THE ULTRASTRUCTURE OF MOUSE LUNG GENERAL ARCHITECTURE OF CAPILLARY AND ALVEOLAR WALLS

The general architecture of capillary and alveolar walls of the mouse lung was studied by means of the electron microscope. In order to minimize tissue damage and to improve the cutting properties of embeddings, several modifications in the tissue processing methods were adopted. These modifications were: fixation by infusion, a prolonged time of dehydration, of impregnation, and of polymerization, the use of acetone for dehydration, ammonium sulfide treatment of the fixed and washed tissue, and an elevated (80°C.) polymerization temperature combined with the use of prepolymerized methacrylate. The generally favorable effects of these modified methods upon preservation and cutting properties of embedded tissue are discussed. Both capillary endothelium and alveolar epithelium were found continuous and without pores. The endothelium was seen to be thinnest in those portions that were adjacent to alveolar air spaces. Two morphological "types" of alveolar epithelial cells were found. One protruded into the alveolar lumen with its thick portion containing the nucleus. The other was often located in a niche of the alveolar wall, and contained peculiar dark inclusions amidst numerous mitochondria. Both were attenuated at their periphery to form the thin epithelial layer. The layer between endothelium and epithelium was designated as basement membrane. It was seen to be generally thin and structureless, but was found thickened in some areas where it also contained collagen fibrils.     

Differential and specific labeling of epithelial and vascular endothelial cells of the rat lung by Lycopersicon esculentum and Griffonia simplicifolia I lectins

In the rat lung, we found that the Lycopersicon esculentum (LEA) lectin specifically binds to the epithelium of bronchioles and alveoli whereas Griffonia simplicifolia I (GS-I) binds to the endothelium of alveolar capillaries. The differential binding affinity of these lectins was examined on semithin (approximately 0.5 microns) and thin (less than 0.1 (microns) frozen sections of rat lung lavaged to remove alveolar macrophages. On semithin frozen sections, LEA bound to epithelial cells lining bronchioles and the alveoli (type I, but not type II epithelial cells). On thin frozen sections, biotinylated Lycopersicon esculentum (bLEA)-streptavidin-gold conjugates were confined primarily to the luminal plasmalemma of type I cells. bGS-I-streptavidin-Texas Red was detected on the endothelial cells of alveolar capillaries and postcapillary venules but not on those of larger venules, veins or arterioles. By electron microscopy, GS-I-streptavidin-gold complexes were localized primarily to the luminal plasmalemma of thick and thin regions of the capillary endothelium. Neither lectin labeled type II alveolar cells, but both lectins labeled macrophages in the interstitia and in incompletely lavaged alveoli.     

Intact alveolar epithelial permeability and transalveolar fluid absorption after thoracic irradiation in rats.

We have addressed the question of how the alveolar space stays relatively free of fluid when thoracic irradiation injures the pulmonary capillary endothelium and plasma fluid leaks into the interstitium. A single dose of 15 Gy to the thorax of rats significantly increased the pulmonary capillary filtration coefficient and the lung wet/dry weight ratio 2 h after irradiation. However, there was no significant increase in the release of lactose dehydrogenase or leaking of Evans blue dye into the alveolar space, indicating that alveolar epithelial permeability remained intact. We found no significant difference in the basal alveolar fluid clearance between control and irradiated animals. There was also no significant difference in blockage of alveolar fluid clearance by amiloride. This indicates that the function of the alveolar epithelial Na(+) channels is not impaired and that alveolar epithelium absorbs fluid normally. Examination of lung tissue by light microscopy demonstrated accumulation of fluid in the perivascular region but not in the alveolar space. Our data appear to indicate that the alveolar epithelial barrier function is more resistant to radiation than that of the pulmonary capillary endothelium. We conclude that intact alveolar epithelial permeability and normal transalveolar epithelial fluid absorption ability are of critical importance in keeping the alveolar space relatively free of fluid during acute radiation lung injury.     

Ultrastructural appearances of pulmonary capillaries at high transmural pressures.

