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Qing-Lin
Long, Dian-Chun Fang, Hong-Tao Shi, Yuan-Hui Luo, Gastroenterology
Research Center, South-west Hospital, Third Military Medical
Univisity, Chongqing 40038, China
Correspondence
to: Dr Dian-Chun Fang, Gastroenterology Research Center, South-west
Hospital, Third Military Medical Univisity, Chongqing, 40038, China.
fangdianchun@hotmail.com
Telephone:
+86-23-68754124
Received:
2003-09-06 Accepted:
2003-10-07
Abstract
AIM:
The pathophysiology underlying gastrointestinal complications of
long-standing diabetes is poorly understood. Recent evidence
suggests an important role of intestitial cells of cajal in
controlling gastrointestinal motility. The aim of this study was to
clarify the changes of ultrastructural characteristics of
interstitial cells of cajal in stomach of diabetic gastro-electric
dysrhythmic rats.
METHODS:
Rats were randomly divided into diabetic group and control group,
the model of diabetic rats was established by peritoneally injection
of streptozotocin. Electrogastrograms were recorded and intestitial
cells of cajal in antrum were observed by electrictelescopy after
diabetic model rat was established for 3 mo.
RESULTS:
In the rats of diabetic group, the gastro-electric dysrhythmia was
increased compared with control group, the abnormal rhythm index and
the cofficient of variation of slow wave frequency were
significantly higher than those of normal rats. The number of the
gap junctions of interstitial cells of cajal in antrum of diabetic
rats was significantly decreased, and the remaining structures were
damaged. The organelles were also damaged, and vacuoles were formed.
CONCLUSION:
It is possible that changes in ultrastructural characteristics of
interstitial cells of cajal in stomach are one of the mechanisms
underlying gastro-electric dysrhythm in diabetic rats.
Long QL, Fang DC, Shi HT, Luo YH. Gastro-electric dysrhythm and lack of
gastric interstitial cells of cajal. World J Gastroenterol 2004; 10(8): 1227-1230
http://www.wjgnet.com/1007-9327/10/1227.asp
INTRODUCTION
Gastric
motility abnormity occurs in up to 30-50% of patients with
long-standing diabetes. Symptoms of diabetic gastropathy can range
from mild dyspepsia to recurrent vomiting and abdominal pain and may
progress to irreversible end-stage gastric failure known as
gastroparesis. Gastroparesis seriously affects quality of life.
There is deterioration in glycemic control and incapacitating
symptoms such as malnutrition, water and electrolyte imbalance, and
aspiration may occur. However, the pathophysiology of diabetic
gastropathy and gastroparesis, including impaired fundic and pyloric
relaxation and impaired electrical pacemaking, is still not
delineated[1-2]. It is generally considered that diabetic
gastropathy and gastroparesis may be due to visceral autonomic
neuropathy, hyperglucose and smooth muscle degeneration.
Interstitial cells of cajal (ICCs) are a unique class of
cells dispersed in gastrointestinal tracts of mammals. In the region
of gastric corpus and antrum, multipolar interstitial cells of cajal
form two dimensional networks, and have been mistaken for neurons,
glial cells, smooth muscle cells, macrophages and fibroblasts. In
fact they are mesenchymal in origin. As interstitial cells of cajal
develop, they assume major gastrointestinal motility. Interstitial
cells of cajal are the pacemaker cells responsible both for
initating slow wave activity in gastrointestinal muscles and for
active propagation of electrical slow wave. These cells also mediate
motor inputs from the enteric nervous system.
Studies have also suggested that many gastrointestinal motor
disorders have changes of number and/or structure of interstitial
cells of cajal[3-9]. In this study, we established the model of
diabetic rats by peritoneal injection of streptozotocin, and
intended to investigate the reason of gastric-electrical
dysrhythmias by recording the gastric electrical activity and
characterizing the ultrastructural features of interstitial cells of
cajal in the antrum.
MATERIALS
AND METHODS
Animals
Fifty
2-mo-old healthy Wistar rats (weighing 100-160 g) of either sex were
obtained from Animal Center of Third Military Medical Univisity and
were divided randomly into control group (n=20) and diabetic
group (n=30). After fasted overnight, diabetic group rats
were injected intraperitoneally with streptozotocin (60 mg/kg) and
control group rats were injected intraperitoneally with saline.
