Download Lack of pyloric interstitial cells of Cajal explains distinct peristaltic

Am J Physiol Gastrointest Liver Physiol 289: G539 –G549, 2005.
First published April 28, 2005; doi:10.1152/ajpgi.00046.2005.
Lack of pyloric interstitial cells of Cajal explains distinct peristaltic motor
patterns in stomach and small intestine
Xuan-Yu Wang,1 Wim J. E. P. Lammers,2 Premysl Bercik,1 and Jan D. Huizinga1
1
Intestinal Disease Research Program and Department of Medicine, McMaster University,
Hamilton, Ontario, Canada; and 2Department of Physiology, Faculty of Medicine
and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates
Submitted 3 February 2005; accepted in final form 14 April 2005
pyloric sphincter; rat; mouse; gastroduodenal junction
ONE OF THE FASCINATING ASPECTS of gastrointestinal (GI) motility
is that each organ has unique motor characteristics and motor
control, yet each organ can synchronize its motor activity with
the adjacent organ to allow for a smooth transfer of content.
The recording of human gastroduodenal motility has shown
that motor patterns from the stomach and duodenum are often
distinct, but peristaltic contractions smoothly crossing the pylorus can also be observed. Complete understanding of control
mechanisms underlying this coordination is important because
abnormal gastroduodenal motor coordination is thought to be
part of many motility disorders (37).
The coordination of motor activities of the stomach and
small intestine was the subject of the earliest studies in GI
Address for reprint requests and other correspondence: J. D. Huizinga,
McMaster Univ., HSC-3N5C, 1200 Main St. West, Hamilton, Ontario, Canada
L8N 3Z5 (e-mail: [email protected]).
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motility. Canon (8) noted in 1911 that “gastric peristaltic
waves do not pass on to the duodenum, but stop at the pylorus.
This separation of the two regions is probably to be accounted
for by the interruption in the continuity of the circular muscle
fibers just beyond the pyloric sphincter.” Indeed, Cai and
Gabella (7) noted that the guinea pig sphincter muscle was
separated by a septum from the duodenal circular muscle but
not from the gastric circular muscle. This was also observed in a
recent study on the cat sphincter (31). Simultaneous recordings
with large arrays of electrodes along the length of the stomach
and proximal intestine of the cat showed that the slow wave
activity was not continuous but abruptly blocked at the level of
the pylorus (31). Because antral and duodenal motor activities
are dominated by slow wave-driven peristalsis, the question
arises how the interstitial cells of Cajal (ICC) network is
organized at the pylorus, how slow wave activity is generated,
and how the peristaltic activities are synchronized to allow for
transport of content across the pylorus. In a landmark paper,
Conklin and Du (10) implied that it was the network of ICC
and not the musculature that was responsible for coordination
of electrical activities along the long axis of the cat colon. This
was confirmed in the canine colon (35, 44) and stomach (21).
To study a possible role of ICC in gastroduodenal coordination, measurements with arrays of electrodes were modified
for use in the rat and mouse, structural studies were carried out
using immunohistochemistry, and electron microscopy and
motor patterns were analyzed based on videofluoroscopy and
image analysis.
METHODS
Electrophysiology. Nine rats and eight mice (all males; weight
338 ⫾ 37 and 35 ⫾ 4 g, respectively) were used for the electrophysiological experiments, approved by the animal ethics committee at
United Arab Emirates University. After an overnight fast, the animals
were anesthetised with chloroform (rats) or urethane (mice, 2 mg/kg
ip). The stomach and duodenum were rapidly removed, opened along
the minor curvature and the mesenteric border, and positioned with
the serosa side upward in a tissue bath. The preparations were
superfused at a rate of 100 ml/min with a modified Tyrode solution
kept at a constant pH (7.35 ⫾ 0.05) and temperature (37 ⫾ 0.5°C) and
saturated with carbogen (95% O2-5% CO2) (31). The intracellular
microelectrode recording displayed in Fig. 1 was obtained using
methods described previously (34).
