Measurement of airway mucosal perfusion and water volume with an inert soluble gas.

The purpose of this study was to develop and validate a new in vivo technique for the measurement of tracheal mucosal blood flow (Qtr) and tissue water volume (VH2O) with an inert soluble gas. The technique was based on the notion that the uptake of dimethyl ether (VDME) from an isolated tracheal segment is governed by VH2O (transient state) and Qtr (steady state). In lightly anesthetized sheep, an endotracheal tube with two cuffs placed 14.5-16.5 cm apart was placed to create a chamber into which dimethyl ether was introduced and from which VDMME into the mucosa was determined with a sensitive pneumotachograph. Mean Qtr was 1.20 ml/min (range 0.87-1.73), and mean VH2O was 1.67 ml (range 1.27-2.26). Qtr correlated with cardiac output but not with body weight or tracheal mucosal surface area determined by He dilution. VH2O did not show a correlation with any of these parameters. The response to selected pharmacological agents suggested that the measurements of Qtr and VH2O are independent of each other and from changes in tracheal diameter. Mean Qtr was 80% of mean tracheal mucosal blood flow measured with radiolabeled microspheres. We concluded that the inert soluble gas method is capable of measuring in vivo the perfusion and a water compartment of the intact tracheal mucosa.

Effect of ozone on the postnatal development of lamb mucociliary apparatus.

We determined whether exposure to O3 early in the postnatal period impairs the normal development of the mucociliary apparatus in lambs and whether such changes lead to prolonged abnormalities in mucociliary function. Lambs were exposed to air (controls) or to 1 ppm O3 for 4 h/day for 5 days during the 1st wk of life. Tracheal mucus velocity (TMV), a marker of lung mucociliary clearance, was measured in vivo at birth (0 wk) and up to 24 wk later, and tracheal secretory function was measured (in vitro) and the morphology of the tracheal mucosa was determined at 0 and 2 wk in both groups. In the control group, TMV increased 94% from 0 to 2 wk (P less than 0.05), continued to increase until reaching a plateau at 8 wk, and then remained constant from 8 to 24 wk. In contrast, O3-exposed lambs showed a 24% decrease in TMV from 0 to 2 wk (P less than 0.05 vs. control), and throughout the remaining time TMV remained below (P less than 0.05) that observed in control lambs. O3 exposure partially prevented the age-dependent decrease in basal secretion of tracheal macromolecules normally observed between 0 and 2 wk. These changes in secretory function were associated with a significant increase in tissue conductance (37%, P less than 0.05 vs. 0 wk), predominantly the result of active chloride secretion. The functional changes induced by O3 were associated with a retardation of the normal morphological development of the tracheal epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Mucociliary interaction in vitro: effects of physiological and inflammatory stimuli.

Mucociliary transport in the airways is governed by the interaction between ciliary activity and the depth and rheological properties of the liquids (mucus) covering the epithelial surface. A change in one of these parameters may not predict the direction and magnitude of a concomitant change in mucociliary transport. We therefore determined the effects of physiological (neurotransmitters) and pathological (inflammatory mediators) stimuli on ciliary beat frequency (CBF), surface liquid velocity (SLV), surface liquid depth (SLD), and viscoelasticity of mucus in pieces of sheep trachea (n = 5 for each treatment) mounted in a chamber such that the submucosal side was bathed with Krebs-Henseleit perfusate (KH) and the luminal side was exposed to conditioned air. SLV, SLD, and CBF were measured with a microscope provided with an electronic micrometer and strobe light. Apparent viscosity and shear elastic modulus were measured with a microcapillary method using mucus collected at the downstream end of the preparation. Control CBF, SLV, and SLD were 11.6 +/- 0.4 (SE) Hz, 91 +/- 8 micron/s, and 33 +/- 5 microns, respectively, at base line and did not change during KH perfusion for 100 min. Perfusion with both acetylcholine and epinephrine (10(-5) to 10(-3) M) produced concentration-dependent increases in mean CBF (maximum increases at 10(-3) M of 16 and 9%, P less than 0.05), whereas only acetylcholine increased mean SLV (+56% at 10(-3) M, P less than 0.05). Perfusion with platelet-activating factor (10(-7) to 10(-5) M) decreased both mean CBF and SLV in a dose-dependent fashion (-6 and -63% at 10(-5) M, P less than 0.05), whereas antigen perfusion (1:60 dilution) increased mean CBF (+10%, P less than 0.05) but decreased SLV (-47%, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental changes in the tracheal mucociliary system in neonatal sheep.

