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DEVELOPMENTAL DYNAMICS 213:271?282 (1998)
Development of Pacemaker Activity and Interstitial Cells
of Cajal in the Neonatal Mouse Small Intestine
LOUIS W.C. LIU,1,2 LARS THUNEBERG,3 AND JAN D. HUIZINGA1,2*
1Intestinal Disease Research Program, Department of Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada
2Department of Electrical and Computer Engineering, McMaster University, Hamilton, Ontario, Canada
3Institute of Medical Anatomy, Section C, The Panum Institute, University of Copenhagen, Copenhagen, Denmark
ABSTRACT
Intestinal motor patterns are
not well developed in premature infants. Similarly, in neonatal mice, irregular motor patterns
were observed. Pacemaker cells, identified in the
small intestine as interstitial cells of Cajal (ICCs)
associated with Auerbach?s plexus (ICC-APs), contribute to the generation of peristaltic movements. The objective of the present study was to
assess the hypothesis that abnormal gut motor
activity in (preterm) newborns can be associated
with underdeveloped ICCs. Specifically, the aim
was to identify at which point the electrical
pacemaker activity is fully developed and
whether or not the development of pacemaker
activity has a structural correlation with the
developmental stage of ICCs. Pacemaker activity
was identified as that component of the slow
wave that is insensitive to L-type calcium (Ca2?)
channel blockers and displays a characteristic
reduction in frequency in the presence of cyclopiazonic acid (CPA), a specific inhibitor of the
endoplasmic reticulum Ca2? pump. In newborn,
unfed neonates, action potentials occurred that
were irregular in frequency and amplitude and
sensitive to verapamil. CPA (5 然) abolished all
action potentials. Quiescent spots were observed
in approximately 50% of impalements. Six hours
after birth, slow-wave activity appeared at a
regular frequency and amplitude, and a welldefined plateau phase was observed. Verapamil
did not affect the frequency, 5 然 CPA decreased
it. The effect of CPA on the pacemaker frequency
2 days after birth was identical to that observed
in adult mice. In 2-hr-old neonates, ICCs could be
identified through selective uptake of methylene
blue, but ultrastructural features were not fully
developed. At 48 hr, a complete ICC network
covering Auerbach?s plexus was formed, confirmed by electron microscopy. In summary, the
pacemaker component of the slow waves can be
identified in neonates as early as 6 hr after birth.
The pacemaker component was fully developed 2
days after birth. These electrophysiological observations correlated with the development of full
network characteristics of ICC-APs and the development of fully differentiated ICC-APs from
r 1998 WILEY-LISS, INC.
??blast-like?? cells. Dev. Dyn. 1998;213:271?282.
r 1998 Wiley-Liss, Inc.
Key words: interstitial cells of Cajal; neonatal
mice; pacemaker development; smooth
muscle; intestinal motility
INTRODUCTION
In preterm human infants, rhythmic contractile activity can often be observed (Morris, 1991) at a dominant
frequency of ?3 cycles per minute (cpm) in the stomach
and ?12 cpm in the small intestine starting from 28
weeks of gestation (Tomomasa et al., 1985). These
characteristic frequencies, which are similar to those in
adult tissue, suggest the presence of electrical pacemaking activity imposing its characteristic rhythm onto the
musculature. However, despite its rhythmic character,
such contractions appear to be predominantly nonpropagating (Tomomasa et al., 1985; Berseth, 1989). A lack of
normal responses to feeding indicates that regulatory
mechanisms for peristalsis are underdeveloped. Such
preterm infants, thus, are said not to ??tolerate?? oral
feed as a consequence of impaired gastrointestinal (GI)
motility (Newell et al., 1993).
The characteristic rhythmic motor activity is driven
by omnipresent electrical activity generated within the
musculature, the so-called ??slow waves,?? which perform
a pacemaker function. Electrophysiological and structural evidence acquired from different isolated and
intact muscle strip preparations suggest that pacemaker activity originates from a network of interstitial
cells of Cajal (ICCs; Thuneberg, 1982; FaussonePellegrini, 1992; Sanders, 1996; Huizinga et al., 1997)
associated with the myenteric plexus of the stomach
(Bauer et al., 1985) and small intestine (Hara et al.,
1986; Ward et al., 1994; Huizinga et al., 1995) and with
the submuscular plexus of the colon (Smith et al., 1987;
Du and Conklin, 1989; Liu and Huizinga, 1993). ICCs
have the Kit tyrosine kinase receptor embedded in their
membranes. Antibodies against the Kit receptor have
greatly aided in the identification of Kit-positive cells.
Correlations with electron microscopy have made it
*Correspondence to: Jan D. Huizinga, Intestinal Disease Research
Program, HSC-3N5C, McMaster University, 1200 Main Street West,
Hamilton, Ontario L8N 3Z5, Canada. E-mail: huizinga@mcmaster.ca
Received 12 January 1998; Accepted 28 July 1998
272
LIU ET AL.
likely that at least most Kit-positive cells within the gut
musculature are ICCs (Komuro and Zhou, 1996). Kitpositive cells are markedly diminished in number in the
afflicted section of the human colon in Hirschsprung?s
disease (Vanderwinden et al., 1996c) and in the circular
muscle layer of the pylorus in infantile pyloric stenosis
(Vanderwinden et al., 1996a). The paucity of ICCs in
these pathological conditions possibly contributes to
the abnormal motility associated with these diseases.
