Optimisation of High frequency Jet Ventilation for the Preterm · Pressure and Frequency during High‐frequency Jet Ventilation in Preterm Lambs. Manuscript in preparation (Chapters

Optimisation of High‐frequency Jet Ventilation for the

Management of Respiratory Distress Syndrome in

Preterm Babies using a Preterm Lamb Model

Gabrielle Christine Musk

BSc BVMS Cert VA Dipl ECVAA

This thesis is presented for the degree of Doctor of Philosophy of

The University of Western Australia

School of Women’s and Infants’ Health

Faculty of Medicine and Dentistry

2011

Thesis Abstract

High‐frequency jet ventilation (HFJV) is a lung protective ventilation strategy used for

the prevention and treatment of ventilator induced lung injury in preterm infants.

Despite its widespread use in neonatal intensive care units there is little data to

support the patient management algorithms that are currently utilised. The strategies

for alveolar recruitment during HFJV rely upon incrementing positive end‐expiratory

pressure (PEEP) and delivering occasional conventional mechanical ventilator (CMV)

breaths, but the impact of these recruitment manoeuvres on pulmonary blood flow,

oxygenation, ventilation and lung injury is largely unknown. This thesis investigates the

parameters that must be selected during HFJV and their impact upon pulmonary blood

flow, physiological changes during ventilation and post mortem lung injury in a

preterm lamb model. The first study examined the effect of incrementing PEEP during

HFJV and found that alveolar recruitment was achieved by incrementing PEEP up to 12

cmH2O without detrimental effects on physiological parameters. The following 3

studies examined the delivery of CMV breaths during HFJV to compare the effect of 2

different CMV breath inspiratory times, peak inspiratory pressures and frequencies. A

shorter inspiratory time CMV breath, a CMV breath delivered to a peak inspiratory

pressure (PIP) above the HFJV breaths and CMV breaths delivered less frequently

provided the most physiological benefit with the least evidence of harm while

adequately ventilating and oxygenating the preterm lambs. The final study compared

what we determined to be optimal HFJV strategy (based upon the results of the

preceding studies) with optimal high‐frequency oscillatory ventilation and an open

lung gentle CMV strategy. In our preterm lamb model of respiratory distress syndrome

we demonstrated little difference in physiological benefit and adverse effects between

these 3 lung protective ventilation strategies.

The results of these studies contribute to the sparse data on HFJV and have provided

fundamental information that will enable a more evidence based approach to clinical decision

making in the neonatal intensive care unit. Future work in this area should focus on the target

population and incorporate randomised controlled trials comparing HFJV to other ventilatory

strategies.

Statement of Candidate Contribution

Chapters 4, 5, 6, 7 and 8 are presented in a manuscript format as they are at various

stages of the publication process. My contribution, and that of my supervisors and co‐

authors, is detailed below:

Chapter 4: High Positive End‐Expiratory Pressure during High Frequency Jet

Ventilation Improves Oxygenation and Ventilation in Preterm Lambs.

Gabrielle C Musk, Graeme R Polglase, J Bert Bunnell, Carryn J McLean, Ilias Nitsos, Yong

Song and J Jane Pillow.

I was involved in the anaesthesia of the pregnant ewe, delivery of the preterm lamb

fetus, and subsequent ventilation. I was also responsible for data collection during the

ventilation period and sample analysis. Graeme Polglase and Ilias Nitsos performed

surgical instrumentation of the fetus prior to delivery, Bert Bunnell assisted in the

ventilator management of the lamb following delivery, Carryn McLean assisted in post

mortem of the lambs and Yong Song performed the q PCR for pro‐inflammatory

cytokines. Graeme Polglase also assisted with pulmonary blood flow waveform

analyses. I was responsible for manuscript preparation. Jane Pillow was involved with

all aspects of the study in a supervisory capacity.

Chapter 5: The Impact of Conventional Breath Inspiratory Time during High‐

frequency Jet Ventilation in Preterm Lambs.

Gabrielle C Musk, Graeme R Polglase, Yong Song, and J Jane Pillow.



ventilation period and sample analysis. Graeme Polglase assisted with delivery of the

preterm lamb fetus and subsequent ventilation. Yong Song performed the q PCR for

pro‐inflammatory cytokines. I was responsible for manuscript preparation. Jane Pillow

was involved with all aspects of the study in a supervisory capacity.

Chapter 6: The Effect of Conventional Breath Peak Inspiratory Pressure during High‐

frequency Jet Ventilation in Preterm Lambs.

Gabrielle C Musk, Graeme R Polglase and J Jane Pillow.







Chapter 7: Alveolar Recruitment with Five or Twenty Conventional Mechanical

Ventilator Breaths per minute during High‐frequency Jet Ventilation in Preterm

Lambs.

Gabrielle C Musk, Graeme R Polglase and J Jane Pillow.







Chapter 8: A Comparison of High‐frequency Jet Ventilation and High‐frequency

Oscillatory Ventilation with Conventional Mechanical Ventilation in Preterm Lambs.

Gabrielle C Musk, Graeme R Polglase, J Bert Bunnell, Ilias Nitsos, David Tingay, J Jane

Pillow.



ventilation period and sample analysis. Graeme Polglase and Ilias Nitsos performed

surgical instrumentation of the fetus prior to delivery, Bert Bunnell and David Tingay

assisted in the ventilator management of the lamb following delivery and data

collection, Yong Song performed the q PCR for pro‐inflammatory cytokines. Graeme

Polglase assisted with pulmonary blood flow waveform analysis. I was responsible for

manuscript preparation. Jane Pillow was involved with all aspects of the study in a

supervisory capacity.

Jane Pillow's supervisory role extended to all aspects of the project including study

design, conduct and critical review of data analysis and written interpretation.

Graeme Polglase and Karen Simmer contributed to critical review of each manuscript.

It is not possible to have each co‐author sign this declaration, in which case, my co‐

ordinating supervisor J Jane Pillow has signed to confirm that these statements are an

accurate reflection of the contributions of each author.

Gabrielle Christine Musk

J Jane Pillow

Publications arising from this Thesis

Musk GC, Polglase GR, Bunnell JB, McLean CJ, Nitsos I, Song Y and Pillow JJ 2011 High

Positive End‐Expiratory Pressure during High‐Frequency Jet Ventilation Improves

Oxygenation and Ventilation in Preterm Lambs. Pediatric Research 69(4):319‐324

(Chapter 4).

Musk GC, Polglase GR, Song Y and Pillow JJ The Impact of Conventional Breath

Inspiratory Time during High‐frequency Jet Ventilation in Preterm Lambs. Submitted to

Neonatology April 2011 (Chapter 5).

Musk GC, Polglase GR, and Pillow JJ The Effect of Conventional Breath Peak Inspiratory

Pressure and Frequency during High‐frequency Jet Ventilation in Preterm Lambs.

Manuscript in preparation (Chapters 6 and 7).

Musk GC, Polglase GR, Bunnel JB, Nitsos I, Tingay D and Pillow JJ A Comparison of High‐

frequency Jet Ventilation and High‐frequency Oscillatory ventilation with Conventional

Mechanical Ventilation in Preterm Lambs. Manuscript in preparation (Chapter 8).

Acknowledgements

This work would not have been possible without the contribution and support of many

people. I have been fortunate to receive scientific guidance, intellectual inspiration

and moral support from my supervisors, colleagues, family and friends. Jane Pillow

had the idea for these studies and a vision for their clinical application in neonatal

intensive care units. She has been an incredible supervisor who leads by example

when it comes to being thorough, concise, articulate, scientifically watertight and

generous with her time. She’s taught me a lot and I will never forget that I am a

passive splitter who sometimes likes to use as many words as possible to simply say

something. Graeme Polglase deserves mention for his unrelenting mission to train me

out of putting 2 spaces after a full stop. If I had a dollar for every time he commented

on that I’d be mortgage free. Karen Simmer made me realise it wasn’t unreasonable to

arrive in another timezone more than 6 hours before a conference is due to start.

Thank you to you all for supervising me – in your own unique ways.

Bert Bunnell developed the ventilator at the centre of this work and is an inspiration to

me. He leads a formidable team of dedicated, enthusiastic and kind people who

mirror his ethos in life. It has been a pleasure and a privilege to be a small part of the

evolution of High‐frequency Jet Ventilation.

Being a vet, working with sheep and based in a maternity hospital took some getting

used to but I very quickly felt settled in at the School of Women’s and Infants’ Health

on the second floor of A block at King Edward Memorial Hospital. Maz Schneider,

Bevelynn Ibrahim and Catherine Arresse in particular have made me feel right at home

and I miss that feeling of family immensely. John Newnham can certainly tell a good

story and I have listened in awe to many of his. Thank you John.

The daily grind of my PhD was filled with humour and friendship thanks to Carryn

McLean, David Cruise, Richard GB Dalton and Joe Derwort. Carryn has great taste in

music (especially Mossimo Park) and necklaces, and was my most constant and reliable

moral support through the frustrating times. Cruisey looks great in undersized overalls

and bakes a mean brownie. I was just sorry that he was only really around for a year.

Richard came from Tasmania to Forrest St and after a road trip to Darkan on day 1 we

became firm friends. Joe couldn’t quite get me out of my cappuccino habit, despite his

efforts in promoting the long macchiato.

Office life was always a giggle with Chris (AKA Toots) and his Style Guide, and Irving.

Irving made sure I didn’t burn down the lab on weekends and Toots made sure that

PRONG’s purse strings were tight.

Ilias has taught me about removing endogenous peroxidase activity, the importance of

blind intubations and has been a stalwart colleague and friend. He’s gone back to

Melbourne now but there’ll never be another Nossie in my life.

My family have all helped make student life less impoverished than it could have been.

My house has become a lovely home thanks to Mum and the pruning jobs are shorter

each year thanks to Dad. My nieces fill it with fun and Harry guards it as his castle.

Harry warrants his own mention – a finer canine companion does not exist.

Lastly, it’s a big thank you to my wonderful friends (that’s you Fraser and Karen) who

have been so supportive. Thank you!

List of Abbreviations

ANOVA analysis of variance

BPD bronchopulmonary dysplasia

CMV conventional mechanical

ventilation

cmH2O centimeters of water

CO2 carbon dioxide

ΔP delta P (PIP-PEEP)

ETT endotracheal tube

FiO2 fractional inspired oxygen

concentration

HFV high-frequency ventilation

HFJV high-frequency jet

ventilation/ventilator

HFOV high-frequency oscillatory

ventilation/ventilator

h hour

Hz Hertz

IgG immunoglobulin G

IL interleukin

iNOS inducible nitric oxide

synthetase

i.v. intravenous

kg kilogram

min minute

mL milliliters

mmHg millimeters of mercury

MPO myeloperoxidase

nm nanometer

O2 oxygen

OI Oxygenation Index

Paw mean airway pressure

PaCO2 partial pressure of carbon

dioxide in arterial blood

PaO2 partial pressure of oxygen in

arterial blood

PAP pulmonary arterial blood

pressure

PBF pulmonary blood flow

PEEP positive end-expiratory

pressure

PIP peak inspiratory pressure

PmvO2 partial pressure of oxygen in

mixed venous blood

psi pounds per square inch (1psi =

70 cmH2O)

PVR pulmonary vascular resistance

RDS respiratory distress syndrome

s seconds

SABP systemic arterial blood

pressure

SD standard deviation

SEM standard error of the mean

SpO2 oxyhemoglobin saturation

measured by a pulse oximeter

tI inspiratory time

tE expiratory time

UVC unventilated control

VT tidal volume

µg microgram

1

TABLE OF CONTENTS

1 LITERATURE REVIEW .......................................................................... 5

1.1 Introduction ........................................................................................................ 5

1.2 Stages of Lung Development .............................................................................. 6

1.2.1 Strategies for Inducing Preterm Lung Maturation .................................... 10

1.3 Respiratory Consequences of Premature Birth ................................................ 12

1.3.1 Respiratory Distress Syndrome ................................................................. 12

1.3.2 Bronchopulmonary Dysplasia ................................................................... 13

1.3.3 Respiratory Outcomes of Bronchopulmonary Dysplasia .......................... 15

1.4 Respiratory Management of Preterm Babies .................................................. 16

1.4.1 Non-invasive Therapies ............................................................................. 17

1.4.2 Indications for Mechanical Ventilation ..................................................... 17

1.4.3 Adjunctive Treatments .............................................................................. 26

1.4.4 Surfactant .................................................................................................. 28

1.4.5 Postnatal Corticosteroids .......................................................................... 30

1.5 Side Effects of Positive Pressure Ventilation .................................................... 32

1.5.1 Ventilator Induced Lung Injury ................................................................. 32

1.5.2 Haemodynamic Consequences of Positive Pressure Ventilation ............. 36

1.5.3 Central Nervous System Consequences of Positive Pressure Ventilation 41

1.6 Assessment of Lung Injury ................................................................................ 44

2

1.6.1 Bronchoalveolar Lavage Fluid ................................................................... 46

1.6.2 Lung Tissue ................................................................................................ 48

1.7 Lung Protective Ventilation Strategies ............................................................. 50

2 HIGH-FREQUENCY VENTILATION ....................................................... 54

2.1 High-frequency Ventilation .............................................................................. 54

2.1.1 Gas Mixing during High-frequency Ventilation ......................................... 54

2.1.2 Mechanical Properties of the Lung and High-frequency ventilation ........ 56

2.1.3 Airway Pressure Waveforms during High-frequency Ventilation ............. 58

2.1.4 Modes of High-frequency Ventilation ...................................................... 60

2.2 Using a High-frequency Jet Ventilator ............................................................. 61

2.2.1 The Role of the Conventional Ventilator .................................................. 64

2.2.2 The Role of the High-frequency Jet Ventilator ......................................... 71

2.2.3 Monitoring during High-frequency Jet Ventilation ................................... 73

2.3 High-Frequency Jet Ventilation in the Clinical Environment ........................... 74

2.3.1 High-frequency Jet Ventilation as a Rescue Therapy ............................... 75

2.3.2 HFJV used Early in the Management of Respiratory Distress Syndrome . 76

2.3.3 Clinical Strategies ...................................................................................... 78

2.4 Summary........................................................................................................... 80

3 GENERAL METHODOLOGY ................................................................ 83

3.1 Animal Breeding and Welfare .......................................................................... 83

3

3.1.1 Nutrition .................................................................................................... 84

3.1.2 General Anaesthesia and Instrumentation ............................................... 84

3.1.3 Caesarean Delivery .................................................................................... 87

3.2 Ventilator Set-up .............................................................................................. 88

3.3 Data Collection ................................................................................................. 89

3.3.1 Pulmonary Arterial Blood Pressure and Blood Flow Measurements ....... 90

3.4 Euthanasia and Post Mortem ........................................................................... 92

3.4.1 Cell Population of Bronchoalveolar Lavage Fluid ..................................... 93

3.4.2 Bronchoalveolar Lavage Protein Assay ..................................................... 94

3.4.3 Immunohistochemistry ............................................................................. 94

3.4.4 Qualitative Polymerase Chain Reaction .................................................... 95

3.4.5 Myeloperoxidase Activity in Lung Tissue .................................................. 96

3.5 Statistical Analyses ........................................................................................... 96

3.6 References ........................................................................................................ 98

4 HIGH POSITIVE END-EXPIRATORY PRESSURE DURING HIGH-

FREQUENCY JET VENTILATION IMPROVES OXYGENATION AND

VENTILATION IN PRETERM LAMBS ................................................. 121

4

5 THE IMPACT OF CONVENTIONAL BREATH INSPIRATORY TIME

DURING HIGH-FREQUENCY JET VENTILATION IN PRETERM LAMBS 153

6 THE IMPACT OF CONVENTIONAL BREATH PEAK INSPIRATORY

PRESSURE DURING HIGH-FREQUENCY JET VENTILATION IN PRETERM

LAMBS ........................................................................................... 181

7 ALVEOLAR RECRUITMENT WITH FIVE OR TWENTY CONVENTIONAL

MECHANICAL VENTILATOR BREATHS PER MINUTE DURING HIGH-

FREQUENCY JET VENTILATION IN PRETERM LAMBS ....................... 207

8 A COMPARISON OF HIGH-FREQUENCY JET VENTILATION WITH HIGH-

FREQUENCY OSCILLATORY VENTILATION AND CONVENTIONAL

MECHANICAL VENTILATION IN PRETERM LAMBS ........................... 233

9 DISCUSSION .................................................................................... 271

10 APPENDIX ....................................................................................... 279

5

1 Literature Review

1.1 Introduction

High-frequency jet ventilation (HFJV) is a novel mode of high-frequency ventilation

offering the potential for lung protective ventilation (1, 2). In the United States of

America, HFJV is a common ventilation modality employed in the clinical setting,

especially for the management of premature babies with respiratory distress

syndrome (RDS). In Australia, only one maternity hospital has access to HJFV.

Babies can be oxygenated and ventilated with HFJV but the incidence of

haemodynamic, respiratory and neurological morbidity is largely unknown. Published

data presents conflicting information with regards to the incidence of adverse

outcomes in a clinical environment and to date there are no controlled studies

exploring the optimal settings for HFJV. Despite 25 years of clinical application, HFJV is

more often employed as a ‘rescue’ ventilation strategy rather than a first line

treatment option. It is possible that if HFJV is used earlier in the clinical course, the

outcome statistics may improve. To use this therapeutic tool to its full potential, a

thorough understanding of the pathophysiology of respiratory diseases, the clinical

indications for HFJV, the unique features of this mode of ventilation, and the

equipment required to safely and effectively apply HFJV is required. This review will

examine information in the scientific literature with relevance to each of these points.

6

1.2 Stages of Lung Development

Human lung development can be subdivided into three chronological periods: early

embryonic, fetal and postnatal (3). The fetal period is further subdivided into three

phases which describe the morphology of the airways and airspaces (pseudoglandular,

canalicular and saccular) as described in Table 1-A. The final phase of lung

development is alveolarisation which commences before birth, and continues

thereafter.

The formation of sufficient gas exchange surface area and pulmonary vasculature

capable of transporting CO2 and O2 through the lungs is vital for survival. Gas exchange

will only occur if inspired alveolar air and pulmonary arterial blood flow are contiguous

to each other and is more efficient if the surface area is greater and the membrane

between air and blood is thinner. Complete development of these components of the

respiratory unit is fundamental to normal postnatal respiratory function. Given the

developmental steps involved in maturation of the lungs, it is no surprise that there is

an inverse relationship between gestational age and the incidence of respiratory

morbidity and mortality. Infants born prior to the alveolar phase of development will

have fewer functional gas exchange units, reduced surfactant, a less compliant chest

wall and an immature pulmonary capillary network. These infants are therefore more

likely to struggle with the transition to air breathing.

7

Table 1-A Stages of prenatal and postnatal structural lung development

Phase Postconceptional Age

Length: terminal bronchiole to pleura

Structure

Embryonic 0-7 weeks <0.1 mm Budding from the foregut

Pseudoglandular 8-16 weeks 0.1 mm Airway division commences and terminal bronchioles formed

Canalicular 17-27 weeks 0.2 mm 3 generations of respiratory bronchioles; primitive saccular formation with type I and type II epithelial cells; capillarisation

Saccular 28-35 weeks 0.6 mm Transitional saccules formed; true alveoli appear

Alveolar >36 weeks 11 mm Terminal saccules formed; true alveoli appear

Postnatal 2 months 175 mm 5 generations of alveolar ducts; alveoli form with septation

Early childhood 6-7 years 400 mm Airways remodelled; alveolar sac budding occurs

Adapted from Bhutani (4).

Aside from maturation of the lung parenchyma, upper airway development is also

essential for normal gas exchange at birth. The patency of the upper airways is under

complex control and allows for conduction, humidification, warming and filtering of air

during inspiration and expiration (4). Airway development is essentially complete by

8

the end of the canalicular period of lung development, so viable preterm babies should

have sufficiently developed conducting airways.

Furthermore, clearance of fetal lung fluid must occur before normal gas exchange can

occur. Fetal lung fluid is produced during the canalicular stage of lung development

and creates a hydrostatic pressure within the respiratory system. The 3 to 5 cmH2O

exerted by the fetal lung fluid contributes to structural development of the lungs and

ensures that lung volume remains approximately equivalent to the functional residual

capacity of the fetal lung (4). Clearance of fetal lung fluid normally occurs during the

transition to air breathing at birth but if a baby is born prematurely, lung fluid may not

clear without intervention.

Recent Australian data reports the mean gestational age for all babies as 38.8 weeks

and the proportion of babies born at term (37-41 w) as 90.9 % (5). The mean

gestational age for all preterm births was 33.2 w (0.9 % of births were at 20-27 w

gestation, 0.8 % were at 28-31 w and 6.5 % were at 32-36 w) (5). In Western Australia

between 2002 and 2004, 8.4 % of births were preterm (< 37 w) (6). The highest

mortality rate occurred in the most immature babies and decreased as gestational age

increased (Table 1-B).

9

Table 1-B Birth and death statistics by gestational age, Western Australia 2002-2004

Gestational

Age (weeks)

Total births

Neonatal

deaths

Post-natal

deaths

Total

deaths

Death rate

(%)

20-27 613 80 8 88 14.4

28-32 933 21 5 26 2.8

33-36 4744 17 15 32 0.7

37-43 68705 48 66 114 0.2

<37 6290 118 28 146 2.3

Adapted from Newnham et al. (6)

Data from a Californian hospital in the 1990s described a similar pattern where mean

hospital stay was longer for infants born at an earlier gestational age (7). Survival

without BPD or retinopathy of prematurity is also strongly associated with maturation:

whereas only 35 % of infants born at 24 w survive without either of these morbidities,

this figure increases to almost 78 % at 26 w (8).

While the statistics describing the incidence of preterm birth reveal that a relatively

small proportion of babies are born at a stage prior to alveolarisation, it is these babies

that require the highest level of care. In 2005, 2.44 % of live births in Australia and

New Zealand were admitted to a neonatal intensive care unit and 91.1 % required

assisted ventilation (9). The major indication for assisted ventilation in babies born

less than 32 w was RDS (72.4 %) and the duration of ventilation increased with

10

decreasing gestational age. From this particular cohort of babies, the median length of

stay in hospital (the survivors) was 133 d if born at 23 w, 118 d at 24 w, and 106 d at 25

w. The length of stay steadily decreased as gestational age increased (9).

1.2.1 Strategies for Inducing Preterm Lung Maturation

Accelerated maturation of the lungs and the impact on the incidence and severity of

BPD have been widely investigated. Exposure to glucocorticoids, chorioamnionitis and

fetal stress in utero all contribute to decreasing the incidence of RDS and therefore

BPD (10, 11).

1.2.1.1 Antenatal Corticosteroids

Clinical and experimental observations of antenatal corticosteroid treatment

demonstrate that endogenous and exogenous glucocorticoids function primarily to

accelerate lung maturation by thinning alveolar walls and increasing lung gas volume

(8, 11). This functional maturation of the preterm fetal lung has led to the

administration of corticosteroids to women at risk of preterm delivery becoming

established therapy (10, 12, 13).

The optimal dose and timing of corticosteroid treatment should aim to stimulate

generalised lung maturation and surfactant synthesis. Human and animal studies have

been performed to investigate the effects of antenatal steroid administration and help

determine when to administer corticosteroids and how much to administer (12, 14-

16). Improvement in lung function and increases in surfactant production both

contribute to improved outcomes in preterm babies whose mothers have received

antenatal corticosteroids (12).

11

1.2.1.2 Chorioamnionitis

Intrauterine inflammation is associated with a reduced risk of RDS in some preterm

babies (17, 18). This realisation has prompted a vast number of animal studies

examining the impact of induced chorioamnionitis on lung maturation and surfactant

production (19-21) and evidence suggests that fetal lung inflammation causes marked

improvements in preterm lung function. These improvements are primarily caused by

increases in pulmonary surfactant but remodelling of the lung parenchyma also occurs

(20). Whether or not chorioamnionitis and corticosteroid therapy work together to

reduce the risk of RDS to an extent greater than either alone is unknown.

Most cases of chorioamnionitis in preterm and term labour are sub-clinical (22) which

makes diagnosis difficult. The most common organism identified is Ureaplasma

urealyticum (22, 23) and a number of animal studies have documented lung

maturation associated with this infection (21). Colonisation of the chorioamnion incites

a fetal inflammatory response which targets a range of organs and tissues in the fetus,

including the lungs (22). Injury subsequent to infection develops and structural

changes to the lungs occur. These changes may in fact be beneficial, at least in the

short term.

1.2.1.3 Fetal stress

The short and long term effects of fetal stress may be both beneficial and adverse. The

beneficial effects result from accelerated organ and tissue maturation which improve

the capacity of a patient to cope with preterm birth (24). This ability to cope may be

interpreted as fetal adaptation to unfavourable circumstances and is likely to be

12

associated with increased circulating cortisol and inflammatory mediators (24). The

manifestations of antenatal glucocorticoid therapy, chorioamnionitis and fetal stress

are comparable, albeit complex, and the balance between the beneficial and adverse

effects will vary according to gestational age and the presence of other pathology (24).

Nevertheless, fetal stress causes an increase in circulating cortisol and the release of

pro-inflammatory mediators which impact upon a number of organs, including the

lungs, to hasten maturation.

1.3 Respiratory Consequences of Premature Birth

1.3.1 Respiratory Distress Syndrome

Respiratory distress syndrome occurs as a result of both structural immaturity and

surfactant deficiency of the premature lung. The symptoms of RDS include

tachypnoea, chest wall retraction, cyanosis and characteristic radiological

abnormalities of the lungs. Clinical management invariably includes oxygen therapy

with or without ventilator support: 96 % of infants born at less than 28 w gestation in

Australasia in 2002, received supplemental oxygen (25). These extremely preterm

infants have a higher incidence of RDS and are hospitalised for longer compared to

those born after 28 w gestation (26). A range of ventilation techniques have been used

to support these preterm infants but mechanical ventilation has itself been implicated

as a cause of lung injury. Further, the combination of immature lung parenchyma and

lung injury may predispose the baby to bronchopulmonary dysplasia (BPD) (27).

13

1.3.2 Bronchopulmonary Dysplasia

Bronchopulmonary dysplasia (BPD) was first defined in 1967 by Northway et al (28) to

describe the clinical, radiological and pathological changes in infants with severe RDS.

The definition applied to babies who had been treated with prolonged mechanical

ventilation and warmed, humidified 80-100 % inspired oxygen concentrations. The

term BPD was coined to emphasise the involvement of all tissues of the developing

lung in the pathological process and represented the toxic manifestations of high

oxygen concentrations and mechanical ventilation on the developing lung

superimposed upon the healing phase of RDS. In 1968 a radiographic description was

published highlighting the distinctive features of this phenomenon (29). The

radiographic features were staged with grade 4 representing the worst case scenario:

symptomatic chronic lung disease with strands of pulmonary parenchymal density;

increased thoracic volume; and cardiomegaly which may gradually clear or progress to

cor pulmonale and death at less than 1 month of age (29). These two publications

marked the beginning of an era.

Despite advances in antenatal glucocorticoid therapy, surfactant treatment and gentle

‘lung protective’ ventilation techniques, the incidence of BPD is increasing (30),

prompting further investigation into its multifactorial pathophysiology. In 2001 a new

NIH consensus definition of BPD was proposed by Jobe and Bancalari (27) to include

categorisation of the severity of BPD (Table 1-C).

14

Table 1-C Diagnostic criteria for defining bronchopulmonary dysplasia

Gestational

Age

<32 weeks ≥ 32 weeks

Time point of

assessment

36 w PMA or discharge to home,

whichever comes first.

>28 d but < 56 d postnatal age or

discharge to home, whichever

comes first.

Treatment with >21 % oxygen for at least 28 d plus.

Mild BPD Breathing room air at 36 w PMA

or discharge, whichever comes

first.

Breathing room air by 56 d

postnatal age or discharge,


Moderate BPD Need* for < 30 % oxygen at 36 w

PMA or discharge, whichever

comes first.

Need* for < 30 % oxygen at 56 d

postnatal age or discharge,


Severe BPD Need* for ≥ 30 % oxygen and/or

positive pressure, (PPV or

NCPAP) at 36 w PMA or

discharge, whichever comes first.

Need* for ≥ 30 % oxygen and/or

positive pressure (PPV or

NCPAP) at 56 d postnatal age or

discharge, whichever comes

first.

Adapted from Jobe and Bancalari (27). BPD = bronchopulmonary dysplasia, NCPAP = nasal continuous

positive airway pressure, PMA = postmenstrual age; PPV = positive pressure ventilation. * A

physiological test confirming that the oxygen requirement at the assessment time point remains to be

defined. This assessment may include a pulse oximetry saturation range.

15

As mechanical ventilation may contribute to the development of an inflammatory

response resulting in altered alveolar and pulmonary vasculature development (27),

the identification of ventilatory strategies that minimise this damage and prevent the

progression of BPD is an important research goal.

1.3.3 Respiratory Outcomes of Bronchopulmonary Dysplasia

In the last 30 years, the chances of surviving the neonatal period as a premature baby

have improved (31). The introduction and evolution of assisted ventilation, surfactant

therapy and antenatal steroid administration are primarily responsible for this

progress (32). Improved survival of extremely low gestational age infants is frequently

followed by significant long term respiratory morbidity and has fiscal implications. The

cost of initial care for this population of neonates in the United States is estimated at

US$10.2 billion each year. Infants born between 24 and 26 weeks gestation account for

11.9 % of this bill (33).

There are limited reports of the long term respiratory complications of preterm birth in

the contemporary era. The survivors of extremely preterm birth (prior to 26 weeks

gestation) are only just reaching adulthood so data are limited to patients less than 20

years of age (34-36). Furthermore, the cohort of patients is small. Despite these

limitations, there is evidence that lung function is reduced as a result of surviving BPD

(36).

Earlier studies present data on patients of a similar age group (gestational age and age

in adulthood at the time of data collection) but from a different era. These patients

16

were born before the routine administration of antenatal corticosteroid therapy and

exogenous surfactant which makes comparisons inappropriate. The definitions of BPD,

the birth weight of the patients and the respiratory parameters that were assessed

vary. The data does however reflect, once again, that in adulthood, the survivors of

preterm birth have some degree of decreased lung function (37, 38).

Overall it appears that a significant number of adult survivors of moderate and severe

BPD may be left with residual functional and characteristic structural pulmonary

abnormalities. The limitation of these studies is a failure to demonstrate a correlation

between perinatal variables and the manifestations in adulthood. This lack of

correlation makes prognostication difficult and emphasises the importance of

exploring management strategies for these preterm babies that will minimise injury to

the preterm lung.

1.4 Respiratory Management of Preterm Babies

The management of preterm babies with RDS aims to reduce the incidence and

severity of BPD. This management goal is challenging given the complexity of the

disease process and the precarious balance between risk and benefit associated with

any therapy. The general treatment goals are to minimise ventilator induced lung

injury, minimise the inspired oxygen concentration, avoid infection and manage

nutritional and fluid requirements (39). Respiratory management of preterm babies

may be non-invasive (e.g. nasal continuous airway pressure (CPAP)) or invasive

(requiring endotracheal intubation). Invasive techniques range from supported

ventilation by CPAP to intermittent positive pressure ventilation (IPPV).

17

1.4.1 Non-invasive Therapies

Endotracheal intubation and the prophylactic administration of surfactant is effective

for the management of RDS in preterm infants (40). A comparison of early versus later

administration of surfactant has demonstrated reduced mortality, frequency of BPD,

and the risk of pneumothorax in ventilated preterm infants with RDS (41).

Endotracheal intubation however, is itself associated with considerable risk and if it

can be avoided, so will the associated complications. Non-invasive respiratory support

may therefore be considered for some infants. This form of respiratory support is a

continuous distending pressure, or CPAP. Continuous positive airway pressure is

applied using a conventional ventilator, bubble circuit or CPAP driver via a face mask,

nasopharyngeal tube, or nasal prongs (40). A Cochrane review published in 2002 found

that these methods of respiratory support reduced the rate of death or the need for

assisted ventilation and reduced the need for IPPV in preterm babies that were able to

breathe spontaneously (40). Subsequent randomised trials have failed to confirm that

CPAP is superior to intubation and mechanical ventilation when outcomes such as BPD

and death are considered (42, 43).

1.4.2 Indications for Mechanical Ventilation

Mechanical ventilation is indicated for any patient unable to breathe spontaneously

and achieve adequate gas exchange. Preterm babies have underdeveloped lungs and

respiratory muscles, insufficient endogenous surfactant production and secretion and

a highly compliant chest wall. Not surprisingly, they may require sophisticated

respiratory support in the form of mechanical ventilation via an endotracheal tube.

18

1.4.2.1 Goals of Mechanical Ventilation

The aims of any ventilation strategy include alveolar recruitment and stabilisation, the

acquisition and maintenance of appropriate arterial blood gas parameters and

minimising the impact of increased intra-thoracic pressure on cardiac output and lung

injury. Achieving just one of these aims may come at the expense of another so it is

essential to strike a balance between oxygenation, CO2 removal and the implications of

higher than normal airway pressures.

1.4.2.1.1 Oxygenation

Hypoxaemia is defined by either a low PaO2, low peripheral or arterial oxyhaemoglobin

saturation (SpO2 or SaO2) or low oxygen content of arterial blood (CaO2). Regardless of

the descriptor, hypoxaemia may result from hypoventilation, inspiration of a hypoxic

gas mixture and venous admixture. Venous admixture refers to the passage of blood

through the pulmonary circulation without oxygenation and occurs in the presence of

ventilation and perfusion mismatch, right to left shunting of blood and diffusion

impairment. Prevention and treatment of hypoxaemia should therefore be targeted to

decrease the impact of each of these factors.

Airway pressure

Airway pressure plays an important role in oxygenation insofar as preventing alveolar

collapse and maintaining an open lung. Lung volume recruitment to reduce shunting of

blood through the lungs is an established concept: in 1959 Mead and Collier showed

that periodic lung inflations were necessary to prevent a progressive fall in compliance

during mechanical ventilation (44). Lung volume recruitment is possible if the lung is

19

inflated beyond the pressure at which atelectatic lung begins to open, and is

maintained at a pressure above its closing pressure (45). In immature or injured lungs

where compliance is poor, these pressures may be high and the detrimental effects of

increased airway pressure must be considered. The balance between sufficient airway

pressure to prevent alveolar collapse and conservative enough airway pressure to

prevent alveolar overdistention may be difficult to achieve. Furthermore, the

haemodynamic implications must also be closely monitored and managed.

