Temporal and spatial distribution of ciliogenesis in the tracheobronchial airways of mice

Little is known about ciliogenesis as it proceeds through the entire airway tree, from the trachea to the terminal bronchioles, especially during the postnatal period. The purpose of this study was to define the spatial and temporal (prenatal and postnatal) pattern of normal cilia development in the mouse. Three airway generations representing the entire airway tree were examined: trachea, lobar bronchi, and terminal bronchiole. Ciliated cells in lung lobe whole mounts were labeled with a fluorescent dye for confocal microscopy, and ciliated cell surface density was measured for each airway generation and age. The same samples were examined by scanning electron microscopy to verify the appearance of ciliated cells among the differentiating epithelium of the airways. Ciliated cells were first detected in the trachea and lobar bronchi at 16 days gestational age (DGA) and in the terminal bronchioles at 18 DGA. Ciliated cell surface density increased with prenatal and postnatal age at all airway levels. However, the ciliated cell surface density of the trachea and lobar bronchi was always greater compared with the terminal bronchiole. In conclusion, the study revealed that in developing tracheobronchial airways of the mouse: 1) Ciliogenesis differs temporally and spatially by airway generation; 2) Ciliated cell surface density increases with age in all airway generations, but density decreases in a proximal to distal direction; and 3) A significant portion of ciliogenesis continues after birth. This study provides a healthy basis for investigations of neonatal pulmonary disease or pollutant toxicity affecting cilia and its functions.     

Identification and Characterization of Epithelial Cells in Mammalian Tissues by Immunofluorescence Microscopy Using Antibodies to Prekeratin

The occurrence of intermediate-sized filaments containing prekeratin-like proteins ('cytokeratins') has been examined in various organs of rat and cow by electron microscopy and by immunofluorescence microscopy on frozen sections using antibodies to defined constitutive proteins of various types of intermediate-sized filaments (prekeratin, vimentin, desmin). Positive cytokeratin reaction and tonofilament-like structures have been observed in the following epithelia: epidermis; ductal, secretory, and myoepithelial cells of sweat glands; mammary gland duct; myoepithelial cells of lactating mammary gland; milk secreting cells of cow; ductal, secretory, and myoepithelial cells of various salivary glands; tongue mucosa; bile duct; excretory duct of pancreas; intestinal mucosa; urothelium; trachea; bronchi; thymus reticulum, including Hassall corpuscles; mesothelium; uterus; and ciliated cells of oviduct. None of the epithelial cells mentioned has shown significant reaction with antibodies to vimentin, the major component of the type of intermediate-sized filaments predominant in mesenchymal cells. The widespread, if not general occurrence of cytokeratin filaments in epithelial cells is emphasized, and it is proposed to use this specific structure as a criterion for true epithelial character or origin.     

The respiratory system in Arctocephalus australis (Pinnipedia, Otariidae). I--Macroanatomy and microanatomy of the trachea and extrapulmonary airways]

This paper presents the results of histological and histochemical organization of trachea and primary bronchi. The presence of a subepithelial lamina and a smooth muscle layer in the superficial corion, probably related with a functional respiratory mechanism are defined. The structure of tracheal and primary bronchi glands, the way they secretes and the type of secretion are also described.     

Expression of carbonic anhydrase isozymes II and III in developing bovine parotid gland

Two cytosolic carbonic anhydrase isozymes (CA-II and CA-III) were studied by immunohistochemistry in bovine parotid glands during fetal development. In a 3-month-old fetus of crown-rump length (CRL) 17 cm, the expression of CA-II in undifferentiated epithelial cells was observed, whereas immunostaining for CA-III remained negative. At 26 cm CRL (4–5 months old), weak expression of CA-III in large ductal epithelial cells was noted. The accumulation of secreted granules in primary acinar cells was initially observed at this stage. In a newborn calf, anti-CA-II reactivity almost disappeared from most duct segments. The time-dependent expression and distribution of the isozymes in parotid glands may reflect different biological functions of these structurally closely related isozymes. Bovine parotid acinar cells of fetuses would thus appear to possess all the cellular structures and immunohistochemical properties at 4 and 5 months of gestation. CA-II subsequently disappeared from duct segments and nearly all acinar cells in adults were present at or just after birth.     

