systemic response to local ischaemia: the evolving … · the evolving concept of remote ischaemic...

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

2

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.

4

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



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


2.6 Assessment of impact of remote ischaemic preconditioning on 52

global cerebral ischaemia in rats


2.6.2 Assessment of hippocampal neurons 53


Chapter 3: RESULTS 55

3.1 Functional responses of human neutrophils to remote 56


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


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.


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


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


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 ).


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.


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 postconditioning 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

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