1. health and fitness
2. disease
3. asthma

MICRO-CT ANALYSIS OF PARAFFIN EMBEDDED LUNG TISSUE:
IS SMALL AIRWAY OBSTRUCTION AN EARLY FEATURE OF COPD?
by
Hyun-Kyoung Koo
MB BCh BAO, Queen‟s University Belfast, 2010
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
in
THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES
(Pharmacology and Therapeutics)
THE UNIVERSITY OF BRITISH COLUMBIA
(Vancouver)
April 2014
© Hyun-Kyoung Koo, 2014
Abstract
Rationale: Airflow obstruction, the hallmark characteristic of Chronic Obstructive Pulmonary
Disease (COPD) has long been attributed to a combination of small airways disease and
emphysematous destruction, however, the relative role of each pathological feature has not been
well understood. McDonough et al., recently reported a significant reduction in terminal
bronchiolar number in end-stage COPD compared to healthy controls. Further, the study reported
that the loss of terminal bronchioles occurred in regions of lung with and without emphysema,
posing the question, which pathological event occurs first?
Hypothesis: Narrowing and obliteration of small airways precedes emphysematous destruction
in COPD.
Methods: Lung samples obtained from patients undergoing surgical treatment for lung cancer
were formalin inflated, sliced and sampled prior to paraffin embedding (FFPE). A Nikon
Metrology micro-CT scanner was used to scan these FFPE cores, and volumetric data sets were
examined to determine the number of terminal bronchioles/ml (TB/ml) and presence of
emphysema using mean linear intercept (Lm). Semi-automatic segmentation enabled 3D
reconstruction of the airways to characterize structure and calibre, and scout for regions of
interest that were sectioned and stained with Movat‟s Pentachrome, for analysis of airway
morphology.
ii
Results: Using a novel combination of multidetector-CT, micro-CT and histology, this study
demonstrates that micro-CT scans of FFPE cores provide adequate contrast to determine Lm
values that were validated by histology. We report that the number of terminal bronchioles/ml is
significantly decreased from 6.2 TB/ml in the controls, to 4.6 TB/ml in mild/moderate COPD
patients. When the number of TB/ml is compared to Lm we demonstrate that terminal
bronchioles are destroyed in tissues where no emphysema is present. These lesions were
confirmed by histology, using the micro-CT images to precisely locate the airway lesions,
enabling efficient sectioning of the FFPE cores for further characterization.
Conclusions: Treatment of COPD has traditionally focused on patients with severe disease and
no current pharmacological therapies have been shown to affect long term, lung function decline.
Our findings suggest that irreversible pathological events occur in the early stages of disease and
emphasize the importance of early diagnosis and intervention to modify the progression of this
debilitating respiratory disorder.
iii
Preface

This study was conducted through collaboration between the Centre for Heart and Lung
Innovation at St Paul‟s Hospital, University of British Columbia and the µ-VIS Imaging
Centre at the University of Southampton, UK.

Dr. Tillie-Louise Hackett was responsible for the conception of this work with
contributions by Dr. Jim Hogg.

Hyun-Kyoung Koo, Dr. Tillie-Louise Hackett and Dr. Dragos Vasilescu contributed to
the design, hypothesis and objectives of this study.

The micro-CT scanning protocol was previously designed and optimized by Dr. Anna
Scott, Dr. Jane Warner and Dr. Ian Sinclair and conducted by Dr. Orestis Katsamenis at
the µ-VIS Imaging Centre, University of Southampton.

Formalin fixed paraffin embedded (FFPE) human lung tissue samples were obtained
from the James Hogg Research Centre Lung Registry at St Paul‟s Hospital. All
specimens were collected over numerous years with informed consent.

Hyun-Kyoung Koo and Dr. Tillie-Louise Hackett prepared the FFPE samples for microCT scanning.

Amrit Samra conducted the histological sectioning and staining with assistance by HyunKyoung Koo and Dr. Tillie-Louise Hackett.

Hyun-Kyoung Koo performed all the data acquisition and analysis under the guidance of
Dr. Tillie-Louise Hackett and Dr. Dragos Vasilescu.

Hyun-Kyoung Koo wrote the manuscript with edits and final approval by Dr. TillieLouise Hackett.
Ethics Approval: This study was approved by the Institutional Providence Research Ethics
Committee (University of British Columbia‟s Research Ethics Board) [certificate # H13-02173].
iv
Table of Contents
Abstract .......................................................................................................................................... ii
Preface ........................................................................................................................................... iv
Table of Contents ...........................................................................................................................v
List of Tables ................................................................................................................................ ix
List of Figures .................................................................................................................................x
List of Abbreviations .................................................................................................................. xii
Acknowledgements .................................................................................................................... xiv
Dedication ................................................................................................................................... xvi
Chapter 1: Introduction ................................................................................................................1
1.1
Overview of chronic obstructive pulmonary disease ...................................................... 1
1.1.1 Definition .................................................................................................................... 1
1.1.2 Clinical features and diagnosis ................................................................................... 2
1.1.3 Epidemiology and risk factors .................................................................................... 3
1.2
Chronic inflammation ..................................................................................................... 5
1.2.1 Inflammatory cells and mediators ............................................................................... 6
1.2.2 Protease-antiprotease imbalance and oxidative stress ................................................ 7
1.3
Anatomical description of the lung ................................................................................. 7
1.3.1 Airway structure.......................................................................................................... 8
v
1.3.2 Pulmonary vasculature .............................................................................................. 10
1.4
Pathophysiology of COPD ............................................................................................ 11
1.4.1 Emphysema ............................................................................................................... 11
1.4.1.1
History............................................................................................................... 11
1.4.1.2
Subtypes of emphysema ................................................................................... 12
1.4.2 Chronic bronchitis ..................................................................................................... 14
1.4.3 Pulmonary hypertension ........................................................................................... 15
1.4.4 Small airways disease ............................................................................................... 16
1.4.4.1
History............................................................................................................... 16
1.4.4.2
Site of airway obstruction ................................................................................. 17
1.4.4.3
Remodeling and narrowing of small airways ................................................... 17
1.5
Management of COPD .................................................................................................. 19
1.6
Imaging techniques ....................................................................................................... 22
1.6.1 Clinical computed tomography ................................................................................. 22
1.6.2 Ultra structure imaging of the lung using micro-CT ................................................ 24
1.6.3 Application of stereology to understand lung morphometry .................................... 25
1.7
The relationship between small airway obstruction and emphysema in COPD ........... 27
1.8
Innovative micro-CT imaging of formalin fixed, paraffin embedded samples to
understand small airway obstruction ........................................................................................ 32
1.9
Hypothesis and specific aims ........................................................................................ 34
vi
Chapter 2: Methods .....................................................................................................................35
2.1
Patient population and lung tissue sampling ................................................................. 35
2.2
Sample preparation for micro-CT ................................................................................. 36
2.3
Micro-CT image acquisition and reconstruction .......................................................... 37
2.4
Image visualization and analysis .................................................................................. 39
2.5
Volume segmentation of clinical MDCT data using stereological methods ................ 41
2.6
Obtaining core volumes ................................................................................................ 42
2.7
Systematic uniform random sampling (SURS) for Lm measurements ........................ 44
2.8
Measurement of mean linear intercept (Lm) ................................................................ 46
2.9
Comparison of Lm in micro-CT versus histological samples ...................................... 48
2.10
Counting terminal bronchioles ...................................................................................... 49
2.11
3D rendering of structures of interest on micro-CT and further characterization by
histology.................................................................................................................................... 51
2.12
Statistical analysis ......................................................................................................... 52
Chapter 3: Results........................................................................................................................53
3.1
Patient characteristics and clinical presentation ........................................................... 53
3.2
Assessment of micro-CT for conducting lung morphometry on FFPE samples .......... 53
3.3
Verification by histology of micro-CT imaging for morphological analysis of FFPE
samples ...................................................................................................................................... 55
3.4
The use of micro-CT to determine emphysematous destruction in diseased tissue ..... 57
3.5
The number of terminal bronchioles is decreased in mild/moderate COPD patients ... 60
vii
3.6
Correlation of data to total lung volumes using MDCT parameters ............................. 62
3.7
The number of respiratory bronchioles is decreased in mild/moderate COPD
patients ...................................................................................................................................... 63
3.8
Tracing and 3D rendering of small airway morphology by micro-CT ......................... 64
3.9
Micro-CT as a scouting tool for histological analysis of regions of interest ................ 66
Chapter 4: Discussion ..................................................................................................................68
4.1
Methodological findings ............................................................................................... 68
4.2
Pathological findings .................................................................................................... 70
4.3
Clinical relevance.......................................................................................................... 73
4.3.1 Diagnosis................................................................................................................... 73
4.3.2 Treatment .................................................................................................................. 75
4.4
Limitations of the study ................................................................................................ 75
4.5
Future directions ........................................................................................................... 77
4.6
Conclusion .................................................................................................................... 78
Bibliography .................................................................................................................................79
viii
List of Tables
1.1 A summary of the therapeutic options available for the management of COPD.................... 21
2.1 Patient demographics .............................................................................................................. 36
2.2 Lung volumes calculated from pre-operative clinical MDCT scans ...................................... 41
3.1 Comparison of the number of terminal bronchioles with previous published data ................ 63
ix
List of Figures
1.1 Fletcher-Peto diagram showing the natural history and rate of decline of FEV1 ..................... 5
1.2 Branching network of the respiratory tree ................................................................................ 9
1.3 Resin cast of the human airway tree ....................................................................................... 10
1.4 Subtypes of emphysema compared to a normal acinus .......................................................... 13
1.5 Histological stains showing remodeling and narrowing of small airways ............................. 18
1.6 Two proposed mechanisms of emphysematous destruction in COPD ................................... 31
2.1 Mass attenuation coefficients versus x-ray energy for paraffin wax and soft tissue .............. 38
2.2 Workflow summary of the study ............................................................................................ 40
2.3 Obtaining core volumes using a semi-automatic segmentation approach .............................. 43
2.4 Systematic uniform random sampling (SURS) method.......................................................... 45
2.5 Lm measurement using a pre-formulated grid mask .............................................................. 47
2.6 Lm measurement of histological sections ............................................................................... 48
2.7 Identification of terminal bronchioles on micro-CT ............................................................... 50
3.1 Comparison of clinical MDCT and micro-CT images ........................................................... 54
3.2 Comparison of Lm by micro-CT and histology ...................................................................... 56
3.3 Lm measurements in FFPE tissue from control and mild/moderate COPD patients ............. 59
3.4 Number of terminal bronchioles per millilitre of resected lung tissue is decreased in
mild/moderate COPD patients ...................................................................................................... 61
x
3.5 Number of respiratory bronchioles per millilitre is decreased in mild/moderate COPD ....... 64
3.6 Three dimensional renderings of small airway morphology .................................................. 65
3.7 Serial micro-CT slices with matched histological sections .................................................... 67
xi
List of Abbreviations
COPD
Chronic Obstructive Pulmonary Disease
FEV1
Forced Expiratory Volume in 1 second
FVC
Forced Vital Capacity
FEV1/FVC
Forced Expiratory Volume/Forced Vital Capacity Ratio
GOLD
Global Initiative for Obstructive Lung Disease
DALY
Disability-Adjusted Life Year
α1AT
Alpha-1 Antitrypsin
TNF- α
Tumour Necrosis Factor- α
ROS
Reactive Oxygen Species
MMP
Matrix Metalloproteinase
TIMP
Tissue Inhibitors of Matrix Metalloproteinase
HRCT
High Resolution Computed Tomography
CT
Computer Tomography
BALT
Bronchus Associated Lymphoid Tissue
2D
2-Dimensional
3D
3-Dimensional
MDCT
Multiple Detector Computer Tomography
DLCO
Diffusing Capacity for Carbon Monoxide
xii
HU
Hounsfield Units
He
Helium
Xe
Xenon
MRI
Magnetic Resonance Imaging
OCT
Optical Coherence Tomography
Micro-CT
Micro-xray Computed Tomography
ATS
American Thoracic Society
ERS
European Respiratory Society
FFPE
Formalin Fixed and Paraffin Embedded
JHRC
James Hogg Research Centre
Lm
Mean Linear Intercept
SURS
Systematic Uniform Random Sampling
FOV
Field of View
ECM
Extracellular Matrix
BOS
Bronchiolitis Obliterans Syndrome
DNA
Deoxyribonucleic Acid
RNA
Ribonucleic Acid
ICS
Inhaled Corticosteroids
FOT
Forced Oscillation Technique
xiii
Acknowledgements
First and foremost, I would like to express my extreme gratitude to my supervisor Dr. TillieLouise Hackett whose dedication to her work and students never fail to amaze. Under her
guidance and expertise, I have gained invaluable research skills and I thank her for this
wonderful project, encouragement and constant support. She has been an inspiration to work
with, a great mentor and friend.
I also express thanks to my co-supervisor Dr. Pascal Bernatchez. I am grateful for his support
through my graduate studies.
I would also like to thank Dr. Dragos Vasilescu for sharing his skills and invaluable teaching,
this project would not have been possible without him.
Thank you to my chair and committee members, Dr. Sastry Bhagavatula, Dr. Jim Hogg and Dr.
André Phillion for their time, expert advice and support. I am privileged to have such
distinguished researchers provide their insight and valuable suggestions to the project.
