Download Sharefah Abdelsalam Morsy Saad_sherifa

Comparative study on the effect of N-acetyl-cysteine versus
sulpiride on experimentally induced stress in ovary of albino rats: a
histological, immunohistochemical and biochemical study
Eman M. Faruk, Sherifa Abd Elsalam Morsy
Department of Histology and Cell Biology,
Faculty of Medicine, Benha University, Benha,
Egypt
Correspondence to Eman M. Faruk, MD,
Department of Histology and Cell Biology,
Faculty of Medicine, Benha University, Benha,
Egypt
Tel: +20 133 221 937;
e-mail: [email protected]
Received 2 October 2014
Accepted 22 October 2015
The Egyptian Journal of Histology
2015, 38: 0000-0000
00 (0000-2015)
commonly used as a safe mucolytic drug. Sulpiride is a selective dopamine
antagonist with antipsychotic and antidepressant activity.
Aim
As the mechanism of stress is not known the aim of this work was to evaluate
the histological and immunohistochemical changes that might occur in rat
ovaries after stress exposure and the possible protective effect of sulpiride and
NAC.
Materials and methods
Thirty-six rats were divided into four groups: group I was the control group
(n=6); group ΙΙ (n=6) was injected with sulpiride at 0.28 mg/kg body weight/day
for 1 month; group ΙIΙ (n=6) was injected with NAC at 150 mg/kg body weight
intraperitoneally for 5 days; group ΙV (the stress group) (n=18) was divided into
three equal subgroups: subgroup ΙVa, in which rats were exposed to crowding
only for 1 month, subgroup ΙVb, in which rats were exposed to crowding along
with sulpiride treatment for 1 month, and subgroup ΙVc, in which rats were
exposed to crowding for 1 month along with NAC treatment for 5 days.
Results
Introduction
Chronic stress is known to produce
significant behavioral,
endocrinological, and neurobiological
changes. N-acetyl-cysteine (NAC) is
Stress resulted in a significant increase in luteinizing hormone, follicle stimulating hormone, and prolactin levels and a significant
decrease in progesterone and estrogen levels. Sulpiride treatment resulted in a significant decrease in progesterone and
estrogen levels and increase in prolactin, with decreased reaction for estrogen receptor. NAC led to a significant increase in the
level of estrogen and progesterone, with no effects on other parameters, and increased reaction for estrogen receptor. The ovary
of both the stress and the sulpiride group revealed cystically dilated follicles lined with granulose cells separated by a proliferation
of ovarian stroma (luteinized stromal cells). NAC-treated group showed decreased number of atretic follicles and thickness of
theca layer.
Conclusion
NAC had a protective role against the induced damage but sulpiride did not counteract the impairing effects of stress. It is
beneficial to use NAC in people exposed to stress.
Keywords:
crowding, N-acetyl-cysteine, sulpiride
Egypt J Histol 38: 000-000
© 2015 The Egyptian Journal of Histology
1110-0559
Stress has become an integral part of human life worldwide and every individual is likely to face stressful situations in everyday
life that seriously disturb physiological and psychological homeostasis [1,2]. Chronic stress represents a state of dyshomeostasis
resulting from the inability of the adaptive stress response to cope with the intensity or the frequency of various stressors [3,4].
Reproductive functions are suppressed under various stress conditions such as restraint, strenuous exercise, malnutrition,
infection, and surgical trauma [5]. Prolonged or chronic stress causes anovulation, which results in infertility because of
suppression of gonadotropic hormones [6]. Crowding stress is a type of psychosocial stress induced by an increased density in
population. Population density may be raised either by increasing the number of species
EHX384-12
Original article
living in the same area and/or by reducing their living space. Crowding stress induces complex changes at the
behavioral, physiological, and molecular levels [7,8]. Sulpiride is the most common drug used to manage stress [9].
Containment of the damage from stress exposure may be achieved through administration of synthetic antioxidant
compounds such as N-acetyl-cysteine (NAC). NAC comes from the amino acid l-cysteine. Amino acids are the
building blocks of proteins and have many uses such as in medicine and it is the acetylated precursor of both amino
acid l-cysteine and reduced glutathione [10,11]. Moreover, NAC is a clinically proven safe dietary supplement that
may be used in radiation therapy to prevent radiation-mediated genotoxicity [10]. Thus, the present study dealt with
the possible protective effect of N-acetyl-cysteine and sulpiride against stress in female albino rats from the
histological, immunohistochemical, and biochemical point of view.
