Influence of in utero inflammation on diaphragm function in preterm lambs

By

Kanakeswary Karisnan, BSc, MSc

This thesis is presented for the degree of

DOCTOR OF PHILOSOPHY

The University of Western Australia

School of Anatomy, Physiology and Human Biology

Submitted: April, 2015

i

Preface

The experimental research presented in this thesis was undertaken in the

School of Anatomy, Physiology and Human Biology, The University of Western

Australia, under the supervision of Dr Gavin Pinniger, Dr Anthony Bakker and

Professor Jane Pillow, with the financial assistance of a Scholarship for

International Research Fees, and Stipend provided by National Health and

Medical Research Council grant. Financial assistance for experimental

research was provided by a National Health and Medical Research Council

grant (Project Grant APP1010665).

The research data presented in this thesis are original. Data collection and

analyses were carried out by myself except where the specific contributions of

other persons are acknowledged. All co-authors have given formal permission

for published experimental work presented in this thesis.

Kanakeswary Karisnan

April 2015

ii

Abstract

Despite recent major advances in neonatal care, preterm birth remains the

leading cause of postnatal morbidity and mortality. The complications of

preterm birth arise from immature organ systems of which the dysfunction of

the respiratory system is the most common clinical problem. Infants require

developed respiratory system to sustain independent ventilation after birth.

Respiratory failure among preterm infant is due to the underdevelopment of the

lungs, however respiratory muscle weakness and/or fatigue is also a major

contributor. The diaphragm is important for lung development in utero as fetal

breathing movements that are mainly driven by diaphragm and intercostal

contraction, stimulate lung cellular proliferation and growth. Thus, the

respiratory system (including muscle pump – diaphragm, as well as gas

exchanger – lungs) is poorly developed in preterm infants. In addition, preterm

infants face an increased work of breathing due to lack of surfactant proteins,

highly compliant chest wall and noncompliant, structurally immature lungs

leading to diaphragm weakness, fatigue and respiratory failure.

Diaphragm function may be compromised further by adverse in utero fetal

exposures such as inflammation. Chorioamnionitis, inflammation of the

placental and fetal membranes, is associated with up to 70 % of preterm births

at < 28 week gestation, the group that is most likely to develop chronic

respiratory illness, such as bronchopulmonary dysplasia (BPD). Notably, little is

known on how diaphragm function in the preterm infant is affected by

chorioamnionitis. Therefore, the overall aim of this thesis was to investigate the

impact of chorioamnionitis induced inflammation on contractile function in the

preterm diaphragm and to elucidate the molecular mechanism underlying

functional alteration. The specific aims of the first study were to establish the

functional changes in the preterm fetal diaphragm after exposure to in utero

inflammation and to elucidate the underlying molecular mechanisms. The

study hypothesis was that acute 2 d and 7 d intra-amniotic (IA) exposure to

lipopolysaccharide (LPS) impairs preterm diaphragm function. The specific

aims of the second study were to assess how gestational age at time of

exposure to IA LPS determines the extent of functional impairment of the fetal

iii

diaphragm and whether weekly inflammatory exposures exacerbate diaphragm

dysfunction. The study hypotheses were that diaphragm weakness persists

after a long duration of LPS exposure (21 d) and that the effects would be more

pronounced after a chronic LPS exposure (weekly LPS injections). The third

study aims were to investigate the effect of acute LPS exposure (2 d and 7 d

prior to delivery) on preterm and term diaphragm function. This study

hypothesis was that the preterm diaphragm was more vulnerable to in utero

inflammation induced contractile dysfunction than term diaphragm. The fourth

study aim was to investigate the role of IL-1 signalling and oxidative stress on

IA LPS induced diaphragm weakness in preterm lambs. This study hypothesis

was that blockade of IL-1 signalling will protect the diaphragm from

inflammation induced contractile dysfunction.

This PhD project used a well-established ovine model of chorioamnionitis

induced by IA injections of LPS. Pregnant Merino ewes received ultrasound

guided sterile IA injections of saline or LPS (10 mg or 4 mg) at different time

points prior to delivery at 121 d (preterm) or at 145 d (term) gestational age

(GA) according to the experimental design of each study. Fetal lamb diaphragm

strips were dissected after terminal anesthesia and mounted in an in vitro

muscle test system for assessment of contractile function. The inflammatory

cytokine response, myosin heavy chain (MHC) fibre composition, protein

synthesis and proteolytic pathways and intracellular molecular signaling were

analysed using qPCR, ELISA, myeloperoxidase (MPO) staining,

immunofluorescence staining, biochemical assays and Western blot.

The first study aimed to determine the effects of acute in utero LPS exposure

on diaphragm function in preterm lambs. In this study, lambs received IA

injections of 10 mg LPS at 2 d and 7 d prior to delivery at 121 d GA. The

maximum specific force produced by the diaphragm following both 2 d and 7 d

LPS exposures was 30 % lower than controls. Diaphragm weakness following

the 2 d LPS exposure was associated with activation of the NF-κB pathway,

increased inflammatory cytokine expression and enhanced 20 S proteasome

activity. In the 7 d LPS exposed lambs, the initial inflammatory response had

subsided however, p70S6K phosphorylation, a key component of the protein

iv

synthesis signalling pathway, was markedly decreased and the proportion of

MHC IIa positive fibres were significantly lower compared to the control group.

These results show that fetal exposure to LPS significantly reduces maximum

diaphragm force generating capacity. The differences in molecular responses

observed in the 2 d and 7 d LPS groups suggest a progressive response to the

initial acute inflammatory exposure that results in persistent impaired

contractility of the preterm diaphragm. However, it was not evident from these

studies how long the diaphragm impairment would persist after the initial

inflammatory exposure, or how the preterm diaphragm would respond to a

chronic inflammatory stimulus.

The second study aimed to: i) compare the effect of acute (7 d) LPS exposure

against a long term (21 d) LPS exposure; and ii) determine the effect of chronic

inflammation on preterm diaphragm function by repeated LPS exposures

administered at 21 d, 14 d and 7 d prior to delivery. The single 21 d LPS

exposure resulted in a significant 40 % decrease in maximum specific force,

whereas the single 7 d LPS exposure reduced maximum specific force by 30

%. Furthermore, the long term LPS exposures (21 d and repeated LPS)

resulted in additional alterations to contractile function including prolonged

twitch contraction times, increased fatigue resistance and elevated protein

carbonyl content, which were not evident following the acute 7 d exposure.

Despite significantly elevated white blood cell counts and IL-6 mRNA

expression following repeated LPS exposures, there were no significant

differences in diaphragm contractile properties between 21 d and repeated LPS

groups suggesting that frequency of inflammatory exposure does not influence

the severity of contractile dysfunction. Rather, these results suggest that the

timing of the initial fetal exposure to LPS critically influences the extent of

inflammation induced diaphragm dysfunction.

Importantly, from the previous study we could not distinguish the relative

importance of the duration of LPS exposure (7 d v 21 d) from the GA at time of

the initial exposure (114 d v 100 d) in determining the severity of diaphragm

dysfunction. Therefore, the third study aimed to specifically examine the effect

of GA at the time of initial exposure on the severity of inflammation induced

v

diaphragm dysfunction. LPS exposures of the same duration (2 d and 7 d) were

administered at different stages of gestation (ie in preterm and term lambs). In

utero LPS exposure impaired diaphragm function in both preterm and term

lambs, however, the severity of diaphragm weakness was greater in preterm

lambs. In the term lambs, 2 d and 7 d LPS exposures resulted in a 20 %

decrease in diaphragm force production compared to a 30 % decrease in

preterm lambs. Relative to the naïve control lambs, the proportional reduction

in peak twitch force was significantly greater in preterm lambs than in term

lambs. In term lambs, LPS exposure was associated with increased fatigue

resistance and a higher proportion of fibres expressing MHCs isoforms.

Whereas both term and preterm lambs displayed significant increases in

inflammatory cytokine expression, preterm lambs demonstrated significant

decrease in the cross sectional area of MHCs and MHCn fibres that were not

present in term lambs. This study suggests that preterm lambs are more

vulnerable to diaphragm dysfunction induced by IA LPS compared to term

lambs. The GA at the time of LPS exposure influences the extent of diaphragm

dysfunction, thus preterm infants are at higher risk than term infants of

experiencing diaphragm weakness and the development of respiratory failure

after birth.

Previous studies have suggested that the fetal inflammatory response to IA

LPS is orchestrated via interleukin 1 (IL-1). Therefore, the final study aimed to

determine if LPS induced contractile dysfunction in the preterm diaphragm is

mediated via the IL-1 pathway. This study examined the effectiveness of IA

recombinant human IL-1 receptor antagonist (rhIL-1ra) injections in preventing

inflammation induced diaphragm dysfunction following an acute 2 d LPS

exposure. Similar to the previous study, the maximum specific force in lambs

exposed to 2 d IA LPS was 25 % lower than in control lambs and was

associated with increased plasma IL-6 protein and diaphragm IL-1β mRNA

expression and elevated oxidised glutathione levels. IA injection of rhIL-1ra

prior to LPS exposure ameliorated the LPS-induced diaphragm weakness and

blocked systemic and diaphragm inflammatory responses, but did not prevent

the rise in oxidised glutathione. These findings indicate that LPS induced

diaphragm dysfunction is mediated via IL-1 and occurs independently of

vi

oxidative stress. Hence, the IL-1 pathway represents a potential therapeutic

target in the management of impaired diaphragm function in preterm infants.

The studies in this thesis demonstrate that in utero exposure to inflammation

impairs preterm diaphragm function. Crucially, diaphragm weakness is

influenced by the timing of LPS exposure during gestation. Evidence from this

thesis also shows that the effects of inflammation on diaphragm function persist

long after the initial inflammatory response has subsided. The initial

inflammatory response is mediated by the IL-1 pathway: nevertheless,

oxidative stress may also contribute to diaphragm weakness following an

inflammatory stimulus. Given the existing structural and functional immaturity of

the naïve preterm diaphragm, it is likely that additional weakness imposed by

inflammatory exposure significantly impairs the contractile capability of the

diaphragm limiting its ability to achieve adequate tidal volumes, thereby

contributing to respiratory failure from inadequate ventilation. As preterm birth

is commonly associated with chorioamnionitis, the findings from this thesis

indicate that the integrity of diaphragm at birth and adverse in utero exposures

may significantly influence the development of postnatal respiratory failure

among preterm infants. Thus, it is critically essential to ensure optimal

diaphragm function in support to lung function when setting up the treatment

and management plan for postnatal respiratory failure in preterm infants.

vii

To my beloved Appa & Amma

viii

Acknowledgments

First and foremost I would like to express my sincere thanks and appreciation

to my PhD supervisors Dr Gavin Pinniger, Dr Anthony Bakker and Professor

Jane Pillow. I am grateful for their guidance, knowledge and advice in helping

me through my PhD project. A special thanks to Gavin for being an amazing

coordinating supervisor.

Many thanks to the members of the Centre for Neonatal Research and

Education (CNRE) especially to Dr. Yong Song and Dr. Peter Noble for

assisting me in teaching new laboratory techniques, problem solving and

general advice.

An extra special thank you to my Amma and Appa, I would not be the person I

am today without your guidance, love and care. Special thanks to my sister,

Sunthary for her love and support during my studies. And I would like to thank

my older sister Seetha and her family Geoff, Holly and Kayla. Last but not least,

I would like to thank James for his advice and support during thesis writing.

Finally I would like to thank and acknowledge the National Health and Medical

Research for their financial support.

ix

Declaration

This thesis contains no material which has been accepted for the award of any

other degree by any other university or tertiary institution to Kanakeswary

Karisnan. To my knowledge this thesis does not contain any material published

by other person except for that which due reference has been made in text.

Permission has been granted by co-authors for any work that has been

published to be included in this thesis.

Signed................................................

Kanakeswary Karisnan

Signed..................................................

Gavin Pinniger

Coordinating Supervisor

x

Table of Contents

Preface i

Abstract ii

Acknowledgments viii

Declaration ix

Table of Contents x

List of Figures xvi

List of Tables xviii

Thesis format xix

Abbreviations xx

Publications arising from this thesis xxii

Conference abstracts and presentations arising from this thesis xxiii

1. Chapter 1: General Introduction 2

1.1. Preterm birth 2

1.2. Respiratory problems in preterm infants 7

1.3. Inspiratory muscles and neural control of breathing 12

1.4. Mechanical properties of diaphragm muscle 13

1.5. Diaphragm muscle development 14

1.6. Diaphragm muscle failure in preterm infants 17

1.7. Antenatal inflammation and its impact on preterm diaphragm function 18

xi

1.8. Potential treatments for inflammation induced diaphragm dysfunction:

Targeting IL-1 signalling 19

1.9. Ovine model of chorioamnionitis 20

1.10. Statement of aims 21

2. Chapter 2: In utero lipopolysaccharide exposure impairs preterm diaphragm contractility 24

2.1 Abstract 25

2.2 Introduction 26

2.3 Materials and methods 28

2.3.1 Animals and experimental design 28

2.3.2 Muscle contractile properties 29

2.3.3 Western blot analysis 29

2.3.4 IL-1β and IL-6 plasma levels 29

2.3.5 RNA isolation, reverse transcription and quantitative PCR 29

2.3.6 Myosin Heavy Chain (MHC) fibre typing and myofibre Cross-

Sectional Area (CSA) 29

2.3.7 Biochemical analysis 30

2.3.8 Data analysis 30

2.4 Results 30

2.4.1 Animal characteristics 30

2.4.2 Contractile measurements 31

xii

2.4.3 MHC protein and fibre CSA 33

2.4.4 Cytokine response 34

2.4.5 Molecular signalling 35

2.4.6 Proteolytic pathways 37

2.5 Discussion 39

3. Chapter 3: Gestational age at initial exposure to in utero inflammation influences the extent of diaphragm dysfunction in preterm lambs 47

3.1. Abstract 48

3.2 Introduction 49

3.3 Methods 50

3.3.1 Animals and experimental design 50

3.3.2 Diaphragm contractile properties 51

3.3.3 Myosin Heavy Chain (MHC) fibre typing and myofibre Cross-

Sectional Area (CSA) 51

3.3.4 Muscle protein extraction 51

3.3.5 Immunoblot analysis 52

3.3.6 Total white blood cell count 52

3.3.7 IL-1β and IL-6 levels in plasma 52

3.3.8 RNA Isolation, Reverse Transcription and Quantitative PCR 52

3.3.9 Protein Carbonyl assay 53

xiii

3.3.10 Data analysis 53

3.4 Results 53

3.4.1 Physiological variables at birth 53

3.4.2 Diaphragm contractile properties 54

3.4.3 MHC isoform composition and fibre CSA 55

3.4.4 Cytokine response 56

3.4.5 Total white blood cell count 57

3.4.6 Anabolic and catabolic pathways 58

3.4.7 Oxidative stress 60

3.5 Discussion 61

4. Chapter 4: Gestational age at time of in utero lipopolysaccharide exposure influences the severity of inflammation-induced diaphragm weakness in lambs 66

4.1. Abstract 67

4.2. Introduction 68

4.3. Methods 70

4.3.1. Experimental design 70

4.3.2. Diaphragm contractile properties 70

4.3.3. Myosin Heavy Chain (MHC) fibre typing and myofibre Cross-

Sectional Area (CSA) 71

4.3.4. Muscle protein extraction 71

xiv

4.3.5. Markers of systemic inflammation 71

4.3.6. RNA isolation, reverse transcription and quantitative PCR 71

4.3.7. Oxidative stress 72

4.3.8. Data analysis 72

4.4. Results 72

4.4.1. Characterisation of lambs 72

4.4.2. Diaphragm contractility 74

4.4.3. MHC composition and myofibre cross-sectional area 75

4.4.4. Markers of systemic and local inflammation 77

4.4.5. Oxidative stress in diaphragm 78

4.4.6. Proteolytic gene expression in diaphragm 80

4.5. Discussion 81

5. Chapter 5: Interleukin-1 receptor antagonist protects against lipopolysaccharide induced diaphragm weakness in preterm lambs 88

5.1 Abstract 89

5.2 Introduction 90

5.3 Methods 92

5.3.1 Animals and experimental design 92

5.3.2 Diaphragm contractile function 93

5.3.3 Muscle protein extraction 94

5.3.4 IL-1β and IL-6 plasma levels 94

xv

5.3.5 Myeloperoxidase (MPO) staining 94

5.3.6 MPO assay 95

5.3.7 Cord blood leukocyte count 95

5.3.8 RNA isolation, reverse transcription and quantitative PCR 95

5.3.9 Biochemical analysis of oxidative stress and proteolysis 96

5.3.10 Data analysis 96

5.4 Results 96

5.4.1 Physiological variables at birth 96

5.4.2 Diaphragm contractile function 97

5.4.3 Systemic inflammation 99

5.4.4 Diaphragmatic inflammation 101

5.4.5 Diaphragm atrophy gene expression and 20 S proteasome activity 102

5.4.6 Oxidative stress 103

5.5 Discussion 104

6. Chapter 6: General Discussion 111

6.1. Study importance and novel findings 113

6.2. Study limitations and implications for future research 121

6.3. Summary and conclusion 122

References 124

Appendices 142

xvi

List of Figures

Figure 1.1 Chorioamnionitis causes an inflammatory cascade

that leads to preterm birth

Page 4

Figure 1.2 Chorioamnionitis is implicated in the pathogenesis

of multiple organ disease of the fetus Page 6

Figure 1.3 Overview of the pro-inflammatory and anti-

inflammatory stimuli that a fetus is exposed to

during preterm birth and postnatal development

Page 7

Figure 1.4 Early growth and developmental phase of the

human lung Page 9

Figure 1.5 Diaphragm muscle Page 13

Figure 2.1 Fetal diaphragm contractile properties, maximal

twitch and tetanic force and force-frequency

relationship

Page 32

Figure 2.2 Susceptibility to fatigue and muscle damage, and

unloaded shortening velocity of fetal diaphragm Page 33

Figure 2.3 Diaphragm muscle fibre type and cross-sectional

area (CSA) measurement

Page 34

Figure 2.4 Systemic and local cytokine response Page 35

Figure 2.5 Activity of signalling molecules after LPS exposure Page 36

Figure s1 Association of IL-1β / MAFbx gene expression, NF-

κB signalling and UPP pathway

Page 37

Figure 2.6 Gene expression of key components in proteolytic

pathways

Page 38

Figure 2.7 Biochemical activity of calpain, caspase-3 and UPP

pathways

Page 39

Figure 3.1 Fetal diaphragm contractile properties Page 55

Figure 3.2 (A) Proportions of slow-twitch MHCs and fast-twitch

MHC IIa fibres and (B) muscle fibre cross-sectional

areas (CSA)

Page 56

Figure 3.3 Local and systemic cytokine response Page 57

Figure 3.4 Cord blood WBC cell count Page 58

xvii

Figure 3.5 Activity of protein synthesis and degradation

pathway signalling molecules in diaphragm after

LPS exposure

Page 59

Figure 3.6 Atrophy gene MAFbx and MuRF1 expression in

diaphragm

Page 60

Figure 3.7 Protein carbonyl content in diaphragm Page 60

Figure 4.1 Diaphragm maximum specific force (A) and twitch

force (B), fatigue measurements represented as

fatigue index (C) and percentage of force deficit

after stretch protocol (D) in term and preterm lambs

Page 75

Figure 4.2 MHC expression (A,B) and fibre cross sectional

area (CSD) (C,D) in diaphragm fibres from term

(A,C) and preterm (B,D) lambs

Page 76

Figure 4.3 Systemic and diaphragm cytokine responses Page 78

Figure 4.4 Oxidative stress genes SOD1 (A), Catalase (B) and

GPX (C) mRNA expression in the diaphragm

Page 79

Figure 4.5 Diaphragm GSH/GSSG ratio presented as mean ±

SEM (A) and protein carbonyl content in diaphragm

presented as Median (range) (B)

Page 80

Figure 4.6 Atrophy gene MAFbx (A) and MuRF1 (B) mRNA

expression in diaphragm for term and preterm

experimental groups relative to its GA control

Page 81

Figure 5.1 Fetal diaphragm contractile properties Page 99

Figure 5.2 Systemic and diaphragm cytokine responses Page 100

Figure 5.3 Diaphragm myeloperoxidase activity Page 102

Figure 5.4 Atrophy related signalling in the diaphragm Page 103

Figure 5.5 Oxidative stress in the diaphragm Page 104

Figure 6.1 Factors contributing to respiratory muscle

dysfunction in preterm infants

Page 112

xviii

List of Tables

Table 2.1 Descriptive data and twitch parameters for saline

and LPS treated preterm lambs Page 31

Table 3.1 Lamb GA, body weight and optimal muscle length

data for saline (Control) and LPS exposed fetal

lambs

Page 54

Table 4.1 Description of lambs and diaphragm twitch

properties Page 73

Table 5.1 Lamb descriptive data and measures of diaphragm

contractile function Page 97

Table 5.2 Cord blood leukocytes counts Page 101

xix

Thesis format

General This thesis is presented as 6 chapters. These chapters are the General Introduction, four manuscripts, and the General Discussion. The Introduction provides an overall perspective of the thesis, including an introduction to the hypotheses. For the General Discussion, the major findings are highlighted and the future research direction is suggested.

Language The majority of this thesis is written according to Australian English. For chapter 2 which is a published manuscript, the language is kept according to Journal of Respiratory Cell and Molecular Biology guidelines.

Presentation of data The data are presented in graphical and tabular format. Figures and tables are placed immediately after the text in which they are cited.

References Text in all chapters is cited according to Harvard (UWA Science) format. That is, author and year of publication are cited in text; where the number of authors exceeds three, only the first author mentioned proceeded by et al. and the year of publication.

xx

Abbreviations

Akt1 Protein kinase B

BPD Bronchopulmonary dysplasia

Ca2+ Calcium ions

CaCl2 Calcium chloride

FI Fatigue index

CSA Cross-sectional area

DAB 3, 3’-diaminobenzidine df/dt Maximum rate of force development DHPR Dihydropyridine

ELISA Enzyme linked immunoabsorbant assay

FIRS Fetal inflammatory response syndrome

FOXO1 Forkhead box protein O1

GA Gestational age

GPX1 Glutathione peroxidase 1

GSH Reduced glutathione

GSSG Oxidised glutathione

IA Intra-amniotic

IGF-1 Insulin like growth factor 1

IL-1 Interleukin-1

Il-1 Interleukin-1

IL-1R1 IL-1 receptor type 1 IL-1ra IL-1 receptor antagonist

IL-6 Interleukin-6

IL-8 Interleukin-8

KCl Potassium chloride

LPS Lipopolysaccharide

MAFbx Muscle Atrophy F-Box

mATPase Myofibrillar adenosine triphosphatase

MCP-1 Monocyte chemotactic protein-1

MgCl2 Magnesium chloride

MHC Myosin heavy chain

xxi

MMPs Matrix metalloproteases

MPO Myeloperoxidase

mTOR Mammalian target of rapamycin

MuRF1 Muscle RING-finger protein-1

NaCl Sodium chloride

NaH2PO4 Sodium dihydrogen phosphate

NaHCO3 Sodium bicarbonate

NF-𝜅B Nuclear factor kappa B

OCT Optimal cutting temperature

P0 Maximal tetanic force

P13K Phosphatidylinositol-3-kinases

PBS Phosphate buffered saline PBST Phosphate buffered saline-triton-x

Pdimax Maximal transdiaphragmatic pressure

PGs Prostaglandins

Pt Maximal twitch force

qPCR, Quantitative polymerase chain reaction

RDS Respiratory distress syndrome

REM Rapid eye movement

ROS Reactive oxygen species

SDH Succinate dehydrogenase

SERCA Sarcoendoplasmic reticulum calcium ATPase

SOD1 Superoxide dismutase 1

TLRs Toll-like receptors

TNF- Tumour necrosis factor-

UPP Ubiquitin–proteasome system

V0 Unloaded shortening velocity

w Week(s)

α-GPDH α-glycerophosphate dehydrogenase

xxii

Publications arising from this thesis

(Published/In preparation for submission)

1. Song Y, Karisnan K, Noble PB, Berry CA, Lavin T, Moss TJ, Bakker AJ,

Pinniger GJ, Pillow JJ. In utero LPS exposure impairs preterm diaphragm

contractility. Am J Respir Cell Mol Biol 2013; 49(5):866-74. (Published)

2. Karisnan K, Bakker AJ, Song Y, Noble PB, Pillow JJ, Pinniger GJ.

Gestational age at initial exposure to in utero inflammation influences the

extent of diaphragm dysfunction in preterm lambs. Respirology, 2015;

12615 (Published)

3. Karisnan K, Bakker AJ, Song Y, Noble PB, Pillow JJ, Pinniger GJ.

Interleukin-1 receptor antagonist protects against lipopolysaccharide

induced diaphragm weakness in preterm lambs. PLOS One, 2015;

pone.0124390.ecollection 2015 (Published)

4. Karisnan K, Bakker AJ, Song Y, Noble PB, Pillow JJ, Pinniger GJ.

Gestational age at time of in utero lipopolysaccharide exposure influences

the severity of inflammation-induced diaphragm weakness in lambs (In

preparation for submission)

xxiii

Conference abstracts and presentations arising from this thesis

1. Karisnan K, Pinniger GJ, Bakker AJ, Song Y, Berry CA, Noble PB, and

Pillow JJ. Gestational age at time of the initial exposure to

lipopolysaccharide determines the severity of diaphragmatic contractile

dysfunction in preterm lambs. 2012. Australian Physiological Society.

Australia. Oral

2. Karisnan K, Pinniger GJ, Bakker AJ, Berry, CA, Noble PB, Song Y and

Pillow JJ. Gestation at onset of chorioamnionitis determines adverse

effect of inflammation on diaphragm function. 2013. Perinatal Society of

Australia and New Zealand meeting. Australia. Oral

3. Karisnan K, Pinniger GJ, Bakker AJ, Berry, CA, Noble PB, Song Y and

Pillow JJ. Antenatal betamethasone does not impair diaphragm muscle

contractility in preterm fetal sheep. 2013. Perinatal Society of Australia

and New Zealand meeting. Australia. Poster

4. Karisnan K, Pinniger GJ, Bakker AJ, Song Y, Berry CA, Noble PB, and

Pillow JJ. Chorioamnionitis impaired preterm lamb diaphragm function.

2013. International Union for Physiological Sciences. Birmingham, UK.

Poster

5. Karisnan K, Pinniger GJ, Bakker AJ, Song Y, Noble PB, and Pillow JJ.

RhIL-1ra protects against LPS induced diaphragmatic weakness in

preterm lambs. 2014. Perinatal Society of Australia and New Zealand

meeting. Australia. Oral

6. Karisnan K, Pinniger GJ, Bakker AJ, Song Y, Noble PB, and Pillow JJ.

RhIL-1ra protects against LPS induced diaphragmatic weakness in

preterm lambs. 2014. Australian Society for Medical Research WA

Scientific Symposium. Australia. Oral

xxiv

7. Karisnan K, Pinniger GJ, Bakker AJ, Song Y, Noble PB, and Pillow JJ.

RhIL-1ra protects against LPS induced diaphragmatic weakness in

preterm lambs. 2014. Thoracic Society of Australia and New Zealand

meeting. Australia. Oral

8. Kanakeswary Karisnan, Anthony J. Bakker, Yong Song, Peter B. Noble,

J. Jane Pillow and Gavin J. Pinniger. Gestational age at time of in utero

lipopolysaccharide exposure influences the severity of inflammation-

induced diaphragm weakness in lambs. Kuala Lumpur International

Neonatology Conference. 2015. Poster

1

Chapter 1

General Introduction

2

Chapter 1: General Introduction

1.1. Preterm birth

Despite major advances in neonatal care over the last two decades, preterm

birth is the leading cause of postnatal morbidity and mortality affecting 15

million infants and comprising over one million deaths every year. Preterm birth

is defined as childbirth occurring at a gestational age (GA) of less than 37

completed week (w) (Goldenberg et al. 2008), but can be further categorised

as: late preterm (34-36 w GA), moderate preterm (32-33 w GA) very preterm

(28-31 w GA) and extremely preterm (<28 w GA) (Tucker & McGuire 2004).

The incidence of extremely preterm and very preterm births is relatively low

(5.2 % and 10.4 % of all preterm births, respectively) with the majority (84.3 %)

being classified as late preterm births (Blencowe et al. 2012).

