Effect of melatonin in the prevention of post-operative adhesion formation in a rat uterine horn adhesion model

Bulent Özçelik1,3, I.Serdar Serin1, Mustafa Basbug1, Semih Uludag1, Figen Narin2 and Mehmet Tayyar1

1 Department of Obstetrics and Gynecology and 2 Department of Biochemistry, Erciyes University Medical Faculty, 38039 Kayseri, Turkey

3 To whom correspondence should be addressed. e-mail: bozcelikmd{at}hotmail.com


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Our main aim was to investigate the effects of melatonin (ME), possibly the most powerful free-radical scavenger, on the prevention of i.p. adhesion formation in rat uterine horn. Our secondary aim was to determine whether different methods of administration of ME were beneficial. METHODS: Animals were randomly assigned into seven groups, each consisting of 13 rats. Measured serosal injury was created using a standard technique. While control and two sham groups were not given ME, two of the remaining four groups were given a single dose of 10 mg/kg (2 mg) of ME i.p. immediately after injury and 30 min prior to injury respectively. In the two other groups, ME treatment was continued daily for 5 days. All animals were killed 2 weeks after surgery and adhesions were determined and scored by a examiner blinded to the test. RESULTS: The extent, severity and total scores of adhesion were found to be significantly reduced in all of the ME treatment groups when compared with control and sham groups. There were no statistically significant differences between the treatment groups. CONCLUSIONS: This study showed that even single dose ME therapy was effective in the prevention of post- operative i.p. adhesion formation.

Key words: adhesion formation/melatonin/prevention/rat


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Adhesion formation after pelvic surgery is still a major cause of several morbidities such as infertility, pain, bowel obstruction and subsequent intraoperative complications (Rodgers et al., 1998Go; Ellis et al., 1999Go). Although numerous adjuvants, including fibrinolytic agents (Hellebrekers et al., 2000aGo), crystalloid solutions (Holtz, 1985Go), corticosteroids (Sanfilippo et al., 1986Go), heparin (Al-Chalabi and Otubo, 1987Go; Basbug et al., 1998Go), hyaluronic acid (Urman et al., 1991Go; Basbug et al., 1998Go), non-steroidal anti-inflammatory agents (Golan et al., 1995Go), calcium channel blockers (Steinleitner et al., 1988Go), progesterone (Blauer and Collins, 1988Go) and barrier methods (Keckstein et al., 1996Go) have been used in the prevention of this complication, no satisfactorily effective adjuvant has yet been found.

The inflammatory response has long been recognized as a common denominator in all pathways for adhesion formation. In a study on this subject, it has been shown that leukocyte-dependent inflammatory reactions may increase cellular and tissue injury through the actions of oxygen-derived free radicals and metabolites (Fantone and Ward, 1982Go). In another study, i.p. superoxide dismutase (SOD) and catalase, known to block the effects of oxygen free radicals and reactive oxygen species, have been shown to reduce the inflammatory reaction and thereby the adhesion formation in an endometriosis animal model (Portz et al., 1991Go).

The hormone melatonin (N-acetyl-5-methoxy-tryptamine) (ME), is synthesized by the pineal gland. There is now evidence that ME may have a role in the biological regulation of circadian rhythms, sleep, mood, and perhaps reproduction, tumour growth and ageing (Brzezinski, 1997Go). In addition to several effects of ME in the human body, a number of authors have recently documented its free radical scavenger activity (Kazez et al., 2000Go; Hara et al., 2001Go). The hydroxyl radical scavenging potency of ME is much greater than that of other well-known free radical scavengers, which have been detected to date (McCord, 1985Go). In this first study based on the facts that oxygen-derived free radicals play a role in adhesion formation and that ME is perhaps the most potent free radical scavenger, we aimed to determine the effectiveness of ME in preventing adhesion formation after a measured unipolar electrocautery injury on the rat uterine horn.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 91 female Wistar-Albino rats, weighing 190–200 g, were divided into seven groups for this experimental study approved by Erciyes University Ethics Committee. All procedures were carried out at the Hakan Cetinsaya Clinical and Experimental Research Center of Erciyes University, Medical Faculty.

ME was obtained as a dry powder (Sigma, St Louis, MO, USA), dissolved in 99% ethanol and then diluted in saline just before injection. The final ethanol concentration was 5% and the amount of ME was 2 mg (10 mg/kg) per 1 ml injectable solution. Vials containing the melatonin solution were covered with aluminum foil and stored at –20°C until dilution.

The rats were randomly assigned into seven groups, each consisting of 13 rats, before being operated on. Because the s.c. ME had to be applied 30 min prior to surgery and ‘blind’ i.p. ME instillation might cause an undesirable bowel or vessel injury, this randomization had to be performed before operation. After the allocation into the study groups, the surgical procedure that we adopted was for a single surgeon to operate on one rat in each of the seven groups in turn. The groups and the treatment procedures are shown in Table I.


