Mucociliary transport determined by in vivo microdialysis in the airways of normal and CF mice

B. R. Grubb,1 J. H. Jones,2 and R. C. Boucher1

1Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7248; and 2Department of Surgery/Radiology, School of Veterinary Medicine, University of California-Davis, Davis, California 95616

Submitted 2 September 2003 ; accepted in final form 13 November 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We report a novel method to measure mucociliary transport (MCT) in both the upper and lower airways of normal and CF mice. The in vivo microdialysis technique involves placing a small quantity of dye on the airway surface and a microdialysis probe a defined distance from the site of dye deposition. The dye is transported toward the probe by ciliary transport and, upon reaching the microdialysis probe, diffuses across the dialysis membrane and is collected in the dialysate leaving the probe. The rate of MCT is calculated from the length of time from dye deposition to recovery. The rate of tracheal MCT in normal mice was 2.2 ± 0.45 (SE) mm/min (n = 6), a value similar to that in reports using other techniques. MCT in CF mice was not different (2.3 ± 0.29, n = 6), consistent with previous observations suggesting that tracheal ion transport properties are not different between CF and normal mice. The rate of MCT in the nasal cavity of normal mice was slower than in the trachea (1.3 ± 0.26, n = 4). MCT in the CF mouse nasal cavity (1.4 ± 0.31, n = 8), a region in which the CF mouse exhibits bioelectric properties similar to the human CF patient, was, again, not different from the normal mouse, perhaps reflecting copious gland secretion offsetting Na+ and liquid hyperabsorption. In conclusion, we have developed a versatile, simple in vivo method to measure MCT in both upper and lower airways of mice and larger animals.

in vivo microdialysis; nasal cavity; trachea; cystic fibrosis


THOUSANDS OF PARTICLES and bacteria are deposited daily on airway surfaces as a result of normal respiration. Several mechanisms are present to protect surfaces of the lung against these airborne contaminants. Macrophages readily engulf the particulates (11), and antimicrobial substances in airway surface liquid (ASL) suppress bacterial growth (9). However, the first line of defense against these environmental insults is the mechanical clearance of the airway surface by mucus transport. The mucus layer and underlying periciliary liquid layer that cover airway epithelia are moved cephalad by the actions of cilia and are eventually swallowed. Consequences of failure of the mucociliary transport (MCT) system are demonstrated in genetic diseases, such as primary ciliary dyskinesia in which dysfunctional cilia result in the absence of MCT (1, 26) and cystic fibrosis (CF) in which MCT is inhibited by a decrease in the depth of the periciliary liquid layer (36). Both diseases are characterized by unremitting pulmonary infection.

With the availability of genetically engineered mouse models of these two diseases (16, 18, 34), it is now possible to study how alterations in MCT compromise lung defense. There have been several reports of mouse models in which either ciliary structure (16, 18) or function (25) has been genetically altered. These alterations result in a range of diminished ciliary function, ranging from the absence of ciliary beat (16) to a 50% decrease in the rate of ciliary beat (25). Some of these mouse models exhibit chronic airways infection (16, 18), whereas airway infections were not reported in others (25). Interestingly, none of these studies reported measurements of the rate of mucociliary transport. Thus correlation between mucociliary function and airway disease could not be determined.

In the CF mouse, the nasal epithelium is the only airway region that exhibits the ion transport defects (hyperabsorption of Na+ and impairment of cAMP-mediated Cl- secretion) that mimic the human disease (12, 15). Thus the nasal region of the CF mouse might be predicted to exhibit reduced ASL volume and hence reduced MCT. Although measurements of MCT have been made in the CF mouse trachea, none have been made in the nasal cavity.

Critical to our understanding of regulation of MCT and its relationship to lung defense is the ability to accurately measure MCT rates in both the upper and lower airways of mouse models. There are published studies on measurement of lower airway MCT in the mouse by particle transport (3, 5, 19, 39) and isotopic clearance (8). Values from these studies vary widely, and there is no method available for in vivo measurement of MCT in the nasal cavity of the mouse. Therefore, we developed a novel method to measure MCT with in vivo microdialysis (IVMD) in both the upper and lower airways of normal and CF mice. IVMD has been widely used to sample the composition of a variety of solutes from small volumes in vivo. We have modified the technique by placing a small amount of dye on the airway and used the microdialysis (MD) probe to detect transport of this dye over a known distance. We have tested this approach in the frog palate and both nasal cavity and lungs of normal and CF mice.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Frog palate MCT. Frog palate MCT measured by particle transport has served as a reference for MCT measurements (7, 31). Therefore, we initiated our studies by comparing MCT measured by IVMD with particle transport in the frog palate to validate the microdialysis technique for MCT measurements. For frog palate MCT measurements, the frog (Xenopus laevis) was killed (cardiac KCl injection) and decapitated, the lower jaw was removed, and the upper jaw (palate) was placed in a humidified Plexiglas chamber and studied at room temperature. When MCT was measured with particles, a small number of carbon particles (~10-100 µm in diameter) were placed in the mucus stream on the palate. With a calibrated eyepiece on a dissecting microscope, MCT was determined from the time required for the carbon particles to be transported over a defined distance. For the MCT using IVMD (see Microdialysis), the MD probe was positioned with the aid of a micromanipulator in the palate mucus stream originating from the internal nares. A small volume of rhodamine dye (250 nl, 5 x 10-3 M, in H2O) was placed on the palate at approximately the same location as the carbon particles were deposited. The dialysate from the probe was collected at 15-s intervals for a 24-min period after dye deposition (see Microdialysis).