We studied the postnatal development of the tracheal epithelium and mucociliary system in neonatal sheep. Secretion of macromolecules (radiolabeled with 35SO4 and [3H]-threonine), unidirectional fluxes of Cl-, Na+, and water (measured with radioactive tracers), and ciliary beat frequency (CBF) were measured in tracheal tissues in vitro. Tracheal mucus transport velocity (TMV) was measured in vivo. Sheep were studied at 0, 2, 4, 8, and greater than 24 (adult) wk after birth. In newborn sheep trachea, secretion of macromolecules was significantly elevated (cf. adults), and there was basal net secretion of Cl- under short-circuit and open-circuit conditions. This induced open-circuit secretion of Na+. Secretion of macromolecules decreased rapidly by 2 wk (by 40-50%) and was not different from adult values by 4 wk. Active Na+ absorption developed rapidly, and from 2 wk onward it predominated under open-circuit conditions, inducing net Cl- absorption. These changes in secretory function were associated with an age-related increase in TMV, whereas inherent tracheal CBF was unchanged. In sheep, therefore, the newborn's trachea has elevated secretion of macromolecules and secretes Cl- and liquid under basal conditions. Normal secretory function (a reduction in secretion of macromolecules coupled with net absorption of ions and presumably of liquid also) approaches adult function by 2-4 wk of age.

Ultrastructural morphometric characterization of alveolar type II cells of perinatal sheep: a comparison with adults.

The quantitative morphologic changes in alveolar type II cells during the perinatal period were characterized morphometrically in the lungs of fetal lambs at 132, 138, and 147 days gestational age (DGA) and in newborns at 2 days postnatal age (2 DPN). Ultrastructural features were compared with those of type II cells of ewes 365 days old. Lamellar body profile number per type II cell profile was highest at term (147 DGA) and 2 DPN. In adults, the number of lamellar body profiles and volume density of lamellar bodies were equal to those of the 132 DGA fetus. Multivesicular bodies were most common at 138 DGA and in adults. The volume density of cytoplasmic glycogen fell dramatically during the latter part of gestation. The volume density of many cellular organelles increased to the level observed in adults by term (147 DGA). Subcellular composition of type II cells of adult sheep differs from that reported for adult rats chiefly by the volume density of lamellar material within the cytoplasm. Plate-like or globe-like inclusions were present only in the type II cells of adults. Cytoplasmic extensions of the type II cell crossing the basal lamina were most abundant in the 132 and 138 DGA fetal sheep. Cytoplasmic extensions were rare in adults. We conclude that morphologic changes of the alveolar type II cell associated with gestational age follow a species-specific time course. In the sheep, this occurs during the later part of gestation and extends into the neonatal period. Morphologic and morphometric changes appear to correspond with cellular interactions between alveolar type II cells and mesenchymal cells of the interstitium.

Tracheobronchial epithelium of the sheep: I. Quantitative light-microscopic study of epithelial cell abundance, and distribution.

Glutaraldehyde-infused tracheas and airways of five castrated sheep were microdissected following the axial airway of the left cranial and caudal lobes. Airway branches were assigned binary numbers indicating their specific location in the tracheobronchial tree. Samples of known airway generation were resin embedded and examined by light-microscopy. Based on differences in cell morphology, staining properties, and distribution, eight major cell groups were recognized and quantified: four mucous cell categories (M1, M2, M3, and M4), ciliated, basal, Clara, and serous cells. The last cell category was restricted to submucosal glands. Tracheal epithelium had the most cells per unit length, primarily due to large numbers of basal cells. Basal cells are found in the epithelium of airways without cartilage or glands. The total mucous cell population (M1, M2, and M3) in proximal airways was relatively constant. M4 mucous cells were present in glands of proximal airways and in the epithelial lining of the airways without glands. The most distal airways were lined by Clara and ciliated cells. A small number of the most proximal noncartilaginous airways had mucous (M1, M2, M3, and M4), basal, and Clara cells sharing the epithelial lining. We conclude that in the sheep lung: (1) epithelial cell distribution does not correlate with airway wall components; (2) more than one type of secretory epithelial cell can share the lining of the same airway; and (3) Clara cell distribution is based on airway generation and proximity to alveoli.

