Heterogeneity of the composition and thickness of tracheal
mucus in rats
David E.
Sims and
Margaret M.
Horne
Department of Anatomy and Physiology, Atlantic Veterinary
College, University of Prince Edward Island, Charlottetown, Prince
Edward Island, Canada C1A 4P3
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ABSTRACT |
Inability to preserve airway mucus in
situ has limited our understanding of its structure and
function. This light- and transmission electron-microscopic study of
rat tracheal mucus used a nonaqueous fixative that retains mucus
(epiphase) over a lucent layer (hypophase). The fixative is a 1%
solution of osmium tetroxide dissolved in a perfluorocarbon. The mean
thickness of rat tracheal epiphase was 5 µm, with significant
variation (0.1-50 µm) around the tracheal circumference.
Tracheal mucus was thickest at the trachealis muscle region and
contained cells, cellular debris, and a variable amount of surfactant
and lipid, estimated at 4-16% of the total epiphase in five rats,
with a mean composition of 9%. Lipid was observed on the surface of
the epiphase, embedded within mucus, and at the epiphase-hypophase
interface. Refined study of developmental, physiological, and
pathological alterations to the airway coat may benefit from this
approach.
ultrastructure; epiphase; microscopy; lipid; glycoprotein
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INTRODUCTION |
UNDERSTANDING OF THE respiratory mucociliary apparatus
has been limited by the inability to reliably preserve mucus in
situ. Epithelial surfaces of the upper and middle
respiratory tract contain many kinociliated cells. Their cilia are
immersed in a watery serous layer (the hypophase) that facilitates the
ciliary return stroke. During their power stroke, cilia stiffen, extend to a mucous layer (the epiphase), and propel mucus toward the pharynx.
The hypophase and epiphase cleanse, moisten, and warm inspired air,
protecting underlying epithelial cells from direct insult. However, the
thicknesses of these supracellular layers has only been estimated, not
quantified.
The reported rate of movement of mucus appears to have considerable
species (and experimental) variation and may be as slow as 2 mm/min or
as fast as 35 mm/min (4, 17, 21). Modeling studies suggest that mucus
does not flow evenly but preferentially concentrates along troughs or
grooves (1). Variable thickness of mucin coats, if real, may be
significant. If mucins are unevenly layered, there may be thin or bare
regions that are more susceptible to injury; thus generalized estimates
of mucus thickness may be misleading. Passage of viruses and bacteria
through the epiphase and hypophase is not well understood due to an
inability to preserve microbes within an intact mucous coat. Peripheral
domains of glycoprotein mucins are able to interact with bacterial
adhesins (12); therefore, bacteria should be retained in tracheal
epiphase in a resolvable manner. In addition, there is considerable
interest and controversy as to the possible contributions of lung- and
airway-derived lipid to the mucous layer (2, 3, 5, 9, 15). Lipids, if present in appreciable amounts, could form an osmotic barrier, thus
protecting the underlying epithelial cells from noxious water-soluble agents such as sulfur dioxide gas. If present as a surface layer, lipids would complicate our perception of the sticky mucous lining of
the airways. However, the current method for most biochemical studies
of normal mucus, airway lavage, dilutes and mixes mucus with cellular
debris, lower airway secretions, and lavage fluid (usually saline) to
such an extent that the composition of tracheal epiphase can only be
estimated.
The purpose of this study was to test the hypothesis that mucus is
unevenly distributed in trachea, based on previous observations of
heterogeneous flow rates of mucus in dogs (8). A recently developed
nonaqueous fixative is reported, and the fixative appears to be ideal
for preserving airway mucus in situ. The fixative consists of osmium tetroxide dissolved in a water-immiscible
perfluorocarbon. Because osmium tetroxide is an effective fixative of
lipids, there was also an opportunity to estimate the lipid content of
mucus.
