Institute of Anatomy, University of Bern, CH-3000 Bern 9, Switzerland
Submitted 29 April 2003 ; accepted in final form 20 August 2003
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ABSTRACT |
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lung; mouse; surfactant; ultrastructure
The surface-lining layer of the airways in normal lungs consists of an aqueous phase and a surfactant film at the air-fluid interface. The continuous aqueous phase lies adjacent to the airway epithelial cells and has a relatively low viscosity. Above this periciliary layer, a gel phase has been postulated to occur, whose thickness, continuity, and very existence in different airway compartments and species are still disputed (12, 24, 33, 40). In small airways, the presence of a thick mucus layer has never been demonstrated (12, 15). However, all available evidence supports the existence of a continuous surfactant film at the air-fluid interface within the alveoli and central airways (3, 12, 15, 16, 19), which plays an important role in the displacement of particles towards the epithelium (13, 30). Although the function and homeostasis of the surfactant film lining the alveoli are well known, those of that in the airways have not been full elucidated (10, 25, 26).
As the dysfunction of the CFTR chloride channel in CF leads to abnormal surface liquids, many studies have been carried out to resolve the homeostasis of the volume and to determine the ion composition of the lung's surface lining layer in health as well as in disease (e.g., 21, 22, 27, 34, 37). Recently, evidence has been furnished of a functional as well as a biochemical disturbance in the surfactant of patients with progressive CF (reviewed by Griese in Ref. 18). Changes in the phospholipid and protein composition of this film may cause dramatic changes in the architecture of the surface-lining layer, which is the first barrier of defense against inhaled and deposited particles of biological or inorganic origin (11). But the ultrastructure of the lung-lining layer in CF is largely unknown and cannot be studied in humans.
With the advent of mouse models for CF in 1992 (8, 36), it became possible to study the mechanisms underlying lung disease in association with this disorder. The Cftrtm1HGU/Cftrtm1HGU mouse, generated by insertional mutagenesis (8), expresses a low percentage of wild-type Cftr mRNA owing to aberrant splicing (9). This lower-than-normal level of CFTR appears to be responsible for the only mild intestinal disturbances manifested by this phenotype and consequently for its improved survival rates, which permits an investigation of the pulmonary aberrations. A similar phenotype is manifested in human compound heterozygotes, who are also predicted to partially retain normal CFTR function (32). Although pancreatic function is improved in these patients, they can still develop severe lung disease. Physiological studies with Cftrtm1HGU/Cftrtm1HGU mutant mice have revealed the cAMP-dependent chloride secretion of lung epithelial cells to be reduced, as is the case in CF-patients (8, 35). Furthermore, these mice develop a more severe form of lung disease than do their non-CF littermates (6), which is pathogen specific, there being no overt clinical signs of pulmonary dysfunction at birth and before bacterial exposure (8). Moreover, the pertinent volumes and surface areas of the airway and alveolar compartments, as well as the thickness of the air-blood barrier, are the same in young adult Cftrtm1HGU/Cftrtm1HGU and non-CF mice before infection with CF-relevant pathogens (14). However, Zahm et al. (41) have demonstrated mucociliary clearance, but not ciliary beat frequency, to be impaired in the trachea of untreated Cftrtm1HGU/Cftrtm1HGU mice. The authors accounted for their observations by a postulated increase in mucus production. Other investigators have reported the phospholipid pool size to be increased in the bronchoalveolar lavage of uninfected Cftrtm1HGU/Cftrtm1HGU mice (5), a defective metabolism of primary phospholipids having been forwarded as a possible explanation for these findings. It would thus appear that structural changes in the surface-lining layer of Cftrtm1HGU/Cftrtm1HGU mice precede infection with CF-relevant pathogens. But so far, the architecture of the surface-lining layer in mice, especially in CF mice, has received very little attention.
In the present study, we compared the ultrastructures of the surface-lining layer of the intrapulmonary conducting airways in untreated young adult Cftrtm1HGU/Cftrtm1HGU and non-CF mice.
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MATERIALS AND METHODS |
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Anesthesia and preparation for lung fixation. Mice were anesthetized by an intraperitoneal injection first of ketamine hydrochloride (Ketalar; Parke-Davis, Walther-Lambert, Baar, Switzerland) and then of pentobarbital (Grogg Chemie, Bern, Switzerland), each at a dose of 50 mg/kg body wt. Under these conditions, mice are deeply anesthetized and in analgesia but still able to breathe spontaneously. Intravascular blood coagulation was inhibited by an intraperitoneal injection of heparin [250 international units/animal (Liquemine; Hofmann-La Roche, Reinach, Switzerland)]. A cannula was inserted into a tracheostoma and tightly affixed in readiness for manual ventilation after the formation of a pneumothorax.