The pleura: a combined light microscopic, scanning, and transmission electron microscopic study in the sheep. I. Normal pleura.

The structural features of the ovine pleura are described using light, scanning, and transmission electron microscopy. Extensive sampling of the visceral and parietal pleura revealed considerable variation in both surface morphology and pleural interstitial anatomy. Variations of mesothelial surface were encountered and these were due to microvillar density and length. The pleural interstitium varied considerably in thickness, being relatively sparse in the anterior lung lobes, where respiratory excursion is least, and much more robust in the caudal lung lobes, where respiratory excursion is greatest. A similar correlation was observed with regard to the abundance and extent of the elastic meshwork embedded in the interstitium. The parietal pleura had openings which connected directly with the lymphatics in the underlying interstitium, thus forming a direct channel between the pleural cavity and the lymphatic system.

Tracheobronchial epithelium of the sheep: II. Ultrastructural and morphometric analysis of the epithelial secretory cell types.

In a light microscopic study we have described the morphology and distribution of six distinct, granule-containing cells in the tracheobronchial epithelium of sheep lung. We designed the present study to determine qualitatively and quantitatively whether these six cell types differ in ultrastructural morphology. Cell height varied from 30.6 micron for mucous cell M1 to 9.6 micron for Clara cells. Cell width varied from 21.2 micron for M1 to 9.3 micron for Clara cells. Nuclear dimensions ranged from 7.5 micron in M3 to 4.0 micron in M1 and M2. Mucous cell M1 had electron-dense granules (1.5 micron in diameter); M2, electron-lucent granules (1.6 micron); M3, nucleated electron-lucent granules (0.51 micron); M4, cored granules (1.1 micron); serous (SC) and Clara cells (CC), electron-opaque granules (0.58 micron and 0.37 micron). The volume fraction of the cell occupied by granules was 63% in M1 and M2, M4 39%, SC 23%, CC 5%, and M3 4.5%. Smooth endoplasmic reticulum was observed only in M3 (33.8%) and CC (49%). Granular endoplasmic reticulum (GER) was most abundant in SC (21%) and least plentiful in M4 (2.2%). We conclude that mucous cells M3 and M4 and serous and Clara cells differ from each other and from M1 and M2 cells. Mucous cells M1 and M2 differ from each other only in amount of GER and secretory granule appearance.

Morphometry of the subepithelial circulation in sheep airways. Effect of vascular congestion.

In order to quantitate the subepithelial microvascular volume and its relation to the airway lumen, we conducted a morphometric analysis of the vascular compartment in the wall of the trachea (within a 55-microns depth from the epithelial basement membrane) and of 1.0 and 0.5-mm bronchioles of sheep. The lungs were fixed by bronchial and pulmonary artery perfusion with glutaraldehyde under three experimental conditions: (1) bronchial artery pressure, 100 mm Hg pulmonary artery pressure, 20 mm Hg (control); (2) bronchial artery pressure, 100 mm Hg, pulmonary artery pressure, 40 mm Hg (pulmonary hypertension, PH); (3) bronchial artery pressure, 100 mm Hg, pulmonary artery pressure, 40 mm Hg (pharmacologic vasodilation with sodium nitroprusside, PH + V). Venous pressures were atmospheric. Under control conditions, the microvascular volume fraction comprised 12, 16, and 15% of the subepithelial tissue in the trachea and 1-mm and 0.5-mm bronchioles, respectively. PH increased the microvascular volume fraction in the bronchioles (p less than 0.05), but it had no effect on the microvasculature in the trachea. PH + V approximately doubled the microvascular volume fraction in the trachea and the bronchioles. PH increased the mean wall thickness, and PH and PH + V decreased the airway cross-sectional area in the 1-mm bronchioles. These observations demonstrate that the microvasculature constitutes a considerable volume fraction of the subepithelial airway tissue and that vascular congestion can narrow the bronchiolar lumen.

