European Journal of Obstetrics & Gynecology and Reproductive Biology 173 (2014) 71–76
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Montelukast prevents ischaemia/reperfusion-induced ovarian damage in rats ¨ zgu¨r Yeniel a, F. Oltulu c, A. Yavas¸og˘lu c, A. Akdemir a, O. Erbas¸ b, M. Ergenog˘lu a, A. O b, D. Taskiran * a b c
Department of Obstetrics and Gynaecology, Ege University School of Medicine, Izmir, Turkey Department of Physiology, Ege University School of Medicine, Izmir, Turkey Department of Histology and Embryology, Ege University School of Medicine, Izmir, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 January 2013 Received in revised form 4 August 2013 Accepted 25 November 2013
Objective: To investigate the efficacy of montelukast for prevention of ischaemia/reperfusion (I/R) injury in rat ovary. Study design: Twenty-four female adult rats were included in the study. I/R injury was induced by CO2 pneumoperitoneum in a laparoscopic rat model. The rats were divided at random into three groups: the sham group was subjected to catheter insertion but was not subjected to pneumoperitoneum; the saline group was subjected to 60 min of pneumoperitoneum and 30 min of reperfusion, with 1 mg/kg physiological saline administered 10 min before pneumoperitoneum; and the montelukast group was subjected to 60 min of pneumoperitoneum and 30 min of reperfusion, with 20 mg/kg montelukast administered 10 min before pneumoperitoneum. Damage to ovarian tissue was scored by histopathological evaluation. Caspase-3 expression was determined immunohistochemically. Ovarian tissue levels of malondialdehyde and glutathione, and plasma total antioxidant capacity were measured biochemically. Results: In comparison with the sham group, ovarian sections in the montelukast group had higher scores for follicular degeneration and oedema (p < 0.001). Montelukast treatment prevented tissue damage in ovaries, and this result was significant. Caspase-3 expression was only observed in ovarian surface epithelium in the saline and montelukast groups. However, the mean caspase-3 expression score was higher in the saline group than the montelukast group (p < 0.001). Tissue levels of malondialdehyde were higher in the montelukast group than the sham group, but plasma total antioxidant capacity and tissue levels of glutathione were significantly lower. Pretreatment with montelukast reduced lipid peroxidation (p < 0.005) and improved antioxidant status in rats (p < 0.001). Conclusion: Montelukast is effective for the prevention of I/R-induced damage in rat ovary. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Montelukast CysLT1 receptor CO2 pneumoperitoneum Laparoscopy Reactive oxygen species Ischaemia/reperfusion injury
1. Introduction Over the last two decades, laparoscopy has become a fundamental technique in gynaecological surgery. The laparoscopic approach has several advantages over open surgery in terms of mortality, postoperative pain, patient mobilization and recovery, and cosmetic results. Although early use was restricted to diagnostic laparoscopy and tubal sterilization, the laparoscopic technique is now recommended for surgical treatment of ectopic pregnancy, and is also applied in fields that have traditionally used an open approach (e.g. myomectomy, hysterectomy,
* Corresponding author at: Department of Physiology, Ege University School of Medicine, 35100 Izmir, Turkey. Tel.: +90 232 3901800; fax: +90 232 3746597. E-mail address: [email protected] (D. Taskiran). 0301-2115/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejogrb.2013.11.021
lymphadenectomy and pelvic floor repair). Despite its benefits, however, CO2 pneumoperitoneum is not free from adverse events, even at low pressures. Experimental and clinical studies have shown that increased intra-abdominal pressure (IAP) associated with pneumoperitoneum reduces blood flow to intra-abdominal organs because of high venous resistance during laparoscopic procedures [1]. This process, known as ischaemia, leads to the production of free radicals [1–5]. Although desufflation of the abdominal cavity at the end of a laparoscopic procedure reduces the IAP and increases the perfusion of intra-abdominal organs, this does not result in the absence of oxidative stress. The ischaemia/ reperfusion (I/R) model leads to the release of various free radicals, which are the most important mediators of oxidative tissue injury and consequential organ dysfunction, especially in the splanchnic organs [2–8]. Recently, both oxidative tissue injury and consequential dysfunction have been shown in ovarian tissue [9].