Mice. Several strains of mice were used in these experiments. The CF (cftrtm1unc) and littermate controls were of mixed strain background (BALB/c, C57BL/6, DBA/2, and 129/SvEv). We also studied wild-type C57BL/6 mice. All mice studied were adults of both sexes and were allowed food and water until the time of study. For the MCT measurements, mice were anesthetized intraperitoneally with avertin (0.4 g/kg tribromoethanol, 0.4 ml/kg amilalcohol). The University of North Carolina's Institutional Animal Care and Use Committee approved all studies.

Murine nasal MCT. For the nasal MCT measurements, the lower trachea of the mouse was surgically exposed and intubated with a short length of PE-50 tubing. Next, a small incision was made in the upper trachea just below the larynx through which the MD probe (see Microdialysis) was inserted. The tip of the probe was positioned in the anterior region of the nasopharynx (NP; in the hard palate region, ~10-12 mm from the insertion site; Fig. 1). We cannot eliminate the possibility that the presence of the MD probe perturbs MCT. However, because the MD probe is placed downstream of the dye deposition site for both nasal and tracheal MCT studies, the presence of the probe should not physically disturb the cells actually clearing the dye. The dye delivery cannula (Fig. 1), preloaded with rhodamine WT dye (20 nl/g body mass, 5 x 10-3 M in H2O), was inserted in the nostril to a depth of ~3 mm. To accurately measure the volume of dye injected on the airway of each mouse, the calculated volume (approximately ±5 nl) was first measured accurately in a 500-nl syringe (SGE). Next, the measured volume of dye was injected into a short piece of PE-50 tubing, from which it was then immediately aspirated into the dye delivery cannula and injected on the airway. Water was used to dissolve the dye rather than a physiological salt solution, because studies have shown a hypotonic solution is absorbed rapidly from the airway surface, whereas a physiological salt solution is absorbed from the airway epithelia much more slowly (23). Thus addition of a small volume of water to the apical airway surface would be expected to change the volume of the ASL less than addition of a physiological solution. Importantly, it has been shown that water added to the apical surface does not appear to damage airway epithelia (33). Samples from the MD probe were usually collected for 24 min (see below). At the end of the experiment, the mouse was killed with an avertin overdose, and the distance between the dye delivery cannula and the MD probe was determined.



View larger version (77K):
[in this window]
[in a new window]
 
Fig. 1. Cross-section of an adult mouse head fixed in neutral buffered formalin (NBF) and stained with hematoxylin & eosin, showing placement and dimensions of the microdialysis (MD) probe in the hard palate region and the dye delivery cannula (colored black to increase visibility) just inside the nasal cavity. The point at which the MD probe enters the trachea (just distal to the larynx) is not shown. In the nasal cavity, mucus (and dye) flow from the nares to the nasal pharynx in the general direction of the arrows. Although it appears that the probe may occlude the pharynx, this is not the case, as the transverse dimension of the anterior nasopharynx is nearly 4x the diameter of the probe. Bar = 1,000 µm.

 

Particle transport was determined in excised nasal preparations by measuring the rate of transport of erythrocytes (present in the ASL postdissection). Excised nasal preparations, housed in a closed chamber, were perfused basolaterally with Krebs-Ringer-bicarbonate buffer gassed with 95% O2-5% CO2 and maintained at 37°C. The apical surface was covered with endogenous ASL.

The rate of MCT was also measured in both the anterior and posterior NP in situ. The rate of MCT on the anterior nasopharyngeal preparations (hard palate region) was measured by transport of particles (carbon particles) placed on the preparation after removing the bone and connective tissue after death. MCT was measured in the posterior NP (soft palate region; posteuthanasia) by timing the rate at which endogenously produced bits of mucus traversed a defined distance. The electrical transepithelial potential difference (PD) was measured in the anterior and posterior regions of the NP as described previously (13). In brief, the outflow line of the MD probe was connected via an agar bridge to a calomel electrode, which in turn was connected to a voltmeter. A second agar bridge (reference bridge), placed subcutaneously in the mouse and connected via a calomel electrode to the voltmeter, completed the circuit when the MD probe was in contact with the nasopharyngeal epithelia. Because a sufficient quantity of amiloride (blocks Na+ absorption) could not be delivered via the dialysis probe, only basal PDs were measured.

Tracheal MCT. For tracheal MCT measurements, the trachea was exposed, a small incision was made in the upper trachea just distal to the larynx, and a fine-bore cannula (~90 µm) preloaded with rhodamine dye (5 nl/g body mass, 5 x 10-3 M in H2O) was introduced in the trachea and advanced to the mainstem (and occasionally secondary) bronchus (~12 mm; Fig. 2). Once the dye cannula was in place, the tip of the MD probe was inserted ~2 mm in the trachea through the same incision (Fig. 2). The incision was made small so that the MD probe and dye cannula fit snugly, thus allowing minimal air exchange through the incision. Because the mouse was breathing through its nose, evaporative water loss through the incision should have been negligible. From the distance between the dye cannula and the MD probe, the distance the dye traveled to reach the MD probe was calculated. The dye deposition cannula was usually left in place during the experiment. The dialysate from the MD probe was collected in individual wells of a 96-well plate (see Microdialysis) for 24 min after dye injection. In a small number of preparations, the dye cannula wicked up the dye so that it reached the probe at the first 15-s sampling period. Data from these preparations were not analyzed. To prevent this problem in subsequent experiments, a small bead of quick-drying silicon (~200 µm in diameter; WPI, Sarasota, FL) was placed ~200-300 µm proximal to the tip of the dye cannula. After this modification of the dye cannula, no further dye-wicking problems were observed.



