Studies of human airways in vitro: A review of the methodology

textabstractThe pathophysiology of human airway narrowing is only partly understood. In order to gain more insight in the mechanisms of human lung diseases and potential beneficial therapeutic agents, adequate models are needed. Animal airway models are of limited value since lung diseases such as asthma and chronic obstructive pulmonary disease (COPD) are unique to humans and because the mechanisms of airway narrowing differ between species. Therefore, it is important to perform studies on human isolated airways. We describe the models that have been developed to study airway function in vitro, emphasizing human airway preparations. The easily prepared airway strip and ring preparations are described first. The potential damage during preparation and the interference with airway structure are important drawbacks in these preparations. Lung parenchymal strips, described next, were designed in order to study responsiveness of small airways. However, parenchymal strips are anatomically complex, and responsiveness is determined by the relative amounts of airway and vascular smooth muscle. The lack of reproducibility between species and even within one animal limits their usefulness. Airway tube preparations, in which luminal and serosal stimulation can be separated, enable us to study the modulatory role of the airways epithelium in vitro. Furthermore, airway compliance can be measured. In the isolated perfused lung preparation, relationships between the airways and the vascular system are preserved and the interaction between these two systems can be studied. Weight gain due to fluid extravasation is a problem in this model which has not been used yet to study human lungs in vitro. Next, methodological aspects such as tissue handling and storage, recording of responses, removal of the epithelium, and electrical field stimulation are discussed in some detail. Although animal airways tissue can be studied immediately after removal, human tissue is often obtained with some delay. However, this seems tenable since electron microscopy of lung tissue obtained at autopsy showed that recovery of the preparation occurs during incubation of carbogenated Krebs-Henseleit (K-H) buffer. Dissected airways can be stored overnight in cooled K-H buffer until up to 55 hr after resection without losing viability. Commonly used physiological salt solutions which bath the tissue contain osmotic molecules, ions important for contractility, glucose as a substrate, and a bicarbonate-carbon dioxide buffer. In studies of isolated perfused lungs, a colloid should be added in order to prevent edema. The responses of isolated airways strips and rings are recorded under isometric or isotonic conditions. Smooth muscle contraction in vivo, however, is auxotonic; the elastic load on the smooth muscle increases during airway narrowing. In perfused airway tubes responsiveness is measured under auxotonic conditions as a change in perfusion pressure or flow. Next, removal of epithelium from isolated airways is discussed. Although mechanical denudation is widely used, more physiological methods that mimic the epithelial damage found in asthma may well be preferable and these methods are described in some detail. Finally, the methodology of electric field stimulation (EPS) is described. EFS is delivered via electrodes suspended in the organ bath. According to the stimulus parameters chosen, autonomic nerves or smooth muscle cells are stimulated. An important side effect of EFS is the generation of oxygen radicals in carbogenated K-H buffer which may alter airway tone directly, or oxidize agonists added to the organ bath. It is concluded that although our knowledge of the pathophysiology of airway disease is rapidly increasing, the role of the bronchial circulation is poorly understood. Therefore, the development of a method to study the interaction between the ventilatory and the vascular systems in the isolated human lung is a major challenge