Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
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ABSTRACT |
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Airway surface liquid (ASL) contains
substances important in mucociliary clearance and airway defense.
Little is known about substance concentrations in ASL because of its
small volume and sampling difficulties. We used in vivo microdialysis
(IVMD) to sample liquid lining the nasal cavity without net volume
removal and incorporated into IVMD a potential difference (PD)
electrode to assess airway integrity. The cystic fibrosis (CF) mouse
nasal epithelia exhibit ion transport defects identical to those in CF
human airways and, thus, are a good model for CF disease. We determined
that nasal liquid [Na+] (107 ± 4 mM normal;
111 ± 9 mM CF) and [Cl] (120 ± 6 mM normal;
122 ± 4 mM CF) did not differ between genotypes. The nasal liquid
[K+] (8.7 ± 0.4 mM) was significantly less in
normal than in CF mice (16.6 ± 4 mM). IVMD accurately samples
nasal liquid for ionic composition. The ionic composition of nasal
liquid in the normal and CF mice is similar.
electrolytes; cystic fibrosis mouse; potential difference electrode; airway surface liquid
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INTRODUCTION |
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THE THIN LAYER OF LIQUID covering the conducting airways is believed to be composed of a periciliary sol phase and a mucus-containing gel phase, both of which are important in mucociliary clearance and airway defense. The volume (depth) of the sol layer is thought to be important for effective ciliary beating, which promotes clearance of the bacteria and particles that have lodged within the mucus blanket. Perturbations in the depth of the sol phase of this liquid layer have been linked to significant changes in mucus clearance in cystic fibrosis (24) and pseudohypoaldosteronism (19). In contrast, it is not certain whether the ionic composition of the sol portion of this liquid layer is important, e.g., with respect to activities of the antimicrobial properties of this liquid layer (3, 8, 9, 25). Because of the small volume of airway surface liquid (ASL) and the difficulties involved in sampling this layer in airways in vivo, it has been very difficult to obtain unperturbed samples of ASL for analysis. Accordingly, there is no consensus in the literature regarding the ionic composition of ASL in normal humans or mice or, indeed, how ASL composition might be perturbed by cystic fibrosis (CF) in either species (4, 13, 15, 17, 27).
We have employed a novel method, in vivo microdialysis, which can be used in the airways of both humans and mice to obtain samples of liquid without net liquid removal from the airway surface. The samples obtained with microdialysis can be assayed for ions as well as a variety of compounds (cytokines, chemokines, defensins, nitric oxide, nucleotides, and glucose). This technique is routinely used in experimental brain research to measure the extracellular concentration of various drugs, neuotransmitters, and other compounds (7). In principle, a probe constructed of dialysis membrane is placed in an extracellular compartment, and the probe is perfused with a small volume of fluid. The solutes in the extracellular liquid diffuse across the semipermeable membrane as a result of the concentration gradient between the extracellular liquid and the perfusate flowing through the probe. The dialysate (solution leaving the probe) exits the probe and is collected and analyzed for the substance of interest. Endogenous compounds in the extracellular liquid will diffuse into the probe, whereas compounds added to the perfusate will diffuse out of the probe in proportion to their respective concentration gradients. Thus this technique can be used to continually measure the substance of interest in the extracellular liquid and to simultaneously deliver drugs and/or other substances to the tissue.
In our murine airway preparation, a small-diameter microdialysis probe
(240-µm outside diameter × 2-mm length) is positioned on the
surface of the nasal cavity and the ionic composition of the dialysate
is determined. We have incorporated a potential difference (PD)
electrode into the probe so that transepithelial PD can be
simultaneously measured and airway integrity assessed. With this
method, we have simultaneously measured Na+,
Cl, and K+ concentrations in the liquid of
the nasal epithelia of normal and CF mice in an attempt to test the
accuracy of this technique and provide new data regarding ASL ionic
composition of the liquid covering the nasal epithelia in normal and CF mice.
