Am J Physiol Gastrointest Liver Physiol
280: G922–G929, 2001.
The adherent gastrointestinal mucus gel layer:
thickness and physical state in vivo
C. ATUMA,1 V. STRUGALA,2 A. ALLEN,2 AND L. HOLM1
1
Department of Physiology, Uppsala University, SE-751 23 Uppsala, Sweden;
and 2Department of Physiological Sciences, University of Newcastle-Upon-Tyne,
Newcastle-Upon-Tyne NE2 4HH, United Kingdom
Received 21 July 2000; accepted in final form 6 December 2000
Atuma, C., V. Strugala, A. Allen, and L. Holm. The
adherent gastrointestinal mucus gel layer: thickness and
physical state in vivo. Am J Physiol Gastrointest Liver
Physiol 280: G922–G929, 2001.—Divergent results from in
vitro studies on the thickness and appearance of the gastro-
intestinal mucus layer have previously been reported. With
an in vivo model, we studied mucus gel thickness over time
from stomach to colon. The gastrointestinal tissues of Inac-
tin-anesthetized rats were mounted luminal side up for in-
travital microscopy. Mucus thickness was measured with a
micropipette before and after mucus removal by suction. The
mucus layer was translucent and continuous; it was thickest
in the colon (;830 mm) and thinnest in the jejunum (;123
mm). On mucus removal, a continuous, firmly adherent mu-
cus layer remained attached to the epithelial surface in the
corpus (;80 mm), antrum (;154 mm), and colon (;116 mm).
In the small intestine, this layer was very thin (;20 mm) or
absent. After mucus removal, there was a continuous in-
crease in mucus thickness with the highest rate in the colon
and the lowest rate in the stomach. In conclusion, the adher-
ent gastrointestinal mucus gel in vivo is continuous and can
be divided into two layers: a loosely adherent layer remov-
able by suction and a layer firmly attached to the mucosa.
rat; intravital microscopy; stomach; small intestine; colon
MUCUS IS SECRETED BY THE epithelial surfaces throughout
the gastrointestinal tract from the stomach to the co-
lon. It is a unique secretion in that it forms a gel
adherent to the surface that provides a protective bar-
rier between the underlying epithelium and the lumen
containing noxious agents, destructive hydrolases, and
microorganisms (2, 4, 16). The protective functions of
this adherent mucus gel layer have mostly been stud-
ied (3, 12) in the stomach and duodenum, where it
provides a stable unstirred layer supporting surface
neutralization of luminal acid by mucosal bicarbonate
secretion. In this way, it maintains a pH gradient from
acid in the lumen to near neutral pH at the mucosal
surface (14, 37, 41). In addition, the mucus layer has
been shown to act as a physical barrier and prevent
access of luminal pepsin to the underlying mucosal
surface (3, 5). Recently, the transport of acid and pep-
sin, secreted by the gastric glands into the lumen, was
Address for reprint requests and other correspondence: C. Atuma,
Dept. of Physiology, Uppsala Univ., PO Box 572, SE-751 23 Uppsala,
Sweden.
G922 0193-1857/01 $5.00 Copyright © 2001 the American Physiological Society
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suggested to occur through restricted channel-like ar-
eas stretching out from the gland openings through the
overlying adherent mucus layer (20, 21, 24). In this
way, newly produced acid and pepsin are also pre-
vented from diffusing freely through the mucus gel and
accessing the mucosal surface.
The functions of the mucus layer in the small intes-
tine and colon are less well defined. Throughout the
gut, the viscous mucus secretion acts as a lubricant
facilitating the passage of digestive matter and protect-
ing the underlying epithelium from excessive mechan-
ical stress. Moreover, within the unstirred layer of the
mucus gel a stable microenvironment is created at the
mucosal surface. The mucus layer provides a protective
barrier against pathogens by acting as a physical bar-
rier, having binding sites for the bacterial adhesins,
maintaining high concentrations of secreted IgA and
lysozyme at the epithelial surface, and acting as a free
radical scavenger (2, 11, 13, 16, 30, 36, 38). At the same
time in the colon, the mucus layer provides an essential
environment for the enteric microflora (4, 28).
Regulation of mucus secretion has been coupled to
neural, hormonal, and paracrine effects (2, 9, 15, 16,
23, 33, 40). Normally, the thickness of the adherent
gastrointestinal mucus layer is a balance between its
secretion rate and its erosion through enzymatic diges-
tion by luminal proteases and mechanical shear (2, 5).
Thus continuous secretion of mucus maintains its pro-