Inspired gas mixture

In preterm infants, oxygen supplementation is a balancing act between the treatment

of tissue hypoxia, pulmonary vasoconstriction, and patency of the ductus arteriosus

with lung injury, retinal damage and oxidative stress. Oxygen has therapeutic and toxic

characteristics and optimal oxygenation, especially in the first few weeks of life is often

under-emphasised. Hypoxanthine accumulates during hypoxia, and during re-

oxygenation, superoxide radicals are produced, leading to cell injury (46). Optimal

oxygen concentrations for resuscitation were reviewed recently and it is

recommended that babies of gestational age greater than 32 weeks should be

ventilated initially with 21 % oxygen. Babies of gestational age less than 32 weeks

should be ventilated initially with 21-30 % oxygen (47). Changes in SpO2 and heart rate

are often used to monitor oxygenation, especially in the acute period following

delivery and alterations to the fractional inspired oxygen concentration (FiO2) should

be made to target a SpO2 range. The consequences of various SpO2 targets have been

investigated and while there may be fewer complications in neonates that have been

20

managed to achieve a lower target range (48), the mortality rate must also be

considered. The current recommendation is a target SpO2 of 91-95% (49).

Automated oxygen delivery is an evolving technology that automatically adjusts the

FiO2 according to feedback information from a pulse oximeter. Preliminary studies

under routine clinical conditions showed this system increased the time in which SpO2

was maintained within a target range, decreased FiO2 and decreased the incidence of

hyperoxaemia without increasing severe hypoxaemia (50). This method of oxygen

delivery may be a useful tool to reduce exposure to hyperoxaemia and hypoxaemia

which may contribute to the development of BPD. Furthermore, this study is pertinent

as the optimal SpO2 range for the extremely preterm or very low birthweight infant

remains elusive (49, 51). There is, however, convincing evidence that there is an

association between oxidative stress and BPD (52).

While hypoxaemia usually stimulates more concern, judicious use of oxygen and

continuous, or at least regular, monitoring of oxygenation to minimise exposure to

high concentrations of oxygen is important. The optimal SpO2 is not known in

premature infants: 2008 data indicated lower mortality if SpO2 was kept below 93 %

and stable (53). More recent data demonstrates increased mortality in a lower SpO2

range (85-89 %) when compared to a higher SpO2 range (91-95%) (49). The incidence

of retinopathy of prematurity was lower in survivors from the lower SpO2 range group

but the higher mortality in this group raises question over the appropriate target range

for SpO2. Further investigations are currently being undertaken to determine the safest

SpO2 target for preterm infants. Results from the Neonatal Oxygenation Prospective

Meta-analysis Collaboration are expected in 2014 (54).

21

1.4.2.1.2 Removal of Carbon Dioxide

Normal cellular metabolism relies upon oxygen supply exceeding oxygen demand and

normal pH. The partial pressure of carbon dioxide in arterial blood (PaCO2) is directly

related to pH and both are maintained within a narrow range. Normal PaCO2 is 35 – 45

mmHg (4.6-5.5 kPa) and normal pH is 7.35 – 7.45.

Carbon dioxide production is a product of the carbonic anhydrase equation:

Equation 1-A

H+ + HCO3- ↔ H2CO3 ↔ H2O + CO2

Carbon dioxide is a byproduct of cellular metabolism and is eliminated by the kidneys

or the lungs. PaCO2 is a balance between carbon dioxide production and elimination.

During conventional mechanical ventilation (CMV), CO2 removal is directly related to

minute volume:

Equation 1-B

V’CO2 α V’min = fx VT

where V’CO2 = rate of carbon dioxide elimination; V’min = minute volume; f = ventilator

frequency or respiratory rate; and VT = tidal volume (alveolar).

A decrease in V’min due to either a decrease in f, VT, or both will decrease the

elimination of carbon dioxide (potentially causing hypercapnia). Conversely, an

increase in V’min accelerates CO2 elimination (potentially causing hypocapnia).

Equipment and physiological dead space will impact upon VT and therefore V’CO2.

22

During high-frequency ventilation, however, CO2 removal is more closely linked to the

VT:

Equation 1-C

V’CO2 α V’min = f x VT2 (55)

The side effects of abnormal CO2 levels are dose and time dependent. However, in

general, hypercapnia leads to cerebral vasodilation, an increase in intracranial

pressure, splanchnic vasodilation and hypotension. Hypocapnia causes cerebral and

splanchnic vasoconstriction and may compromise perfusion of important organs.

During growth and development, the effects of alterations in CO2 are potentially more

profound. Hypocapnia is associated with an increased risk of periventricular

leukomalacia in low birth weight infants (56, 57) while hypercapnia is a predictor of

severe intraventricular haemorrhage in very low birth weight infants (58).

Furthermore, fluctuations in PaCO2 are associated with worse neurodevelopmental

outcomes in extremely low birthweight infants (59).

Permissive hypercapnia refers to an acceptance of PaCO2 levels higher than the normal

range (usually ~ 45-55 mmHg) and has been promoted as a strategy to reduce cyclic

volutrauma and barotrauma associated with mechanical ventilation. Other beneficial

effects include maintenance of cardiovascular performance secondary to hypercapnic

stimulation of the sympathetic nervous system. Despite these potential benefits of

permissive hypercapnia, the impact upon neurological development must be

considered.

23

1.4.2.1.3 Minimising Iatrogenic Injury

Managing preterm babies with RDS is challenging and the outcome data demonstrates

that adverse effects may have implications into adulthood. Minimising iatrogenic injury

is essential to ensure the risk/benefit balance is in favour of the latter. The main risk

factors are IPPV, the administration of supplemental oxygen, the presence of a patent

ductus arteriosus and infection (chorioamnionitis, sepsis, pneumonia, meningitis) (39).

The main therapeutic options which may be associated with adverse side effects are

exogenous surfactant, caffeine, vitamin A, late corticosteroids, IPPV, oxygen, nutrition,

fluid therapy and prophylactic antibiosis (39). The interplay between these factors is

complex and the balance may be precarious.

1.4.2.2 Types of Ventilators

There are 3 main modes of ventilation employed in the management of RDS in

preterm babies. These modes are considered ‘gentle’ if used appropriately in these

patients with immature lungs that are poorly compliant and lacking in surfactant. The

decision making process regarding choice of ventilation strategy should be driven by

the individual patient’s pathophysiology but may also be influenced by availability of

equipment and past experience. Conventional mechanical ventilation (CMV) utilising

high respiratory frequencies and small tidal volumes, high-frequency oscillatory

ventilation (HFOV) and high-frequency jet ventilation (HFJV) have become established

as the ventilator modes most appropriate for the management of RDS in preterm

babies.

24

1.4.2.2.1 Conventional Mechanical Ventilation

There are a number of conventional mechanical ventilators available for use in a

neonatal intensive care unit (NICU). They can be broadly classified according to

whether or not they cycle between breaths when a set pressure or VT has been

delivered. These ventilators have a multitude of settings which include, but are not

limited to: time-cycled pressure-limited ventilation; flow-cycled pressure-limited

ventilation; pressure controlled ventilation; pressure support ventilation; volume

controlled ventilation; volume guarantee; pressure regulated volume control; volume

assured pressure support; synchronised, or patient triggered, ventilation; and assist

control (60). In the context of RDS these ventilators can be used to deliver gas which is

involved in gas exchange by bulk convection and diffusion.

1.4.2.2.2 High-frequency oscillatory ventilation

High-frequency oscillatory ventilation (HFOV) is a form of high-frequency ventilation

where gas is delivered at a rapid rate, with a tidal volume usually less than dead space

volume (61). HFOV generates individual breaths with either a piston and diaphragm

that is electromagnetically driven, a rotating ball and reverse jet, or a vibrating

diaphragm. However the oscillation is generated, both the exhalation and inhalation

phases are active (55). Adjustments to HFOV frequency and breath amplitude (∆P)

alter minute volume while changes to PEEP alter mean airway pressure (Paw), which in

combination with FiO2, determines oxygenation.

High-frequency oscillatory ventilation has increasingly been utilised in the clinical

management of preterm babies with RDS and has evolved over the last 3 decades.

25

Theoretical knowledge and clinical experience led to the recognition by Bryan and

Froese that using higher mean airway pressures than those measured during CMV is

essential to optimise oxygenation (45). Strategies aimed at achieving higher lung

volumes resulted in decreased FiO2 requirements, improved pulmonary mechanics and

less structural injury (62-64). The use of a high volume approach in clinical trials was

shown to be safe and effective and substantially decreased the incidence of BPD (65-

67) compared to earlier trials in which a high volume strategy was not protocolised.

The high volume strategy prioritises alveolar expansion over airway pressure and

creates mean airways pressures higher than those created during CMV (64, 68). Airway

pressure is only decreased after FiO2 is < 0.3 and when decreasing Paw does not

increase the FiO2 requirement. This link between higher Paw and improved

oxygenation formed the basis for studies focused on the high lung volume strategy.

The mechanisms of gas exchange and lung mechanics relevant to HFOV will be

discussed in Chapter 2.

1.4.2.2.3 High-frequency jet ventilation

High-frequency jet ventilation is also a form of high-frequency ventilation that has

been extensively utilised in NICUs. Individual breaths are generated by a flow-

interrupting pinch valve which is located outside the ventilator itself, and close to the

tracheal tube. A detailed description of HFJV and the ways it differs from HFOV will be

presented in Chapter 2.

26

1.4.3 Adjunctive Treatments

Management of preterm babies goes beyond ventilatory support: a multimodal

approach to their care is essential to minimise morbidity and mortality. Respiratory

support is just one aspect of treatment which includes adequate nutrition, appropriate

fluid therapy and pharmacological intervention (39). The overall aims are to limit lung

injury, avoid infection, provide optimal nutrition and carefully control fluid balance.

Caffeine is administered to stimulate spontaneous breathing and seems to be safe and

effective for the prevention of BPD and neurodevelopmental delays. A large

randomised controlled trial published in 2007 concluded that caffeine therapy for

apnoea of prematurity improved the rate of survival without neurodevelopmental

disability at 18 to 21 months in infants with very low birth weight (69). A recent study

assessing children at the age of 5 years, however, concluded that caffeine therapy as a

very low birth weight neonate was not associated with an improved rate of survival

without disability (70).

A number of studies demonstrate that vitamin A intake is inadequate in extremely low-

birthweight infants and that supplementation of vitamin A may reduce the risk of

chronic lung disease (71, 72). Vitamin A deficiency may promote chronic lung disease

by impairing lung healing, increasing the loss of cilia, increasing squamous-cell

metaplasia, increasing susceptibility to infection, and decreasing the number of alveoli

(73). While the short-term benefits of vitamin A supplementation are promising, the

long-term benefits are unclear (72) and as for caffeine treatment, the clinician must

always consider the risk and benefit of such treatment (39).

27

The administration of postnatal corticosteroids has been extensively investigated.

Halliday et al (2009) identified 47 randomised controlled trials of postnatal systemic

corticosteroid administration for the prevention of BPD (74, 75) and found a decreased

risk of death or BPD at 28 d and 36 w post menstrual age when corticosteroids were

used in the first week of life. The adverse effects that were identified included

gastrointestinal bleeding and intestinal perforation, hyperglycaemia, hypertension,

hypertrophic cardiomyopathy, growth failure, developmental delay, cerebral palsy and

abnormal neurological examination results (75). When corticosteroids were

administered later (> 7 days) the risk of BPD and death or BPD was reduced at both 28

d and 36 w (74). Once again, adverse effects included hyperglycaemia, hypertension,

infection and cerebral palsy (if the initial risk of BPD was low). Given these data it has

been suggested that systemic corticosteroids given after 7 d of life should be limited to

infants who cannot be weaned from mechanical ventilation. Furthermore, the dose

and duration of any such course should be minimised (39).

Diuretics have also been administered to preterm infants in an effort to improve lung

compliance. There are no known long term benefits from these therapies so while they

may be appropriate for infants with poor lung compliance or those who are difficult to

wean from a ventilator, it is prudent to use them transiently (39).

Nitric oxide (NO) is a selective pulmonary vasodilator and has been shown to improve

gas exchange and lung maturation in animal models (76-78). Nitric oxide is usually

delivered via the airways in an inhaled form into the inspiratory limb of the breathing

system. In a study investigating 3 doses of NO in term babies it was concluded that a

higher dose (80 ppm) did not seem to have any advantages over either 5 or 20 ppm

28

and caused an increase in methaemoglobin and NO2 levels (79). Its use for preterm

infants, however, has increased as the evidence suggests improved

neurodevelopmental and respiratory outcomes following treatment with inhaled NO

(80-83).

1.4.4 Surfactant

Surfactant is critical for lowering surface tension in the alveoli and prevents alveolar

collapse. It is usually produced by type II pneumocytes in time to promote alveolar

stability at term birth (84, 85). In utero the fetal lung is full of fluid which must be

cleared to enable the transition to air breathing at birth. The amount of fluid in the

lung after birth will be determined by the efficacy of fluid clearance mechanisms, the

length of labour, and whether or not delivery is vaginal or via caesarean section (84).

Delayed clearance of lung fluid will increase the potential for hypoxaemia and

hypercapnia. Furthermore the composition of the fluid also impacts upon the

transition to air breathing as fluid without surfactant, and therefore a high surface

tension, increases the risk of small airway obstruction and the pressure required to

open these airways (84).

In adult animal and human lungs, surfactant is a fluid composed of lipids and proteins,

the action of which is to lower the surface tension of an open air-water interface. It is

the saturated phosphatidylcholine species and surfactant protein B and C components

that give surfactant this unique property (85). Preterm infants with RDS have low levels

of surfactant that is deficient in these lipids and proteins. This deficiency promotes

alveolar collapse, instead of expansion, which contributes to the requirement for

mechanical ventilation.

29

The administration of exogenous surfactant soon after birth has become routine in the

clinical management of neonates with RDS and has yielded significant improvements in

morbidity and mortality (85-87). The most common method of administration is direct

intratracheal instillation (88) and the response to treatment has been described in

three phases: acute, lasting minutes; a more prolonged phase lasting hours; and

chronic, with effects lasting days or even weeks (85). The magnitude of the acute

response depends upon the biophysical properties of the surfactant and the extent of

distribution throughout the lung. Uniform distribution is ideal and whether or not it is

achieved is governed by surface activity, volume of surfactant, gravity, speed of

delivery of surfactant, ventilator settings and the presence of lung fluid within the lung

(85). Attention to these factors will optimise the delivery and efficacy of exogenous

surfactant.

Preterm lungs ventilated without surfactant rapidly develop epithelial disruptions in

the airways and pulmonary oedema as proteins from the vascular space enter the

airspaces (89). Oedema fluid will adversely affect lung mechanics and gas exchange

(90) and potentiate VILI. If surfactant is administered prior to the initiation of

mechanical ventilation it facilitates the movement of fluid out of the small airways and

into the saccules and interstitium, effectively increasing total lung capacity and

decreasing the inflation pressures required to deliver gas volume to the lung (91).

Surfactant is most often delivered together with mechanical ventilation. Ventilatory

mode may influence the distribution of surfactant to the lungs and the clinical

response of the patient. A rabbit study investigating the response to surfactant and

either CMV or HFJV following saline lavage induced lung injury demonstrated

30

improved gas exchange and reduced pulmonary right-to-left shunt in the animals in

which surfactant had been administered prior to HFJV (92). These results support the

hypothesis that the response to surfactant treatment in acute lung injury depends on

the mode of ventilation utilised after surfactant delivery. HFJV may facilitate the

delivery of surfactant to the distal alveoli, decrease alveolar dead space, support open

alveoli and improve gas exchange. This theory is not supported by any data and

warrants investigation in a controlled setting.

A pilot study of 28 newborn infants with RDS, meconium aspiration syndrome or

pneumonia who deteriorated in spite of optimal CMV and exogenous surfactant

therapy were treated with HFJV and continued surfactant therapy (93). The patients

that met the criteria for treatment with HFJV and additional surfactant showed

significant and sustained improvement in several respiratory variables. These results

suggested that the combination of HFJV and exogenous surfactant may be effective in

treating infants with more severe respiratory failure (93). Unfortunately this work has

not progressed and it remains that further investigation into the impact of specific

ventilation methods and strategies on the delivery, distribution and efficacy of

exogenous surfactants is required to optimise this therapy (88).

1.4.5 Postnatal Corticosteroids

The administration of corticosteroids to women at risk of preterm delivery induces

functional maturation of preterm fetal lungs (11) and has become established therapy

to improve the outcome for premature infants (12). The administration of postnatal

corticosteroids however is less encouraging. A recent review of several studies focused

on the risk/benefit balance of postnatal corticosteroids administered to premature

31

babies for prevention and treatment of BPD highlights the risk of long-term adverse

neurodevelopmental outcomes (94). Their recommendations are that systemic

administration of corticosteroids for prevention or treatment of BPD: (i) should not be

used during the first 4 days of life; (ii) is not indicated in the first 3 weeks of life nor (iii)

in extubated infants (nasal ventilation or oxygen therapy) (94). Furthermore, these

authors advise that the systemic administration of steroids should only be considered

after the first 3 weeks of life in very preterm infants to facilitate extubation. In this

scenario corticosteroids may help wean the infant from the ventilator and decrease

the requirement for oxygen supplementation. The justification for these suggestions is

based upon potential unfavourable neurocognitive outcomes associated with the use

of postnatal steroids (94). A policy statement from the American Academy of Pediatrics

was released in 2010 and is in a similar vein, highlighting the deficiency of data to

support the use of corticosteroids in the post natal period without careful

consideration of the potential risks. The Academy did, however, recommend that

consideration of a course of corticosteroids be given if the patient is ventilator-

dependent at 1-2 weeks of age.

The debate regarding the administration of postnatal steroids is not over: in light of

Australian data it appears that low-dose dexamethasone treatment after the first week

of life facilitates extubation thereby decreasing the days of intubation of ventilator

dependent very preterm and extremely low birth weight infants (95). Doyle et al

(2006) reached these conclusions after a randomised controlled trail of either

dexamethasone or saline was administered to 70 infants recruited from 11 centres at a

median age of 23 days. A 2 year follow up on these patients revealed that there was no

32

difference in the incidence of mortality or major disability at 2 years of age between

the treatment and the saline groups (96). Until further data are available it remains

that while dexamethasone administered after the first week of life may have short

term benefits, the long term implications are unknown.

1.5 Side Effects of Positive Pressure Ventilation

Positive pressure ventilation disrupts normal physiological processes and may interfere

with lung structure and cardiac output, and therefore pulmonary and systemic blood

flow. Furthermore, if blood flow to the brain is compromised and cerebral oxygen

delivery does not meet cerebral oxygen demand normal neurological development

may not occur. The side effects of IPPV will be divided into those effects pertaining to

the respiratory (ventilator induced lung injury), haemodynamic (pulmonary blood flow,

persistent pulmonary hypertension of the neonate and systemic blood flow) and

neurological systems.

1.5.1 Ventilator Induced Lung Injury

While mechanical ventilation has decreased mortality rates in neonates since its

introduction in the 1960s, at the same time it has created a new set of potentially life

threatening conditions for these patients (97). Ventilator induced lung injury is

considered an important risk factor for the development of BPD from barotrauma,

atelectatrauma, volutrauma and biotrauma (98, 99) and may have long term

implications for the patient. In preterm infants VILI may be attributed to a plethora of

mechanical ventilation strategies (65, 98, 100). The factors that contribute to the

deleterious effects of mechanical ventilation include the applied airway pressure, the

33

volumes changes associated with that airway pressure, the size of the patient, the

duration of ventilation, whether or not the thorax is open or closed and the presence

of pre-existing respiratory disease (98).

Barotrauma causes the leakage of air due to disruption of the airspace wall (98). The

term is used to describe the impact of pressure related dysfunction resulting from

exposure to persistent, elevated pressures (99). Alveolar distension during IPPV causes

damage to the pulmonary epithelial-endothelial barrier which allows air to shift into

the pulmonary interstitium (101). The consequences of this damage includes:

interstitial airleak; pneumothorax; subcutaneous emphysema; pneumomediastinum;

pneumoperitoneum and pneumopericardium (102). Furthermore, a complex cascade

of physiological events occurs including fluid and protein shifts across the blood-air

interface resulting in a high permeability pulmonary oedema (103). Gas exchange

becomes compromised as ventilation and perfusion (V/Q) mismatch develops and lung

compliance decreases. The mechanical stress associated with barotrauma may activate

humoral and cellular immunological responses leading to the release of pro-

inflammatory mediators and neutrophils (99). Fibrin is then deposited in the alveolar

membrane, increasing V/Q mismatch and decreasing compliance further.

Atelectatrauma refers to lung injury associated with atelectasis and the consequent

shear forces associated with repetitive opening of collapsed alveoli. This creates V/Q

mismatch and increases the inflation pressures required to recruit those areas of the

lung (91). According to LaPlace’s law, the pressure required to inflate a lung unit

depends on the initial radius of that unit. To achieve a certain volume change in larger

alveoli, the necessary pressure changes are much smaller compared to alveoli which

34

are collapsed or have a lower volume. It follows that the pressure needed to keep

alveoli open is lower at a higher functional residual capacity. The critical opening

pressure of a lung unit is therefore inversely proportional to the size of that unit so

higher pressures are required to recruit collapsed lung. The delivery of higher

pressures is potentially dangerous as these pressures may be transmitted to the

normal areas of lung, opening alveoli, promoting stretch, and causing over distension.

Furthermore, the opening of those collapsed units imposes large shear forces which

promotes alveolar disruption (91). Surfactant helps prevent alveolar collapse by

decreasing the surface tension of distal alveoli. The surface area of the alveolus will

however affect the efficacy of surfactant, especially if the surface area is smaller than

the total surface of surfactant molecules. The surfactant molecules will be squeezed

off the alveolar surface towards the alveoli and become inactive. When that lung unit

is inflated again, the surface is replenished with surfactant molecules, a proportion of

which will be ineffectual (104). Opening the lung, and keeping the lung open is the best

strategy for minimising atelectatrauma.

Volutrauma refers to lung injury associated with the static overdistention of the lung

as well as the cyclical delivery of high tidal volumes. By definition, it is independent of

the peak inspiratory pressure required for tidal volume delivery (98, 105). Volutrauma

can occur with only brief exposures to large tidal volumes (106) including as few as 6

consecutive large tidal volume breaths associated with the initiation of ventilation

after birth (107). Thus volutrauma is a risk for the preterm baby receiving IPPV during

postnatal resuscitation if tidal volumes are not monitored. Volutrauma not only causes

acute changes, but increases the injury associated with subsequent IPPV (106). At a

35

microscopic and a molecular level, volutrauma leads to a plethora of abnormalities:

alveolar epithelial cell damage; alveolar protein leakage; altered lymphatic flow;

hyaline membrane formation; inflammatory cell influx; decreased lung compliance and

altered surfactant structure and function (106, 108, 109).

While efforts should be made to minimise volutrauma, there are sparse data in the

literature documenting the effect of predetermined tidal volumes during IPPV. One

prospective randomised controlled trial in preterm infants compared 3 mL kg-1 and 5

mL kg-1 tidal volumes delivered during volume guarantee ventilation. The authors

hypothesised that the lower tidal volume breaths would be associated with less

inflammation and less BPD (110). Their results found the converse, that the lower tidal

volume breaths were associated with greater expression of inflammatory markers,

likely due to progressive atelectasis with the delivery of small tidal volumes in the

absence of an accompanying strategy to optimise distending lung volume. This study

highlights the importance of delivering a targeted ventilation strategy: adequate PEEP

should be used to prevent atelectatrauma and optimal tidal volumes should be used to

prevent volutrauma.

Biotrauma refers to lung injury associated with the release of pro-inflammatory

mediators. This occurs in addition to the lung parenchymal and airway epithelial

changes previously described and results in an increased concentration of cytokines in

the lung tissue, which in turn incites an inflammatory response (102, 111, 112).

Quantification of these pro-inflammatory mediators is particularly useful when

assessing lung injury in an experimental setting as it provides information about the

potential systemic effects of IPPV.

36

Barotrauma, atelectatrauma, volutrauma and biotrauma have been documented in

experimental studies in animals (98, 102, 111, 113) and while they may not be easy to

identify early in the pathogenesis of VILI in a clinical setting they are likely to occur to

some degree in response to mechanical ventilation. Controlled animal studies looking

directly at HFJV are scarce, but a study presented in 1990 was a turning point as it

reported less lung injury in a rabbit model of RDS when comparing high and low

volume strategies during HFJV and HFOV (114). Investigation and documentation of

injury associated with HFJV is therefore warranted to fully understand the impact of

this ventilation strategy on the lungs.

1.5.2 Haemodynamic Consequences of Positive Pressure Ventilation

Lowered right ventricular filling pressure, decreased central blood volume and a

consequent decrease in cardiac output are all side effects of IPPV (115). Animal studies

have demonstrated superior preservation of haemodynamic function during HFJV

when compared to CMV in dogs (115) and when compared to high-frequency positive

pressure ventilation in rabbits (116).

1.5.2.1 Pulmonary Blood Flow

A cat study comparing haemodynamic function during HFJV and HFOV aimed to

investigate the impact of Paw on cardiac output and pulmonary vascular resistance

(117). Despite demonstrating that increasing Paw caused a decrease in cardiac output

and an increase in pulmonary vascular resistance there was no cardiovascular

advantage of one strategy over the other. The major limitation of this study was the

37

range of Paw was low: 2-12 cmH2O. A bigger difference may have been demonstrated

if higher airway pressures were applied.

Kawahito et al (2000) studied HFJV and CMV in adult patients with healthy lungs to

compare the impact of each strategy on pulmonary perfusion and cardiac output

(118). They used transoesphageal echocardiography and found significantly decreased

pulmonary arterial pressure and left atrial pressure during HFJV. These findings

correlated with increases in cardiac output and ejection fraction in their cohort of

healthy patients. Their conclusions that there is a haemodynamic advantage of HFJV

over CMV attributable to lower PIP during HFJV warrant consideration. While the

haemodynamic implications of a ventilation strategy will be affected by the presence

of cardiovascular and pulmonary disease it is still noteworthy that their findings

convincingly demonstrate that HFJV interferes less with venous return and therefore

cardiac output. It is also important to note that the respiratory frequency during HFJV

was 3 Hz in their study. This is considerably lower than would be used in a preterm

infant with RDS.

High-frequency jet ventilation has also been investigated in children. Kocis et al (1992)

studied the impact of pulmonary vascular resistance on cardiac output in the setting of

changing Paw in the transition from CMV to HFJV (7). They found that decreasing Paw

by 50 % caused a 59 % reduction in pulmonary vascular resistance and a 25 % increase

in cardiac output. Their conclusions were that HFJV was a suitable option for their

patient cohort (children with respiratory failure following congenital heart surgery).

While their patients are different to the target population for HFJV in this thesis, these

38

cardiovascular data are nonetheless supportive of the use of HFJV in patients with

severe respiratory dysfunction.

Measurement of pulmonary blood flow is difficult in a clinical setting and given the

paucity of information on the impact of HFJV on pulmonary blood flow, it is prudent to

examine this in a controlled experimental environment. Soon after birth, neonates

undergo the transition from gas exchange across to the placenta to gas exchange

across the alveolar membrane. This transition requires establishment of a low

pressure pulmonary circulation and inflation of the lungs with air. If these patients

require mechanical ventilation, the intervention should not prevent this transition. For

this reason, and in light of the airway pressure required during mechanical ventilation,

animal studies are the only method by which this can be closely examined.

1.5.2.2 Persistent Pulmonary Hypertension of the Neonate

Persistent pulmonary hypertension of the neonate (PPHN) is defined as failure of the

pulmonary vasculature to relax (or dilate) at birth. The transition from fetal to neonatal

life is therefore complicated by right to left shunting of blood, resulting in V/Q

mismatch, venous admixture and hypoxaemia. Persistent pulmonary hypertension

occurs in approximately 1-2 newborns per 1000 live births and despite significant

improvements in treatment it is associated with substantial infant mortality and

morbidity (119). Diagnosis of PPHN can be made with echocardiography but a clinical

impression of the resistance in the pulmonary circulation versus the resistance in the

systemic circulation can be made by comparing pre- and post-ductal SpO2. If

pulmonary vascular resistance is higher than systemic vascular resistance, right-to-left

39

shunting will occur through the ductus arteriosus. Pre-ductal (right forearm) SpO2 will

therefore be higher than post-ductal (lower extremities) SpO2.

Patients with PPHN may have adequate respiratory drive but can remain hypoxaemic

in spite of high FiO2. They may therefore require mechanical ventilation (77). The

suitability of HFJV for these patients has been investigated in a controlled prospective

clinical trial comparing HFJV and CMV. The authors found that in the acute period HFJV

improved oxygenation and ventilation without significantly increasing mortality.

Furthermore, HFJV may be a useful adjunct for stabilisation of the condition in

neonates with severe PPHN (120).

A potential complication of mechanical ventilation of patients with PPHN is a further

increase in pulmonary vascular resistance secondary to positive intrathoracic pressure.

Given the lower airway pressures during HFJV, it may have the least impact on

pulmonary vascular resistance and be the most appropriate ventilation strategy for

patients with PPHN.

Inhaled nitric oxide is widely used to manage PPHN (77, 119, 121). Nitric oxide is a

vasodilator and improves systemic oxygenation by decreasing the right to left shunting

of blood through the ductus arteriosus. It decreases the work of the right side of the

heart and prevents further right ventricular hypertrophy that occurs in response to the

increased pulmonary vascular resistance. The delivery of NO may be facilitated by HFJV

as the flow streaming of the inspired gases helps distribute it to the distal alveoli (122).

40

1.5.2.3 Systemic Blood Flow

While perfusion of the pulmonary vasculature is essential to ensure matching of

ventilation and perfusion and therefore normal gas exchange, changes in systemic

blood flow will determine oxygen delivery to the brain, organs and other tissues.

Assessment of cardiac output is paramount to ensure oxygen supply exceeds oxygen

demand. Animal and clinical studies documenting the impact of HFJV on systemic

blood flow consistently find that there is comparable or better preservation of

systemic arterial blood pressure or cardiac output during HFJV compared to both HFOV

and CMV (7, 123-125).

Intrathoracic pressure during IPPV is higher than during spontaneous ventilation. This

pressure will alter the flow, pressure and resistance of intrathoracic vascular structures

and potentially decrease venous return and cardiac output. These alterations may be

minimised if the increase in intrathoracic pressure is small and short lived.

Furthermore, if respiratory frequency and heart rate are comparable cardiac output

may not decrease (126). Whereas synchronisation of HFJV PIP with ventricular systole

has been proposed for optimisation of ventricular loading in adult patients it is unlikely

to be as applicable to neonates. However, as the lowest respiratory frequency during

HFJV is 3 Hz (though this is determined by the manufacturer) and the normal heart

rate of the neonate is between 2-3 Hz it may be possible to synchronise every second

beat.

Intrathoracic pressure variations during HFJV are smaller than during CMV and it has

been postulated that there will be less cyclical change in cardiac output as a

consequence (127). Sherry et al (1988) measured cardiac output during CMV and HFJV

41

in postoperative elective cardiac surgery patients to determine the impact of CMV or

HFJV at different PEEPs (0, 5, 10 cmH2O) and found that during HFJV with 0 cmH2O

PEEP there was little change in cardiac output. Increasing PEEP increased Paw and

intrathoracic pressure but did not significantly alter cardiac output during HFJV (127).

As premature infants undergo the transition to breathing air systemic arterial blood

pressure will increase. If this increase in blood pressure, and therefore perfusion of the

brain, organs and tissues, is inhibited oxygen delivery may not meet oxygen demand.

While the literature suggests that HFJV has relatively benign affects on cardiac output

it remains important to monitor continuously for evidence of anaerobic metabolism.

1.5.3 Central Nervous System Consequences of Positive Pressure Ventilation

Neurological injury associated with mechanical ventilation is assessed by the presence

of intraventricular haemorrhage (IVH) and periventricular leukomalacia (PVL). The

manifestations of this neurological injury vary in severity but cerebral palsy to some

degree is 25 to 30 times more likely to occur in infants weighing less than 1500 g (128).

Neurodevelopmental follow-up must also be performed in the first few years of life to

determine the consequences of any neurological injury sustained in the first few days

of life (129). The incidence of both IVH and PVL has a direct relationship to cerebral

palsy, intellectual impairment and visual disturbances (55). The causes of these lesions

are unclear but the following factors may predispose to both: asphyxia; severe

haemorrhage; septicaemia; patent ductus arteriosus; low arterial blood pressure;

impaired cardiac function; PaO2 and PaCO2 (130, 131).

42

1.5.3.1 Intraventricular Haemorrhage and Periventricular Leukomalacia

Intraventricular haemorrhage refers to bleeding into the cerebrospinal fluid filled

lateral ventricles of the brain. The majority of IVH occurs in the first few days of life

and the incidence of it is inversely related to gestational age and body weight at birth

(132, 133). It is graded from I to IV by ultrasonography (134) and grades III or IV are

considered severe (135). Periventricular leukomalacia is the most common ischaemic

brain injury in premature infants and occurs in the white matter adjacent to the lateral

ventricles.

Hypocapnia has been investigated as a cause of IVH and PVL but the results of various

studies do not demonstrate an absolute link between hyperventilation (hypocapnia)

and adverse neurological outcomes. Murase and Ishida (2005) found that hypocapnia

(PaCO2 < 3.3 kPa (25 mmHg)) in the first 48 h of life was significantly associated with

cerebral palsy and late-onset PVL. This suggests that any association of cerebral palsy

with early hypocapnia is limited to a minor subtype of PVL, or to infants with cerebral

palsy not related to PVL (130). Fujimoto et al (1994) found that hypocapnia, and other

complications, were associated with PVL and concluded that mechanical ventilation in

premature infants should be managed carefully to avoid a PaCO2 lower than about

2.67 kPa (20 mmHg) (131). Other studies have similarly described periods of

hypocapnia as contributing factors to a poor neurological outcome (56, 136).

The HIFI Study comparing HFOV and CMV demonstrated a significant increase in

severe IVH and PVL (and respiratory and haemodynamic complications) in patients

ventilated with HFOV (137). The authors speculated that the poor outcome results

were a consequence of failure to emphasise volume recruitment and maintenance in

43

the respiratory management of these infants (45). Although this study did not focus on

the PaCO2 targets for these patients and the impact of hypocapnia on cerebral blood

flow, it raised questions about whether the ventilation modality itself, or the blood gas

status of the patient, had an impact on adverse neurological outcome.