View larger version (117K):
[in this window]
[in a new window]
 
Fig. 2. Cross-section of trachea and lower airways of an adult mouse, showing placement and dimensions of the dye delivery cannula (with silicon bead on distal end) and MD probe. To avoid shrinkage of the airways during fixation, the lung preparation (unstained) was inflation fixed in situ with 2% agar in water. After the agar cooled, the mouse was immersed in NBF for 2 h, and the trachea/lung preparation was then removed from the animal and immersed in NBF until being sectioned on a vibratone. Sections were removed from the preparation until a longitudinal airway preparation was achieved. Arrows indicate general direction of mucus and dye flow. Bar = 1,000 µm.

 

Microdialysis. For frog studies, a CMA 7/2 probe (CMA Microdialysis, North Chelmsford, MA) was used. For all murine MCT studies, a CMA 20/4 probe was employed (molecular weight cut-off 20,000). The rate of perfusate flow through each probe was 4 µl/min for all experiments. The perfusate flowing through the probe was PBS for the murine studies and frog Ringer for the Xenopus studies. A previous study demonstrated that there was no net loss of liquid from the probe to the airway (13). The dialysate, i.e., the solution exiting the probe, was collected at 15-s intervals in a 96-well conical-bottom Nunc plate for a 24-min period after dye deposition. After each row of 12 wells was collected (3 min/row), the wells were covered with transparent tape to prevent evaporation of the small samples (1 µl/well). At the end of the 24-min experimental collection period, the plate was immediately inserted in a fluorometric plate reader (Cytofluor; PerSeptive Biosystems, Framingham, MA; excitation wave-length 530 {lambda}, emission 620 {lambda}), and the fluorescence of the dye present in each well was determined. The "relative fluorescence" (the fluorescence measured in each well; see Fig. 3) was directly related to the dye concentration (data not shown). Although the sample volume analyzed was small, good reproducibility was obtained between replicate samples when 1-µl replicates of rhodamine dye (5 x 10-4 M) were pipetted in the bottom of the wells [4.7% coefficient of variation (CV), n = 12]. When the dialysis probe was placed in 5 x 10-3 M rhodamine dye and replicate 1-µl samples collected from the outflow line of the dialysis probe (flow rate 4 µl/min), similar reproducibility (6.3% CV, n = 12) was observed. The conical-bottom plates were found to be crucial to obtain good counting efficiency of the very small sample volumes. When the probe was placed in a beaker containing rhodamine WT, the dye recovery from the CMA 20/4 probe (flow rate 4 µl/min) was ~10-13% and the percent dye recovery were independent of the dye concentration in the beaker. Under these conditions, dye was detectable in the dialysate at the fifth 15-s interval [75 s from time 0]. Thus it took 60 s to flush out the dead space in the probe and outflow lines under the conditions of this study.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3. Typical mucociliary transport curve, from nasal cavity of C57BL/6 mouse. A: plot of raw data points. B: natural log-transformed data, demonstrating the curve-fitting procedure to determine the point at which dye is first visible.

 

Drugs and chemicals. The rhodamine WT dye was a generous gift from Keystone. In some experiments, pilocarpine (0.7 mg/kg; Sigma) was injected subcutaneously 5 min before dye deposition. Also, a group of mice was injected with atropine (5 mg/kg ip; American Pharmaceutical Partners).

Data calculations. The earliest point at which dye was detected in the dialysate was used to calculate the rate of MCT. Several methods can be used to identify this point. First, dye concentrations (fluorometer counts) were natural log transformed and plotted as a function of time. Linear regressions were then fit to 1) the baseline, which was linear both before and after transformation, and 2) the initial linear portion of the curve, where the dye concentration was increasing, which was linearized by the semilog transformation (see Fig. 3). The sum of the error mean squares (MSE) was calculated from the two regressions. Using the same total set of data points, we then determined the summed MSE for regressions, using trial break points that were both faster and slower than the first trial value. We defined the best fit determined with this method as the break point that minimized the sum of the MSE.

We also estimated the value for the break points (i.e., the time the dye was first detectible in the perfusate) with a somewhat simpler method. By averaging the mean background dye concentration (first 8 points before dye injection), we used the first point of four in succession that was three SD above this mean to define the time that dye was first visible.

We compared the accuracy of the method of determining the point at which dye was visible in the dialysate by the three SD method with the value based on the statistical method of conditional error (see above) on 10 randomly selected curves. We found that there was no significant difference between these two methods in defining the time point at which dye was first visible (analyzed by ANOVA).

In all preparations, the distance between the dye deposition site and dye appearance at the MD probe was measured to calculate the rate of MCT. The MCT calculations assume that the dye was transported linearly (the shortest distance) toward the probe. Because the dye (especially in the nasal cavity) may follow a more circuitous route, the values we report may underestimate the rate of MCT.

A Fisher's exact test was used to determine whether there was a significant difference in the number of preparations in which dye was recovered and preparations in which no dye was recovered. A Student's t-test was used for testing differences in the means between two groups. When more than two groups were compared, an ANOVA was used.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Frog palate MCT. To validate the use of IVMD for the determination of MCT, IVMD measurements of MCT were compared with MCT measurements made using particle transport in the frog palate. The rate of MCT determined by IVMD did not differ significantly from that determined by particle transport (Fig. 4). To test whether application of rhodamine dye altered the rate of MCT, particle transport was measured before and after MCT determination by IVMD in some preparations. There were no significant differences in the rate of particle transport when measured either pre- or postdye. Thus dye/volume deposition did not appear to alter the rate of MCT.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4. Mucociliary transport (MCT) determined in the frog palate, using microdialysis (n = 7) or carbon particle transport (n = 7). Carbon particle transport was also determined after rhodamine dye application (n = 5). Data shown are means ± SE.