A special mention is required for Dr. Jim Hogg. It has truly been an inspiration to work
alongside him, a pioneer behind this current study and whose humble passion for research shone
through every encounter and conversation. Without his foresight and dedication, the James Hogg
Research Centre Lung Registry, the source of the valuable human lung tissue samples for this
study would not exist.
xiv
I extend my appreciation to the team at the µ-VIS Imaging Centre of the University of
Southampton, U.K. for providing the facilities and technical assistance necessary for micro-CT
scanning. Thank you especially to Dr. Orestis Katsamenis whose best efforts in scanning our
samples provided us with quality images.
At the Centre for Heart and Lung Innovation, I would like to thank Amrit Samra who provided
excellent technical assistance in histology. Dr. Mark Elliott provided us access to the lung
biobank and sourced important clinical data.
Last but not least, I am grateful to my friends near and far. Thank you to the members of the lab
and the fellow‟s room for creating a warm, friendly and supportive environment to work in. I
especially thank my dearest friend Judith McCluskey, not only for my inspirational quotes sent
faithfully everyday during the writing process but also for always being there for laughs, tears,
stories and life experiences.
xv
Dedication
To my wonderful family who has provided me with unconditional loving support.
I am eternally grateful for all the opportunities, words of wisdom and
encouragement that you have given me in life.
xvi
Chapter 1:
Introduction
1.1 Overview of chronic obstructive pulmonary disease
1.1.1 Definition
Chronic obstructive pulmonary disease (COPD) is defined by „persistent airflow limitation that is
usually progressive and associated with an enhanced chronic inflammatory response within the
lung to noxious particles or gases‟ [1]. COPD is a complex syndrome that encompasses a number
of disease entities, including emphysema, small airways disease, chronic bronchitis and
pulmonary hypertension. Emphysema is a pathological term which is characterized by the
enlargement of airspaces and destruction of alveoli, the gas exchanging surfaces of the lung. The
small airways (<2mm in luminal diameter), are the major site of airflow obstruction in COPD
and involve the narrowing and obliteration of the terminal bronchioles, the last purely conducting
airways of the respiratory tree. In contrast, chronic bronchitis is described clinically as „the
presence of a cough and sputum production for at least three months of two successive years‟
[1], and can occur with or without the presence of airflow limitation which is the defining feature
of COPD. Pulmonary hypertension occurs as a result of pulmonary vascular remodeling,
endothelial dysfunction, hypoxic vasoconstriction and inflammatory cell infiltrates [2]. The
extent and manifestation of each phenotype within COPD patients is highly variable, leading to
significant heterogeneity of the disease. Thus, further understanding of the inter-relationship and
time course of these pathological changes is important for early recognition and successful
management of this common, debilitating respiratory disorder.
1
1.1.2 Clinical features and diagnosis
The three cardinal symptoms of COPD are dyspnea (breathlessness), chronic cough and sputum
production [3]. Wheezing and chest tightness are non-specific and variable symptoms that
patients may also present with. For any patient with one or a combination of the symptoms listed
above, in addition to a history of cigarette smoking or long term exposure to other risk factors, a
clinical diagnosis of COPD should be considered [1, 3, 4].
Lung function measurements, in particular spirometric tests are the gold standard and now a
requirement for the diagnostic evaluation of patients with suspected COPD [1, 5]. This
diagnostic test is performed pre- and post- administration of a short-acting inhaled
bronchodilator and all measurements are assessed by comparison with reference values based on
age, height, sex and race [4]. The patient is requested to take a full breath in until the lungs feel
full. At this point of maximal inspiration, they are instructed to exhale the air as forcefully and as
quickly as possible until their lungs feel empty. The forced expiratory volume in one second
(FEV1) is the volume of air exhaled during the first second of this manoeuvre whilst the forced
vital capacity (FVC) is the total volume of air that can be exhaled. From these two
measurements, the FEV1/FVC ratio is calculated and a post-bronchodilator value of less than 0.7
indicates persistent airflow limitation, thus confirming a diagnosis of COPD [1]. Following a
diagnosis, the severity of COPD is then further characterized by the extent of FEV1 airflow
limitation in that mild GOLD 1 COPD patients have a post-bronchodilator FEV1 ≥ 80%
predicted, moderate GOLD 2 COPD have a 50% ≤ FEV1 < 80% predicted, severe GOLD 3 have
a 30% ≤ FEV1 < 50% predicted, and very severe GOLD 4 have an FEV1 < 30% predicted. It is
important to note that this grading system is now part of a more thorough and global diagnostic
evaluation of COPD severity as the FEV1 only captures a component of this heterogeneous
2
disease. Several tools are now available to assess additional aspects of COPD such as the
severity of symptoms, risk of exacerbations and the presence of co-morbidities as these all
contribute to the patient‟s experience of the disease and prognosis [3].
1.1.3 Epidemiology and risk factors
COPD is predicted to rise from the fourth to the third leading cause of death worldwide over the
next decade, and poses a major public health problem with substantial economic and social
burden [1, 6]. In Canada, COPD is the leading cause of hospital admissions [7] and the total cost
of hospitalizations has been estimated to be $1.5 billion per year [8]. In addition to this direct
impact on the healthcare system, indirect economic costs such as workplace and home
productivity contributes to over a third of the total cost of COPD to society [1, 9]. The DALY
(Disability-Adjusted Life Year) scale is used as an indicator of the burden of disease in a
population [10]. In 2002, COPD was ranked as the eleventh leading cause of DALYs lost
globally and has been projected to become the seventh leading cause by 2030 [6].
This alarming escalation of morbidity and mortality has been attributed to a continued
exposure to COPD risk factors and the world‟s aging population [1]. The major and most well
known risk factor for COPD is inhaled cigarette smoke with the amount and duration of smoking
contributing to disease severity [3]. Exposure to biomass fuels such as coal, wood, straw and
crop residues in poorly ventilated homes play a significant role globally [11, 12]. The chronic
inhalation of other toxic particles and gases such as organic and inorganic dusts, chemical agents
and fumes, and urban air pollution are also recognized risk factors of COPD [1, 13].
Aside from the exposure to noxious particles that induces a chronic inflammatory
reaction characteristic of COPD, additional factors play an important role in the development and
3
progression of this disease. These include genetics, gender, age, lung growth, socioeconomic
status, history of childhood infections, and the presence of other lung disease entities such as
asthma and chronic bronchitis [1, 11]. The most well established genetic risk factor is a
deficiency of alpha-1 antitrypsin (α1AT), a member of the serine proteinase inhibitor family and
a potent inhibitor of neutrophil elastase [14, 15]. Although there are many manifestations of this
disease, one of the key features is the uninhibited proteolytic attack of lung tissue by neutrophil
elastase, predisposing patients to an early emphysematous phenotype [14]. Although COPD has
historically been more prevalent in the male population (thought to be associated with patterns of
smoking and occupational exposures), there is now increasing evidence that the female gender is
more susceptible to the development and progression of this disease [16, 17].
The increased burden of COPD observed in the elderly population, where the prevalence
of disease is two to three times higher in people aged over 60 [18], may be attributed to the
increased cumulative exposures throughout life, or due to age-associated changes in lung
structure and function [1, 18]. Figure 1.1 indicates that although smoking is the major contributor
for the progression of COPD (with smoking cessation providing a beneficial effect at any age),
lung function does decline with normal aging. In addition, there is a susceptible minority of
smokers who progress to the severe stages of disease at a rapid rate. This variation in the
susceptibility of disease between individuals suggests that the gene-environment interaction is
complex, and other unidentified genetic factors are likely to be involved. Studies are ongoing to
identify and further understand the risk factors contributing to the development of this lifethreatening disease.
4
Figure 1.1 Fletcher-Peto diagram showing the natural history and rate of decline of FEV1 in
lifelong non-smokers and smokers who stopped smoking at different years of age. Reproduced
from Pauwels et al. 2004 [4], with permission.
1.2 Chronic inflammation
Chronic inflammation plays a key role in the pathogenesis of COPD. Smoking causes an
inflammatory reaction in all smokers. However, in the susceptible minority of this population
(estimated between 20-40% depending on the study) who progress to COPD, the inflammatory
response appears more amplified and persistent, even after the removal of the insult [19, 20]. The
molecular basis for this enhanced inflammation is not yet fully understood but is postulated to
be, at least in part, genetically determined [21].
5
1.2.1 Inflammatory cells and mediators
The inflammatory process in COPD is heterogeneous and complex, involving both the innate and
adaptive immune responses [21, 22]. In brief, the key cells involved are macrophages,
neutrophils and T lymphocytes (predominantly CD8+ T cells) [23-28].
Macrophages play a central role in orchestrating the chronic inflammatory response. On
activation by cigarette smoke, they secrete pro-inflammatory mediators such as tumour necrosis
factor-α (TNF-α) and CXC chemokines, reactive oxygen species (ROS) and an array of
proteolytic enzymes such as matrix metalloproteinases (MMP), but predominantly MMP-9 and
MMP-12 [21, 29]. Not only are alveolar macrophages increased in number, but have also been
shown to secrete more inflammatory proteins and possess greater elastolytic activity in COPD
patients [30].
Increased neutrophil numbers are associated with COPD disease severity and lung
function decline [26, 31]. They contribute to tissue destruction by the release of key serine
proteases including neutrophil elastase, cathepsin G, proteinase-3, MMP-8 and MMP-9.
Neutrophil elastase, in addition to its elastolytic activity, promotes mucous hypersecretion by
stimulating sub-mucosal glands and goblet cells, contributing to airways obstruction.
T lymphocytes in particular CD8+ cells cause cytolysis and apoptosis of alveolar
epithelial cells through the release of perforins, granzyme-B and TNF-α [32]. Again, there is an
increase in total number of these inflammatory cells in patients with COPD and a correlation
with severity of airflow obstruction [21].
6
1.2.2 Protease-antiprotease imbalance and oxidative stress
The protease-antiprotease imbalance was a long-standing hypothesis explaining the destruction
of the connective-tissue components of the lung, in particular elastin [1, 33, 34]. Proteolytic
enzymes such as neutrophil elastase and MMPs are usually neutralized by their respectful
antiproteases; α1AT and tissue inhibitors of matrix metalloproteinases (TIMPs) [35]. However,
in the susceptible smokers, in addition to an increased activation of proteases, there may be an
impaired function and/or production of these antiprotease proteins causing an imbalance and
consequent uninhibited protease-mediated injury [34, 36].
Increased oxidative stress is a key feature of COPD as smoke exposure both directly
increases the oxidant burden of the lung and indirectly by the production of reactive oxygen
species (ROS). ROS are generated by inflammatory and structural cells including macrophages,
neutrophils and epithelial cells in response to smoke exposure. The oxidant burden within the
lung plays an important pathophysiological role by a variety of mechanisms; amplification of the
inflammatory response, impairment of antiprotease function and induction of endothelial and
epithelial cell apoptosis [21, 36]. However, the exact molecular mechanism of the pathogenesis
of COPD and the reason for such heterogeneous lesions is still unknown.
1.3 Anatomical description of the lung
The respiratory system can be divided into the lungs (right and left), the airways, parenchyma
and associated pulmonary vasculature. Each lung extends from the apex to the base and is
divided into lobes separated by fissures. The right lung is 20% larger in volume, containing three
lobes, whilst the left only contains two.
7
1.3.1 Airway structure
The human airway tree consists of two functional units: the conducting airways, responsible for
transporting air in and out of the lungs, and the respiratory zone where gas exchange occurs. The
airway branching sequence follows a successive dichotomous pattern where each parent airway
bifurcates into two smaller daughter branches. The classic monograph of the human lung
anatomy widely accepted and used today was first produced in 1963 by Weibel who showed that
with each generation of airway branching, there is an exponential increase in the total number
and lumen cross-sectional area of the airways [37, 38].
The conducting unit of the respiratory tree is composed of airway generations 0 to 14
whilst the gas exchange portion extends from generation 15 onwards [39]. The root of this
network is the trachea which measures 1-2cm in diameter in the normal adult lung [40]. As
shown in Figure 1.2A, the trachea branches into the right and left main bronchi, leading then to
the daughter bronchioles and finally the terminal bronchioles. These terminal bronchioles are
therefore the last purely conducting airways and can be defined as „the last bronchioles without
alveolar openings from their walls‟. The transitional and respiratory bronchioles make up the
beginning of the respiratory unit, also known as the pulmonary acinus (a unit of gas exchange
that is ventilated by one single terminal bronchiole) [39, 41]. Hence, from approximately
generation 15, the airway walls begin to be interrupted by the presence of alveolar ducts which
open up into alveolar sacs as demonstrated by the scanning electron microscopy image in Figure
1.2B.
8
After approximately 23 generations of bifurcations, the human lung consists of over 300
million alveoli, each measuring approximately 200µm in diameter at functional residual capacity
(end of quiet expiration) [42]. The total surface area of alveoli within the lung is approximately
130-150m2, an area almost equivalent to a singles tennis court [39, 43].
Figure 1.2 Branching network of the respiratory tree. A: A highly simplified but useful
model of airway branching of the human lung from the trachea (generation 0) to the alveolar
ducts and sacs (generations 20-23). B: A scanning electron microscope image demonstrating the
bifurcation of a terminal bronchiole into two respiratory daughter bronchioles that are interrupted
by alveolar ducts continuing into alveolar sacs. Reproduced and modified from Weibel et al.