There are few clinical trials focusing on the neurological consequences in patients

managed with HFJV, and the results are inconsistent. Keszler et al (1997) documented

a randomised, controlled clinical trial of HFJV and CMV in 130 preterm infants between

700 and 1500 g born before 36 w post conceptional age. They found that HFJV reduced

the incidence of BPD at 36 w, decreased exposure to hypocapnia and reduced the risk

of grade III and IV IVH and/or PVL compared to CMV (138). Wiswell et al (1996) studied

infants born prior to 33 w gestation between 500 and 2000 g. In this randomised

controlled trial they aimed to ventilate infants to normocapnia and found that HFJV

was associated with a greater risk for adverse outcomes (grade IV IVH, PVL or death)

when compared to CMV (139). These authors went on to examine the role of

hypocapnia in the development of IVH and PVL during HFJV and found that the infants

with PVL had been hypocapnic for longer during the first day of life (140).

Direct comparison between HFJV and CMV has been attempted but the populations

are small (100, 120, 141). The results suggest HFJV is associated with lower mortality

but no significant differences were found in the incidence of IVH. There are multiple

limitations to these studies so to determine the cause of IVH and/or PVL the studies

need to target the at-risk population, include enough patients to give the results

power and incorporate long term pulmonary and neurodevelopmental follow up

assessment.

44

The mechanism by which hypocapnia affects the development of IVH and PVL remains

unclear. Whether the PaCO2 level or the duration of time spent at a low PaCO2 has a

causative effect warrants further investigation. Furthermore, it’s possible the blood

gas status created during mechanical ventilation has a greater impact on adverse

neurological outcomes than the ventilation modality itself. Understanding how to

manage a particular ventilator to achieve target blood gas ranges is essential to

maintain a balance between the risks and benefits of mechanical ventilation.

Initial HFJV strategies more often created hypocapnia for periods of time. The focus on

managing Paw to maintain oxygenation and concern about the adverse effects of high

PEEP kept ∆P high enough to create hypocapnia. As the strategies have evolved and

higher PEEP settings are used the incidence of hypocapnia may decrease. Optimal

volume HFJV strategies have been shown to improve oxygenation and decrease

exposure to hypocapnia which in turn reduces the risk of grade III and IV IVH and/or

PVL (142). Future studies should focus on the incidence of adverse neurological effects

in the face of normocapnia.

1.6 Assessment of Lung Injury

The role of inflammation in VILI is important in the pathogenesis of BPD (143, 144). It

may be initiated by a pulmonary fetal inflammatory response and be exacerbated by

mechanical ventilation and exposure to supplemental oxygen. The response is a

complex interaction between proteins that attract inflammatory cells (chemokines),

proteins that facilitate the transendothelial migration of inflammatory cells from blood

vessels (adhesion molecules), proteins that promote tissue damage (pro-inflammatory

45

cytokines and proteases) and proteins that modulate the process (e.g. anti-

inflammatory cytokines, binding proteins and receptor antagonists) (143).

Inflammation as a prelude to injury can be assessed in a number of ways: measuring

the protein content and assessing the cell population of bronchoalveolar lavage (BAL)

fluid; identifying the cellular infiltrate in lung tissue; assessing the expression of

messenger RNA (mRNA) for pro-inflammatory mediators; and measuring the products

of mRNA transcription. Non-invasive tests on BAL fluid are more appropriate in the

clinical setting and help identify infants at risk of BPD. In a research environment,

measurement of biomarkers in lung tissue, and BAL fluid, will further contribute to an

enhanced understanding of the significance of inflammation associated with

mechanical ventilation.

An appreciable increase in biomarkers may take many hours or days (144) which

makes it difficult to identify the initial injury pathways stimulated immediately after

birth. Injury is initiated on commencement of mechanical ventilation, when the lungs

are partially liquid-filled, surfactant deficient and partially aerated. The quantification

of expression of early response genes in the immediate postnatal period revealed that

VILI during the immediate newborn period can initiate changes in gene expression

within 15 minutes. This abnormal gene expression will potentiate inflammation and

promote abnormal lung development (145). Wallace et al concluded that connective

tissue growth factor (CTGF), cysteine-rich 61 (CYR61), early growth response factor 1

(EGR1), interleukin (IL) 1β, IL-6 and IL- 8 are likely to be useful biomarkers for VILI in

the newborn, particularly in the short term (111).

46

Recent work has focused on the identification of mediators of lung injury and

characterisation of their interaction with alveologenesis. This discovery has enabled

identification of the protective mechanisms specific to the mediator of injury which in

turn enables robust protection against lung disease (146). Transforming growth factor

(TGF) β1 was identified in the lungs of preterm infants and is involved in inflammatory

and repair processes encountered in acute and chronic lung disease (147). High initial

levels of TGF β1 persisted over time and were predictive of the need for oxygen

therapy at home. Minoo et al have recently concluded that fibroblast growth factor

(FGF) 10 offers a distinct protective effect by attenuating the TGF β1 pathway and that

FGF 10 treatment strategies may provide protection to neonatal and other forms of

lung diseases caused by TGF β1 (146).

1.6.1 Bronchoalveolar Lavage Fluid

Bronchoalveolar lavage fluid can be collected via a tracheal tube in a clinical setting

and provides information about the permeability of the alveolar membrane and the

infiltration of inflammatory cells into the alveoli.

1.6.1.1 Protein in Bronchoalveolar Lavage Fluid

Two barriers form the alveolar-capillary interface: the microvascular endothelium and

the alveolar epithelium. In the acute phase of lung injury there is an influx of protein

rich oedema fluid into the air spaces as a consequence of increased permeability of the

alveolar-capillary barrier (148). The importance of endothelial injury causing increased

vascular permeability and pulmonary oedema in acute RDS is well established (149).

However, the importance of epithelial injury to recovery from lung injury has become

47

better recognised (150) and the degree of alveolar injury is a predictor of outcome

(149).

The increase in capillary-alveolar permeability to plasma proteins can be quantified by

measuring BAL fluid total protein concentration (19, 106). An increase in BAL fluid

protein concentration is considered a global indicator of lung injury (106). The plasma

proteins that flood the alveoli contribute to the development of fibroproliferation

which in turn contributes to the risk of fibrosis. Efforts to monitor and reduce plasma

protein accumulation in the alveoli could benefit the patient (151).

The BAL procedure may be performed during sedation or anaesthesia, or post-mortem

in animals, and has been included in the overall assessment of lung injury in a number

of studies using a number of species. Measurement of total protein is an indicator of

lung injury but does not characterise the injury unless specific proteins or the actual

size of the protein is determined.

1.6.1.2 Inflammatory Cells in Bronchoalveolar Lavage Fluid

Extravasation of inflammatory cells due to endothelial and epithelial damage occurs as

a result of lung injury. Bronchoalveolar lavage fluid collected ante- or post mortem can

be examined to determine the number and composition of these inflammatory cells to

help determine the degree of lung injury. Bronchoalveolar lavage inflammatory cell

counts (neutrophils and mononuclear cells) increase in ventilated animals compared to

unventilated controls (106, 144, 152).

A recent review by Reynolds (2009) acknowledges that collection and analysis of BAL

fluid is a relatively non-invasive diagnostic test, but that it has limitations as a

48

diagnostic tool. In both a clinical and a research setting BAL fluid analysis contributes

to the understanding of disease processes but the cellular components of BAL fluid are

not strongly correlated with definitive diagnosis (153).

1.6.2 Lung Tissue

Analysis of lung tissue is primarily a research tool. The identification and localisation of

inflammatory cells will characterise alterations in lung tissue associated with

mechanical ventilation. The maturity of inflammatory cells aids in assessing the

duration of the inflammatory process while the location helps characterise the insult.

1.6.2.1 Inflammatory Cells in Lung Tissue

Localisation of inflammatory cells in the microanatomy of the lung is useful to further

characterise an inflammatory response occurring as a prelude to lung injury.

Immunohistochemical staining and histopathological analysis of targeted enzymes

such as inducible nitric oxide synthase (iNOS) and myeloperoxidase (MPO) indicate the

location of inflammatory cells and form part of the overall assessment of inflammation

(112). Positive MPO staining identifies neutrophils and mononuclear cells while

positive iNOS staining identifies macrophages. This differentiation helps age the

inflammatory infiltrate.

1.6.2.2 Gene Expression in Lung Tissue

Describing the pattern of gene expression and activation associated with lung injury

provides the most precise account of the initial mechanisms involved in lung injury. As

the greatest risk of injury may be during the period immediately after birth when the

49

lungs are particularly vulnerable, early response genes and growth factors are of

particular interest (108, 145, 154). The role of early response genes in VILI in the

preterm neonate was elucidated recently (145). Quantitative polymerase chain

reaction (qPCR) of mRNA for CTGF, CYR61, EGR1, IL-1β, IL-6 and IL-8 indicated that

resuscitation and mechanical ventilation at birth with high tidal volumes caused

upregulation of these genes compared to mechanical ventilation with low tidal

volumes. The authors concluded that these findings were indicative of more lung injury

from mechanical ventilation with low tidal volumes.

The precise role of specific mediators in the pathogenesis of VILI is not entirely

understood. The literature regarding the specific role of inflammatory mediators is

expanding, but is not comprehensive (155, 156). Inflammation and injury is a complex

process: initially gene expression is altered and these genes are subsequently

translated to specific proteins. Understanding the temporal properties of specific

genes and proteins enables appropriate analyses to demonstrate inflammation and

injury. Copland et al (2003) demonstrated that altered gene expression occurs before

demonstrable lung injury and that these alterations are time and stretch dependent

with characteristic spatial distributions (109). The choice of specific genes for

examination in the context of a particular study should be made carefully. Factors that

influence the suitability of particular genes include: the duration of the study; the

underlying pathophysiology of the lungs; and the ventilator strategy under

investigation.

50

1.7 Lung Protective Ventilation Strategies

Mechanical ventilation was originally achieved by creating a negative extra-thoracic

pressure to simulate the negative intra-thoracic pressure created during normal

inspiration. These iron lungs mimicked normal spontaneous ventilation insofar as the

respiratory rate, tidal volume and airways pressures were comparable. There were

practical problems unrelated to breathing that led to the development of IPPV. Overall

patient management was a lot easier and ventilation was more efficient with the

ventilator attached to a tracheal tube. The main causes of ventilator associated

morbidity arise from the positive pressure within the chest and the impact on lung

tissue, pulmonary circulation and systemic and cerebral blood flow. These morbidities

may also be described as volutrauma, atelectatrauma, biotrauma and barotrauma with

reference to the impact on the lungs themselves. High-frequency ventilators were

developed as an alternative to conventional positive pressure ventilation as the

concept of smaller breaths more often was believed to be associated with fewer

adverse side effects (157).

Various ventilation strategies have been developed to reduce the prevalence of BPD,

but despite these advances the risk of BPD is still high for very preterm babies (30, 34).

There are 3 gentle ventilation strategies available to the neonatologist in the clinical

realm: gentle CMV with relatively high respiratory rates and small tidal volumes, HFOV

and HFJV. Each of these strategies has evolved to achieve a similar broad aim which is

described by the open lung approach. The open lung approach was first documented

in 1992 and incorporates the following basic treatment principles (91, 158):

51

1. Open the whole lung with the necessary inspiratory pressure

2. Keep the lung open with PEEP above the closing pressure

3. Maintain optimal gas exchange at the smallest possible pressure amplitude to

optimise CO2 removal

This approach has changed the focus of ventilator management from targeting

physiological goals alone to protecting the lung from injury and decreasing the

cytokine modulation of the lung (98, 159, 160). The open lung approach can be applied

during CMV, HFOV and HFJV and aims to recruit and stabilise alveoli, minimise

atelectasis, and maximise gas exchange area without injury to the lung or compromise

to systemic or pulmonary blood flow (158).

Alveolar recruitment refers to the dynamic process of opening previously collapsed

lung units by increasing transpulmonary pressure (158). This pressure change is

primarily responsible for VILI (161, 162) and the effects of pressure should always be

monitored. The choice of recruitment manoeuvre will depend upon the individual

patient and the baseline ventilator mode. The delivery of a sustained high distending

pressure, an increase in PEEP and/or a transient increase in PIP will all facilitate

alveolar recruitment, but may also compromise blood flow and lung structure. Getting

the balance right is essential during lung protective ventilation.

Understanding hysteresis of the pressure-volume curve of lung inflation and deflation

will help attain this balance. The volume achieved on the deflation limb of the

pressure-volume curve is larger for the same distending pressure, compared to that

achieved on the inflation limb. The point of maximal curvature of the deflation

pressure-volume curve is the point at which the lowest pressure achieves optimal lung

52

volume and PaO2 (163). Targeting ventilation to this point by delivering a sustained

inflation followed by small tidal volumes (5-6 mL kg-1) with PEEP above the inflection

point of the pressure-volume curve has been demonstrated to minimise lung injury in

a rat model (164). Alveolar recruitment using a sustained inflation followed by small

breaths with a PEEP below the inflection point will also boost the ventilator to cycle

onto the deflation limb of the pressure-volume curve (165). These findings suggest

that if sufficient lung volume recruitment is achieved with a sustained inflation that

relatively low airway pressures can be used to maintain tidal ventilation with a lower

pressure cost (165).

53

54

2 High-frequency Ventilation

2.1 High-frequency Ventilation

2.1.1 Gas Mixing during High-frequency Ventilation

The mechanisms determining gas flow, gas mixing and airway pressure during high-

frequency ventilation (HFV) are fundamentally different to ventilation at respiratory

frequencies employed during CMV. HFV is characterised by ventilation with tidal

volumes smaller than dead space volume but adequate gas exchange can still occur

because the increased energy of the gas molecules at the high frequencies and flows

leads to augmented mixing of gas in the airways (166). The dynamics of gas flow

distribution during HFV involve a number a different mechanisms including bulk

convection, asymmetric velocity profiles, pendelluft, cardiogenic mixing, Taylor

dispersion and turbulence, molecular diffusion and collateral ventilation (166, 167)

(Figure 2-A).

55

Figure 2-A Gas Transport Mechanisms during High-frequency Ventilation. The gas transport mechanisms responsible for gas exchange during CMV (convection, convection and diffusion, and diffusion) are enhanced during HFV by seven mechanisms: turbulence; direct ventilation of proximal alveoli; turbulent flow with lateral convective mixing; pendelluft; gas mixing due to velocity profiles that cause a central stream of inspiratory gases along the airways and a stream of expiratory gas around this central stream; laminar flow with lateral transport by diffusion (Taylor dispersion); and collateral ventilation through non-airway connections between neighbouring alveoli (166).

High-frequency ventilators do not mimic normal breathing. Much smaller tidal volumes

are delivered at a much higher frequency and gas exchange occurs in a highly efficient

manner similar to that achieved by panting animals whereby:

Equation 2-A

V’CO2 = f x VT2

where V’CO2 = rate of CO2 elimination, f = ventilator frequency, and VT = tidal volume.

56

During normal breathing, effective or physiological dead space must be greater than or

equal to anatomical dead space (157). In the context of the equation above there must

be a lower limit on VT that will effectively provide alveolar ventilation. That lower limit

is related to the effective or physiologic dead space of the lungs according to:

Equation 2-B

VA = f x (VT – VD, physiol)

where VA = alveolar ventilation and VD, physiol = physiological dead space.

As VT approaches VD, physiol, direct ventilation of the alveoli approaches zero at

conventional breathing frequencies. However, when an animal pants, physiological

dead space becomes smaller than anatomic dead space (168). This was demonstrated

in 1915 by Henderson et al in an experiment demonstrating the jet stream of smoke

created during fast and shallow breathing through a tube. Thus, the extent to which a

smaller tidal volume can ventilate alveoli is balanced by an increased in breathing

frequency.

2.1.2 Mechanical Properties of the Lung and High-frequency ventilation

The interaction of resistance, compliance and inertance in the frequency range of high-

frequency ventilators will govern gas exchange. Resistance refers to the opposition to

flow and is determined by the dimensions of the airway, the viscosity of the gas and

whether flow is laminar or turbulent. Compliance is determined by assessing changes

in volume per unit of pressure and inertance is the pressure required to cause a

change in flow. The behaviour of the respiratory system during mechanical ventilation

is determined by these 3 components (169). They therefore give the respiratory

57

system a measure of impedance which will vary according to respiratory frequency.

Impedance is an important determinant of the efficiency of ventilation and is a global

term that encompasses compliance, resistance and inertance. Impedance therefore

represents a mechanical barrier to flow and as impedance increases, greater changes

in pressure are required to generate an equivalent flow (and VT) (170). As the

amplitude of the airway pressure waveform increases, the risk of barotrauma

increases, but if it is too small airway closure and alveolar collapse may ensue. It is

therefore essential to understand impedance throughout the respiratory system (from

the breathing system, the endotracheal tube, the airways and lung tissue) to

determine the magnitude of the pressure required to transmit gas to the alveoli during

high-frequency ventilation safely (170).

In vitro and in vivo studies found that a resonant frequency is observed below which

elastic behaviour of the lungs is dominant (impedance decreases with increasing

frequency) and above which inertial behaviour is dominant (impedance increases with

increasing frequency) (169). The resonant frequency of the lungs will vary according to

maturity and pathology and is inversely related to the square root of the compliance

and the inertance. In the overdamped lung (e.g. preterm lung), the corner frequency

(which is inversely proportional to the resistance and the compliance) defines the

frequency above which the pressure required to ventilate those lungs is minimised

(171). Hence optimal frequency for ventilation most likely falls between the corner

frequency and the resonant frequency. A study of 6 newborn infants with RDS found

the resonant frequency to range from 13 to 23 Hz (169) when an endotracheal tube

was in position. The impedance displayed compliance-like behaviour below the

58

resonant frequency and inertance-like behaviour above the resonant frequency.

Without an endotracheal tube the resonant frequency was higher (169). This study

found that endotracheal tube resistance was equivalent to ~ 50 % of the respiratory

system resistance distal to the tip of the endotracheal tube, whereas virtually all the

respiratory system compliance resided distal to the tip of the endotracheal tube (169).

If the frequency of ventilation is greater than the resonant frequency of a set of lungs,

overriding the resonant frequency requires less pressure and energy compared to a

mechanical frequency that is less than the resonant frequency. The smaller the lungs

are, the higher the resonant frequency: resonant frequency of adult lungs is ~ 4 Hz,

while that of premature infants lungs may be as high as 40 Hz (157). High-frequency

ventilators deliver breaths up to 780 times per minute so come considerably closer to

the resonant frequency of immature lungs than conventional mechanical ventilators

do. This property alone is important when taking into account the potential for side

effects associated with positive pressure ventilation.

2.1.3 Airway Pressure Waveforms during High-frequency Ventilation

In the presence of normal lung compliance, the amplitude of the airway pressure

waveform is greatly attenuated during HFV as gas passes at rapid rates through the

rigid endotracheal tube (Figure 2-B) (55). During CMV, both peak and mean airway

pressures are transmitted to the distal airways and alveoli. During high-frequency

oscillatory ventilation (HFOV) the amplitude of the pressure waveform diminishes but

the Paw is sustained, while during high-frequency jet ventilation (HFJV) both the peak

and mean airway pressure are attenuated at the distal airways and alveoli. This gives

59

HFJV a distinct theoretical advantage when considering the pressure cost of

ventilation.

Figure 2-B Pressure amplitudes generated at the ventilator are attenuated during HFV as high velocity gas passes through the rigid endotracheal tube. At slow respiratory rates during conventional ventilation this does not occur (55).

The impact of lung compliance on ventilator performance has been investigated

extensively for CMV and HFOV (170, 171). In vitro studies of HFOV have demonstrated

that if compliance is reduced, oscillation of alveolar pressure is large and coupled with

a concomitant reduction in VT (170). Furthermore, the clinical significance of changes

in compliance on the inspiratory pressures required to maintain normocapnia are best

assessed as the alveolar pressure cost per unit of ventilation. In a poorly compliant

lung, which has a higher corner frequency than a healthy recruited lung, the lowest

pressure cost of ventilation is achieved at higher frequencies.

60

2.1.4 Modes of High-frequency Ventilation

The important differences between HFOV and HFJV are summarised (Table 2-A). These

differences give rise to the potential for physiological advantages of one strategy over

the other: during HFJV the Paw tends to be lower because less of the respiratory cycle

is spent in inspiration; HFJV enhances mucociliary clearance by combining fast

inspirations with relatively slow, passive exhalations (tI:tE ratio may be as low as 1:12);

and the high velocity and small VT breaths do not penetrate injured areas of lung with

high resistance, allowing for maturation and/or healing.

Table 2-A Differences between HFOV and HFJV.

Device tI:tE ratio Inspiratory time

Waveform Exhalation phase

CMV needed

Endotracheal tube adaptor

HFOV 1:3 to 1:1 0.02-0.1 s Squared or

sinusoidal

Active No No

HFJV 1:12 to 1:1.8

0.02-0.034 s Peaked Passive Yes Yes

The passive exhalation phase during HFJV gives this modality a distinct advantage in

certain scenarios. Gas trapping is less likely if the absolute expiratory time is

lengthened: the longer expiration period facilitates more complete passive exhalation.

Preterm infants with increased airway resistance and long time constants, as might

occur with pulmonary interstitial emphysema, need a longer expiration period to

accommodate exhalation (compared to patients with RDS that have shorter time

constant, less compliant lungs). Otherwise they may develop lung over-inflation and

enter a vicious cycle of progressive gas trapping that can’t be broken unless the

61

absolute expiratory time is shortened. Time in expiration is not the only factor that

impacts upon gas trapping. If exhalation is active, airway collapse may ensue and gas

trapping is exacerbated. Friedlich et al (2003) presented the results of a cross-over

study from HFOV to HFJV in patients with refractory hypoxaemia. Ten patients crossed

over to HFJV and the survival rate was 90 % (2). They attributed this high survival rate

to their exploitation of the low tI:tE ratio and the passive exhalation phase possible

during HFJV.

2.2 Using a High-frequency Jet Ventilator

High-frequency jet ventilators deliver small tidal volume breaths at a very rapid rate.

Breaths may be as small as 0.5-1 mL kg-1 and can be delivered at a frequency of 4-12 Hz

(240-720 breaths/min). The Life Pulse high-frequency jet ventilator (Bunnell

Incorporated, Salt Lake City, U.S.A.) is a pressure limited and time cycled ventilator

with adjustable peak inspiratory pressure (PIP) and inspiratory time (tI). The jet pulse is

delivered via a special jet tracheal tube adaptor (replacing the normal adaptor) which

has a pressure monitoring port and jet port (Figure 2-C). Inspired gases pass through

the jet port from the jet ventilator and expired gases move out passively to the

breathing system. Opposite the jet port a pressure monitoring port feeds back

information to the ventilator.

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Figure 2-C Jet Port Adaptor (Bunnell Inc., Salt Lake City, Utah, U.S.A.)

The Life Pulse high-frequency jet ventilator displays set parameters and monitored

variables. The central control panel details PIP, respiratory rate, tI and calculates the

tI:tE ratio. The monitored variables are PIP, ∆P, PEEP, servo pressure and Paw (Figure

2-D). ∆P and Paw are calculated values.

Figure 2-D The Life Pulse high-frequency jet ventilator. The central control panel displays the current (now) and to be altered (new) PIP, respiratory rate and tI along

63

with the tI:tE ratio. The uppermost panel displays the monitored variables: PIP, ∆P, PEEP, servo pressure and Paw.

Humidified gas travels to the patient through a patient box. This patient box is a flow

interrupter with a pinch valve which controls the breaths delivered to the patient. The

patient box is located in close proximity to the patient to minimise dampening of the

breaths between the flow interrupter and the endotracheal tube. The specialised jet

adaptor which replaces the endotracheal tube adaptor fits a pressure monitoring line

which provides feedback to the ventilator to control the pressure of subsequent

breaths. It also measures PEEP, providing a monitor of PEEP set on the conventional

ventilator (Figure 2-E).

Figure 2-E Configuration of the jet ventilator and conventional ventilator in the preterm lamb model used in the studies presented in this thesis. Gas from the jet ventilator passes through the patient box and a pinch valve acts as a flow interrupter to create the jet breaths. Gas enters the specialised jet adaptor through the green tube and a pressure monitoring line provides feedback to the jet ventilator. Expired gases pass through the expiratory limb of the CMV circuit.

The parameters to set on the jet ventilator are PIP, respiratory rate and tI. PIP is

discussed under ‘The role of the high-frequency jet ventilator’. The respiratory rate can

64

be set between 240 and 720 breath/min (4-12 Hz). Slower rates are chosen if there is

resistance to expiration (e.g. pulmonary hyperinflation, pulmonary interstitial

emphysema). There are no data regarding the optimal respiratory rate during HFJV and

it will vary according to the disease process. An initial rate of 420 breaths/min is most

often chosen (172). The default tI setting is 0.02 s and works best in most situations.

This very short tI provides very small tidal volumes and keeps alveolar pressures low.

Such a short tI prevents the PIP set on the jet ventilator from being completely

transmitted to the alveoli. If the respiratory rate is decreased, and the tI remains the

same, the time in expiration is increased. Since exhalation is passive this facilitates the

movement of gas and secretions along the airways and out of the lungs.

2.2.1 The Role of the Conventional Ventilator

The jet ventilator is set up in tandem with a conventional mechanical ventilator which

provides the bias flow and PEEP, and channels expired gases away from the patient.

The conventional ventilator may be set to continuous positive airway pressure (CPAP)

mode or a ventilation mode where occasional CMV breaths are delivered. The role of

the conventional ventilator during HFJV in providing PEEP, and therefore controlling

Paw, is a major determinant of oxygenation in patients requiring HFJV. The role of

PEEP and Paw in optimising oxygenation is well documented (173-175).

2.2.1.1 Alveolar Recruitment and Positive End-expiratory Pressure

Positive end-expiratory pressure (PEEP) is the positive airway pressure maintained at

the end of expiration. It is used during mechanical ventilation to recruit alveoli and to

prevent alveolar collapse. PEEP is provided by the conventional mechanical ventilator

65

and the jet ventilator monitors PEEP to alert the clinician to a discrepancy between set

and measured values and the development of auto, or inadvertent, PEEP. Choosing the

most appropriate level of PEEP will depend on the need for alveolar recruitment, the

haemodynamic state of the patient and the pulmonary pathophysiology. Excessive

PEEP will compromise cardiac output as the positive intra-thoracic pressure at the end

of expiration will, to some degree, impact upon the low pressure part of the

circulation, the venous return and the right atrium and ventricle. If venous return is

compromised, cardiac output will fall and this may have implications for the systemic

circulation. An algorithm (Figure 2-F) details the decision making process for altering

PEEP and incorporating CMV breaths. This algorithm has been developed from

experience in a clinical setting, however there is no documented evidence to support

the size, shape, frequency and duration of CMV breaths delivered during HFJV.

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Figure 2-F Algorithm for optimising PEEP when changing from CMV to HFJV with or without CMV breaths (176)

The relationship between PEEP and pulmonary blood flow (PBF) is complex and the

clearance of lung liquid during the first few minutes of ventilation will also impact

upon the haemodynamic effects of PEEP. The massive increase in PBF after birth (8

fold) results from the rapid increase in PaO2 at this time, the release of nitric oxide

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from the pulmonary vascular bed and aeration of the lung. Crossley et al (2007) found

that increasing PEEP (to a critical point) improved oxygenation at the same time as

reducing PBF. As increased oxygenation should promote pulmonary vascular

vasodilation it may be that increasing PEEP led to a gradual decrease in the number of

atelectatic regions and therefore improved oxygenation (177). Blood flow through

those recruited regions may have been reduced due to the compression of the

capillaries. A reduction in PBF is reported with increasing levels of PEEP and it is

postulated it may result from an increase in the alveolar capillary transmural pressure

causing capillary compression (177-179). Likewise, too little PEEP may also

compromise PBF, as extra-alveolar vessels lose support. In the preterm lung, however,

increased airway pressures caused by increasing PEEP have less compressive effects on

capillaries than in the mature lung as preterm lungs are less compliant. Increased lung

compliance following antenatal steroids or surfactant administration at delivery may,

however, increase the sensitivity of pulmonary vessels to changes in airway pressure

(177).

Optimising PEEP is essential during mechanical ventilation. Not only can PEEP prevent

alveolar de-recruitment and interfere with cardiac output, it will affect the pressure

cost of ventilation. Changing PEEP settings over a range of just a few cmH2O can

reduce the required PIP by as much as 50 % (171). These authors concluded that

optimising PEEP is especially important given that ventilator parameters (frequency,

PEEP, PIP and VT) that yield adequate ventilation with safe distension of recruited

alveoli are severely limited if lungs have collapsed because of inadequate PEEP (171).

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The algorithm for finding optimal PEEP during HFJV suggests that this is most easily

achieved when HFJV and CMV breaths are combined (Figure 2-F). The PEEP

optimisation process is based upon findings from HFOV and at the present time is not

substantiated by systematic research using HFJV in a controlled environment. The aim

of the algorithm is to assess the response to removing CMV breaths by changing to

CPAP mode on the conventional ventilator. The assessment variable is SpO2 as it is a

continuous non-invasive measurement but arterial blood gas analyses should also

guide decision making. If the SpO2 is stable and in the target range without CMV

breaths then PEEP is deemed to be adequate to maintain end-expiratory lung volume.

However, if SpO2 decreases after CMV breaths are turned off then the current PEEP

level is assumed to be below the closing pressure. In this scenario, CMV breaths should

be re-introduced and PEEP should be increased to improve alveolar stabilisation.

Optimal lung volume may be defined as having achieved satisfactory SpO2 when FiO2 is

approaching 0.21. To achieve this goal in acute atelectatic lung disease, PEEP may be

substantially higher than traditionally used in conventional ventilation. PEEP should be

decreased if cardiac output is compromised or if oxygenation is adequate and SpO2

doesn’t fall with a decrease in PEEP (176).

If PEEP is too low, alveoli will collapse and atelectasis will develop. If PEEP is too high

there is a risk of alveolar overdistension, impedance of pulmonary perfusion,

cardiovascular depression, poor right heart filling and impedance of venous return

(Figure 2-G).

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Figure 2-G During mechanical ventilation PEEP must be optimised to prevent ventilator induced lung injury and haemodynamic compromise (55).

The importance of optimising PEEP and the impact it has on oxygenation was

demonstrated in a study comparing the arterial blood gas and PBF parameters of

preterm lambs delivered by caesarean section at 126 d gestation. The effect of PEEP

was compared in groups of lambs that had received antenatal steroids, exogenous

surfactant, both or neither and ventilated in volume guarantee mode with CMV. When

VT was constant and PEEP was maintained at 8 cmH2O the effect of PEEP on

oxygenation was larger than the effect of antenatal steroid treatment or exogenous

surfactant administration (177). The improvement in oxygenation was not at the

expense of increased PaCO2 or decreased arterial blood pressure. Surprisingly

antenatal steroid treatment and postnatal surfactant had little impact on the effect of

PEEP on PBF.

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During HFJV, PEEP is set on the conventional ventilator and the jet ventilator displays

the measured value. This information is particularly useful if measured PEEP is higher

than set PEEP. This discrepancy indicates inadvertent intrinsic PEEP and most often

reflects an inappropriately high HFJV rate promoting gas trapping. This information is

also useful if a patient is changed from one ventilator to another. The measured PEEP

can be used to ensure that PEEP is maintained in the changeover (180).

2.2.1.2 Oxygenation and Mean Airway Pressure

Mean airway pressure is a measured value that is displayed on the jet ventilator. It is

determined by the relationship between frequency, tI, PIP and PEEP:

Equation 2-C

time cycle

time) exp x PEEP(time) insp x PIP (HFJVaw

P

where Paw = mean airway pressure, HFJV = high-frequency jet ventilation, PIP = peak

inspiratory pressure, PEEP = positive end-expiratory pressure.

Mean airway pressure is considered the primary determinant of oxygenation and is in

the most part manipulated by changing PEEP (174). During HFJV, Paw is closer to PEEP

than PIP as the tI:tE ratio range can be changed (by altering respiratory frequency) from

1:12 up to 1:1.8. Mean airway pressure is attenuated significantly during HFJV so the

pressure in the alveoli is considerably lower than at the endotracheal tube connector

(157). If Paw is too high, airways and alveoli may over distend and rupture. Conversely,

if Paw is too low airways and alveoli may collapse.

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In the clinical setting, the oxygenation index is calculated to determine the impact of

Paw on oxygenation. The oxygenation index describes the relationship between FiO2,

Paw and PaO2 and a lower value implies better arterial oxygenation (175):

Equation 2-D

OI = 2

2

aO

100 x aw xFiO

P

P

where OI = oxygenation index, FiO2 = fractional inspired oxygen concentration, Paw =

mean airway pressure, PaO2 = partial pressure of oxygen in arterial blood.

Calculation of the OI is a useful bedside tool for comparison of oxygenation between

different ventilation strategies that use different mean airway pressures. It is also used

for prognostication (181).

2.2.2 The Role of the High-frequency Jet Ventilator

The parameters that can be set on the Life Pulse high-frequency jet ventilator are PIP,

respiratory rate and tI. Given that carbon dioxide elimination is proportional to

respiratory rate and VT2 (Equation 2-A) and that VT is determined by lung compliance

and ∆P (PIP-PEEP) it is the high-frequency jet ventilator that provides the most control

over PaCO2 (compared to the conventional ventilator).

2.2.2.1 Carbon dioxide Elimination, HFJV Peak Inspiratory Pressure and ∆P

The PIP during HFJV is altered to achieve a target range for PaCO2 according to serial

arterial blood gas analysis. Increasing PIP will increase ∆P at the same PEEP, and

therefore decrease PaCO2. Conversely, decreasing PIP will decrease ∆P at the same

PEEP and increase PaCO2. Non-invasive methods for assessing CO2 elimination include

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capnography and transcutaneous monitoring but neither technique has a fast enough

response time for the frequency of breaths during HFJV. Transcutaneous monitoring is,

however, more commonly used as a trending tool.

While alterations in PIP will primarily affect PaCO2 they may also affect oxygenation if

Paw changes in parallel with PIP, as is the case if PEEP remains the same. At higher

respiratory frequencies during HFJV, PIP has a greater impact on Paw. Thus to maintain

Paw, and oxygenation, but alter PaCO2, PEEP must be changed at the same time as PIP.

The change in PEEP will be in the opposite direction to the change in PIP to maintain

Paw.

When compared to HFOV in an animal model, the PIP required to achieve comparable

pH and PaCO2 was significantly lower during HFJV (123). This difference is probably

greater than the actual values suggest as the jet ventilator measures PIP at the

proximal airway and this pressure will be significantly attenuated at the level of the

alveolus (55). If lung compliance is poor however, pressure attenuation is less marked

(182). High-frequency oscillatory ventilators do not display PIP or PEEP; rather

management of PaCO2 is by altering ∆P (amplitude). Despite the nomenclature, ∆P will

determine PIP, which will be higher during HFOV.