With the rat preparations, a row of 32 electrodes (Teflon-coated
silver wire; 1-mm interelectrode distance) was carefully positioned on
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0193-1857/05 $8.00 Copyright © 2005 the American Physiological Society
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Wang, Xuan-Yu, Wim J. E. P. Lammers, Premysl Bercik, and
Jan D. Huizinga. Lack of pyloric interstitial cells of Cajal explains
distinct peristaltic motor patterns in stomach and small intestine. Am J
Physiol Gastrointest Liver Physiol 289: G539 –G549, 2005. First
published April 28, 2005; doi:10.1152/ajpgi.00046.2005.—The frequency and propagation velocity of distension-induced peristaltic
contractions in the antrum and duodenum are distinctly different and
depend on activation of intrinsic excitatory motoneurons as well as
pacemaker cells, the interstitial cells of Cajal associated with Auerbach’s plexus (ICC-AP). Because ICC are critical for coordination of
motor activities along the long axis of many regions in the gut, the role
of ICC in antroduodenal coordination was investigated. We used
immunohistochemistry, electron microscopy, simultaneous multiple
electrical recordings in vitro, and videofluoroscopy in vivo in mice
and rats. A strongly reduced number of ICC-AP with loss of network
characteristics was observed in a 4-mm area in the rat and a 1-mm
area in the mouse pyloric region. The pyloric region showed a slow
wave-free gap of 4.1 mm in rats and 1.3 mm in mice. Between antrum
and duodenum, there was no interaction of electrical activities and in
the absence of gastric emptying, there was no coordination of motor
activities. When the pyloric sphincter opened, 2.4 s before the front of
the antral wave reached the pylorus, the duodenum distended after
receiving gastric content and aboral duodenal peristalsis was initiated,
often disrupting other motor patterns. The absence of ICC-AP and
slow wave activity in the pyloric region allows the antrum and
duodenum to have distinct uncoordinated motor activities. Coordination of aborally propagating peristaltic antral and duodenal activity is
initiated by opening of the pylorus, which is followed by distentioninduced duodenal peristalsis. Throughout this coordinated motor activity, the pacemaker systems in antrum and duodenum remain independent.
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for 30 min. For the analysis, signals (duration of 60 s) were transferred
to a personal computer, digitally filtered (20-point running average),
and displayed on screen in groups of 16 –32. From these signals, the
spontaneous rate of the stomach and the duodenum was calculated by
measuring the interval between sequential slow waves. The propagation of the slow wave in both organs was calculated by measuring the
difference in time of activation between a proximal and a distal
electrode. Finally, in the recordings that spanned the gastroduodenal
junction, the number of electrodes that did not record a slow wave was
noted.
To determine whether stomach activity had any effect on spontaneous duodenal rhythm, the following analysis was performed. Two
electrograms, one from the antrum and one from the duodenum,
located as close as possible to each other were used for this purpose.
the serosal side of the preparation oriented in the longitudinal direction spanning the corpus, antrum, gastroduodenal junction, and the
upper part of the duodenum. In the murine experiments, a row of 16
electrodes (0.37-mm interelectrode distance) was also positioned
longitudinally on the serosal surface, and recordings were performed
sequentially at three different locations: on the stomach, across the
gastroduodenal junction, and on the duodenum. The recordings were
made unipolarly with a large silver plate in the tissue bath acting as
the indifferent pole. The electrodes were connected to alternatingcurrent preamplifiers (gain 4,000), and the recorded signals were
subsequently filtered (2– 400 Hz), digitized (8 bits, 1 kHz sampling
rate per channel), multiplexed, and stored.
After a 15- to 30-min control period, 1- to 2-min recordings of the
spontaneous rhythm and propagation were performed every 10 min
Table 1. Characteristics of slow wave activity at the gastroduodenal junction
Stomach
Mouse
Rat
Duodenum
n
Width of Slow
Wave-Free Zone, mm
Slow wave frequency,
cycles/min
Conduction
velocity, mm/s
Slow wave frequency,
cycles/min
Conduction velocity,
mm/s
9
8
1.3⫾0.4 (0.0–2.2)
4.1⫾1.0 (0.0–7.0)
6.8⫾0.6 (4.1–9.7)
3.07⫾1.04 (1.3–5.3)
1.1⫾0.4 (0.2–2.5)
1.5⫾0.3 (0.5–2.9)
45.6⫾2.3 (38.5–58.1)
35.5⫾2.4 (29.1–42.1)
14.8⫾4.1 (4.7–40.3)
10.8⫾2.2 (5.6–19.6)
Values are means ⫾ SD with ranges in parentheses; n, no. of animals. Slow wave frequency and conduction velocity were statistically different between antrum
and duodenum (P ⬍ 0.001) both in mice and rats.
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Fig. 1. A: 4-mm gap in slow wave activity is
observed in the pyloric region of the rat.
