This study examined neurons in the medulla oblongata related to gastric mucosal lesions in rats subjected to restraint water-immersion stress (RWIS). The study found that compared to controls, RWIS rats had: 1) increased cholinergic neurons in the dorsal motor nucleus of the vagus and nucleus ambiguous, and increased catecholaminergic neurons in the nucleus of the solitary tract; 2) increased oxytocin receptor- and vasopressin 1b receptor-expressing neurons in the dorsal motor nucleus and nucleus of the solitary tract; but 3) no difference in methionine-enkephalin-expressing neurons in the nucleus of the solitary tract. This suggests hyperactivity of cholinergic and cate

1. 127
OBJECTIVE: Restraint water-immersion stress (RWIS)
can induce a gastric mucosal lesion. Our objective was to
detect the phenotype of activated neurons in the medulla
oblongata of rats subjected to RWIS.
STUDY DESIGN: Male Wistar rats were divided into
2 groups in accordance with the duration of RWIS: a
control group and the RWIS group. The brain sections
were treated with a dual immunohistochemistry of
Fos and choline acetyltransferase (ChAT) or tyrosine
hy­
droxylase (TH) or oxytocin receptor (OTR) or vaso-
pressin 1b receptor (V1bR) or methionine-enkephalin
(M-ENK).
RESULTS: Compared to the control group, the number
of Fos+ChAT-immunoreactivity (IR) neurons in the
dorsal motor nucleus of the vagus (DMV) and nucleus
ambiguous (NA) and that of Fos+TH-IR neurons in the
nucleus of the solitary tract (NTS) were evidently in-
creased in the RWIS group; meanwhile, more OTR-IR
and V1bR-IR neurons in the DMV and NTS were ac-
tivated. A few M-ENK-IR perikarya were observed only
in the NTS, with no difference between the RWIS and
control groups.
CONCLUSION: Data suggest that the hyperactivity of
cholinergic neurons in the DMV and NA and catechol-
amine neurons in the NTS is one of the main mechanisms
of gastric mucosal lesions induced by RWIS; the OT-
sensitive and AVP-sensitive neurons, excepting M-ENK
ones, are involved in the modulation of the gastric mu­
cosal lesion during RWIS. (Anal Quant Cytopathol
Histpathol 2019;41:127–138)
Keywords: acute gastric mucosal lesion; disease
models, animal; gastric mucosa/pathology; gastric
ulcer; medulla oblongata; neurons; neurotransmit-
ter; rats, Wistar; restraint water-immersion stress;
stomach ulcer; stress; stress, psychological; stress
gastric mucosal lesion.
Stress can cause behavior, immune, and neural
responses, and intense and persistent stress re-
sponses can lead to a wide range of physiological
and psychological dysfunctions in the body.1-3 Re-
straint water-immersion stress (RWIS) can induce
a gastric mucosal lesion, one of the most common
visceral complications after wound. Exploring the
pathomechanism of the occurrence and develop-
ment of stress gastric mucosal lesions to find suit-
able treatment measures has become a focus in
Analytical and Quantitative Cytopathology and Histopathology®
0884-6812/19/4104-0127/$18.00/0 © Science Printers and Publishers, Inc.
Analytical and Quantitative Cytopathology and Histopathology®
Neurons in the Medulla Oblongata Related to
Gastric Mucosal Lesion of Rats Subjected to
Restraint Water-Immersion Stress
Dong-qin Zhao, Ph.D., Yan-jiao Bi, M.D., Sheng-nan Gong, M.D., and Peng-fei Li, M.D.
From the Key Laboratory of Animal Resistance of Shandong Province, School of Life Sciences, Shandong Normal University, and Shan-
dong Academy for Environmental Planning, Jinan, Shandong Province, P.R. China.
Dr. Zhao is Professor, Key Laboratory of Animal Resistance of Shandong Province, School of Life Sciences, Shandong Normal University.
Drs. Bi and Gong are Researchers, Key Laboratory of Animal Resistance of Shandong Province, School of Life Sciences, Shandong Nor-
mal University.
Dr. Li is Researcher, Shandong Academy for Environmental Planning.
This research was supported by grants from the National Science Foundation of China (No. 31501861) and the Natural Science Founda-
tion of Shandong Province, China (No. ZR2015CM013).
Address correspondence to: Dong-qin Zhao, Ph.D., College of Life Sciences, Shandong Normal University, Jinan 250014, Shandong
Province, P.R. China (109127@sdnu.edu.cn).
Financial Disclosure: The authors have no connection to any companies or products mentioned in this article.