2009 [39], with permission from EMH Swiss Medical Publishers Ltd.
9
1.3.2 Pulmonary vasculature
In contrast to the airways, the pulmonary branching vasculature is asymmetrical and nondichotomous [44]. The pulmonary arteries are closely associated along the course of the airway
branches and terminate in a dense network of pulmonary capillaries surrounding the epithelial
layer of the alveoli to form the gas exchange unit. The pulmonary venous system does not follow
the path of the arterial system and runs alongside the interlobular or intersegmental septa. Figure
1.3 shows a resin cast of the human lung anatomy illustrating the extensive dichotomous
branching network of the airways and the closely related but non-dichotomous branching of the
vascular tree with the pulmonary arteries in red and pulmonary veins in blue.
Figure 1.3 Resin cast of the human airway tree. The dichotomous branching of the respiratory
tree can be seen in the right lung with a systematic reduction of airway diameter and length with
progressive branching. The non-dichotomous branching of the pulmonary vasculature is
demonstrated in the left lung. Reproduced from Weibel et al. 2009 [39], with permission from
EMH Swiss Medical Publishers Ltd.
10
1.4 Pathophysiology of COPD
Despite significant historical landmark contributions to the knowledge concerning COPD, the
pathophysiology underlying this disease is complex and not yet fully understood. In addition to
the multiple pathological processes involved, the manifestation of each disease entity
encompassed under the umbrella term of COPD is variable between individuals, further
contributing to the significant heterogeneity of this disease. A brief history of the knowledge on
emphysema, chronic bronchitis, pulmonary hypertension and small airways obstruction is
outlined below.
1.4.1 Emphysema
1.4.1.1
History
The word emphysema originated from the Greek term „emphysan‟ which means „to inflate‟ or
„blow into‟ (en meaning “in” and physan “to blow”) [45]. When applied to the lungs, it therefore
implies the presence of excess air within the lung parenchyma. The earliest known references of
parenchymal destruction or emphysema describe the lungs as „voluminous‟ by Swiss physician
Theophile Bonet (1617) and „turgid, particularly from air‟ by Italian anatomist Giovanni
Morgagni (1769) [46]. The first distinct illustration of emphysema as „enlarged air spaces‟ was
however provided by Matthew Baillie in 1799 who recognized that alveoli (“cells”) were full of
air and enlarged beyond their natural size [45]. He illustrated emphysema in what was believed
to be the lung of Samuel Johnson who documented in detail his experiences of respiratory
distress that are parallel with the salient features of COPD (cough, sputum production and
dyspnea) known today; „I had then a cough so violent that I once fainted under its convulsions. I
was afraid of my lungs‟, „a vexatious catarrah‟ and „my lungs seem encumbered and my breath
11
fails me, if my strength is any unusual degree exerted‟ [47]. On autopsy, in 1784, Johnson‟s
lungs were described; „the lungs did not collapse as they usually do when the air is admitted but
remained distended as if they had lost the power of contracting‟. This is believed to represent
emphysematous lungs lacking in elasticity [48]. Rene Laënnec, the French physician known as
the „father of clinical auscultation‟ for his invention of the stethoscope, coined the term
emphysema in 1821 [49]. He recognized that the enlarged air spaces were related with a clinical
syndrome of emphysema and in addition, was associated with chronic bronchitis [46].
Throughout the first half of the twentieth century the understanding of emphysema grew,
predominantly led by direct pathological observation of lungs. After numerous adaptations [50,
51], a workshop in 1984 held by the Division of Lung Disease at the National Heart and Blood
Institute produced the definition of emphysema that still stands today; „A condition of the lung
characterized by abnormal, permanent enlargement of airspaces distal to the terminal bronchiole,
accompanied by the destruction of their walls, and without obvious fibrosis‟ [51, 52].
1.4.1.2
Subtypes of emphysema
There are three anatomic subtypes of emphysema classified according to the portion of the
acinus involved; centriacinar, panacinar and distal acinar emphysema [53], as illustrated in
Figure 1.4. These subtypes can be determined by high resolution computed tomography (HRCT)
scanning and pathological diagnosis.
12
Figure 1.4 Subtypes of emphysema compared to a normal acinus. Centrilobular emphysema
affects the proximal location of the acinus whereas the destruction is more evenly distributed in
panacinar emphysema. Paraseptal emphysema is observed adjacent to the septa. Reproduced and
modified from Thakur et al. [54] with permission.
Centriacinar, more commonly known as centrilobular emphysema is classically
associated with cigarette smoking [55]. Centrilobular emphysema formation begins more
frequently in the upper lung fields [53, 55, 56] and as severity increases, can be observed in all
lung fields by clinical MDCT. During the formation of centrilobular emphysema, the airspaces
surrounding the entrance of the acinus are enlarged and destroyed. In severe disease, the
destruction extends to the periphery of the acinus, making the distinction between centrilobular
and panacinar subtypes more difficult. Furthermore, centriacinar and panacinar emphysema can
occur in the same patient [57].
13
Panacinar, also known as panlobular emphysema is a subtype of emphysema that
uniformly involves the entire acinus starting initially at the lung base and spreading throughout
the lungs. This subtype is generally seen in patients with alpha-one antitrypsin deficiency
(α1AT) as they lack the ability to inhibit neutrophil elastase, leading to destruction of the
connective tissues of the lung. Patients suffering from α1AT account for approximately 3% of
COPD cases [58] and develop emphysema usually during their fourth to fifth decades of life
[59].
The final subtype, distal acinar or paraseptal emphysema affects the peripheral part of the
acinus and is usually located adjacent to the pleural surfaces. It is characterized by single or
multiple bullae and is associated with spontaneous pneumothoraces (lung collapse caused by a
collection of air between the lung and chest wall) in young adults [55].
Although it has been demonstrated that loss of elastic recoil pressure resulting from
emphysema can account for airflow limitation by decreased expiratory force driving air out of
the lung [60-65], abnormalities in both the parenchyma and the airways are present in the vast
majority of patients with symptomatic COPD [33, 52].
1.4.2 Chronic bronchitis
Airway pathology in COPD includes the large and the small airways. As recognized by Laënnec
as early as 1821, mucous hypersecretion is a dominant feature of COPD and caused by mucous
gland enlargement and goblet cell hyperplasia in the epithelial lining of the large cartilaginous
airways [66]. The Reid Index is used to quantify this morphology and measures the ratio of
airway gland to wall thickness [67]. The resulting clinical symptoms of chronic sputum
production, described as chronic bronchitis, became a key component in previous definitions of
14
COPD [33]. It was believed that excess mucous secretion precipitated in bronchial infections
causing obstructive damage to the bronchioles and alveolar tissues. Fletcher and Peto in their
landmark paper; „The natural history of chronic airflow obstruction‟, showed to their own
surprise that this was not the case and concluded that neither mucous hypersecretion or bronchial
infections play a substantial part in causing airflow obstruction [68]. Subsequent work has shown
that there is no consistent relationship between chronic bronchitis and increased airflow
limitation [55, 68, 69].
1.4.3 Pulmonary hypertension
Although the pulmonary vasculature is not the focus of this study, it is important to briefly
describe the pathophysiology of pulmonary hypertension as it is a frequent complication in
COPD, and associated with decreased survival [2, 55, 70, 71]. The exact mechanisms of
pulmonary hypertension have not been eluded to, but pathological changes are believed to occur
as a direct response to cigarette smoking, and indirectly to hypoxia and emphysematous
destruction [55, 72]. The vascular wall is made up of three layers; tunica intima, media and
adventitia, of which intimal hyperplasia is the key feature of pulmonary vascular remodeling [2].
This results in vascular wall thickening with consequent luminal narrowing and increased
resistance. Endothelial cell dysfunction leads to dysregulation of vascular homeostasis with
increased vascular tone and hypoxic vasoconstriction [2]. Increased infiltration of inflammatory
cells has been inversely related to endothelial cell function and directly related to intimal layer
enlargement [73]. Smooth muscle proliferation and extension occurs in pulmonary arterioles
which usually do not contain a muscular layer [72, 74]. Emphysematous destruction leading to a
loss of capillaries may also play a role in the increase of pulmonary vascular resistance.
15
1.4.4 Small airways disease
1.4.4.1
History
Prior to the 1960s, it was believed that even in healthy lungs, the site of most resistance to
airflow occurred in the peripheral airways [75]. The 1963 publication of Wiebel‟s monograph;
„Morphometry of the Human Lung‟, alongside Green‟s subsequent calculations on Weibel‟s new
quantitative data stimulated a paradigm shift to this theory, suggesting that the resistance to flow
at this site was in fact substantially less [41, 75]. In 1967, Macklem and Mead confirmed Weibel
and Green‟s data by providing the first direct measurements of anaesthetized animal and post
mortem human lungs using retrograde catheterization. They showed that the smaller airways
(<2mm) in fact only accounted for less than 20% of the total lower airway resistance in the
healthy lung [76]. This work has subsequently been supported by others [77-79].
In extension to this, Hogg et al., in 1968 demonstrated, again by direct measurement of
airway pressures and flows, that in contrast, these small airways became the major site of airway
obstruction in COPD and the resistance increased 4 to 40 fold compared to normal subjects. This
landmark study; „Site and Nature of Airway Obstruction in Chronic Obstructive Lung Disease‟,
also introduced the concept of „small airways disease‟. This terminology was proposed as it was
illustrated that the pathological processes causing the obstruction was heterogeneous and
appeared to affect both the smallest bronchi and the bronchioles deeming the terms „bronchitis‟
and „bronchiolitis‟ as inadequate [80].
16
1.4.4.2
Site of airway obstruction
The historical findings above stimulated vigorous research on why the major pathological
processes of airflow limitation occur in the small airways, and what these processes are.
Breathing involves a combination of the active work of ventilation and the passive nature of
diffusion. Diffusion occurs around the first orders of the acinus as the airflow velocity
significantly slows in this region [39]. In health, this and the exponential increase in crosssectional area optimizes gas exchange, but also leaves this transitional zone between the
conducting and gas exchange units as a vulnerable target to the deposition of airborne particles
inhaled from cigarette smoke and other environmental substances [75]. The particle size of
cigarette smoke ranges from 0.1 to 1.0 µm [81, 82]. The distance a particle travels by diffusion
decreases with increased particle size and particulate matter greater than 0.1µm are more likely
to be deposited due to sedimentation and impaction in the respiratory tract [83]. As gases also
diffuse 600 times faster than airborne particles [84], the retention of particulate matter in this
particular region is more likely and may continue to stimulate the persistent inflammation that
occurs even after the removal of the inhalational insult.
1.4.4.3
Remodeling and narrowing of small airways
The chronic inflammation in COPD stimulates a repair and remodeling process causing small
airway wall thickening and luminal narrowing. These changes have been strongly correlated with
the extent of airflow obstruction and hence the progression of COPD [85, 86]. The airway wall
contains multiple layers including the epithelium, lamina propria, smooth muscle and adventitia
[87], all of which contribute to wall thickening in various degrees [86]. The repetitive injury and
persistent inflammation caused by smoke exposure leads to an infiltration of the wall tissue by
17
inflammatory immune cells which aggregate into lymphoid follicles. This bronchus associated
lymphoid tissue (BALT) is deposited in the adventitial layer and as shown in Figure 1.5A, can
encroach into the lumen calibre [86]. The small airway epithelial cells play an important role in
the remodeling process. In an attempt to restore the breached epithelium, the cells undergo
squamous metaplasia increasing the thickness of the wall [72, 88, 89]. Squamous epithelial cells
may also secrete a variety of mediators to induce peribronchiolar fibrosis and increased
connective tissue deposition [57, 88, 89], further decreasing airway radius (Figure 1.5B). Figure
1.5C shows the increased accumulation of muco-inflammatory exudates causing narrowing and
obstruction of the airway lumen. This may be caused by an aspiration of the mucus from the
larger central bronchi, but is more likely to be caused by the secretion of mucins from
hyperplastic goblet cells in the small airway epithelium [87, 90].
Figure 1.5 Histological stains showing remodeling and narrowing of small airways . A:
Collection of bronchial lymphoid tissue with a germinal centre (GC) encroaching into the airway
lumen. B: Deposition of connective tissue in and around the small airway wall. C: Thickened
airway wall with the lumen partially obstructed with muco-inflammatory exudates. Reproduced
and modified with permission from Hogg 2004 [90] and Hogg et al. 2004 [86], Copyright
Massachusetts Medical Society.
18
1.5 Management of COPD
Smoking cessation is the most cost-effective [91, 92] and by far the most important intervention
with the greatest capacity to modify the course of the disease in COPD [1, 93, 94].
Pharmacotherapies for tobacco dependence have been shown to be effective in increasing longterm abstinence rates [95-97], and this treatment has been recommended for all patients who
smoke [1].
Once diagnosed, the airflow limitation in COPD is „not fully reversible‟ [4], implying
that an element of reversibility exists and hence is a target for pharmacological treatment.
Bronchodilator medications are the therapeutic mainstay for patients with COPD and include
beta2-agonists, anticholinergic drugs and the less commonly used theophylline [94, 98-100].
