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X-ray microanalysis of airway surface liquid in the mouse

¹Department of Medical Cell Biology, University of Uppsala, Uppsala, Sweden; ²Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany; and ³Physiology Department, University of Cambridge, Cambridge, United Kingdom

Submitted 10 August 2004 ; accepted in final form 22 December 2004

	ABSTRACT

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The ionic composition of airway surface liquid (ASL) has been debated, and, in particular for the mouse, a wide range of values has been published. Two techniques were developed to measure the elemental composition of the ASL. X-ray microanalysis of ASL was carried out at low temperature on trachea removed from isoflurane-anesthetized animals and shock-frozen. In the second technique, dextran beads were placed on top of the epithelium of the trachea removed from pentobarbital-anesthetized animals, left to equilibrate with the ASL, dried, and subjected to X-ray microanalysis. Both techniques showed that mouse tracheal ASL has significantly lower concentrations of Na and Cl (60–80 mM) than serum. Differences between the two techniques were due to different sampling of mucus. CFTR(–/–) mice had significantly higher concentrations of Na and Cl in their ASL than age-matched controls. Pilocarpine or isoproterenol stimulation significantly reduced the ion concentrations in tracheal ASL. ASL was also collected with the dextran bead method from the nasal cavity in situ in pentobarbital-anesthetized animals. In control animals, the elemental composition of nasal fluid was similar to that of tracheal ASL. Pilocarpine stimulation caused a significant increase in Na, Cl, and K; stimulation with isoproterenol or phenylephrine caused a significant increase only in K. It is concluded that mouse ASL under unstimulated conditions is hypotonic, which may be related to the relative paucity of submucosal glands in the mouse trachea.

cystic fibrosis; ion transport

It has been difficult to settle this dispute by determining the exact composition of the ASL. Published data on the composition of the ASL vary considerably (reviewed in Ref. 22). This may, in part, be due to species differences. For the ASL in humans, most reported data for Na and Cl concentrations are in the range of 80–100 mM. For the mouse, however, values for Na varying from 6 to 105 mM and for Cl varying from 1 to 120 mM have been reported (2, 7, 18, 19, 21). It is of interest to have data for the mouse, since several variants of transgenic mice with CF are available.

In the present paper, we have used X-ray microanalysis to determine the elemental composition of the ASL in normal and CF mice. Two methods were used: analysis of frozen-hydrated mouse trachea and analysis of the watery fraction of the ASL collected by dextran beads. Both methods show that the ASL in the mouse is somewhat hypotonic.

	MATERIALS AND METHODS

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Animals. For the physiological experiments, 50 NMRI mice (B&K, Sollentuna, Sweden) of both sexes, 1 mo old, were used. In addition, five NMRI mice of 15–18 mo of age were used as controls for comparison with CFTR(–/–) mice of similar age (see below). The animals were anesthetized with pentobarbital. In some experiments, the anesthetized animals were injected intraperitoneally with pilocarpine (50 mg/kg body wt) or isoproterenol (10 mg/kg body wt), and the trachea was removed 15 min after the injection. In experiments where nasal fluid was collected, the animals received an intraperitoneal injection with pilocarpine, isoproterenol (dose as above), or phenylephrine (10 mg/kg body wt).

The CFTR^tm1Cam(–/–) mice (14) (female, ages 15–18 mo) were raised in Hannover (Germany), transported by air to Uppsala (Sweden), and kept for 1 wk before the start of the experiment on a crude fiber-deficient diet with addition of a laxating salt solution (Oralav; B. Braun, Melsungen, Germany). The mice were anesthetized by spontaneous inhalation of isoflurane (Forene; Abbott Scandinavia, Kista, Sweden). The inhalation gas was administered continuously through a breathing mask (Simtec Engineering, Therwill, Switzerland) and contained a mixture of 40% oxygen, 60% nitrogen, and 2.2% isoflurane. Before harvesting of the trachea, the mice were terminated by spinal translocation. These mice were compared with control NMRI mice of approximately the same age. The experimental protocol was approved by the Regional Committee on Animal Experimentation for Uppsala County.