The difference between PIP (set and measured on the jet ventilator) and PEEP

(determined by the conventional ventilator settings) is equivalent to the amplitude of

the airway pressure waveform. ∆P is therefore a calculated value displayed on the

ventilator and will change as PIP and PEEP are altered. This will determine the VT of

each breath and control PaCO2. During HFJV the VT is as low as 0.1-1.0 mL kg-1 but will

change according to lung and chest wall compliance (183). Regardless of compliance

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the VT during HFJV remains considerably smaller than anatomical and equipment dead

space. The high velocity, central inspiratory flow spike generated during HFJV will

penetrate the dead space and ventilate alveoli (183). Concurrent measurement of VT

will provide information on compliance. For a given ∆P increasing VT indicates relatively

high compliance and decreasing VT indicates relatively poor compliance.

2.2.3 Monitoring during High-frequency Jet Ventilation

The Life Pulse high-frequency jet ventilator monitors airway pressures to provide

continuous feedback information to drive PIP. It also measures servo pressure and

PEEP. The former is the pressure generated within the ventilator that is required to

meet the set PIP and the latter monitors PEEP set on the conventional ventilator.

2.2.3.1 Servo Pressure

The concept of servo pressure is important during HFJV. It refers to the automatically

generated driving pressure that the ventilator itself creates to deliver a breath that

meets the set PIP. Servo pressure will change as lung volume and compliance change

and a decrease in servo pressure may occur as a result of worsening compliance,

endotracheal tube obstruction, the need for airway suctioning, tension pneumothorax,

right maintstem bronchial intubation or a deliberate decrease in ∆P. Servo pressure

will increase with any increase in ∆P, or as compliance improves, the circuit

disconnects or there is an airleak that does not put the lung under tension (184).

Monitoring servo pressure will provide information on lung compliance and contribute

to an understanding of the pathophysiology of lung disease and the progression of this

disease during mechanical ventilation. Servo pressure is measured in pounds per

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square inch (psi) by the Life Pulse high-frequency jet ventilator and will change from

breath to breath giving an early indication of changes within the lungs.

2.2.3.2 Positive End-Expiratory Pressure

The difference between PEEP set on the conventional ventilator and PEEP measured by

the jet ventilator should be negligible. If measured PEEP is higher than set PEEP it is an

indication of auto PEEP. This term refers to the development of end-expiratory

pressure within the patient, most likely due to gas trapping, creating resistance to

expiration. In this instance the VT can’t escape the lungs, servo pressure decreases and

PaCO2 increases. Decreasing the respiratory rate will help minimise the effects of this

inadvertent PEEP. Remaining at a lower respiratory rate is indicated until the set and

monitored PEEP correlate again. If CMV breaths are being delivered at this time,

decrease the frequency of these first, then the frequency of jet breaths.

2.3 High-Frequency Jet Ventilation in the Clinical Environment

Homogeneous restrictive lung disease caused by uniform restriction from extra

pulmonary pathology is difficult to manage with CMV. As a result HFV strategies have

been employed but there are no randomised controlled trials to support the use of

one strategy over another (157). While there are various reports of the use of HFJV in

infants, children and adults, the decision making process during patient management

with HFJV is not evidence based.

High-frequency jet ventilation is theoretically superior for the management of patients

with gas-trapping as the passive exhalation phase and relatively short tI:tE ratio

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facilitates the passage of gas along the periphery of the airways for exhalation. Early

studies report more rapid and more frequent resolution of pulmonary interstitial

emphysema (PIE) with no difference in the incidence of adverse side effects (157). A

rabbit study found that gas trapping was significantly greater during HFJV when

compared to HFOV and attributed the difference to the passive exhalation phase and

relatively short tI:tE ratio (185). This finding contradicts the clinical experience and

highlights the need to compare these ventilation strategies in a controlled setting.

In the 1980s, HFJV was employed in situations where other ventilation modalities had

failed. Early studies document the safety and effectiveness of HFJV in rescue situations

(100, 142, 186, 187). The outcomes of these studies reflected the severity of the

condition of the patients at the time of changeover to HFJV. More recently, however,

attention is shifting to the earlier use of HFJV in infants with RDS that is not yet

complicated by airleak (138).

To date there are 4 randomized controlled trials of HFJV (100, 138, 139, 188). Differing

entry criteria, treatment strategies and the definition of primary outcomes have

complicated interpretation of the results of these trials. The main features of each trial

are summarised in Table 2-B and are evaluated below with respect to their potential

benefits and adverse consequences as both early and rescue therapies in preterm

babies.

2.3.1 High-frequency Jet Ventilation as a Rescue Therapy

A Cochrane review published in 2006 eliminated all but one publication from their

examination of the use of HFJV as a rescue ventilator strategy (141). The study

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reviewed infants who had developed PIE within the first 7 days of life while receiving

CMV (100). Cross-over to HFJV occurred if the patient deteriorated. The results

demonstrated no statistically significant difference in the overall mortality between

the HFJV and CMV groups, no difference in the incidence of BPD in survivors, of new

air leaks after change-over, of total intraventricular haemorrhage, of necrotising

tracheobronchitis at autopsy or of airway obstruction (100). This study only included

144 infants and surfactant administration was not standard practice. Furthermore, the

long term neurological outcomes were not examined. These limitations made it

difficult to conclude that HFJV was a useful rescue therapy in preterm infants.

2.3.2 HFJV used Early in the Management of Respiratory Distress Syndrome

Despite the limited data generated from randomised controlled trials examining the

use of HFJV as a rescue therapy, neonatologists considered HFJV a worthy alternative

therapy when others had failed (100, 142, 186, 187). This acknowledgement led to

investigation of HFJV early in the time-course of patient management (138, 139, 188).

There are only 3 small randomised controlled trials of early HFJV. Carlo et al (1990)

studied 45 infants and found no difference in the incidence of adverse outcomes when

HFJV was compared to CMV in preterm infants. This study used a non-commercially

available high-frequency jet ventilator and none of the infants received surfactant.

Wiswell et al (1996) concluded that the incidence of adverse neurological outcomes

was higher in infants managed with HFJV compared to those managed with CMV (139).

A major factor in this study was the incidence of hypocapnia in the HFJV group of

infants. Despite comparable arterial blood gas values prior to randomisation these

infants had a significantly lower PaCO2 during the study. This complicates

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interpretation of the negative finding for HFJV in this study and is characteristic of the

era of hyperventilation with HFJV. The use of higher PEEP has since decreased the

incidence of hypocapnia during HFJV as higher PEEP allows for a smaller ∆P (at the

same PIP). Lastly, Keszler et al (1997) compared HFJV with CMV in preterm infants.

Despite guidelines for both protocols, during analysis they subdivided the HFJV group

into a low pressure (lung volume) strategy and an optimal volume strategy as some

centres violated the trial protocol. Concern stemming from Wiswell’s earlier study

inadvertently created a unique opportunity to compare 2 different HFJV strategies.

The optimal volume strategy was designed to provide alveolar recruitment, optimise

lung volume and improve V/Q matching, while minimising FiO2 and ∆P. This strategy

also included the delivery of 2 to 5 CMV breaths/min and maintenance of adequate

Paw with relatively high PEEP. They showed that the optimal volume strategy

improved oxygenation, decreased exposure to hypocapnia and reduced the incidence

of high grade IVH and/or PVL (138). This study is the only one in which surfactant was

administered to each infant before entry. They concluded that HFJV reduced the

incidence of BPD at 36 w and the need for home oxygen in preterm infants with

uncomplicated RDS. Further, they surmised that there was no increase in adverse

outcomes compared to CMV. While the results of this study created promise for HFJV

as an early management tool for preterm babies, larger studies have not been

performed.

Table 2-B Summary of randomised controlled trials of high-frequency jet ventilation in preterm babies.

Study n Description Eligibility Surfactant Antenatal Steroids

Ventilators Outcome Measures

Carlo et al 1990

45 Single centre, crossover if failing the assigned therapy, mean randomisation age 14 h (CMV) vs. 15.5 h (HFJV)

Preterm, < 24 h of age, 1000-2000 g, RDS

0 % NR Locally developed, non commercially available jet ventilator with a time-cycled, pressure-limited infant ventilator (Bear Cub) for CMV

Mortality at 28 d, CLD at 28 d, ALS, progression of IVH, success after crossover, days on mechanical ventilation, days on supplemental oxygen, mechanical ventilation at 28 d

Keszler et al 1991

144 (CMV n=70, HFJV n-74)

Multicentre (15), crossover if failing the assigned therapy, mean randomisation age 29.3 w post conceptional age

Preterm, < 7 d of age, < 750 g at birth, PIE

0 % NR Life Pulse (Bunnell) with standard time-cycled, pressure-limited infant ventilators for CMV

Mortality at 28-30 d, success in the original assignment, CLD at 28-30 d, IVH, new airleak, necrotising tracheobronchitis, airway obstruction, CLD in survivors

Wiswell et al 1996

73 (CMV n=36, HFJV n=37)

Single centre, crossover if failing the assigned therapy, mean randomisation age 7.1 h (CMV) vs. 7.3 h (HFJV)

Preterm (< 33 w), 500-2000 g at birth, < 24 h of age at randomisation, chest roentgenographic findings consistent with RDS, requirement for ventilator support with FiO2 > 0.3 and PIP > 16 cmH2O

CMV 97 % HFJV 92 %

CMV 19 % HFJV 22 %

Life Pulse (Bunnell) with a time-cycled, pressure-limited infant ventilator (Bear Cub) for CMV

IVH, periventricular echodensities, cystic PVL, supplemental oxygen at 28 d and 36 w, mortality at 28 d and 36 w, days on mechanical ventilation, days in hospital

Keszler 130 Multicentre (8), Preterm (<36 w), 700- 100 % NR Life Pulse BPD at 28 d and 36 w post-

CMV = conventional mechanical ventilation, HFJV = high-frequency jet ventilation, RDS = respiratory distress syndrome, NR = not recorded, CLD = chronic lung disease, ALS

= airleaks, IVH = intraventricular haemorrhage, PIE = pulmonary interstitial emphysema, BPD = bronchopulmonary dysplasia, PVL = periventricular leukomalacia, PDA =

patent ductus arteriosus, NEC = necrotising enterocolitis, ROP = retinopathy of prematurity.

et al 1997

(CMV n=65, HFJV n=65)

crossover if failing the assigned therapy, mean randomisation age 8.3 h (CMV) vs. 8.1 h (HFJV) HFJV groups subdivided (following analysis) to HF-OPT (optimal volume strategy) and HF-LO (low pressure strategy)

1500 g, requirement for mechanical ventilation with FiO2 > 0.3 at 2-12 h after surfactant administration, received surfactant by 8 h of age, < 20 h of age, and mechanically ventilated for < 12 h

(before entry to study)

(Bunnell) with standard time-cycled, pressure-limited infant ventilators for CMV

conceptional age, survival, gas exchange, airway pressures, airleak, IVH, PVL, pulmonary haemorrhage, PDA, NEC, ROP

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2.3.3 Clinical Strategies

The ventilation strategy employed by the clinician has important physiological

implications for infants treated with HFJV. Optimising lung volume has been

recognised as an important goal during HFJV (138) but as this requires the use of a

relatively high PEEP it may cause a decrease in cardiac output. This decrease in cardiac

output may be less profound in a preterm infant’s non-compliant lung but is

nevertheless a potential adverse side effect. The initial settings chosen for HFJV are

PIP, respiratory frequency, tI, PEEP (on the conventional ventilator). The latter

parameters will determine the tI:tE ratio. Furthermore, a decision must also be made

about whether or not CMV breaths should be delivered.

2.3.3.1 PIP

The optimal PIP during HFJV is determined by PaCO2. The initial setting however has

been made in accordance with the CMV PIP prior to cross-over. Keszler et al (1997)

started the HFJV PIP at the same level as CMV PIP (138) and found that it needed to be

decreased rapidly thereafter to avoid hypocapnia. This comparison to CMV PIP may be

valid, but it is likely that a higher PIP can be used during HFJV for less haemodynamic

expense given that the HFJV airway pressure waveform is attenuated (55). While

changes during patient management are more intuitive (based upon PaCO2) the

starting point is arguably more difficult to determine and there is little evidence in the

literature to support a particular decision making process.

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2.3.3.2 tI:tE ratio

The respiratory frequency and tI determine the tI:tE ratio during HFJV. As the

exhalation phase is passive during HFJV, the potential for gas trapping is decreased at

lower respiratory frequencies. The initial respiratory frequency and tI is rarely

documented in the literature. Wiswell et al (1996) commenced at 420 breaths/min and

0.02 s respectively, giving an tI:tE ratio of 1 : 6.1. Changes to these settings are made if

there is evidence of gas-trapping, in which case respiratory frequency is decreased,

increasing the tI:tE ratio (up to a maximum of 1:12). The advantages of the passive

exhalation phase are greater when respiratory frequency is decreased.

2.3.3.3 PEEP

The initial PEEP has been set at 6-8 cmH2O (138) and 4-5 cmH2O (139) but the key to

maintaining oxygenation is maintaining Paw. PEEP is therefore altered to optimise

oxygenation but the maximum PEEP tolerated has not been investigated. Paranoia

about the expense of high PEEP during CMV has been transferred to HFJV. The use of

low PEEP during HFJV necessitated delivery of CMV breaths and high jet PIP to achieve

adequate Paw, alveolar recruitment and satisfactory oxygenation. A direct

consequence of this approach was the generation of large ∆P, which promoted

hypocapnia. As the attenuation of the airway pressure waveform during HFJV is better

understood, higher PEEPs are being used, but once again, there is little evidence in the

literature to support or refute a suitable PEEP range during HFJV.

Transitioning from HFOV to HFJV may be indicated in some patients, especially in the

instance of airleak or PIE. To maintain open alveoli when changing from one modality

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to the other, maintenance of Paw is essential. Bass et al (2007) investigated the

accuracy of the Life Pulse high-frequency jet ventilator for measuring Paw during HFOV

and then determined a calculation for the PEEP setting required to maintain Paw (180).

In their ex vivo study they found the Life Pulse to be an accurate monitor of airway

pressure during HFOV and predicted PEEP was related to actual PEEP by Equation 2-E:

Equation 2-E

Actual PEEP = (Predicted PEEP x 1.12) – 2.38

The greatest difference was 2 cmH2O (180). This work makes the transition from HFOV

to HFJV less likely to derecruit alveoli, but the same comparison does not exist for

CMV.

2.3.3.4 CMV Breaths during HFJV

The delivery of CMV breaths during HFJV is used to recruit alveoli but the optimal

frequency and characteristics of these breaths is not known. The literature reports

CMV breath rates delivered 2-5 times/min with an tI of 0.5-0.8 s (138) or 5-10

times/min (139). The impact of CMV breath PIP, tI and frequency on oxygenation, CO2

removal, VILI and cardiac output is unknown.

2.4 Summary

The development of HFJV as a clinical tool has been somewhat haphazard. This has led

to a period of HFJV characterised by hypocapnia (most likely due to PEEP paranoia) and

uncertainty regarding the incidence of adverse outcomes (especially neurological). The

recognition of its usefulness and the importance of an open lung ventilation strategy

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have certainly contributed to more recent success with HFJV and it is widely used in

NICUs in the U.S.A. The full potential of HFJV is yet to be realised and with a better

understanding of the impact of different pressures and HFJV and CMV combinations

the management of preterm infants will improve.

The mechanisms of gas transport during HFJV are unique and to exploit this

therapeutic tool to its full potential a thorough understanding of these mechanisms

and the consequences of particular strategies in different clinical scenarios is

necessary. Despite extensive use, and in spite of limited randomised controlled trials,

the intricacies of gas exchange, airway pressure changes along the bronchial tree are

documented, but their impact on lung tissue and the cardiovascular system are for the

most part unknown. This thesis explores HFJV in an experimental setting with

emphasis on alveolar recruitment manoeuvres and their impact on blood flow and

lung tissue.

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3 General Methodology

This chapter provides a detailed description of the materials and methodology relevant

to the studies described in this thesis. Information specific to a particular protocol is

presented within the relevant chapter. All animal procedures were approved by the

University of Western Australia animal ethics committee, according to the guidelines

of the National Health and Medical Research Council of Australia code of practice for

the care and use of animals for scientific purposes.

3.1 Animal Breeding and Welfare

Merino ewes between 5 and 6 years of age were mated over a 24 hour period. Oestrus

and ovulation was synchronised by the insertion of intra-vaginal sponges for the 14

days prior to mating. Pregnancy was confirmed 55-85 days later by transabdominal

ultrasound examination.

Date mated pregnant ewes were transported from the farm of origin in Darkan,

Western Australia at no later than 100 d gestation to either the Large Animal Facility at

the University of Western Australia or the Shenton Park Biomedical Research Facility.

During the week prior to transport, ewes were inspected by a veterinary surgeon to

ensure there was no evidence of disease or injury and that their body condition score

was adequate (minimum 2.5/5). At the Large Animal Facility, ewes were housed in

either single raised pens adjacent to one another or a raised communal pen (4 m x 4.7

m maximum capacity 16 sheep). They were fed a ration of chaff, lupins and pellets

84

with free access to water through a dripper or in a bucket. At the Shenton Park

Biomedical Research Facility, ewes were run in a paddock and supplementary feed was

supplied if necessary.

3.1.1 Nutrition

3.1.1.1 Instrumented Lambs

For studies involving surgical instrumentation of the fetus prior to delivery, food was

withheld from the ewes 24 hours prior to surgery to minimise the potential for

regurgitation and aspiration of rumen contents, to decrease the incidence of bloat and

to reduce compression of the caudal vena cava when positioned in dorsal recumbency.

All ewes had free access to water up until the induction of anaesthesia.

3.1.1.2 Non-instrumented Lambs

Ewes bearing lambs that were not instrumented in utero had food withheld overnight

prior to non-recovery surgical delivery. All ewes had free access to water up until the

induction of anaesthesia.

3.1.2 General Anaesthesia and Instrumentation

3.1.2.1 Lambs Instrumented in utero

At 128-130 days of gestation, anaesthesia was induced with an intramuscular injection

of xylazine (0.2 mg kg-1; Xylazil 20 mg mL-1, Troy Laboratories, Australia) and ketamine

(15 mg kg-1; Ketamil 100 mg mL-1, Troy Laboratories, Australia). A cuffed oral

endotracheal tube (7.5 mm internal diameter, Portex Ltd, England) was positioned and

secured to facilitate maintenance of anaesthesia with isoflurane in 100 % oxygen

85

delivered through a circle breathing system. Throughout surgery the ewe was

ventilated with a volume cycled bag-in-the-bottle mechanical ventilator to maintain

normocapnia (Ohio 7000, Ohio Medical Products, Division of Airco Inc. Madison,

Wisconsin). A side stream capnograph enabled breath to breath non-invasive

assessment of end expiratory CO2 concentration.

Studies involving pulmonary blood flow and pulmonary arterial blood pressure

measurements required instrumentation of the fetus prior to caesarean delivery. The

anaesthetised ewe was positioned in dorsal recumbency and the abdomen was clipped

and prepared for surgery. The uterus was exposed through a ventral midline incision

and the fetal head was located and exposed via hysterotomy at a poorly vascularised

site of the uterine wall. Two incisions were made for twin pregnancies.

The fetus was partially exteriorised to facilitate access to the lateral thorax. An incision

was made at the fourth intercostal space on the left side. Exposure of the heart was

achieved by blunt dissection through the intercostal muscles and pleura and incision of

the pericardium. The left pulmonary artery was isolated by careful blunt dissection and

an ultrasonic flow probe (4R, Transonic Systems, Ithaca, NY) was positioned around it,

upstream of the ductus arteriosus (Figure 3-A). A tapered polyvinyl intravenous

catheter was inserted through the main trunk of the pulmonary artery by direct

puncture and secured so the tip was located in the left main pulmonary artery. A

suture in the wall of the artery secured the catheter in place. The thoracotomy incision

was closed using silk suture material in a simple continuous pattern.

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Figure 3-A Instrumentation of the fetus: Pulmonary artery flow probe and pulmonary artery catheter in situ

3.1.2.2 Non-instrumented Lambs

An intravenous injection of medetomidine (0.02 mg kg-1; Domitor 1 mg mL-1, Pfizer

Animal Health, U.S.A.) and ketamine (10 mg kg-1; Ketamil 100 mg mL-1, Troy

Laboratories, Australia) was administered to induce anaesthesia in pregnant ewes at

128-130 days of gestation. A subarachnoid (spinal) injection of lidocaine (3 mL;

Lignocaine 20 mg mL-1, Troy Laboratories, Australia) was administered with access

through the intervertebral space between the 4th and 5th or 5th and 6th lumbar

vertebrae. A midline laparotomy incision was made, the position of the lamb was

determined by palpation, and the uterus was incised to facilitate exteriorisation of the

entire fetus.

87

3.1.3 Caesarean Delivery

Immediately prior to delivery, a cuffed oral endotracheal tube (4.5 mm internal

diameter, Portex Ltd, England) was positioned under direct vision and secured with an

umbilical tape tie around the back of the lamb’s ears. Lung fluid was suctioned with

gentle negative pressure and 100 mg kg-1 surfactant was administered [either

beractant; (Survanta 25 mg of phospholipids mL-1, Abbott Laboratories, U.S.A.) or

poractant; (Curosurf 80 mg of phospholipids mL-1, Chiesi Pharmaceuticals Ltd, Parma,

Italy)]. The umbilical vessels were clamped and the lamb was delivered, weighed and

commenced immediately on the assigned ventilation protocol. A cord blood sample

was collected from the umbilical artery as the lamb was delivered and analysed

immediately for baseline blood gas status.

After commencement of ventilation, catheters were inserted into an umbilical artery

and an umbilical vein. The arterial catheter was advanced to approximately 15 cm and

the venous catheter to 9 cm. Serial arterial blood gas samples were collected from the

umbilical artery throughout the 2 or 3 hour ventilation period. Anaesthetic and

analgesic drugs were administered through the umbilical venous catheter to ensure

the lamb was unconscious and nonresponsive to the procedures. Propofol (Repose; 10

% Norbrook Laboratories Ltd., Victoria, Australia) and remifentanil (Ultiva; 1 mg vial

requiring reconstitution, Abbott Laboratories, U.S.A.) were infused at 0.1 mg kg-1min-1

and 0.05 µg kg-1min-1 respectively, in the first instance, and the rate adjusted according

to clinical effect.

The ewe was euthanased immediately after delivery of the lamb(s) by an intravenous

injection of pentobaritone (100 mg kg-1) (Valabarb 325 mg mL-1 Jurox, Australia) into

88

the uterine vein or superficial abdominal vein. Death was confirmed by absence of a

heart beat during thoracic auscultation.

3.2 Ventilator Set-up

The high-frequency jet ventilator (Life Pulse High Frequency Ventilator, Bunnell Inc.,

Salt Lake City, U.S.A.) was used in tandem with a pressure limited, time cycled infant

ventilator with a humidifier (MR850 Humidifier, Fisher and Paykel Healthcare,

Auckland, N.Z.). Studies performed in 2007 used a Bourns ventilator (Bourns Life

Systems BP 200 Infant Pressure Ventilator, California, U.S.A.) for CMV. In subsequent

years CMV was delivered via a Drager Babylog (Babylog 8000+, Drägerwerk, Lubeck,

Germany). A LifePort™ Adaptor (Bunnel Inc, Utah, USA) replaced the usual tracheal

tube adaptor (Figure 3-B) to facilitate injection of inspired gas in high velocity spurts

and monitoring of pressure for estimation of the PIP and mean airway pressure (Paw)

at the end of the tracheal tube.

89

Figure 3-B Ventilator Set Up: Life Pulse High-frequency Jet Ventilator with Conventional Mechanical Ventilator. The High-frequency Jet Ventilator (HFJV) is used in tandem with a Conventional Mechanical Ventilator (CMV). Humidified inspiratory gases pass from the HFJV to the Patient box where a pinch valve interrupts the inspiratory gas to create the jet breaths. This is connected to the LifePort adaptor which in turn connects to a standard tracheal tube. At the LifePort Adaptor, the CMV circuit connects and humidified inspiratory gases pass along the inspiratory limb and expired gases pass along the expiratory limb.

3.3 Data Collection

Continuous information regarding the ventilator frequency, PIP, amplitude of the

pressure waveform (ΔP), positive end expiratory pressure (PEEP), servo pressure, Paw,

rectal temperature, pulse rate, oxyhaemoglobin saturation and fractional inspired

oxygen concentration (FiO2) was available. If the lamb was instrumented, pulmonary

blood flow, pulmonary arterial blood pressure and systemic arterial blood pressure

90

were also monitored continuously. Arterial blood samples were collected and data was

recorded at predetermined intervals. Any unexpected or unusual event was noted.

Arterial blood gas samples were collected as planned and analysed immediately to

provide information for decision making. HFJV PIP was adjusted to achieve permissive

hypercapnia (PaCO2 45-55 mmHg): HFJV PIP was increased (to a maximum of 40

cmH2O) if PaCO2 was above and decreased if PaCO2 was below this range. The initial

fractional inspired oxygen concentration (FiO2) was always 0.4 and adjusted according

to the oxyhaemoglobin saturation (SpO2). FiO2 was increased in increments of 0.1-0.2

(to a maximum of 1.0) if SpO2 dropped below 89 %.

3.3.1 Pulmonary Arterial Blood Pressure and Blood Flow Measurements

Left pulmonary arterial blood pressure was measured and recorded in real time using a

digital data acquisition system (Powerlab 8SP, AD Instruments, N.S.W., Australia). The

catheter was connected to a pressure transducer (Maxxim Medical, Tx, U.S.A.) and the

signal was amplified before it was recorded. The sampling frequency was 1 KHz. Mean

pressure values were calculated from the relevant pressure signals.

Left pulmonary arterial blood flow was also recorded in real time using a volume flow

meter (Transonic Systems T108, Neomedix Pty. Ltd., N.S.W., Australia). The transonic

flowmeter incorporates wide beam illumination whereby two transducers pass

ultrasonic signals back and forth, alternately intersecting the flowing blood in

upstream and downstream directions. The flowmeter derives an accurate measure of

the ‘transit time’ it takes for a sound wave to travel from one transducer to the other.

The difference between the upstream and downstream integrated transit times is a

91

measure of blood volume flow and is independent of vessel diameter (Figure 3-C). The

transducers and flowmeter were calibrated every day before recordings began. Zero

and 100 mmHg was calibrated on the transducers and 0 and 400 mL min-1 on the

flowmeter.

Figure 3-C Widebeam Illumination: Schematic view of the perivascular Transonic ultrasonic volume flow-sensor. Using wide beam illumination, two transducers pass ultrasonic signals back and forth, alternately intersecting the flowing liquid in upstream and downstream directions. The flowmeter derives an accurate measure of the ‘transit time’ it takes for the wave of ultrasound to travel from one transducer to the other. The difference between the upstream and downstream integrated transit times is a measure of volume flow rather than velocity (189)

Pulmonary waveform data were recorded continuously throughout the ventilation

period (190). Measurements of mean PBF and pulse-by-pulse minimum values at the

end of diastole and systole were computed from the PBF waveform over 5 consecutive

waveforms at each time point. Mean systolic and diastolic PBF was also calculated at

each time point by including 5 consecutive waveforms. Pulsatility Index, a measure of

downstream resistance to blood flow, was calculated:

Equation 3-A

)cycles econsecutiv 5 over flow systolic peak mean

diastole after flow minimumflow systolic Peak(yIndexPulsatilit

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All data were analysed using a computer software package (Chart v4.2 Powerlab,

ADInstruments, N.S.W., Australia).

3.4 Euthanasia and Post Mortem

The ewes were euthanased immediately following delivery. Lambs were euthanased at

delivery (unventilated controls) or the end of the ventilation protocol. Pentobarbitone

(100 mg kg-1; Valabarb 325 mg mL-1, Jurox, Australia) was administered by intravenous

injection into either the uterine vein (ewe) or umbilical vein (lamb) and death was

confirmed when thoracic auscultation of the heart was negative. Prior to euthanasia

the lungs were ventilated with 100% oxygen for 3 minutes, to promote alveolar

collapse. A third of the dose of pentobarbitone was delivered, the chest cavity was

evacuated, the tracheal tube clamped, and the remainder of the dose of

pentobarbitone delivered to euthanase the lamb.

The thoracic cavity of each lamb was opened. The trachea was isolated and a short

tracheal tube was inserted and secured with the tip positioned 3 cm above the carina.

The gas volume of the lung was measured as the volume used to inflate the lung to 40

cmH20 pressure and maintain it at that pressure for 30 s. The lung was then deflated

sequentially, and the residual volume recorded at 40, 20, 15, 10, 5 and 0 cmH2O

pressure to yield a deflation pressure-volume curve.

Bronchoalveolar lavage (BAL) was performed on the left lung three times with saline

and BAL fluid was snap frozen in liquid nitrogen. Cytospin samples were collected and

prepared immediately for cytology and protein analysis by the Lowry method (191).

93

The right upper lung lobe was fixed to 30 cmH2O in formalin for 24 hours prior to

preparation of tissue for immunohistochemistry. The tissue was washed in 0.1 molar

phosphate buffered saline (3 x 10 minutes), 30% ethanol (1 x 10 minutes), 50% ethanol

(1 x 10 minutes) and 70% ethanol (1 x 10 minutes). Lung sections were processed in

alcohol and xylene overnight then embedded in paraffin wax for cutting 5 µm slices for

histopathology.

Samples of the right lower lobe were collected and snap frozen in liquid nitrogen, then

stored at -80°C for later quantification of mRNA using Real-Time Polymerase Chain

Reaction (RT-PCR).

3.4.1 Cell Population of Bronchoalveolar Lavage Fluid

Cytospin samples of BAL fluid were prepared immediately after collection of BAL fluid.

10 mL of fluid was centrifuged for 10 minutes at 4°C, the supernatant removed to

leave a pellet of cells. This was resuspended and diluted with trypan blue for total cell

counts. Differential cell counts were performed at a later date following Diff-Quik

staining to allow identification and differentiation of inflammatory cells, epithelial cells

and other cells (including junk cells and mucoid cells). Total cell count per milliliter of

BAL fluid enabled calculation of total cell count per lamb based upon the formula:

Equation 3-B

Cell count per kg bodyweight = AWc) x BW)(AWv x (LL

RLLL

where LL = left lung weight (g), RL = right lung weight (g), BW = body weight (g), AWv =

alveolar wash volume (mL), AWc = alveolar wash cell count mL-1 BAL fluid.

94

At 40x magnification 200 cells were counted to identify inflammatory, epithelial and

other cells. Further differentiation of inflammatory cells to mononuclear cells

(including macrophages), neutrophils, lymphocytes, eosinophils and basophils was also

performed. The division of cell numbers within the 200 cell sample was used to

extrapolate to the total cell population.

3.4.2 Bronchoalveolar Lavage Protein Assay

Bronchoalveolar lavage fluid was stored at -80°C until the protein assay was

performed. A standard curve (0-1000 µg mL-1) was created for each set of samples.

Each sample was prepared according to the Lowry method and measured in triplicate

with the Versa Max Microplate Reader Spectrophotometer. Three milliliters of Reagent

1 (Na2CO3 with K+ tartrate, CuSO4 and water) was added to each sample and left to

stand for 10 minutes. Two hundred microlitres of Reagent 2 (Follin’s Reagent and

water) was then added and mixed immediately. After a 30 minute incubation period

(at room temperature) the samples were analysed with the spectrophotometer.

3.4.3 Immunohistochemistry

Immunohistochemical stains were performed for myeloperoxidase (MPO) and

inducible nitric oxide synthetase (iNOS). The dilution of the primary antibody was

1:500 for MPO and 1:250 for iNOS. Biotinylated secondary antibody was diluted 1:200

for each. Anti-rabbit secondary antibody was used for the MPO stain and anti-mouse

for the iNOS stain. A control slide provided by the manufacturer was used for the MPO

staining. Antigen retrieval was performed in NaCitrate buffer (pH 8.45) at 80°C for 30

minutes then room temperature for 60 minutes.

95

Three slices of lung were collected from each lamb, processed overnight and

embedded in paraffin wax. Five micron sections were cut from each block. Each animal

therefore had 3 areas of lung tissue for examination. From each slide 10 fields were

examined giving 30 fields (40x magnification) for each lamb. Each field was

photographed using SPOT insight 4MP, 2048 x 2048 colour mosaic camera with infra

red filter, 14 bit, 20 MHz, C Mount, Firewire, SPOT software through the C Mount

Adaptor 1.2x lens for the Olympus BX Microscope. The number of MPO or iNOS

positive cells were counted in each of the 10 fields/slide and the total cellular area of

those fields was quantified using densitometry software (Image-Pro Plus v4). An

average for each slide was calculated, and then averages for each animal given 3 slides

were examined. The results are expressed as number of positive cells per cellular area

(nm2).

3.4.4 Qualitative Polymerase Chain Reaction

Total RNA was isolated from 30 mg of homogenised lung tissue using the RNeasy Mini

kit (Qiagen, U.S.A.) according to the manufacturer’s instructions. The contaminating

genomic DNA was removed by an on-column DNaseI digestion performed using the

DNaseI digestion kit (Qiagen, U.S.A.). One microgram of RNA was then reverse

transcribed into complementary DNA in a 20 µL reaction with QuantiTect® Reverse

Transcription Kit (Qiagen, U.S.A.). The primers used for amplifying IL-1β and IL-6 (192)

and IL-8, EGR1 and CTGF (111) have been described elsewhere. Amplification and

detection of specific products were conducted on the Rotor-gene 3000 real time PCR

system (Corbett Life Science) with the published cycle profiles using Rotor-gene SYBR

Green PCR Kit (Qiagen, U.S.A.) following the manufacturer’s instructions. The

96

expression levels of genes of interest were normalised into 18S RNA (193) using the 2-

∆∆CT method (194) and presented as expression ratio relative to the unventilated

control group.

3.4.5 Myeloperoxidase Activity in Lung Tissue

Myeloperoxidase (MPO) activity was measured spectrophotometrically using methods

described by McCabe et al in 2001 and Faith et al in 2008 (195, 196). Minor

modifications were made: lung tissue samples were homogenised in 50 nmol L-1

potassium-phosphate buffer (pH 6.0), containing 5 mg mL-1

hexadecyltrimethylammonium bromide. The samples were subjected to 3 cycles of

freeze-thaw, followed by sonication. The suspensions were then centrifuged at 10 000

rpm for 10 minutes. Ten microlitres of supernatant was mixed with 290 µL of 50 mmol

L-1 phosphate buffer (pH 6.0), containing 0.167 mg o-dianisidine dihydrochloride and

0.0005 % hydrogen peroxide in a standard 96 well microtiter plate. The changes in

absorbance at 30 second intervals were recorded at 450 nm. The MPO activity was

then normalised to the total protein content of the tested samples. Activity was

expressed as units of MPO activity per mg of protein, where one unit of MPO was

defined as the amount needed to degrade 1 µmol of hydrogen peroxide per minute at

room temperature.

