Regional and transmural density of interstitial cells
of Cajal in human colon and rectum
ROBERT HAGGER,1 SUSSAN GHARAIE,2 CAROLINE FINLAYSON,2
AND DEVINDER KUMAR1
Departments of 1Surgery and 2Histopathology, St. George’s Hospital,
London SW17 0QT, United Kingdom
gut motility; mast cells; myenteric plexus
THE INTERSTITIAL CELLS OF Cajal (ICC) are a group of
cells found throughout the gut from the esophagus to
the anus. ICC are thought to play an important role in
the control of gut motor activity, acting as pacemakers
(31), intermediaries in the neural control of gut muscular activity (5), spatial coordinators of gut motility (7),
and as stretch receptors (8, 9). These putative roles are
not mutually exclusive; the particular function that
ICC fulfill may be dependent on the location within the
gastrointestinal tract.
The study of ICC in gut tissue has been helped by
immunohistochemical methods using anti-c-kit antibody. ICC express the protooncogene c-kit (15, 36),
which encodes a tyrosine kinase cell surface receptor
(38). Immunohistochemistry using an anti-c-kit antibody is now providing a more selective technique for
identifying ICC in animal (15, 32) and human tissues
(33, 37) at the level of light microscopy; previously
reliable identification of ICC has relied on ultrastructural characteristics seen through electron microscopy
(10, 11, 23–26).
In the human colon, reports of ICC distribution have
been mainly based on electron microscopy studies. ICC
are found within the myenteric muscle sheaths in the
intermuscular plane (11). At the submucosal border of
the circular muscle, ICC are found in association with a
morphologically distinct inner layer of smooth muscle
cells (10, 25). ICC are also seen in the main bulk of the
circular muscle layer and the main intermuscular
septa (25). An immunohistochemical study of the colon
using an anti-c-kit antibody in a small number of
patients showed ICC to be present in the longitudinal
and circular muscle layers and the intermuscular plane
and in association with the submucosal plexus at the
inner margin of the circular muscle layer (34).
The functions of the colorectum include absorption of
water and sodium from the chyme that enters from the
ileum and also the fatty acid products of bacterial
fermentation and then removal of stool from the body
during defecation. The motor activity and gross morphology of the colon and rectum are adapted so that
these processes may proceed efficiently. Segmental
differences are apparent: the right colon acts as a
storage region for ileal effluent so that absorption may
occur, the left colon acts as a conduit for the passage of
feces, and the rectum can act as a temporary storage
region for stool before defecation.
The aim of this study was to evaluate the transmural
and regional pattern of distribution of ICC in the
normal human colorectum, using immunohistochemistry with an anti-c-kit antibody.
MATERIALS AND METHODS
Subjects
Colorectal tissue was obtained from surgical resections
performed for carcinoma of the colon or rectum, from patients
whose previous bowel habit had been normal. Tissue was
defined as normal when the carcinoma was nonobstructing
and the section was not invaded by the tumor. The regions of
interest studied were the right, transverse, and left colon and
the rectum. The specimens were fixed in phosphate-buffered
10% Formalin (pH 7.0) and then processed into paraffin wax.
Thirty-one right colonic, 31 transverse colonic, 53 left colonic,
and 28 rectal tissue sections were obtained from 108 surgical
specimens. There were 52 male and 56 female patients
(median age 74 yr, range 44–91 yr). The mean age of patients
from which specimens were obtained did not differ significantly among the regions.
Immunohistological Staining
Immunohistochemistry was performed using the avidinbiotin-peroxidase method. The primary antibody used was a
commercial rabbit affinity-purified polyclonal anti-c-kit antibody (Oncogene Science, Uniondale, NY). Sections were cut at
4 µm from paraffin-embedded tissue blocks and mounted on
aminoalkylsilane-coated slides (Sigma Chemical, St. Louis,
MO). The sections were deparaffinized in xylene and methanol. Endogenous peroxidase activity was blocked by a 30-min
immersion period in 0.3% hydrogen peroxide in absolute
methanol at room temperature. To unmask antigenic sites,
0193-1857/98 $5.00 Copyright r 1998 the American Physiological Society
G1309
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Hagger, Robert, Sussan Gharaie, Caroline Finlayson, and Devinder Kumar. Regional and transmural density of interstitial cells of Cajal in human colon and rectum.
