SYSTEMIC RESPONSE TO LOCAL ISCHAEMIA: THE EVOLVING … · THE EVOLVING CONCEPT OF REMOTE ISCHAEMIC...
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SYSTEMIC RESPONSE TO LOCAL ISCHAEMIA:
THE EVOLVING CONCEPT OF REMOTE
ISCHAEMIC PRECONDITIONING
PANKAJ SAXENA MBBS, MS, MCh, DNB
This thesis is presented for the degree of Doctor of Philosophy of
The University of Western Australia.
School of Surgery
Year of submission: 2009
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Summary Life on our planet is possible because of oxygen. All mammals, including humans,
evolved with an innate mechanism by which organs and cells may protect themselves
against lack or excess of oxygen to some extent. This lack and excess of oxygen that
occurs during interruption and restoration of blood supply to an organ is referred to as
ischaemia-reperfusion (IR). IR is a ubiquitous phenomenon that accounts for a number
of pathophysiological conditions. A prolonged period of ischaemia followed by
reperfusion causes IR injury and leads to significant damage to the affected organ. The
activation of a systemic inflammatory response is associated with the most detrimental
effects of IR injury.
It has been noticed that brief episodes of intermittent ischaemia applied locally
render significant protection against subsequent prolonged lethal ischaemic insult. This
was termed ischaemic pre-conditioning (IPC). Later, however, it was observed that
similar protection occurs when brief intermittent episodes of ischemia and reperfusion
were applied during or after prolonged ischaemia. These phenomena were termed per-
conditioning and post-conditioning. Thus, protection against prolonged ischaemia by
brief episodes of IR has recently evolved into the unified concept of ischemic
conditioning (IC).
It appears that brief intermittent episodes of ischaemia of the arm or leg can
significantly decrease the systemic inflammatory response triggered by subsequent
prolonged ischaemia of organs located remotely from the site of conditioning, for
instance heart, lung, liver or kidney. Hence, remote ischaemic preconditioning (RIPC)
is a global form of organ protection against ischaemia. The first clinical randomized
controlled trial of RIPC application suggested that clinically relevant protection can be
achieved by RIPC. Despite the proven benefits of the conditioning effects, the overall
application of this organ protective strategy in clinical practice has been limited thus far.
It is known that neutrophils play a key role in IR injury. Bradykinin (BK) is one of
the strongest known activators of neutrophils and the associated inflammatory response.
Interestingly, it has also been demonstrated that BK can induce preconditioning-type
protection against IR injury. It appears that preconditioning decreases the activation of
neutrophils during the IR injury. While it is known that BK activates neutrophils via the
kinin-receptors, the mechanism by which this mediator may induce a preconditioning-
type protection is, as yet, unclear. Furthermore, if neutrophils are crucial to the transfer
3 of RIPC stimulus to distant organs, how is the signal transferred across the blood-brain
barrier? Does RIPC protect the brain? The blood brain barrier creates a unique set of
physiological conditions that allow a glimpse into the mechanisms of RIPC. The aim of
this thesis is to further elucidate the mechanisms responsible for RIPC in order to
facilitate its transition to wider clinical application and to determine promising areas for
future research. Thus, the present work focussed on the effects of RIPC on the
functional responses of human neutrophils and the kallikrein-kinin system (KKS) that
appear crucial to the IR-induced activation of neutrophils and whether the expected
modification of neutrophil function by RIPC would result in cerebral protection.
Experimental work in the present thesis involved studying the changes in the functional
response of neutrophils in human volunteers following forearm preconditioning;
studying the expression of kinin receptors on the surface of human neutrophils
following RIPC and studying the effect of second window of RIPC on the hippocampus
of a murine model following global cerebral ischaemia (GCI).
It is hoped that a better understanding of the mechanisms of RIPC may help define
the potential role of preconditioning. This knowledge of RIPC is currently evolving into
a unifying concept of remote ischemic conditioning. Should the full potential of this
concept be utilized, it may have an immense impact on medical practice in the diverse
clinical scenarios associated with IR injury.
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Acknowledgements I would like to express my deep gratitude and sincere thanks to my friend, Professor Igor
Konstantinov, whose supervision and guidance has made this work possible.
Dr Konstantinov has also introduced me to the world of academic surgery. His inspiration
and invaluable support has been crucial in the completion of these studies and has laid the
background for the future clinical and experimental work in the newly defined area of
remote ischaemic conditioning. His contribution to my learning of operative Cardiac
Surgery is also highly appreciated. I thank Professor Paul Norman, my supervisor, for his guidance and valuable support
with this work. I thank Dr Mikiko Shimizu for her role in designing part of the experimental work related
to this thesis.
Kallikrein kinin assays were performed at the Lung Institute of Western Australia
(LIWA) by Ms Odette Shaw. I acknowledge the support provided by Professor Philip
Thompson with this work. Special thanks are due to all the clinicians and researchers who
were involved in the experimental work and include, Mr Mark Newman, Dr Neil Misso,
Ms Odette Shaw, Dr Arul Bala, Mr Kym Campbell and Mr Bruno Meloni.
I greatly appreciate the help from Professor John Hall, Ms Carleen Ellis, Ms Belinda
Seymour, Ms Elizabeth Doyle, Ms Aggie Jackiewicz and Mr Andrew Davey of the
School of Surgery at University of Western Australia. I also acknowledge the help of Dr
Benjamin Dunne and Dr Arwen Boyle in the final review of this document.
The funding for the kinin receptor study was provided by the National Health and
Medical Research Council (NHMRC) of Australia and the Heart Foundation of Australia.
I acknowledge the support provided by my son Rahul Saxena and my wife Dr Alka Sinha
at home, who are always very patient with my busy clinical and research schedule.
Finally, I appreciate my parent’s contribution to the development of my career who
always taught me to work hard and be sincere in my life.
Pankaj Saxena
Perth, Australia, December, 2009
5 Table of Contents Summary 2
Acknowledgements 4
Table of Contents 5
List of Figures 10
Abbreviations 11
Chapter 1: INTRODUCTION 13
1.1 Clinical importance of ischaemia-reperfusion injury 14
1.1.1 Definitions and key concepts 15
1.1.2 Key elements of ischaemia and reperfusion injury 15
Figure 1.1-1. Diagrammatic representation of the cascade of events 18
that occur in a cell following the onset of ischaemia-reperfusion injury.
Figure 1.1-2. Role of the vascular endothelium in 19
ischaemia-reperfusion injury.
1.1.3 Innate immunity and ischaemia 20
1.1.4 Role of leukocytes in ischaemia-reperfusion injury 20
1.1.5 Role of opiates & kallikrein kinin system in ischaemia-reperfusion injury 21
1.1.6 Summary 21
1.2 Remote ischaemic conditioning 22
1.2.1 The concept of ischaemic preconditioning 22
1.2.2 Remote ischaemic pre-conditioning 23
1.2.3 Remote ischaemic per-conditioning 24
1.2.4 Remote ischaemic post-conditioning 24
1.2.5 Functional response of human neutrophils to remote ischaemic 25
preconditioning stimulus
1.2.6 Expression of kinin receptors on human neutrophils following 25
remote ischaemic preconditioning
1.2.7 Circulating factor of remote ischaemic conditioning 26
1.2.8 Remote ischaemic preconditioning and protection against 27
global cerebral ischaemia
6 1.2.9 Remote ischaemic preconditioning, neuroprotection 27
and blood brain barrier
Figure 1.2-1. Diagram showing hypothetical signal transduction 28
pathway of remote ischaemic preconditioning from plasma
membrane to mitochondria.
1.2.10 Summary 30
1.3 Mechanisms of remote ischaemic conditioning 30
1.3.1 Role of innate immunity 31
1.3.2 Role of gene expression 31
1.3.3 Role of leukocytes 31
1.3.4 Role of signalling pathways 33
1.3.5 Role of mitochondria 34
1.3.6 Summary 34
1.4 Kallikrein-kinin system 35
Figure 1.4-1. Association of kallikrein-kinin system with 36
renin angiotensin aldosterone system.
1.4.1 Kallikrein-kinin system and renin-angiotensin-aldosterone system 38
Figure 1.4-2. A schematic of the components of kallikrein-kinin 39
system.
1.4.2 Kallikrein-kinin system and inflammation 39
1.4.3 Kallikrein-kinin system and cardiovascular system 40
1.4.4 Kallikrein-kinin system in neoplasia 41
1.4.5 Common signalling pathways in neoplasia and protection 42
against ischaemia-reperfusion injury
1.4.6 Kallikrein-kinin system and aprotinin 43
1.4.7 Summary 43
1.5 Rationale, hypotheses, and objectives 44
1.5.1 Rationale 44
1.5.2 Hypotheses 44
1.5.3 Objectives 44
Chapter 2: MATERIAL AND METHODS 46
2.1 Human model of remote ischaemic preconditioning 47
2.2 Rat model of remote ischaemic preconditioning 47
2.3 Rat model of global cerebral ischaemia 48
7 2.4 Assessment of functional responses of human leukocytes to remote 48
ischaemic preconditioning stimulus
2.4.1 Experimental design 48
2.4.2 Isolation of human neutrophils 48
2.4.3 Adhesion 49
2.4.4 Secretion of cytokines 49
2.4.5 Apoptosis 49
2.4.6 Oxidant production 49
2.4.7 Phagocytosis 50
2.4.8 Statistical analysis 50
2.5 Assessment of human kallikrein-kinin system response to remote 50
ischaemic preconditioning stimulus
2.5.1 Experimental design 50
2.5.2 Isolation of human neutrophils 51
2.5.3 Immunoperoxidase labelling 51
2.5.4 Immunofluorescence labelling 51
2.5.5 Confocal microscopy and image analysis 52
2.5.6 Statistical analysis 52
2.6 Assessment of impact of remote ischaemic preconditioning on 52
global cerebral ischaemia in rats
2.6.1 Experimental design 52
2.6.2 Assessment of hippocampal neurons 53
2.6.3 Statistical analysis 54
Chapter 3: RESULTS 55
3.1 Functional responses of human neutrophils to remote 56
ischaemic preconditioning stimulus
3.1.1 Summary 56
3.1.2 Neutrophil adhesion and CD11b surface expression 57
Figure 3.1-1. The effect of RIPC on neutrophil adhesion. 57
3.1.3 Oxidant production 58
Figure 3.1-2. The surface expression of a) CD11b, 58
b) NADPH oxidase production, the surface expression of c) CD63
and d) CD66b.
3.1.4 Exocytosis 58
8 3.1.5 Secretion of cytokines 59
Figure 3.1-3. Cytokine secretion in quiescent and stimulated cells. 59
3.1.6 Apoptosis 60
Figure 3.1-4. The effect of RIPC on neutrophil apoptosis. 60
3.1.7 Phagocytosis 61
Figure 3.1.5. Graphs showing the effect of repeated RIPC stimulus 61
on neutrophil phagocytic activity.
3.2 Human kallikrein-kinin system response to remote 62
ischaemic preconditioning stimulus
3.2.1 Summary 62
3.2.2 Expression of B1 kinin receptors 62
Figure 3.2-1. Representative confocal images showing the expression of 63
B1 receptors on neutrophils.
Figure 3.2-2. Quantitative analysis of kinin B1 receptor 64
immunofluorescence on neutrophils.
3.2.3 Expression of B2 kinin receptor 65
Figure 3.2-3. Representative confocal images demonstrating 65
expression of B2 receptors on neutrophils.
Figure 3.2-4. Quantitative analysis of kinin B2 receptor 65
immunofluorescence on neutrophils.
3.3. Assessment of impact of remote ischaemic preconditioning on 66
global cerebral ischaemia in rats
3.3.1 Summary 66
3.3.2 Delayed hippocampal neuronal death after transient global 66
cerebral ischaemia
Figure 3.3-1. Histological changes in the rat CA1 hippocampus. 67
Figure 3.3-2. Mean hippocampal CA1 neuron counts. 68
Chapter 4: DISCUSSION 69
4.1 Circulating factor of remote ischaemic preconditioning 70
4.2 Functional response of human neutrophils to remote ischaemic 70
preconditioning
4.3 Kinin receptor expression in human neutrophils 73
4.4 Second window of remote ischaemic preconditioning and neuroprotection 74
4.5 Remote ischaemic conditioning and blood brain barrier 76
9 4.6 Clinical applications 77
4.7 Future research 79
Chapter 5: ORIGINAL CONTRIBUTIONS 84
Chapter 6: PUBLICATIONS, PRESENTATIONS AND RESEARCH 86
FUNDING BASED ON THE THESIS
Chapter 7: REFERENCES 89
10 List of figures
Figure 1.1-1. Diagrammatic representation of the cascade of events that occur in a
cell following the onset of ischaemia-reperfusion injury.
Figure 1.1-2. Role of the vascular endothelium in ischaemia-reperfusion injury.
Figure 1.2-1. Diagram showing hypothetical signal transduction pathway of remote
ischaemic preconditioning from plasma membrane to mitochondria.
Figure1.4-1. Association of kallikrein-kinin system with the renin angiotensin
aldosterone system.
Figure 1.4-2. A schematic of the components of kallikrein-kinin system.
Figure 3.1-1. The effect of RIPC on neutrophil adhesion.
Figure 3.1-2. The surface expression of a) CD11b, b) NADPH oxidase production,
the surface expression of c) CD63 and d) CD66b.
Figure 3.1-3. Cytokine secretion in quiescent and stimulated cells.
Figure 3.1-4. The effect of RIPC on neutrophil apoptosis.
Figure 3.1.5. Graphs showing the effect of repeated RIPC stimulus on neutrophil
phagocytic activity.
Figure 3.2-1. Representative confocal images showing the expression of B1 receptors
on neutrophils.
Figure 3.2-2. Quantitative analysis of kinin B1 receptor immunofluorescence on
neutrophils.
Figure 3.2-3. Representative confocal images demonstrating expression of B2
receptors on neutrophils.
Figure 3.2-4. Quantitative analysis of kinin B2 receptor immunofluorescence on
neutrophils.
Figure 3.3-1. Histological change in the rat CA1 hippocampus.
Figure 3.3-2. Mean hippocampal CA1 neuron counts.
11 Abbreviations
AAA Abdominal aortic aneurysm
ACE Angiotensin converting enzyme
AMI Acute myocardial infarction
AOI Area of interest
ATP Adenosine triphosphate
BK Bradykinin
CABG Coronary artery bypass graft surgery
COPD Chronic obstructive pulmonary disease
CPB Cardiopulmonary bypass
CRP C-reactive protein
EEG Electroencephalograph
ECG Electrocardiograph
ELAM Endothelial leukocyte adhesion molecule
GCI Global cerebral ischaemia
GPCR G-protein coupled receptor
HK High molecular weight kininogen
HSP Heat shock protein
IC Ischaemic conditioning
ICAM Intercellular adhesion molecule-1
ICU Intensive care unit
IL Interleukin
IR Ischaemia reperfusion
JG Juxtaglomerular
KATP Potassium adenosine triphosphate
KKS Kallikrein-kinin system
LK Low molecular weight kininogen
LPS Lipopolysaccharide
MDA Malondialdehyde
MMP Matrix metalloproteinase
MnSOD Manganese superoxide dismutase
MPTP Mitochondrial permeability transition pore
NF-κB Nuclear factor kappa B
NOS Nitric oxide synthase
12 PCR Polymerase chain reaction
PI3K Phosphoinositide 3- kinase
PK Protein kinase
RIC Remote ischaemic conditioning
RIPC Remote ischaemic preconditioning
ROS Reactive oxygen species
SNARE N-ethylmaleimide sensitive factor attachment protein receptor
TK Tissue kallikrein
TLR-4 Toll like receptor-4
TNF Tumour necrosis factor
UTR 5’ Untranslated region
VCAM Vascular cell adhesion molecule-1
13
CHAPTER 1
INTRODUCTION
14 1.1 Clinical importance of ischaemia-reperfusion injury
The aerobic function of a cell requires adequate blood flow and oxygen supply.
Ischaemia disrupts steady state oxidative metabolism. If, however, perfusion is restored,
a significant degree of cellular damage occurs due to reperfusion. The injury caused by
ischaemia and reperfusion is known as ischaemia-reperfusion (IR) injury. IR injury can
impair the function of an organ significantly.
IR injury occurs in a number of clinical scenarios. Examples include patients with
coronary artery disease (CAD) and myocardial infarction (MI), treated with
thrombolysis, myocardial revascularization by angioplasty or coronary artery bypass
graft surgery (CABG); organ transplantation and various cerebral and peripheral
vascular procedures requiring transient interruption followed by restoration of blood
flow to the target organ. It has been known since the early days of cardiac surgery that
myocardial injury of variable degree occurs in patients undergoing CABG (1). This
damaging effect is responsible for an increased incidence of MI, myocardial rupture,
myocardial dysfunction and increased mortality following revascularization (2-3). Other
forms of revascularization such as thrombolysis or percutaneous coronary angioplasty
(PCI) may also cause myocardial dysfunction due to IR injury (4-8). CAD is the leading
cause of mortality in the Western world and is responsible for up to one third of all
deaths. Reducing the effects of IR injury may have a major influence on preventing the
morbidity and mortality associated with CAD.
IR injury seems to play an important role following transplantation. Graft
dysfunction occurs in 10-30% patients following transplantation due to reperfusion
injury. Multi-organ dysfunction syndrome (MODS) can occur as a result of
malfunctioning of the transplanted organ (9-11). Moreover, chronic rejection due to the
development of arteriosclerosis may result from the initial IR injury (12).
All organs can be affected by IR injury. IR injury causes cardiac dysfunction,
impairs cerebral function, can be responsible for the breakdown of gastrointestinal
barrier and can lead to systemic inflammatory response syndrome (SIRS) (13). Adult
respiratory distress syndrome (ARDS) can be a manifestation of IR injury and is
mediated by neutrophil activation due to C5a, leukotriene B4 (LTB4) and thromboxane
A2 released from ischaemic tissue (14).
While the pathophysiology of IR injury has been understood for decades, the
development of protective strategies against IR injury is still in their infancy.
15 1.1.1 Definitions and key concepts
IR injury describes an inflammatory process that results from interruption of the blood
supply followed by reperfusion to an organ which causes local and systemic injury.
Deprivation of the oxygen supply to the tissues causes a decrease in cellular respiration
and can cause irreversible damage to the cellular structures unless the blood supply is
restored promptly. Paradoxically, more damage occurs during the reperfusion phase. In
an experimental setting involving cardiomyocytes, the cell death rate was 17% in the
ischaemic group after 4 h of ischaemia in comparison to 73% in the reperfusion group
(15).
Ischaemic preconditioning (IPC) is a phenomenon in which brief cycles of
ischaemia and reperfusion produce a protective response against subsequent prolonged
periods of lethal ischaemia and reperfusion. IPC may provide powerful protection to the
target organs. This concept was first identified by Murry in 1986 using a canine model
(16).
A more clinically useful stimulus is afforded by remote IPC (RIPC) in which a
protective response similar to local IPC is evoked in the target organ by producing
cycles of ischaemia and reperfusion at a distant site. RIPC was first identified by
Przyklenk in 1993 (17). A number of preconditioning stimuli including skeletal muscle,
kidney, liver, mesentery and cerebral circulation, have been studied.
1.1.2 Key elements of ischaemia and reperfusion injury
Several mechanisms may be involved during reperfusion injury (Figure 1.1-1).
Ischaemic injury to a cell causes energy depletion due to defective synthesis of
adenosine triphosphate (ATP) and an increase in degradation of ATP. This energy
depletion results in mitochondrial dysfunction and causes the translocation of bax, a
proapoptotic bcl2 family member protein from the cytosol to the outer mitochondrial
membrane. This contributes to the mitochondrial swelling and induces the efflux of
cytochrome c via opening of the mitochondrial permeability transition pore (MPTP)
into the cytosol where cytochrome c activates effector caspases and initiates apoptosis
(18).
Calcium (Ca) is an important ion that plays a central role in excitation-contraction
coupling. The sodium-calcium pump maintains the haemostasis of Ca in the cells. Ca
ions are transported into the extracellular compartment in exchange for sodium ions. For
three Na ions transported inside the cells, one Ca ion is transported outside the cell (19).
The intracellular concentrations of calcium are maintained at a low level against a high
16 transsarcolemmal gradient by the voltage gated calcium channels that remain closed
(20). However, the sodium-calcium pump is activated during IR injury and causes the
movement of sodium ions from the cytoplasm to the extracellular space and entry of
calcium into the cell. Ischaemia causes the depolarisation of the cell membrane and
enhances calcium movement into the cell. Calcium dependent phospholipases,
endonucleases and proteases are then activated during this process causing a cascade of
events in the cell resulting in cellular dysfunction (21-22). The cell becomes more
permeable to calcium following membrane injury. Calcium overloading of the cell
causes irreversible injury and necrosis. Reducing the accumulation of calcium in the cell
has a cytoprotective effect during reperfusion (23).
Anaerobic metabolism occurs in tissues subjected to ischaemia (24) with
suppression of anti-oxidant activity. Restoration of blood flow causes the oversupply of
oxygen. This releases reactive oxygen species (ROS), generated from hydrogen
peroxide inside the cell, which results in the activation of macrophages and neutrophils.
There is microsomal peroxidation of the phospholipid layer of the cell membrane (25).
These factors cause cell membrane injury. The ultimate result of these changes is
swelling of the cell and intracellular deposition of calcium (26). ROS production
activates nuclear factor kappa B (NF-kB) (27). ROS activate nuclear enzyme poly ADP-
ribose polymerase (PARP). Over activation of PARP due to ROS, consumes ATPs and
can cause cellular dysfunction and death (28).
Proinflammatory cytokines such as tumour necrosis factor (TNF)-α, interleukin-1 β
and interleukin-6 are activated during reperfusion. Activated complement components
have been detected in ischaemic tissues (29). Both the classical and alternate pathways
are involved in IR injury (30). C5a is an important mediator of the complement pathway
in IR injury and causes chemotaxis of neutrophils, release of proteases and production
of ROS, mediating the release of a number of proinflammatory factors such as TNF-α
(13, 31-33). Animal models lacking complement proteins demonstrate resistance to IR
injury (34).
Vascular endothelium represents metabolically active tissue which maintains the
dynamics of the capillary circulation by balancing the levels of vasodilators like nitric
oxide (NO) and vasoconstrictors secreted by endothelial cells. Endothelial cells play an
important role in reperfusion injury (Figure 1.1-2). During IR injury, endothelial
dysfunction causes an abnormal response to the vasoconstrictors and dilators. There is
reduced synthesis of NO via endothelial and inducible nitric oxide synthase (eNOS and
iNOS) contributing to impaired endothelium dependent vasodilatation. There is a
17 marked increase in the release of endothelin-1 following reperfusion which is followed
by vasospasm due to the release of leukotriene B4, activated complement components
and thromboxane A2 (29). The overall result of this vasoconstriction is hypoperfusion.
This reperfusion associated endothelial dysfunction occurs soon after the ischaemic
event is over and lasts for a variable length of time (35-36). Microvascular dysfunction
is due to endothelial cell swelling and increase in the capillary permeability, resulting in
interstitial oedema. Adherence of the activated leukocytes to endothelium and
interstitial oedema of the tissues contributes to the no-reflow phenomenon during
reperfusion. This refers to the failure to restore myocardial blood flow following the
release of coronary obstruction (37). No-reflow reduces myocardial blood flow and
contributes to myocardial injury following AMI with incomplete resoluton of ST-
segment changes (38). The overall result is decreased resting myocardial blood flow and
myocardial hypoperfusion (39).
NO has been associated with both local and remote ischemic preconditioning.
However, the exact role of NO in the preconditioning process has been debated (40, 41).
Preconditioning cycles may activate NOS. The released NO as a result activates protein
kinase C ε, tyrosine kinase and NF-κB - (42-43). The signalling pathways involving NO
leads to the activation of KATP channels, the opening of mitochondrial permeability
transition pore (MPTP) and the release of ROS (44-45).
18
Figure 1.1-1. Diagrammatic representation of the cascade of events that occur in a cell
following the onset of ischaemia-reperfusion injury (Refer to the text, section 1.1.2). IL-
interleukin, TNF-α- tumour necrosis-α, bax- bcl-2–associated X protein, ATP-
adenosine triphosphate, NF-κB- nuclear factor κB, ROS- reactive oxygen species,
PARP- poly (ADP-ribose) polymerase, MPTP- mitochondrial permeability transition
pore.
19
Figure 1.1-2. Role of the vascular endothelium in ischaemia-reperfusion injury (Refer
to the text, section 1.1.2). IL- interleukin, TNF-α- tumour necrosis-α, NO- nitric oxide,
TXA2- Thromboxane A2.
20 1.1.3 Innate immunity and ischaemia
Innate immunity is a non-specific component of the immune system that includes
neutrophils, macrophages and cytokines. Interestingly, it appears that innate immunity
reacts to both infection and IR injury via the same signalling pathways (46). Activation
of neutrophils plays a key role in innate immunity responses to both infections and IR
injury. Moreover, it has been demonstrated that RIPC reduces the neutrophil
sequestration in the lungs following systemic inflammatory response syndrome (SIRS)
(47). A preconditioning-like response may be evoked by cytokines or bacterial cell wall
via the innate immunity pathways.
1.1.4 Role of leukocytes in ischaemia-reperfusion injury
Neutrophils play a central role in IR injury. Activated neutrophils respond by an
increase in the production of ROS and by releasing potent cytotoxic and matrix
degrading proteases (48-49). Following IR injury, a number of events occur that cause
the neutrophils to adhere to the vascular endothelium and extravasation of the cells into
the interstitial space. These actions are mediated by adhesion molecules that are present
on the surface of neutrophils and endothelial cells. The three groups of adhesion
molecules include- selectins, β2-integrin and immunoglobulins. Neutrophil adhesion to
endothelial cells is facilitated by P-selectin (Figure 1.1-2) which is expressed on the
surface of endothelium in response to IR injury or infection (50). Moreover, the rolling
neutrophils become activated and get firmly attached to the endothelium via interaction
of binding proteins of the integrin family, including leukocyte function antigen (LFA-1)
and intercellular adhesion molecule-1 and 2 (ICAM-1 and 2) (28, 51). Upregulation of
ICAM-1 is associated with reperfusion injury (52).