72
A. Akdemir et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 173 (2014) 71–76
An excessive inflammatory response is clearly recognized as a key mechanism of injury during reperfusion. Various chemokines, lipid mediators (e.g. cysteinyl leukotrienes (CysLTs)) and the 5lipoxygenase metabolites of arachidonic acid are potent inflammatory mediators that have been associated with I/R-induced tissue injury [10–12]. Given these findings, it has been hypothesized that pharmacological strategies that limit neutrophil recruitment may also limit the damage induced by reperfusion. Therefore, several anti-inflammatory and antioxidant agents have been used to prevent I/R injury in tissues [13–16]. Montelukast, a selective reversible CysLT1 receptor antagonist, is a new anti-inflammatory agent with antioxidant properties [17]. Montelukast is used for the treatment of asthma as it reduces eosinophilic inflammation in the airways [18]. Additionally, montelukast has been shown to not only reduce I/R-induced oxidative damage in kidney and liver through its anti-inflammatory and antioxidant properties, but also to ameliorate multi-organ damage due to burns and sepsis by a neutrophil-dependent mechanism [15,19–22]. As such, the aim of the present study was to identify the effects of montelukast pretreatment in preventing I/R injury in rat ovary caused by reactive oxygen species (ROS) generated during CO2 pneumoperitoneum. 2. Materials and methods Twenty-four mature, female, non-pregnant Sprague Dawley rats weighing between 200 and 250 g were used in this experimental study. The animals were caged in a controlled environment at 22 8C with 12-h light/dark cycles. Standard rat feed and reverse-osmosis-purified water were provided ad libitum. The study protocol was approved by the University Animal Care and Ethics Committee of Ege University. All rats were anaesthetized by intraperitoneal administration of 50 mg/kg ketamine hydrochloric acid (Ketalar; Eczacibasi WarnerLambert Ilac Sanayi, Levent, Istanbul, Turkey) and 7 mg/kg xylazine hydrochloric acid (Rompun; Bayer Sisli, Istanbul, Turkey). Before pneumoperitoneum, rats were immobilized on a standard rat surgical board, abdominal skin was shaved, antisepsis was obtained with 10% povidone iodine solution, and body temperature was kept constant at 37 8C with a heating blanket. An 18-G Abbocath catheter was inserted into the abdominal cavity below the xiphoid process and connected to an insufflator tube. CO2 was then insufflated to a pressure of 12 mmHg using an abdominal CO2 insufflator (Karl Storz GmbH, Tutlingen, Germany). The rats were allocated at random to one of three groups (eight rats in each group) on the day of the experiment. All investigators responsible for the biochemical and histopathological studies were blinded to the randomization until the end of the study. The sham group was subjected to catheter insertion but was not subjected to pneumoperitoneum; the saline group was subjected to 60 min of pneumoperitoneum and 30 min of reperfusion, with 1 mg/kg of physiological saline (0.9% sodium chloride) administered by oral gavage 10 min before pneumoperitoneum; and the montelukast group was subjected to 60 min of pneumoperitoneum and 30 min of reperfusion, with 20 mg/kg montelukast administered by oral gavage 10 min before pneumoperitoneum. After 60 min of pneumoperitoneum, the CO2 was desufflated and IAP was normalized for 30 min. Immediately after the reperfusion period in the saline and montelukast groups, and 90 min after catheter insertion in the sham group, a midline abdominal incision was made to localize the ovaries. Both ovaries were excised for histopathological (left ovary) and biochemical (right ovary) evaluation. Following rapid fixation in a formaldehyde (10%) solution, ovaries were dehydrated in an ascending alcohol series, cleared in
xylene and embedded in paraffin. Tissue sections (4-mm thick) were prepared and stained with haematoxylin and eosin for general morphological observation. All sections were studied and photographed with an Olympus C-5050 digital camera mounted on an Olympus BX51 microscope (Olympus Corp., Tokyo, Japan). Follicular degeneration and interstitial oedema were scored from 0 to 3 according to injury severity, where 0 represented no pathological findings, and 1, 2 and 3 represented pathological findings of 66% of the ovarian section, respectively [9]. Apoptosis was evaluated through immunohistochemical analysis of caspase-3. Morphological analyses of both investigations were assessed using a computerized image analysis system (Image-Pro Express 1.4.5, Media Cybernetics, Inc., USA) on 10 microscopic fields per section, examined at magnification of 20, with the observer blind to the study group. For biochemical analysis, ovaries were homogenized in ice-cold 150 mM KCl and centrifuged at 5000 g for 10 min. The supernatants were analyzed for malondialdehyde (MDA; lipid peroxidation), glutathione (GSH) and protein levels. Lipid peroxidation was determined in tissue samples by measuring MDA levels as thiobarbituric acid reactive substances [23]. GSH content in tissue samples was measured spectrophotometrically according to Ellman’s method [24]. Total protein concentration in tissue samples was determined according to Bradford’s method [25]. Plasma total antioxidant capacity (TAC) was measured with the ferric reducing antioxidant power assay according to Benzie and Strain [26]. Data analyses were performed using Statistical Package for the Social Sciences Version 15.0 (IBM Corp., New York, USA). All quantitative data were analyzed using the Mann–Whitney U-test. Results are presented as mean standard error of mean. p-Values 0.05 were regarded as statistically significant. 