Am. J. Physiol. 275 (Gastrointest. Liver Physiol. 38): G1309–
G1316, 1998.—The interstitial cells of Cajal (ICC) are thought
to play an important role in the control of gut motility. The
regional and transmural pattern of distribution of ICC in the
normal human colon and rectum was evaluated with immunohistochemistry using an anti-c-kit antibody. The transmural
distribution of ICC was constant throughout the whole colon,
the density of ICC was significantly greater at the myenteric
plexus than at either the longitudinal or circular muscle
layers, and in the rectum the transmural distribution was
more even. Regionally, at the myenteric plexus, the transverse colon had a significantly greater density of ICC compared with the right colon (P 5 0.038), left colon (P 5 0.006),
and rectum (P 5 0.008). The pattern of distribution of ICC
identified in this study is consistent with the proposed roles of
ICC as colorectal pacemakers, intermediaries of the neural
control of muscle activity, and coordinators of colorectal
muscle activity. The highest density of ICC was at the
myenteric plexus of the transverse colon, which is the proposed region of pacemaking activity.
G1310
ICC DENSITY IN HUMAN COLON AND RECTUM
Distribution Analysis
The density of distribution of ICC was assessed in the
longitudinal and circular muscle layers and in the intermuscular plane of the colorectum. The density of ICC was graded
after the evaluation of 10 well-stained and well-orientated
high-power fields (3400 magnification), with the field having
an area of 0.152 mm2. The longitudinal muscle layer, intermuscular layer, and circular muscular layer were each assessed
by 10 different fields. The grades of ‘‘sparse,’’ ‘‘few,’’ ‘‘moderate,’’ and ‘‘many’’ reflected an average count of 0–1, 2–3, 4–8,
and .8 cell bodies per high-power field, respectively. Only
ICC with nuclei were counted. The assessment was made by
two independent observers. If there was disagreement in the
grade assigned, a consensus was reached after joint review.
Interrater agreement gave a k statistic of 0.45, with a
weighted k of 0.59.
Fig. 1. Interstitial cells of Cajal (ICC) (dark stain; arrowheads) in
longitudinal muscle of transverse colon. Original magnification,
3400. Scale bar, 20 µm.
network encasing the myenteric nerve plexus (Fig. 2).
In the circular muscle layer, ICC were again found in
the bulk of the muscle in parallel orientation with the
muscle fibers (Fig. 3) and in association with blood
vessels. ICC were also seen lining the intramuscular
Statistical Analysis
The x2 test was used to compare the transmural variations
of ICC density observed among the circular muscle layer,
myenteric plexus, and longitudinal muscle layer within a
segment of the colorectum. The density of ICC was also
compared among the different segments of the colorectum for
each layer of the muscularis propria.
RESULTS
The immunohistochemical method clearly identified
c-kit-positive cells. ICC and mast cells showed c-kit
immunoreactivity in the colon and rectum. ICC had a
fusiform cell body with a large oval nucleus and two or
more dendritic processes (Fig. 1), and mast cells were
round with a round central nucleus. These characteristics allowed the two cell types to be distinguished.
Colon
The pattern of distribution of ICC in the colon was
the same for the right, transverse, and left colon. In the
longitudinal muscle layer ICC were identified in the
muscle bulk in parallel orientation with the muscle
fibers and also in association with penetrating blood
vessels. In the intermuscular plane, ICC formed a
Fig. 2. ICC (dark stain) encasing myenteric plexus of right colon.
Longitudinal muscle layer (L), myenteric plexus (MP), and circular
muscle layer (C) are indicated. The whole of the field is not visualized,
but the density grade in the myenteric plexus would be ‘‘many.’’
Original magnification, 3250. Scale bar, 40 µm.
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sections were boiled in a microwave for 10 min in citrate
buffer (0.21%, pH 6.0) and then allowed to cool for 20 min. To
prevent nonspecific absorption of immunoglobulin, we incubated the sections for 20 min in 3% BSA (Sigma Chemical) in
Tris buffer (0.05 M, pH 7.6). The sections were then incubated
with the primary antibody, rabbit anti-c-kit antibody 0.1
µg/ml in 3% BSA-Tris for 24 h at 4°C. Sections were then
incubated at room temperature for 30 min with a biotinconjugated swine anti-rabbit immunoglobulin (DAKO, Glostrup, Denmark) diluted 1:250 in Tris buffer, followed by a
30-min incubation period in avidin-biotin-peroxidase complex
(DAKO). Each incubation period was followed by gentle
washing in Tris buffer. The bound complex was visualized by
using the 3,38-diaminobenzidine (DAB)-hydrogen peroxide
reaction according to the method of Graham and Karnovsky
(12). The sections were immersed in DAB (0.5 mg/ml) and
0.03% hydrogen peroxide in Tris for exactly 10 min. The
sections were then washed well in tap water. Counterstaining
was performed with hematoxylin. The sections were dehydrated in methanol and mounted using an automatic Tissue
Tek coverslipping device (Sakura Finetek Europe, Zoeterwoude, The Netherlands).