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MATERIALS AND METHODS |
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Male and female CF mice
(cftrtm1unc) and littermate controls
(both +/+ and +/) were studied. The animal protocol used in this
study was reviewed and approved by the University of North Carolina Institutional Animal Care and Use Committee. A total of 16 CF and 15 normal mice were used in this investigation. The CF mice had a mean
body mass of 22.1 ± 1.2 g (n = 11) (all data
shown are means ± SE; n = no. of animals),
whereas the normal littermates weighed 23.2 ± 0.96 g
(n = 14) (not all the weights of the mice were
recorded). Mice were anesthetized with avertin (0.4 ml/25 g)
intraperitoneally (12), and then a small cannula connected to a syringe containing avertin was placed in the peritoneal cavity so
that supplementary doses of the anesthetic could be administered throughout the experiment as needed (~0.075 ml/25 g each 10 min for
the duration of the study). The mice were positioned on their back with
the head tilted slightly upward. In this study, about half of the mice
breathed through their nose, and the remainder were tracheostomized. In
the mice that breathed through their nose, one nare was sealed with the
microdialysis (MD) probe (see Nasal PD measurements), and
thus they could breathe through only one nare. However, many of
the mice in this group (especially the CF animals) seemed to have
respiratory difficulties, and the mortality rate of CF animals was high
[50% of CF mice (6 of 12) died vs. 8.3% (1 of 12) of normal mice].
Data from these mice were not used. Death usually occurred 30-60
min into the study as a result of respiratory failure. Because the MD
probe in the CF nose was often covered with mucus upon removal, we
hypothesized that the production of thick mucus may have been
obstructing the one open nare in the CF mice. Therefore, we began to
intubate the mice by exposing the trachea, making a small incision, and inserting a short length of polyethylene (PE) 50 or 60 tubing in the
trachea. In the group of mice that were intubated, all mice (4 CF and 3 normal) survived the 3- to 5-h study.
A thermister probe (YSI) was placed in the rectum to continually monitor body temperature, which was maintained at 37 ± 0.5°C by means of a heat lamp on a rheostat. This heat lamp also warmed the perfusate delivered to the MD probe.
Microdialysis probe.
The microdialysis probe used for the mouse study was a CMA 7/2 (CMA
Microdialysis). This probe has a membrane of cuprophane, a molecular
weight cutoff of 6,000 daltons, a 0.24-mm outside diameter, and a
length of 2 mm. Before use, each probe was placed in 70% ethanol for
10 min with a physiological salt solution flowing through the probe at
a rate of 15 µl/min. This procedure removed the glycerol in which the
manufacturer packaged the probe. The inlet tube of the probe was
connected to an infusion pump (Harvard 22, Harvard Instrument), and a
flow rate of 0.5 µl/min was used for all in vivo studies (see
RESULTS). The flow rate of the pump and the pipettes used
in the study was carefully calibrated with the use of 1-µl constant
bore microcapillary tubes (Drummond, Broomall, PA). Before insertion
into the nasal cavity, the flow rate through each probe was calibrated.
After insertion into the nasal cavity, the flow rate through the probe
was checked at the end of each collection period. Because the recovery
of the ions of interest (Na+, K+,
Cl) appeared to decrease when a probe was reused, a new
probe was used for each mouse.
Determination of ASL composition.
To measure the concentration of the ions of interest in the nasal
liquid, an in vivo calibration method was used. The "zero net
flux"(22) method has been described as an elegant method
that yields the most accurate prediction of the extracellular
concentration of the substance of interest (7, 26). With
this method, compound(s) of interest at a number of different known
concentrations are sequentially perfused through the probe at a
constant flow rate, and the dialysate concentration of the compound is
measured. When the concentration difference between the dialysate and
perfusate is plotted as a function of the perfusate concentration, a
straight line with a negative slope should result if the initial
perfusate concentrations are properly selected, i.e., bracketing the
concentration of the substance in the endogenous liquid, and the
extracellular concentration of the substance is constant over the
measurement period (see RESULTS, Fig. 3). Consequently, if
the concentration of the compound in the perfusate is lower than that
in the nasal liquid, the compound in the nasal liquid will diffuse into
the probe, raising the concentration of the substance in the dialysate above that in the perfusate and giving a positive value on the ordinate
(see Fig. 3). If the concentration of the test substance in the
perfusate is greater than that in the nasal liquid, the compound will
diffuse from the probe into the nasal liquid, lowering the
concentration of the compound in the dialysate and giving a negative
value on the ordinate (see Fig. 3). The point at which the compound is
neither lost nor gained from the dialysate [i.e., zero net flux (ZNF),
Y = 0; see Fig. 3] is equal to the concentration of
the compound in the nasal liquid (X value). To construct the ZNF curve, four to five test solutions (Table
1) were perfused through the probe in
random order. The flow rate during all collection periods for all in
vivo experiments was 0.5 µl/min (see RESULTS). With each
new perfusate, the flow rate through the dialysis probe was raised to
15 µl/min for 2 min. This maneuver served to quickly fill the probe
with the new test perfusate and displace any bubbles that occasionally
lodged in the probe. The perfusate flow rate was then decreased to 0.5 µl/min, and an additional 8-min equilibration period was allowed
before the next 10-min collection period was begun. The dialysate was
collected for 10-min intervals, producing 5-µl samples for analysis.