tective mucosal barrier properties. However, the mu-
cus layer can be compromised in pathological states
such as Helicobacter pylori infection, peptic ulcer dis-
ease, and ulcerative colitis, during which both mucus
thickness and structure are major targets (7, 31, 34, 35,
42, 47).
The functional efficacy of the adherent mucus layer
depends on its thickness and stability in vivo as well as
the physical and chemical properties of the gel. Various
methods have been used to visualize and measure the
thickness of the mucus layer in situ. The original
methods for measuring mucus thickness consisted of
observing unfixed sections of fresh mucosa using an
inverted microscope (26) or estimating mucus thick-
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 GASTROINTESTINAL MUCUS
ness over inverted mucosa with a slit lamp and
pachymeter (8). Histological methods employing or-
ganic fixatives and paraffin embedding have proven
unsuccessful because dehydration of the gel causes
severe shrinkage and loss of the adherent mucus layer
(2, 25, 29, 44). A recently developed histological method
(25) has successfully circumvented these problems.
These different techniques have yielded varying re-
sults and have usually only covered a limited region of
the gastrointestinal tract. The potential for loss and
shrinkage of the mucus gel with in vitro techniques
makes it essential that methods are used in which the
mucus gel can be observed in vivo. Extensive studies
(21, 40, 41) have been made using intravital micros-
copy to observe gastric and duodenal mucus in vivo,
and more recently (10), confocal microscopy has been
applied to study the gastric mucus layer in an in vivo
system.
In this study, we used a micropipette technique with
intravital microscopy to study mucus gel thickness and
accumulation rate in each segment of the gastrointes-
tinal tract over time. In this way, we obtained results
that are comparable and project an image of the char-
acteristics and stability of the mucus gel layer in vivo
from the gastric corporal region down to the colon. In
particular, we show for the first time in vivo that there
are two forms of mucus gel: a suction-removable layer
and an underlying firm adherent gel layer.
MATERIALS AND METHODS
Animal Preparation
Male Wistar rats weighing ;200 g were fasted for 18–24 h
before induction of anesthesia but were allowed free access to
drinking water. They were anesthetized by intraperitoneal
administration of thiobutabarbital sodium (Inactin, 120 mg/
kg). Body temperature was maintained at 37–38°C with a
heating pad regulated by a rectal thermistor probe.
The rats were tracheotomized to facilitate spontaneous
breathing. A cannula containing heparin (12.5 IU/ml) dis-
solved in isotonic saline was placed in the right femoral
artery to monitor blood pressure. The femoral vein was can-
nulated for continuous infusion of a modified Ringer solution
at a rate of 1 ml/h.
All experimental procedures in this study were previously
approved by the Swedish Laboratory Animal Ethics Commit-
tee in Uppsala and were conducted in accordance with the
guidelines of the Swedish National Board for Laboratory
Animals. They were also approved by the Home Office
(Dundee, UK) and carried out in accordance with the Home
Office Animal (Scientific Procedures) Act of 1986.
Tissue Preparation
The tissue preparation has been previously described (22,
40) for the stomach and duodenum. Briefly, the stomach or
intestine was exteriorized. The forestomach was opened
along the greater curvature for the corpus studies. For the
antrum studies, an ;7-mm-long incision was made along the
greater curvature in the corpus, starting just proximal to the
junction with the antrum. The intestine was opened along
the antimesenteric border in both the small intestine and the
colon. The rat was placed on its left side on a Lucite micro-
scope stage, and the stomach or intestine was everted
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Copyright © 2001 American Physiological Society. All rights reserved.
G923
through the incision and loosely draped over a truncated cone
with the luminal side up. A mucosal chamber with a hole
(diameter of 1.2 cm2 for the stomach or 0.9 cm2 for the
intestine) in the bottom was placed over the exposed mucosa,
and the junction was sealed with silicone grease (Kebo Lab,
Stockholm, Sweden).
The chamber was filled with a warm (37°C) unbuffered
0.9% saline solution to keep the tissue moist and protect the
mucus gel from dehydration with consequent collapse. In the