ICC DENSITY IN HUMAN COLON AND RECTUM
G1311
cosa, or mucosa, although c-kit-positive cells identified
morphologically as mast cells were present (Fig. 5).
Mast cells were also present in all layers of the muscularis propria.
Rectum
Transmural and Regional Distribution of ICC
Fig. 3. ICC (dark stain; arrowheads) in circular muscle layer of left
colon. The whole of the field is not visualized but the density grade in
the myenteric plexus would be moderate. Original magnification,
3400. Scale bar, 20 µm.
septa. In the muscle layers, ICC were observed to be
connected through dendritic processes to other ICC,
most often in a linear arrangement. ICC were identified
at the inner layer of smooth muscle fibers at the
submucosal border of the circular muscle (Fig. 4). ICC
were not identified in the submucosa, muscularis mu-
Colon. The density of ICC in the muscularis propria
of the colon and rectum, respectively, was graded on the
basis of the number of ICC cell bodies seen in 10
high-power fields (3400). In the colon, x2 distribution
analysis revealed significant differences in the density
of ICC between the myenteric plexus and circular
muscle layer and between the myenteric plexus and the
longitudinal muscle layer in the right colon (Fig. 6A),
transverse colon (Fig. 6B), and left colon (Fig. 6C).
There were no significant differences in the density of
Fig. 4. An ICC at inner border of circular muscle layer (arrowhead). A
mast cell (MC) is also seen. Original magnification, 3400. Scale bar,
20 µm.
Fig. 5. Mast cells (dark stain) in lamina propria of rectum. Original
magnification, 3400. Scale bar, 20 µm.
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In the longitudinal and circular muscle layers of the
rectum, ICC were seen in the muscle bulk and in
association with penetrating blood vessels. The ICC
were mainly in parallel orientation with the muscle
fibers. ICC formed networks in the muscle layers and
dendritic processes ramified between the muscle fibers
more frequently than was observed in the colon. In the
intermuscular plane, ICC were seen in association with
elements of the myenteric plexus. At the inner margin
of the circular muscle, occasional ICC were observed
and were also seen in association with neural elements
of the submucosal plexus. As in the colon, in the lamina
propria and mucosa of the rectum c-kit-positive cells
were present but on a morphological basis were classified as mast cells. Mast cells were again also present in
all layers of the muscularis propria.
G1312
ICC DENSITY IN HUMAN COLON AND RECTUM
ICC between the longitudinal muscle layer and circular
muscle layer in any of the colonic regions.
Rectum. In contrast to the colon, the distribution of
ICC was homogenous throughout the layers of the
muscularis propria in the rectum (Fig. 6D). There were
no significant differences in ICC density between the
circular muscle layer and longitudinal muscle layer,
the circular muscle layer and myenteric plexus, or
between the longitudinal muscle layer and myenteric
plexus.
Longitudinal muscle layer. Segmental variations in
the density of ICC in the layers of the muscularis
propria were apparent. In the longitudinal muscle
layer (Fig. 7A), the only significant differences were
between the rectum and right colon (P 5 0.005) and
between the rectum and left colon (P 5 0.02), with the
rectum having the higher density of ICC.
Intermuscular plane. At the level of the myenteric
plexus (Fig. 7B), the transverse colon had a significantly different ICC density compared with the right
colon (P 5 0.038), the left colon (P 5 0.006), and the
rectum (P 5 0.008), with a higher proportion of sections
with the higher density gradings.
Circular muscle layer. In the circular muscle layer
(Fig. 7C), the rectum had a significantly greater density of ICC than the left colon (P 5 0.004); no other
significant regional differences were apparent in the
circular muscle layer.
DISCUSSION
In this study, immunohistochemistry using a rabbit
anti-c-kit antibody identified two different cell types.
On a morphological basis, these were determined to be
ICC or mast cells. In the lamina propria, muscularis
mucosa, and mucosa, the c-kit-positive cells present
were deemed to be mast cells, which is in agreement
with a previous report as to the nature of c-kit-positive
cells in the human colon (34). Mast cells were also
present in the muscularis propria. The morphological
characteristics of ICC that we identified in human
colonic ICC are similar to those recently described in a
study of the human colon using anti-c-kit immunohistochemistry (34).