Lung fixation. To preserve the airways and alveoli in an air-filled state, we perfusion fixed the lungs in situ. After performing a thoracotomy and removing the ventral portion of the chest wall, we introduced a cannula through the right ventricle into the pulmonary artery and held it firmly in place with surgical gut. The left atrial auricle was then opened, and the circulating blood was flushed out with a plasma substitute (polygeline, Haemaccel; Behring, Marburg, Germany). Three slow inflation-deflation cycles were then set in train, which inflated the lungs to total capacity [TLC (the 25-cm level of a water column)] and prevented atelectasis. The lungs were maintained at 60% TLC by reducing the inflation pressure to 5 cm of water (on the deflation limb), and the intravascular perfusion of fixatives was then begun. The three sequentially applied fixative solutions, 2.5% glutaraldehyde, 1.0% osmium tetroxide, and 0.5% uranyl acetate (1, 20), each contained 3% dextran (T-70, Grogg Chemie) to maintain the oncotic pressure. This triple perfusion fixation has been shown in small rodents to preserve the surface-lining layers of intrapulmonary conducting airways and alveoli, including the phospholipids of the surfactant film at the air-fluid interface (12, 15, 16).
Tissue sampling and processing for microscopic analysis. The lungs were cut into 2-mm-thick slices running perpendicular to the longitudinal axis. Every second slice (with a random start) was processed for transmission electron microscopy [TEM (Fig. 1)]. The other slices were destined for scanning electron microscopy (SEM) or other purposes. Each of the Epon-embedded slices to be used for TEM were sub-sampled, one subslice being taken from the apical region and one from the hilum of the left lobe, and one subslice from the basal region and one from the hilum of the right lobe. Sections 10-µm thick were then cut parallel to the subslice face using a Reichert Jung microtome (2050 Supercut; Leica, Glattbrugg, Switzerland). Airway-containing regions were sampled according to their size with the aid of a WILD M10 stereomicroscope (Leica). The tissue blocks corresponding to the identified regions were sawed out of the Epon subslices and cut into semithin (1- to 2-µm-thick) and ultrathin (60- to 80-nm-thick) sections. Semithin sections, stained with toluidine blue, were used for a general evaluation of the fine pulmonary structures in the light microscope. Ultrathin sections were transferred to uncoated 200-mesh copper grids, stained with uranyl acetate and lead citrate, and examined in a Philips 300 transmission electron microscope (Philips, Zürich, Switzerland) operating at 60 kV. The location of the first micrograph was selected at random. If the lining layer was not preserved at this position, the adjacent optical field was chosen. One micrograph was taken at low magnification (final magnification x9,300) to evaluate the histology of the airway wall. A series of four micrographs, embracing adjacent regions of the airway surface, was then taken at a higher magnification (final magnification x29,700). An additional micrograph was taken at a final magnification of x115,500 to resolve details such as osmiophilic film layer number.
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For SEM, two 2-mm-thick slices, one from the left and one from the right lung, were sampled from each mouse, dehydrated in ethanol, critical point-dried, and sputter-coated with platinum. They were examined in a Philips XL 30-FEG scanning electron microscope (Philips) operating at 10 kV.
TEM analysis. A total of 150 micrographs from each animal group were analyzed according to a standard protocol for the presence of specified structural elements of the airway wall and the surface-lining layer (Tables 1 and 2). The epithelium, the muscle layer, and the connective tissue were evaluated on the low-magnification micrographs, whereas the surface-lining layer was evaluated only on the higher-magnification ones. The aqueous layer adjacent to the epithelial surface, within which the cilia and microvilli of the epithelial cells and the surface macrophages are embedded, was evaluated for its uniformity ("regular" or "irregular" distribution of its components) and electron density ("opaque" or "transparent"). Osmiophilic structures within the surface-lining layer, most of which are linked with the surfactant system, were assessed predominantly by their shape (film-forming, lamellar structures, tubular myelin structures, vesicular forms, or membrane fragments), but also sometimes by their location (at the air-fluid interface or within the aqueous phase).
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SEM analysis. TEM permits a detailed ultrastructural analysis of the surface-lining layer, but the area scored, though representative, is necessarily small. SEM was therefore used to view and investigate the surface-lining layer from above over larger areas.
Statistical analysis. Data were compared by the two-sided Mann-Whitney (Wilcoxon) rank-sum test, the level of significance for differences being set at P < 0.05. First, the group mean values for each parameter were compared. Second, the group mean values for each parameter in large and small airways were compared. Third, the mean values for large and small airways within the same animal group were compared. Within the control group, data pertaining to the two mice heterozygous for Cftr were then excluded, and the statistical analysis was repeated.
