Download file

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. I studie II blev der fundet hhv. 68 og 60 % reduktion i infarktstørrelsen, i forhold til
kontrolgruppen, som resultat af kontinuerlig præ-, per- og post-iskæmisk sevoflurane tilførsel
alene, eller i kombination med kontrolanæstetikum. Der fandtes ingen signifikant effekt af
pentobarbitalinfusion. I studie III fandtes, i venstre ventrikelhypertrofiske dyr, nedsættelse af
myokardieinfarktstørrelsen alene på baggrund af hypertrofien, samt reduktion af sevofluranes
kardioprotektive effekt.
59
REFERENCES
1 World Health Organization: The World Health Report 2004 - Changing History (PDF), 120-4.
ISBN 92-4-156265-X.
2 Mueller RL, Rosengart TK, Isom OW. The History of Surgery for Ischemic Heart Disease. Ann
Thorac Surg 1997: 63; 869-878.
3 Jennings RB, Reimer KA. Factors involved in salvaging ischemic myocardium: effect of
reperfusion of arterial blood. Circulation 1983; 68: I25-I36.
4 Kloner RA, Rezkalla SH. Cardiac protection during acute myocardial infarction: where do we
stand in 2004? Am J Coll Cardiol 2004; 44: 276-286?
5 Murry CE, Jennings RB and KA Reimer. Preconditioning with ischemia: a delay of lethal cell
injury in ischemic myocardium. Circulation 1986; 74: 1124-1136.
6 Cope DK, Impastato WK, Cohen MV, Downey JM. Volatile anesthetics protect the ischemic rabbit
myocardium from infarction. Anesthesiology 1997; 86: 699-709.
7 Cason BA, Gamperl AK, Slocum RE, Hickey RF. Anesthetic-induced preconditioning: previous
administration of isoflurane decreases myocardial infarct size in rabbits. Anesthesiology 1997; 87:
1182-1190.
8 Symons JA, Myles P. Myocardial protection with volatile anaesthetic agents during coronary
artery bypass surgery: a meta-analysis. Br J Anaesth 2006; 97: 127-136. Epub 2006 Jun 21. Review.
9 Fenster JM. Ether Day: The Strange Tale of America's Greatest Medical Discovery and the
Haunted Men Who Made It. New York: Harper Collins. JAMA 2001; 286: 2877-2878.
10 Koblin DD, Chortkoff BS, Laster MJ, Eger AI, Halsey MJ, Ionescu P. Polyhalogenated and
Perfluorinated Compounds that Disobey the Meyer-Overton Hypothesis. Anesth Analg 1994;
79:1043-1048.
11 Koubi L, Biophysical Journal 2000; 78; 800–811.
12 Warltier D, Al-Wathiqui M, Kampine J, Schmeling W. Recovery of contractile function of
stunned myocardium in chronically instrumented dogs is enhanced by halothane or isoflurane.
Anesthesiology 1989; 69: 552–65.
13 Davis R, Sidi A: Effect of isoflurane on the extent of myocardial necrosis and on systemic
hemodynamics, regional myocardial blood flow, and regional myocardial metabolism in dogs after
coronary artery occlusion. Anesth Analg 1989; 69: 575–586.
14 Belhomme D, Peynet J, Louzy M, Launay JM, Kitakaze M, Menasche P: Evidence for
preconditioning by isoflurane in coronary artery bypass graft surgery. Circulation 1999; 100:
II340–344.
60
15 Preckel B, Schlack W, Thämer V: Enflurane and isoflurane, but not halothane, protect against
myocardial reperfusion injury after cardioplegic arrest with HTK solution in the isolated rat heart.
Anesth Analg 1998; 87: 1221–1227.
16 O’Rourke B: Myocardial KATP channels in preconditioning. Circ Res 2000; 87: 845–855.
17 Kersten JR, Gross GJ, Pagel PS, Warltier DC: Activation of adenosine triphosphate-regulated
potassium channels. Anesthesiology 1998; 88: 495–513.
18 Gross GJ, Fryer RM: Sarcolemmal versus mitochondrial ATP-sensitive K_channels and
myocardial preconditioning. Circ Res 1999; 84: 973–79.
19 Toller WG, Gross ER, Kersten JR, Pagel PS, Gross GJ, Warltier DC: Sarcolemmal and
mitochondrial adenosine triphosphate-dependent potassium channels. Anesthesiology 2000; 92:
1731–1739.
20 Tanaka K, Ludwig L, Kersten JR, Pagel PS, Warltier DC. Mechanisms of Cardioprotection by
Volatile Anesthetics. Anesthesiology 2004; 100: 707-721.
