Ischaemia–reperfusion injury of rat ovary and the effects of vitamin C, mannitol and verapamil

Nevin Sagsöz, Üçler Kisa and Alparslan Apan

Kirikkale University Faculty of Medicine, Ankara, Turkey


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: In this prospective controlled study, the aim was to examine the effects of vitamin C, mannitol and verapamil on adnexial ischaemia–reperfusion injury in the rat ovary. METHODS: Thirty-six female Wistar rats were used. In the controls (group 1), only laparotomy was performed. In group 2, ovarian ischaemia was produced and the bilateral ovaries were surgically removed 4 h later. In group 3, an ischaemic period of 4 h was followed by reperfusion for 1 h; the bilateral ovaries were then removed. In groups 4, 5 and 6, after 4 h of ischaemia, either vitamin C, mannitol or verapamil respectively was infused before reperfusion; after 1 h of reperfusion the ovaries were removed. Thiobarbituric acid reactive substance (TBARS) levels were measured in all ovary tissues. RESULTS: TBARS levels of the reperfusion group were significantly higher than those of groups treated with vitamin C or mannitol (P = 0.013 and P = 0.045 respectively), but not of the verapamil group. CONCLUSIONS: Vitamin C and mannitol were found to be effective in reducing ischaemia–reperfusion injury of the ovary during its early stages, but verapamil was ineffective.

Key words: mannitol/ovary/reperfusion/verapamil/vitamin C


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Adnexial torsion is a rare condition, and is the fifth most common gynaecological emergency, with a prevalence of 2.7% in the USA (Hibbard, 1985Go). Adnexial torsion has non-specific signs and symptoms, and so both diagnosis and the treatment are generally delayed. Historically, the preferred treatment was excision of the adnex without releasing the adnexial pedicle (Bayer and Wiskind, 1994Go). However, it has been suggested recently that releasing the pedicle and evaluating the tissue perfusion is a reliable and conservative alternative approach (Mashiach et al., 1990Go; Oelsner et al., 1993Go; Bayer and Wiskind, 1994Go). Intraoperative evaluation of black-purple adnexes does not correlate with viability of the tissue, and detorsion of the adnexes does not increase the risk of thromboembolism (Mashiach et al., 1990Go; Wagaman and Williams, 1990Go; Oelsner et al., 1993Go). Evaluation of tissue perfusion with intraoperative fluorescein dye has also been suggested (McHutchinson et al., 1993Go).

The major aim of treating ischaemia is not only to restore the blood circulation but also to improve tissue perfusion. Following ischaemia, when the circulation and reperfusion is maintained, a new physiopathological process called ‘reperfusion injury’ is encountered, and this causes several degrees of tissue damage. The total damage that the tissue incurs is the sum of that caused by both ischaemia and reperfusion (Sussman and Bulkley, 1990Go; Rangan and Bulkley, 1993Go; Das and Maulik, 1994Go; Zimmerman and Granger, 1994Go); consequently, the prevention of reperfusion injury increases the success of any treatment (Sussman and Bulkley, 1990Go; Rangan and Bulkley, 1993Go). Ischaemia and reperfusion results in the production of reactive oxygen species (ROS) in tissues such as brain, heart and muscle (Yoshida et al., 1980Go; Jolly et al., 1984Go; McCord, 1985Go); subsequently, the ROS (and their products) cause damage to the cell membranes (Fridovich, 1983Go; Slater, 1984Go). Vitamin C, which is water-soluble, not only scavenges hydroxyl radicals (Gutteridge, 1995Go) but also increases vitamin E regeneration (Yoshida et al., 1982Go; Das and Maulik, 1994Go). Mannitol has important physiopathological roles in ischaemia–reperfusion (I–R) injury as it possesses scavenging properties for radicals and also has rearrangement effects on the microcirculation (Shirane and Weinstein, 1992Go; Kariba et al., 1995Go). The major tissue damage that occurs during I–R injury is secondary to calcium influx into the cell. Hence, the calcium-channel blocker verapamil might protect tissues against I–R injury by reducing calcium influx into the cell (Kimura et al., 1998Go). In the present study, the protective effects of vitamin C, mannitol and verapamil against adnexial I–R injury in the rat ovary were evaluated.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A preliminary study was performed to determine the optimum period of ischaemia of the ovary. Following ischaemia of 4 h duration, congestion and oedema at the ovarian stromal tissue were evaluated histologically. The ovaries were seen to be black-purple in colour, but no signs of tissue necrosis were detected. Thus, an ischaemic period of 4 h was considered adequate for subsequent studies.