The pleura: a combined light microscopic and scanning and transmission electron microscopic study in the sheep. II. Response to injury.

The effects of acid-induced injury to the full thickness of the ovine pleura were studied using light, scanning, and transmission electron microscopy. Thirty minutes after injury, the mesothelial layer had disappeared and necrosis extended down to the subjacent alveolar walls. This was followed by an exudative phase, with edema of the pleural interstitium and subjacent alveoli, together with a marked congestion of alveolar capillaries that peaked at 2 days. Subsequently, the edema and congestion subsided and proliferation of fibroblasts and histiocytes was seen in the pleural interstitium. The subjacent alveoli were considerably distorted by interstitial thickening and alveolar type II cell proliferation. Unlike other studies in the peritoneum, no evidence for a diffuse recolonization of the mesothelial surface was found. Our work indicates that the damaged surface is rapidly covered by a layer of fibrin. Cells from the wound edges became plump and their processes projected toward the denuded region, indicating a process of centripetal healing, with migration of cells in irregular wedges to recolonize the surface.

Structure as revealed by airway dissection. A comparison of mammalian lungs.

Microdissection of mammalian pulmonary airways demonstrates branching patterns and provides precisely defined tissue samples for morphologic study. The dissections are performed on lung fixed by airway infusion at standard pressures. Using fine scissors and a high resolution dual-viewing dissecting microscope, extrapulmonary and intrapulmonary airways are dissected down their axial pathways. The plane of dissection is chosen to include as many minor daughter (side) branches as possible. Lungs from 5 species: sheep, goat, cat, rabbit, and bonnet monkey have been dissected, photographed, successive generations numbered, and pieces of tissue processed for LM, TEM, and SEM. Branching patterns differ between lobes (cranial versus caudal) of the same species and between the same lobe in different species. Marked differences in epithelial population distribution within the airway tree are found between the same lobe of different species (i.e., cranial lobes of rabbit and sheep) and between different lobes in the same species (i.e., cranial and caudal lobes of the sheep). The dissection approach to pulmonary airway morphologic studies provides specimens of precisely defined branching history, generation number, and anatomic position within regions of the lung and within specific segments. This allows studies that compare: (1) different airway generations in the same pathway, (2) bifurcation points and the airway segments between them, (3) terminal airways of differing pathway lengths and numbers of branching, (4) terminal airways of different regions of same lobe, (5) same airway generations in different lobes, and (6) same airway generations from animal to animal and species to species.

Ultrastructure of the nonciliated bronchiolar epithelial (Clara) cell of mammalian lung: II. A comparison of horse, steer, sheep, dog, and cat.

Two morphologic characteristics have been used to define the nonciliated bronchiolar epithelial cell: abundant agranular endoplasmic reticulum (AER) and membrane-bound avoid granules. To assess the ultrastructural homogeneity of this cell type in the lungs of large domestic mammals used as experimental models in pulmonary research, we evaluated lungs of horse, steer, sheep, dog, and cat. Bronchioles of known anatomic location were examined by electron microscopy following fixation by airway infusion at standard pressure and processing by selective embedding techniques. Nonciliated bronchiolar epithelium of the horse and sheep had numerous avoid granules (averaging above 15 per cell) and abundant AER. Granules were scarce (averaging less than 2 per cell) in steer and dog and absent in cat. AER was minimal in these species compared to horse and sheep. Glycogen was the dominant cytoplasmic feature in steer, dog, and cat, variable in sheep and rare in horse. Large mitochondria with few cristae and densely staining matrix were present only in cat. We concluded that nonciliated bronchiolar cells of horse and sheep were similar in essential features to this cell type in laboratory mammals, having granules and AER in abundance, while those of steer, dog, and cat were not.

Ultrastructure of the nonciliated bronchiolar epithelial (Clara) cell of mammalian lung: I. A comparison of rabbit, guinea pig, rat, hamster, and mouse.