Microscopic and ultrastructural anatomy of the trachea and bronchi of Melopsittacus undulatus (Aves,Psittaciformes)

Summary The normal microscopic pattern and ultrastructure of the lower trachea and the primary and secondary bronchi of the budgerigar (Melopsittacus undulatus) are described. The trachea is lined by mucociliary pseudostratified columnar epithelium with simple acinar mucous glands; epithelium in primary and secondary bronchi becomes progressively lower and less pseudostratified, and mucous cells less aggregated. The wall structure shows a parallel simplification; tracheal elements are comprised of osseous metaplastic cartilage with bone marrow between cancellous trabeculae, whereas distal secondary bronchial walls are principally comprised of smooth muscle. Mucous cells are similar to those described in mammalian, and other avian respiratory mucosae. Ciliated cells are similar to those known in other avian airways. No brush cells or Clara cells are observed.     

Innervation of lower airways and neuropeptide effects on bronchial and vascular tone in the pig

Summary The occurrence and distribution of peptide-containing nerve fibres [substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), neuropeptide Y (NPY)] and noradrenergic nerve fibres [tyrosine hydroxylase (TH)- and dopamine beta hydroxylase (DBH)-positive] in the airways of the pig were studied by means of immunohistochemistry. SP- and CGRP-immunoreactive (-IR) nerve fibres were present close to and within the lining respiratory epithelium, around blood vessels, within the tracheobronchial smooth muscle layer and around local tracheobronchial ganglion cells. The content of CGRP- and neurokinin A (NKA)-like immunoreactivity (-LI) measured by radioimmunoassay (RIA) was twice as high in the trachea compared to that in the peripheral bronchi. SP was a more potent constrictor agent than NKA on pig bronchi in vitro. CGRP had a relaxant effect on precontracted pig bronchi. On blood vessels CGRP exerted a relaxant effect that was more pronounced on pulmonary arteries than on bronchial arteries. VIP/PHI-IR fibres were seen in association with exocrine glands and in the tracheobronchial smooth muscle layer. VIP-positive nerve fibres were abundant around blood vessels in the trachea but sparse or absent around blood vessels in the peripheral bronchi. This histological finding was supported by RIA; it was shown that the content of peptides displaying VIP-like immunoreactivity (-LI) was 18 times higher in the trachea compared to peripheral bronchi. VIP was equally potent as CGRP in relaxing precontracted pig bronchi in vitro. Both bronchial and pulmonary arteries were relaxed by VIP. NPY was colocalized with VIP in tracheal periglandular nerve fibres and in nerve fibres within the tracheobronchial smooth muscle layer. NPY was also present in noradrenergic (DBH-positive) vascular nerve fibres. The content of NPY was much higher (15-fold) in the trachea compared to small bronchi. NPY caused a contraction of both pulmonary and bronchial arteries. The bronchial smooth muscle contraction to field stimulation in vitro was purely cholinergic. A non-cholinergic relaxatory effect following field stimulation was observed after bronchial precontraction. Capsaicin had no effect on pig bronchi in vitro.     

