THE ROLE OF REACTIVE OXYGEN SPECIES AND CAPSAICIN- SENSITIVE SENSORY NERVES IN THE PATHOMECHANISMS OF GASTRIC ULCERS INDUCED BY STRESS

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2003, 54, 3, 423?437
www.jpp.krakow.pl
S. KWIECIEÑ, T. BRZOZOWSKI, P.C. KONTUREK, M.W. PAWLIK,
W.W. PAWLIK, N. KWIECIEÑ, S.J. KONTUREK
THE ROLE OF REACTIVE OXYGEN SPECIES AND CAPSAICIN-
SENSITIVE SENSORY NERVES IN THE PATHOMECHANISMS
OF GASTRIC ULCERS INDUCED BY STRESS
Department of Physiology, Jagiellonian University School of Medicine, Cracow, Poland
Gastric microcirculation plays an important role in the maintenance of the gastric
mucosal barrier and mucosal integrity. Sensory nerves are involved in the regulation
of mucosal blood circulation and mucosal defense. Therefore, the ablation of these
nerves by neurotoxic doses of capsaicin provides the possibility of determination of
their role in gastric mucosal integrity. Stress ulceration represents a serious gastric
lesions. Results of our previous experiments have indicated that water immersion
and restraint stress (WRS) led to increased oxidative metabolism. Ablation of
sensory nerves by high doses of capsaicin retards healing of gastric ulcers, but the
role of reactive oxygen species (ROS) in the healing process has been little studied.
Therefore, the aim of our present investigations was to determine the participation of
ROS in sensory nerve activity during WRS. Experiments were caried out on 90 male
Wistar rats and the area of gastric lesions was measured by planimetry.Colorimetric
assays were used to determine gastric mucosal levels of malondialdehyde (MDA)
and 4-hydroxynonenal (4-HNE), as well as superoxide dismutase (SOD) activity. We
demonstrated that inactivation of sensory nerves resulted in magnification of gastric
mucosal damage induced by the WRS. In this process, oxidative stress, as reflected
by an increase of MDA and 4-HNE tissue concentrations (an index of lipid
peroxidation), as well as decrease of SOD activity, could play an important role.
Aspirin, applied in a low dose, exerts a protective activity, possibly due to its
metabolites, which possess the anti-oxidant and ROS scavanging properties.
Pentoxyfilline-induced gastroprotection and hyperemia depends upon attenuation of
the oxidative stress. This protection and hyperemia were, at least in part, attenuated
by ASA.
K e y w o r d s : water immersion restraint stress, capsaicin, MDA, SOD, aspirin, pentoxyfilline

---

424
INTRODUCTION
The integrity of the gastric mucosa depends on a variety of different factors.
Gastric microcirculation plays an important role in the maintenance of the gastric
mucosal barrier (1,2). Sensory nerves are involved in the regulation of blood
circulation in gastric mucosa (3,4), that is densely innervated by capsaicin-
sensitive afferent neurons, containing and releasing vasodilator peptides, such as
calcitonin gene-related peptide (CGRP) (5). Capsaicin affects these nerves in two
different ways. Low doses of capsaicin result in the stimulation of sensory nerves,
accompanied by the release of CGRP, whereas high doses of capsaicin lead to
ablation, or functional inactivation, of sensory nerves (6). Therefore, the ablation
of sensory nerves by high doses of capsaicin provides the opportunity to
determine their role in the gastric regulatory processes.
Stress ulceration represents a serious complication in patients under stress
conditions. Experimental studies have demonstrated that the exposure of rat
gastric mucosa to stress produces gastric mucosal lesions (7,8). Results of our
previous experiments indicate that water immersion and restraint stress (WRS) is
capable of raising oxidative metabolism, to the level comparable with that
Fig.1. Mean number of gastric lesions and gastric blood flow (GBF) in rats exposed to 3.5 h of
water immersion restraint stress (WRS) without or with capsaicin denervation (CAP-
DENERVATION) and intragastric (i.g.) pretreatment with aspirin (ASA 40 mg/kg i.g.). Results are
mean ± SEM of 8-10 rats. Asterisk (*) indicates significant change as compared with the respective
values obtained in vehicle control group. Cross (+) indicates significant change as compared with
the respective values obtained in rats exposed to 3.5 h of WRS alone.