The morphologic characteristics have been used to define the nonciliated bronchiolar epithelial: (1) abundance of agranular endoplasmic reticulum (AER) and (2) numerous membrane-bound ovoid granules. To quantitatively and qualitatively assess the ultrastructural homogeneity of this lung cell type among laboratory mammals used in lung research, we examined tissue from adult male rabbits, guinea pigs, rats, hamsters and mice. Following fixation by airway infusion at constant pressure (30 cm H2O), lungs were processed by a selective embedding technique and bronchioles of known anatomic location were examined by electron microscopy. Nonciliated bronchiolar epithelium of all five species contained avoid granules and abundant AER. Granules were most abundant in the rat (11.1 +/- 8.8 per cell) and least in the hamster (4.4 +/- 5.2 per cell). Granules were largest in hamster (0.72 +/- 0.25 micrometer) versus 0.44 micrometer or less in the other species. Granules were of uniform electron density except in the guinea pig. AER spherical mitochondria with few cristae were present only in mice, rabbit, and guinea pig. We concluded that there was considerable interspecies variation in abundance, size, and morphology of the granules, in abundance and distribution of AER; and in mitochondrial morphology.

Ultrastructure of the nonciliated bronchiolar epithelial (Clara) cell of mammalian lung. III. A study of man with comparison of 15 mammalian species.

Two morphologic characteristics have been used to define the nonciliated bronchiolar epithelial cell: (1) abundance of agranular endoplasmic reticulum (AER) and (2) numerous membrane-bound ovoid granules. In this study, we examined lobectomy specimens from three nonsmoking humans: one male (9.5 yr) and two females (62 and 43 yr) for comparison with lung specimens from mammalian species used as experimental models in lung research. Following fixation by airway infusion at constant pressure (20 cm), lung tissue was processed by a selective embedding technique and bronchioles of known anatomic location were studied by electron microscopy. Nonciliated bronchiolar epithelial cells of man contained numerous membrane-bound granules (averaging 6 per cell) in the apical cytoplasm and abundant granular endoplasmic reticulum (GER), AER was not observed. Granules averaged 0.3 micrometer in diameter and contained a fine granular matrix and parallel tubular arrays. After comparing these features in man with those of fifteen other species, we concluded that there is a great deal of interspecies variability in the cellular morphology of the nonciliated epithelial cell of distal conducting airways in mammalian lung. Three categories of this cell were observed: (1) with abundant granules and AER, characteristic of rabbit, guinea pig, rat, hamster, mouse, horse, sheep, and pig; (2) with abundant granules of GER, but no AER, characteristic of man and other primates; and (3) with few granules, little AER, and abundant glycogen, characteristic of steer, dog, cat, and ferret.

Characteristics of bovine lung as observed by scanning electron microscopy.

The surface characteristics of the intrapulmonary airways and alveoli of the lungs from 12 young adult cattle examined by the scanning electron microscope are described. When compared with similar studies of the lungs of various other mammalian species, the bovine lung generally resembles that of other mammals except for several important features: (1) Alveolar pores of Kohn are small and extremely rare; (2) Alveolar macrophages are seldom seen in alveolar air spaces; (3) Interlobular septa completely separate the lung into distinct lobules.

Effect of antigen on the glycoconjugate profile of tracheal secretions and the epithelial glycocalyx in allergic sheep.