Animal studies
Thirty-six female Wistar rats (body weight 250–300 g) were used in the study and maintained in accordance with the National Institutes of Health approved guidelines. The mean age and body weight of all animal groups were identical. Before surgery, the rats were in simultaneous cycle phase. On the day of surgery, each rat was weighed and anaesthetized initially with 40 mg/kg i.m. ketamine hydrochloride (Eczac1basi1, Istanbul, Turkey), with repeat administration as necessary to maintain anaesthesia.

Surgical technique
Each rat was placed in a dorsal recumbent position, and the area of skin to be operated on was cleaned and dressed. A laparotomy was performed using a midline skin incision of 2.5–3 cm.

Group 1 (controls; n = 6) rats underwent laparotomy alone, whilst in group 2 (n = 6) ovarian ischaemia was produced using vascular clamps. The histopathological and biochemical findings of ovarian torsion and vascular clamp usage were shown previously to be similar (Tasik1n et al., 1998). As ovarian torsion is technically more difficult and may inevitably cause damage to the surrounding tissues, atraumatic vascular clamps were used in the present study to produce ovarian ischaemia. The incision was closed with 4/0 nylon sutures, and bilateral ovaries were surgically removed after 4 h for histological examination.

In group 3 (n = 6), a 4 h period of ischaemia was followed by 1 h reperfusion, after which bilateral ovaries were removed for histological examination.

Groups 4, 5 and 6 (all n = 6) were each treatment groups. After a 4 h period of ischaemia, either vitamin C (50 mg/kg), mannitol (3 ml/kg of a 20% solution) or verapamil (0.3 mg/kg) was infused via the inferior caval vein in groups 4, 5 and 6 respectively over a 1 min period. Reperfusion was then continued for 1 h, after which the ovaries were removed for histological examination.

Histology
The ovarian tissues were preserved in 10% buffered formalin solution. Histological examinations were carried out on 7 µm slices, stained with haematoxylin and eosin, and viewed under a light microscope. The tissue samples (the whole ovaries) of each rat were examined in blinded fashion by the same pathologist. As no scoring system relating to ischaemia has been reported previously, the following system was used. Congestion, bleeding, oedema and loss of cohesion (separation of parenchymal cells along with normal ovarian cortex and follicles) were scored from 0 to +3 according to their severity, where 0 = no pathological finding, and scores of 1, 2 and 3 represent pathological findings of <33%, 33-66% and >66% of the ovary respectively. The scores for each parameter were summed and the total tissue damage scores calculated.

Thiobarbituric acid reactive substance (TBARS) measurements
TBARS levels were measured in order to evaluate lipid peroxidation in the tissue homogenate; results were expressed as µmol per gram protein. After washing with 0.9% NaCl, tissue (which had been preserved at –70°C) was homogenized in 1 ml 0.9% NaCl using a tissue homogenizer, and the homogenates were centrifuged at 1500 x g at 4°C for 10 min. A 50 µl aliquot of homogenate was transferred into a 15 ml glass tube to which 1 ml 1,3-diethyl-2-thiobarbituric acid (DETBA) solution (DETBA 10 mmol/l and K2HPO4 75 mmol/l, pH 7.0), 100 µl ethylenediamine tetra-acetic acid (EDTA; 18.75 mmol/l) and 100 µm H3PO4 (3 mmol/l) were added respectively and mixed. The samples were placed in a water bath and heated for 45 min at 95°C. After cooling the samples, 5 ml n-butanol was added and mixed. The butanol phase was separated by centrifugation at 1500 x g for 10 min, and the fluorescence of the butanol extract was measured at 515 nm excitation and 553 nm emission (Perkin-Elmer fluorometer) (Dazhong, 1995Go). The calibration curve was prepared with 1,1,3,3-tetraethoxypropane (TEP; Sigma) standards of 0, 1, 2, 4.8 µmol/l. Protein concentrations were measured using a previously published method (Lowry et al., 1951Go).

Statistical analysis
The data were expressed as mean ± SD. Statistical analyses was performed using the SPSS package programme, including Kruskal–Wallis variance analysis, Mann–Whitney U-test and Spearman correlation analysis. A P-value < 0.05 was considered to be statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Total tissue damage scores were significantly different between the groups (P = 0.003) (Figure 1Go). The I–R group had a significantly higher total tissue damage score than the ischaemia group (P = 0.042), but when the latter group was compared with the vitamin C, mannitol and verapamil groups, total tissue damage scores were not significantly different. Neither was any significant difference seen between the I–R group and the treatment groups. The histology of ovarian samples is shown in Figures 2–7GoGoGoGoGoGo. There were clearly no pathological findings in controls, but the ischaemia group generally showed minimal and moderate oedema, congestion haemorrhagia and loss of cohesion. The I–R group showed more severe pathological findings. Although the vitamin C and mannitol groups showed minimal to moderate ovarian pathological findings, severe changes were identified in the verapamil group.