Blood glucose concentration, weight, appetite and urine volume were
measureed every two weeks. In this test, diabetic group animals were
considered as diabetic if blood glucose levels exceeded 16.9 mmol/L
and were eliminated if blood glucose levels remained <16.9 mmol/L.
Gastric-electrical
activity recorded
Gastric-electrical
activities of control and diabetic group rats were recorded afte 3
mo. After fasted over 12 h, rats were operated under anaesthesia
with 3% soluble pentobarbitone (30 mg/kg) intraperitoneally. A pair
of stainless platinum wires was implanted in deep muscular plexus of
antrum. The wires were arranged in an arching line along the greater
curvature about 0.5 cm apart from pylorus. The distance between
electrodes was about 0.3 cm apart. The electrodes were affixed to
the serosa by nonabsorbable sutures in the seromuscular layer of
stomach. Teflon-insulated wires were brought out from nape through
the anterior abdominal wall percutaneously. After surgery, the rats
were transferred to a recovery cage for a week. After fasted
overnight, rats underwent anaesthesia with 3% soluble pentobarbitone
(30 mg/kg) intraperitoneally. Wires were connected with a
gastro-electrical activity amplifier, and recorded parameters were
adjusted. All recorded signals were displayed and simultaneously
recorded on computer. Gastric-electrical activity recorded lasting
for 60 min. All recorded signals were analyzed by computer. In this
study, the frequency of normal rat gastric slow wave was 4.41±0.91/min and its normal range was 2.63-6.19/min. Gastric slow
wave was defined as bradygastria if its frequency was lower than
2.63/min and had a duration of longer than 1 min and form of slow
wave was ruled and rhythm was in good order. Gastric slow wave was
defined as tachygastria if its freguency was greater than 6.19/min
and had a duration of longer than 1 min and form of slow wave was
ruled and rhythm was in good order. Gastric slow wave was defined as
dysrhythmias if its form of slow wave was not ruled and rhythm was
in bad order and had a duration of longer than 1 min.
Morphological
studies
After
gastric-electrical activity was recorded, the abdomen was opened
immediately and antrum was removed (antrum was cut apart 0.5 cm from
pylorus) and fixed containing 3% glutaraldehyde and 4%
paraformaldehyde. The specimens were rinsed twice in 0.2 mol/L
saccharum-phosphate buffer and post-fixed in 10 g/L osmium tetroxide
for 2 h at 4 °C. The specimens were then rinsed in distilled
water, block-stained with saturated uranyl acetate solution for 3 h,
dehydrated in a grade series of acetone and embedded in epoxy resin
618. Ultrathin sections were cut and double-stained with uranyl
acetate and lead citromalic for observation under JEOL-2000EX
electron microscope.
Statisitical
analysis
Values
in the text were expressed as mean±SEM.
Statistically significant differences were tested using an analysis
of variance. A P value <0.05 was taken as statistically
significant.
RESULTS
Gastric-electrical
activity
Slowform
of normal rats was regular (Figure1), the mean frequency of control
rats was 4.41±0.91
cpm (range: 2.63-6.19 cpm), the abnormal rhythm index was 18.00±4.96%
and the cofficient of variation of slow wave frequency was 10.00±6.46%.
Compared with normal rats, diabetic rats had less chance of normal
rhythm, the mean frequency was 4.03±1.23
cpm (range: 1.62-6.44 cpm),its slowform was irregular (Figure 2).
The abnormal rhythm index was 30.62±7.38%
and the cofficient of variation of slow wave frequency was 23.50±2.98%.
Both of them were significantly higher than those of normal rats (P<0.01).
Figure
1(PDF) Electrgastrogram of
normal rats.
Figure
2(PDF) Electrgastrogram of
diabetic rats.
Ultrastructural
features of interstitial cells of cajal in rat antrum
In
the normal rat antrum (Figure 3), interstitial cells of cajal were
located within the circular muscle layer (ICC-CM) and in the
myenteric region (ICC-AP) and were in closely associated with small
nerve bundles. These cells were interconnected with each other and
neighboring smooth muscle cells via a number of large gap junctions.
