Correspondence to Michael I. Kotlikoff: mik7{at}cornell.edu
The regulation of ventilation and pulmonary blood flow is a function of the diameter of the supply conduits and thus the degree of contraction of the smooth muscle lining these tubes, as determined by bronchomotor and vasomotor nerves, circulating hormones, and local factors such as gas composition. The business end of both systems is the small conduits just upstream of the exchange units, which regulate blood and gas distribution through "small" changes in diameter. How this job is performed (which factors predominate in specific regulatory contexts, what roles do the epithelium and endothelium play in the process, and what goes wrong in, for example, asthma and pulmonary hypertension) is difficult to determine, due in no small measure to the lack of preparations in which these dynamic interactions can be observed. The millions of air/water interfaces within the lung present an overwhelming obstacle to most imaging modalities. Consequently, much of our knowledge of bronchial/pulmonary vascular control derives from isolated airways or vessels, devoid of their surrounding tissues and neural input; much less is known about the critically important small, regulatory bronchioles and arterioles. Moreover, until recently, the application of sophisticated, high speed imaging methods has been largely confined to single cells obtained from large airways or vessels.
Multicellular preparations that preserve dynamic interactions between cells of different lineages have markedly advanced the understanding of complex biological processes; studies in brain slices, for example, have been of great value in illuminating neural processing mechanisms, nerve/glia interactions, and dendritic branching processes, among others. The usefulness of these preparations also has been expanded by the combination of deep fluorescent imaging by multiphoton microscopy and the use of genetic markers that identify specific lineages or even report cell signaling events (Mainen et al., 1999; Feng et al., 2000
; Ji et al., 2004
). In this issue of the Journal of General Physiology (Perez and Sanderson, 2005a
,b
), the laboratory of Michael Sanderson reports a marked improvement in the preparation of lung slices and their use in examining airway and vascular biology in two articles. The importance of this work is that the early lung slice preparations were relatively thick preparations, which were not suitable for high resolution optical imaging (Dandurand et al., 1993
), whereas the previous lung slice preparation developed by the Sanderson lab did not preserve vascular spaces (Bergner and Sanderson, 2002
). Sanderson and colleagues now show how it is possible, by separately perfusing the pulmonary arterial system with gelatin and the airspaces with agarose (followed by cooling the lungs to solidify these materials), to cut 100-µm-thick lung slices that are ideal for optical imaging (Fig. 1). The choice of "filler materials" is critical for their success, as the gelatin dissolves when the lung slice is warmed to room temperature, thereby maintaining vascular integrity, while the agarose remains in the alveolar spaces, thereby preserving lung volume and the parenchymal forces that are critical determinants of airway caliber in vivo. This new preparation therefore is ideal for the study of the relationship between small airways and pulmonary arterioles and the slices reveal a novel view of the dynamic regulation of bronchiolar and arteriolar diameter; cilia beat, vessels twitch, and airways contract in a complex and captivating interplay, and distinct regulatory mechanisms underlying bronchiolar and arteriolar smooth muscle become apparent.
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As important as these results are, what is perhaps most important (for the future) is the development of a preparation in which the physiological relationships between individual lineages and structures in the lung are preserved in all their complexity, as well as the resolution and quality of the data obtained. One can easily imagine how, by careful orientation of sectioning and perhaps the use of slightly thicker sections, key neural elements, such as parasympathetic preganglionic or postganglionic sympathetic nerves, can be preserved within the slice. Genetic tagging of these and other cell lineages with GFP-based reporter transgenes would further facilitate such studies. Moreover, the preparation will be highly valuable also for many studies of complex lung biology in which the full panoply of tools provided by mouse genetics are exploited. Thus one can envision a host of experiments in which specific hypotheses about dynamic regulatory mechanisms are examined through defined manipulations of the mouse genome, and detailed analysis of their effects in dynamic studies within the lung slice. The preparation may also be useful for studies of lung immunology, as tagged immunocytes can be injected and dynamically traced through vascular, interstitial, and airway compartments, using deep fluorescence imaging. Studies of critical airway epithelial cell functions such as coordinated cilia beating and mucous secretion similarly should be facilitated. Thus we can look forward to many laboratories, in addition to the Sanderson laboratory, exploiting this preparation to make important mechanistic advances in our understanding of airway and vascular physiology.
REFERENCES
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