There is no slow wave activity in the fundus
(top part) because of a lack of interstitial
cells of Cajal (ICC) associated with Auerbach’s plexus (ICC-AP). Starting from the
corpus, stomach activity was characterized
by rhythmic oscillatory discharges that propagated in the distal direction before terminating at approximately electrode 15. Each
spindle represents a slow wave as observed
previously using chronically implanted electrodes in the rat (18) and shown in B using
the intracellular recording technique in the
rat corpus. Duodenal slow waves originated
from the upper part of the duodenum (at
approximately electrode 22) and propagated
also in the distal direction. Between the
stomach and the duodenal activity, at least
four electrodes (16 –19) did not register any
slow-wave activity; accounting for a gap
width of 4 mm. Occasional spike activities
(smooth muscle action potentials) were visible in several electrograms.
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With the use of the stomach activity as a reference point, the duodenal
beat-to-beat intervals before, during, and after this stomach discharge
were measured. This analysis was performed 18 times in rats and 18
times in mice. The calculated values were plotted on an individual
basis (as shown in Fig. 4, C and D) and, after normalization, grouped
together as shown in Fig. 4, E and F.
Immunohistochemistry. Twelve Sprague-Dawley rats, 250 –350 g,
and seven CD1 mice (25–35 g, 8 –9 wk old) were used. Animals were
handled according to McMaster University Animal Care Committee
ethical guidelines, and the present study was approved by the Animal
Ethics Research Board at McMaster University. After resection, the
pyloric sphincter region was opened along the lesser curvature and
washed in Krebs-Ringer bicarbonate solution. Both frozen sections
and whole mounts were prepared for c-Kit imunohistochemical staining. For frozen section preparations, fresh tissues were directly embedded in Tissue-Tek and frozen with isopentane emerged in liquid
nitrogen. Frozen longitudinal sections of 8 ␮m were cut from lesser to
greater curvature sides with a cryostat. Musculature whole mounts
were obtained by peeling away the mucosa and submucosa under the
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dissection microscope. Both the section and wholemount preparations
were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH
7.4) for 10 min at 4°C. After being washed for 30 min in 0.05 M PBS
(pH 7.4, with 0.3% Triton X-100), the fixed tissues were processed for
immunohistochemical staining (55). Sections from rat tissues were
incubated with either polyclonal rabbit anti-c-Kit (DAKO, Denmark)
or goat anti-c-Kit (Santa Cruz, CA) after endogenous peroxidase was
quenched, whereas sections from mouse tissues were incubated with
monoclonal rat anti-c-Kit (ACK4, Cedarlane, Canada). All the incubation times were 24 h at 4°C. Secondary immunoreactions were
carried out with either Vectastain ABC kits (with biotinylated antirabbit, anti-goat, or anti-rat IgGs) or FITC-conjugated IgGs (Vector
Laboratories, Burlingame, CA). 3,3-Diaminobenzidine was used as a
peroxidase substrate for the ABC technique. Tissues were examined
with either a conventional microscope with an attached digital camera
(Sony 3CCD, model DXC-930) or confocal microscope (Zeiss LSM
510) with an excitation wavelength appropriate for FITC (494 nm).
Conventional electron microscopy. Tissues were fixed with 2%
paraformaldehyde, 2.5% glutaraldehyde, 3% sucrose, and 1.25 mM
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Fig. 2. A: 1.1-mm gap in slow wave activity
is observed in the pyloric region of the
mouse. Extracellular electrical activity was
recorded in the stomach (A), across the pylorus (B), and in the duodenum (C). Slow
wave activity in the stomach propagated
from corpus to antrum at a low frequency
and slow velocity before dying out in the
gastroduodenal junction. As shown in B, at
least 3 electrodes did not register slow waves
from either the stomach or the duodenum.
Note that spikes (action potentials) did occur
in this area and were synchronous with the
stomach activity.
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CaCl2 in 0.05 M cacodylate buffer (pH 7.4) at 4°C for 2 h. They were
then postfixed in 2% osmium tetroxide (OsO4) for 1 h, stained en bloc
with 2% aqueous uranyl acetate for 40 min, dehydrated, infiltrated,
and embedded in Epon-Araldite resin. Ultrathin sections were cut
parallel to the circular muscle layer and stained with lead citrate for 5
min before viewing with a transmission electron microscope (Jeol
1200EX Biosystem).