3. Results Fig. 1 depicts the histopathological examination scores for the sham, saline and montelukast groups. The sham group demonstrated normal ovarian morphology, such as normal stroma, the presence of all types of follicles in the cortex (primordial, primary, secondary and tertiary), and the presence of capillary vessels in the medullary part of the ovaries. In comparison with the sham group, however, ovarian sections in the saline group had higher scores for follicular degeneration and oedema (p < 0.001). Montelukast treatment prevented the degenerative changes in ovaries, and this result was significant. There were no differences between the sham and montelukast groups (Fig. 2). Caspase-3 expression was only observed in the ovarian surface epithelium in the saline and montelukast groups (Fig. 3). However, the mean caspase-3 expression score was significantly higher in the saline group than the montelukast group (Fig. 1, p < 0.001).
Fig. 1. Comparison of histopathological examination scores and caspase-3 immunoreactivity in ovarian sections. *Different from other groups (p < 0.001).
A. Akdemir et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 173 (2014) 71–76
73
Fig. 2. Histopathological alterations in ovarian sections. (A,B) No pathological changes in sham group. (C,D) Severe follicular degeneration (fd) and oedema (*) in saline group. (E,F) Oedema (*) in montelukast group. sf, secondary follicle; tf, tertiary follicle; s, cortical and medullar stroma; CL, corpus luteum. Haematoxylin and eosin staining. Original magnification 10 for (A), (C) and (D) and 20 for (B), (D) and (F).
As summarized in Table 1, the mean tissue MDA value for the saline group was significantly higher than for the sham group (0.34 0.03 nmol/mg protein vs 0.06 0.01 nmol/mg protein; p < 0.001). Administration of 20 mg/kg montelukast in rats reduced lipid peroxidation significantly compared with the saline group (0.09 0.05 nmol/mg protein; p < 0.005). GSH levels were found to be significantly lower in the saline group than the sham group (5.48 0.28 nmol/mg protein vs 63.64 3.22 nmol/mg protein; p < 0.001). In comparison with the saline group, GSH levels were higher in the montelukast group (8.9 0.18 nmol/mg protein; p < 0.001). Plasma TAC was also significantly lower in the saline group compared with the sham group (80.81 9.1 mM vs 124.76 10.5 mM; p < 0.05). Administration of 20 mg/kg montelukast in rats induced higher plasma TAC compared with the saline group, but the difference was not statistically significant (94.66 20.81 mM; p = 0.26). 4. Comments Laparoscopy is an essential diagnostic and therapeutic procedure in current gynaecological practice all over the world. It is now
acceptable for all surgical procedures, from diagnosis to oncology, to be performed laparoscopically, especially in gynaecology. It has been reported, however, that complications can be encountered in up to 10% of laparoscopic procedures in gynaecology [27]. Some of these complications may be related to pneumoperitoneum and CO2, including metabolic and respiratory acidosis, gas embolism and increased IAP-related organ damage [27–29]. As acidosis and gas embolism due to pneumoperitoneum usually have high morbidity rates, their significance and frequency are well known [28]. Recently, a study in humans demonstrated that CO2 pneumoperitoneum causes systemic ischaemia and releases oxygen radicals, which may be responsible for the detrimental effects of ischaemia such as myocardial ischaemia [29]. Also, Peterson et al. reported three patients who died of myocardial infarction as a result of laparoscopic sterilization [30]; the cause of death for these patients may have been related to systemic oxidative stress induced by CO2 pneumoperitoneum [29,30]. It is well known that the IAP needed for pneumoperitoneum during laparoscopic surgery, which is generally set at 12– 15 mmHg, may lead to decreased organ perfusion [31]. Decreased
74
A. Akdemir et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 173 (2014) 71–76
Fig. 3. Caspase-3 immunoexpression in ovarian sections. (A,B) No expression of caspase-3 in sham group. (C,D) Severe caspase-3 expression in ovarian surface epithelia in saline group. (E,F) Mild caspase-3 expression in ovarian surface epithelia in montelukast group. e, ovarian surface epithelia. Original magnification 20 for (A), (C) and (D) and 100 for (B), (D) and (F).