3.5 Statistical Analyses

Parametric data were analysed with a Student’s t test (for comparisons against one

other group) and one way analysis of variance (ANOVA) for multiple group

comparisons. Non-parametric data were compared with a Rank Sum test for

97

comparisons between two groups or ANOVA on Ranks for multiple comparisons. A

difference was considered significant if p < 0.05. Statistical analyses were performed

using SigmaStat (Version 3.5, Systat Software Incorporated, U.S.A.) and values in the

text are expressed as the mean ± the standard deviation (SD), standard error of the

mean (SEM), if multiple values were collected for each data point or Median (25, 75

centile).

98

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4 High Positive End‐Expiratory Pressure during High Frequency

Jet Ventilation Improves Oxygenation and Ventilation in Preterm

Lambs.

Gabrielle C Musk1, Graeme R Polglase1, J Bert Bunnell2, Carryn J McLean1, Ilias Nitsos1,

Yong Song1 and J Jane Pillow1.

1 School of Women’s and Infants’ Health, University of Western Australia, Perth, Western

Australia, 6009, Australia.

2 Bunnell Inc, Salt Lake City, Utah, USA and Department of Bioengineering, University of

Utah, Salt Lake City, Utah, USA.

This first study investigated the role of positive end‐expiratory pressure for alveolar

recruitment during high‐frequency jet ventilation.

122

Abstract

Increasing positive end‐expiratory pressure (PEEP) is advocated to recruit alveoli during

high‐frequency jet ventilation (HFJV) but its effect on cardiopulmonary physiology and

lung injury is poorly documented. We hypothesised that high PEEP would recruit alveoli

and reduce lung injury but compromise pulmonary blood flow (PBF). Preterm lambs of

anaesthetised ewes were instrumented, intubated and delivered by cesarean section after

instillation of surfactant. HFJV was commenced with a positive end‐expiratory pressure

(PEEP) of 5 cmH2O. Lambs were allocated randomly at delivery to remain on constant

PEEP (PEEPconst, n=6) or to recruitment via stepwise adjustments in PEEP (PEEPadj, n=6) to

12 cmH2O then back to 8 cmH2O over the initial 60 min. Pulmonary blood flow was

measured continuously while ventilatory parameters and arterial blood gases were

measured at intervals. At postmortem, in situ pressure‐volume deflation curves were

recorded, and bronchoalveolar lavage fluid and lung tissue were obtained to assess

inflammation. PEEPadj lambs had lower pressure amplitude, fractional inspired oxygen

concentration, oxygenation index and PBF, and more compliant lungs. Inflammatory

markers were lower in the PEEPadj group. Adjusted PEEP during HFJV improves

oxygenation and lung compliance and reduces ventilator requirements despite reducing

pulmonary perfusion.

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Introduction

High‐frequency ventilation (HFV) is advocated as a lung protective ventilation strategy for

the treatment of respiratory distress syndrome (RDS) in preterm newborn infants. HFV has

proven particularly useful for optimising lung volume, reducing atelectotrauma and

volutrauma, and therefore reducing injurious lung stimuli associated with

bronchopulmonary dysplasia (BPD) (1‐3). High frequency jet ventilation (HFJV) and high‐

frequency oscillatory ventilation (HFOV) are the two main forms of HFV used in neonatal

intensive care units and while there is substantial research on the optimal approach to

lung volume optimisation in preterm RDS using HFOV, data are limited for HFJV. To date

there is one multicentre controlled trial and one single centre controlled trial comparing

the use of HFJV (with a low positive end‐expiratory pressure (PEEP) strategy) to

conventional mechanical ventilation (CMV) for treatment of preterm RDS (4, 5). The

results give conflicting information regarding the respiratory and neurological outcomes of

neonates treated with HFJV. Increased adverse neurological outcomes for HFJV group in

the Wiswell study (1996) were attributed to hypocarbia (6). Subgroup analysis in the

Keszler trial (1997) suggested that the use of low PEEP was associated with an increased

risk for grade III‐IV intraventricular hemorrhage or periventricular leukomalacia. An

important limitation of these trials is that the HFJV groups were not compared to a true

“lung protective” CMV strategy. Further research assessing the pros and cons of optimised

versus low PEEP in HFJV is therefore warranted.

124

HFJV is characterised by a (normally) fixed brief inspiratory time, passive expiration, and

coupling with a conventional ventilator for provision of conventional breaths, positive

end‐expiratory pressure (PEEP) and bias flow. The current recommended strategy for

treatment of RDS with HFJV is to commence HFJV early in the disease process with a peak

inspiratory pressure (PIP) just below that being used during conventional mechanical

ventilation (CMV) (http://www.bunl.com/7%20Steps%20NO%20QT.html). The initial PEEP

is set to achieve a mean airway pressure (Paw) equal to that used prior to commencement

of HFJV. The primary method advocated for optimising lung volume recruitment in HFJV is

by incrementing PEEP until stable peripheral oxyhemoglobin saturation (SpO2) is achieved,

with low‐rate CMV breaths added to HFJV as a supplementary method for recruiting

collapsed alveoli.

Despite the detailed guidelines provided for optimising lung volume during initiation of

HFJV, the evidence basis for this approach is limited. Whereas inadequate PEEP will

encourage airway collapse and atelectasis and initiate the lung injury sequence, excessive

PEEP promotes alveolar overdistension, impedes pulmonary perfusion, decreases venous

return, and depresses cardiovascular function (7‐9).

We hypothesised that a PEEP driven lung volume recruitment protocol would enhance

arterial oxygenation and ventilation without promoting lung injury during the initiation of

ventilation in a preterm ovine model of RDS. Furthermore, we hypothesised that these

effects would be achieved at the expense of pulmonary perfusion. We aimed to compare

125

the effect of an adjusted PEEP strategy on pulmonary blood flow (PBF), blood gases and

lung injury with a constant low PEEP strategy in an instrumented preterm lamb model.

126

Materials and Methods

All animal procedures were approved by the University of Western Australia animal ethics

committee, according to the guidelines of the National Health and Medical Research

Council of Australia code of practice for the care and use of animals for scientific purposes

(10).

Animals, Instrumentation and Delivery

Single and twin‐bearing date‐mated merino ewes were anaesthetised at 128‐130 d

gestation (term is ~ 150 d) with intramuscular xylazine (0.5 mg kg‐1, Troy Laboratories,

N.S.W., Australia) and ketamine (20 mg kg‐1, Parnell Laboratories, N.S.W., Australia), and

intubated (7.5 mm cuffed tracheal tube, Portex Ltd. England). Maternal anaesthesia was

maintained with halothane in 100% O2. The fetus was exteriorised and a right lateral

thoracotomy was performed. A flow probe (4R, Transonic Systems, Ithaca, NY) was

positioned around the left pulmonary artery and a catheter was inserted into the main

pulmonary artery (7). The fetus was intubated orally (4.5 mm cuffed tracheal tube, Portex

Ltd. England), lung fluid was suctioned and intra‐tracheal surfactant (100 mg kg‐1:

Survanta, 25 mg of phospholipids mL‐1, Abbott Laboratories, U.S.A.) was administered

prior to caesarian section delivery of the lamb. Unventilated controls (UVC; n=6) were

euthanased (pentobarbitone 100 mg kg‐1 i.v.) at delivery without instrumentation.

127

Postnatal care

Instrumented lambs were dried, weighed and randomised to one of two ventilation

groups: constant PEEP (PEEPconst; n=6) and adjusted PEEP (PEEPadj; n=6). They were

commenced on HFJV according to a predetermined protocol (Figure 1). Umbilical venous

and arterial catheters were inserted. Propofol (0.1 mg/kg/min; Repose 10%, Norbrook

Laboratories Ltd., Victoria, Australia) and remifentanil (0.05 µg/kg/min; Ultiva, Abbott

Laboratories, U.S.A.) were infused continuously through an umbilical vein for anaesthesia

and analgesia. An umbilical arterial catheter was used for continuous measurement of

systemic arterial blood pressure and intermittent sampling to assess gas exchange and

acid‐base balance. Rectal temperature was monitored continuously and maintained

between 38° and 39° C (normothermic for newborn lambs).

The Oxygenation Index (OI) was calculated as OI=2

2

PaO

100 x Paw xFiO where FiO2 is fractional

inspired oxygen concentration, Paw is mean airway pressure and PaO2 is partial pressure

of oxygen in arterial blood.

High Frequency Jet Ventilation

HFJV (Life Pulse High Frequency Ventilator, Bunnell Inc., Salt Lake City, U.S.A.) coupled to a

pressure‐limited time‐cycled infant conventional ventilator (Bourns Life Systems BP 200

Infant Pressure Ventilator, California, U.S.A.) was commenced immediately following

delivery. HFJV was commenced using a respiratory rate of 420 breaths per minute (7 Hz)

128

and peak inspiratory pressure (PIP) of 40 cmH2O. HFJV PIP was adjusted to a maximum of

40 cmH2O to target moderate permissive hypercapnia (Partial pressure of carbon dioxide

in arterial blood (PaCO2) 45‐55 mmHg). The initial FiO2 of 0.4 was adjusted to maintain

peripheral oxyhemoglobin saturation (SpO2) of 90‐95%. Inspiratory time (tI) was fixed at

0.02 s. No conventional ventilator breaths were applied during the 2 hour study period.

PEEP was maintained at 5 cmH2O in the PEEPconst group for the duration of the study.

Lambs in the PEEPadj group were stabilised on a PEEP of 5 cmH2O for 10 min then PEEP

was incremented at 10 min (8 cmH2O), 15 min (10 cmH2O) and 20 min (12 cmH2O). PEEP

was decreased by 2 cmH2O at 35 min and 60 min and then maintained at 8 cmH2O from

60 min until euthanasia at 120 min.

Continuous measurements of PBF, pulmonary artery blood pressure (PAP) and systemic

arterial blood pressure (ABP) were processed via calibrated pressure transducers (Maxxim

Medical, Tx, U.S.A.). Data were amplified and stored on a digital data acquisition system

(Powerlab 8SP, ADInstruments, N.S.W., Australia). Pulmonary waveform analysis was

performed at regular time points as described previously (11). Pulsatility Index, a measure

of downstream resistance to blood flow, was calculated as (peak systolic flow – minimum

flow after diastolic flow)/mean peak systolic flow over five consecutive cardiac cycles).

Tidal volume (VT) was measured continuously using an electronic flowmeter (Florian,

Acutronics, CH). Ventilator settings (respiratory rate, PIP, PEEP; Paw, Delta P (∆P), Servo

Pressure and ti) were recorded at intervals. After final measurements were obtained, the

129

FiO2 was increased to 1.0 for 2 minutes, the tracheal tube was occluded for 3 min, and the

lamb was euthanased (100 mg kg‐1 pentobarbitone i.v.).

Post‐mortem

The lung was exposed by thoracotomy, and an in situ deflation pressure volume curve was

obtained (12). The right upper lung lobe was inflation fixed (30 cmH2O) in formalin and

samples of the right lower lobe were snap frozen for molecular analyses. Bronchoalveolar

lavage (BAL) was performed on the left lung for cytology and protein analysis by the Lowry

method. Differential cell counts were performed on cytospin samples of the BAL fluid

stained with Diff‐Quik (Fronine Lab Supplies, N.S.W., Australia).

Total RNA was isolated from 30 mg of homogenised lung tissue using the RNeasy Mini kit

(Qiagen, U.S.A.) according to the manufacturer’s instructions. The contaminating genomic

DNA was removed by an on‐column DNaseI digestion performed using the DNaseI

digestion kit (Qiagen, U.S.A.). One microgram of RNA was then reverse transcribed into

complementary DNA in a 20 µL reaction with QuantiTect® Reverse Transcription Kit

(Qiagen, U.S.A.). The primers used for amplifying interleukin (IL) 1β and IL‐6 (13) and IL‐8,

early growth response (EGR) 1, connective tissue growth factor (CTGF) and cysteine rich

61 (CYR 61) have been described elsewhere (14). Amplification and detection of specific

products were conducted on the Rotor‐gene 3000 real time PCR system (Corbett Life

Science) with the published cycle profiles using Rotor‐gene SYBR Green PCR Kit (Qiagen,

U.S.A.) following the manufacturer’s instructions. The expression levels of genes of

130

interest were normalized into 18S RNA (15) using the 2‐∆∆CT method (16) and presented as

expression ratio relative to the unventilated control group (UVC).

Myeloperoxidase (MPO) activity was measured spectrophotometrically using methods

described by McCabe et al in 2001 and Faith et al in 2008 (17, 18). The MPO activity was

normalized to the total protein content of the tested samples. Activity was expressed as

units of MPO activity per mg of protein, where one unit of MPO was defined as the

amount needed to degrade 1 µmol of hydrogen peroxide per minute at room

temperature.

Statistical Analyses

For comparison of 2 groups of ventilated animals at specific time points, a Mann‐Whitney

Rank Sum test (non‐parametric data) or a Student’s t‐test (parametric data) was used.

Comparisons of two ventilated groups against the unventilated controls used one‐way

analysis of variance (ANOVA). A two‐way repeated measure ANOVA was used to

determine the effect of PEEP on PBF and Pulsatility Index and the effect of time on PIP,

Delta P, Paw and Servo Pressure. Data in the text and legends are expressed as mean

(SEM) or median (25,75 centile) unless otherwise stated. Analyses were performed using

SigmaStat (Version 3.5, Systat Software Incorporated, U.S.A.) with p<0.05 considered

statistically significant.

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Results

Baseline characteristics of lambs in each group were not different (Table 1).

Ventilator Settings and Lung Mechanics

Changes in Paw reflected the different PEEP protocols (Figure 2A) but as PIP decreased in

both groups, the amplitude of the airway pressure waveform (∆P) also decreased (Figure

2B). The VT was higher in PEEPadj group (pooled time points: p <0.01) despite a significantly

lower ∆P (Figure 2C). Servo pressure was lower (p = 0.026) in the PEEPadj group at 120 min

(Table 2). Pressure‐volume curves showed a higher volume achieved per unit of pressure

for the PEEPadj lambs compared to the PEEPconst lambs (p = 0.003 at 40 cmH2O) and for

both of the ventilated groups compared to the UVCs (Figure 2D).

Oxygenation

The target SpO2 of 90% to 95% was achieved in both groups within the first 10 minutes

and was maintained throughout the ventilation period. FiO2 and OI were lower in the

PEEPadj group compared to the PEEPconst lambs from 45 minutes until the end of the

ventilation period (Figure 3A and 3B).

Blood gases

The target PaCO2 was achieved within 10 minutes in both groups and was maintained

between 45‐55 mmHg throughout the ventilation procedure (Table 2). The pH, arterial

132

lactate concentration and base excess (BE) were comparable between the two groups for

the duration of the ventilation period (Table 2).

Hemodynamic consequences of different PEEP protocols

PBF increased over the first 10 min of ventilation in both groups and there were no

significant differences between the groups at any time point between 10 and 120 min.

PBF decreased by approximately 8% in the PEEPconst group and by approximately 48% in

the PEEPadj group between 10 and 120 min (p = 0.507 and p = 0.026 respectively) (Figure

4A). After an initial decrease during the transition from fetal to neonatal circulation, the

Pulsatility Index increased in both groups over time and was significantly higher in the

PEEPadj group over much of the first 60 min (Figure 4B). The pulmonary and systemic

arterial blood pressures were no different between the groups at any time point (Table 2).

End diastolic and end systolic pulmonary blood flow were significantly decreased at 15

min in the PEEPadj group (p<0.001 for each parameter). For all pulmonary artery variables,

the difference between PEEPconst and PEEPadj was temporary (Figure 4C and 4D).

Lung Injury

Bronchoalveolar Lavage Fluid

BAL fluid protein concentration was higher in ventilated groups than in the unventilated

controls, but no difference between PEEPadj and PEEPconst was observed. The cell

populations of BAL fluid did not differ between the groups (Table 3).

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Lung Tissue

Compared to unventilated controls, IL‐1β, IL‐6, IL‐8, CTGF and EGR1 mRNA was elevated in

the PEEPconst group compared to UVCs, whereas CYR 61 expression was significantly

elevated in the PEEPadj group compared to UVCs. The expression of IL‐1β, IL‐6, EGR1 and

CTGF was greater in the PEEPconst group compared to PEEPadj. There was no difference in

MPO activity (Table 3) between each of the three groups.

134

Discussion

Alveolar recruitment during HFJV can be achieved by delivering CMV breaths or adjusting

PEEP, or both. To examine the role of PEEP for alveolar recruitment during HFJV, this study

aimed to compare the effect of an adjusted versus a constant PEEP protocol on

oxygenation, ventilation, pulmonary haemodynamics and lung injury during HFJV. We

showed that lambs in the PEEPadj group had better oxygenation and lung compliance but

decreased PBF compared to PEEPconst lambs. Markers of lung injury were higher in the

PEEPconst group in this short‐term study.

The correlation between Paw and oxygenation during HFJV is well understood. As

expected by study design, the Paw of the PEEPadj group was significantly higher than the

PEEPconst group throughout the ventilation period. The benefit of higher Paw was

evidenced by lower FiO2 requirements from as early as 45 min, supporting the concept

that a high Paw strategy enhances arterial oxygenation, decreasing the FiO2 required

during ventilation (19). In a clinical setting, PEEP is increased and FiO2 is decreased until

the SpO2 or PaO2 plateaus or falls. When FiO2 is stable at 0.21 optimal PEEP has been

achieved (20). The OI describes the relationship between FiO2, Paw and PaO2: a lower

value implies better arterial oxygenation (21). After the initial decrease in OI within the

PEEPconst group, the OI increased over the remainder of the study. In the PEEPadj group it

remained relatively low and stable despite the higher Paw, suggesting more efficient gas

exchange due to either airway stenting, or increased gas‐exchange surface due to

effective volume recruitment.

135

Servo pressure is the automatically controlled driving pressure of the ventilator and

changes in response to altered monitored airway pressure, to ensure that the ventilator

will continue to deliver inspiratory gas to meet the set PIP (22). Servo pressure increases

as PIP increases, with increased lung and/or chest wall compliance, a decrease in airway

resistance, or if there is an air‐leak or circuit disconnection. A decrease in servo pressure

however, indicates either a reduction in set PIP, or worsening compliance and increased

resistance to gas flow, obstruction of the tracheal tube, tension pneumothorax, the

requirement for suctioning or right mainstem bronchus intubation

(www.bunl.com/ServoSlidesNEW.html). Our finding of a lower Servo pressure throughout

most of the study period in PEEPadj lambs despite evidence of increased lung compliance,

likely reflects the reductions in PIP required to maintain moderate permissive

hypercapnea and avoid overventilation.

The haemodynamic consequences of the ventilation protocol were assessed by

measurement of PBF, PAP, systemic ABP, pulse rate and calculation of Pulsatility Index.

Pulsatility Index is directly related to resistance to blood flow. An increase in intrathoracic

pressure during any kind of positive pressure ventilation impacts on venous return,

cardiac output, right ventricular end diastolic volume, PBF and pulmonary vascular

resistance (23). Increased PEEP reduces PBF during conventional ventilation of very

premature lambs by increasing pulmonary vascular resistance (PVR) (8). A similar decrease

in PBF in response to Paw driven alveolar recruitment maneuvers during HFOV of up to

69.3% is also reported (7). Importantly, during HFOV, the fall in PBF persisted after Paw

136

was decreased following recruitment (7). The mechanism causing a fall in PBF as PEEP is

increased may include an increase in the alveolar capillary transmural pressure causing

capillary compression (8, 24). The non‐compliant immature lung may be less susceptible

to capillary compression than a mature lung (8, 24). Factors that increase lung compliance

(e.g. antenatal corticosteroids, exogenous surfactant, lung volume recruitment) may

increase the sensitivity of PBF to changes in airway pressure (24). We observed improved

oxygenation at the expense of PBF when PEEP was increased up to 12 cmH2O, as

previously described during HFOV (8). However, this decrease in PBF was relatively small

and of shorter duration relative to the change associated with increasing Paw during

HFOV (7).

The fall in PBF in the PEEPadj group reversed rapidly as PEEP was initially decreased,

suggesting the impact of a PEEP recruitment strategy on PBF during HFJV was not

sustained. During HFOV however, PBF does not recover after a temporary increase in Paw.

(7). Furthermore, the impact of increased PEEP on end diastolic and end systolic blood

flow coincided with the initial increase in PEEP from 5 cmH2O to 8 cmH2O at 10 min. The

difference between the 2 groups was temporary and despite further increases in PEEP in

the PEEPadj group, the blood flow variables did not remain significantly different. The

maintenance of a higher Paw in the PEEPadj group likely contributed to the continued slow

decline in PBF in this group and returning to PEEP of 5 cmH2O (instead of 8 cmH2O) may

have been prudent. Nevertheless, a direct comparison of HFJV and HFOV in a controlled

137

setting is warranted to determine the impact of each of these ventilation strategies on the

extent and duration of effect on pulmonary haemodynamics.

Lung inflammation is a prelude to lung injury. We examined inflammatory markers that

we anticipated would be increased at 120 min in response to lung injury (14, 25‐27). There

was a clear increase in lung injury across the range of inflammatory markers for the

PEEPconst group, compared to the unventilated controls and for the PEEPconst group

compared to PEEPadj. These findings support our hypothesis regarding reduced lung injury

with PEEP recruitment of the lung during HFJV.

Myeloperoxidase activity has been shown to correlate with IL‐6 expression (17) but our

results did not show a clear relationship between these variables. The MPO activity was

not different between the groups, despite a trend for an increase in the ventilated groups.

It is possible that this is a result of maternal anaesthesia, and this finding in itself warrants

further investigation. It is also possible that a Type II statistical error as a result of the

small group sizes prohibited a demonstrable difference between the groups.

There are a number of limitations to our study. We wanted to examine the physiological

changes associated with each PEEP alteration and required at least 10 minutes to

accommodate and document any changes. Consequently, the adjusted PEEP protocol we

studied does not reflect standard clinical practice given that the 60 min period for

increasing and decreasing PEEP is considerably longer than optimally used in a clinical

setting. and potentially masked significant differences between the ventilated groups.

138

Secondly, surfactant was administered to the lambs prior to the commencement of

ventilation. This practice may not be achieved routinely in a clinical setting. However, our

goal was to isolate the effect of PEEP and standardise and optimise all other aspects of

care. Thirdly, the lambs in our study were anaesthetised and underwent an invasive

surgical procedure. The haemodynamic effects of anaesthesia combined with the physical

impact of instrumentation on lung inflation are likely to impact physiological outcomes.

Lastly, the flowmeter used for measuring VT slightly overestimates tidal volume at 7 Hz

(28). However, as HFJV frequency was constant throughout the study, we would not

expect this to affect comparisons between the ventilatory groups.

In conclusion, adjusted PEEP during HFJV improves oxygenation and lung compliance and

reduces ventilator requirements despite reducing pulmonary perfusion. The majority of

markers of injury were higher when PEEP was constant during HFJV. Evaluation of PEEP

recruitment manoeuvers in human patients is indicated to explore the efficacy of the

technique in the target patient population.

139

Acknowledgements

Surfactant was donated by Abbott Australia. The Life Pulse High Frequency Ventilators

were supplied on long‐term loan by Bunnell Incorporated. We would like to express our

sincere appreciation to the members of the Ovine Research Group for technical assistance

and JRL Hall and Co. for provision and early antenatal care of the ewes.

140

Tables

Table 1: Baseline characteristics

UVC PEEPconst PEEPadj

n (male) 6 (3) 6 (4) 6 (4)

Twin (singleton) 4 (2) 6 (0) 6 (0)

Birthweight (kg) 2.9 (0.4) 3 (0.3) 2.8 (0.2)

Gestational Age (d) 127.2 (0.4) 128 (0.8) 128 (0.8)

Cord pH 7.15 (0.1) 7.15 (0.05) 7.09 (0.1)

Cord PaCO2 (mmHg) 81 (13) 73.8 (7.4) 89.7 (20.8)

UVC = Unventilated Control, PEEPconst = Constant Positive End Expiratory Pressure,

PEEPadj = Adjusted Positive End Expiratory Pressure. Values are mean (SEM).

141

Table 2: Cardiovascular and Respiratory Physiological Measurements

PEEPconst PEEPadj

Time 10 min 60 min 120 min 10 min 60 min 120 min

PIP (cmH2O) 40 (0) 34.5 (2.5) 30.2 (2.3) 40 (0) 32.8 (1.9) 27.9 (1)

Paw (cmH2O) 14.1 (0.9) 13.5 (0.6) 12 (0.6) 14.5 (0.2) 16.7 (0.4)† 13.5 (0.3)†

Servo Pressure (psi) 6.6 (0.8) 8.3 (0.3) 7 (0.3) 7.8 (0.4) 6.8 (0.7) 5.9 (0.3)†

PaCO2 (mmHg) 52.0 (4.6) 41.5 (2) 43.3 (2.6) 39.8 (3.4) 38.2 (3.5) 41.4 (3.1)

pH 7.1 (0.1) 7.4 (0.02) 7.4 (0.04) 7.3 (0.1) 7.3 (0.04) 7.4 (0.03)

Lactate (mg dL‐1) 83.9 (13.4) 67.4 (10.7) 48.1 (10.9) 87.2 (22.6) 67.2 (19) 50.4 (20.1)

Base Excess ‐8.8 (2.1) ‐2.2 (1.4) ‐0.9 (2.1) ‐6.8 (3.2) ‐5.4 (1.9) ‐1.5 (2.2)

Left PAP (mmHg) 43.6 (4.3) 35.6 (3.8) 43.2 (6.8) 48.5 (1.6) 37.4 (3.4) 43.8 (5.6)

Systemic ABP (mmHg) 53.5 (6.2) 50.1 (4.4) 60.3 (6.7) 53 (3.2) 50.2 (3.1) 53.3 (3.4)

Pulse rate (bpm) 140 (7) 170 (15) 194 (8)* 145 (8) 181 (15) 210 (8)*

PIP = Peak Inspiratory Pressure, ∆P = Pressure differential (PIP – PEEP), Paw = Mean

Airway Pressure, psi = pounds per square inch, PaCO2 = partial pressure of carbon dioxide

in arterial blood, PAP = pulmonary arterial blood pressure, Systemic ABP = systemic

arterial blood pressure, bpm = beat per minute. * p<0.001 time 120 compared to time 10

in the same group, † p<0.05 PEEPadj compared to PEEPconst at the same time. Values are

mean (SEM).

142

Table 3: Post mortem inflammatory markers

UVC PEEPconst PEEPadj

BAL fluid

Protein concentration (mg mL‐1) 14.7 (7.4) 120.2 (28)* 89.2 (18.5)*

Inflammatory cells (x 103 kg‐1) 0.3 (0.02) 7.5 (3.9)* 2.0 (1.0)

Neutrophils (x 103 kg‐1) 0 4.4 (2.4) 0.4 (0.2)

Mononuclear cells (x 103 kg‐1) 0.3 (0.02) 2.7 (1.4)* 1.5 (0.8)

Lymphocytes (x 103 kg‐1) 0 0.4 (0.3) 0.1 (0.1)

Lung Tissue

IL‐1β fold change 0.6 (0.4, 3.2) 262.9 (159.3, 350)** 37.2 (18.2, 53.7)‡

IL‐6 fold change 1.0 (0.6, 1.6) 95.5 (57.2, 155.4)* 13.4 (3.2, 23.8)‡

IL‐8 fold change 0.7 (0.6, 2.0) 77.6 (26.1, 101.1)** 28.0 (6.3, 53.9)

EGR1 fold change 1.1 (0.3, 2.9) 48.9 (33.5, 94.7)* 15.6 (5.4, 27.1)‡

CTGF fold change 0.8 (0.6, 2.4) 31.2 (13.3, 44.3)* 4.0 (1.97, 5.9)‡

CYR61 fold change 0.9 (0.7, 1.3) 9.7 (5.4, 20.1) 11.0 (5.9, 12.2)*

MPO ac vity † 18.2 (1.4) 29.3 (3.8) 26.6 (6.1)

IL = interleukin, EGR = early growth response, CTGF = connective tissue growth factor, CYR

61 = cysteine rich 61, MPO = myeloperoxidase. † units per mg of protein where 1 unit of

MPO is defined as the amount needed to degrade 1 µmol of hydrogen peroxide per

minute at room temperature. *p<0.05 compared to UVC, **p<0.001 compared to UVC, ‡

p<0.05 compared to PEEPconst. Values are mean (SEM) or median (25,75 centile).

143

Figure 1

144

Figure 2

145

Figure 3

146

Figure 4

147

Figure Legends

Figure 1 Constant and Adjusted PEEP protocols: Lambs were ventilated at either a

constant PEEP of 5 cmH2O (PEEPconst, solid line) or with stepwise increments and

decrements to PEEP (PEEPadj, grey). Values are Mean (SEM).

Figure 2 Ventilator Settings and Lung Mechanics: A: Mean Airway Pressure (Paw), B:

Delta P (ΔP), C: Tidal Volume, D: Deflation limb of the post‐mortem in situ pressure‐

volume curves. ◊ = PEEPconst, ● = PEEPadj, = Unventilated control. * p<0.05 compared to

PEEPconst ** p<0.05 compared to unventilated control. Values are Mean (SEM).

Figure 3 Oxygenation: A: Fractional inspired oxygen concentration (FiO2), B: Oxygenation

index (OI) over the 120 min ventilation period. ◊ = PEEPconst, ● = PEEPadj. * p<0.05

compared to PEEPconst. Values are Mean (SEM).

Figure 4 Pulmonary Perfusion: A: Pulmonary Blood Flow, B: Pulsatility Index over the 120

min ventilation period, C: End Diastolic Blood Flow, D: End Systolic Blood Flow. ◊ =

PEEPconst, ● = PEEPadj , * p<0.05 compared to PEEPadj ** p<0.05 time 120 min compared to

time 10 min. Values are Mean (SEM).

148

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163:1723‐1729

2. Jobe A 1999 The new bpd: An arrest of lung development. Pediatr Res 46:641‐646


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5. Wiswell T, Graziani, LJ, Kornhauser, MS, Cullen, J, Merton, DA, McKee, L, Spitzer,

AR 1996 High‐frequency jet ventilation in the early management of respiratory

distress syndrome is associated with a greater risk for adverse outcomes.

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6. Wiswell TE, Graziani LJ, Kornhauser MS, Stanley C, Merton DA, McKee L, Spitzer AR

1996 Effects of hypocarbia on the development of cystic periventricular

leukomalacia in premature infants treated with high‐frequency jet ventilation.

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7. Polglase GR, Moss TJM, Nitsos I, Allison BJ, Pillow JJ, Hooper SB 2008 Differential

effect of recruitment manoeuvres on pulmonary blood flow and oxygenation

during hfov in preterm lambs. J Appl Physiol 105:603‐610

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20. Groeneveld ABJ, Schneider AJ 2008 The relationship between arterial po2 and

mixed venous po2 in response to changes in positive end‐expiratory pressure in

ventilated patients. Anaesthesia 63:488‐494

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152

153

5 The Impact of Conventional Breath Inspiratory Time during

High‐frequency Jet Ventilation in Preterm Lambs

Gabrielle C Musk,1* Graeme R Polglase,1 Yong Song,1 and J Jane Pillow1

1School of Women’s and Infants’ Health, the University of Western Australia, M550, 35

Stirling Highway, Crawley, 6009, Western Australia, Australia.

This is the first study investigating the role of conventional mechanical ventilator breaths

for alveolar recruitment during high‐frequency jet ventilation. We isolated the effect of

the inspiratory time of the conventional mechanical ventilation breath in this study.

154

Abstract

Background: The delivery of conventional mechanical ventilator (CMV) breaths during

high‐frequency jet ventilation (HFJV) is advocated to recruit and stabilise alveoli.

Objectives: To establish if CMV breath duration delivered during HFJV influences gas

exchange, lung mechanics and lung injury.

Methods: Sedated newborn preterm lambs at 128 d gestational age were studied. HFJV (7

Hz, PEEP 8 cmH2O, PIPHFJV 40 cmH2O, FiO2 0.4) with superimposed CMV breaths (PIPCMV 25

cmH2O, rate 5 breaths/min) was commenced after delivery and continued for 2 h. CMV

breath inspiratory time (tI) was either 0.5 s (HFJV+CMV0.5: n=8) or 2.0 s (HFJV+CMV2.0:

n=8). Age matched unventilated controls (UVC) were included for comparison.

Results: Serial arterial blood gas analyses were performed. PIPHFJV was adjusted to target a

PaCO2 of 45‐55 mmHg. FiO2 was adjusted to target SpO2 90‐95 %. Static deflation

pressure‐volume curves, bronchoalveolar lavage (BAL) and lung tissue samples were

obtained post‐mortem. Gas exchange, ventilation parameters, static lung compliance and

BAL inflammatory markers were not different between HFJV+CMV0.5 and HFJV+CMV2.0.

Both ventilation groups had higher BAL inflammatory markers and increased iNOS positive

cells on histology compared to UVC, whilst lung tissue IL‐1β and IL‐6 mRNA expression was

higher in the HFJV+CMV2.0 group compared to the UVC group.

Conclusions: Preterm lambs were ventilated effectively with HFJV and 5 CMV

breaths/min. CMV breath duration did not alter blood gas exchange, ventilation

parameters ex vivo static lung mechanics or markers of lung injury over a 2 h study,

155

although consistent trends towards increased inflammatory markers with the longer tI

suggest greater risk of injury.

156

Introduction

High‐frequency jet ventilation (HFJV) is a lung protective ventilation strategy for patients

with respiratory distress syndrome (RDS) (1, 2). As with any ventilation strategy, the aim

during HFJV is to recruit, stabilise and maintain open alveoli to facilitate efficient gas

exchange and to prevent lung injury (3, 4). The primary method advocated for optimising

lung volume recruitment during HFJV includes recruiting collapsed units with low‐rate

conventional mechanical ventilator (CMV) breaths superimposed on the HFJV waveform

(1, 5). The peak inspiratory pressure (PIP) of the CMV breath (CMVPIP) is optimally set

above the opening pressure (lower inflection point) on the inflation pressure‐volume

curve. Alveoli are stabilised in expiration by incrementing positive end‐expiratory pressure

(PEEP) until the target oxyhemoglobin saturation (SpO2) is achieved and maintained

without a background CMV rate (6, 7)

While the delivery of CMV breaths during HFJV is advocated, there is little information to

guide the selection of the inspiratory time (tI) of the CMV breath (8). Extending the

duration of the CMV breath beyond that required to complete tidal volume delivery may

promote aeration of alveolar units with long time constants, but will expose aerated lung

regions to unnecessarily high peak inspiratory pressures for extended times.