Numerical investigation of the three-dimensional flow in a human lung model

The flow field at inspiration and expiration in the upper human airways consisting of the trachea down to the sixth generation of the bronchial tree is numerically simulated. The three-dimensional steady flow at a hydraulic diameter-based Reynolds number Re(D)=1250 is computed via a lattice-Boltzmann method (LBM). The simulation is validated by the experimental data based on particle-image velocimetry (PIV) measurements. The good agreement between numerical and experimental results is evidenced by comparing velocity contours and distributions in a defined reference plane. The results show the LBM to be an accurate tool to numerically predict flow structures in the human lung. Using an automatic Cartesian grid generator, the overall process time from meshing to a steady-state solution is <12h. Moreover, the numerical simulation allows a closer analysis of the secondary flow structures than in the experimental investigation. The three-dimensional streamline patterns reveal some insight on the air exchange mechanism at inspiration and expiration. At inspiration, the slower near-wall tracheal flow enters through the right principal bronchus into the right upper lobar bronchus. The bulk mass flux in the trachea is nearly evenly distributed over the left upper, center and lower lobar bronchi and the right center and lower bronchi. At expiration, the air from the right upper lobar bronchus enters the right center of the trachea and displaces the airflow from the lower and center right bronchi such that the tracheal positions of the streamlines at inspiration and expiration are switched. The flow in the left bronchi does not show this kind of switching. The findings emphasize the impact of the asymmetry of the lung geometry on the respiratory air exchange mechanism.     

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.     

Development of mucociliary transport in the postnatal ferret trachea.

Little is known of the developmental aspects of mucociliary transport. Previous studies have documented that newborn ferret trachea has very few ciliated cells but numerous immature secretory cells in the epithelium and only rudimentary submucosal glands. Rapid and complete maturation occurs in the first postnatal month. This study examines mucociliary transport during this period of rapid maturation. We made direct observations of particle movement across the epithelium of ferret tracheas. No mucus transport could be demonstrated on the first day of life. Transport was discernible, although sporadic and slow, by 7 days and reached adult levels (10.7 +/- 3.7 mm/min) by 28 postnatal days. The emergence of transport capability correlated well with previously described developmental changes in ciliation, mucus secretion, and ion permeability and transport. Threshold mucus transport occurred at 1 wk of age when 20-25% of the surface cells are ciliated. The neonatal ferret appears to be a useful model for assessing integrated epithelial structure-function relationships that are important not only during early development but also during repair after airway injury involving deciliation.     

Effect of the larynx on oscillatory flow in the central airways: a model study

Measurements were made of the effect of the larynx on the oscillatory flow profiles in a 3:1 scale model of the human central airways. A fixed glottic aperture corresponding to the shape and size at midinspiration was used. Oscillatory airflows at peak Reynolds numbers, similar to those obtained during spontaneous breathing and panting, were studied. The flow distribution to the five lobar bronchi was maintained by distally placed linear resistors. A hot-wire anemometer probe was used to measure the local velocity along two perpendicular diameters at six stations distributed through the model. Near the proximal end of the trachea, the flat velocity profiles at the beginning of the flow cycle peaked at maximum flow because of the jet created by the glottic aperture. This peaked structure was conserved during the latter half of the inspiratory cycle. Close to the carina, the jet had almost dissipated and the entry conditions into the main bronchi corresponded to those in the absence of the laryngeal model. The effect of the glottic aperture on the mean velocity was not felt beyond the carina, and the characteristic skewed profiles seen in oscillatory flows, in the absence of the larynx, were present in the main and lobar bronchi.     

Tracheobronchial epithelium in the adult rhesus monkey: a quantitative histochemical and ultrastructural study