 

MCT in the murine nasal cavity. We measured the rate of MCT in the murine nasal cavity, comparing results from IVMD in normal mice with in situ measurements of particle transport in the anterior (hard palate region) and posterior (soft palate region) NP and on excised nasal epithelia (septal region). The rate of MCT measured in the nasal cavity with IVMD did not differ significantly from that measured by particle transport in the excised nasal epithelia or the anterior NP (Fig. 5). The rate of MCT measured by particle transport in the posterior NP (soft palate) was significantly greater than that determined for the nasal cavity by IVMD or for the other two regions in which particle transport was measured (Fig. 5).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 5. MCT determined in various regions of the nasal cavity. Data shown by the open bars were obtained by particle transport measurements (n = 4, 10, and 4 for the 3 bars, respectively), and the filled bar represents data obtained using in vivo microdialysis (n = 4). Data are means ± SE. The rate of MCT measured in the posterior nasopharynx was significantly different from that measured in the other regions (*P <= 0.05).

 

Because the MD probe was placed in the NP for the nasal MCT measurements, this region could contribute substantially to the overall rate of MCT determined. Because the anterior NP exhibited a rate of MCT (in situ particle transport) similar to that of the nasal epithelia in vitro (Fig. 5), placing the MD probe in this region would appear to yield a rate of MCT more representative of the nasal epithelia than would be obtained by placing the probe in the anterior NP. However, it was also necessary to determine whether the nasopharyngeal epithelia of the CF mouse exhibit hyperabsoption of Na+, as do the nasal epithelia of this mouse. To estimate nasopharyngeal Na+ transport, we measured the epithelial electrical PD across the anterior NP and the more posterior NP in normal and CF mice. We could detect no significant difference in PD of the posterior NP between the CF and normal mice. However, in the anterior NP associated with the hard palate region (~12 mm cephalad from the incision made just distal to the larynx), the PD was significantly elevated in the CF mouse (Fig. 6).



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 6. Nasopharyngeal potential difference (PD) determined in vivo in the posterior nasopharynx or the anterior nasopharynx. Open bars are data from normal mice and filled bars cystic fibrosis (CF) mice; n = 6 for both genotypes, posterior nasopharyngeal and n = 5 and 7 mice for normal and CF mice, respectively, for the anterior nasopharyngeal measurements. In the anterior nasopharynx the PD values differ significantly between the normal and CF mice (*P <= 0.05).

 

In numerous mice (almost exclusively the heterozygous mixed strain), none of the dye deposited just inside the nostril was recovered in the dialysate from the MD probe placed in the NP. A significant difference was found among genotypes/mouse strains in the number of mice in which dye was recovered (Fig. 7). In the mixed-strain heterozygotes (littermate controls for CF mice), only ~30% of the preparations yielded measurable dye. In the CF mice (same strain background), however, dye was recovered in nearly 90% of the mice (P <= 0.01). In C57BL/6 wild-type mice, dye was recovered in 100% of the mice studied (Fig. 7). When those preparations that failed to transport dye to the NP were visualized by open dissection, it was found that the dye often remained near the site where it had been deposited. However, beating cilia and particle transport were observed in the nasal cavity in postmortem dissection in these mice. Thus it is not apparent why dye failed to be transported in these preparations.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 7. In vivo nasal preparations in which dye was recovered in the dialysate of the MD probe placed in the anterior nasopharynx after injecting rhodamine WT in the nasal cavity. Dye was recovered (and thus successful MCT measurements made) in nearly 100% of all mice except the heterozygous (+/-) mixed-strain mice (n = 13; littermates to the CF mice; n = 9; *P <= 0.05); n = 6 for the C57Bl/6 mice and n = 4 for the heterozygous pilocarpine (pilo) and n = 11 atropine (atrop; both genotypes) mice.

 

When we compared the rate of nasal MCT of CF mice and littermate controls (using data only from those preparations that transported dye from the deposition site), no differences were found (Fig. 8). The rate of nasal MCT measured in the C57BL/6 mice was 30-40% lower than that observed for the mixed-strain mice, but this difference was not significant (Fig. 8).



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 8. Rates of MCT measured using in vivo microdialysis in the nasal cavity of heterozygous (n = 4), CF (n = 8), C57BL/6 (n = 7), and heterozygous pilocarpine (n = 4) and atropine (n = 3) mice; n = 5 atropine-treated CF mice. The pilocarpine mice exhibit a significantly greater rate of MCT (**P <= 0.01) than do the other groups. Data shown are means ± SE.

 

Because there was a very high percentage of heterozygous mice from which we recovered no dye, we hypothesized that the basal rate of secretion in these mice may be lower than that in the CF or C57BL/6 mice. Therefore, we injected a group of heterozygous mice with a very low dose of pilocarpine (a cholinergic stimulant) to induce gland secretion. In these preparations, dye was recovered in the NP in 100% of the mice (Fig. 7), and, in these animals, the rate of MCT was ~3.5-fold greater than that of unstimulated controls (Fig. 8).

