Development of alveolar septa and formation of alveolar pores during the early postnatal period in the rat lung

In order to investigate the formation of alveolar pores, lungs of rats, after intratracheal perfusion of glutaraldehyde, are processed at postnatal days 1, 7, 14, 16 and 21 for light and transmission electron microscopy and at days 7 and 16 for scanning electron microscopy. The initial low secondary crests of day 1 rapidly elongate to pleats subdividing the primary saccules. The ledges of some pleats partly grow toward each other as ring like diaphragms, leaving openings whose boundary is composed of alveolar epithelium separated by a basal lamina from a connective tissue sheath with capillaries. At day 7, in scanning electron microscopy the lumina of some rudimentary alveoli communicate by apertures of different sizes, as a result of the outgrowth of curved alveolar pleats which narrow to a ring-like aperture. The interalveolar openings observed in scanning electron microscopy resemble those investigated by light and transmission electron microscopy. The number of interalveolar pores increases from day 7 on; they become more and more frequent at days 14, 16 and 21, respectively. It appears that alveolar multiplication in newborn rats proceeds not only by segmentation of terminal respiratory units but also by compoundment of septa. The difference between genuine pores and transsections of folds in transmission electron microscopy will be given closer attention in this study. Also, the incidence and location of type II pneumocytes during rapid enlargement of the alveolar surface area is discussed.     

Altered expression of tight junction molecules in alveolar septa in lung injury and fibrosis

The dysfunction of alveolar barriers is a critical factor in the development of lung injury and subsequent fibrosis, but the underlying molecular mechanisms remain poorly understood. To clarify the pathogenic roles of tight junctions in lung injury and fibrosis, we examined the altered expression of claudins, the major components of tight junctions, in the lungs of disease models with pulmonary fibrosis. Among the 24 known claudins, claudin-1, claudin-3, claudin-4, claudin-7, and claudin-10 were identified as components of airway tight junctions. Claudin-5 and claudin-18 were identified as components of alveolar tight junctions and were expressed in endothelial and alveolar epithelial cells, respectively. In experimental bleomycin-induced lung injury, the levels of mRNA encoding tight junction proteins were reduced, particularly those of claudin-18. The integrity of the epithelial tight junctions was disturbed in the fibrotic lesions 14 days after the intraperitoneal instillation of bleomycin. These results suggest that bleomycin mainly injured alveolar epithelial cells and impaired alveolar barrier function. In addition, we analyzed the influence of transforming growth factor-β (TGF-β), a critical mediator of pulmonary fibrosis that is upregulated after bleomycin-induced lung injury, on tight junctions in vitro. The addition of TGF-β decreased the expression of claudin-5 in human umbilical vein endothelial cells and disrupted the tight junctions of epithelial cells (A549). These results suggest that bleomycin-induced lung injury causes pathogenic alterations in tight junctions and that such alterations seem to be induced by TGF-β.     

Polyfunctional role of the alveolar brush cells in the rat lung

By means of scanning and transmission electron microscopy it was demonstrated that the number of vacuoles located in the apical part of cytoplasm in alveolar brush cells of the regenerating rat lung increases, hyperplasia of Golgi-complex takes place and the activation of the protein-synthetising apparatus is evident. The immature surfactant material (osmiophilic lamellar bodies) and secretory dense core vesicles were found in the cytoplasm of alveolar brush cells. Intramuscular injections of colchicin to rats (0.1 mg/100 g body weight) 6 times during 24 hours before decapitation does not influence the number, topography and structure of microfibrilla bundles contained in a sufficient amount by alveolar brush cells. At the same time a part of microvilli of alveolar brush cells undergoes destruction and resorption under the action of colchicin. The data on ultrastructural organization of alveolar brush cells show that they are able to fulfill several functions: absorptive, contractile and secretory.     

Cell differentiation of alveolar epithelium in the developing rat lung: ultrahistochemical studies of glycoconjugates on the epithelial cell surface

Glycoconjugates on the surface of pulmonary epithelial cells were ultrahistochemically examined in the fetal, neonatal and adult rat lung. Lectin and colloidal iron staining procedures were performed in combination with digestion using carbohydrate-degrading enzymes or methylation. The glycoconjugate composition of columnar cells at 16 days gestation was similar to that of cuboidal cells at 19 days gestation. Glycoconjugate differentiation on the cell surface occurred at 20 days gestation, and especially the loss of soybean agglutinin (SBA) binding sites could be detected on type II cells. The contents of Ricinus communis agglutinin-I (RCA-I) and Concanavalin A (Con A) binding sites on type II cells also began to decrease. On the contrary, the content of sulfated saccharides decreased on the surface of type I cells during development. Glycoconjugate differentiation on both type I and II cells was completed with the disappearance of hyaluronic acid and peanut agglutinin (PNA) binding sites; type I and II cells acquired a similar histochemical composition to that on adult type I and II cells at 5 days after birth. Both type I and II cells share a common early precursor cell, that is, the cuboidal epithelial cell at the canalicular stage.     