Prevention and relief of symptoms are the primary aim and single or combination therapies can
be prescribed according to the individual patient‟s need and response [1, 94].
The chronic inflammation associated with COPD provides another target for
pharmacotherapy. Although anti-inflammatory agents, namely corticosteroids play a key role in
the management of COPD, their regular use has not been shown to modify the long-term decline
in FEV1, nor improve mortality rate in these patients [101-105].
Many more treatment options, pharmacologic and non-pharmacologic, are available to
COPD patients and furthermore, new additional therapies are continuously under research or trial
[106]. A detailed description of each is beyond the scope of this introduction and therefore a
brief summary of these therapeutic options are outlined in Table 1.1. While it is evident that a
vast variety of options are available for the management of COPD to date, none of the existing
medications for this disease have been conclusively shown to modify the long-term decline in
lung function [1]. The current GOLD guidelines for the treatment of COPD do not currently
19
recommend therapy until moderate airflow limitation is diagnosed. Thus, further research is
urgently required to identify novel therapeutic targets early on in the disease process in order to
prevent or slow down the disease progression and burden.
20
Table 1.1 A summary of the therapeutic options available for the management of COPD
Pharmacologic
Drug
Beta2-agonists (long-acting and
short-acting)
Anticholinergics
Combination short-acting beta2agonists and anticholinergics
Corticosteroids
Methylxanthines
Phosphodiesterase-4-inhibitors
Vaccines
Alpha-1 Antitrypsin augmentation
therapy
Antibiotics
Mucolytics and antioxidant agents
Antitussives
Narcotics
General Mode of action
Stimulation of beta2 –adrenergic receptors increases cyclic
AMP to relax bronchial smooth muscle.
Antagonizes acetylcholine action on muscarinic receptors of
bronchial smooth muscle preventing an increase in
intracellular calcium concentration.
Combination of the actions above. Indicated when symptoms
are not improved with single agents.
Anti-inflammatory: inhibits inflammatory cells (neutrophils,
macrophages, mast cells, lymphocytes, eosinophils) and
release of inflammatory mediators (histamine, leukotrienes,
cytokines).
Non-selective phosphodiesterase inhibitors which can directly
relax smooth muscle of the respiratory tract.
Reduces inflammation by selectively inhibiting the action of
phosphodiesterase-4 which usually breaks down intracellular
cyclic AMP.
Reduces the incidence of community acquired pneumonia and
hence exacerbations in COPD patients.
Purified human alpha-1-proteinase inhibitor inhibits the serine
protease neutrophil elastase in lungs.
Indicated for treating exacerbations of COPD.
Reduces viscosity of secretions.
Cough suppressant acting centrally on the cough centre in the
medulla.
Used for dyspnea treatment.
Non-pharmacologic
Pulmonary rehabilitation
Components include exercise training, smoking cessation,
nutritional counselling and education with the goals to reduce
symptoms, improve quality of life and increase participation
in everyday activities.
Additional treatments
Oxygen
Ventilatory support
Surgery
Palliative treatment
Long term oxygen therapy is indicated in patients with severe
resting hypoxemia.
Delivery of mechanically assisted breaths without intubation.
Lung volume reduction and lung transplantation.
Prevents and relieves suffering at the end stages of life.
21
1.6 Imaging techniques
1.6.1 Clinical computed tomography
The invention of computed tomography (CT) scanners in 1971 by engineer and Nobel Prize
winner Sir Godfrey Hounsfield is considered as one of the greatest innovations of medical
imaging technology and is one of the most valuable tools for medical diagnosis and management
today. Tomography refers to the imaging of an object through the use of any kind of penetrating
wave, in this case, an x-ray. By taking multiple 2-dimensional (2D) images throughout the object
in sequence, these can then be reconstructed using a computational algorithm to produce a 3dimensional (3D) image of the scanned object. All CT systems are composed of several basic
components: the x-ray unit which functions as a transmitter, a data acquisition unit which
contains the detector and finally the image processing algorithm. Clinical CT scans now use
multiple rows of detectors (MDCT) which permits the acquisition of thin-slice imaging (usually
0.5-1mm thick), allowing true volumetric imaging of the entire lung during a single breath-hold
[107].
Although spirometry is the gold standard diagnostic tool for COPD, this physiological
test only provides a global assessment of lung function and is unable to detect which structural
changes are contributing to the airflow obstruction. Emphysema by its definition is essentially a
pathological diagnosis, limiting accurate examination to post-mortem or resected specimens. Invivo, the diffusing capacity for carbon monoxide (DLCO) provides an index of the degree of
anatomic emphysema with lower DLCO values indicating more severe emphysema. This test is
often used for the pre-operative evaluation for lung resection or lung volume reduction surgery
but cannot detect mild emphysema as it is neither a sensitive nor a specific test.
22
Clinical CTs are currently the best tool to evaluate the presence and extent of emphysema
in-vivo although it is currently not required for diagnosis and not in regular use due to radiation
exposure and cost. The first CT-pathologic correlation in emphysema was reported as early as
1984 by Hayhurst et al. [108]. Since then, the role of CT in diagnosing and quantifying
emphysema has been well established using lung densitometry. Densitometry values are given in
Hounsfield Units (HU) where 0 HU is the radio-density of distilled water at standard pressure
and temperature, and -1000 HU is the radio-density value for air [109]. Patients with pulmonary
emphysema demonstrate low attenuation areas and depending on the type of CT scan and
evaluation utilized, the threshold value for the detection of quantitative pathology is between 850 to -960 HU [110-114].
The advent of high resolution CT (HRCT) introduced the possibility of measuring distal
airway dimensions in-vivo [107, 111, 112]. Nakano et al., were the first group to provide
objective measurements of airway narrowing by CT and correlate these dimensions with
pulmonary function tests and histology [115]. Although small airways could not be visualized
with HRCT, their results indicated that the accurate measurements of thickened large airway
walls correlated favourably with dimensions of the small airways measured on histology and
airway obstruction as measured by spirometry [115, 116]. This suggests that the thickening and
narrowing of the larger airways which are amenable to CT assessment, may be used as a
surrogate marker to quantify small airways disease [107]. This approach however relies on the
assumption that the pathological processes occurring in the larger airways are similar to that of
the small airways. Not surprisingly, Hasegawa et al., demonstrated that airflow limitation
remains more closely related to the luminal and wall areas of the smaller than the larger airways
[117].
23
Numerous studies are now focused on identifying key CT parameter markers to detect
and quantify small airways disease and establish a correlation between CT amenable structures
and lung function that is reliable and reproducible. In addition, other imaging modalities such as
hyperpolarized Helium (He) or Xenon (Xe) Magnetic Resonance Imaging (MRI) and Optical
Coherence Tomography (OCT) are gaining much interest as useful tools in assessing the
morphology of this heterogeneous disease [118-120].
1.6.2 Ultra structure imaging of the lung using micro-CT
The key limitation of MDCT is the inability to accurately image the distal airways and fine lung
parenchyma due to the lack of resolution. Micro-xray computed tomography (micro-CT) works
in a similar concept to clinical CT scanners with the advantage of providing substantially higher
resolution images, with resolutions up to 1µm per voxel. This advantage does however come
with the caveat of significantly high radiation exposures that can damage living tissue and
therefore this valuable imaging tool is used for research purposes only.
Although the first micro-CT imaging results were obtained in the 1980s, the studies of
fine lung architecture using this technique are relatively recent due to the limited contrast of fine
lung parenchyma, tissue preparative difficulties and computer analysis limitations [121]. MicroCT scanners have the same basic components of the clinical CT. The higher resolution is
achieved as the x-ray source and the detectors are not fixed allowing a closer distance to the
tissue sample. In addition, the sample can also rotate, facilitating the multiple views that are
reconstructed to create a 3D image. Micro-CT is therefore able to fill the gap between clinical
imaging and conventional microscopy techniques [121]. Compared to the current challenges of
analyzing and quantifying lung structure by traditional histology, micro-CT has the following
24
advantages: Firstly, samples can be imaged non-destructively maintaining the structural
continuity of the complex vascular and bronchial network at near-microscopic resolution [122].
This is compared to the blind and destructive nature of histological sampling process, leading to
the inevitable loss of regions of interest and discontinuity of 3D information [123]. Secondly, no
special sample preparation such as staining or thin slicing is required. Thirdly, there is less
chance of under-representation and potential for bias with micro-CT scanning as histological
samples are usually significantly smaller than the whole organ and sampled in a manner to
represent pathological lesions for qualitative histological analysis.
Using a combination of micro-CT and MDCT, McDonough et al., were able to identify
the number of terminal bronchioles per millilitre (TB/ml) within a known volume of lung tissue
by micro-CT and calculate the total number of terminal bronchioles per lung with the total lung
volume obtained by MDCT [124]. Their results were in good agreement with previously
published data from four separate studies of individual lung casts, validating the micro-CT
methodology used [41].
1.6.3 Application of stereology to understand lung morphometry
Stereology refers to the application of mathematical methods to determine morphometric
properties of 3D structures using measurements from 2D sections [125, 126]. It was first applied
to quantify lung structure in 1959-1963 [125] and is now the gold standard for lung
morphometry [123]. Although traditionally and principally used for conventional microscopy,
this technique can be applied to any imaging dataset and has recently demonstrated its value in
micro-CT quantitative analysis [124, 127]. This application is particularly useful for
characterizing the 3D geometry of the fine lung architecture with its complex bronchial, vascular
25
and alveolar network. The value of accurate lung morphological research in understanding the
pathophysiology and progression of disease has been recognized by major respiratory bodies
worldwide, prompting the development of an official set of guidelines by the combined task
force of the American Thoracic Society (ATS) and the European Respiratory Society (ERS)
[125].
Stereology is based on two basic but equally important components: sampling and
measurement. A rigorous sampling technique is necessary to account for the heterogeneous
nature of the lung and avoid bias. In addition, the samples analyzed should be representative of
the whole lung. These requirements are met by introducing randomness into the sampling
process [125]. A variety of random, unbiased sampling procedures have been outlined in the
ATS/ERS official standards [125]. For this particular study, a simple and efficient method called
Systematic Uniform Random Sampling (SURS) will be used. In brief, the starting point of the
sample is chosen at random. This determines the position of the following sections which are
sampled systematically at uniform intervals. A more detailed description of how this protocol has
been applied to the lung tissue samples is provided in the methods section.
A coherent set of measurement tools have been developed which comprise of a set of
typical global parameters that can be utilized to characterize any lung component [125]. These
parameters are 3D (volume and size), 2D (surface area), 1D (length or thickness) or 0D
(number). Reference parameters are important in order for stereology to have biological
meaningful data. For example, in order to quantify the number of terminal bronchioles within the
whole lung structure, knowledge of the total lung or lobar volume as a reference value is
important. At the macroscopic level, the non-invasive MDCT scanning of the intact lung
provides an ideal reference volume estimation [128].
26
The most widely used morphometric parameter for the assessment of pulmonary
emphysema is the mean linear intercept (Lm). This technique was first described by Dunnil who
recognized the value of determining quantitative dimensions of the internal surface area or the
air-tissue interface of emphysematous lungs [129]. The Lm is a reflection of the mean air space
diameter and is determined by alveolar wall intersection counting using a pre-formulated test
system of linear probes in 2D sections [125, 130].
1.7 The relationship between small airway obstruction and
emphysema in COPD
It is widely accepted that chronic airflow limitation characteristic of COPD is caused by a
combination of small airways disease and emphysema. The destruction of parenchymal tissue in
emphysema results in the lack of alveolar attachments that usually support and maintain the
patency of small airways, particularly during expiration. In addition, the loss of elastic recoil
decreases the expiratory force driving air out of the lung, contributing to airflow limitation [28,
131-135]. This theory has however been challenged from studies demonstrating a weak
correlation between the physiological measurements of airflow obstruction and the degree of
emphysema in lung tissue obtained post-mortem or post-surgery [65, 132, 136, 137].
Abnormalities in both the lung parenchyma and the small airways are present in the vast majority
of patients with symptomatic COPD. The extent and how each component contributes to
increased airways resistance remains in question.
Hogg et al., identified the need to explore and amalgamate the relationship between the
pathological (emphysema) and physiological (airflow obstruction) entities of COPD back in
27
1968 [80]. At this time, in addition to the discovery that the small airways were the site of major
airflow obstruction in COPD, their data indicated that this small airway obstruction was due to
an organic disease and not from the decreased elastic recoil properties of emphysematous lungs
[80]. Since then, this group and others have built on this foundation by identifying key
pathological lesions and underlying inflammatory mechanisms causing the airflow limitation
characteristic of COPD briefly outlined above. The number and dimensions of small airways
have also been the focus of many studies [124, 138-141]. Matsuba and Thurlbeck reported for
the first time, a small decrease in the number of smallest bronchioles in COPD. Their data was
however insufficient to explain the 4 to 40 fold increase in airflow resistance frequently observed
in COPD patients [142]. The total resistance (RT) of the small conducting airways which are
arranged in parallel is calculated by the sum of the inverse of the resistance of each individual
branch, i.e. RT = 1/R1 + 1/R2 + 1/R3 etc. From this equation, theoretically it would require the
removal of 50% of the airways to double the total resistance [75]. The increase in resistance
varies with the fourth power of the reduction in radius [141]. The theory that the generalized
narrowing of small airways causing the substantial increase in COPD airflow obstruction was
therefore more desirable than the disappearance of small airways [75].