2. current biology research. The expression of c-Fos
in the dorsal motor nucleus of the vagus (DMV),
the nucleus of the solitary tract (NTS), the area
postrema (AP), and the nucleus ambiguous (NA)
were significantly increased with different dura-
tions of RWIS and peaked at 1 hour of the stress,
indicating that the neuronal hyperactivity of the
DMV, NTS, AP, and NA plays important roles in
the gastric dysfunction induced by RWIS.4 Subdi-
aphragmatic vagotomy reduced the degree of the
gastric mucosal lesion upon RWIS, which indicated
that the peripheral regulation mechanism of stress
gastric mucosal lesion is due to the enhancement
of vagal parasympathetic activity.5-7 A variety of
methods such as basic physiology chemistry, elec-
trophysiology, immunocytochemistry, pharmacol­
ogy, and proteomics have been used to explore
the central mechanism of stress gastric mucosal
lesion from neurotransmitters, receptors, agonists,
and blockers.8-11
Synapses are the junctions of information com­
munication between neurons. The chemical syn­
apses in the mammalian nervous system account
for about 99%. Acetylcholine (ACh) is the first
classical neurotransmitter to be found with excite-
ment, and it is widely distributed in the brain. In-
travenous injection of ACh can cause severe gas-
tric mucosal injury in rats.12 Catecholamine, one
of the classical neurotransmitters in the central
nervous system, is the generic term of norepi­
nephrine (NE), adrenaline (E), and dopamine (DA)
and widely exists in the peripheral and central
nervous system. Numerous morphological and
electrophysiological studies have shown that cat-
echolamine is an important endogenous inhibito-
ry neurotransmitter that protects the integrity of
gastric mucosa during stress.13 Studies have found
that oxytocin and arginine vasopressin (AVP)
neurons in the central nervous system have the
effect of inhibiting gastric motility and promot-
ing gastric secretion through the vagus nerve.14,15
The activity of oxytocin and AVP in the central
nervous system is mediated by the oxytocin re-
ceptor (OTR) and AVP receptor (V1aR, V1bR, and
V2).16 Endogenous opioids are neuropeptides with
multifunctionality and widespread distribution.17
Methionine-enkephalin (M-ENK), a member of the
opioid neuropeptides family, is distributed in the
striatum, hypothalamus, interventricular brain,
brain stem, and many parts of the spinal cord. In-
traperitoneal injection of M-ENK analogues could
prevent gastric mucosal injury induced by cold
restraint stress.18 To elucidate the central regulato-
ry mechanism of the gastric dysfunction induced
by RWIS, the phenotype of activated neurons in
the medullary gastrointestinal center of rats dur­
ing RWIS were studied by immunohistochemical
double labeling technique for collocations of cho-
line acetyltransferase (ChAT), tyrosine hydroxy-
lase (TH), OTR, V1bR, and M-ENK with Fos, which
was used as a marker of neuronal activity.19
Materials and Methods
Animals
Male Wistar rats (Experimental Animal Center of
Shandong University, Jinan, China) weighing
220–250 g were housed 2 per cage in a room with
controlled ambient temperature of 22±2°C and hu­
midity of 45–55% under a normal day/night cycle
for at least 7 days with food and water available ad
libitum before initiation of the RWIS. Before stress,
the rats were fasted for 24 hours but allowed free
access to water.
Grouping and Stress Protocols
Rats were randomly divided into 2 groups des-
ignated according to their duration of RWIS: the
RWIS group (n=5) and the control group (n=5).
RWIS was performed as previously described,13
and rats of the control group were not stressed
but were kept under otherwise identical condi-
tions. To avoid the effect of diurnal variations on
the Fos expression, the experiment was performed
between 9:00 and 11:00 a.m. All surgeries and post-
operative care procedures were conducted in ac­
cordance with the National Institutes of Health
Guidelines for the Care and Use of Laboratory
Animals and were approved by the Institution-
al Animal Care and Use Committee at Shandong
Normal University (Jinan, People’s Republic of
China, AEECSDNU2018003).
Evaluation of Gastric Mucosal Damage
At the end of the procedure, rats were sacrificed
with an intraperitoneal pentobarbital sodium in­
jection (100 mg/kg body weight). Stomachs were
removed into 1% formaldehyde solution for 30
minutes, incised along the greater curvature, and
rinsed with normal saline. Gastric mucosal lesions
were identified with a magnifying lens and mea-
sured using the erosion index.20
Tissue Processing
Rats were then perfused via the ascending aorta
128 Analytical and Quantitative Cytopathology and Histopathology®
Zhao et al

3. with 200 mL 0.01 mol/L PBS (pH 7.4) followed by
500 mL 4% paraformaldehyde in 0.1 mol/L phos-
phate buffer (pH 7.4), and brains were removed
and post-fixed at 4°C for 4 hours in the same fix-
ative, and infiltrate with 20% sucrose in 0.1 mol/L
phosphate buffer for 48 hours at 4°C. Series of
frozen coronal sections of the hypothalamus and
medulla oblongata were cut at 30 μm in a cryostat
and collected into 0.01 mol/L PBS.