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RESULTS |
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Aqueous phase. A periciliary aqueous layer was observed in 95-100% of all micrographs (Table 2, Fig. 2). In general, its constituent material tended to be more uniformly (regularly) and less irregularly distributed, as well as more electron dense (opaque) and less transparent in the larger than in the smaller airways of both groups. However, this airway size-related difference reached statistical significance (P < 0.05) only in the Cftrtm1HGU/Cftrtm1HGU group of mice. A comparison of the electron density of the material lining just the small airways in each animal group revealed a greater, though nonsignificant, opacity in the Cftrtm1HGU/Cftrtm1HGU mice.
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The aqueous phase did not usually extend much beyond the uppermost tips of the epithelial cells (<1 µm). A mucus layer lying beneath the osmiophilic surfactant film at the air-fluid interface, which therefore had to be extremely thin, could not be unambiguously distinguished from the rest of the aqueous phase on TEM micrographs in either group.
Epiphase. A second aqueous phase, overlying the first and delimited on both sides by an osmiophilic film, was encountered three times (Table 2, Figs. 2 and 5C). In one instance (large airway of a Cftrtm1HGU/Cftrtm1HGU mouse), the epiphase consisted of electrondense, diffusely distributed floccular material; in the second [large airway of a non-CF (heterozygous) mouse], it contained multilamellar structures and vesicles; and in the third [small airway of a non-CF (wild-type) mouse], it was composed exclusively of vesicular structures.
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Osmiophilic structures. Osmiophilic elements associated with the surface-lining layer and mostly linked with the surfactant system were found in 88-99% of all micrographs (Table 2, Fig. 2). Osmiophilic surfactant films, located at the air-fluid interface (Figs. 3, 4, 5), were observed in 75-88% of all micrographs. Bilayered and multilayered surfactant films were found in the smaller as well as in the larger airways of both animal groups. Films tended to be more diffuse in Cftrtm1HGU/Cftrtm1HGU than in non-CF mice (nonsignificant), especially in the large airways.
Multilamellar (Fig. 5A) and tubular-myelin structures (Fig. 5B) were encountered more often in the smaller than in the larger airways of both animal groups. Extracellular vesicles (Figs. 3 and 4) occurred at the lowest frequency in the large airways of Cftrtm1HGU/Cftrtm1HGU mice. Membrane fragments were more often recorded in the smaller than in the larger airways of both animal groups. Overall, such fragments were more frequently encountered in the aqueous layer of Cftrtm1HGU/Cftrtm1HGU mice. The differences, however, did not attain statistical significance.
When data pertaining to the two control mice that were heterozygous for Cftr were excluded from the analysis, the statistical significances of the aforementioned differences respecting osmiophilic structures remained unchanged.
Surface-lining layer by SEM. The analysis of nine large and seven small airways in Cftrtm1HGU/Cftrtm1HGU mice and of ten large and seven small airways in non-CF ones confirmed the TEM findings. The surface-lining layer covered the cilia and microvilli of epithelial cells and tended to be more compact in the larger than in the smaller airways of both animal groups (nonsignificant, Fig. 6). The thickness of the surface-lining layer beyond the uppermost tips of the epithelial cells, which included the non-SEM-resolvable surfactant film at the air-fluid interface, was constant at <1 µm.
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DISCUSSION |
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Lung fixation in situ. In the present study, the ultra-structure of the surface-lining layer of intrapulmonary conducting airways was investigated for the first time in the lungs of mice that had been fixed by the sequential intravascular perfusion of glutaraldehyde, osmium tetroxide, and uranyl acetate. This triple perfusionfixation technique has been previously shown in small laboratory animals to preserve the surface structures of alveoli and intrapulmonary conducting airways, including the surfactant film at the air-fluid interface (1, 12, 15, 16). Chemical fixation followed by dehydration is still the only technique available for imaging whole lungs in an air-filled state.
Aqueous phase. The aqueous phase, especially that of the small airways, tended to be more electron dense in Cftrtm1HGU/Cftrtm1HGU than in control mice. This appearance might be suggestive of a higher concentration of proteins or other constituents, which would support the hypothesis that surface fluids are underhydrated in CF. However, water depletion of the surface fluids in CF leads to a reduction in the volume of the periciliary phase (37), and there was no evidence of this in the present study.
The aqueous phase of the airways in both animal groups was located between the epithelium and the surfactant film at the air-fluid interface and covered the uppermost extremities (cilia and microvilli) of the epithelial cells. The surface-lining layer has been observed to be thinnest over protruding Clara cells and in positions where macrophages overlie the epithelial surface; it was thickest in ciliated zones (Figs. 3 and 4). The combined height of an epithelial cell (including cilia and/or microvilli) and the surface-lining layer appears to remain constant for a given lung compartment so as to furnish a regular (nonundulating) surface that continues into the alveoli (3, 16). Recently, Bachofen and Schürch (2) have demonstrated that the surface tension exerted by the surfactant film at the air-fluid interface influences the underlying structures in the airways as well as in the alveoli.