Structural features of alveolar wall basement membrane in the adult rat lung

The ultrastructural characteristics of alveolar (ABM) and capillary (CBM) basement membranes in the adult rat lung have been defined using tannic acid fixation, ruthenium red staining, or incubation in guanidine HCl. ABM is dense and amorphous, has 3- to 5-nm filaments in the lamina rara externa (facing the alveolus) that run between the lamina densa and the basal cell surface of the epithelium, has an orderly array of ruthenium red-positive anionic sites that appear predominantly (79%) on the lamina rara externa, and has discontinuities beneath alveolar type II cells but not type I cells that allow penetration of type II cytoplasmic processes into the interstitium of the alveolar wall. The CBM is fibrillar and less compact than ABM, has no lamina rara filaments, and has one fifth the number of ruthenium red- positive anionic sites of ABM that appear predominantly (64%) overlying the lamina densa. Incubation of lung tissue with Flavobacterium heparinum enzyme or with chondroitinase has shown that ABM anionic sites represent heparan sulfate proteoglycans, whereas CBM anionic sites contain this and other sulfated proteoglycans. The CBM fuses in a local fashion with ABM, compartmentalizing the alveolar wall into a thick and thin side and establishing a thin, single, basement-membrane gas-exchange surface between alveolar air, and capillary blood. The potential implications of ABM and CBM ultrastructure for permeability, cell differentiation, and repair and morphogenesis of the lung are discussed.     

Rapid onset of gene expression in lung, supportive of formation of alveolar septa, induced by refeeding mice after calorie restriction

Alveolar regenerative gene expression is unidentified partly because its onset, after a regenerative stimulus, is unknown. Toward addressing this void, we used a mouse model in which calorie restriction produces alveolar loss, and ad libitum access to food after calorie restriction induces alveolar regeneration. We selected four processes (cell replication, angiogenesis, extracellular matrix remodeling, and guided cell motion) that would be required to convert a flat segment of alveolar wall into a septum that increases gas-exchange surface area. Global gene expression supportive of processes required to form a septum was present within 3 h of allowing calorie-restricted mice food ad libitum. One hour after providing calorie-restricted mice food ad libitum, RNA-level expression supportive of cell replication was present with little evidence of expression supportive of angiogenesis, extracellular matrix remodeling, or guided cell motion. Cell replication was more directly assayed by measuring DNA synthesis in lung. This measurement was made 3 h after allowing calorie-restricted mice food ad libitum because translation may be delayed. Ad libitum food intake, following calorie restriction, elevated DNA synthesis. Thus RNA expression 1 h after allowing calorie-restricted mice food ad libitum supported increased cell replication; measurements at 3 h revealed increased DNA synthesis and RNA expression, supportive of the three other processes required to form a septum. These findings identify the first hour after providing calorie-restricted mice ad libitum access to food as the onset of gene expression in this model that supports processes needed for alveolar regeneration.     