Immunohistochemistry
The immunoreaction of Fos plus ChAT, TH,
M-ENK, OTR, and V1b was detected by a dual
SP (streptavidin-biotin-peroxidase) immunohisto­
chemical technique.21 Briefly, the double-labeling
procedure was as follows: free-floating sections
were preincubated in methanolic 3% H2O2 for 30
minutes at room temperature followed by an in­
cubation with blocking buffer containing 5% nor-
mal goat serum and 0.3% Triton X-100 in 0.01
mol/L PBS for 30 minutes. Sections were then
incubated with rabbit anti-c-Fos antibody (sc-52,
Santa Cruz Biotechnology Inc., USA), diluted
1:2000 in 0.01 mol/L PBS containing 3% normal
goat serum (NGS) and 0.3% Triton X-100 for 24
hours at 4°C. After incubating with the biotiny­
lated goat anti-rabbit IgG and streptavidin-biotin-
horseradish peroxidase complex (Zymed Labora­
t­
ories Inc., USA) for 1 hour at room temperature
respectively, the sections were submitted to a dia­
minobenzidine hydrochloride (DAB, Sigma Chem-
ical Co., St. Louis, Missouri, USA), intensified with
0.05% cobalt chloride and 0.05% nickel ammoni-
um sulfate for 4–5 minutes. This method produces
a blue-black nuclear reaction product. The Fos-
immunoreactive sections were incubated for 24–
36 hours with rabbit anti-ChAT (1:1000), M-ENK
(1:2000), V1bR (1:200, International Laboratory USA),
or OTR (1:200, USCN Life Science, Inc., Houston,
Texas, USA), and mouse anti-TH (1:2000, Abcam
plc, Cambridge, UK) in 3% NGS and 0.3% Triton
X-100 for 48 hours at 4°C. Incubated with the bio­
tinylated goat anti-rabbit IgG and streptavidin-
biotin-horseradish peroxidase complex for 1 hour
at room temperature respectively, the visualiza-
tion of the immunoreactive products was obtained
by reaction with unintensified DAB that produces
a brown reaction product. Lastly, the free-floating
sections were mounted on gelatin-coated glass
slides, air-dried overnight, dehydrated in a series
of alcohols, cleared in xylene, and placed under a
coverslip with Permount.
Evaluation of Immunostaining
Pictures of brain sections were taken under iden-
tical conditions with a BX51 Olympus micro-
scope (Olympus Corporation, Japan) coupled to
an Olympus DP70 camera. The nomenclature and
nuclear boundaries defined in the rat brain ste-
reotaxic atlas of Paxinos and Watson22 were used
in this study. For quantitative assessment, the
number of immunoreactive neurons using Image-
Pro Plus 6.0 (Media Cybernetics Inc., USA) was
counted at one level. For the NTS, DMV, and AP:
(Bregma, −13.80–14.04 mm), while NA: (−13.32–
14.52 mm). Fos immunoreactivity (Fos-IR) was
identified as dark blue-black dots deposited in the
nuclei, and others appeared in the cytoplasm as
brown in color, and the Fos and neurotransmitter
double-labeled neurons were the brown perikar­
ya with a dark blue-black nucleus. In each nucle-
us, 3 kinds of neurons were counted, which were
Fos-IR nuclei, V1bR or OTR-IR neurons, and Fos+
V1bR or OTR -IR neurons.
The number of immunoreactive neurons was
counted in 3 adjacent sections per animal, and the
average values of them in 0.01 mm2 are reported
as the number of immunoreactivity.
Statistical Analysis
Counting was performed in 5 rats for each condi-
tion, and the data obtained from each animal were
used to calculate group means±SD. The statistical
procedures were performed with SPSS 20.0 soft-
ware (SPSS Inc., Chicago, Illinois, USA). Statisti-
cal analysis on the data between groups was per-
formed using Student’s t test, while the absolute
value between the 2 groups of a same neurotrans-
mitters variance in different nuclei were analyzed
by ANOVA. The statistical significance level was
set at p<0.05.
Results
Gastric Mucosal Damage Induced by RWIS
Macroscopic analysis showed that there was no
gastric mucosal damage in the control group, while
scattered lesion spots were observed in the muco-
sa of the RWIS group (Figure 1). The damage index
was significantly different between the control
(0.0±0.0) and RWIS groups (12.3±1.1) (p<0.05).
Expression of Fos
Expressions of Fos in DMV, NTS, AP, and NA were
increased significantly between the control group
and RWIS group, but in the different nuclei, en-
Volume 41, Number 4/August 2019 129
Neurons in the Medulla Oblongata

4. hancement degree of Fos had no difference (p>
0.05).