We hypothetized that CF mice may have sustained MCT at levels approximately equal to that of controls by exhibiting a greater rate of basal gland secretion compared with littermate controls. Therefore, we attempted to block gland secretion by the administration of atropine (5 mg/kg ip) 20 min before the determination of MCT. The percentage of heterozygous atropinized mice from which dye was recovered was nearly identical to the untreated heterozygous mice (30.7 vs. 27.3%, respectively; Fig. 7). Although the percentage of atropinized CF mice in which dye was recovered was lower than untreated CF mice (45 vs. 88.8%, respectively), this difference was not significant. There was no tendency for the rates of MCT to differ between the atropinized and untreated mice for either genotype (Fig. 8).

Murine tracheal MCT. In the trachea, no significant difference was found in the rate of MCT between the CF mice and heterozygous littermates (Fig. 9). Again, there was a fairly sizable fraction of the preparations from the normal mice (33.3%) from which we failed to recover dye. This fraction was not significantly different from CF mice (20%). The mice that failed to transport dye were not included in the calculations of the mean MCT.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 9. Rates of MCT measured using in vivo microdialysis in the lower airways of four groups of mice: heterozygous, CF, C57BL/6, and C57BL/6 lavaged (n = 6 for each group). The rates of MCT measured in the latter group of mice differed significantly from the other 3 groups (**P <= 0.01).

 

We also measured the rate of MCT in a group of wild-type C57BL/6 mice. In the C57BL/6 mice, dye was recovered in 100% of the preparations. The rate of MCT was slightly, although not significantly, greater in the heterozygous mice than in the C57BL/6 mice.

In another group of C57BL/6 mice, the lower airways were lavaged with 4 µl PBS, and MCT was measured 20 min later. The rate of MCT was significantly elevated in these mice (Fig. 9), and again dye was recovered in 100% of these C57BL/6 preparations.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The effective functioning of the MCT system requires coordinated ciliary beating in a periciliary layer of optimal depth and composition. In addition, the quantity and rheological properties of the mucus layer are also important in effective mucus transport (37). Much of what is known about the interrelationship between the various components of the MCT system has emanated from in vitro and in vivo studies on large animals. However, it is not yet clear what regulates the basal or stimulated rate of MCT, nor how it may vary within airway regions (28) and between mammalian species (6, 37). The ability to genetically engineer mice in which the mucin composition (27), ciliary function (16, 25), and depth of the periciliary layer (36) are altered will allow us to determine how these components interrelate to determine the rate of MCT in vivo, provided that an accurate method of measuring MCT in both the upper and lower airways of the mouse is available. We have employed IVMD to measure the rate of MCT in the nasal cavity and the lower airways of mice.

The ciliated frog palate has been shown to be a good model for the study of mucus transportability and rheology (7, 31). Thus we employed the mucus-replete frog palate to compare the rate of MCT measured by particle transport with that measured using IVMD. No significant differences in the rate of frog palate MCT were detected between determinations made by particle transport compared with IVMD, suggesting that IVMD is a valid approach for measuring the rate of MCT.

We next used IVMD to measure MCT in the mouse nasal cavity and lower airways (in this paper, we define the trachea and bronchi as "lower airways"). We could locate no data describing MCT measurements in the nasal cavity of the mouse. However, MCT has been measured in the rat nose (in situ postmortem; see Ref. 24), and that study reported on a number of important features. The rate of MCT (particle transport) varied widely between regions in the nasal cavity, ranging from 0.9 mm/min (ethnoid turbinate) to 11 mm/min on the lateral wall. Areas with very slow MCT were also identified just inside the nares (distal to the squamous cells), where ciliated cells were sparse. No measurements were made in the posterior NP (soft palate).

We also found (using both IVMD and particle transport) a wide range of MCT in the mouse nasal cavity, with the highest rate of MCT measured in the posterior NP, the point at which all mucus streams converge. If the MD probe was placed in this distal region of the posterior NP, the mean rate of MCT would have likely been significantly higher because of the contribution of this area (MCT ~11 mm/min, measured with particle transport). The anterior NP (hard palate region) exhibited an MCT rate closer to that measured on the excised nasal septum. We found that the transepithelial electrical PD was significantly elevated in the anterior NP of the CF mouse. An elevated PD, attributed to hyperabsorption of Na+, is a hallmark of the disease both in human CF airways (17) and murine CF nasal epithelia (15). Although we did not deliver amiloride, an Na+ channel blocker, to the nasopharyngeal region, data from the literature suggest that hyperabsorption of Na+ would most likely explain the elevated PD in this region of the nasal cavity of the CF mouse. Thus MD probe measurements in the nasopharyngeal region of the CF mouse should reflect the phenotype of more proximal nasal tissue, which has been demonstrated to exhibit raised Na+ absorption (15).

In our heterozygote mixed-strain mice, we were unable to detect any dye at the dialysis probe in a large fraction of the mice (~70%). These healthy mice clearly had a functional MCT system, since beating cilia and particle clearance could be observed in the dissected nasal cavity. We speculate that the basal rate of secretion over the epithelia close to the nostril in these mice was low; therefore, the dye failed to be transported from the deposition site. When endogenous glandular secretion was cholinergically stimulated in these normal mice, dye was recovered in 100% of the animals, and the rate of MCT was significantly elevated (~3.5-fold increase). These results are similar to those obtained by Ballard et al. (2), in which gland stimulation induced a nearly threefold increase in the rate of MCT in porcine tracheae. Interestingly, we did not experience the problem of no detectable dye transport in the CF mice (littermates to the heterozygotes) nor in the C57BL/6 strain of mice. It may be that the latter two groups of mice had greater basal rates of secretion.

