium. A Gadobutrol solution (Gadovist, Bayer Schering Pharma AG, Berlin, Germany) of 1 mmol/ml was used. To reduce volume related variability, the contrast agent was diluted 1:4 with a standard saline solution. This dilution resulted in an administered weight-adapted volume of 2 µl/g body weight (equivalent dose of 0.5 mmol/kg body weight). The contrast agent was injected intravenously through the tail vein, using a 24-gauge catheter. Histopathology On termination (at 1, 7 and 28 days after MI) the hearts were excised for histological evaluation. A 1.5-mm-thick mid‑axial slice was fixed in 4% formalin and paraffinembedded. Hereafter, 5‑µm‑thick sections were mounted on glass slides and stained with Picrosirius red staining for collagen, Hematoxylin-Eosin (HE) for cellular structures, and Prussian blue staining for iron. The stained sections were photographed with a digital image microscopy using white or polarized light. MR Image analysis T2* maps were generated in Mathematica 7 (Wolfram Research, Champaign, IL, USA) by pixel‑wise fittings of the multi-gradient-echo MR signal intensities to the equation S = S0 e -TE/T2*. The LV myocardium was manually segmented by drawing epicardial and endocardial contours, excluding the papillary muscles. A region of interest in the infarct in the myocardial free wall (infarct ROI) was drawn based on the enhanced region in the LGE images. LGE‑based infarct size is reported as percentage of the LV myocardial cross-sectional area (%LV). A second ROI was drawn in the non‑enhancing remote tissue in the septum (remote ROI). For control mice, an ROI was drawn in the septum only. These ROIs were drawn within the zone free from susceptibility artifacts induced by lungs. T2* values are reported as group average and standard deviation of the mean T2* in the ROIs. Cine MR Images were analyzed with Qmass digital imaging software (Medis, Leiden, the Netherlands). Semi‑automatic segmentation was used to 53 Chapter 3 determine end-diastolic volume (EDV) and end‑systolic volume (ESV). Ejection fraction (EF) was calculated as 100% (EDV‑ESV)/EDV. Statistical analysis All statistical analysis was done using (SPSS 17 for Windows; SPSS). Results are expressed as mean ± standard deviation for each group of animals. Both independent twotailed sample t-test and the Mann-Whitney exact two-tailed U test were performed to compare mean values in subjects for each time point post MI versus baseline. Results were considered significant at P < 0.05. The correlation analysis was performed using Pearson correlation with two‑tailed P values at all time points post MI. 3.3Results In Figure 1 mid-axial T2* maps at baseline are shown for three representative mice. T2* values were homogeneous throughout the left-ventricular myocardial wall and consistent between mice, with a mean T2* value of 15.0±1.1 ms in the septum and 14.6±1.0 ms in the free wall. Figure 1. Representative T2* maps of a mid-ventricle short-axis slice of three healthy mice at baseline. T2* in the left-ventricular myocardium at end‑diastole is colorcoded between 0 and 20 ms according to the pseudo color scale on the left. The background image is a regular gradient echo image with TE = 1.22 ms. 54 Quantitative changes in T2* reflect remodelling of both remote and ischemic myocardium in a murine heart failure model Figure 2. Mid-ventricle short-axis slice of a mouse heart at 1, 7, and 28 days, following permanent ligation of the left coronary artery. (bottom) LGE images. Red arrows point to the location of the infarct core (1) and peri-infarct regions (2). Corresponding T2* maps color-coded from 0 to 20 ms. Note the considerable T2* decrease in the free wall at day 7 and 28 (red arrows). (right) Corresponding Prussian blue staining at day 28. Figure 2 displays LGE and T2* maps at mid-diastole, for two mice at day 1, 7 and 28, following the induction of MI. On day 1, LGE showed a homogeneous enhancement of the infarct tissue in the mid-anterior and mid-anterolateral wall, whereas on days 7 and 28 the enhanced area was smaller, heterogeneous, and difficult to detect. Corresponding quantitative T2* values were dynamic during infarct development and maps showed a good match to the LGE area. T2* values in the mid-anterolateral wall slightly decreased on day 1, and then decreased further on day 7, and 28 post MI. Figure 3 shows the quantitative T2* values in the remote vs. infarct at baseline and throughout infarct development. At day 1, T2* values in the infarcts dramatically 55 Chapter 3 Figure 3. Quantitative T2* values and infarct size Quantitative T2* values in the infarct and remote myocardium as function of days after surgery (A) and the infarct size from LGE-MRI (B). * = significantly different from baseline (P < 0.05), and ** = significantly different from baseline (P < 0.005). decreased to 5.7±0.4 ms (p<0.05, compared to baseline). A slight decrease was observed in remote tissue (13.0±1.5 ms). At day 7 and 28, T2* values in the infarct remained low (5.5±0.5 ms and 5.0±0.5 ms, respectively). T2* values in remote tissue did not normalize but also decreased further at day 7 and 28 towards 9.3±1.3 ms and 8.4±0.3 ms, respectively. In parallel to the decreased T2* values, LGE decreased during follow-up. At day 1, LGE in infarct area was homogeneous and infarct size was 48.4±2.8% of the LV area. After 7 and 28 days post-MI, LGE became heterogeneous and infarct size decreased (28.0±2.6 and 21.0±3.0% of the LV, respectively). Global cardiac function was assessed using left ventricular end-diastolic volume (EDV) and end-systolic volume (ESV) (Figure 4). During post-MI follow-up, EDV and ESV showed a progressive and significant increase, indicative for a significant dilatation of the heart, accompanied by a decline in ejection fraction (EF) from 54.7±2.3 % at baseline to 21.3±10.8 %, 28 days after surgery. A positive correlation was found between LGE-based infarct size and quantitative T2* values in the infarcts (Pearson correlation 0.90, P = 0.045). 56 Quantitative changes in T2* reflect remodelling of both remote and ischemic myocardium in a murine heart failure model Figure 4. Left ventricle functional parameters as function of time (A) EDV and ESV. (B) Ejection fraction. * = significantly different from baseline (P < 0.05) , and ** = significantly different from baseline (P < 0.005). Figure 5. Representative histological images for a mid‑axial slice of a mouse heart at day 1, 7 and 28 post MI. (A) T2* mapping. (B) Hematoxylin-eosin. (C) Picrosirius red under polarized light. (D) Prussian-blue. (magnification is x 200) 57 Chapter 3 We performed immunohistochemistry in heart sections at each time point. Figure 5 shows representative images of 3 mice at baseline, 1 day and 28 days after infarction. Picrosirius staining revealed no collagen deposition in the free wall at baseline and in the infarct at day 1. At day 28, significant collagen deposition was observed. Sections stained with Prussian blue showed scattered spots of iron as early as day 1. Iron deposition became more prominent at day 28. T2* was lower in the peri-infarct zone in comparison to the infarct core. Correspondingly, in the peri-infarct zone more iron was found than in the core (Figure 5). Finally, on sections stained with hematoxylin-eosin (Figures 5D) the infarct area was characterized by infiltration of inflammatory cells. 3.4Discussion We have shown that T2* values in both ischemic and non-ischemic myocardium are dynamic in time in a murine heart failure model. T2* dynamics reflect structural and compositional changes within the tissue. The reduction in T2* with infarct is likely related to the presence of iron and the formation of collagen. Furthermore, the reduction of T2* in the infarct was paralleled by reduced LV function and infarct size. T2* traditionally is used to assess iron deposition in the heart [17]. Despite the large decrease of T2*, however, we did not observe significant amounts of iron and collagen by histology in the infarct at day 1. Nevertheless, iron deposits by hemorrhage may be present in different forms of hemoglobin degradation products [12;18-20], which might have different sensitivity to the histological iron staining. The same is true for the sensitivity of T2* to iron. The reduction of T2* at day 7 and 28 is most likely related to the presence of iron and collagen in the infarct. Lower T2* values were found in the peri-infarct zone in comparison to the core, in agreement with the findings by van den Bos et al. [5]. Interestingly, T2* values in the infarct at day 1 were lower (5.7±0.4 ms) than previously found in I/R infarcts (12.0±1.1 ms) [15], possibly related to the severity of injury at day 1 as a consequence of permanent occlusion. This suggests that the quantitative T2* 58 Quantitative changes in T2* reflect remodelling of both remote and ischemic myocardium in a murine heart failure model values could be used not only to image iron deposition but also to assess the condition of the infarction. Higher T2* values in acutely I/R injured myocardium might also be related to the presence of edema. We also found a significant change of T2* in the remote myocardium, in agreement with finding of Bogaert et al. [21], who observed a dysfunction in the remote area at 5±2 days after reperfusion in patients with transmural anterior MI. Others have also shown that remodeling is initiated in the remote myocardium [22;23] as early as 1 day after injury. LGE showed different degree of enhancement for different mice and at different scan points, reflecting the continuous pathological and cellular changes after infarction. In acute myocardial injury with loss of cell membrane integrity [5;24], inflammatory response, [5;25], necrosis, hemorrhage, micro-vascular obstruction (MVO) and particularly edema, LGE-based infarct size may be overestimated by the presence of edema [5;26]. In the chronic infarction, the formation of scar tissue and the larger extent of vascular obstructions could be responsible for a reduced distribution volume for the extracellular contrast agent, leading to an inhomogeneous enhancement and reduction of LGE-based infarct size. Additionally, the extremely low T2* in the infarct (Figure 3), which locally decreases down to a few milliseconds, could effectively null the signal of the inversion recovery gradient-echo sequence with an echo time of 2.2 ms, obscuring LGE [27]. These findings are in agreement with the observed reduction in LGE infarct size in patients with one week old MI [2]. A drawback of cardiac T2* as a diagnostic marker for myocardial injury is contamination of intrinsic T2* values by the presence of macroscopic susceptibility variation caused by air-lung interfaces [12;13]. Occasionally, we observed lowered T2* values at the lung-heart interface outside areas that could be associated with known locations of myocardial infarction. Also the orientation of the heart with respect to the magnetic field could alter absolute T2* values. Improvements in local shimming techniques might alleviate some of these susceptibility variations. Therefore, use of T2* mapping as a stand-alone technique without the complementary information from LGE might still be undesired. 59 Chapter 3 3.5Conclusions Imaging of the myocardium with a T2* mapping technique in a mouse model of heart failure provides useful and complementary information to LGE and Cine MRI. 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Circulation 109:2411-2416 25. Murphy E, Steenbergen C (2008) Mechanisms underlying acute protection from cardiac ischemia-reperfusion injury. Physiol Rev 88:581-609 26. Schwitter J, Saeed M, Wendland MF, Derugin N, Canet E, Brasch RC, Higgins CB (1997) Influence of severity of myocardial injury on distribution of macromolecules: extravascular versus intravascular gadolinium-based magnetic resonance contrast agents. J Am Coll Cardiol 30:1086-1094 27. Bremerich J, Colet JM, Giovenzana GB, Aime S, Scheffler K, Laurent S, Bongartz G, Muller RN (2001) Slow clearance gadolinium-based extracellular and intravascular contrast media for three-dimensional MR angiography. J Magn Reson Imaging 13:588-593 62 Chapter 4 Lack of circulating Toll-like receptor 2 promotes survival and prevents adverse remodeling after myocardial infarction Eissa N.E. Aguor, Mirjam B. Smeets, Pieter A. Doevendans, Gustav J. Strijkers, Gerard Pasterkamp & Dominique P. de Kleijn, Fatih Arslan In preparation 63 Chapter 4 Abstract Background Excessive inflammation is a known mediator of adverse cardiac remodeling after myocardial infarction. Toll-like receptors (TLRs) have been shown to play a crucial role in inflammatory responses after tissue injury. In the present study, we explored the functional and biochemical consequences of TLR2 absence in left ventricular remodeling after myocardial infarction. Methods and Results C57Bl6 wild-type (WT), TLR2-/- and chimeric mice underwent permanent ligation of the left coronary artery. Cardiac function and geometry were assessed using 9.4T mouse-MRI at baseline, 7 and 28 days after infarction. Despite similar infarct size between WT and TLR2-/- mice (39±1.4% vs. 42±1.5%, respectively; p=.256), the absence of TLR2 promoted survival and prevented left ventricular dilatation during 28 days follow-up. Furthermore, systolic performance of both infarcted and remote myocardium was better in TLR2-/- mice compared to WT animals. Chimeric mice experiments revealed that expansive remodeling after infarction was mediated by bone marrow-derived TLR2 expression. Characterization of peripheral blood cells showed that the absence of TLR2 reduced white blood cell count 7 days after infarction. Peripheral blood monocytes of TLR2-/- mice exhibited similar CD11b expression, but reduced CD49d levels compared to WT animals 7 days after infarction. The functional and cellular changes in TLR2-/- mice were preceded by significant reduction in tissue levels of TNFα, IL-1α, GM-CSF and IL-10 at 3 days after infarction. Conclusions Lack of TLR2 improves survival and cardiac function after myocardial infarction and is determined by leukocytic TLR2 deficiency via reduced inflammation. Hence, TLR2 is a potential therapeutic target to prevent adverse remodeling after myocardial infarction. Keywords: myocardial infarction, heart failure, remodeling, immune system, Toll-like receptor 64 Lack of circulating Toll-like receptor 2 promotes survival and prevents adverse remodeling after myocardial infarction 4.1 Background Optimized reperfusion strategies have resulted in improved survival of patients after myocardial infarction (MI). Unfortunately, quality of life of surviving patients is significantly decreased due to increased morbidity after infarction [1]. Heart failure is one of the most common complications after infarction and inflict an increasing socioeconomic burden on Western societies. Currently, the post-infarct healing process is of increasing importance for therapeutic modulation to enhance cardiac repair and function after MI. Inflammation, proliferation and maturation are chronological processes during infarct healing that can be targeted independently from each other for therapeutic modulation [2]. Inflammation is an interesting therapeutic target, since it is initiated early and has profound effects for the later stages. Cytokine and chemokine production and subsequent leukocyte recruitment are initiated after the ischemic injury. Leukocyte migration is a critical first step in the removal of debris and matrix breakdown and subsequent scar formation [2]. The extent and type of inflammation has great impact on processes like angiogenesis and fibrosis in the (sub)chronic phase after infarction [3-5]. For this reason, modulation of the early inflammatory process may have great therapeutic value to regulate adaptive repair processes hereafter. However, inflammation is not a random binary process rather a well-orchestrated dynamic response to tissue injury. For this reason, therapeutic modulation is challenging and difficult. More importantly, inflammation has shown to have ‘double-edged sword’ characteristics thereby complicating therapeutic interventions [6;7]. Recent studies have clearly demonstrated that Toll-like receptor (TLR) deficiency or inhibition enhance the ‘good’ and blunt the ‘bad’ of the inflammatory reaction in cardiac ischemia. Traditionally known as pathogen recognition receptors, TLRs have been shown to recognize endogenous activators released after cardiovascular injury [8]. TLR2 and 4 have been extensively studied in cardiac ischemia [9]. TLR4 deficiency or inhibition protects against ischemia/reperfusion (I/R) injury and adverse left ventricular remodeling [10-12]. We have recently shown that circulating TLR2 mediates myocardial I/R injury and that its inhibition is cardioprotective. TLR2 inhibition reduces monocytes activation after reperfusion, thereby inhibiting cytokine and chemokine production and subsequent reduction of apoptosis [13]. Furthermore, previous studies 65 Chapter 4 have shown that TLR2 deficiency prevents maladaptive remodeling [14]. However, the relative contribution of parenchymal and hematopoiesis-derived TLR2 expression to cardiac repair responses remain unknown. In the present study, we show for the first time that circulating TLR2 expression mediates adverse remodeling after myocardial infarction. Furthermore, we investigated the functional and biochemical differences between TLR2 deficient and wild type animals during adverse remodeling after infarction. 