Submitted to obtain the degree of

Dissertation
Submitted to obtain the degree of

Docteur de l’Université Louis Pasteur Strasbourg I

Speciality : Life sciences
Molecular and cellular aspects of biology

By François-Xavier BLÉ



Lung magnetic resonance imaging as a
non-invasive alternative to assess experimental
pulmonary diseases in small rodents




rdPublic defense held on December 3 , 2007





Defense committee

Thesis Director Dr Nelly FROSSARD, Faculté de Phamarcie, Illkirch
Thesis Co-director Dr Nicolau BECKMANN, Novartis Pharma AG, Basel
Internal Referee Prof Daniel GRUCKER, IPB, Strabourg
External Referee Prof Vincent LAGENTE, Faculté des sciences, Rennes
External Referee Dr Yannick CRÉMILLIEUX, UCB, Lyon





Thèse
Présentée pour obtenir le grade de

Docteur de l’Université Louis Pasteur Strasbourg I

Discipline: Sciences du vivant
Aspects moléculaires et cellulaires de la biologie

par François-Xavier BLÉ



Imagerie par résonance magnétique des
poumons: un outil non invasif de
caractérisation de modèles expérimentaux des
maladies respiratoires chez le petit rongeur




Soutenue publiquement le 3 décembre 2007





Membres du Jury

Directeur de thèse Dr Nelly FROSSARD, Faculté de Phamarcie, Illkirch
Co-directeur de thèse Dr Nicolau BECKMANN, Novartis Pharma AG, Basel
Rapporteur interne Prof Daniel GRUCKER, IPB, Strabourg
Rapporteur externe Prof Vincent LAGENTE, Faculté des sciences, Rennes Dr Yannick CRÉMILLIEUX, UCB, Lyon Lung MRI as Non-Invasive alternative to assess experimental pulmonary diseases in small rodents

TABLE OF CONTENT

FRENCH SUMMARY............................................................................................. 1
FOREWORD............................................................................................................... 8
GENERAL INTRODUCTION ........................................................................... 11
1. Respiratory diseases and animal models .............................................................. 12
2. Evaluation of pulmonary diseases models ............................................................ 14
2.1. Imaging Pulmonary diseases models .......................................................... 15
2.2. Magnetic Resonance Imaging (MRI) ......................................................... 16
2.2.1. MRI historical.........................................................................................16
2.2.2. MRI basics ............................................................................................... 18
2.2.3. MRI Principle.......................................................................................... 19
2.2.4. MRI of the lung ....................................................................................... 25
3. Investigated models and proton MRI expectations ............................................. 27
3.1. Allergen-induced airway inflammation in sensitized mice........................ 28
3.2. Lipopolysaccharide-induced mucus hypersecretion rat airways ............. 31
3.3. Hypertonicity-induced hydration of the airway secretion in rats ............ 34
3.4. Bleomycin-induced pulmonary fibrosis in mice......................................... 36

OBJECTIVES OF THE THESIS ...................................................................... 39
ORGANISATION OF THE THESIS............................................................... 40
MANUSCRIPT 1: Allergen-induced lung inflammation in actively
sensitized mice assessed by MRI.................................................................................. 41
ADDITIONAL DATA: Time course of lung fluid signals assessed by MRI in
a murine model of allergen-induced inflammation: a comparison of two strains .. 60 Lung MRI as Non-Invasive alternative to assess experimental pulmonary diseases in small rodents

MANUSCRIPT 2: Intranasal instillation of FTY720 reduces lung MRI fluid
signals in a murine model of allergen-induced inflammation: example of a S1P1
agonism ......................................................................................................................... 75
MANUSCRIPT 3: A S1P2 antagonist inhibits allergen-induced increase in
MRI fluid signals in mouse via a non mast cell-dependent mechanism................... 93
MANUSCRIPT 4: In-vivo assessments of mucus dynamics in the lungs using
a Gd-Cy5.5-bilabeled contrast agent......................................................................... 111
MANUSCRIPT 5: ENaC-mediated effects assessed by proton MRI in a rat
model of hypertonic-induced hydration of airways ................................................ 131
PRELIMINARY REPORT: Bleomycin-induced lung injury assessed non-
invasively and in spontaneously breathing mice by proton MRI .......................... 152
GENERAL DISCUSSION.................................................................................. 167
CONCLUSION ....................................................................................................... 175
REFERENCES 178
LIST OF PUBLICATIONS AND PRESENTATIONS ........................... 189
ACKNOWLEDGEMENTS................................................................................ 192 Lung MRI as Non-Invasive alternative to assess experimental pulmonary diseases in small rodents