corpus, the solution was replaced every 10 min, and its acid
content was determined by automatic back titration (ABU 91
autoburette with TIM 90 titration manager; Radiometer,
Copenhagen, Denmark) to the pH of the applied saline solu-
tion.
Mucus Gel Thickness Measuring System
Briefly, glass micropipettes (custom glass tubing; OD, 1.2
mm; ID, 0.6 mm; Rederick Haer, Brunswick, ME) were
pulled to a tip diameter of 1–2 mm (40). They were siliconized
to obtain a nonsticky surface, which facilitates repeated
measurements.
Before the first measurement, the mucus gel was covered
with enough carbon particles (activated charcoal, extra pure,
Kebo Lab) suspended in saline to visualize the surface of the
otherwise near-transparent gel. The micropipettes were held
by a micromanipulator (Leitz, Wetzlar, Germany) and
pushed into the mucus gel at an angle (a) of 35–45° to the cell
surface. The distance (l) from the luminal surface of the
mucus layer to the epithelial cell surface of the mucosa or the
top of the villi was measured with a “digimatic indicator”
(IDC series 543; Mitutoyo, Tokyo, Japan) connected to the
micromanipulator. The mucus gel thickness (T) could then be
calculated from the formula T 5 l 3 sin a. The measurements
were made at three to seven different spots or villi over the
mucosal surface, which were memorized and used on every
measurement occasion throughout the experiment.
Mucus Accumulation Measurement
Mucus accumulation was determined by measuring total
mucus thickness at regular intervals, before and after re-
moval by suction of as much as possible of the adherent
mucus gel layer. Before removal, the mucus gel was covered
with enough carbon particles to properly visualize the entire
mucus gel surface. A vacuum suction pump connected to a
PE-10 cannula was used to suck off the mucus, with care
taken to avoid contact with the underlying cell layer. The
procedure was carried out under observation through the
stereomicroscope.
Experimental Protocol
In all animals, blood pressure and body temperature were
monitored continuously during the entire experiment.
Corpus. Mucus measurements were made in the corpus of
six animals. Each animal was allowed a recovery period of 2 h
after the stomach was mounted to attain a stable blood
pressure and spontaneous acid secretion. Total mucus thick-
ness was measured at 20-min intervals over 80 min to deter-
mine basal mucus accumulation rate. As much as possible of
the mucus gel was then removed by suction, and mucus
accumulation was measured over the next 140 min. Measure-
ments were made immediately after mucus removal, at
20-min intervals over 80 min, and at 140 min after mucus
removal. Acid secretion was followed at 20-min intervals
throughout the experiment.
Antrum. Measurements were made in six animals. Each
animal was allowed 2–3 h to stabilize after the antrum was
G924 GASTROINTESTINAL MUCUS
mounted until a stable blood pressure was attained and a
nonacidic pH in the mucosal chamber was ascertained. The
solution in the mucosal chamber was replaced at 20-min
intervals throughout the experiment. Mucus measurements
were made as described above for the corpus.
Duodenum. The duodenum was studied ;2 cm from the
pylorus in seven animals. Each animal was allowed a recov-
ery period of ;1 h after the duodenum was mounted. The
recovery period for these animals was shorter than for ani-
mals in the corpus and antrum studies because the mucus gel
slowly became opaque over time, making it difficult to per-
form thickness measurements before mucus removal. Total
mucus thickness was measured, to the tops of the villi, at
15-min intervals for 90 min before the first mucus removal.
Subsequently, mucus thickness was measured at 15-min
intervals for another 90 min. The mucus gel was removed by
suction for a second time, and a final mucus thickness mea-
surement was taken immediately.
Jejunum. The mucus gel was studied 15 cm from the
pylorus in six animals. Each animal was allowed to recover
for 1 h after the intestine was mounted, because the mucus
gel became opaque over time similar to that in the duode-
num. Mucus thickness was measured as described above for
the duodenum.
Ileum. The mucus gel in the ileum was studied 5 cm
proximal to the cecum in eight animals. The animals were
allowed a recovery period of 1–2 h. In three animals, the
mucus gel became too opaque to allow accurate measure-
ments before the first mucus removal. In five animals, suc-
cessful measurements were made at 15-min intervals for 45
min. After the first mucus removal, measurements were
