Int J Colorect Dis (1998) 13: 208 – 216
O R I G I N A L A RT I C L E
A. J. Porter · D. A. Wattchow · A. Hunter · M. Costa
Abnormalities of nerve fibers in the circular muscle
of patients with slow transit constipation
Accepted: 14 January 1998
Abstract Abnormalities of the enteric nervous system are
thought to explain the pathophysiology of motility disor-
ders. Our aim was to determine if particular classes of en-
teric neurons are affected in slow transit constipation
(STC). Specimens were taken from the terminal ileum and
ascending, transverse and descending colon of patients
undergoing subtotal colectomy for STC. Immunohisto-
chemistry was performed using antisera to neuron-specific
enolase, tachykinin, leu-enkephalin, choline acetyltransfe-
rase, vasoactive intestinal peptide, nitric oxide synthase,
tyrosine hydroxylase and neuropeptide Y. The density of
nerve fibres labelled with these antibodies in each layer
was compared with age-matched controls. The density of
nerve fibres with tachykinin and enkephalin immunoreac-
tivity was reduced in the colonic circular muscle of the 15
patients with STC, whereas innervation of all other layers
was normal. This reduction of tachykinin-immunoreactive
nerve fibres also occurred in nine of the 12 specimens of
terminal ileum examined. No difference was detected in
the density or distribution of nerve fibres using the other
antisera. Excitatory nerve fibres are present in the circular
muscle in STC but they are deficient in tachykinins and
enkephalin.
Key words Slow transit constipation ·
Immunohistochemistry · Enteric nervous system
A. J. Porter · D. A. Wattchow (½)
Department of Surgery, Flinders Medical Centre,
GPO Box 2100, Adelaide 5001;
Tel.: 0061-8-8204-4140; Fax: 0061-8-8374-0832;
e-mail: pmnajp@pippin.cc.flinders.edu.au
A. Hunter
Department of Surgery, Royal Adelaide Hospital,
Adelaide, Australia
M. Costa
Department of Human Physiology and Centre for Neuroscience,
Flinders University of South Australia, Adelaide, Australia
© Springer-Verlag 1998
Introduction
Patients frequently present to their physicians with symp-
toms of constipation and, in the absence of intestinal ob-
struction, are usually successfully treated by correcting a
fibre-deficient diet. In some cases, no cause is found and
the constipation does not respond to bulking agents, laxa-
tives or prokinetic agents. In these patients, the constipa-
tion may be very severe, with the patients using their bow-
els every 2–3 weeks (most people daefecate between three
times a day and once every 3 days [1]) and having asso-
ciated symptoms of distension and discomfort. Character-
istically these patients are women whose constipation has
commenced in their teenage years, as first described by Ar-
buthnot Lane in 1908 [2]. It is likely that the disease he de-
scribed is the same as the disorder now known as slow tran-
sit constipation (STC). In most cases, the colon and rec-
tum are of normal calibre and, by convention, this condi-
tion excludes patients with megarectum or megacolon [3].
Patients with intractable constipation are classified into
those with slow, generalised passage of faecal content, as
determined by transit studies, and those in whom the tran-
sit is normal but there is a problem with evacuation of con-
tent from the rectum. When patients with severe chronic
constipation are investigated, about 10% have slowed tran-
sit alone and 5% have both slowed transit and pelvic floor
dysfunction [4]. In such patients, the available evidence
indicates that the pelvic floor dysfunction is not respon-
sible for the colonic slow transit [5].
In patients with STC, symptoms are typically worsened
by increasing dietary fibre and they have usually had a con-
siderable period of laxative use for alleviation of their
symptoms. If this is ineffective, subtotal colectomy with
ileorectal anastomosis may be indicated. This operation is
highly effective in relieving the constipation in these pa-
tients, although less effective in relieving problems of
bloating and pain [6].
The colon usually appears normal to routine histologi-
cal evaluation of these subtotal colectomy specimens. Use
of the silver impregnation technique has revealed abnor-
 209

