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Anesthetic Preconditioning in Normal and Hypertrophic Porcine Myocardium The Effects of Sevoflurane Volatile Anesthetic Preconditioning upon Myocardial Infarct Size in a Closed-Chest Ischemia-Reperfusion Model PhD thesis Jens Kjærgaard Rolighed Larsen Faculty of Health Sciences University of Aarhus 2008 Anesthetic Preconditioning in Normal and Hypertrophic Porcine Myocardium The Effects of Sevoflurane Volatile Anesthetic Preconditioning upon Myocardial Infarct Size in a Closed-Chest Ischemia-Reperfusion Model PhD thesis Jens Kjærgaard Rolighed Larsen Faculty of Health Sciences University of Aarhus 2008 Department of Anesthesiology-Intensive Care, Department of Cardiothoracic and Vascular Surgery and Institute of Clinical Medicine Aarhus University Hospital Skejby 1 Preface The Institute of Clinical Medicine (Klinisk Institut), Aarhus University Hospital, Skejby, provided the unique framework for the experimental studies which are included in this PhD dissertation in the period 2005-2008. I owe my gratitude to a number of persons involved in this project, without whom the project would scarcely have become a personal success, let alone come to be realized at all. Permit me to express elaborate thanks to the following; First and foremost I would to thank J. Michael Hasenkam for taking on the role of principal supervisor. He is a person whom it is difficult to get past if you really wish for something to happen, – and probably even more so if you want nothing to happen at all! His personal laudates are many and span widely, but two characteristics of his spring to my mind: Persistence and patience. Thank you for your help and inspiration, Michael, I have learnt much from working with you, -from the beginning and through the entire process. They say: “you can’t beat luck “– but with Michael on your team, you probably could. Søren Aagaard was my accomplice during the initial part of the studies, and he has been an invaluable help and inspiration. A true friend. He and I got ‘our boots wet’ together on many occasion. We dragged ourselves and one another out of the mud on several occasions and ‘lived to tell the tale’. Mutually inspirational and chaotic, but always there for each other. Søren, you were my best colleague. Thank you. Of inspirators and matters of hand; two people have led me to my decision to take on the current project. One was the ignoble Dr. Morten Smerup, who made it possible for me to fathom the initial glimpse of ‘science in action’ and to inspire me to do something about it. Gratitude also goes to Erik Sloth, my practical supervisor, who from the beginning was friendly and supportive and who represented important echocardiographic skills. And to Louise; for love and support. Tabula gratitudinis: Jette Breiner, Morten Smerup, Morten Ølgaard Jensen, Peter Johannesen, J. Michael Hasenkam, Søren Aagaard, Louise Bach-Nielsen, Kim Sivesgaard, Sara Dahl Christensen, Mette Sørensen, Torsten Toftegaard, Bo Løfgren, Annette Strandbo, Rikke Nørregaard, Karin Christensen, Jørgen Frøkiær, Peter Torp, Ian Ortved, Erik Sloth, Rasmus Haarup Lie, Henrik Sørensen, Tanja Thomsen, Jens Christian Djurhuus, Jens Kristensen, Jan M Nielsen, Carsten Riis, Hans Erik Bødtker, Lars Ege Rasmussen, Troels Thim, Per Christensen, Diana, Walther Gyldenløve, Johannes Yde, Annie Sneftrup, Kurt Bruno Sørensen, Klavs Hundebøll, Bente Rolighed Larsen, Anne Sofie Kannerup, Anna Krarup Keller, Else Kirstine Tønnesen, Mads Halbirk, Mads Buhl, Martin Busk, Jesper Hønge Langhoff. 2 CONTENTS Preface … … … … …. … … … … … … … … … …………………………….. … ………... … .…. ….3 Table of contents … … … … … … … … … … … … … … … … … … … … … … .… … …….. …..4 List of Publications… … … …… … … … … … … … …… … … … … …. … … …. …..… …... … 5 Abbreviations.. … … … … … …… … … … … … … … …… … … … … … … … …. … ….…. ..… 6 Abstract…………….… … … … … …… … … … … … … … … … … … … … … … ….…..… …......7 1. Introduction… … … .… … …… … … … … … … … …… … … … … … … ……. …. ….…...…9 2. Background: The Volatile Anesthetics & Ischemia-Reperfusion Injury…….... ....11 3. Objectives… … … … … …… … … … … … … … …… … … … … … … … ……. ….....…..….17 4. Methods: Assessment Of Ischemia-Reperfusion Injury… … … …………………..….. 19 Methods and material… … … … … …… … … …… … … … … …….................…...… …25 The Hypertrophic Left Ventricle … … … … … …… … … ……................ … …..…. … 36 5. Study design… … … … … …… … … …… … … … … …… … … …… … … … … ….. … .…38 6. Summary of Results…… … …… … … … … … … … …… … … … … … … ………..……...42 Sub study I Sub study II Sub study III 7. Discussion… … …… …… … … … … … …… … … … … … ….... ……………………….……..48 8. Conclusions… … …… … … … … … … …… … … … … … … …………………….…....……..55 9. Acknowledgements… … …… … … … … … … …… … … … … … … ……….……....…..…56 10. Dansk resumé… … …… … … … … … … … …… … … … … ………………….……......……57 11. References … … …… … … … … … … … …… … … … … … … ………………….………....…59 12. Appendix … … …… … … … … … … … …… … … … … … ……………………….……….......67 3 LIST OF PUBLICATIONS This PhD-dissertation is comprised of the following papers, which will be referenced by their Roman numerals: I. LARSEN JR, AAGAARD SR, HASENKAM JM, SLOTH E. Pre-occlusion ischaemia, not sevoflurane, successfully preconditions the myocardium against further damage in porcine in vivo hearts. Acta Anaesthesiologica Scandinavica 2007; 51: 402-409. © Blackwell Publishing I I. LARSEN JR, AAGAARD SR, LIE RH, SLOTH E, HASENKAM JM. Sevoflurane Improves Myocardial Ischaemic Tolerance in a Closed-Chest Porcine Model. Acta Anaesth Scand 2008; accepted for publication © Blackwell Publishing I I I. LARSEN JR, SMERUP MH, HASENKAM JM, CHRISTENSEN SD, SIVESGAARD K, TORP P, SLOTH E. Pressure overload hypertrophy remodeled ventricle in young pigs: decreased infarct size from ischemia and sevoflurane. Cardiovascular Research 2008; submitted © Oxford University Press 4 ABBREVIATIONS: APC: Anesthetic preconditioning AAR: Area-at-risk, ischemic risk region [Ca2+]i: Intracellular (cytosolic) calcium concentration ERK: Extracellular signal-regulated kinase GPCR: G protein-coupled receptor IPC: Ischemic preconditioning IS: Infarct size LAD: Left anterior descending coronary artery MAPK: Mitogen-activated protein kinase mPTP: Mitochondrial Permeability Transition Pore PCI/PTCA: Percutaneous coronary intervention/percutaneous transluminal coronary angioplasty PKB/Akt: Protein kinase B [IS/AAR]: Weight-corrected infarct size in relation to the area-at-risk PKC: Protein kinase C IU: International units 2D: second diagonal branch of the LAD KATP-channel: adenosine triphosphatesensitive potassium channel VF: ventricular fibrillation ROS: Radical oxygen species 5 ABSTRACT The effect of volatile anesthetics on cardiomyocyte injury during ischemia and reperfusion is not yet fully understood. Though the efficacy of sevoflurane upon reducing myocardial infarction during the ischemia-reperfusion process is well-validated in some animal models, clinical studies are partially inconsistent with this, and the cardioprotective effect is yet to be translated into an equivocal improvement in patient outcome. We, therefore, used an intact porcine closed-chest animal model with unparalleled anatomic and physiological similarities to man to investigate sevoflurane volatile anesthetic cardioprotection during ischemia and reperfusion. In order to form a comparative base, we initially studied the efficacy of classical ischemic preconditioning upon histologic myocardial infarct size and simultaneously investigated the effects of sevoflurane administered pre-ischemically, which showed only a tendency towards infarct size reduction compared with the effectiveness of ischemic preconditioning. Global cardiac function was estimated with tissue-Doppler echocardiography and was unrelated to sevoflurane administration or size of irreversible ischemic injury. Thus, unable to prove the existence of a ‘trigger’ mechanism for sevoflurane preconditioning, we proceeded to evaluate infarct size mitigation by continuous pre-, per- and post-ischemic sevoflurane administration (=cardioprotection). Sevoflurane inhalation diminished myocardial infarct size by more than 60%. The role of pentobarbital anesthetic infusion was also assessed, both as concomitant and sole sedative during ischemia and reperfusion, showing only an insignificant role on myocardial injury salvage from sevoflurane cardioprotection in the current model. Finally, the pathologic heart condition known as left ventricular hypertrophy (LVH), not uncommonly encountered in cardiac surgery patients, was investigated for its ability to influence sevoflurane cardioprotection in the porcine model. A model of LVH established within our department was used. It comprised a cohort of aortic banded animals which subsequently developed supravalvular aortic stenosis and LV pressure overload with ensuing development of LV hypertrophy. Animals were since allocated to ischemia-reperfusion protocols and this study showed that LVH did not affect sevoflurane cardioprotection. In addition, LVH paradoxically improved tolerance to ischemia in young animals. In conclusion, this dissertation supports previous experimental results from volatile anesthetic cardioprotection and specifies timing and dosages related to sevoflurane administration in an intact large animal model recognized for its comparability to normal human ischemia pathophysiology. Moreover, an unchanged sevoflurane cardioprotective efficacy was found in a model of cardiac (LVH) pathology, offering no explanation for altered clinical capacity for cardioprotection in a typical cardiac surgery cohort. 7 8 1. Introduction Scope: WHO estimated that in 2002, 12.6 percent of deaths worldwide were caused by ischemic heart disease1. Acute coronary occlusion is the most common cause of illness and mortality in this classification. The treatment of acute ischemic heart disease aims primarily at the restoration of adequate myocardial blood supply, termed revascularization. Since its conception in 1880 (Langer), the technique of revascularization has undergone dramatic change2. Today, percutaneous intraluminal coronary intervention and coronary artery bypass surgery are standard procedures throughout the world. Revascularization of an ischemic myocardial area is vital for the survival of the myocardial cells and thereby the cardiac function - whether surgically or by catheter-based intervention. Yet, the restoration of blood supply to a depleted myocardium initiates a complex series of changes which result in cellular damage known as reperfusion injury3. Restoration of adequate blood supply to ischemic myocardium carries with it the risk of reperfusion injury, - a condition in which inflammation and oxidative myocardial, vascular or electrophysiological damage occurs through the induction of oxidative stress - rather than restoration of normal function. However, adjunctive metabolic or pharmacologic strategies may preserve viability of the ischemic and reperfused myocardium, and may represent an important therapeutic target4. Since the therapeutic objective of early recanalization has attained a high degree of effectiveness, further improvements in this direction are expected to yield only minor additional benefits. Therefore, attention was more recently directed to the role of cardioprotective adjunct strategies, pharmacologic or otherwise, to improve survival and quality of life. Cardioprotection is aimed primarily at minimizing ischemia-reperfusion injury and is focused upon the following areas: diminishing reversible myocardial functional losses (‘stunning’); diminishing irreversible cardiomyocyte injury and limiting infarct size; diminishing electrophysiological injury (arrhythmias); and limiting vascular injury to optimize the quality of reflow during reperfusion. Ischemic preconditioning (IPC) is an experimental technique for producing resistance to the loss of blood supply and, thus oxygen, to tissues of many types. Murry and Reimer first described this procedure in 1986 in canine hearts 5 : If the blood supply to an organ or a tissue is halted for a short time (usually less than five minutes) and then restored two or more times so that blood flow is intermittently resumed, the downstream cells of the tissue, or the organ, are robustly protected 9 from a final ischemic insult when the blood supply is cut off entirely. IPC protects the tissue by initiating a cascade of biochemical events that allows for an up-regulation of the energetics of the tissue. Figure 1. Monitor screen-shot illustrating some central features of ischemia-reperfusion injury; tachycardia, dysrhythmia, vasoplegia and myocardial stunning. The locus of this phenomenon is the intracellular organelle, the mitochondrion. A decade later came the discovery of myocardial cardioprotection by limitation of myocardial infarct size in ischemic hearts conferred by inhalation of halogenated volatile anesthetics, and furthermore, by mechanisms which were nearly identical to those of IPC6,7. This was termed anesthetic preconditioning (APC), and has since been the target of considerable research efforts in order to fully clarify the mechanisms underlying this phenomenon and to pioneer clinical benefits. However, clinical trials involving volatile anesthetics to aid in cardioprotection are partially inconsistent with this and controversial8. Using surrogate end-points in small scale clinical investigations has shown reduced cardiac injury biomarkers after bypass surgery, whilst others have not8. Therefore, it appears prudent to study sevoflurane APC in an animal model with great physiologic and anatomic resemblance to the normal human heart- the porcine model. We hypothesized; (i) that exposure of sevoflurane prior to ischemia would mitigate infarct size (preconditioning), (ii) that continuous sevoflurane inhalation would reduce infarct size (cardioprotection), and (iii) that sevoflurane would also mitigate infarct size in the pathologic heart, here the hypertrophied heart. The three studies which constitute the basis for the current Ph.D.