The rate of preterm delivery in developed countries varies from 7 to 12 % of all

births (Abeywardana 2004; Blencowe et al. 2012; Beck et al. 2010) and is

approximately 10 % for Australia and New Zealand (Li et al. 2014). In many

low to middle income countries, however, the incidence of preterm birth is more

than 15 % (Blencowe et al. 2012). The incidence of preterm birth is rising

despite major advances in medical treatment and continues to emerge as a

major public health concern. Infants who are born prematurely often require

special clinical care and face greater risks of serious health complications. The

major clinical problems involving premature infants relate to the under-

development of major organs such as the brain, lungs, kidneys, skin, eyes,

gastrointestinal system, and immune system and that impact on the primary

functions of the human body to support life in the extra uterine environment

(Behrman & Butler 2007). The risk of acute neonatal diseases increases with

decreasing GA.

3

There are a number of known causes of premature birth (Slattery 2002;

Goldenberg 2008), including:

a) spontaneous preterm labour

b) multiple pregnancy

c) assisted reproduction

d) premature prelabour rupture of membranes

e) hypertensive disorders of pregnancy

f) intrauterine growth restriction

g) antepartum haemorrhage

h) intrauterine inflammation/chorioamnionitis

Intrauterine inflammation/chorioamnionitis is one of the most common causes

of preterm birth (Goldenberg et al. 2000). Chorioamnionitis is defined as

histopathologic evidence of inflammation of the amnion and/or the chorion.

Most commonly this inflammation is a consequence of bacterial infection of the

amniotic fluid, fetal membranes, placenta, and/or uterus. Antenatal infection

can trigger intra-uterine inflammation which then promotes preterm labour. In

utero inflammation is one of the most common triggers of preterm births

(Goldenberg et al. 2000; Galinsky et al. 2013). The incidence of clinically

diagnosed chorioamnionitis increases markedly with decreasing gestational

age, with the incidence decreasing from 66 % in infants born at 20–24 weeks’

gestation to 16 % at 34 weeks (Lahra & Jeffery 2004).

Chorioamnionitis stimulates an inflammatory cascade that induces preterm

birth (Goldenberg et al. 2000). Bacterial invasion of the choriodecidual space

results in the release of endotoxins and exotoxins (Goldenberg et al. 2002) that

are recognised by Toll-like receptors (TLRs). TLRs are expressed on the

surface of leukocytes, dendritic, epithelial, and trophoblast cells (Holmlund et

al. 2002). The binding of toxins to TLRs leads to activation of downstream

transcription factors including nuclear factor kappa B (NF-𝜅B), activator protein

1, and signal transducer and activator of transcription which produce cytokines

and chemokines such as interleukin (IL)-6, IL-1, IL-1, IL-8, and tumour

necrosis factor- (TNF-) within the decidua and the fetal membranes

4

(Goldenberg et al. 2000). Inflammatory cytokines stimulate the production of

prostaglandins (PGs) and initiate neutrophil chemotaxis, infiltration, and

activation. Inflammatory cytokines also results in the synthesis and release of

matrix metalloproteases (MMPs) (Kota et al. 2013). PGs stimulate uterine

contractions while MMPs cause cervical ripening and degrade the

chorioamniotic membranes causing them to rupture (Figure 1.1)(Kota et al.

2013).

Figure 1.1 Inflammatory cascade caused by chorioamnionitis leads to preterm birth. FasL: Fas ligand, CRH: Cortico Tropic Hormone, PG:

Prostaglandin, MMP: Matrix Metallo Proteinase (Kota et al. 2013).

In addition to stimulating preterm birth, in utero inflammation also influences

multiorgan function and development (Figure 1.2) (Gotsch et al. 2007). Invasion

of the uterine cavity by bacteria may further infect the fetal compartment

causing production of fetal systemic cytokines or inflammatory response called

5

fetal inflammatory response syndrome (FIRS) (Gotsch et al. 2007). FIRS

causes multi-organ injury and dysfunction including necrotizing enterocolitis,

funisitis, thymic involution, retinopathy of prematurity, brain white matter injury

and cerebral palsy. Inflammation-associated (pro and anti-inflammatory

exposures) changes in lung function are complex (Figure 1.3). After in utero

inflammation, preterm lungs are exposed to multiple injuries due to

resuscitation, mechanical ventilation, hyperoxia and postnatal sepsis leading to

the development of chronic lung disease. Treatments provided antenataly and

postnataly such as steroids and surfactant protein helps to reduce lungs

inflammatory conditions. Changes in lung function due to exposure to an in

utero infection may provide short-term benefits however predisposes the

infants to detrimental long-term effects (Gantert et al. 2010). For example, in

utero inflammation stimulates surfactant production and lung maturation

therefore reducing the incidence of respiratory distress syndrome (RDS),

however inflammation induced lung injury increases the risk of the development

of bronchopulmonary dysplasia (BPD). The long-term development and

outcome of the preterm infants are influenced by the intensity and the time

point of pro-inflammatory and anti-inflammatory exposures.

6

Figure 1.2 Chorioamnionitis is implicated in the pathogenesis of multiple organ disease of the fetus (Gantert et al. 2010).

7

Figure 1.3 Overview of the pro-inflammatory and anti-inflammatory stimuli that a fetal lungs are exposed to during preterm birth and postnatal development (Gantert et al. 2010).

In utero inflammation is implicated in the pathogenesis of lung diseases such

as RDS and BPD, (Figure 1.2) (Behrman & Butler 2007; Ward & Beachy 2003).

Importantly, preterm infants often develop respiratory insufficiency over the first

week of life requiring mechanical ventilation support, corticosteroid treatment

and surfactant delivery as a consequence of immature structure and function of

the lungs.

1.2. Respiratory problems in preterm infants

The respiratory system consists of two parts: a) the lungs, the gas-exchanging

organ, and b) a pump that ventilates the lungs (Roussos & Macklem 1982). The

8

pump includes the chest wall, respiratory muscles, neural respiratory centres,

and the phrenic nerve (Kajekar 2007).

The primary function of the lung is gas exchange, and lung development occurs

mostly in utero. Premature birth disrupts normal prenatal lung development,

which results in significant change in lung function and contributes to postnatal

respiratory disease (Galinsky et al. 2013). Growth and development of the lung

is a continuous process, but can be divided into five overlapping phases

(Figure 1.4). The first phase is embryonic (4–7 w GA), followed by the

pseudoglandular phase (7–17 w GA). Formation of major airways, bronchial

trees and portions of the respiratory parenchyma take place during these first

two phases along with the birth of the acinus. The last generation of the lung

periphery forms during the third, canalicular phase (17–26 w GA) along with

epithelial differentiation and formation of air-blood barrier. Fourth is the saccular

phase (27–36 w GA), during which surfactant production and expansion of air

spaces take place. Last is the alveolar phase (36 w GA to term), whereby

maturation of pulmonary vasculature takes place and this phase continues into

childhood (Rodeck & Whittle 2009; Joshi & Kotecha 2007). Disturbance of the

stepwise process of lung development during any of these phases cause the

lung to be less effective as a gas exchanger, thus preterm infants are more

susceptible to postnatal respiratory disease (Maritz, Morley & Harding 2005).

9

Figure 1.4 Early growth and developmental phase of the human lung (Kajekar 2007)

Extremely preterm birth corresponds with the canalicular phase of the lung

development (Smith et al. 2010). Initiation of mesenchyme vascularisation and

poor differentiation of airway epithelial cells into type I (across which gas

exchange occurs) and type II (the surfactant-generating cells) occur during the

canalicular phase. Very preterm birth parallels with the saccular phase of the

lung development (Smith et al. 2010). The air-blood barrier has only started

thinning, type II epithelial cells are immature and vascularisation is incomplete

at this phase. Infants born during the canalicular and saccular phases of lung

developmental may require mechanical ventilator support. Preterm infants aged

34 to 36 w GA, which are at the saccular stage of lung development, showed

reduced occurrence of neonatal respiratory problems related to surfactant

deficiency (Colin, McEvoy & Castile 2010). Respiratory function of late-preterm

10

infants is affected by RDS, transient tachypnoea of the newborn, pulmonary

hypertension, and pneumonia (Leone et al. 2012; Cheng et al. 2011).

Total lung volume undergoes rapid changes during the last trimester of

gestation. At 30 w GA, the lung volume is only 34 % of the mature birth total

lung volume. At 34 w GA, the lung volume achieves 47 % of the final volume at

maturity (Colin, McEvoy & Castile 2010). At 30 to 32 w GA the surface area

increases from 1.0 to 2.0 m2, increasing further to between 3.0 to 4.0 m2 at

term (Colin, McEvoy & Castile 2010). The challenge of maintaining functional

residual capacity that allows stable gas exchange is likely compounded by

apnoeic events in the preterm infant, which will drive the system to critically low

lung volumes and result in rapid desaturation (Poets et al. 1997). Therefore,

surviving infants of preterm birth face immediate acute respiratory challenges

associated with underdeveloped respiratory system such as RDS or later in life

develop a chronic lung disease such as BPD.

Not surprisingly, respiratory failure among preterm infants is commonly

associated with immature structure and function of the lungs (Behrman & Butler

2007; Kwinta & Pietrzyk 2010; Moss 2006; Sanchez-Solis et al. 2012; Vollsæter

et al. 2013). However, it is likely that the functional immaturity of the preterm

diaphragm also contributes to the development of respiratory failure.

Mechanical stretch from diaphragmatic contraction contributes to lung growth

(Ysasi et al. 2013). Lung cellular proliferation and growth is stimulated by fetal

breathing movements executed by respiratory muscle, primarily the diaphragm

and the intercostal muscles (Jani et al. 2009). Genetically engineered mice

lacking respiratory muscles in utero showed evidence of pulmonary hypoplasia

(Baguma-Nibasheka et al. 2012), which is a precursor for BPD. Pulmonary

development stops in the absence of lung expansion in conditions such as

diaphragmatic hernia or oligohydramnios (Hedrick et al. 1994). Normal in utero

lung development appears to depend upon intact neural innervation of the

diaphragm in humans (Cunningham & Stocks 1978) and animals (Alcorn et al.

1980; Wigglesworth & Desai 1979; Wigglesworth, Winston & Bartlett 1977).

Fetal breathing movements in human infants are observed as early as 11 w

gestation and by 30-40 w. Thus, inefficient diaphragm contractility in utero

11

impairs fetal lung development and increasing the susceptibility to postnatal

lung dysfunction.

For the respiratory pump, there are three major causes of pump dysfunction

(Roussos 1995): i) inadequate output of the centres controlling the respiratory

muscles; ii) a mechanical defect in the chest wall; and iii) dysfunction of

respiratory muscles as force generators even though the central respiratory

drive is consistent with the demand and the chest wall is structurally and

mechanically intact.

Impaired diaphragm function is considered infrequently as a major factor

contributing to development of postnatal respiratory failure in preterm infants,

despite the vital contribution of the diaphragm to self-sufficient breathing.

However, like the lung, the preterm diaphragm is structurally and functionally

immature at birth (Dimitriou et al. 2003; Lavin et al. 2013) and less able to cope

with an increased work of breathing compared to the term diaphragm.

Furthermore, extremely preterm infants are commonly exposed to an

inflammatory environment in utero that affects skeletal muscle function

(Callahan & Supinski 2009). Therefore, the immature diaphragm is vulnerable

to adverse in utero exposures that may contribute to inefficient spontaneous

breathing. The diaphragm (muscle pump) needs to work harder to overcome

the disadvantages imposed by immature lungs function for achieving adequate

tidal volumes for gas exchange. Preterm infants need to generate sufficient

inspiratory force to overcome the mechanical disadvantages imposed by a

highly compliant chest wall, a low level of endogenous surfactant and

noncompliant, structurally immature lungs. The diaphragm, the major

inspiratory muscle, plays an important role in unsupported spontaneous

breathing, and needs to work harder to generate sufficient inspiratory force.

The increased mechanical load on the diaphragm muscles during respiration in

preterm infants may contribute to the development of respiratory insufficiency.

Thus, we propose that the integrity of the diaphragm muscle at birth may

critically influence the development of respiratory failure after birth. Improving

the in utero development of the diaphragm and the structure and function of the

12

diaphragm at birth is crucial to ensure a healthy start to postnatal life for these

extremely vulnerable preterm infants.

1.3. Inspiratory muscles and neural control of breathing

The inspiratory muscles are comprised of the diaphragm and the external

intercostal muscles. The diaphragm is a unique dome-shaped skeletal muscle

separating the thoracic and abdominal cavities (Figure 1.5) (Merrell & Kardon

2013). The oesophagus, and the two main blood vessels, the inferior vena

cava, and the descending aorta pass through the diaphragm. The diaphragm is

composed of three discrete anatomical regions; the costal, sternal and crural

portions (Reid & Dechman 1995). The costal portion ascends from the upper

margins of the lower six rib bones and is closely associated with the sternal

region. The sternal region derives from the posterior aspects of the xyphoid

process. The crural region is thicker and arises from the anterolateral aspect of

the L1-L3 spinal column. Diaphragm muscle fibres from all three regions

radiate inward and attach to the central tendon (Reid & Dechman 1995). The

phrenic nerve that innervates the diaphragm is formed by axons originating

from motor neurons located within the ventral horn of the C3-C5 segment of the

spinal cord. The external intercostal muscles are innervated by the external

intercostal nerves, formed by axons originating from motoneurons located at

the T1-T12 segment of the spinal cord. The rhythmic contraction of the

respiratory muscle is regulated by the respiratory control centre in the

brainstem (medulla and pons).

The diaphragm performs 70 – 80 % of respiratory work during quiet breathing

(Mioxham & Jolley 2009). During inspiration the inspiratory neurons in the

dorsal respiratory group and rostral part of ventral respiratory group discharge

and (Campbell, Agostoni & Davis 1970) activate motor neurons innervating the

diaphragm and external intercostal muscles. Activated diaphragm fibres

contract and shorten, whereas the external intercostals elevate the ribs causing

an increase in volume of the thoracic cavity. As the thoracic cavity enlarges, the

lungs expand, the intra-alveolar pressure drops and air flows down the

pressure gradient into the lungs. At the end of inspiration, the inspiratory

13

muscles relax, the diaphragm assumes its original dome-shaped position and

the elevated rib cage falls. The chest wall and stretched lungs recoil to their

pre-inspiratory size, reducing lung volume and increasing intra-alveolar

pressure forcing air to leave the lungs (Campbell, Agostoni & Davis 1970). The

diaphragm is a skeletal muscle with unique requirements, as it must be

functionally developed at birth to enable spontaneous independent respiration

and is cyclically active throughout the lifespan. Hence, the in utero

development and functional composition are vitally important for proper

postnatal respiratory function.

Figure 1.5 Diaphragm muscle (Aroyan 2015).

1.4. Mechanical properties of diaphragm muscle

Among mammalian species, in vitro measurement of the active force-length

relationship in diaphragm bundles is bell shaped curve that plateaus at lengths

14

ranging from 90 – 110 % maximal muscle length (L0) (McCully & Faulkner

1983; Sieck et al. 1989). The twitch force can be characterised by three

parameters: peak twitch force (Pt), time to peak tension (TTP), and one-half

relaxation time (1/2 RT). These parameters are determined by the muscle fibre

type composition and vary among species and age group. Preterm lamb (121 d

GA) diaphragm TTP is about 151 ms and term lamb (145 d GA) reach Pt 12 ms

faster than preterm diaphragm (Lavin et al. 2013). Similarly diaphragm 1/2 RT

is longer at preterm compared to term lambs. Canine abdominal and intercostal

muscles reach Pt approximately 10-15 ms faster than the diaphragm muscle.

The 1/2 RT follows a species and muscle specific pattern similar to TPT. The

1/2 RT of canine intercostals is similar to the diaphragm. The twitch

characteristics are determined by the rate of cross-bridge attachment and

detachment and intracellular Ca2+ dynamics (Sieck et al. 2013). Skeletal

muscle force increases with an increase stimulus frequency reaching a plateau

that corresponds to maximum tetanic force. The force-frequency relationship of

the diaphragm muscle has a sigmoidal shape in human (Sieck et al. 2013) and

sheep (Lavin et al. 2013). The shape of this relationship is primarily determined

by the rate of force relaxation during a twitch contraction. Tetanic fusion of

force occurs at lower frequencies of stimulation in muscle fibres and motor

units with slower 1/2 RT (Fournier et al. 1988).

1.5. Diaphragm muscle development

The diaphragm muscle is a mixed fibre type skeletal muscle and composed of

both type I (slow twitch) and type II (fast twitch) muscle fibres (Metzger, Scheidt

& Fitts 1985; Sieck et al. 1983). The mixed fibre type composition may reflect a

dynamic capacity of diaphragm muscle to adapt to varying functional demands

(Sieck, Fournier & Enad 1989). The power and endurance of skeletal muscle is

determined by its oxidative capacity and contractile properties. Type I muscle

fibres have a high oxidative capacity and type II fibres are relatively low in

oxidative capacity. Based on histologic staining for metabolic enzyme

(myofibrillar ATPase (mATPase), succinate dehydrogenase (SDH) and α-

glycerophosphate dehydrogenase (α-GPDH)) activities and contractile

properties, muscle fibres are further classified as slow oxidative (SO), fast

15

oxidative, glycolytic (FOG) and fast, glycolytic (FG) (Sieck et al. 2013). Skeletal

muscle fibre type classification is often based on immunoreactivity to antibodies

specific for different myosin heavy chain (MHC) isoforms. Myosin is composed

of heavy and light chains that form the hexameric protein. The major functional

differences in myosin isoforms exist in the heavy chain portion of the myosin

molecule (Maclntosh, Gardiner & McComas 2006). MHC is found in multiple

isoforms that contributes to the functional diversity of muscle fibres (Pette &

Staron 2000). Histochemical staining with specific antibodies categorise muscle

fibres as type I (MHCslow), IIa (MHCIIa), IIb (MHCIIb), and IIx (MHCIIx)

isoforms (Greising et al. 2012). In addition to the four adult MHC isoforms,

there are also embryonic (MHCemb) and neonatal (MHCneo) MHC isoforms

(Sieck et al. 2013) that are found in skeletal muscle fibres during embryonic

and early postnatal development. Histochemical evaluation of fibre type

distribution of the adult rat showed that the diaphragm muscle contained 40 %

type I, 27 % type IIa and 34 % type IIb fibres (Metzger, Scheidt & Fitts 1985).

Adult human diaphragm consist of about 55 % type I, 21 % type IIa and 24 %

type IIb muscle fibres (Lieberman et al. 1973).

During development, the diaphragm undergoes changes in relation to muscle

fibre type composition, size and cross-sectional area, and oxidative capacity.

Most literature on the developmental changes of diaphragm muscle details the

patterns of MHC isoforms expression. Assessment of MHC isoform profiles

using electrophoresis can also yield important quantitative information on

diaphragm development. At 16-24 w gestation the human diaphragm contains

predominantly MHC emb/neo and small proportion of MHCs. At 25-42 w,

MHCIIa, as well as MHC emb/neo and MHCs were evident. From 27 w, four

isoforms including a small fraction of MHC IIb were seen in half of the samples

from these GA groups (Lloyd et al. 1996). Developmental changes in

diaphragm using the ovine model showed that the functional capacity improves

with fetal maturational age, supported by a progressive increase in MHCs) and

MHC II or fast (MHCf) protein content (Lavin et al. 2013).

The proportions of different muscle fibre types are largely determined during

fetal and early postnatal development. Several studies have reported that

16

premature infants have deficient skeletal muscle growth and development

(Keens et al. 1978; Finkelstein et al. 1992; Schloon et al. 1979). Preterm infants

tolerate respiratory load poorly and are at higher risk of respiratory failure

compared with older infants and children (Dimitriou et al. 2003). Impaired

tolerance of respiratory loads was originally thought to be caused by the

relative lack of highly oxidative, fatigue resistant type I fibres in the preterm

diaphragm (Rehan et al. 2001). The rat diaphragm muscle expresses a low

proportion of type I fibres early in development, and a progressive increase in

type I fibre with increasing age (Eddinger, Moss & Cassens 1985). In

premature infants (less than 37 w gestation) the diaphragm muscle is

composed of only 10 % type I fibres while full term newborns have 25 %,

suggesting that the preterm diaphragm muscle is more susceptible to fatigue

than the term infants (Keens et al. 1978). Despite low proportion of type I fibres,

several studies showed contradicting evidence that neonatal diaphragm is

more fatigue resistance than adult diaphragm based on oxidative capacity and

the Burke fatigue test (Maxwell et al. 1983; Watchko & Sieck 1993). Therefore,

muscle fibre type is not the sole contributor to muscle fatigability in immature

skeletal muscle.

In addition to changes in muscle fibre type, there are also developmental

changes in intracellular Ca2+ handling (West et al. 1999), oxidative capacity

(Song & Pillow 2012; Sieck, Cheung & Blanco 1991), and myofilament

structure (West et al. 1999) which impact on force generation and susceptibility

to fatigue. During development, diaphragm myofibres have many large

mitochondria (Maxwell 1989) and poorly developed antioxidant defence system

(Song & Pillow 2012). These developmental changes occur at different rates;

therefore, the level of functional development of the diaphragm will vary with

gestational age and may impact on the resilience of the immature newborn

infant to increased respiratory mechanical loads that occur after birth.

An increase in diaphragm size correlates directly with inspiratory force

(Bottinelli & Reggiani 2000): full-term infants reach adult levels of maximal

transdiaphragmatic pressure (Pdimax) at approximately 6 months of age

(Rochester 1993). Transdiaphragmatic pressure in response to bilateral phrenic

17

nerve stimulation correlated significantly with GA and post conceptional age,

suggesting that diaphragm strength is influenced by ongoing development in

utero and postnatal maturation (Dimitriou et al. 2003). Little evidence is

available on diaphragm weakness and its contribution to respiratory failure

among premature infants.

1.6. Diaphragm muscle failure in preterm infants

A functional diaphragm is critically important to the successful establishment of

unsupported spontaneous breathing (Grinnan & Truwit 2005). The mechanism

for diaphragm failure after birth is likely related to increased mechanical load on

the diaphragm in preterm infants. Many factors may contribute to increased

work of breathing and impaired diaphragm function in the preterm infants. A

highly compliant chest wall, orientation of the diaphragm and poor control over

intercostal muscles may contribute to paradoxical breathing (whereby the lungs

deflate during inspiration and inflation during expiration, the opposite of normal

chest motion) which increases work load of the diaphragm.

The angle of insertion of the diaphragm in newborn infants is considerably

more horizontal than it is in older infants (Muller & Bryan 1979; Levangie &

Norkin 2011; Nichols 1991). The usual dome shape of the diaphragm appears

less pronounced, but this positioning is acceptable for normal breathing.

Nevertheless, during stressed breathing, the diaphragm contracts beyond the

point where the dome is flattened. At this point, the contraction pulls the ribs

inward rather than expanding them thus compromising the increase in volume

of the thoracic cavity and expansion of the lungs. A flatter diaphragm increases

the functional residual capacity, however it is less able to increase the tidal

volume to compensate for changes in respiratory rate or increased oxygen

demand.

Diaphragm performance is likely to be further impaired in infants born

prematurely, in whom growth and development of skeletal muscle is impeded.

Preterm infants spend 50-60% of sleep time with rapid eye movement (REM)

and breathing tends to be irregular in tidal volume and frequency during REM

18

sleep. Chest wall distortion has been suggested to increase the volume

displacement of the diaphragm during inspiration, which may be associated

with muscular fatigue and apnoea in preterm infants (Heldt & McIlroy 1987).

In summary, preterm infants need to generate sufficient inspiratory force to

overcome the mechanical disadvantages imposed by a highly compliant chest

wall, a low level of endogenous surfactant and noncompliant, structurally

immature lungs. Thus, we hypothesise that increased mechanical load on the

intercostal and diaphragm muscles during respiration in premature infants,

induces muscle fibre weakness and diaphragm dysfunction. Additional to

immaturity of structure and function of diaphragm among premature infants, in

utero exposures such as intra-amniotic infection induced inflammation may

further exacerbate preterm diaphragm function.

1.7. Antenatal inflammation and its impact on preterm diaphragm function

Intra-amniotic infection, commonly manifest as chorioamnionitis, is a frequent

cause of premature birth. Chorioamnionitis frequently induces a systemic fetal

inflammatory response syndrome (FIRS) causing multiple organ injury and

adverse neonatal outcomes (Gotsch et al. 2007). FIRS is mediated by pro-

inflammatory cytokines (IL-1, IL-6 and TNF-α) and clinically diagnosed by

increased plasma IL-6 levels and funisitis (Romero et al. 2007; Gomez et al.

1998). Increased cytokine secretion in inflammatory diseases is commonly

linked with the development of muscle weakness (Reid, Lännergren &

Westerblad 2002). Pro-inflammatory cytokines cause reduced muscle

contractility through modulation of Ca2+ transients or sensitivity of myofilaments

to Ca2+ activation (Stamm et al. 2001). Circulating pro-inflammatory cytokines

play an important role in diaphragm weakness in mice after exposure to

intraperitoneal LPS (Labbe et al. 2010). Due to the presence of large numbers

of mitochondria (Demoule 2013) and a less efficient antioxidant defence

system (Song & Pillow 2012), the preterm diaphragm is prone to oxidative

stress. Oxidative stress induces muscle weakness via increased mitochondrial

production of reactive oxygen species (ROS), activation of the proteolytic

19

pathway and myofibre atrophy. Additionally, ROS affects muscle function

adversely by altering myofibrillar Ca2+ and cross-bridge kinetics independent of

muscle proteolysis (Andrade 2001).

Oxidative stress produces ROS that induce changes in protein structure such

as amino acid modification and fragmentation and disrupt redox homeostasis

by activating signalling pathways associated with muscle wasting (Barreiro et

al. 2005b). Thus, in the presence of in utero infection, the preterm fetal

diaphragm may be more susceptible to inflammation and oxidative stress than

the term fetal diaphragm (Callahan & Supinski 2009). A 7 d in utero exposure

to intra-amniotic LPS in lambs increases circulating neutrophils (Kramer et al.

2001), causes systemic oxidative stress (Cheah, 2008) and is likely to

contribute to contractile dysfunction in skeletal muscle. Sepsis is also a major

cause of morbidity and mortality in preterm infants (Kaufman & Fairchild 2004).

The risk for the development of persistent acquired weakness syndromes

increases with sepsis, and affects both the respiratory muscles and the limb

muscles (Callahan & Supinski 2009). Therefore, we hypothesise that the

preterm diaphragm is susceptible to phenotypic alteration induced by antenatal

inflammation. Consequently, inflammatory conditions such as chorioamnionitis

are likely to compromise the integrity of the diaphragm at delivery and may

critically influence the resilience of the infant to developing respiratory failure

after birth.

1.8. Potential treatments for inflammation induced diaphragm dysfunction: Targeting IL-1 signalling

Inflammatory cytokines are increased in amniotic fluid with chorioamnionitis

and interleukin-1 (IL-1) is postulated to play an important role in the fetal

inflammatory response (Kallapur et al. 2009; Berry et al. 2011). IL-1 and IL-1

receptor antagonist (IL-1ra) are co-localised in both normal and inflamed

placentas (Baergen, Benirschke & Ulich 1994). Inhibition of IL-1 signalling

decreases LPS-induced lung and systemic inflammation in the preterm sheep

(Kallapur et al. 2009). IL-1 receptor can be blocked using human recombinant

IL-1ra (commonly used anti-inflammatory drug) (Kallapur et al. 2009) thus

20

inhibition of IL-1 signalling may diminish the inflammation induced muscle

injury. Pre-treatment with rhIL-1ra reduced the damage to alveolar epithelial

cells in a rat model of ventilator induced lung-injury (Frank et al. 2008).

Similarly, deletion of the IL-1 receptor type 1 (IL-1R1) gene in mice attenuated

the pulmonary inflammatory response to aerosolised LPS (Hudock et al. 2012)

suggesting the important role of IL-1 signalling in lung inflammation and injury.

To date, the inhibitory effects of IL-1ra to reduce muscle weakness in LPS

exposed preterm diaphragm are unexplored.

The resilience of infants to developing respiratory failure after birth may be

critically influenced by the integrity of the diaphragm. This PhD project will

examine the influence of clinically relevant intra-uterine inflammation on the

function of the lamb fetal diaphragm muscle. To date, the effect of intra-uterine

inflammation on the development and function of the fetal diaphragm is

unknown.