organ perfusion causes ischaemic injury, and this process may be further aggravated by deflation of the abdomen (reperfusion period), leading to oxidative stress and deleterious effects on cellular function [7,32]. Cells may die during this ischaemic period, which is known as necrosis. After the ischaemic/hypoxic period, during the deflation, visceral organs are perfused with oxygenated blood, which leads to the generation of ROS. These products cause secondary cell damage, leading to cell death by both apoptosis and necrosis [3,4]. This mechanism of action is known as ‘I/R injury’.
ROS are known to be released during reperfusion, and have been proposed to be important mediators of subsequent tissue injury. Additionally, pro-inflammatory cytokines, chemokines and activated complement factors are responsible for neutrophil recruitment and the subsequent neutrophil-induced oxidant stress during reperfusion. CysLTs are potent pro-inflammatory lipid mediators that play a central role in inflammation with their potent chemotactic and chemokinetic properties [33]. Many experimental and clinical studies have shown that increased IAP
Table 1 Comparison of biochemical parameters in rat ovarian tissue between all groups.
Tissue GSH (nmol/mg protein) Tissue MDA (nmol/mg protein) Plasma TAC (mM)
Sham (n = 8)
Saline (n = 8)
Montelukast (n = 8)
63.64 3.22 0.06 0.01 124.76 10.50
5.48 0.28 0.34 0.03 80.81 9.10
8.9 0.18 0.09 0.05 94.66 20.81
GSH, glutathione; MDA, malondialdehyde; TAC, total antioxidant capacity. Data are given as mean standard error of mean. a Mann–Whitney U-test four-group comparison.
p valuea 1 vs 2
2 vs 3
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Montelukast prevents ischaemia/reperfusion-induced ovarian damage in rats ¨ zgu¨r Yeniel a, F. Oltulu c, A. Yavas¸og˘lu c, A. Akdemir a, O. Erbas¸ b, M. Ergenog˘lu a, A. O b, D. Taskiran * a b c
Department of Obstetrics and Gynaecology, Ege University School of Medicine, Izmir, Turkey Department of Physiology, Ege University School of Medicine, Izmir, Turkey Department of Histology and Embryology, Ege University School of Medicine, Izmir, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 January 2013 Received in revised form 4 August 2013 Accepted 25 November 2013
Objective: To investigate the efficacy of montelukast for prevention of ischaemia/reperfusion (I/R) injury in rat ovary. Study design: Twenty-four female adult rats were included in the study. I/R injury was induced by CO2 pneumoperitoneum in a laparoscopic rat model. The rats were divided at random into three groups: the sham group was subjected to catheter insertion but was not subjected to pneumoperitoneum; the saline group was subjected to 60 min of pneumoperitoneum and 30 min of reperfusion, with 1 mg/kg physiological saline administered 10 min before pneumoperitoneum; and the montelukast group was subjected to 60 min of pneumoperitoneum and 30 min of reperfusion, with 20 mg/kg montelukast administered 10 min before pneumoperitoneum. Damage to ovarian tissue was scored by histopathological evaluation. Caspase-3 expression was determined immunohistochemically. Ovarian tissue levels of malondialdehyde and glutathione, and plasma total antioxidant capacity were measured biochemically. Results: In comparison with the sham group, ovarian sections in the montelukast group had higher scores for follicular degeneration and oedema (p < 0.001). Montelukast treatment prevented tissue damage in ovaries, and this result was significant. Caspase-3 expression was only observed in ovarian surface epithelium in the saline and montelukast groups. However, the mean caspase-3 expression score was higher in the saline group than the montelukast group (p < 0.001). Tissue levels of malondialdehyde were higher in the montelukast group than the sham group, but plasma total antioxidant capacity and tissue levels of glutathione were significantly lower. Pretreatment with montelukast reduced lipid peroxidation (p < 0.005) and improved antioxidant status in rats (p < 0.001). Conclusion: Montelukast is effective for the prevention of I/R-induced damage in rat ovary. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Montelukast CysLT1 receptor CO2 pneumoperitoneum Laparoscopy Reactive oxygen species Ischaemia/reperfusion injury