made at 15-min intervals for 30 min and thereafter at 30-min
intervals for another 60 min. Mucus was removed by suction
a second time, and a final measurement was made.
Colon. Measurements were made 1–2 cm distal to the
cecum in 11 animals. The animals were allowed to recover for
1–2 h. Total mucus thickness was measured at 15-min inter-
vals for 90 min before the first mucus removal. Mucus thick-
ness was then measured at 15-min intervals for another 90
min. The mucus gel was removed by suction a second time,
and mucus thickness was measured immediately. The mea-
surements were made to the mucosal surface at the top of the
observed mucosal folds.
Chemicals and solutions
Inactin was purchased from Research Biochemicals Inter-
national (Natick, MA), and heparin was from Kabi Pharma-
cia (Stockholm, Sweden). The modified Ringer solution con-
tained 120 mM NaCl, 2.5 mM KCl, 25 mM NaHCO3, and 0.75
mM CaCl2, which were all purchased from Kebo Lab.
Statistics
All values are presented as means 6 SE. Statistical sig-
nificance was determined by using an ANOVA factorial anal-
ysis [followed by Fischer’s protected least-significant differ-
ence (PLSD) test] to compare the mucus thickness values
between different groups. Differences in accumulation rate
were determined by comparing (using an ANOVA factorial
analysis followed by Fischer’s PLSD test) the accumulation
rates for corresponding time intervals between different
groups. Student’s t-test for unpaired measurements was used
to compare single mucus thickness values. The level of sig-
nificance was set at P , 0.05. All statistical calculations were
performed on a Macintosh computer using Statview-SE and
Graphics software (Abacus Concepts, Berkeley, CA).
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Copyright © 2001 American Physiological Society. All rights reserved.
RESULTS
Corpus
A clear mucus gel covered the corporal mucosa in its
basal state immediately after the stomach was
mounted. In some rats, a loose, sloppy mucus layer
easily detached from the mucus gel due to the shear
forces when the mucosal chamber was filled with sa-
line, leaving a continuous adherent gel layer. Total
mucus thickness (189 6 11 mm) (Fig. 1) did not change
over the first 80 min (Fig. 2 and Table 1). Over one-half
of this mucus layer was removed by suction, leaving a
firmly adherent mucus layer 80 6 5 mm thick attached
to the mucosal surface (Fig. 1). This mucus layer was
continuous with a relatively smooth surface, could not
be removed by further suction or wiping with a cotton
swab (in pilot experiments), and was consistent from
one experiment to another. After removal of the loosely
adherent mucus, there was a progressive increase in
mucus thickness. This increase was rapid during the
first 20 min but decreased with time (Fig. 2 and Table
1). The spontaneous acid secretion at the beginning of
the experiment was 0.14 6 0.05 meq z min21 z cm22 and
remained stable until the last 20-min period (200 min
later) when it significantly increased to 0.5 6 0.18
meq z min21 z cm22.
Antrum
A clear mucus gel 274 6 41 mm thick covered the
antrum (Fig. 1). Again, in some cases, a loose, sloppy
mucus was easily detached from the mucus gel when
the mucosal chamber was filled with saline immedi-
ately after mounting the antrum. There was no change
in the thickness of this mucus layer during the first 80
min (Fig. 2 and Table 1). Suction removed about two-
fifths of the mucus layer, leaving a 154 6 16-mm-thick,
firmly adherent, continuous mucus layer over the mu-
cosal surface (Figs. 1 and 2). A rapid replacement of the
mucus removed by suction ensued with a constant rate
during the first 60 min followed by a more reduced rate
during the last 80 min (Fig. 2 and Table 1). In a few
animals the mucus gel closest to the mucosal surface
became slightly opaque over time.
Duodenum
Immediately after the duodenum was mounted, the
mucus gel was translucent and continuous. It had an
even surface and did not follow the contours of the villi
(Fig. 1). The mucus gel became opaque over time,
starting from the surface of the villi and moving up-
ward with the mucus accumulation. A loose, sloppy
mucus layer up to 600 mm thick was seen in a few
animals. This mucus was easily detached from the
underlying adherent mucus gel, and it attached to the
micropipette used in the measurements or was flushed
off the first time the solution in the chamber was
changed. In general, the adherent mucus layer over the
duodenum was more loosely attached than that in the
stomach.
 GASTROINTESTINAL MUCUS G925