21 Julier K, Da Silva R, Garcia C, Bestmann L, Frascarolo P, Zollinger A, Chassot P-G, Schmid ER,
Turina M, Von Segesser L, Pasch T, Spahn DR, Zaugg M. Preconditioning by Sevoflurane decreases
biochemical markers for myocardial and renal dysfunction in coronary artery bypass graft surgery:
A double-blinded, placebo-controlled, multicenter study. Anesthesiology 2003; 98: 1315-1327.
22 Bouwman RA, Salic K, Padding FG, et al. Cardioprotection via activation of protein kinase Cdelta depends on modulation of the reverse mode of the Na-/Ca2-exchanger. Circulation 2006;
114:I226–I232.
23 Ludwig LM, Weihrauch D, Kersten JR, et al. Protein kinase C translocation and Src protein
tyrosine kinase activation mediate isoflurane-induced preconditioning in vivo: potential
downstream targets of mitochondrial adenosine triphosphate-sensitive potassium channels and
reactive oxygen species. Anesthesiology 2004; 100:532–539.
24 Raphael J, Rivo J, Gozal Y. Isoflurane-induced myocardial preconditioning is dependent on
phosphatidylinositol-3-kinase/Akt signaling. Br J Anaesth 2005; 95:7 56–763.
25 Raphael J, Abedat S, Rivo J, et al. Volatile anesthetic preconditioning attenuates myocardial
apoptosis in rabbits after regional ischemia and reperfusion via Akt signaling and modulation of
Bcl-2 family proteins. J Pharmacol Exp Ther 2006; 318:186–194.
26 Pagel PS, Krolikowski JG, Shim YH, Venkatapuram S, Kersten JR, Weihrauch D, Warltier DC,
Pratt PF Jr. Noble gases without anesthetic properties protect myocardium against infarction by
activating prosurvival signaling kinases and inhibiting mitochondrial permeability transition in
vivo. Anesth Analg 2007; 105: 562-9.
61
27 Obal D, Weber NC, Zacharowski K, et al. Role of protein kinase C-e (PKC-e) in isoflurane
induced cardioprotection. Low, but not high concentrations of isoflurane activate PKC-e. Br J
Anaesth 2005; 94: 166–173.
28 Zhong L, Su JY. Isoflurane activates PKC and Ca(2+) -calmodulin-dependent protein kinase II
via MAP kinase signaling in cultured vascular smooth muscle cells. Anesthesiology 2002; 96: 148–
154.
29 Marinovic J, Bosnjak ZJ, Stadnicka A. Distinct roles for sarcolemmal and mitochondrial
adenosine triphosphate-sensitive potassium channels in isoflurane-induced protection against
oxidative stress. Anesthesiology 2006; 105: 98–104.
30 Wang C, Weihrauch D, Schwabe DA, et al. Extracellular signal-regulated kinases trigger
isoflurane preconditioning concomitant with up regulation of hypoxia-inducible factor-1alpha and
vascular endothelial growth factor expression in rats. Anesth Analg 2006; 103:281–288.
31 Da Silva R, Grampp T, Pasch T, Schaub MC, Zaugg M. Differential activation of mitogenactivated protein kinases in ischemic and anesthetic preconditioning. Anesthesiology 2004;
100:59–69.
32 Toma O, Weber NC, Wolter JI, et al. Desflurane preconditioning induces time-dependent
activation of protein kinase C epsilon and extracellular signal-regulated kinase 1 and 2 in the rat
heart in vivo. Anesthesiology 2004; 101: 1372–1380.
33 Stowe DF, Kevin LG. Cardiac Preconditioning by Volatile Anesthetic Agents: A Defining Role for
Altered Mitochondrial Bioenergetics. Antioxidants & Redox Signaling 2004; 6: 439-448.
34 Matthias L. Riess, Leo G. Kevin, Joseph McCormick, Ming T. Jiang, Samhita S. Rhodes, David
F. Stowe. Anesthetic Preconditioning: The Role of Free Radicals in Sevoflurane-Induced
Attenuation of Mitochondrial Electron Transport in Guinea Pig Isolated Hearts. Anesth Analg
2005; 100: 46-53.
35 Riess M, Camara AK, Novalija E, Chen Q, Rhodes S, Stowe DF. Anesthetic Preconditioning
Attenuates Mitochondrial Ca2+Overload during Ischemia in guinea Pig Intact Hearts: Reversal by
5-Hydroxydecanoic Acid. Anesth Analg 2002; 95: 1540–1546.
