Kirikkale University Faculty of Medicine, Ankara, Turkey
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Abstract |
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Key words: mannitol/ovary/reperfusion/verapamil/vitamin C
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Introduction |
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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, 1990; Rangan and Bulkley, 1993
; Das and Maulik, 1994
; Zimmerman and Granger, 1994
); consequently, the prevention of reperfusion injury increases the success of any treatment (Sussman and Bulkley, 1990
; Rangan and Bulkley, 1993
). Ischaemia and reperfusion results in the production of reactive oxygen species (ROS) in tissues such as brain, heart and muscle (Yoshida et al., 1980
; Jolly et al., 1984
; McCord, 1985
); subsequently, the ROS (and their products) cause damage to the cell membranes (Fridovich, 1983
; Slater, 1984
). Vitamin C, which is water-soluble, not only scavenges hydroxyl radicals (Gutteridge, 1995
) but also increases vitamin E regeneration (Yoshida et al., 1982
; Das and Maulik, 1994
). Mannitol has important physiopathological roles in ischaemiareperfusion (IR) injury as it possesses scavenging properties for radicals and also has rearrangement effects on the microcirculation (Shirane and Weinstein, 1992
; Kariba et al., 1995
). The major tissue damage that occurs during IR injury is secondary to calcium influx into the cell. Hence, the calcium-channel blocker verapamil might protect tissues against IR injury by reducing calcium influx into the cell (Kimura et al., 1998
). In the present study, the protective effects of vitamin C, mannitol and verapamil against adnexial IR injury in the rat ovary were evaluated.
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Materials and methods |
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Animal studies
Thirty-six female Wistar rats (body weight 250300 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 2.53 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, 1995). 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., 1951
).
Statistical analysis
The data were expressed as mean ± SD. Statistical analyses was performed using the SPSS package programme, including KruskalWallis variance analysis, MannWhitney U-test and Spearman correlation analysis. A P-value < 0.05 was considered to be statistically significant.
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Results |
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Discussion |
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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 IR injury, and increases the survival of follicles in ovarian grafts (Nugent et al., 1998). Others (Sugino et al., 1993
) developed an ischaemia and reperfusion model for pregnant rats, and showed that IR 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., 1991
). In another study, it was reported that pentoxiphylline, a methylxanthine derivative, could prevent reperfusion injury after unilateral ovarian torsion (Ciakmak et al., 1999
).
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., 1994), and several studies have indicated such protection against reperfusion injury in lung, brain and skin flaps (Zaccaria et al., 1994
; Henry and Chandy, 1998
; Demertzis et al., 2000
). Ascorbic acid was also found to decrease lipid peroxidation in the cell membrane and protect the myocardium against IR injury (Dingchao et al., 1994
). Ascorbic acid was also shown to prevent IR injury in rat small bowel, in a dose-dependent manner (Nakamura et al., 1997
).
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., 1994). Another group (Magovern et al., 1984
) 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 mannitols myocardial protective effect. Likewise, mannitol was found to be effective in preventing ovarian IR injury in the present study.
After IR, cellular calcium overload may also trigger the release of oxygen free radicals and thereby potentiate oxygen radical-related membrane injury (Malis and Bonventre, 1986). Recently, much attention has been focused on the role of calcium channel blockers in the prevention of IR injury, with encouraging results having been reported for several tissues (Burke et al., 1984
; Kimura et al., 1998
). 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., 1990
). Verapamil may also have an antioxidant effect by reducing mitochondrial calcium overload (Burke et al., 1984
). It has been advocated that verapamil regulates the energy metabolism of the cell by reducing calcium influx, thereby protecting the cell against IR injury (Kimura et al., 1998
). 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., 1992
). 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., 1993
). In the present study, no preventive effects of verapamil on ovarian IR injury were observed, though this may be dose-related as an effective dose in ovarian IR 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 IR 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 IR 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.
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Acknowledgements |
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Notes |
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References |
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Submitted on January 14, 2002; resubmitted on April 15, 2002; accepted on July 24, 2002.