In the mouse, Kit-positive cells are recognized beginning at day 12 of gestation. These cells later differentiate into Kit-positive cells that are associated with
Auerbach?s plexus and Kit-negative longitudinal muscle
cells (Torihashi et al., 1997). Kit-positive ICCs and
smooth muscle myosin heavy chain (SMMHC)-positive
smooth muscle cells both develop from mesodermally
derived mesenchymal pluripotential precursor cells
that express both Kit and SMMHC mRNA markers
(Klu?ppel et al., 1998). Although the location of Kitpositive cells just before birth strongly suggests that
they will develop into ICCs, they cannot be identified as
ICC by using established electron microscopic criteria
(Thuneberg, 1982). Electron microscopic studies have
shown that the cellular differentiation associated with
Auerbach?s plexus is incomplete at birth, and several
candidates for ICC precursor cells were identified up to
2?3 weeks after birth in the mouse small intestine
(Faussone-Pellegrini, 1985) and colon (Faussone-Pellegrini, 1987). The fact that the development of the ICCs
can be interrupted by injection of antibody against the
Kit receptor in the first 4 days after birth, but not after
day 9, is also consistent with ICCs not being fully
developed at birth (Maeda et al., 1992; Torihashi et al.,
1995). ICCs can selectively accumulate methylene blue
(Mikkelsen et al., 1988; Liu et al., 1993), and, subsequently, characteristic changes in ribosomes and chromatin occur (Malysz et al., 1996), so that methylene
blue-positive cells can be recognized and identified with
electron microscopy. Because precursor cells of ICCs
are difficult to identify, we used methylene blue as an
aid in the identification of such cells.
In the mouse, intestinal pacemaker activity appears
to be immature at birth (Maeda et al., 1992). Although
contractile activity has been observed on day 16 of
gestation (Gershon and Thompson, 1973), and electrical activity was seen to start at day 19 of gestation
(Torihashi et al., 1997), a pacemaker component, if it is
present, has not been identified in embryonic or neonatal mice. To determine electrophysiologically the maturity of pacemaker activity, we used specific pharmacological properties of adult pacemaking activity. First,
slow waves have a component that is insensitive to
L-type Ca2? channels blockers (Huizinga et al., 1991;
Ward and Sanders, 1992). Second, the slow-wave activity reacts to cyclopiazonic acid (Liu et al., 1995a), which
is a specific inhibitor of the endoplasmic reticulum Ca2?
pump, with a characteristic and marked reduction in
frequency.
The objectives of this study were to investigate in the
mouse the electrophysiological development of the intrinsic pacemaker activity of the intestinal musculature and the structural development of ICCs, in particular, network formation and ultrastructural characteristics.
An account of these data was presented at the 14th
International Symposium on Gastrointestinal Motility
(Liu et al., 1995b). The expectation was that this might
lead to the formulation of a hypothesis addressing the
mechanism behind certain motor abnormalities in preterm infants.
RESULTS
Electrical Activity of Neonatal Mouse
Small Intestine
The maturity of pacemaker activity in neonatal mice
was investigated by comparing the characteristics of
the recorded activity with pacemaker activity that was
identified previously in adult mice. The pacemaker
activity of the small intestine is the component of the
slow-wave activity that is characterized by 1) insensitivity of amplitude and frequency to L-type calcium channel blockers (Malysz et al., 1995), 2) dose-dependent
reduction in frequency by cyclopiazonic acid (CPA; Liu
et al., 1995a), 3) slow but eventually complete inhibition by the removal of extracellular calcium, and 4)
amplitude inhibition by Ni2?.
Newborn, unfed. All tissues obtained from the
jejunum of newborn, unfed neonatal mice exhibited
spontaneous electrical activity in Krebs solution (Table
1, Fig. 1a). Neonates were discarded if traces of milk
were found in any part of the exposed GI tract. The
electrical activity was irregular in both frequency and
upstroke amplitude (Fig. 1a). Similarly, the isolated but
intact jejunum contracted spontaneously at a low and
irregular frequency. Furthermore, no propagating, ringlike contractions were observed. The plateau phase of
slow waves was not observed in any of 47 stable
impalements from six different muscle strips from five
neonates. In all preparations, many quiescent spots (53
out of 112 impalements) were observed with resting
membrane potentials ranging from ?72 mV to ?58 mV.
The L-type calcium channel blocker, verapamil (1 然)
decreased the frequency, the upstroke amplitude, and
the rate of rise of the electrical oscillatory activity
(Table 2, Fig. 1a).
Within 1 min of perfusion, 5 然 CPA completely
abolished all electrical activity (Fig. 2a, Table 3). The
effects of CPA were completely reversible. Ni2? (1 mM),
with or without verapamil, completely abolished all
electrical activity, with a slight decrease in membrane
potential (2?4 mV). In three additional preparations
obtained from the ileum, the electrical activity was
completely abolished by 1 然 verapamil without a
change in the resting membrane potential; in addition,
there were more frequent encounters of electrically
quiescent cells. In summary, the electrical activity
observed in newborn, unfed neonates does not include a
273
PACEMAKER ACTIVITY AND ICCs IN NEONATAL MOUSE INTESTINE
TABLE 1. Spontaneous Electrical Activity in Neonatal Mouse Small Intestine
Measure
Resting membrane potential
(mV)
Frequency (cpm)
Duration (sec)
Upstroke amplitude (mV)
Plateau amplitude (mV)
Rate of rise (mV/sec)
Newborn, unfed
group (n ? 5)
6?12 Hr group
(n ? 8)a
24?48 Hr group
(n ? 5)
2?7 day group
(n ? 13)
?63.1 ? 2.8
14.1 ? 0.4
0.9 ? 0.2
22.1 ? 3.8
?