36 Kaneda K, Miyamae M, Sugioka S, Okusa C, Inamura Y, Domae N, Kotani J, Figueredo V.
Sevoflurane Enhances Ethanol-Induced Cardiac Preconditioning Through Modulation of Protein
Kinase C, Mitochondrial KATP Channels, and Nitric Oxide Synthase, in Guinea Pig Hearts. Anesth
Analg. 2008; 106:9-16.
37 Kalenka A, Maurer MH, Feldmann RE, et al. Volatile anesthetics evoke prolonged changes in the
proteome of the left ventricular myocardium: defining a molecular basis of cardioprotection? Acta
Anaesthesiol Scand2006; 50: 414–427.
62
38 Lucchinetti E, Aguirre J, Feng J, Zhu M, Suter M, Spahn DR, Härter L, Zaugg M. Molecular
evidence of late preconditioning after sevoflurane inhalation in healthy volunteers. Anesth Analg.
2007; 105: 629-640.
39 Levy D, Garrison RJ, Savage DD, Kannel WB, Castelli WP. Prognostic implications of echo
cardio-graphically determined left ventricular mass in the Framingham Heart Study. N Engl J Med
1990; 322: 1561-1566.
40 Toma BS, Wangler RD, DeWitt DF, Sparks HV Jr. Effect of development on coronary
vasodilator reserve in the isolated guinea pig heart. Circ Res 1985; 57: 538-544.
41 Keane JF, Driscoll DJ, Gersony WM, Hayes CJ, Kidd L, O'Fallon WM, Pieroni DR, Wolfe RR,
Weidman WH. Second natural history study of congenital heart defects. Results of treatment of
patients with aortic valvar stenosis. Circ 1993; 87(2 Suppl): I16-27.
42 Cannon RO 3d, Rosing DR, Maron BJ, Leon MB, Bonow RO, Watson RM, Epstein S. Myocardial
ischemia in patients with hypertrophic cardiomyopathy: filling pressures contribution of
inadequate vasodilator reserve and elevated left ventricular. Circ 1985; 71; 234-243.
43 Rakusan K, Flanagan MF, Geva T, Southern J, Van Praagh R. Morphometry of human coronary
capillaries during normal growth and the effect of age in left ventricular pressure-overload
hypertrophy. Circulation 1992; 86: 38-46.
44 Ritchison A, Smith JM, Engel AM. Gender differences in diabetic patients following coronary
artery bypass graft surgery. Card Surg 2007; 22: 401-405.
45 Jin X, Xia L, Wang LS, Shi JZ, Zheng Y, Chen WL, Zhang L, Liu ZG, Chen GQ, Fang NY.
Differential protein expression in hypertrophic heart with and without hypertension in
spontaneously hypertensive rats. Proteomics 2006; 6: 1948-1956.
46 Takeuchi K, Buenaventura P, Cao-Danh H, Glynn P, Simplaceanu E, McGowan FX, del Nido PJ.
Improved protection of the hypertrophied left ventricle by histidine-containing cardioplegia.
Circulation 1995; 92: II 395-399.
47 Fishbein MC, Meerbaum S, Rit J, Lando U, Kanmatsuse K, Mercier JC, Corday E, Ganz W. Early
phase acute myocardial infarct size quantification: validation of the triphenyl tetrazolium chloride
tissue enzyme staining technique. Am Heart J. 1981; 101: 593-600.
48 Kristensen J, Mortensen UM, Nielsen SS, Maeng M, Kaltoft A, Nielsen TT, Rehling M.
Myocardial perfusion imaging with 99mTc sestamibi early after reperfusion reliably reflects infarct
size reduction by ischaemic preconditioning in an experimental porcine model. Nucl Med
Commun. 2004: 25: 495-500.
49 Apple FS. Plasma 99th Percentile Reference Limits for Cardiac Troponin and Creatine Kinase
MB Mass for Use with European Society of Cardiology/American College of Cardiology Consensus
Recommendations. Clinical Chemistry 2003: 49; 1331-1336.
63
50 Downey JM. Measuring infarct size by the tetrazolium method. 1 July, 2008.
http://www.southalabama.edu/ishr/help/ttc/. University of South Alabama, Mobile.
51 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. Sweden.
52 Zipers DP, Libby P, Bonow RO, Braunwald E. Braunwald’s Heart Disease. Elsevier Saunders;
2005.
53 Swan HJ, Ganz W. Complications with flow-directed balloon-tipped catheters. Ann Intern Med
1979; 91: 494.
54 Herbots L, Maes F, D'hooge J, Claus P, Dymarkowski S, Mertens P, Mortelmans L, Bijnens B,
Bogaert J, Rademakers FE, Sutherland GR. Quantifying myocardial deformation throughout the
cardiac cycle: a comparison of ultrasound strain rate, grey-scale M-mode and magnetic resonance
imaging. Ultras Med Biol 2004; 30: 591-598.