Previous studies of the intrapulmonary conducting airways of sheep and rabbit have demonstrated marked diversity in the epithelial populations lining them. Because studies of trachea and centriacinar regions of macaque monkeys suggested that primates may be even more diverse, the present study was designed to characterize the epithelial population throughout the airway tree of one primate species, the rhesus monkey. Trachea and intrapulmonary airways of the right cranial and middle lobes of glutaraldehyde/paraformaldehyde-infused lungs of five adult rhesus monkeys were microdissected following the axial pathway. Each branch was assigned a binary number indicating its specific location within the tree. The trachea and six generations of intrapulmonary airway from the right cranial lobe were evaluated for ultrastructure and quantitative histology as were those of the right middle lobe for quantitative carbohydrate histochemistry. Four cell types were identified throughout the tree: ciliated, mucous goblet, small mucous granule, and basal. The tallest epithelium lined the trachea; the shortest, the respiratory bronchiole. The most cells per unit length of basement membrane were in proximal intrapulmonary bronchi; the least, in the respiratory bronchiole. The nonciliated bronchiolar epithelial or Clara cell was restricted to respiratory bronchioles. Sulfomucins were present in the vast majority of surface goblet cells in the trachea and proximal bronchi. In proximal bronchi, neutral glycoconjugates predominated in glands and acidic glycoconjugates in surface epithelium. In terminal and respiratory bronchioles the ratio of acidic glycoconjugate to neutral glycoconjugate equaled that in proximal bronchi, although glands were not present. Sulfomucins were minimal in terminal airways. We conclude that the characteristics of the epithelial lining of the mammalian tracheobronchial airway tree are very species-specific. The lining of the rhesus monkey does not have the diversity in cell types in different airway generations observed in sheep and rabbit. Also, the populations lining these airways in the rhesus are very different from either the sheep or rabbit in number, proportions of different cell types, glycoconjugate content, and distribution of specific cell types.     

Neuronal galanin is widely distributed in the chicken respiratory tract and coexists with multiple neuropeptides

Summary The mammalian airways are known to be richly innervated by several types of peptide-containing nerve fibers. Galanin-containing fibers are, however, comparatively few. The results of the present immunocytochemical study indicate that the chicken airways receive a notably dense supply of galanin-storing fibers. Other major neuropeptides were neuropeptide Y, vasoactive intestinal peptide and substance P. Nerve fibers containing these peptides were distributed in the trachea, main bronchi, and the lungs. Minor nerve fiber populations contained calcitonin generelated peptide, enkephalin and gastrin-releasing peptide. In the trachea and main bronchi the majority of peptidecontaining nerve fibers was distributed beneath and sometimes also within the epithelium; fibers were fewer in the lamina propria. In the lungs they occurred both in association with the epithelium of small bronchi and in the septa. Adrenergic nerves (using tyrosine hydroxylase as marker) were predominantly distributed in the lamina propria among bundles of smooth muscle and blood vessels. In the nerve fibers associated with the epithelium and in nerve cell bodies in local ganglia of the tracheal wall, galanin was found to coexist with several other neuropeptides (neuropeptide Y, vasoactive intestinal peptide and substance P) suggesting co-expression of multiple neuropeptide genes in the same population of neurons.     

Degree of mineralization of pulmonary, tracheal and bronchial tissues in healthy persons of different age

In persons died an accidental or sudden death tissues of the lungs, bronchi of various caliber and trachea have been investigated by means of light microscopy and infrared (IR) spectroscopy. The IR spectra of the bronchial and tracheal tissue are identical and differ from the IR spectra of the pulmonary tissues. The mineralization degree (ratio of inorganic to organic components of the tissue) of the pulmonary tissue increases with age, in bronchi and trachea this dependence is expressed not so distinctly. The absolute mineralization degree in the bronchial tissue is 1.3-1.8 times higher than in the lungs. A suggestion is made that mineralization level of the tissue increases with aggravation of sclerosis processes.     

Age changes in the localization and distribution of glycosaminoglycans in human hyaline cartilage