The duration of the inspiratory phase of the CMV breath potentially affects the delivered

CMV tidal volume (depending on the time constant of the lung), and the duration of

exposure of alveoli to the set PIPCMV. When HFJV is applied to poorly compliant, atelectatic

157

lungs, the lungs will inflate completely with a short tI. Whereas a longer tI may enhance

recruitment by facilitating aeration of long time constant acini, this approach may expose

existing aerated units to barotrauma.

We aimed to compare the effect of different duration CMV breaths during HFJV on HFJV

ventilator requirements, gas exchange and markers of lung injury in a preterm lamb model

of respiratory distress syndrome. We hypothesised that in the setting of preterm

respiratory distress syndrome, a CMV breath with a long tI would promote lung injury,

without improvement in arterial oxygenation.

158





(9).

Animals, Instrumentation, and Delivery

An intravenous injection of medetomidine (0.02 mg kg‐1, Pfizer Animal Health, U.S.A.) and

ketamine (10 mg kg‐1, Troy Laboratories, Australia) was administered to induce

anaesthesia in pregnant ewes at a mean (SD) of 128 (0.8) d gestation, prior to spinal

anaesthesia (3 mL lidocaine, 20 mg mL‐1, Troy Laboratories, Australia) and surgical

delivery. Lambs were randomised to either unventilated controls (UVC, n=5) that were

euthanased at delivery (pentobarbitone 100 mg kg‐1 i.v., Valabarb, Jurox, Australia)

immediately prior to postmortem, or to one of two HFJV strategies. For ventilation groups,

the fetus was exteriorised through a uterine incision, intubated orally (4.5 mm ID cuffed

tracheal tube, Portex Ltd. England), suctioned and intratracheal surfactant was instilled

(100 mg kg‐1: Survanta, Abbott Laboratories, U.S.A.) prior to delivery of the lamb.

Postnatal care

Lambs were dried, weighed and commenced on their randomised ventilation strategy:

HFJV with CMV breaths delivered over 0.5 s (HFJV+CMV0.5; n=8); and HFJV with CMV

breaths delivered over 2.0 s (HFJV+CMV2.0; n=8). Propofol (0.1 mg kg‐1 min‐1; Norbrook

159

Laboratories Ltd., Victoria, Australia) and remifentanil (0.05 µg kg‐1min‐1; Abbott

Laboratories, U.S.A.) were infused continuously via an umbilical venous catheter for

anaesthesia and analgesia. An umbilical arterial catheter was sampled intermittently to

assess gas exchange and acid‐base status. Core body temperature was monitored

continuously and maintained between 38° C and 39° C. Oxygenation Index (OI) was

calculated as:

OI=1.36 x aO

100 x aw xFiO

2

2

P

P

where FiO2 is fractional inspired oxygen concentration, Paw is mean airway pressure

(cmH2O) and PaO2 is partial pressure of oxygen in arterial blood (mmHg). The constant

1.36 is for conversion of mmHg to cmH2O.

Mechanical Ventilation

HFJV (Life Pulse High Frequency Ventilator, Bunnell Inc., Salt Lake City, U.S.A.) coupled to

pressure‐limited time‐cycled infant conventional ventilator (Bourns Life Systems BP200

Infant Pressure Ventilator, California, U.S.A.) was commenced immediately following

administration of exogenous surfactant. Initial HFJV settings included a respiratory rate of

420 breaths/min (7 Hz), FiO2 0.4 and a PIPHFJV of 40 cmH2O. PIPHFJV was adjusted to achieve

permissive hypercapnia (partial pressure of carbon dioxide in arterial blood (PaCO2) 45‐55

mmHg) to a maximum of 40 cmH2O. FiO2 was altered to maintain SpO2 at 90‐95 %. HFJV tI

was fixed at 0.02 s. Intermittent CMV breaths using the assigned tI were superimposed on

160

the HFJV waveform to a PIPCMV of 25 cmH2O and a rate of 5 breaths/min. The PEEP was

constant throughout at 8 cmH2O.

High‐frequency jet ventilation measured variables (PIPHFJV, PEEP; Paw,HFJV, pressure

differential (∆PHFJV) and servo pressure) were recorded at regular intervals. After obtaining

final measurements at 2 h, the FiO2 was increased to 1.0 for 2 min followed by a 3 min

tracheal tube occlusion to facilitate lung collapse for post‐mortem pressure‐volume curve

and then euthanasia (pentobarbitone 100 mg kg‐1 i.v. Jurox, Australia).

Post‐mortem

The collapsed lung was exposed by thoracotomy, inflated slowly to 40 cmH2O, and an in

situ deflation pressure volume curve was recorded (10). Bronchoalveolar lavage (BAL) fluid

was collected from the left lung for protein analysis (11, 12) and cytology. Differential cell

counts were performed on cytospin samples of the BAL fluid stained with Diff‐Quik

(Fronine Lab Supplies, N.S.W., Australia). The right upper lung lobe was inflation fixed (30

cmH2O) in 10 % formalin. Immunohistochemical staining for inducible nitric oxide

synthase (iNOS) was performed on 5 µm sections of lung tissue (13). Positive cells were

identified and quantified per field as number of cells per total cellular area (nm2) using

densitometry (Image‐Pro Plus version 4.5, Media Cybernetics, U.S.A.).

RNA was extracted from the left lung and reverse transcribed to cDNA (QuantiTect®

Reverse Transcription Kit, Qiagen, U.S.A.). Expression of IL‐1β and IL‐6 was measured by

qRT‐PCR (14) and normalized to 18S RNA (15) using the 2‐∆∆CT method (16).

161


Kruskal‐Wallis one way analysis of variance was used to compare groups at specific time

points while the effect of CMV strategy on ventilator requirements and physiological

changes over the duration of the study was determined using two‐way repeated measure

analysis of variance. A Holm Sidak post‐hoc test was used to determine significance

(p<0.05). Analyses were performed using SigmaStat (Version 3.5, Systat Software

Incorporated, U.S.A.). Data are expressed as mean (SEM) unless otherwise stated.

162

Results

Baseline characteristics of the lambs were not different between groups (Table 1).

Physiological Measurements

HFJV and CMV monitored pressures (PIPHFJV, Paw,HFJV, ∆PHFJV or servo pressure, Fig. 1),

oxygen requirements (FiO2, Fig. 2A) or indices of arterial oxygenation (PaO2 and OI, Fig. 2B

and 2C) were not different between the two ventilation groups over the 2 h study. PaCO2

was similar for each group throughout the study (Fig. 2D). PaCO2 initially fell rapidly, but

subsequently increased with ventilator adjustment and stabilised within the target range

by 60 min.

Post‐mortem static lung compliance was comparable for HFJV+CMV0.5 and HFJV+CMV2.0.

Lung volume was higher in both ventilated groups compared to the UVC group at 15

cmH2O, 20 cmH2O and 40 cmH2O (Fig. 3).

Lung Injury

The total protein concentration of BAL fluid was higher in both ventilated groups

compared to UVCs, but did not differ between HFJV+CMV0.5 and HFJV+CMV2.0 (p=0.38).

Similarly, there was no difference in the total inflammatory cell population of the BAL fluid

between the ventilated groups (p=0.16), although total inflammatory cell counts were

increased compared to UVCs (Table 2).

163

The number of iNOS positive cells was not different between the ventilated groups. Lung

IL‐1β and IL‐6 mRNA expression was significantly greater in the HFJV+CMV2.0 group (Table

2) compared to UVCs.

164

Discussion

Although low rate CMV breaths are often used to assist alveolar recruitment during HFJV,

few data exist to guide clinicians on the selection of rate, size or duration of such CMV

breaths. We investigated the effect of two different inspiratory times for low rate CMV

breaths during HFJV and found no significant differences in gas exchange, ventilation

parameters, static lung compliance or inflammatory markers (as determined by BAL

protein, tissue iNOS or pro‐inflammatory cytokine mRNA expression) between a CMV tI of

0.5 s and 2.0 s.

The primary purpose of CMV breaths during HFJV is to promote alveolar recruitment via

alveolar exposure to a distending pressure above the opening pressure threshold. In

previous studies using similar gestation naïve lambs, we have consistently observed a

pressure of 25 cmH2O to be above the lower inflection point (17). In the current study we

expected the opening pressure to be lower than our previous experience given that the

ventilated lambs received prophylactic surfactant, thus an initial PIPCMV of 25 cmH2O

should achieve recruitment. Although PIPCMV was weaned alongside PIPHFJV to remain 5

cmH2O lower than PIPHFJV throughout the study period, the PIPCMV did not fall below 20

cmH2O or differ between the two ventilation groups at any time during the study.

Nonetheless, it is possible that the PIPCMV was lower than the opening pressure and that

this may account for the failure to demonstrate differences between the short and long

duration PIPCMV.

165

To isolate the effect of CMV breath tI we kept other CMV variables constant between the

two ventilated groups: a CMV breath rate of 5 breaths/min reflects the recommended

upper limit for common clinical practice (1). Likewise, a constant PEEPCMV of 8 cmH2O was

maintained throughout the study. We showed previously that a constant PEEP of 5 cmH2O

was insufficient to stabilise alveoli (18) during HFJV.

Our selection of 0.5 s for the short tI was guided by our previous experience using CMV in

preterm lambs: in the 128 d preterm lamb, 0.5 s is sufficient to complete inspiration with a

constant ventilator flow of 8 L/min with a waveform pattern comparable to that achieved

using 0.3 s ‐ 0.4 s inspiratory times commonly used in small babies (19). We rationalised

the selection of 2.0 s as the long tI for comparison as it would provide at least 1.5 s of

pressure plateau. If an extended tI for the CMV breath enhanced lung volume recruitment

during HFJV, we would have expected improved oxygenation in the group receiving the

2.0 s CMV breath. The absence of any difference in FiO2, PaO2 or OI between the two

ventilator groups suggests a 2.0 s CMV breath has no volume recruitment advantage over

a 0.5 s CMV breath during HFJV in the preterm lamb over 2 hours in the postnatal

transition period.

Whilst no specific advantage of inspiratory time was demonstrated, there was evidence of

improved compliance for both CMV tI strategies: servo pressure initially stayed high

despite a rapid decrease in PIPHFJV and ∆PHFJV, indicative of increased lung compliance.

Servo pressure is the automatically controlled driving pressure of the jet ventilator which

changes in response to monitored airway pressure to ensure that the ventilator continues

166

to deliver sufficient inspiratory gas flow to meet the set PIPHFJV (20). There were no

differences between the ventilator groups in the servo pressure at any time point,

suggesting that similar improvements in compliance were achieved for both groups during

the initial phases. Interpretation of servo pressure in the latter half of the study is more

complicated: decreases in servo pressure in the latter half of the study paralleled further

reductions in PIPHFJV and ΔPHFJV suggesting that the previously achieved level of

compliance was at least maintained. Reduced servo pressure in the absence of

corresponding reductions in ΔPHFJV would have implied decreased compliance and loss of

lung volume. There was a slow increase in OI towards the end of the study in both

ventilated groups likely indicative of alveolar instability and collapse due to the use of

Paw,HFJV or PEEPHFJV that was too low to maintain end expiratory lung volume.

We deliberately used a constant PEEP strategy throughout the study to isolate the effect

of different CMV tI strategies during HFJV on oxygenation from the potentially

confounding effects arising from altered PEEP. In clinical practice, however, adequacy of

PIPCMV and PEEPCMV settings would be important considerations in assessing response to

changes in the OI. Increasing PEEPCMV rather than decreasing PIPHFJV may have effectively

decreased ΔPHFJV and controlled PaCO2 whilst maintaining Paw,HFJV and optimal lung

volume. Regardless, Paw,HFJV was not different between the two groups over the study

duration despite our rigid protocol.

Low rate CMV has a negligible contribution to ventilation during HFJV, during which CO2

removal is primarily influenced by HFJV frequency and HFJV tidal volume. The absence of

167

difference in HFJV parameters between the two ventilator groups is therefore not

surprising given the same HFJV ventilator strategy was used to target the desired PaCO2

range. The initial rapid fall in PaCO2 values to a level below the target range is most likely

related to the initial PIPHFJV selection.

In the absence of benefits of a specific CMV tI strategy on oxygenation or ventilation, the

question remains whether a long CMV breath tI is more injurious than a shorter breath.

Lung injury is preceded by lung inflammation, evidenced by increased inflammatory cells

in the airspaces and lung tissue that release pro‐inflammatory mediators (21). We

hypothesised that lung injury would be increased in the HFJV+CMV2.0 group compared to a

shorter CMV breath tI, and chose to measure inflammatory markers that we anticipated

would increase within 120 min (22‐25). Whereas HFJV+CMV was injurious in both groups

compared to UVC, there were no statistical differences in mRNA expression or static lung

compliance between the two ventilated groups. Nonetheless, all markers of injury (BAL

protein concentration, BAL inflammatory cells, lung tissue iNOS positive cells and lung

mRNA pro‐inflammatory cytokine expression), showed a consistent pattern of higher

levels in the 2.0 s group compared to the 0.5 s group. The lack of statistically significant

difference is likely related to a type II error. Regardless, any potential increase in injury of

a 2.0 s CMV breath tI compared to 0.5 s, is likely to be subtle at most over a 2 h time

frame. Based on our observed differences in the oxygenation index and IL‐6 and IL‐1β a

group size of 21 would have been required to detect a statistically significant difference

with 80% power.

168

Potential mechanisms promoting injury with a long CMV breath tI likely relate to extended

exposure of distal airways and alveoli to high continuous inflating pressures. Using a

PIPCMV less than the PIPHFJV, we deliberately prevented the CMV breath from interrupting

the HFJV breaths. Thus, theoretically at least, longer exposure to ‘jet‐stacking’ may have

been a consequence of this strategy for the HFJV+CMV2.0 group. However, the amplitude

of the HFJV pulses is markedly damped by the time the HFJV pressure waves are

transmitted to the periphery, and jet‐stacking does not result in PIPHFJV more than a few

cmH2O greater than the preset PIPHFJV. It is more likely that any consequence of longer

CMV breath inspiratory times are related to the PIPCMV (which is fully transmitted to the

periphery) rather than any stacking of HFJV breaths resulting from this approach.

Conclusions

We found no significant differences in gas exchange or markers of injury and inflammation

between HFJV with CMV breaths delivered with an tI of 0.5 s or 2.0 s over 2 h. However, a

consistent tendency for increased inflammatory markers when CMV breaths were

delivered with an tI of 2.0 s compared to a tI of 0.5 s suggests the use of longer CMV

breath inspiratory times may be more injurious if continued over a longer time frame, and

warrants further investigation. Further studies to provide evidence to support strategies

aiming to establish optimal lung volume recruitment during HFJV are recommended.

169

Acknowledgements

We would like to express our sincere appreciation to the members of the Ovine Research

Group, Ilias Nitsos and Carryn McLean, for technical assistance, JRL Hall and Co. for

provision and early antenatal care of the ewes and Professor Karen Simmer for support

and encouragement.

170

Tables

Table 1: Baseline characteristics

UVC HFJV+CMV0.5 HFJV+CMV2.0

n (male) 6 (3) 8 (6) 8 (4)

Twin (singleton) 4 (2) 8 (0) 8 (0)

Birthweight (kg) 2.9 (0.4) 2.9 (0.2) 3.0 (0.2)

Cord pH 7.15 (0.04) 7.15 (0.05) 7.16 (0.05)

Cord PaCO2 (mmHg) 81.0 (5.8) 73.8 (9.2) 80.6 (10.1)

UVC = Unventilated Control, HFJV+CMV0.5 = HFJV with CMV breaths delivered with

an inspiratory time of 0.5 s, HFJV+CMV2.0 = HFJV with CMV breaths delivered with an

inspiratory time of 2.0 s. Values are mean (SEM).

171

Table 2: Post‐mortem inflammatory markers

UVC HFJV+CMV0.5 HFJV+CMV2.0

BAL fluid

Protein concentration (mg mL‐1)

14.7 (7.4) 164.1 (27.2)* 197.8 (32.5)*

Total Inflammatory Cells (cells x 106 kg‐1)

1.9 (0.8) 44.3 (14.8)* 83.5 (22.6)*

Neutrophils (cells x 106 kg‐1)

0.5 (0.5) 31.3 (15.2)* 66.0 (20.9)*

Mononuclear cells (cells x 106 kg‐1)

1.4 (0.4) 12.2 (4.0)* 16.6 (3.2)*

Lymphocytes (cells x 106 kg‐1)

0.03 (0.01) 0.2 (0.2) 0.2 (0.1)

Lung Tissue

iNOS positive cells (cells (nm2)‐1)

0 154.9 (62)* 219.3 (83)*

IL‐1β (fold change)# 1.0 (0.6, 1.6) 64.1 (42.2, 110.9) 82.1 (57.2, 120.8)†

IL‐6 (fold change)# 0.6 (0.4, 3.2) 238.2 (66.3, 295.6) 266.1 (179.9, 490.5)†

UVC = Unventilated Control, HFJV+CMV0.5 = HFJV with CMV breaths delivered with

an inspiratory time of 0.5 s, HFJV+CMV2.0 = HFJV with CMV breaths delivered with an

inspiratory time of 2.0 s. * p<0.01 compared to UVC, † p=0.04 compared to UVC, #

n=7 for analyses. Values are mean (SEM) or median (25th, 75th centile) for parametric

and non‐parametric data respectively.

172

Figure 1

173

Figure 2

174

Figure 3

175

Figure Legends

Figure 1 Ventilator variables: A: Peak Inspiratory Pressure (PIP); B: Mean Airway Pressure

(Paw) calculated by the high‐frequency jet ventilator; C: ∆P (Pressure differential); D: Servo

Pressure (psi = pounds per square inch). ∆ HFJV with CMV breaths delivered over 0.5 s

(HFJV+CMV0.5), о HFJV with CMV breaths delivered over 2.0 s (HFJV+CMV2.0). Solid fill =

PIPHFJV, gray fill = PIPCMV.

Figure 2 Ventilation and Oxygenation: A: Fractional inspired oxygen concentration (FiO2);

B: Partial pressure of oxygen in arterial blood (PaO2); C: Oxygenation Index; D: Partial

pressure of carbon dioxide in arterial blood (PaCO2); E: pH. ∆ HFJV with CMV breaths

delivered over 0.5 s (HFJV+CMV0.5), о HFJV with CMV breaths delivered over 2.0 s

(HFJV+CMV2.0).

Figure 3 Pressure Volume Curve: ∆ HFJV with CMV breaths delivered over 0.5 s

(HFJV+CMV0.5), о HFJV with CMV breaths delivered over 2.0 s (HFJV+CMV2.0),

Unventilated control (UVC). * p<0.05 HFJV+CMV0.5 compared to UVC, ** p<0.01

HFJV+CMV2.0 compared to UVC.

176

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sheep exposed to chorioamnionitis. J Immunol 179:8491‐8499

14. Sozo F, O'Day L, Maritz G, Kenna K, Stacy V, Brew N, Walker D, Bocking A, Brien J,

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17. Pillow JJ, Sly PD, Hantos Z 2004 Monitoring of lung volume recruitment and

derecruitment using oscillatory mechanics during high‐frequency oscillatory

ventilation in the preterm lamb. Pediatric Critical Care Medicine 5:172‐180

18. Musk GC, Polglase GR, Bunnell JB, McLean CJ, Nitsos I, Song Y, Pillow JJ 2010 High

positive end‐expiratory pressure during high‐frequency jet ventilation improves

oxygenation and ventilation in preterm lambs. Pediatr Res Dec 20:Epub ahead of

print

19. Kamlin COF, Davis PG 2003 Long versus short inspiratory times in neonates

receiving mechanical ventilation (review). Cochrane Database of Systematic

Reviews 4:CD004503

20. Bunnell JB 2002 The importance of servo pressure. Bunnell Incorporated

(www.bunl.com/clinical.html) Accessed July 2008, Salt Lake City, Utah

21. Kallapur SG, Jobe AH 2006 Contribution of inflammation to lung injury and

development. Arch Dis Child Fetal Neonatal Ed 91:F132‐F135






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180

181

6 The Effect of Conventional Breath Peak Inspiratory Pressure

during High‐frequency Jet Ventilation in Preterm Lambs

Gabrielle C Musk 1, Graeme R Polglase2 and J Jane Pillow 1.

1School of Women’s and Infants’ Health, University of Western Australia, Perth, Australia.

2The Ritchie Centre, Monash Institute of Medical Research, Monash University,

Melbourne, Australia.

This is the second study investigating the role of conventional mechanical ventilator

breaths for alveolar recruitment during high‐frequency jet ventilation. We isolated the

effect of the peak inspiratory pressure of the conventional mechanical ventilation breath

in this study.

182

Abstract

Alveoli are recruited with conventional mechanical ventilator (CMV) breaths during high‐

frequency jet ventilation (HFJV). We assessed the effect of CMV breath peak inspiratory

pressure (PIP) on gas exchange, ventilator requirements and lung injury during HFJV.

Preterm lambs of anaesthetised ewes were delivered surgically at 128 d gestation

(term=150 d) and randomised to an unventilated control group (UVC) or one of 3

ventilated groups: HFJV; or HFJV with 5 CMV breaths/min to a PIPCMV either 5 cmH2O

below or above PIPHFJV (HFJV+CMVlow and HFJV+CMVhigh). Set PEEP was maintained at 8

cmH2O. PIPHFJV and FiO2 were adjusted to maintain PaCO2 45‐55 mmHg and SpO2 88‐95 %.

Lambs were euthanased after 2 h and a post mortem performed. FiO2 was lowest in the

HFJV+CMVhigh group from 60 min. Oxygenation index increased over time in the HFJV

group. In situ lung volume at 40 cmH2O and bronchoalveolar lavage (BAL) protein content

and inflammatory cell count was higher in all the ventilated groups compared to UVC. BAL

neutrophil count was higher in the HFJV+CMVlow group compared to UVC. Lung tissue IL‐6

mRNA was higher in HFJV+CMVlow compared to UVC. The most physiological benefit with

the least evidence of harm was apparent in the HFJV+CMVhigh group of preterm lambs.

183

Introduction

High‐frequency jet ventilation (HFJV) is a novel mode of high‐frequency ventilation

offering the potential for lung protective low tidal volume ventilation during the

management of respiratory distress syndrome (1, 2). In neonates, HFJV is delivered in

tandem with a conventional ventilator that provides positive end‐expiratory pressure

(PEEP), bias flow for spontaneous breaths, a passage for exhaled gases and a means of

delivering sigh breaths. Alveolar recruitment during HFJV is achieved by altering PEEP or

by delivering conventional mechanical ventilator (CMV) breaths (3). Neonatal clinical

protocols suggest that during HFJV CMV breaths should be delivered at very low rates (0‐3

breaths per minute), with more frequent CMV breaths (5‐10 breaths per minute) used to

recruit collapsed alveoli (4). Despite the widespread use of such protocols in neonatal

units over the last few decades, studies that explore how these breaths should be

delivered are limited.

The peak inspiratory pressure (PIP) of CMV breaths delivered during HFJV will determine

whether or not HFJV breaths are interrupted during delivery of CMV breaths.

Conventional mechanical ventilator breaths delivered to a PIPCMV higher than the HFJV

breaths will interrupt the HFJV breaths while those delivered to a PIPCMV lower than the

HFJV breaths will allow the HFJV breaths to stack on top of the CMV breath, delivering

peak pressures in excess of those set on the ventilator and potential barotrauma.

However, at any given PIPHFJV, a higher PIPCMV will deliver a higher tidal volume, with the

potential of increased volutrauma compared to that resulting from a PIPCMV below the

184

PIPHFJV. Whether a series of CMV breaths that interrupt the HFJV breaths are more

harmful than those that don’t is unknown.

We aimed to investigate the effect of 2 different CMV breath PIPCMV settings during HFJV.

We hypothesised that during HFJV in a preterm lamb model of RDS, CMV breaths

delivered to a PIPCMV greater than the PIPHFJV would promote lung injury.

185





(5).


Anaesthesia was induced in ewes at 128‐130 days of gestation with an intravenous

injection of medetomidine (0.02 mg/kg, Pfizer Animal Health, U.S.A.) and ketamine (10 mg

kg‐1, Troy Laboratories, Australia) followed by a subarachnoid (spinal) injection of lidocaine

(3 mL, 20 mg mL‐1, Troy Laboratories, Australia). The fetus was exteriorised surgically and

intubated orally (4.5 mm cuffed tracheal tube, Portex Ltd. England). Lung fluid was

suctioned and intra‐tracheal surfactant (100 mg kg‐1: 25 mg of phospholipids mL‐1, Abbott

Laboratories, U.S.A.) instilled prior to delivery of the lamb. Unventilated controls (UVC,

negative controls: n=8) were euthanased (pentobarbitone 100 mg kg‐1 i.v. Jurox, Australia)

at delivery.

Postnatal care

Lambs were dried, weighed and randomised to one of three ventilation groups: HFJV only

(HFJV; n=8); HFJV with 5 CMV breaths delivered to a PIP 5 cmH2O below the HFJV PIP

(HFJV+CMVlow, n=8); or HFJV with 5 CMV breaths delivered to a PIP 5 cmH2O above the

HFJV PIP (HFJV+CMVhigh, n=8).

186

Propofol (0.1 mg kg‐1 min‐1, Norbrook Laboratories Ltd., Victoria, Australia) and

remifentanil (0.05 µg kg‐1 min‐1, Abbott Laboratories, U.S.A.) were infused continuously

through an umbilical vein for anaesthesia and analgesia. An umbilical arterial catheter was

sampled intermittently to assess gas exchange and acid‐base status. Rectal temperature

was monitored continuously and maintained at 38 – 39 °C (normothermic for newborn

lambs). Oxygenation Index (OI) was calculated as OI = 2

2

aO

100 xaw xFiO

P

P where FiO2 is

fractional inspired oxygen concentration, Paw is mean airway pressure measured by the

HFJV ventilator and PaO2 is partial pressure of oxygen in arterial blood.



pressure‐limited time‐cycled conventional ventilator (Dräger, Babylog 8000+, Drägerwerk

AG, Lübeck, Germany) was commenced immediately following delivery. Initial HFJV

settings were respiratory rate 420 breaths/min, PIPHFJV 30 cmH2O and inspiratory time (tI)

0.02 s. PIPHFJV was adjusted to target permissive hypercapnia (PaCO2 45‐55 mmHg) to a

maximum of 40 cmH2O. The initial FiO2 (0.4) was adjusted to maintain SpO2 88‐95 %.

CMV breaths were delivered to a PIP (PIPCMV) 5 cmH2O above or below PIPHFJV, and with a

rate of 0 or 5 breaths/min as per randomisation. PIPCMV was adjusted in parallel with

PIPHFJV to maintain the predefined low and high PIP strategy relative to the PIPHFJV. A PEEP

of 8 cmH2O and tI of 0.5 s, were maintained throughout the study.

187

After obtaining final measurements, the FiO2 was increased to 1.0 for 2 minutes. The

tracheal tube was occluded for 3 min to facilitate lung collapse before the lamb was

euthanased (pentobarbitone 100 mg kg‐1 i.v. Jurox, Australia).

Post‐mortem



samples of the right lower lobe were snap frozen for molecular analyses. Bronchoalveolar

lavage (BAL) was performed on the left lung for cytology and protein analysis (7).

Differential cell counts were performed on cytospin samples of the BAL fluid stained with

Diff‐Quik (Fronine Lab Supplies, N.S.W., Australia). Immunohistochemical staining for

myeloperoxidase (MPO) was performed on 5 µm sections of lung tissue (8). Positive cells

were identified and quantified per field as number of cells per total cellular area (nm2)

using densitometry (Image‐Pro Plus version 4.5, Media Cybernetics, U.S.A.) by a blinded

observer (GCM). RNA was extracted from lung tissue and reverse transcribed to cDNA

(Bioscript, Bioline, N.S.W., Australia) for measurement of IL‐1β and IL‐6 mRNA expression

by qRT‐PCR (9).



points. The effect of time on ventilator requirements and physiological changes was

determined using two‐way repeated measure analysis of variance. Analyses were

188

performed using SigmaStat (Version 3.5, Systat Software Incorporated, U.S.A.) with p <

0.05 considered statistically significant. Data are expressed as mean (SEM) unless stated

otherwise.

189

Results


Ventilator Settings

The PIPCMV was 5.3 (0.6) cmH2O higher than PIPHFJV throughout the study in the

HFJV+CMVhigh group and 3.8 (1.5) cmH2O lower than PIPHFJV in the HFJV+CMVlow group.

There were no differences in PIPHFJV, Paw,HFJV, ΔPHFJV and servo pressure between the

groups (Figure 1A, 1B, 1C and 1D).

Gas Exchange

The target SpO2 was achieved in all groups within the first 10 min and maintained for the

duration of the ventilation period. FiO2 was lower in the HFJV+CMVhigh group from 60 min

compared to the HFJV+CMVlow group (p<0.03) and from 90 min compared to the HFJV

group (p<0.03) (Figure 2A). The oxygenation index increased over time in the HFJV group

(p=0.02) but did not change over time in either of the HFJV+CMV groups (Figure 2B).

The target PaCO2 was achieved within 30 min and maintained in all groups (Figure 2C). The

HFJV+CMVhigh group tended to have PaCO2 at the higher end of the target range but it was

not different to the other ventilated groups. There were no differences in arterial pH

(Figure 2D).

Static lung compliance

190

HFJV+CMVlow (p<0.02) and HFJV+CMVhigh (p<0.01) were more compliant than the HFJV

group as determined by a post‐mortem deflation static pressure‐volume curve. All

ventilated groups had a higher static compliance compared to the UVC group. (Figure 3).


The total protein concentration of BAL fluid was higher in each of the ventilated groups

compared to the UVC group, however there were no significant differences between the

ventilatory strategies. There were more inflammatory cells in the BAL fluid from each of

the ventilated groups compared to the UVC group (p<0.01). There were more neutrophils

in the HFJV+CMVlow group compared to UVC (p<0.05) (Table 2).

Lung Tissue

The number of MPO positive cells in the lung tissue of ventilated groups was comparable

between all groups (Table 2). The mRNA expression of IL‐1β was similar between all the

ventilated groups. The mRNA expression of IL‐6 was higher in HFJV+CMVlow compared to

the UVC group (Table 2).

191

Discussion

While the delivery of CMV breaths during HFJV is advocated to recruit alveoli, there are

few data to justify the selection of CMV breath parameters. CMV breaths delivered during

HFJV improved static lung compliance and in the HFJV+CMVhigh group this was associated

with decreased oxygen requirements and a consistent trend towards lower expression of

pro‐inflammatory markers. The use of a PIPCMV higher than the set PIPHFJV appeared more

protective than a PIPCMV lower than the set PIPHFJV. These findings suggest that in the

setting of atelectatic lungs at the initiation of ventilation in preterm lambs with a constant

PEEP, the inclusion of CMV breaths may improve lung volume recruitment. Of the two

strategies including CMV breaths, the strategy using PIPCMV above PIPHFJV appeared to

have the greatest physiological benefit with the least evidence of harm, contrary to our

hypothesis that this approach may invoke barotrauma.

Alveolar recruitment during HFJV can be achieved by adjusting PEEP, delivering occasional

CMV breaths, or both approaches in parallel. PEEP facilitates alveolar stabilisation and

avoidance of collapse. PEEP may also contribute to recruitment after achievement of

partial aeration if PEEP exceeds the opening pressure of atelectatic lung units, or by

assisting airway patency through the action of alveolar‐airway attachments in areas of

recruited lung. The CMV breaths open the lung primarily by exposing the distal alveoli to a

pressure greater than the opening pressure and by the delivery of recruiting volumes. The

PIPCMV determines the delivered volume of inflation above the opening pressure for a

given lung impedance. If the delivered PIPCMV and consequently the delivered tidal volume

192

are excessive, the cost of alveolar recruitment from CMV breaths may outweigh any

benefit of more rapid alveolar recruitment.

We compared HFJV+CMVlow and HFJV+CMVhigh with HFJV alone to investigate the

cost:benefit ratio for inclusion of CMV breaths, and size of those conventional breaths for

effective alveolar recruitment during HFJV. If PIPCMV is above PIPHFJV, the HFJV breaths are

interrupted for the duration of the CMV breath. Provided the PIPCMV is above the opening

pressure of the lung, this breath will act to recruit lung volume. Although this approach

(PIPCMV > PIPHFJV) has been the preferred clinical strategy, CMV breaths may be injurious if

they induce excessive parenchymal stretch (10, 11). As PIPHFJV is primarily determined by

the need to achieve satisfactory clearance of CO2, the situation often arises whereby

PIPHFJV is increased significantly above PIPCMV. In this scenario, (PIPCMV < PIPHFJV), the

phenomenon of ‘HFJV‐stacking’ is observed such that the peak pressure in the central

airways may be higher than either the preset PIPCMV or PIPHFJV. However, as the HFJV

waveform is delivered at high velocity, the PIPHFJV is substantially attenuated such that the

absolute PIP during an HFJV stacked CMV breath is usually only marginally above the

preset PIPHFJV (unpublished observations). Thus it is unlikely that the distal alveoli are

exposed to excessive peak pressures. Therefore, the most important proviso is that the

PIPCMV remains higher than the opening pressure of the lung, such that it recruits lung

volume, without being so high that it promotes cyclic overdistension. As volutrauma is a

more important determinant of lung injury than barotrauma (12), we hypothesised that

the HFJV+CMVhigh strategy would be more injurious than a HFJV+CMVlow approach.

193

Whereas we observed differences in oxygenation outcome variables, there were no

intergroup differences in the pro‐inflammatory markers between the ventilated groups.

There was however a consistent trend towards less injury in the HFJV+CMVhigh group in all

pro‐inflammatory markers studied compared to the HFJV and HFJV+CMVlow group.

Without accurate measurements of lung volume or CMV breath tidal volume (inaccurately

reported by the conventional ventilator during HFJV), we were unable to determine

whether this trend towards less injury in the HFJV+CMVhigh group is a consequence of

interrupted HFJV breaths, or more effective CMV breath related volume recruitment.

Some variability in delivered tidal volumes has beneficial effects in preterm lambs as

lambs managed with a variable ventilation strategy had improved in vivo lung compliance

without increased lung injury (J Pillow, unpublished data). Intermittent delivery of

relatively large breaths to a relatively high pressure is less likely to facilitate alveolar

recruitment more so than uniform breaths delivered repeatedly. The HFJV+CMVhigh group

in this study is likely to have received the most variable set of breaths over 2 h. This

variation may have contributed to the favourable results for this group.