Predictably, the surface-lining layer did usually not extend much beyond the uppermost tips of the epithelial cells (<1 µm) in the intrapulmonary conducting airways of both animal groups (12, 15). A second aqueous phase (epiphase) overlying the first and separated from it by osmiophilic-film structures, which might represent "mucus islands" or "streets" (38), was observed only rarely in the present study. These findings accord with the absence of a substantial mucus layer in the small airway compartments. Although Zahm et al. (41) have reported an accumulation of mucus in their Cftrtm1HGU/Cftrtm1HGU mice, this occurred within the proximal trachea, where goblet cells and submucosal glands are admittedly scarce but nevertheless present.
Air-fluid interface. Preservation of the extremely fragile surfactant film at the air-fluid interface is problematic, and the chemical fixation and dehydration techniques used in the present study are known to induce its artificial rupturing or detachment from the air-fluid interface (16). But such artifacts were readily identified as blunt-ending structures in the transmission electron microscope and as fissures in the scanning electron microscope, and they did not interfere with the analysis.
In the present study, surfactant films of variable thickness were found in the airways of both animal groups. Multilayered surfactant films have been previously observed with the captive-bubble surfactometer and have been interpreted as a "surface-associated surfactant reservoir" (31). Diffuse films, which were more often observed in Cftrtm1HGU/Cftrtm1HGU mice, may be generated by tangential cutting of bilayered or multilayered surfactant films. This being the case, it is not clear why they were encountered more frequently in Cftrtm1HGU/Cftrtm1HGU than in non-CF mice. However, if all of these diffuse films represented tangentially cut multilayers, then the airways of Cftrtm1HGU/Cftrtm1HGU mice, especially the large ones, would have thicker surfactant films than those of non-CF controls.
The phospholipid pool size of fluid derived from bronchoalveolar lavages has been reported to be larger in Cftrtm1HGU/Cftrtm1HGU than in non-CF mice, which indicates that the surfactant pool size is larger. The authors interpreted this finding in terms of a decreased reuptake of phospholipids by the CF epithelium (4, 5).
Osmiophilic structures in the aqueous layer. Osmiophilic elements related to surfactant turnover, such as multilamellar structures, tubular myelin, or vesicles, are commonly encountered in the aqueous layer of alveoli (for review, see Goerke Ref. 17). But we found them also in the intrapulmonary conducting airways of both Cftrtm1HGU/Cftrtm1HGU and non-CF mice, and more frequently in smaller than in larger ones. The occurrence of these structures in the intrapulmonary conducting airways of small rodents has been previously reported (12, 15). Vesicular structures are involved in surfactant film formation and their location at the air-fluid interface has been demonstrated by Schürch et al. (31). In the present study, vesicles were less frequently encountered at the air-fluid interface in the large conducting airways of Cftrtm1HGU/Cftrtm1HGU than in non-CF mice. These vesicles have also been suggested to represent superfluous surfactant drained from the alveoli (23). However, if such were the case, then they would have been expected to occur more frequently in Cftrtm1HGU/Cftrtm1HGU mice, since it is these animals that have the larger surfactant pool size (4, 5).
Airway wall. Nonciliated cells were abundant in the intrapulmonary conducting airways of both animal groups. These cells correspond to the different sub-types of Clara cells, which represent the prevailing secretory cell type in mice (28, 29). There was no evidence of the epithelial metaplasia that has been observed in Cftrtm1HGU/Cftrtm1HGU mice after repeated exposure to CF-relevant pathogens (6).
Zahm et al. (41) have reported a significant increase in the number of inflammatory cells within the lamina propria of the tracheal mucosa of untreated Cftrtm1HGU/Cftrtm1HGU mice. Although the lamina propria tended to be thicker (nonsignificant) in Cftrtm1HGU/Cftrtm1HGU than in non-CF mice, we observed no inflammatory cells within the mucosa of the intrapulmonary airways.
In summary, the present study demonstrates the existence of no major ultrastructural differences in the surface-lining layer of intrapulmonary conducting airways between untreated young adult Cftrtm1HGU/Cftrtm1HGU and non-CF mice. These observations are relevant to the pathogenesis of human CF, firstly because the slightly "leaky" Cftrtm1HGU/Cftrtm1HGU mouse mutant is deemed to serve as a model for the human compound heterozygotes, and secondly because CF is believed to begin in the small-airway compartment.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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