4.2Methods Animals and Experimental Design TLR2-/- animals were backcrossed for 6 generations into a C57Bl6 background. Male C57Bl6/J and C57Bl6-TLR2-/- mice (10-12 wks, 25-30 g) received standard diet and water ad libitum. Myocardial infarction was induced by permanent left coronary artery ligation, just below the left atrial appendage as described previously. Digital images of the infarcts were encrypted before being analyzed by the researcher. Heart function and geometry assessments were done by a technician blinded to treatment. All animal experiments were performed in accordance with the national guidelines on animal care and with prior approval by the Animal Experimentation Committee of Utrecht University. Myocardial Infarction In Vivo Mice were anesthetized with a mixture of Fentanyl (Jansen-Cilag) 0.05 mg/kg, Dormicum (Roche) 5 mg/kg and medetomidine 0.5 mg/kg through an intraperitoneal injection. Core body temperature was maintained around 37°C during surgery by continuous monitoring with a rectal thermometer connected to an automatic heating blanket. Mice were intubated and ventilated (Harvard Apparatus Inc.) with 100% oxygen. The left coronary artery (LCA) was permanently ligated using an 8-0 vicryl suture. Ischemia was confirmed by bleaching of the myocardium and ventricular tachyarrhythmia. In sham operated animals the suture was placed beneath the LCA without ligating. The 66 Lack of circulating Toll-like receptor 2 promotes survival and prevents adverse remodeling after myocardial infarction chest wall was closed and the animals received subcutaneously Antisedan (Pfizer) 2.5 mg/kg, Anexate (Roche) 0.5 mg/kg and Temgesic (Schering-Plough) 0.1 mg/kg. Infarct Size for another 15 minutes. Viable tissue stains red and infarcted tissue appears white. Heart sections were digitally photographed (Canon EOS 400D) under a microscope (Carl Zeiss®). IS, AAR and total LV area were measured using ImageJ software (version 1.34). Infarct size was corrected for the weight of the corresponding heart slice. Generation of Chimeric Mice We generated chimeric mice to study the relative contribution of TLR2 expression in blood and parenchymal cells to cardiac remodeling. Donor bone marrow (BM) cells were collected from wild-type (WT) C57Bl6 and TLR2 knock-out (TLR2-/-) mice by flushing humerus, femurs and tibiae with RPMI-1640 medium. Recipient mice received 5x106 BM cells after receiving a single dose of 7 Gy. Mice recovered for 6 weeks to ensure stable engraftment of the donor bone marrow cells. Hereafter, chimerization was confirmed by phenotyping TLR2 expression on peripheral blood samples with Cytomics FC500 (Beckman Coulter) analysis. Successful chimerization (>95% circulating donor cells) was achieved in all mice (data not shown). Irradiated WT mice with TLR2-/- bone marrow are referred as WT/TLR2 KO BM, and TLR2-/- mice with WT bone marrow as TLR2 KO/WT BM. Magnetic Resonance Imaging Twenty-three mice (n=5 in WT, n=6 in TLR2-/- and n=6 in sham operated mice) underwent serial assessment of cardiac dimensions and function by high resolution magnetic resonance imaging (MRI, 9.4T, Bruker, Rheinstetten, Germany) under isoflurane anesthesia before, 7 and 28 days after MI. Long axis and short axis images with 1.0 mm interval between the slices were obtained and used to compute end-diastolic volume (EDV, largest volume) and end-systolic volume (ESV, smallest volume). The ejection fraction (EF) was calculated as 100*(EDV-ESV)/EDV. Wall thickness (WT) and systolic wall thickening (SWT) were assessed from both the septum (remote myocardium) and 67 Chapter 4 free wall (infarct area). All MRI data are analyzed using Qmass digital imaging software (Medis, Leiden, The Netherlands). Protein Isolation Total protein was isolated from snap frozen infarcted heart sections (infarct and remote area separated) using 40 mM Tris pH 7.4. Flow Cytometry Tumor necrosis factor (TNF)-α, IL-1α, IL-10, and granulocyte macrophage-colony stimulating factor (GM-CSF) levels in isolated tissue protein samples were measured by flow cytometry (Cytomics FC500, Beckman Coulter) using the Th1/Th2 customized multiplex kit (Bender MedSystems, Vienna, Austria). The protein samples were diluted 1:1 in assay buffer, and the protocol is further followed according to the manufacturer’s instructions. CD11b and CD49d expression was assessed on circulating monocytes of EDTA anticoagulated blood by flow cytometry. Whole blood was stained for CD11b (FITC, eBioscience, San Diego, Calif ), and CD49d (Alexa Fluor 488, Serotec, Oxford, UK) and F4/80 for monocytes (Alexa Fluor 647, Serotec, Oxford, UK). Statistical Analysis Data are represented as Mean±SEM. One-way ANOVA with post-hoc LSD test was used for comparison >2 groups. Non-parametric t-test for 2 group comparisons. KaplanMeier survival analysis with log-rank test was used to evaluate mortality differences between groups. All statistical analyses were performed using SPSS 15.1.1. and p<0.05 was considered significant. The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written. 68 Lack of circulating Toll-like receptor 2 promotes survival and prevents adverse remodeling after myocardial infarction 4.3Results Circulating TLR2 Expression Mediates Adverse Remodeling After Infarction There were no differences between WT and TLR2-/- animals in cardiac function and geometry at baseline. Infarct size, determined as a percentage of the left ventricle (IS/ LV), showed no differences between TLR2-/- and WT mice 2 days after infarction (Figure 1A). Despite similar infarct size, TLR2-/- animals showed improved survival compared to WT mice after infarction (Figure 1B). Most animals died between day 4 and 8 postMI due to cardiac ruptures. Serial cardiac MRI assessments revealed a significant preservation of both function and LV dimensions during 28 days follow-up (Figure 1C and D; Table 1). TLR2-/- mice were relatively protected against expansive remodeling of the left ventricle when compared to WT animals. We performed bone marrow transplantation experiments from WT to irradiated knock-out mice and vice versa, in order to dissect the relative contribution to adverse remodeling of parenchymal and circulating TLR2 expression. Chimeric mice experiments revealed that adverse remodeling is relatively prevented in WT mice with TLR2-/- bone marrow (WT/TLR2 KO BM). In contrast, TLR2-/- mice with WT bone marrow (TLR2 KO/WT BM) exhibit similar LV dilatation compared to irradiated WTs with WT bone marrow (WT/WT BM; Figure 1E). 69 Chapter 4 Figure 1. Circulating TLR2 mediates adverse remodeling after myocardial infarction. (A) Infarct size (IS) as a percentage of the left ventricle (LV) 2 days after infarction. (B) Kaplan-Meier survival curve. (C) and (D), End-diastolic and end-systolic volume, respectively (EDV, ESV); p-values see Table 1. (E) EDV and ESV in chimeric mice 28 days after infarction; *p=0.005 †p=0.007 compared to TLR2 KO/WT BM. Each bar represents Mean±SEM, n=5/group for infarct size assessment, n=12 in WT and n=13 in TLR2-/- group for survival analysis. TLR2 KO/WT BM=irradiated TLR2-/- mice with WT bone marrow, WT/TLR2 KO BM= irradiated WT mice with TLR2-/- bone marrow, WT/ WT BM=irradiated WT with WT bone marrow. 70 25.4±4.1 59.1±4.1 0.90±0.01 0.90±0.02 47.1±1.9 52.0±1.9 ESV, μl EF, % WT septum (remote, mm) WT free wall (infarct, mm) SWT septum (remote, %) SWT free wall (infarct, %) -34.0±5.7* 20.6±5.0* 0.60±0.08* 0.67±0.06* 7.2±2.8* 156.7±10.9* 168.6±7.2* -56.6±3.6** 16.1±4.7** 0.45±0.04** 0.75±0.02** 4.3±2.3** 207.4±15.7** 216.3±11.8** 54.2±1.8 47.1±2.1 0.89±0.01 0.85±0.02 57.9±4.3 23.7±4.3 54.1±5.1 -18.6±4.2* 36.9±3.1*† 0.55±0.03* 0.72±0.01* 16.0±1.1* 93.1±6.0*† 110.7±6.6*† 7 days MI NS 0.009 NS NS NS <0.001 <0.001 †p -26.2±4.0**† 35.8±3.2**† 0.45±0.07** 0.71±0.03** 11.9±1.0** 112.4±6.2**† 127.5±6.3**† 28 days MI 0.027 0.012 NS NS NS <0.001 <0.001 †p systolic volume, EF=ejection fraction, WT=wall thickness, SWT=systolic wall thickening One-way ANOVA post hoc Dunnett t-test (2-sided), *p<0.05 compared to baseline, †p compared to WT mice. EDV=end-diastolic volume, ESV=end- 60.9±3.7 Baseline 28 days MI Baseline 7 days MI TLR2-/- C57BL6/J WT EDV, μl Table 1. Lack of circulating Toll-like receptor 2 promotes survival and prevents adverse remodeling after myocardial infarction 71 Chapter 4 Figure 2. Lack of TLR2 reduces tissue cytokine levels and leukocytosis after infarction. Tissue levels of (A) TNFα; *p=0.032 B, (B) IL-1α; *p=0.032 C, (C) IL-10; *p=0.063 and (D) GM-CSF; *p=0.046 compared to WT animals. (E) White blood cell counts (WBC) after infarction; *p=0.05 compared to WT animals. Each bar represents Mean±SEM, n=6/ group/time point. 72 Lack of circulating Toll-like receptor 2 promotes survival and prevents adverse remodeling after myocardial infarction TLR2 Deficiency Reduces Inflammation After Infarction Myocardial infarction is characterized by cytokine and chemokine induction and leukocytosis. In our experiments, TLR2 deficiency resulted in a significant reduction of TNFα, IL-1α, IL-10 and GM-CSF 3 days after infarction (Figure 2A – D). White blood cell counts revealed that TLR2/animals exhibited reduced leukocytosis compared to WT mice after infarction (Figure 2E). TLR2 Mediates Integrin-α4 Expression On Circulating Monocytes After Infarction Monocyte migration to the heart is an integrin-dependent process[15]. The expression of CD11b did not differ between the groups after infarction (Figure 3A). However, compared to WT animals, circulating monocytes of TLR2-/- mice expressed decreased levels of CD49d after infarction (Figure 3B). Figure 3. Lack of TLR2 reduces integrin-α4, but not CD11b expression on monocytes after myocardial infarction. (A) CD11b and (B) CD49d on circulating monocytes; *p=0.023 compared to WT animals. Each bar represents Mean±SEM, n=6/group/ time point. MFI=mean fluorescent intensity 73 Chapter 4 4.4Discussion Toll-like receptors as therapeutic targets may hold great therapeutic value in the near future. We have shown in both murine and porcine (unpublished data) models that TLR2 inhibition is a good candidate for infarct size reduction after ischemia/reperfusion [13]. The question remains whether TLR2 inhibition has adverse effects on remodeling of the scar and remote myocardium in the long-term. There are tragic examples in which inflammatory interventions with beneficial effects in the acute phase turned out to be devastating in the long-term. Corticosteroid administration reduced infarct size in animal experiments, but appeared to result in aneurysm formation and cardiac ruptures in patients after infarction [6;7]. The main issue of down toning inflammation is impaired healing resulting in reduced collagen deposition in the scar and ventricular dilatation. Severe suppression of the immune response is associated with a so-called “mummified myocardium”, characterized by delayed debris removal and decrease infarct collagen content [16]. In addition, reduced infarct collagen is associated with more pronounced LV dilatation and dysfunction [17]. A report by Mersmann J et al. suggests that TLR2 deficiency results in adverse remodeling after infarction, despite smaller infarct size compared to WT animals [18]. Unfortunately, in their study the background of the TLR2-/- and WT mice were different: C57Bl6 vs. C3HeN, respectively. For this reason, it is difficult to draw firm conclusion from these experiments. In the present study, we investigated the healing process after infarction in TLR2/- and WT mice, both on a C57Bl6/J background. Despite similar infarct size, cardiac MRI assessments revealed that LV dilatation and functional deterioration is relatively prevented in TLR2-/- mice. The improved cardiac function and geometry is likely the reason for improved survival in TLR2-/- animals. Moreover, in our experiments lack of TLR2 was relatively protective against bulging of the infarcted myocardium. It is known that bulging of the infarct (i.e. aneurysm formation) is associated with reduced collagen density in the scar [17]. We have previously shown that circulating TLR2 expression mediates ischemia/ reperfusion injury of the heart [13]. In addition, it is well-established that leukocytes migrate to the heart to initiate the process of myocardial healing [2;5]. Hence, we hypothesized that TLR2 expression on circulating blood cells is also the main 74 Lack of circulating Toll-like receptor 2 promotes survival and prevents adverse remodeling after myocardial infarction determinant for adverse remodeling. WT mice with TLR2 knock-out bone marrow were indeed relatively protected against adverse remodeling compared to TLR2-/- mice with WT bone marrow. Findings by Shishido et al. [14] and the present study are in strong contrast to the study by Mersmann et al. in which TLR2 deficiency was associated with impaired cardiac function and geometry. We believe that the different strains used in their experiments (C3HeN background was compared to C57Bl6 background) are the main reason for the conclusions drawn by Mersmann et al. rather than the TLR2 knock-out. It is known that mouse strains strongly differ in myocardial healing after infarction [19]. Since improvement in cardiac function is a relative measure, it is of utmost importance that the intervention (e.g. knock-out) is the only differing variable and that confounding factors like background strain are absent. Furthermore, we demonstrated that TLR2-/- mice exhibit reduced Infarct size (IS) as a percentage of the left ventricle (LV) was determined using Evans’ blue dye injection and TTC staining, 2 days after infarction (n=5/group). By assessing infarct size in the acute phase (at 2 days), one can determine whether differences are present between WT and TLR2-/- mice in myocardial perfusion. Hence, 4% Evans blue dye was injected via the thoracic aorta in a retrograde fashion. By doing so, one can demarcate the area-at-risk (AAR), the extent of myocardial tissue that underwent ischemia (i.e. endangered myocardium). Hearts were rapidly explanted, rinsed in 0.9% saline and put in -20ºC freezer for 1 hour. Hereafter, hearts were mechanically sliced into four 1-mm cross sections. Heart sections were incubated in 1% triphenyltetrazoliumchloride (Sigma-Aldrich) at 37ºC for 15 minutes before placing them in formaldehyde cytokine levels and leukocytosis compared to WT animal after infarction. These findings are in line with the pro-inflammatory actions of TLR2 upon activation. CD49d is an important integrin for leukocyte migration and myofibroblast transdifferentiation [15;20]. Whether leukocyte infiltration and myofibroblast transdifferentiation is altered in TLR2-/- mice remains to be addressed. However, the reduced CD49d expression on circulating TLR2-/- monocytes after infarction suggests that leukocyte migration and myofibroblast transdifferentiation may be altered in TLR2-/- mice. In conclusion, we have shown that circulating TLR2 mediates maladaptive remodeling after myocardial infarction. Lack of TLR2 improves survival and prevents post-infarct heart function deterioration via decreased inflammation and reduced migration 75 Chapter 4 capacity of monocytes. The exact mechanisms by which TLR2 mediates leukocyte migration and myofibroblast transdifferentiation in the healing heart remains to be addressed. Our study demonstrates that chronic absence of TLR2 is beneficial after myocardial infarction and is a good candidate as a therapeutic target in patients after myocardial infarction. Acknowledgements We would like to thank the following persons for their assistance: Ben J. van Middelaar, Cees van der Kolk and Arjan Schoneveld (all from the UMC Utrecht, The Netherlands). Funding Sources This work is supported by research grants from Netherlands Organisation for Scientific Research (NWO) Mozaïek grant (contract 017.004.004 to F.A.), Netherlands Heart Foundation (contract 2010.T001 to F.A.) and Bekales Foundation (P.A.D.) 76 Lack of circulating Toll-like receptor 2 promotes survival and prevents adverse remodeling after myocardial infarction Reference List 1. Juenger J, Schellberg D, Kraemer S, Haunstetter A, Zugck C, Herzog W, Haass M (2002) Health related quality of life in patients with congestive heart failure: comparison with other chronic diseases and relation to functional variables. Heart 87:235-241 2. Frangogiannis NG (2008) The immune system and cardiac repair. Pharmacol Res 58:88-111 3. 