119.2 ? 26.3
?61.5 ? 1.7
17.4 ? 1.1**
1.1 ? 0.1
20.3 ? 3.5
14.9 ? 3.3
146.5 ? 20.9*
?62.0 ? 2.3
19.1 ? 0.9**
1.2 ? 0.1
23.2 ? 3.5
16.7 ? 2.6
136.3 ? 21.6*
?64.8 ? 2.1
19.8 ? 0.8**
1.2 ? 0.1
24.7 ? 2.6
19.6 ? 2.4
157.1 ? 28.9*
aOnly
five animals showed slow wave plateaus.
*Statistically significant difference of different age groups were compared with the newborn, unfed
group (P ? 0.05).
**P ? 0.01.
Fig. 1. Effects of verapamil on the electrical activity in neonatal mouse
small intestine of different age groups. a: In Krebs solution, the spontaneous electrical oscillations in the small intestine of newborn, unfed neonatal
mice were various in amplitude and frequency (top traces). The plateau
phase had not developed. One micromolar of verapamil, an L-type
calcium (Ca2?) channel blocker, decreased both the amplitude and the
frequency without affecting other parameters (bottom). Right traces show
recordings at a faster chart speed. b: In the 6?12 hr group, both the
frequency and the amplitude became steady. The plateau phase of the
slow-wave activity was well defined, which is illustrated better at a faster
chart speed (right top trace). Recordings were made from a an 8-hr-hold
neonate. Verapamil (1 然) decreased the upstroke amplitude and rate of
rise (bottom traces). c: Recordings were made from a 30-hr-old neonate.
In Krebs solution, the electrical activity of this age group was not
significantly different from the 6?12 hr group. Consistent with other age
groups, verapamil (1 然) decreased the upstroke and rate of rise of the
slow waves. d: Recordings were made from a 7-day-old mouse. Spikes
superimposed on the plateau phase of slow waves started to be observed
in 2-day-old mice. To better illustrate the spike activity, traces are shown in
different time scales (note different calibrations). Verapamil (1 然)
abolished all spiking activity and decreased the amplitude and rate of rise
of the slow waves.
pacemaker component of the slow-wave activity corresponding to the one described in adult tissue or neonatal tissue at 48 hr (see below).
Six to twelve hours. Unlike tissues from newborn,
unfed neonates, at 6?12 hr, tissues showed slow wavelike activity with a well-defined upstroke and a plateau
274
LIU ET AL.
TABLE 2. Effects of Verapamil on Electrical Activitya
Measure
Newborn, unfed
group (n ? 4)
Krebs
Verapamil
6?12 Hr group
(n ? 4)
Krebs
Verapamil
24?48 Hr group
(n ? 4)
Krebs
Verapamil
2?7 Days group
(n ? 6)
Krebs
Verapamil
Resting membrane potential (mV)
?59.9 ? 1.4 ?58.8 ? 1.3
?61.3 ? 1.0 ?61.3 ? 1.0
?59.5 ? 2.1 ?58.5 ? 1.5
?59.3 ? 0.5 ?58.8 ? 0.6
Frequency
(cpm)
13.9 ? 0.5
9.9 ? 1.2**
18.4 ? 1.3
18.0 ? 1.1
18.9 ? 1.2
18.4 ? 1.4
20.7 ? 1.7
19.3 ? 1.6
Duration (sec)
1.1 ? 0.2
0.9 ? 0.1
1.2 ? 0.3
1.1 ? 0.2
1.2 ? 0.2
1.1 ? 0.1
1.2 ? 0.1
1.1 ? 0.1
Upstroke
amplitude
(mV)
21.4 ? 5.4
16.3 ? 5.3*
18.5 ? 2.8
16.8 ? 1.8*
15.8 ? 1.1
12.7 ? 1.4*
16.4 ? 1.4
14.3 ? 1.1*
Plateau amplitude (mV)
?
?
11.2 ? 0.6
11.3 ? 0.8
11.1 ? 0.6
9.8 ? 0.8
12.8 ? 0.8
11.2 ? 1.1
Rate of rise
(mV/sec)
103.2 ? 29.1
50.3 ? 16.8** 146.4 ? 34.8 121.9 ? 34.0* 129.3 ? 20.7
98.3 ? 23.3* 134.7 ? 15.7
89.2 ? 13.9**
aActivity in the presence of verapamil (1 然) was compared with the corresponding activity in Krebs solution in the same age
group. Oscillations with amplitude less than 2 mV were neglected.
*P ? 0.05.
**P ? 0.01.
Fig. 2. Effects of cyclopiazonic acid (CPA) on the electrical activity in
neonatal mouse small intestine of different age groups. The beginning of
all tracings show electrical activities in the presence of 1 然 verapamil for
at least 20 min. CPA (5 然), a specific inhibitor of the endoplasmic
reticulum Ca2?-pump, was added to the perfusion solution at arrows.
Wash-out segments show electrical activities after removing CPA from the
perfusion solution for 30 min. Experiments shown were obtained from
impalements of the same cell. The effects of CPA were reversible after
wash out for 30 min. a: In the presence of verapamil, similar to Figure 1a,
action potentials were irregular in amplitude and frequency. CPA first
reduced the frequency and then decreased the amplitude gradually.