Filtration of neutrophils from the circulation during the initial phase of reperfusion
reduces tissue necrosis significantly. Reintroduction of neutrophils to the circulation
causes cellular damage equivalent to the unmodified reperfusion (53). Adhesion of
neutrophils to the vascular endothelium occurs following ischaemic injury to the tissues.
This event precedes the permeation of neutrophils into the extra vascular space. The
release of ROS, myeloperoxidase (MPO) and lysosomal enzymes by the activated
neutrophils causes the plugging of the microvasculature and further tissue damage
during reperfusion (Figure 1.1-2).
21 1.1.5 Role of opiates and kallikrein kinin system in ischaemia-reperfusion injury
The exact mechanism of RIPC is yet to be determined. However, there are a number of
mediators that have been involved in the protective response and include nitric oxide
(NO), opioids, adenosine, protein kinase C (PKC) and kinins. These factors are the
humoral mediators that are released in the circulation following the preconditioning of
tissue. Patel and co-workers reported that opiates released during intestinal ischaemia
were involved in RIPC mediated cardiac protection in an experimental rat model (54).
This effect was abolished by using naloxone. There has been evidence of binding of the
opiate receptors in the coronary effluent of the preconditioned hearts (55). The exact
role of various opiates and their receptors in RIPC has not been clearly identified.
Kallikrein-kinin system (KKS) plays an important role in inflammation, IR injury
and neoplasia. Ischaemic myocardium releases BK that triggers neutrophil activation
and the subsequent inflammatory response. This response appears to be dose-dependent.
This may play a key role in organ protection against IR injury (56). It is of interest that
administration of BK provides cardiac protection in patients undergoing myocardial
revascularization with coronary angioplasty (57). BK acts via B1 and B2 receptors. It is
possible that the initial brief interaction of kinin receptors with BK after the induction of
RIPC stimulus could render neutrophils less sensitive to subsequent large release of
kinins due to prolonged ischaemia. Such interaction could limit the extent of IR injury.
The exact mechanism of interaction between the neutrophils and KKS following the
RIPC stimulus is yet to be determined.
1.1.6 Summary
It appears that neutrophils and KKS are intimately involved in the innate immunity
response to IR injury. More specifically, neutrophil activation via kinin receptors may
play a key role in triggering the IR injury. Rapid and uncontrolled activation of innate
immunity may result in excessive systemic inflammatory response and extensive tissue
damage. Thus, there must be native protective mechanims that limit this non-specific
response of innate immunity. It seems logical to speculate that the natural protective
mechanism against IR injury evolved along with the non-specific innate immunity that
controls an excessive inflammatory response to IR injury. Brief skeletal muscle
ischaemia during the induction of RIPC stimulus provides powerful protection against
IR injury. Thus, it appears logical to assess the effects of RIPC on neutrophil function
and the role of the KKS, and more specifically kinin receptors, in neutrophil activation.
22 1.2 Remote ischaemic conditioning
The mechanism by which all living cells protect themselves from the lack of or an
excess of oxygen which occurs during ischaemia and reperfusion remains a mystery.
Paradoxically, it is reperfusion, rather than ischaemia, that causes major damage to the
tissues. There is, however, a powerful innate protective mechanism against IR injury
that has evolved in all mammalian species. Namely, brief transient episodes of
ischaemia protect against prolonged periods of lethal ischaemia. Prolonged lethal
ischaemia is often referred to as an index ischaemia. Although, the types of brief
transient ischaemia and their timing in relation to index ischaemia may vary greatly,
they appear to render significant protection. These observations have evolved into a
novel concept. My colleagues and I termed this newly defined concept as remote
ischaemic conditioning (RIC) (58). This refers to the global protection of the various
organs against prolonged lethal ischaemia following transient episodes of ischaemia-
reperfusion at a remote site induced before, during and after the index ischaemia. A
clinically applicable concept of ischaemic conditioning has evolved over the last 2
decades from the original description of the IPC. The evolution of the RIC concept is
described below.
1.2.1 The concept of ischaemic preconditioning
In 1984, in an isolated perfused rat heart, it was demonstrated that an initial 10-15 min
period of hypoxia led to better myocardial recovery following 30 min of ischaemic
challenge (59). Subsequently, Murry et al. in 1986 identified the concept of IPC in a
canine model. Four cycles of brief periods of ischaemia and reperfusion were followed
by 40 min of coronary artery occlusion (16). The size of myocardial infarct was reduced
by 75% in the treated animals. This protection was associated with reduced depletion of
high energy phosphates in the myocardium following prolonged ischaemia. Since then,
IPC has been discovered as a universal phenomenon across various species. Similarly,
preconditioning of isolated human atrial myocytes improves the recovery from
subsequent prolonged ischaemia (60). IPC also alleviates ventricular arrhythmias
associated with myocardial dysfunction (61-63). The duration and timing of the
preconditioning stimulus may influence the degree of protection (64-67).
IPC markedly reduces IR injury in most human tissues and has two phases. An early
(also known as classic or first window) IPC effect occurs within several minutes of the
preconditioning stimulus and lasts for approximately 6 h. A late (also known as delayed
or second window) IPC effect occurs within 24 h of the preconditioning stimulus and
23 lasts for up to 96 h. Unlike the early IPC, the late IPC protects the heart not only against
MI but also against reversible post-ischaemic myocardial stunning (68). Because of the
approximately 50-fold longer duration and the more powerful protection of late IPC,
considerable interest has been focused on the late or second window of protection (69).
Whilst the degree of protection against myocardial necrosis is similar, it appears that
protection against myocardial dysfunction is greater with the second window effect.
The studies to date examining the second window effect have been in animal models of
local ischaemia (70). Unfortunately, clinical application of the late phase of local
ischemic conditioning is impossible in most patients. Pharmacologic strategies have
been explored to mimic the powerful protection of the late phase.
1.2.2 Remote ischaemic pre-conditioning
The protection against IR injury by brief episodes of ischaemia at a remote site from the
target organ was first observed in 1993 by Przyklenk (17) and termed remote IPC.
Transient ischaemia of one coronary artery territory in a canine model was shown to
reduce the effects of subsequent potentially lethal ischaemia in the territory of another
coronary artery.
Following the initial identification of the concept of RIPC, the subsequent studies in
rodent models demonstrated that ischaemia of the kidney and intestine may induce
myocardial protection (71-72). Furthermore, a second window of remote protection of
myocardium can be induced in rats and rabbits by applying short periods of
preconditioning ischaemia to the small intestine (41, 54, 73-74). Although providing
proof of the principle, none of these studies has particular relevance to the protection
against IR injury in a clinical setting.
Transient ischaemia of skeletal muscle appears to be a potent preconditioning
stimulus in humans and larger animals (75-77). Four 5-min cycles of occlusion and
reperfusion of the hind limb in a porcine model resulted in significantly decreased size
of MI following subsequent coronary artery occlusion (78). The degree of protection
rendered by brief ischaemia of the arm or leg appears to be similar to local IPC.
Induction of the transient limb ischaemia is a non-invasive procedure and is clinically
relevant.
Significant protection against cardiopulmonary bypass (CPB)-induced tissue injury
has been demonstrated in a porcine model previously (77). The animals were subjected
to 3 h of CPB including 120 min of aortic cross clamping followed by reperfusion. The
parameters monitored were troponin I levels, load independent cardiac indices to assess
24 systolic and diastolic functions, and the measurement of pulmonary resistance and
compliance pre and post-bypass. RIPC was induced by four 5-min cycles of ischaemia
alternating with 5-min reperfusion prior to institution of CPB. The study found that
preconditioning significantly attenuated myocardial and pulmonary injury. Brief
transient limb ischaemia also decreased pulmonary leukocyte sequestration and
attenuated acute lung injury (47). Ischaemic preconditioning by transient limb
ischaemia was also demonstrated to enhance survival of flaps in experimental plastic
surgical procedures (79-81).
1.2.3 Remote ischaemic per-conditioning
In a porcine model, it was demonstrated that brief intermittent limb ischaemia also
provided significant protection during evolving MI (82) and, thus, the concept of
remote ischaemic perconditioning was identified. Four cycles of 5-min ischaemia
alternating with 5-min reperfusion were applied to a limb during the occlusion of left
anterior descending coronary artery for 40 min. This intermittent limb ischaemia
reduced the size of myocardial infarction, preserved global systolic and diastolic
functions, and protected the heart against arrhythmias during the myocardial reperfusion
phase. The process involved adenosine triphosphate dependent potassium (KATP)
channels (82).
1.2.4 Remote ischaemic post-conditioning
Kerendi et al. (83) demonstrated that transient episodes of renal ischaemia-reperfusion
at the end of a prolonged episode of myocardial ischaemia reduced the size of resultant
myocardial infarction. Subsequently, Andreka et al. (84) using a practical stimulus of
transient limb ischaemia applied after the induction of MI, found the protective effects
of remote ischaemic postconditioning. In an isolated rat heart model, Galagudza et al.
found that ischaemic postconditioning had an effective anti-arrhythmic action against
reperfusion-induced persistent ventricular fibrillation (85). Remote ischaemic
postconditioning has been demonstrated in humans and it appears to be as effective as
preconditioning (86). It appears that both forms of conditioning provide equally
effective protection of vascular endothelium. Thus, depending upon the timing of the
application of brief episodes of transient ischaemia, protective strategies have been
classified as pre-conditioning, per-conditioning or post-conditioning. A combination of
all three protective strategies during the different phases of ischaemia and reperfusion,
defined as RIC, may have an additive effect and may increase the degree of organ
25 protection. This beneficial effect may be related to the synergistic response from the
different mechanisms involved. RIC may be more applicable during the controlled
phases of IR injury as in myocardial revascularisation with CABG or PCI and during
organ transplantation. However, further clinical and experimental evidence will be
needed to confirm this postulation.
1.2.5 Functional response of human neutrophils to remote ischaemic preconditioning stimulus
It has been previously shown that transient limb ischaemia provides cardiac protection,
modifies coronary blood flow and resistance (87) and reduces myocardial IR injury
after heart transplantation in porcine models. Importantly, RIPC stimulus decreases the
expression of a portfolio of proinflammatory genes in circulating leukocytes in a human
model (46).
Neutrophils play a key role in IR injury (48, 88). Neutrophil-mediated tissue
damage is dependent on the number of neutrophils infiltrating the post-ischaemic tissue
via a process known as transendothelial migration. Neutrophil transendothelial
migration, in turn, is influenced by the ability of circulating neutrophils to adhere to the
damaged endothelium. Neutrophil adhesion, a crucial element of IR injury, is a two-
stage process of selectin-mediated loose adhesion and integrin-mediated firm adhesion.
One of the key integrins in the firm adhesion is the CD11b receptor. Expression of the
CD11b receptor is directly related to the extent of the post-operative IR injury (89-90).
Interestingly, in a previous experimental study, RIPC attenuated both endothelial
dysfunction (abnormal flow mediated dilation) and increased neutrophil CD11b
expression in humans subjected to 40 min of forearm ischaemia followed by reperfusion
(75). One of the aims of this thesis was to determine if the previously observed effect of
RIPC on neutrophil gene expression correlated with functional changes in the
leukocytes. I therefore examined the effect of repetitive transient human forearm
ischaemia on selected aspects of neutrophil function.
1.2.6 Expression of Kinin receptors on human neutrophils following remote ischemic preconditioning
Although the precise mechanism of RIPC induced organ protection has yet to be
elucidated, it appears that humoral or cellular factors produced in the limb in response
to local ischaemia induce systemic protection against IR injury (91). These humoral
factors significantly modify gene expression (46) and functional responses (92) in
human leukocytes and induce protective responses in remote organs via the adenosine
26 triphosphate dependent potassium (KATP) channels (93-94). It is now apparent that this
remote protection does not involve neuronal pathways but depends on humoral factors
(93-94).
Kinins have been implicated in RIPC mediated protection. Bradykinin (BK) binds to
specific G protein-coupled receptors (GPCR) - B1 and B2 that cause activation of
transduction pathways to mitochondria (95-98). A low dose of BK induces myocardial
protection and this protective effect is abolished by the administration of HOE 140, a
specific B2 receptor antagonist (74, 99). It appears that internalization of BK receptors
is essential for inducing protection after preconditioning (100-101). It has recently been
suggested that interaction of BK with B1 and B2 receptors induces the formation of
signalosomes that interact with mitochondria to open mitochondrial KATP channels
(102-103) (Figure 1.2-1). Neutrophils play a key role in affecting cellular damage in IR
injury (48, 88). We hypothesised that if the signalosome theory (102-103) was correct
and there is indeed internalization of kinin receptors within signalosomes, then RIPC
would significantly decrease the expression of B1 and B2 receptors on the surface of
circulating neutrophils. To date no studies have evaluated this hypothesis or the impact
of preconditioning on the expression of kinin receptors on human neutrophils.
1.2.7 Circulating factor of remote ischaemic conditioning
It appears that a circulating factor may be released following RIPC that prevents
neutrophil activation during IR and renders protection to an ischaemic organ following
reperfusion. RIPC of the recipient provided protection against myocardial IR injury in
the donor heart in a porcine model of orthotopic heart transplantation. This study
provided strong evidence of the existence of a protective factor in circulating blood
(93). It is possible that RIPC creates a protective milieu in the circulating blood that
decreases IR injury in the transplanted organ. Such an environment is likely to be the
result of decreased neutrophil activation. This discovery poses important questions.
What is the mechanism that renders circulating blood “protective”? Could the protection
of the RIPC be transferred across the blood-brain barrier (BBB) ?
27 1.2.8 Remote ischaemic preconditioning and protection against global cerebral
ischaemia
Cell death is the end result of IR injury. The cascade of events following ischemia can
evolve into cell necrosis or apoptosis. The intensity and duration of ischaemia may
determine the termination of these events into necrosis or apoptosis (104). Cell necrosis
results in the loss of cell membrane integrity that causes random deoxyribonucleic acid
(DNA) fragmentation in the nucleus (105). Damage to the cell membrane causes the
release of lysosomal enzymes that digest the cell material. Apoptosis on the other hand
is an energy requiring active process that maintains cell membrane integrity and causes
chromatin condensation and forms specific patterns of fragmentation. Apoptosis
represents IR injury (106). Apoptosis does not necessarily result from cell injury. It
occurs during physiological or pathological processes to eliminate potentially harmful
cells in the tissues.
A significant reduction in the size of cerebral infarction following transient limb
ischaemia has recently been demonstrated (107-109). Studies have previously
demonstrated the role of various modes of protection against both acute necrotic and
delayed apoptotic neuronal death (110-112). Although RIPC induced by transient limb
ischaemia protected against acute necrotic neuronal death, its effect on the delayed
apoptotic neuronal death remains unclear. Although the initial cerebral infarction size
may be decreased, however, delayed neuronal death could still continue to occur during
the first week following ischaemia.
1.2.9 Remote ischaemic preconditioning, neuroprotection and blood brain barrier
There is an indication that local preconditioning stimuli, like episodes of transient
ischaemic attacks (TIA), may be involved in the neuroprotective response against
further episodes of major neurological injury following stroke. This might be
responsible for the limited size of cerebral infarction seen in clinical practice (113-114).
Stroke and neurological injury following cardiac surgery constitute important areas
where the protective strategies may play an important role in preventing associated
morbidity and mortality. It is unclear, however, if RIPC can be applied in cerebral
protection. Recent work has focussed on the role of preconditioning in neuroprotection
given its vast potential applicability in clinical practice (115-119).
28
Figure 1.2-1. Diagram showing hypothetical signal transduction pathway of remote
ischaemic preconditioning from plasma membrane to mitochondria. Bradykinin (BK)
binds to B2 receptor and is incorporated into the cell as a signalosome. Downstream
signalling involves Phosphoinositide 3-kinase (PI3K), which activates the AKT/ERK
pathway and nitric oxide (NO) production by endothelial NO synthase (eNOS). NO
activates cyclic GMP-dependent protein kinase (PKG), which, in turn, leads to opening
of the mitochondrial ATP-dependent potassium (KATP) channel, reactive oxygen
species (ROS) production and inhibition of mitochondrial permeability transition pore
(MPTP), thereby providing protection against ischaemia-reperfusion injury.
29 BBB refers to the anatomic and physiological barrier between the blood and
cerebrospinal fluid (CSF) that controls the exchange of various factors between
systemic circulation and the central nervous system. This barrier protects the brain from
harmful stimuli. It consists of the tight junctions between neurovascular endothelium
and astrocytes. The astrocytes seem to be the key cells that regulate the function of BBB
following neurological injury (120-121). These cells provide protection against
ischaemia and are involved in the storage of glycogen needed for brain metabolism
(122). Neurons, glia cells and endothelium are all involved in the protective effects
against ischaemic injury.
A number of mechanisms are involved in preconditioning induced neuroprotection.
One such factor involves genomic expression that improves the tolerance of cells to
ischaemia (123-125). This response relies on the activation of transcription activator-
hypoxia inducible factor (HIF) (126). HIF has an α and β- subunit (127). This factor is
responsible for the activation of genes coding for the synthesis of erythropoietin,
angiogenesis, endothelial growth factors, vasomotor control and for cell metabolism
(127). During IPC, there is activation of HIF1 and the associated genes (128-131). RIPC
induced neuroprotection involves four mechanisms which include increased delivery of
substrate, reduced energy use due to metabolic downregulation, antagonised mechanims
of damage and improved recovery following ischaemia (132). These signalling cascades
involve a number of sensors, transducers and effectors.
Ischaemic tolerance following a stroke depends upon the preservation of
microvascular circulation and the maintenance of cerebral blood flow to the affected
area (133). This process causes the activation of genes involved in vasoregulation and
angiogenesis (134). Angiogenesis is involved in neuroregeneration following stroke and
depends upon activation of mediators regulated by HIF (135-136). IPC decreases the
extent of post-ischaemic cerebral oedema and hence may improve the cellular function
following neurological injury. This effect relies on the preservation of vascular
endothelium in brain (137). Preconditioning also activates the phosphoinositide 3 (PI3)-
Akt kinase pathway that inhibits post ischaemic apoptosis (138).
Maintenance of adequate glycogen content of the neurons during ischaemia
preserves cellular function and provides cellular protection. IPC in immature brains
increases the glycogen content of various cell types and delays energy depletion caused
by ischaemia (139). The effects of preconditioning seem to extend to the development
of tolerance to extracellular concentration of glutamate. Higher levels of glutamate are
neurotoxic (140). IPC may reduce the release or increase the uptake of glutamate (141).
30 Preconditioning also leads to the release of a number of cytokines that provide
cytoprotection.
The regenerative capacity of neurons has been identified in preliminary studies
following the recent discovery of neural stem cells. IPC promotes cell survival and the
differentiation of neural progenitor cells (142-144). Proliferation and differentiation of
progenitor cells is regulated by growth factors (145-146). A number of mechanisms
may be responsible for the protective effects of these growth factors in cell survival
following IPC. This may include anti-apoptotic and anti-inflammatory effects and the
capacity of these growth factors to cause neurorestoration.
1.2.10 Summary
A combination of various modes of ischaemic conditioning as applied at a remote
location is emerging into a novel concept of RIC. While this concept needs further
experimental and clinical evidence, it seems conceivable that a global organ protective
strategy can be instituted in different clinical scenarios following IR injury. A
combination of remotely located ischaemic stimuli may provide a powerful protective
response which may be more effective as compared to an individual method. While
ischaemic preconditioning has been applicable to almost all organs, the effects of this
phenomenon on neuroprotection have been less clear in the past. Recent work has
identified that the neurons are protected, to some degree, against the detrimental effects
of IR injury following preconditioning and these cells may even have the unique
capability to regenerate following ischaemic injury. Clinical extension of this work may
have significant impact on the protection of the brain following IR injury. This will
open up an entirely new field of remote neural ischaemic conditioning with potential for
new avenues of research and clinical application.
1.3 Mechanisms of remote ischaemic conditioning
Improvement of blood flow following preconditioning may only partially contribute to
organ protection by RIC. Recent studies have attempted to characterize global
molecular response to myocardial IR injury (147) and the RIPC stimulus (46, 148).
Although the precise mechanisms of RIC remain unknown, it is now becoming apparent
that all modes of such conditioning induce profound changes in gene expression and
cellular function, including mitochondrial adaptation to metabolic stress and leukocyte
activation.
31 1.3.1 Role of innate immunity
The mechanisms of IC are multifactorial and the exact interrelationship between the
various signals is not clearly defined. Adenosine, bacterial lipopolysaccharide (LPS),
BK, heat shock proteins (HSP), catecholamines, opioids, ROS, tumour necrosis factor
(TNF)-α, and other triggers may initiate the cascade of conditioning (65, 149-155) and
produce a preconditioning-like effect. It appears that a non-specific innate immunity
may be involved in IC and the key elements of the process are gene expression,
leukocytes, and mitochondria. Preconditioning with bacterial LPS, a component of the
cell wall, can stimulate a powerful anti-inflammatory response and suppress pro-
inflammatory pathways (156).
1.3.2 Role of gene expression
CPB induces a strong genomic response in rat myocardium (157). An early
modification of myocardial gene expression in response to intraoperative ischaemia-
reperfusion in patients undergoing cardiac surgery has been demonstrated (147). It has
been found in mice that transient limb IR modifies genomic responses in remote organs,
specifically, the expression of genes involved in myocardial response to inflammatory
or oxidative stress (148). Although transient limb ischaemia triggered an impressive
global genomic response, the expression of some individual genes was particularly
interesting in this study. For example, expression of an early growth response gene
1(Egr-1) was suppressed. Egr-1 is a master switch that activates the transcription of a
number of genes involved in the process of ischaemic tissue damage (158). Nuclear
factor kappa-B (NF-κB), also involved in preconditioning (159), can be activated via
multiple pathways including innate immunity pathways and a ubiquitous PI3K pathway
(41, 160-161). Changes in gene expression occur in both early and delayed phases of
RIPC (147). Similarly, Li et al. in a murine model demonstrated that delayed
cardioprotection induced by hind limb preconditioning involves signalling through
transcription factor NF-κB and iNOS. RIPC is abolished in mice with targeted deletions
for the p105 subunit of NF-κB or the iNOS (162). Thus, gene transcription appears
crucial for preconditioning (162) and non-specific inhibition of transcription by
actinomycin abolishes the protective effect of IPC (163).
1.3.3 Role of leukocytes
Transient forearm limb ischaemia as a preconditioning stimulus modifies gene
expression in circulating human leukocytes (46). These changes of gene expression
32 correlate with the early (first window) and delayed (second window) phases of RIPC. It
appears that RIPC suppresses leukocyte activation. Genes involved in leukocyte
chemotaxis, adhesion, migration, exocytosis as well as cytokine synthesis and innate
immunity signalling pathways are suppressed. This may, in part, explain previous
observations that brief transient limb ischaemia decreases pulmonary leukocyte
sequestration and attenuates acute lung injury (47). Indeed, innate immunity pathways
in leukocytes are non-specific and can be activated by bacterial LPS, HSP, hypoxia,
hyperoxia, nitric oxide, TNF- α, and many other non-specific stimuli. Many of them
may produce preconditioning-like myocardial protection (164-166). TNF-α plays an
important role in the post-ischaemic injury to various organs. Pre-treatment with TNF-α
results in reduction of IR injury and correlates with an increase in myocardial anti-
oxidant and manganese superoxide dismutase (MnSOD) activity (167-168) via the
activation of the NF-κB pathway, particularly in late preconditioning protection (159,
168-169). Initial TNF-α signalling pathway activation by IPC induces protective
MnSOD synthesis, but also suppresses gene expression responsible for subsequent
TNF-α synthesis and TNF-α signalling pathway restoration. TNF-α plays an important
role in leukocyte function and has been involved in ischaemic conditioning. Responses
to tissue injury by infection, ischaemia and trauma are remarkably similar. These
responses, regardless of the initiating causes may involve innate immunity. Activation
of the innate immunity pathways may, in turn, produce local or systemic inflammatory
response. One of the key players of the innate immunity pathways is toll-like receptor 4
(TLR4). TLR4 is involved in LPS-induced oxidative burst in neutrophils in response to
infection and in a similar response initiated by HSP 70 in IR injury (170-172). For
instance, induction of HSP by glutamine protects against IR injury of local and distant
organs (173).
Multiple stimuli that activate innate immunity pathways may initiate a
preconditioning-like response. This fact suggests that leukocytes play a central role in
preconditioning. The central role of leukocytes is also consistent with the observation of
significant myocardial protection in the donor heart after transplantation into a
preconditioned recipient (93). This study demonstrated that remote conditioning creates
a benign environment in the recipient that protects a denervated donor organ from IR
injury. Because the donor heart was not in the body when the recipient underwent a
transient limb ischaemia, it can be speculated that this protection involves a circulating
factor or suppression of leukocyte activation. Interestingly, the protection of the
transplanted heart was abolished by glibenclamide- a sulfonylurea that blocks KATP
33 channels. Thus, the mechanism of cardiac protection in the transplanted heart can be
abolished by the blockade of the KATP channels.
1.3.4 Role of signalling pathways
Preconditioning causes the release of a number of mediators that activate different
signalling pathways. These pathways require the activation of G-protein coupled
receptors. This leads to alteration in the activity of several important mitochondrial
proteins like KATP channels, mitochondrial permeability transition pore (MPTP) and
components of the bcl-2 family. These processes cause altered cellular metabolism and
help the cells develop resistance against apoptosis.
BK is an important local mediator of preconditioning. There is activation of a
number of kinases following the interaction of BK with its receptors on the cell surface.
These include phosphoinositide-3-kinase (PI3K), protein kinase B (Akt), extracellular
signal-regulated kinases (ERK) and protein kinase G (PKG). Nitric oxide (NO) is also
released during this process. The end effector of these processes is mitochondria. The
final steps in this pathway involve the opening of mitochondrial KATP channels that
causes the generation of ROS. The BK mediated pathway involves an increase in Bcl-2
associated death domain protein (Bad) phosphorylation and inhibition of caspase 3,
thereby making the cells more resistant to hypoxia (174).
Activated PI3K is involved in the protective effects of preconditioning. Agents such
as Wortmannin and LY294002 that inhibit the PI3K pathway also inhibit the effects of
preconditioning (175-176). Hydrogen peroxide has also been found to be responsible
for the activation of PI3K and hence ROS may have a role in the activation of this
pathway in preconditioning (177).
Protein kinase C (PKC) is another important mediator of the preconditioning effect.