-dissertation aim to describe, evaluate and compare three animal experimental investigations into methods of mitigating post- 10 ischemic myocardial injury through the application of volatile anesthetics, in order to put forward recommendations for future clinical work. 2. BACKGROUND WHAT ARE VOLATILE ANESTHETICS ? The first successful public demonstration of reversible loss of consciousness by a volatile anesthetic occurred on October 16, 1846. Administration of diethyl ether allowed the removal of a neck tumor from a quiescent and pain free patient. Not only did this stunning demonstration revolutionize medical practice by changing the scope and frequency of surgery, but it was viewed as a triumph over pain and hailed as a gift to humanity. Oliver Wendell Holmes, who since became the Dean of the Harvard School of Medicine, coined the phrase “anesthesia” in order to give a name to something that had never been conceived as possible by physicians prior to that time 9. The inhaled anesthetics rank among the most important medical advances in our time. SEVOFLURANE: Systematic (IUPAC) name: 1,1,1,3,3,3-hexafluoro-2-(fluoromethoxy)propane Identifiers: CAS number 28523-86-6 Chemical data: Formula C4H3F7O Molecular mass 200.055 g/mol Molecular Weight: 200 u Boiling point: 58.6 °C (at 101.325 kPa) Minimum Alveolar Concentration (MAC): 2 vol% Blood/Gas Partition Coefficient: 0.68 Oil/Gas Partition Coefficient: 47 Sevoflurane, also called fluoromethyl hexafluoroisopropyl ether, is a sweet-smelling, nonflammable, highly fluorinated methyl isopropyl ether used for induction and maintenance of general anesthesia. Alongside desflurane, it is replacing isoflurane, enflurane and halothane in modern anesthesiology. After desflurane it is the volatile anesthetic with the fastest onset and offset. Though desflurane has the lowest blood/gas coefficient of the currently used volatile anesthetics, sevoflurane is the preferred agent for mask induction of anesthesia due to its lesser irritation to mucous membranes. Though it vaporizes readily, it is a liquid at room temperature 11 and is administered via an anesthetic vaporizer attached to an anesthetic machine. It was introduced into clinical practice initially in Japan in 1990. MECHANISMS OF ACTION The mechanism of action of volatile anesthetics remains an enigma, despite their worldwide use. In the early twentieth century, Overton and Meyer independently noted that the more potent drugs were also more soluble in olive oil, and researchers soon verified an impressive correlation between anesthetic potency and oil solubility10. This early work predated our understanding of the composition and structure of the cell membrane and proteins, so conclusions were limited to the probability that anesthetics produce their effect by acting on undefined fatty components of the cell. With the later discovery that biological membranes are constructed largely of lipid and are, therefore, olive oil–like, came the logical extension of Overton and Meyer's working hypothesis that the inhaled anesthetics act by targeting the cell membrane. Moreover, because important cell signaling pathways depend on proteins embedded in the cell membrane, the dissolution of sufficient lipophilic agents, such as the inhaled anesthetics, was predicted to alter some important physical property of the lipid bilayer, which in turn would change the function of embedded proteins. This "unitary hypothesis" launched a host of studies that indeed showed the inhaled anesthetics to change lipid bilayer properties. These changes, however, tended to be small, and were detectable only at anesthetic concentrations many fold higher than those necessary to produce anesthesia. 12 Figure 2: THE LIPOPHILICITY OF INHALED ANESTHETICS. At the turn of the century, Meyer and Overton independently noted the strong correlation between the oil solubility of inhaled gases and their anesthetic potency. Of twelve inhaled agents analyzed, five are identified: 1, nitrous oxide; 2, cyclopropane; 3, diethyl ether; 4, trichloroethylene; 5, thiomethoxyflurane. From: Koblin DD, et al. Polyhalogenated and Perfluorinated Compounds that Disobey the Meyer-Overton Hypothesis. 10. The lateral pressure idea can also be tested via theoretical calculations. With the availability of reliable, high-speed computer models of both saturated and unsaturated phospholipids, researchers should be able to use molecular dynamic simulations to compute lateral pressure profiles and thereby predict anesthetic effects. So far, computer simulations are consistent with an asymmetric distribution of inhaled anesthetics across the bilayer (below) and predict a surprisingly large effect on orientation of the phosphocholine dipole. such surface electrical properties could have profound effects on certain membrane proteins—on voltage gated ion channels, in particular—and could provide additional anesthetic mechanisms based on coupling between membrane proteins and the lipid bilayer. 13 Fig.3: A proposed anesthetic mechanism that includes contributions from both the lipid bilayer and membrane proteins. Halothane (consisting of the larger, shaded spheres), a typical inhaled anesthetic haloalkane, preferentially localizes to the amphiphilic regions (shown as smaller, colored spheres) of the bilayer (in which the acyl side chains are shown as ball-and-stick strands) in this molecular dynamics simulation; the aqueous milieu is represented at the upper and lower peripheries of the box11. W HAT IS ANESTHETIC PRECONDITIONING ? Preconditioning by volatile anesthetics is a promising therapeutic strategy to render myocardial tissue resistant to perioperative ischemia. In 1997 two American research groups, independently of each other6, 7, showed that isoflurane anesthetic gas conferred cardioprotection by reducing myocardial infarct size after prolonged ischemia in rabbits. Volatile anesthetics, which are known to improve post ischemic recovery and to decrease myocardial infarction size, effectively activate protective cellular mechanisms12, 13, 14. Notably, the protective effect of volatile anesthetics occurs even in the presence of already established cardioplegic protection.15 PROPOSED MECHANISM OF CARDIOPROTECTION FROM VOLATILE ANESTHETICS KATP-channels To date, a substantial body of evidence implicates adenosine triphosphate–sensitive potassium (KATP) channels as playing a pivotal role in the acquisition of the preconditioned state in the heart and proposes opening of this channel as the final common step underlying all preconditioned-like 14 states, including those elicited by volatile anesthetics. 16, 17, 18, 19 Although a preponderance of studies point to mitochondrial rather than sarcolemmal channels as likely players in preconditioning, so far it is not clear whether opening of the sarcolemmal KATP (sarcKATP) channel or the mitochondrial KATP (mitoKATP) channel is more important in mediating anesthetic-induced reconditioning. Furthermore, while results from patch clamp experiments demonstrate increased open probability of the sarcKATP channel for a given ATP concentration in response to isoflurane, no such data are available regarding the effects of volatile anesthetics on the activity of the mitoKATP channel, the proposed final effector of preconditioning20. The signalling cascades involve alterations in nitric oxide and free oxygen radical formation and several G-protein-coupled receptors (adenosine and ß-adrenergic receptors), and point to the key role of protein kinase C (PKC) as a signal amplifier and to the KATP channels as the main endeffectors in preconditioning. Laboratory investigations also stress the concept that anaesthetics may precondition endothelial and smooth muscle cells, the main components of blood vessels. As blood vessels are responsible for the supply of nutrients and oxygen to all tissues, anaesthetic preconditioning might beneficially affect a much wider variety of organs, including the brain, spinal cord, liver and kidneys. PKC and sevoflurane In 72 patients 21 scheduled for coronary artery bypass graft surgery under cardioplegic arrest, sevoflurane preconditioning during the first 10 min of complete cardiopulmonary bypass decreased the postoperative release of brain natriuretic peptide (BNP), a biochemical marker for myocardial dysfunction. Translocation of protein kinase C (PKC) was assessed by immunohistochemical analysis of atrial samples. Biochemical markers of myocardial dysfunction and injury (BNP, creatine kinase-MB activity, and cardiac troponin T), and renal dysfunction (cystatin C) were determined. Sevoflurane preconditioning significantly decreased postoperative release of BNP. Pronounced PKC δ and ∈ translocation was observed in sevoflurane-preconditioned myocardium. In addition, postoperative plasma cystatin C concentrations increased significantly less in sevoflurane-preconditioned patients. No differences were found between groups for perioperative ST-segment changes, arrhythmias, or creatine kinase-MB and cardiac troponin T release. Sevoflurane preconditioning thus preserved myocardial and renal function as assessed by biochemical markers in patients undergoing coronary artery bypass graft surgery under cardioplegic arrest. This study demonstrated for the first time translocation of PKC isoforms δ and ∈ in human myocardium in response to sevoflurane. Recently, Bouwman 22 and co-workers showed that activation of PKC δ by sevoflurane depends on modulations of the sodium/calcium exchanger. 