Ischemia is defined as a lack of oxygen in tissues or organs resulting from reduction in arterial or venous blood flow or insufficient perfusion. It causes accumulation of toxic metabolites in tissues or organs, leading to cell death (
Membrane lipids are among the cellular structures that are most sensitive to injury from free radicals (
Moclobemide, the drug used in our study, is an antidepressant drug and a selective inhibitor of the monoamine oxidase-A (MAO-A) (
This experimental study involved a total of 40 albino Wistar female rats weighing between 200 and 215 grams, provided by Ataturk University Medical Experimental Application and Research Centre. The animals were fed and kept at the room temperature (22˚C) in groups. The Rats were divided into the following four groups (10 rats per group): moclobemide (10 mg/kg) + I/R group, moclobemide (20 mg/kg) + I/R group, I/R group, and intact control group without moclobemide treatment. The experiments were performed in accordance with the national guidelines for the use and care of laboratory animals and were approved by the Local Animal Care Committee of Ataturk University.
Of the chemicals used in the study, thiopental sodium was procured from Sigma Co (Munich, Germany) and moclobemide from Deva Drugs (Istanbul, Turkey).
Surgical interventions were performed on the rats in sterile conditions and adequate laboratory environment under intraperitoneal (i.p.) anaesthesia by thiopental sodium (25 mg/kg). Four groups of ten animals were formed: moclobemide groups 1 and 2 were received 10 and 20 mg/kg moclobemide by oral gavage, respectively; the I/R control group and the intact control group were given the vehicle, i.e., distilled water, in the same volume and by the same route as the treatment groups. After thiopental injection, the rats were left for the appropriate time for surgery. When the animals remained motionless on their backs, the surgery was proceeded. At this time, a 2-2.5 cm long vertical incision was made in the lower abdomen of each animal to penetrate the ovaries, then placed a vascular clip in the lower side of the right ovary (in the utero-ovarian contact area) of the rats in the two treatment groups and control group, but not in the healthy animal group. Ischemia took place over three hours, after which the clips were unclamped to provide reperfusion for the next two hours. At the end of the two hours of reperfusion, all rats were killed by high-dose anaesthesia, and their right and left ovaries were taken and subjected to histological and biochemical studies. Observations made about the animals in the treatment groups were compared to those in the I/R control and intact control groups.
All data were subjected to one-way analysis of variance (ANOVA) using Statistical Package for the Social Sciences (SPSS) 18.0 software. The differences among groups were attained using the least significant difference option, and significance was declared at p<0.05. Results were given as mean ± standard deviation.
A sample included both damaged and healthy area of the tissue from the collected ovary was weighed (weight 0.2 mg). The samples were homogenized in ice with 2-ml buffers (consisting of 1.15% potassium chloride solution for malondialdehyde analysis with pH=7.5 and phosphate buffer for the other analyses). Then, they were centrifuged at 4˚C, 10000 rpm for 15 minutes. The supernatant part was used as the analysis sample (
The concentrations of ovarian lipid peroxidation were determined by estimating MDA using the thiobarbituric acid test (
Nitric oxide levels were measured by the Griess reaction (
The amount of Glutathione (GSH) in the total homogenate was measured according to the method of Sedlak and Lindsay, with some modifications (
After the operations, the ovaries were fixed in a 10% neutral buffered formalin solution, and then embedded in paraffin. The serial sections were cut with a microtome at a thickness of 4 μm and stained with haematoxylene-eosin. The histologic sections were examined for the presence of interstitial edema, vascular dilatation, haemorrhage, and polymorphonuclear neutrophilic infiltrations using an Olympus BX-50 microscope, and photographed. The slides were coded, and semiquantative analysis of the ovarian sections was performed without any knowledge of treatment protocol. The observed changes were graded as follows: grade 0, normal; grade I, mild edema, mild vascular congestion, no haemorrhage, and no leukocytic infiltration; grade II, moderate edema, moderate vascular congestion, no haemorrhage, and no leukocytic infiltration; grade III, severe edema, severe vascular congestion, minimal haemorrhage, and minimal leukocytic infiltration; grade IV, severe edema, severe vascular congestion, haemorrhage, and leukocytic infiltration.
The levels of MDA, NOx, and GSH in the ovarian tissues as well as contralateral ovarian tissues in the four experimental groups are separately shown and summarized in
The ovarian tissue of the animals in the intact control group was accepted as being normal; the injury level was estimated as grade 0 (
Comparing MDA, NOx and GSH levels in different dose of Moclobemide (10 mg/kg and 20 mg/kg) and different experimental groups (Intact control, I/R (Ischemia/Reperfusion) group and contralateral group)
|N||MDA (µmol/g protein)||NOx (µmol/g)||GSH (µmol/g protein)|
|10||4.6± 0.36**||10.6 ± 0.23*||2.5± 0.24**|
|10||4.5± 0.21**||9. 5 ± 0.24*||2.7± 0.14**|
|10||4.3± 0.16||8.9± 0.26||2.9± 0.22|
|10||4.2± 0.18||8.6± 0.29||3 ± 0.25|
|10||4.4± 0.24**||8.5± 0.3**||3.1± 0.34**|
|10||10.4 ± 0.24||17.4 ± 1.11||1.03 ± 0.11|
|10||6.5± 0.38||11.7 ± 0.54||1.9± 0.19|
Results were the mean ± standard deviation.
** Refers to p<0.0001, * refers to p<0.001 when compared to I/R control group.
A.The histo-pathological examination of the ovarian tissue of intact control group (Grade 0). B. The histo-pathological examination of the ovarian tissue of Moclobemide 10 mg/kg group (Grade 2). C. The histo-pathological examination of the ovarian tissue of Moclobemide 20 mg/kg group (Grade 1). D . The histo-pathological examination of the ovarian tissue of I/R control group. (Grade 3). E . The histo-pathological examination of the ovarian tissue of I/R control group. (Grade 4). F. The histo-pathological examination of the contralateral ovarian tissue of I/R group (Grade 1). G. The histo-pathological examination of the contralateral ovarian tissue of Moclobemide 10 mg/kg group (Grade 0). H. The histo-pathological examination of the contralateral ovarian tissue of Moclobemide 20 mg/kg group (Grade 0)
This study investigated the levels of MDA, NOx, and GSH in rat ovarian tissue and contralateral ovarian tissue subjected to 3 hours ischemia and 2 hours reperfusion injury followed by the histological evaluation of those tissues. These taking steps were appropriate in inducing damage in ovaries (
NOx production decreased in the ovarian tissue of both moclobemide groups in comparison to the control group after I/R. Endothelial-derived nitric oxide (EDNO) or endothelium-derived relaxing factor (EDRF) may be released into the circulation system in cases of hypoxia, endotoxin discharge, or stress from cellular trauma (
The excessive rise in the intracellular NO concentration initiates the toxic events that lead to cell death (
Cells are not resistant enough to injury by ROS. The harmful effects of the free radicals are not generally seen because of the antioxidant defence mechanisms developed against such effects (
GSH is the most important antioxidant compound. It is synthesized in many tissues from glutamate, cysteine, and glycine. GSH protects the sulphydryl (SH) groups in proteins against oxidation by keeping them in a reduced state, thus preventes the inactivation of functional proteins and enzymes. GSH also plays a role in the detoxification of alien compounds and the membrane transport of amino acids (
While the severe oxidative damage was detected and pathological results showed that in the ovarian tissue of IR control group, the contralateral ovaries showed mild result. In 10 and 20 mg/kg doses, moclobemide repressed oxidative damage in I/R damage, significantly; however, in the contralateral ovaries, it inhibited oxidative damage, totally.