Fig. 1. A schematic figure showing the thicknesses of the 2 mucus gel layers in vivo in the corpus, antrum,
midduodenum, proximal jejunum, distal ileum, and proximal colon of the rat gastrointestinal tract. The mucus gel
layer was continuous and did not follow the contours of the villi in the intestine. However, on removal of the loosely
adherent layer, mucus was also removed between the villi, leaving behind a firmly adherent mucus layer attached
to the mucosa. In the stomach and colon, the firmly adherent layer was continuous, but in the small intestine the
firmly adherent layer had a patchy distribution and was absent on individual villi. Both mucus gel layers were
translucent. The loosely adherent mucus layer was removable by careful suction, whereas the firmly adherent
mucus layer was not. The table presents values for mucus thickness as means 6 SE for each group.

The mean mucus thickness of the mucus layer im-

mediately after the equilibration period was 170 6 38
mm and increased continuously during the first 90-min
period (Fig. 3 and Table 1). With careful suction, prac-
tically the whole mucus gel layer could be removed
down to and between the villi. The opaque mucus
closest to the villi was thicker than the translucent
mucus gel and was somewhat more resistant to re-
moval, especially in the space between villi. The mean
thickness of the remaining firmly adherent mucus gel
varied from 6 to 24 mm (mean thickness, 16 6 3 mm) in
the six animals (Figs. 1 and 3). However, in individual
animals the thickness could vary between different
areas in a patchy manner and be absent on individual
villi immediately after suction. The increase in mucus
thickness after removal by suction was less rapid than
that before removal (Table 1). The second removal of
the mucus gel (at 190 min) was easier, and the Fig. 2. Total mucus thickness over time before and after removal of
the loosely adherent mucus layer in the corpus (F; n 5 6) and antrum
remaining firmly adherent mucus layer was 10 6 3 (E; n 5 6) of the rat stomach. Values are means 6 SE and represent
mm thinner than that after the first removal (at 90 the total mucus thickness at each time point. * P , 0.05, corpus vs.
min) (Fig. 3). antrum.