55 Levy D, Garrison RJ, Savage DD, Kannel WB, Castelli WP. Prognostic implications of
echocardiographically determined left ventricular mass in the Framingham Heart Study.
Prognostic implications of echocardiographically determined left ventricular mass in the
Framingham Heart Study. N Engl J Med 1990; 322: 1561-1566.
56 Larsen JR, Torp P, Norrild K, Sloth E. Propofol reduces tissue-Doppler markers of left ventricle
function: a transthoracic echocardiographic study. Br J Anaesth 2007; 98: 183-188.
57 Gorcsan J 3rd. Assessment of Left Ventricular Systolic Function Using Color-Coded Tissue
Doppler Echocardiography. Echocard 1999; 16: 455-463.
58 Hashimoto H, Imamura S, Ikeda K, Nakashima M. Electrophysiologic Interaction between
Class I Antiarrhythmic Drugs and Volatile Anesthetics in Depressant Effects on Ventricular
Activation in a Canine Myocardial Infarction Model. Japan J Pharmacol 1994: 64; 235-241.
59 Morrison SG, Dominguez JJ, Frascarolo P, Reiz S. A Comparison of the Electrocardiographic
Cardiotoxic Effects of Racemic Bupivacaine, Levobupivacaine, and Ropivacaine in Anesthetized
Swine. Anesth Analg 2000; 90: 1308-1314.
60 Malo O, Desjardins F, Tanguay J, Tardif J, Carrier M, Perrault L. Tetrahydrobiopterin and
antioxidants reverse the coronary endothelial dysfunction associated with left ventricular
hypertrophy in a porcine model. Cardiovascular Research 2003: 59; 501 – 511.
61 Crick SJ, Sheppard MN, Ho SY, Gebstein L, Anderson RH. Anatomy of the pig heart:
comparisons with normal human cardiac structure. J Anat 1998; 193: 105-119.
64
62 Näslund U, Häggmark S, Johansson G, Marklund SL, Reiz S. A closed-chest myocardial
occlusion-reperfusion model in the pig: techniques, morbidity and mortality. Eur Heart J 1992; 13:
1282-1289.
63 Baxter GF. Coronary angioplasty as a model of ischaemic preconditioning: fact or fancy? Eur
Heart J 1996: 17; 812-814. Letter.
64 Kristensen J, Maeng M, Rehling M, Berg JS, Mortensen UM, Nielsen SS, Nielsen TT. Lack of
acute cardioprotective effect from preischaemic erythropoietin administration in a porcine
coronary occlusion model. Clin Physiol Funct Imaging 2005; 25: 305-10.
65 Cabral V, Oliveira F, Machado M, Ribeiro A, Orsi M. Study of Lobation and Vascularization of
the Lungs of Wild Boar (Sus scrofa). Anat Histol Embryol 2001; 30: 205-209.
66 Terp K. Aarhus University Hospital, Skejby: Personal communication.
67 Holmvang L, Jurlander B, Rasmussen C, Thiis JJ, Grande P, Clemmensen P. Use of biochemical
markers of infarction for diagnosing perioperative myocardial infarction and early graft occlusion
after coronary artery bypass surgery. Chest 2002; 121: 103-111.
68 PAPER III: 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
69 PAPER II: 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
70 Kato R, Foëx P. Myocardial protection by anesthetic agents against ischemia-reperfusion injury:
an update for anesthesiologists. Can J Anaesth 2002: 49: 777-791.
71 Mullenheim J, Frassdorf J, Preckel B. Ketamine, but not S-ketamine, blocks ischemic
preconditioning in rabbit hearts in vivo. Anesthesiology 2001; 94: 630–636.
72 Micari A, Sklenar J, Belcik TA, Kaul S, Lindner JR. Automated quantification of the spatial
extent of perfusion defects and viability on myocardial contrast echocardiography. J Am Soc
Echocardiogr 2006; 19: 379-385.
73 S Lunde, Smerup M, Hasenkam M, Sloth E. A Model for Left Ventricular Hypertrophy Enabling
Non-Invasive Assessment of Cardiac Function. Scand Cardiovasc J 2008; in review
74 Thompson K, Wisenberg G, Sykes J, Thompson RT. Similar long-term cardiovascular effects of
propofol or isoflurane anesthesia during ischemia/ reperfusion in dogs. Can J Anaesth. 2002; 49:
978-985.