In all mice, at least three 10-min samples of each test perfusate were
collected, and in some mice four to five 10-min samples were collected.
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Compositional analysis.
The samples were analyzed for Na+ and K+ with a
flame photometer as previously described (21). The
Cl concentration of each sample was analyzed with a
digital chloridometer as described previously (21). Utmost
care was taken in calibrating the instruments to assure accuracy of the
compositional data. Instruments were calibrated as described previously
(21). In addition, with each group of samples for each
mouse, an additional calibration curve was run in which 5-µl
standards were run in triplicate over the concentration range of the
perfusate sample (50-250 mM for Na+ and
Cl
, 4-25 mM for K+). These curves were
always linear and typically had a correlation coefficient of 0.999. These curves were used to calculate the ionic composition of the
samples. As an indication of the accuracy and precision of our
analytical methods, the accuracy and precision of replicate standards
over the concentration range we used is shown in Table
2. Triplicate 5-µl samples of each
perfusate were taken, analyzed, and averaged to obtain the perfusate
ionic composition for each mouse. For each ZNF curve, three to five
samples were obtained to determine the ionic composition in the
dialysate. These values were also averaged to give a mean dialysate
value for each perfusate. When water was used as the perfusate
(perfusate 1) (Table 1), the concentrations of the ions in
the dialysate were often at the lower range of the detection limit of
the assay. Therefore, when water was used as a perfusate, 5 µl of
perfusate 2 (Table 1) were spiked into each sample
(perfusate and dialysate) after collection. This brought the dialysate
samples into a range (~7 mM K+, 70 mM Na+,
and 70 mM Cl
) where both accuracy and precision were
good. The concentration of ions in perfusate 2 was then
subtracted from all the water samples to obtain the true concentration
of ions in the water dialysate. Thus each point on the ZNF curve was
constructed from an average of three perfusate samples (at each test
concentration) and at least three dialysate samples at the same
perfusate concentration.
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Nasal PD measurements. For measuring the PD across the nasal epithelia, PE 10 tubing attached to a 30-gauge needle was filled with agar (3% in 0.9% NaCl) and spliced via small silicon tubing (0.30-mm inside × 0.64-mm outside diameter) into the outflow line of the probe. After insertion of the needle into the splice, it was sealed in place with a quick-drying silicon cement (WPI, Sarasota, FL). The other end of this agar bridge was connected to a calomel electrode, which in turn was connected to a voltmeter (Physiologic Instruments 600) interfaced to a chart recorder (Servogor 124; Fisher) to record the transepithelial PD continuously. A second agar bridge (reference bridge) was placed subcutaneously in the mouse, connected via a calomel electrode to the voltmeter, completing the circuit when the MD probe was in contact with the nasal epithelia. The PD electrode was zeroed in solution 3 (outside) (Table 1) and perfused with the test solution that was first to be perfused through the probe. The probe was inserted so that the midpoint of the dialysis membrane was ~3 mm in the nasal cavity. Quick-drying cement was then placed on the exposed shaft of the probe and the nares, sealing both sides of the nose and holding the probe in place. (The mouse breathed via a tracheotomy tube in these experiments. In early experiments, the mouse was not tracheostomized, and then only the nare in which the probe was placed was sealed.) The PD electrode was very stable; at the end of the experiment the zero was rechecked and was usually within ±0.5 mV of the initial zero reading. The readings were not corrected for junction potentials, which could be as much as several millivolts but should not differ between the CF and normal mice.
Blood collection. Blood was collected at the termination of the experiment by cardiac puncture in a heparinized syringe from the anesthetized mouse. The blood was immediately centrifuged and the plasma aspirated and analyzed (5 µl) for ionic composition as described. We found that it was extremely important that the blood be collected from spontaneously breathing mice, because the plasma [K+] was significantly elevated (up to ~10 mM) immediately after euthanasia.