Summary The localization and distribution of acid glycosaminoglycans, glycoproteins and basic proteins have been studied in human costal cartilage, trachea and primary bronchi from the fetal period through old age. Alcian blue and PAS were used for the staining of acid glycosaminoglycans and glycoprotein and/or sugar-containing compounds respectively. Bromphenol blue and/or bromsulfalein were utilized for the detection of basic proteins. In a further attempt to histochemically identify the various polyanions staining was carried out with AB containing different concentrations of electrolytes. Mild acid hydrolysis, hyaluronidase and proteolytic digestions were also employed. It appeared that the distribution, localization and concentration of the different macromolecules vary according to age, to the type of hyaline cartilage and to the different areas of the same segment. The results also suggested that in developing tissues the areas of appositional growth synthesize mainly chondroitinsulphate while in the territory and interterritory this acidic polyanion is found with keratansulphate. With advancing age the latter slowly disappears from the interterritorial matrix and is nearly exclusively found in the matrix adjacent to the chondrocytes.The susceptibility of these tissues to enzyme digestion also varied according to age and to the type of hyaline cartilage. Proteolytic and hyaluronidase treatment, which were extremely effective in the prenatal period and in young subjects, had practically no effect on mature and old ribs. In the bronchi, instead, the substrates were easily extracted even at an old age. The problems related to the different chemical state of the various macromolecules, their localization and distribution and their possible effect on senescence of hyaline cartilage are discussed.Supported by Research Grant DE-01952(03) of the National Institutes of Public Health, Bethesda, Md.     

Localization of immunoreactive tissue kallikrein in the seromucous glands of the human and guinea-pig respiratory tree

Summary An immunocytochemical study focused on the cellular localization of tissue kallikrein along the human and guinea-pig respiratory tracts is reported. A strong immunoreactivity for tissue kallikrein was observed in the seromucous glands of the nasal mucosa, trachea, and bronchi. In these glands, the immunostaining was restricted to the serous component of the acinus whereas mucous cells showed no staining. Since no immunoreactivity to kininogen was observed in any of the tissue constituents of the human and guinea-pig respiratory tree, transudation of the substrate from plasma was considered to be the preferred mode of delivery of the kininogen into the bronchopulmonary interstitium and lumen. Our results provide morphological evidence for the well documented presence of tissue kallikrein in bronchial lavage fluids and support the hypothesis that kinins may be one of the more important mediators involved during acute episodes of asthma and rhinitis.     

Matrix metalloproteinases are expressed during ductal and alveolar mammary morphogenesis, and misregulation of stromelysin-1 in transgenic mice induces unscheduled alveolar development.

The matrix-degrading metalloproteinases stromelysin-1, stromelysin-3, and gelatinase A are expressed during ductal branching morphogenesis of the murine mammary gland. Stromelysin-1 expression in particular correlates with ductal elongation, and in situ hybridization and three-dimensional reconstruction studies revealed that stromelysin-1 mRNA was concentrated in stromal fibroblasts along the length of advancing ducts. Transgenic mice expressing an activated form of stromelysin-1 under the control of the MMTV promoter/enhancer exhibited inappropriate alveolar development in virgin females. Ultrastructural analysis demonstrated that the basement membrane underlying epithelial and myoepithelial cells was amorphous and discontinuous compared with the highly ordered basal lamina in control mammary glands. Transgenic mammary glands had at least a twofold increase in the number of cells/unit area and a 1.4-fold increase in the percent of cycling cells by 13 wk of age compared with nontransgenic littermates. In addition, transgenic glands expressed beta-casein mRNA, but not protein, and resembled the proliferative and differentiated state of an animal between 8 and 10 days pregnant. An analysis of metalloproteinase expression in the glands of normal pregnant females demonstrated that the same matrix metalloproteinase family members, including stromelysin-1, were expressed in connective tissue cells surrounding epithelial clusters during the time of lobuloalveolar development. These results suggest that metalloproteinases may assist in remodeling ECM during normal ductal and alveolar branching morphogenesis, and that disruption of the basement membrane by an activated metalloproteinase can affect basic cellular processes of proliferation and differentiation.     