23 15 Aside from PKC isoforms, very recent studies revealed that the protein kinase B (AKT)/PI3K pathway also plays a key role in APC24. It was shown that isoflurane reduces apoptosis in rabbits via the AKT pathway25. New findings from Pagel and co-workers 26 showed also that the nonanaesthetic noble gas helium induces cardioprotection by preconditioning and that this effect involves pro survival kinases PI3K, extracellular signal-regulated kinase (ERK1/2), 70 kDa ribosomal s6 kinase (p70s6k) and inhibition of the mitochondrial Permeability Transition Pore (mPTP). A closer look at a cellular level using molecular biology methods revealed that different concentrations of volatile anesthetics may have different effects on the proteins involved in signal transduction: isoflurane at low but not at high concentrations protected the heart by preconditioning, and this effect was mediated via increased phosphorylation and translocation of PKC27. MAPK Further downstream in the signal transduction of APC, mitogen-activated protein kinases (MAPKs) are involved in mediating cardioprotection by anesthetics. ERK 1/2 rather than p38 MAPK is suggested as a down-stream target of PKC after isoflurane administration in a cell culture model28. Marinovic et al. 29 also suggest ERK 1/2 as mediator of isoflurane-induced preconditioning; and these authors also found p70s6k and endothelial nitric oxide synthase responsible for the cardioprotection by isoflurane. It was recently shown that ERK 1/2 acts as a trigger of APC and that its upregulation is correlated with an upregulation in hypoxia-inducible factor 1 (HIF1) and vascular endothelial growth factor in vivo30. Da Silva and co-workers 31 demonstrated a different involvement of MAPK in APC (induced by 1.5 MAC isoflurane) and IPC in the isolated rat heart. The activation of different proteins may follow a certain time course with a rapid return toward normal activity levels for some steps in the signal transduction cascade: desflurane was shown to activate PKC € and ERK 1/2 in a time-dependent manner32. 16 Figure 4. Schematic diagram illustrating the potential role of mechanisms under investigation for both cardioprotection and preconditioning. Volatile anesthetics administered before (preconditioning) or during (cardioprotection) myocardial ischemia are thought to promote blood flow as well as reduce metabolic rate, thus increasing energy stores in ischemic tissue. Protective effects of volatile anesthetics in ischemic tissue could also occur through inhibition of ROS formation from mitochondrial, nuclear, or cytoplasmic sources; scavenging of free radicals; and inhibition of membrane lipid peroxidation. In addition, volatile anesthetics could attenuate ischemia-induced catecholamine release, and inhibit glutamergic transmission. Antagonism of glutamate can subsequently lead to attenuation of ischemiainduced increases in [ca2+]i. Such changes in [ca2+]i can modulate calcium-dependent protective processes in ischemia involving calmodulin, and the MAPK–ERK pathway. Volatile anesthetics are thought to reduce or delay apoptosis through activation of Akt (protein kinase B), an antiapoptic factor downstream of the MAPK-ERK pathway. Adenosine a1 receptor activation may also be involved with the preconditioning effects of volatile anesthetics. Furthermore, adenosine a1 receptor activation may possibly be a trigger for mitochondrial KATP channel opening and activation, which has been linked to the development of ischemic tolerance. It is speculated that KATP channel activation may alter ROS production, blunt intra-ischemic mitochondrial calcium accumulation, and improve postischemic mitochondrial energy production. Inducible nitric oxide synthase and the subsequent generation of NO may be important for volatile anesthetic preconditioning in ischemic tissue as well. Lastly, tolerance induced by volatile anesthetic preconditioning may be mediated through P38 MAPK activation. Reworked from Toma, et al.32 17 ADDITIONAL EVIDENCE Electron transport chain There is strong evidence of altered mitochondrial energetics by anesthetic preconditioning. A central role could be played by volatile anesthetics in uncoupling the trans-matrix electron transfer chain. 33, 34 Ca2+ influx prevention Prevention of cytosolic and indeed mitochondrial calcium overload is thought to play a major role in myocyte contracture, dysfunction (stunning) and cell lysis and apoptosis – and was shown to be attenuated by sevoflurane. This effect could be blocked by 5-HD acid, a KATP-channel blocker35. Inducible NOS Sevoflurane was also shown to induce cardioprotection (diminished infarct size and improved ventricular function) by reducing inducible nitric oxide synthase in ethanol-preconditioned hearts36. Expression of defense proteins - Late preconditioning Kalenka et al. showed alterations in proteomic expression of cellular defense proteins associated with oxidative stress37, and even more recently, Zaugg et al. demonstrated modified response in transcription involving pro-inflammatory adhesion molecules in white blood cells in human volunteers spontaneously breathing sevoflurane at low concentrations38. Similar to the repetitive nature of successful IPC, twice cyclic repetitive sevoflurane administration in rat hearts was previously found to confer greater cardioprotection than a single exposure at double concentration73. The use of functional blockade of enzymes together with molecular biology techniques can provide a far more detailed view of cellular mechanisms of APC. APC induces long-lasting changes in protein phosphorylation and translocation at a cellular level. Further progress in elucidating the underlying mechanisms of APC not only reflects an important increase in scientific knowledge, but may also offer a new perspective of using different anesthetics for targeted intraoperative myocardial protection. 18 THE HYPERTROPHIC LEFT VENTRICLE Left ventricular hypertrophy (LVH) is strongly associated with risk of myocardial ischemia 39 in part due to rarefaction of coronary capillaries and decreased vasodilator responsiveness of the vascular bed 40,41,42. Other myocardial architectural changes include increased fibrosis but, mainly in young growing individuals, there is an increase in angiogenesis, which however, appears to be lagging behind the LVH43. The increased ischemia susceptibility could result in poorer outcome in the coronary surgical patient where ischemia is anticipated. This was previously established in a CABG surgery cohort44. Therefore, this subgroup may require additional cardioprotective benefits from anesthetics, whilst at the same time LVH may itself antagonize the mechanisms involved in anesthetic cardioprotection, and hence at least partly explain the discrepancy between remarkably effective experimental results and less than remarkable clinical results 8. Due to altered proteomic expression45 and reduced vascular reactivity, it is possible that LVH adversely affects the modulation caused by anesthetic and indeed other cardioprotection. The effect of LVH upon cardioprotective strategies was only once reported previously46. Therefore, the current study (paper III) was designed in order to test the hypothesis that reference ischemia would result in increased infarct size in a porcine model of LVH, and secondly, to test if sevoflurane preconditioning would mitigate infarct size to a similar extent as in normal hearts. 19 3. OBJECTIVES The overall objective of this study was to investigate the possibility of reducing post-ischemic reperfusion injury in the myocardium by the use of sevoflurane volatile anesthetic in various clinically feasible application methods. Sub study I: AIM To investigate the protective effect of pre-ischemic double sevoflurane exposure upon reperfusion injury (myocardial necrosis and loss in myocardial function), and furthermore, to investigate the protective effect of ischemic preconditioning in pigs. HYPOTHESIS It is possible to reduce the extent of myocardial necrosis and the extent of myocardial dysfunction caused by 40 minutes of regional, normothermic ischemia by a pre-ischemic double exposure73 to sevoflurane volatile interspersed with washout time, in pigs. It is similarly possible to reduce the extent of myocardial necrosis by ischemic preconditioning by two cycles of 5 minutes of regional coronary artery flow interruption followed by 5 minutes of reflow. Sub study II: AIM To investigate the protective effect of continuous sevoflurane volatile, alone or in conjunction with pentobarbital general anesthesia, upon myocardial necrosis. HYPOTHESIS It is possible to reduce the extent of myocardial necrosis caused by 45 minutes of regional normothermic ischemia and 120 minutes of reperfusion by continuous sevoflurane volatile in anesthetic dosage in pigs. The described effects from sevoflurane are not affected by the use of pentobarbital for general anesthesia. The amendment in ischemia-reperfusion times followed from the results of substudy I 20 73, where considerable variability in infarct sizes led to an extension of the occlusive ischemia period in order to more completely express the myocardial infarct (fig.3). Consequently, reperfusion time was adjusted downward (from 150 min). Sub study III: AIMS To investigate the protective effect of continuous sevoflurane administration upon myocardial necrosis in the hypertrophied heart, and furthermore, to investigate whether the hypertrophied heart is more susceptible to ischemia than the normal heart in a porcine model. HYPOTHESIS Continuous sevoflurane inhalation in anesthetic dosage protects the hypertrophied heart from myocardial necrosis, after 45 minutes of normothermic regional ischemia followed by 120 minutes of reperfusion, to the same extent as in the normal heart in pigs. The hypertrophied heart is as liable to sustain the same extent of post-ischemic myocardial necrosis as the normal heart following regional ischemia during pentobarbital anesthesia. 21 4. METHODS - methodological considerations I. ASSESSMENT OF IRREVERSIBLE ISCHEMIA-REPERFUSION INJURY Ischemia-reperfusion injury can be categorized into; Increased cardiomyocyte death: necrosis, apoptosis (programmed cell death) Loss of functionality; loss of myocardial contractility (‘stunning’) Other: arrhythmia, no-reflow phenomena. Cardiomyocyte death Both clinically and in the laboratory setting it is possible to assess cell necrosis or apoptosis by various methods. Several in vivo and in vitro techniques exist, including a range of histopathology 47, sestamibi-MPI scanning 48 and many intra- and extracellular biomarkers of cardiomyocyte injury or apoptosis, e.g. CK-MB, cardiac troponin-t, -I 49, etc. Frequently, the question arises as to how to estimate the magnitude of the irreversible ischemic injury. The extent of cardiomyocyte necrosis can, for example, be quantified in the excised heart in experimental studies. For that purpose, methods such as tetrazolium chloride staining of necrotic tissue and relating it to the ischemic at risk zone (area-at-risk; AAR) enable quantification of the injury. This is both wellestablished and widely used50. Evaluation of Infarct Size Early phase myocardial 51 infarction can be reliably detected by histochemical methods (fig.5+6). Triphenyl tetrazolium chloride (TTC) salts depend on the enzymatic activity of lactate dehydrogenase (LDH) and co factors in living cells to form a formazan pigment which stains vital cells brick red. No staining is seen in cells lacking LDH, such as dead tissue (fig. 7B). This is used to determine early myocardial infarction size (IS), which can then be measured by planimetric approach. Ischemic Risk Area Area-at-risk (AAR) is a critical determinant of the myocardial infarct size (fig. 4), and when correlated to IS, it constitutes a method of comparing the extent of irreversible ischemia22 reperfusion injury. AAR varies according to the site of the coronary occlusion, and with different species. Comparing AAR to the whole heart or left ventricle (=total area) is therefore also necessary. AAR is delineated during coronary occlusion by injecting dye into the central circulating blood volume (e.g., intra atrially) or into the occluded coronary branch, thus staining non-risk regions (fig. 7A). Dyes include Evans’ Blue and fluorescent particles. Subsequent to area measurements and determination of heart slice mass (g), it is possible to calculate the weightcorrected contribution of each heart slice towards the total infarct. Figure 5: Infarct evolution has been found to occur more slowly in humans than pigs. From Hedström E. Acute myocardial infarction the relationship between duration of ischaemia and infarct size in humans – assessment by MRI and SPECT. Lund university, faculty of medicine doctoral dissertation series 2005:72 doctoral thesis 2005 department of clinical physiology Lund University, Sweden 51. Figure 6: The relationship between area-at-risk (risk zone; AAR) and Infarct Size (IS) in different models of tetrazolium perfusions, showing correlation to histologic necrosis can be obtained from tetrazolium during anesthesia. 