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Figure 1. Total tissue damage scores of all groups (C = controls; I = ischaemia; I–R = ischaemia–reperfusion; Vit C = vitamin C; M = mannitol; V = verapamil). *P = 0.042 versus I–R.

 


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Figure 2. Control group. Photomicrograph showing no pathological changes in ovarian sections. (Haematoxylin and eosin staining; original magnification x100)

 


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Figure 3. Ischaemia group. Photomicrograph showing mild congestion and minimal haemorrhage. (Haematoxylin and eosin staining; original magnification x100)

 


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Figure 4. Ischaemia–reperfusion group. Photomicrograph showing apparent haemorrhage and congestion. (Haematoxylin and eosin staining; original magnification x100)

 


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Figure 5. Vitamin C group. Two ovary tissues; mild congestion and oedema can be seen in one tissue, but there is no clear pathology in the other. (Haematoxylin and eosin staining; original magnification x100)

 


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Figure 6. Mannitol group. Photomicrograph showing minimal congestion and oedema. (Haematoxylin and eosin staining; original magnification x40.)

 


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Figure 7. Verapamil group. Photomicrograph showing apparent haemorrhage and congestion. (Haematoxylin and eosin staining; original magnification x200)

 
Total tissue damage scores were seen to correlate with TBARS levels (r = 0.487, P = 0.003), the latter being significantly different between groups (P = 0.002) (Figure 8Go). The ischaemia group had a significantly higher TBARS level than controls (P = 0.006), whilst the I–R group had significantly higher TBARS levels than both the control and ischaemia groups (P = 0.004 and P = 0.008 respectively). No significant difference was observed when TBARS levels of the ischaemia group were compared with each of the treatment groups. When compared with the I–R group, both the vitamin C- and mannitol-treated groups showed significantly different TBARS levels (P = 0.013 and P = 0.045 respectively), but no difference was seen from the verapamil group.



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Figure 8. Tissue TBARS levels in all groups (C = controls;I = ischaemia; I–R = ischaemia–reperfusion; Vit C = vitamin C; M = mannitol; V = verapamil). *P = 0.013 versus I–R group; **P = 0.045 versus I–R group; ***P = 0.004 and P = 0.008 when comparing I–R group with control and ischaemia groups respectively, and P = 0.006 when comparing the I group with control.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
It is likely that, as with other tissues, ischaemia–reperfusion injury has important consequences on ovarian torsion treated by detorsion. Free radicals are the by-products of monovalent oxygen metabolism, which occurs following an ischaemic event. Under ischaemic conditions, xanthine oxidase metabolizes xanthine and hypoxanthine, with the production of free radicals. However, if these free radicals are not neutralized by endogenous or exogenous antioxidant molecules, then lipid peroxidation will occur at the cell membrane, and this will lead to damage of the cell membrane protein and capillary endothelium. The result is enhanced adherence of platelets to the vessel walls, increased permeability and irreversible tissue damage (Redfors et al., 1984Go; McCord, 1985Go; Rangan and Bulkley, 1993Go; Das and Maulik, 1994Go; Zaccaria et al., 1994Go; Zimmerman and Granger, 1994Go). When the duration of ischaemia is sufficiently long, reperfusion of the tissue results in microvascular dysfunction and there is no reflow phenomenon. When the blood circulation is restored, and even if the subsequent perfusion pressure is adequate, most of the capillary vessels are not reperfused (May et al., 1978Go). As yet, the ischaemic tolerance of the ovary has not been determined, and consequently the response of this organ to reperfusion is not clear. One group (Tasik1n et al., 1998) found no reversible change after 4–24 h of ischaemia and 7 days of reperfusion, though these authors did not measure levels of free radical scavengers within the ischaemic tissue. In the present study, significantly different TBARS levels were observed between the I–R group and the control and ischaemia groups after 4 h of ischaemia and 1 h of reperfusion, and these results were in accordance with the histopathological findings.

Few reports exist concerning ovarian reperfusion injury and its prevention. In a study performed on mice, vitamin E treatment was found to reduce total lipid peroxidase and malondialdehyde concentrations in ovarian grafts. Thus, it was suggested that antioxidant treatment decreases I–R injury, and increases the survival of follicles in ovarian grafts (Nugent et al., 1998Go). Others (Sugino et al., 1993Go) developed an ischaemia and reperfusion model for pregnant rats, and showed that I–R injury increases lipid peroxide levels and decreases levels of superoxide dismutase (SOD), which acts as a scavenger of ROS. Following reperfusion, these authors detected a decrease in serum progesterone levels, but this was not apparent in the groups treated with SOD and catalase. Simultaneous treatment with SOD and catalase was also shown to block the inhibitory effect of xanthine and xanthine oxidase on progesterone production in luteal cells (Gatzuli et al., 1991Go). In another study, it was reported that pentoxiphylline, a methylxanthine derivative, could prevent reperfusion injury after unilateral ovarian torsion (Ciakmak et al., 1999Go).