Quantification of Kit-positive cells within sections at the level of
myenteric plexus of rat pylorus. Quantification of c-Kit positivity was
performed with the KS400 program (Zeiss, Germany). Fifty longitudinal sections cut from lesser to greater curvature sides stained with
c-Kit were chosen. Kit immunopositivity at the level of Auerbach
plexus (mostly ICC-AP) at the points of the pyloric-duodenum junction as well as 1.5 and 3 mm oral and 0 and 1.5 mm caudal were
identified and highlighted using density slicing on color-scale images.
The area of immunopositive cells on each picture was measured and
expressed as percentage of total area. Regions with increased background staining, occasionally found at the borders of the image, were
excluded from the analysis. Due to stronger staining and different
shape, mast cells were easily distinguished from ICC and excluded
before the analysis.
Radiology and image analysis. Mice had access to food and water
ad libidum, and tests were performed between 5:00 and 8:00 PM.
Omnipaque 350 (0.2 ml/mouse; Nycomed Imaging AS, Oslo, Norway) was gavaged into the stomach, and mice were continuously
fluoroscoped (Siemens, Polystar) for 3 min. Video images were stored
on a VCR (JVC BR-S378U, Japan) for off-line analysis and then
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digitized by Power Macinstosh G3 at a frequency of 10 images/s.
Spatiotemporal maps were constructed and analyzed using National
Institutes of Health (NIH) Image software (developed at NIH and
available at http://rsb.info.nih.gov/nih-image) with self-routines as
previously described (3, 13). Frequency and propagation velocity of
contractions in the antrum and duodenum were evaluated. Peristalsis
was defined as aborally propagating waves of muscle wall indentations corresponding to circular muscle contractions. Temporal correlation between peristaltic antral and duodenal contractions was assessed in spatiotemporal maps.
Data were expressed as means(SD). Statistical significance was
determined by a two-tailed t-test. P values ⬍0.05 were considered to
indicate statistically significant differences. The number of animals is
represented by N, and the number of tissue recordings is represented
by n.
RESULTS
Electrical activities in the gastroduodenal region. The gastroduodenal junction of the rat showed a distinct region lacking
any slow wave activity (Fig. 1A). When 32 electrodes were
placed in one line on the stomach, across the pylorus, and into
the small intestine, antral slow waves were seen to propagate
toward the pylorus at a velocity of 1.5 mm/s and a frequency
of 3 cycles/min (Fig. 1A and Table 1). The slow wave stopped
abruptly in the pyloric region. Distally (4 mm), duodenal
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Fig. 3. Propagation of action potentials occurred frequently in the pyloric region of the
mouse associated with the slow wave of the
stomach. A shows, at fast speed, antral oscillations (the slow wave) in electrodes 3–7
and several action potentials propagating
orally. B shows an antral slow wave associated with an aborally and an orally propagating action potential. Electrogram 16 recorded duodenal slow wave activity in both
experiments (open arrowheads).
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slow wave activity started at a propagation velocity of 1.0
mm/s and a frequency of 35 cycles/min (Table 1). Four
electrodes in the pyloric region spaced 1 mm apart did not
record any slow wave activity, although an occasional action
potential was observed. The extracellular recording of the slow
wave activity displayed short, spindle-shaped bursts of electrical oscillatory activity; each spindle representing one slow
wave as shown using the intracellular recording technique in
Fig. 1B.
Slow wave frequency and conduction velocity were statistically different between antrum and duodenum (P ⬍ 0.001)
both in mice and rats [means (SD)].
The gastroduodenal junction of the mouse showed a similar
distinct region without any slow wave activity, albeit shorter in
length (Fig. 2 and Table 1). Slow wave activity of the stomach
propagated distally at a velocity of 10 mm/s and a frequency of
7 cycles/min and stopped abruptly in the oral part of the
sphincter region. After 1.3 mm, the duodenal slow waves
started at 46 cycles/min and a conduction velocity of 15 mm/s.
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Action potentials, originating on the antral slow waves, were
seen to propagate into the sphincter region, on occasion farther
than the slow wave activity (Fig. 3). On 24 occasions where
action potential activity was seen in the pyloric region, 83%
were associated with gastric slow wave activity, propagating
for very short distances (1–3 mm) aborally (66%), orally
(21%), or in both directions (14%).