malities in myenteric neurons [8] but these techniques do
not allow differentiation of the various functional classes
of myenteric neurons that are now known to exist, such as
motor neurons, interneurons or sensory neurons [9]. Sev-
eral studies have reported abnormal innervation patterns
of the bowel wall in patients with STC, although the re-
sults have often been conflicting. This has especially been
true regarding the concentration of vasoactive intestinal
peptide (VIP) and density of VIP-immunoreactive (IR)
nerve fibres in the circular muscle, with decreases [10], in-
creases [11] and no changes [12, 13] being reported. Sev-
eral other abnormalities have been described in patients
with STC, such as an increase in calcitonin gene-related
peptide-IR nerve fibres in the myenteric plexus [12]. Some
of these studies examined only one region of colon, either
the sigmoid or descending colon or the rectum, which may
partially account for the disparity of results. In most stud-
ies, the control group was older than the STC group, as the
bowel was obtained from elderly patients with colon can-
cer. Given that significant changes occur in the enteric ner-
vous system of humans and experimental animals with age-
ing [14–16], it is essential that controls are age-matched.
The present study examines the innervation of the co-
lonic wall of patients with well characterised STC using a
variety of neurochemical markers. The distribution and
density of innervation were compared with age- and sex-
matched control specimens.
Patients and methods
Tissue collection
Fifteen women (median age 36 years; range 23–66 years) underwent
subtotal colectomy and ileorectal anastomosis for the treatment of
STC. All had had constipation for many years unsuccessfully treat-
ed by increasing dietary fibre or fluids, or by the use of laxatives.
Laxative use was common but difficult to quantitate as multiple types
of laxatives had been used for varying lengths of time and the pa-
tients had poor recollection of exact laxative usage. In all of these
patients, the colon was of normal calibre as revealed by barium en-
ema. Patients with megacolon or megarectum were excluded from
this study. Slow transit was defined by slow passage of radiopaque
or radionuclide markers, with retention of more than 20% of the
markers at 96 h being diagnostic of STC. For clinical purposes, these
two tests are equivalent [17].
Once the colon was removed, segments of approximately 2×2 cm
were cut from the terminal ileum (in 12 patients), ascending colon,
transverse colon and descending colon (in all patients). All speci-
mens were cut from the inter-taenial portions of the colonic wall.
Control tissue was taken from six women (median age 35 years);
range 26–44 years undergoing surgery for colonic cancer or polyps
or having colon removed as part of surgery for ovarian cancer or sar-
coma. These six patients were age-matched to the STC group. There
were four specimens of terminal ileum, three of ascending colon, two
of transverse colon and three of descending colon. Histopathologi-
cal examination of these specimens was performed to confirm that
there was no evidence of tumour invasion or inflammation in the
specimens. These patients had no symptoms of bowel obstruction
and none of the patients had systemic disorders affecting the enter-
ic nervous system, motility disorders or inflammatory bowel disease.
All tissue was taken with prior informed consent. This study was ap-
proved by the Flinders Medical Centre Committee on Clinical In-
vestigation.
Table 1 Antisera used for immunohistochemistry
Antigen Antisera code Host Dilution Reference
ChAT AB1582 Sheep 1:1000 [18]
leu-ENK 198B Rabbit 1:400 [19]
NOS K205 Sheep 1:1000 [20]
NPY RMJ263 Rabbit 1:1600 [21]
NSE A 589 Rabbit 1:500 [22]
TK RMSP4 I/II Rabbit 1:2000 [23]
TH LCN1 Mouse 1:1000 [24]
VIP 7913 Rabbit 1:3200 [25]
ChAT, choline acetyltransferase; leu-ENK, leu-enkephalin; NOS, ni-
tric oxide synthase; NPY, neuropeptide Y; NSE, neuron-specific en-
olase; TK, tachykinin; TH, tyrosine hydroxylase; VIP, vasoactive in-
testinal peptide
Immunohistochemistry
Full thickness specimens of intestine were immersed in room tem-
perature phosphate-buffered saline (if operation performed at Flind-
ers Medical Centre) or in ice-cold phosphate-buffered saline (if op-
eration performed at another hospital). After transfer to the labora-
tory, with a delay of about 2 h if transferred from another hospital,
the specimens were pinned to a Sylgard-lined petri dish with suffi-
cient stretch to flatten the preparation. They were then immersed in
a solution of 2% paraformaldehyde with 15% picric acid, in a 0.1 M
phosphate buffer (pH 7.0) overnight at 4 °C.
After fixation, the specimens were cleared by three 15 min wash-
es in dimethyl sulphoxide and then placed in phosphate-buffered sa-
line (pH 7.0) containing 30% sucrose. Frozen sections (12 µm thick)
were cut both in the transverse and longitudinal axes, to provide
more accurate information about nerve fibre density, and collected
on chrome-alum coated glass slides before drying over P 2O5. After
incubating in phosphate-buffered saline containing 10% nonim-
mune serum for 30 min, the sections were incubated in the primary
antibodies (Table 1) overnight at room temperature. The choline
acetyltransferase (ChAT) antiserum (code AB1582; Chemicon,
Temecula, CA) was raised in a sheep using the method described by
Benecke et al. [18] and labels the same neurons in guinea-pig intes-
tine as another well characterised ChAT antibody (code PO 3;
Yeboah, Hannover, Germany; kindly provided by Dr. M. Schemann
[26]). The sections were then washed in phosphate-buffered saline
and incubated for 1 h with secondary antisera. For rabbit antisera,
fluorescein-conjugated sheep anti-rabbit immunoglobulin (IgG)
(Wellcome Diagnostics, Beckenham, England; code 890520) was
used at 1:160; for sheep antisera, Cy5-conjugated donkey anti-sheep
IgG (Jackson, West Grove, PA; code 25324) was used at 1:20
and for the mouse antisera, fluorescein-conjugated donkey anti-
mouse IgG (Jackson; code 34010) was used at 1:100. They were
again washed in phosphate-buffered saline and mounted in buffered
glycerol (pH 8.6)
Analysis
The specimens were viewed under a Leitz epifluorescence micro-
scope (Leitz Inc., Rockleigh, New Jersey) or an AX70 epifluores-
cence microscope (Olympus Optical, Tokyo, Japan) fitted with ap-
propriate filter blocks. The density of nerve fibres in each layer of
the intestinal wall was graded from 0 to 4+ by two independent ob-
servers (AJP and DAW); 0=no nerve fibres seen, 1+=sparse,
2+=moderate, 3+=high, 4+=very high (see examples in figures) [27].
In the rare occurrence that the two observers did not give the same
grade, the average of the two grades was taken. Data obtained from
each patient was grouped into ileum and colon (after averaging re-
sults from the different regions of colon, as there were no differenc-
es in innervation density between the regions of colon examined, ex-
cept where specifically mentioned in the text). The density of nerve
fibres seen with each antibody was expressed as a mean and com-
210
Table 2 Mean density of nerve fibres containing each neurochemical marker in ileum and colon of control and slow transit constipation
(ST) patients