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Biochem Pharmacol 60:1949-1958 78 Part II Sodium MRI 79 Chapter 5 Na Chemical Shift Imaging and Gd Enhancement of myocardial edema 23 Eissa N.E. Aguor, Cees W.A. van de Kolk, Fatih Arslan, Marcel G.J. Nederhoff, Pieter A.F.M. Doevendans, Gerard Pasterkamp, Gustav J. Strijkers, Cees J.A. van Echteld International Journal of Cardiovascular Imaging, in press DOI: 10.1007/s10554-012-0093-6 81 Chapter 5 Abstract Myocardial edema can arise in several disease states. MRI contrast agent can accumulate in edematous tissue, which complicates differential diagnosis with contrast-enhanced (CE)-MRI and might lead to overestimation of infarct size. Sodium Chemical Shift Imaging (23Na-CSI) may provide an alternative for edema imaging. We have developed a non-infarct, isolated rat heart model with two levels of edema, which was studied with 23Na-CSI and CE-MRI. In edematous, but viable tissue the extracellular sodium (Na+e) signal is hypothesized to increase, but not the intracellular sodium (Na+i) signal. Isolated hearts were perfused at 60 mmHg (n=6) and 140 mmHg (n=5). Dimethyl methylphosphonate (DMMP) and phenylphosphonate (PPA) were used to follow edema formation by P-MR Spectroscopy. In separate groups, Thulium(III)1,4,7,10 31 tetraazacyclododecane-N,N /,N //,N ///-tetra(methylenephosphonate (TmDOTP5- )and Gadovist were used for 23Na-CSI (n=8) and CE-MRI (n=6), respectively. PPA normalized signal intensity (SI) was higher at 140 mmHg vs. 60 mmHg, with a ratio of 1.27±0.12 (p<0.05). The (DMMP-PPA)/dry weight ratio, as a marker of intracellular volume, remained unchanged. The mid-heart cross sectional area (CSA) of the left ventricle (LV) was significantly increased at 140 mmHg. In addition, at 140 mmHg, the LV Na+e SI increased with a 140 mmHg/60 mmHg ratio of 1.24±0.18 (p<0.05). Na+i SI remained essentially unchanged. With CE-MRI, a subendocardially enhanced CSA was identified, increasing from 0.20±0.02 cm2 at 60 mmHg to 0.31±0.02 cm2 at 140 mmHg (p<0.05). Edema shows up in both CE-MRI and Na+e. High perfusion pressure causes more edema subendocardially than subepicardially. 23Na-CSI is an attractive alternative for imaging of edema and is a promising tool to discriminate between edema, acute and chronic MI. 82 Na Chemical Shift Imaging and Gd Enhancement of myocardial edema 23 5.1Introduction Myocardial interstitial edema can arise in several disease states or following clinical interventions, including myocardial ischemia and infarction [1-11], arterial [12] and pulmonary hypertension [13], myocarditis [14], cardio-pulmonary bypass and cardioplegic arrest [15] and cardiac transplantation [16]. The causes of edema may vary: in patients with hypertension, differences in hydrostatic pressure may dominate, whereas in patients with coronary artery disease, differences in osmotic pressure and microvascular permeability are probably more important [17]. 1H-based MRI methods such as contrast-enhanced (CE)-MRI and T2-weighted MRI are valuable tools to assess injured myocardium. Indeed, CE-MRI has become the gold standard to delineate chronic myocardial infarction [18;19]. However, the accuracy of CE-MRI to delineate acute myocardial infarction has been questioned because of the accumulation of contrast agent in the expanded extracellular compartment of both infarct and periinfarct regions [1-4;14]. Apparently, in this phase CE-MRI much resembles T2‑weighted MRI, which has shown great promise to delineate the area at risk (AAR) and to quantify myocardial salvage in patients with acute coronary syndromes after events related to the formation of edema [5-11]. However, both these methods depend on relative regional differences in myocardial signal intensity and are perhaps less suitable to identify globally edematous tissue associated with any of the other disease states mentioned above, such as hypertension or myocarditis. An alternative approach to assess myocardial edema is imaging of myocardial sodium with 23Na-MRI, since we expect the extracellular sodium (Na+e) signal to increase with increasing interstitial edema because of the expansion of the extracellular space. In a number of studies an elevated total sodium (Na+t) signal has been observed after myocardial infarction, both in humans and animals [19-22] Particularly, imaging intra(Na+i) and extra- (Na+e) cellular sodium separately can provide more precise information to assess the sodium gradient and cell membrane integrity [23]. This can be achieved with chemical shift imaging (23Na-CSI) in combination with a paramagnetic chemical shift agent to separate Na+i from Na+e signals [23;24]. 83 Chapter 5 We hypothesize that, in viable tissue with cell membranes still intact but with a high level of extracellular edema, Na+i signal intensity is normal because viable cells should be able to maintain a normal Na+ gradient and a normal intracellular volume , whereas Na+e and CE-MRI signal intensities will be increased because of the larger extracellular space. To characterize edema independent from ischemia, this study consisted of two parts. First, using phosphorus-31 magnetic resonance spectroscopy (31P MRS), we developed an isolated heart model with two levels of extracellular edema based on crystalloid perfusion at different pressures. Secondly, we aimed to characterize edema in non-ischemic myocardial tissue with CE-MRI and 23Na-CSI. 5.2Methods Animal and heart preparation Male Wistar rats (n=25, body weight=300 to 350 g) were anesthetized with isoflurane inhalation and given heparin (500 IU/Kg i.v.). Subsequently, the hearts were rapidly excised and placed in modified, ice-cold Krebs-Henseleit (KH) buffer. After cannulation of the aorta, perfusion was initiated according to Langendorff at a constant pressure (60 mmHg) with KH at 37°C. To further prepare the heart, a drain was inserted through the apex via the mitral valve to remove Thebesian flow. A latex balloon was inserted into the left ventricle through the mitral valve and filled with water to reach a left ventricular end-diastolic pressure (LVEDP) of 7.5 mmHg. Two pressure transducers (MP-15, Micron Instruments) were used to monitor perfusion pressure and to measure the isovolumic contractile pressure of the left ventricle via the inserted balloon. Left ventricular developed pressure (LVPD) was determined as the difference between left ventricular systolic pressure (LVSP) and LVEDP. To maintain a heart rate of 300 beats/ min, the outflow tract of the right ventricle was connected with two copper-wire electrodes to a (4-7 V, 0.5 ms) stimulator (model S88, Grass Instruments, Quincey, MA, USA). Heart rate was determined from the pressure curve. Coronary flow was measured with a flow probe (Skalar instruments, Delft, The Netherlands). The data were recorded with a PowerLab Data Acquisition System (ADI Instruments, Australia). Hearts were placed in a 20-mm-diameter NMR tube and positioned inside the MR scanner. The 84 Na Chemical Shift Imaging and Gd Enhancement of myocardial edema 23 experimental protocol was in accordance with the guidelines of the Committee for Animal Experiments of the University Medical Center Utrecht, The Netherlands. Perfusion solutions The modified KH buffer contained (in mmol/L): 119.0 NaCl, 4.7 KCl, 1.0 MgCl2, 24.0 NaHCO3, 1.3 CaCl2, 11.0 glucose, and 5.0 Na-pyruvate. Before perfusion, the KH buffer was filtered through 8 µm (Millipore, Bedford, MA) filters. The solution was equilibrated with 95% O2 and 5% CO2, resulting in a pH of 7.4 at 37°C, prior to the addition of the CaCl2. For 31P-MRS, we included in the buffer 10 mM dimethyl methylphosphonate (DMMP), which distributes evenly in intra- and extracellular spaces, as a marker of total water space and 10 mM phenylphosphonate (PPA), which is non-permeant, as an extracellular space marker [25]. In the 31P spectra, the DMMP and PPA signals can be easily discriminated from endogenous phosphate signals. The difference between DMMP and PPA signals was calculated to determine the intracellular space. After preparations, hearts were allowed to stabilize at 60mmHg. For tetraazacyclododecane-N,N ,N ,N / // Na-CSI, Thulium(III)1,4,7,10 23 -tetra(methylenephosphonate) (TmDOTP5-, 3.5 /// mM) [26] was included in the buffer as a shift reagent (SR), to separate intra and extracellular Na+ signals. TmDOTP5- is also non-permeant and therefore only interacts with Na+e, causing a shift in resonance frequency and leaving the Na+i resonance at its original frequency. To correct for Ca2+ binding by the shift reagent, the total Ca2+ added to the buffer was increased to 3.42 mM, which resulted in a free [Ca2+] of 0.85 mmol/L as measured by a HI 4004-51 Ca2+ Ion Selective Electrode (ISE) connected to a pH/ISE meter (HI 3221, Hanna instruments). For CE-MRI scans, 1.3 mM Gadobutrol (Gadovist, Bayer Schering Pharma AG, Berlin, Germany) was included in the buffer. Modification of Langendorff set up Usually, we aspirate the effluent from a level above the heart to keep the hearts submerged for better magnetic susceptibility matching. However, the effluent generates unwanted extra-cardiac 23Na and 31P signals. Two approaches were used to 85 Chapter 5 reduce these unwanted signals. For the 31P-MRS study, the effluent was removed from the bottom of the NMR tube, sacrificing the susceptibility matching. For the 23Na-CSI study, an additional perfusion line ending at the bottom of the tube was used to flush the tube at a rate of 30 ml/min with a buffer in which Na+ was replaced by Li+. In this case, the hearts were kept submerged. MRS and MRI protocols A vertical 9.4 T, 89 mm diameter bore scanner was used, equipped with a 1500 mT/m gradient system, which was interfaced with an AVANCE 400 DRX spectrometer (Bruker, Germany). All heart preparation including coil tuning and matching and optimization of magnetic field homogeneity was performed at a perfusion pressure of 60 mmHg and took 30-34 min as indicated in Figure 1. P-MRS 31 Phosphorus spectroscopy was performed with a 20 mm multi-nuclear probehead (Bruker, Germany). P spectra were acquired for 10 min by accumulation of 32 31 consecutive free induction decays (FIDs) following 90o pulses, using 2048 data points and 12.9 KHz spectral width (SW). Pulse repetition time was 18.75 s to achieve a fully T1 relaxed signal. CE-MRI and 23Na-CSI The imaging protocol consisted of the following steps. First, 2- and 4-chamber view scout scans were obtained with a 1H 20-mm-diameter birdcage coil (Bruker, Germany) to plan a single mid-ventricular short-axis slice. Next, cine imaging was performed with a self-gated gradient‑echo sequence (TE = 1.9 ms, TR = 5.2 ms, number of movie frames = 15, slice thickness = 2.5 mm, matrix = 256x256, field of view (FOV) = 2x2 cm2). Gated CE-MRI scans were performed with T1-weighted short-axis multi-slice FLASH sequence, with the following parameters: TR = 63 ms, TE = 2.8 ms, FA = 75o, NA=8, 6 slices of 2.5 mm, matrix = 256x256, FOV = 2x2 cm2. To monitor the arrival of Gd containing buffer, eight serial CE-MRI scans were performed. The last scan in this series was used for edema analysis. 86 Na Chemical Shift Imaging and Gd Enhancement of myocardial edema 23 Figure 1: Schematic representation of experimental protocols (A) 31P-MRS, (B) 23Na-chemical shift imaging (23Na-CSI), (C) Contrast-enhanced MRI (CE-MRI). For 23Na-CSI, the 1H birdcage coil was replaced with a 23Na 20-mm-diameter birdcage coil (Bruker, Germany). To verify the heart position, scout images were obtained and subsequently a 5 mm mid-axial ventricular slice was acquired as a reference image with a 23Na self-gated FLASH sequence (TE = 2.4 ms, TR= 25 ms and 128 repetitions, 50o FA and SW = 7714 Hz, matrix = 32x32 and FOV = 2x2 cm2). To monitor the shift of Na+e after switching to the TmDOTP containing buffer, 23Na MR spectra were acquired 23 with a 75o pulse, SW = 5.5 kHz, TR = 105 ms, and 512 data points. Subsequently, acquisition weighted 2D-23Na CSI was performed with the following parameters: 87 Chapter 5 10,000 acquisitions, number of averages (NA) in center of k-space = 55, TR = 30 ms, 90o 0.5 ms sinc pulse, BW = 12420 Hz, FOV = 2x2 cm2, matrix = 16x16 and reconstruction matrix = 32x32. FIDs were collected with 128 complex data points and 100.8 µs dwell time resulting in an acquisition time of 25.8 ms/FID. Gradient switching resulted in an acquisition delay of 0.91 ms. Experimental protocol Two studies were performed. First, we used P-MRS to monitor the formation of 31 edema in 2 groups of isolated hearts. Secondly, edema was characterized with CE-MRI and 23Na-CSI. Schematic diagrams of the experimental protocols for the 2 studies are shown in Fig. 1. P-MRS group 31 After preparing the heart at 60 mmHg perfusion pressure, hearts were continuously perfused with PPA/DMMP-containing buffer for 70 min (Fig. 1A) either at 60 mmHg, n=6 or at 140 mmHg, n=5, to induce different levels of edema. As depicted in figure1A, P MR spectra were acquired as an average over 10 min to track the change of PPA 31 and DMPP signals and to measure the extent of extra-cellular edema during these 70 minutes of perfusion. Na-CSI group 23 Hearts (n=8) were perfused at 60 mmHg and subsequently at 140 mmHg for 60 min (Fig. 1B). After stabilization of the isolated hearts at 60 mmHg, 1H scout images were acquired followed by 2- and 4-chamber cine MRI. Thereafter, a cine scan was acquired for a mid-axial slice. Subsequently, the hearts were perfused with buffer containing SR and 23Na spectra were acquired to monitor the arrival of buffer containing SR. 23Na scout images were obtained to verify the anatomical position of the heart. Then a reference image of a mid-axial slice was acquired, followed by CSI of the same slice. The same scan protocol was repeated after 60 min of perfusion at 140 mmHg. 88 Na Chemical Shift Imaging and Gd Enhancement of myocardial edema 23 CE-MRI group Hearts (n=6) were perfused at 60 mmHg (Fig. 1C) and subsequently at 140 mmHg for 60 min. After stabilization of the isolated hearts at 60 mmHg, 1H scout images were acquired, followed by 2-chamber and 4-chamber cine MRI. Next, a cine scan was acquired for a mid axial slice. Subsequently, the hearts were perfused with buffer containing Gadovist. Eight serial CE-MR scans were acquired every 2 min at 60 mmHg to monitor the arrival of Gadovist containing buffer. The same MR scan protocol was repeated after 60 min of perfusion at 140 mmHg. Data analysis For 31P-MRS, all spectra were analyzed with jMRUI 4.0 software. After a polynomial baseline correction, the spectra were fitted with AMARES, a time-domain based fitting algorithm. The 23Na-CSI data were zero-filled in both the spatial and spectral domain resulting in a 256x64x64 reconstruction size. To optimize peak separation a LorentzianGaussian multiplication was applied. Epi- and endocardial contours of the left ventricle were manually drawn on end-diastolic 1H images to calculate the left ventricular cross sectional area (CSA). Subsequently, these contours were copied to the 23Na-CSI images, to determine the 23Na+e and 23Na+i average signal intensities of the entire left ventricle. For CE-MRI, Epi- and endo-cardial LV contours were drawn on the 1H axial cine images and then copied to CE-MR images. Subsequently, the CSA of the hyperenhanced areas were delineated manually. The CSA and the signal intensities of both the entire LV myocardium and the hyperenhanced areas were determined with Bruker Paravision 4 software. Tissue water content At the end of the MRI measurements, the hearts were cut open and blotted dry with a soft tissue. The hearts were then weighed to determine their wet weights. Subsequently, the dry weights of the hearts were measured after desiccation in an oven at 50 oC for 48 hours. The wet/dry (W/D) weights of the isolated rat hearts were then determined. 89 Chapter 5 Statistical analysis Results were expressed as mean±standard error of the mean for each group of animals. A paired t-test was performed to compare mean values for each time point post perfusion. Results were considered significantly different for a two-tailed p value of less than 0.05. The correlation test of PPA values against W/D ratios was performed using a Pearson correlation with two‑tailed p values. 