---

425
observed in ischemia-reperfusion model. Two parameters are usually useful for
the assessment of oxidative metabolism, namely the tissue levels of
malondialdehyde (MDA) plus 4-hydroxynonenal (4-HNE) and the activity of
superoxide dismutase (SOD). Tissue levels of MDA and 4-HNE are used as
indicators of lipid peroxidation. SOD activity reflects the antioxidative properties
of tissues (9).
Functional ablation of sensory nerves by high doses of capsaicin was shown
to aggravate healing of gastric ulcers. However, the role of reactive oxygen
metabolites in the process of WRS-induced gastric damage has been little studied.
Therefore, the aim of our present investigations was to assess the participation of
reactive oxygen species in sensory nerve action during WRS.
MATERIAL AND METHODS
Experiments were carried out on 90 male Wistar rats, weighing about 200 g and fasted for 24 h
before all studies. Studies were approved by the Ethic Committee for Animal Research of
Jagiellonian University College of Medicine.
Fig.2. Mean number of WRS-induced gastric lesions and gastric blood flow (GBF) without or with
capsaicin denervation (cap-denervation) or pentoxyfilline (10 mg/kg i.p.). Results are mean ± SEM
of 8-10 rats. Asterisk (*) indicates significant change as compared with the respective values
obtained in intact gastric mucosa. Cross (+) indicates significant change as compared with the
respective values obtained in rats exposed to 3.5 h of WRS alone. Double cross (++) indicates a
significant change as compared to the value obtained in rats with capsaicin denervation.

---

426
Fig.3. Mean number of gastric lesions and gastric blood flow (GBF) in rats exposed to 3.5 h of WRS
without or with capsaicin denervation (cap-denervation) or pentoxyfilline (10 mg/kg i.p.) applied
alone or given in the combination with aspirin (ASA 40 mg/kg i.g.). Results are mean ± SEM of 8-
10 rats. Asterisk (*) indicates significant change as compared with the respective values obtained
in intact gastric mucosa. Cross (+) indicates significant change as compared with the respective
values obtained in rats exposed to 3.5 h of WRS alone. Double cross (++) indicates a significant
change as compared to respective values obtained in rats with capsaicin denervation.
Production of gastric lesions
The animals were divided into 6 groups. Groups 1-2 underwent deactivation of sensory
nerves by high doses of capsaicin (capsaicin denervation). For this purpose the animals were
pretreated with capsaicin (Sigma Co., St Louis, USA) injected subcutaneously (s.c.) for 3
consecutive days at a dose of 25, 50 and 50 mg/kg (total of 125 mg/kg) about 2 weeks before
the experiment. All injections of capsaicin were performed under ether anesthesia to counteract
the pain reaction and respiratory impairment associated with injection of this agent. To check
the effectiveness of the capsaicin denervation, a drop of 0.1 mg/ml solution of capsaicin was
instilled into the eye of each rat and the protective wiping movement was counted as described
previously (10).
In groups 3-6 the physiological function of sensory nerves was mantained and capsaicin was not
administered to these animals.
In group 1 the ablation of sensory nerves was accompanied by 3.5 h of WRS in temperature
23°C, using the method originally proposed by Takagi et al. (11). In group 2 aspirin was
administered intragastrically (ASA 40 mg/kg i.g.) to capsaicin-deactivated group of animals, 30
minutes prior to 3.5 h of WRS or pentoxyfilline was applied intragastrically (i.g.) in dose 10 mg/kg

---

427
Fig.4. Concentration of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) in the gastric
mucosa of rats exposed to 3.5 h of WRS without or with capsaicin denervation (cap-denervation)
or aspirin (ASA 40 mg/kg i.g.). Results are mean ± SEM of 8-10 rats. Asterisk (*) indicates
significant change as compared with the respective values obtained in vehicle control group. Cross
(+) indicates significant change as compared with the respective values obtained in rats exposed to
3.5 h of WRS alone.
to rats with deactivated sensory nerves. In group 3 intraperitoneal pretreatment (i.p.) with
pentoxyfilline (10 mg/kg), intragastrical administration of aspirin (40 mg/kg i.g.) was applied in
animals before with intact sensory nerves before 3.5 h of WRS. In group 4 healthy intact rats
underwent 3.5 h of WRS with or without pretreatment with aspirin (ASA 40 mg/kg i.g.) or
pentoxyfilline (10 mg/kg i.p.) applied alone or in combination were administered prior to 3.5 h of
WRS. In group 5 pentoxyfilline (10 mg/kg i.p.) applied in the combination with aspirin (40 mg/kg
i.g.) were given to rats exposed to 3.5 h of WRS. In group 6 the animals served as a control group
and did not undergo any procedures.
Determination of gastric blood flow and number of lesions
The evaluation of gastric lesions and gastric blood flow (GBF) was performed 3.5 hours after
the start of WRS. To measure GBF the laser Doppler flowmeter (Laserflo, model BPM 403A, Blood
Perfusion Monitor, Vasamedics, St. Paul, Minnesota, USA) was employed. The animals were
anaesthetized with pentobarbital 50 mg/kg (Biowet, Pu³awy, Poland), then the abdomen was opened
and the stomach was exposed to determine the GBF. The GBF was measured on the anterior and
posterior walls of the stomach not involving gastric lesions. The mean values of three measurements

