JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2009, 60, Suppl 3, 61-66

www.jpp.krakow.pl

K. GIL1, A. BUGAJSKI1, M. KURNIK1, W. ZARASKA2, P. THOR1

PHYSIOLOGICAL AND MORPHOLOGICAL EFFECTS OF LONG-TERM VAGAL

STIMULATION IN DIET INDUCED OBESITY IN RATS
1

Department of Pathophysiology, Jagiellonian University Medical College, Krakow, Poland; 2Institute of Electron Technology,
Krakow, Poland
Some previous studies have shown suppressive effect of the vagal nerve stimulation (VNS) on long - term feeding
regulation in rats. We assessed body weight, interstitial cells of Cajal (ICC), myenteric plexus neurons, mast cells
in the stomach, duodenum and colon and c-Fos expression in nodose vagal ganglia in the rats with VNS. Male
Wistar rats were implanted with microchip (MC) and kept during the whole study (100 days) on high calorie diet.
Left vagal nerve was stimulated by electrical pulses (10ms, 200mV, 0.05Hz) generated by MC. After finishing the
experiments tissue samples (stomach, duodenum, colon and nodosal vagal ganglia) were taken. Mast cells were
toluidine blue stained and counted in mucosa, muscularis externa and serosa. For immunostaining, antibodies for
ICC (CD117), myenteric plexus neurons (PGP9.5) and c-Fos were used. Positive cells were assessed by image
analysis. Chronic microchip vagal stimulation significantly decreased epididymal fat pad weight, meal size with
effect on decreased weight gain in VNS rat. VNS significantly increased mast cells number in all examined parts of
the gastrointestinal wall, mainly in the muscularis. There were no significant differences in ICC and myenteric
plexus neurons between VNS and control. Expression of c-Fos in nodosal ganglia was higher in VNS group. The
effects observed during long-term VNS concern predominantly mast cells. These data support the theory that VNS
can increase vagal afferent satiety signals leading to reduced food intake and body weight gain and mast cells are
involved in this process.

K e y w o r d s : vagal nerve stimulation, food intake, body weight, fat pad, obesity, mast cells, interstitial cells of Cajal, myenteric
plexus, nodose ganglion

INTRODUCTION
The body weight and body fat content are regulated by
multiple factors (1-6). Body weight remains stable within
relatively narrow range, despite large day-to-day fluctuations in
the amount of food consumed. It is important to recognize, that
short-term and long-term food intake and energy balance are
regulated through different but interacting mechanisms. Shortterm regulation signals have a different function than long-term
that are activated in proportion to both adipose stored and energy
consumed over a long period of time. In short-term regulation
glucostatic hypothesis of regulation of food intake was proposed
by Mayer over 50 years ago (7). Hypoglycemia or inhibition of
glucose metabolism with 2 deoksy-D-glucose not only increases
food intake but also stimulates vagal activity (8). Campfield and
Smith first demonstrated that a vagally mediated spike of plasma
insulin concentration induces small (10-15 mg/dl) transient
decrease of blood glucose level that precedes spontaneous
feeding in rats (9, 10). Eating can be induced by mimicking this
effect by administering small amount of insulin. In addition,
entry of food into the stomach and proximal small intestine
activates stretch- and mechanoreceptors. These signals are
transmitted also via vagal nerves to the hind brain, where they
are integrated and play a major role in short-term regulation, by

limiting the size of single meal (1, 2). These types of signal may
also affect energy intake in subsequent meals.
We previously showed that short-term vagal stimulation
affects volume regulation of food intake and decreases body
weight in rats (11-13). Randich and Cox (14-16) using
extracellular recordings from the vagus nerve showed, that
vagus nerve conducts satiety signal from the jejunum,
activated by fatty acid infusion. On the contrary, in humans
subjected to VNS no changes in body weight were observed.
There is also considerable evidence, that vagal afferent nerves
can be activated by CCK, leptin, and ghrelin (17-24).
The purpose of current work was to evaluate the role of
vagal nerve stimulation in long-term regulation of body weight
and food intake in high fat diet induced obesity in rats. We
examined also the effects of VNS on interstitial cells of Cajal
(ICC), myenteric plexus neurons, mast cells in the stomach,
duodenum and colon and c-Fos expression in nodose vagal
ganglion.
MATERIAL AND METHODS
Eighteen Wistar male rats with mean body weight at the
beginning of the study 374 18 g were used. The animals were