Transgenic mice with cardiac specific PKC or with overexpression of PKC activator
provide endogenous protection against ischemic cardiac injury (178-179). Following the
generation of ROS during IPC, there is activation of PKC (180). It seems that PKC is an
intermediate enzyme in the signaling pathway inhibiting MPTP. PKCε, an isoform of
the enzyme also inhibits Bad which has an antiapoptotic effect (181).
NO is produced in the cells following the application of preconditioning stimulus.
This leads to further activation of PKCε, tyrosine kinase and the NF-κB mediated
signalling pathways (42-43).
There may be an overlap in the activity of the local and remote preconditioning
pathways. It is possible that the local mediators of preconditioning such as
34 acetylcholine, adenosine and BKs are released into the circulation and activate the same
signalling pathways that are activated locally (182).
1.3.5 Role of mitochondria
It seems that the major signalling pathways mediating cell protection following
preconditioning have mitochondria as their end effectors. They have key roles in the
inhibition of cell death by both necrosis and apoptosis. Mitochondria are important in
maintaining the integrity of the cell membrane that is vital for the prevention of cell
necrosis. The opening of mitochondrial KATP channels is crucial to all modes of
ischaemic conditioning. Although both sarcolemmal and mitochondrial KATP channels
appear to be involved, it is the mitochondrial channels that are sine qua non of the
preconditioning effect. It was observed that selective mitochondrial KATP channel
inhibition abolished the cardioprotective effects of both local and remote conditioning
(79, 82, 183). Interestingly, the soluble N-ethylmaleimide-sensitive factor attachment
protein receptor (SNARE) mechanism is not only a key element of exocytosis, but may
also be involved in blocking KATP channels via the sulfonylurea receptor by syntaxin
(184-185). The previous observation of decreased gene expression encoding SNARE
proteins by RIPC (46) may, in part, explain the contributions to functional preservation
of KATP channels. The precise molecular mechanism by which opening of these
channels provides protection is unknown. It is plausible that the opening of the KATP
channels in the target organ prior to or immediately after sustained ischemia as a result
of transient limb ischemia reduces the rate of ATP hydrolysis (186) or mitochondrial
ATPase activity (187-188), thereby decreasing the rate of ATP depletion during
reperfusion. There are other possible mechanisms that are activated by these KATP
channels including the inhibition of mitochondrial calcium uptake, regulation of
mitochondrial volume and modulation of ROS.
1.3.6 Summary
RIC is a complex phenomenon that involves cellular protection against the damaging
effects of ischaemia and reperfusion. There is an interplay of various factors including
components of innate immunity; inhibition of gene expression of key mediators of IR
injury including cytokines, leukocyte activation, innate immunity pathway and
apoptosis; leukocytes and a number of signalling pathways. This process starts from the
plasma membrane and terminates on the mitochondria. The final events involve the
opening of mitochondrial KATP channels which facilitate the preservation of ATPs in
35 the cell and inhibit mitochondrial permeability transition (MPT) pore. This creates a
milieu that protects the ischaemic cells from death.
1.4 Kallikrein-kinin system
KKS plays an important role in activating inflammation, causing neoplasia and
development of IR injury. Furthermore, it is now becoming apparent that KKS may play
a central role in organ protection against IR injury. KKS is ubiquitously involved in the
renin-angiotensin system, the coagulation cascade and the complement activation
pathways. Kinins are formed by plasma and tissue kallikreins. Kallikreins convert
kininogens into vasoactive kinin peptides- BK and lys-bradykinin (lys-BK). There are
two types of kininogens, high and low molecular weight (HK and LK). Plasma
kallikrein is present in hepatocytes and in a number of epithelial and endocrine cells.
Tissue kallikrein is present in endothelium, endocrine cells and in neutrophils. Kinins
play an important role in vascular smooth muscle contraction, dilatation of arterioles,
capillary permeability and also interact with sensory nerve terminal transmitters in pain
response. Kinins act via B1 and B2 receptors (Figure 1.4-1).
Kallikrein
There are two pathways of kinin production, the plasma and tissue kallikrein pathways.
Kallikreins are produced by a family of three genes located on chromosome 19.
Plasma kallikrein-kinin
Plasma kallikrein is a serine protease and is synthesised in hepatocytes as an
inactive molecule called prekallikrein. It circulates in plasma bound to HK. Nearly 80-
90 % of plasma prekallikrein is found as a complex with HK (189-191). The contact of
plasma with a negatively charged surface, as occurs during cardiopulmonary bypass
(CPB) leads to the binding and activation of factor XII (Hageman factor), activation of
prekallikrein to kallikrein by activated factor XII and cleavage of HK by kallikrein to
produce BK (192) (Figure 1.4-1). Factor XII initiates the intrinsic pathway of
coagulation as well as the complement pathway (193). HK binds to platelets,
granulocytes and endothelial cells. Binding of HK to endothelial cells leads to activation
of pre-kallikrein to kallikrein and possibly to the release of BK (193-197).
36
Figure 1.4-1. Association of kallikrein-kinin system with renin angiotensin aldosterone
system.
Tissue kallikrein-kinin
Tissue kallikrein is an acid glycoprotein which differs from plasma kallikrein. It is
widely distributed in the kidney, blood vessels, central nervous system, pancreas, gut,
salivary glands, spleen, adrenal and in neutrophils (191, 198). Tissue Kallikrein is
synthesised as a proenzyme called pro-kallikrein which is activated by plasmin or
plasma kallikrein (199). Tissue kallikrein releases lys-BK from LK (200). The main
substrate of tissue kallikrein is LK, however, it is also capable of cleaving HK and
producing BK (198). In addition to tissue and plasma kallikrein, other serum and tissue
proteases can produce kinins (200).
Kinins
Kinins play a central role in inflammatory response. Kinins are a group of closely
related proteins that include a nonapeptide, BK, decapeptide, lys-BK (also called
37 kallidin) and their carboxy-terminal des-Arg metabolites. Lys-BK can be converted to
BK by aminopeptidases. Kinins cause contraction of visceral smooth muscle cells,
vasodilatation (Figure 1.4-1) by promoting the release of nitric oxide (NO) from
vascular endothelium, increased vascular permeability and chemotaxis of leukocytes.
Kinins lower blood pressure. They are formed during active secretion in sweat glands,
salivary glands, and the exocrine portion of the pancreas. Their vasodilatory action
increases the local blood flow when these tissues are actively secreting their products.
Kininogens
As mentioned above, there are two types of kininogens, high molecular weight
kininogen, H-kininogen (HK) and low molecular weight kininogen, L- kininogen (LK).
The kininogen molecule consists of an amino-acid-terminal heavy chain and a carboxy-
terminal light chain with the kinin moiety interleaved between the two domains.
Kininogens are formed by alternative splicing of a single gene located on chromosome
3. The kininogens are present in the extracellular fluids and have been localised on
human neutrophils, platelets, endothelial cells and the collecting duct of kidneys.
Kininases
Kinin levels depend upon their rate of production and rate of metabolism by a group of
enzymes called kininases. Kinins are destroyed in extracellular fluids, in the circulation
and within cells by two enzymes. Kininase I is a carboxypeptidase which metabolises
kinins by removing carboxy terminal arginine. Kininase II metabolises kinins by
removing Phe-Arg from carboxy terminal and is the same enzyme as angiotensin
converting enzyme that inactivates angiotensin I.
Kinin receptors
There are two types of kinin receptors, B1 and B2. These are the members of a
superfamily of G-protein-coupled rhodopsin-like receptors characterized by seven
transmembrane regions connected by three extracellular and three intracellular loops
which are linked to second messenger signalling systems. The various effects of kinins,
e.g., vasodilatation, increased vascular permeability, stimulation of sensory and
sympathetic nervous systems and smooth muscle contraction are due to the effects of
kinins on the B1 and B2 receptors. These receptors are located on vascular endothelium,
sensory afferent neurons, smooth muscle cells and epithelial cells.
38 B1 receptors
The B1 receptor is rarely expressed in normal tissues. These receptors are involved
in KKS mediated inflammatory response. B1 receptors are rapidly up-regulated during
inflammation and following exposure to bacterial endotoxins and lipopolysaccharides.
There is an increase in the number of B1 binding sites in inflamed tissues, carcinomas,
rheumatoid arthritis, transplant rejection and glomerulonephritis (191, 201-202). The
physiological activity of B1 receptors is regulated by des-Arg9-BK and des-Arg10-
kallidin.
B2 receptors
B2 receptors are present in most of the tissues. These receptors are activated by BK
and lys-BK. These receptors are responsible for causing the majority of kinin mediated
effects. Activation of these receptors has been implicated in hypotension, bronchospasm
and the development of oedema. B2 receptors are involved in the angiotensin
converting enzyme (ACE)-induced prevention of cardiac remodelling following AMI
(203).
1.4.1 Kallikrein-kinin system and renin-angiotensin-aldosterone system
Renin is an enzyme secreted by juxtaglomerular (JG) cells located in the afferent
arteriole of glomerulus. Angiotensinogen is synthesized in liver and circulates in
plasma. Renin acts on angiotensinogen to form angiotensin I. ACE (same as Kininase
II) converts angiotensin I to angiotensin II, which produces powerful vasoconstriction.
This can causes significant rise in systolic and diastolic blood pressure. The angiotensin
converting enzyme inhibitors (ACEI)-mediated antihypertensive effect involves reduced
production of angiotensin II and increase in the levels of BK (Figure 1.4-2).
39
Figure 1.4-2. A schematic of the components of kallikrein kinin system
1.4.2 Kallikrein-kinin system and inflammation
Tissue injury, ischaemia or infections initiate chemotactic migration of neutrophils that
produce the beneficial and harmful effects of inflammation. There is a rapid generation
of kinins following tissue injury. Kinins produce vasodilatation, increase capillary
permeability, cause chemotaxis and produce the associated pain (204, 205). Both kinin
receptors seem to be involved in inflammation.
A high level of B1 receptor endogenous agonists has an important role in causing
an increased expression of B1 receptors (206). Cytokines can cause the rapid induction
of B1 receptors (207-208). Activation of B1 receptors in an area of inflammation leads
to chemotaxis of neutrophils, an effect that can be abolished with the use of B1 receptor
antagonists (209). There is no neutrophil chemotactic response in B1 receptor knockout
mice (210). Stimulation of B2 receptors causes the activation of arachidonic acid-
prostaglandin pathway. This in turn causes the release of c-AMP as a secondary
40 mediator. Prostaglandins are important mediators of pain. BK also causes the release of
NO following the activation of endothelial cells and cellular components of
inflammation (205, 211-213). These signalling pathways seem to be involved in BK
mediated IL-1β production induced by TNF-α (214). BK can also induce the activation
of NF-κB through B2 receptors and can cause the IL-1β gene expression in cultured
human epithelial cells (215). A number of cytokines, including IL-1β, TNF-α, IL-2 and
IL-8, can cause upregulation of B1 receptors (216). Hence, it seems that there is a close
association between the various components of inflammation and KKS pathway.
1.4.3 Kallikrein-kinin system and cardiovascular system
KKS is directly involved in a number of physiological and pathophysiological processes
involving the cardiovascular system that include hypertension, left ventricular
hypertrophy, cardiac failure and myocardial ischaemia (217-225). Hypertension,
myocardial ischaemia and myocardial hypertrophy are associated with a low activity of
the KKS pathway and up regulation of B1 and B2 receptors. Local and systemic
administration of BK can increase coronary blood flow and improve myocardial
metabolism (219).
Hypertension is a common cardiovascular risk factor in the general population and
is also a common co-morbid condition in patients undergoing major surgery including
cardiovascular surgery. BK regulates blood pressure by vasodilatation. This effect
involves reduction of systemic vascular resistance, diuresis and sodium excretion by
kidneys (218, 221, 225). Kinins are also released during myocardial ischaemia (217,
222). Direct infusion of BK into the coronary artery in a canine model of myocardial
ischaemia, reduced the severity of arrhythmias induced by ischaemia (222).
The role of KKS in cardiac protection against ischaemic injury has been identified
in the studies exploring the association of angiotensin II and BK in the ischaemic
myocardium (223). Cardiac protection from angiotensin converting enzyme (ACE)
inhibitors has been linked to BK (Figure 1.4-1). The use of HOE 140, a BK2 receptor
antagonist reversed the myocardial protection induced by BK (224). BK also releases
tissue plasminogen activator and hence might be involved in intrinsic protection against
myocardial ischaemia and infarction by causing local fibrinolysis (220).
In an experimental B2 knockout mouse model, the animals developed hypertension,
left ventricular hypertrophy and cardiac failure. This suggests that an intact KKS is
required for the maintenance of myocardial architecture and function (226). In another
murine model of AMI, there was evidence to suggest the role of TK in promoting
41 myocardial neovascularisation with restoration of regional blood flow and improved
cardiac function (56).
IPC has been studied in experimental and clinical settings in protecting an organ
against IR injury. The cardioprotective effect of local IPC was diminished or abolished
in a rat model lacking the gene coding for B2 kinin receptor (B2 knockout mice) as well
as in rats deficient in HK (227). Hence an intact KKS is also required for the beneficial
effects of IPC. The role of KKS was further explored in a rabbit model of AMI. Use of
HOE 140, a B2 receptor blocker, abolished the beneficial effect of IPC in causing
reduction in the infarct size (228).
Clinical studies have identified the role of BK in IPC. Intracoronary infusion of BK
in patients undergoing PCI provided an effective preconditioning and attenuated
myocardial ischaemia during coronary occlusion from intra coronary balloon inflation
(57). In another study, use of BK preoperatively in patients undergoing standard CABG
using CPB and aortic cross clamping demonstrated less myocardial ischaemia in
comparison to controls (229). In a canine model using a microdialysis technique, Pan
and co-workers demonstrated an increase in the release of BK in myocardial interstitial
space following preconditioning (230).
Unfortunately, clinical application of IPC is limited and not practical in the majority
of clinical settings. Animal studies have found that RIPC can provide cardiac protection
by inducing renal or intestinal ischaemia (72, 231). The cardioprotective effect of
mesenteric ischaemia was related to the release of BK and it could be blocked by using
HOE-140 (Hoechst-140), a BK2 receptor antagonist (99). It appears that activation of
kinin receptors results in internalisation of these receptors within the signalosome to
facilitate protective pathways (102-103).
1.4.4 Kallikrein-kinin system in neoplasia
Tissue kallikrein and plasma kallikrein are distributed in a wide variety of cells through
out the body as a part of KKS. A link between neoplasia and KKS has previously been
demonstrated (232-234). Kinins acting via B1 and B2 receptors cause proliferation and
migration of cells (191, 235). Expression and activation of the various components of
KKS seems to be important in the growth of malignant tumours. Using prostate tissue
from patients with adenocarcinoma, Taub and co-workers stimulated B1 receptors of
carcinoma cells and found an increased growth, migration and invasion of the cells
(236). Similarly in vitro stimulation of B2 receptors caused an increased growth of
breast cancer cells (237). Tissue kallikrein is also involved in local and distant
42 metastasis of breast cancer (238). The ability of malignant tumours to stimulate
neovascularisation is a unique property. This promotes the growth of rapidly
proliferating cells and provides local nourishment. A B2 receptor antagonist suppressed
tumour growth as well as angiogenesis in a murine model of sarcoma (239).
1.4.5 Common signalling pathways in neoplasia and protection against ischaemia-reperfusion injury
PI3K pathway is involved in the development and growth of malignant cells and
facilitates the resistance of cancer cells to apoptosis. Inhibition of epidermal growth
factor receptors caused apoptosis in mesothelioma cell lines (240). This effect was
related to the downregulation of the PI3K signalling pathway. Protection of
cardiomyocytes exposed to lethal ischaemia following IPC is also via PI3K pathway
(241). Cellular protection from BK is dependent on the opening of the mitochondrial
KATP channels and production of ROS which then act as second messengers to activate
protein kinase C (PKC) (242). The interaction of BK with the receptors at the cell
membrane leads to the formation of signalosomes that transport the enzymes involved
in this signalling pathway to mitochondria (103). The final steps in protection from
preconditioning involve the opening of mitochondrial KATP channels (183) that lead to
inhibition of MPTP (Figure 1.2-1). A number of stimuli including HSP, bacterial LPS
and TNF can activate the PI3K pathway (243). In an isolated and perfused murine heart
model, it was demonstrated that BK reduced the size of MI and this protection was
dependent upon PI3K, Akt and endothelial nitric oxide synthase (eNOS) activation
(244). Similarly, BK induced preconditioning of rabbit heart protected the myocardium
against apoptosis with increased Bad phosphorylation and inhibited caspase 3 activation
(174). Bad and caspase 3 are involved in programming cell apoptosis. Hence BK is one
of several important mediators that activate the “survival kinase cascade”.
There appears to be a similarity between the signalling pathways responsible for cell
protective response following RIPC and the anti-apoptotic ability of malignant cells. I
am not aware of any information on the potential of preconditioning in causing
oncogenesis. This has certainly not been detected in the limited clinical studies and the
experimental work done so far. One way of analysing this problem will be doing long-
term preconditioning studies in animal models of tumours and studying the possible
effects of preconditioning on tumour growth or invasion. This also raises the question as
to what is the safe period of preconditioning. I think this discussion highlights the fact
43 that there are several issues in RIPC that need to be understood before this concept can
be applied widely into clinical practice.
Future clinical strategies may rely on KKS and BK for myocardial protection in IR
injury from AMI and cardiac surgery. It also seems conceivable that the treatment of
neoplasms in future clinical trials will be studied using a targeted approach to the
blockade of BK receptors in tumours. This may provide an entirely new approach to the
management of two major global health issues- cancer and cardiovascular diseases.
1.4.6 Kallikrein-kinin system and aprotinin
Royston and co-workers in 1987 accidentally discovered that high-dose aprotinin could
be used to reduce bleeding in patients undergoing cardiac surgery (190). Aprotinin is a
non-specific serine protease inhibitor that is derived from bovine lung. Being a serine
protease inhibitor, aprotinin inhibited plasmin as well as both plasma and tissue
kallikreins. The lysine residue at position 15 in the aprotinin molecule binds to the
active serine residue in proteases and forms an inactive complex. The anti-inflammatory
effects of aproprotin are due to its inhibitory effect on the KKS.
Aprotinin has been widely used in high-risk cardiac surgery patients to reduce
bleeding and decrease systemic inflammatory response until recently. Exposure of
blood to extracorporeal circulation causes activation of the intrinsic pathway of
coagulation, fibrinolysis, KKS and complement system. Activation of these cascades
produces systemic inflammatory response. Kinins have a close relationship to the
coagulation cascade; they activate factor XII and stimulate fibrinolysis via plasminogen
activators (245-246). Plasmin mediated fibrinolysis is suppressed by aprotinin as found
in the lower levels of fibrin degradation and d-dimer products in patients treated with
aprotinin during cardiac surgery (246-247). However, due to the controversy
surrounding an apparent increase in the number of thrombotic complications (248-250),
aprotinin has been withdrawn from the market. A thorough knowledge of KKS and its
protective role against IR injury would be beneficial in avoiding future pitfalls and
allow utilization of the full benefits of the native protective pathways.
1.4.7 Summary
KKS plays an important role in a number of pathophysiological processes. It is possible
that the observed increase in myocardial infarction and stroke with aprotinin use was at
least in part due to the inhibition of KKS and, thus, led to the loss of protective effects
44 of kinins against IR injury. Selective modulation of KKS may be useful in developing
protection against IR injury in a number of clinical situations and in treating some
malignancies.
1.5 Rationale, hypotheses and objectives
1.5.1 Rationale
IR injury is central to the pathophysiology of a number of cardiovascular disorders and
can be responsible for the suboptimal clinical results in patients undergoing cardiac
surgery and organ transplantation. RIPC is emerging as a novel strategy of organ
protection and is supported by the findings of experimental work and preliminary
clinical studies. Further expansion of this concept to per- and post conditioning of a
target organ with a remotely located conditioning stimulus may evolve into RIC.
Further work needs to be done in this area to apply this concept to wider clinical
practice. The present work is based on studies of the functional changes in human
neutrophils following RIPC, the impact of RIPC on the expression of kinin receptors in
human neutrophils and exploration of the effects of the second window of
preconditioning in a murine model subjected to global cerebral ischaemia.
1.5.2 Hypotheses
It is hypothesised that:
1. RIPC causes functional changes in human neutrophils following RIPC that
attenuate the inflammatory response associated with these cells following IR
injury.
2. Kinins play a central role in the RIPC. They activate kinin receptors on the
plasma membrane of human neutrophils and these complexes internalize into
the cytoplasm. This event initiates a cascade that results in cell protection.
3. The delayed phase of the RIPC may not prevent the delayed death of
hippocampal neurons due to apoptosis following global cerebral ischaemia due
to the blood brain barrier.
1.5.3 Objectives
In order to address these hypotheses, the RIPC stimulus was applied to the arm of
human volunteers and to the hind limb of rats. Blood samples were drawn from the
subjects and neutrophils were isolated to study the effects of RIPC in humans. The rat
45 brains were studied to determine the effect of the RIPC on hippocampal neurons. The
following objectives were met during our work:
1. Assessment of the functional changes in human neutrophils following RIPC.
2. Assessment of kinin receptors on the surface of human neutrophils following
RIPC.
3. Assessment of the effects of RIPC on hippocampal cell count in rats subjected
to global cerebral ischaemia.
46
CHAPTER 2
MATERIAL AND
METHODS
47 2.1 Human model of remote ischaemic preconditioning
The forearm was made ischaemic by inflating blood pressure cuff to above systolic
blood pressure for three 5-min periods, separated by 5-min of reperfusion. This protocol
was carried out once (for kallikrein kinin study) and daily for 10 days (neutrophil
function study). Blood flow interruption and restoration was monitored using a standard
pulse-oximeter of the same arm. Venous blood was drawn from the contralateral arm.
Samples were collected in standard sterile tubes with EDTA anticoagulant (Vacutainer;
Preanalytical Solutions, Franklin Lakes, NJ) and transported on ice for immediate
assessment.
The development of protocols for RIPC can be traced back to the protocols used for
IPC in various experimental and clinical studies. The basic concept behind the duration
of cycles of ischaemia and reperfusion is to minimise the duration of adverse effects
associated with ischaemia. However, the duration should be long enough to stimulate a
preconditioning response. During PCI, balloon inflation lasting more than 90 s can
induce myocardial preconditioning but this effect does not occur if the inflation time is
between 60-90 s (251-253). There seems to be no correlation between the degree of
stunning following the application of a preconditioning protocol and the resultant
protection achieved (254). On a similar note, IPC cannot be minimised to avoid the
adverse effects of ischaemia without compromising the degree of protection obtained
(255). The protocol used in the present study was based on a standard protocol
developed in previous studies (46, 75). This protocol was preferred in view of the
documented benefits. Currently, there does not seem to be any consensus on the best
protocol for instituting RIPC (256).
2.2 Rat model of remote ischaemic preconditioning
Animals were anaesthetised with 3% halothane in N2O:O2 (2:1) and maintained at 2%
halothane in the same gas mixture. The rats were ventilated using a rodent ventilator
(Ugo, Basile, Italy), initially at 1ml stroke volume and 90 bpm stroke rate and
positioned supine on a heating pad. A pulse-oximeter was applied to the left hind foot
and local pressure was applied circumferentially to the mid-femur in order to occlude
the femoral artery. Enough pressure was applied to keep the artery occluded during the
period of ischaemia. Occlusion was maintained for a period of 5 minutes, followed by 5
minutes of reperfusion. This cycle was performed 5 times. Interruption and restoration
of blood flow to the limb was confirmed each time by pulse-oximetry, visible cyanosis
and hyperaemia of the limb.
48
2.3 Rat model of global cerebral ischaemia
Transient global cerebral ischaemia was induced 24 h later after initial preconditioning
or sham procedure. Bilateral femoral artery cannulation was carried out using
polyethylene tubing (PE-50) filled with 50 units/ ml of heparinised saline solution. The
right femoral artery was cannulated for blood pressure measurement and the left femoral
artery was cannulated for arterial blood gas analysis and withdrawal of blood to induce
hypotension. Both common carotid arteries were exposed via a ventral midline neck
incision and silk thread with silastic tubing (Dow Corning, Auburn, MI) was loosely
applied. Prior to ischaemia, blood gases were analysed and ventilatory parameters were
adjusted where necessary to ensure that arterial pCO2 at the commencement of
ischaemia was 40 ± 2 mmHg and pO2 was >100 mmHg. Blood glucose levels were also
recorded.
A bipolar electroencephalogram (EEG) was recorded with two active lateral
electrodes and a reference central scalp electrode, which were interfaced with a
bioamplifier (AD instruments, Melbourne, Australia). Global ischaemia was induced by
bilateral common carotid artery occlusion by securing the ligatures accompanied by
exsanguination to maintain arterial BP between 35 and 40 mmHg. GCI time was 8 min
from the moment EEG became isoelectric. The withdrawn blood was then reinfused and
carotid ligatures removed.
2.4 Assessment of functional responses of human leukocytes to remote ischaemic preconditioning stimulus
2.4.1 Experimental design
A longitudinal study using the RIPC protocol as described was carried out in 5 healthy
adult male volunteers (See 2.1). The study was approved by the institutional ethics
committee. Venous blood was drawn from the contralateral arm prior to the ischaemic
stimulus (day 0) and on days 1 and 10 after the stimulus.
2.4.2 Isolation of human neutrophils
Neutrophils were isolated from the whole blood using dextran sedimentation and
discontinuous plasma-Percoll gradients (Amersham Biosciences, Upsala, Sweden) as
described previously (257). The separation procedure was complete within 2 h and the
cells were used immediately after isolation for the experiments described.
49
2.4.3 Adhesion
Adhesion of neutrophils was measured as the percentage (%) of cells that adhered to
tissue culture wells coated with foetal bovine serum. Surface expression of CD11b
(Mac-1), a pivotal adhesion molecule, was assessed by measuring fluorescence intensity
of neutrophils labelled with FITC-conjugated anti-CD11b monoclonal antibodies
(Serotec, Oxford, UK) as described previously (75).