A common finding in studying MCT in mice and larger species is a large variability in the rate of MCT between subjects (3, 10, 39), failure of much of the deposited material to clear, and mucus stasis a short time after particle deposition (3, 6, 10). This may reflect what was observed in many of our nasal preparations from the mixed-strain mice.

We did not detect a difference in the rate of MCT between wild-type and CF mice that have defects in ion transport that lead to hyperabsorption by the superficial nasal epithelia (15, 36). Failure to measure a reduced rate of MCT in the nasal cavity of the CF mouse was surprising, since the volume of the periciliary liquid layer in the nasal cavity of the mouse has been found to be reduced (36). A reduced volume of liquid on the airway surface has been found to result in a significant inhibition in the rate of MCT (2). Furthermore, human CF patients also exhibit hyperabsorption of Na+, and a significant reduction in the rate of MCT in the nasal cavity compared with normal controls is observed (32).

MCT likely is governed in part by the volume of liquid on airway surfaces, which reflects the balance between secretion of liquid from the glands and modulation of surface liquid volume by the superficial epithelia. Compared with the lower airways, where the site of secretion may be the superficial epithelia of the distal airways or the alveolus, the source of liquid in the nasal cavity is most likely from the glands. In the mouse, it is likely that secretion from copious glands in the nasal cavity, rather than the superficial epithelium per se (which exhibits hyperabsorption of Na+ in the CF mice), determines the depth and viscosity of the ASL lining in the nasal cavity. Gland secretion might be disproportionately increased in CF mice if the Ca2+-activated Cl- conductance (secretion) in the nasal glands is upregulated similarly to the superficial epithelium (15). Indeed, Ballard et al. (2) have found that gland secretion is key to maintaining MCT in the porcine trachea. In an attempt to block gland secretion to test effects on MCT, we dosed animals with atropine. However, we failed to decrease the rate of MCT with atropine. There are reports suggesting that atropine is effective in blocking MCT in the lower airways of humans (20) and dogs (4). However, in a study on MCT in the nasal cavity of humans, atropine failed to decrease the rate of clearance of saline labeled with 99mTC (35). It has been suggested that atropine blocks production of respiratory secretions in response to cholinergic stimulation but has no effect on baseline secretions (38). This result may reflect the presence of other receptors that govern gland secretion rates, e.g., NK1 receptors, and failure to block these receptors could account for our failure to block secretion. Because the mice were anesthetized, we also cannot eliminate the possibility that the anesthetic increases glandular secretion rates and thus MCT.

There is a wide range of values reported for MCT in the lower airways of mice, ranging from nearly 0 to ~4.5 mm/min (Table 1). MCT has been measured most often in vivo in the mouse trachea by particle clearance (Table 1). There is also one report (8) of murine lung clearance of a relatively large volume (50 µl tracheal instillation) of radioisotopic colloidal particles. However, absolute rates of MCT were not measured in that study, so they are not included in Table 1. In most of these studies, the tracheae of anesthetized mice were opened, and particles, either dry or suspended in buffer, were deposited on the airway surface. In one report, the lungs were removed and particle transport in the airways was measured ex vivo. The rate of lower airway MCT we measured in mixed-strain mice and C57BL/6 mice is within the range of most of these studies.


View this table:
[in this window]
[in a new window]
 
Table 1. Preparation to determine MCT in the murine lower airways

 

Like other studies of MCT reported in the literature, our method suffers limitations. We cannot eliminate the possibility that the presence of the dye delivery cannula, the airway incision (especially for the tracheal MCT studies), and the presence of the MD probe may perturb basal MCT. In mice, the distance between the dye delivery site and MD probe is small (10-12 mm) because of the small size of the animals. Basal rates of MCT were measured in the range of 1.7-2 mm/min (5- to 6-fold less than the mm distance between dye cannula and MD probe). Diffusion of dye over this distance would be expected to require several hours, and indeed we recovered no dye in tracheal measurements (some carried out for 48 min instead of the usual 24 min) in six mice that had been killed and stored in the refrigerator for 2 days to assure cessation of all ciliary activity. Increasing the MCT rate, as we did with pilocarpine (~4 mm/min), will likely increase the variability of the data, as evidenced by the greater SE (Fig. 8). Our technique would likely be even more suitable to larger species, e.g., rabbits, rats, and dogs, in which the dye cannula and MD would be much further apart.

Brownstein (3) found that the rate of MCT in the lower airways of the mouse depended significantly on the mouse strain. C57BL/6 mice were found to have a significantly faster rate of tracheal MCT than DBA/2J, which had an almost undetectable rate of MCT. Interestingly, the mixed strain of mice we studied was derived in part from DBA/2 mice. There are no data on the effects of mouse strain on the rate of MCT in the nasal cavity.

We detected no difference between normal and CF mice in the rate of MCT in the lower airways. This finding was not surprising, since we have been unable to detect an altered bioelectric phenotype in the trachea of the cftrtm1UNC CF mouse (14). However, in an in vitro study in which the University of North Carolina CF mouse was bred on a C57BL/6 background, the investigators reported a significant decrease in the rate of MCT (particle transport) in CF mice (5). In another study of CF mice (cftrm1HGU), the investigators reported a significant decrease in the rate of MCT (39), although the rates of MCT in both normal and CF preparations were very low (see Table 1). Although some studies fail to demonstrate a difference in the rate of MCT (upper and lower airways) between the CF and normal human (see Ref. 30 for review), radioaerosol measurements of MCT in human CF patients have in general demonstrated a progressive impairment of MCT with increasing disease severity (29, 30).