Activity was completely abolished within 5 min of perfusion with CPA. b:
Recordings were made from a 10-hr-old neonate. Ten minutes of the
recording were omitted between the first and the second traces. CPA
significantly reduced the slow-wave frequency. Unlike in the newborn,
unfed group, the upstroke amplitude reduced only slightly. c: The neonate
used in this experiment was 42 hr old. There was a gap of 17 min between
the first and second traces. CPA slightly depolarized the cells and
subsequently decreased the upstroke amplitude. The frequency was
reduced significantly. d: Experiment performed on a 5-day-old neonate.
Twenty minutes of the recording were omitted between the first and
second traces. Similar to other age groups, CPA significantly decreased
the frequency and slightly depolarized the tissue.
phase. Both the frequency and the amplitude were
regular (Fig. 1b). Consistently, regular rhythmic contractions were observed in the whole intestine of these
neonates. The rate of rise of the slow wave-like activity
in this group was significantly higher than that of
newborn, unfed animals (Table 1). Verapamil (1 然) did
not affect the frequency and plateau amplitude of the
slow wave-like oscillations (Table 2, Fig. 1b) but still
decreased the upstroke amplitude and the rate of rise
(Table 2).
Addition of CPA (5 然) in the presence of verapamil
significantly decreased the frequency of the slow wave-
275
PACEMAKER ACTIVITY AND ICCs IN NEONATAL MOUSE INTESTINE
TABLE 3. Effects of Cyclopiazonic Acid on Electrical Activity
Measure
Newborn, unfed
group (n ? 4)
Verapamil
CPA
6?12 Hr group
(n ? 4)a
Verapamil
CPA
24?48 Hr group
(n ? 4)b
Verapamil
CPA
2?7 Day group
(n ? 5)b
Verapamil
CPA
Resting membrane potential (mV)
?59.8 ? 1.3 ?57.2 ? 0.9* ?62.7 ? 1.3 ?60.0 ? 1.2* ?60.0 ? 0.8 ?59.0 ? 1.0
?59.5 ? 0.6 ?57.8 ? 0.7*
Frequency (cpm)
9.9 ? 1.2
?
18.6 ? 1.2
1.9 ? 0.1**
20.7 ? 0.5
4.8 ? 0.7c**
19.6 ? 1.9
7.8 ? 1.5d**
Duration (sec)
0.9 ? 0.1
?
1.2 ? 0.2
1.0 ? 0.3
1.0 ? 0.1
1.3 ? 0.3
1.2 ? 0.2
1.1 ? 0.1
Upstroke amplitude (mV)
16.3 ? 5.3
?
21.5 ? 3.3
19.0 ? 3.5
15.2 ? 0.4
13.3 ? 0.8*
15.8 ? 1.2
12.2 ? 1.4*
Plateau amplitude (mV)
?
?
12.7 ? 1.5
?
10.1 ? 0.5
10.4 ? 0.3
11.5 ? 1.5
11.2 ? 1.1
Rate of rise (mV/
sec)
50.3 ? 16.8
?
169.2 ? 35.0 102.5 ? 7.5** 110.0 ? 16.7
67.5 ? 27.5** 103.6 ? 21.7
71.2 ? 11.9**
aOnly
one animal exhibited the plateau phase; thus, plateau amplitude is not included in the table.
plateau phase was observed in three animals from each experimental group. Activity in the presence of 5 然 cyclopiazonic
acid (CPA) was compared with the corresponding activity in Krebs solution containing 1 然 verapamil in the same age group.
cSignificantly larger than in the bathing solution in the 6?12 hr group.
dSignificantly larger than in the same bathing solution in the 6?12 hr group and the 24?48 hr group. The effects of CPA were
completely reversible in all experiments.
*P ? 0.05.
**P ? 0.01.
bThe
like activity to 10 % of that in verapamil (Table 3,
Fig. 2b). Both the amplitude and the rate of rise were
also decreased. The effects of CPA were completely
reversible. Ni2? (1 mM) abolished all electrical oscillations accompanied by a depolarization of 2?3 mV.
Twenty-four to forty-eight hours. At 24?48 hr,
electrically quiescent areas were no longer identified.
Verapamil (1 然) decreased only the upstroke amplitude and the rate of rise (Table 2, Fig. 1c) of slow
wave-like oscillations. At 24 hr, regular and forceful
contractions were observed. At 48 hr, spatially coordinated, regular, and forceful contractions had been
developed in the intact intestine, creating propagating
ring contractions. Addition of 5 然 CPA decreased the
slow-wave frequency to 23 % (Table 3, Fig. 2c). In the
presence of verapamil, 1 mM Ni2? abolished all of the
oscillations in two of five preparations; in the remaining
three preparations, periodic oscillations of 2?4 mV in
amplitude were observed at a frequency of 12.1 ? 2.2
cpm (18.2 ? 1.0 cpm in verapamil). To completely abolish
the remaining oscillations, 2 mM Ni2? was required .
Two to Seven Days. Similar to other age groups,
verapamil consistently decreased the upstroke amplitude and the rate of rise of the slow-wave activity (Table
2) but there was no effect on frequency and slowwave
plateau. The effects of CPA in the presence of verapamil
were similar to the 24?48 hr group, except for the
effects on frequency (Table 3). The frequency was
reduced to 40% of that in verapamil alone (Fig. 2d).
