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
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Abstract |
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Key words: adhesion formation/melatonin/prevention/rat
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Introduction |
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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, 1982). 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., 1991
).
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, 1997). 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., 2000
; Hara et al., 2001
). 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, 1985
). 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.
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Materials and methods |
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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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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., 1987). 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 04, 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 Scheffés procedure. The comparison of the number of horns and animals with and without adhesion between the groups was made using the 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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Results |
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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 (2-value = 5.949, P < 0.05), i.p. sham (
2-value = 5.949, P < 0.05) and s.c. sham (
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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Discussion |
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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, 1995). 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., 1999
).
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, 1997).
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 34 min and the anaesthesia increases up to 40 min (Gibbs and Vriend, 1981
). 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., 2000
; Hara et al., 2001
; Lankoff et al., 2002
; Sener et al., 2002
).
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., 1985; Sanfilippo et al., 1995
; Rodgers et al., 1998
). In one of them, Rodgers et al. (1998)
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)
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)
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., 1993), 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., 1995
). 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.
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References |
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Basbug, M., Aygen, E., Tayyar, M., Kaya, E., Narin, F. and Oktem, O. (1998) Hyaluronic acid plus heparin for improved efficacy in prevention of adhesion formation in rat uterine horn model. Eur. J. Obstet. Gynecol. Reprod. Biol., 78, 109112.[CrossRef][ISI][Medline]
Bertuglia, S., Colantuoni, A., Coppini, G. and Integlietta, M. (1993) Effects of leukocytes adhesion and microvascular permeability, capillary perfusion during ischemia reperfusion injury in hamster check pouch. Int. J. Microcirc. Clin. Exp., 13, 128.[ISI][Medline]
Blauer, K.L. and Collins, R.L. (1988) The effect of intraperitoneal progesterone on postoperative adhesion formation in rabbits. Fertil. Steril., 49, 144149.[ISI][Medline]
Brzezinski, A. (1997) Melatonin in humans. N. Engl. J. Med., 336, 186195.
Ellis, H., Moran, B.J., Thompson, J.N., Parker, M.C., Wilson, M.S., Menzies, D., McGuire, A., Lower, A.M., Hawthorn, R.J., OBrien, F. et al. (1999) Adhesion-related hospital readmissions after abdominal and pelvic surgery: a prospective cohort study. Lancet, 353, 14761480.[CrossRef][ISI][Medline]
Fantone, J.C. and Ward, P.A. (1982) Role of oxygen-derived free radicals and metabolites in leukocyte-dependent inflammatory reactions. Am. J. Pathol., 107, 395418.[Medline]
Gibbs, F.P. and Vriend, J. (1981) The half-life of melatonin elimination from rat plasma. Endocrinology, 109, 17961798.[Abstract]
Golan, A., Maymon, R., Winograd, I. and Bukovsky, I. (1995) Prevention of post-surgical adhesion formation using aspirin in a rodent model: a preliminary report. Hum. Reprod., 10, 17971800.[Abstract]
Gutmann, J., Penzias, A.S. and Diamond, M.P. (1995) Adhesions in reproductive surgery. In Wallach, E.E. and Zacur, H.A. (eds) Reproductive Medicine and Surgery. Mosby, St. Louis, Missouri, pp. 681693.
Hara, M., Yoshida, M., Nishijima, H., Yokosuka, M., Ohtani-Kaneko, R., Shimada, A., Hasegawa, T., Akama, Y. and Hirata, K. (2001) Melatonin, a pineal secretory product with antioxidant properties, protects against cisplatin-induced nephrotoxicity in rats. J. Pineal Res., 30, 129138.[CrossRef][ISI][Medline]
Hellebrekers, B.W.J., Trimbos-Kemper T.C.M., Trimbos, J.B.M.Z., Emeis, J.J., Kooistra, T. (2000a) Use of fibrinolytic agents in the prevention of postoperative adhesion formation. Fertil. Steril., 74, 203212.[CrossRef][ISI][Medline]
Hellebrekers, B.W.J., Trimbos-Kemper, T.C.M., Bakkum, E.A., Trimbos, J.B.M.Z., Declerck, P.J., Kooistra, T. and Emeis, J.J. (2000b) Short term effect of surgical trauma on rat peritoneal fibrinolytic activity and its role in adhesion formation. Tromb. Haemost., 84, 876881.