Recently, McDonough et al., provided the first possible explanation to this conundrum
[124]. Using a novel combination of MDCT and micro-CT, they showed that terminal
bronchioles are both narrowed and significantly reduced to a degree of destruction that was
previously not thought possible. Specifically, the study reported a reduction in terminal
bronchiolar number to approximately 10% of the control values in severe centrilobular and 30%
in severe panlobular COPD patient lungs. These results thus explain the previous physiological
data revealing that these airways account for minimal resistance in the healthy lung, but
28
significantly increase their resistance to become the major site of airway limitation in disease
[80, 109].
Furthermore, micro-CT examination of peripheral lung tissue from control and very
severe COPD (GOLD 4) subjects revealed that the reduction in terminal bronchioles occurred in
regions of the lung parenchyma with and without emphysema. This once again raises the key
issue of the relationship and interaction between emphysema and small airways obstruction and
this new data strongly suggests that terminal bronchiolar narrowing and obliteration precedes the
onset of emphysematous destruction [41, 124].
The concept that emphysematous destruction occurs as a result of the outward extension
of inflammation from the bronchioles supplying the acinus was first introduced when
centrilobular emphysema was described by Leopold and Gough in 1957 [56]. Their data showed
evidence of chronic inflammatory cell infiltration in the bronchioles identified as „supplying‟ the
acinus, i.e. the terminal bronchioles. They also reported a loss of elastic and muscle fibres and
these were frequently replaced by fibrous tissue. In addition, no inflammatory changes were
observed in the peripheral emphysematous spaces [56], further supporting the theory that the
inflammation spreads outwards from proximal to the periphery with destruction, repair and
remodeling processes occurring in parallel.
Two possible mechanisms of emphysematous destruction have been postulated regarding
the pathological progression of emphysema in COPD as demonstrated in the modified Figure 1.6
below. Firstly, if alveolar destruction occurs first, inflammation in the alveolar space causes
septal walls to be destroyed with collapse and folding. Subsequently, the tethering between the
alveolar walls and the connective tissue elastic fibres of the terminal bronchioles is lost causing
these airways to recoil and obstruct. In comparison, inflammation in the transitional zone
29
between the conducting and gas exchange units causes destruction of the terminal bronchiole
elastic fibres first with consequent narrowing and obliteration. As a result, the axial support of
the distal acinus is lost causing alveolar walls to collapse and fold [143].
As Mitzner recently suggested in his editorial; „Emphysema - A Disease of Small
Airways or Lung Parenchyma?‟ the compelling evidence from the McDonough et al. study
challenges the long-standing definition of emphysema; „A condition of the lung characterized by
abnormal, permanent enlargement of airspaces distal to the terminal bronchiole, accompanied by
the destruction of their walls, and without obvious fibrosis‟ [143]. Thus, the novel combination
of MDCT and micro-CT opens up the ability to further study, characterize and link the structural
abnormalities of the distal airways and parenchyma that were previously difficult to explore due
to their microstructure and localization within the thoracic cavity [87].
30
Normal Terminal
Bronchiole
Initial alveolar tissue destruction
Initial terminal bronchiole destruction
Figure 1.6 Two proposed mechanisms of emphysematous destruction in COPD. In initial alveolar destruction, inflammation in
the alveolus leads to wall destruction and subsequent loss of the axial tension that links the acinus to the connective tissue elastic
fibres of the terminal bronchiole. The alveolar attachments keeping the airway patent are lost and terminal bronchioles collapse and
obstruct. In initial terminal bronchiole destruction, inflammation in the small airways causes wall thickening and narrowing. The
terminal bronchioles recoil and obstruct causing the distal acinus to lose its tethering support leading to ultimate folding, destruction
and enlargement of the unsupported alveolar walls. Reproduced and modified with permission from Mitzner 2011 [143], Copyright
Massachusetts Medical Society.
31
1.8 Innovative micro-CT imaging of formalin fixed, paraffin
embedded samples to understand small airway obstruction
The chronic airflow obstruction that defines COPD is believed to occur in the small airways with
a diameter of <2mm with no or minimal supporting cartilage. Even with the evolution of clinical
MDCT technology, only macroscopic structures greater than 2mm in diameter can be imaged
with accuracy in-vivo. Histology, although the gold-standard technique for microscopic analysis
of tissue, is destructive in nature and therefore the fine 3D architecture of lung tissue is often
lost. Therefore, micro-CT imaging provides the link between these two limiting techniques as
they have the higher resolution power required to visualize the microscopic structures of the lung
where pathological lesions appear to develop, whilst keeping the tissue samples intact throughout
the whole imaging process.
In order to study tissues under the microscope, they have traditionally been fixed with
formalin and embedded with paraffin wax (FFPE), as infiltration with paraffin allows thin and
precise sectioning for histologic tissue using a microtome. This long-standing, gold standard
method of tissue preservation and storage has led to the establishment of vast archival biobanks
of tissue samples representative of multiple disease states. Previously, archival FFPE samples
were precluded from imaging by micro-CT as paraffin has a similar density to lung tissue. Thus,
earlier micro-CT studies required the use of contrast agents or air-inflation prior to fixation to
maximize contrast. A collaboration of our group with the µ-VIS Imaging Centre at the
University of Southampton has provided access to a new generation micro-CT scanner that has
enabled non-destructive imaging of the archival FFPE samples to visualize the 3D morphometry
of the normal lung, even with the challenging modest tissue attenuation levels. This protocol has
been developed and is under review for publication.
32
In order to further understand the relationship and time-course of the pathophysiological
processes in the progression of small airways disease and emphysema, further investigation in
the early stages of disease is necessary. Whilst mild to moderate COPD patients represent the
majority of patients with COPD, this sub-population is relatively understudied due to the lack of
access to tissue samples as only severe COPD patients are likely to be candidates for lung
transplants. Over the last 35 years, archival FFPE lung tissue from well characterized patients
undergoing lung cancer resection surgery were collected within the James Hogg Research Centre
(JHRC) Lung Registry.
33
1.9 Hypothesis and specific aims
This study will use the novel combination of micro-CT, MDCT and histology to analyze the
microstructure of small airways disease and emphysema in FFPE lung tissue of control patients
that have a similar smoking history and normal lung function, compared to those with mild and
moderate COPD, collected by the JHRC lung registry. With the hypothesis that „narrowing and
obliteration of small airways occur early in mild COPD and precedes the development of
emphysema’, this study has three specific aims:
1.
Assess the feasibility of analyzing fine lung tissue morphometry such as mean linear
intercept (Lm) in FFPE samples using a combination of micro-CT with traditional
stereological methods.
2.
Determine if the significant reduction in terminal bronchioles reported in end stage COPD
begins in mild (GOLD 1) disease.
3.
Define the use of micro-CT as a scouting tool to identify and assess the structural pathology
of obliterated terminal bronchioles using standard histological methods.
34
Chapter 2:
Methods
2.1 Patient population and lung tissue sampling
This study was conducted on lung tissue samples from patients who underwent surgical
treatment for small peripheral lung carcinomas. Ethical approval was obtained by the
Institutional Providence Research Ethics Committee (H13-02173). The specimens were donated
with informed consent to the James Hogg Research Centre Lung Registry between the years
1980 and 2000 for ongoing research. From this archival biobank, ten subjects were selected, five
of which were controls (patients with similar smoking history and normal lung function), and
five patients with mild to moderate COPD (GOLD 1 and 2). For all patients, pre-operative lung
function data, clinical MDCT scans and resected lung tissue were available. Clinical and
demographic characteristics of all donors are presented in Table 2.1
35
Table 2.1 Patient demographics
Controls
n=5
Mild/Moderate COPD
n=5
Gender
(Female:Male)
3:2
1:4
Age (years)
63 ± 11
68 ± 8
Height (cm)
164.6 ± 4.16
169.6 ± 11.50
Weight (kg)
57.8 ± 12.66
86.8 ± 21.93
Smoking history
(pack years)
32 ± 7
37 ± 8
FEV1 (% predicted)
93.6 ± 0.03
81.2 ± 0.05
FEV1/FVC
0.77 ± 0.04
0.64 ± 0.05
2.2 Sample preparation for micro-CT
The collection and preservation of lung tissue by formalin inflation and fixation followed by
paraffin embedding were conducted as previously described by our group [86]. In brief, the
resected lungs or lobes were first inflated and fixed with formalin. After fixation, the lung
specimen was cut into eight 2cm transaxial slices from the apex to the base, but the first and last
slices were not used for sampling. Rules were enforced to ensure non-biased, representative
sampling so that the heterogeneity of disease within the lung specimen was represented. Areas
with disease such as pneumonia or tumour, in addition to blood pooling, large vessels and
airways were avoided. Cores of lung tissue measuring approximately 1.5cm in diameter were
sampled using a cylindrical cutting tool and infiltrated with low melting point paraffin using a
36
Leica Tissue Processor (Model ASP 6025). A total of eight to ten formalin fixed, paraffin
embedded (FFPE) cores from the six transaxial lung slices representative of the lung specimen
from the apex to the base were used per patient case. The FFPE samples were then stacked and
immobilized within a polyethylene tube using wax. The polyethylene tube and wax were chosen
as mounting materials for the samples due to their similar density characteristics to minimize CT
imaging artefacts. Stacking of multiple FFPE samples enabled long duration scans within the
micro-CT scanner to avoid over handling of the processed lung tissue. After preparation, these
specimens were shipped to the µ-VIS Imaging Centre at the University of Southampton, U.K. for
micro-CT scanning.
2.3 Micro-CT image acquisition and reconstruction
The FFPE samples were scanned using a custom built Nikon Metrology HMX micro-CT scanner
with a 225kV X-ray source and resolution capabilities of approximately 1-10µm. A flat panel
detector (Perkin-Elmer 1621 AN) with a high sensitivity cesium iodide (CsI) scintillator, and a
carbon fibre entrance window enabled an improved low energy performance. Scanning time was
approximately 6-8 hours per two sample cores within the tube and a previously optimized
imaging acquisition protocol was applied for all samples [144]. Briefly, an electron accelerating
voltage of 50kV with a reflection target yielding a mean energy of 18KeV was used. This
reflection target was composed of molybdenum which was key to achieving a low keV (below
40keV) for the optimum mass attenuation contrast between tissue and paraffin mounting medium
as shown in Figure 2.1. Other general parameters included: a beam current of 180µA yielding a
37
total beam energy below 9W to avoid tissue damage, no filtration, 1 second exposure, 24dB gain,
3142 projections with 4 frames per projection and shuttling on at 0 pixels. Three dimensional
reconstructions with voxel resolutions of 6.7µm were created using a standard filtered-back
projection algorithm.
Figure 2.1 Mass attenuation coefficients versus x-ray energy for paraffin wax and soft
tissue. Data were based on the National Institutes of Standards and Technology Data [145] for
soft tissue embedded in paraffin or plastic resin. The tolerable range represents the acceptable
contrast in mass attenuation between paraffin and tissue.
38
2.4 Image visualization and analysis
The software package FIJI was used for image processing, 3D visualization and analysis. The
original 32-bit file formats were converted to 8-bit to reduce the volume size, enabling a more
efficient image analysis time. Enhancement techniques were applied to each data set to minimize
the effects of noise and provide optimal images. Each lung tissue core scanned contained
approximately 1000-1200 contiguous micro-CT images. 2D image analysis was performed with
views available in the XY, YZ and XZ planes. These sections were scrolled through and
examined methodologically to explore the lung sample anatomy, allowing the user to follow 3D
airway branching structures and identify regions of interest.
For each lung tissue core, the core volume, the mean linear intercept (Lm), and the
number of terminal and respiratory bronchioles were obtained. Following micro-CT scanning
and image processing to ensure scanning techniques were adequate to obtain the information
above, the FFPE samples were set in paraffin blocks for histological sectioning, allowing a more
comprehensive analysis. A summary of the workflow has been provided in Figure 2.2. To avoid
bias, data collection was performed in a blinded manner, in that all analyses were carried out by
the author without pre-requisite knowledge of the diagnoses and clinical information was
revealed at the end.
39
Figure 2.2 Workflow summary of the study. Pre-operative MDCT and spirometric data were
obtained with the resected lung specimen, which was fixed with formalin inflation and cut into
2cm transaxial slices. Samples were cored from each slice and paraffin embedded. Formalinfixed paraffin embedded (FFPE) lung tissue cores were stacked into polyethylene tubes and sent
for micro-CT scanning. After image acquisition and 3D reconstruction, each micro-CT scan was
traced to determine core volumes, sampled for Lm measurement, terminal bronchiolar number
calculated and regions of interest traced for 3D rendering. These areas of interest were also
located and matched with histological sections for further analysis.
40
2.5 Volume segmentation of clinical MDCT data using stereological
methods
Using pre-operative clinical MDCT scans, we calculated by volume segmentation the total lung
volumes for each individual lung and the lung pair. Volumes were calculated as the product of
the number of voxels multiplied by voxel size and are shown in Table 2.2. Information of the
resected tissue specimen enabled us to correlate the appropriate lung volume (right or left) to
determine the number of terminal bronchioles per total lung.