1. Introduction Over the last two decades, laparoscopy has become a fundamental technique in gynaecological surgery. The laparoscopic approach has several advantages over open surgery in terms of mortality, postoperative pain, patient mobilization and recovery, and cosmetic results. Although early use was restricted to diagnostic laparoscopy and tubal sterilization, the laparoscopic technique is now recommended for surgical treatment of ectopic pregnancy, and is also applied in fields that have traditionally used an open approach (e.g. myomectomy, hysterectomy,
* Corresponding author at: Department of Physiology, Ege University School of Medicine, 35100 Izmir, Turkey. Tel.: +90 232 3901800; fax: +90 232 3746597. E-mail address: [email protected] (D. Taskiran). 0301-2115/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejogrb.2013.11.021
lymphadenectomy and pelvic floor repair). Despite its benefits, however, CO2 pneumoperitoneum is not free from adverse events, even at low pressures. Experimental and clinical studies have shown that increased intra-abdominal pressure (IAP) associated with pneumoperitoneum reduces blood flow to intra-abdominal organs because of high venous resistance during laparoscopic procedures [1]. This process, known as ischaemia, leads to the production of free radicals [1–5]. Although desufflation of the abdominal cavity at the end of a laparoscopic procedure reduces the IAP and increases the perfusion of intra-abdominal organs, this does not result in the absence of oxidative stress. The ischaemia/ reperfusion (I/R) model leads to the release of various free radicals, which are the most important mediators of oxidative tissue injury and consequential organ dysfunction, especially in the splanchnic organs [2–8]. Recently, both oxidative tissue injury and consequential dysfunction have been shown in ovarian tissue [9].
72
A. Akdemir et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 173 (2014) 71–76
An excessive inflammatory response is clearly recognized as a key mechanism of injury during reperfusion. Various chemokines, lipid mediators (e.g. cysteinyl leukotrienes (CysLTs)) and the 5lipoxygenase metabolites of arachidonic acid are potent inflammatory mediators that have been associated with I/R-induced tissue injury [10–12]. Given these findings, it has been hypothesized that pharmacological strategies that limit neutrophil recruitment may also limit the damage induced by reperfusion. Therefore, several anti-inflammatory and antioxidant agents have been used to prevent I/R injury in tissues [13–16]. Montelukast, a selective reversible CysLT1 receptor antagonist, is a new anti-inflammatory agent with antioxidant properties [17]. Montelukast is used for the treatment of asthma as it reduces eosinophilic inflammation in the airways [18]. Additionally, montelukast has been shown to not only reduce I/R-induced oxidative damage in kidney and liver through its anti-inflammatory and antioxidant properties, but also to ameliorate multi-organ damage due to burns and sepsis by a neutrophil-dependent mechanism [15,19–22]. As such, the aim of the present study was to identify the effects of montelukast pretreatment in preventing I/R injury in rat ovary caused by reactive oxygen species (ROS) generated during CO2 pneumoperitoneum. 2. Materials and methods Twenty-four mature, female, non-pregnant Sprague Dawley rats weighing between 200 and 250 g were used in this experimental study. The animals were caged in a controlled environment at 22 8C with 12-h light/dark cycles. Standard rat feed and reverse-osmosis-purified water were provided ad libitum. The study protocol was approved by the University Animal Care and Ethics Committee of Ege University. All rats were anaesthetized by intraperitoneal administration of 50 mg/kg ketamine hydrochloric acid (Ketalar; Eczacibasi WarnerLambert Ilac Sanayi, Levent, Istanbul, Turkey) and 7 mg/kg xylazine hydrochloric acid (Rompun; Bayer Sisli, Istanbul, Turkey). Before pneumoperitoneum, rats were immobilized on a standard rat surgical board, abdominal skin was shaved, antisepsis was obtained with 10% povidone iodine solution, and body temperature was kept constant at 37 8C with a heating blanket. An 18-G Abbocath catheter was inserted into the abdominal cavity below the xiphoid process and connected to an insufflator tube. CO2 was then insufflated to a pressure of 12 mmHg using an abdominal CO2 insufflator (Karl Storz GmbH, Tutlingen, Germany). The rats were allocated at random to one of three groups (eight rats in each group) on the day of the experiment. All investigators responsible for the biochemical and histopathological studies were blinded to the randomization until the end of the