Physiology of the alveolar surface network

The alveolar surface network (ASN) is the totally fluid intraacinar conformation of the alveolar surface liquid (ASL) continuum circulating, both in series and in parallel, through ultrathin (to <7 nm) molecular conduits formed by appositions of unit bubbles of alveolar gas. The ASN is the analogue of foam in vitro. Appositions of unit bubble films, namely foam films, include (a) bubble-to-bubble at the alveolar entrance, across alveolar ducts, and at pores of Kohn ('classical foam films'); (b) bubble-to-epithelial cell surface ('cell-surface foam film'); and (c) bubble-to-open surface liquid layer of the terminal conducting airways ('surface foam film'). These appositions of monolayer bubble films create (a) 'macrochannels' ('pressure points', 'reservoirs') that modulate ASL transfers, volume and flow throughout the acinus and between acinar surface and both the interstitium and the terminal conducting airways surfaces, and (b) 'microchannels' along the broadest surfaces of the appositions. 'Microchannels', which are expectedly bilayer, serve several functions, including (a) virtually frictionless orientation of unit bubbles and ASL to fill the acinar air space; (b) virtually unrestricted diffusion of respiratory gases; (c) architectural support ('infrastructure') against the 'mass' and 'recoil' force of the interstitium; and (d) provision of 'gate' and 'bridge' dynamics that further modulate and direct ASL circulation. The physiological and anatomical boundary between acinar ASN and the bubble-free open liquid surfaces of the conducting airways is marked by the surface foam film. The ASN operates as outlined above in all regions of the lung, at all lung volumes, beginning at the onset of air-breathing at birth and continuing throughout life. Reports of its discovery (Pulmonary Physiology of the Fetus, Newborn and Child (1975) 116; Pediatr. Res. 12 (1978) 1070) and subsequent confirmatory research including the adult lung are summarized in this review by progressive development of each function. These functions, which are normal for a relatively dry foam such as the ASN (where gas:liquid volume ratio is >99:1) cannot be duplicated by the conventional theories and models of an open 'alveolar lining layer'. The unfortunate research technologies upon which these theories and models have been formulated have, indeed, obfuscated recognition of the ASN in vivo. They are also presented and critiqued in this review.     

Cytoskeletal features of alveolar myofibroblasts and pericytes in normal human and rat lung.

Frozen or paraffin-embedded human and rat lung specimens were stained with antibodies against total actin, alpha-smooth muscle (SM) actin, vimentin, desmin, or gelsolin. Alveolar interstitial myofibroblasts [i.e., contractile interstitial cells (CIC)] were labeled by total actin antibody but not by alpha-SM actin antibody. They stained for vimentin and gelsolin and, in rat lungs, most of them for desmin. Pericytes located around venules at the junction of three alveolar septa were always positive for alpha-SM actin and never for desmin. Tissue samples were also immunostained by an alpha-SM actin antibody and studied by electron microscopy. With this technique we confirmed that cells, identified as pericytes on the basis of their location, were intensely labeled by alpha-SM actin antibodies, whereas alveolar myofibroblasts were not. We conclude that in the lung interstitium pericytes and alveolar myofibroblasts have distinct cytoskeletal features, alpha-SM actin antibody staining being a simple method to distinguish between them. Furthermore, it appears that alveolar myofibroblasts have a peculiar pattern of cytoskeletal protein composition which, in the rat, is similar to that previously described for stromal cells in uterine submucosa, liver sinusoids (Ito cells), or the core of intestinal villi.     

Structure and surface energy of the surfactant layer on the alveolar surface

The surface energy of the alveolar surfactant layer is determined in the scope of a modification of the structural model of Larsson et al. [(1999) J Disp Sci Technol 20:1-12], according to which this layer is built up of a lipid monolayer adsorbed at the hypophase/air interface and supported by a network of lipid bilayers immersed into the hypophase, i.e., the alveolar liquid. Formulae are derived for the dependence of the specific surface energy of the surfactant layer on the distance between the bilayers constituting the layer. It is shown that at equilibrium this energy can have values comparable with or less than 1 mJ/m2 needed for normal functioning of the alveolus during the respiration cycle. The specific surface energy of the surfactant layer with monolayer-bilayer structure can have such low values only if the layer is of optimal thickness and if the specific line energy of the monolayer-bilayer contact lines is negative and that of the bilayer-bilayer contact lines is positive. It is found that in dynamic regime the change in the specific surface energy of the alveolar surfactant layer with bilayer-monolayer structure is in qualitative agreement with that determined experimentally during lung inflation and deflation.     

Phosphofructokinase in alveolar type II cells isolated from fetal and adult rat lung