5.3Results P-MRS 31 The addition of PPA and DMMP to the perfusate and the non-submerged state of the heart did not affect the stability of the heart, in terms of steady heart rate and coronary flow. The “dry” setup reduced the shimming quality as expected. The coronary flow was 24.0±0.5 ml/min for the 140 mmHg group and 9.9±0.4 ml/min for the 60 mmHg group. Figure 2 shows examples of P MR spectra of an isolated rat heart (Fig. 2A) still 31 submerged in the NMR tube and (Fig. 2B) after the effluent was removed from the bottom of the tube, both during perfusion with perfusate containing PPA and DMMP. Figure 3A summarizes the PPA signal intensities normalized by heart dry weight (PPA/dry weight), whereas Fig. 3B depicts the difference between DMMP and PPA signal intensities normalized by heart dry weight [(DMMP-PPA)/dry weight], both as function of time for the 60 and 140 mmHg groups. Ten minutes after switching to the phosphonates-containing buffer, PPA/dry weight was 30.6±3.2 and 34.7±2.7 for 60 and 140 mmHg, respectively. From 50 min onward, PPA/dry weight differences between the 60 mmHg and 140 mmHg group were significant (p<0.05). After 70 min PPA/dry weight was 51.5±4.5 at 140 mmHg vs. 40.6±1.4 at 60 mmHg, with a 140 mmHg/60 mmHg ratio of 1.27±0.12. The calculated (DMMP-PPA)/dry weight, as a marker for the intracellular space, remained at approximately baseline values (29.9±2.1) during the entire perfusion period for both groups. 90 Na Chemical Shift Imaging and Gd Enhancement of myocardial edema 23 Figure 2: Typical examples of 31P-MRS spectra of an isolated rat heart, (A) while the heart is submerged in buffer and (B) after removing the effluent. The signal of the three phosphate groups of ATP, phosphocreatine (PCr), phenylphosphonate (PPA) as extracellular space marker, Methylphosphonate (MPA), a solution in a small glass capillary as a reference, and dimethyl methylphosphonate (DMMP) as a marker of total water space are pointed out. MPA reference volume in (B) was different from that in (A). The higher level of edema, deduced from a larger PPA signal at 140 mmHg, was corroborated by an increase in heart wet/dry weight ratio (W/D) of the 140 mmHg group in comparison to the 60 mmHg group (Fig. 3C, 6.5±0.1 vs. 6.0±0.1, respectively, p=0.05). Moreover, a positive, significant correlation was found between W/D and PPA/ dry weight (Fig. 3D, Pearson correlation coefficient 0.71, p=0.02). Na-CSI 23 Baseline cine MRI and 23Na-MRI were performed at 60 mmHg perfusion pressure, after which the perfusion pressure was increased to 140 mmHg (Fig. 1B). The hearts remained stable during perfusion with SR (TmDOTP). However, immediately after switching to SR-containing buffer, the hearts left ventricular developed pressure (LVDP) dropped from 60.0±4.0 mmHg to 34.0±4.3 (p<0.05) at 60 mmHg and from 99.4±6.5 to 48.0±4.3 mmHg (p<0.05) at 140 mmHg as a result of the reduction of the free Ca2+ concentration due to the affinity of the shift reagent to Ca2+ [26]. During perfusion with SR-containing buffer, two 23Na peaks were observed in the acquired spectra (Fig. 4). A large peak 91 Chapter 5 Figure 3: 31P-MRS data on extra- and intracellular space markers and heart wet /dry weights from isolated rat hearts perfused at 60 mmHg and 140 mmHg (A) PPA/heart dry weight as a function of time. (B) (DMMP-PPA)/heart dry weight as a function of time. (C) Normalized heart wet/dry and PPA/heart dry weight ratios 70 min after switching to the phosphonates containing buffer. (D) Heart wet/dry weight ratio vs. PPA/heart dry weight ratio. downshifted by 2.2±0.2 ppm corresponding to Na+e and mainly representing signal from the vasculature and interstitium, and the Na+i intracellular sodium peak at 0 ppm. Similar Na+i and Na+e peaks were seen in the localized spectra of the CSI data. Figure 5 shows 1H longitudinal and axial views of an isolated rat heart and corresponding Na+e and Na+i images at 60 mmHg and 140 mmHg. The balloon in the left ventricle was filled with Na+-free solution and therefore appeared dark in 23Na-CSI images. The right ventricular cavity had very high signal intensity, because it was filled with Na+containing effluent. The Na+i images were of considerable lower intensity than the Na+e images because of the lower Na+i concentrations in the myocardium. As can be readily seen in the cine 1H MR images (and in the 23Na images), the CSA of the mid-axial LV 92 Na Chemical Shift Imaging and Gd Enhancement of myocardial edema 23 myocardium showed a considerable increase at 140 mmHg, going from 0.66 ± 0.03 cm2 at 60 mmHg to 0.91±0.03 cm2 at 140 mmHg (p<0.05) with a 140 mmHg/60 mmHg ratio of 1.40±0.08, in good agreement with the PPA data. Figure 4: Na-NMR spectra acquired with control buffer (A) and with shift reagent (SR) 23 TmDOTP containing buffer. In the absence of TmDOTP (control buffer, A), the on-resonance was seen at 0 ppm. After 10 min of continuous perfusion with TmDOTP, Na+e peak was separated from Na+i peak. Reference: a small glass capillary, which contained a known amount of TmDOTP. In addition to the increase of the LV CSA, also an increase of LV Na+e relative signal intensity was observed, going from 17.4±1.7 at 60 mmHg to 21.6±2.2 at 140 mmHg (p<0.05), with a 140 mmHg/60 mmHg ratio of 1.24±0.18 (p<0.05), in good agreement with CSA and PPA data. We also found an increase in LV Na+i relative signal intensity, going from 1.8±0.2 at 60 mmHg to 2.0±0.2 at 140 mmHg (p<0.05), with a 140 mmHg/60 mmHg ratio of 1.11±0.17. However, the shift reagent resulted in a clear but not complete separation between Na+e and Na+i CSI signals. The Na+e signal is at least an order of magnitude larger than the Na+i signal, and spectral deconvolution 93 Chapter 5 Figure 5: Representative MR images of isolated rat heart perfused at 60 mmHg and 140 mmHg Single frame 1H-cine MRI and extracellular and intracellular Na-CSI. B=balloon, 23 CSA=cross sectional area of the LV, RV=right ventricular lumen. shows that the increase in the Na+i signal can be explained for > 80% by the overlap with the increased Na+e signal. Therefore, the corrected LV Na+i signal shows no change when going from 60 mmHg to 140 mmHg. CE-MRI After switching to the buffer with Gadovist, heart function remained stable. Baseline cine MRI and CE-MRI were performed at 60 mmHg, after which the perfusion pressure was increased to 140 mmHg. Figure 6 shows a series of T1‑weighted MR images every two minutes after switching to Gd-containing buffer at 60 mmHg and 140 mmHg. The Gd started to appear in both groups after 4 min. In addition, CE-MR images showed an increased subendocardial enhanced area compared to the rest of the myocardium at 60 mmHg and even more so at 140 mmHg. From 10 min onwards the signal intensity of this hyper-enhanced area, delineated with a red dashed line, remained constant. Figure 7 shows typical CE-MR images at the beginning of the 60 mmHg perfusion and later at 140 mmHg and corresponding signal profiles across the images. As can be readily seen in the CE-MR image, the CSA of the hearts during perfusion at 140 mmHg was increased. From the cine images, the CSA of the mid-axial slice of the LV 94 Na Chemical Shift Imaging and Gd Enhancement of myocardial edema 23 Figure 6: Representative T1-weighted MR images at 60 mmHg and 140 mmHg of a midaxial slice of an isolated rat heart. Scans were acquired every 2 min after switching to Gadovist containing buffer. B=balloon. myocardium was significantly (p=0.03) increased from 0.74±0.04 cm2 at 60 mmHg to 0.90±0.03 cm2 at 140 mmHg, with a 140 mmHg/60 mmHg ratio of 1.22±0.08. The enhanced subendocardial area (Fig. 7, region outlined by the red dashed line) at 140 mmHg was significantly larger than at 60 mmHg (0.20±0.02 cm2 vs. 0.31±0.02 cm2, p=0.01), with a ratio of 1.55±0.07. Relative signal intensities in these enhanced areas were 15.1±0.7 at 60 mmHg and 16.3±1.0 at 140 mmHg (p>0.05), with a ratio of 1.08±0.05. The relative signal intensities between the subendocardial area and the subepicardial area were significantly different at both 60 mmHg (subendocardial 15.1±0.7 vs. subepicardial 13.4±0.6, p<0.03, ratio 1.13±0.01) and 140 mmHg (subendocardial 16.3±1.0 vs. 14.3±0.9, p<0.03, ratio 1.14±0.01). 95 Chapter 5 Figure 7: Representative CE-MR images for the same mid-axial slice of an isolated rat heart perfused (A) at 60 mmHg and (B) at 140 mmHg. (C) The signal profiles corresponding to the red lines across the images. The enhanced subendocardial area (marked with red dashed line) at 140 mmHg was larger than at 60 mmHg (p=0.01), which can also be appreciated from the signal profile (C). The signal intensity of the subendocardial area was higher than the epicardial area both at 60 mmHg and 140 mmHg (p<0.03). 96 Na Chemical Shift Imaging and Gd Enhancement of myocardial edema 23 5.4Discussion Using P-MRS we have developed a non-infarct model of extracellular edema in 31 isolated rat hearts. With this model, we have demonstrated that CE-MRI can identify edematous myocardial tissue, most likely as a result of an increased distribution volume of Gd. Surprisingly, we found more edema in the subendocadial area than in the subepicardial area. We have demonstrated in this same edema model that the Na+e signal increases with increasing edema, confirming that Na+e is a useful endogenous marker of extracellular volume. Interestingly, a similar transmural distribution of signal intensities as observed in the contrast-enhanced images was also observed in the Na+e images. Na+i essentially remained unchanged in edema. Using 31P-MRS we have shown that perfusion of hearts for an hour with crystalline buffers at high perfusion pressures leads to formation of interstitial edema, as evidenced by an increased distribution volume for PPA (Fig. 3). In addition, we observed a significant correlation between the increased PPA signal and the heart wet/dry weight ratio at the end of experiment. The increased PPA signal and the heart wet/dry ratio in our experiment are in line with findings by Li et al. [27]. The intracellular space remained unchanged, as evidenced by the stability of the difference between DMMP and PPA signals. This is in contrast to the results of Li et al., who observed intracellular edema as well, but in addition to simultaneous arterial/venous perfusion, they have used a hyperkalemic buffer, which may have contributed to the intracellular edema. The presence of edema could also be confirmed by the cine 1H-MRI. The CSA in both Na-CSI (Fig. 5) and CE-MRI (Fig. 7) groups showed a significant increase at 140 mmHg 23 compared to 60 mmHg perfusion pressure, indicating an expanded volume of the extracellular compartment caused by myocardial interstitial edema. The expanded extracellular compartment is in line with the increased PPA signal and the heart wet/ dry weight ratio of the 31P-MRS study. In parallel to the increased CSA, the LV Na+e signal intensity increased at 140 mmHg, corroborating an increased level of interstitial edema. The absence of intracellular 97 Chapter 5 edema in the used model, as found with the 31P-MRS intracellular space measurements, was corroborated by the essentially unchanged LV Na+i signal, after correction for spectral overlap. Previously, we have used 23Na-CSI to study myocardial ischemia and found increases in Na+i signal of 200-300%, where overlap with the Na+e signal had negligible effects on Na+i signal intensity [23;28]. With CE-MRI at 60mmHg and 140 mmHg (Fig. 6), the myocardium appeared dark in the T1 weighted image prior to Gd perfusion, whereas it appeared brighter after Gd perfusion, confirming the arrival of Gd in the myocardium. In addition, higher signal intensities of the mid-axial slice of the LV myocardium and the hyperenhancedsubendocardial area (Fig. 7) were observed at 140 mmHg compared to perfusion at 60 mmHg. One might assume a similar increase in extracellular Gd content as observed for Na+e. However, Gd affects both intra- and extracellular water, and therefore the effects of the increased extracellular Gd content are diluted compared to Na+e. Furthermore, the relationship between [Gd] and signal intensity is complex and non-linear. We consistently observed transmural heterogeneous enhancement of the LV myocardium (Figs. 6-7) after perfusion with Gd. The subendocardial area showed higher signal intensity, suggesting more edema in the subendocardial area than in the subepicardial area. This is most likely related to the intramyocardial pressure gradient, with this pressure being higher in the subendocardial area. In the model used, hydrostatic pressure difference is the most important, if not the only, source of edema, whereas in vivo differences in microvascular permeability and osmotic pressure may also contribute to the formation of edema [17]. The subendocardial area with higher signal intensity was significantly larger in hearts perfused at 140 mmHg than at 60 mmHg, confirming more edema at higher perfusion pressure. This finding might provide an explanation for observed late enhancement in patients with arterial hypertension in the absence of myocardial infarction [29]. Interestingly, a similar transmural distribution of signal intensities as observed in the CE-MR images can also be seen in the Na+e images (Fig. 5). However, since these hearts belong to different groups and, in addition, differences in spatial resolution, slice thickness and cardiac 98 Na Chemical Shift Imaging and Gd Enhancement of myocardial edema 23 gating exist between CE-MRI and 23Na-CSI groups, direct correlation of these patterns is not possible. Whether 23Na-CSI is indeed better capable than CE-MRI to distinguish between normal, edematous and infarcted tissue, in acute and chronic myocardial infarction, requires further experiments in an infarct model. Methodological considerations –– In addition to intracellular and interstitial contributions, the signals from the total water space marker DMMP and the extracellular space marker PPA include also contributions from vasculature, ventricular cavities, and perfusion and suction tubing adjacent to the rat heart. Nevertheless, our results clearly suggest a significant increase of the myocardial extracellular space within an hour of perfusion at higher pressure. –– In biological tissue, 23Na (in Na+ ions) is present in a much lower concentration than 1H in H2O. This explains, together with the lower MR sensitivity of 23Na, the low spatial and temporal resolution of our 23Na-images compared to the 1H-MR images. To increase the SNR, we have chosen a thicker slice and bigger voxel size, and we have used k-space weighted acquisition to avoid Gibbs ringing. Moreover, the fact that 23Na has short relaxation times enabled us to use short repetition times. To partially overcome the low spatial resolution in the 23Na-images, we have used 1H-MR images as anatomical reference. –– In this model, myocardial interstitial edema might already be present at 60 mmHg, resulting in a larger extracellular space than observed in vivo. However, the observed increase of Na+e at 140 mmHg clearly suggests a further expansion of the extracellular compartment, which was corroborated by 31P- and 1H-MR data. –– Na+e mostly shows mono-exponential T2 relaxation (T2 = 38.7 ms, Van Emous et al. [24]) whereas Na+i shows bi-exponential T2 relaxation with usually 60% of the signal fast relaxing (T2f = 2.3 ms [24]) and 40% slow relaxing (T2s =18.9 ms [24]). The CSI method requires an acquisition delay (0.91 ms), which is in the range of the T2f component, causing a non-negligible loss of Na+i signal. However, although Van Emous et al. have observed changes in T2 relaxation during ischemia and 99 Chapter 5 reperfusion, no such changes have been observed during normal perfusion such as used in the current experiments. Therefore, no correction was applied. Similar arguments apply with respect to T1 relaxation. –– It is important to mention that our CE-MRI protocol is different from that used clinically to assess infarct size. We have used continuous perfusion of contrast agent, whereas in the clinic a bolus of contrast agent is administered. Due to the absence of an infarct and due to time constraints caused by the high rat heart rate, nulling of ‘remote’ myocardium is not possible in our model. Clinical implications With a non-infarct model of edema, we have demonstrated that tissue edema shows up in CE-MRI. This suggests that CE-MRI might lead to wrongful diagnosis of infarction in other cardiac disease states exhibiting edema or to overestimation of infarct size [1;2]. 23Na-CSI might be an attractive tool to resolve these issues. We have shown that viable – but edematous myocardium – is characterized by increased Na+e and normal Na+i signals. Previously, we have shown that during global low flow and complete regional ischemia, Na+i signals increase several-fold, with partial recovery upon reperfusion [23]. Furthermore, we have previously observed in a model of chronic infarction after coronary artery ligation, a substantial increase of the Na+e signal and a complete absence of Na+i signal in the infarct area [30;31]. These characteristics make Na-CSI a promising tool to discriminate between edema, acute and chronic MI. So 23 far, only total Na+-MRI has been used clinically. However, to make 23Na-CSI a clinically feasible, high field (7 T) MRI scanners are recommended and a shift reagent safe for human use is required, which is not yet available, but could be developed, since MR contrast agents and MR shift reagents are chemically rather similar. Alternatively, the use of multiple quantum filtered MR methods [32;33] or ultrashort-echo time (UTE) sequences [34] could be investigated. 