The analysis of a possible temporal coordination between
gastric and duodenal activity is shown in Fig. 4. If the gastric
slow wave would influence the timing of the intestinal slow
wave, a premature or delayed intestinal slow wave would
produce shortening or lengthening of the duodenal slow wave
interval. Duodenal slow waves that occurred before and after
an antral slow wave were marked, and the intervals were
calculated (n ⫽ 36 for both rats and mice). The duodenal
intervals in both rats and mice were never significantly lengthened or shortened at any stage before, during, or after antral
activity, suggesting that the duodenal rhythm in both species is
independent of stomach activity.
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Fig. 4. Analysis of a possible temporal coordination between gastric and duodenal activity.
Duodenal slow waves that occurred before and
after an antral slow wave were marked, and the
intervals were calculated to achieve a perievent
time distribution. A and B show the procedure
used to study this relationship for mice and
rats, respectively. Two electrograms were used,
1 from the antrum and 1 from the duodenum,
located as close as possible to each other. All
duodenal slow waves that occurred before and
after an antral slow wave were marked, and the
intervals were plotted [C (related to A) and D
(related to B)]. This procedure was performed
36 times in rats and mice, and the data were
normalized (E and F). The horizontal lines in C
and D indicate the average intervals. As shown
in the individual graphs and in the normalized
plots, the duodenal intervals in both rats and
mice were never significantly lengthened or
shortened at any stage before, during, or after
antral activity.
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Morphological analysis of ICC in the pylorus. The distributions of ICC in the gastroduodenal regions were similar in rat
(Figs. 5 and 6) and mouse tissues (Fig. 7) based on c-Kit
immunohistochemistry. ICC-AP in the pyloric sphincter region
were markedly decreased in a 4-mm region from lesser to
greater curvatures compared with the adjacent antrum and
duodenum. In the distal antrum, Kit-positive ICC were distributed evenly in both muscle layers. In addition a concentration
of cells was observed at the level of Auerbach’s plexus and at
the submucosal border of the circular muscle layer. In the
proximal duodenum, except the first millimeter, Kit-positive
ICC were mainly found concentrated at the level Auerbach’s
plexus (ICC-AP) as well as in the deep muscular plexus area
(ICC-DMP). In the first millimeter caudal to the pylorusduodenum junction, ICC were diffusively distributed in both
muscle layers. In the pyloric sphincter region, there was no
concentration of Kit-positive cells at the level of Auerbach’s
plexus (Figs. 5A and 7A). Although sparse Kit immunoreactivity was visible, no ICC-AP network, as in the antrum and
duodenum, was present in the sphincter region. With Kit
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Fig. 5. Kit immunoreactivity in the musculature of the pyloric sphincter region
of the rat. a: Longitudinal section of the rat pyloric region immunostained with
c-Kit. In the distal antrum (left), ICC-AP are concentrated at the level of
Auerbach’s plexus (arrows) surrounding the ganglia. ICC-IM are scattered in
both circular and longitudinal muscle layers (both sides of Auerbach’s plexus).
There is also a concentration of ICC at the submuscular border (small arrows).
In the pyloric region (middle), ICC-AP are markedly decreased in a segment
⬃4 mm in width. ICC-IM are abundant and diffusively distributed within the
thickened pyloric sphincter. In the proximal duodenum (right), ICC-AP are
present (arrows) except for the first 1–2 mm, where Kit-positive ICC are
evenly distributed in the whole musculature and not concentrated at the level
of Auerbach’s plexus. From 3– 4 mm onward, Kit-positive ICC-DMP are
present in the duodenum at the submucosal border of the circular muscle (small
arrows). Background staining of the mucosa is due to endogenous biotin within
the mucosal glands only. b-d: Whole mount preparation of the rat gastroduodenal junction immunostained with c-Kit. b and d show networks of ICC in the
antrum (b: 3 mm oral to the sphincter) and the duodenum (d: 3 mm caudal to
the sphincter). In both regions, there were abundant ICC-AP (arrows) that were
triangular/multipolar in shape, with processes proceeding in different directions and connecting with each other to form a dense network. In contrast,
there were almost no ICC-AP in the sphincter region (c). Instead, many
bipolar-shaped ICC-IM running parallel to the pyloric sphincter (PS; c, left)
were observed. The right portion of c shows a loose network of ICC in the very
beginning of the proximal duodenum.
antibodies from different sources, similar results were obtained; Kit immunoreactivity at the level of myenteric plexus
was virtually absent. The gap had a width of ⬃4 mm for rats
and ⬃1.2 mm for mice in unstretched tissues, determined from
sections that were taken from the lesser curvature and the
greater curvature. Quantification based on Kit immunoreactivity and image analysis confirmed the significant decrease in
ICC-AP in the pyloric region (Fig. 8). Assessment of whole
mount preparations confirmed the remarkable reduction in
ICC-AP and interruption of ICC-AP networks at the region of
the pyloric sphincter in rat (Figs. 5, B-D) and mouse (Fig. 7B).