Ileum NSE TK ENK ChAT VIP NOS NPY

C ST C ST C ST C ST C ST C ST C ST
n 4 12 4 12 4 12 4 6 4 12 4 7 4 12

LM 3.0 2.6 2.3 1.6 3.0 1.4 1.7 1.0 3.0 1.9 3.0 2.3 1.3 1.3
MP 4.0 4.0 4.0 4.0 4.0 3.0 4.0 3.0 4.0 4.0 4.0 3.7 3.0 2.6
CM 4.0 4.0 4.0 1.5a 3.3 2.3 2.3 2.2 3.7 3.9 4.0 4.0 2.0 1.8
SP 3.3 3.3 2.0 2.7 1.3 1.1 1.7 1.3 3.0 3.0 0.7 0.3 2.3 1.7
MU 4.0 4.0 3.7 3.6 1.0 0.9 0.0 0.0 4.0 4.0 0.0 0.0 2.4 1.7

Colon
n 5 15 5 15 5 15 5 6 5 15 5 7 5 15

LM 1.7 1.2 0.6 0.5 0.4 0.3 0.5 0.4 1.1 0.6 1.3 1.1 0.4 0.3
MP 4.0 4.0 3.9 3.8 3.8 3.3 3.0 2.9 4.0 3.9 4.0 4.0 2.8 2.2
CM 4.0 3.9 3.5 1.2b 3.6 2.0c 2.5 2.3 3.9 3.8 4.0 4.0 2.0 1.5
SP 3.6 3.4 2.7 2.2 0.9 1.3 1.5 1.3 3.0 3.0 0.6 0.4 1.4 1.4
MU 4.0 3.9 3.9 3.3 0.9 0.9 0.0 0.0 4.0 4.0 0.0 0.0 1.9 1.7