---

428
Fig. 5. Concentration of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) in the gastric
mucosa of rats exposed to 3.5 h of WRS without or with capsaicin denervation (CAP-
DENERVATION) or pentoxyfilline (10 mg/kg i.p.). Results are mean ± SEM of 8-10 rats. Asterisk
(*) indicates significant change as compared with the respective values obtained in intact control
group. Cross (+) indicates significant change as compared with the respective values obtained in
rats exposed to 3.5 h of WRS alone without pretreatment with pentoxyfilline.
were calculated and expressed as percent change from value recorded in intact mucosa. The number
of gastric lesions were determined by computerized planimetry (Morphomat, Carl Zeiss, Berlin,
Germany), as described previously (2,9).
Measurement of lipid peroxydation
For lipid peroxydation in the investigated groups, the determination of the levels of
malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) was carried out and their levels were used
as indicators of lipid peroxidation. The procedure of MDA and 4-HNE determination included
excision of about 600 mg of gastric mucosa from intact rats and those exposed to WRS with or
without pretreatment with ASA, pentoxyfilline and their combination. Then 20 ml 0.5 M BHT
(butylated hydroxytoluene) was added in order to prevent sample oxidation. This sample was
subsequently homogenized in 20 mM Tris for 15 s. in pH=7.4. Then the homogenate was centrifuged
(3000 g at 4°C for 10 min). Obtained clear supernatant was stored at -80°C prior to testing.
The colorimetric assay for lipid peroxidation (Bioxytech LPO-586, Oxis, Portland, USA) was
used to determine of MDA and 4-HNE tissue concentrations. This assay is based on the reaction of
a chromogenic reagent N-methyl-2-phenylindole with MDA and 4-HNE at 45°C. This reaction

---

429
Fig.6. Concentration of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) in the gastric
mucosa of rats exposed to 3.5 h of WRS without or with capsaicin denervation (cap-denervation)
or pentoxyfilline (10 mg/kg i.p.) combined with aspirin (ASA 40 mg/kg i.g.). Results are mean ±
SEM of 8-10 rats. Asterisk (*) indicates significant change as compared with the respective values
obtained in vehicle control group. Cross (+) indicates significant change as compared with the
respective values obtained in rats exposed to 3.5 h of WRS alone.
yields a stable chromophore with maximal absorbance at 586 nm. This absorbance was measured
by spectrophotometer Marcel s300 (Warsaw, Poland). Results were expressed as nanomoles per
gram of tissue (nmol/g) (9).
Determination of SOD activity
To determine the activity of superoxide dismutase (SOD), a sample of gastric mucosa was
obtained, as described above. The colorimetric assay for assessment of SOD activity (Bioxytech SOD-
525, Oxis, Portland, USA) was used. This method is based on the SOD-mediated increase in the rate
of autooxidation of tetrahydrobenzofluorene in aqueous alkaline solution to yield a chromophore with
maximum absorbance at 525 nm. This absorbance was measured by spectrophotometer Marcel s300
(Warsaw, Poland) and the results were expressed as units per gram of gastric tissue (U/g) (9).
Statistical analysis
Results are expressed as means ±SEM. Statistical analysis was done using nonparametric
Mann-Whitney test. Differences with p<0.05 were considered significant.