1.9. Ovine model of chorioamnionitis

Various animal models are used for the study of chorioamnionitis. In rats the

surgical route to access the uterus has been used to inject a pro-inflammatory

stimulus into the amniotic cavity (Ikegami et al. 2000). Ultrasound-guided IA

injections have been performed in rabbits, mice and sheep (Bry, Lappalainen &

Hallman 1997; Jobe et al. 2000; Prince et al. 2004). All these models are valid

for chorioamnionitis, however, the underlying developmental biology varies

between species. For example, the alveolar phase of lung development occurs

late in gestation in humans and sheep but postnatally in rodents. Other

differences are present in the timing of myelinisation of the brain and

maturation of the fetal gut. The size of newborn lamb is similar to human

infants, making lambs suitable for testing medical intervention associated with

mechanical ventilation. Therefore, the ovine model of chorioamnionitis has

been widely used to understand the pathogenesis of the lung disease. LPS

from E. coli was used to induce a sterile chorioamnionitis in time-mated

pregnant sheep as an ideal model of chorioamnionitis.

21

1.10. Statement of aims

The aims of this project are to determine the effect of clinically relevant

antenatal inflammatory exposures and the timing of the inflammatory insults on

the function of the preterm diaphragm. In this project the hypotheses to be

tested is that the structure and function of the preterm diaphragm is influenced

by maturation and inflammatory exposure before birth.

The specific aims of the PhD project are:

Aim 1: To determine the effect of timing and frequency of antenatal inflammation during gestation on diaphragm muscle function

Objective: To assess whether gestational age (GA) at the time of exposure

(2d, 7d, 21d before birth) to intra-amniotic (IA) lipopolysaccaride (LPS) and/or

the chronicity of exposure (7d, 14d, 21d before birth) that induces inflammatory

response will alter the function of the diaphragm. We hypothesise that GA at

time of initial exposure to IA LPS would determine the extent of functional

impairment of the fetal diaphragm. Furthermore, we hypothesized that multiple

inflammatory insults would exacerbate the diaphragm impairment resulting from

a single intrauterine fetal exposure to LPS.

Aim 2: To determine the effect of GA at time of antenatal inflammation on diaphragm muscle function in term and preterm sheep

Objective: To determine if the preterm fetal lambs (121 d GA) are more

vulnerable than near-term lambs (145 d GA) to alteration of diaphragm function

induced by an inflammatory stimulus. We hypothesise that preterm diaphragm

is more susceptible to in utero LPS induced inflammation than term diaphragm.

Aim 3: To determine the role of IL- 1 pathway in inflammation induced diaphragm dysfunction in preterm lambs

Objective: To determine how diaphragm injury due to inflammation is

influenced by known modulators of inflammatory and oxidative stress

pathways. The study aims to investigate if IA LPS induced diaphragm

22

weakness is mediated via IL-1 signalling; and/or is dependent on oxidative

stress. We hypothesised that blockade of IL-1 signalling ameliorates diaphragm

dysfunction induced by IA LPS exposure.

23

Chapter 2

In utero lipopolysaccharide exposure impairs preterm

diaphragm contractility

Preface

This study evaluates the functional changes in the preterm fetal diaphragm following in utero inflammation and explores the underlying

mechanisms to explain any change in function

This chapter was published by

Journal of Respiratory Cell and Molecular Biology

Am J Respir Cell Mol Biol, 2013; 49(5):866-74

24

2. Chapter 2: In utero lipopolysaccharide exposure impairs

preterm diaphragm contractility

Yong Song1, 2, Kanakeswary Karisnan1, 2, Peter B Noble1, 2, Clare A Berry1, 2,

Tina Lavin1, 2, Timothy J.M. Moss3 , Anthony J. Bakker1, Gavin J. Pinniger1#, J

Jane Pillow1,2,4#*

1 School of Anatomy, Physiology and Human Biology, The University of

Western Australia, M309, 35 Stirling Highway, Crawley, 6009, Western

Australia, Australia

2 Centre for Neonatal Research and Education, School of Paediatrics and Child

Health, The University of Western Australia, M550, 35 Stirling Highway,

Crawley, 6009, Western Australia, Australia

3 The Ritchie Centre, Monash Institute of Medical Research, and Department of

Obstetrics & Gynaecology, Monash University, 27-31 Wright Street, Clayton,

3168 Victoria, Australia

4 Women and Newborns Health Service, c/-King Edward Memorial and

Princess Margaret Hospitals, 374 Bagot Rd, Subiaco, Perth, Western Australia,

Australia, 6008

# Joint senior author

*Corresponding author

Tel: +61 8 9340 1456; fax: +61 8 9340 1266 (J Jane Pillow); E-mail address:

jane.pillow@uwa.edu.au

Financial and equipment support

This study was supported by NHMRC APP1010665 and a Sylvia and Charles

Viertel Senior Medical Research Fellowship (JJP), WIRF, Ada Bartholomew

25

Medical Research Trust and UWA Research Development Award (YS) and

NHMRC Career Development Fellowship No. 1045824 (PBN).

Authors Contribution: JJP, GJP and AJB obtained the study funding and

were responsible for design of the animal studies. PBN was integral to project

management and together with YS assisted with collection of the muscle

tissues. YS and KK performed experiments and primary data analysis under

supervision of GJP and AJB (physiological measurements) and YS and JJP

(laboratory tissue analysis). All authors contributed to data interpretation; YS

and KK prepared figures and the initial manuscript draft. All authors edited the

manuscript and approved the final version of the manuscript for submission.

2.1 Abstract

Preterm birth is associated with inflammation of the fetal membranes

(chorioamnionitis). We aimed to establish how chorioamnionitis affects

contractile function and phenotype of the preterm diaphragm. Pregnant ewes

received intra-amniotic injections of saline or 10 mg lipopolysaccharide (LPS) 2

d or 7 d prior to delivery at 121 d gestation (term = 150 d). Diaphragm strips

were dissected for assessment of contractile function after terminal anesthesia.

The inflammatory cytokine response, myosin heavy chain (MHC) fibre,

proteolytic pathways and intracellular molecular signalling were analyzed using

qPCR, ELISA, immunofluorescence staining, biochemical assays and Western

blot. Diaphragm peak twitch force and maximal tetanic force were

approximately 30 % lower than control in 2 d and 7 d LPS groups. Activation of

the NF-κB pathway, an inflammatory response and increased proteasome

activity, were observed in the 2 d LPS group relative to control or 7 d LPS

group. No inflammatory response was evident after a 7 d LPS exposure. 7 d

LPS exposure markedly decreased p70S6K phosphorylation but there was no

effect on other signalling pathways. MHC IIa fibre proportion was lower than

control in the 7 d LPS group. MHCs fibre proportions were not different

between groups. Results demonstrate that intrauterine LPS impair preterm

diaphragmatic contractility after 2 d and 7 d exposure. Diaphragm dysfunction

resulting from 2 d LPS exposure was associated with transient activation of

26

pro-inflammatory signalling with subsequent enhanced proteasome activity.

Persistent impaired contractility for 7 d LPS exposure was associated with

down-regulation of a key component of the protein synthesis signalling pathway

and reduction in MHC IIa fibre proportions.

Key words: chorioamnionitis; infant, preterm; diaphragm; contractile

dysfunction; molecular signalling; protein synthesis; proteolysis

2.2 Introduction

Chorioamnionitis, inflammation of the placental and fetal membranes, is

implicated in up to 70 % of preterm births prior to 30 weeks of gestation

(Goldenberg, Hauth & Andrews 2000). Adverse neonatal outcomes of

chorioamnionitis include fetal systemic inflammation; lung, brain, and

gastrointestinal injury (Gomez et al. 1998; Romero et al. 1998; Kramer et al.

2002) and increased risk for bronchopulmonary dysplasia (Hartling, Liang &

Lacaze-Masmonteil 2012).

Little is known about how the structure and function of the preterm diaphragm

is affected by chorioamnionitis despite its obvious role as the primary

respiratory muscle and the incidence of respiratory distress in preterm infants.

In the setting of severe sepsis in adults, diaphragmatic impairment is

acknowledged as a cause of respiratory failure (Hussain, Simkus & Roussos

1985). Because preterm infants often breathe against an increased mechanical

load, diaphragm integrity is particularly critical for self-sufficient ventilation in

preterm infants. Preterm infants exhibit reduced diaphragm contractility

compared to their term gestation counterparts (Lavin et al. 2013; Dimitriou et al.

2003). Additional compromise of the functional and phenotypic integrity of the

weakened immature diaphragm induced by intrauterine exposure to

inflammation could precipitate or accelerate the development of postnatal

respiratory failure.

A marked and rapid reduction is evident in respiratory skeletal muscle strength

with infection in adult animals (Ochala et al. 2011; Supinski et al. 1996;

27

Supinski et al. 2000; Supinski, Vanags & Callahan 2009). Although the precise

mechanisms by which infection impairs muscle function are not fully

understood, accelerated proteolysis and reduced protein synthesis likely

contribute to muscle protein loss during sepsis in adults (Lang, Frost & Vary

2007; Cooney, Kimball & Vary 1997; Attaix et al. 2005). The key factors in the

metabolic response to sepsis include induction of catabolic agents (e.g. TNF-α,

IL-1β, IL-6, cortisol) and suppression of the anabolic factor IGF-1 (Cooney,

Kimball & Vary 1997). The activated ubiquitin–proteasome system (UPP) is a

primary pathway responsible for breakdown of accumulated muscle protein

(Supinski, Vanags & Callahan 2009; Attaix et al. 2005): UPP E3 ligase (atrogin-

1/MAFbx and MuRF1) is up-regulated alongside the induction of skeletal

muscle wasting during infections (Wray et al. 2003; Attaix et al. 2005). The

depression of protein synthesis arises from insulin resistant or impaired IGF-

1/P13K/Akt1 signalling (Lang, Frost & Vary 2007; Cooney, Kimball & Vary

1997).

In addition to inflammation induced muscle wasting, evidence from animal

models suggest that both local (Pinniger, Lavin & Bakker 2012; Bicer et al.

2009) and systemic (Liu et al. 2002) inflammation decreases the intrinsic force-

producing capacity of skeletal muscle (force loss independent of muscle

wasting i.e. reduced contractility). This effect may be mediated by increased

production of reactive oxygen species (ROS) and their effects on myofilaments

and/or altered intracellular calcium homeostasis (Andrade, Reid & Westerblad

2001; Reid, Lannergren & Westerblad 2002).

Previous studies investigating diaphragmatic impairment in the context of

inflammation were undertaken in adult subjects or during the postnatal period

(Bicer et al. 2009; Hussain, Simkus & Roussos 1985; Ochala et al. 2011). The

effect of inflammation on diaphragm structure and function is likely to differ

during critical stages of development occurring prenatally. Since prenatal organ

development is largely under the control of genetic programming (Fowden,

Giussani & Forhead 2006), environmental insults encountered in utero may

alter gene expression, adversely affect the metabolic and endocrine balance of

28

affected individuals and subsequently lead to dysfunction later in life (Fowden,

Giussani & Forhead 2006). Moreover, the premature diaphragm is weaker and

more susceptible to injury compared with that at term owing to differences in

fibre-type (Keens et al. 1978; Lavin et al. 2013), oxidative capacity (Song &

Pillow 2012) and functional immaturity of the immune system response to

bacterial infection (Sadeghi et al. 2007).

The present study sought to evaluate functional changes in the preterm fetal

diaphragm after in utero inflammation and to explore the underlying

mechanisms to explain any change in function. We hypothesised that antenatal

exposure to inflammation promoted structural and physiological changes in the

fetal diaphragm resulting in weakness at birth. To test this hypothesis we used

a well-established preterm ovine model of chorioamnionitis induced by intra-

amniotic (IA) injections of lipopolysaccharide (LPS) (Collins et al. 2012; Berry et

al. 2011) to analyse diaphragm contractile properties, fibre type composition

and activity of protein degradation and synthesis pathways.

2.3 Materials and methods

2.3.1 Animals and experimental design

All experiments were approved by the Animal Ethics Committee of the

University of Western Australia. Date-mated ewes with singleton fetuses were

randomly assigned to a treatment group receiving IA injection of LPS (10 mg

Escherichia coli 055:B5, Sigma Chemical, St. Louis, MO) at 114 d (7 d LPS) or

119 d (2 d LPS) gestational age (GA), or to a control group receiving IA saline

at equivalent time points. Preterm fetal lambs were delivered surgically via a

hysterectomy at 121 d GA (term = 150 d GA), and were killed immediately with

pentobarbitone (150 mg/kg IV, Pitman-Moore, Australia). The right hemi-

diaphragm was removed for analysis of contractile function. The left costal

hemi-diaphragm was used for biochemical and molecular studies or embedded

in optimal cutting temperature (OCT) compound for histological staining.

Plasma was obtained from the umbilical artery to assess systemic response to

IA LPS exposure.

29

2.3.2 Muscle contractile properties

The muscle preparation and contractile property measurement were performed

as described previously (Lavin et al. 2013). The detailed protocol is described

in appendix A1.

2.3.3 Western blot analysis

The whole cell lysate was prepared as described previously (Song & Pillow

2012). Cytosolic and nuclear protein fractions were isolated using NE-PER®

Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Billerica, USA).

Western blot was performed as previously described (Song & Pillow 2012). The

detailed information for antibodies used and quantification and normalization

methods is described in appendix A1.

2.3.4 IL-1β and IL-6 plasma levels

The sheep ELISA assays for IL-1β and IL-6 were developed in our laboratory.

The detailed procedure is described in appendix A1.

.RNA isolation, reverse transcription and quantitative PCR

Detailed methods for RNA purification, reverse transcription and quantitative

PCR condition employed by our laboratory were described previously (Song &

Pillow 2012; Lavin et al. 2013). The proteolytic gene (calpain I, calpain II,

caspase-3, MAFbx, MuRF1, E2, C8 and Ubiquitin) primers used were

optimised and validated by us previously (Song & Pillow 2012). Cytokine gene

(IL-1β, IL-6 and TNF-α) primers were described previously by other

investigators (Zhu et al. 2010; Smeed et al. 2007).

2.3.5 Myosin Heavy Chain (MHC) fibre typing and myofibre Cross-Sectional

Area (CSA)

OCT embedded diaphragm was sectioned and stained with antibodies specific

to laminin (1: 250, Abcam, Waterloo, Australia), MHCs (1:50, Novocastra,

30

Newcastle, UK) or type IIa (1:100, Santa Cruz Biotechnology, Inc, CA, USA).

The detailed protocol is described in appendix A1.

2.3.6 Biochemical analysis

The activities of calpain (Abcam, Waterloo, Australia), caspase-3 (Sigma,

Castle Hill, Australia) and 20S proteasome (The chymotrypsin-like peptidase,

Enzo Life Sciences, Farmingdale, USA) were measured fluorometrically in

crude extracts using commercial kits.

2.3.7 Data analysis

Sigmaplot (version 12.0, Systat Software Inc, San Jose, USA) was used for

statistical analysis. Differences among multiple groups were assessed using

one-way ANOVA with a Tukey honestly significant difference test implemented

as post hoc analysis. Nonparametric data were analysed using ANOVA on

ranks. Pearson correlation index was calculated to determine the association

amongst different variables using linear regression analysis. P < 0.05 was

considered statistically significant. Data are presented as mean (SD) or median

(range) unless specified otherwise.

2.4 Results

2.4.1 Animal characteristics

For the animals used in the physiological assessment of contractile function,

the control group included 3 male and 5 female lambs with a mean gestational

age of 120.7 ± 0.5 d. The 2 d LPS group comprised 3 male and 3 female

lambs, and the 7 d LPS group had 2 male and 4 female lambs, all at 121 d

gestation. There were no significant differences in body weight (Table 2.1) or

condition at delivery.

31

Table 2.1 Descriptive data and twitch parameters for saline and LPS treated preterm lambs.

Saline

(n=8)

2d LPS

(n=6)

7d LPS

(n=6)

Body weight (kg) 2.48 ± 0.34 2.63 ± 0.15 2.50 ± 0.16

Lo (mm) 29.6 ± 3.15 30.3 ± 3.15 28.2 ± 1.87

TTP (ms) 161.9 ± 107.2 115.2 ± 14.4 234.7 ± 143.7

½ RT (ms) 193.1 ± 76.0 220.3 ± 42.9 258.8 ± 60.9

Twitch/Tetanus ratio 0.51 ± 0.06 0.50 ± 0.07 0.53 ± 0.09

Lo, optimal muscle length; TTP, time to peak twitch force; ½ RT, half relaxation

time.

2.4.2 Contractile measurements

Contractile measurements are shown in Table 2.1. There were no significant

differences in optimal muscle length (Lo), time to peak twitch force (TTP), half

relaxation times (½ RT) and twitch/tetanus ratio. Peak twitch force was

decreased by 29 % and 31 % after a 2 d and 7 d LPS exposure, respectively (p

< 0.05) (Figure 2.1A). A similar decrease in maximal tetanic force was also

observed, with 28 % and 33 % reduction in 2 d and 7 d LPS groups

respectively (p <0.05) (Figure 2.1B). The normalised force frequency

32

relationship was not different between the control and LPS treated groups

(Figure 2.1C).

The fatigue protocol reduced maximum tetanic force by approximately 65 %,

although there was no significant difference in the fatigue index between

groups (Figure 2.2A). The stretch induced muscle damage protocol resulted in

a ~15 % decrease in maximum tetanic force which was not altered by prior

Figure 2.1 Fetal diaphragm contractile properties, maximal twitch and tetanic force and force-frequency relationship: Peak twitch force (A), maximal

tetanic force (B) and normalized

force – frequency relationship (C)

for saline (n=8), 2 d LPS (n=6)

and 7 d (n=6) LPS exposure

groups. Values are Mean (SEM). *

and ** indicate p < 0.05 and p <

0.01 respectively, compared with

control group.

33

exposure to LPS (Figure 2.2B). Unloaded shortening velocity (V0) in fetal

diaphragm was also unaffected by LPS exposure (Figure 2.2C).

2.4.3 MHC protein and fibre CSA

MHC fibre typing of preterm diaphragm in the control group showed that 15 %

of total muscle fibres were slow type I and 67 % were fast type IIa fibres (Figure

2.3A). Exposure to LPS 7 d prior to study caused a 19 % decrease in MHC IIa

fibre proportion (p < 0.05) (Figure 2.3B). The apparent lower proportion of type

IIa fibres in the 2 d LPS group did not reach statistical significance (p = 0.190).

The percentage of slow type MHCs fibres was similar in the LPS and control

groups (Figure 2.3B). Total fibre CSA (including all different MHC type fibres)

Figure 2.2 Susceptibility to fatigue and muscle damage, and unloaded shortening velocity of fetal diaphragm: Fatigue index (A),

percentage muscle damage after

stretch protocol (B), and unloaded

shortening velocity (V0) (C) for saline

(n=8), 2 d (n=6) and 7 d (n=6) LPS

exposure groups. Values are Mean

(SEM).

34

was unchanged by LPS exposure (p = 0.136) (Figure 2.3C). Moreover, MHCs

and IIa fibre CSA were not significantly different between the experimental

groups (Figure 2.3C).

2.4.4 Cytokine response

There was a 12-fold increase in plasma IL-6 protein level after 2 d exposure to

LPS (p < 0.05, Figure 2.4A). The IL-6 concentration after 7 d treatment was not

significantly different from control. A similar pattern was observed in systemic

IL-1β, although the difference was not significant (Figure 2.4A).

Consistent with the maximal induction of plasma cytokines after 2 d LPS

exposure, the cytokine mRNA expression in the diaphragm was up-regulated in

the 2 d LPS group, with a 4- and 2-fold change relative to the control group for

Control 2d LPS 7d LPS

Mus

cle

fibre

CSA

(µm

2 )

0

100

200

300

400

500Total MHCMHC I MHC IIa

Figure 2.3 Diaphragm muscle fibre type and cross-sectional area (CSA) measurement: Muscle fibre sections were

stained with laminin (red) or

MHC (green) antibody (A). Graphs show percentage of

MHCs and MHC IIa fibres (B)

and Muscle fibre CSA (C) in the

control (n=4), 2 d (n=4) and 7 d

(n=6) LPS exposure groups.

Values are Mean (SEM). *

indicates p < 0.05. MHC: myosin

heavy chain.

35

IL-1β and IL-6, respectively (p < 0.05) (Figure 2.4B). TNF-α mRNA was not

altered by LPS exposure.

Figure 2.4 Systemic and local cytokine response: A. Plasma IL-1β and IL-6

concentrations in 2 d (n=7) and 7 d (n=7) LPS exposure groups relative to

saline control (n=5). Values are Mean (SEM). * indicate p < 0.05. B. Diaphragm

IL-1β, IL-6 and TNF-α mRNA expression after intra-amniotic LPS exposure.

Values are Median (25th, 75th centile). * # indicates p < 0.05 compared with

control and LPS 7 d group, respectively. Horizontal dashed line indicates mean

or median of reference (saline control) group.

2.4.5 Molecular signalling

To identify signal transduction cascades involved in intra-amniotic LPS-

induced diaphragm dysfunction, we evaluated several key intracellular

mediators of anabolic (Akt, mTOR, p70S6K and 4E-BP1) and catabolic

(FOXO1 and NF-κB) pathways (Figure 2.5). LPS exposure did not change the

phosphorylation state of Akt or 4E-BP1 and total mTOR protein. However, 7 d

36

LPS exposure resulted in decreased phosphorylation of p70S6K (p < 0.05).

The FOXO signalling pathway was not altered in response to LPS. In contrast,

LPS resulted in a significant fall in nuclear NF-κB after 7 d (p < 0.05), although

no effect was evident after 2 d (p=0.136) (Figure 2.5D). There was a significant

association between the dynamic pattern of NF-κB signalling and IL-1β gene

expression in fetal diaphragm (r = 0.481, p < 0.05) (Figure s1).

Figure 2.5 Activity of signalling molecules after LPS exposure: Western

blots illustrate expression of signalling molecules using representative samples

from each group (A). Graphs show p - Akt / total Akt protein (B), mTOR protein

content (C), p – p70S6/total p70S6 kinase (D), p – 4E-BP1/ total 4E-BP1

protein (E), nuclear/cytosolic FOXO protein (F), and nuclear / cytosolic NF-κB

protein (G) in costal diaphragm in controls (n=5) or after exposure to LPS for 2

d (n=7) or 7 d (n=7).Values are Mean (SEM). * indicates p < 0.05. p:

phosphorylated.

37

2.4.6 Proteolytic pathways

The key components of the three major protein degradation pathways (calpain,

caspase-3 and UPP) were analysed for gene expression levels (Figure 2.6)

and the enzyme activities (Figure 2.7). LPS slightly promoted gene expression

of MAFbx, E2, C8 and Ubiquitin after a 2 d exposure, while expression was

reduced after a 7 d LPS exposure. In contrast, a decreasing pattern of gene

expression was observed in MuRF1 in response to increasing duration of LPS

exposure. LPS did not affect caspase-3, calpain I or II mRNA levels.

In accordance with the proteolytic gene expression data, 20S proteasome

activity was markedly higher in 2 d LPS group compared with the control group.

No significant changes in the calpain and caspase-3 activities were detected

across the different groups. Further association analysis demonstrated that

mRNA expression of MAFbx was positively correlated with nuclear: cytosolic

Figure s1 Association of IL-1β / MAFbx gene expression, NF-κB signalling and UPP pathway: A linear relationship

was established between

nuclear / cytosolic NF-κB

protein content ratio and

mRNA levels of IL-1β (solid

line, r = 0.481, p < 0.05) and

MAFbx (dotted line, r = 0.570,

p < 0.05) (A) and between

MAFbx mRNA levels and UPP

pathway activity (r = 0.761, p <

0.001) (B). UPP: ubiquitin–

proteasome system.

38

NF-κB protein content ratio (r = 0.570, p < 0.05) and UPP activity (r = 0.761, p

<0.001)(Figures1).

Figure 2.6 Gene expression of key components in proteolytic pathways: Graphs show calpain I (A), calpain II (B), caspase-3 (C), E2 (D), C8 (E),

Ubiquitin (F), MAFbx (G) and MuRF1 (H) mRNA expression in 2 d (n=7) and 7

d (n=7) LPS exposure groups relative to saline control (n=5). Values are

Median (25th, 75th centile). Horizontal dashed line indicates median of reference

(saline control) group. * p < 0.05 compared with LPS 7 d group.

39

2.5 Discussion

We show that IA LPS impaired preterm (121 d) diaphragmatic contractility after

a 2 d and 7 d in utero exposure. These changes are consistent with the

documented effects on respiratory and limb muscles in animals injected with

LPS (Supinski et al. 2000; Supinski et al. 1996). The phenotypic change was

characterized by preferential reduction in proportion of MHC type IIa muscle

fibres. Short term (2 d) LPS exposure was associated with transient activation

of inflammatory signalling and the NF-κB pathway with increased proteasome

activity. A more prolonged (7 d) exposure involved attenuation of the protein

synthesis pathway. We propose that the difference in regulatory mechanisms

between the 2 d and 7 d LPS exposure groups represent a progressive change

in signalling behaviour associated with either increasing duration of LPS

exposure, or alternatively, the temporal nature of the fetal response to LPS

Figure 2.7 Biochemical activity of calpain, caspase-3 and UPP pathways: Graphs show response of

calpain (A), caspase-3 (B) and UPP

(C) pathways in the control (n=5), 2 d

(n=7) and 7 d (n=7) LPS exposure

groups. Values are Mean (SEM). *

indicates p < 0.05. UPP: ubiquitin–

proteasome system.

40

determined by the gestational age (and diaphragm developmental stage) at the

time of LPS exposure.

Our observation of decreased diaphragm muscle force after 2 d and 7 d in

utero LPS exposure supports our hypothesis that antenatal inflammation

impairs preterm respiratory muscle function. A 2 d and 7 d LPS exposure

reduced both diaphragm peak specific twitch force and maximal specific force

by ~ 30%. This indicates that diaphragm muscle strips from the LPS group are

intrinsically weaker than similar sized strips from the control group. As the strips

varied in size, absolute muscle mass, as an indicator of muscle atrophy, could

not be ascertained in this study. However, decreased specific force is

commonly associated with inflammatory diseases and associated cytokine

secretion (Reid, Lannergren & Westerblad 2002). Cytokines can compromise

muscle contractile function via modulation of calcium transients (Stamm et al.

2001) decreasing the sensitivity of the myofilaments to calcium activation (Reid,

Lannergren & Westerblad 2002), both of which would result in decreased

specific force.

Surprisingly, the reduced force-generating capacity was not accompanied by

increased fatigability. Increased skeletal muscle fatigability has been

demonstrated in LPS-treated adult rats (Goubel et al. 1995) and during sepsis

(Lanone et al. 2005). The discordant results may be due to different

compositions of muscle fibre types in preterm muscles compared to adult

muscles. Preterm diaphragm muscle consists of a majority of oxidative

glycolytic (type I and IIa) fibres that are less fatigable compared to type IIb/x

fibres which are found in a higher proportion in adult muscles (Scott, Stevens &

Binder-Macleod 2001).

Muscle MHC isoform is an important determinant of the contractile properties of

individual myocytes (Bottinelli 2001). Our finding that MHC IIa was the

predominantly expressed isoform (67 %), whereas MHCs represented 15 % of

the total fibres, is consistent with the fibre composition reported in lamb

diaphragm at 127 d GA (Cannata et al. 2011). From late gestation to term, a

41

significant increase in the expression of MHCs and IIa, decrease in MHC IIb/x,

and almost complete loss of embryonic/neonatal MHC expression occurs

(Cannata et al. 2011). After a 7 d LPS exposure, muscle fibre type IIa

proportion was decreased by 19 % although type I remained unchanged.

Altered conformation or reduced absolute number of contractile proteins may

also decrease the number of cross bridges available to generate force and thus

result in a decrease in peak twitch force and maximal tetanic force. However,

the reduced specific force after 2 d and 7 d of LPS exposure preceded any

significant loss of contractile proteins, and was not accompanied by decreased

myofibre CSA. Thus MHC IIa protein loss is not the sole factor accounting for

loss of contractile force deficit, particularly in the 2 d LPS group.

Pro-inflammatory cytokines are primary mediators that trigger the development

of muscle fibre injury (Janssen et al. 2005; Shindoh et al. 1995). Muscle fibre

injury is consistent with our finding that early activation of systemic and local

cytokines (IL-6 and IL-1β) occurs in parallel with impaired contractile function.

Cytokines and other pro-inflammatory mediators released from distant organs

can enter the circulation and act upon the diaphragm in an endocrine fashion in

the setting of sepsis. Local pro-inflammatory cytokine expression may lead to

fibre weakness by promotion of protein degradation (Li et al. 2009) and

suppression of anabolic process (Haddad et al. 2005), induction of oxidative

stress (Jackman & Kandarian 2004). Elevation of circulating cytokines plays a

key role in LPS-induced diaphragm weakness in mice (Labbe et al. 2010).