Interstitial cells of cajal in rat stomach were characterized by
abundant mitochondria. Numerous caveolae were observed on cell
membranes. The nucle were big and irregular, heterochromatins were
often observed on the nuclear membrane and its strucure was clear.
Their cytoplasm was usually less, and one or more processes were
observed. Intermediate filaments, golgi apparatus, rough (RER) and
smooth (RER) endoplasmic reticulum were abundant. Variation in the
ultrastructural features of the intestitial cells of cajal in
different regions was also observed. ICC-CM was characterized by
dense cytoplasm, no basal lamina could be clearly identified. ICC-AP
was characterized by numerous caveolae and a distinct basal lamina.
In the antrum of diabetic rats (Figure 4), interstitial cells of
cajal were fewer. The number of the gap junctions between
interstitial cells of cajal and neuron cells, between interstitial
cells of cajal and smooth muscle cells, and between themselves were
decreased significantly. The remaining structures of those gap
junctions were also damaged. Basal lamina was discontinuous and not
distinct, some were apart from cell membrane and formed cavum. The
organelles-mitochondria and ribosome, for example, were also
significantly decreased. Mitochondria were swollen, vacuoles and
cytoplasm were dissolved, vacuole and myelin figures were formed.
Endoplasmic reticulum was dilated, cytoplasm was dissolving,vacuoles
were formed and distributed along plasma membrane, perinuclear space
was broadened.
Figure
3 Interstitial cells of
cajal of stomach in normal rats.
Figure
4 Interstitial cells of
cajal of stomach in diabetic rats.
DISCUSSION
Despite
reports of diabetic gastroparesis without autonomic neuropath, this
disease, and, particularly, impaired electrical pacemaking, are
usually considered to be the result of systemic and/or enteric
neuropathies. Recent studies in spontaneously diabetic BioBreeding/Worcester
rats and in streptozotocin-diabetic rats have demonstrated decreased
nitric oxide synthase expression and reduced nitrergic motor inputs
to the stomach, as well as impaired intracellular signaling in
response to excitatory neurotransmitters in gastric smooth muscle.
Clearly, these defects may contribute to the many abnormal features
of diabetic gastropathy. However, it is unclear how impaired neural
inputs (especially impaired inhibitory inputs) or impaired smooth
muscle response to cholinergic stimulation could result in the loss
of slow-wave activity, which has been observed in both humans and
animals with diabetes.
Interstitial cells of Cajal were originally described in the
gut more than hundred years ago by Ramóny
cajal[5]. He characterized
“interstitinal neurons” as “primitive accessory components
that perhaps modify smooth muscle contraction, subject themselves to
regulation from principal neurones”. Cajal provided us with
detailed pictures of methylene blue-stained networks of interstitial
cells of cajal, which were described as spindle shaped or stellate
cells with long, ramified cell processes and large, oval, nuclei
with sparse perinuclear cytoplasm, and intercalated between
autonomic nerve endings and smooth muscle cells. Interstitial cells
of cajal were found within discrete locations within the tunica
muscularis throught the gastrointestinal tract and classified into
five types according to different locations, i.e., ICC-AP (Auerbach’s
myenteric plexus) located between the circular and longitudinal
muscle layers, ICC-DMP (deep muscular plexus) located between the
inner thin and outer thick sublayers of the circular smooth muscle,
ICC-SMP (submuscular plexus) located at the submucosal border, ICC-CM
located within the outer thick circular muscle layer; and ICC-LM
located within the longitudinal muscle layer.
Many studies have indicated that each organ manifests a
unique pattern of distribution of interstitial cells of cajal. In
the stomach of rats, we observed interstitial cells of cajal located
in close association with small nerve bundles within the circular
muscle layer (ICC-CM) and in the myenteric region (ICC-AP), no
interstitial cells of cajal were found at the most inner region of
the circular muscle layer, submucosal border and deep muscular
plexus.
Though there are a large number of methods for
indentification of interstitial cells of cajal, such as methylene
blue staining, zinc-iodide osmic acid (ZIO)-staining and
immunohistochemistry using antibodies against kit proteink. The
“gold-standard” for indentification of interstitial cells of
cajal is still a combination of structural features in transmission
electron microscopy. Although some structural varations have been
described to be between species and between various regions of the
gastrointestinal tract, the Interstitial cells of cajal
ultrastructure are characterized by a combination of the following
features: numerouly large and often elongated mitochrondrial
profiles, large bundles of intermediate filaments, absence of thick
filaments, presence of surface caveoli variably developed basal
lamina, synapse-liked contacts between Interstitial cells of cajal
and tertiary nerve bundles, well-developed smooth endoplasmic
reticulum and often also rough endoplasmic reticulum, close
apposition or gap junction contact with smooth muscle cells.