---

430
Fig.7. Superoxide dismutase (SOD) activity in the gastric mucosa of rats exposed to 3.5 h of WRS
without or with capsaicin denervation (cap-denervation) or ASA 40 mg/kg i.g.. Results are mean ±
SEM of 8-10 rats. Asterisk (*) indicates significant change as compared with the respective values
obtained in vehicle control group.
RESULTS
Gastric lesions and gastric blood flow
Figs 1 and 2 show mean ulcer number in the WRS model as well as
accompaning alterations in the GBF. Intact mucosa (control) did not show any
macroscopic lesions and the GBF in this intact mucosa averaged 45±5
ml/min/100 g of tissue, being accepted as the control value (100%). Following 3.5
h of WRS numerous gastric mucosal lesions were produced (mean lesion number
was about 24±1) and GBF was reduced to 56±4 % of control value. Ablation of
sensory nerves by neurotoxic doses of capsaicin resulted in a significant increase
of number of gastric lesion and this effect was accompanied by a decrease of GBF
as compared with that obtained in rats with 3.5 h of WRS alone. Pretreatment
with ASA (40 mg/kg i.g.) tended to increase the number of gastric lesions and
decreased the GBF as compared to the values of lesion number and GBF recorded
after 3.5 h of WRS. Significant changes of these parameters were observed in a
group of animals with capsaicin deactivation and ASA application before 3.5 h of
WRS and these changes were significantly more pronounced as compared with
those in group of capsaicin denervated rats without pretreatment with ASA (Fig.

---

431
Fig.8. Superoxide dismutase (SOD) activity in the gastric mucosa of rats exposed to 3.5 h of WRS
without or with capsaicin denervation (cap-denervation) or pentoxyfilline (10 mg/kg i.p.) applied
alone or in the combination with capsaicin denervation. Results are mean ± SEM of 8-10 rats.
Asterisk (*) indicates significant change as compared with the respective values obtained in non
stressed animals. Cross (+) indicates significant change as compared with the respective values
obtained in rats exposed to 3.5 h of WRS alone.
1). Administration of pentoxyfilline (10 mg/kg i.p.) produced a significant
decrease of gastric lesion number and an increase of GBF, as compared with the
3.5 h of WRS group. The changes occurred in both, the healthy animals and those
with functional ablation of sensory nerves. However, the augmentation of these
changes in group of rats with intact sensory nerves was observed (Fig. 2).
Combination of pentoxyfilline ( 10 mg/kg i.p.) with ASA (40 mg/kg i.g.)
decreased significantly the gastric lesion number and raised significantly the GBF
as compared with WRS group of animals with intact sensory fibers treated with
ASA. The combined pretreatment with pentoxyfilline and ASA in capsaicin-
denervated animals resulted in a significant increase of gastric lesion number as
compared with those in rats exposed to WRS (Fig. 3).
Lipid peroxidation products
Concentration of MDA and 4-HNE in intact mucosa (control) was very low,
almost at the level of analytical limit of detection (5,0±0,1 nmol/g). After 3.5 h of

---

432
Fig.9. Superoxide dismutase (SOD) activity in the gastric mucosa of rats exposed to 3.5 h of WRS
without or with capsaicin denervation (cap-denervation) or pentoxyfilline (10 mg/kg i.p.) applied
without or with the combination with aspirin (ASA 40 mg/kg i.g.). Results are mean ± SEM of 8-10
rats. Asterisk (*) indicates significant change as compared with the respective values obtained in intact
gastric mucosa. Cross (+) indicates significant change as compared with the respective values obtained
in rats exposed to 3.5 h of WRS alone without pretreatment with pentoxyfilline.
WRS the level of MDA and 4-HNE increased by about three times, reaching the
value of 15.8±1.0 nmol/g. Capsaicin deactivation caused larger increment of MDA
and 4-HNE concentration up to the level of 17.4 ± 0.2 nmol/ g of tissue. Application
of aspirin (ASA 40 mg/kg i.g.) resulted in a significant decrease of lipid
peroxidation products. ASA in the same dose given to rats with ablation of sensory
nerves established the concentration of MDA and 4-HNE at the level similar to that
observed in the 3.5 h of WRS group (Fig. 4). Pretreatment with pentoxyfilline (10
mg/kg i.p.) led to a significant fall in lipid peroxidation in rats with or without
capsaicin denervation and further exposed to WRS (Fig. 5). The similar reduction
in MDA plus 4-HNE levels were observed in non-denervated and capsaicin-
denervated rats with the combination of pentoxyfilline and ASA (Fig. 6).
SOD activity
As shown in Fig. 7, SOD activity averaged 352 ± 30 U/g of tissue in intact
gastric mucosa. Following exposure of rats to WRS, a significant decrease of

---