Electronmicroscopic appearances of pulmonary capillaries were studied in rabbit lungs perfused in situ when the capillary transmural pressure (Ptm) was systematically raised from 12.5 to 72.5 +/- 2.5 cmH2O. The animals were anesthetized and exsanguinated, and after the chest was opened, the pulmonary artery and left atrium were cannulated and attached to reservoirs. The lungs were perfused with autologous blood for 1 min, and this was followed by saline-dextran and then buffered glutaraldehyde to fix the lungs for electron microscopy. Normal appearances were seen at 12.5 cmH2O Ptm. At 52.5 and 72.5 cmH2O Ptm, striking discontinuities of the capillary endothelium and alveolar epithelium were seen. A few disruptions were seen at 32.5 cmH2O Ptm (mostly in one animal), but the number of breaks per millimeter cell lining increased markedly up to 72.5 cmH20 Ptm, where the mean frequency was 27.8 +/- 8.6 and 13.6 +/- 1.4 (SE) breaks/mm for endothelium and epithelium, respectively. In some instances, all layers of the blood-gas barrier were disrupted and erythrocytes could be seen moving into the alveolar spaces. In about half the endothelial and epithelial breaks, the basement membranes remained intact. The average break lengths for both endothelium and epithelium did not change significantly with pressure. The width of the blood-gas barrier increased at 52.5 and 72.5 cmH2O Ptm as a result of widening of the interstitium caused by edema. The cause of the disruptions is believed to be stress failure of the capillary wall. The results show that high capillary hydrostatic pressures cause major changes in the ultrastructure of the walls of the capillaries, leading to a high-permeability form of edema.     

Stress failure in pulmonary capillaries

In the mammalian lung, alveolar gas and blood are separated by an extremely thin membrane, despite the fact that mechanical failure could be catastrophic for gas exchange. We raised the pulmonary capillary pressure in anesthetized rabbits until stress failure occurred. At capillary transmural pressures greater than or equal to 40 mmHg, disruption of the capillary endothelium and alveolar epithelium was seen in some locations. The three principal forces acting on the capillary wall were analyzed. 1) Circumferential wall tension caused by the transmural pressure. This is approximately 25 dyn/cm (25 mN/m) at failure where the radius of curvature of the capillary is 5 microns. This tension is small, being comparable with the tension in the alveolar wall associated with lung elastic recoil. 2) Surface tension of the alveolar lining layer. This contributes support to the capillaries that bulge into the alveolar spaces at these high pressures. When protein leakage into the alveolar spaces occurs because of stress failure, the increase in surface tension caused by surfactant inhibition could be a powerful force preventing further failure. 3) Tension of the tissue elements in the alveolar wall associated with lung inflation. This may be negligible at normal lung volumes but considerable at high volumes. Whereas circumferential wall tension is low, capillary wall stress at failure is very high at approximately 8 x 10(5) dyn/cm2 (8 x 10(4) N/m2) where the thickness is only 0.3 microns. This is approximately the same as the wall stress of the normal aorta, which is predominantly composed of collagen and elastin. The strength of the thin part of the capillary wall is probably attributable to the collagen IV of the basement membranes. The safety factor is apparently small when the capillary pressure is raised during heavy exercise. Stress failure causes increased permeability with protein leakage, or frank hemorrhage, and probably has a role in several types of lung disease.     

THE ULTRASTRUCTURE OF MOUSE LUNG : SOME REMARKS REGARDING THE FINE STRUCTURE OF THE ALVEOLAR BASEMENT MEMBRANE

The fine structure of the alveolar basement membrane of mouse lung was discussed on the basis of three electron micrographs. The basement membrane, i.e., the intercellular layer between endothelium and alveolar epithelium, was found to be of variable width. In its thin parts it appeared rather homogeneous, and did not reveal well defined layers of fibrils. In its thicker portions, some of which may be due to oblique sectioning, cell fragments could be seen lying inside the basement membrane layer. Their exact nature was not determined. In other thickened portions of the membrane bundles of slender (about 23 to 25 mµ) fibrils were found and were tentatively interpreted as collagen fibrils, in spite of the fact that a periodicity could not be observed.     