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MATERIALS AND METHODS |
A thin mucous layer situated over a serous layer and attached to
epithelial cells by just the tips of kinocilia is intrinsically fragile. Regardless of the method of preservation, great care has to be
taken to avoid disrupting the mucus. In this study, tracheas were
dissected from rats immediately after death by an overdose of
pentobarbital sodium. Approximately 10-mm lengths from the pharyngeal
swelling to the thoracic inlet were removed and were gently immersed in
one of the following fixatives: aqueous cacodylate-buffered
glutaraldehyde (n = 3), aqueous
buffered glutaraldehyde containing safranin O
(n = 1), aqueous buffered
glutaraldehyde containing ruthenium red
(n = 3), aqueous buffered
glutaraldehyde containing alcian blue
(n = 3), buffered neutral Formalin
(BNF; n = 3), aqueous buffered osmium
tetroxide (n = 3), or osmium tetroxide dissolved
in a perfluorocarbon (n = 7).
After postfixation in buffered aqueous osmium tetroxide, tracheas were
dehydrated and were embedded in plastic resin. Blocks were trimmed to
expose tracheas at approximately the middle of the samples or ~5 mm
from the pharyngeal swelling. Thicker sections for light microscopy
(0.8 µm) and thinner sections for transmission electron microscopy
(silver-gold, ~90 nm) were cut and stained. Mucous thickness was
measured with a ×40 objective lens (final magnification
×400), using an ocular reticule. As indicated in Fig.
1, 12 sites of measurement per trachea were
selected based on placement of site
2 in the center of the trachealis
muscle. Thickness of the mucous coat was further quantified by electron microscopy. Randomly obtained electron micrographs were used for volume-fraction estimation of lipid content of the mucous (epiphase) layer.

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Fig. 1.
Sample sites for measurement of tracheal mucus. Numbers
represent sites of measurement of mucous thickness.
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Methods.
Sprague-Dawley and Long-Evans rats, weighing 250-300 g, were
obtained, cared for, and treated in compliance with guidelines established by the Canadian Council on Animal Care. The two strains and
both sexes were randomly used. Reagents used were 10% BNF (Fisher
Scientific, Orangeburg, NJ); osmium tetroxide (Pelco International, Redding, CA) used as a 1% solution in either aqueous cacodylate buffer
or FC-72 perfluorocarbon (3-M, London, ON, Canada); glutaraldehyde (Pelco International) prepared at a concentration of 2.5% in 0.5 M
sodium cacodylate buffer (JBEM, Dorval, PQ, Canada) and adjusted to pH
7.3 with 1.0 N HCl; ruthenium red (Polysciences, Warrington, PA) used
at 0.1% (wt /vol) concentration; safranin O (Fisher Scientific) used at 0.1% (wt /vol) concentration; and alcian blue (BDH,
Toronto, ON, Canada) used at 0.5% concentration. The protocols for
using ruthenium red, alcian blue, and safranin O were obtained from Hayat (7).
Tracheas were dissected immediately after euthanasia by an overdose of
pentobarbital sodium. They were dabbed with cotton gauze to remove any
blood on the cut ends and were gently immersed in fixative. After 90 min of fixation in the primary fixative at room temperature, including
changes of the fixation solutions at 10 and 60 min, tracheas in aqueous
solutions were briefly rinsed in 0.05 M cacodylate buffer and then were
postfixed in 1% buffered osmium tetroxide for 60 min (with the
exception of the 1 trachea in the aqueous glutaraldehyde section that
was not postfixed to determine if postfixation affected mucus
retention). After postfixation, they were dehydrated in ascending
concentrations of ethanol followed by propylene oxide, infiltrated, and
embedded in epon/araldite. Tracheas fixed in nonaqueous conditions were
rinsed in pure FC-72 to remove any unbound osmium tetroxide and were
then immersed in 100% ethanol. Vials containing tracheas in ethanol
were placed under mild vacuum to remove residual FC-72, which is more
volatile than ethanol, for 3 h. Once in pure ethanol, the
"nonaqueous" specimens were processed in the same manner as the
other tissues.