Relative hysteresis of the airways and lung parenchyma in normal subjects

We evaluated the mechanical properties of the airways sequentially from the glottis toward the main bronchi in 10 normal subjects. Plots of airway cross-sectional area vs. lung volume, measured during inspiration and expiration, were used to determine the relative magnitude of the airways vs. parenchymal hysteresis. Airway cross-sectional area was measured by means of the acoustic reflection technique. We found that the hysteresis of the proximal part of the trachea was greater than that of the lung parenchyma, whereas the hysteresis of the distal trachea and subcarinal segments of the airways was smaller than that of the lung parenchyma. The transition zone between the proximal and the more distal airway properties occurred 8-26 cm distal to the glottis. This transition zone was reproducible in its location on repeated testing in each subject but varied among subjects. To the extent that relative hysteresis of the airways depends on bronchomotor tone, our findings suggest that the bronchomotor tone is inhomogeneous, being maximal at the proximal part of the trachea and gradually decreasing toward the more distal trachea and subcarinal airway segments.     

Surface architecture of the mucosal epithelium of the cat trachea: II. Structure and dynamics of the membranous portion

Bronchoscopic examination of anesthetized cats revealed that the trachea is capable of considerable change in caliber via lateral expansion of the membranous trachea. The morphological basis for this expansile capacity was determined by correlated light microscopy and scanning and transmission electron microscopy. The organization of the membranous trachea differs from that of the cartilaginous trachea. The mucosa is arranged in a series of longitudinal folds that open like an accordion when stretched laterally. These fold are not random, but appear to depend on rows of nonciliated, microvillus-rich cells that form flexure lines in the surface epithelium. The mitochondria in such cell have a condensed configuration, indicating a high level of oxidative metabolism and suggesting that they may participate in transport processes that modify the luminal contents. Goblet cells, which are relatively sparse in the membranous trachea, have mitochondria in which the prominence of matrix granules and degree of mucus storage are inversely related. Mitochondrial morphology allows goblet cells that have discharged their mucin content to be readily distinguished from the microvillus-rich cells, even when their luminal surfaces lie outside the thickness of a section.     

Postnatal development of the respiratory system of the opossum. II. Electron microscopy of the epithelium and pleura.

At birth, the opossum lung is remarkably primitive and consists of a system of branching airways that end in a number of terminal air chambers. From the newborn through the 10 cm stage of development the conducting portion of the lung predominates. The air chambers, which represent portions of the conducting system modified for respiration, are in a constant state of evolution since they are destined to become part of the expanding bronchial system. The airways are devoid of cilia and goblet cells at birth, and are lined by columnar epithelial cells which contain two types of cytoplasmic granules: an electron-dense form and a heterogeneous form. The latter exhibits an electron-dense core surrounded initially by a large halo of flocculent material. This type of granule is not seen beyond the 8 cm stage. The terminal air chambers of the newborn and later stages are lined type I and type II alveolocytes that appear identical to the alveolocytes lining alveoli in the adult. By the 2.5 cm stage, scattered cilia are present in the trachea and bronchi and bands of smooth muscle have differentiated in relation to bronchial epithelium and to proximal areas of the terminal chambers. Citiated cells are separated by ridges composed of light and dark cells which are without cilia and which contain scattered electron-dence granules. Throughout the postnatal period numerous alveolar macrophages and mast cells are noted in relation to the conducting system and pleura. Differentiation of the pleura also occurs during the postnatal period. In the newborn the pleura is simple squamous mesothelium. Later stages develop a thick connective tissue lamina between the pleural mesothelium and lung parenchyma. A large band of elastin is interposed between the mesothelium and underlying bundles of collagen.     

Histo-topography of the lymphoid tissue of children's trachea

By means of certain microscopical methods areas from the middle third of trachea have been examined in children, died from various causes not connected with diseases in the pulmonary pathways. At birth in the mucous membrane of the trachea there is no lymphoid tissue. Its elements in a small amount appear at the initial stage of the suckling period. In two-month-old infants the first signs of lymphoid perinucleoli appear. During the early period of infancy the main regions of the lymphoid cells localization are clearly defined, in the area of some ducts of the tracheal glands including. In children, whose age corresponds to the first period of childhood, lymphoid accumulations have a pattern, resembling similar structures of the trachea in mature persons and the greatest concentration of cells, in comparison to the preceding groups of children in the ages examined.