Biological and Histopathological Investigations of Moclobemide on Injured Ovarian Tissue Following Induction of Ischemia-Reperfusion in Rats

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 (17).

The concentrations of ovarian lipid peroxidation were determined by estimating MDA using the thiobarbituric acid test (18). The rat ovaries were rinsed with cold saline, and the corpus mucosa was scraped, weighed, and homogenized in 10 ml of 100 g/l KCl (Potassium chloride). The homogenate (0.5 ml) was added to a solution containing 0.2 ml of 80 g/l sodium lauryl sulfate, 1.5 ml of 200 g/l acetic acid, 1.5 ml of 8 g/l 2-thiobarbiturate, and 0.3 ml distilled water. The mixture was incubated at 98˚C for 1 hour. Upon cooling, 5 ml of n-butanol: pyridine (15: l) was added. The mixture was vortexed for 1 minute and centrifuged for 30 minutes at 4000 rpm. The absorbance of the supernatant was measured at 532 nm. The standard curve was obtained using 1, 1, 3, 3- tetramethoxypropane (19).

Nitric oxide levels were measured by the Griess reaction (20, 21). Nitric oxide measurement is difficult to achieve because of the chemical short half-life. Nitrate and nitrite levels, which are stable end products of nitric oxide metabolism, were; therefore, used. The measurement mixture [100 μl sample, 100 μl nicotinamide adenine dinucleotide phosphate (NADPH) (50 μmol/l), 100 μl flavin adenine dinucleotide (FAD) (5μmol/l), 20 μl nitrate reductase (200 U/l)] was prepared and incubated for 20 minutes at 37˚C. After this, 25 μl ZnSO4 (300 g/l) was added to this mixture, and in this manner, deproteinization occurred. This mixture was centrifuged for 15 minutes at 1000 rpm. The supernatant part was used as measurement assay. One hundred μl Griess reagent and 100 μl of metaphosphoric acid were added to the supernatant, and a deep purple azo compound was obtained. The Griess reagent consists of 0.5 g sulfanilamide, 12.5 g phosphoric acid, and 0.05 g N-(1-napthyl)- ethylenediamine in 500 ml distilled water. The method is based on a two-step process. In the first step, nitrate is converted into nitrite by nitrate reductase. In the second step, nitrite reacts with the Griess reagent, and at the end of this reaction, a deep purple azo compound is appeared. The absorbance of this deep purple azo compound was measured at 540 nm wavelength by photometric measurement. This azo chromophore accurately determines nitrite concentration as a marker of NOx (17).

The amount of Glutathione (GSH) in the total homogenate was measured according to the method of Sedlak and Lindsay, with some modifications (22). The sample was weighed and homogenized in 2 ml of 50 mM Tris-HCl buffer containing 20 mM ethylenediaminetetraacetic acid (EDTA) and 0.2 mM sucrose at pH=7.5. The homogenate was immediately precipitated with 0.1 ml of 25% trichloroacetic acid. The precipitate was removed after centrifugation at 4200 rpm for 40 minutes at 4˚C, and the supernatant was used to determine GSH level. One thousand five hundred μl of measurement buffer (200 mM Tris-HCl buffer containing 0.2 mM EDTA at pH=7.5), 500 μl supernatant, 100 μl 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) (10 mM), and 7900 μl methanol were added to a tube, vortexed, and incubated for 30 min at 37˚C. DTNB was used as a chromogen, and it formed a yellowtcoloured complex with SH groups. The absorbance was measured at 412 nm using a spectrophotometer. The standard curve was obtained using reduced glutathione (17).

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 table 1, which shows the mean values, standard deviations, and p-values of the comparison with the corresponding level in the control group.

The ovarian tissue of the animals in the intact control group was accepted as being normal; the injury level was estimated as grade 0 (Fig 1A). In the group of rats received a moclobemide dose of 10 mg/kg, the injury level was estimated as grade II (Fig 1B), while the injury level was grade I in the moclobemide 20 mg/kg group (Fig 1C). Histological pictures revealed I/R injury was grade III in the four rats (Fig 1D) and was estimated to be grade IV in the remaining six rats (Fig 1E). While in contralateral ovarian tissue of IR control group, the damage severity was determined as grade I (Fig 1F), in contralateral ovarian tissue of 10 and 20 mg/kg moclobemide rat groups, the severity for both was grade 0 (Fig 1G, H).