The ideal stain for light-microscopic examination of plastic sections
proved to be an azure II-methylene blue-safranin O combination originally described by Laczkó and Lévai (11). Conventional toluidine blue staining enabled resolution of mucus, but contrast and
clarity were inferior, and considerably more time was required for
staining. For electron microscopy, thin sections were mounted on copper
grids and were stained with 5% uranyl acetate in 50% ethanol for 30 min followed by triple lead salts (18) for 2 min in the absence of
carbon dioxide.
Volume fraction of lipid in the mucous epiphase was determined by
taking random micrographs of the epiphase at an initial magnification
of ×20,000. At that magnification, the operator of the electron
microscope cannot easily distinguish subcomponents of the mucous layer
and, hence, is not likely to introduce a bias to the sampling. Ten
micrographs were taken from each of five tracheas. Micrographs were
printed at a final magnification of ×52,000. Regions containing
cells or cellular debris were excluded. A dot-grid transparency was
placed over the micrograph, and dots were counted that lay over
surfactant /lipid vs. other, presumably glycoprotein, matrices.
Mucus presented itself in varying degrees of hydration. Care was taken
to count only those dots directly over electron-dense particles and not
the lucent spaces between the particles. Surfactant was counted with
lipid in this study, but membranes that appeared to be of organellar
origin were excluded.
Statistical analysis was performed with MiniTab and SAS software.
Mucous thickness was compared for intra- and interanimal variance by
the general linear model. Volume fraction of lipid within the epiphase
was subjected to F-test comparison
between animals. Values are presented as means ± SD, with
significance indicated by P < 0.05.
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RESULTS |
Light microscopy.
Aqueous glutaraldehyde, with or without safranin O, aqueous osmium
tetroxide, and BNF yielded no mucous coat (Fig.
2), with one exception in the trachealis
muscle region of a BNF-fixed trachea. Ruthenium red- and alcian
blue-fixed tracheas had sites showing material overlying the hypophase
(3 of 36, and 20 of 36, respectively), but with both fixatives there
was considerable swelling and rupture of mucous cells and preservation
of a flocculant material that was not consistent with a mucous coat
(Fig. 3). In contrast, there was a
discernable mucous epiphase over a clear hypophase in tracheas fixed in
nonaqueous fixative (Figs.
4-7).
Therefore, only the nonaqueous fixed tracheas were subjected to further
analysis. When heterogeneity of mucus thickness and composition became
evident, additional samples were analyzed to increase statistical
confidence. Within seven cross-sectioned tracheas analyzed at 12 sites
each, 66 of 84 possible sites had a discernable mucous epiphase over a
clear hypophase. Sixteen sites had no resolvable mucus (Fig. 4),
several sites had much thicker mucus (Figs. 5-7), and two sites
had a flocculant material indicative of disrupted epiphase (Fig. 6).

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Fig. 2.
Tracheas fixed with aqueous solutions of glutaraldehyde, Formalin, or
osmium showed good preservation of tissue structure but no mucous coat.
Figure is a light micrograph of 0.8-µm plastic section presented at
×600 magnification.
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Fig. 3.
Fixation with ruthenium red or alcian blue added to a glutaraldehyde
solution preserved a flocculant material within the tracheal lumen
along with apical swellings of epithelial cells (arrows). Figure is a
light micrograph of 0.8-µm plastic section presented at ×600
magnification.
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Fig. 4.
Fixation with nonaqueous osmium tetroxide preserved an epiphase
(arrowhead) of variable thickness that, by light microscopy, appeared
to thin down to nonexistence as shown by the left-to-right thinning of
the mucus in this micrograph. Figure is a light micrograph of 0.8-µm
plastic section presented at ×600 magnification.
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Fig. 5.
In the region of the trachealis muscle, mucus was often very thick with
an abundance of cellular debris. A clear hypophase (arrowhead) is
retained around the kinocilia beneath the epiphase. Figure is a light
micrograph of 0.8-µm plastic section presented at ×600
magnification.
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Fig. 6.