In animal studies it has been suggested that the
expression of c-kit by ICC may wane with increased age
(32). In a small group of subjects, with an age range of 1
day to 30 years, the distribution of ICC in the colon was
reported as similar across the age range (34). In this
study, c-kit expression by ICC was present in tissue
taken from patients over 90 years of age. In our
personal experience of ICC in colonic tissue from chil-
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Fig. 6. Transmural distribution of ICC
in right colon (A), transverse colon (B),
left colon (C), and rectum (D). Density
% represents proportion of specimens
that were assigned to a particular density grading in each layer of muscularis
propria. Sparse, few, moderate, and
many represent 0–1, 2–3, 4–8, and .8
ICC cell bodies, respectively, in a highpower (3400) field. In the colon, x2
distribution analysis revealed significant differences in density of ICC between myenteric plexus and circular
muscle layer and between myenteric
plexus and longitudinal muscle layer in
right, transverse, and left colon. In
contrast to colon, in rectum there were
no significant differences in ICC density between circular muscle layer and
longitudinal muscle layer, circular
muscle layer and myenteric plexus, or
between longitudinal muscle layer and
myenteric plexus.
ICC DENSITY IN HUMAN COLON AND RECTUM
G1313
Fig. 7. Regional distribution of ICC in longitudinal muscle layer (A),
myenteric plexus (B), and circular muscle layer (C). See Fig. 6 legend
for explanation of density % and grading scale. In longitudinal
muscle layer, the only significant differences were between rectum
and right colon (P 5 0.005) and between rectum and left colon (P 5
0.02). At the level of myenteric plexus, transverse colon had a
significantly different ICC density compared with right colon (P 5
0.038), left colon (P 5 0.006), and rectum (P 5 0.008). In circular
muscle layer, rectum had a significantly greater density of ICC than
left colon (P 5 0.004); no other significant regional differences were
apparent in circular muscle layer.
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dren in the first decade we have observed a similar
density of ICC to that seen in this adult population. The
use of anti-c-kit immunohistochemistry would therefore appear to be a suitable method for the study of ICC
in all ages; however, it is possible that not all ICC
present are visualized due to diminished c-kit expression with increasing age.
In each segment of the colon, the greatest density of
ICC was observed in the intermuscular plane in which
ICC encased the myenteric plexus. The density of ICC
in the intermuscular plane was significantly greater in
comparison to the circular and longitudinal muscle
layers in the right, transverse, and left colon. Our
finding is in disagreement with a recent report (34) of
an even pattern of distribution of ICC in the muscularis
propria of the colon. Descriptions of the distribution of
ICC in the human colon on the basis of electron microscopic evidence have reported the presence of ICC around
the myenteric plexus (11) and within the bulk of the circularmuscle layer in the right, transverse, and sigmoid
colon (25). We demonstrate that ICC are also present
within the bulk of the longitudinal muscle layer.
We observed ICC throughout the colon, and also in
the rectum, at the inner layer of the circular muscle
layer in association with the submucosal plexus. This is
in agreement with Rumessen et al. (25) who identified
ICC throughout the colon, at the inner layer of the
circular muscle, and contradicts the observations of
Faussone-Pellegrini et al. (10) who identified ICC at
the inner layer of the circular muscle but in the right
colon only. We identified ICC at the inner margin of the
circular muscle layer in association with the submucosal plexus in all regions of the colon and the rectum;
however, these ICC were extremely sparse and certainly did not appear to form a continuous lining as has
been previously described in other animal species (2).
Segmental variations in the density of ICC at various
levels of the muscularis propria were apparent. At the
myenteric plexus, the transverse colon had a higher
proportion of moderate and/or many density gradings
than the rectum or right or left colon. In the circular
muscle layer, the rectum exhibited a significantly higher
density of ICC than the left colon, and the right and
transverse colon tended to have a higher density than
the left colon, but this did not achieve significance. In
the longitudinal muscle layer, the rectum displayed a
significantly higher density of ICC compared with the
right and left colonic regions. Such regional variations
in the human colon have not been previously documented. In a small study (34) of five human colonic
specimens, there were no segmental differences re-
G1314
ICC DENSITY IN HUMAN COLON AND RECTUM
correlates well with our observation that the ICC
density in the myenteric plexus is greatest in the
transverse colon.
ICC may act as intermediaries in the enteric neural
control of gut muscle activity (5). The association of ICC
in the colon and rectum with neural elements of the
myenteric plexus, and to a lesser extent with the
submucosal plexus, would support this hypothesis.