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Copyright © 2001 American Physiological Society. All rights reserved.
G926 GASTROINTESTINAL MUCUS

Table 1. Mucus accumulation rates in gastrointestinal tract before and after

mucus removal by applied suction
After Removal, mm/min
Before Removal
(0–80 min), mm/min 0–20 min 20–40 min 40–140 min

Corpus 20.04 6 0.1 0.9 6 0.1 0.5 6 0.2 0.06 6 0.04

Antrum 20.08 6 0.1 1.3 6 0.3 1.2 6 0.3* 0.3 6 0.08*

Before Removal, mm/min After Removal, mm/min

0–45 min 45–90 min 0–30 min 30–60 min 60–90 min

Duodenum 1.9 6 0.5 2.8 6 0.8 0.9 6 0.3 1.4 6 0.3 1.5 6 0.4
Jejunum 2.5 6 0.3 3.6 6 0.6 2.5 6 0.8 1.7 6 0.3 2.0 6 0.9
Ileum 4.7 6 1.2† ND 1.9 6 0.3 1.5 6 0.4 1.7 6 0.7
Colon 5.2 6 2.1 3.9 6 1.2 6.7 6 1.1 7.1 6 1.4 4.6 6 1.4
Values are means 6 SE for each interval. By multiplying the values by 100 the volume-secretion rate in nl z min21 z cm22 may be obtained.
ND, not determined. * P , 0.05, antrum vs. corpus at same time interval. † P , 0.05, ileum vs. other areas of small intestine at same time
interval. (ANOVA followed by Fischer’s protected least-significant difference test).

Jejunum a 15 6 3-mm-thick, firmly adherent mucus layer, in all

The mucus gel was similar to that in the duodenum,
although no loose, sloppy mucus was observed. With
time, opaque, thick mucus was secreted from the villi,
making mucus measurements difficult. The mean mu-
cus thickness immediately after the equilibration pe-
riod was 123 6 4 mm (Fig. 1), and this increased
continuously during the first 90-min period before mu-
cus removal (Fig. 3 and Table 1). After mucus removal
by suction, a very thin layer of firmly adherent mucus
(mean thickness, 15 6 2 mm) remained, similar to that
in the duodenum (Fig. 1). In the jejunum as well,
individual villi tips could be completely devoid of mu-
cus immediately after suction. Mucus thickness
started to increase immediately after removal and con-
tinued at an even rate for 90 min (Fig. 3 and Table 1).
The second removal of mucus (at 190 min) left behind
aspects similar to that observed after the first mucus
removal (at 90 min).
Ileum
The mucus gel layer in the ileum (mean thickness,
480 6 47 mm) was significantly thicker than in the first
part of the small intestine as described above (Fig. 1).
The mucus gel was initially translucent but became
opaque over time, making measurements difficult and
in some cases impossible. In five animals, a rapid
continuous increase in mucus thickness was observed
for 45 min (Fig. 3 and Table 1). This was a significantly
higher rate of increase than that observed in the duo-
denum and jejunum. In all eight animals, the mucus
gel layer, including the opaque mucus, was easily re-
moved by suction to leave again a thin, uneven, and
discontinuous mucus layer (mean thickness, 29 6 8
mm) firmly adherent to the villi tips (Fig. 1). The
accumulation rate for the last 90 min was not signifi-
cantly different from that in the duodenum and jeju-
num (Fig. 3 and Table 1). A 29 6 4-mm-thick, discon-
tinuous mucus layer remained after the second mucus
removal (at 190 min), which was similar to that after
the first removal (at 90 min).

Fig. 3. Total mucus thickness over time in the rat small intestine
before and after removal of the loosely adherent mucus layer. ■,
Duodenum (n 5 7); h, jejunum (n 5 6); Œ, ileum (n 5 5 before mucus
removal and 8 after mucus removal). Values are means 6 SE and
represent the total mucus thickness at each time point. Mucus
thickness in the ileum could only be measured for 45 min before
mucus removal, because the mucus gel became opaque over time.
Colon
The colonic mucus gel was translucent and very
thick (mean thickness, 830 6 110 mm) with a sloppy
appearance (Fig. 1). Like the corpus but unlike the
small intestine, the mucus gel in the colon remained
clear throughout the experiment and showed no sign of
becoming opaque. There was a rapid increase in mucus
gel thickness during the first 90-min period (Fig. 4 and
Table 1). The loosely adherent upper mucus gel layer
was easily removed by suction (at 90 min). A firmly
adherent mucus gel layer with a mean thickness of
116 6 51 mm remained attached to the mucosal surface
(Figs. 1 and 4). This mucus layer was continuous,
covering the entire mucosal surface, and had a rela-
tively even surface. A rapid increase in thickness of the