Statistical analysis. When more than two groups were being compared, an ANOVA was used to test whether there was a significant difference between the means. A Student-Newman-Keuls test was used for multiple comparisons among groups. When only two groups were being compared, a Student's t-test was used.
For the ZNF curves, some data sets exhibited variability, reflecting several possible problems, including the probe not being properly in contact with the ASL, problems in assay, bubbles in the probe, or movement of the probe during the study. To obtain data that best estimated the true nasal liquid composition, we applied rigid criteria to the ZNF curves for inclusion in the results. For the Na+ and Cl ![]() |
RESULTS |
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Effect of flow rate on percent recovery.
In most applications, the MD probe is used under nonequilibrium
conditions, and therefore the rate of perfusate flow through the probe
will affect the rate of gain or loss of ions from the probe to the
nasal liquid (or bath if done in vitro). Therefore, we determined the
optimal flow rate to use for our in vivo studies. Figure
1 shows an in vitro study demonstrating
that as the rate of perfusate flow is increased above 0.5 µl/min, the
relative recovery of Na+ ([Na+] in
dialysate/[Na+] medium surrounding the probe, ×100 and
expressed as a percentage) is markedly decreased. The greater the flow
rate, the lesser the equilibration time between the perfusate and the
bath, and the lower the concentration of the compound of interest in
the dialysate. Thus recovery is decreased as the flow rate is
increased. See Ref. 7 for a more detailed explanation of
relative recovery. The recovery curves for Cl and
K+ were similar to that of Na+ (data not
shown). With the CMA 7/2 probe at a flow rate of 0.5 µl/min in vitro,
we "recovered" ~65% of the bath [ion]. On the basis of these
data, we chose a flow rate of 0.5 µl/min and a 10-min sample period,
which produced a sample volume of 5 µl for analysis. The recovery of
a compound is independent of the concentration in the bath or ASL (data
not shown). (If the concentration of the compound in the bath is
increased, the concentration in the dialysate will increase
proportionately.)
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Effect of osmolality on dialysate flow rate.
Because we used a wide variety of solutions as test perfusates that
ranged in osmolality from 0 to 540 mosmol (Table 1), it is possible
that when the osmolarity of the bath (or nasal liquid) differed
markedly from that of the probe, the dialysate would gain or lose
water. An in vitro experiment was conducted in which the probe
was placed in a bath of 140 mM NaCl (280 mosmol/l) and water (0 mosmol/l), 50 mM NaCl (100 mosmol/l), or 250 mM NaCl (500 mosmol/l) was
perfused through the probe. As shown in Fig. 2, the perfusate flow rate (inflow) was
exactly matched by the dialysate flow rate (outflow). Thus, in these
experiments, the dialysate neither gained nor lost water.
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ZNF in vitro.
Figure 3 shows a typical in vitro ZNF
curve for Cl. (Very similar curves were obtained for the
Na+ and K+; data not shown.) The perfusate flow
rate was 0.5 µl/min, and each collection period was 10 min.
Triplicate collection periods were run for each perfusate. A regression
analysis was run on each set of data, and the slope was used to
calculate the ZNF value (see MATERIALS AND METHODS). For
each of the ions, the calculated ZNF values were not significantly
different from the concentration of the ion measured in the bath (see
Table 4). Therefore, the ZNF value can be
used to estimate very closely an unknown ion concentration in the
medium surrounding the probe.
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ZNF in vivo.
For the ZNF curves to yield accurate data, it is important that the
concentration of the ion(s) of interest in the dialysate is in a steady
state with respect to the ions in the ASL. Replicate dialysate samples
should by definition exhibit no consistent increase or decrease in the
concentration of the ion of interest as a function of time when a
steady state has been reached. To determine whether a steady state had
been achieved in the dialysate concentration of the ion, we employed up
to four collection periods (10-min each) in initial studies and five
collection periods in some experiments. At the beginning of the
experiment (after the probe was placed in the nose), a 20-min
equilibration period was interposed before the first 10-min sample was
collected. As shown in Fig. 4, the [Na+] in the dialysate was relatively stable, and no
consistent change in the three to four replicates (either an increase
or a decrease) was observed when switching from a high to a low (or low
to high) [salt] perfusate. When the perfusate tested at the beginning
of the experiment was perfused through the probe at ~180 min, the same dialysate concentration resulted (Fig. 4). Therefore, it appears
that the system reaches a steady state rather quickly and that
perfusing with a solution containing a relatively high salt
concentration does not significantly change the composition of the
nasal liquid as evidenced by nearly identical [Na+] in
the dialysate at 180 min (as compared with the initial 25-min value)
following perfusion with a 250 mM Na+ solution (Fig. 4).
