Methods in Pulmonary Research

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 drawbacks of the micropuncture technique.- Micropuncture through the intact parietal pleural window.- References.- 11 Lymphatics.- Basic physiology of the lymphatic system.- Methods.- Common technical problems.- Lymph flow rate measurement.- Lymph protein concentration.- Discussion.- Acknowledgements.- References.- Airway liquid.- 12 Evaluation of secretory and transport processes which determine the composition of airway surface liquid.- Methods.- Studies using isolated trachea.- The ferret isolated whole trachea in vitro preparation.- Protocol for stimulating secretions.- Assay for lysozyme.- Albumin transport.- Measurement of potential difference across the tracheal wall.- Ion transport across the airways.- Measurement of the ionic composition of periciliary fluid.- Electrophysiological methods used in the investigation of ion transport in the airways. Measurement of short-circuit current (ISC) and transepithelial resistance (RT).- Acid/base transport.- Acid/base transport across cell membranes of isolated tracheocytes.- Culture of ovine tracheal submucosal gland cells.- Preparation and characterisation.- Methods for studying the effects of secretagogues on lysozyme release from cultured ovine trachea submucosal gland cells.- Electrophysiology.- Troubleshooting.- Conclusions.- Acknowledgements.- References.- 13 Bronchoalveolar lavage.- Methods.- Endoscopic techniques of BAL in man.- Premedication.- Local anaesthesia.- Site of lavage.- Fluid used to perform lavage.- Methods to instil and recover the fluid.- Volumes of fluid to be used.- Recovery.- Should the first aliquot of lavage be processed separately?.- Handling of the harvested lavage material.- Mucus filtration.- Conventional stains.- Membrane filters.- Cytocentrifuge preparations.- Romanovsky stain.- Papanicolaou stain.- Grocott methenamine silver stain-microwave method.- Gram stain.- Iron stain.- In situDNA hybridization.- Immunocytochemical stains.- Procedure (as one example out of a variety of techniques).- Flow cytometry to quantify lymphocyte subsets.- Principle of flow cytometry.- Preparation of samples.- Analysis by immunofluorescence.- Electron microscopy.- Differential cell counts.- Cultures from BAL.- Microbial culture.- Routine culture.- Fungal culture.- Mycobacterial culture.- Viral culture.- Analysis of soluble components of the epithelial lining fluid.- Attempt to quantify lavage material.- Complications of lavage.- Preparation techniques in animals.- References.- 14 Assessment of surfactant function.- In vitro methods for assessment of surfactant function.- The Langmuir-Wilhelmy balance.- Bubbles on a tube: The pulsating bubble surfactometer according to Enhorning.- Captive bubbles.- Microbubble stability.- Adsorption.- The rate of adsorption.- Adsorption characteristics of pulmonary surfactants.- Spreading.- The measurements of adsorption and spreading.- Adsorption.- Spreading.- Measurement of surface tension in situ.- Choosing a test fluid.- Calibration.- Alveolar miropuncture and surface tension in situ.- Influence of surfactant on static lung pressure-volume characteristics.- Animal models for in vivo evaluation of exogenous surfactants.- Preterm newborn animals.- In vivo lung lavage.- Comments.- Acknowledgements.- References.- Cell culture.- 15 Isolation of type II alveolar epithelial cells.- Why do we need to isolate cells for in vitro studies?.- Strategies for isolation of type II cells.- Methods.- Steps in type II cell isolation.- Removal of blood products and alveolar macrophages.- Dissociating lung tissues with digestive enzymes.- Selecting the enzyme combinations.- Recovery of dissociated cells.- Strategies for selective isolation.- Characterization of isolated type II cells.- Morphology.- Surfactant phospholipid profile.- Immunochemical techniques.- Adhesion and culture on different substrata.- Selection of CO2 atmospheric conditions.- Selection of substrata for culture.- Mn++-enhanced technique for pneumocyte isolation.- Reagents.- Type II Cell Isolation.- Discussion.- Limitation of studies of isolated cells.- Alternatives to cell isolation.- Acknowledgments.- References.- 16 Endothelial cells.- Material and methods.- Tissue culture of pulmonary artery endothelial cells.- Equipment/media and chemicals.- Solutions/media preparation.- Macrovascular endothelial cell isolation procedure.- Tissue culture of pulmonary microvascular endothelial cells: Method 1.- Equipment/media and chemicals.- Solutions/media preparation.- Microvascular endothelial cell isolation procedure.- Endothelial cell procurement.- Tissue culture of pulmonary microvascular endothelial cells: Method 2.- Solutions/media preparation.- Microvascular endothelial cell isolation procedure, lung isolation.- Endothelial cell procurement.- Establishment of primary cultures.- Verification of endothelial cells.- Discussion.- Troubleshooting.- References.- Histology.- 17 Studying lung ultrastructure.- Material and equipment.- List of equipment.- Solutions.- Primary fixative for conventional TEM and SEM.- Primary fixative for immunocytochemistry.- Cryoprotectant for infiltration of specimens.- Postosmication for SEM/TEM.- Postfixation for TEM.- Postfixation for SEM.- Methods.- Modes of fixation.- Fixation by airway instillation.- Fixation by vascular perfusion.- Combined chemical/physical fixation for immunocytochemistry.- Fixation by immersion into a chemical fixative.- Sampling of tissue blocks.- Processing and embedding of tissue blocks.- For conventional LM.- For conventional TEM.- For TEM based immunocytochemistry.- For SEM modified according to an OTOTO method.- Sampling of micrographs.- Discussion.- Selecting the mode of fixation.- Selecting the mode of application.- Selecting the fixing agent.- Selecting the mode of tissue processing.- Stereological analysis.- Interpretation.- Acknowledgements.- References.- 18 Autoradiography in the lung.- Materials and equipment.- Methods.- Slide preparation.- Slide cleaning and subbing.- Preparation of chrome alum/gelatin solution.- Application.- Preparation of emulsion-coated coverslips.- Safelight illumination.- Emulsion coating of coverslips.- Tissue preparation.- Tracheal and bronchial tissue.- Peripheral lung tissue.- Cutting frozen sections.- Radioligand binding procedures.- Attachment of emulsion-coated coverslips.- Emulsion-dipped autoradiograms.- Film autoradiograms.- Exposure of autoradiograms.- Development and staining of autoradiograms.- Preparation for development and tissue staining.- Processing solutions.- Photographic development.- Tissue staining and mounting.- Materials.- Procedure.- Chemography controls.- Discussion.- Controls.- Choosing the appropriate autoradiographic method.- Emulsion-dipped preparations.- Emulsion-coated coverslip preparations.- Film imaging.- Microscopy.- Photography.- Image analysis.- References.- Further methods.- 19 Application of aerosols.- Methods.- Physical characterization of aerosols.- Methods of aerosol generation.- Methods of aerosol measurement.- Exposure methodology.- References.- 20 Cryopreservation of human pulmonary tissues.- Mechanisms of freezing injury.- Cryoprotective agents and cryomedia.- Freezing procedure and storage temperature.- Thawing procedure.- Methods, material and equipment.- Cryopreservation.- Freezing procedure.- Temperature and sample handling during storage.- Thawing procedure.- Inventory control.- Post-thaw functional recovery.- Conclusion.- Safety recommendations.- References.- Appendix I. Physiological data of various mammalian species.- References.- Appendix II. List of suppliers.