ChAT, choline acetyltransferase; ENK, leu-enkephalin; NOS, nitric oxide synthase; NPY, neuropeptide Y; NSE, neuron-specific enolase;
TK, tachykinin; VIP, vasoactive intestinal peptide; LM, longitudinal muscle; MP, myenteric plexus; CM, circular muscle; SP, submucosal
plexus; MU, mucosa.
a
p=0.03, b p=0.003, c p=0.008

parisons of innervation density between STC patients and controls
were made using the Mann-Whitney U test corrected for ties, with a
probability of less than 0.05 considered to be significant. The num-
bers of patients having specimens labelled with each antibody is
shown in Table 2. The presence of nerve cell bodies in the myenter-
ic and submucosal ganglia, mucosal endocrine cells and nerve fibres
surrounding blood vessels was noted with each antibody.
Results
General
Neuron-specific enolase
The same pattern of innervation with neuron-specific en-
olase (NSE)-IR nerve fibres was seen in the control group
and in the patients with STC. There were many nerve fi-
bres in the circular muscle, the myenteric and submucosal
plexuses and the mucosa (Fig. 1). Many nerve cell bodies
were seen in both the myenteric and submucosal ganglia.
In the inter-taenial longitudinal muscle, the density of
nerve fibres varied from none (0) to high (3+) in both
groups of patients.

The mean density of immunoreactive nerve fibres in each Tachykinin

layer of gut is represented in Table 2 for terminal ileum
and large intestine for both the STC and the control group.
There was no apparent difference in innervation pattern
between the different areas of large intestine examined in
the control group, but there was some variation in den-
sity of tachykinin and enkephalin innervation between
different regions of large intestine in five of the patients
with STC. The density of innervation in each layer with
each antibody was very consistent, except for labelling
within the longitudinal muscle layer, which was variable.
Most specimens were from inter-taenial regions, where
only a very thin layer of longitudinal muscle was present.
It is not known if the innervation of this thin layer of lon-
gitudinal muscle is representative of the rest of the lon-
gitudinal layer, the bulk of which is contained in the tae-
nia coli.
The specimens of bowel from patients with STC had
normal histology using standard haematoxylin and eosin
stains. They had no evidence of either muscle hypertrophy
or atrophy, the mucosa was not inflamed and melanosis
coli was present in only one patient.
Patients with STC had a significantly decreased density of
tachykinin (TK)-IR nerve fibres in the circular muscle
layer compared to the control group in the terminal ileum
and large bowel (Fig. 2). TK-IR nerve cell bodies were seen
in the myenteric and submucosal ganglia in both groups,
as were many TK-IR nerve fibres in the myenteric plexus
and mucosa (Fig. 3).
In the control group, the density of TK-IR nerve fibres
in the circular muscle was similar in all regions examined.
However there was more variability in the STC group. Ten
patients had either sparse or no TK-IR fibres in the circu-
lar muscle (0/1+) in all regions examined. In four patients,
the decrease in density was more marked in the ascending
colon, and in one it was more marked in the descending
colon. Of the 12 specimens of terminal ileum examined,
three had normal TK-IR nerve fibre density in the circular
muscle (3+/4+) and nine had a significantly decreased den-
sity (0/1+).

Fig. 1 Transverse section through ascending colon of a patient with
STC labelled with neuron-specific enolase (NSE) immunoreactivity.
A very high density of nerve fibres (4+) was demonstrated in the cir-
cular muscle (closed arrowheads), longitudinal muscle (open arrow-
heads) and myenteric plexus (arrows). Calibration bar, 50 µm
Fig. 2a, b Transverse sections through the descending colon of a
control patient (a) and a slow-transit constipation (STC) patient (b)
showing tachykinin-immunoreactive (TK-IR) nerve fibres. In the
control specimen, there were many TK-IR nerve fibres (4+) in the
circular muscle (closed arrowheads) but in the STC specimen, there
were none (0). There was a very high density of nerve fibres in the
myenteric plexus (arrow) of both patients. The longitudinal muscle
layer had scant innervation (1+) in the control patient (open arrow-
head) and was absent in this specimen in the STC patient. Calibra-
tion bar, 50 µm
211
Fig. 3 Abundant tachykinin-immunoreactive (TK-IR) nerve fibres
(4+) in the mucosa of ascending colon of a patient with slow-transit
constipation (STC) (closed arrowheads). Calibration bar, 50 µm
Fig. 4a, b Ascending colon cut in transverse section in a control
specimen (a) and a slow-transit constipation (STC) specimen (b) la-
belled with leu-enkephalin antiserum. There were many enkephal-
in-immunoreactive (ENK-IR) nerve fibres (4+) in the circular mus-
cle of the control specimen (closed arrowheads) but very few (1+)
in the STC specimen. The myenteric plexus of both specimens had
abundant ENK-IR nerve fibres (arrows), while the longitudinal mus-
cle contained very few (1+) in the control specimen and none in the
STC specimen. Calibration bar, 50 µm
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Leu-enkephalin Nitric oxide synthase