Expression of ChAT
ChAT-IR neurons (per 0.01 mm2) in the DMV were
larger and round or fusiform with a few shorter
processes, while in the NTS they were smaller with-
out processes. ChAT-IR neurons (per 0.01 mm2) in
the AP were round but too small and closely dis-
tributed to count, while in the NA they were larg-
er, fusiform, or polygonal with obvious processes
(Figure 2A–D).
As compared with the control group, Fos+
ChAT-IR neurons (per 0.01 mm2) in the DMV and
NA in the RWIS group were significantly increased
(p<0.05), while there was no difference in the NTS
(p>0.05) (Figure 2E).
The absolute values between the 2 groups of
ChAT-IR, Fos+ChAT-IR, the percentage of Fos+
ChAT-IR in the total Fos-IR, or ChAT-IR neurons
were significantly different in the different nu-
clei (p<0.01) (Figure 3). The percentages of Fos+
ChAT-IR neurons in Fos-IR neurons in the RWIS
group were 64.4% and 89.2%, respectively, in the
DMV and NA. Fos+ChAT-IR neurons in response
to RWIS represented in 21.1% and 36.1% of Fos+
ChAT-IR neurons in the DMV and NA, respec­
tively—significantly different from the control
group (p<0.05) (Table I), but there was no sig-
nificant difference in the NTS (p=0.431). These
data indicate that ACh neurons in the DMV and
NA but not NTS were the major ones related to
RWIS regulation.
Expression of TH
TH-IR neurons (per 0.01 mm2) were small, round,
or fusiform with shorter processes in the DMV,
while in the NTS they were large, round, fusiform,
or triangular with long processes. There were only
processes in the NA, while only perikarya in the
AP (Figure 4A–D). As compared with the control
group, Fos+TH-IR neurons (per 0.01 mm2) in the
DMV, NTS, and AP in the RWIS group were sig-
nificantly increased (p<0.01) (Figure 4E).
The absolute value between the 2 groups of
TH-IR neurons in AP was significantly higher from
the other nuclei (p<0.01) (Figure 3). The percent-
ages of Fos+TH-IR neurons in Fos-IR neurons in
the RWIS group were 17.3%, 12.9%, and 22.4% in
the DM, NTS, and AP. Fos+TH-IR neurons in re-
sponse to RWIS represented in 38.0%, 34.4%, and
18.6% of TH-IR neurons in the DMV, NTS, and AP,
respectively, significantly different from the con-
trol group (p<0.01) (Table I). That indicated that
during the stress process, catecholamine neurons
in the DMV, NTS, and AP were involved in RWIS
regulation.
In the NA, there was a large number of TH-
positive terminals not only in the RWIS group but
also in the control group, which indicated that
neurons in the NA, such as ACh neurons, might
accept the projection of fibers from catecholamine
neurons in other nuclei.
Expression of OTR
OTR-IR neurons in the DMV were large, while
those in the interior part were fusiform profiles
with obvious neuronal processes and those in the
lateral part were round profiles with few num-
bers of neuronal processes. OTR-IR neurons were
smaller but more uniform in size with a scanty
cytoplasm in the NTS, which in the DMV were fu-
130 Analytical and Quantitative Cytopathology and Histopathology®
Zhao et al
Figure 1
Gastric mucosal damage
induced by restraint water-
immersion stress (RWIS).
(A) Control group. (B) RWIS
group.

5. siform profiles without any neuronal processes.
OTR-IR neurons in the NA were larger and round
or fusiform with a few shorter processes, while in
the AP they were small and densely distributed
(Figure 5A–D).
As compared with the control group, Fos+
OTR-IR neurons in the DMV, NTS, AP, and NA
in the RWIS group were significantly increased
(p<0.01) (Figure 5E).
The absolute values between the 2 groups of
OTR-IR and Fos+OTR-IR neurons in AP were sig-
nificantly higher than from the other nuclei (p<
0.01) (Figure 3). Fos+OTR-IR neurons in response
to RWIS represented in 10.4%, 9.7%, 9.7%, and
Volume 41, Number 4/August 2019 131
Neurons in the Medulla Oblongata
Figure 2
Double immunohistochemical
staining of Fos and ChAT
(A–D) and the cell count
(cells per 0.01 mm2) (E) in
the medulla oblongata in the
control (A, C) and RWIS 1h
(B, D). Fos immunoreactivity
was identified as dark blue-
black dots deposited in the
nuclei, and ChAT-IR neurons
appeared in the cytoplasm
as brown in color. 4V = 4th
ventricle, CC = central canal.
Scale bar=100 μm. *p<0.05,
**p<0.01, control vs. RWIS.

6. 18.6% of OTR-IR neurons in the DMV, NTS, AP,
and NA, respectively, significantly different from
the control group (p<0.05) (Table I). The percent-
age of Fos+OTR-IR neurons in Fos-IR neurons
was 55.7%, 47.0%, 95.0%, and 100.0% in the DMV,
NTS, AP, and NA in the RWIS group (p>0.05)
(Table I). These indicated that oxytocin-sensitive
neurons in the DMV, NTS, AP, and NA were one
of the major activated neuron types involved in
RWIS regulation.