Table 2.2 Lung volumes calculated from pre-operative clinical MDCT scans
Controls
(smokers with normal lung function)
Mild/Moderate COPD
1
2
3
4
5
1
2
3
4
5
Gender
F
F
F
M
M
M
M
F
M
M
Surgical
resection
RUL
LUL
RML+RLL
Left
pneumonectomy
LUL
LUL
RUL
LUL
LLL
LLL
Pre-operative
Right lung
volume (cm3)
2520.5
1431.4
2329.6
2564.9
3453.4
3423.5
3690.2
2202.7
2630.2
2715.3
Pre-operative
Left lung
volume (cm3)
2090.0
1193.7
2167.6
2244.0
3186.9
2715.8
2772.3
1577.7
2248.2
2331.9
Pre-operative
Total lung pair
volume (cm3)
4610.5
2625.1
4497.2
4808.9
640.3
6139.2
6462.5
3780.4
4878.4
5047.2
RUL: Right Upper Lobectomy, LUL: Left Upper Lobectomy, RML: Right Middle Lobectomy,
RLL: Right Lower Lobectomy, LLL: Left Lower Lobectomy.
41
2.6 Obtaining core volumes
In order to quantify the number of terminal and respiratory bronchioles per millilitre (ml), the
volume of the core as a reference parameter was obtained. This was achieved using a semiautomatic segmentation tool from the FIJI image processing software and all tracings were
performed with a Wacom Cintiq 22HD graphics tablet which allows the user to draw directly on
the display surface. The micro-CT scan was examined at the XY and the YZ planes to identify
the beginning and end of the core. The outline of the core was traced using a B-spline technique,
where the contiguous sections are scrolled through and each image with a different shape from
the previous is traced. These traced sections were then interpolated to create a best fit curve or a
spline (Figure 2.3). This provided a mask of the core and the total core volume (mm3) was
calculated as the product of the total number of voxels of the segmentation mask multiplied by
the voxel size.
42
Figure 2.3 Obtaining core volumes using a semi-automatic segmentation approach. In the
left panel, lines A, B and C in the YZ plane correspond to their respective images in the XY
plane in the right panel with line A representing the top of the core. The yellow outlines of the
micro-CT XY images represent the tracing of that section which are then interpolated to create a
core mask (shown in white) which represents the volume.
43
2.7 Systematic
measurements
uniform
random
sampling
(SURS)
for
Lm
The systematic uniform random sampling (SURS) method was applied to obtain 10 random,
regularly spaced micro-CT slices throughout the image stack. The contiguous sections of each
micro-CT scan were examined to obtain the beginning and end slice number of each core to
determine the core thickness. In order to avoided sampling artefacts the core thickness was
reduced by 100 slices from the top and the bottom of the core. This number (n) was then divided
by 10 to calculate the „slab‟ thickness and inserted into a random integer generator to establish
the first slice number of the stack. A slice keeper plug-in tool on FIJI software was subsequently
applied to locate the remaining nine slices at regularly spaced intervals and create a sub-stack of
the 10 sections for analysis.
44
Figure 2.4 Systematic uniform random sampling (SURS) method of the micro-CT image
used to obtain 10 regularly spaced sections throughout the core. The core height of the micro-CT
image was reduced by 100 slices at the top and the bottom to produce n. This number is divided
by 10 (n/10) and inserted into a random integer generator to obtain the number of the first slice.
A small image stack consisting of 10 slices was subsequently created by a FIJI slice keeper plugin tool for Lm measurements.
45
2.8 Measurement of mean linear intercept (Lm)
The severity of emphysema or air space enlargement was evaluated by measuring the mean
linear intercept (Lm). To measure the intercepts, a test system in the form of a 4x4 grid mask was
created using Image-Pro which allows the mask to be overlaid onto the cross-sectional image.
Various criteria had to be met when creating the grid mask. First, the grid mask had to cover as
much of the field of view (FOV) as possible whilst avoiding the bright halo of poor resolution
near the edges. The mask was positioned at the centre of the FOV. A segmented grid with an
orthogonal layout and a checkered pattern was chosen. Every horizontal test line was of equal
length (1mm) and evenly spaced. Each image required calibration before projecting the grid
mask to maintain consistency throughout.
Lm is based on point and intersection counting. As shown in Figure 2.5, an intersection
was counted each time a septal wall crossed a test line. Any test line with intercepts from
airways, vessels and artefacts such as air bubbles were not included. Any areas of obvious
collapse or poor inflation were also excluded. Lm was calculated by the following formula: Lm =
L*N/M, where L is the length of the test line, N is the number of lines and M is the sum of the
intersections. The smaller the number of intersections implies less septal walls and hence greater
air space enlargement. Therefore, the greater the Lm, the more severe the emphysema.
46
Figure 2.5 Lm measurement using a pre-formulated grid mask. A: Grid mask overlaid onto a
micro-CT image for Lm measurement. B: Zoomed view of the test area showing the criteria
intercepts counted on grid lines.
47
2.9 Comparison of Lm in micro-CT versus histological samples
To determine the accuracy of the Lm values obtained by micro-CT, the scanned tissue cores
were also sectioned to compare Lm assessed by histological methods which are the traditional
gold standard for stereology. Briefly, after the micro-CT scan, the wax mounted FFPE cores
were separated and embedded into paraffin blocks, then using a microtome, 4µm sections were
cut and mounted onto glass microscope slides. Sections were again selected using SURS method.
These slides were then de-paraffinized and stained using Movat‟s Pentachrome stain, to visualize
the tissue structures. Digital images were obtained using a Nikon light microscope linked to a
digital camera and the imaging software, SPOT advanced. For each image, a flatfield correction
and computed white balance was applied uniformly to all images. As the measurement of Lm is
sensitive to image resolution, the micro-CT and histological images were analyzed at the same
resolution (pixel size 6.7µm), to ensure comparability. After calibration of each image the Lm
was quantified in the histological sections using the same method outlined above and values
compared to matched micro-CT images.
Figure 2.6 Lm measurement of histological
sections. The same grid mask used for microCT analysis is overlaid onto corresponding
histological sections. Images are calibrated to
the same pixel size (6.7µm).
48
2.10 Counting terminal bronchioles
Each branch of a small conducting airway was followed through the volumetric data to
distinguish the point at which the terminal bronchiole divided into respiratory bronchioles, and
subsequent alveolar ducts. Terminal bronchioles were identified as the last purely conducing
airway without alveolar openings. As shown in Figure 2.7, the airways were also visualized in
the XY, YZ and XZ planes within the core volume to ensure that structures were correctly
identified. Each respiratory and terminal bronchiole was marked within the core volume
according to site and slice number in order to avoid duplication. Additionally, terminal
bronchioles were characterized as either normal or obstructed bronchioles according to the lumen
calibre. Each core was assessed blinded on two separate days by the observer. If a discrepancy
between the first and second analysis was observed, an opinion by a second blinded observer was
sought and discussed. The number of airways per millilitre was then calculated using the
reference core volume parameters by dividing the total number of counted airways in the sample
by the total core volume divided by 1000.
No. of airways/ml = No. of airways in the core/(total core volume (mm3)/1000)
49
Figure 2.7 Identification of terminal bronchioles on micro-CT. A: Regions of interest can be
viewed at different planes on micro-CT. B: Contiguous sections of the micro-CT scan are
examined to follow the airway branching structure. From left to right, slice 621 to 691 shows a
terminal bronchiole (red arrow) branching into two respiratory bronchioles (yellow arrows).
50
2.11 3D rendering of structures of interest on micro-CT and further
characterization by histology
Terminal bronchioles were identified as described in section 2.10. Structural changes of airway
lumens were extracted by the semi-automatic segmentation approach. This dataset was used to
reconstruct their 3D-topology using ITK-Snap, a software application used to navigate and
delineate 3D medical images. To further characterize and study the pathological changes in more
detail, the regions of interest were located in the FFPE sample cores using image registration and
spatial co-ordinates from the micro-CT image. Image registration was performed using a FIJI
plug in tool (UnWarpJ elastic registration) by matching at least three or more structural features
of the micro-CT image with the first histological section of the paraffin block. The micro-CT
image was then re-orientated to match the alignment of the embedded core and allow direct
comparison of the histological and micro-CT images. Spatial registration of this re-aligned core
allowed consecutive histological preparation of the specific area of interest only and a detailed
analysis of the connective tissue structures using Movat‟s Pentachrome. This staining technique
is used to highlight various components of the connective tissue. Black represents elastic fibres,
while collagen and reticular fibres stain yellow. Ground substance and mucin are represented by
blue, and red identifies fibrinoid and muscle structures.
51
2.12 Statistical analysis
The number of terminal bronchioles was correlated to Lm to determine if the loss of terminal
bronchioles does precede emphysema in early disease compared to the control group using a
multi-variant ANOVA with a Tukey‟s pairwise comparison. The data will be expressed as the
mean ± SE and was analyzed using Graph pad. A significant difference of P value less than 0.05
will be considered significant.
52
Chapter 3:
Results
3.1 Patient characteristics and clinical presentation
The cohort of lung tissue studied consisted of five controls that were smokers with normal lung
function and five COPD patients, four of which had mild (GOLD 1), and one moderate (GOLD
2) disease as shown in Table 2.1. When comparing the patient characteristics between the two
groups, we found no statistical difference between age, gender, height and smoking history, but a
statistical difference in weight (P<0.01). This may be explained by the increased ratio of males to
females in the mild/moderate COPD group. As to be expected, as COPD is diagnosed by the
presence of persistent airflow limitation as represented by FEV1/FVC ratio, there was a statistical
difference (P<0.05) in the FEV1/FVC ratio between the mild/moderate COPD (0.64) and control
groups (0.77). There was no statistical difference in FEV1, again as expected, as mild disease is
classified as the same FEV1 range (i.e. ≥80%) to normal subjects.
3.2 Assessment of micro-CT for conducting lung morphometry on
FFPE samples
The first aim of this study was to determine if micro-CT can be used to visualize fine lung tissue
morphometry in FFPE samples. Figures 3.1A and C, show representative examples of preoperative clinical MDCT images with a resolution of 1mm compared to Figures 3.1B and D
which show micro-CT images of FFPE lung tissue samples from the same patients with a
53
resolution of 6.7µm. Figures 3.1A and B are from a control patient whereas the bottom Figures
3.1C and D are from a COPD patient with mild (GOLD 1) disease. It is evident that the fine lung
tissue architecture is difficult to differentiate between the two MDCT scans due to the limited
resolution. However in the corresponding micro-CT images, there is a significant attenuation
difference between the tissue and paraffin wax to determine fine lung structures such as the
alveoli which measure approximately 200µm in diameter. As demonstrated in Figure 3.1, with
micro-CT imaging of the FFPE lung tissue it is possible to appreciate the emphysematous
destruction of the alveolar tissue leading to airspace widening in the mild COPD patient (Figure
3.1D) compared to the control patient (Figure 3.1B).
Figure 3.1 Comparison of clinical MDCT and micro-CT images. A and B: Representative
images from a control patient with normal lung function. C and D: Representative images from a
patient with mild COPD. A and C are images of the cross-sectional slices from the pre-operative
clinical MDCT scans and B and D are image slices from the micro-CT scans of the FFPE
resected lung tissue samples from the same patients.
54
3.3 Verification by histology of micro-CT
morphological analysis of FFPE samples
imaging
for
The volumetric, non-destructive images that can be obtained by micro-CT scanning of FFPE
samples have clear advantages over 4µm tissue sections used for histology. Therefore it is
important to determine if micro-CT imaging of FFPE samples provides adequate resolution to
accurately determine morphological measures such as mean airspace size (Lm) compared to the
gold standard which is histology. Figure 3.2A shows a representative slice of a micro-CT stack
compared to its matched histological section (Figure 3.2B), at the same pixel resolution,
following embedding and sectioning of the FFPE core. This figure illustrates that even the fine
anatomical features of the alveolar structures and bronchiole-vascular bundles can be visualized
in the micro-CT images and are comparable to matched histological sections. In order to quantify
this comparison, the FFPE samples from four donors were sectioned and following systematic
uniform random sampling (SURS) of the micro-CT stack and histological sections, Lm was
determined on images of the same pixel resolution. As shown in Figure 3.2C there was no
statistical difference between the two Lm measurements taken by either micro-CT or histology
on the same four lung tissue cores (40 slices/sections in total), verifying that micro-CT imaging
of FFPE samples can be used with traditional stereological methods to analyze fine lung tissue
morphometry.
55
Figure 3.2 Comparison of Lm by micro-CT and histology. A: Micro-CT image of an FFPE
sample, and B: its matched histological section at the same pixel resolution. C: Comparison of
mean linear intercept (Lm) analyzed by micro-CT and histology on the same FFPE cores (n=4).
The solid line depicts an Lm >600µm representative of mild emphysema and the dotted line
denotes the 95% confidence interval (CI) of Lm <489µm in normal lung tissue.
56
3.4 The use of micro-CT to determine emphysematous destruction
in diseased tissue
The second aim of this study was to determine if the loss of terminal bronchioles occurs within
mild/moderate COPD patients prior to the presence of emphysematous destruction. To quantify
air space enlargement in mild/moderate COPD patients (n=5) compared to controls (n=5), the
SURS method was used to select 10 micro-CT slices from the whole image stack of the FFPE
sample for Lm analysis, and this was conducted on eight to ten FFPE cores distributed over six
lung specimen slices per subject (totaling over 800 micro-CT slices for analysis).