2.4.4 Secretion of cytokines
Secretion of primary and secondary granule contents (exocytosis) was assessed by flow
cytometry measuring surface expression of CD63 and CD66b respectively using FITC-
conjugated antibodies (Serotec, Oxford, UK). Secretion of the cytokines TNF-α, IL-1β,
IL-6, and IL-10 were measured using a multiplex fluorescent bead assay (LINCOplex,
LINCO) using a Luminex in response to stimulation with LPS (100ng/ml) (SIGMA) or
vehicle control for 6 h and 24 h at 37°C.
2.4.5 Apoptosis
Apoptosis was assessed using a combination of propidium iodide and annexin V-FITC
(R&D systems, Minneapolis, MN) fluorescence staining with quantification by flow
cytometry as previously described (258).
2.4.6 Oxidant production
Oxidant production (NADPH oxidase) by neutrophils was assessed with flow cytometry
using the oxidant-sensitive fluorescent dye dihydrorhodamine (DHR) 123 as previously
described (259-260). 5x105 cells in suspension were incubated in the presence of 2 µM
DHR for 20 min at 37°C. The cells were fixed with 1.5% paraformaldehyde before
analysis on a FACScan flow cytometer (Becton Dickinson). Peripheral blood
neutrophils were pretreated with cytochalasin B (5 μM) for 10 min followed by
exposure to N-formyl-methionyl-leucyl phenylalanine (FMLP 10-7M) for an additional
10 min. The fluorescence of the cell associated reduction product, rhodamine 1-2-3, was
evaluated by flow cytometry as a measure of oxidant production. FMLP activates
neutrophils by chemotaxis and granule enzyme secretions. Cytochalasin B augments the
response of FMLP stimulted neutrophils.
50 2.4.7 Phagocytosis
IgG-coated prey was constructed as described by Vachon et al. (261). Briefly, 100 µl of
sheep erythrocytes 10% solution (Cappel, West Chester, PA) was washed twice in PBS,
incubated with 2 µl of rabbit anti sheep erythrocyte IgG (INC55806, Cappel) for 1 h,
and washed twice in PBS. Neutrophils were washed twice with Hanks buffered salt
solution followed by addition of the phagocytic prey at a ratio of 20/1 and allowed to
interact and bind to the neutrophils for 5 min at 37ºC. The cells were washed to remove
unbound prey and incubated at 37ºC for an additional 15 min to allow phagocytosis to
proceed. The assays were terminated by cooling the cells by washing with ice cold PBS
without calcium and magnesium. Following incubation, hypotonic lysis of the
extracellular erythrocytes was achieved by addition of water for 30 s, followed by
immediate replacement with calcium and magnesium-free PBS. The coverslips were
mounted on Attofluor® cell chambers (Invitrogen Canada, Inc.) and quantification of
phagocytosis conducted using an inverted microscope (Leica DM-IRB, Wetzler,
Germany).
2.4.8 Statistical analysis
Data were analyzed using a paired t-test for comparison between two conditions on a
same sample, and repeated measure of Analysis of Variance (ANOVA) with post hoc
analysis by Student Newman-Keuls multiple comparison test, or two way ANOVA
using GraphPad Instat or Prism VI (Graphpad Inc., La Jolla, CA) as appropriate for
comparison over the course with three time points (day 0, day 1, and day 10). Statistical
significance was considered for p values of <0.05.
2.5 Assessment of human kallikrein-kinin system response to remote ischaemic preconditioning stimulus
2.5.1 Experimental design
Five healthy male volunteers (mean age 49.6 years, range 38-55 years) who were not on
any medications were enrolled in the study. The study was approved by the institutional
ethics committee. RIPC was carried out according to the previously described protocol
(Section 2.1). Venous blood samples were drawn from the contralateral arm at baseline,
15 min and 24 h following preconditioning.
51 2.5.2 Isolation of human neutrophils
Blood was anticoagulated with 3.8% (weight by volume, w/v) sodium citrate and mixed
with an equal volume of Hanks’ balanced salt solution (HBSS; pH 7.1). The diluted
blood (20 ml) was overlaid on Percoll (density 1.088; pH 7.4; GE Healthcare
Biosciences, Sydney, Australia) and centrifuged at 1000 g for 30 min. Plasma and
lymphocytes were removed and erythrocytes were lysed by resuspension in 20 ml of
ice-cold water for 30s, followed by 20 ml of 2x PIPES buffer (pH 7.4). The erythrocyte
lysis step was repeated and the neutrophils were finally resuspended in HBSS at a
concentration of 2 x 10-6/ ml. Neutrophils were pipetted onto poly-L-lysine coated slides
and fixed (acetone-methanol 1:1, vol/ vol) before immunolabelling.
2.5.3 Immunoperoxidase labelling
The slides were rehydrated in 0.01M phosphate buffered saline (PBS, pH 7.4) and
excess peroxidase activity was inhibited with peroxidase block (Dako, Sydney,
Australia) for 5 min. Non-specific protein binding was blocked with 10% human serum,
20% swine serum and serum-free protein block (Dako, Sydney, Australia) for 15 min
each. Slides were then incubated with one of the following antibodies at a dilution of
1/100 in 0.01 M PBS containing 1% bovine serum albumin (BSA) for 3 h at room
temperature: TK and kininogen (HK) (Abcam, Cambridge, UK), PK, kinin B1 receptor
and kinin B2 receptors. The slides were washed three times (0.01 M PBS, pH 7.4) and
incubated with anti-rabbit (for TK, B1 and B2) or anti-mouse (for PK and HK)
horseradish peroxidase conjugated polymer (Dako, Sydney, Australia) for 30 min at
room temperature. After washing three times (0.01 M PBS, pH 7.4) labelling was
visualized by incubating the slides with 3,3’-diaminobenzidine (DAB), (Dako, Sydney,
Australia) and counter staining with Mayer’s haematoxylin. The specificity of
immunolabelling was verified by negative controls in which the primary antibody was
omitted. A minimum of 200 cells were counted and the number of positively labelled
neutrophils was expressed as a percentage of the total number counted.
2.5.4 Immunofluorescence labelling
Slides were rehydrated, non-specific binding was blocked and incubation was
performed with TK, PK, B1 and B2 antibodies as described for immunoperoxidase
labelling. After washing three times (0.01 M PBS, pH 7.4) slides were incubated for 30
min at room temperature with Alexa-Fluor 488 conjugated goat anti-rabbit IgG
(Invitrogen, Melbourne, Australia) for TK, B1 and B2 or goat anti-mouse IgG for PK.
52 After two washings (0.01 M PBS, pH 7.4) nuclei were stained with Hoechst 33342
(Sigma Chemical Co., St Louis, MO). The specificity of immunolabelling was again
verified by negative controls in which the primary antibody was omitted.
2.5.5 Confocal microscopy and image analysis
Slides were viewed on a Bio-Rad MRC 1000/1024 UV laser scanning confocal
microscope (Bio-Rad, Hercules, CA) and five random fields of view were captured for
analysis. The digitized images were analysed using Image-Pro software. Mean pixel
intensity data was generated using a fixed circular 40x40 μm area of interest (AOI). For
every image, the AOI was placed over a cell and the mean pixel intensity was recorded.
The AOI was then moved to the next cell and the same data recorded. This allowed the
generation of a minimum of 103 and a maximum of 486 data points (n). The mean
intensity of immunolabelling was determined and expressed as pixel x 102/μm².
2.5.6 Statistical analysis
Data are presented as mean ± SEM (standard error of the mean). Comparison of
immunoflourescence labelling at the different time points was performed by one way
analysis of variance with Bonferroni’s post-hoc test. A p value <0.05 was considered
statistically significant.
2.6 Assessment of impact of remote ischaemic preconditioning on global cerebral ischaemia in rats
2.6.1 Experimental design
Our research institution has a previously established model for global cerebral
ischaemia (GCI) (111). All procedures were approved by the Animal Ethics Committee.
Male Sprague-Dawley rats weighing 261-353 g were randomized into 3 groups. Group I
(Control, n = 5) underwent sham procedure, namely 2 general anaesthetics, without
cerebral ischaemia. Group II (GCI, n = 5) was subjected to RIPC induced by transient
left hind limb ischaemia under general anaesthesia prior to GCI. Group III (RIPC + GCI
only, n = 5) underwent sham procedure under general anaesthesia prior to GCI. Twenty
four hours after the RIPC or sham procedure a transient GCI was induced for 8 min in
Groups II and III by means of bilateral common carotid artery occlusion and
hypotension. Hippocampal CA1 neurons were histologically examined at 7 days after
ischaemia.
53 Animals in Group III were first subjected to RIPC. They were anaesthetised with
3% halothane in N2O:O2 (2:1) and maintained at 2% halothane in the same gas mixture.
RIPC was carried out using the previously described protocol (see 2.2). The animals
were then allowed to recover in a warmed room. Sham-operated animals (Group I)
underwent the same anaesthesia and surgery as did experimental animals but were not
rendered ischaemic.
Transient GCI was induced 24 h later. Animals were anaesthetised again as
described above. EEG monitoring was performed. Rectal temperature was monitored
and maintained at 37 ± 0.5 °C. GCI was induced according to the previously described
protocol. Blood gases were analysed using a blood gas analyser (ABL Radiometer,
Copenhagen, Denmark) and ventilatory parameters were adjusted prior to ischaemia
where necessary to ensure that arterial pCO2 at the commencement of ischaemia was 40
± 2 mmHg and pO2 was >100 mmHg. Plasma glucose levels were recorded using a
blood glucose meter (Miles Laboratories Inc., Elkhart, IN). The EEG findings and
arterial blood pressure were recorded using MacLab data acquisition system (AD
Instruments, Melbourne, Australia).
Following GCI, blood gases were again analysed 10 min later. Atropine (3μg) was
administered subcutaneously 10 min before intubation and a total of 0.5 mg of
bupivacaine was infused subcutaneously in the leg wounds after closure. Temperature
was monitored for at least 4 h after surgery to ensure normothermia. All animals had an
uneventful recovery and were monitored for 7 days.
2.6.2 Assessment of hippocampal neurons
Animals were euthanised 7 days after the procedure with an intraperitoneal injection of
pentobarbitone followed by transcardiac perfusion with approximately 200ml 0.9%
NaCl, then approximately 200ml of 4% formalin. Brains were collected and sectioned at
bregma -3.8 according to a standard rat brain atlas (262). The sections were stained with
cresyl violet and examined under 400 x magnification to assess the survival of
hippocampal CA1 neurons. Three 1000μm segments of each hemisphere were assessed
for the number of viable neurons remaining and total cell counts were used as the
results. Neuronal injury in this global ischaemia model is quantified by counting the
number of CA1 neurons in 1000µm segments of the medial, intermediate and lateral
sections of the hippocampal CA1 region for each animal (111).
54 2.6.3 Statistical analysis
The three groups were compared using the Poisson regression model. P-values and
confidence intervals were calculated. The data were expressed as mean ± standard
deviation (SD).
55
CHAPTER 3
RESULTS
56 3.1 Functional responses of human neutrophils to remote ischaemic pre-
conditioning stimulus
3.1.1. Summary Objective: Preconditioning of cells or organs by transient sub-lethal ischaemia, termed
ischaemic preconditioning, protects the cell or organ from a subsequent prolonged
ischemic insult. The mechanisms of this effect are yet to be fully elucidated. It has
recently been reported that IPC of forearm results in alterations in gene expression
profiles of circulating polymorphonuclear leukocytes. The goal of the current study was
to determine if the observed changes in gene expression lead to functional changes in
neutrophils.
Methods: The effect of repetitive transient human forearm ischaemia (3 cycles of 5 min
ischaemia, followed by 5 min of reperfusion) on the function of circulating neutrophils
was examined. Neutrophil functions (with and without lipopolysaccharide stimulation)
were examined before, after 1 day, and after 10 days of daily transient forearm
ischaemia.
Results: Neutrophil adhesion was significantly decreased on day 1 and remained low
on day 10 (p=0.0149) without significant change in CD11b expression. Phagocytosis
was significantly suppressed on day 10 compared to day 0 (p<0.0001). Extracellular
cytokine levels were low in the absence of an exogenous stimulus but stimulation with
LPS induced significant changes on day 10. There was a trend in the reduction of
apoptosis on day 1, and day 10 that did not reach statistical significance (p<0.08).
Conclusions: This study indicates that repetitive ischaemic preconditioning of the
forearm results in alterations in neutrophil function, including adhesion, exocytosis,
phagocytosis, and cytokine secretion. These observations have important implications
for understanding the mechanisms of modulation of the IR injury and its inflammatory
response by remote ischaemic preconditioning.
Effects of RIPC on the functional response of neutrophils
The process of neutrophil separation was completed as described in the methods
section. Neutrophil purity was >98% and viability was > 97% using Trypan Blue
exclusion. The functional integrity and non-activated state of neutrophils isolated has
been validated in previous publications (263). The effects of RIPC on the functional
responses of the neutrophils were as follows:
57 3.1.2 Neutrophil adhesion and CD11b surface expression
Neutrophil adhesion decreased from 13.0% ± 4.3% to 0.81% ± 0.2% at 24 h after the
first RIPC stimulus (Figure 3.1-1) and remained low at day 10 (2.61% ± 0.7%). These
changes were statistically significant (p<0.015). Stimulation with FMLP mirrored the
pattern observed in non stimulated neutrophils and increased adhesion to 34.7% ± 4.7%
in cells studied on day 0 and to 18.6% ± 5.4% on day 1 as compared to 2.9% ± 1.9% on
day 10 (p=0.0167). The surface expression of CD11b, expressed as median fluorescence
intensity (Figure 3.1-2a) did not change on day 1 or day 10 of RIPC (p=0.92) even after
FMLP stimulation.
Figure 3.1-1. The effect of RIPC on neutrophil adhesion assessed as the mean
percentage (%) of cells that adhered to tissue culture wells coated with foetal bovine
serum. Adhesion was significantly suppressed 1 day after the RIPC stimulus and
remained suppressed after 10 days of daily RIPC.
58 3.1.3 Oxidant production
Activation of the NADPH oxidase as assessed by oxidant production, either in resting
or FMLP-activated cells, did not change significantly over the course of the protocol
(p>0.05) (Figure 3.1-2b).
Figure 3.1-2. The surface expression of a) CD11b, b) NADPH oxidase production, the
surface expression of c) CD63 and d) CD66b. RIPC resulted in a significant increase in
FMLP-induced CD63 and CD66b expression (p=0.0012, and p<0.007).
3.1.4 Exocytosis
The surface expression of CD63 in resting cells, a marker of primary granules, did not
change significantly over the 10-day course of the RIPC (p=0.647) (25.4 ± 2.8 on day
0, 21.7 ± 5.0 on day 1, and 26.1 ± 1.8 on day 10 respectively). In contrast, RIPC
resulted in a significant increase in FMLP-induced CD63 expression (25.0 ± 2.2 on day
0, 38.1 ± 8.4 on day 1, and 72.7 ± 9.3 on day 10 (p=0.0012) (Figure 3.1-2c). The level
of surface expression of CD66b, a marker of secondary granules, did not change
significantly in resting cells (61.1 ± 20.6 on day 0, 120.8 ± 26.9 on day 1, and 84.1 ± 4.0
on day 10; p=0.118). Similar to CD63, there was a significant increase in FMLP-
induced CD66b expression in response to RIPC (76.6 ± 18.2 on day 0, 173.2 ± 34.7 on
day1, and 203.2 ± 8.1 on day 10, respectively; p<0.007) (Figure 3.1-2d).
59 3.1.5 Secretion of cytokines
Extracellular levels of cytokines were very low in otherwise quiescent neutrophils. The
cells were incubated with LPS and the extracellular secretion of cytokines assessed by
multiplex analysis at 6 and 24 h. Neutrophil TNF-α secretion increased on day 10 of the
RIPC protocol, 6 and 24 h after LPS exposure (p=0.0048, p=0.0248 one-way repeated
measure of ANOVA, respectively). At both incubation times, LPS-induced TNF-α
secretion was increased significantly as compared with neutrophils examined before the
RIPC stimulus (p=0.0044, and p=0.0085, 2-way ANOVA) (Figure 3.1-3 a). Similarly,
LPS-induced IL-6 secretion at 6 and 24 h was significantly increased in leukocytes
isolated on day 10 of the RIPC protocol as compared to day 0 (one-way repeated
measure of ANOVA) (Figure 3.1-3 b). IL-10 secretion did not change in otherwise
unstimulated cells but increased significantly on day 10 of the RIPC protocol in cells
exposed to LPS for 24 h (p=0.0244, one- way repeated measure of ANOVA) (Figure
3.1-3 c ). Secretion of IL-1β was low in otherwise unstimulated cells and increased
significantly in response to LPS exposure. Interestingly, secretion of IL-1β was
increased significantly in cells on day 10 of the RIPC protocol compared to day 0 and
24 h (p= 0.02, and p=0.01 respectively, 2-way ANOVA) (Figure 3.1-3 d).
A. TNF-α C. IL-10
B. IL-6 D. IL-1b(pg/mL) (pg/mL)
(pg/mL)(pg/mL)
Figure 3.1-3. Cytokine secretion in quiescent (rest) and stimulated (LPS) cells at 2
periods of incubation (6 and 24 h) in cells taken prior to (Day 0), and after 1 day and 10
days of daily RIPC; A.TNF-α, B. IL-6, C. IL-10, D. IL-1β.
60 3.1.6 Apoptosis
There was a reduction of apoptosis after the application of RIPC stimulus from 35.0% ±
12.0% on day 0 (baseline) to 9.3% ± 1.0% on day 1, and 16.1% ± 1.4% on day 10.
However, this change did not achieve statistical significance (p=0.079) (Figure 3.1-4).
Figure 3.1-4. The effect of RIPC on neutrophil apoptosis. The reduction at day 1 and 10
did not reach statistical significance (p=0.08).
61 3.1.7 Phagocytosis
Phagocytosis was significantly suppressed on day 10 compared to day 0 of the RIPC
protocol (p<0.0001) (Figure 3.1-5).
Figure 3.1.5. Graphs showing the effect of repeated RIPC stimulus on neutrophil
phagocytic activity. Phagocytosis was significantly suppressed after 10 days of
application of daily RIPC stimulus compared to day 0 (prior to RIPC) (p<0.0001).
62 3.2 Human kallikrein-kinin system response to remote ischaemic
preconditioning stimulus.
3.2.1 Summary
Objective: RIPC has been shown to reduce ischaemia-reperfusion injury and is induced
by brief forearm ischaemia. Kinins are known to be involved in RIPC and act via the G
protein coupled B1 and B2 receptors. Interaction of the kinins with their respective
receptors causes receptor internalization, thereby reducing the potential for further
activation. This may be critical for the protective effect of RIPC and if so we
hypothesised would significantly decrease the expression of kinin receptors on the
surface of neutrophils.
Methods: The study was performed on 5 healthy human volunteers. The left forearm
was rendered ischaemic for three 5-min periods, each separated by 5 min of reperfusion.
Three venous blood samples were taken from the right arm- one before and two after
RIPC. Neutrophil isolation, immunofluorescence labelling and confocal microscopy
were performed. Mean pixel intensity data was generated using a fixed circular area of
interest (AOI, 40x40 µm). For each image, the AOI was placed over a cell and the mean
pixel intensity was recorded. The mean intensity was expressed as pixel x 102/μm² and
presented as mean ± SEM. Immunofluorescence at the different time points was
compared by one way analysis of variance with Bonferroni’s post-hoc test. A p-value
<0.05 was considered significant.
Results: The mean pixel intensity for kinin B1 receptors was decreased at 24 h after
RIPC when compared with both baseline and 15 min after RIPC (p < 0.001). Similarly
the intensity for B2 receptor labelling on neutrophils was significantly decreased 24 h
after RIPC when compared to the baseline value (p < 0.001).
Conclusions: RIPC decreases the expression of kinin receptors on circulating human
neutrophils and this was evident 24 h after RIPC. The reduction in the number of kinin
surface receptors suggests internalization of receptors and is consistent with the
concepts of kinin receptor activation and their role in RIPC.
The effects of RIPC on the kinin receptors in human neutrophils were as follows:
3.2.2 Expression of B1 kinin receptors
Immunoperoxidase labelling showed that TK, PK, kininogen and the kinin B1 and B2
receptors were expressed on neutrophils at baseline and at 15 min and 24 h after RIPC.
To assess whether there were changes in the numbers of neutrophils that were positively
63 labelled after RIPC, the cells were counted and the number of positively labelled
neutrophils was expressed as a percentage of the total number counted. This analysis
showed that there were no significant differences in the percentages of neutrophils that
were positively labelled for any of the KKS proteins at 15 min or 24 h after RIPC, as
compared to baseline.
Immunofluorescence labelling of neutrophils for kinin B1 and B2 receptors was
assessed by confocal microscopy. There was a qualitative decrease in
immunofluorescence labelling of neutrophils for B1 receptor 15 min after RIPC as
compared to baseline, with a further decrease in immunolabelling 24 h after RIPC
(Figure 3.2-1).
Figure 3.2-1. Representative confocal images from one of the subjects showing the
expression of B1 receptors on neutrophils at baseline (A), 15 min after RIPC (B) and 24
h after RIPC (C). The colour bar indicates the intensity of immunofluorescence
(pixels/μm2) as pseudo-colours applied to the grey-scale images, with red indicating the
highest intensity and black the lowest intensity of immunofluorescence. Scale bar = 10
μm.
64 In order to assess changes in kinin receptor expression quantitatively, image analysis
was performed on the confocal images and mean pixel intensities were calculated for
immunofluorescence labelling of kinin B1 and B2 receptors on neutrophils at baseline,
and at 15 min and 24 h post RIPC for all five subjects. The mean pixel intensity for
kinin B1 receptor labelling on neutrophils was significantly decreased 24 h after RIPC
compared with both baseline and 15 min after RIPC (p < 0.001) (Figure 3.2-2 ).
Figure 3.2-2. Quantitative analysis of kinin B1 receptor immunofluorescence on
neutrophils showing mean pixel intensities for all five subjects at baseline (0 min), 15
min after RIPC and 24 h after RIPC. ***p < 0.001.
65 3.2.3 Expression of B2 kinin receptors
There was a qualitative decrease in immunofluorescence labelling of neutrophils for B2
receptor at 15 min and 24 h after RIPC compared to baseline (Figure 3.2-3). Similarly
the mean pixel intensity for kinin B2 receptor labelling on neutrophils decreased
significantly 24 h after RIPC compared to the baseline value (p < 0.001) (Figure 3.2-4).
Figure 3.2-3. Representative confocal images from one volunteer, demonstrating
expression of B2 receptors on neutrophils at baseline (A), 15 min after RIPC (B) and 24
h after RIPC (C). The colour bar indicates the intensity of immunofluorescence
(pixels/μm2) as pseudo-colours applied to the grey-scale images, with red indicating the
highest intensity and black the lowest intensity of immunofluorescence. Scale bar = 10
μm.
Figure 3.2-4. Quantitative analysis of kinin B2 receptor immunofluorescence on
neutrophils showing mean pixel intensities for all five subjects at baseline (0 min), 15
min after RIPC and 24 h after RIPC. ***p < 0.001.
66 3.3 Assessment of impact of remote ischaemic preconditioning on global
cerebral ischaemia in rats
3.3.1 Summary
Objective: To determine if remote ischaemic preconditioning (RIPC) induced by
transient limb ischaemia is protective against delayed hippocampal neuronal death in
rats undergoing transient global cerebral ischaemia (GCI).
Method: Animals were randomised into 3 groups. Group I (Control, n = 5) underwent
sham procedure, namely, 2 general anaesthetics, without cerebral ischaemia. Group III
(RIPC + GCI, n = 5) was subjected to RIPC induced by transient left hind limb
ischaemia under general anaesthesia prior to GCI. Group II (GCI only, n = 5) underwent
sham procedure under general anaesthesia prior to GCI. Twenty four hours after the
RIPC or sham procedure, transient GCI was induced for 8 min in Groups II and III by
means of bilateral common carotid artery occlusion and hypotension. Hippocampal
CA1 neurons were histologically examined 7 days after ischaemia.
Results: There was no significant difference between the RIPC group and the ischaemia
only group. The number of neurons in the RIPC group were 0.90 (95% CI 0.20, 4.08)
times the number in the ischaemia group (p=0.89). The number of neurons in the RIPC
group were 0.03 (95% CI 0.01, 0.10) times the number in the control group (p=0.0001).
Conclusion: Second window of the RIPC does not prevent hippocampal CA1 neuronal
death at 7 days after transient global cerebral ischaemia.
The effect of RIPC on global cerebral ischaemia in rats was as follows:
3.3.2 Delayed hippocampal neuronal death after transient global cerebral ischaemia
Global cerebral ischaemia significantly decreased the number of hippocampal CA1
neurons (Figure 3.3-1). The mean hippocampal CA1 neuron count was 285 ± 30 in
control (Group I), 10 ± 11 in animals subjected to ischemia only (Group II) and 9 ± 16
in animals subjected to RIPC prior to ischemia (Group III) (Figure 3.3-2). There was
statistically significant difference in CA1 hippocampal neuron counts between Group I
(Control) and the animals subjected to global cerebral ischemia, regardless of whether
preconditioning was applied or not. Both Group II (Global cerebral ischemia) and
Group III (RIPC+Global cerebral ischaemia) exhibited less than 6% CA1 neuronal
survival at 7 days after ischemia. There was no difference in the number of neurons
between the two groups subjected to global cerebral ischaemia. The number of neurons
67 in preconditioned animals (Group III) was 0.90 (95% CI 0.20, 4.08) times the number in
those subjected to ischaemia only (Group II) (p=0.89). The number of neurons in
preconditioned animals (Group III) was only 0.03 (95% CI 0.01, 0.10) times the number
of neurons in control animals (Group I) (p=0.0001).
Figure 3.3-1. Histological changes in the rat CA1 hippocampus. Hippocampal section
(cresyl violet staining) at high (400x) magnification in control (A) animals and those
which underwent cerebral ischaemia (B) and preconditioning 24 hrs prior to cerebral
ischaemia (C). In sham group (A) intact pyramidal neurons are arranged in order with
full nucleus and clear nucleolus. Global cerebral ischaemia for 8 min caused clear
delayed neuronal death (B and C).
68
0
50
100
150
200
250
300
350
Control Ischemic RIPC + Ischemic
Groups
Figure 3.3-2. Mean hippocampal CA1 neuron counts. Bars represent mean deviation in
hippocampal cell count between individual animals.