Likewise, the order in which the various test solutions were perfused
through the probe did not appear to make a difference with respect to
the ZNF values obtained (data not shown).
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Composition of nasal liquid in normal and CF mice.
The Na+, K+, and Cl composition
of the apical liquid layer on nasal epithelia from CF and normal mice
as determined from the ZNF curves is shown in Fig.
6. The [Na+] in the nasal
liquid did not differ between CF or normal mice, but this value was
significantly lower than the [Na+] in mouse plasma (Fig.
6A). The [Cl
] also did not differ between
the genotypes and did not differ from the [Cl
] measured
in the plasma (Fig. 6B). The [K+] in the nasal
liquid was significantly greater than the plasma levels for both
genotypes (Fig. 6C). Furthermore, the [K+] in
the nasal liquid of the CF mice was significantly greater than that in
the normal murine nasal liquid.
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Transepithelial PD measurements.
Because a PD electrode was incorporated in the dialysis probe, in most
experiments a continuous recording of the transepithelial PD was
obtained. In almost all experiments, the PD fell from the initial value
recorded. The nasal epithelia of the CF mice exhibited an initial PD
that was significantly greater than that recorded in the normal mice
[28.9 ± 2.1 mV (12 CF) vs.
22.1 ± 1.1 (11 normal),
P
0.01 (data for all solutions combined)]. The mean PD
at each test perfusate is shown in Table
6. (Data are shown only for mice in which
we obtained a continuous PD reading in every buffer.) There was
variability in the PD readings for both the CF and normal mice. In some
mice (both CF and normal), the PD dropped to fairly low values during
the first 20 min of the experiment and remained low for the duration of
the study. The PD appeared to be better maintained in those mice that
were tracheostomized, likely because the animals had less movement of
the chest (and head) during respiration and thus the probe was
subjected to less movement. A two-way ANOVA showed that the CF mice
exhibited a significantly greater PD across the nasal epithelia for
each of the perfusate solutions tested (Table 6). There was no
significant effect of perfusate solution on electrical PD. However, it
should be stressed that because of uncorrected junction potentials
resulting from the range of ionic composition used in the perfusate,
the PDs reported in Table 6 are meant only to give an indication of
whether the epithelial barrier was intact.
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DISCUSSION |
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The ASL contains a variety of substances that play important roles in airway defense and maintain the homeostasis of the ASL layer. Despite the importance of this liquid layer in vivo, there are few data describing the concentrations of potentially important solutes in this layer. The paucity of accurate information on the composition of ASL reflects the relative inaccessibility of the lower airways and the small volume of liquid present on airway surfaces.
To obtain more insight into the identity and composition of substances in ASL, we employed the technique of in vivo microdialysis to obtain samples that were analyzed for substances of interest and their concentrations. Our results demonstrated that in vivo microdialysis is a very useful technique for collecting samples for determining the ionic composition of the liquid lining the mouse nasal cavity. One major advantage of this method over most others used to sample ASL is that no liquid is actually removed from the airway.
As has been reported by others in a variety of applications (7, 22), we found that the in vitro recovery of solutes is significantly greater than the in vivo recovery (compare slopes of lines in Fig. 3 vs. Fig. 5; see Table 5). This discrepancy may be due in part to differences in diffusion coefficients in vitro and in vivo, reflecting differences in the viscosity in the extracellular fluid and the aqueous fluid used in probe calibration in vitro (7). Also, because recovery of a compound is directly proportional to the exposed surface area of the dialysis membrane, and because the in vivo liquid layer depth is approximately an order of magnitude less than the diameter of the probe, it is possible that immersion depth also limits probe recovery in our studies. However, it has previously been shown in rat airways that when the MD probe was only partially immersed in bronchial surface liquid, the liquid wicked over the entire probe (6). Thus it is not valid to simply use the in vitro bench-top calibration to determine the ASL composition of the compound of interest. Therefore, to obtain an accurate determination of the ionic composition of nasal liquid in vivo, it was necessary to use an in vivo calibration method. In vitro, we found that the ZNF method of probe calibration very closely estimated (within 0.5-2.5%) the ionic composition of the liquid in which the probe was immersed. Others have shown that tissue binding does not appear to alter the ability of this calibration method to predict free concentration of the substance of interest (26), suggesting that the ZNF method will yield the most accurate in vivo characterization of extracellular solute concentration (26).
