MECHANISMS BY WHICH HYPERBARIC OXYGEN THERAPY MAY RESOLVE INFLAMMATION ...

342 Pages·2013·9.2 MB·English

by  Anwar Almzaiel

MECHANISMS BY WHICH HYPERBARIC OXYGEN
THERAPY MAY RESOLVE INFLAMMATION IN CHRONIC
WOUNDS

by
ANWAR JASIB AL-MZAIEL
A thesis submitted to the University of Plymouth in
partial fulfilment for the degree of

DOCTOR OF PHILOSOPHY

School of Biomedical and Biological Sciences
Faculty of Science and Technology
September 2013
I
Copyright Statement

This copy of this thesis has been supplied on the condition that anyone who
consults it is understood to recognise that its copyright rests with its author
and that no quotation from the thesis and no information derived from it may
be published without the author’s prior consent.

II
Dedication
To
my husband, Ahmed
my parents
and
my kids, Azal and Rafal

whose love, interest and encouragement
have steered me to where I am today.

III
MECHANISMS BY WHICH HYYPERBARIC OXYGEN THERAPY
MAY RESOLVE INFLAMMATION IN CHRONIC WOUNDS
ANWAR J AL-MZAIEL
Abstract
Hyperbaric oxygen (HBO) therapy is the intermittent inhalation of 100% oxygen at a
pressure greater than one atmosphere absolute. It is an effective treatment for various
inflammatory conditions, including chronic wounds which are characterized by an
excessive influx of neutrophils and their prolonged persistence at the wound site.
Neutrophil apoptosis and clearance have been shown to be required for resolution of
inflammation. The mechanisms by which HBO aids wound healing are well documented,
but its effects on cellular inflammatory response are not well understood particularly with
respect to neutrophils. The hypothesis presented in this thesis is that increased oxygenation
via HBO assists chronic wound healing by enhancing non-inflammatory neutrophil
defences and cell death through apoptosis.
An investigation was carried out into the effects of HBO on neutrophil antimicrobial
function and apoptosis using differentiated HL-60 cells as an in vitro neutrophil model. The
data clearly showed that a single HBO treatment for 90 min caused an increase in the
oxidative burst activity of neutrophil-like cells as shown by increased NBT staining,
superoxide (cytochrome c reduction) and H O production (Kruskal-Wallis, P < 0.05), and
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phagocytosis of Staphylococcus aureus. HBO treatment displayed a pro-apoptotic effect,
enhancing caspase 3/7 activity both in the presence and absence of a TNF-α stimulus
(Kruskal-Wallis, P < 0.05) and causing morphological changes (observed using Giemsa
IV
and SYBR® Safe staining) associated with apoptosis. Although no consistent pattern was
observed, both hyperoxia and pressure alone seemed to contribute to both the increase in
antimicrobial activity and the increase in apoptosis induced by HBO in these neutrophil-
like cells (Chapters 4 and 5).
HBO-enhanced neutrophil clearance by macrophages was investigated using bovine
neutrophils and monocyte-derived macrophages (MDMФ). A single 90 min HBO exposure
significantly increased the clearance of fresh and 22 h-aged neutrophils by MDMФ (two-
way ANOVA, P < 0.05), suggesting an increase in phosphatidylserine (PS) exposure in
apoptotic neutrophils after HBO treatment (Chapter 6). Importantly, a long-term repetitive
exposure to HBO in patients with chronic wounds caused a significant decrease in the
antioxidant enzyme defence system (one-way repeated measures ANOVA, P < 0.05),
plasma TNF-α and IL-1β after 30 HBO sessions, with down regulation of expression of the
anti-apoptotic factors, NF-B and Bcl-2 (Chapter 7). These findings may go some way
towards explaining the effectiveness of HBO treatment not only for chronic wounds but
also for other inflammatory conditions that may be affected by this treatment.