Origin of the glomerular basement membrane visualized after in vivo labeling of laminin in newborn rat kidneys

To examine the origin and assembly of glomerular basement membranes (GBMs), affinity purified anti-laminin IgG was directly coupled to horseradish peroxidase (HRP) and intravenously injected into newborn rats. Kidneys were then processed for peroxidase histochemistry and microscopy. Within 1 h after injection, anti-laminin bound to basement membranes of nephrons in all developmental stages (vesicle, comma, S-shaped, developing capillary loop, and maturing glomeruli). In S-shaped and capillary loop glomeruli, anti-laminin-HRP labeled a double basal lamina between the endothelium and epithelium. Sections incubated with anti-laminin in vitro showed labeling within the rough endoplasmic reticulum of endothelium and epithelium, indicating that both cell types synthesized laminin for the double basement membrane. In maturing glomeruli, injected anti-laminin-HRP bound throughout the GBMs, and double basement membranes were rarely observed. At this stage, however, numerous knobs or outpockets of basement membrane material extending far into the epithelial side of the capillary wall were identified and these were also labeled throughout their full thickness. No such outpockets were found in the endothelial cell layer of newborn rats (and they normally are completely absent in fully mature, adult glomeruli). In contrast with these results, in kidneys fixed 4-6 d after anti-laminin IgG-HRP injection, basement membranes of vesicle, comma, and S-shaped nephrons were unlabeled, indicating that they were assembled after injection. GBM labeling was seen in maturing glomeruli, however. In addition, the outpockets of basement membrane extending into the epithelium were often completely unlabeled whereas GBMs lying immediately beneath them were labeled intensely, which indicates that the outpockets were probably assembled by the epithelium. Injections of sheep anti-laminin IgG followed 8 d later with injections of biotin-rabbit anti-laminin IgG and double-label immunofluorescence microscopy confirmed that GBM formation continued during individual capillary loop expansion. GBM assembly therefore occurs by at least two different processes at separate times in development: (a) fusion of endothelial and epithelial basement membranes followed by (b) addition of new basement membrane from the epithelium into existing GBMs.     

Immunohistochemical evidence for loss of ICAM-1 by alveolar epithelial cells in pulmonary fibrosis

ICAM-1 is an intercellular adhesion molecule of the immunoglobulin supergene family involved in adherence of leukocytes to the endothelium and in leukocytic accumulation in pulmonary injury. In the current study, the antigen retrieval technique was used to detect ICAM-1 immunohistochemically in paraffin sections of lungs from human, mouse and rat as well as in bleomycin- or radiation-induced fibrotic lungs from rat and human. In normal lung tissue, the expression of ICAM-1 on alveolar type I epithelial cells is stronger than on alveolar macrophages and on endothelial cells. Preembedding immuno-electron microscopy of normal rat, mouse and human lung samples revealed sclective ICAM-1 expression on the surface of type I alveolar epithelial cells and, to a lesser extent, on the pulmonary capillary endothelium and on alveolar macrophages. In fibrotic specimens, both focal lack and strengthening of immunostaining on the surface of type I cells was found. Alveolar macrophages were found focally lacking ICAM-1 immunoreactivity. In some cases, rat type II pneumocytes exhibited positive immunoreactions for ICAM-1. Immunoelectron microscopy with preembedded rat lungs (bleomycin-exposed cases) confirmed the altered ICAM-1 distribution at the alveolar epithelial surface. In the alveolar fluid of fibrotic rat lungs, in contrast to that from untreated controls, soluble ICAM-1 was detected by western blot analysis.     