We were concerned that exchange of salts between the perfusate and the thin liquid layer in the nasal cavity might change the composition of the nasal liquid layer. To test for this possibility, in some mice we collected replicate dialysate samples over a 40-min time period (Fig. 4). Our data indicated that the composition of the nasal liquid layer remained constant, regardless of the solution perfusing the dialysis probe. Though the volume of liquid on the airway surface in which the probe resides is undoubtedly small, this liquid layer is not static; rather, it is constantly flowing, in response to ciliary action, at a relatively rapid rate (~3-10 mm/min; unpublished observations). This constant turnover at the MD probe site is most likely the reason that the composition of this liquid layer was not changed by the salt exchange across the dialysis probe.
In our study we incorporated a PD electrode into the probe to make simultaneous PD measurements across the airway epithelia. There are several advantages to incorporating the PD electrode into the MD probe. Most PD measurements across airway epithelia in humans, mice, and other species employ flowing bridge electrodes (1, 12, 20), which dilute the ASL layer. In our studies, because there was no net liquid flow from the MD probe, there was no dilution of the ASL. By incorporating the PD electrode into the MD probe, the PD is measured across the same region (or site) from which the probe is sampling. Furthermore, by incorporating the PD electrode into the MD probe, rather than positioning the PD electrode beside the probe, the size of the sampling device is kept to a minimum, which is particularly important for measurements in murine airways.
The presence of a significant transepithelial PD across the nasal epithelia on which the MD probe was positioned indicated that the epithelial barrier was still intact. The initial transepithelial PDs measured immediately after the MD probe was placed in the nasal cavity were significantly elevated in CF mice compared with the normal littermates. These PDs are very similar to those that we and others have reported using the flowing bridge electrode (12, 18, 28). This initial PD reading decreased in both the normal and CF mice to a steady-state level that was still significantly elevated in CF mice. PD measurements made across murine nasal epithelia with flowing bridge electrodes exhibited a similar decline (12, 16, 18). The presence of significant steady-state PDs during the course of the MD experiments indicates an intact epithelial barrier. However, we need to point out that any "foreign body" placed on the airway epithelia undoubtedly perturbs the system to some degree. The degree to which the MD probe perturbs the airway epithelia is not known. We can only state that we obtain a measurable PD, significantly greater in CF mice, following probe placement.
Our results demonstrated that the Na+ and Cl
concentration in the liquid on murine nasal airways did not differ
significantly between normal and CF genotypes (Fig. 6). The nasal
liquid was not simply an ultrafiltrate of plasma, because the
[Na+] in the nasal liquid was significantly less and the
[K+] significantly greater than in plasma in both
genotypes. With the exception of data from one group of investigators
in which the [K+] in rat and mouse ASL was found to be
1.7 and 4.7 mM, respectively (4, 5), virtually all studies
on a variety of species report [K+] values greater than
plasma (15-30 mM humans) (2, 17, 21).
The [K+] values in the nasal liquid from CF mice were significantly greater than those in the nasal liquid of normal mice. In the murine airways, the higher PD in the CF airways may play a role in determining the ASL [K+]. In canine bronchial cells, the magnitude of the transepithelial PD was found to be significantly correlated with the luminal [K+], suggesting that the K+ distribution across the epithelia was determined in part by the electrical gradient (11). In the present study, if we calculate the predicted luminal [K+] for a Nernst distribution, using the initially recorded PDs for both genotypes, the predicted luminal [K+] would be 15.1 mM for CF and 12.6 mM for normal mice. These values are fairly close to the measured values (Fig. 6).
The murine nasal epithelia are abundantly supplied with glands. The
basal rate of glandular secretion is not known. We do not know the
extent to which the probe stimulated gland secretion, but undoubtedly
there was some glandular secretion due to the presence of the probe in
the nasal cavity. However, we did find that nasal fluid secretion (and
nasal PD) could be markedly stimulated chemically (pilocarpine) even
when the probe had been in place for several hours. Preliminary results
suggest that in both CF and normal mice, the [K+] is
significantly greater and the [Cl] significantly lower
in the stimulated gland secretions than in the nasal liquid from which
we sampled (unpublished data).