100 Na Chemical Shift Imaging and Gd Enhancement of myocardial edema 23 Acknowledgements We thank Steven S. A. 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Doevendans, Gerard Pasterkamp, Gustav J. Strijkers, Cees J.A. van Echteld † These authors contributed equally to this study Submited 105 Chapter 6 Abstract Using Na-Chemical Shift Imaging (CSI) complemented by 1H-Late Gadolinium 23 Enhanced (LGE)-MRI we aimed to differentiate injured myocardium under three conditions: acute ischemia and reperfusion (I/R) (group1), 2 hours after I/R injury (group2), and chronic infarction (MI) (group3). Isolated rat hearts (n=6) underwent 30, 40 or 50 min of ischemia, followed by 40 min of reperfusion (group1). Seven rats underwent 50 min of in vivo ischemia, followed by 2 hours of in vivo reperfusion (group2). Chronic MI was induced in six rats by 3 weeks of LCA-ligation (group3). Following interventions, group 2 and 3 hearts were also isolated and perfused. During ischemia, Na+i signal intensity (SI) increased to 24.4±1.4, 28.2±1.5, and 33.7±1.4 (a.u.), at 30, 40 and 50 min, respectively, but decreased during 40 min of reperfusion (group1). In group2, Na+e appeared higher in infarct than remote (0.51±0.07 vs. 0.46±0.05, p=0.17), whereas Na+i was equal (0.0138±0.003 vs. 0.0143±0.003, p=0.75). In chronic MI, Na+e SI was elevated in the infarct (0.51±0.05 vs. 0.32±0.03, p=0.002), whereas Na+i was decreased (0.006±0.001 vs. 0.012±0.002, p=0.003). Infarct size (IS) from TTC-staining corresponded to automatically determined IS from LGE and Na+e images, at approximately 5SD thresholds (acute-MI), but in chronic-MI at 2SD thresholds. Na+ tracking is valuable for indentifying the pathophysiological status of the heart: highly elevated Na+i is a unique characteristic of acute ischemia, whereas the absence of Na+i signal in combination with elevated Na+e signal is a marker of scar tissue. The rapid normalization of highly elevated Na+i in infarcted area upon reperfusion provides unique insights in the dynamics of infarct development. Combination of 23Na-CSI and H-LGE-MRI shows added value for differentiation of various pathophysiological states 1 after MI. Keywords Magnetic resonance imaging, sodium MRI, myocardium, myocardial infarction, reperfusion, chemical shift imaging 106 Differentiation of myocardial injury in various infarct models with 23Na Chemical Shift Imaging and Late Gd Enhancement MRI 6.1Background MRI plays an important role in the prognostic assessment of patients with heart disease. It is a powerful noninvasive modality to image cardiac anatomy, regional and global function, myocardial perfusion, and viability. The pathophysiological tissue status alters longitudinal relaxation time (T1) and transverse relaxation time (T2). Thus, an adequate MR protocol may enable identification of the pathophysiological changes after a myocardial infarction (MI). Commonly, these are mainly 1H-MRI techniques which have been extensively investigated and applied to assess edema [1-3], inflammation [4], apoptosis [5], necrosis, fibrosis [6], inflammatory-cell infiltration to myocardial infarction sites [7], and hemorrhage, as well as iron overload in non ischemic heart disease [7;8]. For instance, T1-weighted MRI after the injection of a gadolinium‑based contrast agent is a common method to delineate injured myocardium [9] in so-called Late Gadolinium Enhancement (LGE) MRI. LGE of the injured myocardium occurs because of altered wash-in/wash-out kinetics of the contrast agent in comparison to healthy myocardium. In this respect, the presence of edema is a confounding factor, which may lead to LGE in the absence of necrosis and thus an overestimation of true infarct size [10]. Moreover, selecting a standard signal threshold offset for accurate differentiation between injured but salvageable and non-viable myocardium, both in acute and chronic MI, is often problematic [11]. Accurate distinction between stunned and scar tissue is clinically important to decide whether revascularization may still be beneficial after an ischemic insult. Based on a LGE-MRI scan alone, it is difficult to discriminate between salvageable and scar tissue. Recently, T2-weighted MRI was introduced as a complementary technique to image the salvageable myocardium [12-14]. Although promising, T2-weighted MRI requires further optimization [4]. Viable myocytes sustain a constant gradient between the extra-cellular sodium (Na+e) and the intra-cellular sodium (Na+i) concentrations with an approximate ratio of 150/10 (mM/mM). Alteration of this gradient indicates dysfunction of the Na+/K+ATPase pumps and thus relates to changes in cellular homeostasis occurring during 107 Chapter 6 and after MI [15]. Therefore, measurement of the sodium gradient by 23Na-MRI is of particular interest and provides a direct window on cellular viability [16;17]. Data on 23Na-MRI are scarce due to the relatively lower MR sensitivity and lower in vivo concentration in comparison to 1H-MRI [18]. Still, 23Na-MRI was investigated in a number of studies. In human heart, total 23Na (Na+t) MR signal was significantly elevated in injured myocardium [19]. A more complete way to assess the Na+/K+ATPase pump function for myocardial viability is to monitor Na+i and Na+e separately facilitated by the use of a shift reagent and a chemical shift imaging (CSI) sequence. The Na+i concentration rises shortly after myocardial ischemia because of inhibition of sarcolemmal Na+/K+-ATPase. Na+ may enter the myocytes via many pathways, perhaps most importantly via the Na+ channel, and the Na+-H+ exchanger (NHE) [20]. Therefore, monitoring extracellular and intracellular Na+ separately could be more sensitive to distinguish different pathophysiological changes states after MI than Na+t-MRI and H-MRI alone. 1 We have previously shown that during reperfusion after ischemia induced by application of low coronary flow in an isolated rat heart model, the elevated Na+i signal intensity correlated to the severity of ischemia [16]. Furthermore, we have shown that a higher level of interstitial edema in isolated heart model also increases extracellular volume distribution in LGE-MRI and 23Na-CSI [21]. However, the complex pathophysiological changes in acute and chronic myocardial injury remain to be fully addressed. The aim of this study was therefore to apply 23Na-CSI complemented by 1H-LGE-MRI in isolated rat hearts to characterize the injured myocardium in three different conditions: in acute MI during ischemia and reperfusion, 2 hours after ischemia/reperfusion (IR) injury, and in chronic MI. 108 Differentiation of myocardial injury in various infarct models with 23Na Chemical Shift Imaging and Late Gd Enhancement MRI 6.2 Materials and Methods Experimental protocol All experimental protocols were approved by and performed according to the guidelines of the animal experiments committee of the University Medical Center Utrecht, the Netherlands. Male Wistar rats (body weight=300 to 350 g) were used for this study. Three groups of animals were used. Group 1 was studied during acute MI, group 2 two hours after reperfusion, and group 3 in the chronic phase. Details on the several groups are given below. Group 1: Acute MI Six rats were used for this group. Hearts were excised under anesthesia and perfused in a Langendorff set-up with Krebs-Henseleit (KH) buffer, using a dual perfusion cannula which allowed independent perfusion of the left and right coronary bed [22]. 1H-MRI survey scans were performed in axial and longitudinal orientations, followed by shortaxis cine 1H-MRI. Next, the perfusate was switched to buffer containing the shift reagent (SR) Tm-DOTP5-. Subsequently, the left side of the hearts was subjected to regional ischemia for 30, 40, or 50 min (flow to the other side remained unaltered), followed by 40 min of reperfusion, and in parallel, sequential 23Na-CSI short-axis scans were acquired. After that, the left side of the heart was perfused with buffer containing a Gd contrast agent and a T1-weighted MRI scan was acquired to assess the spatial extent of injured myocardium. A schematic diagram of the experimental protocol is shown in Figure 1. After 2 hours, the right side of the heart was perfused with methylene blue to determine the area at risk for histology. After that, the whole heart was perfused with 1% triphenyl tetrazolium chloride (TTC) to stain the viable tissue. Group 2: Two hours reperfusion Induction of myocardial injury Rats (n=6) were anesthetized with isoflurane in 2:1 mixture of air (0.3 l/min) and oxygen (0.15 l/min). A core body temperature of 37 °C was maintained during surgery 109 Chapter 6 Groups 1: Ctrl SR Gd Buffer Preparation + 1H MRI 25 Ctrl 23Na CSI Ischemia (30-50) SR T1 W MRI 23Na CSI Reperfusion 40 Ctrl 10 Time (min) Gd Buffer 2: Preparation + 1H MRI 25 23Na CSI Washout T1 W MRI 20 Ctrl SR 16 Time (min) Gd Buffer 3: Preparation + 1H MRI 25 23Na 20 CSI T1 W MRI 5 Time (min) Figure 1: Schematic representation of experimental protocols Group 1: during acute MI, group 2: two hours after reperfusion, group 3: chronic MI group. Isolated hearts were perfused with control (ctrl) buffer, with shift reagent (SR) TmDOTP containing buffer, and with contrast agent (Gd) containing buffer. by continuous monitoring with a rectal thermometer and an automatic heating blanket. The rats subcutaneously received Antisedan (Pfizer) 2.5 mg/kg, Anexate (Roche) 0.5 mg/kg, and Temgesic (Schering-Plough) 0.1 mg/kg. Rats were intubated and ventilated (Harvard Apparatus Inc, Holliston, Mass) with 100% oxygen. The left coronary artery was ligated for 50 min with an 8-0 Ethilon (Ethicon) suture with a section of polyethylene-10 tubing placed over the left coronary artery. Ischemia was confirmed by bleaching of the myocardium and ventricular tachyarrhythmia. Reperfusion was initiated by release of the ligature and removal of the polyethylene-10 tubing. Reperfusion of the endangered myocardium was characterized by typical hyperemia in the first few minutes. After 50 minutes of continuous reperfusion in vivo, the rats were injected with heparin (500 IU/Kg i.v.) via the femoral vein. Perfusion After a total of 2 hours of reperfusion, hearts were excised and perfused in a Langendorff set-up with KH buffer, using a single perfusion cannula. Subsequently, 1H-MRI survey 110 Differentiation of myocardial injury in various infarct models with 23Na Chemical Shift Imaging and Late Gd Enhancement MRI scans were performed in axial and longitudinal orientations, followed by short-axis cine H-MRI. Thereafter, hearts were perfused with buffer containing SR and 23Na spectra 1 were acquired to monitor the arrival of SR. 23Na-CSI was performed in a mid-axial slice. Subsequently, the perfusate was switched to control KH buffer to washout the SR for 16 min. Next, the hearts were perfused with buffer containing a Gd contrast agent and eight sequential 1H-LGE-MRI scans were acquired every two minutes to monitor the arrival of contrast agent. Then the perfusion was switched to KH buffer to washout the Gd, monitored by sequential T1-weighted 1H-MRI. At the end of the scan, hearts slices were stained with 1% TTC for viable tissue. A schematic diagram of the experimental protocol is shown in Figure 1. Group 3: Chronic MI Chronic MI of rat hearts (n=6) was induced by ligation of the left coronary artery as described above. After permanent occlusion of the left coronary artery, the thorax was closed and animals were allowed to recover. Three weeks after ligation, hearts were excised and perfused in a Langendorff setup. The remainder of the protocol was the same as for the rats in group 2. A schematic diagram of the experimental protocol is shown in Figure 1. Perfusates and Langendorff setup As perfusate, a modified KH buffer was used containing (in mmol/L): 119.0 NaCl, 4.7 KCl, 1.0 MgCl2, 24.0 NaHCO3, 1.3 CaCl2, 11.0 glucose, and 5.0 Na-pyruvate. Before perfusion, the KH buffer was filtered through 8 µm (Millipore, Bedford, MA) filters. The solution was equilibrated with 95% O2 and 5% CO2, resulting in a pH of 7.4 at 37°C, prior to the addition of the CaCl2. For Na-CSI, Thulium(III)1,4,7,10 tetraazacyclododecane-N,N 23 ,N / ,N // -tetra /// (methylenephosphonate) (Tm-DOTP , 3.5 mM) [23] was included in the buffer as SR 5- to separate Na+i and Na+e. To correct for Ca2+ binding by the SR, the total Ca2+ added to the buffer was increased to 3.42 mM, which resulted in a free [Ca2+] of 0.85 mmol/L as measured by a HI 4004-51 Ca2+ Ion Selective Electrode (ISE) connected to a pH/ 111 Chapter 6 ISE meter (HI 3221, Hanna instruments). For T1-weighted 1H-LGE-MRI, 1.3 mM of Gd contrast agent Gadobutrol (Gadovist) was added to the buffer. For group 2, an additional perfusion line ending at the bottom of the NMR-tube was used to flush the tube at a rate of 30 mL/min with a buffer in which Na+ was replaced by Li+, keeping the extracardiac Na+ signal low and the hearts submerged for good magnetic susceptibility matching. Hearts were rapidly excised and placed in ice-cold KH buffer. The hearts were mounted via the aorta with a single perfusion cannula (groups 2 and 3) or via a home-built dual– perfusion cannula (group 1), which allowed independent perfusion of the left and right coronary beds of the heart. After cannulation, perfusion was initiated according to Langendorff with KH buffer at 37oC. To further prepare the heart, a drain was inserted through the apex via the mitral valve to remove Thebesian flow. A latex balloon was inserted into the left ventricle through the mitral valve and filled with water to reach a left ventricular end-diastolic pressure (LVEDP) of 7.5 mmHg. Two pressure transducers (MP-15, Micron Instruments) were used, one to monitor perfusion pressure and a second one to measure the isovolumetric contractile pressure of the left ventricle via the inserted balloon. Left ventricular developed pressure (LVPD) was determined as the difference between left ventricular systolic pressure (LVSP) and LVEDP. To maintain a heart rate of 300 beats/min, the outflow tract of the right ventricle was connected with two copper-wire electrodes to a (4-7 V, 0.5 ms) stimulator (model S88, Grass Instruments, Quincey, MA, USA). Heart rate was determined from the pressure curve. Coronary flow was measured with a flow probe (Skalar instruments, Delft, The Netherlands). The data were monitored and recorded using data Acquisition System (PowerLab, ADI Instruments, Australia). MRI scans was initiated after placing the hearts in a 20 mm NMR tube diameter that was positioned inside the MR scanner. MR acquisition A vertical 9.4 T, 89-mm-diameter bore magnet was used, equipped with a 1500 mT/m gradient system and interfaced to an AVANCE 400 DRX spectrometer (Bruker, Germany). 112 Differentiation of myocardial injury in various infarct models with 23Na Chemical Shift Imaging and Late Gd Enhancement MRI 1 H-MRI Tuning and global shimming was done using a 20-mm-diameter 1H birdcage coil (Bruker, Germany). Subsequently, scout (2- and 4-chamber view) MR scans were acquired to plan a single mid-ventricular short-axis slice. Cine imaging was performed with a retrospectively‑triggered gradient‑echo (IntraGateFlash), with the following parameters: TE=1.9 ms, TR=5.2 ms, FA=10o, number of movie frames=15, slice thickness=2.5 mm, matrix=256´256, field of view (FOV)=2´2 cm2. 