23 LV AAR Figure 7. (Upper panel) Evans' blue (white = areaat-risk; AAR) and (lower panel) tetrazolium staining (white = infarct size; IS) in transverse heart slices. Total ventricle area = LV. Note: Only a proportion of the area-at-risk becomes necrotic (IS). This proportion, or ratio, [IS/AAR] is the main outcome measure in studies I-III. IS IS 24 II: ASSESSMENT OF REVERSIBLE ISCHEMIC INJURY Cardiac performance depends on four parameters; (1) preload, (2) afterload, (3) heart rate, and (4) contractility52. For that purpose, methods like tissue-Doppler echocardiography57, MRI tracking and sonomicrometry76 can be used for direct assessment of reversible functional ischemic injury (’stunning’). Or, if the above factors 1-3 remain steady, indirect assessment can be made from ventricular blood pressure measurement (e.g. micro-tipped Millar catheter) and cardiac output (CO, using a pulmonary artery catheter)53. Transthoracic Tissue-Doppler Echocardiography Echocardiography, previously ventriculography, and more recently, magnetic resonance imaging (MRI), were in previous investigations used to assess cardiac vitality and functionality of ischemic myocardium54,55. Cardiac MRI could be used successfully in the porcine model, but as the early phase of post-ischemic reperfusion is characterized by exceedingly quickly evolving dynamics, echocardiographic monitoring was preferred. Echocardiography (trans-thoracic, TTE; or Trans esophageal, TEE) (fig. 8) has the possibility of detecting and quantifying time-motion changes in myocardial contraction. Time-velocity changes in myocardial contraction are sensitive markers of myocardial ischemia and can be estimated with tissue-Doppler imaging56,57. Cardiac Output Measurement by Thermodilution Hemodynamic parameters such as blood pressure, heart rate, cardiac output and mixed-venous oxy-hemoglobin saturation in the pulmonary artery provide information about the global cardiac function and thus reflect stunning. In almost all clinical and experimental settings systemic arterial blood pressure is routinely measured. However, several factors influence this assessment of cardiac function, e.g. vascular tone, blood volume, subject positioning, and these may affect results. More direct measurement of cardiac performance can be achieved using cardiac output (CO), either by transit-time flow probe mounted on the pulmonary artery or by a Swan-Ganz catheter passed into the pulmonary artery. This method uses a variant of Fick’s Principle: Flow = (rate of tracer input into an examined system) / (Concentration of tracer 1 – conc. of tracer 2), where 1 and 2 are different time points. The Swan-Ganz technique uses thermodilution, whereby C1 and C2 become differences in temperature. This technique is utilized in the CCOmbo-catheter connected to a monitor (Baxter Vigilance), which allows continuous measurement sampling. 25 Figure 8. Trans-thoracic echocardiograms (TTE) in a clinical study investigating propofol anesthetic effects on left ventricular function. Top panel: Tissue-Doppler myocardial velocity (m/sec) in the lower interventricular septum as a marker of LV global performance. The peak systolic velocity (PSV) is ¨6 m/sec, and declines during myocardial ischemia. Lower panel: tissue-tracking distances (TTD) at six different anatomic locations within the left ventricle. From Larsen JR et al, BJA 2007 56. Additional Parameters Electrophysiologic changes result from prolonged cardiomyocyte ischemia and from exposure to volatile anesthetics. These alterations can affect the functional myocardial syncitium to such an 26 extent that dysrhythmias develop, which may require external DC-cardioversion. Incidence, type, and required conversion frequency of arrhythmia constitute a method for assessing the effects of volatile anesthetics58. Electrocardiographic ST-segment changes similar to those in humans have been described in pigs59, although the heart is dextro rotated compared to in humans, and the EKG axes consequently different to humans. Nevertheless, for the purposes of verifying ongoing occlusive myocardial ischemia this method is well-established, and could disclose differences in ischemia physiology between intervention vs. control groups. The chosen model Based on the considerations discussed in previous sections, we chose a Danish Landrace/Yorkshire pig (female), with a body mass of 25-30 kg as the study subject in all three sub-studies. This is a species commonly used within our department, and furthermore, both the closed-chest model and the left ventricular hypertrophy model were previously established within our research facility. Therefore, the knowledge and handling experience of these animals exists within the core facility. In all three studies, the animal handling complied with the principles stated by the Danish Inspectorate for Animal Experimentations, and this institution approved the present study. THE PORCINE LV HYPERTROPHY MODEL The juvenile porcine LVH model has been established in our department and has previously been described in detail60. 27 III: METHODS AND MATERIALS - assessment of ischemia-reperfusion injury in the porcine model General concepts An experimental animal model offers the possibility of exposing a biological complex individual to surgical or interventional procedures that are not appropriate in humans. One should, however, be aware that results obtained in animal models cannot be directly extrapolated to the clinical setting, because animals are normally healthy at the time of investigation, or they have been subjected to simulated disease. If the factor (parameter) is pertinent to both the animal model and in the clinical situation, then this warrants heightened credibility to the investigation. The culture and knowledge of how to handle a particular species in the institution where the study is conducted, often dictates the choice of experimental animals. Despite this, it is still necessary to consider which animal species best reflects the human condition being investigated. In the domestic farm pig (Sus scrofa domestica), many anatomic and ischemia physiology characteristics show overlap between the normal human heart and that of the pig. These similarities have led to widespread use of the porcine model, in particular in cardiovascular investigations. In the porcine heart the coronary arteries are functional end arteries, like in humans. The gross anatomy and size are also very similar. By contrast, the surface-to-volume ratio in rodents is dissimilar, which may affect metabolic factors, as could elevated resting heart rates in these species, which in fact is dependent upon a very different calcium-handling mechanism in cardiomyocytes – precluding relevant cytosolic calcium studies. Moreover, immunological differences are suggested by the fact that, in rodents, violation of sterile conditions is well tolerated in survival studies, in contrast to human and porcine studies. These differences in inflammation and immunology may be significant in relation to reperfusion injury. Pigs are generally considered not to have any significant collateral coronary blood supply (~0.6%)61. This is of some importance since volatile anesthetics may cause vasodilation of collaterals. Moreover, in occlusive ischemia studies this feature is considered a virtue because a potential confounding factor is excluded. Size matters. The intact animal model tends to yield fewer data than, for example, the isolated heart model (Langendorff-perfusion) – due to its lesser invasive nature. Contrary to the statement on size, this poses an added advantage of operability in terms of the wide range of instrumentation 28 and surgical techniques more readily available as compared to rodents (e.g. pulmonary artery catheter, pulse oximetry etc.). In an effort to reduce external ‘noise’ afflicting ischemia-reperfusion injury, we opted for a minimally invasive ‘closed-chest’ model of coronary occlusion by deploying a standard coronary balloon catheter62 . Although this model may not truly represent acute coronary occlusion63, it is evident that this manoeuvre elicits the expected physiologic reactions. Using smaller animals (2030 kg) than frequently used 64 was conditional to transthoracic echocardiography, because larger pigs develop an accessory pulmonary lobe, precluding echo examination65. Trans Esophageal echo was excluded, as the angle between the esophagus and the basis cordis form a void which does not allow insonation66. Although we subsequently omitted echocardiographic technique, it was decided to retain the format (size) because the setup was ready and the data acquired subsequently would be comparable to existing data. Markers of Cardiac Injury Clinical markers of myocardial infarction include ‘classic’ symptomatology, distinctive ECG findings, and cardiac enzyme release into the blood stream, alongside image diagnostic supplementation. In the anesthetized animal model clinical indicators are irrelevant. Although not infrequently used, the cardiac troponin-t assay is not validated in pigs, and furthermore could prove an unreliable marker of the size of ischemic injury in small sample sizes67. CK-MB is even less specific as an injury marker. Moreover, as the biomarker rise (- and peak value) time extends up to 48 h and the current setup was usually less than 4 h, it was decided to employ a wellestablished method of injury estimate as the primary outcome parameter: triphenyl tetrazolium staining. General Protocol Outline Animals were fed and housed at the institutional facility68. Prior to transportation to the research lab, the animals received midazolam (0.5 mg/kg i.m.) sedation, in order to avoid increased stress levels. Once at the research lab, premedication generally consisted of midazolam and s-ketamine 69 prior to handling, since these do not affect preconditioning70,71. Subsequently, i.v. anesthesia induction was facilitated via an auricular vein I. Induction was according to allocation protocol I-III. No muscle relaxant medications were given. Rapid orotracheal intubation subsequently followed 29 and animals were ventilated by ventilator in 60% oxygen enriched air on a closed circuit system. Anesthesia maintenance was according to allocation group either pentobarbital i.v. infusion or sevoflurane (fig.17) I-III. Monitoring of vital signs consisted of core (rectal) temperature, tail pulse-oximetry and standard ECG. A diagram of the setup is given (fig. 12). Maintenance of temperature at or above 37.5oC, and not exceeding >1 degree temperature change during the experiment was considered mandatory. Electric warming blankets or ice packs were used to accomplish this. Cannulation of the right internal carotid artery and the internal jugular vein was facilitated by a small surgical incision above the sternocleidomastoideus muscle. Through this dissection the vessels were cannulated using Seldinger’s technique to allow placement of 7 Fr (artery) and 8 Fr (vein) intravascular sheaths. Aortic root blood pressure was measured continuously (DatexOhmeda A/5 Avance) via fluid-filled catheter and the vascular sheath facilitated placement of standard percutaneous coronary intervention catheters. The venous access enabled simultaneous fluid infusions, measurement of central venous pressure and placement of Swan-Ganz catheter (continuous CO monitoring, mixed-venous oxy-saturation and PCWP, mPAP measurement). After a stabilization period of at least 15 min, the coronary arteriography procedure commenced, following i.v. injection of 160 IU/kg heparin. Subsequent heparin injections (100 IU/kg) were administered once hourly for the duration of the experiment. Coronary angiography was performed using a standard 6 Fr size 4 JL-type launcher fluoroscopically placed in the left main branch (fig.15). A standard 2.5 mm percutaneous coronary intervention (PCI-) balloon catheter was positioned immediately downstream of the second diagonal branch of the LAD. The balloon was inflated (7 atm) and immediately checked for correct positioning and total blood flow occlusion verified. Occlusion was verified by angiography and electrocardiographic ST-segment changes exceeding 1 mm deflection from baseline. Occlusion was terminated after 45 min (40 min in study II) by balloon deflation, removal and fluoroscopic evidence of good reflow. Pulseless tachycardia or ventricular fibrillation was immediately treated with 200J external DC counter-shock, but no anti-arrhythmic or vasopressors drugs given. Spirometry was used to monitor the levels of respiratory gases (carbon dioxide, oxygen and sevoflurane) inhaled and exhaled in the animal. The purpose of this was two-fold; to calculate the exact amount of sevoflurane dosage (MAC), and to allow low-flow1 anesthesia. 