Expression of V1bR
V1bR-IR neurons in the DMV were smaller than
those in the NTS, mostly round and a few spin-
dled, almost without any processes, while those
in the AP were densely packed to count (Figure
5A–B). V1bR-IR neurons in the NA were large,
while those in the interior part were fusiform
profiles with obvious neuronal processes (Figure
6C–D).
Fos+V1bR-IR neurons (cells per 0.01 mm2) in the
DMV, NTS, AP, and NA in the RWIS group were
significantly increased as compared to those in the
control group (p<0.01) (Figure 6E).
The absolute values between the 2 groups of
V1bR-IR in AP and the percentage of Fos+V1bR-IR
132 Analytical and Quantitative Cytopathology and Histopathology®
Zhao et al
Table I Percentage of Double-Labeled Neurons to Fos or Neurotransmitter Immunoreactivity Ones in the Medulla Oblongata
Gastrointestinal Central (%)
DMV NTS AP NA
Category Control vs. RWIS Control vs. RWIS Control vs. RWIS Control vs. RWIS
Fos+ChAT-IR/Fos-IR 50.3±4.1 vs. 64.4±7.5 38.0±6.4 vs. 9.6±2.2** Too dense to count 82.5±11.8 vs. 79.2±6.8
Fos+ChAT-IR/ChAT-IR 7.2±4.3 vs. 21.1±2.4** 38.3±4.2 vs. 35.6±4.2 Too dense to count 7.5±2.4 vs. 36.1±9.3**
Fos+TH-IR/Fos-IR 6.2±2.2 vs. 17.3±4.5* 4.5±0.6 vs. 12.9±0.8** 16.4±5.8 vs. 22.4±7.4 No perikarya
Fos+TH-IR/TH-IR 14.3±4.5 vs. 38.0±5.3** 9.7±1.6 vs. 34.4±2.4** 4.5±1.3 vs. 18.6±6.1** No perikarya
Fos+OTR-IR/Fos-IR 67.1±10.1 vs. 55.7±6.1 44.0±3.6 vs. 47.0±1.9 98.6±1.5 vs. 95.0±1.0 100.0±0.0 vs. 100.0±0.0
Fos+OTR-IR/OTR-IR 3.1±1.0 vs. 10.4±1.4** 5.3±0.9 vs. 9.7±0.9** 2.3±0.7 vs. 9.7±1.6** 7.4±1.3 vs. 18.6±3.2*
Fos+V1bR-IR/Fos-IR 51.3±10.2 vs. 72.2±3.8* 27.0±2.9 vs. 52.3±4.8** 96.4±2.2 vs. 57.5±11.8* 90.0±10.0 vs. 98.6±1.4
Fos+V1bR-IR/V1bR-IR 2.9±1.1 vs. 10.7±1.6** 3.6±0.4 vs. 8.3±0.7** 2.3±0.6 vs. 4.5±1.1 7.0±1.1 vs. 28.5±3.8**
*p<0.05, **p<0.01 control vs. RWIS.
Figure 3
ANOVA of the absolute value
between the 2 groups of ChAT,
TH, OTR, and V1bR variance
in different nuclei. In the same
neurotransmitter, column with
a same letter, p>0.05; column
with a different lower case
letter, p<0.05; column with
a different upper case letter,
p<0.01.

7. neurons in V1bR-IR neurons in NA were signifi­
cantly higher from the other nuclei (p<0.05) (Fig-
ure 3). Fos+V1bR-IR neurons in response to RWIS
represented in 10.7%, 8.3%, and 3.8% of V1bR-IR
neurons in the DMV, NTS, and NA, respective-
ly—significantly different from the control group
(p<0.01) (Table I). The percentage of Fos+V1bR-IR
neurons in Fos-IR neurons in the RWIS group
was increased in the DMV and NTS, while de-
creased in AP (p<0.05) (Table I). These data in-
dicated that AVP-sensitive neurons in the DMV,
NTS, and NA were the major type of the activated
neuron during RWIS as well.
Expression of M-ENK
M-ENK neurons—large, round, or spindle with
prominent processes—were present in the NTS
but no Fos+M-ENK-IR were found, while only
positive terminals in the DMV, NA, and AP
(Figure 7). That indicated that M-ENK neurons
of the medulla oblongata were not involved in
RWIS regulation.
