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Study of the Mast Cells Distribution and Heterogeneity In Study of the Mast Cells Distribution and Heterogeneity in Experimentally Induced Cystic Ovaries in Rats Razi, Mazdak1 Malekinejad, Hassan2 Nagafi, Gholam-Reza1 Najafpour, Ali-Reza3 Delkhosh, Fatemeh1 Sheykhzadeh, Sanaz1 Ghodraty, Sommayeh1 1Department of Histology and Embryology 2 Department of Pharmacology and Toxicology, Faculty of veterinary medicine, Urmia University, P. O. Box: 1177, Urmia, Iran 3Department of Clinical Science, Faculty of Veterinary Medicine, Azad University, Urmia Branch, Urmia, Iran Corresponding address: [email protected] KEY WORDS: Cystic ovary; mast cells; to the blood vessels in endometrium of the cortex; endometrium; perimetrium uterine and uterine horns. Mast cells were ABSTRACT located in the perimetrium around the blood vessels in the test groups. However, no mast To determine the effect of high serum cell observed in both theca interna and theca concentration of estradiol on mast cell externa of the follicles in control group. The distribution and heterogeneity in experimen- tallyinduced cystic ovary (CO), 56 mature mast cells distribution in the helium of the female rats were subjected to study. Follow- control group was significantly (P≤0.01) less ing CO induction by unilaterally ligation of than that test group. Moreover, no mast cell the ovarian artery, all rats were euthanized demonstrated in the cortex of the control on days 5, 10, 20, 30, 40, 50, and 60, and the group. Hormonal analysis showed that there ovaries were collected. The blood samples are significant decline in the progesterone were collected and serum samples were pre- and FSH concentrations and increase in the pared. The histological sections were stained estrogen and LH levels of the serum in CO with toluidine blue in order to determine the group. This finding confirmed the hormonal mast cell distribution. The observation dem- changes in CO condition and may suggest onstrated that in test group mast cells were that mast cells are involved in CO induction. found in theca externa, theca interna, and It could be also concluded that there should cortex of the cystic ovaries. Observations be a sort of mutual effects on hormonal also showed that mast cells were extensively changes and the mast cells distribution in located in the helium of the treated ovaries. CO cases. Moreover, it might be suggested These cells were in the group form closed that mast cell number increasing in cortex of 124 Vol. 8, No. 2, 2010 • Intern J Appl Res Vet Med. the ovary could be counted as a biomarker system of female rats in both normal and CO indicating a cystic condition. condition to give a new approach in order INTRODUCTION to evaluate CO condition based on mast cell distribution, which in turn related closely to Mast cells, basophiles, platelets, and estrogen level in serum. Moreover, serum endothelial cells are well-known source hormonal and glucose levels also were of histamine in the ovary.1,2 Histamine has measured. been reported to regulate blood flow and vascular permeability in the ovarian tissue, MATERIALS AND METHODS along with an important role in the follicu- Fifty six female 90-days-old rats (Sprague- lar development.2,4 Previous in vitro and in Dawley) were used. The average weight vivo studies have hypothesized that there of the rats was 180 ± 10.1 g. Animals were is an association between the mast cells kept at 22 ± 2° C temperature and 12/12 degranulation, and consequently, activation hours light /dark conditions. They were fed and angiogenesis, and neovascularization.5,8 from standard rat plate and tap water. The This hypothesis is partially supported by the rats were assigned into two groups, ie, the close anatomical association between mast treatment (n = 48) and control group (n = 8), cells, the vasculature and the recruitment which had menstrual cycle of normal length of these cells during tumor growth, wound (4-5 days). The ethical concerns of current healing, and inflammation processes.9 It has study were approved by the institutional been clearly established during the last years animal care and use committee of Urmia that mast cells of the female reproductive University, which is in accordance with system are subjected to cyclical changes NRC Guide for the Care and Use of Labora- during the estrus cycle. For example, in tory Animals. the hamster ovary, mast cells mediate the The main method to determine the vascular response to the gonadotropin surge ovulation day was based on vaginal smear in proestrus stage.2 The Cyclic change in the that was conducted on 200 rats, in order number and the degranulation pattern of the to select 56 rats which that exhibited the cells during the estrus cycle have also been characteristics of ovulation day in light mi- reported in the reproductive system.10,11 It is croscopic analyses of smear slides, as well generally accepted that estradiol influence known method of the ovulation