The number of Leu-enkephalin (ENK)-IR nerve fibres in
the circular muscle of the large intestine of patients with
STC was significantly less than the control group (Fig. 4).
In two patients, the decrease was more apparent in the as-
cending colon, and in one patient it was more marked in
the descending colon. These patients also had correspond-
ing decreases in the density of TK-IR nerve fibres in the
circular muscle between different regions of large bowel.
There was no difference detected in the innervation of the
terminal ileum. The density of innervation in all other
layers of the bowel was the same in both groups of pa-
tients.
The pattern of nitric oxide synthase (NOS)immunoreactiv-
ity was similar in all patients. There were abundant NOS-IR
nerve fibres in both the myenteric plexus and throughout the
circular muscle layer (Fig. 7). Only a few NOS-IR nerve fi-
bres were seen in the submucosa and these were mainly close
to the circular muscle, as has been described previously [28].
Neuropeptide Y
Neuropeptide Y (NPY)-IR nerve fibres surrounding blood
vessels and NPY-IR endocrine cells were abundant in both

Choline acetyltransferase

There was no difference between the number of ChAT-IR

nerve fibres in any layer between the control group and
STC patients. Many ChAT-IR nerve fibres were seen in the
circular muscle in both groups (Fig. 5). The antibody to
ChAT labelled nerve cell bodies much more intensely than
nerve fibres and therefore evaluation of density of nerve
fibres with this antibody was less reliable. Small differ-
ences in the density of ChAT-IR nerve fibres between the
two groups of patients may not have been detected.

Vasoactive intestinal peptide

There was no significant difference in the density of VIP-

IR nerve fibres between control and STC patients in any
layer of the intestine. All layers of the intestine had abun-
dant VIP-IR nerve fibres, except for the longitudinal mus-
cle. VIP-IR nerve fibres appeared to be evenly distrib-
uted throughout the circular muscle in all specimens
(Fig. 6).

Fig. 5 Choline acetyltransferase (ChAT) immunoreactivity in a lon-

gitudinal section of transverse colon from a patient with slow-tran-
sit constipation (STC). There was a very high density (4+) of ChAT-
immunoreactive (IR) nerve fibres in the circular muscle (closed ar-
rowheads) but none in the longitudinal muscle. Many ChAT-IR nerve
cell bodies were seen in the myenteric plexus (arrow). Calibration
bar, 50 µm

Fig. 6 Vasoactive intestinal peptide (VIP) immunoreactivity in as-

cending colon of a slow-transit constipation (STC) patient. A very
high density (4+) of VIP-immunoreactive nerve fibres in the circu-
lar muscle (closed arrowheads) and a high density (3+) in the lon-
gitudinal muscle (open arrowheads) was seen. Many fibres (4+) were
also seen in the myenteric plexus (arrows). Calibration bar, 50 µm

Fig. 7 Transverse section of terminal ileum from a slow-transit con-

stipation (STC) patient labelled with nitric oxide synthase (NOS) im-
munoreactivity. A very large number (4+) of NOS-immunoreactive
nerve fibres were distributed evenly throughout the circular muscle
layer (closed arrowheads), while only scant innervation (1+) of the
longitudinal muscle was seen (open arrowheads). Neurons were seen
in the myenteric plexus (arrow). Calibration bar, 25 µm

