Hypertrophic and hyperplastic changes of mucus-secreting epithelial cells in rat airways: assessment using a novel, rapid, and simple technique.
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abstract
Determination of hyperplastic and hypertrophic changes of mucus-secreting cells in animal airways has been performed in the past by using histologic, immunologic, and/or molecular biologic approaches. Histologic techniques are tedious and time-consuming. The other approaches require specific antibodies and cDNA probes that have proved difficult to develop. Described here is a method for the rapid estimation of hyperplastic and hypertrophic changes of secretory epithelial cells in rat airways. The assay specifically measures acidic and neutral mucoproteins in a linear fashion from 0.5 microgram to at least 10 micrograms. Male Sprague-Dawley rats were exposed to metabisulfite mist (10% wt/vol) for 5 days/wk for 3 wk. The lungs were removed and homogenized in a phosphate-buffered solution containing reducing agents and protease inhibitors. The particulate matter was removed by centrifugation, and the soluble extract was applied to a column packed with Sepharose CL-6B. The material eluting in the void volume was applied to a PVDF membrane and stained for either acidic or neutral mucosubstances using Alcian blue or periodic acid-Schiff (PAS) staining, and the absorbance was read using a 96-well plate reader. Lungs from sodium metabisulfite-exposed animals showed a 7-fold and 3.5-fold increase in PAS-positive and Alcian blue-positive material, respectively. The increase in both PAS and Alcian blue staining was hyaluronidase and chondroitinase insensitive. The observed changes are consistent with morphometric measurements of mucus-containing cells in histologic sections of the tissues. This assay may be useful in determining which neurohumoral mediators might be involved in mucus cell hypertrophy and hyperplasia in animal models of chronic obstructive pulmonary disease.