day diagno- mast cells density and more likely function, sis in rats or other rodents.19 The test group as well.12,14 was anesthetized by using the combination The CO is a common problem in the of ketamin HCl 5% (Iran- Razak), 40 mg/kg reproduction, both in veterinary and medical and xylazine 2% (Germany) 5mg/kg intra- fields. It is associated with insulin resistant, peritoneally. Then, the rats were laparato- hyperinsulinaemia, glucose intolerance, obe- mized from 1/3 caudal end of middle line sity, and altered lipid profile.15,17 High serum and when the site of ligation was exposed, concentrations of androgenic hormones and unilaterally the right ovarian artery was li- increasing of estradiol may be encountered gated by 0-2 silk and blocked completely.20 in these patients.18 The CO could be resulted Also, the rats randomly selected to make a from abnormal hormonal reaching to the sham control group, which surgery process- ovary, as well.7,14,15 It is well known that es were performed on them without ligation reproductive hormones and in particular of any artery or arteriole. On days 5, 10, 20, estradiol has influential effects on mast cells. 30, 40, 50, and 60, the rats were euthanized There is, however, a lack of knowledge as to by using CO2 gas in a special device and how CO condition can affect the mast cells both ovaries were dissected out and cleaned histological distribution and hetrogenesity. by using chilled saline normal. Hence this study carried out to examine the Histomorphological Analysis distribution of mast cells on the reproductive The ovaries were fixed in the isotonic form- Intern J Appl Res Vet Med • Vol. 8, No. 2, 2010. 125 aldehyde acetic solution (IFAA, pH 2.8). antibody position on micro-well plate. The Samples were processed through paraffin limits of detection (LOD) were 0.25mIU/ embedding and cut with rotary microtome ml, 0.3 mIU/ml, 4.5 pg/ml and 0.05 ng/ml and stained with toluidine-blue technique. for FSH, LH, estradiol and progesterone in Distribution of the mast cells was studied in ELISA test, respectively. The intra-assay the different parts of the collected organs. coefficients variance for FSH, LH, estradiol From the above mentioned section series, and progesterone were, 3.56 (for 10 times), 10 sections (each 6 µm thickness) were 2.64 (for 10times), 5.9 (for 10 times) and 4.8 randomly selected as semi- serially. Tissue (for 10 times) respectively and inter-assay samples from rats’ intestine were used as the coefficientsvariances of 8.98 (for 10 times), control for the mucosal mast cells (MMC), 7.52 (for 10 times), 5.9 (for 10 times) and while tissue from the skin of rats was used 9.9 (for 10times) were found for FSH, LH, as the control for the connective tissue mast estradiol and progesterone, respectively. cells (CTMC). Furthermore, the blood glucose was mea- Cell Count sured by an Oncull now set with test strioe 20 A hundred – square ocular micrometer was glucose. In order to measure the blood used for cell count to determine the mast cell glucose, the blood samples were sampled distribution in the preparations stained with from caudal artery and immediately put on toluidine-blue. Mast cells within ocular mi- set and measured. crometer were counted in high power field Statistical Analyses (400X).19 The cells were counted within All results are presented as means ± SD. 18 areas per tissue, selected from different Differences between mast cell numbers, regions of the ovary (cortex, medulla) and hormonal concentrations, and glucose level epithelium, tunica sub mucosa, tunica mas- in various days of treatment were analyzed cularis in the uterus and the uterine horns with a two-way ANOVA followed by a of the test group, and different regions from Bonferroni test, using GraphPad Prism 4.00, the ovaries of control group. Thus, average GraphPad Software. P < 0.05 was consid- mast cell numbers within the area covered ered significant. with 100 square ocular micrometers was RESULTS determined. The area of 100 square ocular micrometers was calculated by means of Mast cells in sections stained with toluidine micrometrical lam by 40 objective en- blue had various size and appearance. They largements. Then, the mast cell density in were oval, flat, or in the form of spindle each site (proposed tissues) was found and shape. Cytoplasms of the mast cells taken recorded as mast cell numbers /mm.2 almost from all samples and stained with metachromatic dye were homogenous. In the Serum Sampling and Hormonal Analysis present, study mast cells were observed in In order to avoid any effect of surgical the theca externa and theca interna (Fig- procedures on hormonal situ, 5 days after ure 1) of the cystic follicles. Observations surgery, the blood samples from correspond- demonstrated that mast cells were abounded ing animals were collected directly from the in the cortex of the ovary that had cystic fol- heart and the serum samples separated by licle (Figure 2).
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