Materials and methods
Experimental animals and grouping
Thirty-six female albino rats weighing 200±30 g were obtained from the animal house, Moshtohor Faculty of
Veterinary Medicine, Benha University. They were given a balanced diet with free access to water. All animal
procedures were performed according to approved protocols and in accordance with the recommendations for the
proper care and use of laboratory animals. The animals were kept under observation for 1 week before the beginning
of the experiment to acclimatize. They were divided into four groups:
(1) Group I (n=6): this group consisted of normal rats that served as negative controls injected with saline for 1
month and kept in a cage measuring (30×30×30 cm).
(2) Group II (n=6): this group consisted of rats injected with sulpiride at 0.28 mg/kg body weight/day for 1 month.
(3) Group III (n=6): this group consisted of rats injected with NAC at 150 mg/kg body weight intraperitoneally for 5
days.
(4) Group IV (the stress group): this group was divided into three equal subgroups (n=18) – subgroup IVa, in which
rats were exposed to crowding without treatment for 1 month; subgroup IVb, in which rats were exposed to
crowding along with sulpiride treatment for 1 month; and subgroup IVc, in which rats were exposed to
crowding for 1 month along with NAC treatment for 5 days.
Application of stresses
Application of crowding
Crowding stress was induced by placing six rats from group IV in a cage measuring 20×20×20 cm [12].
Drugs
N-acetyl-cysteine administration
NAC was injected at in a dose of 150 mg/kg body weight intraperitoneally. The NAC solutions were present at 0.5%
(100 mg/kg) (Sigma Company, Cairo, Egypt) [13].
Sulpiride administration
Sulpiride, dissolved in normal saline, was adiministered orally by means of a gastric tube at a dose of 0.28 mg/kg
body weight/day for 1 month. The dose for the rat was calculated according to Paget’s formula on the basis of the
human dose (Sanofi Company, Cairo, Egypt) [14].
Experimental parameters
Biochemical examination
At the end of experiment, the animals were killed by an overdose of ether and blood samples were collected from
their tail veins. The samples were collected in clean, dry graduated centrifuge tubes and left for 10 min to clot, and
then centrifuged at 5000 rpm for 15 min. Using a pasture pipette about half of the supernatant serum was transferred
into clean dry tubes for subsequent tests. The serum was separated and kept at −20°C until analysis. Serum follicle
stimulating hormone (FSH), luteinizing hormone (LH), serum prolactin, serum progesterone, and serum estradiol
(E2) were measured according to the method described by Tietz [15].
Histological studies
Rats from the control, stressed, and treated groups were sacrificed at around 4 weeks in either proestrus or diestrous
stage, and small specimens of their ovary were taken for histological examination. These specimens were fixed in
10% neutral buffered formol solution for histological and immunochemical studies. Paraffin sections were prepared
at 5 μm thickness and stained with H&E [16].
Immunohistochemical examination
Sections were preincubated for 15 min in a 1% solution of H 2O2 before being incubated overnight at 25°C in 0.3%
Triton X-100 and 0.5 mg/ml bovine serum albumin with the primary Hsp90 polyclonal antibody at 1 : 100 dilution
(Assay Designs and Stressgen; Enzo Life Sciences, Exeter, UK). The sections were incubated for 120 min with antirabbit secondary antibodies (1 : 200 each; Vector Laboratories Inc., Burlingame, California, USA) and then treated
with an avidin–biotin–peroxidase complex for 1 h at room temperature. After several rinses, the sections were
mounted onto slides and counterstained with hematoxylin, dehydrated, and then coverslipped.
To examine the cellular expression of Estrogen receptor ER in the ovaries after treatment, the sections were
processed for immunohistochemical analysis. The primary antibody used wad anti-ER [17,18].
Statistical analysis
Experimental data were analyzed by one-way analysis of variance using SPSS (version 16.0; SPSS Inc., Chicago,
Illinois, USA) software. Significant differences among the treatments were compared by means of the T test. P
values less than 0.05 were considered significant, whereas P values less than 0.01 were considered highly
significant.
Egyptian Journal of Histology 2015, Vol 38 No 4
23