To characterize the glycoconjugate composition of tracheal secretions and the apical glycocalyx of the tracheal epithelium under baseline conditions and after antigen challenge, sheep allergic to Ascaris suum were intubated with a double-balloon nasotracheal tube to create a tracheal chamber. After an initial tracheal lavage, the animals were either exposed to intratracheally nebulized phosphate-buffered saline (PBS) (3 ml, n = 6) or A. suum antigen (251,000 protein nitrogen units in 3 ml of PBS, n = 6). Tracheal lavage was repeated 2 hours later, and the animals were killed. An enzyme-linked lectin assay and lectin histochemical analysis were used to characterize carbohydrate residues in lyophilized, resuspended tracheal secretions and the apical glycocalyx of the tracheal epithelium, respectively. Eight lectins were used to detect GalNAc, alpha-Gal, beta-Gal, alpha-Fuc (1-3)Man, alpha-Man/Glu, alpha-Man, and alpha-(2-3)sialyl residues. The amounts of total nondialyzable solids, proteins, and lipids in tracheal secretions were approximately twice as high after exposure to A. suum than after exposure to PBS. All carbohydrate residues were present in tracheal secretions after exposure to PBS and A. suum, but the reactivity was higher after exposure to A. suum for beta-Gal (+125%), alpha-Man/Glu (+150%), alpha-(1-3)Man (+287%), alpha-(2-3)sialyl (+353%), and alpha-Man (+448%) (p < 0.05). Likewise, the apical glycocalyx contained all carbohydrate residues after exposure to PBS and A. suum; afer exposure to A. suum, the reactivity was greater for alpha-GalNAc (+18%), alpha-(2-3)sialyl (+90%), beta-Gal(1-3)GalNAc (+433%), and alpha-(1-3)Man (+482%) (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Lectin-detectable glycoconjugate profile of the tracheal secretions and epithelial glycocalyx in sheep. Effect of muscarinic stimulation.

Reflex mucus secretion in the airways serves a defense function that includes the binding of bacteria to mucus glycoconjugates thereby preventing bacterial adherence to the epithelium. We therefore compared the lectin-detectable glycoconjugate profile of the epithelial glycocalyx and luminal secretions under baseline conditions and after muscarinic receptor stimulation in the sheep trachea. The sheep were intubated with a double-balloon nasotracheal tube to create a tracheal chamber for collection of secretions. After an initial lavage of the chamber to clear it of secretions, the sheep received an intravenous injection of normal saline, 0.5 mg/kg pilocarpine, or 0.5 mg/kg pilocarpine after pretreatment with 0.2 mg/kg atropine. Tracheal lavage was repeated 2 h later, and the sheep were then killed. An enzyme-linked lectin assay and lectin histochemistry were used to characterize glycoconjugate residues in tracheal secretions and in the apical epithelial glycocalyx, respectively. Eight different lectins were used to detect N-acetyl galactosamine, alpha-galactose, alpha-galactose-N-acetyl galactosamine, beta-galactose, beta-galactose-N-acetyl galactosamine, alpha-fucose, alpha-glucose, alpha-mannose and alpha-(2-3)sialyl residues. After normal saline, reactivity was present for all glycoconjugates in secretions and in the glycocalyx.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphometric estimation of superoxide generation in allergen-induced airway hyperresponsiveness.

BACKGROUND: We previously reported that the airway hyperresponsiveness (AHR) that follows antigen challenge is mediated, in part, by the generation of reactive oxygen species. The purpose of this study was to provide quantitative morphologic evidence of oxygen radical production in the airways during antigen-induced AHR. EXPERIMENTAL DESIGN: Allergic sheep with a history of early and late bronchial responses to inhaled Ascaris suum antigen were used. The sheep were either challenged with antigen (N = 5) or saline (N = 5) and then euthanized 24 hours later when AHR had been documented to occur. Complete transverse sections of the right cranial lobar bronchus were obtained from the animals and were washed three times in Tris buffer and then incubated for 20 minutes in an oxygenated solution of diaminobenzidine which, in the presence of superoxide and manganese, forms an insoluble amber reaction product. Superoxide dismutase (SOD, 2250 units/ml) and phorbol ester (phorbol myristate acetate, 0.5 microgram/ml) were added to some tissues and used as negative and positive controls, respectively. The bronchial samples were then fixed and embedded in paraffin for light microscopy. The diaminobenzidine reaction product was quantified by determining the volume fraction of reaction product by point counting with a differential interference contrast microscope without counterstain. RESULTS: Diaminobenzidine reaction product increased 5-fold (p < 0.05) in challenged animals, and this response was blocked by SOD. The reaction product was localized in and around the airway epithelium. Antigen challenge also resulted in a 2.4- and 2.0-fold increase in eosinophils and metachromatic cells in the airway wall. There were no differences in the number of neutrophils between groups. Pretreatment of animals (N = 2) with the combination of the 5-lipoxygenase inhibitor (zileuton, 10 mg/kg, po) and the platelet-activating factor antagonist (WEB-2086, 3 mg/kg, i.v.) agents, which have been shown to block AHR and antigen-induced inflammation in the sheep model, also blocked the antigen-induced superoxide formation. CONCLUSIONS: These data suggest that superoxide and increased numbers of mediator-containing inflammatory cells are present in sheep airways 24 hours after antigen challenge. The interaction of this reactive oxygen species with these cells could contribute to the AHR seen at this time.