Interstitial cells of cajal have ultrastructural distinction
from fibroblasts or smooth muscle cells. Some interstitial cells of
cajal may have muscle-like ultrastructural features, such as a basal
lamina, caveolae, subsurface cistern and gap junctions. For this
reason, some interstitial cells of cajal have been considered as
modified or specialized smooth muscle cells. Howerer, the
interstitial cells of cajal do not contain the well-organized
contractile apparatus characteristic of muscle cells. Some
interstitial cells of cajal may have an appearance similar to
fibroblasts and lack clear muscle-like features. However, even in
these cases, they are distinguishable from fibroblast-like cells by
a combination of features including a characteristic electron
density of cytoplasm, large gap junctions, abundant intermediate
filaments, numerous mitochondria, well-developed SER and flattened
cisterns of RER. Furthermore, a great number of collagen fibers
often distribute around fibrobalst cells.
Morphological observations have led to a number of hypotheses
on the possible physiological roles of interstitial cells of cajal[10-18]. (1) These cells may be the source of slow electrical
waves in gastrointestinal tract. (2) They participate in conduction
of electrical currents, and (3) They mediate neural signals between
enteric nerves and muscles.These hypotheses have been tested by
experiments. (1) Slow electrical waves in gastrointestinal muscle
strips were absent when interstitial cells of cajal were removed by
dissection or lesioned by cytotoxic chemicals. (2)
Electrophysiological experiments on isolate cells confirmed that
interstitial cells of cajal could generate rhythmic electrical
activity and also respond to messenger molecules known to be
released from enteric nerves. (3) In Ws/Ws mutant rats, or in mice
treated with antibody against the protein c-kit, slow wave activity
was impaired. (4) Slow wave passively decayed as a function of
distance from the pacemaker appeared. (5) After removal of
interstitial cells of cajal, smooth muscle cells continued to be
excitable, but in the absence of interstitial cells of cajal, smooth
muscle produced action potentials rather than slow wave-like
activity. Studies have also suggested that many diseases of
gastrointestinal motor disorders have changes in number and/or
structure of Interstitial cells of cajal[3-9]. In infants with
hypertrophic pyloric stenosis, there was a significant decrease in
the number of interstitial cells of cajal, this decrease was
prominent in the ICC-AP. In some patients with pseudo-obstruction,
there was a marked decrase in the number of interstitial cells of
cajal. Constipation was a very prevalent motility problem, but its
underlying mechanisms were obscure. Studies found that the volume of
interstitial cells of cajal in the colon of patients with slow
transit constipation was significantly decreased compared with
normal controls.
Normal gastric emptying requires the proper function of the
gastric electrical pacemaker system. Gastroparesis has been
associated with electrical abnormalities, and deviations from normal
slow-wave rhythm (dysrhythmias) have been reported to result in
delayed gastric emptying. In this study, we established the model of
diabetic rats by peritoneally injection of streptozotocin, and
recorded the gastro-electrical activity after 3 mo. We discovered
that in the rats of diabetic group, the gastro-electric dysrhythmia
was increased compared with control group,the number of interstitial
cells of cajal in antrum of diabetic rats was significantly
decreased and the number of the gap junctions of interstitial cells
of cajal also was significantly decreased, and the remaining
structures were damaged. The organelles-mitochondria and ribosome,
for example, were also significantly decreased. Mitochondria were
swollen, myelin figures were formed. Endoplasmic reticulum was
dilated, cytoplasm was dissolved, vacuoles were formed and
distributed along plasma membrane, perinuclear space broadened. This
shows that degeneration of interstitial cells of cajal is
responsible for gastro-electrical dysrhythmias of diabetic rats, the
identification of abnormalities in interstitial cells of cajal in
diabetic gastro-electric dysrhythm offers a potential future
therapeurapy.