MCT appeared to be governed primarily by ASL volume. This finding is in agreement with recent studies in porcine tracheae (2). In the nose, increasing gland secretion with pilocarpine accelerated MCT. In the trachea, we found that adding PBS to the lower airways also significantly increased the rate of MCT (Fig. 9). Interestingly, the rate of MCT reported by Look et al. (21) after addition of particles in 5 µl PBS is nearly identical to that which we measured after a 4-µl PBS tracheal lavage. Other studies have reported that hydration of the airways also enhanced the rate of MCT (3, 10, 39). Interestingly, in a mouse model exhibiting hyperabsorption of Na+ and thus a diminished volume of ASL in the lower airways, our IVMD technique has been used to demonstrate that the rate of MCT is significantly (~50%) decreased compared with control mice (22).

In summary, we have developed a method to measure MCT in the upper (nasal cavity) and lower airways of mice, using IVMD. The rate of MCT measured by IVMD in the murine lower airways is similar to that measured in murine lower airways by other techniques. In addition to providing a novel technique for murine models, this method should also be useful for MCT determinations in both airway regions of larger species. Despite the fact that the nasal epithelium of the CF mouse exhibits ion transport defects similar to its human counterpart, we detected no decrease in the rate of MCT in the nasal cavity of the CF mouse. We speculate that, in the CF mouse, copious gland secretions dominate over superficial epithelial Na+ hyperabsorption to maintain MCT in the normal range.


    ACKNOWLEDGMENTS
 
We thank Dr. Beverly Koller for providing CF mice (CFF RDP R-26-CR02). We also thank Kim Burns and the Histology Core for the histological preparations shown in Figs. 1 and 2.

GRANTS

This work was supported by the Cystic Fibrosis Foundation (Pilot & Feasibility Project S880).


