In vivo microdialysis for determination of nasal liquid ion composition

Barbara R. Grubb, James L. Chadburn, and Richard C. Boucher

Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Table 1.   Solute concentrations of perfusates used to construct ZNF curves

The perfusate and dialysate were analyzed for Na+, Cl-, and K+ concentrations (see Compositional analysis), and the ZNF curve was constructed as described above. A linear regression analysis was performed on each set of points to calculate the concentration of the ion of interest in the nasal liquid (the point at which Y = 0).

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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Table 2.   Accuracy and precision of replicate ion determinations

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- data, the correlation coefficient between the perfusate ion concentration and the dialysate-perfusate concentration (see ZNF curve, Fig. 3) had to be -0.95 or greater. In addition, the P value of the regression line had to be <= 0.05 for the data to be included. There tended to be more variability in the K+ data, most likely because a much narrower concentration range of perfusate K+ was used (Table 1) and the absolute differences between the dialysate and perfusate K+ were much less than for the other two ions. Therefore, somewhat less rigid criteria were applied for the acceptance of these data: the correlation coefficient had to be -0.90 or greater, and the P value <=  0.05. Note that in the original paper describing the ZNF method (22), a correlation coefficient of -0.9 or greater was used to accept data. Approximately equal numbers of CF (4 of 10) and normal (3 of 11) data (ZNF curves) were excluded by these criteria. (In addition, in early experiments no data were obtained in three normal mice because of a variety of problems, including bubbles in the probe, probe movement, etc.) All data shown are means ± SE, with n = sample size.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Effect of perfusion flow rate on relative recovery ([Na+] dialysate/[Na+] perfusate, expressed as %) of Na+. The data were obtained by constructing zero net flux (ZNF) curves (with 4-5 different perfusate [Na+] values) at each of the flow rates shown (see Fig. 3), and the slope of each ZNF curve (see Fig. 3) was plotted against the perfusate flow rate for which the data were obtained.

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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Fig. 2.   Effect of salt concentration in the perfusate on the dialysate flow rate (outflow). All data were obtained in vitro. Data shown are means ± SE; n = 3 for each solution. , 50 mM NaCl in perfusate; , 250 mM NaCl in perfusate; triangle , water as perfusate. Microdialysis (MD) probe was placed in a bath of 140 mM NaCl.

This relationship was again checked in vivo in some experiments (where recovery was markedly different from the in vitro recoveries, see below) by measuring the flow rate through the probe at the end of each perfusate period before switching to the next test solution. Again, none of the in vivo dialysate flow rates differed significantly from 0.5 µl/min (perfusate flow rate) with the exception of water (Table 3). When water was used as the perfusate, the dialysate flow rate was significantly (~8%) decreased. To correct for this variable, we added raffinose (200 mosmol/l) to the water perfusate. When water plus raffinose was used as the perfusate, the in vivo flow rate of the dialysate did not differ from that of the other solutions or the flow rate of the perfusate (Table 3). Thus, in in vivo experiments, 200 mosmol/l raffinose was included when the perfusate was water.

                              
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Table 3.   In vivo dialysate flow rate

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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Fig. 3.   In vitro ZNF curve. Five solutions with the [Cl-] shown as X values were perfused through the probe. The dialysate was collected and analyzed (n = 3 at each Y value) and plotted against the perfusate concentration to obtain the ZNF curve. A line was fitted by linear regression, and the point at which Y = 0 is the calculated bath [Cl-]. In this example the bath [Cl-] was measured to be 108.7 mM and the ZNF calculated value was 110.9 mM. The linear equation was Y = 77.23 - 0.696X, R = 0.999, P <=  0.001. The recovery of the ion is calculated from the slope of the line (69.6%). The flow rate was 0.5 µl/min.


                              
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Table 4.   In vitro comparison of ion concentration determined from ZNF value to that measured in bath

The slope of the regression line (Fig. 3) is equal to the percent recovery of each ion from the bath. For the in vitro experiments (as well as in vivo experiments), the recovery for Na+ and Cl- is nearly identical (~70%), whereas that for K+ (~85%) was significantly greater (P <=  0.05; Table 5).

                              
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Table 5.   In vitro vs. in vivo recovery of ions

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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Fig. 4.   Data from in vivo microdialysis. The solid line indicates the perfusate salt concentration, which was changed at the times indicated. The square symbols indicate the replicate dialysate concentrations collected for 10-min intervals over the time period shown. Raising the perfusate salt concentration ([Na+]) to 250 mM did not appear to change the salt concentration in the nasal liquid, because when the perfusate concentration was subsequently lowered to 100 mM (~180 min), the dialysate concentrations were nearly identical to those at the start of the experiment when the perfusate concentration was also 100 mM.

In vivo ZNF curves are shown for Na+, K+, and Cl- (Fig. 5). The in vivo recovery (slope of regression line) of each ion was significantly less than when recovery was determined in vitro (Table 5). For each experiment, however, there was a significant correlation between the recovery of each ion, i.e., when the recovery of Na+ was low, so was the recovery of K+ and Cl- (data not shown). These data demonstrate that large errors could result if one were to use the in vitro recovery to determine the ASL concentration in vivo.


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Fig. 5.   Examples of ZNF curves for in vivo microdialysis. A: ZNF curve for Na+; Y = 21.97 - 0.206X. Nasal liquid [Na+] calculated from the ZNF curve is 106.7 mM. R = -0.99, P <=  0.001. B: ZNF curve of K+; Y = 2.09 - 0.218X. Nasal liquid [K+] is 9.6 mM. R = -0.97, P <=  0.01. C: ZNF curve for Cl-; Y = 18.9 - 0.175X. Nasal liquid [Cl-] is 108 mM. R = -0.98, P <=  0.001.

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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Fig. 6.   A: [Na+] in the nasal liquid determined in samples collected by microdialysis employing the ZNF technique and [Na+] determined directly in plasma samples. Nasal liquid: n = 6 normal, n = 5 CF; plasma: n = 7 normal, n = 5 CF. B: [Cl-] in nasal liquid (obtained by microdialysis) and plasma samples. Nasal liquid: n = 6 normal, n = 6 CF; plasma: n = 7 normal, n = 5 CF. C: [K+] in nasal liquid (obtained by microdialysis) and plasma samples. Nasal liquid: n = 6 normal, n = 4 CF; plasma: n = 7 normal, n = 4 CF. +P <=  0.05 normal vs. CF nasal liquid. *P <=  0.01 compared with values obtained for nasal liquid. Open bars indicate data from normal mice; solid bars indicate data from CF mice.

To estimate osmolarity, we multiplied the sum of the [Na+] and [K+] by two (21). This calculated osmolarity of the nasal liquid did not differ between normal (230 ± 8.6 mosmol/l) and CF (250 ± 12.3 mosmol/l). However, this osmolarity was significantly less than that calculated for plasma (CF 298 ± 5.1 mosmol/l; normal 290.1 ± 7.4 mosmol/l).

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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Table 6.   Electrical PD measured across normal and CF nasal epithelia in vivo


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

We thank Dr. Beverly Koller for providing CF mice and Nicole L. Matkins for technical assistance.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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