As yet, no reports have been made on the prevention of ovarian reperfusion injury with either vitamin C, mannitol or with verapamil. Vitamin C is an endogenous water-soluble compound that is virtually non-toxic and capable of reducing free radicals. Indeed, the reported therapeutic effects of this vitamin may be due to a combination of its antioxidant activity on various free radicals. Vitamin C is known to block lipid peroxidation in the cell membrane and scavenge hydroxyl radicals (Zaccaria et al., 1994Go), and several studies have indicated such protection against reperfusion injury in lung, brain and skin flaps (Zaccaria et al., 1994Go; Henry and Chandy, 1998Go; Demertzis et al., 2000Go). Ascorbic acid was also found to decrease lipid peroxidation in the cell membrane and protect the myocardium against I–R injury (Dingchao et al., 1994Go). Ascorbic acid was also shown to prevent I–R injury in rat small bowel, in a dose-dependent manner (Nakamura et al., 1997Go).

In skeletal muscle, mannitol is known to reduce post-ischaemic oedema mainly by its hyperosmolar properties, whereas the restitution of energy production and reduction of muscle necrosis appears to be an effect of its free radical scavenging. Compartment pressure was also seen to be reduced by a hyperosmolarity effect and free radical scavenging (Oredsson et al., 1994Go). Another group (Magovern et al., 1984Go) reperfused rabbit hearts with equally hyperosmotic solutions of mannitol or glucose or with a standard crystalloid solution, and showed a significantly improved ventricular function with mannitol than with either glucose or crystalloid. These authors suggested that hydroxyl radical scavenging, rather than hyperosmolarity, accounted for mannitol’s myocardial protective effect. Likewise, mannitol was found to be effective in preventing ovarian I–R injury in the present study.

After I–R, cellular calcium overload may also trigger the release of oxygen free radicals and thereby potentiate oxygen radical-related membrane injury (Malis and Bonventre, 1986Go). Recently, much attention has been focused on the role of calcium channel blockers in the prevention of I–R injury, with encouraging results having been reported for several tissues (Burke et al., 1984Go; Kimura et al., 1998Go). The molecular interaction between calcium channel blockers and oxygen radical release or oxygen radical injury is not clear as the effect of calcium may be mediated in different ways. For example, it has been shown that verapamil can block xanthine dehydrogenase to xanthine oxidase conversion in ischaemic rat livers and so prevent or reduce free radical generation (Ishii et al., 1990Go). Verapamil may also have an antioxidant effect by reducing mitochondrial calcium overload (Burke et al., 1984Go). It has been advocated that verapamil regulates the energy metabolism of the cell by reducing calcium influx, thereby protecting the cell against I–R injury (Kimura et al., 1998Go). However, in the rabbit heart, whilst low-dose verapamil (30 µg/kg) prevented ischaemic injury, high-dose verapamil (100 µg/kg) had no such effect (Dikshit et al., 1992Go). A beneficial effect of verapamil was found to occur only in rats sensitized to oxidative injury, suggesting that the calcium channel blocker protected against oxygen radical attack (Stein et al., 1993Go). In the present study, no preventive effects of verapamil on ovarian I–R injury were observed, though this may be dose-related as an effective dose in ovarian I–R has not yet been determined.

Although total tissue damage scores were semi-quantitative, TBARS levels were quantitative and hence may be a more sensitive criterion for statistical analysis. Moreover, in the present study TBARS levels were found to correlate with histological findings.

In conclusion, as options for the conservative treatment of ovarian torsion continue to develop, the importance of protecting the ovary against I–R injury becomes clearer. Although both the effects of antioxidant drugs on the ovary and their effective dose levels are as yet unknown, vitamin C and mannitol were each found to reduce I–R injury of the ovary during its early stages, but verapamil was found to be ineffective. Consequently, an extended and blinded study should be conducted in order to determine whether antioxidant treatment can indeed reduce ovarian ischaemic injury.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank Fatma CiagSlayan and Önder BozdogSan for their support in these studies.


    Notes
 
1 To whom correspondence should be addressed at: Cievreli Caddesi, Kirktasi Sokak, SagSlik Apt. 27/1, Aydinlikevler-Ankara, Turkey; E-mail: nsagsoz{at}isbank.net.tr Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on January 14, 2002; resubmitted on April 15, 2002; accepted on July 24, 2002.





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