The glycolytic enzyme 6-phosphofructokinase (EC 2.7.1.11) was studied in adult and fetal type II pneumocytes which had been isolated from rat lung at different days of development. In addition, the activities of the enzymes hexokinase (EC 2.7.1.1), enolase (EC 4.2.1.11) and pyruvate kinase (EC 2.7.1.40) were assayed. The specific activities of the latter enzymes decrease during perinatal development and reach about adult values shortly after birth. In contrast, 6-phosphofructokinase activity increases slightly until 2 days before birth, and drops sharply afterwards. The 6-phosphofructokinase subunit composition was determined in fetal and adult type II cells. The ratio of the three subunits of 6-phosphofructokinase in type II cells isolated on fetal days 19 and 21 (term is at day 22) and in adult type II cells was identical: the three subunits were present in a ratio of 68: 14: 18 for types L, M and C, respectively. In addition, we investigated some regulatory properties of 6-phosphofructokinase from alveolar type II cells. 6-Phosphofructokinase from alveolar type II cells is strongly inhibited by increasing MgATP concentrations. This inhibition is reflected by an increase in the S0.5 for fructose 6-phosphate. Fructose 2,6-bisphosphate stimulates alveolar type II 6-phosphofructokinase. Half-maximal stimulation occurs at 1.6 and 2.0 microM fructose 2,6-bisphosphate for fetal and adult type II cells, respectively. The level of the most potent positive effector of 6-phosphofructokinase, fructose 2,6-bisphosphate, was also determined. The level of the hexose bisphosphate decreases during prenatal development; however, the level in the adult type II cells is considerably lower. The concentration of fructose 2,6-bisphosphate appears to be sufficient to fully activate 6-phosphofructokinase both in fetal and adult type II cells.     

Architectonics of the surface of alveolar macrophages

The architectonics of the alveolar macrophage surface has been investigated in the raster electron microscope. The material is obtained by means of washing from the lungs of intact noninbred white rats and also 24 h after a single intragastric administration of a cancerogenic agent--nitrosodimethylamine (NDMA)--in a toxic dose (30 mg/kg). The alveolar macrophages are studied both as a suspension and also after 30 min of cultivation. The preparations are dried in the air and by the critical point method. When the latter method is used, the architectonics of the alveolar macrophage surface is much richer. Nevertheless, the former method also gives enough information. NDMA administration produces a damaging effect on the surface architectonics and on the character of the macrophages spreading over the glass. The morphological characteristics of the changes in the surface architectonics of the alveolar macrophages can be used to estimate the cytotoxic effect of different harmful factors of the environment.     

The inside surface of the rat esophagus during ontogenesis]

The surface architecture of rat esophagus during the ontogeny is studied. Single cilia on the cells of the apical surface can be observed with the scanning electron microscope till the day 17 of the fetal period. Ciliogenesis and function of the single cilia are discussed by literature. Based upon results of our investigations we give the following interpretation: The single cilia are responsible for differentiation of the transitional columnar epithelium. The stop of mitosis, which is connected to constitution of single cilia, allows the formation of cell organelles. About the day 21 after conception ciliary cells are found. Their function is still unknown. They are observed on the esophageal surface at the same time, when primary ciliary cells arise on the trachea of the rat. The columnar epithelial cells transform into a squamous epithelium within 48 hours. The keratinisation and exfoliation of the surface cells occur definitely post-partum.     

The luminal surface of the rat trachea during ontogenesis]

To complete the hitherto existing results about the trachea epithelium, the scanning electron microscope was used to get representative statements about the apical surface of the epithelium: First of all the epithelium consists of undifferentiated round cells with cytopodia. The cells rarely carry single cilia at the same time. At the 18. day of pregnancy nearly all of the cells have a single cilium. Until the end of the intrauterine phase the single cilia are retracted again. At the 20. day ciliated cells and cells with protrusions are formed. Mucus granula are secreted into the lumen already before birth. Immediately post partum much mucus appears. Its distribution, viscosity and optical behaviour is different. For ciliated cells and goblet cells no special characteristical distribution was noticed. The building of the cells surface was detailed discussed. The question about function of the single cilia cannot yet be answered.     

Lectin binding patterns to terminal sugars of rat lung alveolar epithelial cells.

We used post-embedding cytochemical techniques to investigate the lectin binding profiles of rat lung alveolar epithelial cells. Sections from rat lung embedded in the hydrophilic resin Lowicryl K4M were incubated either directly with a lectin-gold complex or with an unlabeled lectin followed by a specific glycoprotein-gold complex. The binding patterns of the five lectins used could be divided into three categories according to their reactivity with alveolar epithelial cells: (a) the Limax flavus lectin and Ricinus communis I lectin bound to both type I and type II cell plasma membranes; (b) the Helix pomatia lectin and Sambucus nigra L. lectin bound to type II but not type I cells; and (c) the Erythrina cristagalli lectin reacted with type I cells but was unreactive with type II cells. The specificity of staining was assessed by control experiments, including pre-absorption of the lectins with various oligosaccharides and enzymatic pre-treatment of sections with highly purified glycosidases to remove specific sugar residues. The results demonstrate that these lectins can be used to distinguish between type I and type II cells and would therefore be useful probes for investigating cell dynamics during lung development and remodeling.