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The use of dextrans as particulate tracers in respiratory epithelial permeability studies

University of British Columbia

These experiments were designed to test the suitability of dextrans as alternate light and electron microscopic tracers to horseradish peroxidase (HRP) in studies of respiratory epithelial permeability in control and cigarette smoke-exposed guinea pigs. Nine animals were utilized initially to establish the most effective means of preserving highly water-soluble dextran while adequately maintaining airway cell ultrastructure. The perfusion-nebulization fixation and carbohydrate retention techniques developed were then used on the twenty-six guinea pigs in the permeability study. Five animals served as controls, while others were exposed to the dextran tracer, smoke from 10 cigarettes, or both. Intratracheal tracer instillations of sonicated preparations of unlabelled or either FITC- or iron-labelled dextran T10 or T40 were carried out 1/2-hour after smoke exposure. All dextrans could be visualized light-microscopically along the respiratory epithelium of airways and lung parenchyma by the Mowry-Millican technique, a modified alcoholic periodic acid-schiff (PAS) stain. As witnessed in previous HRP studies, some post-mortem tracer diffusion was seen, and no difference in dextran epithelial penetration was noted between smoked and sham-smoked animals. At the electron microscopic level, treatment of guinea pig trachea and lung with aldehyde-OsO₄-lead citrate fixative cocktail preserved the unlabelled dextran as irregular, highly electron-dense aggregates of particles along the luminal surfaces only of airways of control guinea pigs. One-half of the animals in the smoke-exposed group showed penetration of dextran between the tracheal epithelial cells. Incomplete tracer dissolution prior to instillation, as shown by negative staining of the instillates, is thought to be responsible for this inconsistency. No intrapulmonary or alveolar trans-epithelial tracer transport could be detected. FITC-dextrans presented the additional advantages of qualitative detection by fluorescence-differentiated interference contrast (DIC) microscopy and quantitative detection by spectrophotofluoro-metric means. Iron-dextrans exhibited too large a molecular size for practical use with smoke-exposed guinea pig tracheobronchial epithelium. We conclude that dextrans provide distinct advantages over HRP in terms of low price, inertness, and size versatility, as light and electron microscopic tracers.

Text

2010-03-30

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http://hdl.handle.net/2429/23141

Pathology

University of British Columbia

Graduate