study. The sham group was subjected to catheter insertion but was not subjected to pneumoperitoneum; the saline group was subjected to 60 min of pneumoperitoneum and 30 min of reperfusion, with 1 mg/kg of physiological saline (0.9% sodium chloride) administered by oral gavage 10 min before pneumoperitoneum; and the montelukast group was subjected to 60 min of pneumoperitoneum and 30 min of reperfusion, with 20 mg/kg montelukast administered by oral gavage 10 min before pneumoperitoneum. After 60 min of pneumoperitoneum, the CO2 was desufflated and IAP was normalized for 30 min. Immediately after the reperfusion period in the saline and montelukast groups, and 90 min after catheter insertion in the sham group, a midline abdominal incision was made to localize the ovaries. Both ovaries were excised for histopathological (left ovary) and biochemical (right ovary) evaluation. Following rapid fixation in a formaldehyde (10%) solution, ovaries were dehydrated in an ascending alcohol series, cleared in
xylene and embedded in paraffin. Tissue sections (4-mm thick) were prepared and stained with haematoxylin and eosin for general morphological observation. All sections were studied and photographed with an Olympus C-5050 digital camera mounted on an Olympus BX51 microscope (Olympus Corp., Tokyo, Japan). Follicular degeneration and interstitial oedema were scored from 0 to 3 according to injury severity, where 0 represented no pathological findings, and 1, 2 and 3 represented pathological findings of 66% of the ovarian section, respectively [9]. Apoptosis was evaluated through immunohistochemical analysis of caspase-3. Morphological analyses of both investigations were assessed using a computerized image analysis system (Image-Pro Express 1.4.5, Media Cybernetics, Inc., USA) on 10 microscopic fields per section, examined at magnification of 20, with the observer blind to the study group. For biochemical analysis, ovaries were homogenized in ice-cold 150 mM KCl and centrifuged at 5000 g for 10 min. The supernatants were analyzed for malondialdehyde (MDA; lipid peroxidation), glutathione (GSH) and protein levels. Lipid peroxidation was determined in tissue samples by measuring MDA levels as thiobarbituric acid reactive substances [23]. GSH content in tissue samples was measured spectrophotometrically according to Ellman’s method [24]. Total protein concentration in tissue samples was determined according to Bradford’s method [25]. Plasma total antioxidant capacity (TAC) was measured with the ferric reducing antioxidant power assay according to Benzie and Strain [26]. Data analyses were performed using Statistical Package for the Social Sciences Version 15.0 (IBM Corp., New York, USA). All quantitative data were analyzed using the Mann–Whitney U-test. Results are presented as mean standard error of mean. p-Values 0.05 were regarded as statistically significant. 3. Results Fig. 1 depicts the histopathological examination scores for the sham, saline and montelukast groups. The sham group demonstrated normal ovarian morphology, such as normal stroma, the presence of all types of follicles in the cortex (primordial, primary, secondary and tertiary), and the presence of capillary vessels in the medullary part of the ovaries. In comparison with the sham group, however, ovarian sections in the saline group had higher scores for follicular degeneration and oedema (p < 0.001). Montelukast treatment prevented the degenerative changes in ovaries, and this result was significant. There were no differences between the sham and montelukast groups (Fig. 2). Caspase-3 expression was only observed in the ovarian surface epithelium in the saline and montelukast groups (Fig. 3). However, the mean caspase-3 expression score was significantly higher in the saline group than the montelukast group (Fig. 1, p < 0.001).
Fig. 1. Comparison of histopathological examination scores and caspase-3 immunoreactivity in ovarian sections. *Different from other groups (p < 0.001).
A. Akdemir et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 173 (2014) 71–76
73
Fig. 2. Histopathological alterations in ovarian sections. (A,B) No pathological changes in sham group. (C,D) Severe follicular degeneration (fd) and oedema (*) in saline group. (E,F) Oedema (*) in montelukast group. sf, secondary follicle; tf, tertiary follicle; s, cortical and medullar stroma; CL, corpus luteum. Haematoxylin and eosin staining. Original magnification 10 for (A), (C) and (D) and 20 for (B), (D) and (F).