69
CHAPTER 4
DISCUSSION
70 4.1 Circulating factor of remote ischaemic preconditioning
Wang et al. demonstrated preconditioning of the neonatal rabbit heart on perfusion with
blood taken from RIPC treated rabbits and found that an unspecified humoral factor
preserves mitochondrial structure and function and maintains global cardiac
performance (91). A number of chemical mediators have been suggested to be
responsible for triggering the effect of RIPC. Investigated factors include- adenosine,
BK, calcitonin gene related peptide, opiates, HIF-1α and unspecified mediators (54, 76,
99, 231, 264-266). It appears that these mediators released from local tissues are
transported via the blood stream to the effector organ. Subsequently signalling pathways
are activated and provide cytoprotection.
4.2 Functional response of human neutrophils to remote ischaemic preconditioning
The data from the current study suggest that part of the preconditioning effect may be
due to modulation of the inflammatory response via altered functional responsiveness of
circulating leukocytes. This observation corroborates with the previously reported
finding of marked down-regulation of pro-inflammatory genes in circulating human
leukocytes in response to the RIPC stimulus (78). It is apparent that alterations in gene
expression correlate with functional changes in circulating neutrophils. The present
study also demonstrated that the changes in neutrophils persist when the stimulus is
repeated daily for 10 days. The latter effect was examined in order to elucidate
potential amplification or tachyphylaxis in response to repeated RIPC cycles that might
potentially be relevant to the clinical application of this stimulus. The results from the
current study demonstrate significant alterations in several important functional
responses of neutrophils including adhesion, exocytosis of primary and secondary
granules, and LPS-induced cytokine secretion.
Adhesion of neutrophils to the vascular endothelium is an early event, which
precedes attachment and activation in response to a variety of stimuli, including the
systemic inflammatory reaction to cardiopulmonary bypass (CPB), infection and local
trauma. This is usually followed by a feedback cascade resulting in the local release of
inflammatory mediators such as cytokines and cytotoxic cell-products including ROS,
proteolytic enzymes and antimicrobial peptides contained in specialized granules. This
neutrophil response is primarily a natural host defence mechanism, but, if excessive,
such response can result in inappropriate organ damage. Adhesion of unstimulated
neutrophils decreased significantly on the first day after RIPC stimulus and was still
71 suppressed on day 10. However, cells were still able to react partially to a
physiologically relevant stimulus as demonstrated by some restoration of adhesion in
response to the chemoattractant peptide, FMLP. However, it is noteworthy that even
after exposure to this potent agonist, neutrophil adhesion following RIPC remained less
than control cells. Interestingly, surface expression of CD11b in quiescent and FMLP-
activated cells did not differ over the course of the experiment, suggesting that the
primary effect of RIPC on adhesion was in modulation of integrin affinity. These data
are concordant with another report that there was no change in CD11b expression
before and after RIPC stimulus, although reduced CD11b expression after exposure to a
sustained IR insult was observed (54).
In the current study, cytokine secretion was unaffected by the RIPC stimulus at 24
h, but significantly increased after 10 days of daily RIPC. Furthermore, even after the
10-day period of repetitive RIPC, neutrophils were capable of responding to LPS. TNF-
α secretion did not change in response to the RIPC stimulus alone but when RIPC was
combined with exposure to LPS, TNF-α secretion was significantly augmented.
Although hypothetical, Wang et al. postulated that pro-inflammatory cytokines such as
TNF-α, and IL-6 might contribute to late phase preconditioning (the ‘delayed window’
response that appears 24-72 h after the initial stimulus) in patients with unstable angina
(267). This is supported by observations in TNF-α deficient mice where late phase IPC
is completely abrogated (268). Nonetheless, several different cytokines appear to exert
physiologically important influences during IR injury. For example, the myocardial
infarction-sparing effect of IPC was also completely abrogated in IL-6 deficient mice
(269-270). With regards to mechanism, IL-6 plays an important role in modulation of
oxidant stress in the lung by protecting lung cells from oxidant-induced cell death (270).
An early increase in IL-1β in the lung was observed after (remote) hepatic injury,
implicating this cytokine in the acute systemic inflammatory response (271). Clark et al.
demonstrated that the IL-1 receptor antagonist significantly reduced endothelial-
leukocyte adhesion molecule-1 expression after hypoxia/reoxygenation in cultured
human umbilical endothelium cells (272). The data from the present study demonstrate
that IL-1β release from leukocytes was low 24 h after RIPC, with or without an
exogenous stimulus (LPS), but was significantly increased (in response to LPS) by day
10. Taken together, these observations suggest that daily RIPC for 10 days may induce
a pro-inflammatory milieu and amplify systemic inflammation upon exposure to an
exogenous stimulus such as LPS. However, it was observed that IL-10, a predominantly
anti-inflammatory cytokine, was also significantly increased after the 10 day period of
72 RIPC. This may serve to mitigate against an over-exuberant inflammatory response and
the potential adverse or beneficial effect of any of these changes will need to be studied
in clinically relevant models or scenarios.
The phagocytic ability of neutrophils is a quintessential function in antimicrobial
responsiveness in innate immunity. The phagocytic ability of neutrophils, as assessed by
the ability to ingest IgG-decorated erythrocytes, was maintained at day 1 but was
significantly decreased after 10 days of RIPC (p<0.05, ANOVA) (Figure 3.1.-5). In
contrast, there was no apparent effect of RIPC on activation of the phagocyte NADPH
oxidase. The net effect of these alterations on host defence against microbial pathogens
remains uncertain but it is possible that the phagocytic defect could compromise host
defences.
Finally, is has been previously demonstrated that caspase 8, and caspase 8-
associated protein 2 gene expression, both mediators of apoptosis, were markedly
reduced 24 h after RIPC stimulus (87). There was a tendency to reduction in the
apoptosis of neutrophils following RIPC, however, this did not achieve statistical
significance (p<0.08). This lack of statistical significance was likely due to the small
sample size and the inter-individual variability in the apoptotic response (Figure 3.1-
4).
Another issue that needs to be addressed is the calculation of the appropriate dose of
RIPC. This question has not been addressed specifically in the work done in the past.
However, no studies have shown any increase in adverse effects related to altered
function of neutrophils, for example an increased susceptivity to infections. Close
analysis of the clinical data with regards to the outcome might be able to shed some
light in this area in future studies. Studying the degree of changes in neutrophil
functions following RIPC with different protocols of RIPC may also be helpful in
answering this question.
To conclude, daily RIPC results in significant alterations in physiologically
important functional responses of neutrophils. In particular, there was significant
reduction in adhesion within 24 h, but enhancement in LPS-induced cytokine release
and reduced phagocytotic ability at 10 days. Taken together, these data suggest that
RIPC beneficially modifies inflammatory responses to adverse stimuli within 24 h, but
if repeatedly administered might increase susceptibility to infection. Further studies will
be required to examine the clinical relevance of these observations, but they provide
73 additional evidence for the clinical effectiveness of the multi-organ protection afforded
by RIPC during episodes of predictable IR injury such as CPB.
4.3 Kinin receptor expression in human neutrophils
The RIPC stimulus decreased expression of both B1 and B2 receptors on circulating
human neutrophils for at least 24 h. This supports a model of receptor internalization
and is consistent with the current signalosome hypothesis regarding the benefits of
RIPC in protecting tissues from IR injury.
B1 and B2 receptors are members of a super family of G-protein-coupled
rhodopsin-like receptors characterized by seven transmembrane regions connected by
three extracellular and three intracellular loops, and are linked to a second messenger
signalling system. The various biological effects of kinins result from activation of the
B1 and B2 receptors. The B1 receptor is rarely expressed in normal tissues but is
rapidly upregulated in inflammation and following exposure to bacterial endotoxins and
lipopolysaccharides. There is an increase in the number of B1 binding sites in inflamed
tissues, carcinoma, rheumatoid arthritis, transplant rejection and glomerulonephritis
(191, 202). B2 receptors, on the other hand, are present in most normal tissues and are
responsible for the majority of the biological effects of kinins, including hypotension,
bronchospasm and oedema. B2 receptors are also involved in the angiotensin converting
enzyme induced prevention of cardiac remodelling following acute MI (203).
Activation of B2 receptors in normal tissue may also induce increased expression of B1
receptors (273).
Kinins appear to be directly involved in IPC. Intracoronary infusion of BK in
patients undergoing PCI attenuated myocardial ischaemia during coronary artery
occlusion with balloon inflation (57). In another study, use of BK preoperatively in
patients undergoing standard CABG with CPB and aortic cross clamping, resulted in
less myocardial ischaemia in comparison to controls (229).
The release of BK following RIPC causes the activation of the G protein-coupled
signalling pathway. This cascade activates a number of kinases, including PI3K that is
responsible for activation of Akt, as well as downstream activation of NOS. NO release
leads to activation of mitochondrial PKG. This pathway causes stimulation of
mitochondrial KATP channels and inhibition of MPTP which provides cytoprotection
(Figure 1.2-1) (274-275). The effect of the RIPC stimulus on components of KKS other
74 than the kinin receptors does not appear to be significant, based upon the results of the
present study.
It has been demonstrated from the previous studies and the present work that brief
forearm ischaemia suppresses pro-inflammatory gene expression (46), adhesion and
modifies functional responses in human neutrophils. The exact mechanism behind this
is yet unknown, but clearly clinically relevant and important for the understanding of
post-surgical inflammatory response. As BK is one of the strongest mediators of
inflammation, reduction of B1/B2 receptors on human neutrophils appears consistent
with the studies that demonstrated significant decreases in inflammatory response in
both experimental and clinical scenarios (77, 93). Further studies are needed to assess
molecular changes and downstream pathways of B1/B2 receptors following the RIPC
stimulus. Assessment of global proteomic changes in human neutrophils after the RIPC
may provide an insight into the molecular mechanisms of neutrophil desensitisation. It
should be remembered, however, that kinin-receptor induced changes might be
functional and may not result in any detectable changes in proteins.
The results from the current study strongly indicate that there is a loss of kinin
receptors from the surface of neutrophils following RIPC, and further studies are
required to determine whether this results in intracellular changes that lead to cell
protection, or whether the loss of receptors leads to desensitisation and refractoriness of
neutrophils to activation in the ensuing 24 h thereby providing protection against IR
injury.
4.4 Second window of remote ischaemic preconditioning and neuroprotection
Induction of RIPC by transient limb ischaemia appears to be an attractive, safe and
practical approach in clinical practice. Although beneficial effects of the RIPC have
been demonstrated both in animals and in humans, its effect on neuronal death still
remains controversial (276-279). The hippocampal neurons of the rat brain are
particularly sensitive to IR injury because of their high metabolic rate (280). These
neurons are easy to visualise and, thus, represent an ideal cellular target to study the
effects of RIPC on the brain.
A comprehensive review of several animal models of both focal and global cerebral
ischaemia suggests that significant reduction of cerebral infarction can be achieved by
ischaemic preconditioning (281-282). Both local and remote preconditioning appears to
reduce the size of cerebral infarct (107-110, 283). The mechanism of cerebral ischaemic
tolerance may be reliant on protein synthesis and expression as reflected by the longer
75 onset but also the longer duration of protection. Of clinical interest, Moncayo and
colleagues (284) found that patients with prior ipsilateral transient ischaemic attack
(TIA) lasting no longer than 20 min had a less severe clinical deficit of stroke on
admission and more favourable outcome. Others, however, suggested that duration of
TIA did not influence disability from subsequent stroke (285). TIA would be a clinical
equivalent of a local IPC.
A recent study in rats demonstrated that delayed, or second window RIPC induced
by transient lower limb ischaemia significantly reduced cerebral infarction size
measured 2 days after combined focal and global cerebral ischaemia (107). In this
study, Ren et al. observed a significant decrease in cerebral infarction size by both first
and second window of RIPC at 2 days after cerebral ischaemia (82). However, it
remained unknown if such protection against acute cerebral infarction observed at 2
days after cerebral ischemia would translate into a decreased delayed apoptotic death of
neurons. Two studies examined the effects of RIPC on late apoptotic death of pyramidal
neurons in the CA1 hippocampus (108, 286). Both studies utilised a similar model of 8
min of GCI (108, 286). Zhao et al. (286), demonstrated that RIPC protected against
delayed neuronal death in the CA1 hippocampus at 3 days after 8 min of GCI. Sun et
al., demonstrated that transient limb ischaemia induces brain ischaemic tolerance
manifested by preservation of the CA1 hippocampal pyramidal neurons following GCI
via p38 mitogen-activated protein kinase (MAPK) (108). Inhibition of p38 MAPK by
SB 203580 at 30 min prior to transient limb ischaemia blocked protective effect of
RIPC. It is of interest that p38 MAPK expression peaked on day 1 and 3 after the
transient limb ischaemia, but returned to the baseline level at day 5 (108). Thus,
assessment of delayed death of the CA1 hippocampal pyramidal neurons at day 7, i.e.,
after p38 MAPK level normalisation was performed in the current study. The present
study demonstrated that significant delayed death of the CA1 hippocampal pyramidal
neurons at day 7 may still occur. It is not clear, however, from the current pilot study if
the lack of protection is related to the normalisation of p38 MAPK level or to the fact
that, unlike in other organs, the second window of the RIPC is not effective in brain.
This observation is very interesting by itself, and should stimulate further research into
the mechanisms of RIPC and, hopefully, may identify the role of BBB or other factors
that may render second window of RIPC ineffective in cerebral protection. While it has
been previously demonstrated that the second window of RIPC induces profound
molecular changes in circulating neutrophils and myocardium (46, 148), the lack of
anticipated delayed cerebral protection is intriguing.
76 Finally, is it likely that the degree of brain injury inflicted by 8 min of GCI in the
present study was excessive, rendering any protective method, including the RIPC,
inadequate? There are two reasons why I feel confident that the inflicted brain injury
was not excessive. Firstly, the same protocol has been used by our group before and
demonstrated that administration of magnesium sulphate was very effective in
decreasing hippocampal CA1 neuronal death (111). Secondly, other authors have used
similar protocol for the GCI (107-108, 286). Namely, Ren et al. (107) applied a
significantly longer period of GCI (i.e., 30 min occlusion of bilateral common carotid
arteries compared to 8 min in the current study) combined with additional permanent
ligation of the distal middle cerebral artery. Finally, a virtually identical protocol of 8
min of GCI has been employed by others (108, 286) - both studies demonstrated
attenuation of the apoptotic neuronal death in the CA1 hippocampus for up to 3 days.
The present study demonstrates that second window of RIPC does not appear to
prevent delayed hippocampal neuronal death one week after global cerebral ischaemia.
Further research is necessary, however, to determine if other modes of RIPC may still
provide cerebral protection.
4.5 Remote ischaemic conditioning and blood brain barrier
BBB represents a tightly regulated microenvironment between blood and brain. It
consists of a physical barrier (tight junctions between the cells reducing flux via
intercellular pathways), a transport barrier and a metabolic barrier (287). BBB regulates
the ion movement that maintains the milieu for optimal neuronal function and regulates
the levels of neuroexcitatory transmitters such as glutamine. This barrier also protects
the brain against exogenous and endogenous toxic substances and supports the nutrition
of neurons. BBB is responsible for preventing the entry of macromolecules such as
plasma proteins to CSF. Damaged BBB allows proteins like albumin, pro-thrombin and
plasminogen to penetrate the barrier and cause apoptosis (288-290). Monocytes,
lymphocytes and macrophages are able to enter the central nervous system via BBB
during abnormal pathophysiological conditions and to transform themselves into
immunocompetent microglia cells (291-292). During IR injury or trauma, the activated
neutrophils damage the BBB and enter the central nervous system to initiate an
inflammatory response (293-294).
No specific experiments were performed during the current project to identify the
lack of neutrophil activation in the hippocampal neuronal death model. Future
experiments may identify the mechanism underlying the lack of an anti-apoptotic effect.
77 Herein, I describe a possible example. An animal model of RIPC is selected. Study and
control groups are identified. RIPC is carried out. This is followed by global cerebral
ischaemia. Blood samples from systemic and cerebral circulation are taken while animal
is anaesthetised and neutrophils are isolated from these samples. Animals are euthanized
to study the histopatholgy of BBB and hippocampus of the two groups to identify the
possible changes. Neutrophils are studied for the expression of kinin receptors and the
other components of KKS. Overall, the role of RIPC in neuroprotection remains
unclear. This, undoubtedly, will be an area of fruitful research for many years. The
future research needs to determine as to where BBB, neutrophils, KKS, KATP channels
and mitochondria fit in neuroprotection following preconditioning.
4.6 Clinical applications
Local preconditioning induces ischaemia in the target organ with its potential to cause
detrimental effects. Repeated clamping of the ascending aorta, as has been done to
precondition the heart (295) has the potential to cause thromboembolic phenomena from
dislodgement of atheromatous plaques and is often impractical. Thus, to date, local IPC
has not found wide clinical application (296-298). However, RIPC appears to overcome
these issues and is more practical, safe and potentially clinically applicable.
The first clinical application of RIPC demonstrated significant cardiac and
pulmonary protection and attenuation of systemic inflammatory response in children
undergoing repair of congenital cardiac defects using CPB. Troponin levels and
inotropic scores were assessed and were found to be lower in patients subjected to
RIPC. Dynamic lung compliance was also lower in the same group (299). Hausenloy et
al. (300) has recently demonstrated that RIPC using limb ischaemia significantly
reduced troponin-T release in patients undergoing CABG. It is not surprising that the
role of RIPC in myocardial protection during cardiac surgery is being actively
investigated with ongoing debate regarding its application (301-302). Furthermore, the
same RIPC stimulus was shown to reduce myocardial and renal injury during elective
abdominal aortic aneurysm (AAA) repair (303). The RIPC stimulus in the latter study
was produced by intermittent clamping of the common iliac artery (303).
In a recently conducted randomised controlled study (304) of patients undergoing
coronary angioplasty, RIPC provided significant myocardial protection as reflected by
lower incidence of chest pain, electrocardiographic (ECG) abnormalities and troponin
release. There was a trend to suggest a lower incidence of major cardiac events in the
preconditioned group at 6 months of follow up.
78 Another area of potential clinical application of RIPC is transplantation. A porcine
model of orthotopic heart transplantation (93) was used to study the benefits of this
phenomenon in transplantation. RIPC of the recipient provided significant protection of
the donor heart. Thus, RIPC of the recipient may be, in principle, applicable to
transplantation of any organ.
RIPC as produced in the clinical setting by using limb ischaemia has the potential to
provide a safe, cost-effective and non-invasive strategy of global organ protection. Such
global protection might have far reaching effects in terms of clinical benefits. A better
understanding of the mechanisms of RIPC will facilitate its clinical application in
transplantation, protection against cerebrovascular ischaemia, MI and systemic
inflammatory response (243, 305-306).
Importantly, there may be a role for remote ischaemic conditioning in the setting of
AMI or other ischaemic events which occur without any predictability. This can be
useful in the clinical setting after the onset of organ ischaemia and importantly during
or after the reperfusion process. Myocardial reperfusion can be interrupted during PCI
in the setting of AMI by inflating intracoronary balloon and provide protection with
post-conditioning (307).
Recent advances in cardiac surgery and further refinements of perfusion techniques
may permit surgeons to tackle increasingly complex problems in high risk patients. It
may be beneficial to add a simple and safe procedure of RIPC, perhaps, in combination
with other forms of remote ischemic conditioning, to the existing armamentarium of
cardiac protection such as hypothermia and cardioplegia. It appears that IC may protect
not only the heart but also other organs against systemic inflammatory response. This
may ameliorate multi-organ failure in critically sick patients with ongoing IR injury.
The remote ischaemic conditioning may be clinically applicable, but not limited, to
the protection in the following clinical scenarios:
1. PCI;
2. cardiac surgical procedures requiring CPB;
3. vascular surgery, or any surgery associated with IR injury;
4. acute coronary syndromes;
5. transplantation;
6. systemic inflammatory response to IR injury;
7. in the ambulance in patients with evolving myocardial infarction en route to the
hospital.
79 4.7 Future research
The work of this thesis will, hopefully, further facilitate the transition of RIPC into
clinical randomised controlled trials. Currently, a clinical trial is underway in our
institution to determine the degree of cardiac and pulmonary protection and attenuation
of the systemic inflammatory response to IR injury following preconditioning in
patients undergoing CABG. The details of the background and the protocols of the
proposed clinical study are described below.
Lung injury post cardiac surgery
A number of pulmonary changes occur in patients undergoing cardiac surgery due to the
effects of anaesthesia and CPB. Atelectasis develops due to the resorption of oxygen
from airways, ventilation-perfusion mismatch and relaxation of the diaphragm when the
lungs are not ventilated during extracorporeal circulation. Normally, the surfactant
forms a thin layer over the alveolar surfaces and prevents alveolar and small airway
collapse. CPB may cause changes in surfactant composition and function (308-309).
Atelectasis promotes the production of pro-inflammatory cytokines and reduces the
synthesis of surfactant (310).
CPB activates both innate and acquired immunity. IR injury along with pulmonary
hypoperfusion is responsible for the activation of inflammatory responses following
cardiac surgery.
Endothelial cells are stimulated by surgical trauma, hypoxia and the release of
cytokines such as TNF-α. These events lead to an increased expression of adhesion
molecules, including E-selectin, endothelial leukocyte adhesion molecule (ELAM),
intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule
(VCAM-1). These factors cause chemotaxis and adhesion of leukocytes (50, 311).
Neutrophils become adherent to vascular endothelium in pulmonary capillaries, undergo
aggregation and reduce the microcirculation in the lungs. A high level of neutrophil
elastase is found in patients exposed to CPB and correlates well with clinical indicators
of post-operative pulmonary dysfunction (312). Activated neutrophils migrate to the
areas of inflammation and ischaemia and release the proteolytic enzymes that increase
capillary permeability and aggravate pulmonary injury (312, 313). CPB also increases
the production of neutrophil matrix metalloproteinases (MMP). MMP-9 is associated
with injury to the basement membrane. Leukocytes activated during CPB cause the
release of TNF-α, interleukin (IL)-6 and IL-8 while up-regulation of neutrophil
adhesion molecules causes aggregation of pulmonary parenchymal neutrophils (314).
80
Impact of CPB on pulmonary mechanics
A number of studies have shown the deleterious effects of CPB on chest wall mechanics
(315-316). This can further contribute to impaired ventilation in patients undergoing
cardiac surgery. These effects seem to last for variable periods of time post operatively.
Cardiac surgery using CPB seems to increase airway resistance (317). Avoidance of
CPB, as in off-pump coronary artery bypass surgery in patients with underlying chronic
obstructive pulmonary disease (COPD) may preserve pulmonary function. This
translates to a reduced duration of mechanical ventilation and length of stay in the
intensive care unit (ICU) (318).
Remote ischaemic preconditioning and pulmonary protection
It appears that the activation of neutrophils and endothelial cells is central to the
pulmonary changes resulting from IR injury. There is a decrease in the number of
neutrophils and reduction in the levels of thromboxane B2 and malondialdehyde (MDA)
(a measure of oxidant damage) following local preconditioning in patients undergoing
cardiac valve replacement. On the other hand, the levels of superoxide dismutase (an
antioxidant enzyme) were higher in these patients (319).
Aim
1. To study the effects of early and late RIPC on inflammatory response and
cardio-pulmonary protection in patients undergoing CABG using CPB and
standard myocardial protection.
2. To study the effects of RIPC on the expression of kinin receptors and the various
components of KKS in these patients.
3. To study the effects of forearm preconditioning on the release of markers of lung
injury in patients following CABG.
Methodology
The hypothesis will be tested in patients undergoing CABG with CPB. Sixty patients
will be randomised into 4 groups:
Group I control (n=15);
Group II early preconditioning (n=15);
Group III delayed preconditioning (n=15); and
Group IV early + delayed preconditioning (n=15).
81 Patients with unstabe angina and evolving AMI undergoing urgent or emergency
CABG, diabetic patients receiving sulphonyl urea class of oral hypoglycaemic drugs
and those requiring peri-operative haemodialysis will be excluded from the study.
Remote ischaemic preconditioning protocol
Standard protocol for RIPC will be used. Early RIPC will be performed following the
induction of anaesthesia. Delayed remote preconditioning will be performed between 12
and 24 hours prior to the scheduled surgery.
Coronary artery bypass surgery
CABG will be performed in a standard fashion using antegrade and retrograde cold
blood cardioplegia and CPB.
Blood analysis
Pre-operatively
Blood samples will be collected before and after forearm preconditioning. The
samples will be analysed for the expression of kinin receptors in neutrophils and kinin
levels will be measured. The levels of IL-6, IL-8, IL-10, TNF-α, lactate, troponin I,
creatinine kinase (CK) and C-reactive protein (CRP) will also be measured.
Post-operatively
The levels of the above mentioned cytokines and inflammatory markers will be
measured post-operatively on arrival of the patient in ICU and at 6, 12, and 24 h
following weaning off bypass. Troponin and CRP levels will also be measured at 48 and
72 h post-operatively. Neutrophil activation, oxidant/antioxidant status, surfactant
protein, kinin receptor expression and kinin production will be assessed following
arrival in ICU and at 24 h interval.
Clinical parameters
Alveolar-arterial (A-a) oxygen gradient, cardiac index, respiratory index, PaO2/FiO2
ratio and lung compliance will be measured at the above identified time intervals.
Inotropic support will be calculated using inotropic score (320-321).
82 Expression of kinin receptors
A splice variant of the B1 receptor during mRNA quantification of wild-type B1
receptor in human leukocytes has been detected. Expression of the B1 receptor splice
variant in leukocytes may affect the accumulation of neutrophils at sites of injury and
produce different degrees of inflammation. Furthermore, the expression of this splice
variant in leukocytes may differ between individuals undergoing CPB and this may
influence the effect of remote preconditioning on pulmonary protection.
Measurement of kallikrein-kinin cascade proteins and genes
Neutrophil isolation and immunolabelling
Blood samples will be collected before and after RIPC, and at 30 min and 24 h after
arrival in ICU. A standard method for isolation of the neutrophils will be used. The
harvested neutrophils will be immunolabelled with and without initial fixation.
Confocal microscopy
Slides will be viewed under a confocal microscope. The relative intensity of
immunolabelling in the number of cells will be determined and the values expressed as
pixels x 102/μm². The digitised images will be analysed using the MDS programme.