The ionic composition of the ASL in the murine trachea has been
previously measured by two different techniques. In one study (4), a capillary tube was placed in the distal opened
trachea and the small volume of liquid that collected in the tube after a 30-min period was analyzed by capillary electrophoresis. In that
study, the [Cl] was very low (57 mM), the
[Na+] was slightly higher (87.2 mM), and the
[K+] (4.7 mM) was similar to that in plasma. Limited data
from the tracheae of CF mice were similar (4). However,
because both in vivo and in vitro studies show that airway epithelia
are too water permeable to maintain sustained hypotonic liquid on the surface (23, 24), it is difficult to envision a means by
which the murine epithelial cells could generate or maintain large
osmotic (ionic) gradients predicted by the Cowley study.
In another study, a window was cut in the exposed trachea of
anesthetized mice and, with fluorescent probes, the [Na+]
and [Cl] in the ASL layer were determined by ratio
imaging fluorescence microscopy (15). In that study, the
[Na+] was determined to be 115 ± 4 mM and the
[Cl
] 140 ± 5 mM ([K+] was not
determined). Similar values were found for the CF mice. These values
are closer to what would be predicted for ASL if it were isotonic with
the plasma.
These two studies of tracheal ASL differ markedly in results, one
predicting isotonic ASL (15) and the other markedly
hypotonic ASL (4). No explanation for this discrepancy is
readily apparent. Neither study provided evidence that the epithelia
were not damaged by the collection tube (4) or the
fluorescent dye (15). Although neither study found a
difference in the ASL composition of the normal vs. the CF mouse, the
trachea of the CF mouse is not a good model for CF lung disease,
because it exhibits neither hyperabsorption of Na+ nor a
readily apparent defect in cAMP Cl secretion (see Ref.
10).
There has been no consensus in the literature as to the concentrations
of Na+, Cl, and K+ in ASL of
either normal or CF human patients. However, our data in both normal
and CF mice agree in general with the most recent data from the nasal
cavity of human subjects. The [Na+] in the liquid lining
the human nasal cavity has been determined to be 110-116 mM
(14, 21) and does not differ between CF and normal
(14, 21). The [Cl
] in liquid lining human
nasal epithelia has been determined to be ~115-125 mM (14,
21). Furthermore, neither of these human studies found this
value to differ in CF patients. The [K+] of the liquid on
the nasal epithelia of humans was not determined by Hull et al.
(14), but Knowles et al. (21) found the
[K+] to be 30 mM and not different between normal and
CF ASL.
In summary, we have adapted the microdialysis technique to obtain
samples of the nasal liquid layer without volume removal. Clearly, no
method of sampling the ASL is as yet noninvasive, including in vivo
microdialysis. However, the presence of measurable transepithelial PDs
across the airway epithelia, which were significantly greater in CF
mice, indicated that the epithelial barrier remained intact. By in vivo
calibration of the MD probe, accurate determination of the
concentration of substances of interest in the nasal liquid layer was
possible. Using this technique, we found that the [Na+]
and [Cl] in the nasal liquid exceeded 100 mM and did
not differ between CF and normal mice. Because this technique is only
minimally invasive, it can be used to obtain samples of ASL in upper
and lower airway in humans. Samples of ASL obtained with the use of in
vivo microdialysis can be analyzed for a wide variety of compounds,
including ions, cytokines, metabolites, and drugs, thus providing
better insight into the milieu of ASL in health and disease.
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ACKNOWLEDGEMENTS |
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We thank Dr. Beverly Koller for providing CF mice and Nicole L. Matkins for technical assistance.
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FOOTNOTES |
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This work was supported by Cystic Fibrosis Foundation Grant CFF R026.
Address for reprint requests and other correspondence: B. R. Grubb, Cystic Fibrosis/Pulmonary Research and Treatment Center, 7011 Thurston-Bowles Bldg., CB# 7248, Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599 (E-mail: bgrubb{at}med.unc.edu).
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.
First published February 6;10.1152/ajpcell.00612.2001
Received 26 December 2001; accepted in final form 31 January 2002.
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