Electrical activity was abolished by 2 mM Ni2? in three
out of four preparations; in the remaining preparation,
5 mM Ni2? were needed to completely abolish the
activity, accompanied by a 4-mV depolarization. The
sensitivity of the slow waves to the presence of extracellular calcium was investigated by removing Ca2? salts
from the Krebs solution, leaving only nominal calcium
([Ca2?]nom ? 10?7 M). [Ca2?]nom first decreased the
frequency and then decreased the amplitude to 2?6 mV
(n ? 4; Fig. 3a); in three different preparations all
oscillations were abolished. The resting membrane
potential changed from ?61.8 ? 0.5 to ?59.5 ? 0.6 mV
(P ? 0.05). There was a time delay of 10?26 min for
calcium removal to take effect. Addition of 1 mM of the
calcium chelator EGTA to nominal-Ca Krebs solution
reduced the oscillation amplitude to less than 2 mV in
8?15 min (n ? 4; Fig. 3b). The effects of Ca2? removal
with or without EGTA were completely reversible,
although the wash-out period was longer if the tissue
had been challenged previously with EGTA.
Action potentials superimposed on the plateau phase
of slow waves occurred in 2-day-old mice (11 of 13 mice).
The appearance of action potentials became progressively more frequent with age. The frequency and amplitude of these action potentials were 148.5 ? 21.9 cpm
and 3.5 ? 0.5 mV, respectively (n ? 11). These action
potentials were abolished by 1 mM verapamil (Fig. 1d).
Distribution and Identification of Methylene
Blue-Positive Cells as ICCs Associated
With Auerbach?s Plexus
Light microscopy. Small intestinal wholemounts
from 2-hr-old mice, after methylene blue staining,
revealed scattered ICCs associated with Auerbach?s
plexus (ICC-APs; Fig. 4a). A full network structure had
not yet developed. At 12 hr, the methylene blue-positive
cells formed a partial, incomplete network of increased
density. At 48 hr (Fig. 4b), the staining pattern was
indistinguishable from that in the adult mouse.
Electron microscopy. In newborn mice, a firm identification of ICC-APs based on electron microscopic
criteria alone was not possible, as noted previously
(Faussone-Pellegrini, 1985). Most cell types were undergoing differentiation and frequent divisions and, consequently, had a less differentiated cytoplasm with numer-
276
LIU ET AL.
Fig. 3. Effects of removal of extracellular Ca2? on the slow-wave
activity of small intestine of two 3-day-old neonates. Both experiments
were performed in the presence of 1 然 verapamil. Verapamil had been
perfused for at least 20 min before the perfusion solution was switched to
nominal calcium ([Ca2?]nom). Experiments shown were continuous recordings made from the same cell. a: Removal of extracellular Ca2? from
Krebs solution containing 1 然 verapamil resulted in slow reduction of the
upstroke frequency and amplitude. Small oscillations of approximately
3?4 mV were observed at a lower frequency after an 18-min perfusion
with [Ca2?]nom-Krebs solution containing 1 然 verapamil. The effects were
reversible after a 30-min wash out with Krebs solution containing 1 然
verapamil. b: Addition of 1 mM EGTA to the [Ca2?]nom-Krebs solution
containing 1 然 verapamil accelerated the effects of removal of extracellular Ca2?. Reduction in the frequency was observed after a 4-min
perfusion. In 10 min, oscillations were abolished completely.
ous free ribosomes. In the Auerbach?s plexus region,
interstitial cells were present. These could be identified
tentatively as fibroblast-like or macrophage-like cells.
In addition, we observed occasional, scattered interstitial cells with a higher than usual mitochondrial content (Fig. 5). These cells were always identified at the
interface between the longitudinal and the circular
muscle. Each of these cells was characterized by a large
nucleus, a small amount of perinuclear cytoplasm,
extensive smooth endoplasmic reticulum, and numerous free ribosomes, caveolae, and processes. At the
same stage of development, ICCs at the deep muscular
plexus (ICC-DMPs) could be identified clearly (Fig. 6),
they presented with a pattern and relative density
similar to adult tissue.
After 48 hr postpartum, ICC-APs were easily recognized by cellular characteristics similar to those of
adult ICC (Fig. 7). We then investigated these tissues
after they were prestained with methylene blue. Methylene blue caused characteristic changes in the ultrastructural features of the ICC cytoplasm, namely, fine
granulation of ribosomal material and a nucleus that
showed a pattern of patches of heavily stained heterochromatin (Fig. 8). No other cell type exhibited such a
staining pattern. These data allowed us to investigate
tissues at 12 hr postpartum that were prestained with
methylene blue.
At 12 hr postpartum, scattered interstitial cells in the
Auerbach?s plexus region were positive for methylene
blue (Fig. 9). All of these cells had a higher than usual
mitochondrial content, an abundance of free ribosomes,
and occasional caveolae. These mitochondria-rich cells
and processes were therefore identified as premature
ICC-APs.
PACEMAKER ACTIVITY AND ICCs IN NEONATAL MOUSE INTESTINE
277
Fig. 4. Network characteristics of methylene blue-positive cells. a:
Jejunum of a 2-hr-old neonate. Interstitial cells of Cajal (ICC) associated
with Auerbach?s plexus (ICC-APs) are stained with methylene blue. The
network structure has not been developed (compared with b). Magnification: ?210. b: Jejunum of a 48-hr-old neonate. Note the change from
scattered, not visibly connected ICC-APs at 2 hr to a fully developed
network structure of ICC-APs. Magnification: ?210.
DISCUSSION
The main findings of the present study are that
ICC-APs are not fully differentiated at birth, although
their precursor cells can be identified by specific structural alterations due to selective uptake of methylene
blue. The ICC-AP precursor cells are mitochondrionrich interstitial cells with occasional caveolae. These
precursor cells are scattered and do not form a complete
network. Forty-eight hours after birth, ICC-APs can be
identified without the aid of methylene blue, and their
network structure is complete. Electrophysiologically,
at birth, the adult-type pacemaker activity is not
present. Forty-eight hours after birth, the pacemaker
activity is stable and has attained adult characteristics.