Volume 41, Number 4/August 2019 133
Neurons in the Medulla Oblongata
Figure 4
Double immunohistochemical
staining of Fos and TH (A–D)
and the cell count (cells per
0.01 mm2) (E) in the medulla
oblongata in the control (A, C)
and RWIS 1h (B, D). Fos
immunoreactivity was
identified as dark blue-black
dots deposited in the nuclei,
and TH-IR neurons appeared
in the cytoplasm as brown in
color. 4V = 4th ventricle,
CC = central canal. Scale
bar=100 μm in A and B, while
50 μm in C and D. *p<0.05,
**p<0.01, control vs. RWIS.

8. Discussion
The vagus nerve fibers regulating the function of
the stomach originate from the DMV, NA, NTS,
the nucleus intercalatus, and the nucleus retro­
ambigualis.23 RWIS evoked marked neuronal acti-
vation in the DMV, NTS, AP, and NA assessed by
134 Analytical and Quantitative Cytopathology and Histopathology®
Zhao et al
Figure 5
Double immunohistochemical
staining of Fos and OTR (A–D)
and the cell count (cells per
0.01 mm2) (E) in the medulla
oblongata in the control
(A, C) and RWIS 1h (B, D).
Fos immunoreactivity was
identified as dark blue-black
dots deposited in the nuclei,
and OTR-IR neurons appeared
in the cytoplasm as brown in
color. 4V = 4th ventricle,
CC = central canal. Scale
bar=100 μm. *p<0.05,
**p<0.01, control vs. RWIS.

9. Volume 41, Number 4/August 2019 135
Neurons in the Medulla Oblongata
The Phenotypic Nature of Activated Neurons in the
Medullary Visceral Zone Were AVP-Sensitive or
Oxytocin-Sensitive Neurons
Oxytocin, AVP neurons within the central ner-
vous system, have the effect of inhibiting gas-
tric motility, promoting gastric secretion, and this
effect is achieved through the vagus nerve. By in-
fecting rat paraventricular hypothalamic nucleus
(PVN) and supraoptic nucleus (SON) hypotha-
lamic nuclei with a virus construct, an extensive
network of oxytocin fibers in various areas of
the brain was revealed including NTS, DMV, and
NA.28 When oxytocin receptor blockers are in-
jected into the DMV, gastric acid secretion is
inhibited and gastric motility is strengthened,
while when oxytocin is injected into the DMV,
the opposite effect is observed.29 Unilateral in-
jection of AVP V1 receptor blockers into the NTS
can eliminate the damage to gastric ischemia-
reperfusion injury by electrical stimulation of
PVN, and microinjection of AVP into the DMV
can inhibit gastric motility and promote gastric
acid secretion.30 In our study the occurrence of
OTR and V1bR was nearly evenly on the mem-
brane of neurons of the DMV, NTS, AP, and NA.
The significant difference of the ratio of Fos+
OTR-IR, Fos+V1bR-IR neurons to OTR-IR, Fos+
V1bR-IR between the RWIS and control groups
indicates that the oxytocin-sensitive and AVP-
sensitive neurons are involved in the modula-
tion of the gastric dysfunction during RWIS. In
addition, in the RWIS group the percentages of
Fos+OTR-IR and Fos+V1bR-IR neurons in Fos-IR
nuclei were more than 58% and 72% in the DMV
and 45% and 52% in the NTS, which indicated
that oxytocin and AVP mediate the activity of
these neurons mainly by OTR and V1bR, particu-
larly the V1b receptor.
M-ENK Neurons Have No Obvious Relation with
Gastric Mucosal Injury Caused by RWIS
Previous studies found that peripheral intravenous
injection of M-ENK analogue could attenuate gas-
tric mucosal injury induced by cold-restraint stress.
At the observation of the hypothalamus and me­
dulla oblongata, M-ENK-IR perikaryon was only
found in the NTS, while only M-ENK nerve end-
ings in the DMV and NA and no M-ENK–positive
neurons and fiber terminals in the PVN and SON
were observed, the M-ENK neurons are not obvi-
ously related to gastric mucosal injury caused by
RWIS, which is consistent with previous studies.18
Fos expression.4 DMV and NA is the visceral para-
sympathetic efferent nucleus, so the gastric mu-
cosal lesion induced by RWIS was predominantly
because of the hyperactivity of vagal parasympa-
thetic efferents.
Hyperactivity of Cholinergic Neurons in the DMV
and NA but Not the NTS Might Be One of the
Primary Central Mechanisms of the Gastric
Dysfunctions Induced by RWIS
ChAT-IR neurons were all distributed in the
DMV, NTS, AP, and NA. In the DMV and NA, the
proportion of Fos+ChAT-IR neurons to ChAT-
IR neurons in the RWIS group was significantly
higher than that in the control group, and 64.4%
and 89.2% Fos-IR neurons were ChAT-IR, indi­
cating that the hyperactivity of cholinergic neu­
rons in the DMV and NA is one of the main
mechanisms of gastric mucosal injury induced
by RWIS, which is consistent with the results of
continuous intravenous injection of ACh to cause
severe gastric mucosal injury in rats.12 In the
NTS, there was no significant difference in the
ratio of Fos+ChAT-IR neurons to ChAT-IR neu-
rons, which indicates that cholinergic neurons
in the NTS may not participate in RWIS regu-
lation. However, studies have shown that the
injection of ACh into the NTS also can signifi-
cantly increase the protective effect of electro­
acupuncture on gastric mucosal injury.24 Possibly
it is the different regulation mode of different
stress models, but the specifics need to be further
studied.