Thus, the local up-regulation of IL-6 and IL-1β expression we observed was

likely induced by circulating pro-inflammatory mediators in a synergistic

manner. IL-6 and IL-1β function as catabolic factors to stimulate muscle

weakness and induce contractile dysfunction (Spate & Schulze 2004; Schaap

et al. 2006; Haddad et al. 2005).

In addition to IL-6 and IL-1β, TNF-α is an important mediator of adult muscle

dysfunction. However, we found that local TNF-α mRNA was unchanged by

antenatal fetal exposure to LPS. IA LPS does not induce a TNF-α response in

the liver, placental membranes and jejunum of fetal lambs (Kallapur et al.

42

2001). Furthermore, Ikegami et al (Ikegami et al. 2003) showed that preterm

lambs did not develop an inflammatory response to TNF-α, known as a potent

inducer of inflammation in adult sheep. This unique differential response of the

fetus to TNF-α is probably due to poorly developed innate immune system in

the preterm compared to adult subject (Kramer et al. 2010; Hillman et al. 2008).

It follows that the predominant signalling pathways in the development of fetal

diaphragm dysfunction after IA LPS exposure may diverge from those defined

in the adult diaphragm muscle.

Aberrant NF-κB signalling is triggered by inflammatory stimuli and implicated in

muscle atrophy (Haegens et al. 2012; Yamaki et al. 2012). Normally, NF-κB is

sequestered into the cytoplasm of non-stimulated cells and is subsequently

translocated into the nucleus to promote gene expression once the NF-κB

pathway is activated. Indeed, we observed a consistent change between NF-κB

signalling activity and cytokine response in fetal diaphragm after antenatal LPS

exposure. The mechanistic link between NF-κB signalling activity and cytokine

response was supported further by the significant correlation between IL-1β

mRNA expression level and nuclear:cytoplasmic NF- κB protein ratio (Figure

s1).

NF-κB is a transcriptional factor that accelerates muscle protein loss by

regulating expression of multiple atrophic genes and by activating the

proteasome system (Haegens et al. 2012; Wu, Kandarian & Jackman 2011).

We observed upregulation of multiple atrophy genes (MAFbx, E2, C8 and

Ubiquitin) associated with increased activity of the UPP pathway after a 2 d

LPS exposure in comparison to a 7 d LPS group. Moreover, the expression of

MAFbx, the gene specific to muscle atrophy, correlated with both NF-κB

signalling and UPP activity. Other proteolytic enzyme systems such as

caspase-3 and calpain may also contribute to muscle proteolysis under

catabolic conditions by breaking down the contractile proteins and releasing the

protein elements to be targeted by UPP. As we showed no detectable change

in gene expression and activity of caspase-3 and calpain, we propose that UPP

activation within the preterm diaphragm is mainly responsible for loss of muscle

43

protein in response to 2 d LPS exposure in utero. Thus, UPP activation is

probably mediated by inflammatory cytokine and subsequent NF-κB signalling,

similar to the regulatory mechanism in an adult animal model of inflammation-

associated diaphragm muscle weakness (Haegens et al. 2012).

Compared to the 2 d LPS group, a 7 d LPS exposure resulted in a marked

reduction in cytokine response, NF-κB signalling and UPP activity. The

diminished response of the cellular events beyond 2 d LPS exposure

suggested an ability of the preterm diaphragm to resolve the inflammation.

Although the precise mechanisms are unclear, negative regulators such as

SOCS1, IRAK-M and SHIP are proposed to play a key role along with the

down-regulation of TLR4 on cell surface and gene re-programming (Biswas &

Lopez-Collazo 2009) .

The administration of LPS could also reduce skeletal muscle protein synthesis

in neonatal animals (Orellana et al. 2002; Kimball et al. 2003) through

suppression of the anabolic cascade Akt/mTOR and its downstream effectors

(p70S6K and 4E-BP1) to impede efficiency of translation initiation (Orellana et

al. 2011; Tarabees et al. 2011). A 7 d LPS exposure in utero did not change

Akt/mTOR activity, but significantly decreased activity-related phosphorylation

of p70S6K. The activation of p70S6 kinase is essential to maintain normal

muscle fibre mass in vivo, whilst the attenuation of p70S6K signalling could

interfere with the translation of mRNAs into 5' terminal oligopyrimidine tract and

accretion rates of protein synthesis (Ruvinsky & Meyuhas 2006). These data

may imply that the alteration of p70S6K activity in response to fetal

inflammation contribute to muscle protein loss, independently of Akt/mTOR

regulation. However, direct measurement of in vivo protein synthesis rate is

needed to support this possibility. Additionally, nuclear translocation of

cytoplasmic FOXO is a common mechanism in disused muscle atrophy via

decreased activity of Akt (Crossland et al. 2008). Unsurprisingly, the level of

translocated FOXO remained unchanged, which is consistent with its upstream

regulator Akt, further excluding the role of Akt / FOXO in cell signalling and

increased protein breakdown. Of note, however, the reduced specific force

44

after 2 d and 7 d of LPS exposure preceded any significant loss of contractile

proteins, and was not accompanied by signs of atrophy. Thus, contractile

protein loss is not the sole factor accounting for loss of contractile force deficit,

particularly in the 2 d LPS group.

Using a sheep model of chorioamnionitis, we showed that IA LPS caused

systemic oxidative stress at 7 d after in utero exposure, but not after a 2 d

exposure (Cheah et al. 2008). Cytokines and LPS are well known to prime the

increase in reactive oxygen species (ROS)-induced production of neutrophils

through activation of NADPH (DeLeo et al. 1998; Mitchell, Albright & Caswell

2003). The increase in systemic and local cytokine response after a 2 d LPS

exposure in the current study contrasts with our previous observation that a 7 d

LPS exposure was required to increase oxidant activity (Cheah et al. 2008).

Together, these data suggest that the oxidant response after LPS is mediated

by inflammation. Redox disturbance is a known modulator of disused muscle

atrophy through activating multiple proteolytic systems (Powers, Kavazis &

McClung 2007). A recent in vitro study also revealed that oxidants depressed

protein synthesis by reducing the phosphorylation of mTOR substrates (4E-

BP1 and p70S6K) (Zhang et al. 2009). Moreover, ROS has a direct effect on

muscle contractile function via altering myofibrillar Ca2+ sensitivity and cross-

bridge kinetics, leading to muscle weakness (Andrade, Reid & Westerblad

2001). It is therefore feasible that ROS also contributed to fetal diaphragm

weakness in the current study, particularly in the 7 d LPS group by direction

modulation of muscle function and /or indirect activation of signalling pathways.

We argue that the difference in regulatory mechanisms between the 2 d and 7

d LPS exposure groups represent a progressive change in signalling behaviour

associated with either increasing duration of LPS exposure, or the temporal

nature of the fetal response to LPS determined by the gestational age (and

diaphragm developmental stage) at the time of LPS exposure.

45

Summary

In conclusion, a brief (2 d and 7 d) in utero exposure to an inflammatory

stimulus impairs the function of preterm diaphragm. The dysfunction resulting

from 2 d LPS is strongly related to pro-inflammatory signalling, activated NF-κB

pathway and 20S proteasome system. In contrast, 7 d LPS exposure directly

affects the key component of signal transduction pathways regulating protein

synthesis. Overall, IA LPS appears to trigger a complex series of effects

consisting of impaired contractile function, an early inflammatory response

accelerating proteolysis and secondary changes to protein synthesis pathway,

leading to muscle weakness. The contribution of diaphragm dysfunction to

respiratory insufficiency in the preterm infant after a pro-inflammatory exposure

warrants further investigation.

46

Chapter 3

Gestational age at initial exposure to in utero inflammation

influences the extent of diaphragm dysfunction in preterm

lambs

Preface

This study examines the effect of gestational age at the time of IA LPS

exposure and the frequency of exposure on the preterm fetal diaphragm

This chapter was published by Respirology

Resp, 2015; 12615

47

3. Chapter 3: Gestational age at initial exposure to in utero

inflammation influences the extent of diaphragm dysfunction

in preterm lambs

Kanakeswary Karisnan1 MSc, Anthony J. Bakker1, 2 PhD, Yong Song1 PhD,

Peter B. Noble1 PhD, J. Jane Pillow1,2 PhD and Gavin Jon Pinniger1 PhD *

Affiliations:

1 School of Anatomy, Physiology and Human Biology, University of Western

Australia, 35 Stirling Hwy, Crawley, WA 6009, Australia.

2 Centre for Neonatal Research and Education, School of Paediatrics and Child

Health, University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009,

Australia.

* Corresponding author: Gavin J. Pinniger, School of Anatomy, Physiology and

Human Biology, University of Western Australia, 35 Stirling Highway, M309,

Crawley 6009, Western Australia. Email: gavin.pinniger@uwa.edu.au. Phone:

+61 8 6488 3380. Fax: +61 8 6488 1025

Authors Contribution: JJP, GJP and AJB obtained the study funding and

were responsible for design of the animal studies. PBN was integral to project

management and together with YS assisted with collection of the muscle

tissues. KK performed experiments and primary data analysis under

supervision of GJP and AJB (physiological measurements) and YS and JJP

(laboratory tissue analysis). All authors contributed to data interpretation; KK

prepared figures and the initial manuscript draft. All authors edited the

manuscript and approved the final version of the manuscript for submission.

Summary at a glance: We investigated the effect of timing and frequency of in

utero LPS exposure on diaphragm function in preterm lambs. LPS exposure

earlier in gestation caused more extensive alterations to diaphragm function

regardless of frequency. Inflammatory exposures during development cause

48

diaphragm dysfunction that may contribute to the development of chronic

respiratory failure in preterm infants.

3.1. Abstract

Background and objective: In utero infection may critically influence

diaphragm development and predispose preterm infants to postnatal

respiratory failure. We aimed to determine how frequency and gestational age

(GA) at time of intra-amniotic (IA) lipopolysaccharide (LPS) exposure affects

preterm diaphragm function.

Methods: Pregnant ewes received IA injections of saline or 10 mg LPS at 7 d

or 21 d or weekly injections at 7 d, 14 d, and 21 d prior to delivery at 121 d GA.

Fetal lambs were killed with pentobarbitone (150mg/kg; IV). Diaphragm

contractile function was measured in vitro. Muscle fibre type, activation of

protein synthesis and degradation pathways, pro-inflammatory signalling and

oxidative stress were evaluated using immunofluorescence staining, RT-qPCR,

ELISA, Western blotting and biochemical assay.

Results: In utero LPS exposure significantly impaired diaphragm contractile

function. After 7 d LPS exposure maximum specific twitch and tetanic forces

were 30% lower than controls. When the initial LPS exposure occurred 21 d

before delivery (ie for the 21 d and repeated LPS groups) maximum specific

forces were 40 % lower than controls. Earlier exposure to LPS was also

associated with prolonged twitch contraction time, increased fatigue resistance

and elevated protein carbonyl content. Despite increased white blood cell

counts and IL-6 mRNA expression following repeated LPS exposure, there

were no significant differences in contractile properties between 21 d and

repeated LPS groups suggesting that frequency of inflammatory exposure does

not influence the severity of contractile dysfunction.

Conclusion: Our results suggest that GA at time of initial fetal LPS exposure,

rather than frequency of exposure, influences the extent of inflammation

induced diaphragm dysfunction.

49

Keywords: chorioamnionitis; inflammation; preterm diaphragm; contractile

dysfunction

3.2 Introduction

Impaired diaphragm function is considered infrequently as a major factor

contributing to development of postnatal respiratory failure, despite the vital role

of the diaphragm to self-sufficient breathing. However, like the lung, preterm

diaphragm is structurally and functionally immature at birth (Lloyd et al. 1996;

Dimitriou et al. 2003) and less able to cope with an increased work of breathing

compared to term diaphragm (Hammer & Eber 2005). Furthermore, many

extremely preterm infants are exposed to an inflammatory environment in utero

that affects skeletal muscle function. Therefore, the immature diaphragm is

vulnerable to adverse in utero exposures that may contribute to inefficient

spontaneous breathing and development of respiratory failure requiring

mechanical ventilator support.

In utero exposure to inflammation, as occurs in chorioamnionitis, is associated

with adverse respiratory outcomes after birth (Watterberg et al. 1996).

Subclinical/histologic chorioamnionitis is present in up to 70 % of extremely

preterm births (Goldenberg 2000) and the incidence of in utero inflammation

increases with decreasing GA (Sweet et al. 2010). Chronic chorioamnionitis is

more common in spontaneous preterm births (Goldenberg et al. 2008),

suggesting that prolonged or repeated exposure to in utero infection may

exacerbate the inflammatory response. Chorioamnionitis induces a systemic

fetal inflammatory response that affects multiple organ systems (Gotsch et al.

2007 ; Galinsky et al. 2013). However, there is limited understanding of the

effect of systemic fetal inflammation on preterm diaphragm function.

Functional and structural maturity of the diaphragm is associated with

developmental changes in myosin heavy chain (MHC) composition (Keens et

al. 1978), intracellular Ca2+ handling (West 1999), oxidative capacity (Song &

Pillow 2012; Sieck 1991), protein metabolism (Song & Pillow 2013) and

myofilament structure (West 1999). In fetal sheep, the establishment of regular

50

and episodic diaphragm contractions (fetal breathing movements) and

development of the hypothalamo-pituitary-thyroid axis occur at ~100 d GA and

are critical in the maturational changes in muscle phenotype (Dawes 1984;

Finkelstein et al. 1991; Finkelstein et al. 1992). Because these developmental

changes occur at different rates, the functional impact of adverse in utero

exposures may vary depending on the GA at time of exposure. Furthermore,

the impact of chronic chorioamnionitis or repeated acute pro-inflammatory

stimuli on the functional development of preterm diaphragm is currently

unknown.

This study examined the effect of GA at the time of IA LPS exposure and the

frequency of exposure on the preterm fetal diaphragm. We hypothesized that: i)

GA at time of exposure to IA LPS determines the extent of functional

impairment of the fetal diaphragm; and ii) repeated inflammatory exposures

exacerbate diaphragm impairment.

3.3 Methods

3.3.1 Animals and experimental design

Animal experiments were approved by the institutional Animal Ethics

Committee (RA/3/400/1023; RA/3/100/1000). Date-mated Merino ewes were

randomly assigned to a treatment group receiving IA injection of LPS (10 mg

Escherichia coli 055:B5, Sigma Chemical, St. Louis, USA) at 114 d (7 d LPS,

n=6) or 100 d (21 d LPS; n=7) or at 100 d, 107 d and 114 d (repeated LPS;

n=5) GA. Control ewes received IA saline at equivalent time points (n=8). At

121 d GA, ewes were killed with pentobarbitone (150 mg/kg; IV, Pitman-Moore,

Australia). Fetal lambs were delivered via caesarean section after maternal

euthanasia and immediately killed with pentobarbitone (150 mg/kg; IV, Pitman-

Moore, Australia). The right hemi-diaphragm was removed for contractile

function measurements and the left hemi-diaphragm was immediately snap

frozen in liquid nitrogen for molecular and biochemical studies or embedded in

optical cutting temperature (OCT) medium and frozen on dry ice for histological

staining. Plasma was obtained by centrifugation (3 000 RPM, 10 min, 4 C) of

51

blood samples from the umbilical artery to assess the systemic response to IA

LPS exposure. All samples were stored at -80 C prior to analysis.

3.3.2 Diaphragm contractile properties

Diaphragm contractile measurements were performed using an in vitro muscle

test system (model 1205, Aurora Scientific In., Canada) as previously

described (Song et al. 2013a). Muscle strips were adjusted manually to the

optimal length (Lo) at which maximum twitch force (Pt) was recorded.

Contractile measurements included maximum tetanic force (Po), maximum

twitch force (Pt), time to peak (TTP), half relaxation time (1/2 RT), maximum

rate of force development (df/dt) of twitch contractions, and fatigue index (FI)

(Song et al. 2013a). The detailed protocol is described in appendix A1 (page

139-140).

Molecular and biochemical assays used in the current study focused on

pathways involved in LPS-induced inflammation that includes inflammatory

cytokines (IL-1 and IL-6), protein synthesis (AKT) and degradation (FOXO-1,

muscle atrophy; MuRF1 and MAFbx) and oxidative stress (protein carbonyl).

3.3.3 Myosin Heavy Chain (MHC) fibre typing and myofibre Cross-Sectional

Area (CSA)

OCT embedded diaphragms were sectioned and stained with laminin and MHC

I or MHC IIa as previously described (Song et al. 2013a). The proportion of

MHC neonatal fibres was not evaluated due to unavailability of suitable

antibody. Images were captured using fluorescence microscope (Nikon, NY,

USA) and mean CSA and proportion of MHC I and IIa fibres were measured

using the NIS elements software (Nikon, NY, USA). Refer to appendix A1

(page 140-141) for detailed protocol.

3.3.4 Muscle protein extraction

NE-PER® Nuclear and Cytoplasmic Extraction kit (Thermo Scientific, USA)

with inclusion of protease inhibitor cocktail (Roche, USA) was used to isolate

52

the diaphragm cytoplasmic and nuclear protein fractions. Total protein

extraction was carried out as described previously (Song et al. 2013a). Protein

concentration was determined by Bradford protein assay (Biorad, Australia) for

cytoplasmic, nuclear and total protein extracts.

3.3.5 Immunoblot analysis

Immunoblot was performed as described previously (Song et al. 2013a).

Primary antibodies included Akt, phosphorylated (p) Akt (Ser473), FOXO1 and

α-Tubulin (1:2000 dilution) (Cell Signalling Technology, Carlsbad, USA). The

activity of Akt was represented as p-Akt/total Akt ratio. FOXO1 activity was

expressed as nuclear/cytosolic ratio. The detailed protocol is described in

appendix A1 (page 141-142).

3.3.6 Total white blood cell count

Total white blood cell count of cord blood was carried out using an automated

cell analyser (VetScanHM5, Abaxis, USA).

3.3.7 IL-1β and IL-6 levels in plasma

Plasma IL-1β and IL-6 concentration were measured using a sandwich ELISA

assay according to Song et al (Song et al. 2013a). Refer to appendix A1 (page

142) for detailed protocol.

3.3.8 RNA Isolation, Reverse Transcription and Quantitative PCR

RNA purification, reverse transcription and quantitative PCR were carried out

as described previously (Song et al. 2013a). Cytokine genes (IL-1, IL-6) and

proteolytic genes (MAFbx, MuRF1) were evaluated. The fluorescence signal of

samples was normalised against the average of 18S RNA and GAPDH.

Relative expression levels were calculated using the 2-ΔΔCT method (Livak &

Schmittgen 2001) and presented as fold change relative to control. Refer to

appendix A1 (page 142-145) for detailed protocol.

53

3.3.9 Protein Carbonyl assay

Protein carbonyl content in diaphragm was determined using a colorimetric

assay kit (Cayman 10005020, USA).

3.3.10 Data analysis

Data are presented as mean ± SEM or median (range). Differences among

multiple groups were assessed using one-way ANOVA with Tukey post hoc

analysis. Nonparametric data were examined using ANOVA on ranks.

Statistical significance was accepted at p < 0.05.

3.4 Results

3.4.1 Physiological variables at birth

Descriptive characteristics for each group are presented in Table 3.1. There

were no significant differences in GA at birth, body weight or optimal muscle

length between any groups.

54

Table 3.1: Lamb GA, body weight and optimal muscle length data for saline (Control) and LPS exposed fetal lambs.

Control

(n=8)

7 d LPS

(n=6)

21 d LPS

(n=7)

7,14,21 d LPS

(n=5)

a GA (d) 120.7 ± 0.5 121.0 ± 0.0 120.3 ± 0.3 121.4 ± 0.2

Sex ratio (M:F)

3:5 1:5 3:4 2:3

Body weight (kg)

2.48 ± 0.14 2.50 ± 0.06 2.30 ± 0.10 2.31± 0.15

b L0 (mm) 29.6 ± 1.29 28.2 ± 0.76 27.8 ± 0.85 27.2 ± 1.77

c FI 0.34 ± 0.04 0.42 ± 0.04 0.51 ± 0.04* 0.54 ± 0.03*

aGA, gestational age; bL0, optimal muscle length; cFI, fatigue index (lower value

indicates increased fatigability).Values are mean ± SEM. * p<0.05 compared

with Control.

3.4.2 Diaphragm contractile properties

IA exposure to LPS significantly impaired diaphragm contractile function in

each group (Figure 3.1A). Compared to control, maximum specific force (Po)

was significantly lower by 32 %, 40 % and 39 % in the 7 d, 21 d and repeated

LPS exposed groups, respectively. Similarly, peak twitch forces (Pt) were 31 %,

33 % and 34 % lower than controls for the corresponding groups (Figure 3.1B).

Importantly, the time to peak (TTP) and half relaxation times (1/2 RT) for twitch

contractions were significantly longer in 21 d and repeated LPS exposed lambs

compared with control, but were not affected in 7 d LPS lambs (Figure 3.1C,D).

The fatigue index (FI) was significantly higher in 21 d and repeated LPS

55

exposed lambs compared with control and 7 d lambs (Table 3.1), indicating

increased fatigue resistance. There were no significant differences in

contractile properties between 21 d and repeated LPS exposure groups.

Figure 3.1 Fetal diaphragm contractile properties. (A) Maximum specific

force (P0); (B) Peak twitch force (Pt); (C) Time to peak twitch force (TTP); (D)

Half-relaxation time (1/2 RT) for control (n=8), 7 d LPS (n=6), 21 d LPS (n=7)

and 7,14,21 d LPS (n=5) exposure animals. Values are Mean ± SEM. * significantly different to control, p<0.05.

3.4.3 MHC isoform composition and fibre CSA

The proportion of slow-twitch MHC I fibres was 13-15 % in all groups, and was

unaffected by LPS exposure. The percentage of fast-twitch MHC IIa fibres in 7

d LPS lambs (54 %) was significantly lower than control (66 %; Figure 3.2A).

There was no significant difference in the proportion of MHC IIa fibres in 21 d

(59 %) and repeated LPS (58 %) groups compared with control. CSA of MHC I

and Ia fibres were not significantly affected by LPS exposure (Figure 3.2B).

P0 Pt

56

Figure 3.2 (A) Proportions of slow-twitch MHCs and fast-twitch MHC IIa fibres and (B) muscle fibre cross-sectional areas (CSA). Images of

immuno-stained muscle fibres with (C) MHCs and (D) MHC IIa antibodies in the

control (n = 4), 7 d (n = 4), 21 d (n = 6) and 7,14,21 d (n=5) LPS exposure

animals. Green fluorescence indicates positively stained muscle fibres and red

fluorescence indicates basal lamina. Magnification 200x, scale bar = 100µm.

Values are mean ± SEM. * significantly different to control, p<0.05.

3.4.4 Cytokine response

IL-1β and IL-6 mRNA expression in diaphragm from LPS exposed lambs was

not significantly different to control (Figure 3.3A,B respectively). However, IL-6

mRNA expression in the repeated LPS exposure group was significantly higher

compared to the 7 d LPS group. Consistent with the mRNA expression of local

cytokines, the plasma protein concentrations of IL-1β and IL-6 for LPS groups

were not significantly different to control (Figure 3.3C,D respectively). Cytokine

57

(IL-1 and IL-6) protein content in the diaphragm was not measured due to low

detection levels.

Figure 3.3 Local and systemic cytokine response. (A) Diaphragm IL-1β and

(B) IL-6 mRNA expression in 7 d (n=6), 21 d (n=7) and 7,14,21 d (n=5) LPS

exposure groups relative to saline control (n=5). Values are median (with 10th

and 90th centiles). (C) Plasma IL-1β and (D) IL-6 protein content in 7 d (n=6),

21 d (n=7) and 7,14,21 d (n=5) LPS exposure groups relative to saline control

(n=6). Values are mean ± SEM. significantly different to 7 d LPS, p<0.05.

3.4.5 Total white blood cell count

The concentrations of neutrophils and monocytes in cord blood were

significantly greater following repeated LPS exposures compared to control and

the 21 d LPS group (p<0.05; Figure 3.4). Neutrophil and monocyte counts in 7

d and 21 d LPS groups were not significantly different to control. There were no

significant differences in lymphocytes count between groups.

58

Figure 3.4 Cord blood WBC cell count. Neutrophil, lymphocyte and

monocyte cell count/L in the control (n = 8), 7 d (n = 6), and 21 d (n = 7) and

7,14,21 d (n=5) LPS exposure animals. Values are mean ± SEM. * significantly

different to control and 21 d LPS; p<0.05.

3.4.6 Anabolic and catabolic pathways

Akt and FOXO1 protein content in the diaphragms of LPS exposed groups

were not significantly different to control (Figure 3.5A-C). The mRNA

expression of atrophy related E3 ligases (MuRF1 and MAFbx) in preterm

diaphragm were not significantly different after IA LPS exposure compared to

control (Figure 3.6A,B respectively).

59

Figure 3.5 Activity of protein synthesis and degradation pathway signalling molecules in diaphragm after LPS exposure. (A) Western blots

display protein content of signalling molecules for each experimental group. (B)

p-Akt/total Akt protein and (C) nuclear/cytoplasmic FOXO1 protein in control

(n=5) or after exposure to LPS for 7 d (n=6), 21 d (n=7) or 7,14,21 d (n=5).

Values are mean ± SEM.

60

Figure 3.6 (A) Atrophy gene MAFbx and (B) MuRF1 expression in diaphragm in control (n=5) or after exposure to LPS for 7 d (n=6) or 21 d (n=7)

or 7,14,21 d (n=5). Values are median (with 10th and 90th centiles).

3.4.7 Oxidative stress

Protein carbonyl content was significantly higher in 21 d and repeated LPS

exposure groups compared to 7 d and control lambs (p<0.05; Figure 3.7A).

Figure 3.7 Protein carbonyl content in diaphragm. Values are mean ± SEM.

* significantly different to control, p<0.05.

61

3.5 Discussion

Our primary findings are that an acute in utero exposure to inflammation

significantly impairs diaphragm contractile function and that the timing of initial

inflammatory exposure has a greater impact on diaphragm function than the

frequency of exposure. Although exposure to LPS reduced maximum specific

force in all groups, exposure at the earlier GA resulted in more extensive

alterations to contractile function that persisted until birth at 121 d gestation.

Our findings are important given the pivotal role of the diaphragm in

maintaining independent respiration: impaired postnatal diaphragm function

resulting from fetal inflammatory conditions may contribute to respiratory failure

among premature infants.

The earliest inflammatory exposure in the present study occurred at 100 d GA

(for 21 d and repeated exposure groups) at which time, development of ovine

respiratory muscles is characterised by the establishment of fetal breathing

movements and the hypothalamo-pituitary-thyroid axis (Dawes 1984;

Finkelstein et al. 1991). The time course of these changes coincide with

morphological development of the diaphragm including increases in myofibre

size and density (Ashmore et al. 1972). Previously we showed significant

increases in MHC content and maximum specific force with increasing GA

(Lavin et al. 2013) and reduced proteolytic signalling activity at GA > 100 d in

fetal sheep diaphragm (Song & Pillow 2012). The marked disruption of

contractile function that we observed with IA LPS administered at 100 d GA

suggests that in utero exposure to inflammation at this critical period may

disrupt the biochemical and morphological development of the diaphragm,

resulting in persistent functional impairment after birth. Further studies in which

initial IA LPS exposure occurs at GA <100 d would be necessary to fully

characterise the GA dependence of LPS induced diaphragm dysfunction.

IA LPS exposure induces an acute inflammatory response in preterm lambs

that is associated with impaired mitochondrial function, oxidative stress (Song

et al. 2013b) and reduced diaphragmatic specific force production (Song et al.

2013a). The fetal inflammatory response to IA LPS is characterised by elevated

62

plasma and local (diaphragm (Song et al. 2013a) and lung (Kallapur et al.

2007) cytokine concentrations that are evident 2 d after LPS exposure, but

return to control levels by 7 d. Despite the resolution of the inflammatory

response by 7 d, diaphragm specific force remains significantly depressed

indicating that the initial inflammation has persisting adverse effects on

diaphragm function. These observations are consistent with the present study

in which diaphragmatic force remained significantly lower than control, that we

now show persists for up to 21 d after the initial LPS exposure.