Fig. 5. Identification in an unfed, 2-hr-old neonate of a mitochondria
rich cell in the Auerbach?s plexus region of the proximal jejunum. a: In the
Auerbach?s plexus region, the typical ICCs of the adult, differentiated type
were absent. Furthermore, it was not possible to identify with certainty a
precursor cell, because many cells contained a high number of mitochondria and free ribosomes. Nevertheless, scattered throughout the region,
ICC-like cells were present (ICC) that had several structural characteristics of adult ICC-AP cells, in particular, a higher than average number of
mitochondria in the nuclear region. The ICC-like cells, similar to the
smooth muscle cells, contained more free ribosomes than the adult type.
Magnification ?14,300. CM, circular muscle; LM, longitudinal muscle. b:
Enlargement of part of a. Note the mitochondria (m) in both ICC-like cells
and smooth muscle cells, free ribosomes (R), and caveolae (arrows).
Magnification ?20,000. Scale bars ? 1 痠 in a, 0.5 痠 in b.
Ontogenesis of the Pacemaker Activity
in Neonatal Mouse Small Intestine
Electrophysiology. In newborn mice, the mature
pacemaker activity is not present, but action potentials
are generated, as noted previously (Torihashi et al.,
1997). To characterize the action potentials, reference
can be made to adult tissue. In the mouse small
intestine, action potentials are identified as superimposed on the slow-wave activity and are very sensitive
to L-type calcium channel blockers (Malysz et al.,
1995). Action potentials are further reported in W
mutant mice, which do not have ICC-APs (Malysz et al.,
1996). In these mice, the action potentials 1) occur at
variable frequencies of 0?50 cpm, 2) consist of a slow
component with superimposed spikes, and 3) are completely abolished by L-type calcium channel blockers.
278
LIU ET AL.
Fig. 7. Identification of ICC-APs at 48 hr. Jejunum was first preincubated with lysolecithin, which selectively destroys the mesothelium (M).
Thereafter, incubation with methylene blue was followed by uptake into
ICC-APs (ICC); note the changed nuclear morphology, seen as an
increased contrast of condensed chromatin in these two cells. LM,
longitudinal muscle layer. Magnification ?3,000. Scale bar ? 2 痠.
Fig. 6. Identification in an unfed, 2-hr-old neonate of ICCs at the deep
muscular plexus (ICC-DMPs). a: A long process of ICC-DMPs (asterisk)
occupies its characteristic position between the main outer layer of the
circular muscle (oCM) and the inner layer of circular muscle (iCM)
opposite the deep muscular plexus. The ICC-DMP process is closely
contacted by a nerve fascicle of the DMP (N). ICC-DMP cells and their
cellular processes were present at a cellular density that was comparable
to the adult pattern. SUB, submucosa. Magnification ?14,300. b: Enlargement of part of a. Note the numerous caveolae (small arrows), close
contact with the outer circular muscle layer (arrowhead) but not with the
inner circular muscle layer, and close contact with bundles of varicose
axons of the DMP (N; large arrow). Magnification ?20,000. Scale bars ?
1 痠 in a, 0.5 痠 in b.
Similar action potentials have been described in the
canine colonic circular smooth muscle, when devoid of
the submuscular pacemaker ICC network (Liu and
Huizinga, 1994). The electrical activity in newborn
mice is highly sensitive to L-type calcium channel
blockers and is abolished by Ni2?. This is in contrast to
the adult slow waves, which possess a pacemaker
component that is insensitive to L-type calcium channel
blockers and which always have an Ni2?-insensitive
component (up to 2 mM). The lack of pacemaker
activity in newborn mice is illustrated further by the
abolition of the electrical activity by CPA (1 然). In
adult mice, CPA (1 然) causes a reduction in frequency
but not abolition. These observations are all consistent
with the hypothesis that the electrical activity exhibited in the newborn mouse small intestine is generated
primarily by smooth muscle cells with little influence
from ICCs. During the first 2 days after birth, the
pacemaker component in the electrical activity becomes
stronger; by 48 hr, the pharmacology of the slow-wave
activity is similar to that in adult tissue.
Morphology. Faussone-Pellegrini (1985) identified
two possible cell types in neonates as candidates for
precursors of ICC-APs. ??Blast-like cells?? were elongated
in shape with short, lateral branches and possessed a
large, ovoid, electron-lucent nucleus; numerous free
ribosomes; abundant, large mitochondria; poorly differentiated cytoplasm; and a close association with nerve
fibers and nerve endings. The second candidate was
described as a fibroblast-like cell, which presented with
bundles of filaments in their peripheral cytoplasm. In
the present study, the methylene blue-positive cells
that developed into ICC-APs, as identified by electron
microscopy, were very similar to the blast-like cells. The
absence of caveolae in early stages of development was
also noted in smooth muscle cells (Gabella, 1989).
Therefore, we propose that the mitochondrion-rich interstitial cells identified in the present study are the
PACEMAKER ACTIVITY AND ICCs IN NEONATAL MOUSE INTESTINE
279
Fig. 9. Neonate at 12 hr. Only mitochondria-rich, ICC-like cells, as
identified in Figure 7, were methylene blue positive. The region shown is
between the longitudinal muscle layer (LM) and the circular muscle layer
(CM). Only the mitochondria-rich processes show the cytoplasmic change
associated with methylene blue uptake: The common ribosomal appearance (as in the muscle cell marked LM) changes to a finely granular or
flocculent appearance. R, ribosomes; m, mitochondria. Magnification
?20,000. Scale bar ? 0.5 痠.