Frozen-hydrated specimens. The trachea was removed and immediately frozen in liquid propane cooled by liquid nitrogen to avoid compression during dissection. The trachea was then dissected into tracheal rings under liquid nitrogen. The pieces were stored in liquid nitrogen until analysis. For analysis, the tracheal rings were placed onto a specially designed holder and transferred to a Philips 525 scanning electron microscope (Philips Electron Optics, Eindhoven, The Netherlands) equipped with a Bio-Rad (Hemel Hempstead, UK) Polaron 7400E cold stage. The samples were coated with a thin carbon layer in the cold stage, at a temperature of –190°C, and kept at this temperature throughout analysis (Fig. 1).

View larger version (105K): [in this window] [in a new window]   Fig. 1. Scanning electron micrograph of frozen-hydrated mouse trachea (at –190°C) as mounted for X-ray microanalysis. The airway lumen (black) is to the left. The thin layer of airway surface liquid (ASL) is seen as a dark gray layer. The outer layer of the tracheal wall consists of cartilage (C). Bar = 100 µm.

Ion-exchange beads. Dextran (Sephadex G-25) beads (diameter 20–40 µm; Pharmacia, Uppsala, Sweden) were equilibrated for 10 min with the ASL in the trachea in the following way. A small amount of beads was placed in the opening at the base of a Microlance 3 needle (0.8 x 40 mm; Becton Dickinson, Dublin, Ireland). The needle was connected to a syringe previously filled with air, and the beads were sprayed evenly over the tracheal surface by pressure on the syringe. Earlier experiments have shown that saturation of the beads with a salt solution is obtained after 5 min (20). After absorption of the ASL, the beads were recovered by being flushed with hydrophobic volatile silicone oil (Dow Corning 200/1cS; BDH, Poole, UK) and collected in a watch glass (12). Under a preparation microscope, all adhering fluid and debris were removed from the beads, and single beads were transferred onto nylon electron microscopy grids (Agar Scientific, Stansted, UK), which had been submerged into the oil. The grid with beads was slowly lifted out of the oil bath and mounted onto an aluminum holder covered with carbon adhesive tape and left at room temperature for evaporation of the oil.

Nasal fluid was collected in Sephadex beads as follows. The Sephadex G-25 beads were applied to double-sided tape (3M, Minneapolis, MN) attached to a filter paper (width 1–2 mm, length 5 mm; Schleicher and Schuell, Dassel, Germany) (20). The filter paper with the beads was inserted into one or both nostrils of the mouse, with beads facing the nasal septum, and were kept there for 10 min. Then, the filter paper with saturated beads was removed from the nostril and carefully washed in silicon oil to ascertain that no fluid was left on the outside of the beads. In some cases, the beads were separated and each bead was individually moved to a nylon electron microscopy grid until the grid contained 10–15 beads. The grid was then carefully removed from the oil, dried by evaporation of the oil at room temperature, and mounted on a specimen holder. In other cases, when it was difficult to separate the beads from the tape, the filter paper with the beads was carefully washed in the silicon oil and mounted on a specimen holder with the beads facing upwards.

Venous and arterial blood was collected from the mice, and both plasma and serum (after clotting for 24 h at 4°C) were collected. Sephadex beads were equilibrated with serum or plasma for 10 min. After that, the beads were recovered by being flushed with silicon oil and transferred to grids as described above.

Grids with Sephadex beads were carbon coated before analysis. X-ray microanalysis of the beads was carried out at room temperature with the instrumentation described above at 20 kV for 100 s with a beam size of 100 nm. Typically, 10–12 beads were analyzed from each sample. For quantitative analysis, the data were compared with the results obtained on beads soaked in salt solutions of different concentrations (50–250 mM) (20).

Morphological studies. For morphological studies, tissue was removed from the anesthetized animal and immediately fixed in 2.5% glutaraldehyde in water or different concentrations of sodium cacodylate buffer (0.025, 0.05, 0.1, or 0.15 mM). The tissues were kept in fixative for 24 h at 4°C and then postfixed with osmium tetroxide, dehydrated in a graded ethanol series, and embedded in epoxy resin. Ultrathin sections were cut for electron microscopy, contrasted with uranyl acetate and lead citrate, and viewed at 75 kV in a Hitachi 7100 transmission electron microscope.

Statistics. Data are presented as means ± SE. Differences between more than two groups were determined by ANOVA, and differences between two groups were determined using Student's t-test.