We deliberately kept PEEP constant for the duration of this study to isolate the effect of

including CMV breaths and the magnitude of those breaths on oxygenation, ventilation

and lung injury. PEEP during HFJV is used primarily to stabilise alveoli and prevent alveolar

derecruitment (13‐16) although it can contribute to lung volume recruitment (17). We

selected a PEEP of 8 cmH2O as previous studies (18) found a PEEP of 5 cmH2O insufficient

to stabilise alveoli (18). Nonetheless, the increased OI in the HFJV group and the

comparable trend in the HFJV+CMV groups over the 2 h suggests progressive atelectasis

194

and inadequate PEEP stabilisation of the lung at end expiration. In clinical practice, an

increment in PEEP as PIPHFJV and ∆PHFJV are weaned may be essential to avoid alveolar

collapse.

In earlier studies we had observed the lower inflection point of similar gestation preterm

lambs as 20‐25 cmH2O (19) and hence expected that an initial PIPCMV of 25 cmH2O (in the

HFJV+CMVlow group) would be above the critical opening pressure. Nonetheless, it seems

likely that the HFJV+CMVlow group generated insufficient pressures and tidal volumes to

achieve effective recruitment and hence to provide the same benefit as the HFJV+CMVhigh

group.

We chose inflammatory markers of ventilation induced lung injury that typically increase

within 120 min (20‐23), and confirmed previous observations using CMV strategies that

any ventilation, even using HFJV alone, increases inflammatory markers in the premature

lung (17, 24). With only 8 lambs per group we cannot exclude a type II statistical error for

detection of significant differences between the ventilated groups. Even so, the lack of

significant differences between the different ventilated groups suggests that such

differences, if present, are likely to have minimal significance over a 2 h ventilation period.

Nonetheless, the trends towards increased BAL protein and inflammatory cells, and lung

tissue MPO and interleukin expression in the HFJV+CMVlow group compared to other

ventilated groups and the UVC group are suggestive of increased lung injury in this group.

A possible explanation may be the trend to lower mean airway pressures in the

HFJV+CMVlow group compared to the other two ventilated groups, resulting from the fall

195

in ΔP over the study period. In clinical practice, a drop in mean airway pressure with

weaning of the PIPHFJV could be offset by increases in PEEP, however by design, PEEP was

maintained at a constant level throughout the study duration, in all groups.

A higher static compliance in the HFJV+CMVhigh group was expected given the improved

oxygenation and likely increased lung volume in vivo protecting the lung from injury.

Whereas the OI increased over time in the HFJV group, it is unlikely that there was

significant alveolar recruitment in either the HFJV only or the HFJV+CMVlow group as with

the exception of the 15 min time point, their oxygenation indexes did not differ

subsequently and FiO2 remained elevated throughout the study. It is intriguing therefore,

that the static lung compliance was lower in the HFJV group compared to the

HFJV+CMVlow group given the trends towards increased lung injury in the latter group.

One explanation for this finding may be that the intermittent CMV breaths in the

HFJV+CMVlow group stimulated surfactant production and release. Further studies to

quantify surfactant protein mRNA may help to elucidate the answer to this question.

In summary, CMV breaths delivered to a PIPCMV 5 cmH2O above the PIPHFJV provided the

most physiological benefit with the least evidence of harm over a 2 h period following

initiation of ventilation in preterm lambs. Our results support the judicious use of

infrequent, but appropriately targeted, CMV breaths for alveolar recruitment during

initiation of HFJV in acute neonatal respiratory distress syndrome. Further studies

investigating CMV breath parameters during HFJV in the target patient population are

indicated.

196

Acknowledgements



sincere appreciation to the members of the Ovine Research Group, Ilias Nitsos, Carryn

McLean, Richard Dalton and Yong Song for technical assistance and JRL Hall and Co. for

provision and early antenatal care of the ewes.

197

Tables

Table 1: Baseline lamb data

UVC HFJV HFJV+CMVlow HFJV+CMVhigh

n (male) 5 (3) 8 (5) 8 (3) 8 (4)

Birth weight (kg) 2.6 (0.3) 2.6 (0.6) 2.8 (0.6) 3.1 (0.3)

Gestational age (d) 129.6 (0.5) 129.1 (0.8) 129.1 (0.6) 129.5 (0.5)

Cord pH 7.11 (0.18) 7.15 (0.09) 7.09 (0.04) 7.11 (0.13)

Cord PaCO2 (mmHg) 88.9 (28) 77.1 (14.3) 99.3 (24.1) 84.0 (37.0)

Term = 150 d. UVC = unventilated control; HFJV = high‐frequency jet ventilation

(HFJV) alone; HFJV+CMVlow = HFJV with 5 conventional mechanical ventilation

(CMV) breaths/min to a peak inspiratory pressure 5 cmH2O below HFJV peak

inspiratory pressure; HFJV+CMVhigh = HFJV with 5 CMV breaths/min to a peak

inspiratory pressure 5 cmH2O above HFJV peak inspiratory pressure. Values are

mean (SD). Cord samples were collected from the umbilical artery.

198


UVC HFJV HFJV+CMVlow HFJV+CMVhigh

BAL fluid

Protein (mg mL‐1) 153.0 (85.9) 209.8 (90.5)* 353.3(212.1)* 273.(169.7)*

Total Inflammatory Cells

(x 106 kg‐1) 1.9 (0.8) 43.0 (13.9)* 90.2 (28.3)* 36.8 (15.0)*

Neutrophils (x 106 kg‐1) 0.5 (0.5) 14.2 (5.9) 34.7 (18.1)* 12.1 (6.4)

Mononuclear cells (x 106 kg‐1) 1.4 (0.4) 25.5 (11)* 53 (33.2)* 24.5 (10.9)*

Lung Tissue

MPO positive cells (cells (nm2)‐1) 0 (0, 1.5) 0.8 (0, 14.2) 1.1 (0, 4.4) 2.5 (0, 6.8)

IL‐1β (fold change) 1.0 (0.5, 1.4)

4.6 (1.6, 8.8)

5.6 (2.1, 9.4)

2.4 (1.7, 8.1)

IL‐6 (fold change) 1.0(0.7, 1.5)

19.4(1.0, 29.8)

21.5*(4.6, 38.1)

10.7 (6.9, 22.0)

*p<0.05 compared to UVC. Values are Mean (SD) or median (25, 75 centile).

199

Figure 1

200

Figure 2

201

Figure 3

202

Figure Legends

Figure 1 Ventilator Parameters: A: High‐frequency jet ventilation inspiratory pressure

(PIPHFJV) (cmH2O)was adjusted to target PaCO2 45‐55 mmHg; B: HFJV Mean Airway

Pressure (cmH2O); C: HFJV Delta P (HFJVPIP ‐ HFJVPEEP) (cmH2O); D: HFJV Servo Pressure

(psi). Grey diamond = HFJV, open circle = HFJV+CMVlow, solid circle = HFJV+CMVhigh.

Figure 2 Gas Exchange: A: FiO2 * p<0.03 HFJV+CMVhigh compared to HFJV+CMVlow, # p

<0.03 HFJV+CMVhigh compared to HFJV B: Oxygenation Index * p = 0.02 time 120

compared to time 5 for HFJV C: PaCO2 (mmHg); D: pH. Grey diamond = HFJV, open circle =

HFJV+CMVlow, solid circle = HFJV+CMVhigh.

Figure 3 Pressure‐Volume Curve: # p<0.02 HFJV+CMVhigh compared to HFJV, ## p<0.02

HFJV+CMVlow compared to HFJV, * p<0.01 compared to unventilated controls (UVC). Grey

diamond = HFJV, open circle = HFJV+CMVlow, solid circle = HFJV+CMVhigh, open triangle =

UVCs.

203

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frequency jet ventilation in neonates with hypoxaemia refractory to high‐

frequency oscillatory ventilation. Journal of Maternal‐Fetal and Neonatal Medicine

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207

7 Alveolar Recruitment with Five or Twenty Conventional

Mechanical Ventilator Breaths per minute during High‐frequency

Jet Ventilation in Preterm Lambs.

Gabrielle C Musk1, Graeme R Polglase2 and J Jane Pillow 1.

1School of Women’s and Infants’ Health, University of Western Australia, Perth, Australia.

2The Ritchie Centre, Monash Institute of Medical Research, Monash University,

Melbourne, Australia.

3Centre for Neonatal Research and Education, University of Western Australia, Perth,

Australia.

This is the third study investigating the role of conventional mechanical ventilator breaths

for alveolar recruitment during high‐frequency jet ventilation. We isolated the effect of

conventional ventilator breath frequency in this study.

208

Abstract

Alveoli are recruited with conventional mechanical ventilator (CMV) breaths during high‐

frequency jet ventilation (HFJV). We assessed the impact of CMV breath frequency on gas

exchange, ventilator requirements and lung injury during HFJV.

Preterm lambs of anaesthetised ewes were delivered surgically at 128 d gestation

(term=150 d) and randomised to an unventilated control group (UVC) or one of 3

ventilated groups including HFJV alone or HFJV with either 5 or 20 CMV breaths/min to a

PIPCMV 5 cmH2O below PIPHFJV (HFJV+CMV5 or HFJV+CMV20). Set PEEP was maintained at 8

cmH2O. PIP and FiO2 were adjusted to maintain PaCO2 45‐55 mmHg and SpO2 88‐95 %.

Lambs were euthanased after 2 hours and a post mortem performed.

Physiological variables and ventilator requirements did not differ between groups.

Compared to other ventilated groups the HFJV+CMV20 group had higher measured PEEP

(auto PEEP) from 15 min and higher FiO2 from 30 min. In situ lung volume at 40 cmH2O

was higher in the HFJV+CMV5 group compared to all other groups. Bronchoalveolar lavage

(BAL) fluid inflammatory cell count was higher in HFJV+CMV groups and BAL protein

concentration was higher in all ventilated groups compared to UVCs. Surprisingly, lung

tissue IL‐1β and IL‐6 mRNA was lowest in HFJV+CMV20 compared to other ventilated

groups. Whereas the improved static compliance in the HFJV+CMV5 group suggests

potential benefit of low‐rate CMV breaths during HFJV during the initiation of HFJV in

acute neonatal respiratory distress syndrome, the mixed inflammatory outcomes in the

209

HFJV+CMV groups suggests further studies to clarify the optimal frequency of CMV

breaths are required.

210

Introduction

High‐frequency jet ventilation (HFJV) is considered a lung protection ventilation strategy

suitable for ventilatory support of preterm infants with respiratory distress syndrome

(RDS) (1, 2). A high‐frequency jet ventilator is set up in tandem with a conventional

ventilator that provides positive end‐expiratory pressure (PEEP), bias flow for

spontaneous breaths, a passage for exhaled gases and a means of delivering sigh breaths.

This tandem arrangement facilitates alveolar recruitment manoeuvers during HFJV which

may be achieved by altering PEEP or by delivering conventional mechanical ventilator

(CMV) breaths (3). Neonatal clinical protocols suggest that during routine HFJV, CMV

breaths should be delivered at very low rates (0‐3 breaths per minute), with more

frequent CMV breaths (5‐10 breaths per minute) used to recruit collapsed alveoli (4).

Whereas protocols are based on the presumption that excessive CMV breaths are

injurious, there are no experimental data that define the optimal frequency for CMV

breaths during HFJV in the setting of acute neonatal RDS.

Compared to the small volume HFJV breath, CMV breaths are more likely to cause cyclic

stretch of the lung parenchyma and contribute to volutrauma (5). The frequency of CMV

breaths during HFJV may therefore influence volume recruitment. However, whereas

occasional sigh breaths may promote recruitment, repetitive cyclic stretch of the lung

parenchyma may also promote injury. The CMV breath parameters that must be selected

include the inspiratory time (tI), the peak inspiratory pressure (PIP) and the respiratory

frequency.

211

We aimed to investigate the effect of 2 different CMV breath frequencies during HFJV. We

hypothesised that during HFJV in a preterm model of RDS, 20 CMV breaths/min would be

more injurious to the lung than 5 CMV breaths/min.

212



committee, according to the guidelines issued by the National Health and Medical

Research Council of Australia (6).


Pregnant ewes at 128‐130 days of gestation were anaesthetised with an intravenous

injection of medetomidine (0.02 mg/kg, Pfizer Animal Health, U.S.A.) and ketamine (10 mg

kg‐1, Troy Laboratories, Australia) followed by a subarachnoid (spinal) injection of lidocaine

(3 mL, 20 mg mL‐1, Troy Laboratories, Australia). The fetus was exteriorized surgically and

intubated orally (4.5 mm cuffed tracheal tube, Portex Ltd. England). Lung fluid was

suctioned and intra‐tracheal surfactant (100 mg kg‐1: 25 mg of phospholipids mL‐1, Abbott

Laboratories, U.S.A.) instilled prior to delivery of the lamb. Unventilated controls (UVC,

negative controls: n=8) were euthanased at delivery with pentobarbitone (100 mg kg‐1 i.v.

Jurox, Australia).

Postnatal care

Lambs were dried, weighed and randomised to one of three ventilation groups: HFJV only

(HFJV; n=8); HFJV with 5 or 20 CMV breaths delivered to a PIP 5 cmH2O below the PIPHFJV

(HFJV+CMV5, n=8; HFJV+CMV20, n=8).

213

Propofol (0.1 mg kg‐1 min‐1, Norbrook Laboratories Ltd., Victoria, Australia) and

remifentanil (0.05 µg kg‐1 min‐1, Abbott Laboratories, U.S.A.) were infused continuously

through an umbilical vein for anaesthesia and analgesia. An umbilical arterial catheter was

sampled intermittently to assess gas exchange and acid‐base status. Rectal temperature

was monitored continuously and maintained between 38° and 39° C (normothermic for

newborn lambs). Oxygenation Index (OI) was calculated as OI = 2

2

aO

100 x aw x FiO

P

P where FiO2

is fractional inspired oxygen concentration, Paw is mean airway pressure measured by the

jet ventilator and PaO2 is partial pressure of oxygen in arterial blood.



pressure‐limited time‐cycled conventional ventilator (Dräger, Babylog 8000+, Drägerwerk

AG, Lübeck, Germany) was commenced immediately following delivery. Initial HFJV

settings were: respiratory rate 420 breaths/min; PIPHFJV 30 cmH2O; and inspiratory time (tI)

0.02 s. PIPHFJV was adjusted to achieve permissive hypercapnia (PaCO2 45‐55 mmHg) to a

maximum of 40 cmH2O. The initial FiO2 (0.4) was adjusted to maintain SpO2 88‐95 %.

CMV breaths were delivered to a PIPCMV 5 cmH2O below the PIPHFJV, and with a rate of 0, 5

or 20 breaths/min as per randomisation. PIPCMV was adjusted in parallel with PIPHFJV to

maintain PIPCMV 5 cmH2O below the PIPHFJV. A PEEP of 8 cmH2O and tI of 0.5 s for CMV

breaths, were maintained throughout the study.

214

After final measurements were obtained, the FiO2 was increased to 1.0 for 2 minutes,

after which the tracheal tube was occluded for 3 min to facilitate lung collapse before the

lamb was euthanased (100 mg kg‐1 i.v. Jurox, Australia).

Post‐mortem



samples of the right lower lobe were snap frozen for molecular analyses of IL‐1β and IL‐6

mRNA expression by qRT‐PCR (8). Bronchoalveolar lavage (BAL) was performed on the left

lung for cytology and protein analysis (9). Differential cell counts were performed on

cytospin samples of the BAL fluid stained with Diff‐Quik (Fronine Lab Supplies, N.S.W.,

Australia). Immunohistochemical staining for myeloperoxidase (MPO) was performed on 5

µm sections of lung tissue (10). Positive cells were identified and quantified per field as

number of cells per total cellular area (nm2) using densitometry (Image‐Pro Plus version

4.5, Media Cybernetics, U.S.A.) by a blinded observer (GCM).



points. The effect of time on ventilator requirements and physiological changes was

determined using two‐way repeated measure analysis of variance. Analyses were

performed using SigmaStat (Version 3.5, Systat Software Incorporated, U.S.A.) with p<0.05

215

considered statistically significant. Data are expressed as mean (SEM) unless stated

otherwise.

216

Results

Baseline characteristics of lambs in each group, including weight and cord blood gas

status, were not different (Table 1).

Ventilator Settings

The PIPHFJV, Paw,HFJV, ΔPHFJV and servo pressure were similar between the 3 ventilated

groups during the 2 h ventilation period (Figure 1A, 1B, 1C and 1D). The measured PEEP

was higher than set PEEP in HFJV+CMV20 animals at all time points (p=0.03; Figure 1E).

Gas Exchange

The target SpO2 was achieved in all groups within the first 10 minutes and maintained for

the duration of the ventilation period (data not shown). PaO2 was not different between

groups for the duration of the study (data not shown). From 90 minutes, FiO2 and OI were

significantly higher in the HFJV+CMV20 group compared to HFJV+CMV5 and HFJV (Figure

2A and 2B). The target PaCO2 was achieved within 15 minutes and maintained in all

groups (Figure 2C). There were no differences in arterial pH (Figure 2D).

Static lung compliance

The lungs of animals in the HFJV+CMV5 group were more compliant compared to HFJV

and HFJV+CMV20 (p<0.02) as determined by a post‐mortem static pressure‐volume curve.

As expected, unventilated control lambs had the lowest compliance compared to all

ventilated groups (p<0.01; Figure 3).


217

The total protein concentration of BAL fluid was higher in all the ventilated groups

compared to the UVC group. The total inflammatory cell counts, and the differential

neutrophil counts, were higher in the BAL fluid from HFJV+CMV5 and HFJV+CMV20 groups

compared to the UVC group (p<0.01; Table 2) but were not different to HFJV alone. The

differential mononuclear cell counts were higher in each of the ventilated groups

compared to the UVC group.

Lung Tissue

The number of MPO positive cells in the lung tissue of ventilated groups was low and

comparable between all groups (Table 2). The expression of IL‐1β mRNA was higher in all

the ventilated groups compared to HFJV+CMV20 (p<0.05). The expression of IL‐6 mRNA

was higher in the HFJV+CMV5 group compared to the UVC group and higher in both HFJV

alone and HFJV+CMV5 compared to HFJV+CMV20 (p<0.05) (Table 2).

218

Discussion

While the delivery of CMV breaths during HFJV is advocated to recruit alveoli, there are

few studies justifying the selection of CMV breath parameters, including breath frequency.

In the current study, we showed that 20 CMV breaths/min increased oxygen requirements

and decreased static lung compliance compared to a strategy using 5 CMV breaths/min,

but resulted in less up‐regulation of pro‐inflammatory markers in the lung.

PIPHFJV was comparable between groups and between the commencement and the end of

the study within each group. The PIPCMV was maintained 5 cmH2O below the PIPHFJV in this

study. Recent work from this group compared PIPCMV in relation to PIPHFJV and found that

the most physiological benefit, with the least evidence of harm, was apparent if the PIPCMV

was 5 cmH2O above the PIPHFJV when 5 CMV breaths/min were delivered during HFJV

(unpublished data – see Chapter 6). These results were not available at the time the

current study was performed, and it may be that we have hindered our ability to

demonstrate a difference between our HFJV+CMV groups by using insufficient CMVPIP to

recruit the lung effectively in either HFJV alone, or HFJV+CMV5.

ΔPHFJV was not different during the study but there was a trend towards a higher ΔPHFJV in

the HFJV only group for the second hour of the study. Given that during high‐frequency

ventilation, ventilation is proportional to the product of respiratory frequency and tidal

volume (VT)2 (11), this finding demonstrates how little the CMV breaths contribute to

overall minute ventilation during HFJV. Even when 20 CMV breaths/min were delivered,

219

the contribution to overall minute volume did not translate to a lower ΔPHFJV. This trend

may be due to a low VT of the CMV breaths and despite delivering 20 breaths/min, the

impact on PaCO2 was negligible.

PEEP during HFJV is provided by the conventional ventilator and used primarily to stabilise

alveoli and prevent alveolar derecruitment (11‐14). To avoid confounding the

interpretation of CMV breath strategy on outcomes, PEEP was kept constant for the

duration of this study. We selected a PEEP of 8 cmH2O as a previous study found a PEEP of

5 cmH2O insufficient to stabilise alveoli (15). Despite our attempts to maintain a constant

PEEP between and within groups there was a negative difference between PEEP set on the

conventional ventilator and PEEP measured by the jet ventilator for the duration of the

study in the HFJV+CMV20 group. This discrepancy is indicative of auto PEEP, or gas

trapping, and might be expected at higher CMV breath frequencies. This auto PEEP should

translate to a higher Paw but there was no difference in Paw between the groups. Gas

trapping, however, will compromise oxygenation and may explain the higher FiO2

requirements and OI in the HFJV+CMV20 group. The value for Paw on the high‐frequency

jet ventilator is an estimate of the mean pressure at the tip of the tracheal tube and may

not reflect intrapulmonary pressure. It is therefore possible that mean pressure at the

parenchymal level was higher and compromised pulmonary blood flow and oxygenation.

We assessed oxygenation by recording the FiO2 required to achieve a target SpO2 and by

calculating OI. Paw is a determinant of oxygenation (16), but was similar for all groups,

leaving differences in oxygenation parameters attributable to variables other than Paw.

220

There was a higher inspired oxygen requirement and OI in the HFJV+CMV20 group which

suggests either inferior alveolar recruitment, or shunting due to impaired pulmonary

blood flow and increased pulmonary vascular resistance. Although Paw was not different

between groups, it is estimated by the high‐frequency jet ventilator at the tip of the

tracheal tube and it is conceivable that inadvertent PEEP translated to a higher mean

pressure at the parenchyma level that may have impaired pulmonary blood flow and

oxygenation.

In the absence of a clear advantage of higher CMV breath frequency for lung volume

recruitment and arterial oxygenation, we expected the HFJV+CMV20 approach to be

associated with increased potential for lung injury due to increased frequency of distal

alveolar exposure to the PIPCMV (which is transmitted along the airways) and volutrauma

from the associated cyclic tidal volume. Despite choosing inflammatory markers of

ventilation injury that typically increase within 120 min (17‐20), the results provided

conflicting evidence with a trend towards higher BAL protein and inflammatory cell counts

in the HFJV+CMV groups compared to the HFJV only group, but a higher compliance in the

HFJV+CMV5 group compared to either the HFJV+CMV20 and the HFJV only group on the

post‐mortem static pressure‐volume curve. The finding of reduced pro‐inflammatory

markers in the lung tissue for the HFJV+CMV20 group was similarly unexpected.

Inadvertent PEEP may have protected the HFJV+CMV20 lungs from atelectatrauma at the

expense of impaired oxygenation.

221

The limitations of this study may contribute to the lack of significant differences in lung

injury markers. With 8 animals per group we cannot exclude a type II statistical error for

detection of small significant differences between groups. It is also possible that the

strategies were not injurious enough to elicit a strong inflammatory response. The

combination of endogenous surfactant administered immediately after delivery, the short

duration of the study and a modestly high level of PEEP throughout the study may have

limited injury in all three ventilated groups. Furthermore, we have a single snapshot of

inflammation in the lungs and captured a higher BAL neutrophil population in the

HFJV+CMV groups along without an increase in IL‐1β and IL‐6 mRNA in the HFJV+CMV20

group. It is possible that a longer ventilation period may have resulted in an increase in

these cytokines in all groups.

In summary the use of a high CMV breath rate during HFJV strategy increased oxygen

requirements and decreased static lung compliance over a 2 h study compared to a low

CMV breath frequency strategy. The increased compliance of the post‐mortem static

deflation curve in the absence of a significant increase in pro‐inflammatory markers in the

low‐rate CMV breath HFJV strategy over HFJV alone supports the judicious use of

infrequent CMV breaths during initiation of HFJV in acute neonatal respiratory distress

syndrome. The lack of significantly enhanced oxygenation in the HFJV+CMV5 group

compared to HFJV alone suggests that specific targeting of the PIPCMV to achieve

recruitment may be essential to derive further benefit from inclusion of low‐rate CMV

breaths during HFJV. Further studies investigating the use of CMV breaths during HFJV

222

that measure the effect of these breaths on lung volume, gas exchange and lung injury

during neonatal RDS are indicated.

223

Acknowledgements



sincere appreciation to the members of the Ovine Research Group, Ilias Nitsos, Carryn

McLean, Richard Dalton and Yong Song for technical assistance and JRL Hall and Co. for

provision and early antenatal care of the ewes.

224

Tables


UVC HFJV HFJV+CMV5 HFJV+CMV20

n (male) 5 (3) 8 (5) 8 (3) 8 (4)

Birth weight (kg) 2.6 (0.3) 2.6 (0.6) 2.8 (0.6) 3.0 (0.5)


Cord pH 7.11 (0.18) 7.15 (0.09) 7.09 (0.04) 7.14 (0.12)

Cord PaCO2 (mmHg) 88.9 (28) 77.1 (14.3) 99.3 (24.1) 86.0 (22.3)

Term = 150 d. UVC = unventilated control; HFJV = high‐frequency jet

ventilation (HFJV) alone; HFJV+CMV5 = HFJV with 5 conventional

mechanical ventilation (CMV) breaths/min to a peak inspiratory pressure

5 cmH2O below HFJV peak inspiratory pressure; HFJV+CMV20 = HFJV with

20 CMV breaths to a peak inspiratory pressure 5 cmH2O below HFJV peak

inspiratory pressure. Values are mean (SD). Cord samples were collected

from the umbilical artery.

225


UVC HFJV HFJV+CMV5 HFJV+CMV20

BAL fluid

Protein (mg mL‐1) 83.2 (23.) 306.5 (27)* 437.4 (105)* 411.4 (37)*


(x 106 kg‐1) 1.93 (0.8) 43.0 (13.9) 90.2 (28.3)* 81.3 (30.6)*

Neutrophils (x 106 kg‐1) 0.5 (0.5) 14.2 (5.9) 34.7 (18.1)* 39.3 (18.1)*

Mononuclear cells (x 106 kg‐1) 1.4 (0.4) 25.5 (11)* 53 (33.2)* 41.8 (15.2)*

Lung Tissue

MPO positive cells (cells (nm2)‐1) 0 (0, 1.5)

0.8 (0, 14.2)

1.1 (0, 4.4)

3.0 (0, 7.0)

IL‐1β (fold change) 1.0 (0.5, 1.4)

4.6† (1.6, 8.8)

5.6† (2.1, 9.4)

0.7 (0.2, 1.4)

IL‐6 (fold change) 1.0 (0.7, 1.5)

19.4† (1.0, 29.8)

21.5*† (4.6, 38.1)

2.5 (0.6, 5.7)

*p<0.05 compared to UVC, †p<0.05 compared to HFJV+CMV20. Values are Mean (SEM) or

median (25, 75 centile).

226

Figure 1

227

Figure 2

228

Figure 3

229

Figure Legends

Figure 1 Ventilator Parameters: A: High‐frequency jet ventilation peak inspiratory

pressure (PIPHFJV) (cmH2O)was adjusted to target PaCO2 45‐55 mmHg; B: HFJV Mean

Airway Pressure (cmH2O); C: HFJV Delta P (HFJVPIP ‐ HFJVPEEP) (cmH2O); D: HFJV Servo

Pressure (psi); E: The difference between set PEEP and measured PEEP (cmH2O). *p<0.05

HFJV+CMV20 compared to HFJV+CMV5 and HFJV. Grey diamond = HFJV, open circle =

HFJV+CMV5, solid circle = HFJV+CMV20.

Figure 2 Gas Exchange: A: FiO2 * p<0.03 HFJV+CMV20 compared to HFJV+CMV5, # p<0.03

HFJV+CMV20 compared to HFJV; B: Oxygenation Index * p=0.04 HFJV+CMV20 compared to

HFJV+CMV5, # p=0.05 HFJV+CMV20 compared to HFJV; C: PaCO2 (mmHg); D: pH. Grey

diamond = HFJV, open circle = HFJV+CMV5, solid circle = HFJV+CMV20.

Figure 3 Pressure‐Volume Curve: * p<0.02 HFJV+CMV5 compared to HFJV, # p<0.02

HFJV+CMV5 compared to HFJV+CMV20, ** p<0.01 unventilated controls (UVC) compared to

all ventilated groups. Grey diamond = HFJV, open circle = HFJV+CMV5, solid circle =

HFJV+CMV20, open triangle = UVCs.

230

References

1. Plavka R, Dokoupilova M, Pazderova L, Kopecka P, Sebroa V, Zapadlo M, Keszler M

2006 High‐frequency jet ventilation improves gas exchange in extremely immature

infants with evolving chronic lung disease. American Journal of Perinatology

23:467‐472

2. Friedlich P, Subramanian, N, Sebald, M, Noori, S, Seri, I. 2003 Use of high‐

frequency jet ventilation in neonates with hypoxaemia refractory to high‐

frequency oscillatory ventilation. Journal of Maternal‐Fetal and Neonatal Medicine

13:398‐402





Utah

5. Dreyfuss D, Saumon G 1998 Ventilator‐induced lung injury. Lessons from

experimental studies. Am. J. Respir. Crit. Care Med. 157:294‐323



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dose high‐frequency ventilation work? Critical Care Medicine 22:S49‐S57

13. Bass A, Gentile M, Heinz J, Craig D, Hamel D, Cheifetz I 2007 Setting positive end‐

expiratory pressure during jet ventilation to replicate the mean airway pressure of

oscillatory ventilation. Respiratory Care 52:50‐55

14. Crossley KJ, Morley CJ, Allison BJ, Polglase GR, Dargaville PA, Harding R, Hooper SB

2007 Blood gases and pulmonary blood flow during resuscitation of very preterm

lambs treated with antenatal betamethasone and/or curosurf: Effect of positive

end‐expiratory pressure. Pediatr Res 62:37‐42



oxygenation and ventilation in preterm lambs. Pediatr Res 69:319‐324

16. Keszler M 2006 High frequency jet ventilation. In Donn SM, Sinha SK (eds) Manual

of neonatal respiratory care. Mosby Elsevier, Philadelphia, pp 232‐233

232






52:356‐362








233

8 A Comparison of High‐frequency Jet Ventilation and High‐

frequency Oscillatory Ventilation with Conventional Mechanical

Ventilation in Preterm Lambs

Gabrielle C Musk 1, Graeme R Polglase 1,2, J Bert Bunnell 3, Ilias Nitsos 1, David Tingay

4, J Jane Pillow 1,5

1 School of Women’s and Infants’ Health, The University of Western Australia, Perth,

Australia.

2 The Ritchie Centre, Monash Institute of Medical Research, Monash University,

Clayton, Australia.

3 Bunnell Inc, Salt Lake City, Utah, USA and Department of Bioengineering, University of

Utah, Salt Lake City, Utah, USA.

4 Murdoch Children’s Research Institute, Melbourne, Australia

5 Centre for Neonatal Research and Education, The University of Western Australia,

Perth, Australia

This is the final study in this thesis. We compared an optimal high‐frequency jet

ventilation strategy against a high‐frequency oscillatory ventilation and a gentle

conventional mechanical ventilation strategy in our preterm lamb model of respiratory

distress syndrome. The high‐frequency jet ventilation strategy was developed based

upon the results of the previous study chapters.

234

Abstract

Conventional mechanical ventilation (CMV), high‐frequency oscillatory ventilation

(HFOV) and high‐frequency jet ventilation (HFJV) are accepted ventilatory strategies

for respiratory distress syndrome (RDS) in preterm babies. We hypothesised these

strategies would successfully manage oxygenation and ventilation, but that HFJV and

HFOV would cause the least lung injury. Furthermore, we hypothesised that HFJV

would have the least impact on pulmonary blood flow. Preterm lambs of anaesthetised

ewes were instrumented, intubated and delivered by caesarean section after

intratracheal suction and instillation of surfactant. Each lamb was managed for 3 hours

according to a predetermined algorithm for ventilator support consistent with the

open lung approach. Pulmonary blood flow was measured continuously and pulsatility

index was calculated, while ventilatory parameters and arterial blood gases were

measured at intervals. At postmortem, in situ pressure‐volume deflation curves were

recorded, and bronchoalveolar lavage fluid and lung tissue were obtained to assess

inflammation. There was no difference in arterial oxygenation despite lower mean

airway pressure during CMV for most of the study. HFOV animals had a higher PaCO2

at multiple time points. The lack of significant differences in end systolic and end

diastolic PBF for the majority of the study, lung injury data and static lung compliance

demonstrate that in the absence of airleaks each of these strategies can be employed

in the clinical setting with a comparable pressure cost of ventilation.

235

Introduction

The lungs of preterm infants are vulnerable to ventilation induced lung injury (1).

Preterm newborns often require invasive ventilatory support which exposes them to

positive intrathoracic pressures, the risk of lung injury and compromised cardiac

output. The three contemporary ventilatory strategies most frequently used for

preterm babies with respiratory distress syndrome (RDS) are synchronised, volume‐

targeted/guaranteed conventional mechanical ventilation (CMV), high‐frequency jet

ventilation (HFJV) and high‐frequency oscillatory ventilation (HFOV).

A number of clinical trials have compared HFOV and CMV for the management of

preterm infants with acute RDS but the results are conflicting (2, 3). Cools et al (2010)

found in their systematic review and meta‐analysis HFOV and CMV equally effective (2)

while Bhuta and Henderson‐Smart (1998) found little difference other than a decrease

in new pulmonary airleak following treatment with HFOV (3). Variation in study design,

ventilator management and outcome measures between studies make it difficult to

substantiate a clear benefit of one strategy over another. Perhaps the greatest

limitation to these studies was failure to pursue a protective lung ventilation approach

which relies upon opening the lung and keeping it open to optimise ventilation and

perfusion (4, 5). HFJV and CMV have also been compared in a number of studies but

similar issues exist in that an open lung strategy was not always employed, or achieved

(6‐8).

A direct comparison of all three of these ventilatory strategies for neonatal RDS has

not been performed in a controlled setting. Each modality has unique features which

236

provide justification for its use in certain scenarios. A CMV strategy delivers breaths of

a similar size and at a similar frequency to spontaneous ventilation while high‐

frequency strategies deliver breaths smaller than dead space volume at frequencies

varying from 3‐20 Hz depending on the specific ventilator used. High‐frequency

ventilation offers potential lung protective ventilation (9‐14) as the low tidal volumes

reduce the risk of cyclic volutrauma. The airway pressure waveform is attenuated

along the airways during HFJV and HFOV which may decrease the pressure and flow

cost of ventilation induced lung injury (15).