1 Fresh gas flow usually less than 1.5 l /min due to the cost of volatile anesthetics. 30 Fluid balance was maintained constant by infusion of 0.9% NaCl at 12 ml/kg/hr throughout the experiment. Electrolyte balance, blood acid-base levels and fluid maintenance were monitored by heparinized arterial blood samples analyzed using an ABL-600 (Radiometer, Copenhagen) analyzer. Additionally, potassium chloride in 10% glucose was given as 1 miliequivalent/kg/hr. At the end or the reperfusion period the heart was exposed via a standard midline thoracotomy, allowing histochemical staining procedure and harvesting the heart (see following sections). A general protocol outline is given below (fig.9). Figure 9. Global LV Function Echocardiography Pigs begin to develop an additional pulmonary lobe65 when they attain a body mass of around 25 kg, which in turn obstructs internal (TEE) view finding from this size and upwards. This limited our choice to small pigs and TTE. The echocardiograms were recorded from standard parasternal and sub xiphoid positions and included standard short-axis and long-axis (2-, 4-chamber) views. We chose to employ this to monitor global cardiac function by assessing contraction velocity and tissue tracking indices in the interventricular septum in study I I. Quantification of myocardial contractile performance indices was obtained using EchoPac® software in off-line analysis of the digitally stored echocardiograms. Using this software, peak systolic velocity (PSV) and time-topeak (TTP) were determined at predetermined measurement time points and at a predetermined anatomic location in the interventricular septum 1.0 cm above the atrioventricular plane. Additionally, tissue-tracking distances (TTD) were measured at several locations in the LV. Cardiac Output In subsequent studies II, III, global LV function was determined using continuous measurements of cardiac output by a Swan-Ganz catheter (S-G). This alteration was due to technical reasons; (1) we 31 found no appreciable difference in LV function despite significant differences in infarct size in substudy I I, (2) the rapidly evolving hemodynamic changes that occurred, especially during the first minutes of reperfusion made repeated echocardiographic recordings difficult. The S-G catheter has the added advantage of continuous direct measurement of mixed-venous oxygen saturation (SvO2) and replaced echocardiography as the preferred measure of global cardiac performance. Infarct Size Measurement The reperfusion period was terminated after 120 min. At the end of reperfusion, the heart was harvested by the following method; a standard midline sternotomy was performed and the pericardium was opened. The LAD is typically visible on the anterior surface of the heart. The site of occlusion was identified by inspection and the previously recorded cinematic fluoroscopy, and subsequently, a ligature was passed around the artery at this site. In the event of pericarditis in the animal, standard fluoroscopy was reperformed to relocate the ligature site. The ligature was closed tightly and Evans’ blue dye (500 mg in 8 ml NaCl) was injected by bolus directly into the left atrial appendage. The heart was then quickly excised after epicardial appearance of the dye, and placed in cold saline. The heart preparation was subsequently sectioned into 3-5 mm slices in the shortaxis plane and transferred to a photo-scanner (Epson, USA) for recording of high resolution digital images (800 dpi; 24-bit color) (fig. 10A). Following this, slices were incubated for 10 min at 37 °C in 2,3,5-triphenyl tetrazolium chloride 1% in phosphate buffer 0.1 M solution and subsequently transferred for recording of a second scanned image and weighed (fig.10B). Slices were consistently incubated for 9-12 minutes in this until a sharp contrasting edge between necrotic and adjacent areas developed. Figure 10. 32 IMAGE ANALYSIS USING COMPUTER-ASSISTED PLANIMETRY Owing to the painstaking, often inaccurate, and rarely reproducible task of hand tracing IS or AAR areas other methodological approaches were explored. Bias could be eliminated and reproducibility improved by employing computer software-assisted techniques72. The National Institutes of Health (Bethesda, DC, USA) have made public web based free-ware,’ Image J © ’, a JAVA-based platform which enables macro functions within the general areas of image analysis. After some early developmental work, we decided to generate our own unique macro application using this software. The general outline of the function is given (fig.11) and the description is as follows: (a) the scanned photo image (digitized) is blinded, and a region-of-interest (ROI) (=slice) outlined. The ROI is enhanced by removing obvious artifacts, such as the ventricular lumen (b). The ROI image is threecolor split into component images (c); Red, Green and Blue, each consisting of grey-scale images where each pixel is assigned a value between 1 and 256 (center images below). Figure 11. The Red-filtered image is used for AAR planimetry, whilst the Green-filtered image is selected for IS planimetry (d). For each of these images (right hand images), an automated thresholding function which uses a histogram to automatically determine the lowest point in the grey-scale spectrum where the separation between intensity peaks is greatest. 33 This determines the delineation between the surrounding area and the IS or AAR, respectively (e). Subsequently, the thresholded pixels are counted and summated (f). Subsequent to planimetry and determination of heart slice mass (g), the weight-corrected contribution of each heart slice towards the total infarct was calculated as; 34 Experimental Setup: A schematic illustration (fig.12) of the experimental setup showing monitoring, anesthesia equipment, measuring devices and image acquisition equipment is given, and a photograph shown (fig.13). Figure 3. Diagram of the experimental setup at a glance. 35 Figure 13. The experimental setup. Note attention paid to environment temperature. THE LEFT VENTRICULAR HYPERTROPHY MODEL Briefly, infant pigs weighing around 5 kg (~10 days) were fitted with aortic banding (nonrestricting silicone tubing around the aorta) via a left sided thoracotomy. Normal growth of the animal gradually results in supravalvular (and supra coronary) restriction in the ascending aorta. Subsequently, this leads to progressive pressure overload in the left ventricle and hypertrophy gradually developed. The animals are housed and fed under normal conditions. LVH is monitored by assessment of cardiac dimensions and pressures during ambulatory transthoracic echocardiography (TTE) examinations (fig. 14). Post-mortem measurements of heart mass and wall thickness were used to verify LVH and compared to TTE findings. A pressure drop across the stenosis >60 mm Hg is generally considered sufficient to attain LVH in these animals, and is found most frequently to occur ~2 months after the banding procedure73. Animals subsequently entered into allocation for the ischemia-reperfusion protocol. FIGURE 14. H19 and siblings at day 7 after Aortic banding procedure. The shaved left thoracic surface and incisional scar in the axillary fold indicate recent surgery. Postoperative analgesia was in part provided by Durogesic®-fentanyl adhesive 36 Aortic valve FIGURE 15: (Left panel) Left main coronary arteriogram in a normal animal. Right panel: Diagram of PCI-catheter (“JL”-type) via carotid artery sheath and into the left coronary ostium. Note placement of launcher (cf. fig 14). From sub study III. The position of the silicone aortic band, and the ensuing aortic wall adaptation to the new structure, gave rise to alterations in the intravascular pathway followed during the procedure of PCI-catheter placement. The normal selection of “JL”-type launcher (fig.15) was substituted for “J”-type launchers (fig. 16). Moreover, coronary angiography gave visual evidence of increased coronary blood flow. FIGURE 16: Left main stem coronary angiography in LVH animal after aortic banding. The position of the band precludes use of JL-type; alternatively J-type launcher was used. Note also vascular enlargement of coronary arteries due to aortic stenosis. 37 STATISTICS All data was tested for normal distribution by Komolgorov-Smirnoff test. Comparison of histochemistry area data (infarct size, area-at-risk size and total area) was performed by MannWhitney rank sum test for independent samples for two group comparisons in sub-study I. In substudies II and III, a three group comparison was made by one-way analysis of variance (ANOVA), and in the event of significance a Mann-Whitney rank sum test was performed. Comparisons between group functional measurements repeated over time (heart rate, MAP, CO, SvO2, ST-segment displacement, end-tidal spirometry measurements, and temperature) were made using univariate repeated measures two-way ANOVA with time repeated. In the event of significant difference between groups over time, a post hoc estimated difference was made using a t-test. Comparison between groups of arrhythmia events requiring DC-conversion was made by analysis of frequencies and Chi-square table. P<0.05 was considered to reflect a significant difference. MedCalc© release 9.4.2.0 (Mariaklerke, Belgium) and Intercooled STATA© 9.2 (College Station, TX, USA) were used in analysis and presentation of the results. 38 5. STUDY DESIGN RANDOMIZATION Each of the three sub studies was performed as a randomized, controlled investigation. Randomization was performed by stable personnel unrelated to the study, who chose the animal for delivery on a given experimental day. The intervention form (intervention or control) was decided after drawing a sealed envelope by a person unrelated to the study. INDUCING ISCHEMIA -REPERFUSION INJURY In all three experimental investigations, acute myocardial infarction was induced in the animals, labeled control ischemia, by occluding flow in the left anterior descending coronary artery distal to the 2D terminal branch with a standard balloon-tipped PCI catheter for 45 min (40 min in sub study I 27). Choosing this location for coronary artery occlusion provided an ischemic risk area large enough to assess by planimetry in heart slices, whilst being small enough to avoid global heart failure and risk of dysrhythmia 64. The ischemic period was lengthened to 45 min in order to more completely express infarction by the TTC-method (fig. 4). The reperfusion period was short (120150 min) in contrast to the normal course of myocardial infarction, which may take days-weeks. However, studies of infarct evolution in a canine study stretched over 4 days of anesthesia revealed only marginal alteration in infarct evolution74. Infarct evolution is faster in pigs than it is in humans 51. Reperfusion was also curtailed to reduce the length, and thereby the adverse effects of extended anesthesia. GROUPS, NUMBERS AND MEASURING TIME -POINTS Sub study I: This study 75 comprised three groups of animals; 1. CON: Controls (n=10). Pentobarbital infusion. 2. IPC: Ischemic preconditioning (n=12); 2 x 5 min LAD occlusions, interspersed with 5 min reflow, prior to LAD occlusion. 39 3. APC: Anesthetic preconditioning (n=11); 2x 5 min sevoflurane (4%vol/vol) inhalation, interspersed with 5 min washout time, prior to LAD occlusion. This study was designed to set up the model and assess sevoflurane APC efficacy in the human-like porcine model, which was not previously published. Control anesthetic was pentobarbital infusion. Ischemia time was 40 min followed by 150 min reperfusion. An ischemic preconditioning group was added to evaluate the efficacy of the model and as a reference against other similar studies. Ischemic preconditioning was performed with the PCI-balloon catheter. Transthoracic echocardiographic assessment of LV global function was performed at baseline, early, and late ischemia, and at several time-points during early and late reperfusion. Sub study II: In this study, based upon the standard deviation (SD) in infarct size in sub study I (calculated sample size=15), 45 animals were randomized to three groups; 1. CON: Controls (n=15) Pentobarbital infusion. 