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housed in individual cages and all were fed the same high fat diet
to induce obesity (DIO) (Bento Kronen Products, Belgium)
during whole experiment. The caloric distribution of the DIO
was: protein 29.5%, fat 45.6%, carbohydrates 24.9%, and
metabolizable energy was 4.34 Kcal/g. All animals were housed
in the same optimal conditions of the lifestyle with food and
water ad libitum and at 23 2C temperature on a 12:12-hour
dark/light cycle. The Jagiellonian University Bioethical
Committee approved the care and use of the animals. The rats
were randomly divided into three groups: 1. rats with active
microchip (MC) connected by electrodes with the left vagal
nerve (MC group, n=6), 2. animals with inactive MC without
electrodes on the vagal nerve (Sham, n=6), 3. intact rats -without
MC and electrodes (Control, n=6). To eliminate effects of
surgical procedures and microchip implantation on
morphological parameters (mast cells count, interstitial cells of
Cajal, and myenteric plexus neurons) control group of
unoperated animals (intact group) was included in the study.
Sham operated group served as the most important reference
group for the assessment of the food intake, body weight and
epididymal fat pad weight. After about 3 weeks (day 25th) of
adaptation to new environmental conditions and fat diet rats with
the average body mass 478 46 g were starved for 12 hours and
operated in general anesthesia induced with sodium
pentobarbital given intraperitoneally (Vetbutal, 0.25mg/kg, i.p.,
Biowet, Pulawy, Poland). The MC for chronic vagal stimulation
(Institute of Electron Technology, Cracow, Poland) was placed
into the subcutaneous pocket and the ends of the MC platinum
electrodes were wrapped around the subdiaphragmatic left vagal
nerve; cathode and anode were positioned at 0.5 cm distance. In
the second group inactive MC was implanted and laparotomy
was performed (sham group). Food was restored on the day after
operation. The parameters of the impulses generated by MC
were: unipolar rectangular pulses duration -10 ms, amplitude 200 mV, frequency - 0.05 Hz. The parameters of MC stimulation
were based on our previous studies (11, 13). Daily food intake
and body weight were measured each morning. The amount of
daily food intake was determined by subtracting the amount of
food remaining from that given 24 h before.
At the end of the experiment (day102nd) all the rats were
killed by decapitation and weighed. Both epididymal fat pads,
located between the cauda epididymis and the distal extremity of
the testis, were dissected from the animal and weighed.
Epididymal fat pad/body weight ratio was calculated by dividing
the fat pad weight by the total body weight. Tissue samples
(stomach, duodenum, proximal colon and vagal nodose ganglia)
were taken for further analyses. All tissue fragments were
formalin fixed and paraffin-embedded 5 m slices were made.
For mast cells assessment specimens were toluidine blue stained.
Mast cells were counted in mucosa, muscularis externa and
serosa (total number and percentage of degranulated cells were

assessed). For immunostaining heat induced antigen retrieval was

carried out to unmask antigen determinants. Primary polyclonal
antibodies as markers for ICC (rabbit CD117 DAKO, 1:200)
and myenteric plexus neurons (rabbit PGP 9.5DAKO; 1:200)
were used with LSAB2 (DAKO) visualization system using
diaminobenzidine - DAB - (DAKO) as a chromogen. The area of
positive cells was assessed by image analysis system (Multiscan
8.01, CSS). Data were expressed as positively stained surface of
Cajal cells (or myenteric plexus cells respectively) per 1 mm of
length of the analyzed tissue section. At least 5 serial slides for
every part of gastrointestinal tract examined in each animal were
analyzed. For c-Fos primary antibodies (rabbit, polyclonal, Santa
Cruz Biotechnology) were used. Cells positive for c-Fos staining
were counted and data were expressed as a percentage of total
neurons in nodose ganglion.
Data are expressed as mean and standard deviation (SD).
Results were analyzed by one-way analysis of variance
(ANOVA), followed by post-hoc LSD test with STATISTICA
6.0 software package (StatSoft, Tulsa). Statistical significance
was set at p<0.05.
RESULTS
Food intake and body weight
Electrical stimulation of the left vagal nerve reduced the
daily and total food intake in MC group compared with sham
group, but the differences were not statistically significant
(p=0.37). No differences between MC and control group were
observed. (Table 1).
Consequently to reduced food intake, final body weight was
lower, but not significantly, in group with VNS (MC) compared
with no stimulated group (sham) (p=0.30) but not with control
animals (Table 1).
Epididymal fat pad weight
Fat pad weight reflecting the total body fat content was
significantly higher in sham group compared to MC group
(p<0.05). For control group fat pad weight was slightly higher
than in MC group, but these data were not significant. Mean
epididymal fat pad weight relative to body weight (fat pad/body
weight ratio) was significantly lower (27.4%) for rats with active
MC as compared to sham animals (p<0.01), but not to control
group (Table 1).
Mast cells
In the stomach wall VNS significantly increased total mast
cells number compared to sham and control groups (282.2 168