groups of patients. The density of NPY fibres was the same
in the control group as in the patients with STC, with a
moderate number of nerve fibres in the circular muscle and
myenteric plexus and sparse innervation of the longitudi-
nal muscle.
Tyrosine hydroxylase
The pattern of tyrosine hydroxylase (TH) immunoreactiv-
ity was the same in both groups. TH-IR nerve fibres were
abundant around myenteric ganglia and surrounding blood
vessels, especially in the submucosa. No nerve fibres were
seen in the muscle layers.
Discussion
General
There were no significant differences between controls and
STC patients using the NSE antiserum. The unchanged
density of nerve fibres labelled with NSE, which is a
marker of all enteric neurons, indicates that there is no gen-
eral neuronal reduction in these patients in any layer or re-
gion of intestine.
Excitatory nerve fibres
In this study, a reduction of TK-containing nerve fibres was
demonstrated in the circular muscle of patients with STC.
While all STC patients had this abnormality in at least one
region of colon, several had regions of large bowel which
were relatively spared. The reduction of TK-IR circular
muscle nerve fibres was also present in the terminal ileum
of some patients. This indicates that the reduction of ta-
chykinins in the circular muscle of these patients may be
part of a generalised disorder, with regions of the gastroin-
testinal tract showing variable degrees of this abnormality.
Indeed, this may explain why patients with STC often have
other motility disorders, such as reflux oesophagitis and
abnormalities of gastric and small bowel transit [7, 29, 30].
Long-term follow-up of these patients may indicate
whether the presence of abnormal innervation of the ter-
minal ileum is a predictor of a poor long-term prognosis
following subtotal colectomy. Evidence in rats indicates
that substance P levels are not altered in the colon after
long-term treatment with laxatives, although VIP levels are
reduced [31], suggesting that the reduction of TK-IR fi-
bres in our study is unlikely to be due to laxative treatment
in these patients.
Another study found that the concentration of substance
P and density of substance P-containing nerve fibres in the
circular muscle of patients with STC was not different to
that of control patients [13]. However, their control group
was significantly older than the STC group, so a direct com-
parison between the two groups is not possible, given the
213
reports of significant changes in the enteric nervous system
in humans and small animals with ageing [14–16].
No difference was demonstrated in the density of TK
innervation in any of the other layers of intestine, suggest-
ing that the decrease in tachykinins in these patients is spe-
cific to the circular muscle and is not a reduction of all TK-
containing nerve fibres. As the number of nerve fibres in
the myenteric plexus is so great, large reductions would
need to occur to be detected in frozen sections. The den-
sity of TK-IR nerve fibres in the submucosa and mucosa
from the terminal ileum to the descending colon was the
same in controls and STC patients, although another study
has shown that the concentration of substance P in the rec-
tal mucosa and submucosa of patients with STC is reduced
[32]. There may have been a subtle decrease in the amount
of tachykinins in the mucosa and submucosa which our
methodology was unable to detect. The finding in our study
that there was no detectable change in the mucosa suggests
that performing mucosal biopsies and examining for the
markers we used on STC patients would not reveal any ab-
normality.
The tachykinin antisera used was raised against the C-
terminal peptide of substance P and so did not differentiate
between the different members of the tachykinin family,
i.e. substance P, neurokinin A and neurokinin B. All three
peptides have been localised to the myenteric and submu-
cosal plexuses, the circular and longitudinal muscle layers
and the mucosa of human bowel [33, 34]. As selective anti-
sera or radioimmunoassay techniques were not used, we
were unable to determine if all three tachykinins are re-
duced in the circular muscle by the same extent or if there
is a selective decrease in STC.
The number of ENK-IR circular muscle nerve fibres in
the large bowel of patients with STC was also decreased
compared with the control group, although this decrease
was not significant in the terminal ileum. There was a cor-
relation between the reduction of TK- and ENK-IR nerve
fibres at the different regions of large bowel in the patients
in whom the decrease occurred predominantly in one re-
gion. This close correlation between TK- and ENK-IR
nerve fibres is to be expected, given that the coexistence
of tachykinins and enkephalin in nerve fibres in circular
muscle of human colon is well recognised [35]. The den-
sity of ENK-IR nerve fibres in all other layers of the gut
was unchanged compared with the control group, indicat-
ing that the reduction in the circular muscle was not part
of a general decrease in all ENK-containing nerve fibres.
Much recent work, both in experimental animals and
humans, indicates that different functional classes of neu-
rons can be identified by the combinations of neurocher-
nicals within them, a concept known as “chemical coding”