1H-LGE-MRI was performed with (group 2) an ECG-gated, short axis multi-slice FLASH sequence, using TR=63 ms, TE=2.8 ms, FA=75o, NA=8, 6 slices of 2.5 mm, matrix=256´256, FOV=2´2 cm2 or (groups 1 and 3) with a multi-slice MSME sequence, using TR=1003 ms, TE=6.1 ms, NA=1, 2 slices of 2.5 mm, matrix=256´256, FOV=2´2 cm2. Na-MRS and CSI 23 For Na MR scans, the 1H birdcage coil was replaced with a 23 Na birdcage coil 23 (Bruker, Germany). A 5 mm mid-axial slice was acquired as a reference image using a retrospectively gated 23Na FLASH sequence, with TE=2.4 ms, TR=25 ms and 128 repetitions, FA=50o, matrix=32x32, and FOV=2´2 cm2. To monitor the shift of Na+e after switching to the buffer with SR, 23Na MR spectra were acquired using a pulseand-acquire sequence, with TR=105 ms, FA=75o, SW=5.5 kHz, and 512 data points. Acquisition-weighted Na-CSI was performed with 10,000 acquisitions, NA=55 23 in center of k-space, TR= 30 or 27 ms, FA=90o, pulse=0.5 ms Sinc, BW=12420 Hz, matrix=16x16, reconstruction matrix=32x32, and FOV=2´2 cm2. FIDs were collected with 128 complex data points and 100.8 µs dwell time resulting in an acquisition time of 25.8 ms/FID. The 23Na-CSI data were zero-filled in both the spatial and spectral domain resulting in a (256x32x32) reconstruction size. To optimize peak separation, a Lorentzian-Gaussian multiplication was applied. Analysis To determine average Na+ signal intensities, regions-of-interest (ROIs) were manually drawn to delineate the myocardial free wall (injured myocardium) and septal wall (remote) in the 1H-MR images and subsequently copied into the 23Na images. The 113 Chapter 6 average signal intensities in the ROIs for myocardial Na+i and Na+e were calculated with Paravision 4 software (Bruker, Germany). To semi-automatically determine infarct size in groups 2 and 3, the 1H and Na 23 signal intensities in the remote myocardium were used. The enhanced areas in the Na-CSI and 1H-LGE-MR images were quantified with a semiautomatic algorithm 23 (Segment, Medviso AB, Sweden) based on signal-intensity thresholds (2, 4, and 6 standard deviations (SDs) above remote myocardium) as proposed by Kim et al. [24]. To avoid contamination by extra-cardiac and intra-ventricular signals, epicardial and endocardial contours of the left ventricle were manually drawn on end-diastolic 1HMR images. Subsequently, these contours were copied to the 23Na-CSI and 1H-LGE-MR images. TTC stained tissue sections were photographed under a microscope with a digital image camera. Infarct size (IS) and total LV area were manually delineated and quantified using ImageJ software (version 1.42). The IS was then expressed as percentage of the total LV area. Statistical analysis Results were expressed as mean ± standard error of the mean. A paired t-test was performed to compare mean values. Results were considered significantly different for a two-tailed p value < 0.05. The correlation test of the infarct size for TTC vs. LGE was performed using a Pearson correlation with two‑tailed p values. 114 Differentiation of myocardial injury in various infarct models with 23Na Chemical Shift Imaging and Late Gd Enhancement MRI 6.3Results Group 1: Acute MI Figure 2A shows a collection of short-axis 23Na-CSI and 1H-LGE-MR images, and the corresponding TTC stained section for an isolated rat heart subjected to 40 min of ischemia of the left side, followed by 40 min of reperfusion. As expected, regions with high sodium concentration such as the balloon, the right ventricular cavity and the heart-surrounding fluid appeared bright in Na+e-CSI. During ischemia, Na+i MR image intensity increased significantly at the left side (infarct) whereas that of the right side (remote) remained unaltered. The hyperenhanced region of Nai at the end of ischemia Figure 2: Representative 23Na CSI and 1H MR images of a mid-axial slice of an isolated rat heart of the acute MI (group 1) and intra- and extracellular Na+ signal in the infarct, before ischemia, during ischemia and reperfusion (A) Example of extracellular and intracellular 23Na CSI, LGE MRI and corresponding TTC staining of isolated rat heart, and the corresponding 23Na CSI signal intensities (B) before ischemia, during ischemia and reperfusion 115 Chapter 6 appears to correlate better to the TTC unstained region, than the hyperenhanced region of Nai at the end of reperfusion. As expected, Na+e image intensity in the infarct showed a slight decrease during ischemia whereas it increased again during reperfusion. TTC infarct areas also corresponded to 1H-LGE-MRI infarct areas. Quantitative analysis of the 23Na-CSI data revealed the dynamic changes of the Na+i and Na+e signals over time (Figure 2). Prior to ischemia, relative Na+i and Na+e signal intensity values, derived from magnitude images, were in remote tissue 6.5±0.5 and 286±23, respectively, and 6.4±0.6 and 265±28 in the region that would become infarcted at later time points. Values in the remote region did not change during the course of the experiment. During ischemia, Na+i increased linearly (R=0.97) with relative signal intensities of 24.4±1.4, 28.2±1.5, and 33.7±1.4, at 30, 40 and 50 min, respectively. 40 min after reperfusion, Na+i values had decreased towards baseline values of 7.6±0.1 and 5.3±2.3, respectively, for the 30 and 40 min hearts, whereas for the heart that suffered 50 min of ischemia Na+i remained elevated at 13.10, suggesting more severe myocardial damage or poor reflow. Relative Na+e signal intensities in the infarct (Figure 2B) decreased to 201±58, 183±39, and 173, at 30, 40, and 50 min of ischemia, respectively. After 40 min of reperfusion, the Na+e signal intensities had decreased but remained slightly higher than baseline values, suggesting the presence of extracellular edema or cell damage. Group 2: Two hours reperfusion The isolated rat hearts that underwent 50 min of in vivo ischemia followed by 2 hours of reperfusion had a heart rate of 305.96±0.05 beats/min during the whole experiment. The left ventricular developed pressure (LVDP) decreased from 29.1±1.4 mmHg for control buffer to 22.1±2.7 mmHg for buffer with SR. the LVDP partly recovered to 25.7±2.4 mmHg after switching back to control buffer. For the three conditions, the coronary flow rates were 7.87±0.45, 7.01±0.86, 6.73±1.03 mL/min, respectively. Figure 3 depicts a time-series of T1‑weighted MR images every two minutes during control perfusion (A), after switching to Gd-containing buffer (B) and again after 116 Differentiation of myocardial injury in various infarct models with 23Na Chemical Shift Imaging and Late Gd Enhancement MRI washout with control buffer (J). The enhancement in T1‑weighted MRI confirmed Gd arrival in the myocardium. The enhancement was more obvious in the mid-anterior and mid-anterolateral wall. After switching to the control buffer, the Gd enhanced area started to decrease until it finally disappeared, indicating that the dynamics of Gd wash in and wash out influence LGE MRI. The best contrast of infarct vs. remote was observed at the 3rd MR scan (L) (approx. 6 minutes) after Gd washout. Figure 3: Representative T1-weighted MR images during infusion and washing-out of Gd containing buffer of a mid-axial slice of an isolated rat heart (group 2). Scans were acquired every 2 min after switching to Gd containing buffer. A good contrast between infarct and remote is observed after washout. Figure 4A presents a representative example of corresponding mid short-axis 23Na-CSI, H-LGE-MRI, and TTC slices, after in vivo ischemic injury and 2 hours in vivo reperfusion. 1 The balloon in the left ventricle contained a Na+-free solution, thus appeared dark in Na-CSI. Regions of high sodium concentration such as in the right ventricular cavity, 23 which filled with effluent, had high signal intensity. The Na+i images displayed lower SNR than those of Na+e because of the lower Na+i content. The Na+e signal was hyperenhanced in the mid-anterior and mid-anterolateral wall in the same region of 1H-LGE and the unstained region in TTC, indicating the presence of myocardial injury in this area. 117 Chapter 6 Figure 4: Representative example of a mid-axial slice of an isolated rat heart with acute inferior myocardial infarction (group 2). (A) Extracellular and intracellular Na CS images, LGE image and corresponding TTC staining. (B) Normalized signal intensity of extracellular and intracellular Na+ and LGE in infarct vs. remote. (C) Infarct size delineation based on signal intensity thresholds 2, 4, and 6 SDs above remote normal myocardium. The infarct region is marked with a red dashed line in the enhanced area of the LGE MRI. We have specifically evaluated the changes of the Na+e and Na+i signal intensity in the infarct and remote tissue (Figure 4B). The signal intensity was normalized to the buffer Na+ signal. Na+e was slightly higher in infarct than remote tissue (0.51±0.07 vs. 0.46±0.05, p=0.17), whereas Na+i values were equal (0.0138±0.003 vs. 0.0143±0.003, p=0.75). 1H-LGE-MRI corresponded to Na+e, with higher intensity in the infarct ((6.3±0.5) x104 vs. (4.3±0.9)x104, p<0.05). The infarct size determined from the TTC (percentage of the LV) was 17.8±3.2%. To further characterize the MRI enhancement of the injured myocardium in more detail, we determined the Infarct size based on 2, 4, and 6 SD cut-off thresholds above remote 118 Differentiation of myocardial injury in various infarct models with 23Na Chemical Shift Imaging and Late Gd Enhancement MRI signal intensity (Figure 4C). The best agreement between TTC determined infarct size, on the one hand, and infarct size from LGE and Na+e, on the other hand, was observed for thresholds of 4 and 6 SD. The high correspondence between LGE and Na+e confirms that these two readouts are both markers of the extracellular volume fraction. Figure 5: Representative example of a mid-axial slice of an isolated rat heart with chronic myocardial infarction (group 3). (A) Extracellular and intracellular Na CS images, LGE image and corresponding TTC staining. (B) Normalized signal intensity of extracellular and intracellular Na+ in the infarct vs. remote. (C) Infarct size delineation based on signal intensity thresholds 2, 4, and 6 SDs above remote normal myocardium. The scar region is marked with red dashed line in the enhanced area of the LGE MRI and in the hypo-enhanced area of the Nai CSI Group 3: Chronic MI Axial 23Na-CSI, 1H-LGE-MRI, and corresponding TTC slices of isolated rat heart 3 weeks after LAD ligation are shown in Figure 5A. 23Na+e and LGE revealed similar enhanced 119 Chapter 6 infarcts corresponding well to the TTC infarct areas. Quantitative analysis (Figure 5B) revealed that the normalized Na+e signal intensity was significantly elevated in the infarct versus remote tissue (0.51±0.05 vs. 0.32±0.03, p=0.002), indicating a significant increase in the extracellular volume fraction in the infarct scar. Na+i was significantly decreased in the infarct as compared to remote tissue (0.006±0.001 vs. 0.012±0.002, p=0.003). Similar to group 2, the infarct size estimation based on signal intensity was determined using (2, 4, and 6 SD) threshold above the remote signal intensity for LGE and Na+e (Figure 5C). TTC based infarct size (as percentage of the LV) was 25.4±3.7%. Best agreement with TTC staining was obtained for 2 SD. For LGE the estimated infarct size rapidly decreased for higher thresholds. The estimation of infarct size based on Na+i failed because of the very low Na+i signal intensity in the chronic infarct. 6.4Discussion We have investigated the dynamic behaviour of intra- and extra-cellular sodium using cardiac Na CSI, combined with 1H LGE MRI under different myocardial 23 pathophysiological conditions, including acute ischemia, reperfusion, and chronic infarcts using in vivo and in vitro rat models of myocardial ischemia and infarction. We have demonstrated that Na CSI is a powerful tool to identify sarcolemmal 23 dysfunction related to myocardial pathophysiological status in the time-course after MI. Acute, totally ischemic myocardium is characterized by highly elevated Na+i levels, caused by Na+/K+ ATPase dysfunction and continued Na+ influx, and concomitant decrease of Na+e (group 1). In contrast, chronic MI (group 3) was characterized by an almost complete absence of Na+i, indicating the loss of viable myocardium and replacement by scar tissue, corroborated by the observed increased Na+e. However, the situation in acutely reperfused myocardium (groups 1 and 2) is less clear. The (partial) recovery of Na+i may signify recovery of Na+/K+ ATPase function, but could be equally well explained by heterogeneous reperfusion with areas of no reflow and 120 Differentiation of myocardial injury in various infarct models with 23Na Chemical Shift Imaging and Late Gd Enhancement MRI persistent high Na+i in combination with areas showing advanced cell deterioration and sarcolemmal dysfunction/rupture with loss of cellular content. The slightly elevated Na+e could signfy edema, but could also be explained by loss of sarcolemmal integrity. These findings are summarized below: Status of injured myocardium Na+i Na+e Acute Ischemia (without reperfusion) +++ - Acute reperfusion -/0/+ 0/+ --- +++ Chronic infarct (scar tissue) Group 1: Acute MI In the acute MI animals (group 1) the Na+i infarct signal increase during ischemia was directly proportional to the ischemia duration. The linear Na+i increase during ischemia is in line with previously published data from our group [20;25;26]. During reperfusion after 30 or 40 minutes of ischemia, Na+i levels normalized. However, reperfusion after 50 minutes of ischemia resulted in a persistently elevated Nai+, which could be interpreted as more severe injury caused by the prolonged ischemia. In parallel, Na+e levels decreased during ischemia and were slightly higher than remote after reperfusion. We have demonstrated before [25] that recovery of Na+i levels after a substantial ischemic rise, is not necessarily correlated with recovery of function and that post-ischemic Na+-H+ exchange blockade is not cardioprotective, since most of the rise of Na+i has already taken place during ischemia, resulting anyway in detrimental reverse Na+-Ca2+ exchange and subsequent Ca2+ overload. In view of the good correspondence of TTC infarct area and the area of end-ischemic enhanced Na+i and the poor correspondence with the smaller area of end-reperfusion enhanced Na+i, a more likely interpretation of the observed changes in Na+i is that the recovery of Na+i signifies detrimental Ca2+ overload and the persistently elevated Na+i in the core of the infarct represents an area of poor reflow. 121 Chapter 6 Group 2: Two hours reperfusion We have examined isolated rat hearts with 1H-LGE-MRI and 23Na-CSI following in vivo coronary ligation and in vivo reperfusion. We did not observe a significant difference of the Nai signal intensity between the injured myocardium and the remote region. This might be construed as full recovery, but in view of the discussion on group 1, it is more likely that the dynamics of 2 hours of reperfusion are such that the entire infarct area has been reperfused, leading to recovery of Na+i at the expense of Ca2+ overload, setting the stage for further infarct development. Alternatively, the findings may also be explained by a combination of myocardial cells in which Ca2+ overload has resulted in sarcolemmal disintegration, with myocardial cells exhibiting persistently elevated Na+i due to poor reflow, resulting in no net change in Na+i. However, we have to keep in mind that, in contrast to group 1, in case of areas of complete no reflow in group 2, the shift reagent would not be able to reach these areas, which would result in Na+e resonating at the Na+i position and a concomitant strong increase in signal intensity, which we obviously have not observed. H-LGE-MRI was performed during continuous infusion of Gd-based contrast agent 1 as well as during (delayed) wash-out (Figure 3). Higher contrast between infarct and remote tissue was observed in the wash-out phase, supporting the notion that LGE is very sensitive to timing of the experiment [10;27;28]. Infarct size was estimated with 23Na-CSI and 1H-LGE-MRI using different signal threshold levels and compared to TTC staining(Figure 4B). Different values of infarct size based on different cutoff threshold values have been also observed by others [29]. Although 1H-LGE-MRI has obvious advantages in terms of SNR and spatial resolution, infarct size from 23Na-CSI, compared well to both LGE and TTC. Na+e and 1H signals were elevated in the infarct, but, as mentioned above, Na+i in the infarct area was not different from remote tissue, and could not be used to estimate infarct size. Interestingly, the best correspondence between TTC infarct size and both LGE and Nae was found for areas defined by approx. 5 SD’s above remote, indicating an area of lower enhanced LGE and Nae signal intensities, not corresponding to the true infarct. Edema is pathognomonic for IR injury [30] and we have demonstrated before in a non-ischemic model of edema that both LGE and 122 Differentiation of myocardial injury in various infarct models with 23Na Chemical Shift Imaging and Late Gd Enhancement MRI Nae signal intensities are elevated in edema. Therefore, the current results could be well explained by edema in a peri-infarct zone. Group 3: Chronic MI In chronic infarcts, 3 weeks after MI, Na+i was very low and Na+e levels were significantly increased. These findings confirm the absence of viable cells and increased interstitial space in fibrotic scar (Figure 5A-B). Interestingly, 2-3 SD’s thresholds seem to have a better match to the TTC infarct area in this group, which could be explained by lower or absent edema levels. Clinical perspective LGE MRI has become the current gold standard to determine chronic infarct size, but Na-CSI has not yet been used clinically. To make 23Na-CSI a clinical reality, high field 23 (7 T) MRI scanners are recommended. Futhermore, a shift reagent suitable for use in human is required, which is not yet available, but, given the similarity between contrast agents and shift reagents, could be developed. Alternatively, multiple quantum filtered MR methods [31;32] or ultrashort-echo time (UTE) sequences [33] could be used, but further investigations are still needed for clinical implementation of these methods. Na-CSI of normal Na+i suffers from poor SNR and low spatial resolution, but the 23 absence of Na+i signal in combination with strongly elevated Na+e signal unambiguously indicates a chronic infarct, unlike LGE, which would also show enhancement with edema. Highly elevated Na+i levels indicate ischemia, which requires immediate reperfusion, preferably accompanied by interventions to prevent reverse Na+-Ca2+ exchange . 