In the human colon and rectum, contractile activity
is characterized by propagated and nonpropagated
contractions. The nonpropagated motor activity may
reflect pacing of the smooth muscle by sparsely distributed ICC in the bulk of the muscle layers. Propagated
motor activity in the form of colonic motor complexes,
rectal motor complexes, or high amplitude propagated
contractions would require communication between
different regions of the colon and/or rectum through
neural or ICC networks. Similarly, transmural differences in ICC density may explain the initiation of
segmental contractions in the colon and the lack of
segmenting activity in the rectum. Data from animal
studies suggest that ICC networks mediate communication between different areas of the gut, whether between different muscle layers (17) or along the long axis
of the gut (13, 19). The arrangement of ICC in linear
chains or branching networks would be consistent with
this role. ICC in the intermuscular plane may coordinate the activity of the circular and longitudinal muscle
layers and may allow neural networks to modify muscular activity through alteration of ICC activity.
In summary, in the muscularis propria of the colon,
ICC density was found to be greatest in the intermuscular plane compared with the muscle layers, but in the
rectum the distribution of ICC was more even (Fig. 8).
Fig. 8. Schematic representation of ICC density in colon and rectum.
ICC density in colon is greatest at myenteric plexus and is maximal
in transverse colon. In rectum ICC density is more even through
muscularis propria.
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ported. In animal studies (4), regional variations of
density of ICC in the colon have been described. In the
cat, dog, rabbit, and opossum, ICC were sparse in the
cecum, reached maximal density in the middle part of
the colon, and became progressively less dense toward
the rectum (4); this pattern is similar to that which we
observed in humans. However, in the animals (4) this
regional variation was observed at the submucosal plexus,
whereas we observed the regional variation at the myenteric plexus. In these animals (4), few ICC were observed
in association with the myenteric plexus, in which the
density of ICC was proportional to the density of the
plexus itself, and ICC were less conspicuous in the
cecum and rectum compared with the rest of the colon.
Proposed functions for ICC include acting as pacemakers (21, 31, 32, 35, 36), intermediaries in the neural
modulation of muscle activity (5), spatial coordinators
of muscle activity (7), and as stretch receptors (8, 9).
These roles are not mutually exclusive. The evidence
that ICC act as pacemakers of gut muscular activity is
derived from animal studies. In the dog, colonic ICC at
the inner margin of the circular muscle layer in association with the submucosal plexus generate slow waves
(1, 18, 20, 28) and thus act as pacemakers; a second
pacemaker region at the myenteric plexus is also
present (29). Our study would suggest that the ICC at
the inner layer of the circular muscle layer would not
form a sufficiently dense network to act as the main
pacemaker region. A more plausible candidate for the
main pacing region would be the intermuscular plane,
in which the density of ICC is greatest. The electrical
activity of the longitudinal muscle of the colon is
similar in nature across the species (3). In the human
longitudinal muscle layer, electrical activity is characterized by oscillatory activity in a narrow frequency
range [24–36 counts/min (cpm)] with superimposed
spiking activity with or without periods of quiescence.
The electrical activity in the circular muscle layer
shows interspecies variation: in the dog slow waves
with constant amplitude and frequency are observed
(3) and in the human colon a range of electrical
frequencies is apparent (3, 14). In the human circular
muscle layer, the slow wave frequency is very variable
and lies within the range of 4–60 cpm (14). The
frequency and amplitude are both variable and change
with stimulation, and periods of electrical quiescence
are apparent (14). The variable slow wave frequency
suggests that multiple pacemakers exist. Pacing of the
circular muscle in the human may not primarily reside
in the submucosal ICC as suggested by the canine
model (6, 30), and another pacing region in the intermuscular region may be present and may play the more
dominant role, as suggested by our observations.
It has been suggested from animal studies of the cat,
dog, ferret, guinea pig, opossum, rabbit, and rat that
the density of ICC in the stomach and colon roughly
parallels the prominence of slow waves (4). In the
human colon, regional variations in slow wave activity
have been reported, and in vivo studies have demonstrated that the overall dominant frequency of contractions is highest in the midcolonic region (27). This
ICC DENSITY IN HUMAN COLON AND RECTUM
Segmental density variations of ICC were identified;
in the intermuscular plane the greatest density of
ICC was observed in the transverse colon, which is
the region of proposed pacemaking activity. Abnormal
distribution of ICC has been reported in the affected
tissue in gut dysmotility disorders, such as infantile
pyloric hypertrophic stenosis (16, 33) and Hirschsprung’s disease (34, 37), and abnormal structure
of ICC has been identified in patients with ulcerative
colitis (22). Labeling ICC using an anti-c-kit antibody
will help in the understanding of normal human
colonic physiology and pathophysiology of diseases in
which abnormal gut motor activity is an underlying
feature.
Received 3 June 1997; accepted in final form 2 September 1998.
14.
15.
16.
17.
18.
19.
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