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Copyright © 2001 American Physiological Society. All rights reserved.
 GASTROINTESTINAL MUCUS
Fig. 4. Mucus gel thickness in the rat colon over time (n 5 11).
Values are means 6 SE and represent the total mucus thickness at
each time point. * P , 0.05, mucus thickness after the first and
second mucus removals.
mucus gel layer followed during the next 90-min period
(Fig. 4 and Table 1). After the second mucus removal
(at 190 min), a 67 6 1-mm-thick, firmly adherent layer
remained (Fig. 4). This layer was significantly thinner
than that after the first mucus removal (at 90 min).
DISCUSSION
This study measures mucus thickness and secretion
after its partial removal, from stomach to colon, in the
rat in vivo by intravital microscopy. Key findings for all
regions of the gut studied are as follows: 1) the mucus
layer is continuous and substantially thicker than in
previous measurements made in vitro (2, 6, 25, 29, 39,
43, 44) and 2) the mucus layer consists of two compo-
nents, a loosely adherent gel that can be removed by
suction and an underlying firmly adherent gel that
remains. The relative thickness of the two component
layers of the mucus gel varies for different regions of
the gut. Thus in the stomach, the thickness of the
firmly adherent mucus component is 80 and 154 mm for
the corpus and antrum, respectively, with an overlying
layer, of similar thickness, of loosely adherent mucus
that can be removed by suction. In contrast, in the
small intestine, practically all of the mucus gel could be
removed by suction to leave a very thin discontinuous
mucus gel layer, with several tops of the villi appar-
ently free of mucus. In these studies, the mucus be-
tween villi was also removed during the suction proce-
dure, which otherwise would offer an ;500-mm-thick
layer covering the crypts and the basal villus epithe-
lium. In the colon (mean mucus thickness, 830 mm),
and to a lesser extent the ileum, the mucus layer was
very thick (up to 4-fold thicker) compared with that in
other regions of the gut. Most of the difference in
mucus thickness was due to the loosely adherent mu-
cus. Thus in the colon, 86% of the mucus layer was
removed by suction to leave a firmly adherent gel of
similar thickness to that in the stomach.
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G927
During resting conditions, mucus thickness was con-
stant over time in the stomach. A most interesting
observation was that removal of the loosely adherent
mucus layer by suction stimulated the accumulation of
new mucus in the stomach. The rate of increase in
thickness was higher in the antrum compared with the
corpus, although there was no difference in the amount
of loosely adherent mucus gel present in the two re-
gions before mucus removal. This increase in mucus
thickness was solely a renewal of the loosely adherent
mucus layer, because the thickness of the firmly ad-
herent mucus layer was the same after a second mucus
removal as that following the initial mucus removal by
suction. In another study, using the same experimental
setup, the gastric pathogen Helicobacter pylori has
been shown to reduce the rate of renewal of the mucus
layer removed by suction, as well as an acid-induced
increase in its renewal rate (7).
Unlike the stomach, the small intestine had a con-
tinuous increase in basal mucus thickness with the
lowest rate in the duodenum and the highest in the
ileum. After suction, the remaining mucus layer in the
intestine was very thin and discontinuous, but mucus
renewal was again observed. Shear forces in vivo there-
fore might be expected to reduce much of the mucus
layer and enhance uptake of nutrients from chyme.
However, an increased mucus secretion could occur in
response to local factors, e.g., irritation. Sababi et al.
(40) have shown that luminal acid in the duodenum
increases the rate of mucus accumulation after mucus
removal by suction. In the same study, inhibition of
nitric oxide (NO) and prostaglandin synthesis reduced
basal mucus accumulation by 20% and 35%, respec-
tively, after removal by suction.
In the colon, the mucus gel was thickest and the rate
of mucus accumulation both in the basal state and
after mucus removal was greatest. Similar to the stom-
ach, only the loosely adherent mucus layer was re-
newed. This high rate of mucus accumulation in the
