............................................................................................................................................... i Acknowledgements ....................................................................................................................... iii Table of contents ............................................................................................................................. v List of figures ................................................................................................................................ viii List of tables ..................................................................................................................................... xi List of abbreviations .................................................................................................................... xii Introduction ...................................................................................................................................... 1 1. The pulmonary circulation ........................................................................................................................ 2 2. Modulation of pulmonary vascular tone ............................................................................................. 3 2.1. External modulation by the sympathetic nervous system ................................................................. 4 2.2. Intrinsic modulation of the pulmonary vasculature .............................................................................. 6 3. Hypoxic pulmonary vasoconstriction ................................................................................................... 7 3.1. The role of pulmonary vascular smooth muscle ..................................................................................... 7 3.2. The role of pulmonary vascular endothelium ....................................................................................... 10 3.3. The role of the sympathetic nervous system ......................................................................................... 12 3.3.1. Regulation during acute hypoxia ...................................................................................................... 12 3.3.2. Regulation during chronic hypoxia .................................................................................................. 13 4. Pulmonary Hypertension ......................................................................................................................... 14 4.1. Epidemiology ....................................................................................................................................................... 15 4.2. Animal models for pulmonary hypertension. ........................................................................................ 16 4.2.1. Chronic hypoxia induced pulmonary hypertension ................................................................. 16 5. Obesity ............................................................................................................................................................. 18 5.1. Epidemiology of the obesity paradox ........................................................................................................ 18 5.2. Increased sympathetic nerve activity in obesity .................................................................................. 19 6. Synchrotron radiation microangiography ........................................................................................ 21 6.1. Uses of synchrotron radiation microangiography .............................................................................. 22 7. Summary ......................................................................................................................................................... 24 Objectives of this study .............................................................................................................. 25 1. Hypothesis ...................................................................................................................................................... 25 2. Aims ................................................................................................................................................................... 25 Methods ........................................................................................................................................... 26 1. Animals ............................................................................................................................................................ 26 vi 1.1. Inducing pulmonary hypertension ............................................................................................................. 26 1.2. Animals used in different protocols ........................................................................................................... 28 2. General surgical techniques .................................................................................................................... 29 2.1. Anaesthesia and surgical preparation ...................................................................................................... 29 2.2. Tracheotomy ........................................................................................................................................................ 29 2.3. Femoral artery and vein catheterisation ................................................................................................. 30 3. In vivo electrophysiology surgical preparation .............................................................................. 32 3.1. Left thoracotomy ................................................................................................................................................ 32 3.1.1. Isolating the stellate ganglion and pulmonary sympathetic nerve .................................... 33 3.2. In vivo electrophysiology experimental protocol ................................................................................ 35 3.3. In vivo electrophysiology data collection and analysis. ........................................................... 36 4. Microangiography ....................................................................................................................................... 38 4.1. Surgical preparation ......................................................................................................................................... 38 4.1.1. Right jugular vein catheterisation .................................................................................................... 38 4.2. Principles of synchrotron radiation microangiography ................................................................... 41 4.3. Positioning of the experimental rat ........................................................................................................... 41 4.4. Microangiography experimental protocol .............................................................................................. 44 4.4.1. Priming of the jugular catheter .......................................................................................................... 44 4.4.2. Baseline imaging ...................................................................................................................................... 44 4.4.3. Imaging during the acute hypoxia challenge ............................................................................... 45 4.4.4. Imaging after β1-‐adrenoceptor blockade ...................................................................................... 45 4.4.5. Imaging after β1-‐ and β2-‐adrenoceptor blockade ..................................................................... 46 4.4.6. Terminating the protocol ..................................................................................................................... 46 4.5. Microangiography data acquisition. .......................................................................................................... 48 4.5.1. Heamodynamic data acquisition ....................................................................................................... 48 4.5.2. Image data acquisition ........................................................................................................................... 48 4.6. Microangiography data analysis ................................................................................................................. 49 4.6.1. Microangiography morphological analysis .................................................................................. 49 4.6.2. Microangiography haemodynamic data analysis ....................................................................... 49 4.6.3. Microangiography image enhancement ......................................................................................... 49 4.6.4. Microangiography image analysis .................................................................................................... 52 5. Equipment calibration .............................................................................................................................. 55 5.1. Blood pressure calibration ............................................................................................................................ 55 5.2. Nerve activity calibration ............................................................................................................................... 55 5.3. Microangiography scale calibration .......................................................................................................... 55 5.3.1. Reproducibility of microangiography vessel measurements ............................................... 55 6. Western blotting ...........................