Expression of kinin receptor genes in neutrophils
The mRNA expression of kinin receptor genes in neutrophils will be assessed
quantitatively using real time RT-PCR. RNA will be extracted from isolated neutrophils
(Rneasy Kit, QIAGEN) and reverse transcribed to cDNA (Omniscript RT, QIAGEN).
Kinin levels in blood
Kinin concentrations in the plasma samples will be measured using a commercially
available ELISA.
Measurement of biomarkers of lung injury
Plasma will be separated from whole blood (5 ml) prior to neutrophil isolation, and
will be stored at -80°C. The levels of neutrophil elastase and alpha1-protease inhibitor
(protease/anti-protease), glutathione and glutathione peroxidase (oxidant/antioxidant
status) and surfactant proteins will be measured.
83 Other areas of research
Large clinical randomised controlled trials are needed in patients undergoing surgery or
endovascular interventions associated with IR injury to determine further clinical
application of the RIC phenomena. The optimal protocols and the timing of
conditioning are yet to be defined in order to obtain the maximum benefit of this
protection strategy. The most efficient protocol is likely to combine early and late RIPC
with combinations of per- and post- conditioning to optimise protection.
Similarly, the exact mechanisms of the ischemic conditioning are yet to be
determined. Does the conditioning stimulus change a global proteomic profile or is it
driven by non-proteins? Are the neutrophils the messengers or the key players or both?
How does the conditioning optimise mitochondrial function? Identification of the exact
mechanisms of ischaemic conditioning may open new avenues for possible
pharmacologic augmentation of this phenomenon. Thus, in time, we may be able to
enhance this naturally evolved protection against the lack or excess of oxygen that
occurs during clinically relevant ischaemia and reperfusion. Should the full potential of
remote ischaemic conditioning be utilised, it may have an immense impact on medical
and surgical practice in diverse clinical scenarios.
Finally, it seems that there are many questions to be answered on the subject of
remote conditioning. What will be the extent of clinically relevant protection? Shall we
be able to fully comprehend the simplicity and complexity of this innate mechanism by
which all living cells protect themselves from the lack of oxygen or an excess of it?
Thus, I would like to summarize the work presented herein with a quote from Albert
Einstein (1879-1955): “The most incomprehensible thing about the universe is that it is
comprehensible!”
84
CHAPTER 5
ORIGINAL
CONTRIBUTIONS
85 5. Original contributions
The present study has resulted in the following original contributions to the
understanding of the mechanisms and organ protection by RIPC:
1. Definition of the concept of remote ischaemic conditioning for global organ
protection against ischaemia reperfusion injury;
2. Demonstration of suppressed adhesion and phagocytic function of human
neutrophils by the RIPC stimulus induced by forearm ischaemia in humans;
3. Demonstration of a reduction in the expression of kinin receptors on human
neutrophils by the RIPC stimulus induced by forearm ischaemia;
4. Demonstration that the second window of remote ischaemic preconditioning in
a rat model does not reduce the hippocampal neuronal death 7 days following
global cerebral ischaemia.
86
CHAPTER 6
PUBLICATIONS,
PRESENTATIONS AND
RESEARCH FUNDING
BASED ON THE THESIS
87 6. Publications, presentations and research funding based on the thesis
Publications
1. Saxena P, Newman MA, Shehatha JS, Redington AN, Konstantinov IE. Remote
ischemic conditioning: Evolution of the concept, mechanisms and clinical
application. J Cardiac Surg. 2010;25(1):27-34.
2. Shimizu M, Saxena P, Konstantinov IE, Cherepanov V, Cheung MM, Wearden
P, Zhandong H, Schmidt M, Downey GP, Redington AN. Remote ischemic
preconditioning decreases adhesion and selectively modifies functional
responses of human neutrophils. J Surg Res. 2010;158(1):155-61.
3. Saxena P, Bala A, Campbell K, Meloni B, d’Udekem Y, Konstantinov IE. Does
remote ischemic preconditioning prevent delayed hippocampal neuronal death
following transient global cerebral ischemia in rats? Perfusion. 2009;24(3):207-
11.
4. Saxena P, Shaw OM, Misso NL, Naran A, Shehatha J, Newman MAJ,
d’Udekem Y, Thompson PJ, Konstantinov IE. Remote ischemic preconditioning
stimulus decreases the expression of kinin receptors in human neutrophils. J
Surg Res. 2010, in press.
5. Saxena P, Thompson PJ, d’Udekem Y, Konstantinov IE. Kallikrein-kinin
system: A surgical perspective in post-aprotinin era. J Surg Res. 2010, in press.
Presentations
1. Does remote ischaemic preconditioning prevent delayed hippocampal neuronal
death following transient global cerebral ischaemia in rats? Presentation at 46th
Surgical Research Society meeting, Adelaide, Australia on November 20, 2009.
2. Remote ischaemic preconditioning stimulus decreases expression of kinin
receptors in human neutrophils. Presentation at 46th Surgical Research Society
meeting, Adelaide, Australia on November 20, 2009.
88 Research Funding
Heart Foundation (Australia) grant:
1. 2008: Role of Kallikrein-Kinin system in remote ischaemic preconditioning.
National Health and Medical Research Council (Australia) grants:
2. 2008: Delayed phase of remote ischaemic preconditioning: clinical application
and the role of kallikrein-kinin pathway.
3. 2009: Identification of a plasma factor of remote ischaemic preconditioning and
its effect on the proteome after heart surgery.
4. 2009: Does remote ischaemic preconditioning induce protective mitochondrial
function in congenital heart defect surgery?
89
CHAPTER 7
REFERENCES
90 7. References
1. Bulkely BH, Hutchins GM. Myocardial consequences of coronary artery bypass
graft surgery. The paradox of necrosis in areas of revascularization. Circulation.
1977;56(6):906-13.
2. Abbo KM, Dooris M, Glazier S, O'Neill WW, Byrd D, Grines CL, et al. Features
and outcome of no-reflow after percutaneous coronary intervention. Am J
Cardiol. 1995;75(12):778-82.
3. Morishima I, Sone T, Mokuno S, Taga S, Shimauchi A, Oki Y, et al. Clinical
significance of no-reflow phenomenon observed on angiography after successful
treatment of acute myocardial infarction with percutaneous transluminal coronary
angioplasty. Am Heart J. 1995;130(2):239-43.
4. Christian TF, Gitter MJ, Miller TD, Gibbons RJ. Prospective identification of
myocardial stunning using technetium-99m sestamibi-based measurements of
infarct size. J Am Coll Cardiol. 1997;30(7):1633-40.
5. Kloner RA, Jennings RB. Consequences of brief ischemia: stunning,
preconditioning, and their clinical implications: part 2. Circulation.
2001;104(25):3158- 67.
6. Kloner RA, Jennings RB. Consequences of brief ischemia: stunning,
preconditioning, and their clinical implications: part 1. Circulation.
2001;104(24):2981-9.
7. Sheiban I, Tonni S, Marini A, Trevi G. Clinical and therapeutic implications of
chronic left ventricular dysfunction in coronary artery disease. Am J Cardiol.
1995;75(13):E23-E30.
8. Wijns W, Serruys PW, Slager CJ, Grimm J, Krayenbuehl HP, Hugenholtz PG, et
al. Effect of coronary occlusion during percutaneous transluminal angioplasty in
humans on left ventricular chamber stiffness and regional diastolic pressure-
radius relations. J Am Coll Cardiol. 1986;7(3):455-63.
9. Margarit C, Asensio M, Dávila R, Ortega J, Iglesias J, Tormo R, et al. Analysis of
risk factors following pediatric liver transplantation. Transpl Int. 2000;13 Suppl
1:S150-3.
10. Pokorny H, Gruenberger T, Soliman T, Rockenschaub S, Längle F, Steininger R.
Organ survival after primary dysfunction of liver grafts in clinical orthotopic liver
transplantation. Transpl Int. 2000;13 Suppl 1:S154-7.
91 11. Clavien PA, Harvey PR, Strasberg SM. Preservation and reperfusion injuries in
liver allografts. An overview and synthesis of current studies. Transplantation.
1992;53(5):957-78.
12. Fellström B, Akϋyrek LM, Backman U, Larsson E, Melin J, Zezina L.
Postischemic reperfusion injury and allograft arteriosclerosis. Transplant Proc.
1998;30(8):4278-80.
13. Collard CD, Gelman S. Pathophysiology, clinical manifestations, and prevention
of ischemia-reperfusion injury. Anesthesiology. 2001;94(6):1133-8.
14. Klausner JM, Paterson IS, Mannick JA, Valeri R, Shepro D, Hechtman HB.
Reperfusion pulmonary edema. JAMA. 1989;261(7):1030-5.
15. Becker LB. New concepts in reactive oxygen species and cardiovascular
reperfusion physiology. Cardiovasc Res. 2004;61(3):461-70.
16. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of
lethal cell injury in ischemic myocardium. Circulation. 1986;74(5):1124-36.
17. Przyklenk K, Bauer B, Ovize M, Kloner R, Whittaker P. Regional ischemic
'preconditioning' protects remote virgin myocardium from subsequent sustained
coronary occlusion. Circulation. 1993;87:893-9.
18. McCully JD, Wakiyama H, Hsieh YJ, Jones M, Levitsky S. Differential
contribution of necrosis and apoptosis in myocardial ischemia-reperfusion injury.
Am J Physiol Heart Circ Physiol. 2004;286(5):H1923-35.
19. Hinata M, Kimura J. Forefront of Na+/Ca2+ exchanger studies: stoichiometry of
cardiac Na+/Ca2+ exchanger; 3:1 or 4:1? J Pharmacol Sci. 2004;96(1):15-8.
20. Meldrum DR, Cleveland JC, Jr., Sheridan BC, Rowland RT, Banerjee A, Harken
AH. Cardiac surgical implications of calcium dyshomeostasis in the heart. Ann
Thorac Surg. 1996;61(4):1273-80.
21. Ernster L. Biochemistry of reoxygenation injury. Crit Care Med.
1988;16(10):947-53.
22. Seal JB, Gewertz BL. Vascular dysfunction in ischemia-reperfusion injury. Ann
Vasc Surg. 2005;19(4):572-84.
23. Langer GA. Sodium-calcium exchange in the heart. Annu Rev Physiol.
1982;44:435-49.
24. Carden DL, Granger DN. Pathophysiology of ischaemia-reperfusion injury. J
Pathol. 2000;190(3):255-66.
25. Bolli R. Causative role of oxyradicals in myocardial stunning: a proven
hypothesis. Basic Res Cardiol. 1998;93(3):156-62.
92 26. Jennings RB, Sommers HM, Smyth GA, Flack HA, Linn H. Myocardial necrosis
induced by temporary occlusion of a coronary artery in the dog. Arch Pathol.
1960;70:68-78.
27. Hensley K, Robinson KA, Gabbita SP, Salsman S, Floyd RA. Reactive oxygen
species, cell signaling, and cell injury. Free Radic Biol Med. 2000;28(10):1456-
62.
28. Szabó G, Liaudet L, Hagl S, Szabó C. Poly(ADP-ribose) polymerase activation in
the reperfused myocardium. Cardiovasc Res. 2004;61(3):471-80.
29. Hill JH, Ward PA. The phlogistic role of C3 leukotactic fragments in myocardial
infarcts of rats. J Exp Med. 1971;133(4):885-900.
30. Arumugam TV, Shiels IA, Woodruff TM, Granger DN, Taylor SM. The role of
the complement system in ischemia-reperfusion injury. Shock. 2004;21(5):401.
31. Goldstein IM, Weissmann G. Generation of C5-derived lysosomal enzyme-
releasing activity (C5a) by lysates of leukocyte lysosomes. J Immunol.
1974;113(5):1583-8.
32. Sacks T, Moldow CF, Craddock PR, Bowers TK, Jacob HS. Oxygen radicals
mediate endothelial cell damage by complement-stimulated granulocytes. An in
vitro model of immune vascular damage. J Clin Invest. 1978;61(5):1161-7.
33. Shin HS, Snyderman R, Friedman E, Mellors A, Mayer MM. Chemotactic and
anaphylatoxic fragment cleaved from the fifth component of guinea pig
complement. Science. 1968;162(3851):361-3.
34. Weiser MR, Williams JP, Moore Jr FD, Kobzik L, Ma M, Hechtman HB, et al.
Reperfusion injury of ischemic skeletal muscle is mediated by natural antibody
and complement. J Exp Med. 1996;183(5):2343.
35. Kaeffer N, Richard V, Francois A, Lallemand F, Henry JP, Thuillez C.
Preconditioning prevents chronic reperfusion-induced coronary endothelial
dysfunction in rats. Am J Physiol. 1996;271(3 Pt 2):H842-9.
36. Lefer AM, Lefer DJ. The role of nitric oxide and cell adhesion molecules on the
microcirculation in ischaemia-reperfusion. Cardiovasc Res. 1996;32(4):743-51.
37. Kloner RA, Ganote CE, Jennings RB. The "no-reflow" phenomenon after
temporary coronary occlusion in the dog. J Clin Invest. 1974;54(6):1496-508.
38. Claeys MJ, Bosmans J, Veenstra L, Jorens P, De R, Vrints CJ. Determinants and
prognostic implications of persistent ST-segment elevation after primary
angioplasty for acute myocardial infarction: importance of microvascular
reperfusion injury on clinical outcome. Circulation. 1999;99(15):1972- 7.
93 39. Kloner RA, Ganote CE, Jennings RB. The “no-reflow” phenomenon after
temporary coronary occlusion in the dog. J Clin Invest. 1974;54(6):1496- 508.
40. Petrishchev NN, Vlasov TD, Sipovsky VG, Kurapeev DI, Galagudza MM. Does
nitric oxide generation contribute to the mechanism of remote ischemic
preconditioning? Pathophysiology. 2001;7(4):271-4.
41. Xiao L, Lu R, Hu CP, Deng HW, Li YJ. Delayed cardioprotection by intestinal
preconditioning is mediated by calcitonin gene-related peptide. Eur J Pharmacol.
2001;427(2):131-5.
42. Bolli R. Cardioprotective function of inducible nitric oxide synthase and role of
nitric oxide in myocardial ischemia and preconditioning: an overview of a decade
of research. J Mol Cell Cardiol. 2001;33(11):1897-918.
43. Oldenburg O, Qin Q, Sharma AR, Cohen MV, Downey JM, Benoit JN.
Acetylcholine leads to free radical production dependent on K(ATP) channels,
G(i) proteins, phosphatidylinositol 3-kinase and tyrosine kinase. Cardiovasc Res.
2002;55(3):544-52.
44. Qin Q, Yang XM, Cui L, Critz SD, Cohen MV, Browner NC, et al. Exogenous
NO triggers preconditioning via a cGMP-and mitoKATP-dependent mechanism.
Am J Physiol Heart Circ Physiol. 2004;287(2):H712-8.
45. Shahid M, Tauseef M, Sharma KK, Fahim M. Brief femoral artery ischaemia
provides protection against myocardial ischaemia-reperfusion injury in rats: the
possible mechanisms. Exp Physiol. 2008;93(8):954-68.
46. Konstantinov IE, Arab S, Kharbanda RK, Li J, Cheung MM, Cherepanov V, et al.
The remote ischemic preconditioning stimulus modifies inflammatory gene
expression in humans. Physiol Genomics. 2004;19(1):143-50.
47. Harkin D, Barros D’Sa A, McCallion K, Hoper M, Campbell F. Ischemic
preconditioning before lower limb ischemia-reperfusion protects against acute
lung injury. J Vasc Surg. 2002;35:1264-73.
48. Park JL, Lucchesi BR. Mechanisms of myocardial reperfusion injury. Ann Thorac
Surg. 1999;68(5):1905-12.
49. Weiss SJ. Tissue destruction by neutrophils. N Engl J Med. 1989;320(6):365-76.
50. Springer TA. Traffic signals for lymphocyte recirculation and leukocyte
emigration: the multistep paradigm. Cell. 1994;76(2):301-14.
51. Diamond MS, Springer TA. A subpopulation of Mac-1 (CD11b/CD18) molecules
mediates neutrophil adhesion to ICAM-1 and fibrinogen. J Cell Biol.
1993;120(2):545-56.
94 52. Kukielka GL, Hawkins HK, Michael L, Manning AM, Youker K, Lane C, et al.
Regulation of intercellular adhesion molecule-1 (ICAM-1) in ischemic and
reperfused canine myocardium. J Clin Invest. 1993;92:1504-16.
53. Rubin BB, Chang G, Liauw S, Young A, Romaschin A, Walker PM.
Phospholipid peroxidation deacylation and remodeling in postischemic skeletal
muscle. Am J Physiol Heart Circ Physiol. 1992;263(6):H1695-702.
54. Patel HH, Moore J, Hsu AK, Gross GJ. Cardioprotection at a distance: mesenteric
artery occlusion protects the myocardium via an opioid sensitive mechanism. J
Mol Cell Cardiol. 2002;34:1317-23.
55. Dickson EW, Tubbs RJ, Porcaro WA, Lee WJ, Blehar DJ, Carraway RE, et al.
Myocardial preconditioning factors evoke mesenteric ischemic tolerance via
opioid receptors and K(ATP) channels. Am J Physiol Heart Circ Physiol.
2002;283(1):H22-8.
56. Noda K, Sasaguri M, Ideishi M, Ikeda M, Arakawa K. Role of locally formed
angiotensin II and bradykinin in the reduction of myocardial infarct size in dogs.
Cardiovasc Res. 1993;27(2):334-40.
57. Leesar MA, Stoddard MF, Manchikalapudi S, Bolli R. Bradykinin-induced
preconditioning in patients undergoing coronary angioplasty. J Am Coll Cardiol.
1999;34(3):639-50.
58. Saxena P, Newman MAJ, Shehatha JS, Redington AN, Konstantinov IE. Remote
ischemic conditioning: Evolution of the concept, mechanisms, and clinical
application. J Card Surg.25(1):127-34.
59. Neely JR, Grotyohann LW. Role of glycolytic products in damage to ischemic
myocardium. Dissociation of adenosine triphosphate levels and recovery of
function of reperfused ischemic hearts. Circ Res. 1984;55(6):816-24.
60. Lawson CS. Does ischaemic preconditioning occur in the human heart?
Cardiovasc Res. 1994;28(10):1461-6.
61. Yellon DM, Downey JM. Preconditioning the myocardium: from cellular
physiology to clinical cardiology. Physiol Rev. 2003;83(4):1113-51.
62. Taggart P, Yellon D. Preconditioning and arrhythmias. Circulation.
2002;106:2999-3001.
63. Li YW, Whittaker P, Kloner RA. The transient nature of the effect of ischemic
preconditioning on myocardial infarct size and ventricular arrhythmia. Am Heart
J. 1992;123(2):346-53.
95 64. Barbosa V, Sievers RE, Zaugg CE, Wolfe CL. Preconditioning ischemia time
determines the degree of glycogen depletion and infarction size reduction in rat
hearts. Am Heart J. 1996;131(2):224-30.
65. Liem DA, van den Doel MA, de Zeeuw S, Verdouw PD, Duncker DJ. Role of
adenosine in ischemic preconditioning in rats depends critically on the duration of
the stimulus and involves both A(1) and A(3) receptors. Cardiovasc Res.
2001;51(4):701-8.
66. Alkhulaifi A, Pugsley W, Yellon D. The influence of the time period between
preconditioning ischemia and prolonged ischemia on myocardial protection.
Cardioscience. 1993;4:163-9.
67. Burckhartt B, Yang X, Tsuchida A, Mullane K, Downey J, Cohen M. Adenosine
extends the window of protection afforded by ischemic preconditioning in
conscious rabbits. Cardiovasc Res. 1995;29:653-7.
68. Bolli R. The early and late phases of preconditioning against myocardial stunning
and the essential role of oxyradicals in the late phase: an overview. Basic Res
Cardiol. 1996;91(1):57-63.
69. Bolli R. The late phase of preconditioning. Circ Res. 2000;87(11):972-83.
70. Marber MS, Latchman DS, Walker JM, Yellon DM. Cardiac stress protein
elevation 24 hours after brief ischemia or heat stress is associated with resistance
to myocardial infarction. Circulation. 1993;88(3):1264-72.
71. Pell TJ, Baxter GF, Yellon DM, Drew GM. Renal ischemia preconditions
myocardium: role of adenosine receptors and ATP-sensitive potassium channels.
Am J Physiol Heart Circ Physiol. 1998;275(5):H1542-7.
72. Gho BC, Schoemaker RG, van den Doel MA, Duncker DJ, Verdouw PD.
Myocardial protection by brief ischemia in non-cardiac tissue. Circulation.
1996;94:2193-200.
73. Wang Y, Xu H, Mizoguchi K, Oe M, Maeta H. Intestinal ischemia induces late
preconditioning against myocardial infarction: a role for inducible nitric oxide
synthase. Cardiovasc Res. 2001;49(2):391-8.
74. Wolfrum S, Schneider K, Heidbreder M, Nienstedt J, Dominiak P, Dendorfer A.
Remote preconditioning protects the heart by activating myocardial PKCε-
isoform. Cardiovasc Res. 2002;55(3):583-9.
96 75. Kharbanda RK, Peters M, Walton B, Kattenhorn M, Mullen M, Klein N, et al.
Ischemic preconditioning prevents endothelial injury and systemic neutrophil
activation during ischemia-reperfusion in humans in vivo. Circulation.
2001;103(12):1624-30.
76. Tang Z, Dai W, Li Y, Deng H. Involvement of capsaicin-sensitive sensory nerves
in early and delayed cardioprotection induced by a brief ischemia of the small
intestine. Naunyn Schmiedebergs Arch Pharmacol. 1999;359:243-7.
77. Kharbanda RK, Li J, Konstantinov IE, Cheung MMH, White PA, Frndova H, et
al. Remote ischaemic preconditioning protects against cardiopulmonary bypass-
induced tissue injury: a preclinical study. Heart. 2006;92(10):1506-11.
78. Kharbanda RK, Mortensen UM, White PA, Kristiansen SB, Schmidt MR,
Hoschtitzky JA, et al. Transient limb ischemia induces remote ischemic
preconditioning in vivo. Circulation. 2002;106(23):2881-3.
79. Moses MA, Addison PD, Neligan PC, Ashrafpour H, Huang N, Zair M, et al.
Mitochondrial KATP channels in hindlimb remote ischemic preconditioning of
skeletal muscle against infarction. Am J Physiol Heart Circ Physiol.
2005;288(2):H559-67.
80. Küntscher MV, Schirmbeck EU, Menke H, Klar E, Gebhard MM, Germann G.
Ischemic preconditioning by brief extremity ischemia before flap ischemia in a rat
model. Plast Reconstr Surg. 2002;109(7):2398-404.
81. Moses MA, Addison PD, Neligan PC, Ashrafpour H, Huang N, McAllister SE, et
al. Inducing late phase of infarct protection in skeletal muscle by remote
preconditioning: Efficacy and mechanism. Am J Physiol Reg Integr Comp
Physiol. 2005;289(6):R1609-17.
82. Schmidt MR, Smerup M, Konstantinov IE, Shimizu M, Li J, Cheung M, et al.
Intermittent peripheral tissue ischemia during coronary ischemia reduces
myocardial infarction through a KATP-dependent mechanism: first demonstration
of remote ischemic perconditioning. Am J Physiol Heart Circ Physiol.
2007;292(4):H1883-90.
83. Kerendi F, Kin H, Halkos ME, Jiang R, Zatta AJ, Zhao ZQ, et al. Remote
postconditioning. Basic Res Cardiol. 2005;100(5):404-12.
84. Andreka G, Vertesaljai M, Szantho G, Font G, Piroth Z, Fontos G, et al. Remote
ischaemic postconditioning protects the heart during acute myocardial infarction
in pigs. Heart. 2007;93(6):749-52.
97 85. Galagudza M, Kurapeev D, Minasian S, Valen G, Vaage J. Ischemic
postconditioning: brief ischemia during reperfusion converts persistent ventricular
fibrillation into regular rhythm. Eur J Cardiothorac Surg. 2004;25:1006-10.
86. Loukogeorgakis SP, Williams R, Panagiotidou AT, Kolvekar SK, Donald A, Cole
TJ, et al. Transient limb ischemia induces remote preconditioning and remote
postconditioning in humans by a K(ATP)-channel dependent mechanism.
Circulation. 2007;116(12):1386-95.
87. Shimizu M, Konstantinov IE, Kharbanda RK, Cheung MH, Redington AN.
Effects of intermittent lower limb ischaemia on coronary blood flow and coronary
resistance in pigs. Acta Physiol. 2007;190(2):103-9.
88. Gross GJ, Kersten JR, Warltier DC. Mechanisms of postischemic contractile
dysfunction. Ann Thorac Surg. 1999;68:1898-904.
89. Rinder CS, Fontes M, Mathew JP, Rinder HM, Smith BR. Neutrophil CD11b
upregulation during cardiopulmonary bypass is associated with postoperative
renal injury. Ann Thorac Surg. 2003;75(3):899-905.
90. Healy DG, Wood AE, O’Neill A, McCarthy JF, Fitzpatrick JM, Watson RW. Can
preoperative modelling of individual neutrophil adhesion responses predict renal
morbidity? Eur J Cardiothorac Surg. 2007;31(6):1088-93.
91. Wang L, Oka N, Tropak M, Callahan J, Lee J, Wilson G, et al. Remote ischemic
preconditioning elaborates a transferable blood-borne effector that protects
mitochondrial structure and function and preserves myocardial performance after
neonatal cardioplegic arrest. J Thorac Cardiovasc Surg. 2008;136(2):335-42.
92. Shimizu M, Saxena P, Konstantinov IE, Cherepanov V, Cheung MM, Wearden P,
et al. Remote ischemic preconditioning decreases adhesion and selectively
modifies functional responses of human neutrophils. J Surg Res. 2008 Nov 12.
93. Konstantinov IE, Li J, Cheung MM, Shimizu M, Stokoe J, Kharbanda RK, et al.
Remote ischemic preconditioning of the recipient reduces myocardial ischemia-
reperfusion injury of the denervated donor heart via a Katp channel-dependent
mechanism. Transplantation. 2005;79(12):1691-5.
94. Kristiansen SB, Henning O, Kharbanda RK, Nielsen-Kudsk JE, Schmidt MR,
Redington AN, et al. Remote preconditioning reduces ischemic injury in the
explanted heart by a KATP channel-dependent mechanism. Am J Physiol Heart
Circ Physiol. 2005;288(3):H1252-6.