Tracheobronchial epithelium of the sheep: III. Carbohydrate histochemical and cytochemical characterization of secretory epithelial cells.

We examined histochemically (light microscopy-LM) and cytochemically (electron microscopy-EM) the secretory epithelial cells in the tracheobronchial mucosa of sheep. Six morphologically distinct, granule-containing cells have been described, on the basis of their morphology and airway distribution: four mucous (M1-M4), serous (SC), and Clara (CC). Stereological and morphometric data indicated that M3, M4, SC, and CC were distinctly different from each other and from M1 and M2 cells. Mucous cells M1 and M2 differed in granule morphology. Samples of tracheas, sixth-generation bronchi, distal bronchi, and terminal bronchioles of 18 adult sheep were examined. At the LM level, methacrylate sections were reacted with an alcian blue (pH 2.5), periodic acid Schiff (PAS) sequence to differentiate neutral from acidic glycoconjugates (GC), and a high-iron diamine (HID), alcian blue sequence to differentiate sulfated from nonsulfated (sialylated) GC. At the EM level the periodic acid-thiocarbohydrazide localized hexose-rich, neutral GC. Dialyzed iron (DI) and high-iron diamine localized carboxylated and sulfated GC, respectively. Granules of all but Clara cells were PAS-positive. All mucous cells contained acidic groups, but only M1 and M4 cells had LM-detectable sulfated GC. At the ultrastructural level, minimal but discernible HID and LID reaction product was observed on granule profiles of M2, M3, and SC, indicating acidic and sulfated GC not detected at the LM level. Histochemically, the sheep tracheobronchial epithelium was more similar to that of humans than some other examined mammalian species.

Effects of ozone on lamb tracheal mucosa. Quantitative glycoconjugate histochemistry.

Whether or not the previously reported O3-induced abnormality in the postnatal development of tracheal secretory function in lambs is accompanied by changes in epithelial cell populations and their glycoconjugate composition was determined. Six lambs were killed at birth and 12 lambs at age 2 weeks. Of the latter 12, six were exposed to O3 (1 ppm, 4 hours daily for 5 days during the 1st week of life) and five had air-sham exposures (controls). Tracheal glycoconjugates were localized in situ with lectins to detect N-acetyl-galactosamine (galNAc), alpha-D-galactose (alpha-gal), beta-D-gal(1----3)-galNAc (beta-gal), and fucose (fuc). Mean (+/- SD) epithelial cell density (cells/mm basal lamina) was 418 +/- 57 in the newborns, 385 +/- 63 in controls (P was not significant), and 342 +/- 47 in O3-exposed lambs (P less than 0.05). Mucous cell density was 87 +/- 12 in newborns, 63 +/- 10 in controls (P less than 0.05), and 76 +/- 10 in O3 exposed lambs (P was not significant). Ciliated cells remained unchanged from birth to 2 weeks (P was not significant), but decreased (P less than 0.05) in O3-exposed lambs. All counted mucous cells contained fuc and galNAc at birth and retained these residues after sham and O3 exposure. The alpha-gal-containing mucous cells declined from 97 +/- 13 to 7 +/- 1 (P less than 0.05) and beta-gal containing cells from 39 +/- 5 to 25 +/- 4 in controls. In contrast, cells containing alpha-gal 71 +/- 10 remained at newborn levels (97 +/- 13) and beta-gal-containing cells increased from 40 +/- 5 at birth to 58 +/- 8 in O3-exposed animals (P less than 0.05). It was concluded that early postnatal exposure of lambs to O3 causes a decrease in epithelial cell density, but retards the developmental decrease in the number of tracheal mucous cells and alters the lectin detectable carbohydrate composition of mucus in these cells. These developmental defects were interpreted to be the morphologic correlates of the previously shown effects of O3 on the maturation of secretory function and mucus transport.