[9]. These studies suggest that human colonic circular
muscle motor neurons contain NOS±VIP±NPY or ChAT±
TK±ENK, and these two classes of neurons are likely to
represent inhibitory and excitatory motor neurons respec-
tively [35–40]. Our study found that ChAT-IR nerve fibres
were present in normal numbers in the circular muscle of
patients with STC, although a small reduction was possible
given the relatively faint labelling of nerve fibres with the
214
ChAT antibody. Thus it appears that while excitatory nerve
fibres are present in STC, they are deficient in tachykinins
and enkephalin.
Tachykinins have been shown to be involved in excit-
atory neurotransmission to human intestinal circular mus-
cle, mainly via NK2 receptors [41, 42]. A reduction in the
release of tachykinins from nerve fibres to the circular
muscle would lead to a reduction of excitatory transmis-
sion to the circular muscle. This would affect the ascend-
ing excitatory reflex, as tachykinins have been shown to
be involved in this pathway in human intestine [43]. Inter-
estingly, propagating contractions have been found to be
reduced in number and amplitude in patients with STC
[44, 45].
The role of opioid-containing nerve fibres in the circu-
lar muscle is less clear. In the circular muscle of the hu-
man colon, leu-ENK and met-ENK can act on prejunctional
δ-opioid receptors to produce inhibition of nonadrenergic,
noncholinergic inhibitory neuromuscular transmission
[461 and κ agonists also inhibit neuromuscular transmis-
sion [47]. Naloxone, the opioid antagonist, has been shown
to ameliorate constipation in patients with STC [48], al-
though its site of action is unknown. Thus, the effect of re-
duced opioid containing nerve fibres in the circular mus-
cle is unclear.
There is some evidence regarding functional abnormal-
ities in patients with STC. There is a reduction in the re-
lease of acetylcholine from the taenia following electrical
field stimulation in patients with STC compared with con-
trols [49], suggesting that fewer cholinergic nerves are
functional. In addition, the circular muscle of STC patients
was found to be hypersensitive to the application of car-
bachol [50]. These results support the theory that there is
a reduction of excitatory nerve fibres supplying the circu-
lar muscle in these patients. To date, no studies have ex-
amined neuromuscular transmission from excitatory or in-
hibitory motor neurons in STC.
Inhibitory nerve fibres
The distribution and density of NOS- and VIP-IR nerve fi-
bres were the same in both groups. Nerve fibres which
contain NOS and/ or VIP are likely to be inhibitory nerve
fibres to the circular muscle [36, 52]. As we found no ev-
idence of differences in control and STC patients in circu-
lar muscle fibres with these markers, it is likely that there
are no abnormalities of inhibitory innervation to the circu-
lar muscle in STC patients. Another study found an in-
creased number of NOS-IR neurons and a decreased num-
ber of VIP-IR neurons in STC patients in both the submu-
cosal and myenteric plexuses but incongruously reported
an increased number of VIP-IR nerve fibres in the circu-
lar muscle [11]. Some studies have shown a decreased con-
centration of VIP and density of VIP-IR nerve fibres in the
circular muscle [10], while other studies have reported nor-
mal concentrations of VIP [13] and density of VIP-IR fi-
bres in the circular muscle [12, 13]. It appears that even in
the complete absence of neuronal NOS in mice, due to the
targeted disruption of the gene, there is no obvious disrup-
tion to colonic function, although pyloric stenosis results
[53].
Extrinsic nerve fibres
The finding that nerve fibres with TH, which is a marker
of noradrenergic fibres, surrounding myenteric ganglia and
blood vessels were not different in STC patients suggests
that sympathetic innervation is preserved in these patients.
This is reinforced by the observation that NPY-IR nerve fi-
bres around blood vessels, shown to be sympathetic nerve
fibres [51], are still present in STC patients.
Conclusion
The finding of a deficiency in tachykinins and enkeph-
alin in excitatory nerve fibres in the circular muscle in
STC raises several questions. For example, it would be
important to address whether excitatory transmission is
impaired in these patients, especially whether tachykiner-
gic excitation alone is abnormal or if cotransmitters such
as acetylcholine are also affected. It would also be inter-
esting to investigate potential abnormalities of the pro-
jections and neurochemistry of circular muscle motor
neurons of patients with slow transit constipation using
retrograde tracing combined with immunohistochemis-
try. These further lines of investigation may provide cru-
cial information regarding this poorly understood motil-
ity disorder.
Acknowledgements Anthony Porter is the recipient of a National
Health and Medical Research Council Medical Postgraduate Re-
search Scholarship. We would like to thank Prof. P. O’Brien, Mr. R.
Sarre, Mr. G. Otto and Mr. J. Sweeney for contributing patients to
this study and Janine Falconer-Edwards for expert technical assis-
tance. This study was supported by a Flinders Medical Centre 2000
Research Foundation Grant.
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