Within the nonaqueous fixation group, 2 of 84 sites for measurement
showed little or no mucus, with flocculant material (*) replacing
normal epiphase (right). There is an
abrupt transition between the disrupted region and the presumably
normal mucus. These are interpreted as sites of artifactual damage to
the epiphase. Figure is a light micrograph of 0.8-µm plastic section
presented at ×600 magnification.
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Fig. 7.
Normally smooth tracheal surface was indented or grooved at the sides
of the trachealis muscle. At these sites, epiphase mucus was thickest.
Figure is a light micrograph of 0.8-µm plastic section presented at
×600 magnification.
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A heterogeneous distribution of mucus was observed, with a tendency for
thicker mucus in the region of the trachealis muscle. Entire cells
could be resolved within the mucus, most often along the sides of the
trachealis muscle (sites
1 and
3), where a troughing or grooving of
the epithelium occurred. Overall mean thickness of the mucus was 6 ± 9 µm (n = 66) if sites without
apparent mucus were excluded and was 5 ± 8 µm
(n = 82) if they were included as zero
values. There was significant variation between animals, indicating a
lack of uniformity in the thickness of mucous epiphase.
Electron microscopy.
When sites of tracheas determined above to have no discernable mucus
were examined by electron microscopy, they were found to have a layer
of mucus that was too thin for resolution by light microscopy (Fig.
8). The mucus at those sites was between
0.1 and 0.3 µm thick. The surface layer of mucus was smooth between thicker and thinner regions, suggesting that variations were
physiological, not artifactual (Fig. 4).

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Fig. 8.
Within the nonaqueous fixation samples, sites determined to have no
epiphase by light microscopy were observed by transmission electron
microscopy to have a thin (~0.2 µm) coat (arrowheads) with a clear
hypophase region beneath. Magnification, ×14,000; bar, 1 µm.
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Adjacent to the trachealis muscle, epiphase was observed to be thicker,
often containing cellular debris (Fig. 9),
whereas epiphase on epithelia opposite the trachealis muscle usually
did not (Figs. 8 and
10-12).
Based on this observation, the mean thickness of mucus as determined by
light microscopy was recalculated, with 0.2 µm replacing zero values
for nonresolvable mucus. Then 82 of 84 possible test sites had mucus
with a mean thickness of 5 ± 8 µm, the same as determined by
light microscopy. Interanimal differences of mucous thickness remained
significant.

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Fig. 9.
In regions with thicker epiphase, cells and cellular debris
(arrowheads) were commonly observed within the mucous coat.
Magnification, ×3,500; bar, 1 µm.
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Fig. 10.
Thin epiphase includes a stack of lipid sheets (arrow) but little or no
lipid on the surface and bottom of the mucus. Magnification,
×26,000; bar, 1 µm.
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Fig. 11.
Region of epiphase with extensive coverage by a monolayer of lipid
(arrow; note contrast between this epiphase surface and that shown in
Fig. 10) along with numerous lipid profiles within the mucus.
Magnification, ×46,000; bar, 1 µm.
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Fig. 12.
Higher magnification of part of Fig. 8. Surfactant is shown with its
characteristic lattice-like profile. Lipid bi- and trilayers cover a
significant portion of the surface and about one-half of the bottom of
the epiphase. Magnification, ×46,000; bar, 1 µm.
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Noting a tendency for thicker mucus in the region of the trachealis
muscle, the observation points were reconfigured such that the
circumference of the trachea was divided into 4 regions instead of 12. These were named the dorsal (adjacent to the trachealis muscle),
ventral, and left and right lateral regions. Sampling points
1, 2,
and 3 were merged into the dorsal
region, points 4, 5,
and 6 were merged into right lateral,
etc. The thickness of mucus in these regions based on 21 or 22 sample
sites per region was as follows: dorsal, 11 ± 15 µm; ventral, 2 ± 3 µm; and left and right lateral, 4 ± 6 and 3 ± 4 µm,
respectively. Mucous was significantly thicker in the dorsal region.