Immuno- and lectin histochemistry of epithelial subtypes and their changes in a radiation-induced lung fibrosis model of the mini pig

Cell types of lung epithelia of mini pigs have been studied using a panel of monoclonal and polyclonal antibodies against cytokeratins (CKs) and vimentin and three lectins before and after radiation-induced fibrosis. In normal tissues, CK18 specific antibodies reacted above all with type II alveolar epithelial cells, while CK7 and pan CK-specific antibodies stained the whole alveolar epithelium. In bronchial epithelial cells, CKs 7, 8, 18 and focally CKs 4 and 13 as well as vimentin were found. Cell specificity of the CK pattern was confirmed by double label immunofluorescence using type II cell-specific Maclura pomifera (MPA) lectin, type I cell specific Lycopersicon esculentum (LEA) lectin and capillary endothelium-binding Dolichos biflorus (DBA) lectin. In experimental pulmonary fibrosis, enhanced coexpression of CK and vimentin was observed in bronchial epithelium. Subtypes of alveolar epithelial cells were no longer easily distinguishable. CK18 was found to be expressed in the entire alveolar epithelium. The gradual loss of the normal alveolar epithelial marker, as seen by the binding of MPA to type I-like cells, of LEA to type II-like cells and the partial loss of MPA-binding to type II cells, was paralleled by the appearance of CK4, typical for squamous epithelia, and the occurrence of DBA-binding in epithelial cells. Implications of these results for general concepts of intermediate filament protein expression and lectin binding in the fibrotic process are discussed.     

Microbial flora of the mouse ileum mucous layer and epithelial surface.

We have developed new methods to minimize fluid shear during preparation of specimens for electron microscopy and to retain the mucous blanket that covers the tissue surface of the ileum in mice. We also used general stabilization by nonspecific antibodies to minimize the collapse of the mucous layer during dehydration for electron microscopy. These methods allowed us to visualize the gradual progression of the mucous blanket from a thin diaphanous layer in newborn animals to a very thick (ca. 50 micrometers), coherent structure in older animals that contained a mixed population of bacteria and protozoa. Some bacteria, notably filamentous forms, were patently anchored to the epithelial tissue but projected into the mucous blanket, whereas others clearly existed within the mucous blanket and were unattached to the epithelial surface. Similarly, some protozoa were firmly attached to the tissue surface, whereas others were suspended in the viscous mucous blanket. In an adult animal, the mucous blanket was a very thick layer which actually occluded most of the tissue surface and contained a rich variety of bacteria and protozoa.     

Lungs of Polypterus and Erpetoichthys

The wall of the asymmetrical saclike lungs of the fishes Polypterus and Erpetoichthys consists of several functionally different tissue layers. Their lumen is lined by a surface epithelium composed of (1) highly attenuated cells, termed pneumocytes I; (2) pneumocytes II with lamellar bodies, presumably indicating surfactant production; (3) mucous cells; and (4) ciliated cells. Underlying the pneumocytes I is a dense capillary net. The thin continuous endothelium of this net, together with the pneumocytes I, constitute the very thin blood-air barrier. The basement membrane of epithelium and endothelium fuse in the area of the blood-air barrier (thickness 210 m?m). Secretory and ciliary cells form longitudinal rows in the epithelium. Below the zone with a gas-exchanging tissue, a layer of connective tissue containing collagen and special elastic fibers occurs. The blood vessels that give rise to or drain the superficial capillary plexus are located in this connective tissue. The outermost layer of the lung consists of muscle cells, a narrow inner zone with smooth muscle cells, and an outer, broader zone with cross-striated muscle cells. The lung is innervated by myelinated and nonmyelinated nerve fibers. The morphology of the gas-exchange tissue in the lungs of these primitive bony fish is fundamentally very similar to that of the lungs of tetrapod vertebrates. The morphologic observations are in close agreement with physiologic data, disclosing well-developed respiratory capacities. Structural simplicity can be regarded as a model from which the lungs of the higher vertebrates derived. In addition to respiratory function, the lungs seem also to have hydrostatic tasks.     