The increased protein carbonyl content we observed in the 21 d and repeated

LPS groups is consistent with oxidative stress related contractile dysfunction

(Song et al. 2013b). Oxidative stress contributes to the diaphragm contractile

dysfunction in animal models of sepsis and endotoxemia (Callahan & Supinski

2009; Sun et al. 2006) and protein carbonyl content is increased in respiratory

muscles after mechanical ventilation (Zergeroglu et al. 2011 ; Falk et al. 1996)

and in response to sepsis (Fagan et al. 2008 ; Barreiro et al. 2005). Hence,

LPS induced oxidative stress may contribute to the impaired contractile function

by carbonylation of key myofilament proteins in the diaphragm including actin,

myosin light and heavy chains, desmin, and tropomysin (Barreiro et al. 2005).

In addition to diaphragm weakness, we also noted significantly longer twitch

contraction times and increased fatigue resistance following 21 d and repeated

LPS exposures. Although these changes are suggestive of a slower muscle

phenotype, the proportion of MHC I positive fibres was not affected by LPS

exposure. Similarly, Akt and FOXO1 activity and expression of atrophy related

genes, MuRF1 and MAFbx, are not affected by LPS. Together, these

observations indicate that myofibre atrophy or changes in MHC composition do

not contribute to the LPS-induced alterations in diaphragm contractile function.

An alternative explanation for the increased fatigue resistance may be a

reduction in metabolic activity due to the significantly (40 %) lower maximum

specific force. Reduced force production and cross-bridge cycling would lower

metabolic demand and consequently reduce the build-up of contraction-

induced metabolites such as inorganic phosphate and reactive oxygen species

which are associated with the development of muscle fatigue (Allen 2008). In

63

support of this hypothesis, there was a significant correlation between P0 and

fatigue index (r = -0.649; p=0.001, Pearson’s correlation): diaphragm strips that

produced lower maximum specific force had the greatest fatigue resistance.

However, as measurements of mitochondrial activity or metabolic by-products

of muscle contraction were not conducted in the current study we cannot be

certain of the mechanism underlying the change in fatigue resistance.

Contrary to our hypothesis that repeated pro-inflammatory exposure would

exacerbate the contractile dysfunction, the impairment of diaphragm contractile

function following repeated LPS exposures (7 d, 14 d and 21 d) was similar to

the single 21 d LPS exposure. The lack of additional force deficits due to

repeated LPS exposure is consistent with an altered immune response to LPS

after the initial exposure (Kramer et al. 2009; Kallapur et al. 2007). Repeated

exposure to IA endotoxin reduces cytokine (IL-6) responsiveness in cultured

monocytes (Kramer et al. 2005) and inhibits lung cytokine (IL-1β, IL-6 and IL-8)

mRNA expression in preterm lambs (Kallapur et al. 2007). Our results are

consistent with these observations. Cytokine levels in plasma and diaphragm

were unaltered when compared with control group suggesting that the fetal

immune system is hypo-responsive to repeated LPS challenges.

Very premature male infants have markedly higher rates of adverse pulmonary

neonatal outcomes compared to females (Peacock et al. 2012). Although each

of our experimental groups contained a mix of male and female lambs, we did

not have an equal male:female ratio. Therefore, we cannot exclude the

possibility that our results were influenced by a male disadvantage in relation to

the severity of LPS induced diaphragm weakness.

The immature diaphragm is vulnerable to adverse in utero exposures such as

chorioamnionitis. We show that in utero LPS exposure in preterm lambs has

persistent effects on diaphragm function and the GA at time of initial exposure

influences the extent of alterations to diaphragm function. We speculate that

persistent diaphragm dysfunction resulting from early inflammatory exposures

may contribute to inefficient spontaneous breathing and the development of

late-onset respiratory failure in premature infants. However, the contribution of

64

diaphragm dysfunction on postnatal respiratory failure among preterm infants

warrants further investigation.

Acknowledgements

The authors gratefully acknowledge the assistant of Clare Berry, Tina Lavin,

Stephen Gray (animal breeding) and staff from Animal Care Services at the

University of Western Australia.

Funding

This study is supported by National Health and Medical Research Council

(NHMRC) Project Grant APP1010665, Women and Infants Research

Foundation, a Sylvia and Charles Viertel Senior Medical Research Fellowship

(JJP) and a NHMRC Career Development Fellowship (PNB, 1045824).

65

Chapter 4

Gestational age at time of in utero lipopolysaccharide exposure

influences the severity of inflammation-induced diaphragm

weakness in lambs

Preface

This study investigates the influence of gestational age on IA LPS induced diaphragm weakness in lambs

This chapter is a manuscript prepared for submission to a suitable Journal

66

4. Chapter 4: Gestational age at time of in utero

lipopolysaccharide exposure influences the severity of

inflammation-induced diaphragm weakness in lambs

Kanakeswary Karisnan1, Anthony J. Bakker1, Yong Song1,2, Peter B. Noble1,2 J.

Jane Pillow1,2 and Gavin J. Pinniger1

School of Anatomy, Physiology and Human Biology1 and Centre for Neonatal

Research and Education, School of Paediatrics and Child Health2, University of

Western Australia, 35 Stirling Hwy, Crawley, WA 6009, Australia.

Correspondence: Gavin J. Pinniger, School of Anatomy, Physiology and

Human Biology, University of Western Australia, 35 Stirling Highway, M309,

Crawley 6009, Western Australia. Email: gavin.pinniger@uwa.edu.au. Phone:

+61 8 6488 3380. Fax: +61 8 6488 1025

Authors Contributions: JJP, GJP and AJB obtained the study funding and

were responsible for design of the animal studies. PBN was integral to project

management and together with JJP responsible for animal care and treatment.

KK performed experiments and primary data analysis under supervision of GJP

and AJB (physiological measurements) and YS and JJP (laboratory tissue

analysis). All authors contributed to data interpretation; KK prepared figures

and the initial manuscript draft. All authors edited the manuscript and approved

the final version of the manuscript for submission.

Running title: Preterm vulnerability to inflammation-induced diaphragm

dysfunction

67

4.1. Abstract

Background: The preterm diaphragm is structurally and functionally immature

compared with its term counterpart. In utero inflammation further exacerbates

diaphragm dysfunction. We hypothesised that preterm lambs are more

vulnerable to in utero inflammation induced diaphragm dysfunction compared

to term lambs.

Methods: Pregnant ewes received intra-amniotic (IA) injections of saline or 10

mg LPS 2 d or 7 d prior to delivery at 121 d (preterm) or ~145 d (term)

gestational age. Lambs were killed immediately after caesarean delivery and

diaphragm contractile function was assessed in vitro. The muscle fibre myosin

heavy chain (MHC) isoforms, inflammatory cytokine response and oxidative

stress were evaluated using immunofluoresence staining, qPCR, ELISA and

biochemical assay.

Results: In utero LPS exposure impaired both preterm and term diaphragm

function to differing degrees. Relative to naive control lambs, the proportional

decrease in peak twitch force was significantly greater in preterm lambs (40%)

compared to term lambs (10%; p < 0.05). A similar trend was observed for the

proportional reduction in maximum specific force for preterm (30%) and term

lambs (20%; p = 0.058). Despite similar inflammatory cytokine responses to

LPS exposure, term lambs also displayed a significant increase in the

proportion of slow MHC positive fibres and increased fatigue resistance;

whereas preterm lambs showed a significant decrease in cross sectional area

of fibres positive for slow and neonatal MHC.

Conclusions: Preterm lambs are more vulnerable to diaphragm dysfunction

induced by IA LPS compared to term lambs. The gestational age at the time of

LPS exposure determines the severity of inflammation-induced diaphragm

dysfunction and may contribute to the development of respiratory failure after

birth.

Keywords: in utero inflammation, preterm and term infants, diaphragm,

contractile dysfunction, oxidative stress, chorioamnionitis

68

4.2. Introduction

The diaphragm is the primary inspiratory muscle and performs the majority of

the work of breathing. From birth, the diaphragm muscle must generate

adequate force to sustain ventilation. Therefore, a functional diaphragm is

critically important for successful establishment of unsupported spontaneous

breathing. The structural and functional immaturity of the preterm diaphragm

may increase the vulnerability to additional in utero exposures that are strongly

associated with very preterm birth such as chorioamnionitis. In utero, a

weakened diaphragm may reduce effectiveness of fetal breathing movements

and contribute to reduced lung growth (Jani 2009; Ysasi 2013). Postnatally,

reduced diaphragm contractility may contribute to inefficient respiratory efforts

(Bissonnette 2011).

The respiratory system must be fully functional at birth. However, like the lung,

the growth and development of the diaphragm are incomplete in infants born

prematurely. The muscle fibre type composition differs markedly between the

immature and mature diaphragm. For example, the fetal diaphragm has a very

low proportion of fibres expressing slow myosin heavy chain (MHC) isoforms

(10 %) (Keens et al. 1978), and is dominated by neonatal MHC expression

(Maxwell et al. 1983). In the adult diaphragm, however, approximately 50 % of

fibres express slow MHC isoforms (Keens et al. 1978). Intracellular Ca2+

handling (West et al. 1999), oxidative capacity (Song & Pillow 2012; Sieck,

Cheung & Blanco 1991), protein metabolism (Song & Pillow 2013) and

myofilament structure (West et al. 1999) are also not fully developed in the

preterm diaphragm. Furthermore, the immune system of the premature infant is

functionally immature thus reducing its capacity to adequately respond to

bacterial infection in utero (Lang, Frost & Vary 2007; Pinniger, Lavin & Bakker

2012).

Our previous study characterising the functional development of the ovine

diaphragm showed that the maximum specific force increased by twofold from

69

128 d to 145 d gestational age (GA) (term = 150 d) (Lavin et al. 2013). In

contrast, fatigue resistance decreases significantly over this period (Lavin et al.

2013). Furthermore, the susceptibility of the diaphragm to stretch-induced

damage was significantly greater at lower GA, which may reflect the poorly

defined cytoskeleton and sarcomeric structures in the immature diaphragm

(Ashmore et al. 1972). Because these developmental changes occur

progressively with growth, the response to an inflammatory exposure and the

subsequent functional impact on the developing diaphragm may vary

depending on the GA at time of exposure.

In utero inflammation, commonly manifest as chorioamnionitis, is associated

with up to 70 % of preterm births (Goldenberg, Hauth & Andrews 2000). Earlier

studies investigating diaphragmatic impairment in the context of inflammation

were undertaken in adult subjects: chronic obstructive pulmonary disease,

acute respiratory distress syndrome and sepsis are accompanied by impaired

respiration related to diaphragm dysfunction (Levine et al. 2013; Finkelstein et

al. 1991; Kallet 2011). The above clinical conditions are accompanied by local

and systemic inflammation, suggesting that inflammatory cytokines may

contribute to diaphragm weakness. Importantly, structural and functional

deficits in the developing diaphragm are poorly understood. The effects of

inflammation on diaphragm structure and function are likely to differ during

critical stages of prenatal development. Acute intra-amniotic (IA)

lipopolysaccharide (LPS) exposure impairs diaphragm function in preterm

lambs (Song et al. 2013a); however, the molecular responses differed when the

exposure occurred at 2 d or 7 d before delivery. It is unclear if the different

duration of LPS exposure or the gestation at time of exposure determines the

mechanisms and severity of diaphragm dysfunction. This study investigated the

effect of acute LPS exposure (2 d and 7 d prior to delivery) on preterm and

term diaphragm function. We hypothesised that the preterm diaphragm is more

vulnerable to in utero inflammation induced contractile dysfunction than term

diaphragm. To test this hypothesis, we used a well-established ovine model of

chorioamnionitis induced by IA injections of LPS at preterm (121 d) or term

70

(~145 d) GA. We examined the effects of IA LPS on diaphragm contractile

function, MHC isoform composition, inflammatory markers and oxidative stress.

4.3. Methods

Animal experiments were performed at the University of Western Australia with

approval from the institutional Animal Ethics Committee. Methodology is

summarised below, and detailed in appendix A1 (page 139-146).

4.3.1. Experimental design

Pregnant Merino ewes were randomised to ultrasound guided IA injection of

LPS (10 mg in 2 mL saline: Escherichia coli 055:B5, Sigma-Aldrich, St Louis,

MO) or an equal volume of saline (Sal) at 2 d or 7 d prior to delivery at 121 d

GA (preterm) or 145 d (term) gestation. Lambs were delivered via caesarean

section and immediately euthanised by pentobarbitone (150 mg/kg IV, Pitman-

Moore, New South Wales, Australia). Longitudinal strips of muscle fibres were

dissected from the right hemi-diaphragm for in vitro assessment of contractile

function. The left hemi-diaphragm was removed for molecular and biochemical

studies. Plasma was obtained by centrifugation (3,000 RPM, 10 min, 4 C) of

umbilical arterial blood to assess the systemic response to IA LPS exposure.

All tissue samples were immediately snap frozen in liquid nitrogen and stored

at -80 C prior to analysis.

4.3.2. Diaphragm contractile properties

Diaphragm contractile measurements were performed according to Song et al

(Song et al. 2013a). The contractile parameters measured include maximum

isometric twitch force (Pt) and maximum tetanic force (P0). Time to peak (TTP)

and half relaxation time (1/2 RT) of twitch contractions were determined using

the Dynamic Muscle Analysis (DMA) software (Aurora Scientific, Ontario,

Canada). Susceptibility to fatigue was evaluated from a series of 150 tetanic

contractions. The fatigue index (FI) was determined from the ratio of the force

71

produced during the 150th contraction relative to the 1st contraction (Javen et al.

1996), in which a higher number indicates a greater fatigue resistance.

Susceptibility to muscle damage was determined as percentage of force deficit

from five lengthening (eccentric) contractions at 2 min intervals: For each

lengthening contraction, a stretch of 10 % of optimal muscle length (L0) was

applied during the isometric plateau phase of a maximal tetanic contraction.

4.3.3. Myosin Heavy Chain (MHC) fibre typing and myofibre Cross-Sectional

Area (CSA)

OCT embedded diaphragm samples were sectioned at 8 m thickness and

stained according to Song et al (Song et al. 2013a). The sheep MHC slow

(MHCs; 2400101), MHC fast (MHCf; 2400107) and MHC neonatal (MHCn;

2400104) antibodies were produced by Mimotopes Australia and used at 1:25

for preterm and 1:50 for term diaphragm of 1 mg/mL stock.

4.3.4. Muscle protein extraction

Diaphragm tissue was homogenised in cold lysis buffer followed by 6 cycles of

freeze-thaw (Song et al. 2013a). Total protein extract was harvested after

centrifugation at 10,000 g for 25 min at 4 C. Supernatant was collected and

protein concentration was determined using Bradford assay (Bradford 1976).

4.3.5. Markers of systemic inflammation

Plasma IL-1β and IL-6 concentrations were measured using indirect ELISA

assay (Song et al. 2013a).

4.3.6. RNA isolation, reverse transcription and quantitative PCR

RNA purification, reverse transcription and quantitative PCR used the protocol

described by Song et al (Song et al. 2013a). Gene expression of inflammatory

cytokines (IL-1β and IL-6), proteolysis (muscle RING-finger protein-1, MuRF1;

Muscle Atrophy F-Box, MAFbx) and antioxidant genes (Superoxide dismutase

1, SOD1; Glutathione peroxidase 1, GPX1; and Catalase) were evaluated in

diaphragm tissue. The fluorescence signal of samples was normalised against

72

the average of 18S and GAPDH RNA. The 2-ΔΔCT method (Livak & Schmittgen

2001) was used to calculate relative expression levels and expression is

presented as fold increase relative to controls.

4.3.7. Oxidative stress

The amount of reduced (GSH) and oxidised (GSSG) glutathione in the

diaphragm were measured using glutathione fluorescent detection kit (DetectX

K006-F1, Arbor Assays, USA) and expressed as GSH/GSSG ratio. The protein

carbonyl content in diaphragm was measured using commercially available

protein carbonyl colorimetric kit (Cayman, Ann Arbor, MI, USA).

4.3.8. Data analysis

Data are presented as mean ± SEM or median (centiles). Statistical analyses

were performed using Sigmaplot (version 12.0, Systat Software Inc, San Jose,

USA). To examine our hypothesis that the preterm diaphragm is more

vulnerable to LPS-induced contractile dysfunction than the term diaphragm, all

data were normalised to the mean of the gestation matched control data and

expressed as a relative measure. The relative data were compared using two-

way ANOVA with factors of GA (121 d and 145 d) and LPS exposure (2 d and 7

d), and post hoc analyses were performed using the Duncan's method. If there

were no significant differences between 2 d and 7 d LPS within same GA, the

data were pooled to generate single LPS group to enable comparisons

between different GA groups using a t-test. Nonparametric data were analysed

using Mann-Whitney U Test. Statistical significance was accepted at p < 0.05.

4.4. Results

4.4.1. Characterisation of lambs

The gestation, body weight and optimal muscle lengths (L0) were significantly

lower in preterm lambs, compared to term lambs. However, within each age

group, there was no significant effect of LPS exposure on these measures

(Table 4.1).

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Table 4.1 Description of lambs and diaphragm twitch properties

Term Preterm

Control 2d LPS 7d LPS Control 2d LPS 7d LPS

GA (d) 144 ± 0.3 141 ± 0.9 142 ± 0.2 121 ± 0.2* 121 ± 0.0 121 ± 0.2

Body weight kg) 4.8 ± 0.3 4.5 ± 0.2 4.7 ± 0.2 2.5 ± 0.1* 2.6 ± 0.1 2.5 ± 0.1

L0 (mm) 35.2 ± 2.1 36.5 ± 2.2 34.5 ± 1.3 29.6 ± 1.3* 30.3 ± 1.3 28.2 ± 0.8

Pt (N/cm2) 9.09 ± 0.83 8.17 ± 0.95 8.03 ± 0.84 7.97± 2.10* 5.66 ± 1.97# 5.52 ± 0.39#

P0 (N/cm2) 20.12 ± 1.23 15.49 ± 1.32# 16.39 ± 1.35 15.58 ± 1.45* 11.20 ± 1.30# 10.50 ± 0.25#

TTP (s) 0.28 ± 0.04 0.37 ± 0.01 0.37 ± 0.01 0.16 ± 0.04* 0.12 ± 0.01 0.23 ± 0.06

1/2 RT (s) 0.22 ± 0.03 0.22 ± 0.01 0.23 ± 0.02 0.19 ± 0.03 0.22 ± 0.02 0.26 ± 0.02

FI 0.27 ± 0.04 0.42 ± 0.02# 0.38 ± 0.02# 0.34 ± 0.04 0.34 ± 0.03 0.42 ± 0.03

Force deficit (%) 8.55 ± 1.12 8.51 ± 2.52 11.54 ± 1.26 16.55 ± 2.39* 16.30 ± 2.21 14.38 ± 1.42

GA, gestational age; L0, optimal muscle length; P0, maximum specific force; Pt, peak twitch force; TTP time to peak twitch force; 1/2 RT, half relaxation time; FI, fatigue index; Values are mean ± SEM. * significantly different to term control; # significantly different to GA matched control

74

4.4.2. Diaphragm contractility

In utero LPS exposure caused diaphragm contractile dysfunction in both term

and preterm lambs. The maximum specific force (P0) of term lambs exposed to

LPS 2 d and 7 d before delivery was significantly reduced by about 20 %

compared with controls (Figure 4.1A). In preterm lambs, P0 from 2 d and 7 d

LPS exposures were about 30 % lower than controls (relative P0 = 0.72 and

0.68, respectively; Figure 4.1A). Within each GA, there was no significant

difference between 2 d and 7 d LPS exposures, therefore these data were

pooled to compare the effect of LPS exposure between different GA’s. There

was no significant difference between term and preterm lambs when P0 was

expressed relative to controls (p = 0.058).

The peak twitch force (Pt) in term lambs following 2 d and 7 d LPS exposures

was not significantly different to controls (relative Pt = 0.89 and 0.88

respectively; Figure 1B). In preterm lambs, however, Pt from 2 d and 7 d LPS

exposures were significantly lower than controls by approximately 40 %

(relative Pt = 0.54 and 0.35, respectively; Figure 4.1B). When Pt data were

pooled for each GA, the relative Pt for preterm lambs was significantly lower

than for term lambs (p = 0.002).

In term lambs, the fatigue index (FI) was significantly increased by 53 % and 39

% after 2 d and 7 d LPS exposures, respectively, relative to controls (Figure

4.1C). In contrast, the FI in preterm lambs was unaltered after LPS exposure.

When pooled for each GA, the relative FI was significantly greater for term than

preterm lambs after LPS exposure (p = 0.003).

There were no significant differences in the twitch contractions times, TTP, 1/2

RT (Table 4.1), or the susceptibility to stretch-induced muscle damage (Force

deficit; Figure 4.1D) between the control and LPS groups in term and preterm

lambs. However, in comparison to the term naïve control lambs, the preterm

control lambs had significantly shorter TTP (p=0.005 and significantly greater

stretch-induced muscle damage (p = 0.004; Table 4.1)

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Figure 4.1 Diaphragm maximum specific force (A) and twitch force (B), fatigue

measurements represented as fatigue index (C) and percentage of force deficit

after stretch protocol (D) in term and preterm lambs. Term control (n=9), Term

2 d LPS (n=5), Term 7 d LPS (n=7), Preterm control (n=8), Preterm 2 d LPS

(n=6), Preterm 7 d LPS (n=6). Values are mean ± SEM of proportional change

relative to its GA matched controls. * Significantly different when compared

between term and preterm (p < 0.05).

4.4.3. MHC composition and myofibre cross-sectional area

The term diaphragm consists of predominantly MHCf (72 %) positive fibres

and smaller proportion of MHCs fibres (21 %). The preterm diaphragm,

however, consists of predominantly MHCn positive fibres (~68%) with a smaller

proportion of MHCs (19 %) and MHCf (16 %) positive fibres. After fetal

exposure to LPS, the proportion of MHCs positive fibres in the term diaphragm

increased by 36 % and 66 % after 2 d and 7 d LPS exposures, respectively.

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There is a significant difference in MHCs proportional increase relative to

controls between term 2 d and 7 d LPS groups (p=0.001; Figure 4.2A). In the

preterm diaphragm LPS exposure had no significant effect on the proportion of

MHCs positive fibres. In comparison, the proportion of MHCf fibres in term and

preterm diaphragms were not altered by either 2 d or 7 d LPS exposures

(Figure 4.2B), nor was the proportion of MHCn fibres affected by LPS exposure

in preterm lambs (Figure 4.2C).

The cross-sectional area of MHCs and MHCf fibres were not altered by LPS

exposure in term lambs (Figure 4.2D, 4.2E respectively). Importantly, CSA for

MHCs and MHCn fibres was significantly lower in 7 d LPS exposure compared

to 2 d LPS (p = 0.009, p=0.006; Figure 4.2D, 4.2F, respectively). There was no

change in CSA for preterm MHCf positive fibres.

Figure 4.2 MHCs, MHCf and MHCn percentage (A,B,C) and fibre cross

sectional area (CSA) (D,E,F) in diaphragm from term and preterm lambs. Term

control (n=4), Term 2 d LPS (n=4), Term 7 d LPS (n=4), Preterm control (n=5),

Preterm 2 d LPS (n=4), Preterm 7 d LPS (n=4). Values are mean ± SEM of

proportional change relative to its GA matched controls. * Significantly different

when compared between 2 d and 7 d LPS (p < 0.05).

77

4.4.4. Markers of systemic and local inflammation

Plasma IL-1 concentration was not significantly affected by LPS exposure in

term or preterm lambs (Figure 4.3A). However, in both term and preterm lambs

plasma IL-6 concentrations following 2 d LPS exposure were significantly

greater than their respective GA controls (p = 0.024, p < 0.001, respectively;

median: control term = 0.040; 2 d LPS term 0.139; control preterm = 0.050, 2 d

LPS preterm = 0.390). In the term lambs, plasma IL-6 protein concentrations

were not significantly different between 2 d and 7 d LPS exposures (p = 0.052).

In preterm lambs, however, plasma IL-6 protein concentration in the 2 d LPS

group was significantly higher compared with 7 d LPS group (p<0.001, Figure

4.3B).

IL-1 and IL-6 mRNA expression was measured as an indicator of active local

inflammation in the diaphragm: in the diaphragm of both term and preterm

lambs, IL-1 mRNA expression in the 2 d LPS groups was significantly greater

than their respective controls (p = 0.007, p = 0.019, respectively; median; term

3.70; preterm 2.63; Figure 4.3C). IL-1 mRNA expression was not different in

the 7 d LPS for term and preterm lambs (Figure 4.3C). There was no difference

in the pooled (2 d + 7 d) IL-1 mRNA expression between term and preterm

lambs.

In term lambs, diaphragm IL-6 mRNA levels were significantly increased in 2 d

LPS but not in the 7 d LPS in comparison with controls (p = 0.04; median;

2.56). In preterm lambs, diaphragm IL-6 mRNA levels after 2 d or 7 d LPS

exposures were not significantly different to controls (Figure 4.3D). There was

no difference in the pooled (2 d + 7 d) IL-6 mRNA expression between term

and preterm lambs.

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Figure 4.3 Systemic and diaphragm cytokine response. Plasma IL-1β (A) and

IL-6 (B) protein content relative to its gestation matched control. Term control

(n=4), Term 2 d LPS (n=5), Term 7 d LPS (n=7), Preterm control (n=6),

Preterm 2 d LPS (n=7), Preterm 7 d LPS (n=6) exposure groups. Box plots

show median (10th, 90th centiles). Diaphragm IL-1β (C) and IL-6 (D) mRNA

expression in term and preterm experimental groups relative to its gestation

matched control. Box plots show median (10th, 90th centiles). * Significantly

different when compared between 2 d and 7 d LPS (p < 0.05).

4.4.5. Oxidative stress in diaphragm

LPS exposure had no significant effect on SOD1, catalase and GPX1 gene

expression for term and preterm lambs (Figure 4.4A, 4.4B, 4.4C). In term

lambs, the diaphragm GSH/GSSG ratio was unaltered by LPS exposure

(Figure 4.5A). In the preterm lambs, the GSH/GSSG ratio after a 7 d LPS

exposure was significantly greater than controls (p <0.001) and the relative

change in GSH/GSSG ratio after a 7 d LPS exposure was significantly greater

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than 2 d LPS (p = 0.003; Figure 4.5A). Protein carbonyl content was unaltered

by LPS exposure in both term and preterm lambs (Figure 4.5B).

Figure 4.4 Oxidative stress genes SOD1 (A), Catalase (B) and GPX (C) mRNA

expression in the diaphragm relative to its gestation matched control. Term

control (n=6), Term 2 d LPS (n=5), Term 7 d LPS (n=7), Preterm control (n=5),

Preterm 2 d LPS (n=6), Preterm 7 d LPS (n=5) exposure groups. Box plots

show median (10th, 90th centiles).

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Figure 4.5 Diaphragm proportional change in GSH/GSSG ratio relative to its

GA matched controls presented as mean ± SEM (A). Proportional change in

protein carbonyl content relative to GA matched controls in diaphragm. Box

plots show median (10th, 90th centiles) (B) for Term 2 d LPS (n=5), Term 7 d

LPS (n=6), Preterm 2 d LPS (n=4), Preterm 7 d LPS (n=6) exposure groups. * Significantly different when compared between 2 d and 7 d LPS (p < 0.05).

4.4.6. Proteolytic gene expression in diaphragm

Muscle atrophy gene (MAFbx and MuRF1) expression (Figure 4.6A, 4.6B) was

unaltered by LPS exposure in the term and preterm lambs relative to its GA

controls. When pooled for each GA, the relative MAFbx mRNA level was

significantly greater for term than preterm lambs after LPS exposure (p <0.05).

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Figure 4.6 Atrophy gene MAFbx (A) and MuRF1 (B) mRNA expression in

diaphragm for term and preterm experimental groups relative to its GA control.

Box plots show median (10th, 90th centiles). * Significantly different between

term and preterm (p < 0.05).

4.5. Discussion

We used an established ovine model of chorioamnionitis and fetal inflammatory

response syndrome to determine the effect of in utero LPS exposure on

diaphragm function in term and preterm lambs. Our results indicate that

preterm lambs are more vulnerable to inflammation induced diaphragm

dysfunction than term lambs. Appropriate in utero development and postnatal

function of the diaphragm is vital for maintaining independent respiration. The

absence of diaphragm contractility in utero contributes to lung hypoplasia

(Baguma-Nibasheka et al. 2012); and postnatally, impaired diaphragm function

leads to inefficient respiratory efforts. Impaired lung growth, and/or promotion of

atelectasis may then create a scenario for chronic lung disease postnatally.

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Therefore, impaired postnatal diaphragm function due to fetal inflammatory

conditions may contribute to the development of chronic respiratory disease

among premature infants.

