The role of nitric oxide synthases in the pathophysiology of chronic obstructive pulmonary disease [Elektronische Ressource] / by Nirmal Parajuli

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• Gesundheit

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2009
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

The role of nitric oxide synthases in the pathophysiology of
chronic obstructive pulmonary disease




Inaugural Dissertation
Submitted to the
Faculty of Medicine
in partial fulfilment of the requirements
for the PhD Degree
of the Faculty of Veterinary Medicine and Medicine
of the Justus Liebig University Giessen



by

Nirmal Parajuli
of
Bhojpur, Nepal


Giessen 2009
From the Medical Clinic II, Excellence Cluster of Cardio-Pulmonary System,
University of Giessen Lung Centre
Chairman: Werner Seeger, Prof., M.D.
of the Faculty of Medicine of the Justus Liebig University Giessen







First Supervisor and Committee Member: Prof. Dr. Norbert Weißmann
Second Supervisor and Committee Member: Prof. Dr. Paul T. Schumacker

Committee Members: Prof. Dr. Martin Diener
PD Dr. Christian Mühlfeld





thDate of Doctoral Defence: 9 December, 2009CONTENT I
CONTENT
CONTENT .................................................................................................................................I
LIST OF FIGURES................................................................................................................. V
LIST OF TABLES...............................................................................................................VIII
LIST OF ABBREVIATION..................................................................................................IX
1. INTRODUCTION 1
1.1 Definitions........ 1
1.2 Epidemiology.... 3
1.3 Risk factors........ 4
1.4 Pathology.......................................................................................................................... 4
1.4.1 Chronic bronchitis ..................................................................................................... 4
1.4.2 Emphysema ............................................................................................................... 5
1.4.3 Systemic effects......................................................................................................... 5
1.4.4 Pulmonary vascular changes ..................................................................................... 5
1.5 Functional changes.. 5
1.6 Cigarette smoke................................................................................................................ 6
1.7 Pathogenesis...... 8
1.7.1 Inflammation ............................................................................................................. 8
1.7.2 Protease–antiprotease imbalance............................................................................... 9
1.7.3 Oxidative stress ....................................................................................................... 10
1.7.4 Apoptosis................................................................................................................. 11
1.7.5 Systemic oxidative stress......................................................................................... 12
1.7.6 Nitrosative stress ..................................................................................................... 12
1.7.7 Hypoxia ................................................................................................................... 13
1.8 Animal models ............................................................................................................... 14
2. AIMS OF STUDY 17
3. MATERIALS...................................................................................................................... 18
3.1 Solutions and substances ................................................................................................ 18
3.2 Consumables .................................................................................................................. 18
3.3 Systems and machines for animal experiments.............................................................. 19
3.4 Histology ........................................................................................................................ 19
3.5 Antibodies ...................................................................................................................... 21
3.6 Systems and software for morphometry......................................................................... 21
3.7 Smoke generating system............................................................................................... 21
4. METHODS.......................................................................................................................... 22 CONTENT II
4.1 Animals .......................................................................................................................... 22
4.2 Experimental design and tobacco smoke exposure........................................................ 22
4.3 Mice preparation............................................................................................................. 23
4.3.1 Alveolar morphometry ............................................................................................ 23
4.3.2 Vascular m 23
4.3.3 Lumen morphometry............................................................................................... 24
4.3.4 Ratio of the number of alveoli / number of vessels................................................. 24
4.4 Isolated perfused mouse lung experiment ...................................................................... 24
4.5 In vivo hemodynamic measurement............................................................................... 25
4.6 Heart ratios ..................................................................................................................... 26
4.7 Localization of eNOS, iNOS, and nitrotyrosine............................................................. 26
4.8 Non-isotopic in situ hybridization (NISH) combined with immunofluorescence on
mouse lung sections.............................................................................................................. 27
4.9 Laser-assisted microdissection ....................................................................................... 27
4.10 RNA isolation, pre-amplification, cDNA synthesis and real-time polymerase chain
reaction ................................................................................................................................. 28
4.11 Western blots................................................................................................................ 29
4.12 Patient characteristics ................................................................................................... 30
4.13 Statistical analyses........................................................................................................ 30
5. RESULTS............................................................................................................................ 31
5.1 Lung emphysema development in wild-type mice exposed to tobacco smoke.............. 31
5.2 Lung functional changes in wild-type mice exposed to tobacco smoke ........................ 32
5.3 Pulmonary hypertension development in wild-type mioke... 32
5.3.1 Hemodynamics, heart ratio and number of alveoli / number of vessels ................. 32
5.3.2 Degree of musculariazation and vascular lumen area ............................................. 33
5.4 Regulation of iNOS and eNOS expression in the pulmonary vasculature of wild-type
mice after exposure to tobacco smoke ................................................................................. 35
5.4.1 Localization of iNOS and eNOS in mRNA and protein level ................................ 35
5.4.2 Expression of iNOS and eNOS on mRNA and protein level.................................. 36
5.5 iNOS but not in eNOS deficient mice are completely protected from lung emphysema
development upon tobacco smoke exposure ........................................................................ 37
5.6 iNOS but not in eNOS deficient mice are completely protected from lung functional
changes upon tobacco smoke exposure................................................................................ 39
CONTENT III
5.7 iNOS but not in eNOS deficient mice are completely protected from pulmonary
hypertension development upon tobacco smoke exposure .................................................. 39
5.7.1 Hemodynamics, heart ratio and number of alveoli / number of vessels ................. 39
5.7.2 Degree of musculariazation and vascular lumen area ..................................