Electron microscopic (EM) examination of the sphincter
region rarely encountered ICC-AP cell bodies (Fig. 6B). In
contrast, in the antrum (Fig. 6A) and duodenum (Fig. 6C), a
large number of ICC-AP was found around and in between
myenteric ganglia. Connections including gap junctions between ICC-AP were commonly observed in antrum and duodenum, indicating their network structure (insets in Fig. 6, A
and C). In the pyloric region (Fig. 6B), only occasional ICC
processes were found between the profiles of nerve bundles.
Ganglia were not commonly present in the myenteric plexus of
the pyloric sphincter region. Instead, many large enteric nerve
bundles with varying density of varicosities were present. In
contrast to the sparse number of ICC-AP, intramuscular ICC
(ICC-IM) were found in abundance and in close contact with
enteric nerves and smooth muscle cells within the sphincter
(Fig. 6D).
Antral and duodenal peristaltic activities in mice in vivo.
Videofluoroscopic image analysis showed that immediately
after contrast material was introduced into the stomach,
aborally directed peristaltic contractions occurred in the
stomach at a frequency of 5.1 ⫾ 1.5 cycles/min and propagated at a velocity of 1.02 ⫾ 0.16 mm/s (Fig. 9). Concomitantly, contrast material entered the duodenum in a pulsating manner. On its entry into the small intestine, contractions occurred at a frequency of 44.5 ⫾ 2.9 cycles/min; they
propagated in an aboral direction at a velocity of 10.4 ⫾ 2.1
mm/s and orally at 4.9 ⫾ 3.0 mm/s. Regular aborally
directed peristalsis prevailed in the first minute after gavage
of contrast material into the stomach (Fig. 9, left). Thereafter, regular aboral peristalsis in the stomach continued, but
segmental contractions and retroperistalsis dominated the
motor patterns of the duodenum, interrupted at regular
intervals by one or more aborally propagating contractions
(Fig. 9, middle). This allowed us to answer the question
whether a temporal relationship between the antral and
duodenal peristaltic contractions existed. Video images and
spatiotemporal maps showed that one to several duodenal
aborally propagating peristaltic contractions appeared most
of the time temporally related to the antral peristaltic wave
(Fig. 10). On average, duodenal contractions started 2.4 ⫾
2.8 s before the front of an antral contraction reached the
pylorus, pushing content ahead of them. This coincided with
the opening of the pylorus and passage of the gastric luminal
contents into the proximal duodenum (Fig. 10). On rare
occasions, independent and noncoordinated patterns coexisted in antrum and duodenum (Fig. 9, right). This corresponded to periods when the pylorus remained closed and
there was no mechanical communication between the stomach and duodenum.
ICC AND GASTRODUODENAL COORDINATION
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DISCUSSION
The present study used narrowly spaced surface electrodes
to monitor for the first time simultaneously the electrical
activity of the distal antrum, pylorus, and proximal small
intestine in the mouse and rat. Slow wave activity was not
generated in and did not spread into the pylorus; consistently,
we show that no pacemaker ICC network was present. The
absence of slow wave activity in the pyloric region allowed for
distinct independent slow wave activities in stomach and duodenum with distinct frequencies and propagation velocities
and hence independent motor activities. Indeed, our in vivo
experiments showed predominantly independent motor activities, in particular during periods of pyloric closure. However,
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when a single antral wave of contraction was followed by
opening of the pylorus, gastric content arrived into the duodenum a few seconds ahead of the antral contraction reaching the
pylorus, and a few synchronized aborally propagating contractions in the duodenum were evident. Presumably, the luminal
content evoked peristaltic contractions in the duodenum either
by distending the muscle wall (5, 29) or by shear stress induced
by luminal flow (15, 28, 38, 39). Distention and/or shear stress
cause activation of enteric excitatory motoneurons providing
the final stimulus for contraction (11). The slow waves allow
the musculature to be depolarized only periodically such that
contractions are periodic and propagate aborally. The role of
the pylorus is not to transmit electrical slow waves or action
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Fig. 6. ICC ultrastructure in the musculature of the pyloric sphincter region of the rat. Ultrastructure of the Auerbach’s plexus region in the rat antrum, pylorus,
and duodenum. In both antrum (a) and duodenum (c), an abundant presence of ICC-AP (arrows) was observed surrounding myenteric ganglia (G). Processes
of these ICC-AP were in close contact (insets). In contrast, there were very few ICC-AP around nerve bundles (N) in the pyloric region (b), where ganglia were
rare. Collagen and elastic fibers (*) surrounded the nerve bundles. In the pyloric sphincter (d), an enteric nerve varicosity (N) formed a synapselike connection
(inset) with an intramuscular ICC (ICC-IM), whose membrane was lined with many caveoli (arrows). SM, smooth muscle cells; BV, blood vessel. The small
arrows in the insets show gap junctions between ICC-AP (a and c) or a synapse-like junction between ICC-IM and nerves (d) at higher magnification.