The cytokine response to IA LPS exposure was similar for both term and

preterm lambs and is consistent with previous observations in our own and

others studies (Song et al. 2013a; Kramer et al. 2009; Kallapur et al. 2007).

Plasma IL-6 and diaphragm IL-1 mRNA expression were significantly elevated

in the 2 d LPS group but return to control levels in the 7 d LPS lambs, reflecting

the time-course of a transient inflammatory response to LPS. In spite of the

similar inflammatory response, the subsequent impact of LPS exposure on

diaphragm contractile function differed between term and preterm lambs.

Although IA LPS exposure reduced the maximum diaphragm force production

in all groups, the severity of diaphragm dysfunction was more prominent in

preterm compared to term lambs. The reduction in peak twitch force relative to

the naïve state (control) was significantly greater in preterm compared to term

lambs. A similar trend was observed for the relative decrease in maximum

specific force. Considering the maximum force production in the naïve preterm

is significantly lower (by 20%) than in naïve term lambs, an additional 30 %

decrease in maximal force production after in utero LPS exposure in

comparison with naïve preterm, would severely compromise the functional

capacity of the diaphragm and negatively impact on the ability to overcome the

increased intrinsic work of breathing in the preterm infant.

Despite the decrease in maximum force production, in utero LPS exposure

reduced the susceptibility to fatigue (increased FI) in the term diaphragm.

Reduced fatigability was associated with muscle fibre type remodelling: we

showed an increase in the proportion of fibres expressing MHCs after LPS

exposure. A transition from fast to slow MHC isoforms in the diaphragm has

been observed in other clinical inflammatory conditions such as systemic

inflammation, chronic obstructive pulmonary disease, chronic hyperinflation,

and emphysema (Levine et al. 2013; Clanton & Levine 2009; Nguyen et al.

2000 ). Unlike the term diaphragm, the reduction in maximal (P0) and

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submaximal (Pt) force production in preterm diaphragm occurred without any

change in MHC fibre type proportion. In contrast to term diaphragm, there was

a significant decrease in mean CSA of MHCs and MHCn fibres in the

diaphragm of 7 d LPS preterm lambs which may reflect inflammation induced

diaphragm atrophy.

Activation of ubiquitin-proteasome pathway (UPP) is mainly responsible for

breakdown of muscle protein and UPP E3 ligases (MAFbx and MuRF1) are up-

regulated in infection induced skeletal muscle atrophy (Callahan & Supinski

2009; Supinski et al. 2000; Song & Pillow 2013). However, MURF1 and MAFbx

gene expression were not significantly elevated by LPS exposure in the present

study, and when expressed relative to control levels, MAFbx expression in

preterm lambs was significantly lower than in term lambs. Unfortunately we

were unable to evaluate MURF1 or MAFbx protein expression due to the lack

of ovine specific antibodies, however, these mRNA data suggesting transient

inactivation of protein the degradation pathway in the 7 d LPS preterm

diaphragm in the current study. Importantly, in our previous study, we did

observe a significant increase in 20 S proteasome activity after a 2 d LPS

exposure and a significant reduction in the protein synthesis pathway (p-

p70S6K activity) after 7 d LPS exposure in preterm lambs (Song et al. 2013a).

Thus, the MHCs and MHCn fibre atrophy observed at 7 d LPS exposure in the

preterm diaphragm in the current study may reflect a transient increase in UPP

and decrease in protein synthesis activity that were not reflected by mRNA

expression..

During development, diaphragm myofibres have many large mitochondria

(Maxwell et al. 1989) and poorly developed antioxidant defence system (Song

& Pillow 2012), which renders the preterm diaphragm susceptible to oxidative

stress. Oxidative stress may induce muscle weakness via the activation of

proteolytic pathway and myofibre atrophy (Song et al. 2013b; Callahan &

Supinski 2009; Callahan et al. 2001) or via direct effects on Ca2+ handling and

myofilament force production (Callahan & Supinski 2009). Previous studies

reported an increase in protein carbonyl content in respiratory muscles after

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mechanical ventilation (Falk et al. 1996; Zergeroglu et al. 2011 ) and in

response to sepsis (Barreiro et al. 2005; Fagan et al. 2008 ). Cheah et al

showed that a 7 d exposure to IA LPS increased protein carbonyl content in

bronchoalveolar lavage fluid and plasma protein however there was no

increase in the lung tissues of fetal lambs (Cheah et al. 2008). Therefore, it was

surprising that we did not observe any significant increases in protein carbonyl

content or changes in antioxidant gene expression in the diaphragm after IA

LPS exposure in the current study. We did however, observe a significant

increase in the GSH:GSSG ratio after 7 d LPS exposure, suggesting an

increase in antioxidant capacity in preterm lambs. These observations may

reflect an adaptive response to an earlier oxidative stress. The reason for

differential regulation of LPS induced diaphragm dysfunction at different

gestations is unclear. We used constant exposure duration for our term and

preterm groups; hence propose that diaphragm responses to LPS may be

critically dependent on the gestation at initial exposure. For preterm lambs, the

LPS exposure occurred at 114-119 d GA, whereas LPS exposure was at 138-

143 d for term lambs. These gestations may represent distinct phases in the

maturation of the diaphragm: development of ovine respiratory muscles is

characterised by the establishment of fetal breathing movements and the

hypothalamo-pituitary-thyroid (HPT) axis (Finkelstein et al. 1992; Finkelstein et

al. 1991) beyond ~100 d GA. The time course of the HPT axis and fetal

breathing movement changes coincide with morphological development of the

diaphragm including increases in myofibre size and density (Ashmore et al.

1972). Previously, we showed significant increases in MHC content and

maximum specific force with increasing GA (Lavin et al. 2013) and reduced

proteolytic signalling activity at GA > 100 d in fetal sheep diaphragm (Song &

Pillow 2012). The more extensive disruption of contractile function that we

observed in the preterm lambs suggests that in utero exposure to inflammation

at this critical period may disrupt the biochemical and morphological

development of the diaphragm, resulting in persistent functional impairment

after birth.

85

Infants have a high work of breathing, and a proportion of the force generated

by diaphragm contraction is dissipated in distortion of the highly compliant

chest wall rather than drawing sufficient air into the lungs. The dead-space to

alveolar volume ratio is higher in the infant compared to the adult, so a

proportion of the energy expended in generating tidal volume is wasted as it

doesn’t participate in gas exchange (Currie et al. 2011). Therefore, relative to

body size, greater diaphragmatic contraction is required to sustain tidal volume

in the infant compared to the adult. We propose that the preterm diaphragm is

less able to cope with the increased respiratory load due to reduced contractile

force generating capacity. Previously, we showed that the adverse effect of

immature diaphragm development on contractile function is compromised

further by a pro-inflammatory exposure in utero (Song et al. 2013a). The

structural integrity, MHC composition and oxidative defence system is impaired

in the preterm compared to term diaphragm (Song & Pillow 2012; Lavin et al.

2013). Histological chorioamnionitis is present in up to 70 % of preterm births

and further compromises diaphragm contractile function. Thus, we

hypothesised that the magnitude and nature of adverse effects of in utero LPS

exposure on diaphragm function will differ with gestation at time of LPS

exposure. An increased impact of inflammatory stimulus on more immature

diaphragm has clinical relevance, as there is an inverse relation between the

incidence of chorioamnionitis and fetal inflammatory response syndrome with

gestation. Up to 70 % of placentas from extremely preterm pregnancies show

evidence of histologic chorioamnionitis: therefore, it is reasonable to expect that

impaired diaphragm function may exacerbate postnatal breathing difficulties

and ultimately contribute to chronic respiratory failure in preterm infants.

In conclusion, we show that the preterm diaphragm is particularly vulnerable to

inflammation induced contractile dysfunction. Although the term diaphragm was

also susceptible to inflammation induced diaphragm weakness, it appears to

undergo muscle remodelling resulting in increased fatigue resistance. In

contrast, the preterm diaphragm experienced a more severe reduction in

muscle force following LPS exposure and was associated with myofibre

atrophy. Consequently, preterm infants are at increased risk of respiratory

86

muscle weakness that may impede adequate ventilation and contribute to the

development of postnatal respiratory failure.

Acknowledgements

The authors gratefully acknowledge the assistant of Clare Berry, Tina Lavin,

Steven Gray (animal breeding) and staff from Animal Care Services at the

University of Western Australia.

Disclosures

No conflicts of interest, financial or otherwise, are declared by the author(s).

Funding

This study is supported by National Health and Medical Research Council

(NHMRC) Project Grant APP1010665, Women and Infants Research

Foundation, a Sylvia and Charles Viertel Senior Medical Research Fellowship

(JJP) and a NHMRC Career Development Fellowship (PNB, 1045824).

87

Chapter 5

Interleukin-1 receptor antagonist protects against

lipopolysaccharide induced diaphragm weakness in preterm

lambs

Preface

This study investigates the role of IL-1 signalling and oxidative stress on IA LPS induced diaphragm weakness in preterm lambs

This chapter was published by PLoS One

PLoS One, 2015; Pone.0124390.ecollection 2015

88

5. Chapter 5: Interleukin-1 receptor antagonist protects against

lipopolysaccharide induced diaphragm weakness in preterm

lambs

Kanakeswary Karisnan1, Anthony J. Bakker1, Yong Song1,2, Peter B. Noble1,2 J.

Jane Pillow1,2 and Gavin J. Pinniger1*

1School of Anatomy, Physiology and Human Biology, University of Western

Australia, Perth, WA, Australia.

2Centre for Neonatal Research and Education, School of Paediatrics and Child

Health, University of Western Australia, Perth, WA, Australia.

*Corresponding author

Email: gavin.pinniger@uwa.edu.au (GJP)

Authors Contributions: JJP, GJP and AJB obtained the study funding and

were responsible for design of the animal studies. PBN was integral to project

management and together with JJP responsible for animal care and treatment.

KK performed experiments and primary data analysis under supervision of GJP

and AJB (physiological measurements) and YS and JJP (laboratory tissue

analysis). All authors contributed to data interpretation; KK prepared figures

and the initial manuscript draft. All authors edited the manuscript and approved

the final version of the manuscript for submission.

89

5.1 Abstract

Chorioamnionitis (inflammation of the fetal membranes) is strongly associated

with preterm birth and in utero exposure to inflammation significantly impairs

contractile function in the preterm lamb diaphragm. The fetal inflammatory

response to intra-amniotic (IA) lipopolysaccharide (LPS) is orchestrated via

interleukin 1 (IL-1). We aimed to determine if LPS induced contractile

dysfunction in the preterm diaphragm is mediated via the IL-1 pathway.

Pregnant ewes received IA injections of recombinant human IL-1 receptor

antagonist (rhIL-1ra) (Anakinra; 100 mg) or saline (Sal) 3 h prior to second IA

injections of LPS (4 mg) or Sal at 119d gestational age (GA). Preterm lambs

were killed after delivery at 121d GA (term = 150 d). Muscle fibres dissected

from the right hemidiaphragm were mounted in an in vitro muscle test system

for assessment of contractile function. The left hemidiaphragm was snap frozen

for molecular and biochemical analyses. Maximum specific force in lambs

exposed to IA LPS (Sal/LPS group) was 25% lower than in control lambs

(Sal/Sal group; p=0.025). LPS-induced diaphragm weakness was associated

with higher plasma IL-6 protein, diaphragm IL-1β mRNA and oxidised

glutathione levels. Pre-treatment with rhIL-1ra (rhIL-1ra/LPS) prevented the

LPS-induced diaphragm weakness and blocked systemic and local

inflammatory responses, but did not prevent the rise in oxidised glutathione.

These findings indicate that LPS induced diaphragm dysfunction is mediated

via IL-1 and occurs independently of oxidative stress. Therefore, the IL-1

pathway represents a potential therapeutic target in the management of chronic

respiratory failure in preterm infants.

Keywords: preterm, diaphragm, chorioamnionitis, inflammation, IL-1, oxidative

stress, respiratory failure

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5.2 Introduction

A functioning diaphragm is critically important for the initiation and sustainment

of spontaneous unsupported breathing. However, like the lung, the preterm

diaphragm is structurally and functionally immature at birth and therefore is

poorly equipped to meet the mechanical demands of breathing. Inadequate

diaphragm development may also enhance the vulnerability to additional in

utero exposures that are strongly associated with very preterm births such as

chorioamnionitis. IL-1 has a critical role in the inflammatory pathway associated

with pulmonary responses to chorioamnionitis (Kallapur et al. 2009).

Identification of a similar pathway as a determinant of an adverse impact of

antenatal inflammation on diaphragm function would support treatment

strategies targeting inhibition of the IL-1 pathway.

The preterm diaphragm needs to generate sufficient inspiratory force to

overcome the mechanical disadvantages imposed by a highly compliant chest

wall, low levels of endogenous surfactant and noncompliant, structurally

immature lungs. However, preterm infants have significantly lower twitch trans-

diaphragmatic pressure (Dimitriou et al. 2003), a low proportion of type I and a

high proportion of immature, neonatal muscle fibres compared to term infants

(Keens 1978; Maxwell et al. 1983). Furthermore, the maximum force producing

capacity of the diaphragm in preterm sheep is significantly lower than the term

counterparts (Lavin et al. 2013). These characteristics suggest functional

impairment of the diaphragm that may impede adequate ventilation.

Additionally, as weak muscles need to work closer to maximum contractile

capacity, preterm infants may be predisposed to the development of respiratory

muscle dysfunction, thereby contributing to postnatal respiratory failure.

Data from a number of species indicate that the immature diaphragm contains

a low proportion (<10 %) of type I fatigue resistant muscle fibres, compared to

the adult diaphragm (50-60% type I fibres) (Javen et al. 1996; Keens et al.

1978; Maxwell et al. 1983). Although this suggests the preterm diaphragm is

highly susceptible to fatigue, numerous studies report the opposite finding of a

high fatigue resistance in the diaphragm of newborn rats (Watchko & Sieck

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1993), cats (Sieck, Fournier & Blanco 1991) and baboons (Maxwell et al.

1983). For the most part, these studies have evaluated muscle fatigue under in

vitro conditions using repeated isometric contraction of isolated diaphragm

tissue. The fatigue resistance of the immature diaphragm under in situ

conditions is less widely studied, but may differ from the in vitro condition

(Lesouef et al. 1988). Maxwell et al reported that primate immature diaphragm

has high proportion of immature muscle fibres that are highly oxidative and high

mitochondrial content that may contribute to fatigue resistance (Maxwell et al.

1983). These findings suggest that the relationship between MHC expression,

contractile function and fatigability is less robust in fetal muscle compared to

adult muscle.

In addition to immaturity, antenatal inflammation may further exacerbate

diaphragm dysfunction in preterm infants. About 70 % of preterm births are

associated with intra-uterine infection which commonly manifests as

chorioamnionitis (Romero et al. 2007). Chorioamnionitis frequently induces a

systemic fetal inflammatory response syndrome (FIRS) causing multiple organ

injury and adverse neonatal outcomes (Gotsch et al. 2007). FIRS is mediated

by pro-inflammatory cytokines (IL-1, IL-6 and TNF-α) and diagnosed clinically

by increased plasma IL-6 levels and funisitis (Romero et al. 2007; Gomez et al.

1998). Increased cytokine secretion in inflammatory diseases is commonly

linked with the development of muscle weakness (Reid, Lännergren &

Westerblad 2002). Circulating pro-inflammatory cytokines play an important

role in diaphragm weakness in mice after exposure to intraperitoneal

lipopolysaccharide (LPS) (Labbe et al. 2010). Pro-inflammatory cytokines may

reduce force production directly through disruption to Ca2+ handling or altered

sensitivity of myofilaments to Ca2+, or indirectly via myofibre atrophy or

increased production of reactive oxygen species (ROS) (Callahan & Supinski

2009; Callahan 2001; Supinski, Wang & Callahan 2009). Developing

diaphragm myofibres contain large numbers of mitochondria (Maxwell et al.

1983) and have a less efficient antioxidant defence system (Song & Pillow

2012), suggesting that the preterm diaphragm is also prone to oxidative stress.

Increased mitochondrial production of ROS may contribute to muscle

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weakness via activation of proteolytic pathways and myofibre atrophy (Reid,

Lännergren & Westerblad 2002) or by altering excitation-contraction coupling.

Previously we showed that a two day intra-amniotic (IA) exposure to LPS

causes diaphragm weakness in preterm lambs and is associated with

mitochondrial oxidative stress and electron chain dysfunction (Song et al.

2013b). However, IA LPS exposure also increases IL-1 expression in the

diaphragm and increases systemic IL-6 protein (Song et al. 2013a).

Importantly, IL-1 signalling plays a key role in IA LPS induced lung and

systemic inflammation in fetal lambs (Kallapur 2009; Berry et al. 2011).

Blockade of IL-1 signalling in the amniotic cavity using rhIL-1ra inhibits both

lung and systemic inflammatory responses (Kallapur et al. 2009). It is unknown

whether IA LPS induced diaphragm weakness is mediated primarily via IL-1

signalling or is associated with oxidative stress.

This study investigates the role of IL-1 signalling and oxidative stress on IA LPS

induced diaphragm weakness in preterm lambs. We hypothesised that

blockade of IL-1 signalling ameliorates diaphragm dysfunction induced by IA

LPS exposure.

5.3 Methods

5.3.1 Animals and experimental design

All experiments were conducted in accordance with the guidelines of the

National Health and Medical Research Council Code of practice for the care

and use of animals for scientific purposes and were approved by the University

of Western Australia Animal Ethics Committee (Approval Number: 3/400/1023).

Pregnant Merino ewes were randomised to ultrasound guided IA rhIL-1ra (100

mg; Kineret® (Anakinra); Amgen, CA, USA) or saline (Sal) injections 3 h prior to

a second IA injection of LPS (4 mg; Escherichia coli 055:B5, Sigma Chemical,

St. Louis, MO) or Sal at 119 d gestational age (GA) generating two

experimental groups (Sal/LPS, n = 7; rhIL-1ra/LPS, n = 8) and two control

groups (Sal/Sal, n = 7; rhIL-1ra/Sal, n = 8). Preterm lambs were delivered at

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121 d GA (term = 150 d) via caesarean section and killed immediately with

pentobarbitone (150 mg/kg IV, Pitman-Moore, NSW, Australia). Longitudinal

muscle fibre strips were dissected from the right hemidiaphragm and used for

assessment of contractile function. The left hemidiaphragm was immediately

snap frozen in liquid nitrogen for molecular and biochemical analyses. Blood

samples collected from the umbilical artery were centrifuged (3 000 RPM, 10

min, 4 C): the systemic response to IA LPS exposure was determined from the

plasma supernatant. All samples were snap frozen in liquid nitrogen and stored

at -80 C prior to analysis.

5.3.2 Diaphragm contractile function

Contractile measurements were performed according to Song et al (Song et al.

2013a). A longitudinal strip of diaphragm muscle fibres (3-5 mm wide) was

isolated with a portion of the central tendon at one end and rib attachment on

the other end. The ends were tied with surgical silk thread and the preparation

was mounted in an in vitro muscle test system (model 1205, Aurora Scientific

In., Aurora, Canada) containing Krebs physiological salt solution (in mM: NaCl,

109; KCl, 5; MgCl2, 1; CaCl2, 4; NaHCO3, 24; NaH2PO4, 1; sodium pyruvate,

10). The organ bath was maintained at 25 C and continuously bubbled with 95

% O2 /5 % CO2.

The muscle strip was manually adjusted to the optimal muscle length (L0) upon

which maximum isometric twitch force (Pt) was recorded. L0 was measured

using a digital calliper. Time to peak (TTP), half relaxation time (1/2 RT) and

maximum rate of force development (df/dt) of twitch contractions were

determined using the DMA software (Aurora Scientific In., Aurora, Canada).

The fatigue resistance of the diaphragm was assessed by a series of 150

tetanic contractions (300 ms contraction times at 60 Hz once every second).

The fatigue index (FI) was determined from the ratio of the force produced

during the 150th contraction relative to the 1st contraction (Javen et al. 1996), in

which a higher number indicates a greater fatigue resistance. P0 and Pt were

normalised for cross-sectional area (CSA) and expressed as specific force

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(N/cm2). CSA was estimated as muscle mass (g) / (L0 x muscle density (1.056

g/cm3)).

5.3.3 Muscle protein extraction

Total protein extraction of the diaphragm and analysis of protein concentration

were performed as described previously (Song et al. 2013a).

5.3.4 IL-1β and IL-6 plasma levels

Plasma IL-1β and IL-6 protein concentrations were measured using a sandwich

ELISA assay (Song et al. 2013a). The wells in 96-well microplate (High binding,

Microlon Greiner Bio-One, Frickenhausen, Germany) were coated with 100 μL

of capture antibodies from SeroTec (5 μg/mL; MCA1658 for IL-1β and

MCA1659 for IL-6, East Brisbane, Australia) in 0.1 M carbonate buffer (pH 9.6)

at 4 °C overnight. The wells were blocked with 3 % skim-milk solution in

phosphate buffered saline (PBS: pH 7.2) for 1 h, then washed three times with

PBS containing 0.05 % Tween 20 (PBST). Plasma samples were added and

incubated for 2 hours at room temperature. After washing three times with

PBST, the detection antibodies from SeroTec (2 μg/mL; AHP423 for IL-1β and

AHP424 for IL-6, East Brisbane, Australia) were added into the wells and

incubated for 2 hours at room temperature. The wells were subsequently

washed as above and the bound antigen was detected with goat anti-rabbit

IgG-HRP (1:2000; 7074S Cell Signalling Technology, Carlsbad CA, USA).

Colour development was initiated by adding 3,3’,5,5’-tetramethyl-benzidine

liquid substrate (Sigma, Castle Hill, Australia) and was stopped after 15 min by

adding 0.5 M sulphuric acid. The optical density (OD) was measured at 450 nm

on a microplate reader (Labtec Multiskan, Wals, Austria).

5.3.5 Myeloperoxidase (MPO) staining

To quantify intra-muscular neutrophil infiltration, diaphragm sections (8 µm

thickness) were incubated with anti-myeloperoxidase polyclonal antibody

(CMC28917023, Cell Marque, CA, USA) 1:100 dilution overnight at 4C.

Preterm lamb liver sections were used as positive controls. VECTASTAIN®

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ELITE ABC kit (PK-6200, Vector Laboratories, Burlingame, CA, USA) and

ImmPACT™ 3, 3’-diaminobenzidine (DAB) peroxidase substrate (SK-4105,

brown, Vector Laboratories, Burlingame, CA, USA) were used to identify the

MPO positive cells. Sections were counterstained with haematoxylin for 45

seconds and cover slip applied using VectaMount AQ mounting medium

(Vector Laboratories, Burlingame, CA, USA). Sections were imaged using a

light microscope (Nikon, NY, USA) at 400X magnification.

5.3.6 MPO assay

Analysis of diaphragm MPO activity was obtained from total protein extract: 50

μL protein extract was pipetted into microplate wells and 200 μL phosphate

buffer (pH 6.0 containing 0.167 mg/mL O-dianisidine dihydrochloride and

0.0005 % hydrogen peroxide) were added to each sample well. Lysis buffer for

protein extraction was used for negative control wells. After three minutes

incubation, the optical density was measured at 450 nm using a microplate

reader (Labtec Multiskan, Wals, Austria). MPO activity was normalised to total

protein content of the protein extract and expressed as units of MPO

activity/mg protein.

5.3.7 Cord blood leukocyte count

Cord blood was collected before lamb euthanasia and analysed for neutrophils,

lymphocytes and monocytes counts using an automated cell analyser

(VetScanHM5, Abaxis, CA, USA).

5.3.8 RNA isolation, reverse transcription and quantitative PCR

RNA purification, reverse transcription and quantitative PCR were performed as

described by Song et al (Song et al. 2013a). Diaphragm mRNA expression was

measured to evaluate changes in cytokine genes (IL-1β and IL-6), proteolytic

genes (muscle RING-finger protein-1 (MuRF1) and Muscle Atrophy F-Box

(MAFbx) and anti-oxidant genes (Superoxide dismutase 1 (SOD1), Glutathione

peroxidase 1 (GPX1) and Catalase). The fluorescence signal of samples was

normalised against the average of 18S RNA and GAPDH. The 2-ΔΔCT method

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(Livak & Schmittgen 2001) was used to calculate relative mRNA expression

levels and presented as fold increase relative to controls.

5.3.9 Biochemical analysis of oxidative stress and proteolysis

The activities of reduced (glutathione, GSH) and oxidised (glutathione

disulphide, GSSG) glutathione in the diaphragm were measured using

glutathione fluorescent detection kit (DetectX K006-F1, Arbor Assays, MI, USA)

and expressed as GSH:GSSG ratio.

The 20 S proteasome levels in the diaphragm were measured fluorometrically

in total protein extracts using an assay kit (BML-AK740 assay kit, Enzo Life

Sciences, NY, USA). The specific activity of the proteasome was calculated

according to kit instructions and normalised against total protein concentration.

The protein carbonyl content in diaphragm was measured using a commercially

available protein carbonyl colorimetric kit (Cayman, Ann Arbor, MI, USA).

5.3.10 Data analysis

Data are presented as mean (SEM) or median (range). Statistical analyses

were performed using Sigmaplot (version 12.5, Systat Software Inc, USA).

Differences among multiple groups were assessed using one-way ANOVA with

post hoc analysis using Tukey honestly significant difference (HSD) test.

Nonparametric data were examined using ANOVA on ranks. Statistical

significance was accepted at p < 0.05.

5.4 Results

5.4.1 Physiological variables at birth

Descriptive characteristics for each group are presented in Table 5.1. There

were no significant differences in gestational age at birth, body weight or

optimal muscle length between any of the groups.

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Table 5.1 Lamb descriptive data and measures of diaphragm contractile function.

Sal/Sal

(n=7)

Sal/LPS

(n=7)

rhIL-1ra/LPS

(n=8)

rhIL-1ra/Sal

(n=8)

Gestational age

(d) 122.5 ± 0.2 121.7 ± 0.5 122.0 ± 0.3 121.8 ± 0.3

Body weight (kg) 2.7 ± 0.1 2.9 ± 0.1 2.8 ± 0.1 3.0 ± 0.0

L0 ( mm) 28.6 ± 0.9 28.8 ± 0.9 29.4 ± 0.6 28.7 ± 1.0

TTP (s) 0.21 ± 0.02 0.28 ± 0.02 0.30 ± 0.02 0.26 ± 0.02

1/2 RT (s) 0.29 ± 0.01 0.28 ± 0.03 0.29 ± 0.01 0.26 ± 0.01

Max df/dt (g/s) 653 ± 46 573 ± 62 779 ± 41# 727 ± 35

TTP/Pt (s/N.cm-2) 0.023 ± 0.003 0.045 ± 0.004 0.035 ± 0.003 0.026 ± 0.002

Twitch/Tetanus

ratio 0.60 ± 0.02 0.63 ± 0.01 0.55 ± 0.02 0.64 ± 0.01

Fatigue index (FI) 0.58 ± 0.02 0.59 ± 0.02 0.57 ± 0.02 0.60 ± 0.02

L0 - optimal muscle length; TTP – time to peak and 1/2 RT – half relaxation

time of twitch contraction; df/dt - rate of force development; (FI index; lower

value indicates increased fatigability). Values are mean ± SEM. - significantly

different to Sal/Sal (p < 0.05). # - significantly different to Sal/LPS (p < 0.05)

5.4.2 Diaphragm contractile function

Intra-amniotic (IA) LPS exposure two days prior to delivery significantly

impaired diaphragm contractile function in preterm fetal lambs (Figure 5.1).

Maximal specific force (P0) and twitch force (Pt) in Sal/LPS lambs were 25 %

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and 31 % lower, respectively, compared to Sal/Sal control lambs. RhIL-1ra

treatment three hours prior to IA LPS injection prevented the LPS induced

decrease in P0 and Pt. P0 and Pt in the rhIL-1ra/LPS group were not

significantly different to Sal/Sal, but were significantly greater than Sal/LPS

lambs (p = 0.044; p = 0.009, respectively). The rhIL-1ra treatment alone did not

alter diaphragm contractile function as P0 and Pt were not significantly different

between Sal/Sal and rhIL-1ra/Sal lambs.

The maximum rate of force development (df/dt) for twitch contractions was

significantly greater for rhIL-1ra/LPS compared to Sal/LPS lambs (p < 0.05).

Although there were no significant differences in TTP between any groups

(Table 5.1), when TTP was normalised to Pt (to account for differences in the

amplitude of twitch contractions) the normalised TTP values were significantly

higher in Sal/LPS lambs compared to Sal/Sal lambs (p < 0.001; Table 5.1).