123 Chapter 6 6.5Conclusion We have demonstrated the feasibility of cardiac imaging of intra- and extra-cellular sodium to assess myocardial viability in different rat models of myocardial infarction. Na+ tracking is a valuable tool to indentify the pathological status of the heart: highly elevated Na+i is a unique characteristic of acute ischemia, whereas the absence of Nai signal in combination with elevated Na+e signal is a marker of scar tissue. The rapid normalization of highly elevated Na+i in infarcted area upon reperfusion provides unique insights in the dynamics of infarct development. The combination of Na-CSI and 23 H-LGE-MRI shows added value for accurate evaluation, understanding and distinction 1 of different pathophysiological states after myocardial injury and helps to interpret results mutually. Competing interests None. Acknowledgements This research is supported by the Netherlands Heart Foundation (NHS) grant 2006B066 (CvE) and 2010T001 (FA), as well as by the Netherlands Heart Institute (ICIN). The work of GJS is supported by the Dutch Technology Foundation STW, Applied Science Division of NWO and the Technology Program of the Ministry of Economic Affairs (grant number 07952). We would like to thank Maringa Emons for biotechnical assistance. Reference List 1. Walls MC, Verhaert D, Min JK, Raman SV (2011) Myocardial edema imaging in acute coronary syndromes. J Magn Reson Imaging 34:1243-1250 2. Beyers RJ, Smith RS, Xu Y, Piras BA, Salerno M, Berr SS, Meyer CH, Kramer CM, French BA, Epstein FH (2012) T(2) -weighted MRI of post-infarct myocardial edema in mice. Magn Reson Med 67:201-209 124 Differentiation of myocardial injury in various infarct models with 23Na Chemical Shift Imaging and Late Gd Enhancement MRI 3. Edwards NC, Routledge H, Steeds RP (2009) T2-weighted magnetic resonance imaging to assess myocardial oedema in ischaemic heart disease. Heart 95:1357-1361 4. Eitel I, Friedrich MG (2011) T2-weighted cardiovascular magnetic resonance in acute cardiac disease. 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Navon G, Werrmann JG, Maron R, Cohen SM (1994) 31P NMR and triple quantum filtered 23Na NMR studies of the effects of inhibition of Na+/H+ exchange on intracellular sodium and pH in working and ischemic hearts. Magn Reson Med 32:556-564 33. de Jong S, Zwanenburg JJ, Visser F, van der Nagel R, van Rijen HV, Vos MA, de Bakker JM, Luijten PR (2011) Direct detection of myocardial fibrosis by MRI. Journal of Molecular and Cellular Cardiology 51:974-979 127 Part III 129 Chapter 7 Summarizing discussion 131 Chapter 7 Ischemic heart disease is a leading cause of morbidity and mortality globally. Many studies have demonstrated the potential of proton based cardiac MR in fundamental and clinical research. Still, very limited data are available on sodium cardiac MRI, although the technique potentially provides very promising imaging readouts of myocardial tissue status. In this thesis, we have assessed myocardial viability using novel cardiac MRI techniques based on proton (1H) MRI in the first part of this thesis and on sodium (23Na) combined with 1H MRI in the second part. Different models were used to induce changes in myocardial tissue status, e.g. ischemia,in mouse heart in vivo and in rat heart under perfused conditions. In this final chapter, the results will be summarized and further possible improvements in experimental research and possibilities for translating some of the imaging techniques into the clinic will be discussed. Part I Late gadolinium enhancement MRI cannot discriminate between necrotic, fibrotic and edematous tissue[1;2]. Therefore, acute myocardial infarction assessment with LGE MRI is complicated. The aim of the first part of this thesis was to explore the potential value of quantitative T2* mapping in combination with commonly used cine and LGE MRI methods in mouse myocardium with different models of myocardial infarction. T2* MRI traditionally is used to assess iron deposition in the heart [3]. In chapter 2, quantitative T2* mapping was compared to cine MRI and LGE MRI in a mouse model of ischemia reperfusion (I/R) injury, starting from 1 day to 28 days post MI. The reason for selecting this I/R infarct model was not only to induce a relatively small infarct, but also to induce myocardial edema, known to accompany I/R injury [4;5]. This model was meant to mimic a similar myocardial status as found in patients after coronary revascularization, in which edema can complicate the diagnosis. We found that quantitative T2* values in the infarcted tissue decreased progressively during infarct development [6]. Indeed, for acute infarcts at day 1 we found a large LGE-based infarct size accompanied by increased wall-thickness, suggesting the presence of edema. Surprisingly, we did not observe a transient increase in T2* at day 1 accompanying this edema, as previously observed with T2‑weighted imaging [7]. Apparently, the 132 Summarizing discussion effect of edema on T2* is less than the decrease of T2* induced by iron or collagen. This observation is critical in the clinical setting where early assessment of viability is significantly hampered by the presence of infarct-induced edema. T2*-imaging may be a tool to circumvent this inevitable problem after acute MI. The changes of T2* were associated with infarct age, reversibility of the injury, and gave an indication of the structural variation in the myocardium throughout infarct maturation. The local decrease of T2* in the infarct was accompanied by globally and locally reduced ventricular function. In addition, the reduction in T2* with infarct age was associated also with the presence of iron and the formation of significant amounts of collagen. Following the assessment of small, myocardial I/R injury in mice with T2* mapping (chapter 2), we aimed in chapter 3, to further explore quantitative changes in T2* after more severe MI in a mouse heart failure model, induced by permanent ligation of the left coronary artery. In comparison to the results of the mild I/R injury (chapter 2), T2* in infarcted areas exhibited a more dramatic decrease starting from day 1, but did not decrease further on day 7 and 28. In contrast to the I/R injury with relatively small infarcts, T2* in remote tissue also decreased significantly and progressively from baseline accompanied by adverse ventricular remodeling of non-infarcted areas after MI, in agreement with findings of Bogaert et al. [8], who observed dysfunction in remote myocardium in patients with transmural anterior MI. Serial LGE scans only revealed changes in the infarct area and not in the remote myocardium. Future research will include the quantification of fibrosis in the remote area, to see if T2* MRI can identify interstitial fibrosis in non-ischemic remote tissue. We concluded that, quantitative T2* assessment provides a valuable readout, not only for infarct, but for remote areas as well. Also the more classical Cine MRI for assessing cardiac function can provide valuable information on left ventricular remodeling after myocardial infarction. In Chapter 4, this approach was used to assess cardiac function after myocardial infarction in TLR2/- mice. Many studies have suggested that Toll-like receptors (TLRs) play a crucial role in inflammatory responses after tissue injury [9]. Cardiac MRI analysis showed that the lack of TLR2 in a mouse model of MI indeed improved survival and cardiac function. 133 Chapter 7 Hence, TLR2 is a potential therapeutic target to prevent adverse remodeling after myocardial infarction. Part II The second part of this thesis was dedicated to sodium MRI in comparison to proton MRI methods. Accumulation of edema is not only a result of myocardial I/R injury, but may arise from many causes and with different extent, such as arterial hypertension [10] and myocarditis [11], which complicates differential diagnosis with LGE MRI. To address this problem, a non-infarct model of different levels of edema in isolated rat hearts based on different perfusion pressures was developed in chapter 5. Subsequently, the influence of myocardial edema was investigated with 23Na CSI in combination with a shift reagent and 1H MRI in combination with a contrast agent. With this model, we have demonstrated that contrast enhanced (CE) MRI with a Gd contrast agent can identify edematous myocardial tissue, most likely as a result of an increased distribution volume of Gd. Surprisingly, we found more edema in the subendocardial area than in the subepicardial area. We have also demonstrated in this same edema model that the Na+e signal increases with increasing edema, confirming that Na+e is a useful endogenous marker of extracellular volume. Interestingly, a similar transmural distribution of signal intensities as observed in the contrast-enhanced images was observed in the Na+e images. Na+i essentially remained unchanged in edema. We concluded that edema shows up both in CE MRI and Na+e, confirming that CE MRI may overestimate infarct size. In addition, we could conclude that high perfusion pressure causes more edema subendocardially than subepicardially. After assessing myocardial edema in edematous but viable myocardium, in chapter 6, we used 23Na CSI to study cellular integrity and ion homeostasis during and after myocardial ischemia in rat hearts to differentiate injured myocardium under three conditions: in acute ischemia and reperfusion (I/R), 2 hours after I/R injury, and in chronic infarction (MI). In this study, we demonstrated that 23Na CSI is a powerful tool to identify sarcolemmal dysfunction related to the myocardial pathophysiological status in the time-course after MI. Acutely, the ischemic myocardium was characterized by highly elevated Na+i levels, caused by Na+/K+ ATPase dysfunction and continued Na+ influx, and 134 Summarizing discussion concomitant decrease of Na+e. In contrast, chronic MI was characterized by an almost complete absence of Na+i, indicating the loss of viable myocardium and replacement by scar tissue, corroborated by the observed increased Na+e. However, the situation in 2 hours reperfused myocardium was less clear. The (partial) recovery of Na+i may signify recovery of Na+/K+ ATPase function, but could be equally well explained by heterogeneous reperfusion with areas of no reflow and persistent high Na+i in combination with areas showing advanced cell deterioration and sarcolemmal dysfunction/rupture with loss of cellular content. The slightly elevated Na+e could signify edema, but could also be explained by loss of sarcolemmal integrity. It is known that infarct size estimation with LGE is influenced by threshold selection [12]. Similarly, the determination of infarct size was very much dependent for both CE MRI and Nae MRI on threshold selection, which is not surprising since both techniques monitor the same compartment. Infarct size determination based on Nai images is also similarly dependent on threshold selection. Furthermore, we have demonstrated that wash-out of contrast agent from edematous tissue occurs much faster than from infarcted tissue, providing further explanation for the threshold dependence of infarct size determination. Further development and clinical translation Part I MRI is steadily becoming the preferred imaging tool for characterizing the heart in health and disease. Several new cardiac imaging methods, focusing on quantitative readouts of heart function and physiology as well as tissue MR relaxation time parameters, are under development As shown in this thesis T2* may have added value in the assessment of infarct and remote tissue. A serious limitation to the use of T2* at 9.4 Tesla as a diagnostic marker for myocardial injury is contamination of intrinsic T2* values by the presence of macroscopic susceptibility differences, mainly induced by the lung-air interfaces. Using a lower magnetic field scanner should reduce such an artifact. MRI scanners with lower magnetic field are commonly available in the clinic, where T2* mapping is 135 Chapter 7 commonly used for thalassemia patients. Moreover, using shorter echo times and using different excitation pulses might solve or at least reduce this problem. In addition, application of local shimming techniques might alleviate some of these susceptibility contaminations. We showed here that T2* mapping is not sensitive to edema in acute MI, unlike LGE MRI, which makes T2* mapping a valuable tool to assess myocardium,in acute MI. Furthermore, in contrast to LGE MRI, T2* mapping does not require any contrast agent injection, which may be risky for some patients [13]. Recently, it was demonstrated that T2* has the potential to image fibrosis in the infarct zone using an ultra-short echo time (UTE) technique [14]. In vivo application of such a technique should be possible in the clinic using radial and spiral k-space acquisitions. A particular application of T2* mapping is the tracking of superparamagnetic iron oxide labeled stem cells after delivery in cardiovascular disease [15]. Methods utilizing magnetization transfer have also been explored for assessing edema in infarcted areas [16] This method may give valuable information on specific pathological conditions of the myocardium, however, optimizing for small animals with a fast beating heart is not trivial. Part II The studies conducted in this part of this thesis were performed to get more insights in the potential of 23Na CSI in evaluating myocardial injury. Although the techniques have provided valuable information on myocardial tissue status, their transition to clinic is a challenge. For instance, 23Na CSI of normal Na+i suffers from poor SNR and low spatial resolution. To make 23Na CSI a clinical reality, high field (7 T) MRI scanners are recommended [14]. Furthermore, a shift reagent suitable for use in human is required, which is not yet available, but, given the similarity between contrast agents and shift reagents, could be developed. Alternatively, multiple quantum filtered MR methods [17;18] or ultrashort-echo time (UTE) sequences [14] could be used, but further investigations are still needed for clinical implementation of these methods. 136 Summarizing discussion Conclusion Quantitative relaxation time measurement by MR is an emerging tool in the assessment of myocardial infarction, offering a quantitative standard MRI scale related to specific pathological conditions. Our approach using T2* mapping promises to be a powerful MRI method to monitor remodeling post-infarction and provides useful and complementary information to LGE and Cine imaging. In addition, we have demonstrated the feasibility of cardiac imaging of intra- and extra-cellular sodium to assess myocardial viability. 23Na-CSI is an attractive alternative for imaging of edema. In addition, Na+ tracking is a valuable tool to indentify the pathological status of the heart: highly elevated Na+i is a unique characteristic of acute ischemia, whereas the absence of Nai signal in combination with elevated Na+e signal is a marker of scar tissue. The rapid normalization of highly elevated Na+i in infarcted area upon reperfusion provides unique insights in the dynamics of infarct development. The combination of 23Na-CSI and 1H-LGE-MRI shows added value for accurate evaluation, understanding and distinction of different pathophysiological states after myocardial injury and helps to interpret results mutually. Further development of both sodium and proton MRI will allow better understanding of myocardial viability. Reference List 1. 