As summarized in Table 1, the mean tissue MDA value for the saline group was significantly higher than for the sham group (0.34 0.03 nmol/mg protein vs 0.06 0.01 nmol/mg protein; p < 0.001). Administration of 20 mg/kg montelukast in rats reduced lipid peroxidation significantly compared with the saline group (0.09 0.05 nmol/mg protein; p < 0.005). GSH levels were found to be significantly lower in the saline group than the sham group (5.48 0.28 nmol/mg protein vs 63.64 3.22 nmol/mg protein; p < 0.001). In comparison with the saline group, GSH levels were higher in the montelukast group (8.9 0.18 nmol/mg protein; p < 0.001). Plasma TAC was also significantly lower in the saline group compared with the sham group (80.81 9.1 mM vs 124.76 10.5 mM; p < 0.05). Administration of 20 mg/kg montelukast in rats induced higher plasma TAC compared with the saline group, but the difference was not statistically significant (94.66 20.81 mM; p = 0.26). 4. Comments Laparoscopy is an essential diagnostic and therapeutic procedure in current gynaecological practice all over the world. It is now
acceptable for all surgical procedures, from diagnosis to oncology, to be performed laparoscopically, especially in gynaecology. It has been reported, however, that complications can be encountered in up to 10% of laparoscopic procedures in gynaecology [27]. Some of these complications may be related to pneumoperitoneum and CO2, including metabolic and respiratory acidosis, gas embolism and increased IAP-related organ damage [27–29]. As acidosis and gas embolism due to pneumoperitoneum usually have high morbidity rates, their significance and frequency are well known [28]. Recently, a study in humans demonstrated that CO2 pneumoperitoneum causes systemic ischaemia and releases oxygen radicals, which may be responsible for the detrimental effects of ischaemia such as myocardial ischaemia [29]. Also, Peterson et al. reported three patients who died of myocardial infarction as a result of laparoscopic sterilization [30]; the cause of death for these patients may have been related to systemic oxidative stress induced by CO2 pneumoperitoneum [29,30]. It is well known that the IAP needed for pneumoperitoneum during laparoscopic surgery, which is generally set at 12– 15 mmHg, may lead to decreased organ perfusion [31]. Decreased
74
A. Akdemir et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 173 (2014) 71–76
Fig. 3. Caspase-3 immunoexpression in ovarian sections. (A,B) No expression of caspase-3 in sham group. (C,D) Severe caspase-3 expression in ovarian surface epithelia in saline group. (E,F) Mild caspase-3 expression in ovarian surface epithelia in montelukast group. e, ovarian surface epithelia. Original magnification 20 for (A), (C) and (D) and 100 for (B), (D) and (F).
organ perfusion causes ischaemic injury, and this process may be further aggravated by deflation of the abdomen (reperfusion period), leading to oxidative stress and deleterious effects on cellular function [7,32]. Cells may die during this ischaemic period, which is known as necrosis. After the ischaemic/hypoxic period, during the deflation, visceral organs are perfused with oxygenated blood, which leads to the generation of ROS. These products cause secondary cell damage, leading to cell death by both apoptosis and necrosis [3,4]. This mechanism of action is known as ‘I/R injury’.
ROS are known to be released during reperfusion, and have been proposed to be important mediators of subsequent tissue injury. Additionally, pro-inflammatory cytokines, chemokines and activated complement factors are responsible for neutrophil recruitment and the subsequent neutrophil-induced oxidant stress during reperfusion. CysLTs are potent pro-inflammatory lipid mediators that play a central role in inflammation with their potent chemotactic and chemokinetic properties [33]. Many experimental and clinical studies have shown that increased IAP
Table 1 Comparison of biochemical parameters in rat ovarian tissue between all groups.
Tissue GSH (nmol/mg protein) Tissue MDA (nmol/mg protein) Plasma TAC (mM)
Sham (n = 8)
Saline (n = 8)
Montelukast (n = 8)
63.64 3.22 0.06 0.01 124.76 10.50
5.48 0.28 0.34 0.03 80.81 9.10
8.9 0.18 0.09 0.05 94.66 20.81
GSH, glutathione; MDA, malondialdehyde; TAC, total antioxidant capacity. Data are given as mean standard error of mean. a Mann–Whitney U-test four-group comparison.
p valuea 1 vs 2
2 vs 3