Holtz, G. (1985) Current use of ancillary modalities for adhesion prevention. Fertil. Steril., 44, 174176.[ISI][Medline]
Kagoma, P., Burger, S.N., Seifter, E., Levenson, S.M. and Demetriou, A.A. (1985) The effects of vitamin E on experimentally induced peritoneal adhesions in mice. Arch. Surg., 120, 949951.[Abstract]
Kazez, A., Demirbaº, M., Ustundag, B., Ozercan, I.H. and Saglam, M. (2000) The role of melatonin in prevention of intestinal ischemia-reperfusion injury in rats. J. Pediatr. Surg., 35, 14441448.
Keckstein, J., Ulrich, U., Sasse, V., Roth, A., Tuttlies, F., Karageorgieva, E. (1996) Reduction of postoperative adhesion formation after laparoscopic ovarian cystectomy. Hum. Reprod., 11, 579582.[Abstract]
Lankoff, A., Banasik, A. and Nowak, M. (2002) Protective effect of melatonin against nodularin-induced oxidative stress. Arch. Toxicol., 76, 158165.[CrossRef][ISI][Medline]
Lay, K. and Arfors, K.E. (1982) Changes in macromolecular permeability by intravascular generation of oxygen-derived free radicals. Microvasc. Res., 24, 2533.[CrossRef][ISI][Medline]
Linsky, C.B., Diamond, M.P., Cunningham, T., Constantine, B., De-Cherney, A.H. and diZerega, G.S. (1987) Adhesion reduction in a rabbit uterine horn model using an absorbable barrier TC-7. J. Reprod. Med., 32, 1720.[ISI][Medline]
McCord, J.M. (1985) Oxygen derived free radicals in postischemic tissue injury. N. Engl. J. Med., 312, 159163.[Abstract]
Portz, D.M., Elkins, T.E., White, R., Warren, J., Adadevoh, S. and Randolph, J. (1991) Oxygen free radicals and pelvic adhesion formation: I. Blocking oxygen free radical toxicity to prevent adhesion formation in an endometriosis model. Int. J. Fertil., 36, 3942.[ISI][Medline]
Reiter, R.J. (1995) The role of the neurohormone melatonin as a buffer against macromolecular oxidative damage. Neurochem. Int., 27, 453460.[CrossRef][ISI][Medline]
Reiter, R.J., Tan, D.X., Cabrera, J., DArpa, D., Sainz, R.M. and Mayo, J.C. (1999) The oxidant/antioxidant network: Role of melatonin. Biol. Signal Recept., 8, 5663.[CrossRef][ISI]
Rodgers, K.E., Girgis, W., St Amand, K., Campeau, J. and diZerega, G.S. (1998) Reduction of adhesion formation in rabbits by intraperitoneal administration of lazaroid formulations. Hum. Reprod., 13, 24432451.[Abstract]
Sanfilippo, J.S., Booth, R.J. and Burns, C.D. (1995) Effects of vitamin E on adhesion formation. J. Reprod. Med., 40, 278282.[ISI][Medline]
Sanfilippo, J.S., Cox, J.G., Nealon, N.A. and Barrows, G.H. (1986) Comparison of corticosteroid therapy in the prevention of pelvic tissue reaction and adhesion formation. Int. J. Fertil., 30, 5761.[Medline]
Sener, G., Sehirli, A., Satiroglu, H., Keyer-Uysal, M. and Yegen, C.B. (2002) Melatonin improves oxidative organ damage in rat model of thermal injury. Burns, 28, 419.
Steinleitner, A., Lambert, H., Montoro, L., Kelly, E., Swanson, J. and Sueldo, C. (1988) The use of calcium channel blockade for the prevention of postoperative adhesion formation. Fertil. Steril., 50, 818821.[ISI][Medline]
Urman, B., Gomel, V. and Jetha, N. (1991) Effect of hyaluronic acid on postoperative intraperitoneal adhesion formation in the rat model. Fertil. Steril., 56, 563567.[ISI][Medline]
Vipond, M.N., Whawel, S.A., Thompson, J.N. and Dubley, H.A. (1990) Peritoneal fibrinolytic activity and intra-abdominal adhesions. Lancet, 335, 11201122.[CrossRef][ISI][Medline]
Submitted on January 24, 2003; accepted on May 7, 2003.