The fine structure of the intra-uterine epithelium during late gestation in the blue shark,Prionace glauca

The fine structure of the intra-uterine epithelium of the pregnant blue shark,Prionace glauca, was examined. The intra-uterine epithelium was bilaminar and the underlying epithelial cell was extremely reduced in cytoplasm. Two cytological characteristics were shown in the outer epithelial cell; open inter-cellular spaces closed in the apical portion by a junction complex and, numerous mitochondria distributed in the basal and lateral portions of the cytoplasm. Secretive characteristics were not recognized in the outer epithelial cell, although few regions composed only of mucous cells were seen. The flattened endothelium of the capillary lay closely beneath the epithelium. These structures are thought to facilitate the water-solute transport and gaseous exchange. It suggests that the intra-uterine epithelium is involved in the osmoregulation of the uterine fluid and the exchange of respiratory gases between mother and fetus.     

Chloride and potassium channel function in alveolar epithelial cells

Electrolyte transport across the adult alveolar epithelium plays an important role in maintaining a thin fluid layer along the apical surface of the alveolus that facilitates gas exchange across the epithelium. Most of the work published on the transport properties of alveolar epithelial cells has focused on the mechanisms and regulation of Na(+) transport and, in particular, the role of amiloride-sensitive Na(+) channels in the apical membrane and the Na(+)-K(+)-ATPase located in the basolateral membrane. Less is known about the identity and role of Cl(-) and K(+) channels in alveolar epithelial cells, but studies are revealing important functions for these channels in regulation of alveolar fluid volume and ionic composition. The purpose of this review is to examine previous work published on Cl(-) and K(+) channels in alveolar epithelial cells and to discuss the conclusions and speculations regarding their role in alveolar cell transport function.     

Type I cell-like morphology in tight alveolar epithelial monolayers

The pulmonary alveolar epithelium separates air spaces from a fluid-filled interstitium and might be expected to exhibit high resistance to fluid and solute movement. Previous studies of alveolar epithelial barrier properties have been limited due to the complex anatomy of adult mammalian lung. In this study, we characterized a model of isolated alveolar epithelium with respect to barrier transport properties and cell morphology. Alveolar epithelial cells were isolated from rat lungs and grown as monolayers on tissue culture-treated Nuclepore filters. On Days 2-6 in primary culture, monolayers were analyzed for transepithelial resistance (Rt) and processed for electron microscopy. Mean cell surface area and arithmetic mean thickness (AMT) were determined using morphometric techniques. By Day 5, alveolar epithelial cells in vitro exhibited morphologic characteristics of type I alveolar pneumocytes, with thin cytoplasmic extensions and protruding nuclei. Morphometric data demonstrated that alveolar pneumocytes in vitro develop increased surface area and decreased cytoplasmic AMT similar to young type I cells in vivo. Concurrent with the appearance of type I cell-like morphology, monolayers exhibited high Rt (greater than 1000 omega.cm2), consistent with the development of tight barrier properties. These monolayers of isolated alveolar epithelial cells may reflect the physiological and morphological properties of the alveolar epithelium in vivo.     

Isolation of inclusion bodies from rabbit lung parenchyma

The mitochondrial-plus-lysosomal fraction of rabbit lung parenchyma was studied by equilibrium density centrifugation in continuous sucrose density gradients (specific gravity 1.035 to 1.250). High concentrations of lysosomal marker enzymes were found both in a broad band at density 1.15–1.18, a density typical for lysosomes, and in a band at density 1.06–1.07. This light density band also had the highest specific activity of phospholipid, which thin layer and gas-liquid chromatography showed to be primarily lecithin with a high content of palmitic acid residues. Electron microscopy of material from the light density band showed a homogeneous array of particles which bear a strong resemblance to the inclusion bodies of the type II alveolar epithelial cell as seen in electron micrographs of rabbit lung tissue sections. These data suggest that the light density band is an isolation of intact type II alveolar epithelial cell inclusion bodies, which previous studies have implicated as the storage site of the phospholipid moiety of pulmonary surfactant.     