	RESULTS

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X-ray microanalysis of the ASL layer in frozen-hydrated mouse trachea (Fig. 1) showed a Na concentration of 80 mM and a Cl concentration of 50 mM (Fig. 2). The concentration of K was considerably higher than expected for an extracellular fluid. The fluid also contained large amounts of P and S. There was no significant difference in elemental composition between the young (1 mo) control animals and the old (15–18 mo) control animals (Fig. 2). The ASL in CF mice had significantly higher concentrations of Na and Cl compared with their age-matched controls (Fig. 2). Data for Mg, P, S, and Ca were not significantly different among any of the groups (Mg, P, and S not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 2. X-ray microanalysis of frozen-hydrated mouse trachea from young control (contr) mice (1 mo old; 5 animals), old control mice (15–18 mo old; 5 animals), and CFTR(–/–) mice (3 animals). Data are in mmol/kg wet weight, means ± SE. Significant differences between age-matched controls and CFTR(–/–) animals are given: ##P < 0.01, ###P < 0.001; significant differences between young animals and CFTR(–/–) animals are given: *P < 0.05, ***P < 0.001. CF, cystic fibrosis.

View larger version (132K): [in this window] [in a new window]   Fig. 3. Transmission electron micrograph of CFTR(–/–) mouse trachea, showing a concretion (arrow) in a gland duct. Bar = 2 µm.

View larger version (14K): [in this window] [in a new window]   Fig. 4. X-ray microanalysis of ASL from the trachea collected with the Sephadex bead method. Data (in mM, means ± SE) are given for control (6 animals), pilocarpine-stimulated (pilocarpine stim; 4 animals), and isoproterenol-stimulated (isoproterenol stim; 4 animals) mice, as well as for serum/plasma (4 animals). The data for serum and plasma did not differ significantly and were pooled. Significant differences between stimulated and control trachea are indicated, *P < 0.001.

Transmission electron microscopy of the surface epithelium of mouse trachea fixed in a buffer with a strength of 200 mosmol/kgH₂O (equivalent to 100 mM NaCl) did not show any damage to the cells, compared with tissue fixed in a buffer with a strength of 300 mosmol/kgH₂O (Fig. 5). In surface epithelium fixed in a buffer with a strength of 100 mosmol/kgH₂O, minor damage was observed in the form of small vacuoles in the apical part of some of the cells (not shown).

View larger version (157K): [in this window] [in a new window]   Fig. 5. Transmission electron micrograph of surface epithelial cells from mouse trachea fixed in 100 mM sodium cacodylate buffer. No damage can be observed. Bar = 2 µm.

View larger version (16K): [in this window] [in a new window]   Fig. 6. X-ray microanalysis of nasal liquid collected with the Sephadex bead method. Data (in mM, means ± SE) are given for control (5 animals), pilocarpine-stimulated (4 animals), isoproterenol-stimulated (4 animals), and phenylephrine-stimulated mice (4 animals). Significant differences between stimulated and control trachea are indicated, **P < 0.01, ***P < 0.001.

	DISCUSSION

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There is a significant difference between the data from the frozen-hydrated tracheae and the data from the Sephadex beads. The data on the frozen-hydrated ASL show higher levels of, e.g., P and K, elements associated with cellular contents rather than with extracellular fluid. Because the ASL in the mouse is only 10 µm thin (2), measurements in the frozen-hydrated state are technically difficult. It could be proposed that the electron beam would penetrate the thin ASL layer and excite the underlying cells, including their cilia. Theoretically, the penetration of the electron beam in ice at an accelerating voltage of 10 kV can be calculated to 2–3 µm (15). This is not sufficient for overpenetration of the ASL layer. Although it cannot be completely excluded that the tips of the cilia could be excited by the electron beam, it is not likely that the high K and P content of the ASL is due to excitation of the underlying cells. If the Na in the X-ray spectra were (mainly) due to the fluid, and K (mainly) to the underlying cells, one would expect a negative correlation between Na and K, with the Na:K ratio depending on the extent of overpenetration. It was, however, found that there was no negative correlation between Na and K in the individual measurements (Fig. 7).

View larger version (9K): [in this window] [in a new window]   Fig. 7. Correlation between Na and K in measurements of frozen-hydrated trachea, showing that the Na concentration is independent of the K concentration.