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Table I. Treatment procedures for different study groups
 
In the operative procedure, each rat was anaesthetized with ketamine hydrochloride (40 mg/kg i.v.). Before incision, the abdomen was shaved and prepared with a povidine iodine solution. Using sterile technique, the abdominal cavity was entered via a 3 cm vertical midline incision. A model following a previously published system (Basbug et al., 1998Go) was performed on the rats for the creation of a standard lesion and a 2 cm segment of each uterine horn was devascularized by creating a window and traumatized in 10 spots on the antimesenteric surface using unipolar cautery. Handling of other tissues was minimized and care was taken to avoid gross bleeding from injured sites. After the creation of standard lesions, 1 ml NaCl dilution vehicle containing 5% ethanol and 5% ethanol plus 2 mg ME was instilled i.p. onto uterine horns (0.5 ml onto each uterine horn) in i.p. sham and i.p. ME groups respectively. At the end of the procedure, the abdomen was closed in double layer using 5–0 monofilament delayed absorbable suture in a continued fashion.

All rats were housed under controlled temperatures (22 ± 2°C) and 12/12 h light/dark cycle with food and water ad libitum. After completion of the 2-week recovery period, the animals were killed and evaluated for adhesion formation and grading. The investigators who participated in scoring the adhesions had no prior knowledge as to which group the rats belonged. The extent and severity of adhesions in the operation site for each uterine horn were evaluated using an established scoring system (Linsky et al., 1987Go). According to this system the extent of adhesions was evaluated as follows: 0, no adhesion; 1, 25% of traumatized area; 2, 50% of traumatized area; 3, total involvement. Fractional scores were given for extent of adhesions between the above grades. The severity (tenacity) of the adhesions was measured as follows: 0, no resistance to separation; 0.5, some resistance (moderate force was required); 1, sharp dissection needed. The total grade was additive, giving a range of adhesion scores from 0–4, which represented both extent and severity.

The statistical comparison of the groups’ adhesion scores was made using one-way ANOVA. Post-hoc comparisons on parameters were performed using Scheffé’s procedure. The comparison of the number of horns and animals with and without adhesion between the groups was made using the {chi}2-test. Power analysis indicated the sufficiency of the data number when the alpha value was taken as 0.05 and 0.01 in the evaluation of all data (Power >0.90).


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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
All rats recovered without incident after operation and resumed their pre-operative physical activity and feeding patterns post-operatively. In the scoring laparotomies, no finding of residual viscous solution or ascites in the peritoneum was found, and no adhesion or intra-abdominal tissue abnormalities were detected except on cauterized horn sites.

The numbers of the horns without adhesion in control, i.p. sham, s.c. sham, single dose i.p. ME, single dose s.c. ME, i.p. ME plus 5 days s.c. ME and s.c. ME plus 5 days s.c. ME groups were 1, 1, 0, 3, 4, 7 and 7 respectively. When the groups were compared with respect to these numbers, i.p. ME plus 5 days s.c. ME and s.c. plus 5 days s.c. ME groups were significantly different from control ({chi}2-value = 5.949, P < 0.05), i.p. sham ({chi}2-value = 5.949, P < 0.05) and s.c. sham ({chi}2-value = 3.693, P < 0.01) groups. The numbers of the animals without adhesion were two in i.p. ME plus 5 days s.c. ME group and two in s.c. ME plus 5 days s.c. ME group, and there was no statistical difference between study groups.

The mean ± SD extent, severity and total scores of the adhesions are given in Table II. As shown in the table, single dose i.p. ME, single dose s.c. ME, i.p ME plus 5 days s.c. ME and s.c. ME plus 5 days s.c. ME treatments significantly reduced the adhesion scores compared with the control and sham groups. Although the statistically significant difference was higher in the 5 day treatment groups (P < 0.01) than in the single dose groups (P < 0.05) compared with control and sham groups, no statistically significant difference was found between the four different treatment groups.


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Table II. Extent, severity and total scores of adhesions in study groups
 

    Discussion
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 Introduction
 Materials and methods
 Results
 Discussion
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ME has received much attention in recent years due to its direct radical scavenging and antioxidant properties. In the present study, which is the first on the prevention of post-operative pelvic adhesion formation by ME, we wanted to assess the efficacy of ME in vivo. Although the exact aetiology of pelvic adhesion formation is not completely understood, there are several well-known risk factors for their formation. One of these factors is tissue ischaemia (Gutmann et al., 1995Go). The source of superoxide and other radicals may be cytochrome oxidase in mitochondria and possibly xanthine oxidase in endothelial cells in different tissues (Lay and Arfors, 1982Go). Notably, during the first 5 min of ischaemia, there is already a significant production of free radicals, which are able to react rapidly with oxygen, exacerbating the oxygen deficit (Bertuglia et al., 1993Go). Both superoxide anion and hydrogen peroxide have been shown to be toxic to a wide variety of eukaryotic cells, including endothelial cells, erythrocytes, platelets and fibroblasts, by extracellular cytolysis of host cells. As a result of cytolysis and lipid peroxidation of the cellular membranes, vascular permeability increases, which leads to the formation of serosanguineous exudate, which in turn initiates adhesion formation. These adhesions are generally lysed within 72 h after formation by the endogenous fibrinolytic activity and much of the healing is complete within 5 days (Gutmann et al., 1995Go; Hellebrekers et al., 2000aGo). If there is an imbalance between fibrin deposition and fibrin dissolution, deposited fibrin may persist and fibrinous adhesions may develop (Vipond et al., 1990Go; Hellebrekers et al., 2000bGo).