Lipid profiles were seen throughout the epiphase (Figs. 10-12),
taking the appearance of mono- and bilayers, sheets and whorls, and
surfactant-like complexes. Lipid was observed on the luminal surface of
the epiphase (Fig. 11), within the epiphase (Fig. 10), and at its base
(Figs. 11 and 12). A volume fraction of surfactant and lipid in the
epiphase was calculated based on point counting from 10 randomly
selected micrographs from each of five animals. The epiphase was
composed of 9 ± 9% surfactant and lipid. Means of lipid
composition for individual animals ranged from 4 to 16%, with
interanimal variance indicating significant difference.
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DISCUSSION |
Nonaqueous fixatives offer significant advantages over conventional
aqueous fixatives. They may retain more protein (14) and lipid (13).
Perfluorocarbons are also desirable for their oxygenating properties
(10, 16). Lacking in osmotic or pH-related forces, nonaqueous solvents
interfere with cell physiology and structure to a minimal extent (19,
20). As is shown in this report, osmium tetroxide dissolved in a
perfluorocarbon preserves more of the components of tracheal mucus in
situ than has previously been possible.
Ruthenium red and alcian blue likely impart a labeling effect on
carbohydrates (7). As mucus dissolves during an aqueous fixation
process, some label is bound to the cell surface and some to mucin,
possibly creating a cross-linking effect as well as a flocculant label.
However, in this study, neither ruthenium red nor alcian blue preserved
a mucous layer consistent in appearance with what might be expected for
a predominantly glycoprotein substance, and both caused unacceptable
cellular swelling of tracheal epithelium. Osmium tetroxide presented in
a nonaqueous manner reliably fixed mucus, whereas aqueous
glutaraldehyde (with or without additives), osmium tetroxide, and
Formalin did not. Glutaraldehyde and formaldehyde do not
dissolve in perfluorocarbon, so the remainder of the possible solvent-fixative combinations are not testable. The relatively small
size of osmium atoms and the appearance of retained mucus suggest that
the micrographs of this report reflect the biological nature of mucus
and not a label attached to it.
Two possible caveats that must be considered during interpretation of
these data are the position of the trachea immediately before fixation
and the randomness with which the tracheas were sampled. The rats used
in this study were placed in sternal recumbency after onset of
anesthesia. However, during the surgical removal of tracheas, animals
were laid on their backs; hence, there could have been some
displacement of epiphase toward the trachealis muscle region that was
caused by gravity. Viscous properties of mucus and the natural grooving
of tracheal epithelium would tend to limit this effect. Heterogeneity
of the epiphase along the length of the trachea was not examined in
this study and may play a role in the interanimal variations of
thickness and lipid content observed.
Heterogeneity of epiphase in rat trachea applies to both its thickness
and composition. The normal mucous coat of rat trachea varied from thin
(~0.2 µm) to thick (~50 µm), with estimates of epiphase
thickness by light and electron microscopy being comparable. Thinner
regions of epiphase, particularly along the ventral aspect of the
trachea, may indeed be more susceptible to insult. Grooving or
troughing of mucus adjacent to the trachealis muscle region appears to
occur. Another variable of physiological significance is the extent of
hydration of the epiphase. A given amount of glycoprotein may have
increased thickness by addition of water. Hydration states of tracheal
mucus, a random event within this study, likely contributed to the
reported heterogeneity.
In disease states, the lipid profile of mucus may be altered to include
glycolipids (2); their ultrastructural interpretation awaits further
study. Lipid and surfactant are observed in all parts of the epiphase
and need to be considered in modeling studies of gas diffusion and
particle penetration of the epiphase. Investigations of developmental,
physiological, and pathological processes of the entire mucociliary
apparatus, which have previously been compromised (6), will benefit
from the use of nonaqueous fixation.
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FOOTNOTES |
Address for reprint requests: D. E. Sims, Dept. of Anatomy and
Physiology, Atlantic Veterinary College, Univ. of Prince Edward Island,
Charlottetown, PE, Canada C1A 4P3 (E-mail: sims{at}upei.ca).
Received 21 February 1996; accepted in final form 12 August 1997.
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