Both techniques agree, however, on the fact that the ASL in mouse is hypotonic. The data also show that neither age nor the method of anesthesia affect the composition of the ASL. Our results agree reasonably well with data obtained by Cowley et al. (7), who found 87 mM for Na and 57 for Cl. However, our concentrations are lower than those published by Caldwell et al. (5), Song et al. (18), Tarran et al. (19), and Verkman (21), who found chloride concentrations of 115–120 mM. On the other hand, our values are much higher than those given by Baconnais et al. (2), who found concentrations <10 mM. It may seem unreasonable that airway surface epithelial cells could be continuously exposed to a hypotonic fluid, but ultrastructural investigations appear to confirm that the cells are not noticeably damaged by a fluid with a salt concentration of 100 mM (200 mosmol/kgH₂O), which is close to the 90 mM Na plus K measured.

The relatively low ionic concentrations in mouse ASL are in contrast with our findings in the human (20) and the pig (10). It is well known that the mouse airway has much fewer submucosal glands than pig and human airways (6). Light microscopy of the control mouse tracheae used in the present study failed to find a noticeable number of submucosal glands. It could be hypothesized that much of the ASL is produced by glands as an isotonic fluid and that the surface epithelium absorbs ions from the ASL (3). In animals with few glands relative to the extent of surface epithelium, this would result in lower ionic concentrations in the ASL compared with airways with many glands, such as pig and human airways.

CF mice have significantly higher concentrations of Na and Cl in their ASL than the controls. This appears to disagree with findings of Zahm et al. (24), who found no significant difference between ASL in control and CF mice, but since those data are presented as mmol/kg dry weight, whereas our data are in mmol/kg wet weight, these studies are difficult to compare. One should be careful, however, in extending data from the mouse to the human, because of the differences in airway architecture.

In one of the CFTR(–/–) mice, a concretion in a duct of a submucosal gland was observed. In general, transgenic mice with CF have been reported to show no evident signals of airway disease that would be comparable to the human airway with dilated gland ducts and inspissated mucus (9), but a recent report found that in long-living CFTR(–/–) mice, airway pathology developed that resembled that in CF patients (8). Our findings can thus be related to the age of the mice.

The ionic composition of the ASL can be changed by pharmacological treatment. The effect of stimulation on the ionic composition of tracheal and nasal fluid, respectively, is markedly different. The effects on the nasal fluid can be explained by assuming secretion by glands in the nose, where the cholinergic stimulation gives rise to secretion of a NaCl-rich fluid, whereas the -adrenergic agonist isoproterenol mainly causes secretion of protein-rich secretory granules, to which, apparently, K is bound. The -adrenergic agonist phenylephrine has only small effects. In the trachea, however, both pilocarpine and isoproterenol cause a significant decrease in ionic concentrations. This remarkable effect can only be explained by assuming that the ASL is diluted due to secretion of water (fluid secretion can be observed macroscopically). The origin of this water (the tracheal wall or the distal airways or alveoli) remains to be elucidated. In the trachea, different from the situation in the nose, submucosal glands would not contribute significantly to the fluid under stimulated conditions. Admittedly, the doses of the agonists used in the present study are very high and some unspecific effects may be present. However, the data provide "proof of principle" that the ionic concentrations in the ASL can be manipulated by pharmacological treatment. The experimental system used in the present study offers the possibility to directly test the effect of drugs on the ionic composition and water content of the ASL, which may be helpful for research on diseases where one wishes to increase the hydration of the fluid lining the airway wall.

	GRANTS

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This study was sponsored by the Cystic Fibrosis Foundation, the Swedish Science Research Council, the Swedish Heart Lung Foundation, and the Swedish Asthma-Allergy Association.

	ACKNOWLEDGMENTS

 
The authors thank Leif Ljung, Anders Ahlander, and Marieanne Ljungkvist for excellent technical help.

	FOOTNOTES

 

Address for reprint requests and other correspondence: G. M. Roomans, Dept. of Medical Cell Biology, Univ. of Uppsala, Box 571, SE-75123 Uppsala, Sweden (E-mail: godfried.roomans{at}medcellbiol.uu.se)

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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This Article Abstract Full Text (PDF) All Versions of this Article: 288/5/L874    most recent 00303.2004v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (6) Citing Articles via Google Scholar Google Scholar Articles by Kozlova, I. Articles by Roomans, G. M. Search for Related Content PubMed PubMed Citation Articles by Kozlova, I. Articles by Roomans, G. M.