ME is the most potent scavenger of the highly toxic hydroxyl radical and other oxygen centred radicals, and its action is not mediated by receptors (Reiter, 1995Go). This protective effect may be due to the ME indole structure which has been shown to scavenge free radicals, in particular the hydroxyl radical, thereby giving rise to new structures which are resonance-stabilized and no longer toxic to macromolecules. In addition, ME may stimulate several antioxidative enzymes and inhibit a pro-oxidative enzyme by intracellulary binding to calmodulin (Reiter et al., 1999Go).

ME is normally found in the human circulation but the antioxidant effects of melatonin in humans probably occurs only at pharmacological concentrations. No serious side effects or risks have been reported in association with the use of ME. The dose-dependent physiological effects of the hormone, however, have not yet been properly evaluated in people who take large doses for prolonged periods of time (Brzezinski, 1997Go).

The half-life of ME injected intra-arterially is ~20 min and is related to the amount injected and to the presence of anaesthesia. The higher dosage of ME reduces the half-life to 3–4 min and the anaesthesia increases up to 40 min (Gibbs and Vriend, 1981Go). Therefore, taking into account this complex interference, the pharmacological dose of the ME and the time of the s.c. administration prior to injury was determined in the light of previous studies, in which ME was found to be effective in the prevention of ischaemic injury and tissue damage (Kazez et al., 2000Go; Hara et al., 2001Go; Lankoff et al., 2002Go; Sener et al., 2002Go).

Although a large number of studies have been published regarding the scavenger effect of ME in the prevention of oxidative damage in different tissues, we were unable to find (using Medline) any previous studies designed to examine the use of ME in the prevention of pelvic adhesion formation. Furthermore, the number of studies on the effects of antioxidants on i.p. adhesion formation is very limited (Kagoma et al., 1985Go; Sanfilippo et al., 1995Go; Rodgers et al., 1998Go). In one of them, Rodgers et al. (1998)Go demonstrated that pre- and post-operative i.p. administration of either a steroidal or non-steroidal lazaroid (a lipid peroxidation inhibitor) reduced the post-operative adhesion formation and reformation in three rabbit models. However, the other two studies investigating the effects of vitamin E on adhesion formation and fibrosis have conflicting results. In the study of Kagoma et al. (1985)Go they investigated whether dietary supplementation with either 65 IU/kg or 300 IU/kg vitamin E would decrease peritoneal adhesion formation following peritoneal ligation in mice. At the end of the study they determined a statistically significant decrease in the incidence and degree of adhesion formation in the two vitamin E-supplemented groups in comparison with the control animals. On the other hand, the results of Sanfilippo et al. (1995)Go did not correlate with the preceding study despite the same doses of vitamin E having been used.

In our study, we found a significant reduction in post-operative adhesion formations in rats treated with ME, regardless of application procedure and duration of the agent (Table II). Although daily administration of ME for 5 days in the post-operative period seemed to be much more effective in the reduction of adhesion, no statistically significant difference was detected in the adhesion scores in comparison with the groups treated with a single dose of ME. In our opinion, because the significant production of free radicals occurs within 5 min of endothelial tissue damage (Bertuglia et al., 1993Go), the ME may be essentially effective in this period. Oxygen-derived free radical inhibitors are mentioned under the title of anti-inflammatory agents (Gutmann et al., 1995Go). Because most of the members of this group are effective in the phase of inflammatory response exposed to the initial cellular membrane damage, the hypothesis that antioxidants and free radical scavengers are essentially effective in the first step of the histiogenesis of adhesions is not wrong.

In conclusion, this experimental study is the first on the prevention of post-operative i.p. adhesion formation by ME. Our results show that ME can act as an endogenous antioxidant and even single dose treatment with ME provides a significant reduction in i.p. adhesion formation. Further studies are necessary with ME on human subjects and comparative studies with other preventive agents that have been found effective in the prevention of adhesion formation in preceding studies. In addition, studies must be carried out to ascertain whether or not ME affects adhesion reformation after lyses.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on January 24, 2003; accepted on May 7, 2003.