98 95. Costa ADT, Garlid KD, West IC, Lincoln TM, Downey JM, Cohen MV, et al.
Protein kinase G transmits the cardioprotective signal from cytosol to
mitochondria. Circ Res. 2005;97(4):329-36.
96. Costa ADT, Jakob R, Costa CL, Andrukhiv K, West IC, Garlid KD. The
mechanism by which the mitochondrial ATP-sensitive K+ channel opening and
H2O2 inhibit the mitochondrial permeability transition. J Biol Chem.
2006;281(30):20801- 8.
97. Costa ADT, Pierre SV, Cohen MV, Downey JM, Garlid KD. cGMP signalling in
pre- and post-conditioning: the role of mitochondria. Cardiovasc Res.
2008;77(2):344-52.
98. Oldenburg O, Qin Q, Krieg T, Yang XM, Philipp S, Critz SD, et al. Bradykinin
induces mitochondrial ROS generation via NO, cGMP, PKG, and mitoKATP
channel opening and leads to cardioprotection. Am J Physiol Heart Circ Physiol.
2004;286(1):468-76.
99. Schoemaker RG, Van Heijningen CL. Bradykinin mediates cardiac
preconditioning at a distance. Am J Physiol Heart Circ Physiol
2000;278(5):H1571-6.
100. Pizard A, Blaukat A, Muller-Esterl W, François A-G, Rajerison RM. Bradykinin-
induced internalization of the human B2 receptor requires phosphorylation of
three serine and two threonine residues at Its carboxyl tail. J Biol Chem.
1999;274(18):12738-47.
101. Tong H, Rockman HA, Koch WJ, Steenbergen C, Murphy E. G protein-coupled
receptor internalization signaling is required for cardioprotection in ischemic
preconditioning. Circ Res. 2004;94(8):1133-41.
102. Murphy E, Wong R, Steenbergen C. Signalosomes: delivering cardioprotective
signals from GPCRs to mitochondria. Am J Physiol Heart Circ Physiol.
2008;295(3):H920-2.
103. Quinlan CL, Costa ADT, Costa CL, Pierre SV, Dos Santos P, Garlid KD.
Conditioning the heart induces formation of signalosomes that interact with
mitochondria to open mitoKATP channels. Am J Physiol Heart Circ Physiol.
2008;295(3):H953-61.
104. Daemen MA, de Vries B, Buurman WA. Apoptosis and inflammation in renal
reperfusion injury. Transplantation. 2002;73(11):1693-700.
99 105. Wang NP, Bufkin BL, Nakamura M, Zhao ZQ, Wilcox JN, Hewan-Lowe KO, et
al. Ischemic preconditioning reduces neutrophil accumulation and myocardial
apoptosis. Ann Thorac Surg. 1999;67(6):1689-95.
106. Fliss H, Gattinger D. Apoptosis in ischemic and reperfused rat myocardium. Circ
Res. 1996;79(5):949-56.
107. Ren C, Gao X, Steinberg GK, Zhao H. Limb remote-preconditioning protects
against focal ischemia in rats and contradicts the dogma of therapeutic time
windows for preconditioning. Neuroscience. 2008;151(4):1099-103.
108. Sun XC, Li WB, Li QJ, Zhang M, Xian XH, Qi J, et al. Limb ischemic
preconditioning induces brain ischemic tolerance via p38 MAPK. Brain Res.
2006;1084(1):165-74.
109. Zhao HG, Sun XC, Xian XH, Li WB, Zhang M, Li QJ. The role of nitric oxide in
the neuroprotection of limb ischemic preconditioning in rats. Neurochem Res.
2007;32(11):1919-26.
110. Meloni BP, Majda BT, Knuckey NW. Evaluation of preconditioning treatments to
protect near-pure cortical neuronal cultures from in vitro ischemia induced acute
and delayed neuronal death. Brain Res. 2002;928(1-2):69-75.
111. Miles AN, Majda BT, Meloni BP, Knuckey NW. Postischemic intravenous
administration of magnesium sulfate inhibits hippocampal CA1 neuronal death
after transient global ischemia in rats. Neurosurgery. 2001;49(6):1443-50;
discussion 50-1.
112. Zhu HD, Martin R, Meloni B, Oltvolgyi C, Moore S, Majda B, et al. Magnesium
sulfate fails to reduce infarct volume following transient focal cerebral ischemia
in rats. Neurosci Res. 2004;49(3):347-53.
113. Moncayo J, de Freitas GR, Bogousslavsky J, Altieri M, van Melle G. Do transient
ischemic attacks have a neuroprotective effect? Neurology. 2000;54(11):2089-94.
114. Weih M, Kallenberg K, Bergk A, Dirnagl U, Harms L, Wernecke KD, et al.
Attenuated stroke severity after prodromal TIA: a role for ischemic tolerance in
the brain? Stroke. 1999;30(9):1851-4.
115. Chan MT, Boet R, Ng SC, Poon WS, Gin T. Effect of ischemic preconditioning
on brain tissue gases and pH during temporary cerebral artery occlusion. Acta
Neurochir Suppl. 2005;95:93-6.
116. Dirnagl U, Meisel A. Endogenous neuroprotection: mitochondria as gateways to
cerebral preconditioning? Neuropharmacology. 2008;55(3):334-44.
100 117. Kirino T. Ischemic tolerance. J Cereb Blood Flow Metab. 2002;22(11):1283-96.
118. Obrenovitch TP. Molecular physiology of preconditioning-induced brain
tolerance to ischemia. Physiol Rev. 2008;88(1):211-47.
119. O'Duffy AE, Bordelon YM, McLaughlin B. Killer proteases and little strokes--
how the things that do not kill you make you stronger. J Cereb Blood Flow
Metab. 2007;27(4):655-68.
120. Abbott NJ, Revest PA, Romero IA. Astrocyte-endothelial interaction: physiology
and pathology. Neuropathol Appl Neurobiol. 1992;18(5):424-33.
121. Chen Y, Swanson RA. Astrocytes and brain injury. J Cereb Blood Flow Metab.
2003;23(2):137-49.
122. Dringen R, Gebhardt R, Hamprecht B. Glycogen in astrocytes: possible function
as lactate supply for neighboring cells. Brain Res. 1993;623(2):208-14.
123. Bernaudin M, Tang Y, Reilly M, Petit E, Sharp FR. Brain genomic response
following hypoxia and re-oxygenation in the neonatal rat. Identification of genes
that might contribute to hypoxia-induced ischemic tolerance. J Biol Chem.
2002;277(42):39728-38.
124. Stenzel-Poore MP, Stevens SL, Xiong Z, Lessov NS, Harrington CA, Mori M, et
al. Effect of ischaemic preconditioning on genomic response to cerebral
ischaemia: similarity to neuroprotective strategies in hibernation and hypoxia-
tolerant states. Lancet. 2003;362(9389):1028-37.
125. Tang Y, Pacary E, Fréret T, Divoux D, Petit E, Schumann-Bard P, et al. Effect of
hypoxic preconditioning on brain genomic response before and following
ischemia in the adult mouse: identification of potential neuroprotective candidates
for stroke. Neurobiol Dis. 2006;21(1):18-28.
126. Ran R, Xu H, Lu A, Bernaudin M, Sharp FR. Hypoxia preconditioning in the
brain. Dev Neurosci. 2005;27(2-4):87-92.
127. Kaelin WG, Jr., Ratcliffe PJ. Oxygen sensing by metazoans: the central role of
the HIF hydroxylase pathway. Mol Cell. 2008;30(4):393-402.
128. Bergeron M, Gidday JM, Yu AY, Semenza GL, Ferriero DM, Sharp FR. Role of
hypoxia-inducible factor-1 in hypoxia-induced ischemic tolerance in neonatal rat
brain. Ann Neurol. 2000;48(3):285-96.
129. Marti HJH, Bernaudin M, Bellail A, Schoch H, Euler M, Petit E, et al. Hypoxia-
induced vascular endothelial growth factor expression precedes
neovascularization after cerebral ischemia. Am J Pathol. 2000;156(3):965.
101 130. Bernaudin M, Nedelec AS, Divoux D, MacKenzie ET, Petit E, Schumann-Bard P.
Normobaric hypoxia induces tolerance to focal permanent cerebral ischemia in
association with an increased expression of hypoxia-inducible factor-1 and its
target genes, erythropoietin and VEGF, in the adult mouse brain. J Cereb Blood
Flow Metab. 2002;22(4):393-403.
131. Prass K, Scharff A, Ruscher K, Lowl D, Muselmann C, Victorov I, et al.
Hypoxia-induced stroke tolerance in the mouse is mediated by erythropoietin.
Stroke. 2003;34(8):1981- 6.
132. Dirnagl U, Simon RP, Hallenbeck JM. Ischemic tolerance and endogenous
neuroprotection. Trends Neurosci. 2003;26(5):248-54.
133. Dawson DA, Furuya K, Gotoh J, Nakao Y, Hallenbeck JM. Cerebrovascular
hemodynamics and ischemic tolerance: lipopolysaccharide-induced resistance to
focal cerebral ischemia is not due to changes in severity of the initial ischemic
insult, but is associated with preservation of microvascular perfusion. J Cereb
Blood Flow Metab. 1999;19(6):616-23.
134. Gustavsson M, Mallard C, Vannucci SJ, Wilson MA, Johnston MV, Hagberg H.
Vascular response to hypoxic preconditioning in the immature brain. J Cereb
Blood Flow Metab. 2007;27(5):928-38.
135. Sun Y, Jin K, Xie L, Childs J, Mao XO, Logvinova A, et al. VEGF-induced
neuroprotection, neurogenesis, and angiogenesis after focal cerebral ischemia. J
Clin Invest. 2003;111(12):1843-51.
136. Wang X, Deng J, Boyle DW, Zhong J, Lee WH. Potential role of IGF-I in
hypoxia tolerance using a rat hypoxic-ischemic model: activation of hypoxia-
inducible factor 1alpha. Pediatr Res. 2004;55(3):385-94.
137. Andjelkovic AV, Stamatovic SM, Keep RF. The protective effects of
preconditioning on cerebral endothelial cells in vitro. J Cereb Blood Flow Metab.
2003;23(11):1348-55.
138. Zhang Y, Park TS, Gidday JM. Hypoxic preconditioning protects human brain
endothelium from ischemic apoptosis by Akt-dependent survivin activation. Am J
Physiol Heart Circ Physiol. 2007;292(6):H2573-81.
139. Brucklacher RM, Vannucci RC, Vannucci SJ. Hypoxic preconditioning increases
brain glycogen and delays energy depletion from hypoxia-ischemia in the
immature rat. Dev Neurosci. 2002;24(5):411-7.
140. Rothman SM, Olney JW. Excitotoxicity and the NMDA receptor-still lethal after
eight years. Trends Neurosci. 1995;18(2):57-8.
102 141. Dirnagl U, Becker K, Meisel A. Preconditioning and tolerance against cerebral
ischaemia: from experimental strategies to clinical use. Lancet Neurol.
2009;8(4):398-412.
142. Lee SH, Kim YJ, Lee KM, Ryu S, Yoon BW. Ischemic preconditioning enhances
neurogenesis in the subventricular zone. Neuroscience. 2007;146(3):1020-31.
143. Naylor M, Bowen KK, Sailor KA, Dempsey RJ, Vemuganti R. Preconditioning-
induced ischemic tolerance stimulates growth factor expression and neurogenesis
in adult rat hippocampus. Neurochem Int. 2005;47(8):565-72.
144. Theus MH, Wei L, Cui L, Francis K, Hu X, Keogh C, et al. In vitro hypoxic
preconditioning of embryonic stem cells as a strategy of promoting cell survival
and functional benefits after transplantation into the ischemic rat brain. Exp
Neurol. 2008;210(2):656-70.
145. Kuhn HG, Winkler J, Kempermann G, Thal LJ, Gage FH. Epidermal Growth
Factor and Fibroblast Growth Factor-2 Have Different Effects on Neural
Progenitors in the Adult Rat Brain. J Neurosci. 1997;17(15):5820-9.
146. Jin K, Minami M, Lan JQ, Mao XO, Batteur S, Simon RP, et al. Neurogenesis in
dentate subgranular zone and rostral subventricular zone after focal cerebral
ischemia in the rat. Proc Natl Acad Sci U S A. 2001;98(8):4710-5.
147. Arab S, Konstantinov IE, Boscarino C, Cukerman E, Mori A, Li J, et al. Early
gene expression profiles during intraoperative myocardial ischemia-reperfusion in
cardiac surgery. J Thorac Cardiovasc Surg. 2007;134(1):74-81, e1-2.
148. Konstantinov IE, Arab S, Li J, Coles JG, Boscarino C, Mori A, et al. The remote
ischemic preconditioning stimulus modifies gene expression in mouse
myocardium. J Thorac Cardiovasc Surg. 2005;130(5):1326-32.
149. Liu GS, Thornton JBS, van Winkle DM, Stanley AWH, Olsson RA, Downey JM.
Protection against infarction afforded by preconditioning is mediated by A1
adenosine receptors in rabbit heart. Circulation. 1991;84:350-6.
150. Thornton JD, Liu GS, Olsson RA, Downey JM. Intravenous pretreatment with
A1-selective adenosine analogues protects the hearts against infarction.
Circulation. 1992;85:659-65.
151. Liu GS, Richards SC, Olsson RA, Mullane K, Walsh RS, Downey JM. Evidence
that the A3 receptor may mediate the protection afforded by preconditioning in
the isolated rabbit heart. Cardiovasc Res. 1994;28:1057-61.
103 152. Schultz JEJ, Hsu AK, Gross GJ. Ischemic preconditioning in the intact rat heart is
mediated by delta-1 but not mu- or kappa-opioid receptors. Circulation.
1998;97:1282-9.
153. Hu K, Nattel S. Mechanisms of ischemic preconditioning in rat hearts:
Involvement of alpha 1B-adrenoreceptors, pertussis toxin-sensitive G proteins,
and protein kinase C. Circulation. 1995;92:2259-65.
154. Baines CP, Goto M, Downey JM. Oxygen radicals released during ischemic
preconditioning contribute to cardioprotection in the rabbit myocardium. J Mol
Cell Cardiol. 1997;29:207-16.
155. Goto M, Liu Y, Yang XM, Ardell JL, Cohen MV, Downey JM. Role of
bradykinin in protection of ischemic preconditioning in rabbit hearts. Circ Res.
1995;77(3):611-21.
156. Stenzel-Poore MP, Stevens SL, King JS, Simon RP. Preconditioning reprograms
the response to ischemic injury and primes the emergence of unique endogenous
neuroprotective phenotypes: a speculative synthesis. Stroke. 2007;38(2):680-5.
157. Podgoreanu MV, Michelotti GA, Sato Y, Smith MP, Lin S, Morris RW, et al.
Differential cardiac gene expression during cardiopulmonary bypass: ischemia-
independent upregulation of proinflammatory genes. J Thorac Cardiovasc Surg.
2005;130(2):330-9.
158. Yan SF, Fujita T, Lu J, Okada K, Zou YS, Mackman N, et al. Egr-1, a master
switch coordinating upregulation of divergent gene families underlying ischemic
stress. Nat Med. 2000;6(12):1355-61.
159. Xuan Y-T, Tang X-L, Banerjee S, Takano H, Li RCX, Han H, et al. Nuclear
factor-kB plays an essential role in the late phase of ischemic preconditioning in
conscious rabbits. Circ Res. 1999;84:1095-109.
160. Hausenloy DJ, Tsang A, Mocanu MM, Yellon DM. Ischemic preconditioning
protects by activating prosurvival kinases at reperfusion. Am J Physiol Heart Circ
Physiol. 2005;288:H971-6.
161. Tsang A, Hausenloy DJ, Mocanu MM, Yellon DM. Postconditioning: a form of
"modified reperfusion" protects the myocardium by activating the
phosphatidylinositol 3-kinase-Akt pathway. Circ Res. 2004;95:230-2.
162. Li G, Labruto F, Sirsjo A, Chen F, Vaage J, Valen G. Myocardial protection by
remote preconditioning: the role of nuclear factor kappa-B p105 and inducible
nitric oxide synthase. Eur J Cardiothorac Surg. 2004;26(5):968-73.
104 163. Strohm C, Barancik M, von Bruehl M-L, Strniskova M, Ullmann C,
Zimmermann R, et al. Transcription inhibitor actinomycin-D abolishes the
cardioprotective effect of ischemic reconditioning. Cardiovasc Res.
2002;55(3):602-18.
164. Zeeuw SD, van den Doel MA, Duncker DJ, Verdouw PD. New insights into
cardioprotection by ischemic preconditioning and other forms of stress. Ann N Y
Acad Sci. 1999;874:178-91.
165. Tähepôld P, Valen G, Starkopf J, Kairane C, Zilmer M, Vaage J. Pretreating rats
with hyperoxia attenuates ischemia-reperfusion injury of the heart. Life Sci.
2001;68:1629-40.
166. Bolli R, Dawn B, Tang X-L, Qiu Y, Ping P, Xuan Y-T, et al. The nitric oxide
hypothesis of late preconditioning. Basic Res Cardiol. 1998;93(5):325-38.
167. Nelson SK, Wong GHW, McCord JM. Leukemia inhibitory factor and tumor
necrosis factor induce manganese superoxide dismutase and protect rabbit hearts
from reperfusion injury. J Moll Cell Cardiol. 1995;27:231-42.
168. Yamashita N, Hoshida S, Otsu K, Taniguchi N, Kuzuya T, Hori M. The
involvement of cytokines in the second window of ischemic preconditioning. Br J
Pharmacol. 2000;131(3):415-22.
169. Eddy LJ, Goeddel DV, Wong GHW. Tumor necrosis factor-α pre-treatment is
protective in a rat model of myocardial ischemia-reperfusion injury. Biochem
Biophys Res Commun. 1992;184(2):1056-9.
170. Asea A, Rehli M, Kabingu E, Boch JA, Baré O, Auron PE, et al. Novel signal
transduction pathway utilized by extracellular HSP70: role of toll-like receptor
(TLR) 2 and TLR4. J Biol Chem. 2002;277(17):15028-34.
171. Oyama J, Blais C, Jr., Liu X, Pu M, Kobzik L, Kelly RA, et al. Reduced
myocardial ischemia-reperfusion injury in toll-like receptor 4-deficient mice.
Circulation. 2004;109(6):784-9.
172. Remer KA, Brcic M, Jungi TW. Toll-like receptor-4 is involved in eliciting an
LPS-induced oxidative burst in neutrophils. Immunol Lett. 2002;85(1):75-80.
173. Murphy CG, Chen G, Winter DC, Bouchier-Hayes DJ. Glutamine
preconditioning protects against tourniquet-induced local and distant organ injury
in a rodent ischemia-reperfusion model. Acta Orthop. 2007;78(4):559-66.
174. Feng J, Bianchi C, Sandmeyer JL, Sellke FW. Bradykinin preconditioning
improves the profile of cell survival proteins and limits apoptosis after
cardioplegic arrest. Circulation. 2005;112(9 Suppl):I190-5.
105 175. Mocanu MM, Bell RM, Yellon DM. PI3 kinase and not p42/p44 appears to be
implicated in the protection conferred by ischemic preconditioning. J Mol Cell
Cardiol. 2002;34(6):661-8.
176. Tong H, Chen W, Steenbergen C, Murphy E. Ischemic preconditioning activates
phosphatidylinositol-3-kinase upstream of protein kinase C. Circ Res.
2000;87(4):309-15.
177. Qin S, Chock PB. Implication of phosphatidylinositol 3-kinase membrane
recruitment in hydrogen peroxide-induced activation of PI3K and Akt.
Biochemistry (Mosc). 2003;42(10):2995-3003.
178. Cross HR, Murphy E, Bolli R, Ping P, Steenbergen C. Expression of activated
PKC Epsilon (PKC ε) protects the ischemic heart, without attenuating ischemic
H+ production. J Mol Cell Cardiol. 2002;34(3):361-7.
179. Ping P, Song C, Zhang J, Guo Y, Cao X, Li RCX, et al. Formation of protein
kinase C ε-Lck signaling modules confers cardioprotection. J Clin Invest.
2002;109(4):499-507.
180. Baines CP, Goto M, Downey JM. Oxygen radicals released during ischemic
preconditioning contribute to cardioprotection in the rabbit myocardium. J Mol
Cell Cardiol. 1997;29(1):207-16.
181. Baines CP, Zhang J, Wang G-W, Zheng Y-T, Xiu JX, Cardwell EM, et al.
Mitochondrial PKC(epsilon) and MAPK form signaling modules in the murine
heart: enhanced mitochondrial PKC(epsilon)- MAPK interactions and differential
MAPK activation in PKC(epsilon)- induced cardioprotection. Circ Res.
2002;90(4):390-7.
182. Hausenloy DJ, Yellon DM. Preconditioning and postconditioning: underlying
mechanisms and clinical application. Atherosclerosis. 2009;204(2):334-41.
183. O’Rourke B. Evidence for mitochondrial K+ channels and their role in
cardioprotection. Circ Res. 2004;94:420-32.
184. Pasyk EA, Kang Y, Huang X, Cui N, Sheu L, Gaisano HY. Syntaxin-1A binds
the nucleotide-binding folds of sulphonylurea receptor 1 to regulate the KATP
channel. J Biol Chem. 2004;279(6):4234-40.
185. Leung YM, Kwan EP, Ng B, Kang Y, Gaisano HY. SNAREing voltage-gated K+
and ATP-sensitive K+ channels: tuning β-cell excitability with syntaxin-1A and
other exocytotic proteins. Endocr Rev. 2007;28(6):653-63.
106 186. Dos Santos P, Kowaltowski AJ, Laclau MN, Seetharaman S, Paucek P, Boudina
S, et al. Mechanisms by which opening the mitochondrial ATP- sensitive K+
channel protects the ischemic heart. Am J Physiol Heart Circ Physiol.
2002;283:H284-95.
187. Vander Heide RS, Hill ML, Reimer KA, Jennings RB. Effects of reversible
ischemia on the activity of the mitochondrial ATPase: relationship to ischemic
preconditioning. J Mol Cell Cardiol 1996;28(1):103-12.
188. Vuorinen K, Ylitalo K, Peuhkurinen K, Raatikainen P, Ala-Rämi A, Hassinen IE.
Mechanism of ischemic preconditioning in rat myocardium. Circulation.
1995;91:2810-8.
189. Mandle RJ, Colman RW, Kaplan AP. Identification of prekallikrein and high-
molecular-weight kininogen as a complex in human plasma. Proc Natl Acad Sci
U S A. 1976;73(11):4179-83.
190. Royston D, Taylor KM, Bidstrup BP, Sapsford RN. Effect of aprotinin on need
for blood transfusion after repeat open-heart surgery. Lancet.
1987;330(8571):1289-91.
191. Bhoola KD, Figueroa CD, Worthy K. Bioregulation of kinins: kallikreins,
kininogens, and kininases. Pharmacol Rev. 1992;44(1):1-80.
192. Kaplan AP, Joseph K, Shibayama Y, Reddigarri S, Ghebrehiwet B, Silverberg M.
The intrinsic coagulation/kinin-forming cascade: assembly in plasma and cell
surfaces in inflammation. Adv Immunol. 1997;66:225-72.
193. Merlini PA, Cugno M, Rossi ML, Agricola P, Repetto A, Fetiveau R, et al.
Activation of the contact system and inflammation after thrombolytic therapy in
patients with acute myocardial infarction. Am J Cardiol. 2004;93(7):822-5.
194. Lin Y, Pixley RA, Colman RA. Kinetic analysis of the role of zinc in the
interaction of domain 5 of high-molecular weight kininogen (HK) with heparin.
Biochemistry (Mosc). 2000;39(17):5104-10.
195. Motta G, Rojkjaer R, Hasan AAK, Cines DB, Schmaier AH. High molecular
weight kininogen regulates prekallikrein assembly and activation on endothelial
cells: a novel mechanism for contact activation. Blood. 1998;91(2):516-28.
196. Nishikawa K, Shibayama Y, Kuna P, Calcaterra E, Kaplan AP, Reddigari SR.
Generation of vasoactive peptide bradykinin from human umbilical vein
endothelium-bound high molecular weight kininogen by plasma kallikrein.
Blood. 1992;80(8):1980-8.
107 197. Zhao Y, Qiu Q, Mahdi F, Shariat-Madar Z, Røjkjær R, Schmaier AH. Assembly
and activation of HK-PK complex on endothelial cells results in bradykinin
liberation and NO formation. Am J Physiol Heart Circ Physiol.
2001;280(4):H1821-9.
198. Mahabeer R, Bhoola KD. Kallikrein and kinin receptor genes. Pharmacol Ther.
2000;88(1):77-89.
199. Kaplan AP, Joseph K, Shibayama Y, Nakazawa Y, Ghebrehiwet B, Reddigari S,
et al. Bradykinin formation. Plasma and tissue pathways and cellular interactions.
Clin Rev Allergy Immunol. 1998;16(4):403-29.
200. Dobrovolsky AB, Titaeva EV. The fibrinolysis system: regulation of activity and
physiologic functions of its main components. Biochemistry (Mosc). 2002
Jan;67(1):99-108.
201. Bhoola KD, Fink E. Kallikrein-kinin cascade. In: Laurent G, Shapiro SD, editors.
Encyclopaedia of respiratory medicine. Oxford, U K: Elsevier Ltd; 2006. 483-93.
202. Dray A, Perkins M. Bradykinin and inflammatory pain. Trends Neurosci.
1993;16(3):99-104.
203. Wollert KC, Drexler H. The kallikrein-kinin system in post-myocardial infarction
cardiac remodeling. Am J Cardiol. 1997;80(3A):158A-61A.
204. Hall JM, Morton IKM. The pharmacology and immunopharmacology of kinin
receptors. In: Farmer SG, editor. The handbook of immunopharmacology: the
kinin system: London: Academic press; 1997. p. 9-43.
205. Marceau F, Bachvarov DR. Kinin receptors. Clin Rev Allergy Inflamm.
1998;16(4):385-401.
206. Schanstra JP, Bataille E, Marin Castano ME, Barascud Y, Hirtz C, Pesquero JB,
et al. The B1-agonist [des-Arg10]-kallidin activates transcription factor NF-
kappaB and induces homologous upregulation of the bradykinin B1-receptor in
cultured human lung fibroblasts. J Clin Invest. 1998;101(10):2080-91.