TABLE OF ABBREVIATIONS
Abbreviation Description
-6µM Micro (x10 ) molar
2DFT Two-dimensional Fourier Transformation
AB/PAS Alcian blue/ periodic acid Schiff’s staining
ADC Apparent diffusion coefficient
ASL Airway surface liquid
ASM Airway smooth muscle
B Homogeneous magnetic field 0
BAL Bronchoalveolar lavage
BLM Bleomycin
BN Brown Norway
b.w. Body weight
CCD Charged coupled device
CF Cystic Fibrosis
CFTR Chloride Cystic Fibrosis Transepithelial Receptor
Compound Condensation product of p-methoxphenethyl methylamine with
48/80 or c48/80 formaldehyde. Mixture of low molecular weight polymers
having a degree of polymerization between 3 and 6.
COPD Chronic obstructive pulmonary disease
DMSO Dimethylsulphoxide
DSCG Di-sodium-cromoglycate
EDTA Ethylenedaminetetraacetic acid
EGFR Epithelial growth factor receptor
EIPA Ethylisopropyl amiloride
ENaC Epithelial Natrium Channel
EPO Eosinophil peroxidase
et al. et alia; and others
High affinity receptors for IgE FcεRI
fMRI Functional magnetic resonance imaging
FOV Field of view Lung MRI as Non-Invasive alternative to assess experimental pulmonary diseases in small rodents

g Gram
Gd-DTPA Gadolinium and diethylenetriaminepentaacetic acid complex
h Hour
HBSS Hank's balanced salt solution
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid
HS Hypertoninc saline
Hz Hertz
i.p. Intraperitoneally
i.t. Intratracheally
i.v. Intravenously
Ig Immunoglobulin
IL- Interleukin-
INF Interferon
kg Kilogram
l Litre
LPS Lipopolysaccharide
LT Leukotriene
Microgram µg
mg Milligram
min Minute
ml Millilitre
-3mM Milli (x10 ) molar
MCC Mucociliary clearance
MPO Myeloperoxidase
MR Magnetic resonance
MRI Magnetic resonance imaging
mRNA Messenger ribonucleic acid
n Number of replicates
ºC Degrees centigrade
OPD O-phenylenediamine
OVA Ovalbumin
p.o. Per os (by gavage) Lung MRI as Non-Invasive alternative to assess experimental pulmonary diseases in small rodents

per se By its very nature; intrinsically
PET Positron-emission tomography
PG Prostaglandin
pH -log hydrogen ion concentration 10
RF Radiofrequency
R Airway resistance L
ROI Region of interest
rpm Revolutions per minute
s Second
S1P Sphingosine-1-Phosphate
S1P Sphingosine-1-Phosphate receptor subtype X
SEM Standard error of the mean
T Tesla (1T = 10 000 gauss) defines intensity of a magnetic field
T1 Longitudinal relaxation time
T2 Transversal relaxation time
T2* al relaxation time, taking into account microscopic
magnetic field inhomogeneities
TE Echo time (The time between the RF pulse and the echo signal)
TNF Tumour necrosis factor
TR Repetition time (the time interval with which the RF pulse
sequence is repeated)
TRIS Tris(hydroxymethyl)-amnithan
U Units
UTP Uridine Tri-Phosphate
Alpha α
Beta β
Magnetic moment µ
Larmor frequency ω
Gyromagnetic ratio γ
RF flip angle θ
Lung MRI as Non-Invasive alternative to assess experimental pulmonary diseases in small rodents

FRENCH SUMMARY

Les maladies respiratoires et pulmonaires incluant l’asthme, les bronchopathies
chroniques obstructives (BPCO), la fibrose, la pneumonie, le cancer du poumon et
bien d’autres, comptent parmi celles dont l'incidence et la morbidité ont le plus
consi