    FOOTNOTES
 

Address for reprint requests and other correspondence: B. R. Grubb, Cystic Fibrosis/Pulmonary Research and Treatment Center, The Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7248 (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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Afzelius BA. A human syndrome caused by immotile cilia. Science 193: 317-319, 1976.[ISI][Medline]
  2. Ballard ST, Trout L, Mehta A, and Inglis SK. Liquid secretion inhibitors reduce mucociliary transport in glandular airways. Am J Physiol Lung Cell Mol Physiol 283: L329-L335, 2002.[Abstract/Free Full Text]
  3. Brownstein DG. Tracheal mucociliary transport in laboratory mice: evidence for genetic polymorphism. Exp Lung Res 13: 185-191, 1987.[ISI][Medline]
  4. Chopra SK. Effect of atropine on mucociliary transport velocity in anesthetized dogs. Am Rev Respir Dis 118: 367-371, 1978.[ISI][Medline]
  5. Cowley EA, Wang CG, Gosselin D, Radzioch D, and Eidelman DH. Mucociliary clearance in cystic fibrosis knockout mice infected with Pseudomonas aeruginosa. Eur Respir J 10: 2312-2318, 1997.[Abstract/Free Full Text]
  6. Felicetti SA, Wolff RK, and Muggenburg BA. Comparison of tracheal mucous transport in rats, guinea pigs, rabbits, and dogs. J Appl Physiol 51: 1612-1617, 1981.[Abstract/Free Full Text]
  7. Festa E, Guimaraes E, Macchione M, Saldiva PHN, and King M. Acute effects of uridine 5'-triphosphate on mucociliary clearance in isolated frog palate. J Aerosol Med 10: 25-39, 1997.[ISI]
  8. Foster WM, Walters DM, Longphre M, Macri K, and Miller LM. Methodology for the measurement of mucociliary function in the mouse by scintigraphy. J Appl Physiol 90: 1111-1117, 2001.[Abstract/Free Full Text]
  9. Ganz T. Antimicrobial polypeptides in host defense of the respiratory tract. J Clin Invest 109: 693-697, 2002.[Free Full Text]
  10. Gatto LA. Cholinergic and adrenergic stimulation of mucociliary transport in the rat trachea. Respir Physiol 92: 209-217, 1993.[CrossRef][ISI][Medline]
  11. Gehr P, Geiser M, Im Hof V, Schuerch S, and Cruz-Orive LM. Stereological estimation of particle retention and clearance in the intrapul-monary conducting airways of the hamster lungs. J Aerosol Sci 21: 362-368, 1990.[CrossRef][ISI]
  12. Grubb BR and Boucher RC. Pathophysiology of gene-targeted mouse models for cystic fibrosis. Physiol Rev 79: S193-S214, 1999.[Medline]
  13. Grubb BR, Chadburn JL, and Boucher RC. In vivo microdialysis for the determination of ariway surface liquid ion composition. Am J Physiol Cell Physiol 282: C1423-C1431, 2002.[Abstract/Free Full Text]
  14. Grubb BR, Paradiso AM, and Boucher RC. Anomalies in ion transport in CF mouse tracheal epithelium. Am J Physiol Cell Physiol 267: C293-C300, 1994.[Abstract/Free Full Text]
  15. Grubb BR, Vick RN, and Boucher RC. Hyperabsorption of Na+ and raised Ca2+-mediated Cl- secretion in nasal epithelia of CF mice. Am J Physiol Cell Physiol 266: C1478-C1483, 1994.[Abstract/Free Full Text]
  16. Ibanez-Tallon I, Gorokhova S, and Heintz N. Loss of function of axonemal dynein Mdnah5 causes primary ciliary dyskinesia and hydrocephalus. Hum Mol Genet 11: 715-721, 2002.[Abstract/Free Full Text]
  17. Knowles M, Gatzy J, and Boucher R. Increased bioelectric potential difference across respiratory epithelia in cystic fibrosis. N Engl J Med 305: 1489-1495, 1981.[Abstract]
  18. Kobayashi Y, Watanabe M, Okada Y, Sawa H, Takai H, Nakanishi M, Kawase Y, Suzuki H, Nagashima K, Ikeda K, and Motoyama N. Hydrocephalus, situs inversus, chronic sinusitis, and male infertility in DNA polymerase lambda-deficient mice: possible implication for the pathogenesis of immotile cilia syndrome. Mol Cell Biol 22: 2769-2776, 2002.[Abstract/Free Full Text]
  19. Kurosawa H, Wang CG, Dandurand RJ, King M, and Eidelman DH. Mucociliary function in the mouse measured in explanted lung tissue. J Appl Physiol 79: 41-46, 1995.[Abstract/Free Full Text]
  20. Lippmann M, Albert RE, Yeates DB, Berger JM, Foster WM, and Bohning DE. Factors affecting tracheobronchial mucociliary transport. Inhaled Part 1: 305-319, 1975.
  21. Look DC, Walter MJ, Williamson MR, Pang L, You Y, Sreshta JN, Johnson JE, Zander DS, and Brody SL. Effects of paramyxoviral infection on airway epithelial cell Foxj1 expression, ciliogenesis, and mucociliary function. Am J Pathol 159: 2055-2069, 2001.[Abstract/Free Full Text]
  22. Mall M. Overexpression of ENaC in mouse airways: a novel animal model for chronic bronchitis and cystic fibrosis lung disease (Abstract). Pediatr Pulmonol Suppl 25: 121, 2003.
  23. Matsui H, Grubb BR, Tarran R, Randell SH, Gatzy JT, Davis CW, and Boucher RC. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 95: 1005-1015, 1998.[ISI][Medline]
  24. Morgan KT, Jiang XZ, Patterson DL, and Gross EA. The nasal mucociliary apparatus. Correlation of structure and function in the rat. Am Rev Respir Dis 130: 275-281, 1984.[ISI][Medline]
  25. Neesen J, Kirschner R, Ochs M, Schmiedl A, Habermann B, Mueller C, Holstein AF, Nuesslein T, Adham I, and Engel W. Disruption of an inner arm dynein heavy chain gene results in asthenozoospermia and reduced ciliary beat frequency. Hum Mol Genet 10: 1117-1128, 2001.[Abstract/Free Full Text]
  26. Palmblad J, Mossberg B, and Afzelius BA. Ultrastructural, cellular, and clinical features of the immotile-cilia syndrome. Annu Rev Med 35: 481-492, 1984.[CrossRef][ISI][Medline]
  27. Parmley RR and Gendler SJ. Cystic fibrosis mice lacking Muc1 have reduced amounts of intestinal mucus. J Clin Invest 102: 1798-1806, 1998.[Abstract/Free Full Text]
  28. Phipps RJ. The airway mucociliary system. Int Rev Physiol 23: 213-260, 1981.[Medline]
  29. Regnis JA, Robinson M, Bailey DL, Cook P, Hooper P, Chan HK, Gonda I, Bautovich G, and Bye PT. Mucociliary clearance in patients with cystic fibrosis and in normal subjects. Am J Respir Crit Care Med 150: 66-71, 1994.[Abstract]
  30. Robinson M and Bye PTB. Mucociliary clearance in cystic fibrosis. Pediatr Pulmonol 33: 293-306, 2002.[CrossRef][ISI][Medline]
  31. Rubin BK, Ramirez O, and King M. Mucus-depleted frog palate as a model for the study of mucociliary clearance. J Appl Physiol 69: 424-429, 1990.[Abstract/Free Full Text]
  32. Rutland J and Cole PJ. Nasal mucociliary clearance and ciliary beat frequency in cystic fibrosis compared with sinusitis and bronchiectasis. Thorax 36: 654-658, 1981.[Abstract]
  33. Smith JJ, Travis SM, Greenberg EP, and Welsh MJ. Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 85: 229-236, 1996.[ISI][Medline]
  34. Snouwaert JN, Brigman KK, Latour AM, Malouf NN, Boucher RC, Smithies O, and Koller BH. An animal model for cystic fibrosis made by gene targeting. Science 257: 1083-1088, 1992.[ISI][Medline]
  35. Takeuchi K, Suzumura E, Majima Y, and Sakakura Y. Effect of atropine on nasal mucociliary clearance. Acta Otolaryngol 110: 120-123, 1990.[ISI][Medline]
  36. Tarran R, Grubb BR, Parsons D, Picher M, Hirsh AJ, Davis CW, and Boucher RC. The CF salt controversy: in vivo observations and therapeutic approaches. Mol Cell 8: 149-158, 2001.[ISI][Medline]
  37. Tomkiewicz RP, Albers GM, De Sanctis GT, Ramirez OE, King M, and Rubin BK. Species differences in the physical and transport properties of airway secretions. Can J Physiol Pharmacol 73: 165-171, 1995.[ISI][Medline]
  38. Wanner A. Effect of ipratropium bromide on airway mucociliary function. Am J Med 81: 23-27, 2003.
  39. Zahm JM, Gaillard D, Dupuit F, Hinnrasky J, Porteous D, Dorin JR, and Puchelle E. Early alterations in airway mucociliary clearance and inflammation of the lamina propria in CF mice. Am J Physiol Cell Physiol 272: C853-C859, 1997.[Abstract/Free Full Text]