Methods in Pulmonary Research

"Methods in Pulmonary Research" presents a comprehensive review of methods used to study physiology and the cell biology of the lung. The book covers the entire range of techniques from those that require cell cultures to those using in vivo experimental models. Up-to-date techniques such as intravital microscopy are presented. Yet standard methods such as classical short circuit techniques used to study tracheal transport are fully covered. This book will be extremely useful for all who work in pulmonary research, yet need a practical guide to incorporate other established methods into their research programs. Thus the book will prove to be a valuable resource for cell biologists who wish to use organs in their research programs as well biological scientists who are moving their research programs into more cell related phenomena.

Contenu

Airways.- 1 Measurement of lung function in rodents in vivo.- Spontaneous respiration.- Pulmonary manoeuvres.- Material and equipment.- Lung function laboratory.- Methods.- Preparation and calibration.- Pulmonary function testing.- Examples for applications.- Discussion.- Troubleshooting.- References.- 2 The isolated perfused lung.- Advantages and disadvantages of perfused lungs.- Theoretical background.- Vascular resistance.- Respiratory mechanics.- Material and equipment.- Artificial thorax chamber and ventilation.- Perfusion.- Weight measurement.- Gas exchange.- Methods.- Surgery and setting up the lung.- Criteria for viability.- Cleaning the apparatus.- An example application.- Discussion.- Interpretation of the results.- Constant flow (CFP) versus constant pressure perfusion (CPP).- Negative or positive pressure ventilation.- Choice of perfusate.- Recirculating versus non-recirculating perfusion.- Additional experimental options.- Troubleshooting.- Final comments.- References.- 3 Lung explants.- Material and equipment.- Preparation of culture media.- Preparation of agarose.- Preparation of animals.- Preparation of explants.- Image acquisition.- Variations on this technique.- Applications.- Effects of bronchoconstriction.- Measurements of mucociliary clearance.- Measurements of pulmonary vasculature.- Long term explant culture techniques.- Investigations of protein and gene expression.- Troubleshooting.- Discussion.- Acknowledgements.- References.- 4 Tracheal preparations.- Methods.- Guinea pig tracheal preparations.- Immersion techniques.- Tracheal chain.- Spirally cut trachea.- Zig-zag tracheal strip.- Tracheal tube preparations.- Superfusion techniques.- Electrically stimulated trachea.- Epithelium-denuded trachea.- Conclusion.- References.- Vessels.- 5 Intravital microscopy: Airway circulation.- Materials and equipment.- Microscope.- Video equipment.- Peripheral equipment.- Ventilation.- Solutions.- Methods.- Surgery.- Experimental procedure.- Species differences.- Discussion.- References.- 6 The bronchial circulation.- Importance and role of the bronchial circulation.- Postobstructive pulmonary vasculopathy (POPV) and principles of the techniques.- Material and equipment.- Production of POPV in dogs, rats and guinea pigs: Ligation of the left main pulmonary artery.- In situ perfused LLL preparation.- Morphological assessment of the bronchial and pulmonary vasculature using light microscopy and morphometry.- Methods.- Surgical ligation of the left main pulmonary artery in dogs, rats and guinea pigs.- Canine model.- Rat and guinea pig model.- In situ perfused LLL preparation to measure pulmonary and bronchial vascular flows, pressures and resistances using modified AO and VO and bronchial vascular micropuncture.- Procedure for the in situ perfused LLL preparation.- AO and VO measurements.- Modified in situ perfused LLL preparation for bronchial collateral.- vascular pressure measurements by micropuncture.- Morphological assessment of the bronchial and pulmonary vasculature, using light microscopy and morphometry.- Measurement of pulmonary vascular medial thickness and muscularization in lungs injected with pigmented gelatin-barium mixtures.- Fixation and preparation.- Morphometry.- Assessment of proliferation in the bronchial vasculature.- Bronchial vessel number per airway.- Assessment of bronchial vascular endothelial proliferation using bromodeoxyuridine (BrdU) labeling.- Discussion and troubleshooting.- Production of POPV.- In situ perfused left lower lobar preparation.- Morphological assessment of the bronchial and pulmonary vasculature.- Acknowledgements.- References.- 7 Segmental vascular resistance and compliance from vascular occlusion.- Methods.- The lumped parameter RCR model.- The continuous RC distribution.- More distributed lumped parameter models.- The 3C4R model.- The 3C2R model.- Arterial occlusion in vivo.- Acknowledgements.- References.- Edema.- 8 Experimental and clinical measurement of pulmonary edema.- Definitions.- Lung water filtration and clearance.- Lung water filtration.- Lung water clearance.- Edema formation = filtration ? clearance.- Lung protein filtration and clearance.- Protein filtration.- Protein clearance.- Protein accumulation = filtration ? clearance.- Mechanisms of pulmonary edema.- Material and methods.- Quantifying pulmonary edema formation and clearance in the experimental setting.- Lung microvascular filtration rate.- Isolated lung.- Intact lung.- Lung water clearance.- Lymph flow.- Airway fluid clearance.- Pleural fluid clearance.- Lung water = filtration ?clearance.- Lung weight (isolated lung).- Indicator dilution.- Gravimetry.- Pathology.- Starling equation components.- Microvascular pressure.- Indirect measurements.- Interstitial liquid pressure.- Plasma protein osmotic pressure.- Interstitial colloid osmotic pressure.- Filtration coefficient (Kf,c).- PS.- Sigma (?).- The filtered volume method.- Quantifying pulmonary edema formation and clearance in the clinical setting.- Lung water and edema.- Indicator dilution technique: Extravascular thermal lung volume.- Imaging techniques.- Transthoracic bioimpedance.- Solute filtration: Capillary-alveolar macro-molecule transport.- External radioflux detection.- Positron emission tomography (PET).- Magnetic resonance imaging (MRI).- Edema fluid protein and Bronchoalveolar Lavage fluid.- Starling equation components.- Microvascular pressure.- Small solute PS.- Lung water clearance.- Epithelial permeability: DTPA clearance.- Identifying pulmonary edema and its mechanism in the clinical setting.- Present.- Diagnosis and quantification of pulmonary edema.- Identification of mechanisms.- Identification of an imbalance in Starling forces.- Identification of an altered transvascular permeability.- Future.- Role of edema clearance.- Role of exchange surface area.- Macromolecule transport.- Anatomic distribution.- Conclusion.- References.- 9 Neurogenic inflammation in the airways: Measurement of microvascular leakage.- Material and equipment.- Methods.- Anaesthesia.- Surgery.- Experimental procedure.- Direct electrical stimulation of the vagus nerve.- Chemical stimulants.- Capsaicin.- Bradykinin.- Cigarette smoke.- Sodium metabisulphite.- Other stimulants.- Quantification.- Evans blue dye technique.- Monastral dyes as tracers.- [125?]-albumin.- Application.- Species differences.- Discussion.- Troubleshooting.- Difficulty in cannulating veins.- Difficulty in cannulating arteries.- Poor blood pressure trace.- Acknowledgements.- References.- 10 Intravital microscopy: Surface lung vessels and interstitial pressure.- Material and equipment.- Pipette preparation.- Methods.- Surgery.- Cannulation of left atrium and pulmonary artery.- Preparation of the intact parietal pleural window.- Video imaging analysis.- Experimental procedure.- Application.- Physiological conditions.- Transition to edema.- Lung fluid balance in the newborn.- Mechanical behavior of interstitial matrix.- Video image analysis of the superficial lung structures.- Species differences.- Discussion.- Troubleshooting.- Technical problems.- General drawback…