As previously described in the McDonough et al. study which used air inflated and
critically point dried lung tissue cores for micro-CT, mild emphysema was determined as Lm
>600µm (shown as the solid bars), which is the absolute limit of the frequency distribution for
Lm in normal lung tissue. Normal tissue was determined as an Lm <489µm (shown as dashed
bars), which is the upper 95% confidence interval value for Lm in normal lung tissue [124]. The
results presented in Figures 3.3A and B show the distribution of Lm in µm according to the slice
number of the lung specimen where 1 is the top and 6 is the bottom of the resected tissue, and all
Lm values were plotted for each core. In the control group, we found no evidence of emphysema
as the Lm ranged between 210 to 395µm in all FFPE core samples (Figure 3.3A). Interestingly,
in the mild/moderate COPD group, we observed a much greater variation in Lm ranging from
214 to 793µm within the FFPE tissue cores (Figure 3.3B). However, it is important to note that
the mean value (328µm) of Lm for the mild/moderate COPD group was well beneath the Lm
emphysematous score (>600µm). Lastly, as the total number of terminal bronchioles are counted
throughout an entire FFPE tissue core it is important to compare these morphological counts
between the control and disease groups using the average Lm of the entire core. Thus, in Figure
57
3.3C, using the mean Lm value for each FFPE core we demonstrate that although there is a
statistical difference in the average Lm in mild/moderate COPD FFPE samples compared to
controls (P<0.05), there is no evidence of emphysematous destruction in mild/moderate COPD
patients.
58
Figure 3.3 Lm measurements in FFPE tissue from control and mild/moderate COPD
patients. A: Lm distribution in control patients by lung specimen slice number of resected tissue.
B: Lm distribution in mild/moderate COPD patients by lung specimen slice number of resected
tissue. C: Mean Lm per core and lung specimen slice in controls (filled circles) and
mild/moderate COPD (filled squares). The dotted line indicates an Lm <489µm which represents
the upper 95% confidence limit for Lm in normal lung tissue. The solid line indicates Lm
>600µm which represents mild emphysema.
59
3.5 The number of terminal
mild/moderate COPD patients
bronchioles
is
decreased
in
Terminal bronchioles, identified as the last purely conducting airways without alveolar openings
cannot be detected by MDCT as the limited resolution only allows visualization of airways
greater than 2mm. In contrast, micro-CT imaging of FFPE samples allowed the complex 3D
small airway branching network to be followed by scrolling through and examining the
contiguous sections of each scan. Each micro-CT scan can provide 3D visualization of the same
region of interest, in the XY, YZ and XZ planes which is important for the correct identification,
tracing and counting of terminal bronchioles. As shown in Figure 3.4A we found that the number
of terminal bronchioles per millilitre (TB/ml) is significantly reduced to 4.6 TB/ml in
mild/moderate COPD FFPE cores compared to control patients (mean 6.2 TB/ml, P<0.01).
Furthermore, of the counted terminal bronchioles within the tissue, we found a significant
increase in the number of obstructed terminal bronchioles in patients with mild/moderate COPD
(mean 1.98 TB/ml), compared to controls (mean 0.45 TB/ml, P<0.0001, Figure 3.4B). Although
not measured, obstructed terminal bronchioles were identified as those with >50% narrowing of
the lumen by either exudate, thickening or collapse of the airway wall. Importantly, to test our
hypothesis we wanted to determine if loss of terminal bronchioles occurs in mild/moderate
COPD patients prior to emphysematous destruction. As shown in Figure 3.4C, the mild/moderate
COPD patients had significantly lower numbers of terminal bronchioles compared to the control
group and this occurred in FFPE lung tissue with the average Lm ranging from >210 to <600
µm, and therefore in the presence of no emphysematous destruction. All of the terminal
bronchioles counted within the FFPE cores from control patients were within FFPE lung tissue
with an average Lm below 489µm.
60
Figure 3.4 Number of terminal bronchioles per millilitre of resected lung tissue is decreased
in mild/moderate COPD patients. A: The mean number of terminal bronchioles per millilitre
(TB/ml) in mild/moderate COPD (grey bars) compared to control patients (open bars, P<0.01).
B: The mean number of obstructed TB/ml in mild/moderate COPD patients (grey bars)
compared to controls (open bars, P<0.0001). C: The mean number of TB/ml in patients with
mild/moderate COPD (grey bars) compared to controls (open bars) segregated by Lm (<489<600µm = no emphysema, P<0.01).
61
3.6 Correlation of data to total lung volumes using MDCT
parameters
Table 2.2 shows the pre-operative MDCT total right, left and lung pair volumes of all patients,
calculated by volume segmentation using stereological methods. These reference parameters
were then used to correlate the number of terminal bronchioles/ml to the number of terminal
bronchioles per total lung volume, allowing for comparison with previously published studies.
As shown in Table 3.1, McDonough et al., previously report for healthy control lung tissue 6.9 ±
1.2 TB/ml which equates to 22,300 ± 3900 per total lung. In comparison, for the control groups
in our study we calculated 6.2 ± 1.1 TB/ml, which equated to 14,004 ± 3,276 per lung. We also
observed a further decrease in terminal bronchioles in mild/moderate COPD patients which had
4.6 ± 1.0 TB/ml equating to 11,410 ± 3,294 per total lung. When comparing the tabulated mean
values between the two studies, these data indicate that there is a reduction in the total number of
terminal bronchioles per lung in our control group (patients with similar smoking history and
normal lung function) and mild/moderate COPD patients compared to the healthy control group
in the McDonough et al. study. This is in keeping with the hypothesis that terminal bronchioles
are lost prior to the onset of emphysematous destruction in mild/moderate COPD subjects as we
observe a 49% reduction in terminal bronchioles/lung compared to the 89% and 72% reduction
previously documented by McDonough et al., for end stage centri- and pan- lobular emphysema
patients respectively.
62
Table 3.1 Comparison of the number of terminal bronchioles with previous published data
Mild/Moderate
COPD
End stage COPD
Patients with
centrilobular
emphysema
(McDonough et al.)
End stage COPD
patients with
panlobular
emphysema
(McDonough et al.)
n=5
n=5
n=4
n=8
6.9 ± 1.2
6.2 ± 1.1
4.6 ± 1.0
0.7 ± 1.2
1.6 ± 1.2
Total number of
Terminal
bronchioles per
total lung volume
22,300 ± 3,900
14,004 ± 3,276
11,410 ± 3,294
2,400 ± 600
6,200 ± 2100
% reduction from
Healthy Controls of
McDonough et al.
-
37%
49%
89%
72%
Healthy Controls
(McDonough et al.)
Controls
(patients with
normal lung
function)
n=4
Terminal
bronchioles per ml
of lung volume
COPD: Chronic Obstructive Pulmonary Disease. Values are the mean ± SE.
3.7 The number of respiratory bronchioles is decreased in
mild/moderate COPD patients
In addition to counting the number of terminal bronchioles, we also counted the number of
respiratory bronchioles within the FFPE tissue cores. As shown in Figure 3.5, we demonstrated a
significant decrease (<0.0001) of 10.0 respiratory bronchioles/ml of FFPE lung tissue for
mild/moderate COPD patients compared to the control group (16.7 respiratory bronchioles/ml).
These data are in keeping with the hypothesis that the obstruction and obliteration of small
airways occur early in mild/moderate COPD, and spreads through the respiratory bronchiole into
the alveoli leading to the development of emphysema.
63
Figure 3.5 Number of respiratory bronchioles per millilitre is decreased in mild/moderate
COPD. The mean number of respiratory bronchioles per millilitre in mild/moderate COPD (grey
bars) compared to controls (open bars, P<0.0001).
3.8 Tracing and 3D rendering of small airway morphology by
micro-CT
The final aim of this study was to define the use of micro-CT as a scouting tool to identify and
assess the structural pathology of obstructed and obliterated terminal bronchioles using standard
histological methods. 3D segmentations can be used to identify changes in directional course
along the length of the airways and their relation to surrounding structures such as vessels and
parenchyma. In addition, quantification of airway diameter, patency and length can also be
obtained. A key advantage of obtaining 3D information is the ability to observe the pathological
lesion of interest in perspective to the branching airway network. Figure 3.6 shows the image
views of the micro-CT stack from a control patient with a normal terminal bronchiole, and two
mild/moderate COPD patients with obstructed and obliterated terminal bronchioles in relation to
its surrounding structures. Semi-automatic manual tracing was then used to obtain the 3D
renderings of the small airways (orange) and alveolar ducts (purple).
64
Figure 3.6 Three dimensional renderings of small airway morphology. Figures 3.6A and B
demonstrate the 3D rendering of a normal terminal bronchiole generated from semi-automatic
tracing. In comparison to controls, in mild/moderate COPD patients we identified obstructed
terminal bronchioles (green) as shown in Figures 3.6C and D, and obliterated terminal
bronchioles (red arrow) as shown in Figures 3.6E and F.
65
3.9 Micro-CT as a scouting tool for histological analysis of regions
of interest
Using micro-CT imaging of FFPE sections, the exact location of areas of interest can be scouted
and applied for precise histological sectioning. This enables the potential to first re-embed the
FFPE sample so that specific structures of interest can be sectioned in a more appropriate
orientation for a desired analysis. Thus, instead of the traditional laborious and blinded cutting of
sections, there is no risk of losing important pathological structures and information. This
application of micro-CT is demonstrated in Figure 3.7 which shows the corresponding serial XY
micro-CT images of the lesions of interest from the 3D renderings of Figure 3.6, with their
matched histological sections stained with Movat‟s Pentachrome. Figure 3.7A shows serial
sections of an intact terminal bronchiole of a control patient (corresponding to Figures 3.6A and
B) demonstrating normal regular walls of the terminal bronchiole. Figure 3.7B demonstrates the
serial sections of the obstructed terminal bronchiole from Figures 3.6C and D, where the airway
wall appears more thickened, and become narrowed and obstructed in the middle of the terminal
bronchiole before re-opening up into a respiratory bronchiole. Figure 3.7C, shows the obliterated
terminal bronchiole from Figures 3.6E and F where the terminal bronchiole is obliterated before
the patency and lumen calibre is resumed before entering the respiratory bronchiole. It is clear
that although these can be easily visualized on micro-CT, histology provides significant
advantage using histological stains to provide a more comprehensive analysis of the airway
tissue structures and pathological features at the cellular level.
66
Figure 3.7 Serial micro-CT slices with matched histological sections. All pictures are
described from left to right. A. Normal terminal bronchiole where lumen calibre remains patent
and intact throughout the core. B. An obstructed terminal bronchiole showing a patent terminal
bronchiole that becomes narrowed and obstructed then widens again before opening into a
respiratory bronchiole. C. An obliterated terminal bronchiole where the patency of the small
airway is intact but suddenly disappears before reappearing again.
67
Chapter 4:
Discussion
This novel combination of MDCT, micro-CT and histological analysis of FFPE human lung
tissue shows that the number of terminal bronchioles is reduced (P<0.01) in the earliest stages of
COPD, and confirmed that narrowing and destruction of terminal bronchioles precedes the onset
of emphysematous destruction in COPD. These new data have important implications for the
treatment of COPD in that it suggests the majority of the clinical trials have shown little effect on
long-term lung function decline because interventions were not introduced until after the
substantial narrowing and reduction in numbers of terminal bronchioles had already occurred.
4.1 Methodological findings
In contrast to previous micro-CT studies on human lung tissue which have used either air
inflated and air dried cores [124, 146] or formalin steam inflation [121], this study demonstrates
that micro-CT can be used to effectively image and analyze routinely prepared FFPE human lung
tissue samples, without the use of contrasting agents. Specifically, our micro-CT protocol
involved the use of a molybdenum reflection target, a key advantage over the standard tungsten
reflection targets as it was able to achieve the low flux, low keV setting required to exploit the
narrow window of x-ray attenuation contrast that exists between soft tissue and paraffin wax.
This provided the resolution for optimal visualization of the fine lung microstructure and
furthermore, the images acquired were comparable to conventional histology for stereological
analysis. Subsequently, this enabled us to conduct multiple image-based analyses including:
68
1) Visualization of the distal airways, vasculature and alveolar septum, not amenable by MDCT;
2) Measures of lung structures such as Lm; 3) Semi-automatic segmentation of airways to
perform 3D rendering; and 4) Scouting for specific regions of interest within the tissue samples.
The capacity to conduct micro-CT imaging on FFPE lung tissue provides numerous
benefits in addition to the fact that FFPE samples generated over the years by tissue registries
can now be used for valuable cross-sectional cohorts to understand tissue pathology in disease.
Most importantly, the non-destructive nature of micro-CT is a significant advantage over
standard histology, in that it provides the ability to identify terminal bronchioles anatomically
and identify the lesions within them before the histological examination begins. This makes it
possible to orient the sample and process it in a way that allows the histology of the airway to be
observed in cross-section, in exactly the same orientation as the micro-CT image. Moreover it
makes it possible to use semi-automatic segmentation to trace and follow airway lumens within
the micro-CT stack to identify the obliteration of terminal bronchioles within networks of patent
small airways, respiratory bronchioles and alveolar ducts. Such 3D analysis would not be
possible with histological sections due to the complex branching structure of the airway tree,
which cannot be visualized alone with cross-sectional 2D views. In contrast, the acquisition of
3D imaging enables virtual sectioning of the sample at any orientation for a better understanding
of the intricate lung architecture. However, one limitation of micro-CT is that the image
resolution is still not at the cellular level, and therefore only microscopy enables comprehensive
analysis of cellular and extracellular matrix (ECM) structures with the use of histological stains.
