J. A. St George

Regional differences in quantities of histochemically detectable mucosubstances in nasal, paranasal, and nasopharyngeal epithelium of the bonnet monkey.

Inhaled irritants induce secretory cell hyperplasia in nasal epithelium of animals. To characterize this response histochemically it is first important to know the histochemical character and distribution of epithelial mucosubstance in the normal nasal cavity. An automated image analyzing method was used to detect and quantitate acidic, neutral, and sulfated mucosubstances in the epithelium lining the nasal and paranasal airways of eight bonnet monkeys. Tissue sections 2 micron thick from defined regions of these airways were stained with either alcian blue/periodic acid-Schiff to demonstrate acid and neutral mucosubstances or high iron diamine to demonstrate sulfated mucins. Respiratory epithelium covering maxilloturbinates had the largest volume of stainable mucosubstance per unit surface area of basal lamina, whereas the maxillary sinus epithelium had the least. There was a general anteroposterior increase in the quantity of total epithelial mucosubstance along the septal and lateral walls of the nasal cavity, and there was more acidic than neutral mucosubstance in the posterior nasal airway than in the anterior. Epithelial mucosubstance in the maxillary sinus was predominantly neutral. Therefore, we conclude that there are substantial regional quantitative differences in stainable mucosubstances in the primate nasal epithelium which must be considered when examining nasal mucosa for irritant-induced changes in epithelial mucins.

Carbohydrate cytochemistry of tracheobronchial airway epithelium of the rabbit.

Three types of nonciliated secretory epithelial cells contribute to the mucous lining of pulmonary airways: mucous cells, serous cells, and Clara cells. Contrary to observations in other species, airways of the rabbit have very few mucous cells. In the rabbit, the predominant secretory cell throughout the entire airway tree, including the trachea, appears to be one cell type, the Clara cell. While these cells share the same ultrastructural features throughout the tree, the nature of their contribution to the mucous blanket is not clear. This study was designed to characterize the carbohydrate components of secretory granules in tracheal Clara cells, and to compare that carbohydrate with that of tracheal mucous (goblet) cells and with Clara cells of more distal airway generations. Trachea and lungs of six adult male rabbits were fixed by airway infusion, the conducting airways of the right cranial lobe dissected and tissue selected from the trachea and five distal airway generations. For light microscopy (LM), sections of paraffin-embedded tissues were stained with Alcian blue-periodic acid-Schiff (AB/PAS), dialyzed iron (DI), and high iron diamine-Alcian blue (HID-AB). For electron microscopy (EM), fixed tissues were incubated with DI, HID, MgCl2, or buffer, postosmicated, embedded in epoxy resin, and thin sections stained with periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP). By LM, most Clara cells did not react with PAS, AB, HID, or DI. A few in trachea and bronchi had PAS-positive apical margins. Mucous goblet cells were positive with PAS, AB, and HID, indicating sulfated glycoproteins. By EM, a small number of Clara cells had PA-TCH-SP-positive luminal granules, a few luminal granules had DI-positive rims. Almost all Clara cell granules were negative with PA-TCH-SP, HID, and DI. The granules of mucous goblet cells had a finely granular core surrounded by a meshwork of variable density. The meshwork was positive with PA-TCH-SP, DI, and HID. The cores were not. We concluded that: 1) the Clara cell does not contribute carbohydrates to the airway mucous lining; 2) mucous goblet cells secrete predominantly sulfated glycoprotein; and 3) the contribution to mucous carbohydrates by Clara cells does not vary with the airway level in which they are located.

Quantitative histochemistry of mucosubstance in tracheal epithelium of the macaque monkey.

Experimentally applied irritants and chronic respiratory diseases appear to alter the amount and composition of secretory cell product in surface epithelium and submucosal glands of pulmonary airways. Previous methods used to quantify these changes have been very time-consuming or have not measured the same components of the airway wall. The present study describes a rapid, reproducible, and standardized automated method for quantifying secretory products. The tracheas from eight macaque monkeys were fixed with glutaraldehyde-paraformaldehyde, embedded in glycol methacrylate, serially sectioned at 2 microns, and histochemically stained to demonstrate neutral, sialylated, and sulfated mucosubstances in the cartilaginous, intercartilaginous, and membranous regions of both proximal and distal trachea. Volume densities were determined using an image analyzer and are expressed as volume of stained mucosubstance per unit surface area of epithelial basal lamina. Comparison of the automated method to manual point counting and evaluation of internal variance showed that the automated method had a twelve-fold increase in efficiency with no significant differences in measurements. After weighting the values of each region according to their anatomical contribution, the total secretory product (TSP) for the entire trachea was determined. Periodate-reactive acid material predominated (73%) in luminal surface epithelium, and neutral material predominated (78%) in submucosal glands. Surface epithelium contained 66% of the TSP. The greater contribution by surface epithelium and predominance of acid mucins there resulted in a TSP from the trachea that consisted of 59% acid material (most of which was sulfated) and 41% neutral material. The method proved to be a valid, reproducible, and rapid technique for evaluating variability in abundance of mucosubstances within airway epithelium.

Ozone-induced structural changes in monkey respiratory system

Abstract The principal sites of ozone-induced damage in the respiratory tracts of monkeys are the anterior nasal cavity and respiratory bronchioles. Intermittent exposures (8 h/day) for 6 or 90 days to 0.15 or 0.30 ppm ozone resulted in ciliated cell necrosis, shortened cilia, and secretory cell hyperplasia with less stored glycoconjugates in the nasal region. Respiratory bronchiolitis was also observed in these monkeys at 6 days and persisted to 90 days of exposure. Even at the lower concentration of 0.15 ppm O s nonciliated bronchiolar cells appeared hypertrophied and increased in abundance in respiratory bronchioles. The response of respiratory bronchioles to intermittent (8 h/day), long-term ozone exposure of 0.4 or 0.64 ppm included the following morphometric changes: 1) a thicker wall and narrower lumen, 2) thicker epithelial compartment and a much thicker interstitial compartment, 3) shifts in epithelial cell populations with many more nonciliated bronchiolar epithelial cells and fewer squamous type I epithelial cells, 4) larger nonciliated bronchiolar epithelial cells with a larger compliment of cellular organelles associated with protein synthesis, 5) greater volumes of interstitial fibers and amorphous ground substance and 6) greater numbers of interstitial smooth muscle cells, neutrophils, macrophages and mast cells per surface area of epithelial basal lamina. Following cessation of exposure there was persistence of some degree of the hyperplastic and metaplastic epithelial changes, but worsening of interstitial fibrosis. The principal lesion in response to intermittent (8 h/day) O s exposure of 0.25 ppm daily or to cyclical exposures (9 cycles of 1 month of O s followed by 1 month of filtered air) for 18 months was respiratory bronchiolitis. Cyclically exposed monkeys, but not those exposed daily, had significantly increased total lung collagen content, chest wall compliance and inspiratory capacity. The conclusions of this study are: 1) there is persistent epithelial injury in the anterior nasal cavity and respiratory bronchiole by as low as 0.15 ppm 0 3 , 2) there is worsening of the respiratory bronchiole lesion in monkeys in the post-exposure period and 3) cyclical exposures cause more severe injury than continued daily exposures.