2. COMBINED: Pentobarbital infusion combined with sevoflurane (2.1%) inhalation (n=15). 3. SEVO only: Sole anesthetic was sevoflurane inhalation 3.2% endtidal concentration (n=15). This study tested the maximal APC-response of sevoflurane (1.5 MAC) inhalation before and throughout ischemia-reperfusion. Circulatory collapse with higher MAC values during reperfusion prevented higher sevoflurane dosage. Pentobarbital infusion was chosen as reference; however, as the observed difference could thus be ascribed to negative conditioning from control anesthetic, this necessitated a combined group. Ischemia time was prolonged to 45 min; whereas reperfusion time was reduced to 120 min. At interim analysis, the SD was half of the expected value, and hence, the study twice the necessary size. For ethical reasons the investigation was therefore terminated after 35 experiments, at which time study actually comprised; combined =14 animals, sevo=9, and con=12 animals. Seven time-points were included for the assessment of functional data: baseline, 10 and 40 min of ischemia, 2, 5, 15 and 110 min of reperfusion. 40 Sub study III: In this study, we randomized 40 animals to two groups, 1. NORMAL Controls (n=20) 2. LVH (n=20) Group 2 underwent aortic banding and group 1 received no treatment. At 9 weeks post-banding, both groups were further subdivided into four groups, to which animals (by group) were randomly allocated; 1. NORMAL CON (n=12) Healthy age- and gender-matched controls receiving standard pentobarbital infusion. 2. NORMAL + SEVO (n=8) Healthy age matched female pigs receiving 3.2% vol/vol (et-conc.) sevoflurane throughout the duration of the experiment. 3. LVH CON (n=9) LV Hypertrophy animals receiving standard pentobarbital infusion. 4. LVH + SEVO (n=7) LV Hypertrophy animals receiving 3.2% sevoflurane. The purpose was to evaluate the efficacy of sevoflurane APC in a model of left ventricular hypertrophy. Four LVH animals died prior to allocation to ischemia-reperfusion protocol. The development of LVH was monitored by ambulatory in vivo echocardiographic examinations, and was verified by post-mortem measurement of LV free wall thickness in heart slices and measurement of heart weight-to-body weight ratioIII. Seven time-points were included for the assessment of functional data: baseline, 10 and 40 min ischemia, 2, 5, 15 and 110 min of reperfusion. 41 cardiac exvisceration STUDY 1 - STUDY 2 - STUDY 3 - Figure 17. Experimental study protocols at an overview; Sub-studies I (top), II (center) and III (bottom). 42 6. SUMMARY OF RESULTS STUDY I: A possible ‘trigger’ effect from sevoflurane APC was previously postulated, and demonstrated in rat hearts76. The aim of the current study was to establish a closed-chest porcine ischemia-reperfusion model, and in this to assess sevoflurane anesthetic preconditioning referenced to control ischemia and ‘classic’ ischemic preconditioning. The design is given in fig. 17 (top). The primary end-point was a comparison of infarct size assessed by tetrazolium staining. Secondary end-points were differences in functional parameters; hemodynamics and echocardiographic markers of LV contractility. Myocardial infarct size [IS/AAR], was reduced by 29% by pre-ischemic sevoflurane (P=0.38), and by 53% by ischemic preconditioning (P=0.038). Figure 18. Primary result of study I: Histochemical area (means±SEM), by group. No difference was found between groups with respect to functional parameters (HR, blood pressure, echocardiographic variables: PSV, TTP or TTD; or temperature, DC-cardioversion frequencies) (fig. 19). DC counter shock generally occurred most frequently at approx. 20 min ischemia in controls I-III, or later during reperfusion in sevoflurane treated animals. No statistical difference in distribution between study groups was found I-III. The current result is explained by 1) 43 insufficient sevoflurane dosage or possibly washout prior to ischemia, and/or 2), no ‘trigger’-effect of sevoflurane. However, there was a trend towards effect of pre-ischemic bolus of sevoflurane. All functional group means were modified over time. The lack of functional differences between groups was explained by the fact that the contribution of differing infarct sizes within the ischemic risk area was too small to cause deleterious effects in global LV function. Lack of effect of sevoflurane upon LV function in a porcine model was previously described77,78. Figure 19. Hemodynamic and echocardiographic data in substudy I. STUDY II: The aims were to document efficacy of continuous sevoflurane inhalation (cardioprotection) in anesthetic dosage (1.5 MAC) in order to elicit the maximum practical protective response without causing circulatory collapse, and to assess the antagonistic preconditioning effect caused by pentobarbital infusion given at a specified rate in controls. This was done to ensure that any observed difference in effect was not simply due preconditioning antagonism by pentobarbital, and this was achieved by adding a third group; combined sevoflurane and pentobarbital. [IS/AAR] was reduced 68% (P=0.002) by sevoflurane alone (3.2% et-conc.), and by 60% by sevoflurane (2.2%) combined with pentobarbital (P=0.001). Risk area comparison showed no difference between groups (fig.20). 44 * Ϯ Figu re 20. Box-and-whisker plots of infarct size and risk area comparisons in study II. Box delineations: median with lower to upper quartile [box], the horizontal line extends from the minimum to the maximum value, excluding "outside” values which are displayed as separate points. * P=0.0001 vs. controls; Ϯ P =0.0002 vs. controls. Model control of group functional data showed that all functional measurements were modified by time, but only heart rates showed differences between groups over time. This difference appeared at early reperfusion (2 min) and subsequently subsided (fig.21). This leads to the speculation that the difference in infarct size could be caused by HR differences, since higher HR requires more oxygen when the heart is very susceptible to ischemic damage during reperfusion. A ‘beta-blockade’ theory of action could be formulated, whereby the action of sevoflurane is similar to beta-blockers, i.e. favors oxygen supply-demand relations. The finding supports the efficacy of sevoflurane APC in the porcine model which is characterized by little or no collateral coronary blood supply, and this suggests that the mechanism of action is independent of augmented collateral flow ‘bypassing’ an occlusion 79. Figure 21. Heart rate was significantly higher in controls at 2 min reperfusion (47’ mark), which could be associated with reduced infarct size from sevoflurane. * P=0.0047. 45 STUDY III: The previously demonstrated efficacy of sevoflurane cardioprotection in normal porcine hearts II was evaluated in left ventricular hypertrophied (LVH) pigs which were pretreated with aortic banding resulting in gradual stenosis and LV hypertrophy. Sevoflurane was given at 1.5 MAC * ** throughout the ischemia-reperfusion protocol in both LVH and in healthy animals. LVH and normal animals receiving pentobarbital infusion acted as controls. A study design outline is given in fig. 17 (bottom). Figure 22. Box-plot demonstrating LVH development after 9 weeks (median) postbanding showing a 40% increase in HW:BW-ratio. Heart weight- to- body weight ratio in 4 study groups (from left); LVH controls, LVH+sevo, normal controls and normal + sevo. Aortic banding resulted in increased heart weight- to body-weight ratio in operated animals (P < 0.0001) in post-mortem analysis, indicating >40% increase in this parameter (fig.22). LVH appeared to reduce the sevoflurane cardioprotective efficacy relative to normal, healthy ageand gender-matched controls (from 68 to 57%), whereas the absolute infarct sizes were equally sized (15% vs. 17% in normal+sevo) (P=ns) (fig.23). Moreover, LVH reduced the size of the reference infarcts from 55% [IS/AAR] to 34% (P=0.0034). This unexpected association between LV hypertrophy and decreased infarct size is contrary to previous findings (in adults). However, as the animals in this study were young there may be an association to a previous post-mortem survey in LVH patients, in which it was found that children with LVH had compensatory angiogenesis and lower incidence of unexpected death than adults with failed compensatory angiogenesis and Figure 23. Infarct size results from 46 sub study III. a higher incidence of unexpected death. Functional parameters showed the same pattern of modification over time but not by group, except in two instances; HR was higher in normal animals receiving pentobarbital infusion compared to the other groups (fig.25), and temperature was lower in both pentobarbital groups. HR increases could explain larger infarcts but lowered temperature is normally associated with smaller infarcts (fig.24). Figure 24. Functional parameters at baseline in Study III. Figure 25. HR in 4 groups during ischemia-reperfusion in sub study III (means, error bars±SEM) 47 * ** ** *** LVH Isch Prec # # LVH with Pento LVH Figure 26. Summary of infarct size results in the current three investigations. Regarding efficacy, sevoflurane reduces myocardial infarct size after prolonged ischemia (40-45 min) and up to 2.5 hr reperfusion, as determined by post-mortem histologic tetrazolium staining and planimetry. Means±SEM. * p=0.03 vs control ** p<0.002 vs. control *** p<0.0034 vs. control # p<0.05<control and LVH control. 48 7. DISCUSSION Improving outcome after ischemia-reperfusion injury remains a challenge in all disciplines of cardiac medicine. New approaches to targeted reperfusion strategies may provide the answer. Compelling experimental data in multiple animal models showing the protective effects of volatile anesthetics still remain to be translated into therapeutic approaches to reduce morbidity and mortality in patients with ischemic heart disease. The current thesis supports that conjoint treatment with volatile anesthetics before and during ischemia-reperfusion is likely to reduce myocardial injury in normal and, to a lesser extent, pathologic hearts. ANESTHETIC CARDIOPROTECTION IN NORMAL HEARTS Clinical results are controversial and partially inconsistent8 with compelling experimental evidence, but are nevertheless sufficiently clear in studies of human atrial tissue80. However, experimental results are yet to be translated into veritable improvements in clinical mortality and morbidity outcome. There is a general lack of large-scale, well designed clinical trials to counteract the confounding influence of a low number of surgeons and using surrogate endpoints such as cardiac troponins. The establishment of sevoflurane APC in a large mammal must therefore be considered important for future work. Sevoflurane APC is well-established in several species, however, not in pigs. There are many reasons why it is important in pigs. Firstly, the similarities between human and porcine hearts allow comparison and modeling on an unprecedented level of comparability. Second, it is possible to obtain information which is not as easily obtainable, or even impossible, in humans. Or it is not ethical to expose humans to or to acquire control tissue from. Third, other species or experimental models may not sufficiently represent a reference to human ischemia pathophysiology, and thus not adequately cover the translation into pre clinical results upon which future work can rest. This is possible only in intact organisms and probably in very few species, including the porcine model61. Sevoflurane cardioprotection was established in the current model by demonstrating significant reduction in ischemic injury. We found 30-68% reduction in histological myocardial infarct size after sevoflurane was administered either before (preconditioning)I or throughout prolonged ischemia and reperfusion (cardioprotection) II,III. This order of magnitude is similar to what was previously found in other species, e.g. canine (~30%) 13, rabbit (30-35%) 6, 7, rats (30-50%) 15, 19, 20 and even in isolated heart models. Conversely, it appears as if there could be an inverse relationship between the degree of coronary collateralization and the degree of salvage from cardioprotection. This might be explained by augmented innate ischemic preconditioning in species/individuals with prolific collateralization. 