缝隙连接蛋白43 研究进展

缝隙连接蛋白(Connexin,Cx)组成缝隙连接通道发挥其通讯功能,其本身也对细胞的生长、分化、凋亡、肿瘤具有调节作用。缝隙连接蛋白43(Cx43)在肺泡上皮和肺血管内皮高表达,在多种肺损伤的病理过程中起重要作用,因此成为研究热点。Cx43的表达和功能受多种因素的调节,丝裂原激活的蛋白激酶(MAPKs)是主要的调节途径。本文就Cx43在肺的病理生理过程中的作用及MAPK对其调节作用进行综述。     

Fatal pneumonia with terminal emaciation in nude mice caused by pneumonia virus of mice

Athymic nude mice used as sentinel animals in a mouse holding room died of pneumonia 17 to 32 weeks after being placed in the room. Lesions in the pulmonary parenchyma consisted of monocytic exudate, epithelial cell necrosis, hemorrhage, fibrin deposition and interstitial fibrosis. Septal edema, septal cell necrosis and septal capillary stasis were common, but there was limited sloughing of bronchial lining epithelium. Indirect fluorescence microscopy (IFA) of lung sections using pneumonia virus of mice (PVM) antibody was positive. The pneumonia and IFA results were reproduced in euthymic mice inoculated experimentally with lung suspension from naturally infected mice or with tissue culture fluid from cultures infected with American Type Culture Collection PVM. The lungs of a naturally infected nude mouse were studied by transmission electron microscopy. Virus growth was found on Type II alveolar epithelium and on poorly differentiated replacement alveolar epithelium. Virus particles appeared as long exophytic filaments containing one to six linearly arranged nucleocapsids. Inclusion bodies and intracellular virus structures were not observed.     

Regional variations of cell surface carbohydrates in human oral stratified epithelium

The distribution of blood group carbohydrate chains with antigen A, B, H type 2 chain (A and B precursor), and N-acetyllactosamine (H type 2 precursor) specificity was studied in human oral epithelium from different anatomical regions. These represented various epithelial differentiation patterns such as non-keratinized, parakeratinized, and orthokeratinized stratified squamous epithelium. The material included buccal and palatal epithelium from 20 persons with blood group A or O, gingival, and alveolar epithelium from 10 persons with blood group A or B, and buccal metaplastically keratinized epithelium from nine blood group A, two blood group B, and nine blood group O individuals. The blood group carbohydrate chains were examined in tissue sections by immunofluorescence microscopy. The A and B blood group antigens were detected by human blood group sera, and antigen H type 2 chains and N-acetyllactosamine by murine monoclonal antibodies. Each antigen showed a similar staining pattern in buccal and alveolar epithelium (non-keratinized) which differed considerably from that seen in palatal and gingival epithelium (ortho- and parakeratinized). The expression of blood group antigens A or B and the precursor antigen H type 2 chains in metaplastically keratinized buccal epithelium was found to differ significantly from that seen in normal non-keratinized buccal epithelium. The regional variations demonstrated in cell surface carbohydrates are suggested to reflect differences in tissue differentiation.     

The response of subpleural pulmonary capillary endothelium to hydrothorax in rats

The principal focus of this study was to evaluate the hypothesis that increased interstitial fluid pressures served to stimulate de novo vesicle formation in pulmonary capillary endothelium. Direct measurements of interstitial fluid pressures within the alveolar septa pose great technical difficulty. The pleural space and subpleural capillaries are easily accessible, and thus, provide a more feasible model to test this hypothesis. After hydrostatic pressure of pleural space fluid was increased by periodic saline infusions into the pleural cavity, vesicle numerical densities were significantly increased in portions of the subpleural capillary endothelium. Those segments of the endothelium that directly apposed the interstitium of the visceral pleura displayed de novo vesicle formation. The endothelial segments located immediately adjacent to the alveolar epithelium were not affected by the elevated interstitial fluid pressures. In addition to the increased vesiculation, those same segments of the endothelium were characterized by increased attenuation of their cytoplasmic compartments. These conformational changes in the plasmalemma of portions of the subpleural capillary endothelium provide support to the tentative hypothesis, however, whether the increased numbers of vesicles contribute to a potential transendothelial transport system or expand a possible static network of membrane invaginations remains uncertain.