Together these data reflect a relative slowing of the twitch contraction time in

LPS exposed lambs which were attenuated by rhIL-1ra treatment. Other

physiological contractile parameters (1/2 RT, twitch/tetanus ratio, and FI) were

not significantly different between groups (Table 5.1).

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Figure 5.1 Fetal diaphragm contractile properties. Maximum specific force

(A); and peak twitch specific force (B) for Sal/Sal (n=7), Sal/LPS (n=7), rhIL-

1ra/LPS (n=8) and rhIL-1ra/Sal (n=8) exposure lambs. Values are mean ±

SEM. * p < 0.05 compared to Sal/Sal; # p < 0.05 compared to Sal/LPS.

Samples sizes are the same for subsequent figures.

5.4.3 Systemic inflammation

There were no significant differences in the plasma IL-1 concentration

between any of the experimental groups (Figure 5.2A). However, plasma IL-6

protein levels in the Sal/LPS group were significantly higher compared to the

Sal/Sal group (p = 0.019) reflecting a systemic inflammatory response to IA

LPS (Figure 5.2B). RhIL-1ra treatment prior to IA LPS injection inhibited the

fetal systemic inflammatory response. Plasma IL-6 protein levels in the rhIL-

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1ra/LPS group were not significantly different to the Sal/Sal controls, but were

significantly lower than in the Sal/LPS group (p = 0.023). Total white blood cell

counts from cord blood were not significantly different between groups (Table

5.2).

Figure 5.2 Systemic and diaphragm cytokine responses. Plasma IL-1β (A)

and IL-6 (B) protein content. Values are mean ± SEM. Diaphragm IL-1β (C) and

IL-6 (D) mRNA expression. Values are median (with 10th and 90th centiles). * p

< 0.05 compared to Sal/Sal; # p < 0.05 compared to Sal/LPS.

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Table 5.2 Cord blood leukocytes counts

Group Neutrophils

(x 109/L)

Monocytes

(x 109/L)

Lymphocytes

(x 109/L)

Sal/Sal 0.10 ± 0.02 0.019 ± 0.002 3.58 ± 0.03

Sal/LPS 0.24 ± 0.11 0.014 ± 0.002 2.41 ± 0.04

rhIL-1ra/LPS 0.24 ± 0.09 0.013 ± 0.002 2.13 ± 0.27

rhIL-1ra/Sal 0.15 ± 0.06 0.018 ± 0.003 3.48 ± 0.52

Values are mean ± SEM

5.4.4 Diaphragmatic inflammation

Diaphragm IL-1 mRNA expression was significantly higher in the Sal/LPS

group compared to the Sal/Sal control group (p < 0.05) (Figure 5.2C). Again,

pre-treatment with rhIL-1ra inhibited the local diaphragmatic inflammatory

response. Diaphragm IL-1 mRNA levels in the rhIL-1ra/LPS and rhIL-1ra/Sal

groups were not significantly different to the Sal/Sal controls. There were no

significant differences in the diaphragm IL-6 mRNA levels between any groups

(Figure 5.2D). Histological and biochemical analyses of MPO revealed no

significant difference in the number of inflammatory cells in the diaphragm after

IA LPS exposure (Figure 5.3).

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Figure 5.3 Diaphragm myeloperoxidase activity. MPO assay (A) (values are

mean ± SEM) and images of MPO positive neutrophils in diaphragm

cryosections from Sal/Sal (B) and Sal/LPS (C) lambs. Fetal liver (Sal/Sal; D)

sections were used as positive control. MPO positive cells are stained brown.

Magnification 400x, scale bars = 100 µm.

5.4.5 Diaphragm atrophy gene expression and 20 S proteasome activity

There were no significant differences in mRNA expression of muscle atrophy

genes MuRF1 and MAFbx (Figure 5.4A, B) or in 20 S proteasome activities

(Figure 5.4C) between groups.

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5.4.6 Oxidative stress

IA LPS exposure caused oxidative stress in the diaphragm as reflected by a

significantly higher oxidised glutathione (GSSG) level (p = 0.003) and

consequently, a significantly lower GSH:GSSG ratio (p = 0.002) in Sal/LPS

compared to Sal/Sal lambs (Figure 5.5A). In contrast to the inflammatory

response, prior treatment with rhIL-1ra did not prevent the LPS induced

increase in oxidative stress. GSSG was also significantly higher (p < 0.001) and

GSH:GSSG significantly lower (p = 0.004) in rhIL-1ra/LPS lambs compared to

control lambs. There was no significant difference in GSSG between Sal/Sal

Figure 5.4 Atrophy related signalling in the diaphragm.

Atrophy gene MuRF1 (A) and

MAFbx (B) mRNA expression in

diaphragm. Values are median

(with 10th and 90th centiles). 20

S proteasome activity (C)

normalised against total protein

concentration. Values are mean

± SEM.

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and rhIL-1ra/Sal groups. Furthermore, there were no significant differences in

protein carbonyl levels or mRNA expression of antioxidative genes (catalase,

GPX1, SOD1) between groups (Figure 5.5 B-E).

Figure 5.5 Oxidative stress in the diaphragm. Free and oxidised glutathione

activity and GSH:GSSG ratio (A). Values are mean ± SEM. Protein carbonyl

content (B). Values are mean ± SEM. Antioxidant genes catalase (C), GPX1

(D), SOD1 (E) mRNA expression in the diaphragm. Values are median (with

10th and 90th centiles). * p < 0.05 compared with Sal/Sal.

5.5 Discussion

Chorioamnionitis is associated with increased IL-1 levels in the amniotic fluid

and IL-1 is the major contributor to lung proinflammatory activity and injury. IA

LPS induced chorioamnionitis causes diaphragm muscle weakness (Song et al.

2013a). We show that blocking IL-1 signalling via IA rhIL-1ra treatment

ameliorates the diaphragm muscle weakness in preterm lambs. Blocking IL-1

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also attenuates the LPS induced increase in systemic IL-6 levels and

diaphragm IL-1 mRNA expression. These findings suggest that rhIL-1ra

treatment protects against IA LPS induced diaphragm dysfunction by blocking

the systemic and local inflammatory responses to in utero infection.

Previous animal studies show IL-1 pathway inhibition ameliorates inflammation

related respiratory dysfunction. Pre-treatment with rhIL-1ra reduces the IL-1

induced damage to alveolar epithelial cells in a rat model of ventilator induced

lung-injury (Frank et al. 2008). Similarly, deletion of the IL-1 receptor type 1 (IL-

1R1) gene in mice attenuates the pulmonary inflammatory response to

aerosolised LPS (Hudock et al. 2012) suggesting IL-1 signalling has an

important role in lung inflammation and injury. Furthermore, blocking IL-1

signalling using IA rhIL-1ra injections reduces the pulmonary and systemic

inflammation induced by IA exposure to LPS in preterm lambs (Kallapur et al.

2009). Importantly, we show that the proinflammatory cytokine IL-1 is also

implicated in IA LPS induced diaphragm dysfunction in preterm lambs.

Diaphragmatic weakness leading to acute respiratory failure is associated with

increased expression of pro-inflammatory cytokines resulting from a systemic

inflammatory response syndrome (Callahan & Supinski 2009; Labbe et al.

2010; Supinski & Callahan 2014). Cytokine levels in amniotic fluid and fetal

cord blood increase in response to chorioamnionitis in both clinical and animal

studies (Yoon et al. 1997; Viscardi et al. 2004; Kallapur et al. 2009; Berry et al.

2011). Monocyte chemotactic protein-1 (MCP-1) is a leukocyte chemoattractant

and key regulator of the cytokine response to inflammation. Inhibition of MCP-1

with antibody neutralisation prevents diaphragm weakness in endotoxin treated

mice (Labbe et al. 2010) which suggests a coordinated cytokine response is

critical in the development of inflammation related respiratory disorders.

We measured systemic and local (diaphragm) cytokine expression two days

after IA LPS exposure. At this time plasma IL-6 protein level and diaphragm IL-

1 mRNA expression are significantly elevated and blocking IL-1 signalling with

rhIL-1ra prevented the increase in systemic and diaphragm inflammatory

markers. The time-course of cytokine release initiated by IA LPS exposure

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suggests that IL-1 secretion occurs rapidly at the chorion/amnion (Kallapur et

al. 2001; Kramer et al. 2001) and precedes the release of secondary cytokines

including IL-6. Numerous studies show that activating the IL-1 pathway

stimulates IL-6 production in cultured mouse skeletal muscle cells (Luo et al.

2003), human lung fibroblast (Elias & Lentz 1990), endothelial cells (Jirik et al.

1989) and neutrophils (Oishi & Machida 1997). Although we could not measure

cytokine levels at earlier time points in this study, we propose that the increase

in systemic IL-6 is regulated by initial IL-1 secretion at the site of LPS

exposure and leads to the induction of cytokine expression in the diaphragm.

Previously we showed that local and systemic inflammatory responses to IA

LPS are resolved within seven days, reflecting a progressive change in

cytokine expression after LPS exposure (Song et al. 2013a). These

observations are consistent with the time course of cytokine expression

characterised by Kallapur et al following IA LPS injection in preterm sheep

(Kallapur et al. 2001).

Proinflammatory cytokines can impair contractile function by disrupting

excitation-contraction coupling (Reid, Lännergren & Westerblad 2002) and

reducing muscle mass (Reid & Moylan 2011) via atrophy related signalling. Our

previous study in preterm lambs (Song et al. 2013a) showed that IA LPS (10

mg) exposure initiated a complex response, characterised by an early (2 d)

increase in pro-inflammatory cytokine expression and 20S proteasome activity,

followed by a significant decrease in protein synthesis activity and atrophy

related gene expression at 7 d after the initial LPS exposure. In the current

study, using a lower dose of LPS (4mg) we show that 20S proteasome enzyme

activity and MURF1 and MAFbx gene expression after IA LPS exposure were

not different to control levels suggesting that the diaphragm weakness that we

observed at 2 d after a low dose IA LPS exposure was not due to muscle

wasting. However, because sampling at two days after LPS exposure may

have failed to identify a transient increase in gene expression, and a lack of

ovine specific antibodies prevented us from measuring MuRF1 or MAFbx

protein expression in these samples, we cannot exclude the possible

involvement of atrophy signalling in the current study. We believe this is

107

unlikely for two reasons: Firstly, our previous study showed that at two days

after LPS exposure there was no significant difference in the proportion or

cross-sectional area of MHCI or MHCII positive fibres in the diaphragm (Song

et al. 2013a). Secondly, the diaphragm weakness that we observed in this

study was reflected by a significantly lower maximum specific force in the 2 d

LPS group compared to controls. Because the specific force is a measure of

force production relative to the amount of muscle tissue, it is unlikely to be

affected by muscle wasting. Therefore, it is likely that the initial inflammation

related diaphragm weakness that we observed was mediated by alterations to

excitation-contraction coupling (Reid, Lännergren & Westerblad 2002; Callahan

& Supinski 2009).

Our analysis of the time course for twitch contractions suggest that IA LPS

exposure slows the twitch contraction times and this slowed contraction is

prevented by blocking the IL-1 pathway. These findings are consistent with

LPS-induced alteration to the calcium release mechanism. This proposed

mechanism is supported by the IL-1 associated decrease in sarcoplasmic

reticulum Ca2+ release, achieved by altering L-type Ca2+ channel (El Khoury,

Mathieu & Fiset 2014) and ryanodine receptor (Duncan et al. 2010) function in

cardiac muscle. In skeletal muscle, IL-1α (that binds to the same receptor as IL-

1) directly inhibits sarcoplasmic reticulum Ca2+ release by inhibiting ryanodine

receptor activation (Friedrich et al. 2014 ).

Interestingly, rhIL-1ra treatment does not protect the diaphragm against LPS

induced oxidative stress. IA LPS was associated with elevated GSSG levels

and consequently, a reduced GSH:GSSG ratio, and this response was not

altered by rhIL-1ra treatment. These findings suggest that the LPS induced

preterm ovine diaphragm dysfunction is not mediated by oxidative stress.

However, it is worth noting that there were no changes in other measures of

oxidative stress (protein carbonyl content, or mRNA expression of antioxidant

genes SOD1, GPX1 or Catalase) after a two day IA LPS exposure: therefore,

the overall level of oxidative stress may be relatively low at this time point.

Further, our observation that MPO activity and MPO positive inflammatory cells

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in the diaphragm were unaltered two days after LPS exposure is consistent

with previous reports showing LPS induced diaphragm weakness can occur

without changes in the intramuscular levels of neutrophils or macrophages

(Labbe et al. 2010). The modest changes in monocytes and oxidative stress

markers may reflect the lower dose of LPS used here (4 mg) compared to

previous studies (10-20 mg) (Kallapur et al. 2009; Song et al. 2013a; Kallapur

et al. 2001). Although an IA LPS dose response study showed that 4 mg and

10 mg IA endotoxin caused similar inflammation in the lung and chorioamnion

and lung maturation (Kramer et al. 2001), it is possible that the inflammatory

response is somewhat weaker in the more distal diaphragm muscle. While IL-1

signalling is an important contributor to LPS induced inflammation, other

pathways downstream of toll-like receptor 4 activation also contribute to fetal

inflammation and oxidative stress (Kallapur et al. 2009).

Although our results indicate that IA LPS exposure did not alter the fatigue

resistance of the preterm diaphragm, extrapolation of these results to the

clinical setting should be made with caution. The in vitro fatigue protocol used

in this study involved maximal isometric contractions of isolated diaphragm

fibres. Although this technique may be adequate for evaluating the decrease in

maximum force producing capacity over time, it is unlikely to accurately reflect

the in vivo function in which the diaphragm is activated at submaximal levels

and is required to contract against a compliant rib cage. Respiratory fatigue in

the clinical setting reflects the balance between the work performed by the

diaphragm during breathing, and the functional capacity of the diaphragm. Due

to the significant (25%) reduction in the force producing capacity, the

diaphragm of LPS exposed animals is more likely to be operating closer to

maximal functional capacity and therefore any level of fatigue may result in the

development of insufficient spontaneous respiratory effort and respiratory

failure.

In conclusion, IA LPS exposure causes diaphragm weakness in preterm lambs

and blockade of IL-1 signalling protects the diaphragm from inflammation

induced contractile dysfunction. We suggest that the IL-1 pathway is implicated

109

in diaphragm weakness following LPS induced chorioamnionitis and IL-1 may

directly affect excitation-contraction coupling. Diaphragmatic dysfunction due to

immature muscle function, increased work load, inflammatory insult and/or

fatigue may contribute to postnatal respiratory failure in preterm infants.

Therefore, IL-1 may be an attractive therapeutic target in chorioamnionitis

induced diaphragm dysfunction.

Acknowledgements

The authors gratefully acknowledge the assistant of Steven Wainewright

(animal breeding) and staff from Animal Care Services at the University of

Western Australia.

Disclosures

No conflicts of interest, financial or otherwise, are declared by the author(s).

110

Chapter 6

General Discussion

111

6. Chapter 6: General Discussion

Background

The work presented in this PhD thesis represents pioneering research into the

impact of in utero inflammation on preterm diaphragm function. To date there is

limited information on preterm diaphragm function despite the major

contribution of the diaphragm to sustaining respiratory function. The diaphragm

is the primary respiratory muscle and contributes approximately 70 – 80 % of

the work of breathing (Reid & Dechman 1995). Preterm infants typically

breathe against an increased mechanical load due to non-compliant immature

lungs, insufficient surfactant protein and a highly compliant chest wall (Figure

6.1). Lung cellular proliferation and growth is stimulated by fetal breathing

movements (Leone et al. 2012) executed by respiratory muscle, primarily the

diaphragm and the intercostal muscles. Therefore, diaphragm integrity is

critically important for self-sufficient respiratory function in preterm infants.

Importantly, chorioamnionitis, inflammation of the fetal and placental

membranes, is implicated with up to 70 % of preterm birth before 30 w of

gestation (Goldenberg 2008). Chorioamnionitis together with the associated

fetal inflammatory response syndrome (FIRS) have been shown to affect

multiple organ systems including the cardiopulmonary, cerebral,

gastrointestinal and renal systems (Gotsch et al. 2007). Importantly, exposure

to inflammation in utero may further compromise the functional and phenotypic

integrity of immature diaphragm. However, little is known about the impact of

chorioamnionitis on the structure and function of the preterm diaphragm. This

research examined the hypothesis that exposure to inflammation in utero

further compromises the functional and phenotypic integrity of immature

diaphragm. The weakened immature diaphragm could contribute to the

development of postnatal chronic respiratory failure among premature infants

(Figure 6.1).

112

Figure 6.1 Factors contributing to respiratory muscle dysfunction and respiratory failure in preterm infants.

Main study focus and experimental approach

The main aims of this project were to study the effects of common, clinically

relevant antenatal exposure to inflammation and the timing of the inflammatory

insults on the functional and structural phenotype of the fetal and newborn

diaphragm. Increased cytokine secretion in inflammatory conditions such as

chorioamnionitis and FIRS may be linked with the development of skeletal

muscle weakness. Thus, it was hypothesised that the functional and structural

integrity of the newborn diaphragm is influenced by maturation and

113

inflammatory exposures before birth. This hypothesis was examined using a

well-established ovine model of chorioamnionitis induced by intra-amniotic (IA)

lipopolysaccharide (LPS) injections. Chapter two of this thesis describes the

experiments examining the effects of acute in utero LPS exposure (2 d, 7 d

LPS) on physiological and molecular changes in the preterm fetal diaphragm.

The experiments presented in chapter three examined the effect of gestational

age (GA) at the time of IA LPS exposure and the frequency of exposure on the

preterm fetal diaphragm. Chapter four further examined the significance of GA

at the time of the inflammatory exposure by comparing the effects of 2 d and 7

d IA LPS exposures on diaphragm function in preterm and term lambs. Finally,

chapter five investigated the role of IL-1 signalling and oxidative stress on IA

LPS induced diaphragm weakness in preterm lambs. Collectively, these studies

provide critical new information on how impaired postnatal diaphragm function

resulting from in utero fetal inflammatory conditions may contribute to the

development of chronic respiratory disease and late-onset respiratory failure

among premature infants. The effect of in utero inflammation on diaphragm

function in human infants, the duration of diaphragmatic impairment and

relation with late onset respiratory failure or chronic lung disease warrants

further investigation.

6.1. Study importance and novel findings

Chapter 2: In utero lipopolysaccharide exposure impairs preterm diaphragm contractility

The aims of this study were to establish the functional changes in the preterm

fetal diaphragm after exposure to in utero inflammation and to elucidate the

underlying molecular mechanisms. The results of this study supported the

hypothesis that acute 2 d and 7 d IA exposure to LPS significantly impairs

preterm diaphragm function. IA LPS exposure 2 d and 7 d prior to preterm

delivery caused a 30 % reduction in diaphragm maximum twitch and tetanic

force when compared with controls. The phenotypic change was characterised

by a reduction in the proportion of MHC type IIa muscle fibres. A 2 d (short

term) LPS exposure induced a transient activation of inflammatory signalling

114

and the NF-B pathway, with increased proteasome activity. Meanwhile a more

prolonged 7 d exposure reduced activity of the protein synthesis pathway.

Overall, in utero LPS exposure triggers a complex series of effects leading to

the impairment of preterm diaphragm function. The acute response to

inflammatory exposure was an increase in proteolysis and this was followed by

a secondary attenuation of the protein synthesis pathway that contributes to

diaphragm weakness. Therefore, we provided evidence that in utero

inflammation impairs preterm diaphragm function. We proposed that

inflammation induced diaphragm dysfunction may contribute to chronic

respiratory insufficiency in preterm infants.

In support of this study, other studies showed consistent diaphragm muscle

weakness when adult animals were injected with LPS (Supinski et al. 1996;

Supinski et al. 2000; Aimbire et al. 2006) and in patients with chronic

inflammatory diseases (Hammond 1990; Levine et al. 2013). However, in the

adult settings of inflammatory conditions, tumor necrosis factor-alpha (TNF-α)

is a potential mediator of contractile dysfunction (Wilcox, Osborne & Bressler

1992). We found that diaphragm TNF-α mRNA levels were unchanged after in

utero LPS exposure in preterm lamb diaphragm. Similarly, other studies

showed little change in TNF-α mRNA levels in preterm lambs exposed to IA

LPS (Kallapur et al. 2001; Kallapur et al. 2009). Furthermore, fetal lamb lung

and blood cells respond minimally to IA injection of TNF-α (Ikegami et al. 2003).

The lack of TNF-α response of preterm lambs may be due to underdeveloped

innate immune functions compared to adults (Kramer et al. 2010; Hillman et al.

2008). Importantly, the immune system of preterm infants has a smaller pool of

monocytes, lymphocytes and neutrophils compared to term infants (Currie et al.

2011). Monocytes from preterm neonates have reduced production of TNF-α

compared to term infants (Kramer et al. 2010; Hillman et al. 2008).

This study showed that IL-1 and IL-6 play a key role in the inflammation

induced diaphragm weakness in preterm lambs. Multiple inflammatory

cytokines and chemokines are elevated in amniotic fluid with chorioamnionitis,

however IL-1 plays the central role in progression of preterm labor and FIRS

115

(Goldenberg, Hauth & Andrews 2000; Genc et al. 2002). IA LPS induced lung

and systemic inflammation in fetal lambs also showed that IL-1 signalling plays

a key role (Kallapur et al. 2009, Berry et al. 2012). The time-course of cytokine

release initiated by IA LPS exposure suggests that IL-1 secretion occurs

rapidly at the chorion/amnion (Kallapur et al. 2001) and precedes the release of

secondary cytokines including IL-6. IL-1β and IL-6 function as catabolic factors

or directly altering excitation contraction coupling to stimulate muscle weakness

and induce contractile dysfunction (Haddad et al. 2005; Schaap et al. 2006).

In summary, this study provided evidence that acute IA LPS exposure triggers

a complex series of effects on preterm diaphragm consisting of impaired

contractile function, an early inflammatory response accelerating proteolysis

and secondary changes to protein synthesis pathway, leading to muscle

weakness. As IL-1 played a central role in the inflammation induced diaphragm

contractile dysfunction, we targeted IL-1 pathway for therapeutic intervention

for the final study (chapter 5) in this thesis. The contribution of diaphragm

dysfunction to respiratory insufficiency in the preterm infant after a pro-

inflammatory exposure warrants further investigation. Crucially, the impact of

chronic chorioamnionitis or repeated acute pro-inflammatory stimuli on the

functional development of preterm diaphragm is unknown.

Chapter 3: Gestational age at initial exposure to in utero inflammation influences the extent of diaphragm dysfunction in preterm lambs

This study examined the hypotheses that: i) gestational age at time of exposure

to IA LPS determines the extent of functional impairment of the fetal

diaphragm; and ii) repeated inflammatory exposures exacerbate diaphragm

dysfunction. In the clinical setting, chronic exposure to chorioamniotis is more

common than acute in utero inflammation (Goldenberg, Hauth & Andrews

2000). The previous study showed a single acute inflammatory exposure

significantly impaired preterm diaphragm function, and that diaphragm

weakness was observed up to 7 d after a single LPS exposure. Thus, the

current study examined if diaphragm weakness still persists after a long

116

duration of LPS exposure (21 d) and if the effects were more pronounced after

a chronic LPS exposure (weekly LPS injections).

After 7 d LPS exposure maximum specific twitch and tetanic forces were 30 %

lower than controls. When the initial LPS exposure occurred 21 d before

delivery (ie for both the 21 d and repeated (7 d, 14 d and 21 d) LPS groups)

maximum specific forces were 40 % lower than controls. Furthermore, the

earlier exposure to LPS (21 d before delivery) was associated with prolonged

twitch contraction times, increased fatigue resistance and elevated protein

carbonyl content. Although exposure to LPS reduced maximum specific force in

all groups, exposure at the earlier GA resulted in more extensive alterations to

contractile function that persisted until birth at 121 d gestation. These findings

suggest that an acute in utero exposure to inflammation significantly impairs

diaphragm contractile function and that the timing of initial inflammatory

exposure has a greater impact on diaphragm function than the frequency of

exposure.

Transition to a slow muscle fibre phenotype may contribute to the increased

TTP, 1/2 RT and fatigue resistance in the diaphragm exposed to LPS 21 d prior

to delivery. However, there was no significant change in the proportion of

MHCs positive muscle fibres after LPS exposure in the current study. Several

studies report an increased proportion of slow muscle fibres in diaphragm

under inflammatory conditions (Levine et al. 2013; Grassino & Macklem 1984;

Cohen et al. 1982). We used immunohistochemistry with antibodies against

human/rabbit MHC isoforms that cross-reacts with sheep (MHCs and MHC IIa)

to examine the effect of LPS exposure on the MHC phenotype in the

diaphragm. Ovine specific MHC antibodies and antibodies for neonatal MHC

were not available at the time of analysis of these samples but may provide a

more accurate measure of the effects of LPS exposure on MHC expression in

the current study.

The decreased specific force, slowed twitch contraction times and increased

protein carbonylation level suggest that prolonged oxidative stress may alter

contractile function through effects on the excitation contraction coupling

117

system, such as the Ca2+ sensitivity of the myofilaments and sarcoplasmic

reticulum Ca2+ handling (Powers 2008; Callahan 2001). A reduction in

metabolites build up as a result of the significantly (40 %) lower maximum

specific force may explain the increased fatigue resistance. Reduced force

production and cross-bridge cycling would lower metabolic demand and

consequently reduce the build-up of contraction-induced metabolites such as

inorganic phosphate and ROS which are associated with the development of

muscle fatigue (Allen, Lamb & Westerblad 2008). Despite increased white

blood cell counts and IL-6 mRNA expression following repeated LPS exposure,

there were no significant differences in contractile properties between 21 d and

repeated LPS groups suggesting that frequency of inflammatory exposure does

not influence the severity of contractile dysfunction.

Not surprisingly, there was no evidence of inflammatory markers at the time of

sampling for lambs exposed to LPS 21 d prior to delivery, suggesting the initial

inflammatory exposure has a persistent effect on the preterm diaphragm

muscle weakness. The previous study showed that inflammatory cytokines

resolved to control values after a 7 d LPS exposure, thus the lack of evidence

of inflammation after 21 d LPS exposure is expected.

Importantly, oxidation of contractile proteins may alter muscle force generation

capacity. The slowing of the twitch contraction time, as reflected by increased

TTP and 1/2 RT may reflect altered Ca2+ release from the sarcoplasmic

reticulum or direct oxidation of contractile proteins like actin, myosin and

tropomyosin. Defects in the excitation-contraction (EC) coupling pathway may

include a delayed arrival of the action potential in t-tubules, altered

dihydropyridine and/or ryanodine receptors function and dysfunction of

sarcoendoplasmic reticulum calcium ATPase (SERCA) pump (Callahan &

Supinski 2009; Ríos & Gonzalez 1991). The above alterations in EC coupling

processes may lead to impaired Ca2+ release and incomplete activation of

contractile proteins (Morgan 1991). Importantly, IL-1 the major cytokine

involved in LPS induced diaphragm weakness in preterm lambs, is known to

alter Ca2+ signalling in skeletal muscle (El Khoury, Mathieu & Fiset 2014;

118

Duncan et al. 2010; Friedrich et al. 2014). Due to large size and length of the

fibres found in the fetal lamb diaphragm, it was not possible to evaluate the

effect of LPS exposure on Ca2+ signalling in the current study. Future studies to

quantify the effect of LPS exposure on intracellular Ca2+ signalling is important

to identify if there is any defect in the EC coupling pathway. Identification of any

alteration in Ca2+ signalling due to inflammation may help to further elucidate

the molecular mechanism underlying diaphragm weakness in the current study.

The results of this study did not support the hypothesis that chronic in utero

exposure to inflammation would exacerbate the diaphragm dysfunction that

was observed after a single exposure. However, a single exposure to

inflammatory stimulus 21 d prior to delivery resulted in persistent diaphragm

weakness and associated with oxidative stress and altered twitch kinetics that

were not observed after the acute LPS exposures (2d, 7d). It is unclear whether

the more extensive changes were due to the duration of the exposure (21 d v’s

2-7d) or the GA at the time of the initial exposure (100 d v’s 114,119d).

In summary, we showed that in utero LPS exposure in preterm lambs has

persistent effects on diaphragm function and the timing of the initial exposure

critically influences the extent of diaphragm dysfunction. We speculate that

persistent diaphragm dysfunction resulting from early inflammatory exposures

may contribute to inefficient spontaneous breathing and the development of

late-onset respiratory failure and chronic respiratory disease in preterm infants.