Fig. 8. Changes in ultrastructure due to accumulation of methylene
blue. a: Increased nuclear contrast due to condensed chromatin in ICC (at
right). Fibroblast nuclei (at left) retain the normal contrast. Cells were
identified during scanning of the whole cell. b: Normal ribosomal particles
in the fibroblast (at left) in contrast to the finely granular or flocculent
ribosomal material in the ICC (at right).
precursor cells of ICC-APs, consistent with the tentative conclusion of Faussone-Pellegrini.
A close spatial relationship was observed between
ICC-AP precursor cells and nerves in newborn neo-
nates. However, neural interaction should not be interpreted as essential for ICC development. ICCs in the
mouse (Young et al., 1996) and chicken (Lecoin et al.,
1996) have been shown to develop in the absence of the
enteric nervous system. The growth factor, ??steel factor,?? is essential for maturation of the ICC-AP network
in the mouse small intestine (Ward et al., 1994; Klu?ppel
et al., 1998). Steel factor in the mouse may be produced
by enteric nerves (Torihashi et al., 1996), although this
apparently is not the case in humans (Vanderwinden et
al., 1996c).
In the present study, the identification of ICC-DMPs
was unequivocal when using electron microscopic characteristics, even in neonates. Faussone-Pellegrini (1984)
was more cautious, in that she also referred to these
cells as ??ICC blast-like cells,?? which could not be
identified with certainty. Torihashi and coworkers (Willenbucher et al., 1992; Torihashi et al., 1997) reported
that such ICC blast cells were not observed at day 18 of
gestation. Thus, in combination with our observation, it
is suggested that a significant development of this ICC
network is undertaken in the last 3 days before birth. It
is noteworthy that pacemaker activity was not present
in newborn neonates, even with well-developed ICCDMPs. This is consistent with the existing data, suggesting that the intestinal pacemaker activity is associated
with ICC-APs rather than with ICC-DMPs.
Functional Implications of an Underdeveloped
ICC-AP Network
In preterm infants, dominant, nonpropagating, rhythmic, contractile activity can be associated with food
280
LIU ET AL.
intolerance. We showed in the normal mouse that the
slow-wave activity plays a major role in normal peristaltic activity of the proximal small intestine (DerSilaphet et al., 1998). In W mutant mice, in which
ICC-APs as well as slow-wave activity were absent,
nonpropagating, rhythmic, contractile activity was also
dominant. Hence, the presence of underdeveloped ICCAPs, as observed in newborn mice, may lead to abnormalities in peristaltic, propagating, contractile activity.
It would lead to inadequate signal transmission among
ICCs, nerves, and smooth muscle cells: hence the
hypothesis is that, in preterm infants with a dominant
pattern of nonpropagating motor activity, the network
of ICCs is not fully developed. Consistent with this
hypothesis is a recent finding that a premature infant
without peristaltic activity in the colon was found to
have no ICCs. Muscle cells as well as distribution of
neuronal tissue were normal (Kenny et al., 1998). It is
noteworthy that ICC networks can mature normally
after such developmental delays (Vanderwinden et al.,
1996b; Kenny et al., 1998). It is also important to note
that, in the colon, the ICC network is responsible for
coordinating motor activities along the long axis of the
colon, across circular muscle lamellae. These data
encourage further studies in preterm infants into the
development of ICCs, both in relation to ultrastructural
maturation and the forming of a network structure.
Pacemaker Ion Channel in Neonatal Mouse
Small Intestine
The slow wave in the adult mouse small intestine is
likely initiated by a Ca2?-dependent, nonspecific cation
channel (Malysz et al., 1995; Thomsen et al., 1998). In
the canine colon, a similar pacemaker channel has also
been proposed (Huizinga et al., 1991; Ward and Sanders, 1992). More specifically, it has been demonstrated
recently that the frequency of activation of the pacemaker channel is entrained with the calcium-refilling
cycle in the endoplasmic reticulum associated with the
plasma membrane (Liu et al., 1995a). The observations
with CPA in the present study show that a similar
coupling mechanism is likely operating in the mouse
small intestine.
In summary, this is the first documentation of the
development of the pacemaker component of the electrical activity in the small intestine. 1) Methylene blue
was employed successfully to identify ICC precursor
cells. 2) At birth, the absence of slow-wave activity was
correlated with ICCs that were not fully differentiated
and were not organized yet as a network These data
provide the hypothesis that, in preterm infants, dominant patterns of nonpropagating motor activity may be
due to an underdeveloped network of ICCs.
EXPERIMENTAL PROCEDURES
Tissue Acquisition and Preparation
Neonatal mice (birth to 7 days) were decapitated.