REFERENCES
1
Qi HB, Luo JY, Dai XG, Wang XQ. A study on motility in
patients with diabetic gastroparesis. Clin J New Gastroenterol
1999;
5: 661-662
2 Quigley EMM. The evaluation of gastrointestinal function in
diabetic patients. World J Gastroenterol 1999; 5: 277-282
3 Long QL, Fang DC. Function of interstitial cells of cajal in
gastrointestinal tract. Shijie Huaren Xiaohua Zazhi
2002; 10:
352-355
4
Huizinga JD, Berezin I, Sircar K, Hewlett B, Donnelly G,
Bercik P, Ross C, Algoufi T, Fitzgerald P, Der T, Riddell RH,
Collins
SM, Jacobson K. Development of interstitial cells of Cajal
in a full-term infant without an enteric nervous system.
Gastroenterology 2001; 120: 561-567
5
He CL, Soffer EE, Ferris CD, Walsh RM, Szurszewski JH,
Farrugia G. Loss of interstitial cells of cajal and inhibitory
innervation in insulin-dependent diabetes. Gastroenterology 2001;
121: 427-434
6
Der T, Bercik P, Donnelly G, Jackson T, Berezin I, Collins
SM, Huizinga JD. Interstitial cells of cajal and
inflammation-induced motor dysfunction in the mouse small intestine.
Gastroenterology 2000; 119: 1590-1599
7
He CL, Burgart L, Wang L, Pemberton J, Young-Fadok T,
Szurszewski J, Farrugia G. Decrased intestitial cell of cajal
volume
in patients with slow-transit constipation. Gastroenterology 2000;
118: 14-21
8
Hudson N, Mayhew I, Pearson G. A reduction in interstitial
cells of Cajal in horses with equine dysautonomia (grass
sickness).
Auton Neurosci 2001; 92: 37-44
9
Sandgren K, Larsson LT, Ekblad E. Widespread changes in
neurotransmitter expression and number of enteric neurons
and
interstitial cells of cajal in lethal spotted mice: an explanation
for persisting dysmotility after operation for
Hirschsprung’s
disease? Dig Dis Sci 2002; 47: 1049-1064
10
Komuro T, Seki K, Horiguchi K. Ultrastructural
characterization of the interstitial cells of Cajal. Arch Histol
Cytol
1999; 62: 295-316
11
Koh SD, Kim TW, Yan JY, Glasgow NJ, Ward SM, Sanders KM.
Regulation of pacemaker currents in interstitial cells of
Cajal from
murine small intestine by cyclic nucleotides. J Physiol
2000; 527(Pt1): 149-162
12
Ordög T,
Takayama I, Cheung WKT, Ward SM, Sanders KM. Remodeling of networks
of interstitial cells of Cajal in a
murine model of diabetic
gastroparesis. Diabetes 2000; 49: 1731-1739
13
Dickens EJ, Edwards FR, Hirst GD. Selective knockout of
intramuscular interstitial cells reveals their role in the
generation
of slow waves in mouse stomach. J Physiol 2001; 531(Pt3):
827-833
14
Daniel EE, Thomas J, Ramnarain M, Bowes TJ, Jury J. Do gap
junctions couple interstitial cells of Cajal pacing and
neurotransmission to gastrointestinal smooth muscle?
Neurogastroenterol Motil 2001; 13: 297-307
15
Salmhofer H, Neuhuber WL, Ruth P, Huber A, Russwurm M,
Allescher HD. Pivotal role of the interstitial cells of Cajal in
the
nitric oxide signaling pathway of rat small intestine. Morphological
evidence. Cell Tissue Res 2001; 305: 331-340
16
Horiguchi K, Sanders KM, Ward SM. Enteric motor neurons form
synaptic-like junctions with interstitial cells of Cajal in
the
canine gastric antrum. Cell Tissue Res 2003; 311: 299-313
17
Jain D, Moussa K, Tandon M, Culpepper-Morgan J, Proctor DD.
Role of interstitial cells of Cajal in motility disorders of
the
bowel. Am J Gastroenterol 2003; 98: 618-624
18
Suzuki H, Ward SM, Bayguinov YR, Edwards FR, Hirst GD.
Involvement of intramuscular interstitial cells in nitrergic
inhibition in the mouse gastric antrum. J Physiol 2003; 546(Pt3):
751-763
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