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potentials from the stomach to the duodenum but to control the
flow of luminal contents into the duodenum, also observed in
humans (47). The pylorus opens according to complex regulatory systems involving extrinsic and intrinsic nerves. Antral
contractile activity can result in the opening of the pylorus (42)
mediated by intrinsic descending inhibitory nerves (1, 33). On
arrival of each antral peristaltic wave in the pyloric region,
there are 6 – 8 duodenal slow waves in the mouse and 10 –14
slow waves in the rat available for coordinated motor activity;
hence, the smooth transition of contents into the small intestine. Recently, simultaneous assessment of antral and duodenal
peristalsis and video analysis as performed in the present study
were carried out in humans, whereas for each antral slow wave,
three to five slow waves occur in the duodenum (4).
The ICC-AP are the pacemaker cells that generate the
primary dominant pacemaker activity (25, 49). Tissues that are
rich in ICC-IM but lack ICC-AP, such as the esophagus and the
fundus, do not generate omnipresent slow wave activity. When
pacemaker ICC are removed from tissues in the intestine (24,
56) or stomach (2, 21) or colon (34, 48), the primary slow wave
activity disappears. It is likely, however, that the ICC-IM do
play a role in modifying and propagating slow waves once they
are generated. In the mouse antrum, the ICC-IM contribute to
the secondary regenerative component of the slow wave (14)
and may be involved in slow wave propagation (20). Furthermore, they can provide secondary peristalsis in the absence of
the primary pacemaker activity (22, 24, 40). The present study
shows that slow waves are not generated in the pylorus despite
the presence of ICC-IM. Similarly, strong slow wave activity
in the very distal part of the canine antrum quite suddenly
disappears when entering the pylorus (45). Hence, ICC-IM
may not be capable of regenerating slow wave activity that
originates from ICC-AP over long distances without specific
stimulation.
Action potentials were observed in the pyloric region, both
independent of and associated with gastric slow wave activity.
Contractile activity in the pylorus related to the slow wave
activity of the stomach or duodenum will result from action
potentials originating at the crest of a stomach or duodenal
slow wave being able to propagate into the pyloric muscle as
shown in this study and in the cat sphincter (30). This indicates
that lack of slow wave propagation into the pylorus is not due
to a barrier to electrical conduction but likely due to the
strongly reduced number of ICC-AP. In the canine pylorus, one
Fig. 8. Quantification of Kit-positive ICC-AP
in different regions at the gastroduodenal junction. Bottom: examples of areas used for quantification. Kit positivity as a percentage of total
area of the sections chosen was calculated and
shown as a histogram (top). Kit positivity included both cell bodies and processes. Kitpositive ICC-AP were significantly decreased
within a segment (4 mm wide in unstretched rat
tissue) at the pyloric sphincter compared with
the adjacent antrum (P ⬍ 0.0001) and proximal
duodenum (P ⬍ 0.003); 50 sections were examined in each region.
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Fig. 7. Kit immunoreactivity in the musculature of mouse pylorus. a: Kit immunoreactivity in the longitudinal section of mouse
pyloric region. A similar distribution of ICC
was found in mouse as in the rat gastroduodenal area (Fig. 5a), except that the segment
devoid of ICC-AP was shorter in the mouse,
by ⬃1 mm. Arrows, ICC-AP in both antrum
(left) and duodenum (right). b: Whole mount
preparation of the mouse gastroduodenal
junction immunostained with c-Kit. Serial
pictures were taken from the distal antrum
(left) through the pyloric sphincter (middle)
to the proximal duodenum (right). Abundant
ICC-AP were found in both antrum and duodenum; these ICC-AP networks were interrupted at the region of the pyloric sphincter
(PS). The tissue was slightly stretched, making assessment of the width of the region
with a reduced number of ICC inaccurate.