Catecholaminergic Neurons in the Medulla
Oblongata Gastric Nerve Center May Be Related to
the Stress-Responsive Signal Transduction
Numerous morphological and electrophysiolog-
ical studies have demonstrated that NE and DA
are important endogenous inhibitory neurotrans-
mitters that play a protective role in the integrity
of gastric mucosa during stress.25 Central admin­
istration of DA antagonists aggravates stress-
induced gastric mucosal injury, whereas DA ag-
onists attenuate stress-induced gastric mucosal
injury.26,27 The higher proportion of Fos+TH-IR
neurons to TH-IR or Fos-IR neurons in the RWIS
group than that in the control group indicates
that catecholamine neurons in the medulla oblon-
gata participate in the regulation of gastric dys­
function induced by RWIS, though maybe not the
major neuron phenotype.

10. 136 Analytical and Quantitative Cytopathology and Histopathology®
Zhao et al
sensitive or oxytocin-sensitive neurons, while cat-
echolamine neurons in the medulla oblongata are
intermediate neurons.
In summary, activated ACh neurons in the
medulla oblongata are the vagal presympathet-
ic preganglionic neurons, most of which are AVP-
Figure 6
Double immunohistochemical
staining of Fos and V1bR
(A–D) and the cell count
(cells per 0.01mm2) (E) in the
medulla oblongata in the
control (A, C) and RWIS 1h
(B, D). Fos immunoreactivity
was identified as dark blue-
black dots deposited in the
nuclei, and V1bR-IR neurons
appeared in the cytoplasm
as brown in color. 4V = 4th
ventricle, CC = central canal.
Scale bar=100 μm. *p<0.05,
**p<0.01, control vs. RWIS.

11. Volume 41, Number 4/August 2019 137
Neurons in the Medulla Oblongata
water‑immersion stress. Mol Med Rep 2019;20:2303-2315
10. Fan F, Li L, Liu W, Yang M, Ma X, Sun H: Astrocytes and neu-
rons in locus coeruleus mediate restraint water immersion
stress-induced gastric mucosal damage through the ERK1/2
signaling pathway. Neurosci Lett 2018;675:95-102
11. Xu Y, Jia J, Xie C, Wu Y, Tu W: Transient receptor potential
Ankyrin 1 and Substance P mediate the development of gas-
tric mucosal lesions in a water immersion restraint stress rat
model. Digestion 2018;97:228-239
12. Gatón J, Fernández de la Gándara F, Velasco A: The role
of the neurotransmitters acetylcholine and noradrenaline
in the pathogenesis of stress ulcers. Comp Biochem Physiol
1993;106(1):125-129
13. Zhao DQ, Lu CL, Ai HB: The role of catecholaminergic neu-
rons in the hypothalamus and medullary visceral zone in re-
sponse to restraint water-immersion stress in rats. J Physiol
Sci 2011;61(1):37-45
14. Pirnik Z, Kiss A: Fos expression variances in mouse hypo-
thalamus upon physical and osmotic stimuli: Co-staining
with vasopressin, oxytocin, and tyrosine hydroxylase. Brain
Res Bull 2005;65:423-431
15. Llewellyn-Smith IJ, Kellett DO, Jordan D, Browning KN,
Travagli RA: Oxytocin-immunoreactive innervation of iden-
tified neurons in the rat dorsal vagal complex. Neurogastro-
enterol Motil 2012;24(3):e136-46
16. Young WS, Li J, Wersinger SR, Palkovit MR: The vasopres-
sin 1b receptor is prominent in the hippocampal area CA2
where it is unaffected by restraint stress or adrenalectomy.