Table 1. Food intake, body weight and epididymal fat pad weight in microchip left vagus nerve stimulation (MC), sham operated and
control (intact) rats
Food intake during experiment (g)
Initial body weight day 1 (g)
Final body weight - day 102 (g)
Body weight gain (g)
Epididymal fat pad weight-EFP (g)
EFP/body weight ratio
EFP/body weight ratio increment
over MC rats (%)

Sham
1997 203
373.3 14.1
688.7 76
315.8 27.4
13.8 3.5
0.020

MC
1896 171
376.5 12.9
651.7 63
291.2 29.2
10.2 2.5
0.0157

Control
1876 215
370.1 21.4
660.9 57
290,8 19.7
11.0 2.6
0.0166

127.4

100

105.7

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in MC group, 192.3 103.7 in sham group and 188.7 57.2 in
control). The highest values were observed in muscularis externa
(180.8 98.9 in MC group, 131.5 67.8 in sham group and
124.3 41.1 in control) (Fig.1).
In the duodenum mast cells number after VNS (16.0 9.05)
remained unchanged compared to sham (18.0 8.6) and control
(23.7 22.8) as well as in proximal colon (MC 18.7 13.4;
sham 17.3 11.6; control 22.3 22.1). No significant
differences between VNS group and control groups were
observed.
Interstitial cells of Cajal
The area of the ICC in myenteric plexus region stained with
CD117 differs in each region of gastrointestinal tract of the rats
(for control group: stomach 6688 2467 m2; duodenum
6534 3545 m2; colon 10590 4464 m2, for sham group:
stomach 7008 2645 m2; duodenum 7212 3639 m2;
colon 8801 3406 m2, and for MC group: stomach - 7428
2804 m2; duodenum 7690 3983 m2; colon 10742 3790
m2). No significant differences between VNS group and control
groups in each part GI tract were observed (Fig. 2).
Myenteric plexus

Fig. 2. Interstitial cells of Cajal (ICC) area in stomach,

duodenum and proximal colon of the rat after left vagus nerve
stimulation (MC), in sham and control groups (n=6 for each
group), expressed as area of c-kit positive cells in myenteric
plexus region. Data are presented as mean and standard
deviation.

The area of the myenteric plexus stained with PGP 9.5 was
different in each region of gastrointestinal tract of the rats (for
control group: stomach 11215 5210 m2; duodenum 6745
2185 m2; colon 9744 4910 m2, for sham group: stomach
10809 5437 m2; duodenum 5740 3043 m2; colon
7456 3533 m2, and for MC group: stomach 9039 5669
m2; duodenum 6102 3412 m2; colon 9351 6180 m2).
However, no statistical differences between VNS group and
control groups in each part GI tract were observed (Fig. 3).
c-Fos positive cells in nodose ganglion
Assessment of c-Fos positive neurons in nodose ganglia of
vagus nerve showed significant increase in percentage of

Fig. 3. Myenteric plexus area in stomach, duodenum and

proximal colon of the rat after left vagus nerve stimulation
(MC), in sham and control groups (n=6 for each group) assessed
by image analysis in PGP 9.5 stained specimens. Data are
presented as mean and standard deviation.

positive cells in the left nodose ganglion (3.86%) and right

nodose ganglion (3.24%) compared to control (1.53 %) after
VNS stimulation. In our experiment c-Fos positive neurons
number was significantly higher in left vagal ganglion compared
to the right ganglion and control.
DISCUSSION
Fig. 1. Mast cells count in the stomach wall of the rat after left
vagus nerve stimulation (MC), in sham and control groups (n=6
for each group). Cells were counted in 10 area of view with
magnification 200x in toluidine blue stained specimens in
mucosa, muscularis externa and serosa. Data are presented as
mean and standard deviation. * p<0.05 as compared to sham and
control group.

Our experimental study was performed using high fat diet

because obesity induced by high-fat diet mimics obesity in
humans (25). Effect of short-term vagal neuromodulation in
standard diet fed rats was studied previously (11-13). Vagal
nerve stimulation by MC leads to a decrease in food intake in
rats combined with a decrease in body mass when compared to
sham operated animals with inactive microchip implanted, but