Pregnant mice (CD1) were purchased from Charles
River Laboratories (Wilmington, MA) at 15?16 days of
gestation and monitored for delivery (time zero) after
19?20 days of gestation. The GI tract, starting from the
lower esophagus to the colon, was removed with the
intact mesenteric vascular bed to minimize stretch
when the gut was transferred to a dissecting dish,
which was filled with prewarmed Krebs solution. After
releasing the gut from the mesenteric vascular bed, the
gut was mounted without stretching onto a Sylgard
(184 silicone elastomer; Dow Corning Corporation, Midland, MI) surface with insect pins (Fine Science Tools
Inc.; 0.1 mm in diameter) at the stomach and the
ileocaecal junction. The musculature of the proximal
small intestine (20?50% of the entire length, as measured between the pylorus and the ileocaecal junction)
was carefully dissected from the submucosa without
opening the gut under a dissection microscope (Zeiss,
Thornwood, NY) at a magnification of ?40. Electron
microscopic examination of the dissected tissue revealed that the musculature cleaved along the deep
muscular plexus, leaving the outer circular muscle
layer, the myenteric plexus, and the longitudinal muscle
layer intact.
The isolated musculature was mounted with the
serosal surface facing up in between two pieces of
Sylgard that were anchored with insect pins. The top
piece of Sylgard had a circular opening approximately 1
mm in diameter, through which microelectrodes were
able to access the tissue. Before experimentation began,
all preparations were equilibrated for at least 2 hr at
37.0 ? 0.5蚓 in a tissue chamber with continuously
aerated (95% O2 and 5% CO2) Krebs solution perfusing
at a rate of 500 ml/hr (P-1; Pharmacia LKB, Uppsala,
Sweden).
Electrophysiological Measurements
Intracellular recordings were made by microelectrodes (50?80 M?) filled with 3 M KCl. A microelectrode
was inserted into a microelectrode holder, which was
connected to an electrometer (Duo773; World Precision
Instruments, New Haven, CT). Microelectrodes were
driven into the tissue vertically by using a micromanipulator (MN-151; Narishige). The output of the electrometer was displayed on a Gould oscilloscope (1421;
Gould, Inc., Cleveland, OH) and recorded on a Gould
ink-writing recorder (2400S).
Drugs and Solutions
All solutions perfused into the partition chamber
were prewarmed to 37.0 ? 0.5蚓 and equilibrated with
95% O2 and 5% CO2. The composition (in mM) of the
Krebs solution was NaCl, 120.3; KCl, 5.9; CaCl2, 2.5;
MgCl2, 1.2; NaHCO3, 20.2; NaH2PO4, 1.2; and glucose,11.5. The [Ca2?]nom Krebs solution was prepared
by omitting CaCl2 in the Krebs solution formula. CPA
(Sigma, St. Louis, MO) was dissolved in dimethyl
sulfoxide (DMSO; Sigma) to prepare a stock solution of
10 mM. Verapamil (verapamil hydrochloride; Sigma)
stock solution (1 mM) was prepared in deionized,
PACEMAKER ACTIVITY AND ICCs IN NEONATAL MOUSE INTESTINE
distilled water. Vehicles in the concentrations applied
did not have any effect on the electrical activity.
Result Presentation and Statistical Analysis
All data were expressed as mean ? standard error of
the mean. ?n? represents the number of mice used in
each set of experiments. Statistically significant differences between data sets were determined by one-way
repeated measures analysis of variance (KWIKSTAT 4;
TexaSoft, Cedar Hill, TX). The slow-wave duration was
measured at the half maximum of the slow-wave plateau amplitude. Because of the temporal variation of
the slow-wave activity in the presence of CPA, representative slow-wave parameters were obtained from analyses over periods of at least 5 min.
Light and Electron Microscopy
Unfed 12-hr-old, 24-hr-old, and 48-hr-old neonatal
mice (n ? 4 mice of each age) were decapitated. After
exposing the intestines, half of the animals were incubated for methylene blue staining, as described below,
followed by fixation, whereas the other half of the
animals were directly immersed in fixative. The primary fixative was glutaraldehyde 2%, formaldehyde
2%, picric acid 0.2%, and phosphate buffer 0.1 M, pH
7.5.
Methylene Blue Staining
The decapitated animals, after removal of the abdominal wall, were preincubated for 30 sec in calcium-free,
phosphate-buffered saline containing 0.7 mM lysolecithin (Sigma) to permeabilize the mesothelial cell layer.
They were transferred to and incubated for 45 min at
room temperature under subdued light in a wellaerated (95% O2 and 5% CO2) Krebs solution supplemented with 50 然 methylene blue B (Merck, Darmstadt, Germany; Mikkelsen et al., 1988). The animals
were transferred to the aldehyde fixative, which contained picrate to precipitate the methylene blue in the
tissue. The exposed parts of the intestines were well
stained and were selected under the magnification of a
stereomicroscope. Wholemounts of intestinal segments
as well as the isolated external muscle were studied
and photographed under a Leitz Orthoplan microscope
(Wetzlar, Germany).
Processing for Thin and Ultrathin Sectioning
After an overnight aldehyde fixation at 4蚓, samples
of duodenum, jejunum, and ileum from the methylene
blue-stained and the directly fixed intestines were cut
in millimeter-sized pieces, washed with 0.1 M phosphate buffer, postfixed in 2% osmic acid/0.1 M phosphate buffer for 1 hr, dehydrated in a graded series of
ethanol with block-staining for 1 hr in 1% uranyl
acetate in absolute ethanol, followed by propylene
oxide, and Epon embedding. One-micrometer-thick sections were stained with toluidine blue and examined
under a Leitz Orthoplan microscope. Ultrathin sections
were poststained with alcoholic uranyl acetate and lead
281
citrate and were examined under a Philips 300 electron
microscope (Eindhoven, The Netherlands).
ACKNOWLEDGMENTS
The Medical Research Council (MRC) of Canada
provided operating grants and an MRC Scientist Award
to J.D.H. and a studentship to L.W.C.L. NATO provided
travel funds for L.T.
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