An advantage of micro-CT scanning of FFPE samples is imaged volumes can be used as a
scouting tool to precisely identify structures of interest. Using image registration we can then
align the micro-CT stack with a histological section from the FFFE sample, to locate regions of
69
interest within it, to efficiently section, or if required re-orientate the sample, for enface
sectioning of airways of interest. This technique of matching histology to micro-CT has
previously been demonstrated in a recent study of Bronchiolitis Obliterans Syndrome (BOS)
[146], however they used lung samples that were rapidly frozen and critically point dried which
causes significant artefact in the tissue histology due to the ice crystal formation during the
freezing process. The advantage of our study using FFPE tissue is the preservation of cellular
structures within the sample, allowing for detailed analysis by microscopy. Micro-CT imaging
of FFPE samples therefore provides an extremely useful tool for future implementation that will
enable the next level of histological evaluation to understand the cellular, ECM, and molecular
components that are present during the destructive process within these terminal bronchioles,
with the hope of eluding to the pathological mechanisms of COPD.
4.2 Pathological findings
The aforementioned cohort of surgical resected lung tissue within the JHRC lung registry and
new innovative methods in micro-CT scanning of FFPE tissue described in this study have
permitted re-analysis of lung tissue samples from mild/moderate COPD [86], a cohort that has
been relatively understudied due to the difficulties in obtaining such pathological samples. We
report that the number of terminal bronchioles/ml (TB/ml) is decreased from 6.2 ± 1.1 TB/ml in
the control group with similar smoking history and normal lung function to 4.6 ± 1.0 TB/ml
(P<0.01) in mild/moderate COPD patients. In addition, as the total number of terminal
bronchioles is decreasing in mild/moderate disease, the number of obstructed terminal
bronchioles/ml was found to increase suggesting that the narrowing of terminal bronchioles may
70
occur prior to total obliteration. Furthermore, when the numbers of terminal bronchioles/ml are
compared to Lm the result confirms the findings of McDonough et al., in end-stage COPD.
These findings support the hypothesis that „narrowing and obliteration of small airways
precedes emphysematous destruction in mild to moderate COPD’. Lastly, we demonstrate that
respiratory bronchioles are significantly decreased in patients with mild/moderate disease
compared to controls who have smoked similar amounts but maintain normal lung function. This
is again in keeping with our theory that emphysema begins at the terminal bronchiole and reiterates Leopold and Gough‟s first description of centrilobular emphysema as a peripheral
extension of bronchial inflammation to the respiratory bronchioles with consequent destruction
[56]. The previously published data by McDonough et al., demonstrated that in end-stage COPD
patients being treated with lung transplantation, there is approximately a 90% and 70% loss of
terminal bronchioles/ml in centrilobular and panlobular phenotypes respectively [124]. Based on
these findings we postulate that the rapid decline in FEV1 that leads to severe (GOLD 3) and
very severe (GOLD 4) COPD resulting in disability and premature death is caused by a process
that narrows and obliterates terminal bronchioles.
The various mechanisms underlying small airways disease have been postulated and
outlined in the introductory chapter of this thesis. The persistent and exuberant inflammatory and
immune responses characteristic of COPD are thought to affect both the small airways and
alveoli, resulting in small airway remodeling/obliteration and alveolar destruction, however the
precise mechanisms are unknown. We postulate that the initial loss of terminal bronchioles could
precipitate the onset of emphysema as the acinar walls distal to the terminal bronchiole would
lose their tethering support leading to subsequent collapse and destruction. In these proposed
chain of events, the remaining intact small airway wall would lose its parenchymal radial support
71
through the loss of elastic recoil, causing further closure of the terminal bronchiole as it lacks
cartilage support unlike the more proximal and larger airways. In contrast, emphysema can
contribute to increased airflow limitation by varying mechanisms which include: 1) Loss of
elastic recoil which is the driving force of expiratory airflow; and 2) loss of the opening support
for intra-parenchymal airways through the lack of airway-alveolar attachments predisposing
airways to collapse during expiration [147]. However, numerous studies have documented the
poor correlation of functional spirometry assessment with emphysematous severity, whereas it
has been well established that small airways disease is the major site of airflow obstruction in
COPD.
The above mechanisms outlined are theoretical and have not been proven. Furthermore,
they do not explain the heterogeneous nature of COPD and why some patients develop either a
more emphysematous or airway dominant phenotype, whilst others develop both. Our data
demonstrates that the average Lm value for mild/moderate COPD patients is 328µm which is
within the normal range for air space size (<489 µm). Interestingly, on closer examination of this
group, it was noted that one patient case showed signs of emphysema by micro-CT, and when we
assessed all Lm values for this case, we found a range of Lm values from 455 to 792µm which
are within the lower range of mild emphysema. However, despite the presence of widespread
emphysema, this patient‟s post-bronchodilator FEV1 was 81% with an FEV1/FVC ratio of 0.68,
which classifies his disease as mild GOLD 1 COPD (FEV1/FVC ratio <0.7, FEV1 ≥80%).
Importantly, although this patient case demonstrated greater emphysematous tissue destruction
we also observed decreased numbers of terminal bronchioles (4.35 TB/ml). We therefore
postulate that the heterogeneous picture of terminal bronchiole reduction with widespread
emphysema, as demonstrated by this particular patient case presented above, may represent the
72
early stages of tissue pathology that is representative of the population of patients that are „rapid
decliners‟ that progress to end-stage disease. To test this hypothesis, future studies on these
archival FFPE tissues would require ethical consent to the subsequent 10-20 years of postoperative patient information if available for these subjects, to determine by spirometry if this
correlation is true.
4.3 Clinical relevance
The findings of this study demonstrate that patients in the early phases of COPD show evidence
of pathological destruction and the initial event appears to be the narrowing and obliteration of
terminal bronchioles, before the development of emphysematous destruction. It is therefore not
surprising that numerous population studies have shown that compared to healthy individuals,
symptomatic patients from this early category have a more rapid annual FEV1 decline [148],
increased mortality [11, 149], hospitalizations and exertional (activity related) dyspnea, and
decreased daily physical activity and quality of life [150-155].
4.3.1
Diagnosis
COPD is currently under-diagnosed [156] and under-treated due to the silent nature of early
disease [157]. Spirometry is currently the gold-standard investigation for COPD as it is
accessible and reproducible. However, it is evident that irreversible structural damage in the
small distal airways may have already occurred despite a preserved FEV1. This is due to the
parallel organization of the respiratory tree and hence, a significant loss of peripheral airways
would be required before global lung function deterioration is detected [41]. A more sensitive
73
clinical test to detect the presence of small airways disease is therefore required. The importance
of the quantification of small airways disease by clinical CT has recently gained much attention
and there have been significant developments in CT technology to facilitate this. Most of the
techniques available involve the use of mathematical algorithms of attenuation levels [158], and
these measurements have been accurately correlated with pathological airway specimens as
small as 2mm in luminal diameter [115, 159, 160]. However, the major site of airways
obstruction in COPD, are the small airways <2mm in diameter, which cannot be reliably
measured by clinical MDCT due to limits of resolution [118]. Early attempts to overcome this
problem used algorithmic reconstruction of the distal airways from measurements of the larger
proximal airways based on the anatomy of the respiratory tree [115]. Additional techniques in
quantifying small airways disease have focused on densitometry measurements of expiratory CT
scans. This is based on the theory that gas trapping and hyperinflation occurs during expiration in
the presence of small airways obstruction [161, 162]. Although CTs are a useful and valuable
resource for the potential diagnosis of early disease, radiation exposure, lack of resources and
cost limit their availability as a regular diagnostic test. Other physiological measurement tools to
measure small airways disease such as the forced oscillation technique (FOT) and ventilation
distribution tests have been proposed but have shown differing degrees of sensitivity and
specificity [163, 164].
74
4.3.2 Treatment
There are currently no available pharmacological therapies that are capable of either slowing or
reversing the rate of decline in FEV1 that leads to severe and very severe COPD [1]. The most
recent GOLD guidelines recommend that anti-inflammatory treatment, namely inhaled
corticosteroids (ICS) are not commenced until patients are within category C of the new
combined assessment, which is equivalent to GOLD grade 3. As our findings show that the
irreversible destruction of terminal bronchioles has already begun in mild/moderate COPD, this
emphasizes the importance of early recognition and intervention. The effective role of current
anti-inflammatory agents in stable COPD has remained controversial [1, 165]. This may be due
to the relative inaccessibility to the distal location of the terminal bronchioles by large ICS
particles, but recent studies investigating the effects of small particle ICS have shown promising
but inconclusive results [166, 167]. Secondly, it has been shown that CD8+ cells that are
elevated in COPD are less sensitive to steroid treatment, suggesting that the inflammatory
processes specific to COPD may be resistant to this particular treatment strategy [81, 156].
Further research is therefore required to gain a greater understanding of the inflammatory
mechanisms that occur in the early stages of COPD and those specifically involved in small
airways disease.
4.4 Limitations of the study
While there are key advantages to the micro-CT imaging of FFPE tissue samples, the presence of
tissue infiltration processing-induced artefacts in <10% of samples analyzed must be
acknowledged. Poor and inconsistent paraffin infiltration can lead to the formation of fine air
75
bubbles which exclude these areas from analysis due to poor visualization of these regions.
Areas of tissue collapse were also observed and this may have been due to organic causes or
poor inflation. However, exclusion criteria were determined to ensure that these artefacts were
not included for analysis.
In order to achieve the resolution required for lung tissue and paraffin differentiation, the
average micro-CT scanning time was 6 to 8 hours per two cores which may have induced some
movement of the tissue samples within the tube, producing movement artefact. Additionally, a
bright halo artefact which outlines the micro-CT images was caused during the reconstruction
algorithm stage as the processed samples were generally larger than the field of view (15mm). It
is also recognized that the imaging of FFPE samples does not provide as good a contrast as lung
tissue that is air-inflated, fixed and dried. Additionally, it is important to note that the high and
long radiation exposures may induce some tissue damage and the quality of DNA, RNA and
protein following scanning will need to be determined in future studies.
Another limitation of this study is that the lung tissues used were from resection surgery
and not the whole lung, and therefore may not represent the full disease heterogeneity throughout
the lung. Additionally, McDonough et al., used lungs that were air inflated and rapidly frozen,
then cores were fixed in 1% gluteraldehyde in pure acetone at -80oC [124], whereas the lung
samples in our study were fixed by inflation with formalin. As formalin does not cross link
elastin whereas glutaraldehyde does, there is the possibility of tissue shrinkage. This difference
could explain the discrepancy in values for control patients between the two studies.
Finally, our early disease cohort included a combination of mild to moderate COPD (four
GOLD 1 and one GOLD 2) which may have influenced our overall results. One of the future
goals of this study is to expand on this cross-sectional cohort to contain FFPE samples from
76
GOLD 1, GOLD 2 and GOLD 4 categories. Further, as COPD patients do not have the same rate
of disease progression, it would be ideal to initiate a prospective study where FFPE samples
could be taken at the time of surgery, analyzed, and the predicted prognosis of lung function
followed longitudinally.
4.5 Future directions
This study demonstrates that small airways disease manifest in the early phases of COPD
through a destructive and remodeling process. Further investigation into the molecular and
cellular mechanisms underlying these processes is therefore required. With micro-CT imaging
and scouting of specific regions of interest in FFPE samples, it will be possible to carry out
morphological analysis by immuno-histochemistry to understand structural tissue changes, and
correlated gene expression signatures with nanostring technology. These future studies will
reveal potential molecular determinants of disease providing insight into why only a susceptible
minority of smokers develop COPD, and point to potential therapeutic targets.
To date, nearly all clinical trials investigating the efficacy of pharmacotherapy treatments
available have precluded patients with mild disease [102, 168]. Guenette et al., showed for the
first time that in mild to moderate COPD patients, a combination of steroid and bronchodilator
therapy (fluticasone/salmeterol) was associated with significant improvements in airway function
at rest and during exercise [164]. This study was however limited to 6 weeks and the long-term
efficacy was not evaluated. The importance of including this subset population must be
recognized and incorporated into future, longitudinal study designs.
77
4.6 Conclusion
This study has used a novel combination of MDCT, micro-CT and histology to demonstrate for
the first time that terminal bronchioles are significantly decreased in FFPE lung tissue of patients
with mild/moderate COPD, and that this process occurs prior to the onset of emphysematous
tissue destruction. COPD continues to be a global and expanding public health burden and
despite substantial advances in diagnostic and management approaches, a curative treatment has
not yet been achieved. Current pharmacotherapies have not shown benefit in modifying the
progression of disease and this may be because treatment is commenced at a stage when
irreversible damage has already occurred. The key findings of this study support the wellestablished theory that peripheral small airways are the key therapeutic target in the treatment of
COPD, and emphasize the importance of earlier recognition and intervention to slow down or
prevent the progression of this debilitating disease.
78
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