colon could provide good lubrication, crucial to its func-
tion, and be important in providing a suitable environ-
ment for the endogenous microflora. Regulation of mu-
cus secretion in the colon has been shown (33) to be
mediated by NO, prostaglandins, and a wide range of
other neurotransmitters and hormones.
In all regions of the gut, total thickness of the mucus
layer was considerably greater than measurements
reported for in vitro studies, up to 40-fold in the case of
the colon. Standard histological methods employing
organic fixatives, extensive dehydration, and paraffin
embedding result in severe shrinkage of the adherent
mucus layer and frequently its complete loss from the
mucosal surface (2, 6). For example, in one study (39)
using a modified unfixed section technique, values of
39, 18, and ;20 mm were reported for the rat stomach,
cecum, and colon, respectively. However, in another
study (44), using celloidin fixation of cryostat sections,
approximate values for mucus thickness in the rat
duodenum, jejunum, ileum, and proximal colon were
28, 93, 101, and 53 mm, respectively. A recent (25, 43)
histological procedure for cryostat sections, which min-
G928
imizes the shrinkage of the adherent mucus gel layer,
gives values for mucus thickness in the rat antrum,
corpus, and colon of 166, 184, and 45 mm, respectively.
These values are reasonably close to those for the
firmly adherent gel seen in the present study and
compatible with mainly the loosely adherent gel, re-
movable by suction in vivo, being lost during the his-
tological procedures.
Mucus thickness in the rat stomach in vivo has been
measured as 118 mm and in the duodenum as 136 mm
(1, 45). With a confocal imaging system, rat corpus
mucus thickness was found to have a median value in
the interval 50–75 mm but with ;23% of the values in
the interval 0–25 mm (10). These values are somewhat
lower than the values reported here, which may largely
be explained by methodological differences, with the
former studies (1, 10, 45) using a perfusion system in
contrast to the unstirred conditions in the present
study.
The relationship of the loosely adherent gel remov-
able by suction to the firmly adherent gel is not clear.
The possibilities range from a continuum of the same
secretion, which reflects a gradual diluting out of the
gel the further one proceeds from the mucosa, to two
different mucus secretions. It is generally accepted
that there is one major mucin gene product, MUC2,
in the mucus secretion by goblet cells of the colon and
small intestine in humans and its equivalent in rats
(27, 46). This would tend to argue against two dif-
ferent mucin secretions in colon and small intestine
being responsible for the firmly adherent and loosely
adherent gels, although the pattern of glycosylation
could be different. Interestingly, two different mucus
secretions from the goblet and columnar cells of
human colonic crypts, with control mechanisms re-
stricting total mucus release, have recently been
reported (17). In the stomach, there are two mucin
gene products: MUC5AC from the surface epithelia
and MUC6 secreted by the neck cells around the pits
(19). Therefore, in the stomach, it is possible that the
two layers of gel, the firmly adherent and that re-
moved by shear, are two different mucin gene prod-
ucts or different mixtures of two gene products, also
perhaps with differing glycosylation patterns. The
possibility of a nonblended lamina-ordered gastric
gel has been reported for carbohydrate staining (32)
and recently for MUC5AC and MUC6 (18).
In summary, this study demonstrates the presence
of two types of mucus gel secretion, one that is firmly
adherent to the mucosal surface and one that is rela-
tively sloppy and can be removed by suction. The firmly
adherent gel, which forms a thick layer over the gastric
and colonic mucosal surfaces, would be expected to act
as a relatively stable protective barrier, whereas the
sloppy mucus, which is rapidly replaced after its re-
moval, might have more lubricative properties in vivo.
This study was supported by Grant 08646 from the Swedish
Medical Research Council.
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Copyright © 2001 American Physiological Society. All rights reserved.
GASTROINTESTINAL MUCUS
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