V
Table of contents
Abstract ............................................................................................................................... IV
Table of contents ................................................................................................................ VI
List of figures ................................................................................................................... XIV
List of tables ..................................................................................................................... XIX
Acknowledgements ............................................................................................................ XX
Author’s Declaration ...................................................................................................... XXI
Platform presentations................................................................................................... XXII
Publications ................................................................................................................... XXIII
Abbreviations ............................................................................................................... XXIV
CHAPTER ONE .................................................................................................................. 1
GENERAL INTRODUCTION ........................................................................................... 1
1.1 Introduction .................................................................................................................... 2
1.2 Normal wound healing ................................................................................................... 3
1.2.1 The inflammatory phase ................................................................................................ 3
1.2.2. The proliferative phase ................................................................................................. 6
1.2.3 The remodelling phase .................................................................................................. 8
1.3 Chronic wounds .............................................................................................................. 9
1.3.1 Pathological conditions in chronic wounds ................................................................ 11
1.4 Immune cells in wound healing ................................................................................... 12
VI
1.4.1 Neutrophils .................................................................................................................. 13
1.4.2 Neutrophils in wound healing ..................................................................................... 15
1.4.3 Macrophages in wound healing .................................................................................. 17
1.4.4 Interactions between neutrophils and macrophages .................................................... 20
1.5 Role of oxygen in wound healing ................................................................................ 25
1.6 Hyperbaric oxygen (HBO) therapy ............................................................................ 27
1.6.1 Side effects and counter indications ............................................................................ 29
1.6.2 Physical mechanisms of HBO ..................................................................................... 31
1.6.3 Evidence for the effectiveness of HBO in the treatment of chronic wounds .............. 33
1.6.4 Effects of HBO on wound healing .............................................................................. 37
1.6.4.1 Effect of HBO on the immune system ................................................................... 38
1.6.4.2 HBO, growth factors and cytokines in wound healing ......................................... 41
1.7 Hypotheses and aims .................................................................................................... 44
1.7.1 Hypotheses .................................................................................................................. 44
1.7.2 Aims and objectives .................................................................................................... 45
CHAPER TWO .................................................................................................................. 47
2.1 Material and methods .................................................................................................. 48
2.1.1 Hyperbaric oxygen and pressure control treatments ................................................... 48
2.1.2 Measurement of NBT reduction.................................................................................. 50
2.1.3 Assay of extracellular hydrogen peroxide................................................................... 50
2.1.4 Assay of phagocytic activity ....................................................................................... 51
2.1.5 Assay for cell viability ................................................................................................ 53
2.1.6 MTT assay ................................................................................................................... 53
VII
2.1.7 Gene expression .......................................................................................................... 54
2.1.7.1 RNA extraction ..................................................................................................... 54
2.1.7.2 DNA digestion ...................................................................................................... 55
2.1.7.3 Quantitation of RNA ............................................................................................. 55
2.1.7.4 Reverse Transcription for synthesis of complementary DNA (cDNA) ................. 56
2.1.7.5 Real-time quantitative polymerase chain reaction (Q-PCR) ............................... 60
2.1.7.6 Stability of β-actin following different treatments ................................................ 61
2.1.8 Measurements of pro-and anti-inflammatory cytokines using multiplex Luminex bead
assays ................................................................................................................................... 63
2.1.9 Neutrophil isolation ..................................................................................................... 65
2.1.10 Statistical analysis ..................................................................................................... 67
CHAPTER THREE ........................................................................................................... 68
CHARACTERISATION OF DIFFERENTIATED HL-60 CELLS AS A
NEUTROPHIL MODEL FOR IN VITRO STUDIES..................................................... 68
Abstract ............................................................................................................................... 69
3.1. Introduction ................................................................................................................. 70
3.2 Materials and methods ................................................................................................ 73
3.2.1 Cell culture .................................................................................................................. 73
3.2.2 Experimental design .................................................................................................... 74
3.2.3 Assessment of differentiation ...................................................................................... 75
3.2.4 Quantification of TNF- and IL-10 by ELISA ........................................................... 76
3.2.5 Induction of apoptosis ................................................................................................. 78
3.2.6 Statistical analysis ....................................................................................................... 79
3.3 Results ........................................................................................................................... 80
VIII
3.3.1 Cell proliferation in response to ATRA, 9-cis RA and DMSO .................................. 80
3.3.2 NBT reduction during differentiation of HL-60 cells by retinoids and DMSO .......... 84
3.3.3 Morphological alterations during HL-60 cells differentiation .................................... 86
3.3.4 Phagocytosis of Staphylococcus aureus NCIMB 6571 by differentiated HL-60 cells 88
3.3.5 TNF-α production by neutrophil-like cells produced by differentiation of HL-60 cells
.............................................................................................................................................. 89
3.3.6 TNF-α and IL-10 production in response to oxygen exposure at different
concentrations ...................................................................................................................... 91
3.3.7 Induction of apoptosis in differentiated HL-60 cells .................................................. 93
3.4 Discussion ...................................................................................................................... 96
3.4.1 Conclusion ................................................................................................................ 100
CHAPTER FOUR ............................................................................................................ 101
THE EFFECT OF HBO ON ANTIMICROBIAL DEFENCE MECHANISMS IN
NEUTROPHILS ............................................................................................................... 101
Abstract ............................................................................................................................. 102
4.1 Introduction ................................................................................................................ 103
4.2 Materials and methods .............................................................................................. 107
4.2.1 Experimental design .................................................................................................. 107
4.2.2 Measurement of superoxide production via cytochrome c reduction ....................... 109
4.2.3 Assay of NO -/ NO - ................................................................................................. 109
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4.2.4 Myloperoxidase (MPO) assay ................................................................................... 111
4.2.5 Measurement of total glutathione.............................................................................. 113
4.2.6 Statistical analysis ..................................................................................................... 114
4.3 Results ......................................................................................................................... 114
IX
4.3.1 HBO enhances respiratory burst activity by differentiated HL-60 cells ................... 114
4.3.2 Effect of HBO on NO production from differentiated HL-60 cells ........................ 120
x
4.3.3 Effects of HBO on degranulation of differentiated HL-60 cells ............................... 123
4.3.4 Effect of HBO on redox status .................................................................................. 124
4.4 Discussion .................................................................................................................... 126
4.4.1 Conclusions ............................................................................................................... 133
CHAPTER FIVE .............................................................................................................. 134
THE EFFECTS OF HBO ON NEUTROPHIL VIABILITY AND APOPTOSIS ..... 134
Abstract ............................................................................................................................. 135
5.1 Introduction ................................................................................................................ 136
5.2 Material and methods ................................................................................................ 138
5.2.1 Experimental design .................................................................................................. 138
5.2.2 Morphological assessment of cell apoptosis ............................................................. 140
5.2.3 Assessment of apoptosis by fluorescence microscopy.............................................. 141
5.2.4 Preparation of nuclear protein extracts ..................................................................... 141
5.2.5 Protein assay ............................................................................................................. 142
5.2.6 Sodium dodecyl sulphate polyacrylamide gel electophoresis ................................... 143
5.2.7 Western blotting ........................................................................................................ 146
5.2.8 Statistical analysis ..................................................................................................... 147
5.3 Results ......................................................................................................................... 147
5.3.1 Effects of treatments on neutrophil-like cell viability............................................... 147
5.3.2 Effects of treatments on apoptosis of neutrophil-like cells ....................................... 149
5.3.3 Effects of HBO on morphological changes relating to cell apoptosis ...................... 151
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