207. Ni A, Chao L, Chao J. Transcription factor nuclear factor κB regulates the
inducible expression of the human B1 receptor gene in inflammation. J Biol
Chem. 1998;273(5):2784-91.
208. Yang X, Taylor L, Polgar P. Mechanisms in the transcriptional regulation of
bradykinin B1 receptor gene expression. Identification of a minimum cell-type
specific enhancer. J Biol Chem. 1998;273(17):10763-70.
108 209. Ahluwalia A, Perretti M. Involvement of bradykinin B1 receptors in the
polymorphonuclear leukocyte accumulation induced by IL-1 beta in vivo in the
mouse. J Immunol. 1996;156(1):269-74.
210. Pesquero JB, Araujo RC, Heppenstall PA, Stucky CL, Silva JA, Jr., Walther T, et
al. Hypoalgesia and altered inflammatory responses in mice lacking kinin B1
receptors. Proc Natl Acad Sci U S A. 2000;97(14):8140-5.
211. Pan ZK, Christiansen SC, Ptasznik A, Zuraw BL. Requirement of
phosphatidylinositol 3-kinase activity for bradykinin stimulation of NF-κB
activation in cultured human epithelial cells. J Biol Chem. 1999;274(15):9918-22.
212. Pyne NJ, Tolan D, Pyne S. Bradykinin stimulates cAMP synthesis via mitogen-
activated protein kinase-dependent regulation of cytosolic phospholipase A2 and
prostaglandin E2 release in airway smooth muscle. Biochem J. 1997;328 ( Pt
2):689-94.
213. Ritchie RH, Marsh JD, Schiebinger RJ. Bradykinin-stimulated protein synthesis
by myocytes is dependent on the MAP kinase pathway and p70S6K. Am J Physiol
Heart Circ Physiol. 1999;276:H1393-98
214. Modéer F, Andurén I, Yucel-Lindberg T. Bradykinin synergistically stimulates
interleukin 6 production in human gingival fibroblasts challenged with interleukin
1 or tumor necrosis factor α. Cytokine. 1998;10(1):26-31.
215. Pan ZK, Ye RD, Christiansen SC, Jagels MA, Bokoch GM, Zuraw BL. Role of
the Rho GTPase in bradykinin-stimulated nuclear factor-κB activation and IL-1β
gene expression in cultured human epithelial cells. J Immunol. 1998;160(6):3038-
45.
216. Campos MM, Souza GEP, Calixto JB. In vivo B1 kinin-receptor upregulation.
Evidence for involvement of protein kinases and nuclear factor κB pathways. Br J
Pharmacol. 1999;127(8):1851-9.
217. Britos J, Nolly H. Kinin-forming enzyme of rat cardiac tissue. Subcellular
distribution and biochemical properties. Hypertension. 1981;3(6 Pt 2):II-42-5.
218. De Freitas FM, Faraco EZ, de Azevedo DF. General circulatory alterations
induced by intravenous infusion of synthetic bradykinin in man. Circulation.
1964;29:66-70.
219. Linz W, Wiemer G, Gohlke P, Unger T, Schölkens B. Contribution of kinin to the
cardiovascular actions of converting-enzyme inhibitors. Pharmacol Rev.
1995;47(1):25-49.
109 220. Linz W, Wiemer G, Schölkens BA. Bradykinin prevents left ventricular
hypertrophy in rats. J Hypertens. 1993;11(5):S96-7.
221. McGiff JC, Itskovitz HD, Terrango NA. The action of bradykinin and eledocin in
the canine isolated kidney: relationship to prostaglandins. Clin Sci Mol Med.
1975;49(2):125-31.
222. Nolly H, Carretero OA, Scicli AG. Kallikrein release by vascular tissue. Am J
Physiol Heart Circ Physiol. 1993;265(4 Pt 2):H1209-14.
223. Oza NB, Goud HD. Kininogenase of the aortic wall in spontaneously
hypertensive rats. J Cardiovasc Pharmacol. 1992;20 Suppl 9:S1-3.
224. Sharma JN, Uma K, Yusof APM. Left ventricular hypertrophy and its relation to
the cardiac kinin-forming system in hypertensive and diabetic rats. Int J Cardiol.
1998;63(3):229-35.
225. Webster ME, Gilmore JP. Influence of kallidin-10 on renal function. Am J
Physiol. 1964;206:714-8.
226. Schölkens BA. Kinins in the cardiovascular system. Immunopharmacology.
1996;33(1-3):209-16.
227. Brown NJ, Gainer JV, Stein CM, Vaughan DE. Bradykinin stimulates tissue
plasminogen activator release in human vasculature. Hypertension.
1999;33:1431-5.
228. Emanueli C, Maestri R, Corradi D, Marchione R, Minasi A, Tozzi MG, et al.
Dilated and failing cardiomyopathy in bradykinin B2 receptor knockout mice.
Circulation. 1999;100(23):2359-65.
229. Wei M, Wang X, Kuukasjärvi P, Laurikka J, Rinne T, Honkonen E-L, et al.
Bradykinin preconditioning in coronary artery bypass grafting. Ann Thorac Surg.
2004;78(2):492-7.
230. Pan H-L, Chen S-R, Scicli GM, Carretero OA. Cardiac interstitial bradykinin
release during ischemia is enhanced by ischemic preconditioning. Am J Physiol
Heart Circ Physiol. 2000;279:H116-21.
231. Pell TJ, Baxter GF, Yellon DM, Drew GM. Renal ischemia preconditions
myocardium: role of adenosine receptors and ATP-sensitive potassium channels.
Am J Physiol Heart Circ Physiol. 1998;275(5 Pt 2):H1542-7.
232. Chee J, Naran A, Misso NL, Thompson PJ, Bhoola KD. Expression of tissue and
plasma kallikreins and kinin B1 and B2 receptors in lung cancer. Biol Chem.
2008;389(9):1225-33.
110 233. Chee J, Singh J, Naran A, Misso NL, Thompson PJ, Bhoola KD. Novel
expression of kallikreins, kallikrein-related peptidases and kinin receptors in
human pleural mesothelioma. Biol Chem. 2007;388(11):1235-42.
234. Singh J, Naran A, Misso NL, Rigby PJ, Thompson PJ, Bhoola KD. Expression of
kallikrein-related peptidases (KRP/hK5, 7, 6, 8) in subtypes of human lung
carcinoma. Int Immunopharmacol. 2008;8(2):300-6.
235. Leeb-Lundberg LMF, Marceau F, Müller-Esterl W, Pettibone DJ, Zuraw BL.
International Union of Pharmacology. XLV. Classification of the kinin receptor
family: from molecular mechanisms to pathophysiological consequences.
Pharmacol Rev. 2005;57(1):27-77.
236. Taub JS, Guo R, Leeb-Lundberg LMF, Madden JF, Daaka Y. Bradykinin
receptor subtype 1 expression and function in prostate cancer. Cancer Res.
2003;63(9):2037-41.
237. Greco S, Elia MG, Muscella A, Romano S, Storelli C, Marsigliante S. Bradykinin
stimulates cell proliferation through an extra-cellular-regulated kinase 1 and 2-
dependent mechanism in breast cancer cells in primary culture. J Endocrinol.
2005;186:291-301.
238. Wolf WC, Evans DM, Chao L, Chao J. A synthetic tissue kallikrein inhibitor
suppresses cancer cell invasiveness. Am J Pathol. 2001;159(5):1797-805.
239. Ishihara K, Hayashi I, Yamashina S, Majima M. A potential role of bradykinin in
angiogenesis and growth of S-180 mouse tumors. Jpn J Pharmacol.
2001;87(4):318-26.
240. Rascoe PA, Cao X, Daniel JC, Miller SD, Smythe WR. Receptor tyrosine kinase
and phosphoinositide-3 kinase signaling in malignant mesothelioma. J Thorac
Cardiovasc Surg. 2005;130(2):393-400.
241. Hausenloy DJ, Tsang A, Mocanu MM, Yellon DM. Ischemic preconditioning
protects by activating prosurvival kinases at reperfusion. Am J Physiol Heart Circ
Physiol. 2005;288(2):H971-H6.
242. Cohen MV, Yang X-M, Liu GS, Heusch G, Downey JM. Acetylcholine,
bradykinin, opioids, and phenylephrine, but not adenosine, trigger
preconditioning by generating free radicals and opening mitochondrial KATP
channels. Circ Res. 2001;89:273-8.
111 243. Konstantinov IE, Li J, Redington AN. From mesothelioma to cardiovascular
protection via the phosphoinositide-3 kinase pathway: a new vista in
cardiothoracic surgery. J Thorac Cardiovasc Surg. 2006;131(2):509-10; author
reply 10.
244. Bell RM, Yellon DM. Bradykinin limits infarction when administered as an
adjunct to reperfusion in mouse heart: the role of PI3K, Akt and eNOS. J Mol
Cell Cardiol. 2002;35(2):185-93.
245. Kluft C, Dooijewaard G, Emeis JJ. Role of the contact system in fibrinolysis.
Semin Throm Hemost. 1987;13(1):50-68.
246. van der Salm TJ, Ansell JE, Okike ON, Marsicano TH, Lew R, Stephenson WP,
et al. The role of epsilon- aminocaproic acid in reducing bleeding after cardiac
operation: a double- blind randomized study. J Thorac Cardiovasc Surg.
1988;95(3):538-40.
247. Dietrich W, Jochum M, Schramm W, Blümel G, Richter JA. Reduction of
homologous blood requirement in cardiac surgery using high dose aprotinin.
Anesthesiology. 1989;71(3A):A 7.
248. Fergusson DA, Hebert PC, Mazer CD, Fremes S, MacAdams C, Murkin JM, et
al. A comparison of aprotinin and lysine analogues in high-risk cardiac surgery. N
Engl J Med. 2008;358(22):2319-31.
249. Mangano DT, Miao Y, Vuylsteke A, Tudor IC, Juneja R, Filipescu D, et al.
Mortality associated with aprotinin during 5 years following coronary artery
bypass graft surgery. JAMA. 2007;297(5):471-9.
250. Mangano DT, Tudor IC, Dietzel C. The risk associated with aprotinin in cardiac
surgery. N Engl J Med. 2006;354(4):353-65.
251. Deutsch E, Berger M, Kussmaul WG, Hirshfeld JW, Jr., Herrmann HC, Laskey
WK. Adaptation to ischemia during percutaneous transluminal coronary
angioplasty. Clinical, hemodynamic, and metabolic features. Circulation.
1990;82(6):2044-51.
252. Inoue T, Fujito T, Hoshi K, Sakai Y, Yamaguchi H, Takayanagi K, et al. A
mechanism of ischemic preconditioning during percutaneous transluminal
coronary angioplasty. Cardiology. 1996;87(3):216-23.
253. de Jong JW, de Jonge R, Marchesani A, Janssen M, Bradamante S. Controversies
in preconditioning. Cardiovasc Drugs Ther. 1997;10(6):767-73.
112 254. Miura T, Goto M, Urabe K, Endoh A, Shimamoto K, Iimura O. Does myocardial
stunning contribute to infarct size limitation by ischemic preconditioning?
Circulation. 1991;84(6):2504-12.
255. Cohen G, Shirai T, Weisel RD, Rao V, Merante F, Tumiati LC, et al. Optimal
myocardial preconditioning in a human model of ischemia and reperfusion.
Circulation. 1998;98(19 Suppl):II184-94; discussion II94-6.
256. Hausenloy DJ, Yellon DM. Remote ischaemic preconditioning: underlying
mechanisms and clinical application. Cardiovasc Res. 2008;79(3):377-86.
257. Downey GP, Chan CK, Lea P, Takai A, Grinstein S. Phorbol ester-induced actin
assembly in neutrophils: role of protein kinase C. J Cell Biol. 1992;116: 695-706.
258. Vermes I, Haanen C, Reutelingsperger C. Flow cytometry of apoptotic cell death.
J Immunol Methods. 2000;243(1-2):167-90.
259. Rothe G, Oser A, Valet G. Dihydrorhodamine 123: a new flow cytometric
indicator for respiratory burst activity in neutrophil granulocytes.
Naturwissenschaften. 1988;75(7):354-5.
260. Waddell TK, Fialkow L, Chan CK, Kishimoto TK, Downey GP. Potentiation of
the oxidative burst of human neutrophils. A signaling role for L-selectin. J Biol
Chem. 1994;269(28):18485.
261. Vachon E, Martin R, Plumb J, Kwok V, Vandivier RW, Glogauer M, et al. CD44
is a phagocytic receptor. Blood. 2006;107(10):4149-58.
262. Paxinos G, Watson C. The rat brain in stereotaxic co-ordinates. 4th ed. San
Diego: Academic press; 1998.
263. Haslett C, Guthrie LA, Kopaniak MM, Johnston RB, Jr., Henson PM. Modulation
of multiple neutrophil functions by preparative methods or trace concentrations of
bacterial lipopolysaccharide. Am J Pathol. 1985;119:101-10.
264. Serejo FC, Rodrigues LF, Jr., da Silva Tavares KC, de Carvalho ACC,
Nascimento JH. Cardioprotective properties of humoral factors released from rat
hearts subject to ischemic preconditioning. J Cardiovasc Pharmacol.
2007;49(4):214-20.
265. Hajrasouliha AR, Tavakoli S, Ghasemi M, Jabehdar-Maralani P, Sadeghipour H,
Ebrahimi F, et al. Endogenous cannabinoids contribute to remote ischemic
preconditioning via cannabinoid CB2 receptors in the rat heart. Eur J Pharmacol.
2008;579(1-3):246-52.
113 266. Kant R, Diwan V, Jaggi AS, Singh N, Singh D. Remote renal preconditioning-
induced cardioprotection: a key role of hypoxia inducible factor-prolyl 4-
hydroxylases. Mol Cell Biochem. 2008;312(1-2):25-31.
267. Wang YY, Yin BL. Pro-inflammatory cytokines may induce late preconditioning
in unstable angina patients. Med Hypotheses. 2006;67(5):1121-4.
268. Dawn B, Guo Y, Rezazadeh A, Wang O-L, Stein AB, Hunt G, et al. Tumor
necrosis factor-α does not modulate ischemia/reperfusion injury in native
myocardium but is essential for the development of late preconditioning. J Mol
Cell Cardiol. 2004;37(1):51-61.
269. Dawn B, Xuan Y-T, Guo Y, Rezazadeh A, Stein AB, Hunt G, et al. IL-6 plays an
obligatory role in late preconditioning via JAK-STAT signaling and upregulation
of iNOS and COX-2. Cardiovasc Res. 2004;64(1):61-71.
270. Kida H, Yoshida M, Hoshino S, Inoue K, Yano Y, Yanagita M, et al. Protective
effect of IL-6 on alveolar epithelial cell death induced by hydrogen peroxide. Am
J Physiol Lung Cell Mol Physiol. 2005;288(2):L342-9.
271. Glasgow SC, Ramachandran S, Csontos KA, Jia J, Mohanakumar T, Chapman
WC. Interleukin-1β is prominent in the early pulmonary inflammatory response
after hepatic injury. Surgery. 2005;138(1):64-70.
272. Clark ET, Desai TR, Hynes KL, Gewertz BL. Endothelial cell response to
hypoxia- reoxygenation is mediated by IL-1. J Surg Res. 1995;58(6):675-81.
273. Phagoo SB, Poole S, Leeb-Lundberg LMF. Autoregulation of bradykinin
receptors: agonists in the presence of interleukin-1ß shift the repertoire of
receptor subtypes from B2 to B1 in human lung fibroblasts. Mol Pharmacol.
1999;56(2):325-33.
274. Griffiths EJ, Halestrap AP. Protection by Cyclosporin A of ischemia/reperfusion-
induced damage in isolated rat hearts. J Mol Cell Cardiol. 1993;25(12):1461-9.
275. Halestrap AP, Clarke SJ, Javadov SA. Mitochondrial permeability transition pore
opening during myocardial reperfusion--a target for cardioprotection. Cardiovasc
Res. 2004;61(3):372-85.
276. Heurteaux C, Lauritzen I, Widmann C, Lazdunski M. Essential role of adenosine,
adenosine A1 receptors, and ATP-sensitive K+ channels in cerebral ischemic
preconditioning. Proc Natl Acad Sci U S A. 1995;92(10):4666-70.
277. Matsuyama K, Chiba Y, Ihaya A, Kimura T, Tanigawa N, Muraoka R. Effect of
spinal cord preconditioning on paraplegia during cross-clamping of the thoracic
aorta. Ann Thorac Surg. 1997;63(5):1315-20.
114 278. Ondrejcák T, Vanický I, Gálik J. Ischemic preconditioning does not improve
neurological recovery after spinal cord compression injury in the rat. Brain Res.
2004;995(2):267-73.
279. Sakurai M, Hayashi T, Abe K, Aoki M, Sadahiro M, Tabayashi K. Enhancement
of heat shock protein expression after transient ischemia in the preconditioned
spinal cord of rabbits. J Vasc Surg. 1998;27(4):720-5.
280. Ergin MA, Uysal S, Reich DL, Apaydin A, Lansman SL, McCullough JN, et al.
Temporary neurological dysfunction after deep hypothermic circulatory arrest: a
clinical marker of long-term functional deficit. Ann Thorac Surg.
1999;67(6):1887-90; discussion 91-4.
281. Schaller B, Graf R. Cerebral ischemic preconditioning. An experimental
phenomenon or a clinical important entity of stroke prevention? J Neurol.
2002;249(11):1503-11.
282. Steiger H-J, Hänggi D. Ischaemic preconditioning of the brain, mechanisms and
applications. Acta Neurochir (Wien). 2007;149(1):1-10.
283. Glazier SS, O'Rourke DM, Graham DI, Welsh FA. Induction of ischemic
tolerance following brief focal ischemia in rat brain. J Cereb Blood Flow Metab.
1994;14(4):545-53.
284. Moncayo J, de Freitas GR, Bogousslavsky J, Altieri M, van Melle G. Do transient
ischemic attacks have a neuroprotective effect? . Neurology. 2000;54:2089-94.
285. Blanco M, Lizasoain I, Sobrino T, Vivancos J, Castillo J. Ischemic
preconditioning: a novel target for neuroprotective therapy Cerebrovasc Dis.
2006;21:38-47.
286. Zhao H-G, Li W-B, Li Q-J, Chen X-L, Liu H-Q, Feng R-F, et al. Limb ischemic
preconditioning attenuates apoptosis of pyramidal neurons in the CA1
hippocampus induced by cerebral ischemia-reperfusion in rats. Sheng Li Xue
Bao. 2004;56(3):407-12.
287. Abbott NJ, Patabendige AAK, Dolman DEM, Yusof SR, Begley DJ. Structure
and function of the blood-brain barrier. Neurobiol Dis.In Press, corrected proof.
288. Gingrich MB, Junge CE, Lyuboslavsky P, Traynelis SF. Potentiation of NMDA
receptor function by the serine protease thrombin. J Neurosci. 2000;20(12):4582-
95.
289. Gingrich MB, Traynelis SF. Serine proteases and brain damage - is there a link?
Trends Neurosci. 2000;23(9):399-407.
115 290. Nadal A, Fuentes E, Pastor J, McNaughton PA. Plasma albumin is a potent
trigger of calcium signals and DNA synthesis in astrocytes. Proc Natl Acad Sci
USA. 1995;92:1426-30.
291. Bechmann I, Goldmann J, Kovac AD, Kwidzinski E, Simbürger E, Naftolin F, et
al. Circulating monocytic cells infiltrate layers of anterograde axonal
degeneration where they transform into microglia. FASEB J. 2005;19(6):647-9.
292. Hess DC, Abe T, Hill WD, Studdard AM, Carothers J, Masuya M, et al.
Hematopoietic origin of microglial and perivascular cells in brain. Exp Neurol.
2004;186(2):134-44.
293. Bechmann I, Priller J, Kovac A, Böntert M, Wehner T, Klett FF, et al. Immune
surveillance of mouse brain perivascular spaces by blood-borne macrophages. Eur
J Neurosci. 2001;14(10):1651-8.
294. Scholz M, Cinatl J, Schädel-Höpfner M, Windolf J. Neutrophils and the blood-
brain barrier dysfunction after trauma. Med Res Rev. 2007;27(3):401.
295. Przyklenk K, Kloner RA. Ischemic preconditioning: exploring the paradox. Prog
Cardiovasc Dis. 2009;40(6):517-47.
296. Yellon DM, Dana A. The preconditioning phenomenon: a tool for the scientist or
a clinical reality? Circ Res. 2000;87(7):543-50.
297. Ghosh S, Galinanes M. Protection of the human heart with ischemic
preconditioning during cardiac surgery: role of cardiopulmonary bypass. J Thorac
Cardiovasc Surg. 2003;126(1):133-42.
298. Perrault LP, Menasche P, Bel A, de Chaumaray T, Peynet J, Mondry A, et al.
Ischemic preconditioning in cardiac surgery: a word of caution. J Thorac
Cardiovasc Surg. 1996;112(5):1378-86.
299. Cheung MMH, Kharbanda RK, Konstantinov IE, Shimizu M, Meng HF, Li J, et
al. Randomized controlled trial of the effects of remote ischemic preconditioning
on children undergoing cardiac surgery. J Am Coll Cardiol. 2006;47(11):2277-82.
300. Hausenloy DJ, Mwamure PK, Venugopal V, Harris J, Barnard M, Grundy E, et
al. Effect of remote ischemic preconditioning on myocardial injury in patients
undergoing coronary artery bypass graft surgery: a randomised controlled trial.
Lancet. 2007;370:575-9.
301. Rahman I, Bonser RS. Remote ischaemic preconditioning: the current best hope
for improved myocardial protection in cardiac surgery? Heart. 2009;95(19):1553-
5.
116 302. Venugopal V, Hausenloy DJ, Ludman A, Di Salvo C, Kolvekar S, Yap J, et al.
Remote ischaemic preconditioning reduces myocardial injury in patients
undergoing cardiac surgery with cold-blood cardioplegia: a randomised
controlled trial. Heart. 2009;95(19):1567-71.
303. Ali ZA, Callaghan CJ, Lim E, Ali AA, Nouraei SAR, Akthar AM, et al. Remote
ischemic preconditioning reduces myocardial and renal injury after elective
abdominal aortic aneurysm repair: a randomized controlled trial. Circulation.
2007;116(11 Suppl):I98-105.
304. Hoole SP, Heck PM, Sharples L, Khan SN, Duehmke R, Densem CG, et al.
Cardiac Remote Ischemic Preconditioning in Coronary Stenting (CRISP Stent)
Study: a prospective, randomized control trial. Circulation. 2009;119(6):820-7.
305. Kloner R. Clinical application of remote ischemic preconditioning. Circulation.
2009;119(6):776-8.
306. Konstantinov IE, Redington AN. Linking gene expression, nuclear factor kappa
B, remote ischemic preconditioning, and transplantation: a quest for an elusive
Holy Grail or a road to an amazing discovery? J Thorac Cardiovasc Surg.
2006;131(2):507-9.
307. Staat P, Rioufol G, Piot C, Cottin Y, Cung TT, L'Huillier I, et al. Postconditioning
the human heart. Circulation. 2005;112(14):2143-8.
308. Friedrich B, Schmidt R, Reiss I, Günther A, Seeger W, Müller M, et al. Changes
in biochemical and biophysical surfactant properties with cardiopulmonary
bypass in children. Crit Care Med. 2003;31(1):284-90.
309. Griese M, Wilnhammer C, Jansen S, Rinker C. Cardiopulmonary bypass reduces
pulmonary surfactant activity in infants. J Thorac Cardiovasc Surg.
1999;118(2):237-44.
310. Pryhuber GS, Bachurski C, Hirsch R, Bacon A, Whitsett JA. Tumor necrosis
factor-alpha decreases surfactant protein B mRNA in murine lung. Am J Physiol
Lung Cell Mol Physiol 1996;270(5 Pt 1):L714-21.
311. Patel KD, Cuvelier SL, Wiehler S. Selectins: critical mediators of leukocyte
recruitment. Semin Immunol. 2002;14(2):73-81.
312. Tönz M, Mihaljevic T, von Segesser LK, Fehr J, Schmid ER, Turina MI. Acute
lung injury during cardiopulmonary bypass. Are the neutrophils responsible?
Chest. 1995;108(6):1551-6.
313. Ng CSH, Wan S, Yim APC, Arifi AA. Pulmonary dysfunction after cardiac
surgery. Chest. 2002;121(4):1269-77.
117
314. Sander M, von Heymann C, von Dossow V, Spaethe C, Konertz WF, Jain U, et
al. Increased interleukin-6 after cardiac surgery predicts infection. Anesth Analg.
2006;102(6):1623-9.
315. Andersen NB, Ghia J, Moffitt EA. Pulmonary function, cardiac status, and
postoperative course in relation to cardiopulmonary bypass. J Thorac Cardiovasc
Surg. 1970;59:474-83.
316. Ghia J, Andersen NB. Pulmonary function and cardiopulmonary bypass. JAMA.
1970;212(4):593-7.
317. Babik B, Asztalos T, Petak F, Deak ZI, Hantos Z. Changes in respiratory
mechanics during cardiac surgery. Anesth Analg. 2003;96(5):1280-7.
318. Güler M, Kirali K, Toker ME, Bozbuga N, Ömeroglu SN, Akinci E, et al.
Different CABG methods in patients with chronic obstructive pulmonary disease.
Ann Thorac Surg. 2001;71(1):152-7.
319. Li G, Chen S, Lu E, Luo W. Cardiac ischemic preconditioing improves lung
preservation in valve replacement operations. Ann Thorac Surg. 2001;71:631-5.
320. Shore S, Nelson DP, Pearl JM, Manning PB, Wong H, Shanley TP, et al.
Usefulness of corticosteroid therapy in decreasing epinephrine requirements in
critically ill infants with congenital heart disease. Am J Cardiol. 2001;88(5):591-
4.
321. Wernovsky G, Wypij D, Jonas RA, Mayer Jr JE, Hanley FL, Hickey PR, et al.
Postoperative course and hemodynamic profile after the arterial switch operation
in neonates and infants: a comparison of low-flow cardiopulmonary bypass and
circulatory arrest. Circulation. 1995;92(8):2226-35.