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region close to the antrum had variable intrinsic oscillatory
activity that could be paced by antral slow waves (45). The
results from the present study make it likely that this region in
the canine pylorus with variable slow wave-like activity is a
region with an irregular and sparse ICC network. Some motor
patterns originating from this region might be referred to as
intrinsic pyloric activity. Interestingly, this region was gener-
ally more excitable consistent with distinct neural input (12,
27). It is clear that the pylorus does not have a primary
pacemaker system in connection with the networks of ICC-AP
in stomach and duodenum. Pyloric phasic contractile activities
(52) rely on 1) excitation of the pyloric muscle cells and
ICC-IM by efferent nerve supply specific to the pylorus (59) or
2) propagation of action potentials from the gastric or duodenal
Fig. 10. Temporal association between antral and duodenal
peristaltic contractions in the mouse. A: video images of mouse
stomach and duodenum showing an antral contraction (arrow)
that propagates from the proximal antrum (image 2) to the
pylorus (image 5). The pylorus opens (arrowhead) when the
antral contraction reaches midantrum (image 3). B: a spatiotemporal map constructed from the above video sequence is
shown. The numbers indicate time points corresponding to
single video images. Time lag between the end of an antral
contraction, defined as the contraction reaching the pylorus
(dotted line) and the beginning of a duodenal contraction, was
measured. Note that opening of the pylorus (arrowhead) with
passage of gastric content coincides with the origin of the
duodenal contraction. C: most frequently, duodenal contractions started a few seconds before the arrival of the antral
contraction at the pylorus.
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Fig. 9. In vivo motor patterns in mice. Spatiotemporal maps of contractions in the stomach and duodenum (x-axis ⫽ position along
the gut, y-axis ⫽ time). Darker regions contain
more contrast medium and represent relatively
relaxed areas, whereas lighter regions are associated with contractions. Any propagating
contraction appears in a map as an oblique
light line due to changes in optical density
associated with displacement of contrast material. Antral peristaltic contractions (double
arrows) propagated up to the pylorus (dotted
line). Duodenal peristaltic contractions (single
arrows) appeared either continuously (left) or
coordinated with (but preceding) the antral
contractions (middle; most common). Occasionally, there was continuous retroperistalsis
in the duodenum (single arrow, right) despite
the presence of regular aborally propagated
contractions in the antrum (double arrows).
This appeared on occasions when the pylorus
remained closed and no gastric contents entered the duodenum.
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through the ICC pacemaker network (10). The present study
shows that slow wave propagation does not happen through the
pylorus despite a continuous longitudinal muscle, clearly due
to the absence of a pacemaker ICC network.
The human pylorus has Kit-positive ICC-associated Auerbach’s plexus, which has extensions that are short and
branched (53) and possess ICC-IM, scattered throughout the
musculature that are bipolar with relatively long branches (53).
Ultrastructurally, the ICC of the human stomach have typical
features (50) and have close contact with smooth muscle cells
and nerve varicosities (17) very similar to the rat and mouse
(26, 43). The electrical pacemaker activities in both human
(16) and mouse (6, 14) stomach and human (9) and mouse (36)
small intestine are not fully elucidated but at this point do not
appear to be fundamentally different. Hence, the rat and mouse
appear to be suitable models to gain insight into human
gastroduodenal motility (43).
In conclusion, the motor patterns of the stomach and duodenum are largely independent due to absence of slow waves
and absence of an ICC-AP network in the pyloric region.
Aborally propagating peristaltic contractions of the stomach
are coordinated with peristaltic contractions of the duodenum
on flow of luminal content through a transiently relaxed pyloric
sphincter. These results have implications for development of
new therapies designed to improve gastropyloroduodenal coordination, which could focus on pyloric function (41) and
duodenal mechanosensitivity.
ACKNOWLEDGMENTS
The authors thank B. Stephen for performing the electrophysiological
experiments and S. Dhanasekaran for constructing the recording electrodes.
Discussions with Dr. Natalia Zarate were highly appreciated. Dr. L. Liu
provided Fig. 1B.
GRANTS
This study was supported by the Canadian Institutes of Health Research.
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