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not intact group. These effects may be explained by imitation
of the physiological vagal input associated with gastric
mechanoreceptors activation by food. VNS may mimic the
physiological input associated with gastric mechanoreceptors
and jejunal chemoreceptors activation by food and leads to
decrease in food intake and subsequently decrease in weight gain
(3, 26). Vagal nerve has unique abilities, consists mainly of
afferent fibers conducting signals from gut mechano- and
chemoreceptors (26, 27), but also on the vagal afferents are
located numerous receptors for cytokines, CCK (17-19), ghrelin
(23, 24) and peptides released by meal. Some authors postulated
that vagal fibers transfer hunger signals that promote feeding
(28). Results of vagal stimulation obtained in humans are
contradictory. In humans treatment of depression or epilepsy by
VNS did not affect body weight (29-31). Opposite results were
published by Burneo et al., who observed weight loss in patients
with epilepsy treatment by VNS (32). This discrepancy may be
caused by different parameters of vagal stimulation or by side
effects of VNS (32). Peripheral effects of VNS cannot be
excluded such the release of pancreatic polypeptide and insulin.
Release of these hormones depends probably upon the frequency
of stimulation. These signals are transmitted also via vagal
nerves to the hind brain where they are integrated and have
major role in short-term regulation of food intake by limiting the
size of meal (33). These types of signal may also affect energy
intake in subsequent meals. However, when the amount of
ingested energy was decreased over long period of time by more
frequent but smaller meals consumed, energy intake remains
constant (34). Thus volume detection does not appear to have a
major role in the long-term regulation of energy homeostasis.
We previously showed that low frequency vagal stimulation
affects short-term volume regulation of food intake and
decreases body weight in rats (11-13). Current data show
decrease in body weight and food intake in long-term VNS rats.
In obesity increase in adipose tissue mass is observed. In
rodents adipose tissue is localized in different depots. A particular
depot of visceral fat is localized in the epididymal fat pad, which is
well delimitated and easy to excise. Epididymal fat pads represent
only a small part of total body weight but previous studies have
shown that the epididymal fat pad weight as a proportion of total
body weight is highly correlated with total body fat in mice and rats
(35-37). The additional reason for including the caudal epididymal
fat pad weight as one of the parameters was that fat pads reflects
total body fat mass and are under hormonal and nervous regulation
(22, 38-41). Body composition was altered by vagus nerve
stimulation (VNS) as expected: epididymal fat pad weight relative
to body weight was significantly lower for rats with VNS
compared with sham operated rats.
To confirm afferent character of VNS, we assessed c-Fos
positive neurons in nodose vagal ganglia. In both, right and left
nodose ganglion we found an increase in cell number
expressed c-Fos protein, which is a reliable marker of neuronal
stimulation (42). Interestingly, we observed increased number
c-Fos positive neurons also in the right nodose ganglion. We
presume, that efferent signal from the left vagus nerve is
transmitted to the right vagus by muscle fibers or by mediators
released by mast cells.
In current study VNS significantly increased mast cells
number compared with sham and control groups in every part of
the gastric wall. The highest values were observed in muscularis
externa. The number of degranulated mast cells was elevated
mainly in the muscularis externa and serosa; however the
percentage of degranulated mast cells remained unchanged. The
role of vagus nervemast cells interaction in the stomach was
previously described (43, 44). There is even morphological
evidence, that mast cells and endings of vagal fibers are colocalized in the stomach wall (44). The vagus nerve and the mast

cells relationship was demonstrated by Stead in a series of

experiments showing that vagal afferents penetrate the small
intestinal mucosa and contact interstitial mucosal mast cells,
moreover, vagotomy causes a reduction in mast cells density,
suggesting a trophic effect, and stimulation of the cervical vagus
causes an increase in histamine and serotonin in mast cells (45).
That is consistent with our data showing an increase in mast cell
number after VNS and demonstrates an important role of mast
cells in the transmission of the satiety signals by afferent vagal
fibers, which needs to be elucidated. Further understanding the
mast cellvagus axis may be important in the development of
treatments for various human diseases, including functional
bowel disorders.
In the present study we examined the interstitial cells of
Cajal localized in myenteric plexus region in the stomach,
duodenum and proximal colon and the myenteric plexus neurons
as well. We did not find quantitative differences caused by VNS.
Previously, we have shown that chronic vagal stimulation
significantly decreased ICC number in the proximal colon in rats
(46). Current data did not confirm those results. We assume that
damage of ICC in proximal colon was caused by higher voltage
(0.55 V) and longer duration of impulse (100 ms) generated by
previously used microchip.
The present study demonstrates that food intake and body
weight gain may be decreased by long-term vagus nerve
stimulation in the high-fat diet induced obesity in rats. These
data support our hypothesis that artificial electrical signals
generated by MC and conducted by vagal afferents as satiety
signals, can modify central regulation of food intake and body
fat content. Peripheral mechanisms, concerning mast cells are
also involved in this process. Vagal neuromodulation seems to
be a promising method for obesity treatment (47-49).
Acknowledgements: The authors would like to thank to Mrs.
Ewa Zieba-Nytko and Mrs. Beata Skowron for their precious
technical help with staining of the specimens.
Conflict of interest statement: None declared.
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R e c e i v e d : October 8, 2008
A c c e p t e d : April 23, 2009
Authors address: Dr Krzysztof Gil, Department of
Pathophysiology, Jagiellonian University Medical College, 18
Czysta Street, 31-121 Krakow, Poland; Phone: + 4812 4210993;
Fax: + 4812 4210993; e-mail: mpgil@cyf-kr.edu.pl