There are important differences between HFJV and HFOV which give rise to potential

physiological advantages of one strategy over another: optimal mean airway pressure

(Paw) is lower during HFJV compared to HFOV because less of the respiratory cycle is

spent in inspiration; HFJV enhances mucociliary clearance by combining fast

inspirations with relatively slow, passive exhalations (I:E ratio may be as low as 1:12);

and the use of small tidal volume (VT) breaths during HFJV at low frequencies, in

combination with low inspiratory to expiratory ratios, make HFJV especially useful in

patients with gas trapping. Both strategies deliver high velocity and small VT breaths

that do not penetrate injured areas of lung with high resistance, allowing for

maturation and healing (15). HFOV, however, has an active expiratory phase which

makes it useful at higher respiratory frequencies. This feature is advantageous for

lungs that are poorly compliant and that have a higher corner frequency, as it

facilitates gas exchange but avoids unnecessary barotrauma (16). HFOV has been

investigated in and ex vivo and gas flow and gas exchange is more extensively

documented (16, 17). This extensive data from HFOV has facilitated its widespread use

237

in neonatal intensive care units. Furthermore, during HFOV, a conventional mechanical

ventilator is not required and a standard endotracheal tube adaptor is suitable.

Early recruitment of the functional residual capacity immediately after birth may

facilitate the commencement of ventilation on the deflation limb of the pressure‐

volume curve. The delivery of a sustained inflation before commencing a particular

ventilation strategy has consistently decreased the requirement for subsequent

aggressive alveolar recruitment manoeuvres in both animal and human studies (18‐

20).

The aim of this study was to compare HFJV and HFOV with a moderate lung protective

CMV strategy in a preterm lamb model of RDS. We hypothesised that each strategy

would facilitate acceptable oxygenation and ventilation, but that the low delivered

tidal volumes of HFJV and HFOV would result in less evidence of ventilation induced

lung injury than a CMV strategy. Furthermore, based upon the findings of Polglase et al

(2008) (21) we hypothesised that compared to HFOV, HFJV would cause less reduction

of pulmonary blood flow due to a proportionately greater duration of the cycle spent

at the positive end expiratory pressure with less impairment of venous return.

238


All animal procedures were approved by the University of Western Australia animal

ethics committee, in accordance with guidelines of the National Health and Medical

Research Council of Australia (22).


Twin‐bearing date‐mated merino ewes were anaesthetised at 128‐130 d gestation

(term ≈ 150 d) with intramuscular xylazine (0.5 mg kg‐1, Troy Laboratories, N.S.W.,

Australia) and ketamine (20 mg kg‐1, Parnell Laboratories, N.S.W., Australia) and

intubated (7.5 mm cuffed tracheal tube, Portex Ltd. England). Maternal anaesthesia

was maintained with isoflurane in 100 % O2. The fetus was exteriorized via

hysterotomy and a right lateral thoracotomy was performed. A flow probe (4R,

Transonic Systems, Ithaca, NY) was positioned around the left pulmonary artery and a

catheter was inserted into the main pulmonary artery (21). The fetus was intubated

orally (4.5 mm cuffed tracheal tube, Portex Ltd. England), lung fluid was suctioned and

intra‐tracheal surfactant (100 mg kg‐1, Abbott Laboratories, U.S.A.) was administered

prior to delivery of the lamb. Unventilated controls (UVC; n=6) were euthanised

(pentobarbitone 100 mg kg‐1 i.v. Jurox, Australia) at delivery without instrumentation,

suctioning or surfactant. Remaining lambs were randomized to one of three ventilation

groups including conventional mechanical ventilation (CMV: n=6), high‐frequency jet

ventilation (HFJV; n=8) and high‐frequency oscillatory ventilation (HFOV; n=8) as

outlined below.

239

Ventilation

Instrumented lambs were dried, weighed. Functional residual capacity was established

by delivering 2 sustained inflations to 30 cmH2O (20 s and 10 s duration respectively)

with an infant T‐piece resuscitator (NeopuffTM, Fisher & Paykel Healthcare, Auckland,

New Zealand), immediately before ventilation was commenced using the assigned

ventilation strategy as detailed below. The initial FiO2 was 0.4 for all groups.

Conventional Mechanical Ventilation

Initial settings for positive pressure ventilation (PPV) with volume guarantee (VG)

included: respiratory rate 50 breaths/min; tI 0.5 s; VG 5 mL/kg; positive end‐expiratory

pressure (PEEP) 7 cmH2O; and a peak inspiratory pressure limit (PIPlimit) of 30 cmH2O

(Babylog 8000+, Drägerwerk, Lubeck, Germany). After 5 min, the VG was increased to 7

mL/kg and PIPlimit was increased to 40 cmH2O. FiO2 was altered to target SpO2 88‐94 %,

VT was altered to target PaCO2 45‐55 mmHg and PEEP was altered according to the

response of SpO2 to changes in FiO2 (Figure 1A).

High‐frequency Jet Ventilation

High‐frequency jet ventilation (Life Pulse™, Bunnell Inc., Salt Lake City, U.S.A.) coupled

to a pressure‐limited time‐cycled infant conventional ventilator (Babylog 8000+,

Drägerwerk, Lubeck, Germany) was commenced with the following initial settings:

respiratory rate 420 breaths/min (7 Hz); peak inspiratory pressure (PIPHFJV) 40 cmH2O;

PEEP 8 cmH2O; FiO2 0.4 and tI was fixed at 0.02 s. PIPHFJV was adjusted to achieve

permissive hypercapnia (PaCO2 45‐55 mmHg) to a maximum of 40 cmH2O. FiO2 was

240

altered to target SpO2 88‐94 % and CMV breaths were delivered according to the

protocol algorithm (Figure 1 B) to target lung volume recruitment.

High‐frequency Oscillatory Ventilation

High‐frequency oscillatory ventilation (3100A, Care Fusion, CA, U.S.A.) was

commenced as follows: frequency 12 Hz (720 breaths/min); Paw 16 cmH2O; tI 33 %

(tI:tE 1:2) and an amplitude (ΔP) of 30 cmH2O. The amplitude was adjusted to achieve

permissive hypercapnia (PaCO2 45‐55 mmHg) to a maximum of 50 cmH2O. The Paw

was adjusted to optimise oxygenation, preceding adjustments to FiO2 to target SpO2

88‐94 % (Figure 1 C).

Postnatal care

Propofol (0.1 mg/kg/min; Norbrook Laboratories Ltd., Victoria, Australia) and

remifentanil (0.05 µg/kg/min; Abbott Laboratories, U.S.A.) were infused continuously

through an umbilical vein for anaesthesia and analgesia. An umbilical arterial catheter

was used for intermittent sampling to assess gas exchange and acid‐base balance.

Rectal temperature was monitored continuously and maintained between 38° and 39°

C (normothermic for newborn lambs). Ventilator settings and physiological data were

recorded at intervals. After final measurements were obtained, the FiO2 was increased

to 1.0 for 2 min after which the tracheal tube was occluded for 3 min to facilitate lung

collapse prior to euthanasing the lamb (pentobarbitone 100 mg kg‐1 i.v. Jurox,

Australia).

241

Physiological Analyses

Continuous measurements of pulmonary blood flow (PBF) and pulmonary artery blood

pressure (PABP) were processed via calibrated pressure transducers (Maxxim Medical,

Tx, U.S.A.). Data were amplified and digitally recorded (Powerlab 8SP, ADInstruments,

N.S.W., Australia). Pulmonary waveform analysis was performed at regular time points

as described previously (23) to quantifiy changes in pulmonary blood flow throughout

the cardiac cycle. Pulsatility Index (PI), a measure of downstream resistance to blood

flow, was calculated as (peak systolic flow – minimum flow after systolic peak)/mean

peak systolic flow and averaged over five consecutive cardiac cycles.

The Oxygenation Index (OI) was calculated as OI=2

2

aO

100 x aw xFiO

P

P where FiO2 is fractional

inspired oxygen concentration, Paw is mean airway pressure and PaO2 is partial

pressure of oxygen in arterial blood. An increase in the OI suggests deterioration in

arterial oxygenation.

Post‐mortem

The lung was exposed by thoracotomy, and an in situ deflation pressure volume curve

was obtained (24). The right upper lung lobe was inflation fixed (30 cmH2O) in formalin

and samples of the right lower lobe were snap frozen for molecular analyses.

Bronchoalveolar lavage (BAL) was performed on the left lung for cytology and protein

analysis by the Lowry method (25, 26). Differential cell counts were performed on

cytospin samples of the BAL fluid stained with Diff‐Quik (Fronine Lab Supplies, N.S.W.,

Australia).

242

RNA was extracted from the left lung and reverse transcribed to cDNA (QuantiTect®

Reverse Transcription Kit, Qiagen, U.S.A.). Expression of IL‐1β and IL‐6 was measured

by qRT‐PCR (27) and normalized to 18S RNA (28) using the 2‐∆∆CT method (29).


Kruskal‐Wallis one way analysis of variance on ranks was used to compare groups at

specific time points while the effect of ventilator strategy on ventilator requirements

and physiological changes over the duration of the study were determined using two‐

way repeated measure analysis of variance. Posthoc comparisons were performed

using the Holm‐Sidak method. Analyses were performed using SigmaStat (Version 3.5,

Systat Software Incorporated, U.S.A.) with p<0.05 considered statistically significant.

Data are expressed as mean (SEM) unless otherwise stated.

243

Results


Ventilation and Oxygenation

PaCO2 was higher (and above the target range) in the HFOV group compared to both

HFJV and CMV at 45, 60, 120 and 180 min (Figure 2A). The ∆P was highest for HFOV

from 20 min (Figure 2B).

FiO2 commenced at 0.4 in all ventilated groups. At 60 and 75 min, FiO2 was higher in

the HFOV group compared to HFJV and CMV groups. At 75 min FiO2 was higher in the

HFJV group compared to the CMV group (Figure 2C). The alveolar‐arterial difference in

partial pressure of oxygen (AaDO2) was higher in the HFOV group at 60 and 75 min

(Figure 2D). Paw was lowest in the CMV group for most of the study while HFJV Paw

was lower than HFOV Paw at 45, 120, 150 and 180 min (Figure 2E).

Pulmonary Blood Flow

End systolic and end diastolic pulmonary blood flows were comparable until 150 min

from which time HFJV lambs had lower flows (Figure 3A and 3B respectively).

Pulsatility Index was comparable between all groups for the duration of the study

(Figure 3C).

Post‐mortem

BAL fluid protein concentration and cell populations were similar between all groups.

The expression of IL‐1β and IL‐6 were not different between groups (Table 2). There

244

was no difference in the static lung compliance, as assessed by the deflation limb of

the post‐mortem pressure‐volume curve, between the ventilated groups. The UVC

group had lower static lung compliance (Figure 4A).

245

Discussion

This study aimed to compare a CMV strategy that included moderate PEEP (7 cmH2O)

with both HFJV and HFOV over a 3 hour ventilation study using a preterm lamb model

of RDS. Gas exchange was comparable for both CMV and HFJV lambs, however, HFOV

did not achieve oxygenation and ventilation targets within the algorithm utilised.

Pulmonary vascular resistance was similar for all groups though HFJV caused shunting

of blood at the end of the ventilation period. Despite these differences in gas exchange

and pulmonary blood flow there were no differences in the markers of lung injury.

Each lamb was managed identically immediately after delivery and then according to a

predetermined ventilation strategy specific algorithm that aimed to open the lungs,

keep the lungs open and maintain optimal oxygenation as described by the open lung

approach (4, 5). The initial sustained inflations were performed as a specific lung

volume recruitment manoeuvre for all animals so the subsequent ventilation strategy

followed a lung volume recruitment process which was less aggressive than it would

have been otherwise. In the CMV group, the initial PEEP was chosen on the basis of

previous observations in the preterm lamb model to maintain end‐expiratory lung

volume above the closing volume as PEEP below the inflection point will increase the

potential for atelectotrauma (30). During HFOV, we used a stepwise recruitment

strategy as standardly performed in clinical practice (31, 32). The maximum Paw in this

group was limited to, but did not reach 26 cmH2O as we expected that the prophylactic

administration of surfactant and the delivery of sustained inflations immediately prior

to HFOV would establish ventilation above the critical closing pressure of the lungs

246

(33). For the HFJV group we chose to use a combination of intermittent CMV breaths

and incrementing PEEP to recruit and stabilise alveoli. The maximum PIPHFJV was set at

40 cmH2O as we have previously successfully ventilated lambs without needing to

exceed this limit (34). The number of CMV breaths was limited to 5 breaths/min in line

with clinical recommendations and our previous experience that this CMV breath rate

was sufficient to provide physiological benefit at initiation of ventilation (unpublished

data ‐ see Chapter 7). Despite our previous experience that PIPCMV 5 cmH2O above

PIPHFJV provides greater physiological benefit with the least evidence of harm

(unpublished data – see Chapter 6), the PIPCMV was kept 5 cmH2O below PIPHFJV as

results of the previous study were not fully available at the time of the study. All

ventilation protocols aimed to minimise cyclic stretch within the lung using a

permissive hypercapnia approach.

The comparison of 3 different ventilation strategies is a challenge as ventilator settings

and displayed measurements vary between modalities. During CMV and HFJV, Paw is

determined by PIP, PEEP, respiratory frequency, and the tI:tE ratio (35). During CMV,

PIP, ∆P and Paw are fully transmitted to the distal airways so pressure measurements

at the airway opening closely approximate alveolar pressures (15). During high‐

frequency ventilation (both HFJV and HFOV), however, the ∆P is attenuated in the

distal airways and alveoli. During HFJV, pressure monitored at the airway opening via

the custom designed tracheal tube adaptor closely approximates the mean pressure at

the distal tip of the tracheal tube (36). In contrast, during HFOV the mean pressure is

measured at the airway opening and hence does not reflect mean pressure drop

across the tracheal tube.

247

One of the aims of this study was to compare the physiological responses to different

ventilation strategies in a standardised model of RDS. Oxygenation and ventilation

variables were recorded to determine whether or not the different strategies achieved

similar physiological outcomes. In the HFOV group we were unable, without exceeding

the preset limits of Paw and ∆P, to approach optimal oxygenation and ventilation and

there were times when FiO2 and PaCO2 were higher in this group. Oxygen

requirements followed a similar trend between the groups with a gradual increase in

FiO2 and AaDO2 over the 3 h study indicating progressive ventilation‐perfusion

mismatching. Nonetheless, FiO2 requirements and AaDO2 were increased from 60‐75

min ventilation in the HFOV group compared to both HFJV and CMV lambs. The Paw

during HFOV, however, was highest throughout the study but the indices of

oxygenation were worst for this group. Given the correlation of Paw and oxygenation it

is surprising that this higher Paw did not translate to a lower FiO2 in this group. FiO2

tended to be higher in the HFOV group from soon after initiation of ventilation hence it

is conceivable that the limit of 26 cmH2O placed on Paw may have limited achievement

of an open lung. Secondly, while an tI:tE ratio of 1:2 during HFOV may minimise gas

trapping, it may also create a mean pressure drop across the tracheal tube that

becomes clinically significant (37). If alveolar pressure is lower than Paw displayed on

the ventilator, the benefits to oxygenation may not manifest. Conversely, it is also

feasible that Paw remained too high for too long during HFOV and the associated

barotrauma caused the deterioration in oxygenation in this group. Another

explanation is that the sustained inflations may have been more effective at volume

recruitment than we appreciated and our subsequent ventilation strategy caused

overinflation of the lungs in the HFOV lambs.

248

There were differences in ventilation which may be attributed to the upper limits set

on the parameter primarily responsible for carbon dioxide elimination (VT/kg for CMV,

∆P for HFJV and ∆P for HFOV). The ∆P measurements during HFOV reflect measured

pressure at the proximal end of the tracheal tube, while ∆P for HFJV are calculated (by

the ventilator algorithm) to estimate the airway pressure at the distal end of the

tracheal tube. The pressure transmitted along the airway will be affected by lung

compliance where reduced lung compliance will increase transmission of ∆P from the

airway opening to the alveoli (38) The ∆P recorded during HFOV may be attenuated by

between 40 and 80 % when measured just distal to the tracheal tube (32). This means

the highest ∆P of 50 cmH2O during HFOV is likely to have created between 20 and 40

cmH2O of pressure in the trachea, which creates a range which includes the highest ∆P

during HFJV of 27.5 cmH2O. Nevertheless we did not achieve the target PaCO2 for the

HFOV animals and in hindsight a higher limit on ∆P may have been appropriate.

There was a predictable initial increase in PBF in all groups with the transition from

fetal to neonatal circulation as the pulmonary vasculature dilates. Pulmonary blood

flow subsequently steadily became more negative over the remainder of the study in

all groups. Given the fall in PBF was mirrored by a steady increase in pulsatility index,

particularly in the first 90 min after birth, these findings are most likely explained by an

increase in pulmonary vascular resistance and right to left shunting of blood through

fetal vascular channels (39).

The significant fall in end systolic and end diastolic pulmonary blood flow in the HFJV

animals over the last 30 min of the study compared to both CMV and HFOV groups was

unexpected and is difficult to explain. We had expected the magnitude of change in

249

pulmonary blood flow to mirror changes in Paw in all the ventilated groups as it has

been reported that increasing Paw decreases PBF and increases pulmonary vascular

resistance during CMV (40), HFJV (34) and HFOV (21). With a Paw consistently lower in

HFJV than that observed in the HFOV group, we would have expected that HFJV would

be less likely to negatively impact pulmonary blood flow and to have a lower pulsatility

index compared to HFOV. Although the pulsatility index increased in the HFJV group

over the last 30 min of the study, it was not significantly higher than the pulsatility

index in either the CMV or HFOV groups throughout the study and hence does not

explain the late fall in PBF in the HFJV animals.

Given that we followed an algorithm for optimal ventilation with each strategy, and

there were not marked differences in ventilator parameters it is not surprising that we

did not find a difference in lung injury markers. A comparison such as ours has not

been performed before but there are a multitude of studies assessing lung injury in

response to mechanical ventilation in neonates and animals (41‐44). The results of

studies comparing lung injury following HFOV and CMV vary and demonstrate either

little difference in the alveolar leakage and systemic inflammation in neonates (45),

probable attenuation of early activation of inflammation and clotting in preterm lambs

during HFOV when compared to CMV (46) and reduced pro‐inflammatory cytokines in

HFOV treated neonates compared to CMV (47). There are fewer studies comparing

lung injury during HFJV but a comparison of HFJV and HFOV in rabbits showed a clear

reduction in lung injury following HFJV (48). The lack of difference in the markers of

lung injury we chose to study suggests that the strategies were equivalent in their

ability to produce inflammation in the lung.

250

There are a number of limitations to this study. We aimed to compare three different

ventilation strategies utilising an open lung approach but failed to achieve our target

arterial blood gas parameters in all the groups. The basis for the higher PaCO2 in the

HFOV group is unknown and the higher FiO2 required to achieve equivalent SpO2 is

difficult to interpret given that the limit of Paw was reached. Furthermore, the

ventilation period was short which may have prevented a difference in lung injury

markers. The changes in end systolic and end diastolic blood flow are also difficult to

interpret in light of the short duration of the study. It is also important to acknowledge

that surfactant was administered to the lambs immediately after delivery, which may

not always be achieved in clinical practice. The lambs in our study were anaesthetised

and underwent an invasive surgical procedure. The hemodynamic effects of

anaesthesia combined with the physical impact of instrumentation on lung inflation

are likely to impact physiological outcomes. Nevertheless, these limitations were for

the most part standard across each group and this model provides the first direct

comparison of these three lung protective ventilation strategies in an animal model.

In conclusion, the open lung approach to CMV, HFJV and HFOV were all suitable for the

respiratory management in the context of our model of RDS in preterm lambs. The lack

of significant differences in end systolic and end diastolic PBF for the majority of the

study, lung injury data and static lung compliance demonstrate that in the absence of

airleaks each of these strategies can be employed in the clinical setting with a

comparable pressure cost of ventilation.

251

Acknowledgements

We would like to express our sincere appreciation to the members of the Ovine

Research Group, Ilias Nitsos and Carryn McLean, for technical assistance and JRL Hall

and Co. for provision and early antenatal care of the ewes.

252

Tables


UVC CMV HFJV HFOV

n (male) 6 (3) 6 (3) 8 (4) 8 (6)

Birth weight (kg) 3.9 (0.2) 3.5 (0.2) 3.6 (0.2) 3.5 (0.2)


Cord pH 7.16 (0.03) 7.21 (0.03) 7.19 (0.03) 7.20 (0.04)

Cord PaCO2 (mmHg) 75.7 (3.6) 75.4 (6.7) 75.6 (5.2) 81.9 (10.5)

UVC = Unventilated Control, CMV = Conventional Mechanical Ventilation, HFJV = High‐

frequency Jet Ventilation, HFOV = High‐frequency Oscillatory Ventilation. Values are

mean (SEM).

253

Table 2: Post mortem inflammatory markers

UVC CMV HFJV HFOV

BAL fluid

Protein (mg mL‐1) 183.2 (37.9) 363.3 (71.4) 272.3 (37.9) 275.9 (28.1)


(x 106 kg‐1) 1 (0.3) 17.6 (18.9) 6.7 (1.7) 6.9 (2.6)

Neutrophils (x 106 kg‐1) 0.6 (0.3) 14.4 (18) 4.9 (1.4) 4.9 (2.1)

Mononuclear cells (x 106 kg‐1) 0.3 (0.1) 3.2 (3) 1.5 (0.4) 1.7 (0.8)

Lung Tissue

IL‐1β (fold change) 1.4

(0.7, 2.2)

4.8

(3.2, 18.0)

7.9

(3.1, 15.4)

10.9

(3.1, 53.1)

IL‐6 (fold change) 1.2

(0.5, 2.4)

3.0

(2.2, 33.5)

6.8

(2.2, 19.8)

11.8

(2.4, 40.4)

UVC = Unventilated Control, CMV = Conventional Mechanical Ventilation, HFJV = High‐

frequency Jet Ventilation, HFOV = High‐frequency Oscillatory Ventilation, IL‐1β =

interleukin 1 beta, IL‐6 = interleukin 6. Values are mean (SEM) or median (25th, 75th

centile) for parametric and non‐parametric data respectively.

254

Figure 1A

255

Figure 1B

256

Figure 1C

257

Figure 2

258

Figure 3

259

Figure 4

260

Figure Legends

Figure 1 Ventilation strategies:

A: Conventional mechanical ventilation. PPV = Positive pressure ventilation, VG =

volume guarantee, tI = inspiratory time, VT = tidal volume, PIP = peak inspiratory

pressure, PEEP = positive end‐expiratory pressure SpO2 = oxyhaemoglobin saturation

measured by the pulse oximeter, FiO2 = fractional inspired oxygen concentration,

PaCO2 = partial pressure of carbon dioxide in arterial blood.

B: High‐frequency jet ventilation strategy. HFJV = high‐frequency jet ventilation, tI =

inspiratory time, PIP = peak inspiratory pressure, CMV = conventional mechanical

ventilation, PEEP = positive end‐expiratory pressure, CPAP = continuous positive

airway pressure, SpO2 = oxyhaemoglobin saturation measured by the pulse oximeter,

FiO2 = fractional inspired oxygen concentration, PaCO2 = partial pressure of carbon

dioxide in arterial blood.

C: High‐frequency oscillatory ventilation strategy. Paw = mean airway pressure, tI =

inspiratory time, I:E = inspiratory to expiratory time ratio, SpO2 = oxyhaemoglobin

saturation measured by the pulse oximeter, FiO2 = fractional inspired oxygen

concentration, PaCO2 = partial pressure of carbon dioxide in arterial blood.

Figure 2 Ventilation and Oxygenation: A: Partial pressure of carbon dioxide in arterial

blood (PaCO2), B: Airway pressure differential (∆P) (cmH2O), C: Fractional inspired

oxygen concentration (FiO2), D: Alveolar ‐ arterial difference in the partial pressure of

oxygen (AaDO2) (mmHg), E: Mean airway pressure (Paw). *p<0.05 HFJV compared to

261

CMV, #p<0.05 HFOV compared to CMV, ^ p<0.05 HFJV compared to HFOV. Closed

circle = CMV, closed square = HFJV, closed triangle = HFOV.

Figure 3 Pulmonary Blood Flow: A: End Systolic Pulmonary Blood Flow (PBF), B: End

Diastolic Pulmonary Blood Flow, C: Pulsatility Index. *p<0.05 HFJV compared to CMV, ^

p<0.05 HFJV compared to HFOV. Closed circle = CMV, closed square = HFJV, closed

triangle = HFOV.

Figure 4 Static Lung Compliance: Deflation limb of the post‐mortem in situ pressure‐

volume curves, * p<0.05 HFJV compared to CMV, ^ p<0.05 HFJV compared to HFOV, **

p<0.05 UVC compared to HFJV, HFOV and CMV. Closed circle = CMV, closed square =

HFJV, closed triangle = HFOV, open diamond = UVC.

262

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271

9 Discussion

The overall aim of this collection of studies was to systematically investigate high‐frequency

jet ventilation (HFJV), in a preterm lamb model of respiratory distress syndrome (RDS), to

establish an evidence base to support clinical HFJV strategies in neonatal intensive care

units. Although HFJV has been available to clinicians for over 20 years, extensive in vivo

studies exploring HFJV for RDS have not been performed. This has led to the development of

clinical strategies that lack an evidence based structure. Furthermore, the results of

randomised controlled trials have not demonstrated convincingly a clear benefit of HFJV

over other ventilatory strategies in preterm infants. In order to elucidate optimal ventilator

settings during HFJV the impact of positive end‐expiratory pressure (PEEP) and the size,

duration and frequency of conventional mechanical ventilator (CMV) breaths delivered

during HFJV were examined. The results of these controlled studies led to the composition

of an algorithm which was used to compare HFJV with high‐frequency oscillatory ventilation

(HFOV) and a gentle CMV strategy in the preterm lamb model.

We hypothesised that increasing PEEP would recruit alveoli and improve oxygenation at the

expense of pulmonary blood flow and lung injury. Further, we hypothesised that CMV

breaths delivered at a longer inspiratory time, to a higher peak inspiratory pressure and

more frequently would recruit alveoli and improve oxygenation at the expense of lung

injury. To this end we investigated and documented the effect of HFJV on ventilation

(elimination of CO2), oxygenation, pulmonary blood flow, static lung compliance and lung

272

injury. These investigations were undertaken by categorising the ventilator parameters

responsible for alveolar recruitment and maintenance of an open lung during HFJV into

PEEP and CMV breaths. The CMV breaths delivered during HFJV were then compared by

isolating the effect of 2 different CMV breath inspiratory times, peak inspiratory pressure

settings and frequencies. We chose a preterm lamb model for these studies as this non‐

primate animal model has been extensively utilised in the past. This widespread application

of the preterm lamb model has enabled correlation of lung developmental stages between

Ovis aries and Homo sapiens. Furthermore, ventilatory equipment used for human babies

can be used in the laboratory without modifications as the size of a preterm lamb is close to

a term baby.

The high‐frequency jet ventilator we used must be set up in tandem with a conventional

ventilator. The conventional ventilator, amongst other functions, delivers PEEP. PEEP is

utilised during mechanical ventilation to prevent alveolar collapse at the end of expiration,

but also to recruit and stabilise alveoli. PEEP is usually employed during mechanical

ventilation of preterm babies but the consequences of PEEP may outweigh the benefit. The

first study in this thesis: “"High Positive End‐Expiratory Pressure during High‐Frequency Jet

Ventilation Improves Ventilation in Preterm Lambs" investigated PEEP recruitment

manoeuvres and found that incrementing PEEP up to 12 cmH2O facilitated lung volume

recruitment without significant adverse effects on pulmonary blood flow, ex vivo lung

compliance and lung injury. The premise of this study challenged common clinical practices

which rarely increased PEEP to this level. The results were presented the following year

(2008) and clinical strategies have been altered to increase PEEP to a higher level for

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alveolar recruitment manoeuvres based upon the results of that first study (personal

communication: Rob Graham, R.R.T./N.R.C.P., N.I.C.U., Sunnybrook Health Sciences Centre

Toronto,Ontario,Canada).

The subsequent studies examined the parameters of the CMV breaths that can be delivered

during HFJV using a ventilated control group and an unventilated control group for

comparison. A comparison of CMV breaths delivered over 0.5 s and 2 s was made over a 2 h

study: “The Impact of Conventional Breath Inspiratory Time during High‐frequency Jet

Ventilation in Preterm Lambs”. Consistent with our hypothesis for this study we concluded

that a longer CMV breath inspiratory time was more injurious to the lung given the trend for

increased inflammatory markers in this group. To follow this study a comparison of CMV

breaths delivered to a peak inspiratory pressure (PIP) 5 cmH2O above and below the HFJV

PIP was made: “The Effect of Conventional Breath Peak Inspiratory Pressure during High‐

frequency Jet Ventilation in Preterm Lambs”. The results of this study were contrary to our

hypothesis that CMV breaths delivered to a PIP 5 cmH2O above the HFJV PIP would be more

injurious. In both of the aforementioned CMV breath studies we delivered 5 CMV

breaths/min so our final study compared CMV breaths delivered 5 or 20 times/min:

“Alveolar Recruitment with Five or Twenty Conventional Mechanical Ventilator Breaths per

minute during High‐frequency Jet Ventilation in Preterm Lambs”. The CMV breaths in this

study were delivered to a PIP 5 cmH2O below the HFJV PIP and while more frequent CMV

breaths increased oxygenation requirements and compromised static lung compliance there

was no clear evidence of greater lung injury in this group. An issue with this study was the

lack of a third treatment group. There was an unventilated control group, a HFJV only

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ventilated control group and 2 HFJV+CMV groups. In hindsight the addition of another group

in which 20 CMV breaths were delivered to PIP 5 cmH2O above the HFJV PIP would have

been interesting, especially in light of the results of the CMV breath PIP comparison study.

We attributed the differences in the CMV breath PIP comparison study to less effective lung

volume recruitment when 5 CMV breaths/min were delivered to a PIP 5 cmH2O below HFJV

PIP. This finding left us comparing a strategy of sub optimal lung volume recruitment (5 CMV

breaths/min to a low PIP) with a strategy that we hypothesised would cause greater lung

injury (20 CMV breaths/min) Whether optimal lung volume recruitment can be achieved

when CMV breaths are delivered to PIP higher than HFJV breaths or when CMV breaths are

delivered at a faster rate (>5 CMV breaths/min) is difficult to say without comparing a high

CMV PIP, high CMV breath rate group to the groups we already have. However, the high

CMV breath rate group did demonstrate convincing inadvertent PEEP and this should

increase Paw which, to a point, may improve oxygenation, at the expense of lung injury. The

combination of inadvertent PEEP when CMV breaths are delivered more often with a CMV

breath PIP higher than HFJV PIP may cause greater lung injury. The data we have suggest

CMV breaths delivered to a PIP 5 cmH2O above HFJV PIP is better, but the optimal frequency

for these breaths is unknown and warrants further investigation.

The final study: “A Comparison of High‐frequency Jet Ventilation and High‐frequency

Oscillatory Ventilation with Conventional Mechanical Ventilation in Preterm Lambs”

provided an opportunity to compare the 3 strategies most commonly utilised in the

neonatal intensive care unit. This direct comparison has not been performed previously and

gave us a unique opportunity to investigate the ventilatory efficacy of these 3 strategies in a

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controlled environment. Our hypothesis was not supported by our results insofar as the

cardiovascular side effects of HFJV were not substantially different to the other strategies.

At best, we demonstrated that when an open lung approach drives the decision making

process there was no clear benefit of one strategy over another in our preterm lamb model

of RDS.

There are a number of limitations to the studies in this thesis. The short duration of

ventilation prevented extensive temporal data collection, which limits the relevance of the

information to the clinical setting as babies requiring HFJV will be ventilated for days,

potentially weeks. Nevertheless, the acute effect of changes in airway pressures is still

relevant to the neonatal intensive care unit environment. An animal model will always

introduce species specific characteristics which may or may not be recognized. Furthermore,

the use of animals in research is bound by the Australian code of practice for the care and

use of animals for scientific purposes. Working within the code requires anaesthesia of the

pregnant ewe for caesarean delivery, and therefore transplacental transfer of anaesthetic

drugs to the fetus, and the administration of additional anaesthesia and analgesia to the

lamb following delivery. The influence of anaesthetic drugs was controlled as it was

standard throughout the studies, however, it remains that these drugs will affect the

cardiovascular system and the target population is unlikely to be subject to the same

pharmacological restraint.

A major limitation of these studies was the sample size. Our largest study group had 8

animals which, in light of the variability of some data, decreased the power of these studies.

Larger groups would have increased study power, but the financial and animal welfare

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expense was too great to accommodate this. This limitation is particularly applicable to the

final study: “A Comparison of High‐frequency Jet Ventilation and High‐frequency Oscillatory

Ventilation with Conventional Mechanical Ventilation in Preterm Lambs” where the decision

making process was governed by the physiological status of the lamb at preset time points.

We wanted to manage this group as though they were individual patients where survival

was imperative. This was in contrast to the preceding studies where a predetermined

ventilatory strategy was set without aiming for survival, but rather, aiming to document the

effects of individual ventilator settings. It is also noteworthy that comparing the efficacy of

ventilation of one strategy compared to another was difficult as we targeted a tight PaCO2

and SpO2 range. This meant that our primary outcome variables were other indicators of

physiology (not including PaCO2 and SpO2) and injury.

It is also important to note that our preterm lamb model of RDS represents just one of the

many clinical presentations that may require ventilatory support. Preterm babies are at risk

of a number of different lung diseases and may not be commenced on a ventilator protocol

immediately after delivery. With this in mind, the results of these studies cannot be directly

translated to babies with heterogeneous lung disease or those requiring ‘rescue’ from

another ventilatory strategy. Nonetheless these data are still useful for more evidence

based decision making in the context of an individual patient.

The results of these studies contribute to the sparse data on HFJV and have provided

fundamental information that will enable a more evidence based approach to clinical

decision making in the neonatal intensive care unit. Future work in this area should focus on

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the target population and incorporate randomised controlled trials comparing HFJV to other

ventilatory strategies.

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Appendix

Musk GC, Polglase GR, Bunnell JB, McLean CJ, Nitsos I, Song Y and Pillow JJ 2011 High

Positive End‐Expiratory Pressure during High‐Frequency Jet Ventilation Improves

Oxygenation and Ventilation in Preterm Lambs. Pediatric Research 69(4):319‐324

See attached pdf

Documents

Optimisation of High frequency Jet Ventilation for the Preterm · Pressure and Frequency during High‐frequency Jet Ventilation in Preterm Lambs. Manuscript in preparation (Chapters