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Stacul F, van der Molen AJ, Reimer P, Webb JA, Thomsen HS, Morcos SK, Almen T, Aspelin P, Bellin MF, Clement O, Heinz-Peer G (2011) Contrast induced nephropathy: updated ESUR Contrast Media Safety Committee guidelines. Eur Radiol 21:2527-2541 14. de JS, Zwanenburg JJ, Visser F, der NR, van Rijen HV, Vos MA, de Bakker JM, Luijten PR (2011) Direct detection of myocardial fibrosis by MRI. J Mol Cell Cardiol 51:974-979 15. Kedziorek DA, Kraitchman DL (2010) Superparamagnetic iron oxide labeling of stem cells for MRI tracking and delivery in cardiovascular disease. Methods Mol Biol 660:171-183 16. Henkelman RM, Stanisz GJ, Graham SJ (2001) Magnetization transfer in MRI: a review. NMR Biomed 14:57-64 138 Summarizing discussion 17. Navon G, Werrmann JG, Maron R, Cohen SM (1994) 31P NMR and triple quantum filtered 23Na NMR studies of the effects of inhibition of Na+/H+ exchange on intracellular sodium and pH in working and ischemic hearts. Magn Reson Med 32:556-564 18. Dizon JM, Tauskela JS, Wise D, Burkhoff D, Cannon PJ, Katz J (1996) Evaluation of triplequantum-filtered 23Na NMR in monitoring of Intracellular Na content in the perfused rat heart: comparison of intra- and extracellular transverse relaxation and spectral amplitudes. Magn Reson Med 35:336-345 139 Korte samenvatting in het Nederlands Wanneer een deel van het hart niet goed samentrekt is het cruciaal te weten of een bypass operatie of een dotterbehandeling dit zou kunnen verhelpen. Is dit deel van het hart nog levensvatbaar of is het al littekenweefsel? Veelal wordt hiervoor MRI met contrastmiddel (DCE-MRI) gebruikt. Een nadeel hiervan is dat de infarctgrootte vaak overschat wordt, waarschijnlijk vanwege vocht rondom het infarct. M.b.v. proefdieren zijn alternatieve MRI methoden onderzocht. Gebleken is dat een van de beeldparameters van het op water gebaseerde MRI beeld, de T2*, bij een klein infarct in de loop der tijd daalt als gevolg van afzetting van collageen en ijzer, terwijl de T2* in het gezonde hartspierweefsel normaliseert. Bij een groot infarct daalt de T2* in het infarct sneller, terwijl nu in het ‘gezonde’ weefsel T2* ook daalt door remodelleren (hartfalen). Hartfunctie MRI onderzoek liet zien dat de ‘Toll-like receptor2’ in het bloed een belangrijke rol speelt bij dit remodelleren. M.b.v. een ‘shift reagens’ is het ook mogelijk het natrium in (Na+i) en buiten (Na+e) de hartspiercellen af te beelden. Door (bloed) drukverhoging werd vochtophoping in de hartspier veroorzaakt, waarmee bevestigd werd dat deze vochtophoping inderdaad door DCE-MRI wordt afgebeeld en ook door Na+e-MRI, maar niet door Na+i-MRI. Aan de binnenkant van de hartspier was er meer vochtophoping dan aan de buitenkant. Tenslotte bleek dat Na+i sterk verhoogd is bij acute infarcten, terwijl in het chronische infarct Na+e verhoogd is en Na+i nagenoeg verdwenen. Voor toepassing van deze nieuwe MRI-methoden bij patiënten is nader onderzoek vereist. 141 List of publications Manuscripts Na chemical shift imaging and Gd enhancement of myocardial edema Aguor EN, 23 van de Kolk CW, Arslan F, Nederhoff MG, Doevendans PA, Pasterkamp G, Strijkers GJ, van Echteld CJ Int J Cardiovasc Imaging. 2012 Jul 12. [Epub ahead of print] Quantitative T2 * assessment of acute and chronic myocardial ischemia/reperfusion injury in mice Aguor EN, Arslan F, van de Kolk CW, Nederhoff MG, Doevendans PA, van Echteld CJ, Pasterkamp G, Strijkers GJ MAGMA. 2012 Feb 11. [Epub ahead of print] MicroRNA-155 inhibits cell migration in human cardiomyocyte progenitor cells (CMPCs) via targeting MMP-16 Liu J, van MA, Aguor EN, Siddiqi S, Vrijsen K, Jaksani S, Metz C, Zhao J, Strijkers GJ, Doevendans PA, Sluijter JP J Cell Mol Med. 2012 Feb 20 Faraday Effect Position Sensor for Interventional Magnetic Resonance Imaging Bock M, Umathum R, Sikora J, Brenner S, Aguor EN, Semmler WA Phys Med Biol. 2006 Feb 51[4], 999-1009. Differentiation of myocardial injury in various infarct models with 23Na Chemical Shift Imaging and Late Gd Enhancement MRI Aguor EN, van de Kolk CW, Arslan F, Nederhoff MG, Doevendans PA, Pasterkamp G, Strijkers GJ, van Echteld CJ, submitted MSC-derived exosomes enhance myocardial viability after ischemia/reperfusion injury Arslan F, Ruenn CL, Choo A, Aguor EN, Smeets M b, Akeroyd L, van Rijen HV, Timmers L, Doevendans PA, Pasterkamp G, Lim KS , de Kleijn DP, submitted 143 Molecular Imaging in Stem Cell therapy Siddiqi S, Aguor EN, Sluijter JPG, Chamulaeu SA, Doevendans PA, submitted Quantitative T2* assessment of acute and chronic myocardial permanent ligation injury in mice Aguor EN, van de Kolk CWA, Nederhoff MGJ, Doevendans PA, van Echteld CJA, Pasterkamp G, Strijkers GJ, Arslan F, Submitted Lack of circulating Toll-like receptor 2 promotes survival and prevents adverse remodeling after myocardial infarction Aguor EN, Arslan f, Smeets MB, Doevendans PA, Strijkers GJ, Pasterkamp G, de Kleijn DP, In preparation Abstracts and Conference Proceedings 1st author only Na Chemical Shift Imaging and Late Gadolinium enhanced (LGE) MRI of acute ischemia 23 reperfusion myocardial injury Aguor EN, Arslan F, Strijkers GJ, Nederhoff MGJ, van de Kolk CW, Pasterkamp G, Doevendans PA, van Echteld CJA 20th Intl. Soc. Mag. Reson. Med. (ISMRM), Annual meeting and exhibition Intl. 2012 May 8, Melbourne, Australia. “Presentation” Na Chemical Shift Imaging and Late Gadolinium enhanced (LGE) MRI of acute 23 ischemia reperfusion myocardial injury Aguor EN, Arslan F, Strijkers GJ, Nederhoff Marcel GJ, van de Kolk CW, Pasterkamp G, Doevendans PA, Van Echteld CJA 4th annual meeting of the ISMRM Benelux, 2012 Jan 16, the Leuven Institute for Ireland in Europe, Leuven, Belgium. “Presentation” Na Chemical Shift Imaging of Myocardial Edema Aguor EN 23 ImagO Promovendimiddag, 2011 Dec 9, presentation, University medical centre, Utrecht, the Netherlands. “Presentation” Assessment of myocardial viability with Magnetic Resonance Imaging techniques Aguor EN 2nd ICIN- Netherlands Heart Institute Masterclass, 2011 Sep 6, Utrecht, the Netherlands “Presentation” 144 Longitudinal assessment of T2* changes in mouse myocardium following ischemiareperfusion injury Aguor EN 3rd annual meeting of the ISMRM Benelux, 2011 Jan 19, Roosendaal, the Netherlands. “Presentation” Na Chemical Shift Imaging of Myocardial Edema Aguor EN, van de Kolk CW, 23 Nederhoff MGJ, Doevendans PA, Pasterkamp G, Strijkers GJ, Arslan F, van Echteld CJA 3rd annual meeting of the ISMRM Benelux, 2011 Jan 19, Roosendaal, the Netherlands. “Presentation” Na Chemical Shift Imaging of Myocardial Edema Aguor EN 23 Masterclass, 2010 Dec 15, Biomedical NMR (TU/e), Eindhoven Technical University, Eindhoven, the Netherlands. “Presentation” Lack of Toll-like receptor 2 promotes survival and preserves cardiac function and geometry after acute MI Aguor EN, Arslan F, van de Kolk CW, Doevendans PA, de Kleijn, G. Pasterkamp. de Kleijn DP Eur Heart J 2010;31 (Suppl 1): 9(abstract 186), Stockholm, Sweden. “Presentation” Assessment of myocardial viability with Magnetic Resonance Imaging techniques Aguor EN Bruker MRI /MRS User’s Meeting 2008 Oct 13, Germany. “Presentation” A Reflection Mode Fiber-optic Faraday Sensor for Device Localization in Interventional MRI Aguor EN, Brenner S, Umathum R, Semmler W, Bock M. Proc. 14th Scientific meeting of ISMRM 2006, Seattle, USA A Reflection Mode Fiber-optic Faraday Sensor for Device Localization in Interventional MRI Aguor EN, Brenner S, Umathum R, Semmler W, Bock M . Proc. ISMRM. 14 (2006), Washington, USA. “Presentation” 145 Na Chemical Shift Imaging of Myocardial Edema Aguor EN, van de Kolk CW, Arslan F, 23 Nederhoff MG, Doevendans PA, Pasterkamp G, Strijkers GJ, van Echteld CJ Proc. ISMRM, 19(2011), (Abstract 1353), Montréal, Canada. “Poster” Longitudinal assessment of T2* changes in mouse myocardium following ischemiareperfusion injury Aguor EN, Arslan F, van de Kolk CW, Nederhoff MG, Doevendans PA, van Echteld CJ, Pasterkamp G, Strijkers GJ Proc. ISMRM,19(2011), (Abstract 1363), Montréal, Canada. “Poster” Assessment of myocardial viability with Magnetic Resonance Imaging techniques Aguor EN Heart foundation the Netherlands (NHS), 2008 Sep, Amsterdam, the Netherlands, “Poster” 146 Acknowledgment After all this years of ups, downs, doubts, deadlines, mice and MR scans it’s time to look back and think of everyone in this long, exciting, challenging journey. Still I am glad I have come to the Netherlands and decided to start a PhD study in Utrecht. First I want to thank Prof. dr. C.J.A. van Echteld. Dear Cees, thank you for accepting me as a member in your group and thanks for giving me the freedom to investigate on this “Sodium MRI”. Just a few months later, you went to Switzerland. Being busy on a new carrier and in a different place, you were still able to support me in the project. Committed as you were, you always found a moment for consultation via Skype or phone, you even came to Utrecht on a regular base. I am grateful for your scientific criticisms and honest opinion about the results. I have always enjoyed discussions with you about science and numerous other topics. Without your help in all aspects of this project, it would never have resulted in the nice thesis as it lies here in front of you. The next to thank is Prof. dr. P.A.F.M. Doevendans. Dear Pieter, after Prof. van Echteld left, you took over the supervision of our MRI group and became my promoter. It has been good to know that you were always there to support me after the big changes in our lab and all the obstacles I met during this journey. Thanks for your trust and for giving me the opportunity to continue this research. It was always nice meeting you to discuss the progress of my project. You always kept asking me about the articles I was writing. It was very important to have you on my side all the time. You won’t have to worry any more about this project Prof. dr. G. Pasterkamp, dear Gerard, you became the supervisor of our lab and I am very thankful for your fast and efficient action whenever things went wrong. Thanks for your assistance on my research. Your group “the Experimental Cardiology” provided me a stimulating scientific and social environment. I learned a lot from the variety of fellow researchers within your group. It was always nice to discuss my research and what was missing from a clinical point of view. Dr.ir. G.J. Strijkers, dear Gustav, you were the savior, you showed up in a critical moment. I remember the first time I met you giving a lecture in Wageningen University about MRI. I was truly amazed about your 147 lecture and your Cardiac-MRI-dedicated group. Your contribution certainly speeded up things. Thank you for the critical remarks, suggestions and corrections. Not to forget you presenting my abstracts at ISMRM. Also thanks for preparing the nice fresh coffee in the early morning at our lab. Dr. Fatih Arslan, already involved in so many projects, you still found the time to step into a joint study with me. Thanks for the excellent surgery on my mice and rats, for revising the articles and for all the good advise during the years. Dear Marcel Nederhoff, thanks for your support and tricks with the Langendorff, but also for your tricks for inserting the catheter and these tiny tubes. I enjoyed very much our conversation, it was always interesting and special to know your opinion about topics such as God, martial arts, dancing, music, culture and many other. I liked the way you think about things. Thanks for your advice, discussion and humors both in science but also in personal level. It was great to have you in the lab for all these years. Thanks for sharing my thoughts and feeling when many hearts did not work, you were still positive and saying “of course that is normal in science”!! Who knows maybe one day you will have your own company brewing your own beer. I am glad I do not have to see strange photos in my drawer anymore. Drs. Ing. Kees van de Kolk, dear Kees, you were the MRI-operator as well as the computer system manager, therefore always there. I still remember calling you at night and having you remotely-connected to the MR scanner. Thanks for your support also for the buffer recycling and analysis, especially at the end when I was really running out of time. Thanks for your commitment. You were the man I could talk to about MRI. Thanks for the good time. I really wish you lots of success in the coming years. Sandra and Bernard, we were the last PhD students in the MRI-lab, it was nice to have you in the lab and I wish you good luck too. Krista den Ouden, thank you very much for all the imaging work. Nice to find someone already in the lab made coffee when I arrived in the morning. It was great to have you in the lab. Sanne Willems, it was great to have you as my neighbor in the Lange Nieuwstraat. We always had so many topics to talk about and you were the first person I could think to choose to be my Paranymph. Thanks so much for your friendship and for all these educational Tennis matches we had. I wish you lots of success in your PhD too. 148 Thanks for other members in the lab. Arjan, Sander, Loes, Noortje, Chaylendra, Joost S., Evelyn, Cees, Joyce, Marlijn, Ben, Joost F. Bas, Joyce, Vince, Paul, Vincent, Pleunie, Dave, Verena, Esther, Roberto, Sailay, Ellen, Geert, Marten, Krijn, Alain, Dries, Zhiyong, Hamid, Claudia, Marish, Diana, Frebus, Hendrik, Imo, Karlijn, Mirjam, Jiongwei, Frederieke, and all the interns, thanks for making my project here at the Experimental Cardiology a very pleasant times. Dear Maringa, special thanks for your help in completing the imaging and analysis of the last groups. I wish you all success. Special thanks of course, to Marjolijn and Ineke, Margo, Tamara, Jantine and Sylvia for your pro-activity, you’re always being there when necessary. It was also nice to be working next to the in vivo lab, Lissette, Geralda, Annette, Gerhard, Erwin, Ivo, Maurits, Kaio, Rick, Mark, Umesh, Pavel. For you who did not graduate yet, go for it. And thanks for the “borrels” that I sadly enough were not able to attend all of them. From the GDL, thanks for, Helma, Anja, Willeke, Romy, Sabine, Gerard, Kiki and all the others. Many thanks to ICIN, starting from Jan Weijers, Eelco Soeteman, Rachida Tallahi and Marijan de jonge. Thank for all your help for the management of my project. You were always there when it was necessary and thanks for all the nice social activity. Special thanks to my very dear friends, Raffa, Tina, Gianpiero. It all began in the kitchen next to my room in biltstraat’s house. Raffa: great to see progressing in your scientific carrier ‘even more to the north’ in the UK as a married but still stylish lady. Great to see you with Hazem. Good luck Hazem with your new life in Italy, it was great to met you in Siegen! Tina: I would say you mad it very clear that you love Germany very much being there for the second time. So you guys stop complaining. Good luck also to Gianpie: it was always nice talking to you and the nice food you always cooked. I can say you’re doing fine now since Raffa is next to you to you now. You were the most popular guy in Biltstraat house. Gene was also amazing to get to know you, a happy, polite, generous person. Well finally I am the one who stayed here and your guys were 149 gone to different places now, but remarkably we are still friends. Thanks also to Edu and his food specialties and the good time. I hope now you are a bit faster in cooking in Switzerland. Thanks for the friendship and good time for the people I met in Lange Nieuwstraat house, Tobias, Emma, Setfiee, Natasha, Pradeep, Wouter, Metta, Astrid, Gudrun, Susana, Ariana, Jeroen and Eneda. Michaela and Augustine, thanks for the good time and the nice trips to the black forest, it is nice to have you here too, I wish you all much success. Thanks for my Salsa gang: Axel, Alex Juko, Natasja, Trevor, Sandra, Marleen, Arwen, Ellen, Valendard, Emrah, Glenda, Marjolein, Amy, Karin, Michille and Loes. Gerhard, thanks for your friendship and I am very glad I met you, we have the similar opinion most of the time, thanks for your friendship. Marijke Coster and Els thanks for your friendship and for the support you offer. I always think of you as a family here. Neeltje thanks for everything and thanks for your support and sharing many things together. Finally, thanks for the support of my family from far away. It was always difficult to meet, but you were always in my soul and heart. I dedicate this thesis to my mother and my late father ‘may his soul rest in peace’. 150 Curriculum Vitae Eissa N. E. Agour was born on January 16, 1979 in Riyadh, Saudi Arabia. After graduating from higher secondary school in, Riyadh, Saudi Arabia in 1996, he obtained his Higher Diploma’s degree in Electronic and Electrical Engineering with specialization in computer science at the Islamic Institute of Technology, Dhaka, Bangladesh in 2000 (currently Islamic University of Technology). Hereafter he worked as a service engineer/ imaging application specialist for Hitachi medical imaging systems in Saudi Arabia. From 2004-2006 he worked as a research assistant at the interventional MRI division of the German Cancer Research Center (DKFZ), Heidelberg, Germany. He obtained his Master’s degree in Imaging Physics at the University of Siegen, Germany in 2006. In 2007 he started as a PhD candidate at the Department of Cardiology of the University Medical Center Utrecht (UMCU), the Netherlands under the supervision of prof. dr. C.J.A. van Echteld and prof. dr. P.A.F.M. Doevendans. The results of his PhD research are described in this thesis. Recently he joined Medis medical imaging systems bv, Leiden, the Netherlands as an Imaging product specialist. 151