Neuroscience 2006;143(4):1031-1039
References
1. Yang G, Guo H, Li H, Shan S, Zhang X, Rombout JH, An L:
Molecular characterization of LEAP-2 cDNA in common
carp (Cyprinus carpio L.) and the differential expression
upon a Vibrio anguillarum stimulus; indications for a sig-
nificant immune role in skin. Fish Shellfish Immunol 2014;
37:22-29
2. Rombout JH, Yang G, Kiron V: Adaptive immune responses
at mucosal surfaces of teleost fish. Fish Shellfish Immunol
2014;40:634-643
3. Yang HT, Zou SS, Zhai LJ, Wang Y, Zhang FM, An LG, Yang
GW: Pathogen invasion changes the intestinal microbiota
composition and induces innate immune responses in the
zebrafish intestine. Fish Shellfish Immunol 2017;71:35-42
4. Zhang YY, Cao GH, Zhu WX, Cui XY, Ai HB: Comparative
study of c-Fos expression in the rat dorsal vagal complex
and nucleus ambiguous induced by different durations of
restraint water-immersion stress. J Chin Physio 2009;52(3):
143-150
5. Liu M, Xie S, Zhou J: Use of animal models for the imaging
and quantification of angiogenesis. Exp Anim 2018;67:1-6
7. Kobayashi K, Kashimak K, Higuchi K, Arakawa T: The mech-
anisms of gastro-intestinal mucosal injury repair. Nippon
Rinsho 1998;56(9):2215-2222
8. Gong SN, Zhu JP, Ma YJ, Zhao DQ: Proteomics of the medi-
odorsal thalamic nucleus of rats with stress-induced gastric
ulcer. World J Gastroenterol 2019;25(23):2911-2923
9. Zhao DQ, Gong SN, Ma YJ, Zhu JP: Medial prefrontal cor-
tex exacerbates gastric dysfunction of rats upon restraint
Figure 7
Immunohistochemical
staining of M-ENK-IR neurons
in the NTS and DMV induced
by control (A, B) and RWIS
for 1h (C, D). M-ENK
immunoreactivity neurons
appeared in the cytoplasm as
brown in color. NTS = nucleus
of solitary tract, DMV = dorsal
motor nucleus of the vagus,
4V = 4th ventricle.
Magnification ×200, scale
bar=100 μm in A and C while
×400, scale bar=50 μm in
B and D.

12. 138 Analytical and Quantitative Cytopathology and Histopathology®
Zhao et al
17. Arida RM, Gomes da Silva S, de Almeida AA, Cavalheiro EA,
Zavala-Tecuapetla C, Brand S, Rocha L: Differential effects
of exercise on brain opioid receptor binding and activation
in rats. J Neurochem 2015;132(2):206-217
18. Chen QS, Dai YL: Role of morphine and methy-enkephalin
in stress-induced gastric mucosal lesions. Journal of Nanjing
Medical University 1990;10(3):196-198
19. Herrera DG, Robertson HA: Activation of c-Fos in the brain.
Prog Neuro Bio 1996;l50:83-107
20. Guth PH, Aures D, Paulsen G: Topical aspirin plus HCl gas-
tric lesions in the rat: Cytoprotective effect of prostaglandin,
cimetidine, and probanthine. Gastroenterology 1979;76:88-93
21. Ding NZ, Qi QR, Gu XW, Zuo RJ, Liu J, Yang ZM: De novo
synthesis of sphingolipids is essential for decidualization in
mice. Theriogenology 2018;106:227-236
22. Paxinos G, Watson C: The Rat Brain in Stereotaxic Coordi-
nates. Fifth edition. Burlington, Massachusetts, Elsevier Ac-
ademic Press, 2005
23. Hayakawa T, Takanaga A, Tanaka K, Maeda S, Seki M: Cells
of origin of vagal motor neurons projecting to different parts
of the stomach in the rat: Confocal laser scanning and elec-
tron microscopic study. Anat Embryol (Berl) 2003;207:289-297
24. Zhou YU, Wang Pan, Tan XQ, Li CW, Huang BL: The effects
of microinjection of acetylcholine into nucleus tractus soli-
tarious on electroacupuncture against stress gastric muco-
sa injury in rats. Journal of Xianning College (Medical Sci)
2007;21(3):34-36
25. Glavin GB, Szabo S: Dopamine in gastrointestinal disease.
Dig Dis Sci 1990;35:1153-1155
26. Glavin GB: Central dopamine involvement in experimental
gastrointestinal injury. Prog Neuro-psychopharmacol Biol
Psychiatry 1992;16(2):217-221
27. Glavin GB: Dopamine: A stress modulator in the brain and
gut. Gen Pharmacol 1992;23(6):1023-1026
28. Knobloch HS, Charlet A, Hoffmann LC, Eliava M, Khrulev S,
Cetin AH, Osten P, Schwarz MK Seeburg PH, Stoop R, and
Grinevich V: Evoked axonal oxytocin release in the central
amygdala attenuates fear response. Neuron 2012;73:553-566
29. Rogers RC, Hermann GE: Oxytocin, oxytocin antagonist,
TRH, and hypothalamic paraventricular nucleus stimula-
tion effects on gastric motility. Peptides 1987;8:505-513
30. Zhu J, Chang L, Xie J, Ai H: Arginine vasopressin injected
into the dorsal motor nucleus of the vagus inhibits gastric
motility in rats. Gastroenterol Res Pract 2016;2016:4618672