49 On the contrary, in the current studies we found no firm evidence of irreversible ischemic injury abrogation by sevoflurane. Previously, other authors have published enhanced ventricular recovery times81 but in a porcine model no beneficial effects upon left ventricular performance or recovery could be demonstrated from halothane 75 or preischemic sevoflurane 76. Despite reduced infarct size in study II, we found no functional benefits (by echocardiography) during ischemia or reperfusion. The most likely reason for this is that the contribution of infarcted area towards global LV function is negligible compared to the many times larger ischemic area-at-risk, which again, is only a third of the total LV. No obvious sparing from stunning, or improved recovery times were seen in several studies I, II, 75, 76. This has, however, been demonstrated in clinical studies81. Sevoflurane, however, preserved global outflow despite contractility depression due to reduced afterload82. We addressed the issue of timing of sevoflurane administration; and found clear evidence of myocardial infarct size abrogation by continuous (pre-, per-, and post-ischemic) sevoflurane 1.5 minimum alveolar concentration throughout ischemia and reperfusion in a closed-chest porcine experimental model II. In study I in normal hearts, the administration of two brief cycles of sevoflurane anesthetic prior to onset of ischemia did not significantly lower infarct sizes, although a tendency towards this was observed (ns). Given a larger sample size or a volatile anesthetic with less rapid washout profile, e.g. isoflurane, this result may have attained significance. It does, however, support the notion that duration and concentration of exposure is important, as well as the time from exposure to time of ischemic injury assessment, and thus, indirectly supports the phosphorylated kinases theory 21-32, as well as up-regulation of defense proteins 37, 38. Alternatively, it is also possible that infarct size mitigation as a result of sevoflurane, as our results indicate, was the result of blunted cardio acceleration during early reperfusion. This is apparent from the tachycardia seen in controls in study II and III. Volatile anesthetics depress cell membrane activity 12, 17,83 and a diminished cardio acceleratory response via efferent cardiac nerves could play a part. Significant differences in heart rates would, of course, lead to different oxygen consumption rates and supply of coronary blood flow, and could therefore have caused the observed difference in infarct size. Furthermore, this would putatively mean that sevoflurane’s actions, in this case, were similar to short-acting beta-blockade84 and this could advantageously be studied in the present model by administering sevoflurane to limit excessive tachycardia during reperfusion. In general, sevoflurane is known to reduce ischemia-reperfusion by initiating complex intracellular signal transduction 20, but the current studies indicate that alternative mechanisms may also be involved. The role of different anesthetics and adjuvant medications is the topic of some general debate and during ischemia-reperfusion modeling in particular20, 70. Barbiturates are generally viewed with some disdain as they appeared to antagonize preconditioning85, 86 even though these were used in 50 laboratory concentrations far exceeding clinical doses. Pentobarbital was demonstrated to inhibit mitochondrial respiration, but its effect upon infarct size is unknown. Substudy II was partly conceived to clarify the relative significance of pentobarbital infusion’s position in abrogating preconditioning. The interest in a closer examination of the effects of pentobarbital is spurned by the need for a neutral anesthetic in control animals. Since coronary occlusion by PCI-catheter in conscious pigs is possible, it is ethically impractical, and therefore a neutral, neither preconditioning nor antagonistic sedative is desirable in many investigations. Because pentobarbital is lipid soluble and it acts slightly myocardially depressively and this effect tends to accumulate with time unless the dosage is gradually reduced. Currently, however, infarct size was not found to be reduced from pentobarbital infusion although a small difference (ns) was seen. However, this could also be due to the differences in sevoflurane concentration between the combined group (2.2%) and the sevoflurane group (3.2%). Over time, temperature drop caused by accumulating barbiturate ought to provide smaller infarcts. In the present range of dosage and in the particular model we conclude the effect is negligible. ISCHEMIC PRECONDITIONING Ischemic preconditioning (IP) was investigated as a part of the armamentarium in order to have a suitable reference. IP consisted of ‘classical’ multiple brief coronary artery occlusions by percutaneous transluminal coronary angioplasty balloon catheter inflations upstream of the targeted myocardial region causing intermittent ischemia and reflow. Its efficacy was significant (>50% infarct size reduction) during the investigations and confirmed previous findings63. In other species ischemic preconditioning is well established 5, and demonstration of IP efficacy in the current closed-chest porcine model lends credibility to the model. CARDIOPROTECTION IN HYPERTROPHIED HEARTSIII The results from the present experimental studies demonstrate that LV hypertrophy caused by pressure overload in young pigs is associated with reduced efficacy of sevoflurane cardioprotection. Furthermore, that LVH appears to attenuate the impact of myocardial ischemia. The unexpected finding of reduced infarct size after ischemia in LVH pigs warrants further clarification, because it contrasts most previous experience. Traditionally, hypertrophy of the left ventricle is viewed as increasing susceptibility to ischemia. This is partly due to rarefaction of coronary capillaries in LVH remodeled myocardium, but also because of compromised response to vasodilator challenge in these capillaries (due to increased tissue pressure and thickening of capillary walls) 39-45. However, in a previous post-mortem study of LVH hearts, a lowered incidence of sudden death was found in children with LVH secondary to congenital heart disease, as compared to adults with LVH 43. The authors concluded based on careful morphometric analyses in these hearts, that hypertrophy in children was associated with proportional coronary capillary 51 angiogenesis, whereas in adults LVH appeared to be associated with failure of compensatory angiogenesis. As increased angiogenesis would instigate lowered ischemia susceptibility, this could explain the results from the present study III. Moreover, in the present investigation we examined infarct size mitigation in a LVH model where the dynamic parameters (including the stenosis gradient) are unstable and in progression, which is likely to place the animals’ myocardium under the continued stimulus of stressors (=ischemic preconditioning). Add to this the fact, that animals are juvenile and thus retain their ability to compensate through angiogenesis and growth in cardiomyocyte diameter 40 and the environment is dominated by ‘innate’ preconditioning simultaneously with the ability to compensate. An expression of this compensatory ability in the young was also found in a study in aortic banded young pigs, where the largest difference (vs. normal) in ventricular free wall thickness existed at 4-6 weeks post-banding, after which the difference attenuated and stabilized 73. An experimental LVH model in chronic pressure overloaded guinea pigs’ hearts showed that in the short term (4 wk) LVH was associated with concentric ventricular enlargement (=increased cardiomyocyte diameter), whereas elongation in cardiomyocytes was associated with dilatation and congestive failure at 6 months 40. Consequently, there is a clear distinction, not only between juvenile and adult LVH conditions, but also between early (compensated) and late (decompensated) pressure overload, and the present findings should therefore not immediately be disregarded. The present results may also explain why LVH is typically associated with increased ischemic susceptibility in the form it is found in adults, and why reduced myocardial infarction was not previously found in adult study populations55. Thus, it is important to underline that extrapolations from the present animal model to an adult clinical population should be done with due care, whereas it may be better related to LVH in juveniles. The development of LVH in animal models alters proteomic expression and microvascular function 44. However, it is also associated with myocardial ischemia in spite of normal coronary blood supply. Episodic myocardial ischemia could lead to ischemic preconditioning I, and in the context of the present results this is a possible further cause of attenuated infarct size in LVH controls. Similarly, this might reflect preconditioning in patients who underwent cardiac surgery8, 44, a proportion of who may have had abnormal LVH, and this would explain why in these patients APC efficacy is low 8. This, however, is speculative. Contrasting previous findings of elevated arterial blood pressures in experimental porcine LVH models 87 we found normal blood pressures. This discrepancy may be explained by the facts that, in this study the LVH model was based on chronic unilateral renal artery clamping causing hypertension, and in unsedated animals. Furthermore, blood pressures measured in the present study were made invasively distal to the aortic stenosis and may thus not represent cardiac pressures. This could have been avoided by employing micro-tipped pressure catheters inserted in 52 retrograde fashion via the carotid access into the LV but no such room could be accommodated due to the relatively small size of the stenosed lumen and the position of the PCI balloon catheter. We found clear evidence of myocardial infarct size abrogation by continuous sevoflurane 1.5 MAC inhalation in a closed-chest porcine experimental model III. It is apparent from the present results that, although sevoflurane reduced infarct size to largely the same levels in normal and in LVH animals, the sevoflurane APC efficacy was diminished in LVH (68% to 57%). Conversely, the results obtained by a direct comparison of infarct sizes (15 vs. 18% of AAR, P=ns) indicate that sevoflurane largely retained its APC efficacy in the LVH group. No previous studies of anesthetic cardioprotection in LVH models exist. The possible influence of the extent of hypertrophy development upon the ischemia risk is less established than functional restrictions from LVH, which are well described both experimentally and clinically 55, 88. Thus, we analyzed this relationship by correlating infarct size to the LVH markers, heart weight-to-body weight ratio (HW: BW) and LV posterior free wall ventricle thickness. Though HW: BW-ratio was found to be the more sensitive marker of LVH, it showed no immediate correlation to infarct size, whereas LV posterior free wall thickness showed a nearsignificant (P=0.08) trend towards the existence of such a relationship. To our knowledge, this was not previously disclosed. A relationship between growth in myocardial wall thickness and time lag to angiogenesis was previously described in young animals 45. LIMITATIONS Since the present investigations were performed in young animals, the results should not be interpreted into clinical work without due caution. The extrapolation from young healthy individuals to the clinical setting and from one species to another seems overtly inane, but for ethical, practical and other reasons it appears sensible to bridge the translation gap between molecular research and the clinical setting. The validity of the closed-chest model as a representation of acute myocardial ischemia may also be drawn into the line of questioning 63, but nevertheless probably represents an improvement compared with the ‘traditional’ open-chest model due to its lesser invasive nature 62 and subsequent minimized influence of systemic inflammatory response. Moreover, the possibility of a more thorough Electrophysiologic documentation is possible in a closed chest62. The porcine heart comparison to the human heart was discussed in earlier sections. Additionally, determination of coronary blood flow was not undertaken for technical reasons, but may have lent some insight into mechanisms of sevoflurane’s preconditioning action, and furthermore provided additional evidence of level of comparability between groups. Distribution of coronary flow in conjunction with reperfusion would be of widespread interest when examining 53 cardioprotective approach and could in future investigations in the model be attained by cardiac MRI. The current model is a short-term investigation which precludes us from obtaining definitive results regarding final infarct size. However, as was previously shown 51, pigs develop myocardial infarction much more rapidly than humans. This implies that the obtained results are more closely related to final infarct size than a similar time-course would be in man. Technically, longer anesthesia and intervention times could also pose the problem of disrupting infarct evolution processes. Moreover, in a 4 day reperfusion study in canines, insignificant additional development in myocardial infarct size occurred after the initial hours evaluated by cardiac MRI 74. Variability: In spite of the porcine model benefitting from smaller genetic diversity than a given human cohort, variability in the size of myocardial infarction remains a major drawback. On one hand it seems that it advantageously and correctly represents the impact of any given intervention on the biological individual but on the other hand, it wreaks havoc on scientific methods such as calculating sample size, statistics etc. The prompt answer would be to include more individuals in each study group, but some factors causing variation in infarct size could potentially be positively influenced58. For instance, some researchers advocate the use of anti-arrhythmic drugs (e.g. amiodarone, lidocaine) in the porcine model in order to avoid this model’s preponderance to develop ventricular fibrillation (VF)89. Although a lower VF incidence is sometimes found to follow from this, the variability in infarct size is undeniably lower90. The problem facing this intervention is that these medications antagonize preconditioning, and thus, longer reperfusion periods are required and possibly longer ischemia time also. This is not always possible when pentobarbital infusion is the control anesthetic as it tends to accumulate in fatty tissue and eventually becomes cardiodepressive causing circulatory collapse. When we became aware of the problem surrounding VF during the course of the present investigations, it was decided that the time had passed to revise the setup, as it would render comparison with the initial data impossible. In addition, some investigators lean towards using collateral coronary flow measurement to exclude outliers on the basis of errant blood flow which causes unpredictable flow distribution once sevoflurane is introduced in the model. This appears to be a good idea; however, it is technically difficult and eliminates the possibility of comparison to an adult human population, a number of whom have significant collateral development. Biomarkers (e.g. Tn-T, CK-MB) have been used successfully as predictors of outcome after acute myocardial infarction 67 and after CABG91. However, these were not used in the current investigations, mainly for two reasons; firstly, the peak plasma value required for significant diagnostic can be as long as 48 hr (Tn-T), and secondly, the statistical difference in biomarkers between study groups is poor when sample size is small. Moreover, in a very recently published porcine investigation, cardiac troponin-T (a novel porcine assay) was inversely correlated to 54 infarct size which was probably confounding dependent of coronary reflow differences during reperfusion 90. The current studies would probably be rightly accused of being solely observational and not offering sufficient mechanistic insight into the resultant myocardial injury alleviation, however some limitations to resources and time should be acknowledged. Moreover, the (as yet) relative scarcity of porcine molecular probes was also a limiting factor, which would have necessitated a disproportionate amount of resources allocated to identifying, using and not least validating commercially available antibodies. However, we anticipate that in the near future more validated porcine molecular probes and gene arrays will become available. The perspective that underlies the current investigations includes putting forward recommendations to aid in clinical work. However, wider perspectives must be considered. The possibilities of acquiring new insight into mechanisms of volatile anesthetic cardioprotection via molecular probes, gene arrays and into coronary artery flow redistribution via MRI in the existing model seem apparent. Additionally, the model may afford the opportunity to study similar mechanisms in other vital organs. In conclusion, we aimed the present study at determining sevoflurane’s ischemia-reperfusion injury-mitigation in normal and LV hypertrophied porcine hearts, and report sevoflurane cardioprotection efficacy to be reduced, but more interestingly, that LVH in juvenile animals is associated with reduced infarct size after ischemia. Sevoflurane cardioprotection was retained in normal hearts, and showed effective reduction of I-R injury on a similar scale of magnitude as ischemic preconditioning, the as yet most powerful infarct-mitigator. 55 CONCLUSIONS The three individual investigations in porcine experimental models lead to the following conclusions: I. In normothermic ischemia/reperfusion, ischemic preconditioning by percutaneous intraluminal coronary angioplasty catheter leads to >50% reduction in histological verified myocardial infarct size. II. A double, pre-ischemic 5 min 4% dose of sevoflurane inhalation shows a nonsignificant trend towards myocardial infarct size reduction. A ‘triggering’-effect with a memory phase is not seen from sevoflurane in the porcine model. III. Hemodynamic and cardiodynamic changes are not seen to follow from pre-ischemic 4% sevoflurane administration. IV. Sevoflurane continuously administered in anesthetic concentration (1-1.5 MAC) before the onset of ischemia, and throughout ischemia and reperfusion, significantly ameliorates infarct size compared to neutral anesthetic. V. Myocardial infarct size mitigation by sevoflurane inhalation is independent from concomitant administration of pentobarbital in anesthetic doses in pigs. VI. Hemodynamic response (attenuated reperfusion tachycardia) concomitant with sevoflurane cardioprotective administration could be partially responsible for the observed beneficial effects upon the extent of cardiomyocyte necrosis. VII. Sevoflurane cardioprotection, expressed as reduced myocardial infarct size relative to the ischemic area-at-risk, is possible in the porcine heart, which distinguishes itself by lack of a significant collateralization and is therefore likely to be independent of pharmacologic actions on coronary arteries evoking increased collateral flow. VIII. Continuous sevoflurane (1.5 MAC) administration during ischemia and reperfusion in a porcine model of the initial phase of left ventricular hypertrophy remodeling provides a similar infarct-mitigating response as in healthy animals; however, the efficacy appears to be reduced. IX. The initial phase of the development of left ventricular hypertrophy is seen to result in decreased capacity to express myocardial infarction as compared to healthy animals, and is therefore consistent with ischemic preconditioning. X. In the porcine model of left ventricular hypertrophy, the ratio between heart weight and body weight serves a better marker of the degree of hypertrophy (greater sensitivity) than does the left ventricular posterior free wall thickness. 56 ACKNOWLEDGEMENTS The present investigations were financially supported by grants from: The Danish Heart Foundation Aarhus University Hospital Research Initiative Abbott, Denmark Helga and Peter Kornings Fond 57 DANSK RESUMÉ Denne ph.d.-afhandling er udført under min ansættelse som klinisk assistent på AnæstesiologiskIntensiv afd. I, Århus Universitetshospital, Skejby fra juli 2005 til 2008. Arbejderne er udført på Klinisk Institut under samme ansættelse. Vejledere var professor, dr. med. J. Michael Hasenkam, T-forskningsafsnittet, Hjerte- Lunge- Karkirurgisk afd., Århus Universitetshospital, Skejby samt overlæge, dr.med. Erik Sloth, Anæstesiologisk-Intensiv afd. I, Århus Universitetshospital, Skejby. Afhandlingen er baseret på følgende tre arbejder: I. Larsen JR, Aagaard SR, Hasenkam JM, Sloth E. Pre-occlusion ischaemia, not sevoflurane, successfully preconditions the myocardium against further damage in porcine in vivo hearts. Acta Anaesthesiologica Scandinavica 2007; 51: 402-409. © Blackwell Publishing. I I. Larsen JR, Aagaard SR, Lie RH, Sloth E, Hasenkam JM. Sevoflurane Improves Myocardial Ischaemic Tolerance in a Closed-Chest Porcine Model. Acta Anaesthesiologica Scandinavica 2008; accepted for publ. © Blackwell Publishing. I I I. Larsen JR, Smerup MH, Hasenkam JM, Christensen SD, Sivesgaard K, Torp P, Sloth E. Pressure Overload Hypertrophy Remodeled Ventricle in Young Pigs: Decreased Infarct Size from Ischemia and Sevoflurane. Cardiovascular Research 2008; in review © Oxford University Press. Formål med studiet: Halogenerede gasanæstetikas effekter på myokardieceller under iskæmi og reperfusion er endnu ikke fuldt ud klarlagt, men flere eksperimentelle studier har vist, at endogene cellulære forsvarsproteiner aktiveres på linje med ’klassisk’ iskæmisk prækonditionering, under indflydelse af udvalgte gasanæstetika. Effekten af dette er slående, eksempelvis er sevoflurane i stand til markant at reducere størrelsen i myokardieinfarkt størrelse under iskæmi-reperfusion i eksperimentelle studier i dyr. Dog er kliniske undersøgelser inkonsistente med dette, og der savnes endnu et samlet entydigt bevis på forbedret klinisk resultat som følge af denne kardioprotektive effekt. Arbejdet, som ligger til grund for afhandlingen, havde til formål at belyse om sevoflurane, et hyppigt klinisk anvendt universelt anæstetikum, kunne reducere graden af irreversible iskæmiske myokardieskader i forbindelse med langvarig koronarokklusion og reperfusion. Ved anvendelsen af en intakt dyremodel i gris, som anses for at være i besiddelse af de bedste komparative hjerteanatomiske og -fysiologiske egenskaber i forhold til mennesket, udnyttedes disse egenskaber til at vurdere effekten af de tidligere påviste mekanismer for sevofluranes kardioprotektion på størrelsen af myokardieinfarkt efter koronarokklusion, forårsaget af ballon-kateter aflukning. 58 Hermed indhentes viden om den hjertebeskyttende effekt af sevoflurane under iskæmi, som kan forekomme under bypasskirurgi og ballon-udvidelser. Hensigten var at indhente ny viden til støtte for kliniske rekommandationer. Kapitel 1-2 i afhandlingen er en introduktion til iskæmi-reperfusionsskader, halogenerede gasanæstetika og disses mekanismer, som de i dag formodes at aktivere organismen selvforsvar. Derefter følger en kort beskrivelse af patologisk-anatomiske forhold ved hypertrofisk venstre ventrikel (LVH), som kan gøre sig gældende ved forskellige former for prækonditionering med anæstesimidler. Efterfølgende beskrives målsætninger, samt i detaljeret grad de i arbejderne anvendte metoder, herunder histokemiske farvemetoder (TTC), vurdering af global ventrikelfunktion, hæmodynamik og computerplanimetri i grisen som forsøgsmodel. Endvidere beskrives den hypertrofiske ventrikel model hos grisen. I kapitel 6-7 beskrives resultaterne af de tre arbejder; I studie I etableredes modellen og der fandtes signifikant nedsættelse af infarktstørrelsen ved iskæmisk prækonditionering, hvorimod en ikkesignifikant nedsættelse af infarktstørrelsen på 30 % fandtes som følge af præ-iskæmisk sevoflurane tilførsel. 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