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:
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: [email protected]. 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: [email protected]. 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).
73
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.
76
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
79
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).
80
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).
81
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.
82
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
83
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
84
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: [email protected] (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
96
(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-
100
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.
101
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
105
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
106
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
108
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
150
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