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
ultrastructure; epiphase; microscopy; lipid; glycoprotein
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 waterimmiscible perfluorocarbon. Because osmium tetroxide
is an effective fixative of lipids, there was also an
opportunity to estimate the lipid content of mucus.
UNDERSTANDING OF THE
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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 5 3), aqueous buffered glutaraldehyde containing safranin O (n 5 1), aqueous buffered glutaraldehyde
containing ruthenium red (n 5 3), aqueous buffered glutaraldehyde containing alcian blue (n 5 3), buffered neutral
Formalin (BNF; n 5 3), aqueous buffered osmium tetroxide
(n 5 3), or osmium tetroxide dissolved in a perfluorocarbon
(n 5 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 340 objective lens
(final magnification 3400), 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.
1040-0605/97 $5.00 Copyright r 1997 the American Physiological Society
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Sims, David E., and Margaret M. Horne. Heterogeneity
of the composition and thickness of tracheal mucus in rats.
Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17): L1036–
L1041, 1997.—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 epiphasehypophase interface. Refined study of developmental, physiological, and pathological alterations to the airway coat may
benefit from this approach.
PRESERVATION OF TRACHEAL MUCUS
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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 3600 magnification.
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
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 3600 magnification.
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Fig. 1. Sample sites for measurement of tracheal mucus. Numbers
represent sites of measurement of mucous thickness.
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 320,000. 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 352,000. 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
L1038
PRESERVATION OF TRACHEAL MUCUS
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 3600 magnification.
comparison between animals. Values are presented as
means 6 SD, with significance indicated by P , 0.05.
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).
A heterogeneous distribution of mucus was observed,
with a tendency for thicker mucus in the region of the
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
3600 magnification.
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 6 9 µm (n 5 66) if sites without apparent
mucus were excluded and was 5 6 8 µm (n 5 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).
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 6 8 µm, the same as
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 3600 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
3600 magnification.
PRESERVATION OF TRACHEAL MUCUS
L1039
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, 314,000;
bar, 1 µm.
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, 326,000; bar, 1
µm.
determined by light microscopy. Interanimal differences of mucous thickness remained significant.
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 6
15 µm; ventral, 2 6 3 µm; and left and right lateral, 4 6
6 and 3 6 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 com-
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Fig. 9. In regions with thicker epiphase, cells and cellular debris (arrowheads) were commonly observed within
the mucous coat. Magnification, 33,500;
bar, 1 µm.
L1040
PRESERVATION OF TRACHEAL MUCUS
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,
346,000; bar, 1 µm.
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 pre-
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 onehalf of the bottom of the epiphase.
Magnification, 346,000; bar, 1 µm.
serves 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
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plexes. 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 6 9% surfactant and lipid. Means of lipid composition for individual animals ranged from 4 to 16%, with
interanimal variance indicating significant difference.
PRESERVATION OF TRACHEAL MUCUS
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: [email protected]).
Received 21 February 1996; accepted in final form 12 August 1997.
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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.
L1041