65
75 PAPER 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 Anaesthesiol Scand. 2007; 51: 402-409.
76 Riess ML, Kevin LG, Camara AK, Heisner JS, Stowe DF. Dual exposure to sevoflurane improves
anesthetic preconditioning in intact hearts. Anesthesiology 2004; 100: 569-574.
77 Conradie S, Coetzee A, Coetzee J. Anesthetic modulation of myocardial ischemia and
reperfusion injury in pigs. Comparison between halothane and sevoflurane. Can J Anaesth 1999;
46: 71–81.
78 Aagaard S, Larsen JR, Berg JS, Sloth E, Hasenkam JM. Does the pre-ischaemic administration
of sevoflurane reduce myocardial stunning? A porcine experimental model. Acta Anaesthesiol
Scand. 2007; 51: 577-581.
79 Sill J, Bove A, Nugent M, Blaise G, Dewey J, Grabau C. Effects of isoflurane on coronary arteries
and coronary arterioles in the intact dog. Anesthesiology 1987; 66: 273-279.
80 Hanouz JL, Zhu L, Lemoine S, Durand C, Lepage O, Massetti M, Khayat A, Plaud B, Gérard JL.
Reactive oxygen species mediate sevoflurane- and desflurane-induced preconditioning in isolated
human right atria in vitro.Anesth Analg 2007; 105:1534-1539.
81 De Hert SG, Cromheecke S, ten Broecke PW, Mertens E, De Blier IG, Stockman BA, Rodrigus
IE, Van der Linden PJ. Effects of propofol, desflurane, and sevoflurane on recovery of myocardial
function after coronary surgery in elderly high-risk patients. Anesthesiology 2003; 99: 314-323.
82 Preckel B, Müllenheim J, Hoff J, Obal D, Heiderhoff M, Thämer V, Schlack W. Haemodynamic
changes during halothane, sevoflurane and desflurane anaesthesia in dogs before and after the
induction of severe heart failure. Eur J Anaesthesiol 2004; 21: 797-806.
83 Wang J, Lei B, Popp S, Meng F, Cottrell JE, Kass IS. Sevoflurane immediate preconditioning
alters hypoxic membrane potential changes in rat hippocampal slices and improves recovery of CA1
pyramidal cells after hypoxia and global cerebral ischemia. Neuroscience 2007; 145: 1097-1107.
84 Sleight P. Interventions during and after acute myocardial infarction. Postgrad Med J 1983; 59:
80-88.
85 Kohro S, Hogan QH, Nakae Y, Yamakage M, Bosnjak ZJ. Anesthetic effects on mitochondrial
ATP-sensitive K channel. Anesthesiology 2001; 95: 1435-1440.
86 Zaugg M, Lucchinetti E, Spahn DR, Pasch T, Garcia C, Schaub MC. Differential effects of
anesthetics on mitochondrial K(ATP) channel activity and cardiomyocyte protection.
Anesthesiology 2002; 97: 15-23.
66
87 Rodriguez-Porcel M, Zhu XY, Chade AR, Amores-Arriaga B, Caplice NM, Ritman EL, Lerman A,
Lerman LO. Functional and structural remodeling of the myocardial microvasculature in early
experimental hypertension. Am J Physiol Heart Circ Physiol 2006; 290: H978-984.
88 Verdecchia P, Carini G, Circo A, Dovellini E, Giovannini E, Lombardo M, Solinas P, Gorini M,
Maggioni AP; MAVI (MAssa Ventricolare sinistra nell'Ipertensione) Study Group. Left ventricular
mass and cardiovascular morbidity in essential hypertension: the MAVI study. J Am Coll Cardiol
2001; 38: 1829-1835.
89 Finance O, Manning A, Chatelain P. Effects of a new amiodarone-like agent, SR 33589, in
comparison to amiodarone, D,L-sotalol, and lignocaine, on ischemia-induced ventricular
arrhythmias in anesthetized pigs. J Cardiovasc Pharmacol 1995; 26: 570-576.
90 Hein M, Roehl A, Bantes B, Baumert J, Bleilevens C, Bernstein N, Steendijk P, Rossaint R.
Establishment and evaluation of a porcine right ventricular infarction model for cardioprotective
and anti-inflammatory actions of xenon and isoflurane. Acta Anaesthesiologica Scandinavica 2008;
in review.
91 Hausenloy DJ, Mwamure PK, Venugopal V, Harris J, Barnard M, Grundy E, Ashley E, Vichare S,
Di Salvo C, Kolvekar S, Hayward M, Keogh B, MacAllister RJ, Yellon DM. Effect of remote
ischaemic preconditioning on myocardial injury in patients undergoing coronary artery bypass
graft surgery: a randomised controlled trial. Lancet 2007; 370: 575-579.
67