Chapter 4: Gestational age at time of in utero lipopolysaccharide exposure influences the severity of inflammation-induced diaphragm weakness in lambs

The effects of inflammation on diaphragm structure and function are likely to

differ during critical stages of prenatal development. Mechanisms of

dysfunction in term and preterm diaphragm may vary due to differences in the

maturational stage of immune responses and diaphragm structure. IA LPS

exposure significantly impaired diaphragmatic force production in the preterm

lambs, however the functional and molecular responses differed when the

119

exposure occurred at 2 d, 7 d or 21 d before delivery. What remained unclear

was whether it is the duration of LPS exposure, or the timing of initial exposure

that determines the severity of diaphragm dysfunction. Therefore, this study

tested the hypothesis that GA at time of LPS exposure determines the severity

of LPS induced diaphragm weakness. This hypothesis was examined by

comparing the effects of acute IA LPS exposures (2 d and 7 d prior to delivery)

on diaphragm contractile function in term (150 d GA) and preterm (121 d GA)

lambs.

The preterm diaphragm showed a more significant reduction in force (about 30

%) and diaphragm weakness persisted after 7 d LPS exposure. The loss of

diaphragm force was accompanied by a progressive change in the regulatory

mechanism for 2 d and 7 d LPS exposures. After a 2 d LPS exposure, the

reduction in diaphragm force was accompanied by increased inflammatory

cytokines in plasma and diaphragm. The inflammatory response subsided by 7

d, but was associated with subsequent increase in proteolysis and atrophy of

MHCn and MHCs fibres. In term lambs, 2 d LPS exposure reduced maximum

diaphragm force production by 20 % and was accompanied by a moderate

increase in proportion of MHCs fibres, increased fatigue resistance and

increased inflammatory cytokines and atrophy gene expression. Most

importantly, after a 7 d LPS exposure, the diaphragm force generating capacity

was not significantly different to control levels, suggesting term diaphragm is

able to recover from an inflammatory insult. The 7 d LPS lambs showed a

significant increase in MHCs fibres in diaphragm.

Hence, this study suggested that in the term lambs, the diaphragm undergoes

muscle remodelling in response to an inflammatory insult. In contrast, the

preterm diaphragm suffered a more severe reduction in force producing

capacity and the muscle force reduction persisted after a 7 d LPS exposure

when compared with term diaphragm. Therefore, this study provided evidence

that preterm lamb is more vulnerable to diaphragm dysfunction when compared

to term lamb and inflammation-induced diaphragm weakness may contribute to

postnatal respiratory failure among premature infants.

120

Evidence from this study showed that inflammatory exposure has a higher

impact on preterm diaphragm when compared with the term diaphragm.

Although the term diaphragm was also susceptible to inflammation induced

diaphragm weakness, it appears to undergo muscle remodelling, as reflected

by changes in MHC composition, resulting in increased fatigue resistance. In

contrast, the preterm diaphragm experienced a more severe reduction in

muscle force following LPS exposure and was associated with oxidative stress

and myofibre atrophy.

Chapter 5: IL-1 receptor antagonist protects against LPS induced diaphragm weakness in preterm lambs

All the previous studies in this thesis identified that inflammatory cytokines and

oxidative stress play a major role in inflammation induced diaphragm

dysfunction. Thus, this study investigated the role of IL-1 signalling and

oxidative stress on IA LPS induced diaphragm weakness in preterm lambs.

This study tested the hypothesis that blockade of IL-1 signalling will protect the

diaphragm from inflammation induced contractile dysfunction. Pre-treatment

with rhIL-1ra ameliorated the LPS-induced diaphragm weakness and blocked

systemic and local inflammatory responses, but did not prevent the rise in

oxidised glutathione. These findings indicate that acute LPS induced

diaphragm dysfunction is mediated via IL-1 and occurs independently of

oxidative stress. We suggest that the IL-1 pathway is implicated in diaphragm

weakness following LPS induced chorioamnionitis and IL-1 may directly affect

excitation-contraction coupling. Thus, IL-1 may be an attractive therapeutic

target in chorioamnionitis induced diaphragm dysfunction and chronic

respiratory failure in preterm infants.

Several researchers showed the direct effect of IL-1 in skeletal muscle

weakness is due to alteration of Ca2+ channels and ryanodine receptors

reducing the intracellular Ca2+ concentration (El Khoury, Mathieu & Fiset 2014;

Friedrich et al. 2014). As we did not analyse the Ca2+ signalling in the

diaphragm from the current study, it would be important to further characterise

121

the direct effect of IL-1 on Ca2+ signalling in the preterm diaphragm in future

studies.

In addition to examining the direct impact of inflammatory cytokines on

diaphragm function, it is also essential to further evaluate the oxidative

pathways that may be associated with inflammation-induced diaphragm

dysfunction. We measured the glutathione activity and gene expression level of

selected oxidative markers (SOD1, catalase, GPX1) as indicators of oxidative

stress. However, it is critical to further analyse other oxidative markers such as

mitochondrial respiratory complex activity and protein levels of SOD1, catalase,

GPX1 and gene and protein level of voltage-dependent anion selective channel

protein 1 (VDAC1) and erythroid 2-related factor 2 (Nrf2) mediated antioxidant

signalling pathway. Nrf2 plays an important role in mitochondrial stress and the

VDAC1 protein couples with ROS-induced apoptosis in skeletal muscle. The

LPS exposure early at 100 d GA (chapter 3) for a duration of 21 d showed

increased protein carbonylation in the diaphragm, suggesting oxidative stress

does occur after prolong inflammatory exposure. A combination treatment to

combat inflammation and oxidative stress may be beneficial with chronic

inflammatory conditions. Providing IL-1ra plus anti-oxidants such n-acetyl

cysteine (NAC) treatment to infants exposed to inflammatory conditions such

as chorioamnionitis may be beneficial to reduce the risk of developing chronic

respiratory failure among preterm infants.

6.2. Study limitations and implications for future research

There were some unavoidable study limitations in this thesis. First the sample

number is small. As we lost some sample number due to fetal death or

spontaneous delivery, we were unable to repeat the lost experiments due to the

practical limitations associated with sheep experiments (sheep breeding

season, planned twice yearly experiments and high cost). Secondly the

distribution of male to female lambs is unequal among experimental groups.

Male premature infants have markedly higher rates of adverse pulmonary

neonatal outcomes compared to females, therefore we cannot exclude the

possibility that our results were influenced by a male disadvantage in relation to

122

the severity of LPS induced diaphragm weakness. Future research should

consider a larger sample number and an equal distribution of male:female

samples.

6.3. Summary and conclusion

Preterm birth is among the leading causes of postnatal morbidity and mortality

in infants worldwide. Up to 70 % of very preterm births are associated with

inflammation of the fetal membranes that commonly manifests as

chorioamnionitis. In utero infection may critically influence diaphragm

development and predispose preterm infants to postnatal respiratory failure.

Respiratory failure in the preterm infant has a multitude of causes of which

respiratory muscle weakness and/or fatigue is a major contributor. The

diaphragm of premature infant’s has reduced force generation capacity

compared with term infants and in utero inflammation may further compromise

diaphragm force production. The findings presented in this thesis provide novel

evidence on the effect of inflammation on diaphragm dysfunction among

preterm lambs. Acute in utero inflammatory exposures induced diaphragm

muscle weakness. Importantly, when the inflammatory exposure occurred

during the critical stages of diaphragm and immune system development in the

lambs, as in the 21 d group, the diaphragm weakness was more pronounced

compared to acute inflammatory exposure. Repeated inflammatory exposure

does not induce an additive effect on diaphragm dysfunction in comparison with

single inflammatory exposure. Gestational age at the time of inflammatory

exposures critically influences the susceptibility to diaphragm weakness.

Inflammatory cytokines plays an important role in diaphragm dysfunction in

preterm lambs.

Given the pivotal role of the diaphragm in maintaining independent respiration,

the optimal function of the diaphragm is essential for normal respiration.

Impaired postnatal diaphragm function resulting from fetal inflammatory

conditions may contribute to the development of chronic respiratory disease

and late-onset respiratory failure among premature infants. However, further

investigations are required on preterm diaphragm dysfunction and the relation

123

with late onset respiratory failure or chronic lung disease in human infants.

Collectively, diaphragm dysfunction may further impact the preterm vulnerability

to other common exposures such as postnatal steroids, mechanical ventilation

and malnutrition. Thus, it is critically important to ensure optimal diaphragm

function in support to lung function when setting up the treatment and

management plan for respiratory failure among premature infants.

124

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Appendices

A1: Detailed protocols

Diaphragm contractile function

A longitudinal strip of diaphragm muscle fibres (3-5 mm wide) was isolated with

a portion of the central tendon at one end and rib attachment on the other end.

The ends were tied with surgical silk thread and the preparation was mounted

in an in vitro muscle test system (model 1205, Aurora Scientific In., Aurora,

Canada) containing Krebs physiological salt solution (in mM: NaCl, 109; KCl, 5;

MgCl2, 1; CaCl2, 4; NaHCO3, 24; NaH2PO4, 1; sodium pyruvate, 10). The

organ bath was maintained at 25C and continuously bubbled with 95 % O2 /5

% CO2.

The muscle strip was manually adjusted to the optimal muscle length (L0) at

which maximum isometric twitch force (Pt) was recorded. L0 of the muscle strip

was measured using a digital caliper. The muscle was stimulated by a 701B

stimulator (Aurora Scientific Inc.) delivering 0.2 ms square wave pulses via two

platinum electrodes running parallel on either side of the suspended muscle.

Time to peak (TTP) and half relaxation times (1/2 RT) of maximum twitch force

and maximum rate of force development (df/dt) were determined using the

DMA software (Aurora Scientific In., Aurora, Canada). The parameters for

force-frequency measurements are: electrical stimulation from 0.2 – 0.7

seconds at different frequencies (5, 10, 20, 40, 60, 80, 100 Hz) and recorded

for a total duration of 1 minute. The force-frequency relationship (5-100 Hz)

was plotted from which the maximum tetanic force (P0) was recorded. Max

twitch/tetanus ratio was calculated manually. The fatigue resistance of the

diaphragm was assessed by a series of 150 tetanic contractions. The

parameters for fatigue protocols are tetanic contraction stimulated at 80 Hz,

total of 150 tetanic contractions once every second. The fatigue index (FI) was

calculated from the ratio of the tetanic force produced during the 150th

contraction relative to the 1st contraction (Javen et al. 1996); a lower ratio

representing a greater susceptibility to fatigue. The susceptibility to muscle

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damage was determined from a series of five lengthening (eccentric)

contractions at 2 min intervals. For each lengthening contraction, a stretch of

10% of L0 was applied during the isometric plateau phase of a maximal tetanic

contraction. P0 was recorded before and at 2, 5, and 10 min after the

lengthening contraction protocol. The severity of damage was determined by

the mean reduction in P0 after the stretch. Finally, the rib and tendon were

removed from the diaphragm strip and the wet muscle weight was recorded. To

account for slight differences in the size of the dissected diaphragm strips, the

absolute force was normalised for cross-sectional area and expressed as

specific force (N/cm2).

CSA and specific force (normalised for CSA; N.cm2) was calculated using the

following formula.

CSA = muscle mass (g) / (optimal fibre length x muscle density; 1.056)

Specific force = force (g) / CSA

Myosin Heavy Chain (MHC) fibre typing and myofibre Cross-Sectional Area (CSA)

Optimal cutting temperature (OCT) compound embedded diaphragm was

transversely orientated and serially sectioned with a Leica CM1900 cryostat

(Meyer Instruments, Houston, USA) to a thickness of 8 µm. The sections for

muscle fibre typing were dried at RT for 30 min and snap fixed in a 1:1 solution

of ice cold acetone: methanol then rinsed in PBS solution containing 0.1% triton

X-100. The sections were blocked with 1% normal goat serum in PBS for 2

hours at 4ºC and then incubated with primary antibodies specific to laminin (1:

250, Abcam, Waterloo, Australia), MHC slow (1:50, Novocastra, Newcastle,

UK) or type II (1:100, Santa Cruz Biotechnology, Inc, CA, USA) or sheep MHC

slow (MHCs; 2400101), MHC fast (MHCf; 2400107) and MHC neonatal (MHCn;

2400104) (preterm 1:25; term 1:50) in a humid chamber at 4ºC overnight. The

optimum primary antibody concentration was determined after trial runs at 1:10;

1:25; 1:50; 1:100 and 1:200 antibody dilutions. Sections were subsequently

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rinsed 3 times in PBS solution containing 0.1% triton X-100 (PBST) and then

incubated with a cocktail of DyLight™ 488 anti-mouse or anti-rabbit IgG (1:

500, Biolegend, London, UK) and Dylight™ 549 anti-rabbit or anti-chicken IgG

(1:1000, KPL, Gaithersburg, USA) for 2 hours at RT. The optimum secondary

antibody was determined after trial runs at 1:500, 1:1000. After washing 3

times in PBST, the slides were mounted with an aqueous mounting medium

containing DAPI (H-1200, Vector Laboratories, CA, USA). Slides were cover

slipped and sealed for viewing via a fluorescence microscope (Nikon Eclipse

Ti-U Nikon, Nikon Instruments, New York, USA). Images were analysed using

Nikon BR 3.0 software (NIS Elements, New York, USA). Mean CSA for total,

MHC slow and MHCII fast fibres were measured by tracing the perimeters of

each fibre using the software. The proportion of slow and fast fibres were

determined by counting the positively stained fibres and divided by total

number of fibres stained with laminin within the same section.

Immunoblot analysis

Diaphragm frozen samples were homogenised in ice-cold lysis buffer

containing 20 mM HEPES pH 7.7, 2.5 mM MgCl2, 0.1 mM EDTA, 20 mM b-

glycerophosphate, 100 mM NaCl, 0.1% Triton 100, 500 mM DTT, 100 mM

Na3Vo4, 100 mM PMSF, 0.01% NP40 and protease inhibitor cocktail tablet

(Roche, Castle Hill, Australia). Homogenates were subjected to six cycles of

freeze–thaw then centrifuged at 10,000g, for 25 min at 4C. Total protein

concentration in the supernatant was measured by Bradford protein assay.

Equal amounts of total protein lysates (50 µg) were separated by 12% SDS–

PAGE and transferred to nitrocellulose membranes. After blocking in PBS

containing 5% non-fat dry milk, the membranes were incubated with primary

antibodies. The primary antibodies used in 1:1 000 dilutions were purchased

from Cell Signalling Technology (Carlsbad CA USA) including Akt,

phosphorylated (p) Akt (Ser473), FOXO1 and α-Tubulin. Bound antibodies

were detected with 1:1,000 dilutions of either anti-rabbit or anti-mouse (Cell

Signalling) immunoglobulin conjugated with horseradish peroxidase (HRP). The

blots were developed by adding a SuperSignal West Pico Chemiluminescent

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Substrate (Thermo Scientific, Billerica, MA) and quantified by computerised

image analysis (ImageQuantTM 350; GE Healthcare, Little Chalfont, UK). To

avoid the variation across membranes arising from different exposure time and

transferring/blotting efficiency, a same control sample was added in each test.

The values for each protein were standardised with the control sample and

then normalised into a-tubulin abundance. The activities of Akt, was

represented as phosphorylated / total protein ratio. Immunoblots with anti-

phosphorylated specific antibodies were stripped and reprobed with the

corresponding antibodies against the total proteins for normalization. FOXO1

activity was expressed as nuclear/cytosolic ratio after normalising into nuclear

or cytosolic α-Tubulin abundance.

The control sample for immunoblot analysis was selected after optimisation

during the pilot study and was checked on 3 controls (α-Tubulin, β-Tubulin and

β-actin). By observing the protein levels against whole (total) cell protein and

comparing data obtained from both gene and protein expression on the same

molecules such as myosin heavy chain and sarcoendoplasmic reticulum Ca2+

ATPase during development as well as basing on the literature reports, we

determined that the most suitable quality control sample for this study as α-

Tubulin, which showed stability during the study.

IL-1β and IL-6 levels in plasma

Plasma IL-1β and IL-6 concentrations were measured using a solid-phase

sandwich ELISA. The ELISA protocol was standardised using standard sheep

recombinant IL6 and IL1β proteins, which were used to determine the ELISA

specificity and sensitivity. The wells in 96-well microplate (High binding,

Microlon Greiner Bio-One, Frickenhausen, Germany) were coated with 100 μl

of capture antibodies from SeroTec (5 μg/ml; MCA1658 for IL-1β and

MCA1659 for IL-6, East Brisbane, Australia) in 0.1 M carbonate buffer (pH 9.6)

at 4 °C overnight. The wells were blocked with 3 % skim-milk powder in

phosphate buffered saline (PBS: pH 7.2) for 1 h, then washed three times with

PBS containing 0.05 % Tween 20 (PBST). Plasma samples were added and

incubated for 2 hours at room temperature. After washing three times with

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PBST, the detection antibodies from SeroTec (2 μg/mL; AHP423 for IL-1β and

AHP424 for IL-6) were added into the wells and incubated for 2 hours at room

temperature. The wells were subsequently washed as above and the bound

antigen was detected with goat anti-rabbit IgG-HRP (1:2000). Colour

development was initiated by adding 3,3’,5,5’-tetramethyl-benzidine liquid

substrate (Sigma, Castle Hill, Australia) and was stopped after 15 min by

adding 0.5 M sulphuric acid. The optical density (OD) was measured at 450 nm

on a microplate reader (Labtec Multiskan, Wals, Austria).

Quality controls for ELISA were sheep recombinant IL6 and IL1β proteins,

which were used to determine the ELISA specificity in the pilot study.

Additionally this ELISA assay was developed in-house and validated using

standards and intra-assay precision (CV of <5%).

RNA isolation, reverse transcription and quantitative PCR

Total RNA was isolated from 30 mg homogenised diaphragm tissue using the

RNeasy Mini kit (Qiagen Pty Ltd., Doncaster, Australia) according to

instructions detailed in RNA extraction protocol (page 144). Contaminating

genomic DNA was removed by an on-column DNaseI digestion (DNaseI

digestion kit; Qiagen Pty Ltd.). RNA purity determined using 260/280

absorbance measurements using nanodrop. RNA purity of 1.8-2.0 was used for

reverse transcription. Isolated RNA was reverse transcribed into

complementary DNA (cDNA) in a 20 ml reaction (QuantiTect1 Reverse

Transcription Kit; Qiagen Pty Ltd.) detailed in Reverse transcription protocol

(page 146). Specific products were amplified and detected on the Rotor-gene

3000 real time PCR system (Corbett Life Science, Mortlake, Australia) using

Rotor-Gene SYBR Green PCR Kit (Qiagen Pty Ltd.) following the PCR

protocol. The cycling conditions for all genes were as follows: 3 min at 95C,

35–40 cycles of 5 sec at 95C, 20 sec at 60C annealing temperature and 20

sec at 72C. The expression levels of genes of interest were normalised into

18S RNA and GAPDH using the 2-CT method (Livak & Schmittgen 2001) and

presented as a fold change relative to the control group.

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Identification of suitable house-keeping genes is crucial for accurately

evaluating gene expression data and had been carried out in the preliminary

study. During the selection stage, a panel of well documented house-keeping

genes were assessed including 18S RNA, β-actin and GAPDH for qPCR. 18S

RNA and GAPDH were determined to be the most suitable house-keeping

genes based on rigorous comparisons of gene expression against whole

mRNA; comparing data obtained from both gene expression and protein levels

on the same molecules such as myosin heavy chain and sacroendoplasmic

reticulum Ca2+ ATPase throughout development; and based on previous

literature reports. These genes showed stable expression across the studies

and therefore were considered suitable housekeeping genes.

RNA extraction protocol for lamb diaphragm

[RNeasy mini RNA extraction kit (Qiagen)] 14.3 M β-mercaptoethanol (β-ME) (commercially available solutions are usually 14.3 M) must be added to Buffer RLT before use. β-ME is toxic; dispense in a fume hood and wear appropriate protective clothing. Add 10 μl β-ME per 1 ml Buffer RLT. Buffer RLT is stable for 1 month after addition of β-ME.

1. Excise the ~30mg tissue sample from the animal or remove it from

storage.

2. Place the samples into 2 ml microcentrifuge tubes containing 2 stainless

steel bead, add 300 ul Buffer RLT and homogenize immediately using

TissueLyser LT according to the protocol of Homogenization for RNA.

3. Transfer the samples into a new 1.5 tube (not provided).

4. Add 590 μl double-distilled water to the homogenate. And then add 10 μl

QIAGEN Proteinase K solution and mix thoroughly by pipetting. Do not vortex.

5. Incubate at 55°C for 10 min.

6. Centrifuge for 3 min at 10,000 x g at room temperature.

7. Pipet the supernatant (approximately 900 μl) into a new tube (not

provided).

148

8. Add 0.5 volumes (usually 450 μl) of ethanol (96–100%) to the cleared

lysate. Mix well by pipetting. Do not centrifuge.

9. Pipet 700 μl of the sample, including any precipitate that may have

formed, into an RNeasy mini column placed in a 2 ml collection tube.

Centrifuge for 15 s at 11,000 rpm. Discard the flow-through.

10. Repeat step 8, using the remainder of the sample. Discard the flow-

through.

11. Pipet 350 ul Buffer RW1 into the RNeasy mini column, and centrifuge

for 15 s at 11,000 rpm to wash. Discard flow-through.

12. Add 10 μl DNase I stock solution to 70 μl Buffer RDD. Mix by gently

inverting the tube. Note: DNase I is especially sensitive to physical

denaturation. Mixing should only be carried out by gently inverting the

tube. Do not vortex.

13. Pipet the DNase I incubation mix (80 μl) directly onto the RNeasy silica-

gel membrane, and place on the benchtop (20° to 30°C) for 15 min.

Note: Make sure to pipet the DNase I incubation mix directly onto the

RNeasy silica-gel membrane.

14. Pipet 350 μl Buffer RW1 into the RNeasy mini column, and centrifuge for

15 s at 11,000 x rpm. Discard flow-through and collection tube.

15. Transfer the RNeasy column into a new 2 ml collection tube (supplied).

Pipet 500 μl Buffer RPE onto the RNeasy column. Close the tube gently,

and centrifuge for 15 s at 12,000 rpm to wash the column. Discard the

flow-through. Note: Buffer RPE is supplied as a concentrate. Ensure

that ethanol is added to Buffer RPE before use.

16. Add another 500 μl Buffer RPE to the RNeasy column. Close the tube

gently, and centrifuge for 2 min at 12,000 rpm to dry the RNeasy silica-

gel membrane.

17. Place the RNeasy column in a new 2 ml collection tube (not supplied),

and discard the old collection tube with the flow-through. Centrifuge in a

microcentrifuge at full speed (15,000 rpm) for 1 min.

18. Transfer the RNeasy column to a new 1.5 ml collection tube (supplied).

Pipet 30 μl RNase-free water directly onto the RNeasy silica-gel

149

membrane. Stay at room temperature for 1 min. Close the tube gently,

and centrifuge for 1 min at 11,000 rpm) to elute.

Reverse transcription protocol

(QuantiTect1 Reverse Transcription Kit 205311)

1. Thaw template RNA on ice. Thaw gDNA Wipeout buffer, Quantiscript

Reverse Transcriptase, Quantiscript RT buffer, RT Primer Mix and

RNase-free water at room temeperature (15-25°C).

2. Prepare the genomic DNA elimination reaction on ice according to Table

1.

Table 1

gDNA Wipeout Buffer 2 µl

Template RNA Variable (1000 ng concentration)

RNase-free water Variable

Total volume 14 µl

3. Incubate for 2 min at 42°C. The place immediately on ice.

4. Prepare the reverse transcription master mix on ice according to Table 2

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Table 2: Reverse transcription reaction components

Quantiscript Reverse Transcriptase 1 µl

Quantiscript RT Buffer 5x 4 µl

RT Primer Mix 1 µl

Entire genomic DNA elimination

reaction (step 3)

14 µl (add at step 5)

Total volume 20 µl

5. Add template RNA from step 3 (14 µl) to each tube containing reverse

transcription master mix. Mix and then store on ice.

6. Incubate for 30 mins at 42°C.

7. Incubate for 3 mins at 95°C to inactivate Quantiscript Reverse

Transcriptase

151

A2: Raw data table-example data for Chapter 3

Table A: Raw data for Chapter 3

Lamb ID Gp

ExpDur GA Sex

IL1B prot

RELATIVE To ctrl MEAN

IL1B prot (abs)

IL6 prot

RELATIVE To ctrl MEAN

IL6 Prot (abs)

IL1 mRNA

IL6 mRNA

MuRF mRNA

MAFbxmRNA

CAT mRNA

SOD1 mRNA

GPX mRNA

V11-67 CTRL 0 121 F 0.501 0.091

0.939 1.010 1.000 2.282 V11-69 CTRL 0 121 F 0.706 0.128 1.080 0.048 0.915 0.990 0.758 1.000 1.569 1.094 1.464

V11-70 CTRL 0 121 F 0.872 0.158 1.148 0.051 0.897 2.420 2.282 1.000 1.000 0.497 0.877 09-97 CTRL 0 121 F 1.276 0.231

0.852 0.793 2.204 0.086 0.871 1.000 1.000

V12-01 CTRL 0 120 M 1.506 0.144 0.580 0.026 0.905 0.278 0.611 0.301 0.314 0.337 0.444 V12-15 CTRL 0 121 M 1.138 0.206 1.193 0.053

1.723

3.138 1.558 1.385 2.313

V11-76 7d LPS 7 121 F 1.127 0.204 1.602 0.071 0.478 2.667 1.329 0.241 0.448 0.547 1.057 V11-77 7d LPS 7 121 F 0.545 0.099 1.011 0.045 0.132 0.710 1.765 0.551 0.629 0.507 0.940 V11-78 7d LPS 7 121 F 0.878 0.159 1.398 0.062 0.071 0.509

0.227 0.337 0.363 0.532

V12-02 7d LPS 7 120 M 1.526 0.276 0.693 0.031 0.523 0.375 0.574 0.304 0.540 0.460 0.747 V12-16 7d LPS 7 121 F 0.401 0.073 1.011 0.045 0.635 0.162 2.497 1.149 0.334 0.586 0.578 V12-17 7d LPS 7 121 F 0.579 0.105 0.852 0.038 1.479 0.534 2.751 1.905 0.532 0.497 0.883 V12-08 21d LPS 21 119 F 0.828 0.150 0.966 0.043 1.117 1.244 1.636 0.646 1.395 1.474 1.790 V12-09 21d LPS 21 120 F 0.656 0.119 1.080 0.048

0.732 0.655 0.768

V12-10 21d LPS 21 120 F 0.911 0.165

1.023

2.848 1.932 1.495 1.021 2.428 V12-11 21d LPS 21 120 M 1.016 0.184 0.784 0.035 3.204 2.630 1.133 0.664 2.099 1.537 1.932 V12-12 21d LPS 21 121 M 0.490 0.089 1.330 0.059 0.295 0.428 0.555 0.438 1.079 0.807 0.940 V12-13 21d LPS 21 121 F 0.590 0.107 0.489 0.022 0.563 0.798 0.801 0.547 0.768 0.914 0.966 V12-14 21d LPS 21 121 M 0.407 0.074 1.511 0.067 0.301 0.481 1.537 0.859 0.651 0.841 0.688

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V12-03 7+14+21

d LPS 21 121 M 1.354 0.245

0.304 1.815 2.858 1.347 0.871 1.366 1.526 2.071

V12-04 7+14+21

d LPS 21 121 F 0.540 0.098 1.443 0.064 4.000 4.070 2.639 1.485 0.959 0.946 1.206

V12-05 7+14+21

d LPS 21 121 M 0.446 0.081 1.898 0.084 1.647 3.021 1.853 1.057 1.434 1.206 1.474

V12-06 7+14+21

d LPS 21 122 F 0.468 0.085 1.057 0.047 0.768 2.166 1.815 0.877 1.840 1.647 2.362

V12-07 7+14+21

d LPS 21 122 F 0.429 0.078 1.534 0.068 2.189 0.626 1.310 0.382 0.973 1.181 1.181

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A3: Example of recorded traces for stretch protocol

Figure A: Physiology traces for stretch induced muscle damage protocol.

A. prestretch max force; B. Post stretch max force; C. Corresponding force and

length traces during stretch. Force deficit is calculated as the difference in

maximum isometric force (Po) after stretch compared with before stretch,

expressed as a percentage of the pre-stretch maximum isometric force.

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A4: Reverse Transcription qPCR _example melt curve and log fluorescence signal for chapter 5

Figure B: Melt curve

Figure C: Raw data for cycling A green

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Figure D: Quantification data for Cycling A green